Anim

al Biodiversity and C

onservation, 38.1 2015

2015

and

AnimalBiodiversity Conservation38.1

2Editor en cap / Editor responsable / Editor in Chief Joan Carles Senar

Editors temàtics / Editores temáticos / Thematic EditorsEcologia / Ecología / Ecology: Mario Díaz (Asociación Española de Ecología Terrestre – AEET)Comportament / Comportamiento / Behaviour: Adolfo Cordero (Sociedad Española de Etología y Ecología Evolutiva – SEEEE)Biologia Evolutiva / Biología Evolutiva / Evolutionary Biology: Santiago Merino (Sociedad Española de Biología Evolutiva – SESBE)

Editors / Editores / EditorsPere Abelló Inst. de Ciències del Mar CMIMA–CSIC, Barcelona, SpainJavier Alba–Tercedor Univ. de Granada, Granada, SpainRussell Alpizar–Jara Univ. of Évora, Évora, PortugalMarco Apollonio Univ di Sassari, Sassari, ItalyXavier Bellés Inst. de Biología Evolutiva UPF–CSIC, Barcelona, SpainSalvador Carranza Inst. Biologia Evolutiva UPF–CSIC, Barcelona, SpainLuís Mª Carrascal Museo Nacional de Ciencias Naturales–CSIC, Madrid, SpainAdolfo Cordero Univ. de Vigo, Vigo, SpainMario Díaz Museo Nacional de Ciencias Naturales–CSIC, Madrid, SpainJosé A. Donazar Estación Biológica de Doñana–CSIC, Sevilla, SpainArnaud Faille Museum National histoire naturelle, Paris, FranceJordi Figuerola Estación Biológica de Doñana–CSIC, Sevilla, SpainGonzalo Giribet Museum of Comparative Zoology, Harvard Univ., Cambridge, USASusana González Univ. de la República–UdelaR, Montivideo, UruguaySidney F. Gouveia Univ. Federal de Sergipe, Sergipe, BrasilGary D. Grossman Univ. of Georgia, Athens, USAJacob Höglund Uppsala Univ., Uppsala, SwedenJoaquín Hortal Museo Nacional de Ciencias Naturales-CSIC, Madrid, SpainDamià Jaume IMEDEA–CSIC, Univ. de les Illes Balears, SpainJennifer A. Leonard Estación Biológica de Doñana-CSIC, Sevilla, SpainJordi Lleonart Inst. de Ciències del Mar CMIMA–CSIC, Barcelona, SpainJorge M. Lobo Museo Nacional de Ciencias Naturales–CSIC, Madrid, Spain Pablo J. López–González Univ. de Sevilla, Sevilla, SpainJose Martin Museo Nacional de Ciencias Naturales–CSIC, Madrid, SpainSantiago Merino Museo Nacional de Ciencias Naturales–CSIC, MadridJuan J. Negro Estación Biológica de Doñana–CSIC, Sevilla, SpainVicente M. Ortuño Univ. de Alcalá de Henares, Alcalá de Henares, SpainMiquel Palmer IMEDEA–CSIC, Univ. de les Illes Balears, SpainJavier Perez–Barberia Estación Biológica de Doñana–CSIC, Sevilla, SpainOscar Ramírez Inst. de Biologia Evolutiva UPF–CSIC, Barcelona, SpainMontserrat Ramón Inst. de Ciències del Mar CMIMA –CSIC, Barcelona, SpainIgnacio Ribera Inst. de Biología Evolutiva UPF–CSIC, Barcelona, SpainAlfredo Salvador Museo Nacional de Ciencias Naturales–CSIC, Madrid, SpainDiego San Mauro Univ. Complutense de Madrid, Madrid, SpainConstantí Stefanescu Museu de Ciències Naturals de Granollers, Granollers, SpainJosé L. Tellería Univ. Complutense de Madrid, Madrid, SpainFrancesc Uribe Museu de Ciències Naturals de Barcelona, Barcelona, SpainJosé Ramón Verdú CIBIO, Univ de Alicante, Alicante, SpainCarles Vilà Estación Biológica de Doñana–CSIC, Sevilla, SpainRafael Villafuerte Inst. de Estudios Sociales Avanzados (IESA–CSIC), Cordoba, SpainRafael Zardoya Museo Nacional de Ciencias Naturales–CSIC, Madrid, Spain

Secretària de Redacció / Secretaria de Redacción / Managing EditorMontserrat Ferrer

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Animal Biodiversity and Conservation 38.1, 2015© 2015 Museu de Ciències Naturals de Barcelona, Consorci format per l'Ajuntament de Barcelona i la Generalitat de CatalunyaAutoedició: Montserrat FerrerFotomecànica i impressió: Inspyrame PrintingISSN: 1578–665 XeISSN: 2014–928 XDipòsit legal: B. 5357–2013

AnimalBiodiversityandConservationes publica amb el suport de: l'Asociación Española de Ecología Terrestre, la Sociedad Española de Etología y Ecología Evolutiva i la Sociedad Española de Biología Evolutiva

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Dibuix de la coberta:Canismesomelas, xacal de llom negre, chacal de lomo negro, Black–backed jackal (Jordi Domènech)

1Animal Biodiversity and Conservation 38.1 (2015)

© 2015 Museu de Ciències Naturals de BarcelonaISSN: 1578–665 XeISSN: 2014–928 X

Ejigu, D., Bekele, A., Powell, L. & Lernould, J.–M., 2015. Habitat preference of the endangered Ethiopian walia ibex (Capra walie) in the Simien Mountains National Park, Ethiopia. Animal Biodiversity and Conservation, 38.1: 1–10.

AbstractHabitat preference of the endangered Ethiopian walia ibex (Capra walie) in the Simien Mountains National Park, Ethiopia.— Walia ibex (Capra walie) is an endangered and endemic species restricted to the Simien Mountains National Park, Ethiopia. Recent expansion of human populations and livestock grazing in the park has prompted concerns that the range and habitats used by walia ibex have changed. We performed observations of walia ibex, conducted pellet counts of walia ibex and livestock, and measured vegetation and classified habitat characteristics at sample points during wet and dry seasons from October 2009 to November 2011. We assessed the effect of habitat characteristics on the presence of pellets of walia ibex, and then used a spatial model to create a predictive map to determine areas of high potential to support walia ibex. Rocky and shrubby habitats were more preferred than herbaceous habitats. Pellet distribution indicated that livestock and walia ibex were not usually found at the same sample point (i.e. 70 of ua70 of uadrats with walia pellets were without livestock droppings; 73 of uadrats with livestock droppings did not have walia pellets). The best model to describe probability of presence of walia pellets included effects of herb cover (β = 0.047), shrub cover (β = 0.030), distance to cliff (β = –0.001), distance to road (β = 0.001), and altitude (β = 0.004). Walia ibexes have shifted to the eastern, steeper areas of the park, appearing to coincide with the occurrence of more intense, human–related activities in lowlands. Our study shows the complexities of managing areas that support human populations while also serving as a critical habitat for species of conservation concern.

Key words: Endemic, Ethiopia, Habitat preference, Simien Mountains, Walia Ibex

ResumenPreferencia de hábitat del íbice de Etiopía (Capra wallie), en peligro de extinción, en el Parque Nacional de las Montañas Simien, en Etiopía.— El íbice de Etiopía (Capra wallie) es una especie en peligro de extinción endémica del Parue Nacional de las Montañas Simien, en Etiopía. La reciente expansión de las poblaciones humanas y el pastoreo de ganado en el parue han suscitado preocupación por ue los límites y los hábitats utilizados por el íbice de Etiopía hayan cambiado. Se realizaron observaciones del íbice de Etiopía y conteos de excrementos de íbice y de ganado, asimismo, se describió la vegetación y se clasificaron las características del hábitat en los puntos muestrales durante las estaciones seca y húmeda, desde octubre de 2009 hasta noviembre de 2011. Se evaluó la influencia de las características del hábitat en la presencia de excrementos de íbice y posteriormente se utilizó un modelo espacial para crear un mapa predictivo de las zonas con mayor probabilidad de albergar a esta especie. Los hábitats preferidos fueron los rocosos y arbustivos en comparación con los herbáceos. La distribución de los excrementos indicaba ue el ganado y el íbice de Etiopía no solían encontrarse en el mismo punto muestral (el 70 de los cuadrados ue contenían excrementos de íbice carecían de defecaciones de ganado y el 73 de los cuadrados con defecaciones de ganado no contenían excrementos de íbice). El mejor modelo para describir la probabilidad de presencia del íbice tomaba en consideración el efecto de la cubierta herbácea (β = 0,047), la cubierta arbustiva (β = 0,030), la distancia a un acantilado (β = –0,001), la distancia a una carretera (β = 0,001) y la altitud (β = 0,004). Los íbices de Etiopía se han trasladado hacia

Habitat preference of the endangered Ethiopian walia ibex (Capra walie) in the Simien Mountains National Park, Ethiopia

D. Ejigu, A. Bekele, L. Powell & J.–M. Lernould

2 Ejigu et al.

las zonas más orientales y abruptas del parue, lo ue parece estar relacionado con la concentración de actividades humanas más intensas en las tierras bajas. Nuestro estudio pone de manifiesto la complejidad de gestionar zonas habitadas por poblaciones humanas y ue a la vez constituyen un hábitat fundamental para las especies en conservación.

Palabras clave: Endémico, Etiopía, Preferencia de hábitat, Montañas Simien, Íbice de Etiopía

Received: 30 IX 13; Conditional acceptance: 18 XII 13; Final acceptance: 13 I 15

Dessalegn Ejigu, Dept. of Biology, College of Sciences, Bahir Dar Univ., P. O. Box 79, Bahir Dar, Ethiopia.– Afework Bekele, Dept. of Zoology, College of Natural Sciences, Addis Ababa Univ., Ethiopia.– Larkin Powell, School of Natural Resources, Univ. of Nebraska–Lincoln, Lincoln, Nebraska, USA.– Jean–Marc Lernould, Conservation des Especesetdes Populations Animales, Schlierbach, France.

Corresponding author: Dessalegn Ejigu. E–mail: dessalegn_ejigu@yahoo.com

Animal Biodiversity and Conservation 38.1 (2015) 3

Introduction

Walia ibex (Capra walie Ruppell, 1835) is a species of conservation concern and one of the Palearctic ibex species in Ethiopia (Nievergelt, 1981; Last, 1982). Distribution of Caprinae has been influenced mainly by rapid environmental changes caused by glaciation (Geist, 1971). Simien Mountains National Park (SMNP) is the southern limit of the natural range of ibexes in the world and the only place where walia ibex occurs (Nievergelt, 1981; Gebremedhin et al., 2009). Walia ibex lives at higher altitudes and is adapted to partial forest life in the SMNP. Thus, it lives in areas with different habitats compared to other ibex species occurring in the other regions of the world (Nievergelt, 1981; Fiorenza, 1983; Yalden & Largen, 1992).

Ibexes, in general, prefer areas with steep slope and cliffs and avoid grasslands and flat hillsides (Feng et al., 2007). The presence of livestock usually has a negative effect on their relative abundance and distribution. Livestock act as a disturbance, and ibex retreat to less suitable habitats (Namgail, 2006; Pelayo et al., 2007). The behavioural responses are key to understanding animal–habitat interactions; the way individuals obtain food, seek shelter, escape from predators, find mates, and care for the young can provide clues to the effect of disturbances (Hickman et al., 1993).

Walia ibex has a restricted habitat, and its main distribution range is in the steep, rocky and topographically heterogeneous habitats of the mountains of Ethiopia (Nievergelt, 1981; Yalden & Largen, 1992).The walia ibex is an outstanding rock climber on steep cliffs, and it prefers to live in mountainous areas, sub–afroalpine grasslands, and areas with low vegetation cover (Last, 1982; Yalden & Largen, 1992; Hurni & Ludi, 2000).The distribution of walia ibex in the SMNP has shift ed towards the east since the 1970s, and intensified use of the park for livestock grazing has contributed significantly to such changes in walia ibex distribution (Hurni & Ludi, 2000). Low protection efficiency of wild life habitat is the main conservation problem in the park (Ludi, 2005).Thus, walia ibex prefer areas with little or no disturbances and occupy the most remote and inaccessible habitats (Hurni & Ludi, 2000). Simien Mountains National Park is heavily affected by livestock grazing, fuel wood collection and timber cutting, and crop cultivation (Hurni & Ludi, 2000; Ludi, 2005).

Habitat preference models for a species can be used effectively in their conservation and management (Krausman & Morrison, 2003; Doswald et al., 2007).Such models provide information to determine the species’ ecological niche through the relationship between observed species locations and habitat variables that restrict or drive their distribution (Hirzel & Le Lay, 2008). Factors such as competition, predation, human disturbances and the type of habitat patches can affect the species' habitat preference (Ottaviani et al., 2004; Rhodes et al., 2005). Habitat loss is a critical threat to most endangered species and the problem becomes significant in the SMNP where walia ibex occurs. Identification of suitable habitats is an essential step to ensure sustainable conservation of species such as walia ibex (Huettmann & Calgary, 2003; Jean Desbiez et al., 2009).

Unless resources are abundant, two populations cannot occupy the same niche at the same place and time (Hardin, 1960). Some degree of competition can occur in natural populations (Namgail, 2006). Thus, negative interactions will increase the extinction probabilities of a species and result in population size reduction (Hickman et al., 1993). A similar scenario can also occur in SMNP, where the original habitats of walia ibex, especially in the lowlands, have been occupied by livestock. Thus, the walia ibex population is confined to relatively inaccessible areas within gorges and escarpments towards the eastern part of the park (Hurni & Ludi, 2000).

The goal of our research was to determine areas of potential habitats for walia ibex in the SMNP to support sustainable conservation and management plans. Our specific objectives were to: (1) use presence data based on direct observations to describe habitat used by walia ibex; (2) use presence/absence data from pellet counts to assess and compare habitat useand preference of walia ibex and livestock; and (3) develop a descriptive, spatial model of habitat preference to highlight areas of SMNP that are critical for protection and management of walia ibex.

Material and methods

Study area

Simien Mountains National Park is located in the Amha located in the Amhara National Regional State within the North Gondar Administrative Zone (UTM 376047 E to 444522 E, and 1458552 N to 1467230 N; fig. 1). The SMNP is composed of a broad undulating plateau and the highest point is Ras Dejen (altitude: 4,543 m). The park is known for its impressive escarpments (Nievergelt, 1981). The SMNP borders were established in 1966 (Hurni & Ludi, 2000; ANRSPDPA [Amhara National Regional State Park Development and Protection Authority], 2009). UNESCO declared SMNP as a World Heritage Site in 1978 based on its importance as a refuge for rare and endemic animals and plants, as well as its exceptional natural beauty (Yalden & Largen, 1992; Hurni & Ludi, 2000; Puff & Nemomissa, 2001; Debonnetet al., 2006). However, regulations adopted during the park’s establishment allowed livestock grazing, agriculture, and human settlement in 80 of the park (Debonnet et al., 2006). The total area of the park is 412 km2, and walia ibex habitats (mountainous regions and sub–afroalpine grasslands) are restricted to 94.1 km2.

From 2000 to 2009, the mean annual rainfall at SMNP was 1,054 mm. The mean annual minimum and maximum temperatures were 8.7oC and 19.9oC, respectively (National Meteorological Agency, Addis Ababa, Ethiopia: http://www.ethiomet.gov.et/). Seasonal differences in temperature are minimal due to Ethiopia's proximity to the euator (Nievergelt, 1998).

The Simien Mountains form a contact zone between the Palearctic region in the north and the Ethiopian region in the south. This makes the flora and fauna of the area representative of both regions (Nievergelt, 1981). The mountain’s geographical position and the presence of

4 Ejigu et al.

altitudinal belts as well as different topographic features in the SMNP results in a mosaic pattern of different habitats that can promote species diversity and richness (Puff & Nemomissa, 2001). The altitudinal variations in the SMNP can determine variations in the natural vegetation. The vegetation of the park consists mainly of Erica arborea, Lobelia rhynchopetallum, Hypericumrevolutum, Helichrysum spp., Rosa abyssinica and Solanum spp.

Endemic large mammals include walia ibex, Ethiopian wolf (Caniss imensis) and gelada baboon (Theropithecus gelada). The distribution of walia ibex in SMNP extends from Buyetras in the western parts of the park to the southeastern end of Sebatminch, which is 94.1 km2 of the area. The density of walia ibex in its current range is 7.99 individuals/km2, and counts of walia ibex during the last ten years (2002–2011) have indicated a gradual increase (ANRSPDPA, 2009).

Uncontrolled human use of natural resources in the park is the greatest threat to biodiversity. More than 75 of the SMNP is used by local human communities for grazing, agriculture and settlements, leaving only the highest peaks and inaccessible cliffs relatively undisturbed (Hurni & Ludi, 2000; Ludi, 2005). Barley is the main crop type in the area, and livestock species grazing in the park are cattle (7.49 individuals/km2), sheep, goat and euine.

Data collection

We conducted our study of walia ibex at SMNP over 15 days every second month from October 2009 to November 2011. Our observations covered wet and dry seasons (wet: May–October; dry: November–April) and all hours of daylight. Data were collected by the primary investigator and two well–trained field assistants. Our study design was affected by the logistic hurdles presented by the rough terrain and remote locations of SMNP. First, we used a series of transects through portions of the park deemed most likely to contain walia ibex, as judged by anecdotal evidence and/or habitat characteristics. We followed transects to locate and observe walia ibex herds to document the sizes of groups and assess the habitats in which walia ibex were found. We used GPS to record the location and habitat classifications of individuals or herds, and morphological features —particularly horn shape of males, uniue skin colour of some groups of individuals, and deformities such as swelling belly or broken horn— were used to identify the herd. We identified the topography (open plateau, bushy plateau, top of plateau, escarpment, or gorge/cave), and we visually scored the density of vegetation (sparse: < 25 cover, moderate: 25–50, dense: 51–75, very dense: > 75) in the area of each individual or herd.

Second, we used randomly distributed, systematic transect surveys to describe habitat that was available for walia ibex. We established a total of 637 (319 during wet and 318 during dry seasons) uadrats to characterize vegetation along transects at 200 m intervals. We separated the parallel transects by 500 m in areas that allowed multiple transects (e.g., plains and plateaus); we used single transects in gorges and escarpments and the direction of the transect was constrained by

topography. Quadrats were suare, and the boundaries of the 400 m2 were marked by rope while we collected data. We visited each transect during wet and dry seasons, and each transect was visited every other month for two consecutive years. We determined the aspect and slope of each uadrat using a Clinometer (Gillen et al.,1984); we recorded the slope at each corner of the uadrat and used the mean of the four samples to represent the slope of the uadrat. The availability of water and food influences the distribution of animals (Kauffman & Krueger, 1984; Knight et al., 1988); thus, we visually estimated the distance from each uadrat to a cliff, water source, and nearest road and settlement (Rondinini et al., 2004). Ground cover (grass, herbs, shrubs, trees, rocks) was described as the percent cover of the uadrat.

We also used the vegetation transects and uadrats as sample locations to conduct observations of pellets of walia ibex and livestock. We identified pellets within each uadrat. Walia pellets were identified from livestock pellets (goat or sheep) based on their colour, shape and size. Sutherland (1996) suggested that pellet counts are the best indication of animal abundance when species are not easily observed. We used the presence of pellets rather than the number of pellets in our analysis, thus avoiding any problems (e.g., unknown defecation rates or decay rates) that may arise from trying to determine relative abundance from pellet counts.

Statistical analyses

We used Chi–suare (x²), independent sample t– tests and means of samples to compare distribution between wet and dry seasons. We used α = 0.05 for statistical significance, and data were analysed using SPSS software version 16.0.

Habitat preference is the measure of a species’ disproportional use, relative to availability (Krausman, 1999). To evaluate habitat preference, we used two methods to compare levels of habitat use (locations of sightings or pellets) and habitat availability (uadrats, as representative of SMNP) to assess habitat preference of walia ibex following Harrett (1982) and Steinein et al. (2005). First, we compared the vegetation cover of uadrats in which walia ibex pellets were found with the cover of uadrats without pellets. Second, we used data from pellet surveys to describe the potential of habitat characteristics to predict the probability (with the ranges of 0–1) of detecting walia pellets in a uadrat. We summarized the pellet counts at each sample point as presence/absence data (success: pellet found, failure: pellet not found) for use in logistic regression models that predicted the probability (Ψ) of detecting walia pellets in a uadrat:

Ψ(pellet) = 1 / (1+ e–z )

where z is a linear model with intercept ß0 and factors ß1–ßn:

Z = ß0 + ß1(x1) + ß2(x2) + ß3(x3) + … + ßn(xn)

We determined the best linear model to describe the probability of detecting walia pellets by a priori

Animal Biodiversity and Conservation 38.1 (2015) 5

describing 11 potential habitat variables: ground cover (grass cover, herb cover, shrub cover, tree cover and rock cover), altitude, slope, and distance from cliffs, water sources, roads and settlements. We used a backwards stepwise variable removal procedure to form the final model from the initial 11 variables. Prior to the analyses, we assessed colinearity (r > 0.6) among the variables under consideration.

We then applied the predictive model to our spatial data from the uadrats to visually determine areas of SMNP that had a high probability of walia presence. Quadrat–specific characteristics were then applied to the final regression model to calculate Ψ at each uadrat and characterize the probability of pellets of walia ibex across the study site. We assumed that areas of high probability of presence of pellets were also areas of high habitat preference; conversely, areas with a low probability of presence of pellets were areas of low habitat preference for walia ibex within their range at SMNP (Conroy, 1996). Finally, we used the ordinary kriging method with a spherical

semi–variogram model in ArcMap (ESRI, Redlands, CA, USA) to spatially extrapolate habitat preference of walia ibex across points not sampled at SMNP. We displayed the probability of presence of pellets in the park as a gradient from low to high probability of presence as adopted by Rondinini et al. (2004).

Results

Herd size was variable (range 1–32 ibex per observation) and increased by approximately seven ibex per observation as altitude increased from our lowest observation (3,543 m) to our highest (4,361 m; F1,261 = 9.9, slope = 0.007, P < 0.01, r2 = 0.04, n = 263).Similarly, we observed more walia pellets in uadrats at higher altitudes (F1,381 = 19.9, slope = 0.003, P < 0.0001, r2 = 0.05) than uadrats at lower altitudes.

Most of our 267 independent observations (wet season: 132, dry season: 135) of walia ibexes were in open plateaus (42) and escarpments (32; fig. 2).

Ethiopia

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Fig. 1. The location of Simien Mountains National Park in Ethiopia, and Park boundary with associated towns, local campsites, and roads.

Fig. 1. La ubicación del Parque Nacional de las Montañas Simien en Etiopía y los límites del parque con las poblaciones, campamentos y carreteras cercanas.

6 Ejigu et al.

Use of open plateaus, bushy plataus, and gorges was higher during wet season, and the use of escarpments and tops of plateaus was higher in the dry season (x² = 10.1, df = 4, P = 0.03). More observations of walia ibex tended to be from areas with dense vegetation cover (34.2 of observations) than other vegetation densities (sparse: 26.7, moderate: 21.8, very dense: 17.3), and these observations did not vary by season (x² = 5.94, df = 3, P = 0.11).

We collected samples from 319 and 318 uadrats during wet and dry seasons, respectively. Most of our uadrats (323, 50.7) had west–facing aspects (north: 103, 16.2; south: 94, 14.8; east: 75, 11.8; southwest: 16, 2.5; northwest: 15, 2.4; southeast: 7, 1.1; northeast: 4, 0.6). The mean slope at our uadrats was 23.3º (SE = 3.0) or 43.1 slope. Walia ibex and livestock pellets were not typically found in the same uadrat. In fact, 70 of uadrats with walia pellets had no livestock droppings, and 73 of uadrats with livestock droppings had no walia pellets.

Our uadrat samples were dominated by grass and rock cover. Quadrats with walia ibex pellets had less grass and trees but more rocks and shrubs than uadrats without pellets. In contrast, uadrats with livestock pellets had less grass and rocks and more trees than uadrats without livestock pellets (fig. 3). The only difference in habitat use between wet and dry seasons was that uadrats with pellets of walia ibex had 28.7 of rock cover in the wet season but only 14.4 in the dry season. Livestock habitat use varied

only in tree cover; uadrats with livestock pellets had 13.2 tree cover during the wet season and 9.1 during the dry season.

Prior to the regression analysis, we removed 'tree' from the variable set, as altitude and tree were correlated (r = 0.64, P < 0.001). No other variable pairs were correlated above the level r = 0.6. The logistic regression analysis suggested that the habitat characteristics of herb cover, shrub cover, altitude, and distance of the sampled habitat from cliffs and roads were the best factors to describe the probability of walia pellets (Ψ(pellet)). The values of the associated intercept and regression coefficients were: β0 = –15.991, βherb = 0.047, βshrub = 0.030, βdist to cliff = –0.001, βdist to road = 0.001, βaltitude = 0.004. The probability of presence of pellets ranged from 0.17 to 0.93 throughout the park. The spatial descriptive map indicated that the portion of the park with the highest probability of presence of walia ibex (as measured by pellets) occurred from Chenek–Buahit to Mesareri towards the eastern portion of the park (fig. 4).

Discussion

Walia ibex and livestock in SMNP

Ludi (2005) documented that walia habitats within SMNP had been affected by the increase of the human population within and around the park, which had

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Fig. 2. Percentage of observations of walia ibex individuals or herds according to habitat type during wet and dry seasons at Simien Mountains National Park, Ethiopia, during 2009–2011.

Fig. 2. Porcentaje de observaciones de individuos o rebaños de íbice de Etiopía según el tipo de hábitat durante las estaciones seca y húmeda en el Parque Nacional de las Montañas Simien, en Etiopía, entre los años 2009 y 2011.

Animal Biodiversity and Conservation 38.1 (2015) 7

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resulted in heavier pressure from grazing of livestock. We found that herd size of walia ibex increased with altitude. Our data suggest that walia ibex use shrubby areas of open plateaus extensively and use rocky escarpments preferentially. Nievergelt (1981) reported that walia ibex prefer rocky terrains with no human related disturbances.

We suggest that walia ibex are responding to the impacts documented by Ludi (2005) by moving farther from human populations and into habitats that are less likely to overlap with livestock grazing. Indeed, we found that pellets of walia ibex and livestock did not tend to be found in the same sampling uadrat, which may indicate ibex are avoiding livestock areas. As expected, livestock in the SMNP tend to use areas with more grass cover. Many species of wildlife are threatened by intensification of agriculture and overgrazing of livestock on their habitats (Jean Desbiez et al., 2009).

Hurni & Ludi (2000) also suggested that severe human–related disturbance at lowlands force the movements of walia ibex towards inaccessible habitats. Currently, the distribution of walia ibex is towards Sebatminch in the eastern portion of the park (fig. 1), which has more highlands available. Walia were previously found further west in the park, and more forage plants are available in the lowlands. But human disturbance and livestock grazing at lowlands, especially in the Gich area (fig. 1), may have contributed to displacement of walia ibex to the east and to the highest, steepest areas of the park (Hurni & Ludi, 2000). Our study was not designed to determine the relative uality of habitats for walia ibex, but we encourage biologists to consider the possibility that presence of livestock in former ibex range within the park has forced walia ibex to select habitats of lesser uality (Pelayo et al., 2007). We concur with

Fig. 3. Mean (95 confidence interval) percentage of types of ground cover in uadrats with and without pellets of livestock (A) and walia ibex (B) in Simien Mountains National Park, Ethiopia, during 2009–2011.

Fig. 3. Porcentaje medio (95 % intervalo de confianza) de los tipos de cubierta vegetal en los cuadrados con y sin excrementos de ganado (A) y de íbice de Etiopía (B) en el Parque Nacional de las Montañas Simien, en Etiopía, entre los años 2009 y 2011.

A

B

8 Ejigu et al.

0 1 2 4 6 8 km

Relative preference High Moderate Low

N

previous suggestions that assessment of habitat preference for species can provide critical information for problems in conservation and management of a species (Morris, 2003).

As most mammals are highly selective in their habitats, degradation of habitat by domestic animals is a common problem (Jean Desbiez et al., 2009). Human activities interfere with animal distribution and anticipate access to critical habitat change (Williamson et al., 1988), and the animal’s behavioural response to the presence of humans has often been used as an indication to its susceptibility to disturbance (Beale & Monaghan, 2004). As described by Brown (1971), coexistence between pastoralists and wildlife reuires the maintenance of a very low–density of livestock and human populations. However, it is not achievable in developing countries as the local people raise a large number of livestock and practise free grazing; such is the case in the SMNP.

Seasonal trends of habitat use and preference

Identification of potential suitable habitats for wildlife is an essential step to insure sustainable conservation of

species (Huettman & Calgary, 2003; Jean Desbiez et al., 2009). Human activity and wildlife food availability vary in space and uantity at the SMNP between wet and dry seasons. We observed only minor differences in the topography used by walia ibex during wet and dry seasons (fig. 2). However, these minor shifts may reveal important factors in the ecology of walia ibex. Regardless of season, ibex were found extensively in open plateaus. Nievergelt (1981) also reported that open areas with L. rhyncopethalum and Festucama-crophylla were used by ibex. We found that ibex were more often found on bushy plateaus during the wet season than the dry season. This may be because herbaceous plants such as Thymus, Alchemillarothi and Simeniaacaulis, the principal diet for walia ibex, become available in the bushy plateau during the wet season.

Similarly, we found that slopes and troughs had Alchemilla rothi, Arabis alpine, Simenia acaulis and Festuca macrophylla, all of which are used by walia ibex (Nievergelt, 1981). During the dry season, the abundance of herbs decreases. Thus, walia ibexes may have been forced to switch their diet to other plant species,

Fig. 4. Spatial representation of relative preference of walia ibex within Simien Mountains National Park (SMNP), Ethiopia, defined as the probability of finding a walia ibex pellet at a sample point. The predictive model was applied, a posteriori, to sample locations (dots) and then extrapolated from the sample points to the portion of SMNP in which samples were collected during 2009–2011. Dark areas indicate a higher preference for sample locations and landscape.

Fig. 4. Representación espacial de la preferencia relativa del íbice de Etiopía en el Parque Nacional de las Montañas Simien, en Etiopía, definida como la probabilidad de encontrar excrementos de íbice de Etiopía en un determinado punto muestral. El modelo predictivo se aplicó, a posteriori, a los lugares muestreados (puntos) y después se extrapoló a la porción del Parque en las que se recogieron muestras durante 2009 y 2011. Las áreas oscuras indican una preferencia alta para los lugares muestreados.

Animal Biodiversity and Conservation 38.1 (2015) 9

causing them to move to escarpments and the top of the plateau. Indeed, herds of walia ibex were observed in less dense areas during the dry season.

Livestock pellets were found in areas with more tree cover during the wet season than during the dry season. We observed that livestock were restricted to tree–covered areas during the wet season because crops were grown in open areas. After harvesting, livestock were able to range into open fields to forage on leftover crops.

Predictive maps for conservation planning

We used pellet counts to survey habitat use of walia ibex, allowing us to assess vast spatial areas of the park at lower costs than radio–telemetry or other individual–based methods of habitat assessment. Walia ibex in the SMNP are a small population, and individuals are difficult to detect and capture. Furthermore, to determine whether they had used a specific sample location, pellet counts were more reliable than visual surveys of animals because individuals are constantly moving to new locations; detection of use at a given sample point would therefore be very low (Gu & Swihart, 2004). Pellet counts were more cost–effective and more logistically realistic than remote cameras. We designed our pellet survey to allow us to consider spatial processes, which would have been much more difficult logistically to conduct with direct observation of animals (Rhodes et al., 2005; Skarin, 2006). Conseuently, we suggest that biologists consider pellet counts for similar assessments of habitat use and preference in remote areas that present logistical difficulties.

Walia ibex were previously described to be found in areas of the SMNP with altitudes of 2,800 to 3,400 m a.s.l. (Nievergelt, 1981). However, we found walia ibex to commonly occur at altitudes about 4,000 m a.s.l. and that their habitats have been shifted towards the eastern part of the park. Our predictive model shows the potential spatial extent of the park that could be defined as providing the remaining suitable habitat for walia ibex, and we believe this is a key piece of information in the process to establish a conservation plan for the species (Owen, 2009). We encourage biologists and managers of SMNP to consider further collection of demographic information, such as breeding success and survival. Such information can further refine and target management efforts, especially if demographic patterns can be related to habitat use (Peek, 1986).

The SMNP is a complex system that includes sensitive wildlife species and human activities, and management decisions to support the conservation of species of wildlife such as walia ibex are also complex. Our surveys of habitat use and preference serve to document recent changes in the range of walia ibex. Indeed, much of the process of habitat selection for a species may depend upon several limiting factors (Peek, 1986; Ottaviani et al., 2004).The disturbances within the park caused by human activities could be minimized by identifying zones to allow tourism, human use, and protection of critical biodiversity. Information provided by habitat use and selection data should enable more informed decisions about the conservation status of walia ibex and help to ensure its long–term survival.

Acknowledgements

We thank the field assistants and scouts in the Simien Mountains National Park for their help during data collection. D. Ejigu was a Visiting Scholar at the School of Natural Resources at the University of Nebraska–Lincoln, and L. Powell’s contributions were supported by Hatch Act funds through the University of Nebraska Agricultural Research Division, Lincoln, Nebraska. The authors are greatly indebted to Addis Ababa University Postgraduate Program Office, CEPA, the Mohamed bin Zayed Species Conservation Fund, Chicago Zoological Society and Lleida University for funding. The authors are greatly indebted to the anonymous reviewers for their constructive comments and suggestions while reviewing the manuscript.

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Rocha, G. & Quillfeldt, P., 2015. Effect of supplementary food on age ratios of European turtle doves (Streptopelia turtur L.). Animal Biodiversity and Conservation, 38.1: 11–21.

AbstractEffect of supplementary food on age ratios of European turtle doves (Streptopelia turtur L.).— Many farmland birds have difficulties finding sufficient food in intensely managed agricultural ecosystems, and in more extensively worked landscapes they are often attracted to human–induced dietary sources. European turtle doves Strepto-pelia turtur feed on seeds collected on the ground, and are readily attracted to supplementary provided grain at feeding stations. Supplementary feeding is a common management practice on hunting estates around the world. This study was conducted in 40 hunting estates located in central west Spain: 20 sites where supplementary food was provided to attract turtle doves and 20 control sites without feeding stations. At sites with supplemen-tal feeding, the field age ratio was 20% higher and the hunted age ratio was 33% higher than at control sites, indicating a positive effect of the food supplementation of the breeding success around supplemented sites. Both the amount of food provided per day and the amount of time where supplemental food was given (20–120 days) were positively correlated with the field age ratio and, less strongly, with the hunted age ratio. These data suggest that providing extra food can increase the breeding success of this species when the amount provided is sufficiently large and when supplementary food is provided early in the breeding season. However, hunting pressure was also higher at supplemented sites. Future studies should therefore closely monitor the positive and negative effects in order to ascertain which management practices will ensure the viability of these important European turtle dove populations.

Key words: Age–ratio, Hunting, Management, Streptopelia turtur, Food supplementation

ResumenEfecto de la alimentación complementaria en la razón de edad de la tórtola europea (Streptopelia turtur L.).— Son numerosas las aves de los hábitats agrícolas que tienen dificultades para encontrar suficiente alimento en los ecosistemas agrícolas intensivos y que, en los hábitats explotados de forma más extensiva, suelen ser atraídas por las fuentes antrópicas de alimento. La tórtola europea, Streptopelia turtur, se alimenta de semillas que se hallan en el suelo y es atraída inmediatamente por los cereales que se aportan como complemento a los comederos. El aporte complementario de alimento es una práctica habitual en la gestión de los cotos de caza de todo el mundo. Este estudio se realizó en 40 cotos de caza ubicados en el centro y el oeste de España: 20 zonas en las que se aportó alimentación complementaria para atraer a las tórtolas y 20 zonas de control sin comederos. En las zonas con alimentación complementaria, las razones de edad en el campo y en las aves cazadas fueron, respectivamente, un 20% y un 33% más elevadas que en las zonas de control, lo que indica que la alimentación complementaria tiene un efecto positivo en el éxito reproductivo en torno a las zonas con aporte de alimento complementario. Tanto la cantidad de alimento suministrado por día como el período en el que se aportó (20-120 días) se correlacionaron positivamente con la razón de edad en el campo y, con menos intensidad, con la razón de edad en las aves cazadas. Estos datos sugieren que el suministro de alimento extra puede aumentar el éxito reproductivo de esta especie si la cantidad aportada es suficientemente abundante y si se empieza a proporcionar a principios de la temporada de cría. No obstante, la presión cinegética también fue mayor en las zonas con aporte de alimento complementario, por lo que sería necesario analizar minuciosamente los efectos positivos y negativos de dicho aporte con vistas a determinar qué prácticas de gestión garantizarán la viabilidad de estas importantes poblaciones de tórtola europea.

Effect of supplementary food on age ratios of European turtle doves (Streptopelia turtur L.)

G. Rocha & P. Quillfeldt

12 Rocha & Quillfeldt

Palabras clave: Razón de edad, Caza, Gestión, Streptopelia turtur, Alimento complementario

Received: 24 VI 14; Conditional acceptance: 17 X 14; Final acceptance: 18 I 15

Gregorio Rocha, Dept. of Agro–forestry Engineering, Univ. of Extremadura, Avda. Virgen del Puerto 2, 10600 Plasencia, Cáceres, Spain.– Petra Quillfeldt, Inst. für Tierökologie und Spezielle Zoologie, Justus Liebig Univ. Gießen, Heinrich–Buff–Ring 38, 35392 Gießen (Germany).

Corresponding author: Gregorio Rocha. E–mail: gregorio@unex.es

Animal Biodiversity and Conservation 38.1 (2015) 13

Introduction

The availability of food is a principal parameter deter-mining breeding success in animals. The lack of an adequate food supply often leads to birds abandoning their brood, brood reduction, and poorer condition of offspring (Martin, 1987). Food supplementation there-fore has consistent effects on parameters of breeding success in birds, such as earlier laying, larger clutch size, and accelerated population growth (reviews by Boutin, 1990; Schoech & Hahn, 2007). The use of supplemental feeding as a positive tool in species conservation has been consistently recommended (Schoech et al., 2008), although some unwanted effects may appear (Martínez–Abrain & Oro, 2013). In the case of some gallinaceous game species in Europe (pheasant Phasianus colchicus and quail Coturnix coturnix), supplementary feeding at hunting estates can influence the proportion of juveniles to adults, through an increase in reproductive success (Draycott et al., 2005; Díaz–Fernández et al., 2013). However, this practice may be negative if the hunting bag is not thoroughly controlled, because it can increase the hunting pressure excessively (Rocha & Hidalgo, 2001).

Although the distribution area of European turtle doves Streptopelia turtur is wide, they are restricted to warm, lowland areas, which are often agricultural areas (Cramp, 1985). In recent decades, the European turtle dove has experienced a widespread decrease both in population density and in its area of distri-bution (Tucker & Heath, 1994; Jarry, 1997; Browne et al., 2005). This decline has led to its inclusion as Vulnerable in the Red Book of Vertebrates of Spain (Blanco & González, 1992; Madroño et al., 2004). As a result of this, and being a hunted species in an unfavorable demographic situation, this species is the subject of a management plan by the European Commission (Boutin, 2001; Lutz & Jensen, 2007). On the Iberian peninsula, the breeding population has declined significantly, by 29.3% between 1998 and 2012 (SEO/BirdLife, 2013), and this decline has been particularly marked since 2008.

European turtle doves feed primarily on the seeds of weeds (Murton et al., 1964; Dias & Fontoura, 1996), especially at the start of the breeding season when seeds of cultivated plants are not yet available (Jiménez et al., 1992). Some studies suggested that late in the breeding season cereal seeds become available and then play a larger role (Jimenez et al., 1992; Browne & Aebischer, 2003).

On the Iberian peninsula, the European turtle dove is hunted from the second half of August to the first half of September, i.e. the late breeding season and the post–breeding migration. The hunting of this species is carried out using the 'fixed location' method, which takes advantage of birds passing to feeding areas such as crops and natural pastures, where they are shot at by a row of hunters. The European turtle dove plays an important role in Extremadura, where it is considered one of the main game bird species of this hunting season (Hidalgo & Rocha, 2001). However, there are as yet no studies on the economic value of this activity.

Many hunters and estate managers use supplemen-tary food to attract and concentrate the birds (Rocha & Hidalgo, 2001). Such supplementation consists of seed mixes of various oleaginous or leguminous cereals scattered throughout the crops or natural pastures. The feeding stations usually occupy an area of between 0.2 and 5.0 hectares, although they can be larger. Currently, over 70% of the estates that hunt European turtle doves during August–September in Extremadura operate this kind of hunting management (Rocha, own data).

Food supplementation on the hunting estates can start 1 to 4 months before the hunting season (Rocha, own data). At sites with early supplementation, this co-vers most of the breeding season of the European turtle dove. Thus, food supplementation could influence the population dynamics of this species during the breeding season, including changes in abundance, breeding success, feeding ecology and migratory phenology.

It is known that a greater amount of food available on the estates attracts the European turtle dove since they are killed in greater numbers when extra food is provided (Rocha & Hidalgo, 2002). However, it is unknown to what extent supplemental food affects the productivity of the populations.

The main objectives of this work were to summarize data on quantity and duration of the food supplemen-tation at hunting estates and to study how this game management practice influences reproductive success. Specifically, we tested whether the breeding success of the European turtle doves was influenced by the supplemental feeding at hunting estates by comparing the age ratio of populations of post–breeding aggrega-tions in the second half of August. Observations of age ratios are a widely used method to estimate breeding success in birds (e.g., Wagner & Stokes, 1968; Newton, 2001; Flanders–Wanner et al., 2004; Peery et al., 2007). Although observations of age ratios in the field do not provide a direct measure of productivity, they have proven to be a useful technique because they are relatively easy to apply, yet they avoid time–consuming and potentially harmful nest searches.

Methods

Study species

The European turtle dove is a migratory species that winters in the African Sahel and breeds in large parts of Europe, Asia and North Africa (Cramp, 1985).

In the Iberian peninsula, its main habitats are the areas populated by holm oaks (Quercus ilex L.) and cereal cultivation, where they present densities of some 2.3 birds/10 ha (Muñoz–Cobo, 2001). In the central and western part of the Iberian peninsula they nest among small to medium sized oaks, in a mix of natural pasture and cultivated habitats (Peiró, 1990; Rocha & Hidalgo, 2002). In these areas, they can rea-ch densities of up to 10.5/10 ha (Santamaría, 2007). They also inhabit open areas with scattered trees and shrubs, riverine forests and orchards, and they are very scarce in coniferous woodlands, scrubland and all across the thermo–Mediterranean (Díaz et al.,

14 Rocha & Quillfeldt

1996). The study area has traditionally been one of the principal nesting territories of the European turtle dove in Spain (Rocha & Hidalgo, 2002).

Study sites

The study area is located in the region of Extrema-dura, in the central–western Iberian peninsula (fig. 1). This region has a rich biodiversity. More than 30% of the territory is included in the Natura 2000 ecological network (Fernández, 2004; Junta de Extremadura, 2012) and 80% of the territory is subject to hunting rights (Lázaro, 2004).

The predominant habitat on the hunting estates is the dehesa: open managed parkland used for livestock grazing within a savanna–like woodland of evergreen Quercus trees, mainly Q. ilex (holm oak) and Q. suber (cork oak). The dehesa is intermixed with cropland and Mediterranean woodland and scrub (Díaz et al., 1997).

The use of feeding stations for hunting had been banned in Extremadura since 2007 (included) (Junta de Extremadura, 1991, 2007, 2008), but not in the surrounding regions. Despite being banned, 62% of estate managers where hunting takes place added supplementary food during the season 2004/2005 (Hidalgo & Rocha, 2006). This hunting management is frequently used because it increases the number of birds hunted (Hidalgo & Rocha, 2001).

During the spring and summer of 2009, 40 hunting estates with dehesa habitats were selected throughout the region where the European turtle dove has been hunted traditionally. These estates were split in two groups according to the provision or not of supple-mental food. In the first group, hunting management involved consistently adding supplementary food year after year, dispersed throughout the crops and natural pastures (group with extra food added). Supplemental food was composed of mixtures of crop seeds: wheat (Triticum aestivum L.), sunflower (Helianthus annuus L.) and vetch (Vicia sativa L.). The estates with feeding stations always provided the same amount of food every year, and for the same duration of time. In the control group, no supplementary food was added to the environment and the birds fed on seeds from crops and natural pastureland (group without extra food added).

The size of the estates did not differ between supple-mented and control sites (mean ± sd: 661.5 ± 132.9 ha; t–test: t38 = –0.84, P = 0.41). The mean number of hunters per estate (± sd) was 11.8 ± 2.6, and did not differ between supplemented and control sites (t–test: t38 = –0.71, P = 0.48). There are no data on movements of turtle doves in Spain, but in two English populations, the mean foraging distances for radio–tagged doves were 450 m and 1,400 m (Browne & Aebischer, 2003). The sites in our study area were over 10 km distant from each other (fig. 1). We thus assume that the population recorded at each site was mainly local.

Data collection

The following data were collected at supplemented sites: the amount of food added (in kilos) and the duration (in days) of the food supplementation until

the beginning of the hunt. These data were freely provided by estate staff in charge of the food supple-mentation. Age ratios were estimated as an indicator of the breeding success by counting the number of young birds observed after the breeding season (in the second half of August), compared to the total number of adults (young/adults), just before the beginning of the migratory season (Cramp, 1985).

Post–breeding age ratios are a common tool to estimate the reproductive productivity in monitoring programmes. Age ratios can be obtained easily and economically, while avoiding biases due to disturbance at the nest in sensitive species such as turtle doves. In the present study, age ratios were measured using two different methods based on the proportion of young to adult birds among live birds observed in the field (termed 'field age ratio') and birds killed during the hunt (termed 'hunted age ratio'), respectively.

The field age ratio was obtained by observation, from a hidden fixed position, of live birds perched, where the difference between young and adult birds could be directly ascertained as they gathered in post–reproduc-tion groups or 'aggregates'. Field age ratios have been successfully used in work on this species in Andalucía (SEO/BirdLife, 2002). Field age ratios are important parameters when trapping methods influence the age ratio sample of the population (e.g., Domènech & Senar, 1997). Hunted age ratios of turtle doves were determined in Morocco, where they were found to depend largely on reproductive productivity and hunting pressure (Hanane, 2009). To determine the 'field age ratio', we conducted observations once in each estate, from between 2 and 10 days before the hunt started. Post–reproduction groups were observed and counted in all 20 supplemented sites. However, in the control group, data were obtained from 11 of the 20 sites because post–reproduction groups were not located in the remaining nine estates. On the 11 estates, post–reproduction groups were found at feeding sites such as harvested fields, where food can be found but is not as concentrated as at supplemented sites. On observation days, one person per estate occupied an observation point (hidden) before the birds began to arrive (at dawn). This point was located near the feeding area, so that the birds were seen at a distance of 20–40 m. The observer remained there for 30 min recording the birds that came with a telescope 20–40 x, differentiating young and adult birds by identifying the presence or absence of the 'collar'. The 'collar' is a dis-tinctive feature of the neck plumage of adult European turtle doves. It is composed of feathers that form black and white bands (Cramp 1985; Sáenz de Buruaga et al., 2001). Young European turtle doves usually have no collar, although some juveniles birds hatched from early broods may have a partial collar. Thus, birds with partial collars were always considered juveniles. Double counting was avoided by using the data of the birds recorded in the peak of simultaneous concentration. A total of 982 birds were observed (773 at supplemented sites and 209 at control sites).

The 'hunted age ratio' was determined at supple-mented and control sites during the first hunting weekend in 2009 (22–23 August), in order to ensure that all of the hunted individuals belonged to the local

Animal Biodiversity and Conservation 38.1 (2015) 15

breeding population and not to passing migratory po-pulations which appear from the north from September onwards and mix with the local populations (Fernán-dez & Camacho, 1989; Rocha & Hidalgo, 2002).

Each estate was visited on the day of the hunt in order to count the young and adult birds shot, as well as the number of hunters involved. The ratio of young to adult birds, obtained at the end of the hunt, is a simple parameter used previously on the Iberian peninsula (Gutiérrez, 2001). In total, 4,132 killed birds were observed (3,154 in the group with extra food and 987 in the group without extra food).

Data analysis

Data analyses were carried out in the R environment (R Development Core Team, 2013). Normality was tested using Kologorov–Smirnoff tests and Q–Q plots. Means were compared using unpaired Student’s t–tests. Ge-neral linear models were used to analyze the influence of the quantity of supplementary food and the time it was available on the field on hunted age ratios. This relationship was visualised with contour plots produced with the function vis.gam from the R package mgcv. A general lineal model was used to compare the correla-tions between field and hunted age ratios between the supplemented and control sites, by defining hunted age ratio as dependent, supplement (yes/no) as factorial predictor and field age ratio as covariate. The models were initially carried out with two–way interaction terms, but these were removed as they did not reach statistical significance. We used Mann Whitney U–tests to com-pare the number of turtle doves killed per hunter and day on the estates with extra food and those without extra food. All tests were two–tailed.

Results

Differences between supplemented sites and controlsites

The mean field age ratio (fig. 2) based on the obser-vation of post–breeding aggregates was significantly higher at supplemented sites (mean ± sd: 1.61 ± 0.19) than in control sites (1.43 ± 0.27; t–test: t29 = –2.13, P = 0.04). The mean hunted age ratio at supplemen-ted sites (1.84 ± 0.22) was also significantly higher (t–test: t38= –6.83, P < 0.001) than at control sites (1.38 ± 0.19).

The hunted age ratio was correlated with the field age ratio, but the regression functions differed bet-ween supplemented sites and controls (fig. 3; linear model: F1,28 = 21.8, P < 0.001; effect of field age ratio: F1,28 = 7.9, P = 0.009).

At control sites, the hunted age ratio did not vary significantly from that obtained from the killed bird count (paired t–test: t10 = 0.8, P = 0.422). At supple-mented sites, in contrast, the field age ratio was lower than the hunted age ratio (1.61 ± 0.19 vs. 1.84 ± 0.22; paired t–test: t19 = –3.52, P < 0.001).

The total number of birds killed per hunter varied significantly between groups (Mann–Whitney U–test: Z = –4.17, P < 0.001). At supplemented sites, an average of 12.74 ± 9.52 European turtle doves per hunter per day were killed (n = 3,154), while the average was 3.91 ± 4.54 at control sites (n = 978).

Age ratio variation within supplemented sites

The quantity of supplementary food added annually ranged from a minimum of 300 kg to a maximum of

N

Control sitesSupplemented sites10 km

Extremadura region

Iberian peninsula

100 0 100 200 km

Fig. 1. Location of the region of Extremadura on the Iberian peninsula and distribution of the estates.

Fig. 1. Localización de la región de Extremadura en la península ibérica y distribución de los cotos.

16 Rocha & Quillfeldt

Birds observed inpost–breeding aggregates

Birds hunted

2

1.75

1.5

1.25

1

0.75

0.5

0.25

0Age

rat

io (

juve

nile

s/ad

ults

)Control sitesSupplemented sites

go, 2001). In the present study, in contrast, age ratio was analyzed before the onset of migration.

The hunted and field age ratios at supplemented sites in the present study were higher than in Andalusia, where they ranged from 1.15 to 1.25 (Gutiérrez, 2001). This could be explained by the positive effect of the increased availability of food, availability being a limiting factor in the dry ecosystems of these latitudes from May to September, i.e. during the breeding season. In this respect, Rocha & Hidalgo (2002) found that European turtle doves largely depend on the dehesa zones of cereal cultivation during the breeding season. This type of habitat is highly suitable because it provides abundant food and quiet and protected nest sites for the birds (Santamaria, 2007). In recent decades, the acreage used in the region for cereal cultivation has decreased notably (e.g., 1,500,000 ha since the 1980s across Spain; Olona, 2014), while cereal production has increased due to the agricultural intensification put in place by the Common Agricultural Policy of the European Union (Alés, 1996; Naredo, 1996; Robson, 1997). The loss has especially affected less intensi-vely managed and marginal areas, which were a very suitable habitat for European turtle doves. Therefore, the increased age ratio of the European turtle dove in areas where supplementary food was added could be considered a response to the lack of naturally available food due to the scarcity of crops.

Several studies highlight the susceptibility of this species to agricultural changes (increased intensity, changes in crops, pesticide use, etc.), both in breeding areas and wintering quarters (Browne & Aebischer, 2004; Browne et al., 2005; Wilson & Cresswell, 2006; Eraud et al., 2009). Supplementary feeding could be used as a management tool to contribute to mitiga-

1,300 kg (mean ± sd: 785 ± 275 kg). The contribution of food, from when it was added until the beginning of the hunt, had a range of 100 days, with a minimum of 20 to a maximum of 120 days (72 ± 30 days). The amount of food supplied was greater when food was supplied over a longer time (R2 = 0.567, P < 0.001). Therefore, daily supplement rates were calculated and included as predictor variables in GLMs.

The daily amount of food provided and the duration of the supplementation correlated positively with the field and hunted age ratios (table 1, fig. 4). Together, the amount and duration of the supplementation explained 47 and 31% of the variation in field and hunted age ratios, respectively (fig. 4). The correlation coefficient was higher for field age ratios (R2 = 0.408) than for hunted age ratios (R2 = 0.227).

Discussion

The present data suggested a positive influence of food supplementation on the number of juveniles pre-sent at the end of the breeding season, suggesting a higher breeding success. A higher percentage of juveniles hunted, compared to field observations clo-se–by, suggests that juveniles behave less cautiously at feeding sites or have a slower escape response than the more experienced adult birds.

Effects of food supplementation on post–breedingage ratios

The effect of artificial feeding on the turtle dove age ratio had previously been investigated over a longer period including postnuptial migration (Rocha & Hidal-

Fig. 2. Field age ratio (observed in post–reproduction aggregates) and hunted age ratio in estates with food supplementation and control sites in Extremadura, Spain, in late August 2009 (mean and standard error).

Fig. 2. Razón de edad obtenida en el campo (observada en agregados posreproductores) y razón de edad en las aves cazadas, en cotos con aporte de alimento complementario y en las zonas de control en Extremadura, en España, a finales de agosto de 2009 (media y error estándar).

Animal Biodiversity and Conservation 38.1 (2015) 17

1.0 1.2 1.4 1.6 1.8 2.0 Field age ratio

2.2

2.0

1.8

1.6

1.4

1.2

1.0

Hun

ted

age

rati

o

YesNo

Supplemented

Fig. 3. Relationship between field age ratio and hunted age ratio of European turtle doves in 20 estates with food supplementation and 11 control sites in Extremadura, Spain, in late August 2009. No field age ratio could be established for the remaining nine control sites because no post–breeding aggregations were located at these sites in the days preceding the late–August hunt.

Fig. 3. Relación entre la razón de edad en el campo y en las aves cazadas de tórtola europea en 20 cotos con aporte de alimento complementario y 11 zonas de control en Extremadura, en España, a finales de agosto de 2009. No se pudieron obtener las razones de edad en el campo para las nueve zonas de control restantes porque no se localizaron agregados postreprodutores en estos sitios en los días precedentes a la caza de finales de agosto.

Table 1. Influence of food supplementation on the field and hunted age ratio, assessed using generalized linear models.

Tabla 1. Influencia del aporte de alimento complementario en la razón de edad en el campo y en las aves cazadas, utilizando modelos lineales generalizados.

GLM predictor F1,19 t P

Dependent: field age ratio

Days supplemented 15.1 5.0 0.001

Supplement/day (kg) 6.4 2.5 0.022

Dependent: hunted age ratio

Days supplemented 4.7 2.2 0.043

Supplement/day (kg) 7.2 2.7 0.016

ting the decline of biodiversity produced by recent changes in European agricultural and livestock uses (Potts, 1997; Krebs et al., 1999; Donald et al., 2000). Likewise, planting cereal crops has been proposed as a management tool in addition to supplementary food supply, both in spring and in summer, in order to guarantee sufficient food during the breeding season and to increase productivity of the species (Rocha, 2007; Gutiérrez–Bermejo, 2009; Rocha et al., 2009). The survival rate during the first year of life of this species is very low, around 36% (Calladine et al., 1997); therefore, such measures would be effective provided they are not over–compensated by too high a hunting pressure. Mortality from hunting on breeding populations should not exceed the breeding capacity of the species. This would ensure the sustainability of the population in spite of hunting, since it would allow the annual return of birds to their breeding quarters because of the possible breeding philopatry of this species (Cramp, 1985).

Field vs. hunted age ratio

Regarding the methodology used to assess age ratios, the field and hunted age ratios did not differ signifi-cantly at control plots, while at supplemented sites, a greater proportion of young birds were counted when using data from the hunt as opposed to data from direct observation. Thus, supplementation resulted

in about 20% higher field age ratios, but up to 33% higher hunted age ratios. One plausible explanation for these differences may lie in the poorer escape response and lack of experience of the juveniles,

18 Rocha & Quillfeldt

which are not so skilled in flight and encounter the shots of the hunters for the first time. Thus, juveniles would be killed more easily than the adults, artificially raising the ratio of young to adult birds. However, in areas with supplemental feeding, an increased hunting pressure could also partially explain the results here presented. Since the hunted age ratio was consistently larger than the field age ratio (a more reliable but more time–consuming method), an adjustment factor could be established to estimate breeding success from hunting bags. According to the present data of supplemented sites, hunted age ratios should be adjusted by a factor of 0.87 to obtain values similar to field age ratios. This correction factor would be useful for future studies.

A previous study in Extremadura reported a higher proportion of young to adult birds killed on estates with supplementary food than in control sites during the migratory period (Rocha & Hidalgo, 2001). The larger proportion of young birds shot at supplemented sites might have a negative effect on the renewal of the population, leading to an ageing population and therefore the disappearance of breeding populations in the medium to long term (Rocha & Hidalgo, 2001).

At 11 control sites, a total of 121 juveniles and 81 adults were observed (a mean of 11.0 juveniles and 8.0 adults per estate). In comparison, a total of 477 juveniles and 296 adults were observed at 20 supplemented sites (a mean of 23.9 juveniles and 14.8 adults per estate). These observation numbers are 2.2 times higher on supplemented sites for juveniles and 1.9 times higher for adults, suggesting a positive effect on breeding turtle dove populations.

However, this positive effect would be counteracted if the hunting pressure at the supplemented estates was even higher in relative terms. At the 11 control sites with observation data, a total of 410 juveniles and 281 adults were hunted (a mean of 37.3 juveniles and 25.5 adults per estate). In comparison, a total of 2021 juveniles and 1,133 adults were observed at 20 supplemented sites (a mean of 101.1 juveniles and 56.7 adults per estate). These numbers for the hunting pressure were 2.7 times higher on supple-mented sites for juveniles and 2.2 times higher for adults. Based on these numbers, an increase of 2.2 (positive effect of supplementary feeding) would be counteracted by a decrease of 2.7 (negative effect of increased hunting pressure) for juveniles, thus suggesting a stronger negative effect on breeding turtle dove populations than the gain by supplemental feeding. A similar reasoning applies to adults, where an increase of 1.9 (positive effect of supplementary feeding) would be counteracted by a decrease of 2.2 (negative effect of increased hunting pressure). It has been mentioned in previous studies that increased pressure from hunting could cause serious problems for the species (Lucio & Purroy, 1992; Purroy, 1995, 1997; Rocha, 2007; Gutiérrez–Bermejo, 2009). The present data support this point of view. The average number of birds killed per hunter per day was 3 times higher on supplemented sites than on control sites. Similar figures have been recorded previously in Extremadura, where the average in other years has reached up to 4 times more (Rocha & Hidalgo, 2001). However, our methods (a single observation period per estate) were not ideal, and the relative population numbers given here are therefore tentative and should

25

20

15

10

25

20

15

10

Field age ratio (juveniles/adults) Hunted age ratio (juveniles/adults)

20 40 60 80 100 120 20 40 60 80 100 120 Days supplemented Days supplemented

Sup

plem

ent/

day

(kg)

Sup

plem

ent/

day

(kg)

Fig. 4. Contour plot of fitted values for the age ratios.

Fig. 4. Gráfico de los valores ajustados para las razones de edad.

2.5

2.42.3

2.22.1

2

1.91.8

1.71.6

1.5

2.2

2.1

21.9

1.8

1.7

1.6

1.5

1.41.3

Animal Biodiversity and Conservation 38.1 (2015) 19

be monitored more closely to ascertain which of the two opposing effects exerts a greater influence.

Additional factors may also need to be taken into account. For example, the species is also hunted in non–breeding zones, such as the open fields of cereals and sunflowers in the southern half of Ex-tremadura (and other areas of Iberian peninsula), where the European turtle dove is only hunted in migratory passage (Puerta, 2011). A negative effect through overhunting of young birds is expected on the migratory populations from western and central Europe (Rocha & Hidalgo, 2001).

Effects of the annual amount of food added and theduration of its availability

On the estates where a greater amount of extra food was added, a higher age ratio was observed, and this relationship was stronger for the field age ratio than for the hunted age ratio (table 1).

The field age ratio was especially strongly explained by the duration of the addition of food, suggesting that supplementation early in the breeding season had a particularly positive effect on the breeding success. European turtle doves can have up to three successive breeding attempts, and a longer supplementation would thus support early and late breeding attempts alike. In contrast, less variability was explained in the hunting age ratios by the amount of food added. This suggests that more variability in the hunting age ratios was ex-plained by unknown factors that may also impact on the ratio of young to adult birds killed. These unknown factors could be related, among other things, to the variation in the hunting pressure applied on the esta-tes, as occurs with other hunted species, such as the Woodcock (Scolopax rusticola) (Fadat, 1981). Hunting pressure may vary from estate to estate depending on variables such as the distance between hunters and the distance from the posts to the feeding zones, (the shorter the distance, the higher the pressure). These distances could facilitate or hinder the capture of young and adult birds, thereby affecting a greater variability in the proportions obtained. A further issue is the variety in the levels of marksmanship (shooting efficiency) between hunters from estate to estate.

By providing food from the beginning of June to the end of August, managers and hunters would assure the existence of food readily available for most of the fee-ding period of the chicks, from the eggs being hatched to their first flight and their preparation for migration.

These measures could also serve to avoid a pos-sible reduction in the breeding season as occurred in the UK, decreasing the number of broods and causing a drop in the species' reproductive rates (Browne & Aebischer, 2004). These authors consi-der that agricultural intensification over the second half of the 20th century has caused a clear change in feeding habits of turtle doves by decreasing the availability of wildflower seeds (probably due to ex-tended herbicide use, disappearance of uncultivated land, degraded field edges, etc.). This could be the ultimate reason why food supplementation could be successful in increasing productivity.

Where turtle doves are hunted, however, over–ex-ploitation of the breeding populations of the species is possible despite, or even helped by, supplementation due to its effects as bait. This will depend on the level of extraction that the hunt exerts on the populations: we thus suggest that hunting pressure needs to be carefully controlled for this migratory species due to its vulnerability (Madroño et al., 2004). It is also possible that migratory populations suffer from the negative effects of increased hunting at supplemented sites. In any case, we do not know to what extent the positive effects on productivity can be offset or even reversed by the effect of increased hunting pressure at supplemented sites for both breeding and migratory populations.

To limit the adverse effects of this practice in Ex-tremadura, since 2008, the addition of extra food is permitted only when the hunters are situated more than 200 meters from the edge of the area where the food is added (Junta de Extremadura, 2008). Furthermore, the distance between hunting posts has been limited to 50 meters and a quota of 15 European turtle doves/hunter/day (reduced to 10 European turtle doves/hun-ter/day since 2011) has been established. In this study we did not analyse the distances between hunters and the distance between hunters and feeding stations but it would be interesting to know if these variables effectively reduce the hunting pressure.

Acknowledgments

We thank the owners of the hunting estates for kindly allowing us to work on their property. We also ack-nowledge the assistance of the guards and hunters who participated in recording data at the observation posts and on the hunts.

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23Animal Biodiversity and Conservation 38.1 (2015)

© 2015 Museu de Ciències Naturals de BarcelonaISSN: 1578–665 XeISSN: 2014–928 X

Prieto, M., Mederos, J. & Comas, J., 2015. A new species of Laemostenus Bonelli, 1810 (Coleoptera, Carabidae) from Els Ports Natural Park (Catalonia, northeastern Iberian peninsula). Animal Biodiversity and Conservation, 38.1: 23–30.

AbstractA new species of Laemostenus Bonelli, 1810 (Coleoptera, Carabidae) from Els Ports Natural Park (Catalonia, northeastern Iberian peninsula).— Laemostenus (Antisphodrus) portsensis n. sp. is described from five caves at Els Ports Natural Park. The new taxon can be distinguished from its geographical neighbours, L. (A.) levantinus (Bolívar, 1919) and L. (A.) lassallei Mateu, 1989, by the shape of its head and pronotum, and particularly by the morphology of the male genitalia. The study includes some remarks about the habitat and ecology of the new species.

Key words: Coleoptera, Carabidae, Laemostenus (Antisphodrus), New species, Iberian peninsula, Catalonia, Cave habitat

ResumenUna nueva especie de Laemostenus Bonelli, 1810 (Coleoptera, Carabidae) del Parc Natural dels Ports (Cataluña, nordeste de la península ibérica).— Se describe Laemostenus (Antisphodrus) portsensis sp. n., localizada en cinco cavidades del Parc Natural dels Ports. Se compara con sus vecinos geográficos, L. (A.) levantinus (Bolívar, 1919) y L. (A.) lassallei Mateu, 1989, de los que se distingue por la forma de la cabeza y el pronoto, y especialmente por la morfología de la genitalia masculina. Se completa el estudio aportando datos sobre el hábitat y la ecología de la nueva especie.

Palabras clave: Coleoptera, Carabidae, Laemostenus (Antisphodrus), Especie nueva, Península ibérica, Cataluña, Hábitat cavernícola

Received: 14 XI 14; Conditional acceptance: 7 I 15; Final acceptance: 5 II 15

M. Prieto, J. Mederos & J. Comas, Museu de Ciències Naturals de Barcelona, Passeig Picasso s/n., 08003 Barcelona, Spain.

Corresponding author: M. Prieto. E–mail: m.primanz@gmail.com

A new species of Laemostenus Bonelli, 1810 (Coleoptera, Carabidae) from Els Ports Natural Park (Catalonia, northeastern Iberian peninsula)

M. Prieto, J. Mederos & J. Comas

24 Prieto et al.

Introduction

The genus Laemostenus Bonelli, 1810 is represented by 19 species in the Iberian peninsula (Casale & Co-mas, 2012; Serrano, 2013), most of them belonging to the subgenus Antisphodrus Schaufuss, 1865, with several species showing morphological adaptations to the subterranean environment (Casale, 1988). The Iberian species of Antisphodrus belong to the group of L. (A.) navaricus (Vuillefroy, 1893), and are divided into three subgroups. The subgroup of L. (A.) navaricus is distributed in the northern part of the Peninsula, mainly in the northwestern third, while the subgroups of L. (A.) villardi (Antoine, 1948) and L. (A.) kabylicus (De Miré, 1958) are confined to the southeastern quadrant. The most recently described species (Fernández–Cortés, 1995; Mateu, 1996; Carabajal–Márquez et al., 2002) belong to the kabylicus and villardi subgroups, and are found widely in the Betic–Riffian area (Casale, 1988).

Although the subterranean fauna of the Iberian pen-insula is considered one of the richest and best known in Europe (Sendra et al., 2011), new data about species as conspicuous as those of the genus Laemostenus have recently been published, the latest provided by Casale & Comas (2012) (including the description of a new species) and Ramos–Abuin (2013).

In 2012, with the collaboration of the Associació Catalana de Bioespeleologia (Catalan Association of Biospeleology, BIOSP), the Department of Arthropods of the Museu de Ciències Naturals de Barcelona (MCNB) performed a first biospeleological survey of the arthropod fauna from Els Ports Natural Park (Caballero–López & Masó–Ros, 2013) and identified a new species of Laemostenus (Antisphodrus) which we describe herein.

Material and methods

Study area

The caves where the new species was found are lo-cated in the mountain massif of Els Ports, in Els Ports Natural Park and surrounding areas, southwest of the Tarragona province (fig. 1). This massif faces NE–SW and is situated at the confluence of the Catalan Pre–Coastal Range with the Iberian System. The substrate consists of limestone and dolomites of the Mesozoic, mainly Triassic and Jurassic, except at the SW end of the massif, dominated by Cretaceous outcrops.

The prospected caves were:1. Avenc dels Ermets de Passamonte (31T–278852,

4535748; 990 m): located in the municipality of Prat de Comte, in the Barranc d’Engrilló. The opening is on the right side of the track leading to Tossal d’Engrilló (northern end of the Natural Park).

2. Cova del Conill (31T–276201, 4535632; 425 m): in the municipality of Horta de Sant Joan, located near the Barranc del Closet, between Reguers and Bosc d’Horta, at the northern end of the Natural Park.

3. Avenc de la Barcina (31T–277230, 4521575; 1,300 m): cave located at the peak of Barcina, about 2 km NE of the peak of Caro (municipality of Roquetes).

4. Avenc dels Mamelons (31T–270981, 4520902;

1,225 m): municipality of Tortosa, located in the Mola de Catí.

5. Forat the Rastre (31T–270829, 4520086; 1,240 m): also located in the Mola of Catí, but within the municipality of Roquetes.

UTM coordinates (ETRS89 Datum) and official toponymy from the Institut Català de Cartografía (ICC) are used.

Collection and study of specimens

A total of 32 specimens were obtained by direct capture and with pitfall traps installed according to the survey design (Caballero–López & Masó–Ros, 2013). Several sectors of the caves, from the penumbra zones to the innermost chambers, were prospected using two sets of traps. In the first set, bait consisted of a mixture of cheese and dried meat, and propyleneglycol was used as preservative. In the second set, beer and salt were used as attractant and preservative.

Fifteen specimens were preserved in 70° ethanol and one in 100° ethanol. The remaining specimens were dry–preserved and mounted on cards. Male ge-nitalia were fixed in dimethyl–hydantoin–formaldehyde (DMHF) and mounted on a transparent card attached to the pin bearing the respective specimens.

The criteria used for the species description were basically those established by Casale (1988) and the morphometric parameters were those as defined by Casale & Comas (2012): TL. Body total length, taken from the anterior margin of the epistome to the apex of the elytra, measured along the suture; PW/PL. Ratio between the width of the pronotum (PW) and length of the pronotum (PL), taking respectively the greatest transverse distance and the distance from the basal to the anterior margin along the midline; EL/EW. Ratio between the elytra length (EL), taken from the basal margin to the apex along the suture, and the elytra width (EW), taken as the greatest transverse distance.

All types are housed in the entomological collection of the MCNB, identified by their corresponding register numbers (MZB acronym). The information associated with each one has been documented and recorded in the database of the collection.

The comparison of the new species with L. (A.) lassallei is based on the original description by Mateu (1989) and nine specimens from the private collections of Eduard Vives and Achille Casale (6 and 3, F. Baget leg.), collected at several localities (including the type locality) in Els Ports and Beceite (Tarragona and Teruel provinces). Examined material of L. (A.) levantinus is from the type locality (Bolívar, 1919) and is housed in the MCNB collection (register numbers MZB 76–9239, 76–9240 and 76–9267).

Results

Laemostenus (Antisphodrus) portsensis n. sp. (figs. 2, 3A, 4A, 5A)

Type materialHolotype: , Avenc dels Ermets de Passamonte, Prat

Animal Biodiversity and Conservation 38.1 (2015) 25

Fig. 1. Location of Els Ports Natural Park (Tarragona province) in the Iberian peninsula, showing location of the caves (see text).

Fig. 1. Localización del Parc Natural dels Ports (provincia de Tarragona) en la península ibérica y ubicación de las cavidades (véase el texto).

12

45 3

Els Ports Natural Park0 10 km

del Comte, P. N. Els Ports, Tarragona, Catalunya. 18 IV–29 VIII 2012 (pitfall trap), MZB 2012–0520.

Paratypes: Avenc dels Ermets de Passamonte, Prat del Comte (pitfall trap): 18 IV–29 VIII 2012, 3 (MZB 2012–0521 to 2012–0523); 21 X–28 XI 2012, 4 and 8 (MZB 2012–1076 to 2012–1081); 28 XI 2012–7 II 2013, 2 (MZB 2013–1244); 7 I–13 III 2013, 1 (MZB 2013–1245). Cova del Conill, Horta de Sant Joan (pitfall trap): 18 IV–31 VIII 2012, 2 (MZB 2012–0746); 31 VIII–28 XI 2012, 1 (MZB 2012–1082); 28 XI 2012–7 II 2013, 2 and 3 (MZB 2013–1239 to 2013–1243). Avenc de la Bar-cina, Roquetes: 24 V 2012 (direct capture), 1 and 1 (MZB 2012–0329, 2012–0398); 24 V–8 X 2012 (pitfall trap), 1 (MZB 2012–0836). Avenc dels Ma-melons, Tortosa: 7 IX 2012 (direct capture), 1 (MZB 2012–0726). Forat del Rastre, Roquetes: 22 XI 2012 (direct capture), 1 (MZB 2012–1075).

DiagnosisTotal length: 11.70–16.30 mm (holotype 12.60). Body slender and depressed, with dark reddish brown integuments; large and ovoid head, with small eyes; transverse–cordiform pronotum, with prominent anterior angles; elongate–ovate elytra, with well–defined striae and elytral intervals flat or moderately convex; median lobe of the edeagus, in lateral view, elongated and widened towards the middle with prominent apical lamina; dorsal surface of head and pronotum shiny and almost smooth, and elytra intervals with a microsculpture conferring a dull appearance.

DescriptionBody slender and depressed, with dark, reddish brown integuments (morphometry, table 1).

Head: big, ovoid, longer than wide, and somewhat narrower than the base of the pronotum. Eyes re-duced and slightly convex, not protruding from the contour of the head, as long as 1/2 the temple, the latter slightly arched, with two suborbital setiger-ous pores. Neck constriction well impressed. Front smooth and slightly convex, with frontal impressions short and shallow. Antennae long, surpassing first third of the elytra.

Pronotum: transverse–cordiform (PW/PL: 1.12 to 1.22), maximum width at the anterior fourth, lateral sides slightly sinuated at posterior third and cons-tricted posteriad, basolateral angles right. Anterior margin concave, with anterolateral angles rounded and prominent; base slightly concave at middle. Disc depressed, with shallow transversal wrinkles; lateral edges slightly raised, especially at base. Basal im-pressions elongated and extended beyond basal third. Median groove deep. Anterolateral and basolateral setigerous pores present.

Mesosternum: unarmed in front of mesocoxae.Elytra: elongate–ovate, maximum width at apical

third, with lateral margins less curved and convergent in basal third (EL/EW: 1.62 to 1.77). Shoulders almost effaced, humeral tooth barely visible. Disc depressed. Striae well impressed and shallowly punctuate. Elitral intervals flat in the anterior half of the disc, moderately convex towards the apex, with distinct isodiametric microsculpture.

Iberian peninsula

26 Prieto et al.

Legs: elongate and slender. Profemora with ventral side flat. Protibiae with pubescence on the apical third, reaching backwards to the cleaning organ. Mesotibiae and metatibiae straight in both sexes. Tarsomeres with dorsal pubescence. Tarsal claws smooth on the inner margin.

Male genitalia: median lobe of aedeagus in lateral view elongate and slighty constricted basally, with basal bulb well–developed and apex attenuated (fig. 3A); apical lamina in dorsal view broadly curved, nonscooped–out at middle (fig. 4A).

EtymologyThe specific epithet derives from name of the masiff Els Ports, where the species is located.

Discussion

Comparative notes

According to Casale (1988), the group of Laemostenus (Antisphodrus) navaricus (Vuillefroy, 1893) is divided into four subgroups. The closely related subgroups of L. kabilicus (De Miré, 1958), L. prolixus (Fairmaire, 1875) and L. villardi (Antoine, 1948) comprise Be-tic–Riffian species confined in the Iberian peninsula to the southeastern quadrant, with northern limit at

Fig. 2. Habitus of Laemostenus (Antisphodrus) portsensis n. sp. (male holotype).

Fig. 2. Habitus de Laemostenus (Antisphodrus) portsensis sp. n. (holotipo macho).

2 m

m

Fig. 3. Aedeagus in lateral view: A. Laemostenus (Antisphodrus) portsensis n. sp., holotype; B. L. (A.) lassallei, from Beceite (Teruel); C. L. (A.) levantinus, specimen MZB 76–9240 from the Cueva de las Maravillas (Castellón).

Fig. 3. Edeago en visión lateral: A. Laemostenus (Antisphodrus) portsensis sp. n., holotipo; B. L. (A.) lassallei, de Beceite (Teruel); C. L. (A.) levantinus, ejemplar MZB 76–9240 de la Cueva de las Maravillas (Castellón).

1 mm

A

B

C

Animal Biodiversity and Conservation 38.1 (2015) 27

Fig. 4. Apex of aedeagus in dorsal view: A. Laemostenus (Antisphodrus) portsensis n. sp., holotype; B. L. (A.) lassallei, from Beceite (Teruel); C. L. (A.) levantinus, specimen MZB 76–9240 from the Cueva de las Maravillas (Castellón).

Fig. 4. Ápice del edeago en visión dorsal: A. Laemostenus (Antisphodrus) portsensis sp. n., holotipo; B. L. (A.) lassallei, de Beceite (Teruel); C. L. (A.) levantinus, ejemplar MZB 76–9240 de la Cueva de las Maravillas (Castellón).

0.25

mm

A B C

Fig. 5. Head and pronotum in dorsal view: A. Laemostenus (Antisphodrus) portsensis n. sp., holotype; B. L. (A.) lassallei, from Beceite (Teruel); C. L. (A.) levantinus, specimen MZB 76–9240 from the Cueva de las Maravillas (Castellón).

Fig. 5. Cabeza y pronoto en visión dorsal: A. Laemostenus (Antisphodrus) portsensis sp. n., holotipo; B. L. (A.) lassallei, de Beceite (Teruel); C. L. (A.) levantinus, ejemplar MZB 76–9240 de la Cueva de las Maravillas (Castellón).

1 m

m

A B C

28 Prieto et al.

the Sierra de Alcaraz (province of Albacete). The subgroup of L. navaricus (Vuillefroy, 1893) includes four species in the Iberian peninsula, distributed in its northern half. Two of them, L. navaricus (Vuillefroy, 1893) and L. peleus (Schaufuss, 1861), are spread in the Pyrenees and the Cantabrian Cornice, whereas the other two, L. levantinus and L. lassallei, are lo-cated in the north of the Valencian region (provinces of Castellón and Valencia) and the mountain massif of Els Ports–Beceite (southern of the provinces of Tarragona and Teruel), respectively. The records of Els Ports–Beceite attributed to L. levantinus by Vives & Vives (1983) and Ortuño (1989) must refer to L. lassallei, according to Ortuño (2006).

Laemostenus lassallei, very similar in morphology to the new species, was included by Mateu (1989) in the subgroup of L. kabylicus, in particular, for its transverse pronotum. In a later review of Spanish Sphodrina (Mateu, 1996), the same author transferred L. lassallei to the subgroup of L. navaricus. By sharing the main features of the subgroup of L. navaricus stated by Casale (1988), as well as for geographical considerations, L. portsensis n. sp. should be placed in that subgroup, with L. levantinus and L. lassallei, its geographical neighbours.

Despite the known variability within the Laemostenus species, the shape of the head and the pronotum are within a range that distinguishes the new species from L. lassallei, even considering population variations from different localities. The pronotum is slightly less transverse and clearly less constricted basally in L. portsensis n. sp. (fig. 5), and both especies show significant diferences in the male genitalia (fig. 3). The median lobe in L. lassallei is markedly curved in its basal third, with geniculated bulb and shorter apex than L. portsensis n. sp.; there are also differences in the contour of the apex, in the dorsal view (fig. 4).

The new species differs from L. levantinus by its less elongate and slender body, shorter appendages (antennae not reaching half of elytra), more bulky head and pronotum (fig. 5) with anterolateral angles mar-

Table 1. Morphometry of Laemostenus (Antisphodrus) portsensis n. sp. Mean values and ranges of variation are indicated.

Tabla 1. Morfometría de Laemostenus (Antisphodrus) portsensis sp. n. Se indican los valores promedio y los intervalos de variación.

Dimensions Holotype Total sample

TL (mm) 12.60 13.22 (11.70–14.40) 14.10 (12.90–16.30) 13.82 (11.70–16.30)

PL (mm) 2.45 2.59 (2.30–2.85) 2.70 (2.40–3.00) 2.66 (2.30–3.00)

PW (mm) 2.90 3.08 (2.75–3.45) 3.23 (2.85–3.65) 3.18 (2.75–3.65)

PW/PL 1.18 1.19 (1.13–1.22) 1.19 (1.12–1.22) 1.19 (1.12–1.22)

EL (mm) 7.20 7.65 (6.70–8.40) 7.96 (7.20–9.10) 7.86 (6.70–9.10)

EW (mm) 4.30 4.53 (4.10–4.80) 4.68 (4.10–5.20) 4.63 (4.10–5.20)

EL/EW 1.67 1.69 (1.62–1.75) 1.70 (1.62–1.77) 1.70 (1.62–1.77)

kedly more prominent, and basolateral angles being right instead of acute. For differences in aedeagus, see figures 3 and 4.

A key that allows to distinguish the three northeastern Iberian species of the Laemostenus navaricus subgroup is given.

Distribution and ecology

Most specimens of L. portsensis n. sp. were captu-red with pitfall traps in the caves of the Avenc dels Ermets Passamonte and the Cova del Conill. The traps were placed at the entrance to these caves, along a path of 30 m, inclined about 45° and 30°, respectively. No specimens were located beyond a distance of 40 m from the entrance. However, a few specimens were captured in relatively deep zones of vertical caves (second chamber of Avenc dels Ma-melons and Avenc de la Barcina), walking on damp stalagmites. Some related species are considered troglobitic, with L. levantinus and L. navaricus being the most modified species within the subgenus (Ca-sale, 1988; Ortuño, 2006), the last species termed as 'quasi aphaenopsian' by Casale (1988). Most Iberian species of Antisphodrus have been located in deep subterranean habitat, although the presence of certain species in the superficial subterranean habitat (MSS) or even in epigean environments (Novoa et al., 1989; Ortuño, 1989; Fernández–Cortés, 1995; Peláez & Salgado, 2006; Ramos–Abuin, 2013) indicates a wide ecological range for the group.

At the time of placement and removal of traps, temperature ranged from 9.1 to 16.3°C (lower temperatures corresponded to deeper sectors of the vertical caves). The relative humidity reached values close to saturation in most prospected ca-ves. Invertebrates cohabiting with L. portsensis n. sp. were chilopods (Lithobius sp.), aracnids (e.g. Tegenaria and Metellina), crickets (Petaloptila sp.) —very abundant—, and several species of dipterans (Mycetophilidae, Phoridae, Heleomyzidae and Limo-

Animal Biodiversity and Conservation 38.1 (2015) 29

Identification key for the three northeastern Iberian species of the Laemostenus navaricus subgroup

Clave de identificación para las tres especies del subgrupo de Laemostenus navaricus del nordeste ibérico.

1 Head elongate and narrow, with temples barely convex and neck constriction feebly marked. Pronotum elongate, with anterolateral angles less prominent (fig. 5C) L. levantinus (Bolívar, 1919)Head wider, with temples more convex. Neck constriction well marked. Pronotum transverse, with prominent anterolateral angles 2

2 Pronotum more transverse (PW/PL: 1.23 to 1.28), with lateral sides markedly sinuated (fig. 5B) L. lassallei Mateu, 1989Pronotum less transverse (PW/PL: 1.12 to 1.22), with lateral sides less sinuated (fig. 5A) L. portsensis n. sp.

Fig. 6. Distribution area of Laemostenus (Antisphodrus) portsensis n. sp., L. (A.) lassallei, and L. (A.) levantinus (showing only the northern part of the province of Castellón).

Fig. 6. Área de distribución de Laemostenus (Antisphodrus) portsensis sp. n., L. (A.) lassallei, y L. (A.) levantinus (se muestra solo el norte de la provincia de Castellón).

L. (A.) portsensis n. sp.L. (A.) lassalleiL. (A.) levantinus

Teruel (Aragón)

Castellón (Valencia)

Tarragona (Catalonia)

0 20 km

nidae) (Caballero–López & Masó–Ros, 2013). The presence of the beetle Paraphaenops breuilianus Jeannel, 1916 (family Carabidae), a remarkable troglobitic endemism of Els Ports massif (Español, 1979; Bellés, 1987), was detected in the Forat del Rastre and Avenc dels Mamelons.

The known distribution of L. lassallei is limited to the locality of Beceite and Els Ports massif, showing a slight overlap with the distribution area of L. portsensis n. sp. in el Mascar, adjacent to Mola del Catí (fig. 6). Unlike the new species, L. lassallei has been located so far under stones, in deeply fissured limestone areas (Ortuño, 1989; Eduard Vives, pers. com.), and it is considered an inhabitant of the MSS (Ortuño, 2006). The survey that led to the discovery of

the new species was limited to the exploration of the deep subterranean domain. Therefore, the possibility of L. portsensis n. sp. also inhabiting the MSS cannot be disregarded.

The separation of the two species could be due to ecological rather than geographical barriers. More interesting is the fact that the occurrence of both taxa in the same area is a strong argument for supporting their genetic distinctness and their status of isolated species. Within Laemostenus, there are other docu-mented cases of sympatric species of Antisphodrus which are morphologicaly closely related, as occurs with L. (A.) elongatus (Dejean, 1828) and L. (A.) cavi-cola (Schaum, 1858), distributed in the Balkan region (Casale, 1988).

30 Prieto et al.

Acknowledgements

This study was conducted within the framework of a project to study the subterranean arthropod diversity of Els Ports Natural Park, supported by the Direcció General del Medi Natural i Biodiversitat (Departament d’Agricultura, Ramaderia, Pesca, Alimentació i Medi Natural, Generalitat de Catalunya). We wish to thank Glòria Masó and Berta Caballero, curators of the Arthropods Department of the Museu de Ciències Naturals de Barcelona and coordinators of the pro-ject, and also Jordi Agulló, Neus Brañas and Mireia Nel·lo (MCNB) for their assistance. We are indebted to Floren Fadrique for his essential role in the explo-ration of caves, and to other speleologists from the Associació Catalana de Bioespeleologia, especially Josep Pastor, for their field assistance. Our thanks also to Joan Mestre and other technical staff at Els Ports Natural Park for the facilities provided during field work, and to Achille Casale (Università di Sassari, Itàlia) and Eduard Vives (MCNB) for their invaluable comments and loan of material of L. lassallei.

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García–Feria, L. M., Ureña–Aranda, C. A. & Espinosa de los Monteros, A., 2015. Minimally invasive blood sampling method for genetic studies on Gopherus tortoises. Animal Biodiversity and Conservation, 38.1: 31–35.

AbstractMinimally invasive blood sampling method for genetic studies on Gopherus tortoises.— Obtaining good quality tissue samples is the first hurdle in any molecular study. This is especially true for studies involving management and conservation of wild fauna. In the case of tortoises, the most common sources of DNA are blood samples. However, only a minimal amount of blood is required for PCR assays. Samples are obtained mainly from the brachial and jugular vein after restraining the animal chemically, or from conscious individuals by severe handling methods and clamping. Herein, we present a minimally invasive technique that has proven effective for extracting small quantities of blood, suitable for genetic analyses. Furthermore, the samples obtained yielded better DNA amplification than other cell sources, such as cloacal epithelium cells. After two years of use on wild tortoises, this technique has shown to be harmless. We suggest that sampling a small amount of blood could also be useful for other types of analyses, such as physiologic and medical monitoring.

Key words: Blood extraction, DNA source, Tortoises

ResumenMétodo de extracción de sangre mínimamente invasivo para estudios genéticos en tortugas terrestres del género Gopherus.— La obtención de muestras de tejido de buena calidad es la primera dificultad en cualquier estudio molecular. Esto es especialmente cierto en los estudios de gestión y conservación de la fauna silvestre. En el caso de las tortugas terrestres, la fuente más habitual de ADN son las muestras de sangre obtenidas principalmente de las venas braquial y yugular por contención química, o de individuos conscientes mediante métodos de manipulación y sujeción que pueden causar estrés en el animal. Sin embargo, se requiere una cantidad mínima de sangre para los ensayos del PCR. A continuación, presentamos una técnica mínimamente invasiva que ha resultado eficaz para extraer pequeñas cantidades de sangre apropiadas para realizar análi-sis genéticos. Además, las muestras obtenidas producen una amplificación de ADN mejor que otras fuentes celulares, como las células epiteliales cloacales. Después de dos años de aplicación en tortugas terrestres silvestres, esta técnica ha demostrado ser inofensiva. Sugerimos que el muestreo de pequeñas cantidades de sangre con esta técnica podría ser útil para otro tipo de análisis, como el seguimiento fisiológico y médico.

Palabras clave: Extracción de sangre, Fuente de ADN, Tortugas terrestres

Received: 24 X 14; Conditional acceptance: 15 I 15; Final acceptance: 6 II 15

Luis M. García–Feria, Red de Biología y Conservación de Vertebrados, Inst. de Ecología A. C., carretera an-tigua a Coatepec 351, El Haya, CP 91070, Xalapa, Veracruz, México.– Cinthya A. Ureña–Aranda & Alejandro Espinosa de los Monteros, Lab. de Sistemática Filogenética, Red de Biología Evolutiva, Inst. de Ecología A.C., carretera antigua a Coatepec 351, El Haya, CP 91070, Xalapa, Veracruz, México.

Corresponding author: L. M. Gracía–Feria. E–mail: luis.garcia@inecol.mx

Minimally invasive blood sampling method for genetic studies on Gopherus tortoises

L. M. García–Feria, C. A. Ureña–Aranda &A. Espinosa de los Monteros

32 García–Feria et al.

Introduction

Many behavioral, ecological, physiological, and medici-nal studies for conservation purposes require the use of blood samples. To reduce physical risk during animal handling, a minimal invasive method has been tested to obtain blood samples for these as well as for other kinds of studies (e.g., genetic, systematic, toxicological and stable isotope analyses). In many cases, the use of molecular markers is currently one of the prime tools in wildlife research, management, and conservation (DeWoody, 2005, DeYoung & Honeycutt, 2005). Ga-thering good quality samples has sometimes been a problem under field conditions. In the past, collectors used to sacrifice individuals for museum collections and sometimes for obtaining accessory material such as tissue samples. The rapid development of PCR methods and kits for DNA extraction has made it possible to obtain suitable genetic material from mi-nuscule samples (e.g., one hair). In many instances; however, collecting samples requires restraining the animals by physical or chemical means (i.e., invasive techniques), resulting in prolonged stress and even injuring the subject. New non–invasive methods have been developed for specific taxa groups (García–Feria, 2008); nonetheless, for many species or for field con-ditions, these techniques are not an option. Isolation of DNA from stool samples is among the common non–invasive methods used in many wildlife studies (Dalén et al., 2004; Lukacs & Burnham, 2005), with the DNA source being the epithelial cells. However, these cells are usually scarce, and the feces may contain PCR inhibitory substances. Besides, there is a high risk of contamination from alien DNA (Taberlet et al., 1999; Broquet et al., 2007).

For reptiles, particularly for live turtles and tortoises, DNA has been extracted by means of oral scrapes and cloacal swabs (Nagy & Medica, 1986; Mautino & Page, 1993; Van der Kuyl et al., 2005; Wendland et al., 2009). Even so, whole blood is the best source, and several blood extraction techniques have been deve-loped (Gandal, 1958; Avery & Vitt, 1984; Gottdenker & Jacobson, 1995; Knotková et al., 2002; López–Olvera et al., 2003; Rohilla & Tiwari, 2008). The sample is usually extracted either from the main veins (e.g., jugular, brachial, femoral, iliac vein) (Mader, 2005), the subcarapacial venous plexus (Hernández–Divers, et al., 2002), or the occipital sinuses, or by cardiac puncture (Mautino & Page, 1993; Fowler, 1995). Nonetheless, the anatomy and behavior of tortoises (Testudinidae) makes blood extraction rather difficult by any of these methods. The thick scales on the skin and the characteristic retraction of the limbs and the head within the shell block access to the veins. Sometimes the use of forceps and anesthetics for safe handling of the animals is necessary (Fowler, 1995). Additionally, lymphatic vessels running beside the main body veins may be damaged (Wendland et al., 2009). These methods are therefore excessive for PCR purposes. Turtles, like other reptiles, have nucleated erythrocytes (Knotková et al., 2002), so small quantities of blood are sufficient to obtain good quality genomic DNA.

Material and methods

When disturbed, Gopherus flavomarginatus (Bolson tortoise) can strongly retract its head and limbs, bloc-king access to the vessels used to draw blood. While in the retracted position, the exposed soft parts are covered with dense and thick scales (fig. 1). Howe-ver, between the fingers and the dorsal area of the forelegs there is a characteristic thin line of almost naked skin (fig. 2). This is the recommended area for obtaining small amounts of blood. We have used the following method for over two years in a study that assesses the genetic variations of 76 wild individuals of the Bolson tortoise (Ureña–Aranda & Espinosa de los Monteros, 2012). According to Germano (1994), maturity is attained once the carapace reaches at least 28 cm; therefore all the handled specimens can be considered adults. Before the present field work, the blood sampling method was tested on two species of captive tortoises, G. berlandieri (n = 1) and G. agassizii (n = 5), also sampled by means of cloacal swabs. Blood was sampled as follows. First, we cleaned the dorsal side of the hand with a cotton swab soaked in 75% ethanol to avoid any possible infection or contamination of organic material. Then, a puncture was performed using a sterile hypodermic needle (27G x 13 mm; Becton, Dickinson & Company, Franklin Lakes, NJ) in the bare line of skin located at the distal edge of the hand just before the fingers. The needle should be introduced between the second and third fingers in an angle of 45º (approximately) toward the third finger. There may be no bleeding if the needle is introduced elsewhere or at a different angle. Without practice, no more than three puncture attempts were required to obtain the blood sample. Immediately after removing the needle, the blood can be collected in a borosilicate glass capillary tube that does not contain heparin. Finally, we placed a cotton swab with 75% ethanol on the puncture for a few seconds, applying little pressure as to stop any extra bleeding. The cloacal swabs were taken after cleaning the cloacal area with a cotton swab soaked in 75% ethanol. We then introduced and softly spun a rayon swab (Medical Wire and Equipment, 100–100 MW, Biomerieux) in the cloaca to obtain the epithelium cells.

Results

In the field, we usually collected up to 30 μl of whole blood and transferred this to 500 μl vials containing 100 μl lysis buffer (Longmire et al., 1997); the ani-mals were released immediately after manipulation. The samples preserved in this buffer do not require refrigeration, which is an advantage for field condi-tions. However, if the genetic study involves protein analyses, blood samples must be stored by different means (e.g., liquid nitrogen). Once in the laboratory, we extracted DNA from the cloacal swabs, and dried tissue from shells samples and from the small aliquots of the blood–buffer mixture (10–20 μl) using Chelex 100© resin (Walsh et al., 1991). We obtained greater yields of high molecular weight DNA from the blood

Animal Biodiversity and Conservation 38.1 (2015) 33

samples than from other tested tissue sources (i.e., cloacal swabs, and dry tissue from shells; fig. 3). DNA amplification was conducted in Peltier–effect thermocyclers (ABI GeneAmp PCR system 2400) using the following parameters: one initial cycle at 95º for 120 s, followed by 32 cycles of 95º for 20 s, 47º for 20 s, 74º for 60 s, with one final cycle at 72º for 180 s.

This minimally invasive blood sampling method used on Gopherus species has been extremely useful. Performing the whole procedure takes no more than two minutes, even for those people who have been trained only once, and have little or no experience in animal handling. Using this technique, stress for the animal is kept to a minimum, and there is practically no risk of injuring the tortoise.

Fig. 1. Bolson tortoise in retracted position. Note the inaccessibility to the blood vessels commonly used to draw blood.

Fig. 1. Tortuga del Bolsón en posición retraída. Nótese la imposibilidad de acceder a los vasos sanguí-neos que habitualmente se utilizan para la extracción de sangre.

Fig. 2. Suggested puncturing site for the minimally invasive blood sampling method.

Fig. 2. Lugar sugerido para la punción como método de extracción de sangre mínimamente invasivo.

34 García–Feria et al.

1,353

1,078

872

603

310

MW 1 2 3 4 5 6 7 8

A remarkable characteristic of this species is its burrowing behavior. Several nerves and ligaments are located in the hand area, and some concerns may result from puncturing around this area. We have used this method for two years (i.e., between 2009 and 2011), to collect blood samples from 76 individuals of the Bolson tortoise. This species is highly philopatric, and the adults do not have natural predators. In recent visits to the study area, we have been able to verify the health status of every manipulated individual, and none of them showed any apparent problem. Therefore, we are confident that this technique of foot puncture is harmless compared to other inva-sive blood sampling methods (e.g., Gandal, 1958; Avery & Vitt, 1984; Fowler, 1995). Several analyses for physiological and medical surveys require larger volumes of blood than those extracted with the foot puncture method (from 0.5 ml to microtainer until 1.8 ml or more). However, the amount of blood that is obtained with the suggested method (≈ 30–40 μl, approximately two–thirds to three–quarters of a capi-llary tube; Kerr, 2002) is adequate for implementing laboratory and clinical analyses other than PCR. This minimally invasive method can be applied to obtain blood for a morphological characterization of peripheral blood cells from blood smears (Knotková et al., 2002), packed cell volume (PCV) measure-ment by microhaematocrit, refractometry to assess

the total protein level, specific gravity and refractive index of serum, glucose level by blood glucose strips or pocket glucose meter (Kerr, 2002).We therefore recommend the use of this minimally invasive method before attempting more aggressive blood extraction techniques for genetic analysis or for any other survey that requires only small amounts of blood.

Acknowledgements

The Bolson tortoise individuals were captured with collecting permits from the Secretaría de Me-dio Ambiente y Recursos Naturales (No. SGPA/DGVS/04690/09). We thank Gustavo Aguirre–León for his advice on the biology of the Bolson tortoise, the Centro Ecológico del Estado de Sonora, CEDES, for permission to conduct captive tortoise sampling. Rolando González Trapaga and Francisco Herrera for providing invaluable assistance during field work, and Marco A. L. Zuffi and three anonymous review-ers for their pertinent comments.

References

Avery, H. W. & Vitt, L. J., 1984. How to get blood from a turtle. Copeia, 1984: 209–210.

Fig. 3. Ethidium bromide stained 2% agarose gel showing PCR products from two regions of the mitochondrial D–loop gene. Amplifications from several specimens were attempted from forelimb puncture (lines 1, 2, 5 and 6), cloacal swab (lines 3 and 7), and carcass tissue (lines 4 and 8): MW indicates the molecular weight bands in base pairs.

Fig. 3. Productos de PCR de dos regiones del gen mitocondrial D–loop mostrados en un gel de agarosa teñido con bromuro de etidio al 2%. Se intentó realizar amplificaciones de diferentes muestras obtenidas a partir de la punción de la pata delantera (líneas 1, 2, 5 y 6), hisopos cloacales (líneas 3 y 7) y tejidos de carcasas (líneas 4 y 8): MW indica las bandas del peso molecular en pares de bases.

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Broquet, T., Ménard, N. & Petit, E., 2007. Noninvasive population genetics: A review of sample source, diet, fragments length and microsatellite motif effects on amplification success and genotyping error rates. Conservation Genetics, 8: 249–260.

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Van der Kuyl, A. C., Ballasina, D. L. P. & Zorgdrager, F., 2005. Mitochondrial haplotype diversity in the tortoise specie Testudo graeca North Africa and the Middle East. BMC Evolutionary Biology, 5: 29–36.

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Matos, M., Alves, M., Ramos Pereira, M. J., Torres, I., Marques, S. & Fonseca, C., 2015. Clear as daylight: analysis of diurnal raptor pellets for small mammal studies. Animal Biodiversity and Conservation, 38.1: 37–48.

AbstractClear as daylight: analysis of diurnal raptor pellets for small mammal studies.— Non–invasive approaches are increasingly investigated and applied in studies of small mammal assemblages because they are more cost–effective and bypass conservation and animal welfare issues. However, pellets of diurnal raptors have rarely been used for these purposes. We evaluated the potential of marsh harrier pellets (Circus aeruginosus) as a non–invasive method to sample small mammal assemblages, by comparing the results with those of sampling using Sherman live–traps and pitfalls. The three methods were applied simultaneously in an agricultural–wet-land complex in NW Portugal. Estimates of species richness, diversity, evenness, abundance, and proportion of each species within the assemblage showed significant differences between the three methods. Our results suggest that the use of marsh harrier pellets is more effective in inventorying small mammal species than either of the two kinds of traps, while also avoiding any involuntary fatalities associated with the sampling of small non–volant mammals. Moreover, the analysis of pellets was the most cost–effective method. Comparison of the two trapping methodologies showed involuntary fatalities were higher in pitfalls than in Sherman traps. We discuss the advantages and flaws of the three methods, both from technical and conservational perspectives.

Key words: Animal welfare, Circus aeruginosus, Pitfalls, Pellets, Sherman traps, Small mammals

ResumenClaro como el agua: análisis de las egagrópilas de aves rapaces diurnas para los estudios sobre pequeños ma-míferos.— Los métodos no invasivos se investigan y se aplican cada vez más en los estudios de comunidades de pequeños mamíferos, ya que son más rentables en cuanto a sus costos y evitan los problemas relacionados con la conservación y el bienestar animal. Sin embargo, las egagrópilas de aves rapaces diurnas rara vez se han utilizado para estos fines. En este trabajo se evaluó el potencial que tienen las egagrópilas del aguilucho lagunero (Circus aeruginosus) como un método no invasivo para estudiar las comunidades de pequeños mamíferos, me-diante la comparación de los resultados con los obtenidos en las trampas de tipo Sherman y las de caída (pitfall). Los tres métodos se utilizaron simultáneamente en un complejo formado por tierras agrícolas y humedales en el noroeste de Portugal. Las estimaciones de la riqueza, la diversidad, la uniformidad y la abundancia de especies y la proporción de cada una de ellas dentro de la comunidad mostraron diferencias significativas entre los tres métodos. Nuestros resultados sugieren que la utilización de las egagrópilas del aguilucho lagunero es más eficaz para inventariar las especies de pequeños mamíferos que cualquiera de los dos tipos de trampas, al mismo tiempo que evita la muerte involuntaria de animales asociada con el muestreo de pequeños mamíferos no voladores. Además, el análisis de las egagrópilas fue el método más rentable. Entre los dos métodos de captura, la muerte involuntaria de animales fue mayor en las trampas de caída que en las trampas de tipo Sherman. Se discuten las ventajas y los inconvenientes de los tres métodos tanto desde una perspectiva técnica como conservacionista.

Palabras clave: Bienestar animal, Circus aeruginosus, Trampas de caída, Egagrópilas, Trampas de tipo Sherman, Pequeños mamíferos

Received: 2 IX 14; Conditional acceptance: 8 XII 14; Final acceptance: 9 II 15

Milena Matos, Michelle Alves, Maria João Ramos Pereira, Inês Torres, Sara Marques & Carlos Fonseca, Dept. of Biology & CESAM, Univ. of Aveiro, 3810–193 Aveiro, Portugal.

Corresponding author: Michelle Alves. E–mail: michellealves@ua.pt

Clear as daylight: analysis of diurnal raptor pellets for small mammal studies

M. Matos, M. Alves, M. J. Ramos Pereira, I. Torres, S. Marques & C. Fonseca

38 Matos et al.

Introduction

Species inventories are usually the very first step towards biodiversity conservation in a certain region and the basis for integrative and effective manage-ment strategies (Begon et al., 2005).

Small, non–volant mammals are highly diverse and play a major role in ecosystem structure and function worldwide. With a wide range of reproductive, loco-motion and foraging strategies, small mammals are responsible for the maintenance of several interactions among wildlife communities, namely by promoting seed dispersal (Adler, 1995), or by constituting key prey for several groups of vertebrates (Carey & Jo-hnson, 1995). Additionally, due to their sensitivity to environmental changes (Pardini et al., 2005), non–volant small mammals are excellent models for the study of ecosystem processes and patterns, and an important group to consider where the protection of ecological values is a concern (Converse et al., 2006).

Studying small mammals usually requires an effec-tive capture plan to achieve a realistic assessment of the assemblies in accordance with the purpose of the work (Voss et al., 2001). Snap–trapping and live–trapping, with Sherman or Tomahawk traps, are the most commonly used methods to capture most small mammal species (Gurnell & Flowerdew, 2006), and supplementary surveys, such as pitfall trapping or active search are used for insectivorous or burrowing species (Voss et al., 2001). Differences in behavior, habitat use, diet, body size and use of vertical strata seem to significantly influence the effectiveness of traps (Sealander & James, 1958; Williams & Braun, 1983). It therefore seems that no single method will effectively yield an adequate sample of the species richness in an area (Voss et al., 2001). Besides effi-ciency and techniques, trapping small mammals also raises concerns related to ethics and animal welfare (Powell & Proulx, 2003; Putman, 1995), and to the ecological effects of involuntary or voluntary fatali-ties, the latter in removal–trappings. Acknowledging the usefulness of small mammals as bioindicators in terrestrial ecosystems, research and inventories of small mammal populations and assemblies have significantly increased in recent years. The potential associated fatality rates caused by hypothermia, discomfort or distress (Putman, 1995) may disrupt local populations and consequently, metapopulations (Sullivan & Sullivan, 2013), which taken to an extreme could result in conservation issues, such as monitoring in programmes dealing with sensitive or rare species.

Attending to all these constraints, and adding to the logistics and costs associated with trapping schemes, non–invasive approaches are increasingly investigated and applied (De Bondi et al., 2010; DeSa et al., 2012; Torre et al., 2013). When studies seek to examine aspects of assemblage composition, the most common non–invasive methods have long been the analysis of owl pellets, particularly from widespread and common species, such as Tyto alba (Torre et al., 2004, Rocha et al., 2011) and Strix alu-co (Balčiauskienė, 2005; Petty, 1999), due to their generalist diets and close foraging ranges (Torre et

al., 2013). However, pellets of diurnal raptors have rarely been used for these purposes, perhaps due to the relative difficulty in finding suitable amounts of pellets or in identifying prey remains, as many raptor species decapitate their prey before ingestion (Balfour & Macdonald, 1970) or are able to digest the skeletal parts of mammalian prey (Glue, 1970). Pellets of diurnal raptors were used by Santos et al. (2009) and Scheibler & Christoff (2007) but in both cases only as a complementary method for the inventory of small mammals in their study areas, specifically salt ponds of Aveiro, Portugal, and in agricultural areas of southern Brazil.

The marsh harrier (Circus aeruginosus) is a diurnal, medium–sized bird of prey whose distribution ranges from Europe and central Africa to central Asia, the northern parts of the Middle East and the Indian subcontinent (BirdLife International and NatureServe, 2014). It occurs in a wide range of habitats, from wetlands to agricultural areas (Cardador et al., 2011) and other human–shaped environments (Vandermeer, 2010). Marsh harriers mostly roost (Moreno et al., 2014) and nest (González, 1991) in paludal vegeta-tion. They also use perches (Kitowski, 2007), under which it is common to find pellets.

Marsh harriers usually forage in open agricultural areas, particularly on the edge of ponds of fresh or brackish water, using the raid as the main hunting technique (Clarke et al., 1993). However, they have a high foraging plasticity, allowing them to use ha-bitats that are not accessible to other birds of prey (Kitowski, 2007). The diet of marsh harriers is usually characterized as generalist (Strandberg et al., 2008) and influenced by seasonal and local conditions (Wi-tkowski, 1989; Cardador et al., 2012), but most studies list small mammals as their primary prey (González, 1991; Alves, 2013). Lagomorphs (Schipper, 1973) and birds (Mateo et al., 1999; Clarke et al., 1993) may also be important prey.

Here we aimed to evaluate the potential of the analysis of marsh harrier pellets as a non–invasive method to determine the composition of small mammal assemblages, by comparing the results with those of two other methods, Sherman live–trapping and pitfalls, applied simultaneously in the same mosaic of habitats. We discuss advantages and drawbacks of the three methods, both from technical and conservational perspectives. Although other authors have made some considerations about the viability of diurnal raptor pe-llets as a technique to sample small mammals (e.g., Andrews, 1990), to our knowledge this is the first study specifically designed to compare the efficiency of this method with that of other widely used capture methods.

Methods

Study area

This study was developed in Baixo Vouga Lagunar, located approximately between 8º 32' 57'' W and 8º 41' 32'' W; and 40º 49' 43'' N and 40º 41' 32'' N, in NW Portugal. The study area occupies about 12,205 ha

Animal Biodiversity and Conservation 38.1 (2015) 39

and encompasses important ecosystems integrated in the Natura 2000 Network (PTZPE0004, PTCON0061), featuring one of the most important Portuguese wetlands (Ria de Aveiro). The climate is Mediterranean with strong Atlantic influences, and it has an annual mean temperature of about 15ºC. The average annual humidity is 77% and the average annual rainfall is 1,387 mm, with a shortage of rainfall in the summer. The system represents a complex agriculture–wetland mosaic, inte-grating a variety of natural and human–altered habitats (fig. 1), such as pastures (2.35% of the area), rice fields Oryza sativa (0.98%) and maize Zea mays (28.13%); and 'Bocage' (7.09%), a typical and rare landscape unit which consists of hedgerows of trees (e.g., Salix alba), shrubs (e.g., Rubus ulmifolius) and ditches that compartmentalize farmlands and pastures. The wetlands are composed of reedbeds of Phragmites australis (4.50%), saltmarshes of Spartina maritima (12.79%) and rushes of Juncus maritimus (6.67%) (Alves et al., 2014). This region also houses high faunal richness (e.g. amphibians, birds (Special Protection Area for Birds PTZPE0004), and bats (Mendes et al., 2014). According to national bird censuses, the study area shelters 11 to 12 resident pairs of breeding marsh harriers, which corresponds to about 17% of the breeding population in the country (Rosa et al., 2006). The biological richness of Baixo Vouga Lagunar attracts ecotourists, and the region receives 25000+ visitors per year.

Sampling and identification of small non–volant mammals

Once a month, we collected marsh harrier pellets (n = 75) near nesting sites and under perches used by the species, during the breeding season of 2012, i.e. from February to August. The closest distance between a pellet collecting site and a nest was ca. 200 m. Our collecting procedures did not seem disturbing to the birds, especially considering the regular touristic visits to the area. Using a telescope, we spotted the collecting sites through direct observation of the birds from nine observation points each covering a circular area with a radius of 1.5 km. Observations took place monthly in the first three months and lasted for two hours per observation point. Regular flooding around perches and roosts prevented us from collecting pellets throughout the whole year. We oven–dried the pellets at 60°C for a day and the dry content was then separated after moisturizing. Food items were identified and quantified through the presence of non–digestible remains. Since harriers tend to rip the meat off their prey rather than swallow the entire animal (Hosking, 1943; Balfour & Macdonald, 1970), mammals were identified based on cranial structures described in the literature (Gállego & López, 1982; Gállego & Alemany, 1985; Blanco, 1998a; Blanco, 1998b) but also on detailed features of the fur: cuticular print, core and cross section (Teenrik, 1991; Quadros & Monteiro–Filho, 2006; Valente, 2012). Talpa occidentalis was the only species identified so-lely through cranial structures; all other species were identified by both cranial and hair structures. We used the 'minimum number of individuals' analysis in order to reduce possible erroneous counting of the number of prey (Lyman et al., 2003).

Simultaneously we sampled small non–volant mammals in the study area using Sherman and pitfall traps. For each habitat in the study area (reedbeds, rushes, saltmarshes, Bocage and rice and maize fields) and whenever possible, three replicate of small mammal sampling sites were randomly distri-buted within the nine 1.5 km radius harriers sampling areas, as long as 1 km of minimal distance between sampling sites was assured, to maintain spatial inde-pendence. Small mammals sampling sessions took place every two months, in a total of three sampling rounds for the study period. Each small mammal sampling site consisted of a line of 30 Sherman live traps (17.5 x 6 x 6 cm) separated 10 m from each other and baited with a mixture of canned sardines and hamster food, and a line of four pitfalls (buckets ca. 30 cm deep, 5 L capacity) connected with a drift fence buried to prevent animals from passing under it. In the Sherman traps, cotton was provided as nesting material. Whenever possible, traps were set under the cover of stones, shrubs or herbs to provide camouflage and some thermal insulation. Both stan-dardized methodologies were applied simultaneously, in order to minimize the effect of the selectivity of each method and collect more representative data on the composition of the small mammal assemblage. At each sampling round, traps were active for five consecutive nights and visited every early morning as was previously tested (Gurnell & Flowerdew, 2006). At each trap check, we provided dry bedding material and a new food supply. We ringed the collected animals individually and released them after identification.

Statistical analysis

Since the trapping methods did not allow the identifi-cation of all small mammals to the species level, we used genera accumulation curves to assess patterns of genera richness in the incidence matrix obtained with each sampling method (Gotelli & Colwell, 2001). We calculated Mao Tau and Chao 1 richness estima-tors (Chao et al., 2009; Torre et al., 2013) using the software EstimateS 9.0 for Windows (Colwell, 2011). The completeness of the inventory made with each method was assessed by fitting the Clench equation to the observed genera accumulation curve, using the quasi–Newton method equation (Soberón & Llorente, 1993). We used the same procedures to estimate species richness with the pellet sampling method.

For each method we assessed the assemblage structure using the number of identified genera, abun-dance (measured as the number of individuals captured in 100 night–traps or in 100 pellets [Mills et al., 1991]), and the proportion of each taxa within the assemblage, measured through the percentage of occurrence of each taxa, calculated as %Oi = ni / N+ × 100, where ni is the number of individuals of species i and N+ is the total number of individuals identified or captured with a given method. We also calculated indices of evenness (Pielou index) and diversity (Shannon–Wiener and Simpson index) for all three methods. We calculated two diversity indices to ascertain possible effects of rarely recorded taxa: Simpson’s index is less sensiti-

40 Matos et al.

ve to presence, giving more weight to common taxa (Simpson, 1949); Shannon–Wiener’s index is more sensitive to rare taxa (Magurran, 2004). To calculate evenness and diversity indices, due to a high number of zeros, pellet collecting sites (n = 8; see map in fig.1) and habitat replicates (for trappings; n = 13) were considered as samples.

We searched for differences between methods in assemblage composition and abundance of small ma-mmals, as well as in evenness and diversity indices, using analysis of variance (ANOVA) and controlling for the effects of sample size (Rahbek, 1997).

Cost comparison

For each sampling method we calculated the asso-ciated costs for total working hours and expenses. Working hours for each trapping method considered two people working in the field, and included all steps of the monitoring programme (installation, checking, animal handling and trap removal), totaling on ave-rage 24 six–hour–days per person and per sampling round for Sherman traps and 24 three–hour–days per person and per sampling round for pitfall traps. As for pellets, field work was performed by two people, and included spotting of pellet collection sites (on avera-ge five five–hour–days per person and per month), and pellet collection (on average one four–hour–day per person and per month). Pellets (lab work) were analysed by the same person and took on average of 20 five–hour–days per month. Expenses included fuel for field trips and supplies, such as bait and cotton for the traps and cover slips, and microscope slides for pellet analysis. The price of traps and lab equipment was not included in the budget as these items were already available in the research facilities.

Results

In total, 429 small mammals of 11 species were re-corded: seven rodents and four insectivore species (table 1). All eleven species were detected in marsh harrier pellets. Sherman traps captured five rodent and one insectivore taxa, and pitfall traps captured three rodent and two insectivore taxa. Six taxa were identified to species level only with pellets: Arvicola sapidus, Mus musculus, Mus spretus, Sorex granarius, Sorex minutus and Talpa occidentalis. Traps did not add any distinct species. Overall, pellets presented higher scores for the number of identified genera and species, evenness and diversity (table 1) than either of the two trapping techniques. The total number of captured individuals was highest with Sherman trap sampling (table 1).

The estimated species richness of small mam-mals using the pellet sampling was 11.33 ± 0.93; (n = 75; fig. 2A). The Clench equation showed strong adjustment to the species accumulation cur-ve (r2 = 0.9999), with a slope of 0.029, showing the proximity to an asymptote, and thus indicating that the sampling of small mammals with this method was quite complete and reliable (fig. 2A). Estimates of the

number of identified genera (Sherman 5.00 ± 0.45, n = 39; pitfalls 5.00 ± 0.17, n = 39; pellets 8.00 ± 0.25, n = 75), Clench model adjustment to the genera accu-mulation curve (Sherman r2 = 0.954; pitfalls r2 = 1.000; pellets r2 = 0.998) and slopes of the obtained curves (Sherman 0.004; pitfalls 0.033; pellets 0.015) showed that further field efforts would not result in a relevant increase of detected genera (fig. 2B, 2C, 2D). All eva-luated assemblage composition parameters showed significant differences between methods: number of genera (F = 54.424, P < 0.0001), abundance (F = 30.548, P < 0.0001), and the proportion of each genus within the assemblage (F = 4.112, P = 0.017). Tukey post–hoc tests showed there were differences between pairwise comparisons, in the number of genera and abundance between Sherman and pitfall traps and between Sherman traps and pellets; and percentage of occurrence between Sherman traps and pitfalls. Sherman traps detected a greater number of genera per sample (2.103 ± 1.046 genera/sample) than pellets (0.7475 ± 0.617 genera/sample) or pitfalls (0.462 ± 0.482 genera/sample) (results expressed as mean ± standard deviation).

Indices presented significant differences between methods: Pielou (F = 15.685, P < 0.0001), Shan-non–Wiener (F = 11.009, P < 0.0001), and Simpson (F = 3.742, P = 0.037), with pellets consistently scoring the highest values.

Involuntary fatalities associated with trapping me-thods were 0.51 and 1.79 individuals per 100 trap–nights with Sherman traps and pitfalls, respectively. The species showing highest fatality rates were Crocidura russula and Microtus lusitanicus.

Cost estimations showed that pitfall trapping was the fastest method, although pellet analysis was the cheapest. Sherman trapping was the most time–con-suming and the most expensive method (table 2).

Discussion

The combination of sampling methods used in this study identified 11 species of small non–volant mam-mals in the study area, where 12 species are known to be present (considering rodents and shrews, and excluding squirrels, hedgehogs and bats; Bandeira et al., 2013). Comparing our results with the indepen-dent and long–term mammal study of Bandeira et al. (2013), pellets only failed to detect Rattus rattus, a scavenger species that prefers to live around human settlements (Ewer, 1971). We did not sample within or around urban areas, but based on previous obser-vations, it is plausible to assume that areas in such close proximity to humans are avoided by the marsh harriers (Alves et al., 2014).

In the pellets we found remains of small non–volant mammal species known to be less common or rare, such as Sorex minutus and Arvicola sapidus, an aquatic species. The two Iberian endemisms, Sorex granarius and Talpa occidentalis, were also only recorded in pellet samples. When comparing the three methods alone, pellet analysis seemed to be most cost effective and efficient method for inventorying small mammals

Animal Biodiversity and Conservation 38.1 (2015) 41

Land use classes Urban tissue Riparina gallery Industrial tissue Saltmarshes Temporary crops / Maize field Reedbeds Pastures / Fallow land Rushes Bocage Inner waters (Vouga River) Rice fields Coastal and marine waters Forests Marsh harrier observation points Pellet collecting points

SpainP

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Fig. 1. Study area: A. Location of the study area in the Iberian peninsula; B. Land cover of the study area with marsh harrier observation points and pellet collecting sites.

Fig. 1. Área de estudio: A. Ubicación del área de estudio en la península ibérica; B. Cobertura del suelo en el área de estudio, con los puntos de observación de aguilucho lagunero y los puntos de recolección de las regurgitaciones.

N

0 0.5 1 2 km

in our study area. However, if we consider the two trapping schemes together the differences begin to fade. Nonetheless, in terms of species inventorying, our results indicate that even by analyzing a reduced number of pellets, information on the number of taxa detected can be significantly higher than that retrieved with large trapping efforts. Indeed, the species accumu-lation curve for pellet analysis indicates that 11 pellets

were sufficient to reach five small mammals species, which is equivalent to the entire species count allowed by Sherman traps and pitfalls altogether throughout the whole study. At the genus level, trapping methods altogether yielded six genera, a score achieved with the analysis of 22 pellets.

Our field trapping did not always allow identifica-tion of some individuals to the species level, such

42 Matos et al.

Table 1. Type of activity (N, nocturnal; D, diurnal, according to Blanco 1998a, 1998b), Portuguese conservation status (PT), international conservation status (IUCN), number of individuals (N), percentage of occurrence (%O), and abundance (A, measured as the number of individuals captured in 100 night–traps or in 100 pellets) of all species recorded with Sherman and pitfall trapping and with the analysis of marsh harrier pellets. Number of identified genera and species, number of captured individuals, abundance, Pielou’s evenness index, Shannon–Wiener’s diversity index and Simpson’s diversity index are presented for each method. Indices were calculated with data to the genus level, allowing comparison between methods. Numbers in bold highlight the highest value obtained per considered parameter: * Iberian endemism.

Tabla 1. Tipo de actividad (N, nocturna; D, diurna, de acuerdo con Blanco 1998a, 1998b), estado de conservación (PT); estado de conservación internacionañ (UICN), número de individuos (N), porcentaje de presencia (%O) y abundancia (A, medida como el número de individuos capturados en 100 trampas/noche o en 100 regurgitaciones), de todas las especies registradas con trampas de tipo Sherman y de caída y mediante el análisis de regurgitaciones de aguilucho lagunero. Para cada método evaluado se presentan el número de géneros y de especies identificados, el número de individuos capturados, la abundancia, el índice de uniformidad de Pielou, el índice de diversidad de Shannon–Wiener y el índice de diversidad de Simpson. Los índices se calcularon con los datos a nivel de género, lo que permitió comparar los métodos. Los números en negrita son los valores más altos obtenidos en los parámetros indicados: * Endemismo ibérico.

Conservation status Sherman Pitfalls Pellets

Taxa Activity PT IUCN N %O A N %O A N %O A

Rodentia

Apodemus sylvaticus N LC LC 79 22.44 1.35 1 5.26 0.13 8 13.79 10.67

Arvicola sapidus N/D LC VU – – – – – – 2 3.45 2.67

Microtus agrestis N LC LC 2 0.57 0.03 – – – 15 25.86 20.00

Microtus lusitanicus N/D LC LC 33 9.38 0.56 – – – 7 12.07 9.33

Microtus sp. – – – – – – 9 47.37 1.15 – – –

Mus musculus N LC LC – – – – – – 4 6.90 5.33

Mus spretus N LC LC – – – – – – 6 10.34 8.00

Mus sp. – – – 167 47.44 2.85 2 10.53 0.26 – – –

Rattus norvegicus N NA LC 1 0.28 0.02 – – – 4 6.90 5.33

Eulipotyphla

Crocidura russula N/D LC LC 70 19.89 1.20 5 26.32 0.64 7 12.07 9.33

Sorex granarius N/D DD* LC – – – – – – 3 5.17 4.00

Sorex minutus N/D DD LC – – – – – – 1 1.72 1.33

Sorex sp. – – – – – – 2 10.53 0.26 – – –

Talpa occidentalis N/D LC* LC – – – – – – 1 1.72 1.33

No. identified genera 5 5 8 No. identified species 5 2 11 Total captures 352 19 58

Abundance 6.02 2.44 77.33 Pielou eveness index 0.78 0.83 0.84 Shannon–Wiener index 1.26 1.33 1.75 Simpson diversity index 0.68 0.72 0.80

Animal Biodiversity and Conservation 38.1 (2015) 43

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Observed species richnessClench equationChao1 estimator

Observed species richnessClench equationChao1 estimator

0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Samples (pellets) Samples (pellets)

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Observed species richnessClench equationChao1 estimator

Observed species richnessClench equationChao1 estimator

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Fig. 2. A. Species accumulation curve and estimated number of species (Chao 1 estimator) for the small mammal assemblage preyed by marsh harriers; B–E. Genera accumulation curve and estimated number of genera identified through marsh harrier pellets (B), Sherman traps (C), pitfalls (D), Sherman and pitfall traps altogether (E). Observed data were fitted to the Clench equation to evaluate the completeness of the inventories.

Fig. 2. A. Curva de acumulación de especies y riqueza de especies estimada (estimador Chao 1) para el agregado de pequeños mamíferos cazado por los aguiluchos laguneros; B–E. Curva de acumulación de géneros y riqueza estimada de los géneros identificados a través de las regurgitaciones de aguilucho lagunero (B), las trampas de tipo Sherman (C), las trampas de caída (D) y las trampas de tipo Sher-man y de caída juntas (E). Los datos observados se ajustaron a la ecuación de Clench para evaluar la exhaustividad de los inventarios.

as members of the genus Crocidura, which, when wet and anxious, may be difficult to carefully ob-serve and distinguish. It is important to emphasize that no animals were intentionally sacrificed for later

identification in the lab, so all morphometric analyses were done in the field and in live individuals, which were ultimately released. In fact, due to conservation constraints, the sacrifice of terrestrial vertebrates is

44 Matos et al.

even in studies evaluating indirect and non–invasive sampling methods (Jaksić et al., 1981). Marsh harrier pellets, for instance, proved to be particularly efficient for inventorying species richness. However, population parameters such as abundance are probably more accurate when calculated with direct approaches, such as capture–recapture schemes (Hopkins & Kennedy, 2004). Also, due to the large home range and ecolo-gical preferences of raptors, pellets may fail to provide accurate information on the microhabitat preferences of small mammals.

Very few studies have used pellets of a diurnal raptor to study the assemblages of small non–volant mammals (but see Santos & Fonseca, 2009; Scheibler & Christoff, 2007). Most studies use pellets of common and widespread nocturnal birds of prey, such as Tyto alba, a generalist species that presents a relatively narrow home range, pellets that are very easy to find and collect, and prey remains that are easy to identify through cranial structures. However, owls mainly prey on small nocturnal mammals and may over or under sample some particular type of prey present in specific habitats that they may prefer or avoid, respectively (Torre et al., 2004). The same is true for marsh ha-rriers, but these birds seem to forage on habitats that are inaccessible to other birds of prey, such as high crops and reedbeds (Kitowski, 2007). The predator’s hunting time may bias the sampling results towards more nocturnal or diurnal species, but our data suggest this may not be a major issue, because though marsh harriers are mostly diurnal, their pellets contained rele-vant amounts of mammals described as predominantly nocturnal (e.g., Apodemus sylvaticus and Microtus agrestis; table 1). This may be due to the general activity patterns of small mammals, which are rarely exclusively nocturnal or diurnal. Even short periods of diurnal activity may represent hunting opportunities for fast raptors, such as the marsh harrier, that use the raid hunting technique. Furthermore, in wetlands and other open areas that lack nesting sites for owls —such as the Baixo Vouga Lagunar— owl pellets are not even an option. On the other hand, the marsh harrier is a widespread species in the region, easy to identify and spot for pellet deposit sites. Studies developed in the study area (Alves, 2013) confirmed the diet of marsh harrier as generalist and mostly (68%) consti-tuted of small mammals. It was also confirmed that marsh harriers forage on reedbeds and saltmarshes —wetlands that can be quite difficult to sample with traps, due to regular flooding, tides (in salt marshes) and vegetation density— but also on crops, providing a general sampling of the small mammals of a vast number of habitats. Our results also show that marsh harrier pellets provided more complete information on small non–volant mammal richness, and potentially of evenness and diversity. Table 3 summarizes further advantages and disadvantages of the three methods assessed in our study.

Finally, it should be highlighted that when designing any study on the structure of animal assemblages the choice of methods must carefully consider not only the technical limitations, but also the purposes of the study and ethical and legal questions. The

neither ethically acceptable for the purpose of this study, nor legally permitted, so the identification of those individuals remained ambiguous. Conversely, all mammal remains present in marsh harrier pellets were identified to the species level, based on cranial and/or fur features.

Sherman traps tend to capture species with high capturability, oversampling these species and under-sampling trap–shy species (Iriarte et al., 1989). Trap size and layout, among many other factors, influence their effectiveness (Smith et al., 1975). According to Boonstra & Krebs (1978), pitfalls are more efficient in sampling individuals of all ages among the small ma-mmal assemblage but may fail to capture species with greater body size (M. Alves, personal observation). In our study area, it is possible that Sherman traps may have oversampled small mammal species that are more active on the surface, such as Apodemus sylvaticus and Crocidura russula, though presenting higher capturability rates than fossorial species, such as Microtus rodents, which may be undersampled. On the other hand, pellet sampling may be biased by the predator’s ecological habits and preferences. Indeed, previous studies in the area have shown that during the breeding season, marsh harriers significantly pre-fer to forage on reedbeds (Alves et al., 2014), and to feed on Microtus species over other more abundant taxa (Alves, 2013). Also, marsh harriers may range over distances of up to 5,000 m from the nest during the breeding season (Cardador et al., 2009). Pellets may thus reflect a larger foraging area than that surveyed by trapping techniques; this may be useful when the objectives are to sample wide study areas.

Consistent differences in the estimates of species richness, abundance and proportion of species bet-ween the three methods suggest that supporting an assemblage study using only one method may lead to seriously biased results. Using various sampling methods combined is a way to overcome the biases of each method and obtain more complete information on the non–volant small mammal assemblage present in the study area. This is a well–established and recurring conclusion (Smith et al., 1975; Williams & Braun, 1983),

Table 2. Summary of the estimated cost for each sampling method (total working hours and total expenses): S. Sherman traps; Pt. Pitfall traps; Pl. Pellets.

Tabla 2. Resumen de la estimación de costos para cada método de muestreo (total de horas de trabajo y gastos totales): S. Trampas de tipo Sherman; Pt. Trampas de caída; Pl. Regurguitaciones.

S Pt Pl

Total working (hours) 1,008 504 856

Total expenses (€) 1,596 1,211 365

Animal Biodiversity and Conservation 38.1 (2015) 45

Pellets are hard (or sometimes impossible) to collect, especially in flooded or hard–reaching areasCollection is more time–consuming than pitfallsNot suitable to estimate densityThe quality of the results depends on the diet of the birds, which is influenced by environmental constraints (e.g. landscape features and prey availability)Does not provide information on the spatial ecology of small mammals.May underestimate nocturnal and overestimate diurnal prey species

Time–consuming and expensive. High logistical requirementsFrequently sprung–but–emptyNot usable in all kind of habitats (e.g. wetlands)Lower potential to detect rare speciesResults biased towards trap–prone speciesMay oversample species that live at the surfaceTrapping success largely depends on external conditions that influence animal activity

Time–consuming and expensiveHigh logistical requirementsVery conspicuous apparatus, subject to vandalism or theftLower potential to detect rare speciesBiased towards common speciesMay oversample species that live at the surfaceMay fail to capture larger animalsHigh mortality rates, not suited for studies with endangered speciesTrapping success largely depends on external conditions that condition animal activity

Low logistical requirementsCost–effectiveLess time–consuming than Sherman trapsMore positive identification of speciesHigher completeness and effectiveness in species inventory, especially in heterogeneous and wide areasDetection of species occupying habitats where trapping is not possibleHigher potential to detect rare speciesProvides more accurate information at the species level, allowing better diversity and evenness calculationsNon–invasive method for small mammals

Table 3. Comparative list of advantages and disadvantages of using Sherman traps, pitfalls and the analysis of marsh harrier pellets for small mammal studies, according to our study.

Tabla 3. Lista comparativa de las ventajas e inconvenientes de utilizar trampas de tipo Sherman, trampas de caída y el análisis de las regurgitaciones de aguilucho lagunero para los estudios sobre pequeños mamíferos, según nuestro estudio.

Advantages Disadvantages

Marsh harrier pellets

Sherman traps

Pitfalls

Lower mortality than pitfallsAllows the observation of gender, age and physical conditionAllows capture–mark–recapture techniquesSuitable for density estimationProvides information on the spatial ecology of small mammals

Less time–consuming than Sherman traps or pellet analysisAllows simultaneous and sequential multiple capturesAllows the observation of gender, age and physical conditionAllows capture–mark–recapture techniquesSuitable for density estimationMore successful in sampling trap–shy species than Sherman traps

46 Matos et al.

efficiency of marsh harrier pellets —and also other non–invasive methods— showed that, in some ca-ses, and depending on the objectives (for instance, presence–absence data), trapping is unnecessary, thereby avoiding disturbance and fatalities among small mammals (Powell & Proulx, 2003; Sullivan & Sullivan, 2013). Collecting pellets, in particular near nests, may disturb the birds (Fernández & Azkona, 1993), but if the study is well planned and takes the reproductive and spatial ecology and the habits of the species into consideration, impact can be minimized. In our study, pitfalls presented considerable involun-tary fatality rates for small mammals, mostly due to the humidity in the buckets, causing the animals to die from hypothermia. This sampling approach cannot therefore be recommended for small non–volant ma-mmals in wetlands or areas with high humidity levels. In regions that harbour endangered or rare species, we encourage researchers to seek for non–invasive methodologies, or, at least, to previously determine the least detrimental research protocol for wildlife, safeguarding animal and ecosystem welfare.

Acknowledgements

This work was co–supported by European Funds through COMPETE and by National Funds through the Portuguese Science Foundation (FCT) within project PEst–C/MAR/LA0017/2013. The authors would like to thank Câmara Municipal de Estarreja and OHM Estarreja for logistical and financial support. We also thank Eduardo Mendes for support in field work, Rita Rocha, Eduardo Ferreira and Victor Bandeira for their help identifying food items, and two anonymous reviewers for their helpful comments on a previous version of the manuscript. Milena Matos and Maria João Ramos Pereira were financed by post–doctoral grants from Fundação para a Ciência e Tecnologia (SFRH/BPD/74071/2010 and SFRH/BPD/72845/2010, respectively). All animals were captured and handled in accordance with Portuguese law (licenses 385/2011/CAPT and 99/2012/CAPT issued by ICNF–Institute for the Conservation of Nature and Forests). Pel-lets were collected under the ICNF license 95/2012/PERTURBAÇÃO.

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Vieira, B. P., Fonseca, C. & Rocha, R. G., 2015. Critical steps to ensure the successful reintroduction of the Eurasian red squirrel. Animal Biodiversity and Conservation, 38.1: 49–58.

AbstractCritical steps to ensure the successful reintroduction of the Eurasian red squirrel.— Wildlife reintroduction strategies aim to establish viable long–term populations, promote conservation awareness and provide economic benefits for local communities. In Portugal, the Eurasian red squirrel (Sciurus vulgaris) became extinct in the 16th century and was reintroduced in urban parks in the 1990s, mainly for aesthetic and leisure purposes. We evaluated the success of this reintroduction in two urban parks and here described the critical steps. We assessed habitat use, population density and abundance, and management steps carried out during reintroduction projects. Reintroductions have been successful to some extent given squirrels are present 20 years after release. However, populations in both parks are declining due to the lack of active management and poor quality habitat. Successful reintroduction of Eurasian red squirrel in areas without competition of alien tree squirrels involves three critical main stages. The pre–project stage includes studies on habitat quality, genetic proximity between donors and closest wild population, and health of donor stocks. In the release stage, the number of individuals released will depend on resource variability, and the hard release technique is an effective and economically viable method. Post–release activities should evaluate adaptation, mitigate mortality, monitor the need for supplementary feeding, provide veterinary support, and promote public awareness and education.

Key words: Conservation, Management, Release, Rodentia, Sciurus vulgaris, Urban park

ResumenPasos fundamentales para garantizar la eficacia de la reintroducción de la ardilla roja.— El objetivo de las estrategias de reintroducción de fauna silvestre es establecer poblaciones viables a largo plazo, fomentar la concienciación con respecto a la conservación y aportar beneficios económicos para las comunidades locales. La ardilla roja (Sciurus vulgaris), que estaba extinta en Portugal desde el s. XVI, fue reintroducida en varios parques urbanos en la década de los años 90, principalmente con fines estéticos y recreativos. Evaluamos la eficacia de esta reintroducción en dos parques urbanos y describimos los pasos fundamentales de la misma. Se evaluaron la utilización del hábitat, la densidad y abundancia de la población y las medidas de gestión adoptadas durante los proyectos de reintroducción. Las reintroducciones fueron relativamente eficaces dado a que las ardillas seguían presentes 20 años después de la liberación. No obstante, las poblaciones en ambos parques están disminuyendo debido a la falta de una gestión activa y a la mala calidad del há-bitat. La reintroducción eficaz de la ardilla roja en zonas donde no hay ardillas arborícolas exóticas conlleva tres etapas fundamentales. La etapa previa al proyecto comprende estudios sobre la calidad del hábitat; la proximidad genética entre los donantes y la población silvestre más cercana, y la salud de las poblaciones donantes. En la etapa de liberación, el número de individuos liberados dependerá de la variabilidad de los recursos disponibles; asimismo, se ha observado que la técnica de liberación dura es un método eficaz y viable desde el punto de vista económico. Las actividades posteriores a la liberación deberían analizar la adaptación, mitigar la mortalidad, hacer un seguimiento de la necesidad de aportar alimentación complementaria, prestar apoyo veterinario y fomentar la sensibilización pública y la educación.

Palabras clave: Conservación, Gestión, Liberación, Rodentia, Sciurus vulgaris, Parque urbano

Received: 2 V 14; Conditional acceptance: 18 XI 14; Final acceptance: 9 II 15

Bianca P. Vieira, Post–graduate Research Program, Inst. of Biodiversity, Animal Health and Comparative Medicine, Univ. of Glasgow, G12 8QQ, Glasgow, U. K.– Carlos Fonseca & Rita G. Rocha, Depto. de Biologia & CESAM, Univ. de Aveiro, Campus Santiago, 3810–193, Aveiro, Portugal.

Corresponding author: Rita G. Rocha. E–mail: rgrocha@ua.pt

Critical steps to ensure the successful reintroduction of the Eurasian red squirrel

B. P. Vieira, C. Fonseca & R. G. Rocha

50 Vieira et al.

Introduction

Animal translocation is an ancient process used by humans to relocate species from one place to another (Griffith et al., 1989; Hodder & Bullock, 1997; Arms-trong & Seddon, 2007; Seddon et al., 2007; Ewen et al., 2012). Griffith et al. (1989) defined animal trans-location as the intentional release to establish, re–es-tablish or increase the population of a given species. Reintroduction is currently one of the most popular translocation strategies used in the management of species (Armstrong & Seddon, 2007; Seddon et al., 2007; Ewen et al., 2012). Wildlife reintroductions are conducted to establish viable populations, enhance long–term survival of a given species, settle long–term economic benefits for local communities, and to promo-te conservation awareness (IUCN, 1998). Reintroduc-tions should be carefully planned by a multidisciplinary team, and follow a three–step protocol, focusing on the pre–project activities, release stages and post–released activities (IUCN, 1998). Such projects require complex planning, implementing and monitoring species and habitats according to their biology, socio–economic impact on local communities, and legal requirements (Caughley & Gunn, 1996; IUCN, 1998; Armstrong & Seddon, 2007; Seddon et al., 2007; Ewen et al., 2012; Harrington et al., 2013). Parameters of success chan-ge in each project but should follow the principles of long–term survival of species while providing benefits for the local community and fostering conservation awareness (IUCN, 1998, 2012). The potential positive impact of reintroductions depends on temporal, spa-tial, and taxonomic factors (Ewen et al., 2012), and if reintroductions are not properly carried out they can damage both donor and receptor populations as well as ecosystems (Hodder & Bullock, 1997; Armstrong & Seddon, 2007; Seddon et al., 2007; Ewen et al., 2012). Therefore, publication and dissemination of successful and unsuccessful cases contribute to improve current reintroduction protocols (Armstrong & Seddon, 2007; Seddon et al., 2007; Ewen et al., 2012; IUCN, 2012).

Mammals, together with birds, are the most fre-quently chosen groups for releases with conservation purposes (Griffith et al., 1989; Seddon et al., 2005, 2007). Although most of reintroductions focus on ungulates and carnivores (Seddon et al., 2005), ro-dents such as the edible dormouse Glis glis in Poland (Jurczyszyn, 2006) and the European ground squirrel Spermophilus citellus in Central Europe (Matějů et al., 2010) have also been released in the last 20 years. The most commonly reported reintroductions among rodents are those concerning the Eurasian red squirrel Sciurus vulgaris reintroductions, with a considerable number of programmes being imple-mented in Europe over the last 30 years (Swinnen, 1988; Fornasari et al., 1997; Wauters et al., 1997a, 1997b; Poole & Lawton, 2009).

Although the Eurasian red squirrel is a widespread Palearctic species (Lurz et al., 2005; Shar et al., 2008; Bosch & Lurz, 2012), some of its populations, particularly in the United Kingdom and Italy, are threatened or extinct due to habitat loss, hunting, disease and competition with alien tree squirrels

Sciurus carolinensis, Callosciurus erythraeus and C. finlaysonii (Gurnell, 1987; Wood et al., 2007; Bosch & Lurz, 2012; Bertolino & Lurz, 2013). In Portugal, the Eurasian red squirrel became extinct in the 16th century due to significant habitat loss, and it only reappeared in extreme northern areas around the 1980s (Mathias & Gurnell, 1998; Ferreira et al., 2001). One decade later, isolated reintroductions occurred in some urban parks, but no monitoring has been conducted since then to understand population dynamics and their status or to evaluate manage-ment and success. In order to determine whether Eurasian red squirrel reintroductions carried out in Jardim Botânico da Universidade de Coimbra and in Parque Biológico de Gaia were successful or not, we estimated population viability through density, abundance, and habitat use in released sites. We also evaluated stepwise reintroduction in both urban parks, based on the IUCN guidelines (IUCN, 1998, 2012) to highlight critical steps and suggest actions ensure the long–term persistence and viability of these Eurasian red squirrel populations.

Material and methods

Study area

The Parque Biológico de Gaia (PBG), which was created in 1983, initially covered 2 ha but has been extended to include 35 ha (Oliveira, 2013). It is situated in Vila Nova de Gaia, northern Portugal (41º 05' N and 8º 33' W; fig. 1A) and it is managed by a municipal company (Oliveira, 2013). This urban park is composed of open areas, a wildlife rehabili-tation center and monospecific forests of black alder Alnus glutinosa, oak Quercus robur or cork oak Q. suber (fig. 1A). It also has enclosures distributed throughout open areas in the park containing wil-dlife that could not be rehabilitated or that provide examples of native and exotic fauna. The aim is to promote environmental education, as the case of the Eurasian red squirrel.

The Jardim Botânico da Universidade de Coimbra (JBUC) was created in 1772 as part of the Museu de História Natural da Universidade de Coimbra (UC, 2013). It covers 13 ha in Coimbra city, central Portugal (40º 12' N and 8º 25' W; fig. 1B). This park comprises mainly gardens and forests of alien flora (fig. 1B).

Eurasian red squirrel survey

From 7th October to 11th November 2013, 15 walking transects of 100 m were established in each study site. All transects were surveyed in the morning and afternoon for seven days to avoid biases from squi-rrel behavior (Gurnell et al., 2001). Transects were performed one after other to avoid double counting of individuals moving from one transect to another. Transects were selected to include all habitat types and to cover most of the area at each of the two ur-ban parks, but with at least 20 m distance from each

Animal Biodiversity and Conservation 38.1 (2015) 51

other also to avoid double counting (fig. 1; Gurnell et al., 2001). We counted squirrels using the distance sampling method with direct observation using bino-culars 8–16 x 40 (Gurnell et al., 2001), given that both parks had great visibility with small and clear forests. Squirrel surveys were conducted between 8:00 and 16:00 in autumn when higher numbers of squirrels can be found (Tonkin, 1983; Wauters et al., 1992; Bosch & Lurz, 2012). The distance from the observer to the squirrel was measured using a telemeter, and compass bearings were taken to determine the angle between the animal and the transect line (Buckland et al., 1993; Gurnell et al., 2001). We measured

the distance of squirrels once and did not consider individuals again after moving to a different position.

Population density and abundance were estimated using Distance Sampling 6.0 software (Thomas et al., 2010). Estimates were stratified and based on Conventional Distance Sampling. Half–normal, hazard and negative exponential rate models for the detection function were fixed against the records using a cosi-ne function (Thomas et al., 2010). Models assumed certainty of detection and measurements (Thomas et al., 2010). The selection of the best model and adjustment term were based on the lowest Akaike information criterion (AIC).

Oak forestBlack alder forestOpen areasCork oak forestMaritime pine forestAlien conifer gardenEurasian red squirrell drey

Portugal

0 65 130 m

0 45 90 m

40º

05' 5

1'' N

40º

06'

04'

' N40

º 12

' 17'

' N

40

º 12

' 24'

' N

8º 25' 26'' W 8º 25' 13' W

8º 33' 38'' W 8º 33' 12'' W

Fig. 1. Study area in northern and central Portugal with inset showing vegetation type and distribution of dreys (white circles) in the Parque Biológico de Gaia at Vila Nova de Gaia (A), and in the Jardim Botânico da Universidade de Coimbra at Coimbra (B).

Fig. 1. Zona de estudio situada en el norte y el centro de Portugal con recuadros que muestran el tipo de vegetación y la distribución de los nidos de ardilla (círculos blancos) en el Parque Biológico de Gaian en Vila Nova de Gaia (A) y en el Jardín Botánico de la Universidad de Coimbra, en Coimbra (B).

N

B

A

Atlantic Ocean

Mountain ash forestBamboo forestNarrow–leafed ash gardenPalm gardenCentral squareMixed alien forest

52 Vieira et al.

Habitat use

Eurasian red squirrels prefer mature native forests that can provide them with an abundant supply of food (Bosch & Lurz, 2012). We assessed vegetation type, location of dreys (i.e. squirrel nests) and food availability to understand habitat use in both urban parks. The survey was conducted in the PBG in October 2013 and in the JBUC in November 2013. Vegetation type (fig. 1) was mapped with a geogra-phic information system in ArcView GIS 9.2 software (ESRI, 2008). The geographical limits of forests and gardens having the same composition and dominance were confirmed in the field. The number of trees to determine dominant species was verified in 10 x 10 m quadrats randomly within the study sites. Due to di-fferent area sizes, 80 quadrats were located in PBG and 60 in JBUC.

Dreys were mapped to determine preferences in relation to vegetation type (fig. 1). Drey counts were obtained by direct observation in a 3 km transect at each site. Transects to count dreys were larger than transects to count individuals because dreys were fixed and double counting was unlikely. We determined the position of dreys, tree species chosen and drey height (Cagnin et al., 2000; Kopij, 2009). Old or abandoned dreys were excluded from counts (Wauters & Dhondt, 1988; Cagnin et al., 2000; Kopij, 2009). The significance of the distribution of dreys in relation to height was measured using a one–way ANOVA in Bioestat 5.0 software (Ayres et al., 2007). Tukey’s post hoc test (F) was applied to determine the significance of any differences (Zar, 1999).

Food availability focused on three aspects: number of feeders, relative abundance and richness of edible mushrooms, and energetic content of natural seeds (cones, acorns, hackberries, and nuts). Feeders with supplementary food were counted directly. Relative abundance of Basidiomycota was estimated by counting fungal bodies or remains with characteristic squirrel bites in stipe and cap on the ground in the same 10 x 10 m quadrats where the vegetation type was measured. Only mushrooms eaten by squirrels during the surveys or reported in the literature were

considered as a component of Eurasian red squirrel diet (Fogel & Trappe, 1978; Bertolino et al., 2004). Fungi identification and nomenclature follows Crous et al. (2004).

In each 10 x 10 m quadrat, the number of trees with fruits of each species was counted. Only tree species already reported in the literature (Lurz et al., 2005; Bosch & Lurz, 2012) or those seen being consumed during fieldwork were considered as a component of the Eurasian red squirrel diet. A quadrat of 5 x 5 m was placed below every tree bearing fruit inside the 10 x 10 m quadrat to count fallen cones, acorns, hac-kberries or nuts. The remains of fruits consumed by Eurasian red squirrels were also recorded and identified by characteristic squirrel bites. Only natural sources of seeds were evaluated given that the composition of seeds offered in feeders varied widely. Seed counts provided an estimate of seed availability (calculated as 103 seeds / ha, Bosch & Lurz, 2012). We used data on seed production and calorific content obtained from the literature (Grodziński & Sawicka–Kapusta, 1970; Demir et al., 2002; Wauters et al., 2002; Bosch & Lurz, 2012; Stock et al., 2013) to measure the mean energy value (103 kJ / ha–1) and standard deviation (± SD) related to seed counts per habitat.

Reintroduction management

Reintroductions were considered successful as viable populations were established, long–term benefit for local communities were achieved, and improvements in conservation awareness were made, in accor-dance with IUCN guidelines (IUCN, 1998, 2012). Qualitatively data on management were assessed by unstructured interviews with park managers and employees, and by consulting official documents. We investigated release histories according to motiva-tion, year of release, reintroduction technique (e.g., soft release in which animals are first acclimatized with new habitat in enclosures before release, or hard release in which individuals are directly released into the new environment; see Ewen et al., 2012), supplementary feeding, veterinary support, choice of donor population, number of individuals released,

Table 1. Best–fitting models according to Akaike information criterion (AIC) and degree of freedom (df) values to estimate the population density of Eurasian red squirrels at the Parque Biológico de Gaia (PBG) and at the Jardim Botânico da Universidade de Coimbra (JBUC), Portugal, in autumn 2013.

Tabla 1. Los mejores modelos según el criterio de información de Akaike (AIC) y los valores del grado de libertad (df) para estimar la densidad de la población de ardilla roja en el Parque Biológico de Gaia (PBG) y en el Jardín Botánico de la Universidad de Coimbra (JBUC), en Portugal, en otoño de 2013.

PBG JBUC

Model Negative exponential Half–normal Hazard Negative exponential Half–normal Hazard

AIC 378.09 378.28 376.65 17.51 17.56 19.56

df 60 59 59 3 3 2

Animal Biodiversity and Conservation 38.1 (2015) 53

Table 2. Estimation of seed production in each habitat at the Parque Biológico de Gaia (PBG) and at the Jardim Botânico da Universidade de Coimbra (JBUC), Portugal, in autumn 2013: S. Seed (103 seeds/ha); Sec. Seed energetic content (103 kJ/ha–1). Habitats: Of. Oak forest; Baf. Black alder forest; Oa. Open area; Cof. Cork oak forest; Mpf. Maritime pine forest; Ecg. Exotic conifer garden; Maf. Mountain ash forest; Bf. Bamboo forest; Nag. Narrow–leafed ash garden; Pg. Palm garden; Cs. Central square; Mef. Mixed exotic forest.

Tabla 2. Estimación de la producción de semillas en cada hábitat en el Parque Biológico de Gaia (PBG) y en el Jardín Botánico de la Universidad de Coimbra (JBUC), Portugal, en otoño de 2013: S. Semillas (103 seeds/ha); Sec. Contenido energético de las semillas (103 kJ/ha–1). Hábitats: Of. Robledal; Baf. Alisal; Oa. Zona despejada; Cof. Alcornocal; Mpf. Pinar de pino negral; Ecg. Plantación de coníferas exóticas; Maf. Bosque de eucalipto regnans; Bf. Bosque de bambú; Nag. Plantación de fresno de hoja pequeña; Pg. Palmeral; Cs. Cuadrado central; Mef. Bosque mixto exótico.

Urban parks Measured parameters

Habitat S Sec PBG

Of 3.6 ± 2.2 96.4 Baf 143.8 ± 163.6 9,524 Oa – – Cof – – Mpf 74.6 ± 35.2 1,859

JBUC Of 974.0 ± 534.9 20,779 Oa – – Ecg – – Maf – – Bf – – Nag – – Pg – – Cg – – Mef 56.3 ± 19.5 3,821

an important food source in JBUC, with a relative abundance of 30.4% for R. foetens in the diet, which together with Amanita gemmata represented 91.2% of available edible mushrooms in the JBUC (table 3). Supplementary feeding was not recorded in this urban park during the study.

reinforcements, population expansion or decrease, presence/absence of dispersal into surrounding areas (distances greater than 500 m and less than 35 km from release sites), and the presence/absence of squirrels killed on roads.

Results

Eurasian red squirrel population

During the surveys, we observed 61 individuals in the PBG and four in the JBUC. The best relative fit model and adjustment term for the population in the PBG was a hazard–rate cosine based on the lowest AIC score (table 1). In contrast, the best fit for the JBUC population was a negative exponential cosine model based on the lowest AIC score (table1). Estimated abundance and density were higher in PBG (N = 47 squirrels, D = 1.33 squirrel/ha) than in JBUC (N = 2 squirrels, D = 0.17 squirrel/ha). The detection proba-bility in the PBG was 44% whereas in the JBUC it was 51.5%. The encounter rate was 56% and 48.5% for the PBG and JBUC, respectively.

Habitat use

Seven squirrel dreys were found placed in the oak and cork oak forest at PBG (fig. 1B). Squirrels placed a significant portion of dreys in the height of 13 m in forests dominated by Q. robur and Castanea sativa (F = 8.35, df = 11, P < 0.01). Seven dreys were found around 14 m high in the oak forest in the JBUC (F = 15.74, df = 14, P < 0.01). One drey was found on Pseudotsuga menziesii in the alien conifer garden (fig. 1B).

During autumn 2013, only three tree species were fruiting in the PBG: Q. robur, C. sativa and P. pinaster (fig. 1A, table 2). The black alder forest had higher seed productivity (143.8 ± 163.6 x 103 seeds/ha) and energetic content (9,524 x 103 kJ/ha–1) due to the high concentration of fruiting C. sativa (table 2). We coun-ted 471 fungal bodies from 28 species in the PBG. Only 16% of mushrooms were edible to the Eurasian red squirrel (table 3). Russula spp. showed significant relative abundance of edible fungi in the PBG, with R. cyanoxantha and R. decipiens together accounting for 59.4% (table 3). Ongoing supplementary feeding in the PBG consisted of five feeders daily supplied with birdseed to attract birds, but these were also used by squirrels. Squirrels were observed eating mainly sunflower seeds.

Only the oak and mixed forests had fruiting trees in the JBUC during surveys (table 2), namely Pinus pinea, Quercus robur, and Celtis australis. Fruits of this last tree were seen being eaten by squirrels during fieldwork. The oak forest had higher seed productivity (974.0 ± 534.9 x 103 seeds/ha) and energetic content (20,779 x 103 kJ / ha–1) due to the high productivity of seeds per cone of P. pinea (table 2). In the JBUC, we counted 33 fungal bodies of seven species and 70% of them were edible to the Eurasian red squirrel (table 3). As in PBG, the genus Russula was also

54 Vieira et al.

Reintroduction management

Table 4 summarizes reintroductions attendance to IUCN stepwise. Both reintroductions in PBG and JBUC aimed at enhancing parks aesthetics and enable people to become familiar with this species (table 4). Park managers used the Eurasian red squirrel historical population observations of Antunes (1985) as proving of the species historical range in Portugal. Both urban parks acquired squirrels from commercial creators with veterinary control and support which lowered possibi-lities of diseases or parasites.

The PBG released 12 squirrels In 1997, and a further 40 couples between 1998 and 2001 using a hard release approach. The animals were from Azé (France). The squirrels in the PBG have continuous veterinary support, because a wildlife rehabilitation center is located therein (table 4), and continuous feeding is provided through bird feeders. Two main failures were detected in the reintroduction project in PBG: the absence of genetic comparison between donors and the closest wild population, and a lack of long–term, technical monitoring.

Twelve squirrels from Madrid (Spain) were hard released at JBUC in 1994. Four squirrel feeders in the forest were active only during the first year (ta-ble 4). As in the PBG, the reintroduction project at

Table 3. Relative abundance of mushrooms (Basidiomycota) recorded in the diet of Eurasian red squirrel (Sciurus vulgaris) and found at the Parque Biológico de Gaia (PBG) and at the Jardim Botânico da Universidade de Coimbra (JBUC), Portugal, in autumn 2013: * Consumption seen during fieldwork.

Tabla 3. Abundancia relativa de hongos (Basidiomycota) observada en la alimentación de la ardilla roja (Sciurus vulgaris) y encontrada en el Parque Biológico de Gaia (PBG) y en el Jardín Botánico de la Universidad de Coimbra (JBUC), en Portugal, en otoño de 2013: * Consumo visto durante el trabajo de campo.

Urban park RelativeTaxon Fungal bodies abundance (%) Source

PBG

Boletus aestivalis 1 1.3 Fogel & Trappe (1978)* Cantharellus cibarius 12 16.2 Fogel & Trappe (1978) Pholiota alnicola 9 12.1 Fogel & Trappe (1978) Amanita rubescens 1 1.3 Fogel & Trappe (1978) Russula cyanoxantha 30 40.5 * Russula decipiens 14 18.9 * Xerocomus chrysenteron 7 9.4 Fogel & Trappe (1978)*Total 74 100

JBUC

Agaricus campestris 2 8.7 Fogel & Trappe (1978) Amanita gemmata 14 60.8 Fogel & Trappe (1978) Russula foetens 7 30.4 *Total 23 100

this park did not consider genetic comparison between donors and the closest wild population. This project ended one year after releases and no post–project management measures and/or long–term monitoring were conducted (table 4).

Discussion

To date, reintroductions of Eurasian red squirrels in Portugal have been successful to some extent given that squirrels are still present in the urban parks almost 20 years later. However, the populations of squirrels are decreasing in both urban parks. Studies found densities from 0.03 to 1.80 squirrels/ha in mixed woodlands (Wauters & Dhondt, 1988; Cagnin et al., 2000; Magris & Gurnell, 2002; Vilar, 1997), figures that are similar to our estimate (0.17 squirrels/ha in JBUC and 1.33 squirrels/ha in PBG). Considering urban parks of limited area and resources, the density in the PBG is similar to densities found in Belgium (Wauters et al., 1997a) and Spain (Vilar, 1997, while the density in the JBUC is lower. The difference in density of the reintroduced populations is mainly related to post–release management since the PBG had squirrel population reinforcements but the JBUC did not. Habitat quality also regulates species

Animal Biodiversity and Conservation 38.1 (2015) 55

Table 4. Conditions of Eurasian red squirrel (Sciurus vulgaris) reintroductions in the Parque Biológico de Gaia (PBG) and in the Jardim Botânico da Universidade de Coimbra (JBUC), Portugal.

Tabla 4. Condiciones de las reintroducciones de ardilla roja (Sciurus vulgaris) en el Parque Biológico de Gaia (PBG) y en el Jardín Botánico de la Universidad de Coimbra (JBUC), en Portugal.

Phase

Aspect PBG JBUC

Pre–project Main motivation Aesthetic, leisure and Aesthetic, leisure and environmental education environmental education Origin of donor population Azé (France) Madrid (Spain) Captive or wild squirrels Captive Captive Subspecies of donor population Sciurus vulgaris fuscoater Sciurus vulgaris infuscatus Study of historical range Antunes (1985) Antunes (1985) of extinct populations Study of genetic individual variability No No of donor population Governmental permits Not required Not required Veterinary certification of health Yes Yes and absence of parasites Other certifications Origin and transportation Origin and transportation

Release Year of release November 1997 June 1994

Number of squirrels released 12 (6 and 6) 12 (6 and 6) Method (soft or hard) Hard release Hard release Supplementary feeding Five feeders for birds, Four squirrel feeders but used by squirrels Kind of supplementary feeding Birdseed with Walnuts, hazelnuts, sunflower seed and others

Post–project Population monitoring No No Veterinary support Yes No Continuity of supplementary feeding Yes, to date Stopped after one year Manager’s general feeling about Decrease and need Population explosionsquirrels abundance for reinforcement in next three years Population reinforcement Yes (three) No Subspecies used to reinforcement Sciurus vulgaris fuscoater – Number of individuals (origin, 10 couples (Epe, Netherlands, X 1998) – and year of population 15 couples (Epe, Netherlands, VII 2001) – reinforcement) 15 couples (Azé, France, VIII 2001 – Manager’s general feeling about Population explosion squirrel abundance after reinforcement in next five years – Squirrels seen in nearby areas Yes Yes (> 500 m and < 35 km from release site) Name of five localities where Sermonde, Vila Chã, Mata do Buçaco, Serra da squirrels were seen Serra da Agrela, Serra da Freita, Lousã, Alfarelos, and Marco de Canaveses Serra do Sicó, and Soure First year of squirrels seen in these 2010 2001 nearby localities Squirrels killed on nearby roads Yes Yes

56 Vieira et al.

abundance and density and is of great importance for the success or failure of reintroductions (Ewen et al., 2012). Squirrel dreys in both urban parks were predominantly placed in native oak forests and near food sources, reinforcing the need for high quality habitat and food diversity for the maintenance of these populations. The studied parks had few fruiting trees compared with other studies (Bosch & Lurz, 2012). Forests in the JBUC had a higher energetic content than those in the PBG but the diversity of native food items was poorer. In contrast, forests in the PBG had less energetic content, but they presented richer and more abundant additional food items, such as edible mushrooms. Additionally, the PBG had continuous supplementary food, mainly through bird feeders also used by squirrels, whereas the JBUC only had feeders in the year following the reintroduction.

In terms of species identity for conservation purpo-ses, genetic proximity was only adequately considered in the JBUC where the subspecies Sciurus vulgaris infuscatus was reintroduced, while in the PBG the subspecies S. v. fuscoater was released. Although both subspecies occur in the Iberian Peninsula, only S. v. infuscatus occurs naturally in Portugal (Mathias & Gurnell, 1998; Lurz et al., 2005; Bosch & Lurz, 2012). Further studies on Eurasian red squirrel distribution, taxonomy and genetic diversity in the Iberian Penin-sula should consider the influence of S. v. fuscoater presence in Portugal, as has been done to other subspecies in the United Kingdom (see Hale & Lurz, 2003; Hale et al., 2004).

Post–project monitoring was not explicitly conside-red in either park. It is there not fully understood whe-ther dispersal to vicinity (table 3) was natural or due to stress of limited resources. Deficiency in post–release actions, such as monitoring health and abundance, is responsible for the long–term decrease in Eurasian red squirrel populations in both urban parks but acti-ve adaptive management could improve the current situation (Ewen et al., 2012; Runge, 2013). Future actions should consider improving habitat quality by means of specific feeders for Eurasian red squirrels, and replacement of alien trees for native oak forest. Monitoring population health, adaptation and demo-graphic variation will endorse the long–term success of the reintroductions. In addition, managers should ensure active human community involvement so that effective education would not only foster knowledge of species but also concern for its needs (IUCN, 2012).

Critical steps for successful reintroduction of Eura-sian red squirrels in areas without competition of alien tree squirrels should follow three stages, consisting of pre–project activities, release stages and post–release activities (IUCN, 1998, 2012). Pre–project activities should include studies on (1) habitat quality, (2) ge-netic proximity between donors and the closest wild population, and (3) the health of donor stocks.

In the release stage, (1) the number of individuals released should consider 35 to 85 individuals to achieve a long–term viable population in an area of high resource variability, and 55 to 175 individuals in areas of low resource variability (Wood et al., 2007) and (2) hard release technique proved to be a good

and cheaper method to Eurasian red squirrel rein-troductions (Swinnen, 1988; Fornasari et al., 1997). Finally, post–release activities should (1) evaluate population adaptation, (2) mitigate mortality, (3) mo-nitor the need for supplementary feeding, (4) provide veterinary support, and (5) promote continuous public awareness and education (IUCN, 1998, 2012).

Reintroductions for aesthetic and leisure purposes are not usually concerned about strictly following conservation protocols unless required by law. Howe-ver, these reintroductions for aesthetic and leisure purposes have significant effects on wildlife mana-gement and conservation (Hodder & Bullock, 1997). Therefore, we strongly suggest that reintroductions with aims other than conservation should also have standardized international guidelines, regulations and monitoring.

Acknowledgments

We thank Maria A. Neves for helping with mushroom identification, and Paulo Trincão (Jardim Botânico da Universidade de Coimbra) and Nuno G. Oliveira (Parque Biológico de Gaia) for permits, institutional support, and data on reintroductions. We also thank the reviewers for improving this manuscript. Bianca P. Vieira had support from the Brazilian National Council for Scientific and Technological Development (CNPq) under a Science without Borders fellowship (nº 221.575/2012–0). The project was partially supported by European Funds through Operational Program for Competitiveness Factors and by National Funds through the Portuguese Science Foundation (PEst–C/MAR/LA0017/2013).

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Ruiz–García, A. & Ferreras–Romero, M., 2015. El estado ecológico de las pequeñas cuencas de cabecera en las serranías béticas húmedas (parque natural Los Alcornocales, sur de España) según la Directiva Marco del Agua: ¿su aplicación garantiza la conservación? Animal Biodiversity and Conservation, 38.1: 59–69.

AbstractEcological status of headwaters in the wet Betic Mountains (Los Alcornocales Natural Park, southernmost Spain) according to the WFD: does the application of this Directive ensure conservation?— In compliance with the Euro-pean Water Framework Directive, member states have had to develop a method to assess the quality of aquatic ecosystems by comparing the current situation regarding near–natural reference conditions for each river type. In 2008, the Spanish Ministry of Environment approved the Order of Water Planning Statement. This statement sets out reference conditions and ecological status class change limits for the different types of rivers in Spain for which sufficient data are available. In the present study, we established reference conditions and quality class thresholds for streams classified as wet Betic mountain rivers from 24 reaches of streams located in Los Alcorno-cales Natural Park, using two qualitative indices based on macroinvertebrates (IBMWP and IMMi–L). The results for the IBMWP index indicate that from the standpoint of management of the ecological state, the watercourses studied show more affinity with the types of the Spanish Atlantic siliceous slope than with those of the Mediter-ranean siliceous slope when we consider EQR values. Considering the threshold values, the index resembles siliceous low Mediterranean mountain rivers (type 8). However, the EQR values do not match those calculated in this study. These results suggest that it is necessary to use an index adapted to the characteristics of these watercourses. Application of the quality criteria contained in the Guadalete–Barbate and Mediterranean–Anda-lusian Basin Plans to the management of these waterways is discussed, because it is unlikely that they ensure the maintenance of good ecological status. We thus propose a new calibration of the IBMWP index that ensures the maintenance of good environmental status of watercourses in this natural area, and the use of the IMMi–L index as an effective management tool. However, as our study area represents only a part of the wet headwa-ters in the southern Iberian peninsula, analysis of other basin types is necessary to complete such information.

Key words: WFD, Reference values, Quality class boundaries, IBMWP, IMMi–L, Wet–Betic Mountains

ResumenEl estado ecológico de las pequeñas cuencas de cabecera en las serranías béticas húmedas (parque natural Los Alcornocales, sur de España) según la Directiva Marco del Agua: ¿su aplicación garantiza la conserva-ción?— El cumplimiento de la Directiva Marco del Agua (DMA) de la Unión Europea (UE) ha obligado a los Estados miembros a elaborar una metodología para evaluar la calidad de los ecosistemas acuáticos a partir de la comparación de la situación actual respecto a unas condiciones de referencia casi naturales para cada tipo de río. El Ministerio de Medio Ambiente, y Medio Rural y Marino aprobó en 2008 la instrucción de planifi-cación hidrológica donde se recogen las condiciones de referencia y los límites de cambio de clase del estado ecológico de los distintos tipos de ríos españoles de los que se dispone de datos suficientes. En el presente estudio se establecen las condiciones de referencia y los umbrales de las clases de calidad para los cursos catalogados como ríos de serranías béticas húmedas, a partir de 24 tramos incluidos en el parque natural Los Alcornocales, mediante el empleo de dos índices de calidad basados en macroinvertebrados (IBMWP e IMMi–L). Los resultados obtenidos para el índice IBMWP indican que, desde el punto de vista de la gestión del estado ecológico, los cursos de agua estudiados muestran más afinidad con los tipos silíceos de la vertiente atlántica española que con los tipos silíceos mediterráneos cuando comparamos sus valores EQR (ecological

El estado ecológico de las pequeñas cuencas de cabecera en las serranías béticas húmedas (parque natural Los Alcornocales, sur de España) según la Directiva Marco del Agua: ¿su aplicación garantiza la conservación?

A. Ruiz–García & M. Ferreras–Romero

60 Ruiz–García & Ferreras–Romero

quality ratio). Si tenemos en cuenta los umbrales, el índice se asemeja al de los ríos de baja montaña mediter-ránea silícea (tipo 8). Sin embargo, los valores EQR no coinciden con los calculados en este estudio. Estos resultados parecen indicar que es necesario utilizar un índice adaptado a las características de estos cursos de agua. Si se aplican a la gestión de estos cursos de agua los criterios de calidad recogidos en los planes de las cuencas Guadalete–Barbate y Mediterránea Andaluza, el mantenimiento de su buen estado ecológico probablemente no estaría asegurado. Por este motivo, se propone volver a calibrar el índice IBMWP para que asegure el mantenimiento del buen estado ecológico de los cursos de agua de este espacio natural, así como utilizar el índice IMMi–L como un instrumento eficaz de gestión. No obstante, nuestra área de estudio solo representa uno de los tipos de cabeceras húmedas existentes en el sur de la península. Sería necesario estudiar el resto de los tipos de cuencas para tener la información completa.

Palabras clave: DMA, Valores de referencia, Clases de calidad, IBMWP, IMMi–L, Serranías béticas húmedas

Received: 8 V 14; Conditional acceptance: 3 VII 14; Final acceptance: 3 III 15

Antonio Ruiz–García & Manuel Ferreras–Romero, Depto. de Sistemas Físicos, Químicos y Naturales, Univ. Pablo de Olavide, A –376 km 1, 41013 Sevilla, España (Spain).

Corresponding author: Antonio Ruiz–García. E–mail: aruigar@upo.es

Animal Biodiversity and Conservation 38.1 (2015) 61

Introducción

La Directiva Marco del Agua, DMA (DOCE, 2000) constituye el marco legislativo comunitario en el ámbito de la política del agua. La Directiva supone un enfoque novedoso en el contexto europeo al con-templar la calidad del agua como uno de sus objetivos principales, mediante la obligatoriedad de conseguir el "buen estado ecológico" de los ecosistemas acuáticos de la UE. Este compromiso fue fijado originalmente para el año 2015, prorrogable hasta diciembre de 2027 (BOE, 2007, Real Decreto 907/2007).

Para determinar el buen estado ecológico de las masas de agua, hay que seguir una serie de indi-caciones especificadas en los anexos II y V de la Directiva. En el caso de los ríos, para conseguir los objetivos ambientales propuestos en la Directiva, es fundamental definir los diferentes tipos de ríos con características ambientales homogéneas y establecer un sistema de estaciones de referencia lo suficien-temente amplio para cada tipo. A partir de los datos anteriores podemos clasificar el estado ecológico de una masa de agua en relación con los valores de referencia del tipo al que pertenece (EQR) en cinco categorías (muy bueno, bueno, moderado, deficiente y malo) y cinco colores (azul, verde, amarillo, naranja y rojo) (Ortiz Casas, 2004).

Por otro lado, la DMA permite a los Estados miem-bros utilizar o elaborar sus propios métodos para calcular el estado ecológico, lo que puede dificultar saber si el “buen estado ecológico” es lo mismo en toda la UE (Friberg et al., 2011). Por este motivo, la Comisión Europea ha promovido un ejercicio de intercalibración para dar homogeneidad a los cortes de calidad usados por los distintos Estados miembros, de forma que los grados de calidad biológica sean parecidos en toda Europa (Munné & Prat, 2009).

De acuerdo con la DMA, la clasificación de los cursos de agua europeos puede llevarse a cabo mediante dos métodos diferentes. Por un lado (Sis-tema A), la diferenciación de clases tipológicas, que debe realizarse utilizando tres descriptores ambien-tales: altitud, tamaño y geología de la cuenca. Por otro (Sistema B), la diferenciación, que se basa en la utilización de cinco variables obligatorias (latitud, longitud, altitud, tamaño y características geológicas) y algunas opcionales. En un estudio llevado a cabo por Munné & Prat (2004) sobre varias cuencas medi-terráneas, se concluyó que el Sistema B refleja mejor las condiciones ambientales de la península ibérica, ya que toma en consideración variables ambientales clave, como las hidrológicas y climáticas.

En España ha habido algunos intentos de tipifi-cación, como los llevados a cabo por Vidal–Abarca et al. (1990) en la cuenca del Segura, Munné & Prat (1999) en la cuenca del Ebro o la propuesta de ca-racterización jerárquica de todos los ríos españoles de González del Tánago & García de Jalón (2006). Pero sin lugar a dudas, la mayor tentativa la ha constituido el proyecto GUADALMED (fases primera y segunda), uno de cuyos objetivos fue elaborar una tipología óptima a partir de la cual calcular el estado ecológico de los ríos mediterráneos españoles (Prat,

2004). En una primera fase, se estableció una tipo-logía preliminar, donde se reconoce que el Sistema B muestra una mayor coherencia ecológica con la biología de las comunidades características de cada ecotipo (Bonada et al., 2004), que fue mejorada en una segunda fase con la inclusión de nuevas localidades de referencia y variables ambientales (Sánchez–Montoya et al., 2009).

Según la tipología oficial elaborada por el Centro de Estudios y Experimentación de Obras Públicas (CEDEX) y posteriormente publicada en la instruc-ción de planificación hidrológica (BOE, 2008, Orden ARM/2656/2008) se establecen dos ecorregiones denominadas Pirineos y Región iberomacaronésica, en las que las masas de agua superficiales de la categoría de los ríos se clasifican en 32 tipos di-ferentes, y se especifican los objetivos de calidad que tendrá que cumplir cada tipo de río. Los ríos y arroyos de cabecera de la serranía bética occidental parecen constituir una excepción dentro del entorno mediterráneo al que pertenecen debido a su elevada aportación específica media anual (CEDEX, 2005), de tal modo que el organismo encargado de la gestión de la cuenca del Guadalquivir los incluye en un tipo específico: ríos de serranías húmedas (Confedera-ción Hidrográfica del Guadalquivir, 2005) y según la Orden ARM/2656/2008 corresponden al tipo 20 (ríos de serranías béticas húmedas), que incluye, básicamente, las cabeceras de los ríos Guadalete, Barbate, Palmones, Hozgarganta y Genal.

Una vez establecida la tipología, el estado o po-tencial ecológico de cada tipo de río se clasificará de acuerdo con las cinco categorías antes citadas, en función de determinados elementos de calidad biológicos, hidromorfológicos o fisicoquímicos. Uno de los elementos más utilizados para evaluar el estado ecológico de los ríos es la fauna bentónica de invertebrados, mediante la utilización de índices calibrados específicamente para cada tipo de río. En este estudio se calibran dos índices biológicos cua-litativos basados en macroinvertebrados, el IBMWP (Iberian Biomonitoring Working Party) y el IMMi–L (Iberian Mediterranean Multimetric Index), según las normas marcadas por la DMA, a partir de la riqueza biológica que alberga una serie de localidades de referencia localizadas en el interior del parque natural Los Alcornocales. El principal objetivo es cuestionar la idoneidad de los instrumentos de gestión actuales (índices y umbrales) y proponer un cambio en el valor de referencia y en los umbrales de las clases de calidad. Asimismo, comparamos los resultados obtenidos con los propuestos para los diferentes tipos de ríos de la península ibérica.

Material y métodos

Área de estudio

El estudio fue realizado en algunos cursos fluviales del parque natural Los Alcornocales, que es un es-pacio protegido ubicado en el sistema montañoso más suroccidental de Europa, cerca del estrecho

62 Ruiz–García & Ferreras–Romero

de Gibraltar (fig. 1). Es un sistema de mediana altitud, con cumbres que alcanzan alrededor de 800 m s.n.m. El material litológico predominante son areniscas silíceas (areniscas del Aljibe) con un alto contenido de cemento ferruginoso. La temperatura es la típica del clima mediterráneo, pero atenuada por la influencia del Atlántico y el Mediterráneo. La humedad relativa del aire es elevada, con un valor medio mensual próximo al 75% (Almazán, 1991) y las nieblas persisten incluso en verano, espe-cialmente en los valles altos conocidos localmente como canutos, lo que produce un microclima que minimiza la sequía estival. Las lluvias son abundan-tes, incluso torrenciales, y superan los 1.400 mm anuales (Capel–Molina, 1987). La vegetación está dominada por bosques de alcornoque (Quercus suber), bosques de quejigo andaluz (Quercus ca-nariensis) en las umbrías y matorral mediterráneo en las cumbres (p. ej. Erica sp. y Cistus sp.). En los valles altos se ha desarrollado un bosque de ribera único en Europa, compuesto por vegetación relicta del Terciario: Laurus nobilis, Rhododendron ponticum, Frangula alnus, Ilex aquifolium y especies poco comunes de helechos como Psilotum nudum y Culcita macrocarpa, entre otras.

En este espacio natural estos cursos están ocu-pados por especies de macroinvertebrados de gran interés por su singularidad o su reducida área de dis-

tribución, como lo demuestran los estudios realizados sobre odonatos, tricópteros y coleópteros (Burmeister, 1983; Fery & Fresneda, 1988; Ruiz García, 1994; Agüero–Pelegrín et al., 1998; Castro & Delgado, 1999; González & Ruiz, 2001; Ferreras–Romero & Cano–Villegas, 2004; Ruiz–García et al., 2013).

Métodos

Los macroinvertebrados fueron muestreados en 24 localidades correspondientes a cinco cuencas diferentes: Barbate, Guadalete, Hozgarganta, Pal-mones y Jara (tabla 1). Las localidades seleccio-nadas fueron muestreadas tres veces: en invierno (10 II–6 III), al inicio de verano (24 VI–8 VII) y en otoño (17 XI–16 XII) de 2003; en cinco localidades de la vertiente norte de la sierra de Ojén la muestra de invierno fue tomada el 9 de marzo de 2004. Para la extracción de la entomofauna se utilizó una red de mano con una abertura de 0,25 x 0,25 m y un tamaño de malla de 250 µm. En primer lugar, se seleccionó un tramo de 100 m en el que se hacía un recorrido inicial para localizar todos los tipos de há-bitat presentes, incluida la vegetación de macrófitos acuáticos, e identificar los diferentes microhábitats que debían muestrearse. En cada toma de muestras, gran parte de la extracción de macroinvertebrados fue realizada mediante la agitación del sustrato

Fig. 1. Localización del área de estudio en la península ibérica, donde se muestra la red hidrográfica que drena el parque natural Los Alcornocales. La denominación de cada localidad muestreada está indicada en la tabla 1.

Fig. 1. Location of the study area in the context of the Iberian peninsula, showing the river system that drains Los Alcornocales Natural Park. The designation of each sampling site is indicated in table 1.

Río Majaceite

Río Hozgarganta

Río Guadarranque

Río PalmonesRío B

arbate

9

8

12

65

1213

11

10 15

14

201819

17 162221

2324

43

N

14 km

7

Animal Biodiversity and Conservation 38.1 (2015) 63

con los pies (kicking). El muestreo terminó cuando dejaron de detectarse nuevos taxones (Alba–Ter-cedor, 1996). Por último, el material recolectado se fijó en alcohol al 70% para su identificación en el laboratorio a nivel de familia, que es el nivel taxo-nómico necesario para la aplicación de los índices biológicos empleados.

La DMA establece la necesidad de calcular el estado ecológico de una masa de agua a partir de la desviación de la calidad con respecto a unas condiciones de referencia muy parecidas al estado natural para cada tipo (DOCE, 2000). Para estable-

cer la condición de referencia de las 24 localidades seleccionadas a priori hemos seguido el método propuesto por Sánchez–Montoya et al. (2009) para ríos mediterráneos de la península ibérica. Para calcular la calidad se utilizó el índice IBMWP (Alba–Tercedor et al., 2004), ampliamente utilizado en España, y el índice multimétrico IMMi–L, diseñado para los ríos mediterráneos españoles, especial-mente para los temporales (Munné & Prat, 2009). El valor de referencia para cada índice se obtuvo calculando la mediana de los 24 valores medios, obtenidos a partir de los valores registrados en los

Tabla 1. Coordenadas y algunas características de las 24 localidades estudiadas (sistema de referencia: ETRS89; huso: 30S): UTM. Coordenadas UTM; Cf. Cuenca fluvial; Eh. Estado hidrológico (P. Permanente, I. Intermitente, E. Efímero); D. Área de drenaje, en km2; A. Altitud; OS. Orden Strahler.

Table 1. Coordinates and characteristics of the 24 sampling sites (reference system: ETRS89; huso: 30S): UTM. UTM coordinates; Cf. River basin; Eh. Hydrological status (P. Permanente, I. Intermittent, E. Ephemeral); D. Drainage area, in km2; A. Altitude; OS. Strahler Order.

UTM Lugar de muestreo X Y Cf Eh D A OS

Canuto de Puerto Oscuro (1) 264113 4044674 Barbate P 2,57 580 1

Canuto del Montero (2) 265470 4041028 Barbate P 5,38 550 1

Río Rocinejo 1 (3) 262479 4038591 Barbate I 20,65 100 2

Río Rocinejo 2 (4) 260529 4038041 Barbate E 26,32 70 2

Canuto Albina de las Flores (5) 271406 4053202 Guadalete P 3,14 330 1

Gª Albina de las Flores (6) 271496 4054207 Guadalete P 23,39 250 2

Canuto del Aljibe (7) 264526 4047071 Guadalete P 4,72 440 1

Canuto del Caballo (8) 263422 4046834 Guadalete P 0,72 410 1

Gª del Caballo (9) 264525 4051431 Guadalete P 12,49 140 2

Canuto de los Sauces (10) 267405 4044314 Hozgarganta P 0,23 742 1

Canuto del Moral (11) 266894  4046282 Hozgarganta P 0,20 800 1

Gª La Sauceda (12) 268087 4045122 Hozgarganta P 2,46 590 2

Gª Pasada Llana (13) 268459  4045811 Hozgarganta P 3,67 501 2

Diego Duro (14) 274200 4044258 Hozgarganta P 111,72 165 4

Puente de Las Cañillas (15) 274281 4042984 Hozgarganta P 115,21 160 4

Canuto del Gandelar (16) 269988 3999640 Palmones P 1,28 530 1

Canuto Pinito (17) 267004 4001010 Palmones P 0,17 370 1

Gª del Gandelar (18) 268311  4002361 Palmones P 13,45 220 2

Gª de Cebrillo (19) 267654  4003272 Palmones I 7,46 200 2

Gª del Tiradero (20) 267917  4004953 Palmones P 26,67 180 3

Aº de los Molinos (21) 269242  3997679 Jara P 3,15 380 1

Aº de la Verruga (22) 268213 3997893 Jara P 1,52 330 1

Aº del Chivato (23) 267506  3998308 Jara P 1,52 260 1

Gª de los Molinos (24) 268111  3997103 Jara P 8,59 206 2

64 Ruiz–García & Ferreras–Romero

tres muestreos estacionales efectuados en cada una de las 24 estaciones de referencia seleccionadas. El valor de los umbrales de calidad depende de la dispersión de los valores de referencia y de la re-lación de cada índice con el gradiente de alteración ambiental. El percentil 25 de los valores utilizados para calcular el valor de referencia se considera un valor representativo de estado poco alterado

con respecto a las condiciones de referencia y se utiliza como umbral entre las clases de calidad muy bueno y bueno (European Commission, 2005; Pollards & Van den Bund, 2005). En el caso del índice multimétrico, hemos supuesto la existencia de una relación lineal entre los valores del índice y el gradiente de alteración ambiental, por lo que el resto de umbrales de calidad se han obtenido dividiendo los valores de referencia por debajo del percentil 25 en bandas iguales. El índice IBMWP no tiene una relación lineal con los gradientes de alteración que se refleje en los intervalos de corte que definen las distintas clases del estado ecoló-gico (Alba–Tercedor et al., 2004). La relación es polinómica y también la han descrito Munné & Prat (2009). Sin embargo, aplicando la metodología del modelo predictivo MEDPACS, Poquet et al. (2009) observaron una relación lineal entre el gradiente de presión y los EQR (valores observados y esperados de IBMWP) en los ríos mediterráneos. A pesar de ello, el Ministerio de Medio Ambiente, Rural y Marino español, en la instrucción de planificación aprobada por la Orden Ministerial ARM/2656/2008, asumió que el índice IBMWP tiene una relación lineal con la alteración ambiental. Por ello, la acotación de los umbrales de calidad por debajo de muy bueno y bueno se ha realizado de dos modos diferentes: polinómico y lineal. Cuando consideramos que la relación del índice IBMWP con el gradiente de alteración no es lineal, los umbrales de las clases de calidad se establecieron según Alba–Tercedor et al. (2004). En caso contrario, se siguió el mismo protocolo que para el índice multimétrico.

Tabla 2. Cumplimiento de los criterios de referencia en las 24 localidades seleccionadas, según la metodología propuesta por Sánchez–Montoya et al. (2009): C. Criterios cumplidos; N. Número de localidades.

Table 2. Compliance with the reference criteria in the 24 selected localities according to the methods proposed by Sánchez–Montoya et al. (2009): C. Achieved criteria; N. Number of locations.

Grados de alteración C NTramos no alterados o mínimamente alterados 20 24

Tramos poco alterados 16–19 0

Tramos alterados ≤ 15 0

Localidades IBMWP IMMi–L

Canuto de Puerto Oscuro 175,6 19,59

Canuto del Montero 198,3 23,31

Río Rocinejo 1 137 17,59

Río Rocinejo 2 138 15,75

Canuto Albina de las Flores 116,6 13,72

Gª Albina de las Flores 137 16,07

Canuto del Aljibe 226,3 26,92

Canuto del Caballo 222,3 24,44

Gª del Caballo * 183,6 24,53

Canuto de los Sauces 126,3 18,70

Canuto del Moral 101,6 14,51

Gª La Sauceda 166,6 19,59

Localidades IBMWP IMMi–L

Gª Pasada Llana 166 20,63

Diego Duro 166 19,10

Puente de Las Cañillas * 133,3 18,21

Canuto del Gandelar 165,6 18,26

Canuto Pinito 173,3 20,59

Gª del Gandelar 162 17,45

Gª de Cebrillo 172,3 19,38

Gª del Tiradero 131,3 14,99

Aº de los Molinos 162 17,19

Aº de la Verruga 168,6 19,45

Aº del Chivato 143,6 15,24

Gª de los Molinos 173,6 18,60

Tabla 3. Valores medios de los índices IBMWP y IMMi–L calculados a partir de los tres muestreos estacionales: * Presencia de especies exóticas (P. clarkii).

Table 3. Mean values of IBMWP and IMMi–L indices calculated from three seasonal samplings: * Presence of exotic species (P. clarkii).

Animal Biodiversity and Conservation 38.1 (2015) 65

Resultados

Las 24 localidades seleccionadas cumplieron todos los criterios de referencia (tabla 2). Los valores medios obtenidos para los dos índices utilizados en cada una de las localidades estudiadas están recogidos en la tabla 3. Todas las localidades mostraron valores de IBMWP superiores a 100 en todos los muestreos, con la excepción de tres cau-ces de pequeñas dimensiones en los que en una ocasión (invierno en los tres casos) no se alcanzó este valor. Tampoco se ha detectado la presencia generalizada de especies exóticas, con la excepción de dos localidades donde fue recolectado Procam-barus clarkii, con escasa abundancia. El alto valor de los índices analizados indica que la presencia del cangrejo rojo no tiene efectos destacables en la fauna de macroinvertebrados de estas localidades.

El análisis estadístico de los valores medios ob-tenidos para los índices está recogido en la tabla 4. A partir del valor de la mediana y el percentil 25, y siguiendo la metodología indicada en el apartado de métodos, se han calculado los umbrales de las dis-tintas clases de calidad (tabla 5) para ambos índices, expresados en valores de los índices y valores EQR; en el caso del IBMWP, tanto aquellos obtenidos con gradiente polinómico (*) como lineal.

Los umbrales de las clases de calidad del índice IBMWP fueron más bajos que los del índice IMMi–L debido a la alta dispersión de los valores de referen-cia. Por otro lado, la variabilidad de los valores EQR de referencia respecto a la mediana fue parecida en ambos índices y su distribución, casi simétrica en el caso del índice IMMi–L (fig. 2).

Al comparar los umbrales de calidad obtenidos en este estudio con los propuestos en otras publicaciones para distintos ríos españoles (tabla 6), apreciamos que los valores EQR de IBMWP* se corresponden perfectamente con las clases de calidad propuestas para los ríos de cabecera silíceas (Alba–Tercedor et al., 2004) y los pequeños ríos mediterráneos temporales de baja altitud (Munné & Prat, 2009),

Tabla 4. Resumen estadístico obtenido a partir de los valores medios de los índices IBMWP e IMMi–L.

Table 4. Statistical summary obtained from the average values of the IBMWP and IMMi–L indices.

Estadístico IBMWP IMMi–L

N 24 24

Mínimo 101,6 13,72

Máximo 226,3 26,92

Media 160,28 18,91

Desviación estándar 30,26 3,32

Mediana 165,8 18,65

Percentil 25 137 16,35

Percentil 75 173,52 20,34

Tabla 5. Umbrales para las clases de calidad de los índices IBMWP e IMMi–L calculados para los cursos de agua del parque natural Los Alcornocales: * Clases de calidad de IBMWP suponiendo la existencia de una relación no lineal con el gradiente de alteración ambiental calculado según Alba–Tercedor et al. (2004).

Table 5. Thresholds for quality classes of IBMWP and IMMi–L indices calculated for the waterways of the Los Alcornocales natural park: * Quality classes of IBMWP assuming a nonlinear relationship with the disturbance gradient calculated according to Alba–Tercedor et al. (2004).

Clases de calidad IBMWP* EQR IBMWP EQR IMMi–L EQR

Bueno–Muy bueno 137 0,83 137 0,83 16,35 0,88

Moderado–Bueno 83,57 0,50 102,75 0,62 12,26 0,66

Deficiente–Moderado 49,32 0,30 68,5 0,41 8,17 0,44

Malo–Deficiente 20,55 0,12 34,25 0,21 4,09 0,22

excepto para el umbral de calidad entre bueno y muy bueno, cuando consideramos la existencia de una relación polinómica entre este índice y el gradiente de alteración ambiental. Sin embargo, cuando esta relación es lineal, la calibración del índice coincide con la propuesta para los ríos de tipo 24 (gargantas de Gredos–Béjar) y 25 (ríos de montaña húmeda silí-cea). Por otro lado, los valores EQR obtenidos para el índice IMMi–L coinciden con los umbrales entre muy bueno y bueno y entre bueno y moderado calculados por Munné & Prat (2009) para el tipo R–M1. Además, desde el punto de vista de la gestión, es interesante diferenciar entre los valores del índice y los EQR, puesto que un mismo EQR se traducirá en un valor distinto del índice en función del valor de referencia.

66 Ruiz–García & Ferreras–Romero

En este sentido, observamos que los umbrales de las clases de calidad del índice IBMWP* polinómico obtenidos en este estudio son más elevados que los propuestos para los ríos de cabeceras silíceas (Sil/cab). Por el contrario, cuando la calibración es lineal, los umbrales del índice son más bajos que los propuestos para los ríos 24 y 25, y son parecidos a los valores de los ríos tipo 8 (ríos de baja montaña mediterránea silícea).

Discusión

El territorio contenido en el parque natural Los Alcor-nocales constituye una unidad ambiental homogénea desde los puntos de vista climático y geológico, con una composición litológica que comprende casi ex-clusivamente areniscas silíceas (areniscas del Aljibe) y una precipitación media anual superior a 1.000 mm que ha permitido el desarrollo de una densa cubierta vegetal en muy buen estado de conservación. Es-tas altas precipitaciones permiten la formación de pequeños cursos de agua que, a pesar de la fuerte estacionalidad, mantienen agua corriente durante todo el año. Aunque en la DMA no se han considerado las cuencas menores de 10 km2 de superficie, pen-samos que estos pequeños cursos de agua deben

tenerse en cuenta en la gestión, ya que constituyen enclaves únicos para la fauna acuática que podrían explicar la mayor parte de la biodiversidad presente en la cuenca (Clarke et al., 2008; Morrissey & De Kerckhove, 2009), de tal modo que la conservación deficiente de uno de estos cauces implicaría la pérdida de una diversidad única en toda la red fluvial (Finn et al., 2011; Mùrria et al., 2013).

Los umbrales de las clases de calidad del índice IMMi–L calculados en este estudio son mayores que los del IBMWP, lo que significa que el primero es más exigente en cuanto al acotamiento de las clases de calidad. Estos resultados coinciden con los obtenidos por Munné & Prat (2009) en la evaluación de la ca-lidad del agua en los ríos mediterráneos españoles mediante índices multimétricos. Además, los umbrales de las clases de calidad obtenidos en el área estu-diada coinciden con los propuestos por estos autores para otros ríos mediterráneos (especialmente R–M1), lo que parece confirmar la idoneidad de este índice como instrumento de gestión del estado ecológico del agua en esta zona.

Si consideramos los umbrales entre muy bueno y bueno y entre bueno y moderado para el índice IBMWP, podemos apreciar que los valores EQR ob-tenidos se aproximan más a los tipos atlánticos (tipos 24 y 25) que a los mediterráneos (tipos 6, 8 y 11); sin embargo, los valores de referencia y los umbrales de calidad acotados para estos ríos atlánticos son demasiado elevados para aplicarlos a los cursos de agua estudiados. Por otro lado, cuando consideramos los valores del índice IBMWP y no los EQR, encon-tramos que los umbrales son parecidos a los de los ríos de baja montaña mediterránea silícea (tipo 8), sin embargo los valores EQR propios de esos ríos no coinciden con los calculados en este estudio. Además, al comparar la relación entre el valor de referencia y los umbrales del índice obtenemos unos cocientes ligeramente inferiores para el índice calibrado en este estudio con respecto a los del tipo 8, lo que implica que nuestro índice es ligeramente más exigente en el establecimiento de las clases de calidad. No obstante, el principal inconveniente para aplicar la calibración del tipo 8 en nuestra área de estudio es el elevado valor de referencia propuesto para los ríos de baja montaña mediterránea silícea.

Estos resultados parecen indicar que las cuencas estudiadas necesitarían una gestión diferenciada, en cuanto a criterios de calidad, con respecto al resto de las cuencas mediterráneas circundantes. Los futuros planes hidrológicos de las subcuencas andaluzas Guadalete–Barbate y Mediterránea, basándose en el borrador de informe sobre la interpolación del IBMWP en los tipos de masas de agua en los que no se dispone de información de estaciones de referencia (versión 5.2), asignarán a los ríos del tipo 20 un valor de referencia de 115 para dicho índice, valor muy inferior al calculado para estos cursos en el presente estudio. Según nuestros datos, este valor de referencia sería claramente insuficiente para mante-ner la calidad ecológica óptima de los ríos y arroyos estudiados, pues todas las localidades consideradas en este estudio, que tienen una superficie de drenaje

Fig. 2. Diagrama de cajas que representa la distribución de los valores de referencia EQR para los índices IBMWP e IMMi–L. Cada caja muestra la mediana, los percentiles 75 y 25 y los valores máximo y mínimo. La localidad Canuto del Aljibe (31) presentó un valor EQR anormal-mente elevado con respecto a las demás.

Fig. 2. Box plot diagram representing the distribution of the EQR reference values for the indices IBMWP and IMMi–L. Each box shows the median, the percentiles 75 and 25 and the maximum and minimum values. The locality Canuto del Aljibe (31) showed an outlier value.

1,6

1,4

1,2

1

0,8

0,6

EQ

R

IBMWP IMMi–L Índices

31

Animal Biodiversity and Conservation 38.1 (2015) 67

Tabla 6. Comparación de los umbrales de clases de calidad calculados en el presente estudio con los obtenidos por otros autores para ríos españoles. Entre paréntesis se muestran los valores EQR. * Clases de calidad de IBMWP suponiendo la existencia de una relación no lineal con el gradiente de alteración ambiental calculado según Alba–Tercedor et al. (2004): Valor. Valores de referencia; Mb / B. Muy bueno / bueno; B / M. Bueno / moderado;; M / D: Moderado / deficiente; D / M. Deficiente / malo; Sil / cab. Pequeños arroyos silíceos de cabecera; MEDPACS. Mediterranean prediction and classification system; Tipo 6. Ríos silíceos del piedemonte de Sierra Morena; Tipo 8. Ríos de la baja montaña mediterránea silícea; Tipo 11. Ríos de montaña mediterránea silícea; Tipo 18. Ríos costeros mediterráneos; Tipo 24. Gargantas de Gredos–Béjar; Tipo 25. Ríos de montaña húmeda silícea; R–M1. Pequeños ríos de mediana altitud; R–M5. Pequeños ríos temporales de baja altitud; SBH. Pequeños ríos de las sierras béticas húmedas.

Table 6. Comparison of quality class thresholds calculated in this study with those obtained by other authors for Spanish rivers. The EQR values are shown in parenthesis. * Quality classes of IBMWP assuming a nonlinear relationship with the disturbance gradient calculated according to Alba–Tercedor et al. (2004): Valor. Reference values; Mb / B. Very good / good; B / M. Good / moderate; M / D. Moderate / deficient; D / M. Deficiente / poor; Sil / cab. Siliceous headwater streams; MEDPACS. Mediterranean prediction and classification system; Tipo 6. Siliceous rivers in the foothills of Sierra Morena; Tipo 8. Mediterranean low mountain siliceous rivers; Tipo 11. Mediterranean mountain siliceous rivers; Tipo 18. Mediterranean coastal rivers; Tipo 24. Gredos–Béjar gorges; Tipo 25. Wet, silicious mountain rivers; R–M1. Mid–altitude small rivers; R–M5. Low altitude, temporal rivers; SBH. Small rivers and streams in the wet Betic mountains.

Índice Tipo Valor Mb / B B / M M / D D / M Fuente

IBMWP* Sil / Cab 130 109(0,84) 66(0,51) 39(0,30) 16(0,12) Alba–Tercedor et al. (2004)

IBMWP MEDPACS – (0,91) (0,68) (0,45) (0,22) Poquet et al. (2009)

IBMWP Tipo 6 147,5 115(0,78) 87(0,59) 57,5(0,39) 29,5(0,20) ORDEN ARM/2656/2008

IBMWP Tipo 8 171 135(0,79) 100,9(0,59) 68,4(0,40) 34,2(0,20) ORDEN ARM/2656/2008

IBMWP Tipo 11 180 140,4(0,78) 106,2(0,59) 70,2(0,39) 36(0,20) ORDEN ARM/2656/2008

IBMWP Tipo 18 112 103(0,92) 77,3(0,69) 53,8(0,48) 25,8(0,23) ORDEN ARM/2656/2008

IBMWP Tipo 24 210 178,5(0,85) 134,4(0,64) 88,2(0,42) 44,1(0,21) ORDEN ARM/2656/2008

IBMWP Tipo 25 178 149,5(0,84) 112,1(0,63) 74,8(0,42) 37,4(0,21) ORDEN ARM/2656/2008

IBMWP* R–M1 – (0,78) (0,48) – – Munné & Prat (2009)

IBMWP* R–M5 – (0,80) (0,50) (0,30) (0,10) Munné & Prat (2009)

IBMWP* SBH 166 137(0,83) 83,6(0,50) 49,3(0,30) 20,5(0,12) Este trabajo

IBMWP SBH 166 137(0,83) 102,8(0,62) 68,5(0,41) 34,2(0,21) Este trabajo

IMMi–L R–M1 – (0,89) (0,66) – – Munné & Prat (2009)

IMMi–L R–M5 – (0,90) (0,70) (0,50) (0,20) Munné & Prat (2009)

IMMi–L SBH 18,65 16,3(0,88) 12,2(0,66) 8,2(0,44) 4,1(0,22) Este trabajo

superior a 10 km2, muestran un valor del IBMWP superior a 115. No obstante, el área estudiada solo representa una parte de las cabeceras húmedas del sur de la península. Sería necesario extender la red de referencia al resto de las cuencas (Guadalete y Genal) para disponer de la información correspondien-te a los otros tipos de cuencas existentes en estas sierras húmedas del suroeste peninsular.

El tipo 20 (BOE, 2008, Orden ARM/2656/2008) constituye un conjunto muy heterogéneo de ríos y arroyos de cabecera cuya única afinidad es la elevada precipitación registrada en estas áreas, pero no tiene en cuenta que la composición lito-

lógica es muy diferente. En un estudio sobre la caracterización fisicoquímica de las localidades de referencia en cinco ecotipos de ríos mediterráneos, Sánchez–Montoya et al. (2012) encontraron que la conductividad y el pH eran los únicos parámetros significativamente más bajos en las cabeceras silí-ceas que en el resto de los ecotipos. Además, estos arroyos de cabecera silíceos albergan comunidades de macroinvertebrados significativamente diferentes del resto (Sánchez–Montoya et al., 2007). Si la conductividad es un parámetro tan decisivo en la tipificación de los ríos mediterráneos, resulta difícil aceptar un tipo único para los ríos de las serranías

68 Ruiz–García & Ferreras–Romero

béticas húmedas, donde se incluyen tramos marca-damente silíceos (p. ej., las sierras del Campo de Gibraltar y Aljibe) y otros marcadamente calcáreos (p. ej., la Sierra de Grazalema).

En resumen, hemos mostrado que el valor de referencia adoptado por la normativa para la ela-boración de los planes hidrológicos de las cuencas mediterráneas andaluzas y la demarcación hidrográfica Guadalete–Barbate, que comprenden los ríos de las serranías béticas húmedas, es claramente insuficien-te para asegurar el mantenimiento del buen estado ecológico de los sistemas fluviales del parque natural Los Alcornocales. Para gestionar este espacio natural debidamente, proponemos elevar dicho valor de refe-rencia a 166 y adoptar los umbrales de las clases de calidad del índice IBMWP calculados en este trabajo. Por último, queremos destacar que el índice multimétrico IMMi–L, tal como lo hemos calibrado aquí, y utilizado junto con el anterior, puede ser un instrumento de gestión eficaz para asegurar el mantenimiento del buen estado ecológico de estos cursos de agua.

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71Animal Biodiversity and Conservation 38.1 (2015)

© 2015 Museu de Ciències Naturals de BarcelonaISSN: 1578–665 XeISSN: 2014–928 X

Confirmation of the presence of Ischnura senegalensis (Rambur, 1842) on the Canary Islands

R. A. Sánchez–Guillén & A. Cordero–Rivera

Sánchez–Guillén R. A. & Cordero–Rivera, A., 2015. Confirmation of the presence of Ischnura senegalensis (Rambur, 1842) on the Canary Islands. Animal Biodiversity and Conservation, 38.1: 71–76.

AbstractConfirmation of the presence of Ischnura senegalensis (Rambur, 1842) on the Canary Islands.— The presence of one or two species of damselflies of the genus Ischnura in the Canary Islands has been a matter of debate in recent years. The first published records listed I. senegalensis as the only zygopteran inhabiting the archipelago, but this proved to be wrong, and until recently, all specimens of Ischnura captured in the islands were unanimously regarded as belonging to I. saharensis. Recent photographic evidence, however, is compatible with the presence of I. senegalensis. In this study, we give morphological and genetic evidence of the presence of I. senegalensis in the Canary Islands, and we discuss the importance of voucher specimens to correctly identify very similar species.

Key words: Odonata, Ischnura, Genetic identification, Island, Voucher specimens, Macaronesia

ResumenConfirmación de la presencia de Ischnura senegalensis (Rambur, 1842) en las islas Canarias.— La presencia de una o dos especies de zigópteros del género Ischnura en las islas Canarias ha sido objeto de debate en los últimos años. Los primeros registros publicados señalaban que I. senegalensis era el único zigóptero presente en el archipiélago; pero resultó no ser correcto y hasta hace poco, todos los especímenes del género Ischnura capturados en las islas se clasificaban sin excepción como I. saharensis. No obstante, las recientes pruebas fotográficas son compatibles con la presencia de I. senegalensis. En el presente estudio aportamos pruebas morfológicas y genéticas de la presencia de I. senegalensis en las islas Canarias, y analizamos la importancia de los especímenes de referencia para identificar correctamente especies muy parecidas.

Palabras clave: Odonatos, Ischnura, Identificación genética, Isla, Especímenes de referencia, Macaronesia

Received: 20 X 14; Conditional acceptance: 26 I 15; Final acceptance: 11 III 15

R. A. Sánchez–Guillén, Genome Integrity and Instability Group, Inst. de Biotecnologia i Biomedicina (IBB), Univ. Autònoma de Barcelona, Campus UAB, 08193 Barcelona, Espanya (Spain).– A. Cordero–Rivera, Depto. de Ecología y Biología Animal, Univ. de Vigo, España (Spain).

Corresponding author: R. A. Sánchez–Guillén. E–mail: rguillenuvigo@hotmail.com

72 Sánchez–Guillén & Cordero–Rivera

Introduction

The genus Ischnura is one of the most speciose genera of the family Coenagrionidae, with around 70 species with a worldwide distribution (Dumont, 2013). The Mediterranean is home to nine species: I. elegans (Vander Linden, 1820); I. evansi (Morton, 1919); I. fountaineae (Morton, 1905); I. genei (Rambur, 1842); I. graellsii (Rambur, 1842); I. intermedia (Dumont, 1974); I. pumilio (Charpentier, 1825); I. saharensis (Aguesse, 1958); and I. senegalensis (Rambur, 1842) (Boudot et al., 2009). Given the inherent dynamic nature of animal distributions, the species found at particular localities are expected to change over time, especially in a scenario of global warming and range expansions (Hickling et al., 2005; Sánchez–Guillén et al., 2013, 2014b).

To date, 14 species of Odonata (three Zygoptera and 11 Anisoptera) have been recorded in the Canary Islands, including one Macaronesian endemic genus (Sympetrum nigrifemur, also found in Madeira) (Weihrauch, 2013). However, only one zygopteran has been reported regularly on the islands. It was first identified as Ischnura senegalensis by Valle (1955), but this identification was questioned by Hämäläinen (1986), who examined Valle’s specimens preserved in the Zoological Museum of the University of Turku, and confirmed that they belong to Ischnura saharensis. For many years, the Canarian Ischnura was considered to be represented by only one species, namely I. saharensis, and consequently the monograph by Báez (1985) lists it as the only species of Zygoptera in the Archipelago.

In most of the Mediterranean area, I. elegans and I. pumilio occur in sympatry. Other species overlap locally: I. elegans and I. graellsii in Spain, I. elegans and I. genei in the islands of Elba and Giglio (Italy), and I. graellsii and I. saharensis in Morocco (Boudot et al., 2009). Additionally, I. fountaineae, I. saharensis

and I. graellsii overlap in Morocco, while I. elegans, I. evansi, I. fountaineae, I. pumilio and I. senegalensis overlap in the Middle East (Boudot et al., 2009). Given that I. senegalensis has been recorded from Mauritania (Boudot et al., 2009) and Cape Verde Islands (Aistleitner et al., 2008), its presence in the Canary Islands is possible due to the frequent winds coming from the desert. These winds have been invoked as the explanation for the only record of Platycnemis subdilatata for the Canaries (Kalkman & Smit, 2002).

In recent years, there has been an on–going discussion about the presence of I. senegalensis in the Canaries (Weihrauch, 2013), particularly because several pictures uploaded by amateurs to web servers suggested its presence. Nevertheless, scientific evidence needs voucher specimens (Corbet, 2000), because this is the only way to re–exam data in the light of future taxonomic knowledge. Our initial goal, therefore, was to obtain a sample of Canarian I. saharensis for molecular analyses, but we only found specimens which morphologically resembled I. senegalensis. The main purpose of this work was therefore to determine whether mtDNA sequencing would confirm that our Ischnura specimens were I. senegalensis. AdditionAdditionally, we investigated the phylogenetic relationships of seven Mediterranean Ischnura species (I. elegans, I. fountaineae, I. genei, I. graellsii, I. pumilio, I. saharensis and I. senegalensis) and discuss our findings in the light of the recent review by Dumont (2013).

Material and methods

We collected five Ischnura samples on 20 V 2007 near San Andrés (Santa Cruz de Tenerife, Canary Islands) (28° 30' 51.26'' N and 16° 11' 31.53'' W). Three males and two gynochrome females, which we identified as

Table 1. Taxon sampling of Ischnura genus: * Data not available.

Tabla 1. Muestreo de taxones del género Ischnura: * Información no disponible.

Species N samples Locality Country Date CollectorI. asiatica 3 * Japan 2006 Yuma TakahasiI. elegans 3 Kaiserslautern Germany 2006 Jürgen OttI. fountaineae 1 Za Morocco 2009 Sánchez–GuillénI. genei 3 Coginas Sardinia, Italy 2008 Sánchez–GuillénI. graellsii 2 Ribeira de Cobres Portugal 2005 Sánchez–GuillénI. graellsii 2 Saïdia Morocco 2009 Sánchez–GuillénI. saharensis 3 Ouarzazate Morocco 2007 Sánchez–GuillénI. senegalensis 3 San Andrés, Tenerife Canary Islands 2007 Sánchez–GuillénI. senegalensis 3 Reservoir Van Bach Namibia 2007 Sánchez–GuillénI. senegalensis 2 * Japan 2006 Yuma TakahasiI. pumilio 1 Kaiserslautern Germany 2006 Jürgen OttI. pumilio 1 São Miguel Azores 2003 Cordero–Rivera

Animal Biodiversity and Conservation 38.1 (2015) 73

Ischnura senegalensis, were captured and preserved in absolute ethanol for further DNA studies.

For the molecular identification, we combined two mitochondrial markers, cytochrome oxidase II and cytochrome b, because the use of several genetic markers improves species identification (Bergmann et al., 2013). DNA of three samples of putative Ischnura senegalensis from the Canary Islands, and one to three samples of each of the seven Mediterranean Ischnura species: I. elegans, I. fountaineae, I. genei, I. graellsii, I. pumilio, I. saharensis, and I. senegalensis was extracted from the thorax using a standard phenol/chloroform extraction protocol (Sambrook et al., 1989). Additionally, we included three samples of I. asiatica and one of Enallagma basidens, E. cyathigerum

and Telebasis salva as outgroups (Gene bank accession numbers: AF067669.1, AF067670.1, AF067681.1, AF067689.1, AF067690.1 and AF067701.1). Table 1 gives the localities of capture of these samples and the accession numbers. Extracted DNA was amplified by PCR for part of cytochrome oxidase II (673 bp), with the primers TL2–J–3037 and C2–N–3494 and C2–J–3400 and TK–N–3785 (Simon et al. 1994) (Gene bank accession numbers KC430114–KC430232), and cytochrome b (457 bp) with the primers CB–J–10933 and TS1–N–11683 (Simon et al., 1994) (Gene bank accession numbers KC430114–KC430232). The PCR program had an initial cycle of 95ºC for 3 min, followed by 34 cycles at 95ºC for 30 s, with annealing for 45s, an elongation phase at 72ºC for 45s, and

Fig. 1. Posterior view of the abdomen, showing the anal appendages, all at the same magnification. The specimens of I. senegalensis from Tenerife (young male, A; mature male, B) are indistinguishable from I. senegalensis from Namibia (C). All show the upper appendages in close contact, and the lower appendages convergent, findings that contrast with I. saharensis from Morocco (D), whose upper appendages cross, and whose lower appendages are divergent. Pictures taken with LAS software (Leica Microsystems).

Fig. 1. Vista posterior del abdomen donde se observan los apéndices anales. Todas las imágenes tienen el mismo aumento. Los especímenes de I. senegalensis de Tenerife (macho joven, A; macho maduro, B) son indistinguibles de los de I. senegalensis de Namibia (C). Todos presentan los apéndices superiores en estrecho contacto y los inferiores, convergentes, lo cual difiere de I. saharensis de Marruecos (D), cuyos apéndices superiores se cruzan y los inferiores, divergen. Imágenes tomadas con el programa informático LAS (Leica Microsystems).

A B

C D

0.5 mm

74 Sánchez–Guillén & Cordero–Rivera

elegans Germanyelegans Germanyelegans Germanysaharensis Moroccograellsii Portugalgraellsii Portugalelegans Germanyelegans Germanyelegans Germanygenei Sadiniagenei Sadiniasaharensis Moroccograellsii Morocco graellsii Moroccogenei Sardiniasaharensis Moroccofountaineae Moroccosenegalensis Canary Islandssenegalensis Canary Islandssenegalensis Canary Islandssenegalensis Namibiasenegalensis Namibiasenegalensis Japansenegalensis Japansenegalensis Namibia

asiatica Japanasiatica Japanasiatica Japanpumilio Azorespumilio GermanyEnallagma basidensEnallagma cyathigerumTelebasis salva

63

80

6899

82

98

100

99

100

100 68

79

I. e

lega

ns g

roup

Cla

de e

lega

ns C

lade

pum

ilio

100

a final extension phase at 72ºC for 10 min. It was performed in 10 ml and amplification conditions were as follows: 1–2 ng of DNA (2 mL), 5.0 mL of 2X Ready MixTM PCR Master Mix (1.5 mM MgCl2), 1mL 10Î of BSA, 0.3 mL of MgCl2 (50 mM), 1.1µL of distilled water, and 0.3 mL of each primer (10 pmol) in a 'GeneAmp PCR system 2700' thermocycler (Applied Biosystems). Bidirectional sequencing reactions were conducted using the BigdyeTM terminator cycle sequencing kit

(Applied Biosystems) using the automatic sequencer ABI3100. Forward and reverse sequences were edited and aligned with Clustal X (Thompson et al., 1997) implemented in Mega version 6 (Tamura et al., 2013). Variable positions were revised by eye, and only high quality sequences were considered.

We generated a neighbour–joining tree using Mega version 6 (Tamura et al., 2013) by using the consen(Tamura et al., 2013) by using the consen by using the consensus sequence of mtDNA cytochrome oxidase II and

Fig. 2. Consensus neighbour–joining tree based on Kimura 2–parameters (K2P) with the rate of variation between sites being modelled with a gamma distribution (shape parameter = 1). Branches corresponding to partitions reproducing more than 50% of bootstrap replicates are shown.

Fig. 2. Árbol de consenso producido mediante el método del “vecino más cercano” (neighbour–joining) y basado en el modelo de Kimura con dos parámetros (K2P) donde la tasa de variación entre los sitios se define con una distribución gamma (parámetro de forma = 1). Se muestran las ramas correspondientes a las particiones que reproducen más del 50 % de las réplicas por remuestreo con reemplazamiento (bootstrap).

Animal Biodiversity and Conservation 38.1 (2015) 75

cytochrome b (n = 33 sequences, 668 informative positions). The tree was based on Kimura 2–parameters (K2P) (Kimura, 1980) with the rate of variation among sites being modelled with a gamma distribution (shape parameter = 1). The confidence probability of each interior branch was multiplied by 100.

Results

All five samples we collected were morphologically identified as I. senegalensis. Figure 1 shows the male anal appendages in the posterior view of an immature (A) and a mature male (B), both from Tenerife, and samples of I. senegalensis from Namibia (C) and I. saharensis from Morocco (D) for comparison.

For the DNA analysis, after deleting gaps and missing data, we obtained a total of 660 informative positions in the final dataset. The resulting neighbour joining tree (fig. 2) grouped our sample of Ischnura species in two main clusters. In the first cluster, one branch included all species of the I. elegans group and I. foun founfountaineae. The second cluster includes all the samples of I. senegalensis, irrespective of their geographical origin. Therefore, our morphological identification was confirmed by the analyses of DNA sequences.

Discussion

The five Canarian samples were similar to the samples of I. senegalensis from Namibia although they were larger. The easiest way to distinguish between I. saharensis and I. senegalensis is the different orientation of lower appendages, which are clearly divergent in saharensis (as is typical of the Ischnura elegans group, to which it belongs; Dumont, 2013; see also below) and convergent in senegalensis (fig. 1).

The neighbour–joining tree (fig. 2) of the eight Ischnura species showed two main clusters, with all species of the I. elegans group (I. elegans, I. genei, I. graellsii and I. saharensis) and I. fountaineae in the first cluster. However, not all species formed monophyletic groups in our analysis. This is expected for taxa which have recently diverged, and is consistent with the hybridization processes between I. elegans and I. graellsii in Southern Europe (Sánchez–Guillén et al., 2011), I. graellsii and I. saharensis in Morocco (Sánchez–Guillén et al., 2014a), and I. elegans and I. genei in the Tyrrhenian Islands Elba and Giglio (Sán Elba and Giglio (Sán (Sán(Sánchez–Guillén et al., 2014a). The second group of the first cluster included all I. senegalensis samples, those from Namibia and Japan, and also from the Canary Islands. Finally, the second cluster included I. pumilio samples from the Azores and Germany, and I. asiatica from Japan. Our results are consistent with the tree constructed by Dumont (2013) using cytochrome oxi(2013) using cytochrome oxi using cytochrome oxidase I and ITS DNA fragments. Ischnura pumilio and I. elegans appear separated in the two main clades that form the genus, and I. elegans, I. genei, I. graellsii, and I. saharensis appear as closely related species.

In a recent paper, Peels (2014) presented pho(2014) presented pho presented photographs of Ischnura individuals from the Canary

Islands, morphologically identified as I. senegalensis. He also provided useful characters that allow the two Ischnura species found in the islands to be separated. Our research highlights the importance of voucher specimens in taxonomy and ecology (Corbet, 2000). Had no specimens been captured by Håkan Lindberg in 1949, studied by Valle (1955) and preserved in a museum, their identity would never have been confirmed. We agree that restraint should be exercised when collecting insects, or other animals, and that photography is preferable for identification purposes. This is indeed the safe approach for some species, particularly when the number of possible species is low (Peels, 2014). For instance, the first record of Erythemis vesiculosa for the Galapagos islands is based on an unpublished picture examined by expert taxonomists (Muddeman, 2007). Photo–identification is also possible for species that have a unique appearance, like Ischnura nursei, and Indian species recently found in the United Arab Emirates, but even in this case a voucher specimen was collected (Feulner & Judas, 2013). However, as photographs can not be used to extract DNA or to conduct a detailed study of morphology, they are of limited use in science. We do not know where I. senegalensis came from, although it likely colonized from northwestern Mauritania where the nearest populations of the species are found (Boudot et al., 2009). This question might be answered by a detailed genetic analysis, including samples from different geographical regions. Again, voucher specimens would be needed to do so.

Acknowledgements

We thank Marcos Báez (University of La Laguna) for his help and suggestions for field work, and María Casal Nantes and Guillermo Velo Antón for their help with sampling in the Canary Islands. Yuma Takahasi provided the samples of I. senegalensis and I. asiatica from Japan and Jürgen Ott those of I. pumilio and I. elegans from Germany. We also thank Jesús Ramsés Chavez Ríos for his comments on earlier versions of the manuscript, and Fons Peels for sending us his paper before it had been published. RAS–G was supported by a postdoctoral grant from 'Alianza 4 Universidades'. Funding was provided by grants from the Spanish Ministry with competence in Science, including FEDER funds (CGL2008–03197–E and CGL2008–02799).

References

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© 2015 Museu de Ciències Naturals de BarcelonaISSN: 1578–665 XeISSN: 2014–928 X

Jugovic, J., Praprotnik, E., Buzan, E. V. & Lužnik, M., 2015. Estimating population size of the cave shrimp Troglocaris anophthalmus (Crustacea, Decapoda, Caridea) using mark–release–recapture data. Animal Biodiversity and Conservation, 38.1: 77–86.

AbstractEstimating population size of the cave shrimp Troglocaris anophthalmus (Crustacea, Decapoda, Caridea) using mark–release–recapture data.— Population size estimates are lacking for many small cave–dwelling aquatic in-vertebrates that are vulnerable to groundwater contamination from anthropogenic activities. Here we estimated the population size of freshwater shrimp Troglocaris anophthalmus sontica (Crustacea, Decapoda, Caridea) based on mark–release–recapture techniques. The subspecies was investigated in Vipavska jama (Vipava cave), Slovenia, with estimates of sex ratio and age distribution. A high abundance of shrimps was found even after considering the lower limit of the confidence intervals. However, we found no evidence of differences in shrimp abundances between summer and winter. The population was dominated by females. Ease of capture and abundant popula-tion numbers indicate that these cave shrimps may be useful as a bioindicator in cave ecosystems.

Key words: Mark–release–recapture, Population size, Dinarides, Vipavska jama, Sex ratio

ResumenEstimación del tamaño de la población del camarón cavernícola Troglocaris anophthalmus (Crustacea, Decapoda, Caridea) mediante la utilización de datos de marcaje, liberación y recaptura.— Se desconoce el tamaño de la población de numerosos invertebrados acuáticos cavernícolas que son vulnerables a la contaminación de las aguas subterráneas provocada por las actividades antropogénicas. En este estudio estimamos el tamaño de la población del camarón de agua dulce Troglocaris anophthalmus sontica (Crustacea, Decapoda, Caridea) me-diante las técnicas de marcaje, liberación y recaptura. La subespecie se estudió en la Vipavaska jama (cueva de Vipava), en Eslovenia, y se calcularon la proporción de sexos y la distribución por edad. Incluso tras considerar el límite inferior de los intervalos de confianza, se halló un gran abundancia de camarones. No obstante, no se encontraron indicios de que haya diferencias en cuanto a la abundancia de camarón entre verano e invierno. La población estaba formada predominantemente por hembras. La facilidad de la captura y las elevadas cifras de población indican que estos camarones podrían utilizarse como bioindicadores en los ecosistemas cavernícolas.

Palabras clave: Marcaje, liberación y recaptura, Tamaño de población, Dinárides, Vipavska jama, Proporción de sexos

Received: 25 III 14; Conditional acceptance: 20 V 14; Final acceptance: 17 III 15

Jure Jugovic, Elena V. Buzan & Martina Lužnik, Dept. of Biodiversity, Fac. of Mathematics, Natural Sciences and Information Technologies, Univ. of Primorska, Glagoljaška 8, 6000 Koper, Slovenia; and Institute for Biodi-versity Studies, Science and Research Centre, Univ. of Primorska, Garibaldijeva 1, 6000 Koper, Slovenia.– Eva Praprotnik, Dept. of Biodiversity, Fac. of Mathematics, Natural Sciences and Information Technologies, Univ. of Primorska, Glagoljaška 8, 6000 Koper, Slovenia

Corresponding author: Jure Jugovic. E–mail: jure.jugovic@upr.si

Estimating population size of the cave shrimp Troglocaris anophthalmus (Crustacea, Decapoda, Caridea) using mark–release–recapture data

J. Jugovic, E. Praprotnik, E. V. Buzan & M. Lužnik

78 Jugovic et al.

Introduction

The freshwater shrimp genus Troglocaris (Dormitzer, 1853) consists of four subgenera distributed in the Western Balkans (fig. 1A; Sket & Zakšek, 2009; Matjašič, 1956; Babić, 1922; Jugovic et al., 2011) and the Caucasus (Sadovsky, 1930; Sket & Zakšek, 2009; Marin & Sokolova, 2014) and inhabits underground karst waters flowing to the surface.

Not much is known about the biology, ecology, dis-tribution or habitat requirements of the European cave shrimp (Gottstein Matočec, 2003; Juberthie–Jupeau, 1974, 1975; Jugovic et al., 2010a). In this study we aimed to estimate population size for a population of Troglocaris anophthalmus sontica Jugovic et al., 2012, a subspecies of a type species of the genus. This subspecies is currently recorded from four sub-terranean localities (two in Slovenia and two in Italy) belonging to the Vipava–Soča River System (Jugovic et al., 2012). This river system is located on the north westernmost border of the distribution of subgenus Troglocaris s. str. (sensu Sket & Zakšek, 2009; fig. 1A). The Vipava River is a major water resource for the Vipava valley (SW Slovenia), with its headwaters arising from springs in Vipavska jama. The landscape behind the spring is an area of fractured and dissolved carbonate bedrock that stores significant quantities of groundwater.

As variation in daily and annual insolation is lac-king in cave habitats, there is a significant regression of circadian (e.g. locomotor activity) and circaanual (e.g. reproduction) patterns in troglobionts (Langec-ker, 2000; see also examples in Juberthie & Decu, 1994). Despite this, egg production of two species of Amphipods (Amphipoda), Niphargus virei and N. rhenorodanensis, exhibit reproductive patterns timed with hydrological fluxes that reach a maximum in summer and decrease in autumn. Similar annual variation in egg production has also been recorded in Troglocaris planinensis (Juberthie–Jupeau, 1975, 1974). Timed reproductive patterns are not clear for N. rhenorhodanensis living in pools of percolated water, where the water does not flow, or for interstitial populations of the same species (Mathieu & Turquin, 1992). Fluctuations of population size were reported to be highly dependent upon water discharge rates that often vary considerably in correlation with conditions on the surface. In the case of N. rhenorhodanensis, increased water discharge rates subject animals to drift and change population abundance (Essafi et al., 1992).

Population size estimates provide important infor-mation about rare and endangered species (Bueno et al., 2007). They are also used to identify species that may occur in sufficient numbers for use as bioin-dicators in environmental monitoring (Knapp & Fong, 1999). Mark–release–recapture (MRR) techniques are a popular choice for population size estimates; several methods have been developed to take into account the aim of the analysis and the type of species under investigation (Sutherland, 2006; Krebs, 1999; Seber, 1982). Non–commercial decapod crustaceans are rarely the object of population research (Rabeni

et al., 1997; Bueno & Bond–Buckup, 2000; Bueno et al., 2007) and to our knowledge, few MRR studies have been conducted in stygobionts (Hobbs, 1978, 1981; Culver, 1982; Simon, 1997; Knapp & Fong, 1999; Cooper & Cooper, 2009; Venarsky et al., 2012). Such studies are also rare in troglophilic terrestrial invertebrates (Carchini et al., 1982, 1994; Bernardi-ni et al., 1996). However, simple Lincoln–Petersen calculations have often been applied to cave beetles (Fejér & Moldovan, 2013).

The aim of our study was to estimate population size and sex ratios in a population of T. a. sontica from Veliko jezero (Large lake) in Vipavska jama. Using MRR techniques, we estimated population size and sex ratio in summer and winter.

Material and methods

Study site and field work

Veliko jezero is accessed through a 239 m man–made passage (fig. 1B), originally excavated for mercury ore. The lake is ellipsoid, with a surface area of approximately 180 m2 and maximum dimensions of approximately 10 X 18 m. The lake is surrounded by steep, almost vertical walls and is accessible at only one point. The sampling area covered by the current study was approximately 6 m2. Four square meters were reached in the lake from the access point; a further two square meters were accessible from the same point but situated along a narrow crevice (fig. 1B).

As these cave shrimps are omnivorous/detritivorous animals (Gottstein Matočec, 2003) and little is known about their diet, the development and testing of baits was outside the scope of the current study. Hence, animals were caught by hand–net. Hand nets of 1 mm mesh size were used for both capture and recapture. A net with a 1.5 m handle was used for specimens collected in deeper water. Although animals are always present in the lake, long periods of time (≥ 8 hours) were spent in the field as much patience was needed to catch the shrimps.

During the summer estimate, four sampling sessions were carried out, from 22–29 IX 2012. Three additio-nal sampling sessions were carried out in winter from 18–24 II 2013 (table 1). Sampling was carried out by two people on a single day, over a period of 8–10 hours. If fewer than 25 animals were caught on a single day, the session was extended to the following day to ensure sufficient sample sizes. When an extended sampling session was required, animals caught on the first day were kept in a plastic tank with water over night. This approach was adopted in order to strengthen the equality of sampling among the sampling sessions. For both normal and extended sampling, a single day was left before the next sampling session.

By cutting off the tips of the uropods, telson or rostrum, animals were occasion–specifically marked (see an example of broken telson, fig. 1C). The pos-sible negative impact of the marking procedure has been investigated previously on rostra (Jugovic et al., 2010a), revealing no noticeable impact on survival

Animal Biodiversity and Conservation 38.1 (2015) 79

Fig. 1. A. Approximate geographic distribution of Troglocaris sg. Troglocaris showing T. anophthalmus sontica distribution area and geographic position of Vipavska jama; B. Sketch of Vipavska jama from entrance to second artificial channel (redrawn after JDDR; http://www.jddr.org/kataster/vipavska/) with depiction of Veliko jezero and access point to the lake where shrimp sampling was conducted; C. A shrimp with broken telson; this specimen was kept alive in a laboratory for over two years (photo: J. Jugovic).

Fig. 1. A. Distribución geográfica aproximada de Troglocaris sg. Troglocaris donde se aprecia el área de distribución de T. anophthalmus sontica y la localización geográfica de la cueva de Vipara; B. Es-quema de la cueva de Vipara desde la entrada hasta el segundo canal artificial (modificación del dibujo de la sociedad espeleológica eslovena JDDR, http://www.jddr.org/kataster/vipavska/) donde se muestra el lago Veliko y el punto de acceso al mismo en el que se llevó a cabo el muestreo del camarón; C. Un camarón con un telson roto; este espécimen se mantuvo vivo en un laboratorio durante dos años (fotografía: J. Jugovic).

Distribution area:

Troglocaris sg. Troglocaris

Troglocaris anophthalmus sontica

Vipavska jama

Slovenia

Croatia

Bosnia & Herzegovina

50 km

Adriatic Sea

Europe

Italy

50 m Side view

Entrance 1st artificial passage 2nd artificial passage (leading to natural parts of the cave)

Floor view(enlarged)

1st passage

2nd passage

Access pointLake

A

B

C

N

Siphon

80 Jugovic et al.

of marked animals. During the past samplings (for other studies), individuals with broken parts of telson, uropods or other appendages were commonly found alive and in good condition (field observations, see also Jugovic et al., 2010a). Moreover, one shrimp with a heavily injured telson (fig. 1C) remained alive in a laboratory for over two years.

Capture history for each animal was made in the following order by the subsequent markings for four sampling sessions during the summer: (1) left uropod exopodite, (2) right uropod exopodite, (3) left uropod endopodite, (4) rostrum and for two sampling sessions during the winter, (5) right uropod endopodite, and (6) the telson. Animals were not marked in the last sampling session.

Whenever samples were taken, the temperature of the air and water was recorded. For each indivi-dual, sex and age group (adult male, adult female, ovigerous female, juvenile, see Jugovic et al., 2010b for age groups) were determined.

Data analysis

Different models assume either an open or closed population. When the assumptions of closed popula-

tion models are met, they may provide more precise estimates of population size than open models can. Care must be taken when choosing the best models. Knowledge of the biology and ecology of the target population should guide the choice of appropriate models, and methods exist for relaxing some of the common assumptions of MRR (Greenwood & Robin-son, 2006). MRR models can provide more reliable estimates than simple sightings or once–off counts (Knapp & Fong, 1999; Cooper & Cooper, 2011). Two different approaches to population size estimation were used within the Programme MARK (White & Burnham, 1999) to test closed population models.

Closed models were tested within the module 'Closed captures' and with 'Capture' available within MARK. Data were coded as individual encounter histories and entered into the input file (.inp). The two approaches differ in model selection. Models within MARK were selected based on AICc criterion. Four models were tested for each season, with different parameterization of capture probability (p), recapture probability (c), and the mixture parameter (π): Mt, Mb, Mh and M0 (Chao & Huggins, 2005). Presence of individual heterogeneity within the population could cause non–identifiability of population size (Link, 2003;

Table 1. Data on shrimp sampling during late summer (September 2012) and winter (February 2013): ni. Number of animals caught in the i–th sampling occasion caught; mi. Number of recaptures in the i–th sampling occasion; ui = ni – mi. Number of unmarked animals in the i–th sampling occasion; Mi. Number of animals marked prior to the i–th sampling occasion; mi/ni. Ratio of marked animals in the i–th sampling occasion; * 47 animals were caught but only 44 were released (three were kept for morphometrics); no deaths regarding the manipulation of animals were observed.

Tabla 1. Datos sobre el muestreo de camarones a finales de verano (septiembre de 2012) y a finales de invierno (febrero de 2013): ni. Número de animales capturados en cada muestreo; mi. Número de recapturas en cada muestreo; ui = ni – mi. Número de animales no marcados en cada muestreo; Mi. Número de animales marcados antes de cada muestreo; mi/ni. Proporción de animales marcados en cada muestreo. * Se capturaron 47 animales, pero solo se liberaron 44 (tres se utilizaron para realizar estudios morfométricos); no se observaron muertes relacionadas con la manipulación de los animales.

Sampling occasion ni mi ui Mi mi/ni

Summer (September 2012)22 IX * 44 0 44 0 0.00024 IX 53 2 51 44 0.03826–27 IX 60 6 54 95 0.10029 IX 42 4 38 149 0.095Sum (summer) 199 12 187 0.060

Winter (February 2013)18 II 27 0 27 0 0.00020–21 II 25 1 24 27 0.04223–24 II 29 1 28 51 0.034Sum (winter) 81 2 79 0.025

Sum (total) 280 14 266 0.050

Animal Biodiversity and Conservation 38.1 (2015) 81

Table 2. Analysed close population models in MARK closed population module for summer and winter with model selection criteria. Selected model is in bold: ML. Model likelihood; NP. Number of parameters; D. Deviance; N ± SE. Estimated population size; CI. Confidence interval;* Model Mb was ranked high but parameter estimates, SEs and CIs were unrealistic; ** Model Mh with two mixture groups was especially considered, but estimates of π were not reliable.

Tabla 2. Modelos de población cerrada analizados en el módulo de MARK para las poblaciones cerradas durante el verano y el invierno con criterios de selección de modelos. El modelo seleccionado se destaca en negrita: ML. Modelo de probabilidad; NP. Número de parámetros; D. Desviación; N ± SE. Tamaño poblacional estimado; CI. Intervalo de confianza; * El modelo Mb obtuvo buenos resultados pero la estimación de los parámetros, las desviaciones estándar y los intervalos de confianza no fueron realistas; ** El modelo Mh con dos grupos de mezcla se estudió exhaustivamente, pero las estimaciones de π no fueron fiables.

Model AICc ∆AICc WAICc ML NP D N ± SE 95% CISummer

M0 –960.564 0 0.55966 1 2 14.3276 1,196 ± 322 735–2,043Mt –958.837 1.7268 0.23602 0.4217 5 9.9896 1,188 ± 319 731–2,029

Mb* –958.548 2.0153 0.20432 0.3651 3 14.3268 1,245 ± 1848 291–10,939

Mh** –956.526 4.0377 0.06919 0.1328 4 14.3276 1,196 ± 322 735–2,043

Winter

M0 –336.129 0 0.61097 1 2 8.2864 1,064 ± 727 350–3,668Mb* –334.091 2.0376 0.22058 0.3610 3 8.2724 2,802 ± 40,789 107–260,905

Mt –332.312 3.8168 0.09062 0.1483 4 7.9822 1,062 ± 726 349–3,662

Mh** –332.008 4.1211 0.07783 0.1274 4 8.2864 1,065 ± 727 350–3,668

Holzmann et al., 2006). We have therefore especially considered modelling the population with heterogenei-ty in capture probability (model Mh). Capture models were tested with the selection of the 'Appropriate' function, which is based on a discriminant function analysis procedure.

Equality of frequencies of males and females (ovigerous and non–ovigerous pooled together) was tested by x2–test. Juveniles were excluded from the test. For the calculations, Excel 2007 was used.

Results

Population size estimates

All marks on the recaptured animals were clearly visible and easily distinguished from injuries of other origin. On four sampling occasions during the sum-mer period, 199 animals were marked. On the first occasion, we caught but did not release an additional three animals that were kept for laboratory analysis. Only 81 animals were marked during the three winter sampling occasions. The average number of marked individuals recaptured was low, 6.0% for summer and 2.5% for winter, with no animals marked during the summer recaptured in the winter period (table 1).

Water temperature was almost constant, at 10ºC in winter and 11ºC in summer; air temperature was constant across seasons at 9ºC.

Both approaches, MARK and Capture, selected M0 model (with constant and equal capture and recapture probabilities, p = c) over other models for summer and winter capture periods (tables 2, 3; fig. 2). Most proba-bly, this model was selected due to sparse data in terms of recaptures. Results based on models Mt and Mh were also comparable (table 2), despite our data showing less statistical support for those models. Noticeable differences in abundance estimates, with unrealistic standard errors and confidence intervals, resulted for Mb model, and were not taken into account as relevant (table 2). The summer M0 estimate of population size was 1,196 individuals (SE = 322; 95% CI = 735–2043), with p = 0.042 (SE = 0.012; 95% CI = 0.024–0.071) within MARK’s 'Closed captures' (table 2, fig. 2). In winter the population size estimated was 1,064 indivi-duals (SE = 727; 95% CI = 350–3668), with p = 0.025 (SE = 0.018; 95% CI = 0.006–0.095; table 2, fig. 2). Capture estimates with model M0 for summer were 1,195 individuals (SE = 320; 95% CI = 736–2,038), with p = 0.042 (no SE or CI reported in Capture), while in winter the estimates were 1,064 individuals (SE = 720; 95% CI = 353–3628), and p = 0.0254 (no SE or CI reported in Capture; fig. 2).

82 Jugovic et al.

Sex ratio and distribution of age groups

In all sampling occasions within both seasons, the pro-portion of females (mean = 76.5%; range = 55.1–86.8%) was much higher than that of males (mean = 19.6%, range = 9.4–37.9%) and unsexed juveniles (mean = 3.1%, range = 0–4.8%; fig. 3). Adult females were more numerous than adult males (x2 = 37.463, p < 0.001). Only two ovigerous females were caught, one in each season, each carrying approximately 10 eggs (counted in vivo).

Discussion

Population size and structure are basic demographic parameters that allow ecologists to evaluate the current status of a species, and may also serve for bioindication (Knapp & Fong, 1999; Praprotnik et al., 2013). In crustaceans, several MRR approaches have been used to estimate population size (Knapp & Fong, 1999; Bueno et al., 2007). We chose to apply the MRR technique for cave shrimps from Veliko jezero in Vipavska jama for the following reasons: (1) the

Table 3. Models analysed in programme Capture and model selection criteria (model selected has maximum value). The selected model is in bold.

Tabla 3. Modelos analizados en el programa Capture y criterios de selección de modelos (el modelo seleccionado tiene el valor máximo). El modelo seleccionado se destaca en negrita.

Model Mo Mh Mb Mbh Mt Mth Mtb Mtbh

Summer criteria 1.00 0.81 0.18 0.52 0.00 0.35 0.26 0.65

Winter criteria 1.00 0.83 0.38 0.68 0.00 0.45 0.32 0.73

2,000

1,800

1,600

1,400

1,200

1,000

800

600

400

200

0

2,043

1,196

735

2,038

1,195

736

Pop

ulat

ion

size

est

imat

ion

Summer Winter

Fig. 2. Summer and winter population size estimates derived from model M0 with upper (above the estimates) and lower (below the estimates) 95% confidence limits.

Fig. 2. Estimaciones del tamaño de la población en verano y en invierno obtenidas con el modelo M0 con los límites de confianza del 95% superior (por encima de las estimaciones) e inferior (por debajo de las estimaciones).

3,668

1,065

350

3,628

1,064

353

MARK

Capture

Animal Biodiversity and Conservation 38.1 (2015) 83

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%

M

Fov

F

Juv

22nd 24th 26th– 29th 18th 20th– 23rd– Total 27th 21st 24th September 2012 February 2013

Fig. 3. Population structure (adult males (M), adult ovigerous females (Fov), adult non–ovigerous females (F) and juvenile animals with no sex identified (Juv)) for Troglocaris anophthalmus sontica. The data were collected on seven sampling occasions in summer (September 2012, five sampling days, four occasions) and winter (February 2013, five sampling days, three occasions). In total, 283 individuals were recovered across all sampling occasions.

Fig. 3. Estructura de la población (machos adultos (M), hembras adultas ovígeras (Fov), hembras adultas no ovígeras (F) y animales jóvenes de sexo no identificado (Juv)) de Troglocaris anophthalmus sontica. Los datos se recogieron en siete muestreos llevados a cabo en verano (septiembre de 2012, cinco días de muestreo, cuatro muestreos) y en invierno (febrero de 2013, cinco días de muestreo, tres muestreos). En todos los muestreos se recuperaron 283 individuos en total.

chosen site was appropriate for its relatively static nature compared to underground streams or rivers; the calmer waters in this lake environment are a preferred habitat to study organisms as they can reach high population densities (Jugovic, personal observations; see also Praprotnik, 2014) and (2) Troglocaris cave shrimps with adult sizes ranging from 15–25 mm are large enough to be marked using occasion–specific identifiers. Although injuries made in marking pro-cedure can disrupt the shrimps’ movement to some extent, we assumed (3) that these small injuries do not significantly affect their survival, considering that shrimps can successfully survive injuries of the chitinous parts of the body due to cave salamander (Proteus anguinus Laurenti 1768) attacks (Jugovic et al., 2010a). Salamanders were present during our field work in Vipavska jama. We are also aware that markings could be lost through successive moults, but (4) laboratory observations over a three–year period indicated that moulting occurs only rarely (Praprotnik, 2014; Jugovic, unpublished data).

We obtained the first rough estimates of T. a. sontica population size in the ground water system of Vipavska jama. In spite of the large confidence intervals, our estimates appear relatively high for such a small sampling area. As access to most cave ecosystems is difficult, it is not easy to estimate po-pulation sizes of cave taxa, and no such sampling procedures that are needed for multi–occasion MRR analyses have been conducted so far in such small stygobitic invertebrates. Nevertheless, some attempts have been made in larger stygobionts or troglophilic terrestrial invertebrates. Simple Lincoln–Petersen calculations have often been applied to cave beetles (see references in Introduction).

Although there are no data of cave shrimp li-fespan in the literature, we assumed low mortality rates between sampling seasons. These organisms are k–strategists with low metabolic rates, and they produce a small number of relatively large eggs. They are capable of depositing extensive extracellular lipids (oleospheres) as a reserve for at least two years of

84 Jugovic et al.

starvation (Jugovic, pers. observ. in laboratory; see also Vogt & Štrus, 1999). Despite recorded longevity, we did not have recaptures from the first season. Hence we treated these two seasonal samples as separate populations.

The population size estimates were mostly similar across the two seasons and their wide confidence intervals overlapped. The low rate of recaptures should not be neglected, indicating a large population, along with the presence of many places for shrimps to avoid capture; shrimps are also capable of movement be-yond the surveyed area (Zakšek et al., 2009). Although low capture probabilities generally tend to overestimate population size due to the estimator structure (Chao & Huggins, 2005), this is true for the surveyed area of 6 m2 exclusively. Therefore, we believe our estimations represent the minimum number of shrimps living in the wider area of the suitable habitat within the lake. Individuals present in samples may thus comprise only a small portion of a large population that cannot be easily detected. A larger part of a population in Vipavska jama is probably present in other parts of the lake, or in the siphon itself from where shrimps can move to other waters of the cave system. In the cave amphipod Stygobromus emarginatus (Hubricht, 1943), researchers indicated that the pool habitat represents a window into the epikarst zone, and the low recapture rates indicate a large hidden population in the epikarst (Knapp & Fong, 1999).

We found no evidence of statistical differences bet-ween summer and winter regarding shrimp’ abundan-ce. Moreover, we detected the presence of ovigerous females during both seasons, but their low frequency of occurrence together with only two sampling periods did not allow for conclusions about possible annual rhythmicity for egg laying. Juveniles were also present during both seasons. According to the literature, ovi-gerous females are present in Troglocaris planinensis throughout the year, with a peak in late autumn (Jubertie–Jupeau, 1975). It should be noted that the estimated number of eggs in T. a. sontica from Vipa-vska jama is much lower and not consistent with data given by Juberthie–Jupeau (1974) for its closer relative Troglocaris planinensis (20–45 eggs). The estimated number instead resembles data for Gallocaris inermis (Fage, 1937) (8–12 eggs, Jubertie–Jupeau, 1974), another European cave dwelling shrimp species from southern France.

The small proportion of males contradicts the expected equal frequencies of males and females in invertebrates, but numerous exceptions have been reported (see Hodgson in EOLSS). The observed proportion of approximately 20% males is even lower than was estimated from random samples collected over the past years (i.e. approx. one male per three females; Jugovic et al., 2010b). A low proportion of juveniles may be the result of either heterogeneity in capture probabilities or the relatively small proportion of a lifespan that can be recognized as juvenile (i.e. excluding larval stages that were not sampled). The short period of the juvenile stage in many cave dwell-ing animals has been reported previously (for cave shrimps, see Matjašič, 1958). The accelerated juvenile

development is considered to be a result of a rather small number of large eggs with lots of nutrients (Ju-berthie–Jupeau, 1975; Matjašič, 1958).

Acknowledgements

We would like to thank Fabio Stoch (Trieste, Italy), for valuable samples of T. a. sontica from Italy and Valerija Zakšek (Ljubljana, Slovenia) for molecular analyses, and contribution to the known distribution of the subspecies. We also thank Alenka Mihelčič (Bled, Slovenia) and Sara Zupan (Celje, Slove-nia) for help on the field. Summer estimation was carried out during the Biocamp Rakitovec 2012 organised by Društvo varstvenih biologov – Bio-diva (Conservation Biologists Society – Biodiva). Scott Mills (Adelaide, Australia) and Tilen Genov (Koper, Slovenia) kindly reviewed the manuscript and improved the English. We thank Tilen Genov (Koper, Slovenia) for useful discussions on MRR techniques. The comments of anonymous reviewers and editor greatly contributed to improvement of the manuscript. Field work was financed in the frame of CBC Programme Italy–Slovenia 2007–2013 (Project title: BioDiNet – Network for biodiversity and cultural landscape conservation).

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87Animal Biodiversity and Conservation 38.1 (2015)

© 2015 Museu de Ciències Naturals de BarcelonaISSN: 1578–665 XeISSN: 2014–928 X

Una nueva especie troglobiomorfa de Trechus Clairville, 1806 y evidencias de colonizaciones múltiples del medio subterráneo de los montes cantábricos (Coleoptera, Carabidae, Trechinae)

J. Fresneda, C. Bourdeau & A. Faille

Fresneda, J., Bourdeau, C. & Faille, A., 2015. Una nueva especie troglobiomorfa de Trechus Clairville, 1806 y evidencias de colonizaciones múltiples del medio subterráneo de los montes cantábricos (Coleoptera, Carabidae, Trechinae). Animal Biodiversity and Conservation, 38.1: 87–100.

AbstractA new troglobiomorphic Trechus Clairville, 1806 and evidence of multiple colonizations in the subterranean environment of the Cantabrian mountains (Coleoptera, Carabidae, Trechinae).— We describe Trechus (Trechus) valenzuelai n. sp. from the Cantabrian area of Spain: Sierra de Cuera, Asturias. The species was collected in the subterranean environment, in caves and dolines. Morphological examination revealed that the new species is sister to Trechus escalerae Abeille de Perrin, 1903 within the T. saxicola clade. A synapomorphy of the male genitalia, shared by T. valenzuelai n. sp. and T. escalerae, is described and illustrated: the endophallus has a sclerotised piece shaped like a club, elongated, robust, strongly sclerotised and with a membranous sac covered with small spicules at the base. We discuss the taxonomy of the new species and provide illustrations of struc-tures showing the differences between T. escalerae and T. valenzuelai n. sp., along with biogeographical and distributional data and hypotheses regarding the speciation events based on previously pubished molecular data, and the geological structure and the palaeoclimatology of their geographical area. We hypothesize that in this clade, the colonization of the subterranean environment was the result of multiple, independent and simultaneous colonization processes. A lectotype is designated for T. escalerae.

Key words: Carabidae, Trechini, Trechus n. sp., Speciation, Colonization subterranean environment, Cantabrian area, Spain

ResumenUna nueva especie troglobiomorfa de Trechus Clairville, 1806 y evidencias de colonizaciones múltiples del medio subterráneo de los montes cantábricos (Coleoptera, Carabidae, Trechinae).— Se describe Trechus (Trechus) valenzuelai sp. n. de los montes cantábricos en España: Sierra de Cuera, en Asturias. La especie se ha en-contrado en el medio subterráneo, en cuevas y dolinas. El estudio morfológico revela que se debe situar como especie hermana de Trechus escalerae Abeille de Perrin, 1903, en el clado de T. saxicola Putzeys, 1870. Se ha encontrado una sinapomorfía en los edeagos de T. escalerae y T. valenzuelai sp. n. que se describe e ilustra: el endofalo tiene una fanera con forma de porra, alargada, robusta y muy esclerotizada, en cuya base se en-cuentra un saco membranoso recubierto por espínulas. Se discute la taxonomía y la sistemática, y se aportan ilustraciones de las estructuras que muestran las diferencias entre T. escalerae y T. valenzuelai sp. n.; también se proporcionan datos relativos a la distribución y la biogeografía, así como hipótesis sobre los procesos de especiación, tomando como base de argumentación los datos moleculares previamente publicados, la estruc-tura geológica de la región y los acontecimientos paleoclimáticos; se postulan procesos de colonización activa, simultáneos e independientes, del medio subterráneo. Se designa el lectotipo de T. escalerae.

Palabras clave: Carabidae, Trechini, Trechus sp. n., Especiación, Colonización del medio subterráneo, Área cantábrica, España

Received: 20 II 15; Conditional acceptance: 12 III 15; Final acceptance: 17 III 15

Javier Fresneda, Ca de Massa, 25526 Llesp, El Pont de Suert, Lleida, España (Spain) i Museu de Ciències Naturals de Barcelona, Passeig Picasso s/n., 08003 Barcelona, España (Spain).– Charles Bourdeau, 5 chemin Fournier–Haut, F–31320 Rebigue (France).– Arnaud Faille, Zoologische Staatssammlung München, Münchhau-senstraße 21, 81247 Munich (Germany).

Corresponding author: J. Fresneda. E–mail: ffresned@gmail.com

88 Fresneda et al.

Introducción

El panorama sistemático de los Trechus Clairville, 1806 pirenaico–cantábricos es complejo. Los diversos grupos de Jeannel (1927) presentes en la región están poco caracterizados. El descubrimiento de nuevas especies ha motivado que hubieran de redefinirse los grupos con el fin de encajar las novedades y ha conllevado además diversas transferencias de especies entre ellos (Coiffait, 1952, 1974; Español, 1970; Casale & Laneyrie, 1982; Pham, 1987; Dupré, 1991; Toribio, 1992, 2013; Toribio & Rodríguez, 1997; Salgado & Ortuño, 1998; Sciaky, 1998; Ortuño & Toribio, 2005; Ortuño & Arribas, 2010; Quéinnec & Ollivier, 2011; Toribio, 2014). En los trabajos más recientes se ha venido dando un tratamiento más filogenético a esos grupos, que se han organizado en clados. Incluso los Apoduvalius Jeannel, 1953 figuran en el árbol filogenético de los Trechus, que aparece en dos ramas distintas del árbol propuesto por Faille et al. (2013: fig. 3b) para numerosos Trechini. Entre los Trechus cantábricos se encuentran formas ápte-ras, microftalmas y despigmentadas, es decir, que presentan cierto grado de troglobiomorfía, en algunos casos bastante avanzado, como T. apoduvalipenis Salgado & Ortuño, 1998. Lo curioso es que los datos moleculares (Faille et al., 2012, 2013) muestran que esas especies troglobiomorfas forman parte de clados que cuentan también con especies no adaptadas al ambiente subterráneo. ¿Se podría estar observando un proceso de colonización activa del medio? ¿Se podría interpretar que se están produciendo colonizaciones simultáneas e independientes del medio? En relación con otros coleópteros hipogeos, algunos datos previos muestran que ha habido una única colonización del medio subterráneo y una diversificación posterior: las radiaciones pirenaicas de Leptodirini (Ribera et al., 2010) (Coleoptera, Leiodidae, Cholevinae) y de Trechini (Faille et al., 2010, 2011) (Coleoptera, Carabidae, Trechinae), ambos sin representantes epigeos. La tendencia a seguir estrategias demográficas de tipo K, frecuente en coleópteros hipogeos, es una de las adaptaciones al medio subterráneo más llamativas y confirma el modelo de colonización única (Cieslak et al., 2014). Faille et al. (2013, 2014) muestran que la colonización múltiple se da en diversos clados de Trechini: en Duvalius Delarouzée, 1859 o en los Trechus del grupo «fulvus». Los datos que se aportan en este estudio en relación con el clado de los Trechus cantábricos apuntan en ese mismo sentido.

En este artículo se describe una nueva especie troglobiomorfa descubierta en cavidades subterráneas de la Sierra de Cuera, una prolongación septentrional de los Picos de Europa, en Asturias (España), que posiblemente forme parte del clado de T. saxicola Put-zeys, 1870 (sensu Faille et al., 2013) y que muestra las mismas tendencias evolutivas que las propugnadas para otros grupos de Trechus en otras zonas geográficas.

Material y métodos

Los ejemplares se recolectaron a vista con un aspirador entomológico y se introdujeron en tubos con acetato de etilo; se conservaron en viales con alcohol de 70º

para el estudio morfológico, si bien algún ejemplar se conservó en alcohol absoluto para realizar estudios mo-leculares. También se recolectaron en trampas de caída con diferentes cebos y conservantes. La genitalia del macho se extrajo, se sumergió en una solución acuosa de KOH al 10% durante seis horas y posteriormente se pasó por una serie alcohólica (60º–96º) durante unos 15 minutos para deshidratarla y, por último, por un baño de xilol durante unas 12 horas. Finalizados estos procesos, las estructuras se incluyeron en bálsamo del Canadá sobre una plaquita rectangular de acetato transparente que se conserva insertada en la misma aguja que el ejemplar al que pertenece, el cual se ha montado sobre una cartulina rectangular. La genitalia de algunos ejemplares se montó en DMHF. Las fotografías de los habitus y de otras estructuras de la morfología externa se hicieron con un microscopio estereoscópico Olympus SZX16; las del edeago, con un microscopio de transmisión Olympus CH; en ambos casos se usó una cámara Olympus C5060WZ. Las series de fotografías se montaron con el programa Combine ZP y posteriormente se procesaron con Adobe Photoshop CS. La longitud del cuerpo se midió entre el borde anterior del labro y el ápice de los élitros.

Para la elaboración de este estudio se dispuso de numerosos ejemplares procedentes de localidades dispersas por gran parte del área de distribución del complejo. Algunos se encuentran depositados en instituciones públicas y otros forman parte de archivos entomológicos privados; la lista de las co-lecciones estudiadas se proporciona a continuación, en el apartado de abreviaciones.

Abreviaciones: IBE. Institute of Evolutionary Bio-logy, Barcelona (España); MNCN. Museo Nacional de Ciencias Naturales, Madrid (España); MNHN. Muséum National d´Histoire Naturelle, París (Fran-cia); MZB. Museu de Ciències Naturals de Barcelona (Zoologia), Barcelona (España); ZSM. Zoologische Staatssammlung, Múnich (Alemania); CAC. Col. A. Ca-sale, Turín (Italia); CAF. Col. A. Faille, París (Francia); CCB. Col. C. Bourdeau, Rebigue (Francia); CEV. Col. E. Valenzuela, Puerto de Vega (España); CFL. Col. Fresneda–Lagar, Llesp (España); CMT. Col. M. Toribio, Tres Cantos (España); CZULE. Colección Zoológica de la Universidad de León, León (España). Proporciones corporales: LE. Longitud del élitro; LP. Longitud del pronoto; WE. Anchura de los élitros; WH. Anchura de la cabeza; WP. Anchura del pronoto; WPB. Anchura de la base del pronoto.

Resultados

Trechus valenzuelai Fresneda, Bourdeau & Faille sp. n.(figs. 1, 3, 5 y 10–12)

Localidad típicaEspaña, Asturias, El Mazucu, Pozu’l Fresnu, UTM (WGS 84): 30T 349 4804, 360 m.

Serie tipoHolotipo (): España, Asturias, El Mazucu, Pozu’l Fresnu, 4 VII 2013, Bourdeau & Fresneda leg., genitalia

Animal Biodiversity and Conservation 38.1 (2015) 89

montada en una etiqueta transparente que se conserva insertada en la misma aguja que el ejemplar (MZB).

Paratipos: con los mismos datos de recolección que el holotipo, 3 y 3; misma localidad, 1979, P. Chapman leg., 1 (MZB 74–3926); misma localidad, 17 IV 2014, E. Valenzuela leg., 11 y 6; misma localidad, 8 V 014, E. Valenzuela leg., 13 y 7; misma loca-lidad, 6 XI 2014, E. Valenzuela leg., 1 y 2. España, Asturias, El Mazucu, Surgencia Las Bolugas, UTM (WGS 84), 30T 350 4804, 30 X 2014, E. Valenzuela leg., 4 y 3; misma localidad, 31 X 2014, E. Valenzuela leg., 3 y 1. España, Asturias, El Mazucu, cueva cerca de Surgencia Las Bolugas, UTM (WGS 84), 30T 350 4804, 6 VII 2014, E. Valenzuela leg., 2. España, Asturias, El Mazucu, cueva Callao Ruviera, UTM (WGS 84), 30T 351 4804, 28 X 2014, E. Valenzuela leg., 1. España, Astu-rias, El Mazucu, dolina cerca de la cueva Callao Ruviera, UTM (WGS 84), 30T 350 4804, 6 VII 2014, E. Valenzuela leg., 4 y 6. España, Asturias, Caldueñín, cueva de Caldueñín, UTM (WGS 84), 30T 348813 4804606, 6 VII 2014, E. Valenzuela leg., 3 y 2. España, Asturias, Rioseco–Llanes, cueva la Zurra, UTM (WGS 84), 30T 347 4807, 30 XI 2013, E. Valenzuela leg., 1. España, Asturias, Villa–Llanes, Sumidoriu H.ou Collau, UTM (WGS 84), 30T 346 4805, 30 XI 2013, E. Valenzuela leg., 1; misma localidad, 8 I 2014, E. Valenzuela leg., 1 y 2; misma localidad, 4 II 2014, E. Valenzuela leg., 3 y 2; misma localidad, 8 III 2014, E. Valenzuela leg., 2 y 3; misma localidad, 30 X 2014, E. Valenzuela leg., 3. España, Asturias, Bricia–Llanes, Jou la Legua, UTM (WGS 84), 30T 350 4810, 5 II 2014, E. Valenzuela leg., 2 y 8 (1: especímen de referencia ZSM L986); misma localidad, 5 XI 2014, E. Valenzuela leg., 2 y 1. Alícuotas de ADN conservadas en las colecciones de tejidos y ADN de ZSM, MNHN e IBE, paratipos con-servados en CZULE, MNCN, MNHN, ZSM, CAC, CAF, CCB, CEV, CFL, CMT.

DiagnosisEspecie de gran tamaño, de poco más de 5 mm de longitud, de aspecto oblongo, deprimida, con los apén-dices alargados, despigmentada, de color amarillento–rojizo, microftalma y áptera (fig. 1). El lóbulo medio del edeago en visión lateral (fig. 10) se reduce desde la base hasta el ápice, que es estrecho y redondeado; en vista dorsal (fig. 5) las dimensiones son regulares hasta el sector terminal y la punta es ancha y redon-deada. El endofalo tiene una fanera robusta, alargada y muy esclerotizada, en cuya base se encuentra un saco membranoso recubierto por espínulas (fig. 12).

Descripción del holotipo ()El habitus se muestra en la figura 1. El aspecto es oblongo, deprimido, despigmentado, con los apéndi-ces alargados. El tegumento presenta una microrre-ticulación densa que forma unas mallas poligonales alargadas, de disposición transversa en el pronoto y los élitros, con las mallas redondeadas que se marcan más profundamente en la cabeza. Áptero. La longitud tomada entre el labro y el ápice de los élitros es de 5,1 mm.

Color. La superficie dorsal es amarillenta–rojiza, moderadamente brillante. Las antenas, los palpos y

las patas son del mismo color pero algo más claro.Quetotaxia (fig. 1). Los élitros son glabros con la

excepción de una seta periescutelar. Tiene dos setas discales en la tercera estría; cuatro setas humerales, de las cuales las dos primeras están menos sepa-radas que las otras; cuatro setas agrupadas de dos en dos a lo largo del margen lateral, y tres formando el triángulo apical. Se encuentran cuatro setas mar-ginales en el pronoto, dos en el tercio anterior y las basales situadas antes del ángulo posterior. El clípeo tiene 2 + 2 setas y el labro 3 + 3. La pubescencia ventral se limita a una seta en cada medio ventrito.

Cabeza. Es más larga que ancha y más estrecha que el borde anterior del pronoto; las mandíbulas son agudas y pertenecen al tipo «tridentatae» de Jeannel (1926). El último artejo de los palpos maxilares es algo más largo (0,25 m) que el penúltimo (0,21 mm); el dien-te labial es bífido; los surcos frontales son profundos, bordean el área ocular y se prolongan por el clípeo; el labro tiene el margen anterior escotado; los ojos son diminutos, blanquecinos, se ven a través del tegumento que los recubre, que es de la misma naturaleza que el del resto de la cabeza, son muy reducidos, despig-mentados, planos, dos veces más largos que anchos y están dispuestos verticalmente (fig. 3); las sienes son largas, algo convexas y con pilosidad minúscula; existen en cada lado dos setas supraoculares: la anterior se sitúa al nivel del borde posterior del ojo y la posterior está muy alejada y casi pegada al surco frontal; se en-cuentran otras dos setas contiguas en las proximidades de la antena. La reticulación del tegumento la forman polígonos tan largos como anchos. Las antenas son proporcionalmente largas (3,3 mm) y los antenómeros están densamente recubiertos de setas salvo el primero; el tercer antenómero es más largo que el segundo e igual que el cuarto.

Pronoto. Es transverso, con su mayor anchura en el primer tercio a la altura de la seta pronotal anterior, con la base algo sinuosa y ligeramente más ancha que el borde anterior; el borde lateral está fuertemente curvado hasta poco más allá de la mitad de su longitud y sigue recto hasta los ángulos posteriores, donde se sitúa la seta pronotal posterior; los vértices posteriores son divergentes y agudos; el canal lateral disminuye su anchura desde la base hasta el margen anterior y los ángulos anteriores son redondeados; el surco medio no alcanza el borde anterior ni el posterior; las fosetas basales son anchas y profundas. Proporciones del pro-noto: WP/LP = 1,31; WP/WPB = 1,31; WP/WH = 1,38 y WE/WP = 1,40.

Élitros. Son convexos, alargados, de márgenes subparalelos, con los hombros caídos, el ápice re-dondeado y su mayor anchura en la región media; la terminación del borde basal acaba en el inicio de la quinta estría; las interestrías son algo convexas y el canal lateral es ancho; las estrías están bien impresas y punteadas: la primera y la quinta están unidas en su extremo y recorren el borde apical, la octava termina cerca del ápice, la tercera y la cuarta tienen los extremos unidos y acaban en el quinto apical; la sexta y la séptima acaban en el cuarto apical. Proporción: WE/LE = 0,60.

Patas. Las protibias son glabras en su cara in-terna anterior. Las patas son largas, sobre todo las

90 Fresneda et al.

posteriores. Los dos primeros protarsómeros tienen una robusta espina en su margen interior.

Edeago. El lóbulo medio es esbelto, con el lóbulo basal poco diferenciado y un alerón sagital bien desa-rrollado. En vista lateral (fig. 10) se reduce de forma rápida y progresiva desde la base hasta el ápice, que es redondeado. En vista dorsal (fig. 5), las dimensiones son regulares hasta el sector apical, que tiene la punta ancha y redondeada. Los parámeros son esbeltos y cada uno tiene insertadas cuatro setas en el ápice. La pieza copulatriz está formada por una fanera robusta y muy esclerotizada en cuya base se encuentra un saco membranoso recubierto por espínulas (fig. 12).

HembraLos dos primeros protarsómeros son simples y carecen de espina en su margen interior. Proporciones del prono-to: WP/LP = 1,30; WP/WPB = 1,36; WP/WH = 1,34 y WE/WP = 1,40. Proporciones de los élitros: WE/LE = 0,65.

TallaLongitud media tomada entre el labro y el ápice elitral (5 ejemplares): 5,4 mm () y 5,2 mm ().

EtimologíaLa nueva especie se dedica a nuestro amigo Enrique Valenzuela (Puerto de Vega, Asturias), bioespeleólogo asturiano. Sustantivo en genitivo singular.

AfinidadesLos datos morfológicos muestran que Trechus va-lenzuelai sp. n. tiene una estrecha afinidad con T. escalerae. Son dos especies de aspecto muy similar (figs. 1, 2), de gran tamaño, microftalmas (figs. 3, 4), despigmentadas, deprimidas y con los apéndices alargados; es decir, muestran un cierto grado de tro-globiomorfía. Coinciden, y esto es una sinapomorfía, en la estructura de la pieza copulatriz, que presenta una robusta fanera muy esclerotizada con forma de porra en cuya base se encuentra un saco membranoso recubierto por espínulas (figs. 9, 12, 16 y 17). En Tre-chus saxicola la pieza copulatriz es más simple y solo está formada por una lámina esclerotizada triangular que se dobla hasta formar un hemicono cuya punta se prolonga formando un pedúnculo; en T. jeannei es más compleja, pues consta de varias faneras esclero-tizadas engarzadas en un saco membranoso. Trechus valenzuelai sp. n. y T. escalerae se distinguen por los siguientes caracteres:

1. Los vértices de los ángulos posteriores del pronoto son divergentes y muy puntiagudos en T. escalerae, mientras que en T. valenzuelai sp. n. prolongan el margen lateral hacia atrás, lo que los hace moderadamente divergentes. El pronoto es más transverso en T. valenzuelai sp. n.

2. Los élitros tomados en su conjunto son marcadamen-te ovales en T. escalerae, mientras que en T. valenzuelai sp. n. son proporcionalmente más estrechos, con un aspecto más alargado, y los márgenes, subparalelos.

3. Las antenas son proporcionalmente más largas en T. escalerae.

4. En T. escalerae, la forma del lóbulo medio del edeago en visión lateral es robusto hasta las proximi-

dades del ápice, donde mengua formando un nódulo (figs. 13–24); en cambio, en T. valenzuelai sp. n. se reduce de forma regular desde la base hasta el ápice (figs. 10–12). En visión dorsal, en T. escalerae se engro-sa fuertemente en las proximidades del ápice, de modo que este tiene forma triangular (figs. 6–9), mientras que en T. valenzuelai sp. n., la disminución de tamaño es regular y la punta es ancha y redondeada (fig. 5).

Faille et al. (2012) muestran que el clado de estas dos especies comprende además a T. saxicola y T. jeannei, las cuales tienen afinidad con Apoduvalius alberichae Español, 1971, más el grupo de Trechus «brucki». Todo el conjunto forma parte del gran clado pirenaico–cantábrico donde además se imbrican es-pecies de los grupos «angusticollis» (sensu Jeannel, 1927) y «bonvouloiri» (sensu Dupré, 1991).

Distribución (fig. 25) y ecologíaEn España, Asturias, entre la Sierra de Peña Villa y el mar Cantábrico. Esta sierra pertenece a la unidad geológica de Cuera y se encuentra en el extremo occidental de la sierra. En su vertiente norte, está separada de esta por el valle cárstico de Viango. La ribera oriental del río Bedón marca el límite occidental de su distribución; en los relieves de su ribera oeste se encuentra T. escalerae. Habita en cavidades subte-rráneas con las características del medio subterráneo profundo: ausencia de luz y fotoperiodo y temperatura y humedad relativa constantes. También se ha en-contrado debajo de piedras en el fondo de dolinas. Convive con Apoduvalius aphaenopsianus Español & Vives, 1983; con Pterostichus (Lianoe) drescoi dres-coi Nègre, 1957; con Laemostenus (Anthisphodrus) peleus (Schaufuss, 1861) (Carabidae) y con Breuilia triangulum (Sharp, 1872) (Leiodidae). Collado (1977) lo había citado en el Pozu’l Fresnu como T. escalerae.

Trechus escalerae Abeille de Perrin, 1903(figs. 2, 4, 6–9 y 13–24)

Trechus (Anophtalmus) escalerae Abeille de Perrin, 1903: 299Trechus escalerai Abeille de Perrin: Jeannel, 1921: 178

Localidad típica«Acquis de M. de la Escalera, qui l’avait étiqueté: Bejas, grotte de la Armioña, 21 août 1903 (Espag-ne).» (Abeille de Perrin, 1903). No se sabe porqué Jeannel (1921: 178) indica: «type : ? cueva de la Armioña (en réalité le type provient de la cueva de la Loja).» y además no incluye la Armioña en la lista de localidades. Los autores han comprobado que la especie se encuentra en las cavidades subterráneas de Bejes (Cantabria) y el lectotipo está inequívoca-mente etiquetado como procedente de la localidad indicada en la descripción.

Designación del lectotipo de Trechus (Anophtalmus) escalerae Abeille de Perrin, 1903

Serie tipoLectotipo (MNHN), designación: «Bejes C. de la Armioña 21.8.903 / TYPE / anophtalmus Escalerae

Animal Biodiversity and Conservation 38.1 (2015) 91

1 2

1 m

m

ab. Bull.so.Ent.Fr. 1903 p. 299», «Lectotypus Trechus (Anophtalmus) escalerae Abeille de Perrin Fresneda, Bourdeau & Faille des. 2014» (etiqueta rectangular roja [impresa]), genitalia extraída y montada en una etiqueta separada insertada en la misma aguja que el ejemplar.

Paralectotipos (MNHN): 1 y 2: «Bejes C. de la Armioña 21.8.903 / ESCALERA.», «Paralec-totypus Trechus (Anophtalmus) escalerae Abeille de Perrin Fresneda, Bourdeau & Faille des. 2014»

(etiqueta rectangular roja [impresa]); 1: «Bejes C. de la Armioña 21.8.903 / TYPE / Escalera ab. Typ.», «Paralectotypus Trechus (Anophtalmus) escalerae Abeille de Perrin Fresneda, Bourdeau & Faille des. 2014» (etiqueta rectangular roja [impresa]); 1 y 1: «Bejes C. de la Armioña 21.8.903 / MUSEUM PARIS COLL. H. MARMOTTAN 1914», «Paralectotypus Trechus (Anophtalmus) escalerae Abeille de Perrin Fresneda, Bourdeau & Faille des. 2014» (etiqueta rectangular roja [impresa]).

Figs. 1–2. Habitus: 1. Trechus valenzuelai sp. n., holotipo (Pozu’l Fresnu); 2. Trechus escalerae (minas de Áliva). Quetotaxia: círculos negros.

Figs. 1–2. Habitus: 1. Trechus valenzuelai n. sp., holotype (Pozu’l Fresnu); 2. Trechus escalerae (Minas de Aliva). Chetotaxy: black circles.

92 Fresneda et al.

3

4

Distribución (fig. 25)Material estudiado y datos bibliográficosAsturias

1. Amieva (Serrano, 2013), Carbes, cueva del Collao de la Cueva, 16 VI 1984, Salgado leg., 2 y 1 (Salgado, 1986); Salgado (1997); 12 IX 1984, J.M. Salgado leg., 1 (CZULE401750).

2. Benia de Onís, cueva de la Pruneda (Salgado, 1997); VI 1990, C. Bourdeau, 5 ejs. (CCB); 5 V 1990, J.M. Salgado leg., 1 (CZULE401755).

3. Benia de Onís, cueva del Osu (Salgado, 1997).4. Cabielles, cueva de la Huelga, 24 IX 1984,

Salgado leg., 1 y 3 (Salgado, 1986, 1997).

5. Cabrales (Serrano, 2013), cueva de Tresmial-ma, UTM (WGS84): 30T 354 4793, 15 III 2014, E. Valenzuela leg., 29 y 12 (CEV, CFL).

6. Cabrales, Puertas, cueva de los Canes (=cueva de los Perros, Salgado, 1997), 26 I 2014, E. Valen-zuela leg., 9 y 15 (CEV, CFL, ZSM 1: L984).

7. Cadenava, cueva Cotazosa II, 7 IX 1980, J. M. Salgado–E. Samartino leg., 1 (CZULE401757).

8. Cangas de Onís (Serrano, 2013).9. Cangas de Onís–Covadonga, cueva de Porro Co-

vañona (Collado, 1977 y Salgado, 1997), 15 VII 1952, 2 y 1 (ex Coll. Nègre MNHNP «Cueva del Porro de Covañona Covadonga 15.VII.52»); 28 VII 1962, F. Español leg., 1 (ZSM 2009–Coll. Daffner «Hispania Covadonga C. Porro Cobariona 28.7.1962 Leg. F. Español / Trechus escalerae Ab. Det. Daffner 84 / Coll Daffner ZSM 2009»); 28 VII 1962, 1 (ex Coll. Nègre MNHNP «C Porro Covañona Covadonga 28.VII.62 ES. NE. Leg.»); 28 VIII 1967, F. Español leg., 2 ejs. (MZB 74–3919); 28 VIII 1967, F. Español leg., 1 ej. (MZB 74–3920); VIII 1967, F. Español leg., 1 (MZB 74–3922); Español (1965).

10. Cangas de Onís–Covadonga, 3 (ex Coll. Nègre MNHNP «Covadonga perte sans nom»).

11. Cangas de Onís–Covadonga, cueva del Bustio, C. Cardin leg. (Jeannel, 1927; Español, 1965; Collado, 1977), 3 ejs. (MNCN_Ent 108625, MNCN_Ent 108634 y MNCN_Ent 108635).

12. Cangas de Onís–Covadonga, refugio de Vega-rredonda, 10 VII 1974, C. Bourdeau leg., 1 (CCB).

13. Cangas de Onís–Covadonga, sistema Burdió–la Peña, 11 VIII 1978, O. Escolà leg., 2 restos, élitro + pronoto (MZB 74–3924); cueva de Burdió, 16 IV 1981, O. Escolà leg., 1 (MZB 74–39252).

14. Cangas de Onís–Covadonga, cueva de la Vega de Teón (Collado, 1977; Salgado, 1997).

15. Cangas de Onís–Covadonga, cueva de Uber-dón (Collado, 1977; Salgado, 1997).

16. Cangas de Onís–Covadonga, cueva del Infier-no (Salgado, 1997).

17. Cangas de Onís–Covadonga, cueva del Re-guerín (Salgado, 1997).

18. Cangas de Onís–Covadonga, cueva del Can-tiellu (Salgado, 1997).

19. Cangas de Onís–Covadonga, Pozo Palomero (Collado, 1977; Salgado, 1997).

20. Cangas de Onís–Covadonga, cueva entre dos dolinas, Nègre leg. (Collado, 1977).

21. Cuerres, cueva cerca de Collao de Fonta-ninas (probablemente Cueva Negra), UTM (WGS 84): 30T 337 4809, 6 XI 2014, E. Valenzuela leg., numerosos ejemplares.

22. Cuerres, pequeña cueva al oeste de cueva de Lledales cerca de la cueva Tinganón, UTM (WGS 84): 30T 335 4809, 6 XI 2014, E. Valenzuela leg., 5 y 4.

23. El Mazu–Panes, cueva de la Loja, IX 1915, C. Bolivar leg., 10 ejs. (MNCN_Ent 108219, MNCN_Ent 108220, MNCN_Ent 108617, MNCN_Ent 108618, MNCN_Ent 108619, MNCN_Ent 108620, MNCN_Ent 108621, MNCN_Ent 108622, MNCN_Ent 108623 y MNCN_Ent 108624); sin datos, C. Bolívar leg., 1 ej. (MZB 74–3923); IX 1915, C. Bolívar leg., 1 (ex Coll.

Figs. 3–4. Vista lateral de la cabeza: 3. Trechus valenzuelai sp. n. (Pozu’l Fresnu); 4. Trechus escalerae (minas de Áliva).

Figs. 3–4. Lateral view of the head: 3. Trechus valenzuelai n. sp. (Pozu’l Fresnu); 4. Trechus escalerae (Minas de Aliva).

Animal Biodiversity and Conservation 38.1 (2015) 93

5 6 7 8 9CP

0,5

mm

Nègre MNHNP «Loja El Mazo–Panes / Asturias C. BOLIVAR / IX–1915 / Anophthalmus Escalerai Ab. R. JEANNEL det.»); C. Bolívar leg., 1 (ex Coll. Nègre MNHNP «Loja El Mazo–Panes / Asturias C. BOLIVAR / Anophthalmus Escalerai Ab. / Ex coll. Ch. Fagniez»); C. Bolívar leg., 2 (Coll. Générale MNHNP «Loja El Mazo–Panes / Asturias C. BOLIVAR»); H. Breuil leg. (Jeannel, 1921, 1927); 25 IX 94, M. de la Escalera leg., 1 y 1 (Coll. Générale MNHNP «C. de Loja. El Mazo. 25.9.94 / Escalera.»); VII 1913, Ch. Alluaud leg., 1 (Coll. Générale MNHNP «Cueva de la Loja / Oviedo Alluaud. VII–13 / MUSEUM PARIS COLL. R. JEANNEL 1931 / R. Jeannel escalerai Ab.»); 1911, H. Breuil leg., 1 (Coll. Générale MNHNP «Cueva de la Loja / Oviedo Breuil 1911 / MUSEUM PARIS COLL. R. JEANNEL 1931 / R. Jeannel escalerai Ab.»); 30 VIII 1895, M. de la Escalera leg., 1 (Coll. Générale MNHNP «C. del Mazo Panes 30.8.95 / Escalera.»); Español (1965); Collado (1977).

24. La Franca, cueva Mazaculos, 16 IX 1995, J. M. Salgado leg., 1 (CZULE, preparación del edeago sobre portaobjetos de vidrio, separado del ejemplar).

25. La Molina, cueva Pompedro, 25 I 2014, E. Valenzuela leg., 1.

Figs. 5–9. Edeago en vista dorsal: 5. Trechus valenzuelai sp. n., paratipo (Pozu’l Fresnu). Trechus escalerae: 6. Bodega los Trillos; 7. Minas de Áliva; 8. Cueva de Samoreli; 9. Cueva de Burdió; CP. Pieza copulatriz.

Figs. 5–9. Aedeagus in dorsal view: 5. Trechus valenzuelai n. sp., paratype (Pozu’l Fresnu). Trechus escalerae: 6. Bodega los Trillos; 7. Minas de Aliva; 8. Cueva de Samoreli; 9. Cueva de Burdió; CP. Copulatory piece.

26. Oceño, El Cuevu, 8 III 2014, E. Valenzuela leg., 1 y 1 (CEV).

27. Oceño, cueva Calluenga, UTM (WGS84): 30T 361 4794, 4 II 2014, E. Valenzuela leg., 2 (ZSM: L991).

28. Onís (Serrano, 2013), Villar, cuevas de Villar (Salgado, 1997).

29. Orlé, Xerra Buceñao, Conforcos, cueva cerca de Foz Melordaña, 30T 314 4784, 19 VII 2014, E. Valenzuela leg., 2 y 1 (CFL); 26 VII 2014, E. Valenzuela leg., 7 y 5 (CFL).

30. Ortiguero, cueva Los Hoos, UTM (WGS84): 30T 344 4798, 24 I 2014, E. Valenzuela leg., 3 y 1 (CEV, CFL); VII 1956, 1 (CFL); 1 y 1 (ex Coll. Nègre MNHNP «C. de los Joos Ortiguero As-turias – 7.56»).

31. Panes, cueva de los Torcos, 20 VIII 1927, J. Royo–C. Bolívar leg., 1 ej. (MNCN_Ent 108636).

32. Panes, «Petite grotte sur la Peña Mellera au Puerto de las Llaves» (Jeannel, 1921), H. Breuil leg. (MNHNP); Jeannel (1927); Español (1965); Collado (1977); VIII 1909, R. Jeannel leg., 1 (Coll. Générale MNHNP «Cueva de P. melliera / asturies VIII 09 / Tre-chus Escalerai Ab. / Dr Jeannel»); VIII–1909, 1 (Coll.

94 Fresneda et al.

Générale MNHNP «Cueva de P. melliera / asturies VIII 09 / MUSEUM PARIS COLL. L. BEDEL 1922 / Escalerai Ab.»); VIII 1909, 2 (Coll. Générale MNHNP «Cueva de P. melliera / asturies VIII 09 / MUSEUM PARIS COLL. R. JEANNEL 1931 / R. Jeannel escalerai Ab.»); VIII 1909, 1 ej. (MNCN_Ent 108626 «Cueva de L. Mellieza»).

33. Pimiango, cueva del Pindal, H. Breuil leg. (Jeannel, 1921); H. Breuil–Ch. Alluaud leg. (Jean-nel, 1927); VII 1913, H. Breuil leg., 1 y 1 (Coll. Générale MNHNP «Cueva de Pindal / Oviedo Breuil VII 13 / MUSEUM PARIS COLL. R. JEANNEL 1931 / R. Jeannel escalerai Ab.»); 1 IX 1924, M. de la Es-calera leg., 2 ejs. (MNCN_Ent 108630 y MNCN_Ent 108632); sin datos, M. de la Escalera leg., 1 ej. (MNCN_Ent 108633); IX 1915, C. Bolívar leg., 1 ej. (MNCN_Ent 108628 y MNCN_Ent 108631); sin datos, C. Bolívar leg., 1 ej. (MNCN_Ent 108629); VII 1913, Ch. Alluaud leg., 1 ej. (MNCN_Ent 108627), 2 y 2 (Coll. Générale MNHNP «Cueva de Pindal / Pimianso Alluaud VII 13 / MUSEUM PARIS COLL. R. JEANNEL 1931 / R. Jeannel escalerai Ab.»); Franz leg., 1 (Coll. Générale MNHNP «C. Pindal Franz»); 1 (ex Coll. Nègre MNHNP «D. escalerai / Pimiango C. del Pindal»); Español (1965); Collado (1977).

34. Rales, cueva de Samoreli, 14 I 2012, E. Valenzuela leg., 1 y 2 (CEV); 18 XII 2013, E. Valenzuela leg., 2 y 1 (CEV, CFL).

35. Viego, cueva del Aljibe, 19 IX 1982, Salgado leg., 5 y 2 (Salgado, 1986).

Cantabria36. Bejes, cueva de la Armioña (véase la serie tipo).37. Bejes, Bodega los Trillos, UTM (WGS84): 30T

366 4788, 9 III 2014, E. Valenzuela leg., 4 y 1 (CEV, CFL).

38. Camaleño, minas de Áliva, 24 VII 1999, M. Toribio leg., 1 (CMT).

39. La Liébana, A. Kricheldorff leg., 1 (ZSM 2009–Coll. Daffner «Picos de Europa La Liebana A: Kricheldorff / Zool Mus Berlin / Trechus escalerae Ab. Det. Daffner 84 / Coll Daffner ZSM 2009»); A. Kricheldorff leg., 1 (ex Coll. Nègre MNHNP «Picos de Europa La Liebana A. Kricheldorff»).

León40. La Uña, cueva del Castillo, IV 1976, J.M.

Salgado leg., 1 ej. (MZB 74–39212).41. Sajambre (Serrano, 2013), Soto de Sajambre,

cueva de Llagos (Salgado, 1997).42. Sajambre, Soto de Sajambre, cueva de Soto-

rriza (Salgado, 1997).43. Valdeón (Serrano, 2013), Cordiñanes, cueva

de los Moros (Salgado, 1997).

No se conoce la situación de la cueva del Sell en Llanes, Asturias, donde H. Breuil recolectó T. escalerae (Jean-nel, 1921, 1927; Collado, 1977); estos ejemplares se encuentran depositados en Coll. Générale MNHN y están etiquetados como sigue: «Cueva del Sell Pañes / VIII 09 asturies / MUSEUM PARIS COLL. R. JEAN-NEL 1931 / R. Jeannel escalerai Ab.». En Suarías, muy cerca de Panes, se encuentra un lugar llamado "El Sel" (UTM WGS 84: 30T 372768 4795545, 370 m), pero no se tiene la certeza de que sea el lugar donde H. Breuil tomó las muestras. Esta localidad no se ha incluido en el mapa de distribución. Tampoco se han incluido en el mapa unos ejemplares de Asturias (1 y 1) ex Coll. Nègre MNHN etiquetados «Rio Dobres» ni el dato que aporta Collado (1977) «dolina entre dos cuevas (Negre)». Collado (1977) cita T. escalerae de la cueva del Covarón en La Pereda y Vives (1980) también de la cueva de Balmori: dada la situación de estas localidades, probablemente se trate de T. valenzuelai sp. n., aunque no se han podido estudiar ejemplares de esas procedencias.

Trechus escalerae se encuentra entre 50 y 100 m de altitud en las localidades próximas a la costa (La Franca, Pimiango) y hasta los 1.900 m de los puertos de Áliva en los Picos de Europa (fig. 25). Aunque responde al mismo modelo general, se encuentra una gran variabilidad de formas de edeago (figs. 13–24): desde las formas robustas de Cabrales (fig. 13) o Covadonga (fig. 14), pasando por las más sinuosas de La Uña (fig. 15), Rales (fig. 16), Benia de Onís (fig. 17), Ortiguero (fig. 18) o Puertas (fig. 19), hasta las más gráciles y regulares de las minas de Áliva (fig. 20), Bejes (fig. 21), Panes (figs. 22, 23) o Pimiango (fig. 24). No se ha encontrado un patrón claro de distribución de estas formas, por lo que todas se incorporan al intervalo de variabilidad de T. escalerae.

10 11 12

CP

0,5

mm

Figs. 10–12. Edeago en vista lateral de Trechus valenzuelai sp. n.: 10. Holotipo (Pozu’l Fresnu). Paratipos de: 11. Cueva la Zurra; 12. Sumidoriu h.ou Collau. CP. Pieza copulatriz.

Figs. 10–12. Aedeagus in lateral view of Trechus valenzuelai n. sp.: 10. Holotype (Pozu’l Fresnu); Paratypes from: 11. Cueva la Zurra; 12. Sumidoriu h.ou Collau. CP. Copulatory piece.

Animal Biodiversity and Conservation 38.1 (2015) 95

13 14 15 16 17 18

19 20 21 22 23 24

CP

0,5

mm

Discusión

Tal como se ha apuntado en la introducción, los grupos de Trechus con distribución cantábrica de Jeannel (1927) no están suficientemente caracte-rizados. Los caracteres morfológicos que han de examinarse son pocos, pero no se han sistematizado convenientemente; asimismo, hay que pensar en la posibilidad de que las similitudes morfológicas puedan ser homoplásicas. No resulta nada fácil hacer agrupaciones basadas en la morfología.

Además, para complicar aún más el asunto, Faille et al. (2010, 2011, 2012, 2013, 2014) muestran que Apoduvalius (Trichapoduvalius) alberichae y otros Apoduvalius como A. anseriformis Salgado & Pelaez, 2004 pertenecen al clado de los Trechus pirenaico–cantábricos y que además se encuentran en posiciones distantes dentro de él. Anteriormente, Ortuño & Jiménez–Valverde (2011) habían estableci-do la sinonimia entre Trichapoduvalius Vives, 1976 y Apoduvalius. Faille et al. (2012) definen el grupo «brucki» (= grupo «uhagoni» Jeannel, 1927 partim)

Figs. 13–24. Edeago en vista lateral de Trechus escalerae: 13. Cueva de Tresmialma; 14. Cueva de Porro Covañona; 15. Cueva del Castillo; 16. Cueva de Samoreli; 17. Cueva la Pruneda; 18. Cueva los Ho; 19. Cueva los Canes; 20. Minas de Áliva; 21. Bodega los Trillos; 22. Cueva los Torcos; 23. Cueva de la Loja; 24. Cueva del Pindal: CP. Pieza copulatriz.

Figs. 13–24. Aedeagus in lateral view of Trechus escalerae: 13. Cueva de Tresmialma; 14. Cueva de Porro Cobañona; 15. Cueva del Castillo; 16. Cueva de Samoreli; 17. Cueva la Pruneda; 18. Cueva los Hoos; 19. Cueva los Canes; 20. Minas de Aliva; 21. Bodega los Trillos; 22. Cueva los Torcos; 23. Cueva de la Loja; 24. Cueva del Pindal: CP. Copulatory piece.

96 Fresneda et al.

incorporando especies del grupo «angusticollis». El grupo «brucki» está basado tanto en criterios mole-culares como morfológicos, como la existencia de una pieza copulatriz secundaria. Al árbol filogenético que elaboran estos autores se añaden posteriormen-te diversos taxones de cladogénesis basales de la filogenia, si bien la topología del clado no cambia de forma sustancial (Faille et al., 2013). De acuerdo con Faille et al. (2013), la distribución sistemática obtenida a partir de datos moleculares muestra que existe un clado pirenaico–cantábrico bien fundamen-tado que comprende diversos subclados, algunos de los cuales están bien definidos morfológicamente y otros no. El clado está constituido por un grupo externo formado por ((T. aubryi Coiffait, 1953 + T. distinctus Fairmaire & Laboulbène, 1854) (T. abeillei Pandellé, 1872 (T. bonvouloiri Pandellé, 1867 (T. bordei Peyerimhoff, 1909 + T. navaricus Vuillefroy, 1867))))); un grupo hermano de A. alberichae + Tre-chus: el grupo «brucki», formado por ((T. escalerae + T. valenzuelai sp. n.) (T. saxicola + T. jeannei)); una especie hermana del grupo «brucki», que es Apoduvalius alberichae; y el grupo «brucki», el único bien caracterizado morfológicamente, que está for-mado por (T. bouilloni Faille, Bourdeau & Fresneda, 2012 (T. uhagoni Crotch, 1869 + T. grenieri Pandellé, 1867) (T. brucki Fairmaire, 1862 (T. beusti Schau-fuss, 1863 + T. pieltaini Jeannel, 1920)))). Todavía no se han publicado los datos moleculares de otras especies que presuntamente pertenecen a esos gru-pos o están presentes en la región: T. angusticollis Kiesenwetter, 1850; T. apoduvalipenis; T. arribasi Jeanne, 1988; T. baztanensis Dupré, 1991; T. carrilloi Toribio & Rodríguez, 1997; T. cifrianae Ortuño & Jiménez–Valverde, 2011; T. gloriensis Jeanne, 1971; T. kricheldorffi Wagner, 1913; T. marcilhaci Pham, 1987; T. ortizi Español, 1970; T. pecignai Toribio, 1992; T. pisuenensis Ortuño & Toribio, 2005; y T. triamicorum Ortuño & Jiménez–Valverde, 2011; ni de diversas subespecies. Tampoco hay datos mo-leculares de las últimas aportaciones al conjunto: Ortuño et al. (2014) describen T. arrecheai Ortuño, Gilgado & Cuesta, 2014 del macizo del Moncayo, en el Sistema Ibérico, e indican que debe incluirse entre las especies del grupo «angusticollis» al igual que T. pilonensis Toribio, 2014, que es una especie braquíptera, microftalma y despigmentada que habita en MSS en la sierra del Sueve, en Asturias. Ortuño et al. (2014) también indican que a la vista de los resultados que aporta Faille et al. (2012), de las doce especies que Ortuño & Toribio (2005) incluían en el grupo «angusticollis» se deben extraer cuatro. Ortuño et al. (2014) incluyen a T. meregallii Casale, 1981 en el grupo «angusticollis».

De todos estos datos cabe inferir la existencia de un proceso de colonización activa del medio, al igual que se ha postulado para otros grupos afines de Trechus (Faille et al., 2013: figs. 6a y b). Para los organismos de los que en general solo se sabe de su existencia y cuyo acceso a los hábitats potencialmente colonizables viene condicionado por la higrometría, lla troglobiomorfía puede ser un buen indicador de su grado de dependencia del ambiente hipogeo

estricto o, al menos, un reflejo de la evolución en este entorno. Pues bien, en los relieves cantábricos se encuentran en el mismo clado especies epigeas oculadas junto a especies hipogeas marcadamente troglobiomorfas. Trechus escalerae y T. valenzuelai sp. n. podrían constituir la primera cladogénesis de un proceso de radiación al medio subterráneo, tal como parece haber sucedido con el grupo de T. fulvus (Faille et al., 2014) o en Apoduvalius, si bien en otras ocasiones la colonización del medio no ha desembocado en un fenómeno de radiación como en el caso de varios Trechini fuertemente adapta-dos al ambiente hipogeo: Paraphaenops Jeannel, 1916; Sardaphaenops Cerruti & Henrot, 1956; y Typhlotrechus G. Müller, 1913. Trechus escalerae y T. valenzuelai sp. n. presentan un cierto grado de troglobiomorfía, una marcada microftalmia y despig-mentación, y habitan preferentemente en cavidades subterráneas, mientras que las dos especies de su grupo hermano, cuya cladogénesis se sitúa en 7–8 millones de años (Faille et al., 2013) son epigeas y oculadas: T. jeannei está fuertemente pigmentada, mientras que T. saxicola es despigmentada. Su grupo hermano no presenta grandes adaptaciones al me-dio subterráneo, ya que todas son oculadas, y solo la pigmentación presenta distintos grados, a saber:

Subclado 1: T. bonvouloiri y T. bordei están pig-mentadas, aunque algunas poblaciones de esta última presentan un cierto grado de despigmentación, y T. navaricus y T. abeillei están incipientemente despig-mentadas.

Subclado 2: T. aubryi y T. distinctus están incipien-temente despigmentadas.

De igual forma, se podría interpretar que se están produciendo colonizaciones simultáneas e indepen-dientes del medio. Los elementos troglobiomorfos se encuentran distribuidos por los distintos subclados del clado pirenaico–cantábrico a pesar de que se concentran en los de distribución cantábrica (Faille et al., 2013). Las peculiaridades paleoclimáticas que, entre otros factores, pueden haber impulsado la colonización del medio subterráneo han afectado a la cornisa cantábrica, pero no al macizo pirenaico donde no se encuentran Trechus troglobiomorfos; solo en el extremo occidental del macizo (distrito bio-espeleológico vasco de Bellés [1987]) se encuentran formas incipientemente troglobiomorfas: T. beusti y T. pieltaini. En cambio, existen Trechini hipogeos troglobiomorfos en los Pirineos, pero los procesos de colonización del medio y especiación son otros: Faille et al. (2011) sitúan la cladogénesis de Aphae-nops Bonvouloir, 1861; Geotrechus Jeannel, 1919; e Hydraphaenops Jeannel, 1926 con el clado en el que se encuentran los Trechus cantábricos en 37,85 millones de años, así que la radiación de los Trechini hipogeos pirenaicos ha de situarse en otro contexto geológico y climático.

Biogeografía y especiación (fig. 25)

Para este estudio se ha usado el mapa geológico de la región del Cuera y los Picos de Europa publicado por Marquínez (1989) y los paleoclimas definidos por

Animal Biodiversity and Conservation 38.1 (2015) 97

Fig. 25. Mapa y corte geológico. Distribución de Trechus escalerae y T. valenzuelai sp. n. (material es-tudiado: símbolos con cruz). El mapa y el corte geológico se han modificado a partir de los publicados por Marquínez (1989).

Fig. 25. Geologic map and profile. Distribution of Trechus escalerae and T. valenzuelai n. sp. (material studied: symbols with cross). Geologic map and geologic profile modified after Marquínez (1989).

Trechus Trechus valenzuelai sp. n. escalerae

UnidadPisuerga—Carrión

UnidadCuera

UnidadPicos de EUropa

A B

Cabalgamiento básico

Caba

lgam

ient

o b

ásic

oUni

dad

Cue

raU

nida

dP

icos

de

EU

ropa

Trechus escalerae

Trechus valenzuelai sp. n.

Material estudiado

N

0 1 2 3 4 km

A

B

a. Gamonedo: calizas, lutitas y margasb. Puentelles: calizasc. Cavandi: rocas clásticasCalizas de la unidad Picos de Europa

a. Areniscas, limolitas y lutitas de la formación Beleñob, c. Calizas de la unidad Cuera

Calizas de la formación Griotte y areniscas del Devónico

Formaciones Barrios y Oville: cuarcitas

Formación Lancara: calizas y dolomitas

Carbonífero

Ordovícico

Cámbrico

Unidad Pisuerga–Carrión Mesozoico Cuaternario

Rocas cásticas:a. Formación Amivea b. Formación Lebeña

98 Fresneda et al.

Uriarte (2003). La nueva especie se distribuye por las calizas del carbonífero de la unidad geológica del Cuera. Esta unidad está separada de los Picos de Europa (láminas de Gamonedo–Panes, imbrica-do principal del macizo y sus láminas frontales) por cuarcitas ordovícicas no carstificables; por las rocas calizas de la unidad de los Picos de Europa y las del mesozoico se distribuye T. escalerae; esas cuarcitas ordovícicas aíslan la unidad del Cuera de la de los Picos de Europa.

La existencia de una sinapomorfía del conjunto Trechus escalerae–T. valenzuelai sp. n. (la peculiar estructura de la pieza copulatriz del edeago) parece indicar que son especies hermanas y por lo tanto su cladogénesis ha de ser mucho más reciente que la que propone Faille et al. (2013) para T. escalerae–T. saxicola. Dado que Faille et al. (2013) postulan, aunque con pocos apoyos, que esta última se debe situar entre los siete y ocho millones de años, en-tonces la de T. escalerae–T. valenzuelai sp. n. ha de ser más reciente: habría que situarla en algún momento entre los siete y ocho millones de años y la actualidad. Los acontecimientos paleoclimáticos de este lapso de tiempo ya se han esgrimido para explicar la dispersión y especiación de las especies del grupo de T. brucki (Faille et al., 2012) o del género Troglocharinus Reitter, 1908 (Leiodidae, Cholevinae y Leptodirini) (Rizzo et al., 2013); estos episodios son los siguientes:

Episodio 1. El clima cálido de la primera mitad del mioceno es sustituido por un desplome brusco de las temperaturas y una importante disminución de la precipitación; en este escenario, la cladogénesis del ancestro de T. jeannei (oculado) y T. saxicola (con los ojos algo reducidos pero pigmentados) con el ancestro de T. escalerae y T. valenzuelai sp. n. (microftalmos e hipogeos), que durante ese periodo debe acceder al medio subterráneo, debe de producirse entre los siete y ocho millones de años.

Episodio 2. Se considera probable que se haya aprovechado el período cálido del plioceno medio (entre 3,3 y 3 millones de años), en el que la tempe-ratura y la precipitación medias eran más altas que en el presente (unos 3 ºC y entre 400 y 1.000 mm, respectivamente), para distribuirse por toda la región y desplazarse por la superficie (suelo y humus en el ambiente forestal).

Episodio 3. La transición al pleistoceno (2,7 millo-nes de años) es el inicio de una marcada variabilidad climática. El aislamiento y, por tanto, la reducción o la interrupción del flujo génico entre poblaciones, debe haber ocurrido durante este periodo al ritmo de la alternancia de periodos fríos y cálidos y secos y húmedos: las variaciones climáticas del pleistoceno produjeron cambios drásticos en la composición de los biomas y limitaron o favorecieron las posibilidades de dispersión. Para el género Troglocharinus, Rizzo et al. (2013) consideran que el incremento de la estacionali-dad y la aparición del clima mediterráneo, con veranos secos y cálidos que impedirían los desplazamientos por superficie, son un factor fundamental. Debido a la proximidad del océano, en la cornisa cantábrica la estacionalidad no debió ser tan marcada y quizá

por eso algunos grupos están menos modificados y se han producido menos radiaciones estrictamente subterráneas. En este escenario climáticamente cam-biante, el ancestro de T. escalerae y T. valenzuelai sp. n. penetraría de nuevo en el dominio subterráneo y de este modo se podría explicar el proceso de es-peciación: los materiales silícicos aíslan la formación del Cuera de la unidad de los Picos de Europa e interrumpen el contacto entre las poblaciones. Otras fronteras de materiales no carstificables (cuarcitas, pizarras y areniscas) aíslan a las numerosas esca-mas de calizas del carbonífero de la unidad de los Picos de Europa, donde la variabilidad observada en T. escalerae parece ser la consecuencia de un flujo génico reducido. No parece pues plausible que la dispersión de estos taxones se haya producido por desplazamiento por el medio subterráneo, sino por la superficie durante el episodio 2. De este modo, la divergencia entre Trechus escalerae y T. valenzuelai sp. n., así como también el origen de la divergencia entre las diversas formas de T. escalerae, se deberían situar entre el inicio del pleistoceno y la actualidad. Los estudios moleculares en curso precisarán más esta aproximación.

Agradecimientos

Por el préstamo de ejemplares para el estudio a T. Deuve (MNHN), A. Taghavian (MNHN), M. Balke (ZSM), M. París (MNCN), G. Masó (MZB), L. M. Fer-nández (CZULE), M. Toribio (España, Tres Cantos) y J. M. Salgado (España, Vigo). A dos revisores que han contribuido sustancialmente a clarificar diversos apartados del artículo. Un agradecimiento muy especial para el bioespeleólogo E. Valenzuela (España, Puerto de Vega) y para I. Ribera (IBE) y J. M. Salgado por la lectura crítica del artículo. A. Faille desarrolla un proyecto de la Deutsche Forschungsgemeinschaft (FA 1042/1–1).

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– 1997. Estado actual de la coleopterofauna troglobia de “Picos de Europa” (España). Zoologica baetica, 8: 85–94.

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Toribio, M., 1992. Un nuevo Trechus Clairville, 1806 del norte de España (Coleoptera: Trechidae). Elytron, 6: 87–90.

– 2013. Datos sobre algunos Carábidos de la Pe-nínsula Ibérica (Coleoptera). Revista gaditana de Entomología, 4(1): 1–5.

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Saavedra, S., Maraver, A., Anadón, J. D. & Tella, J. L., 2015. A survey of recent introduction events, spread and mitigation efforts of mynas (Acridotheres sp.) in Spain and Portugal. Animal Biodiversity and Conservation, 38.1: 121–127.

AbstractA survey of recent introduction events, spread and mitigation efforts of mynas (Acridotheres sp.) in Spain and Portugal.— The common myna Acridotheres tristis is listed among the world’s 100 worst invasive alien species. We combined previous records with a field survey to update the extent and fate of myna introductions in Spain and Portugal. Results suggest that there have been at least 22 independent accidental introductions of three myna species throughout the Iberian peninsula and three archipelagos since the early 1990s. While bank mynas (A. ginginianus) did not become established elsewhere, common mynas reached breeding populations on four islands. Eradication efforts allowed the extirpation of these breeding island populations, but common mynas continue to breed in the Tagus Estuary (continental Portugal). In this region, there is also a breeding population of crested mynas (A. cristatellus), which was undergone an exponential population growth in the last decade. To avoid further accidental introductions, eradication campaigns should be combined with preventive actions aiming to stop the trade of these species in Europe.

Key words: Bank myna, Common myna, Crested myna, Eradication, Introduction pathways

ResumenUn estudio sobre los recientes episodios de introducción, la propagación y las iniciativas de mitigación de los minás (Acridotheres sp.) en España y Portugal.— El miná común, Acridotheres tristis, está catalogado entre las 100 especies más invasoras del mundo. En el presente artículo combinamos las observaciones ya existentes con un estudio de campo para determinar los procesos de invasión de tres especies de minás en España y Portugal. Los resultados sugieren que hubo al menos 22 introducciones accidentales e independientes desde comienzos de los años 90 en la península ibérica y en tres archipiélagos. Si bien el miná oscuro (A. ginginianus) no ha llegado a establecerse, hay poblaciones reproductoras de miná común en cuatro islas. Las iniciativas de erradicación permitieron eliminar esas poblaciones insulares, pero la especie se mantiene en el estuario del Tajo (Portugal). En esta región existe también una población reproductora de miná crestado (A. cristatellus), que ha crecido exponencialmente en la última década. Es necesario combinar las campañas de erradicación con acciones preventivas, cuyo objetivo sea detener el comercio de estas especies en Europa, para evitar nuevas introducciones accidentales.

Palabras clave: Miná oscuro, Miná común, Miná crestado, Erradicación, Vías de introducción

Received: 19 XI 14; Conditional acceptance: 14 I 15; Final acceptance: 7 IV 15

S. Saavedra, INBIMA–Invasive Bird Management, P. O Box 6009, 38008 S/C de Tenerife, España (Spain).– A. Maraver & J. L. Tella, Dept. of Conservation Biology, Estación Biológica de Doñana (CSIC), Avda. Amé-rico Vespucio s/n., 41092 Sevilla, España (Spain).– J. D. Anadón, Queens College, City Univ. of New York, 65–30 Kissena Blvd., Flushing NY 11367, USA.

Corresponding author: José L. Tella. E–mail: tella@ebd.csic.es

A survey of recent introduction events, spread and mitigation efforts of mynas (Acridotheres sp.) in Spain and Portugal

S. Saavedra, A. Maraver, J. D. Anadón & J. L. Tella

122 Saavedra et al.

Introduction

Mynas (Family Sturnidae) are medium–sized, om-nivorous passerine birds native to south east Asia (Feare & Craig, 1998). The common myna (Acri-dotheres tristis Linnaeus 1976) has been traded worldwide as a cage–bird species and was often deliberately introduced to control insect plagues. It has been introduced in at least 15 continental countries and 17 archipelagos and is considered among the most invasive birds in the world (Lever, 2005). The introduction of common mynas is of great concern. Besides being considered a crop pest, it spreads invasive plant species, and may contribute to the decline and even extinction of native bird species through competition for food and nest sites, predation of eggs and nestlings, and transmission of parasites and diseases (Feare & Craig, 1998; Lever, 2005). These ecological and economic im-pacts have led to the common myna being listed among the world’s 100 worst invasive alien species (Lowe et al., 2004). Although much less intensively, up to six other myna species have been introduced outside of their native ranges during the last two centuries, in many cases resulting in established populations (Lever, 2005). Some introductions are relatively recent, such as the introduction of crested mynas (Acridotheres cristatellus Linnaeus 1766) in 1982 in Argentina, an event which resulted in an established and further spread population (Bassó et al., 2012).

While the fate of early introductions of mynas and other bird species has been reported in detail (Lever, 2005), continuing introductions and chan-ges in vectors of transport mean that catalogues of introduced bird species are becoming increasingly outdated (Blackburn et al., 2010). Accounts of recent introduction events and established populations are thus needed to avoid biases when inferring patterns and processes of avian invasions (Blackburn et al., 2010). Such information is crucial when dealing with species like mynas that potentially cause serious impacts because early detection of newly introdu-ced populations makes them easier to control and eradicate (Edelaar & Tella, 2012). Information on myna species introduced in Europe is scarce and outdated (Lever, 2005), and there is a continuous risk of accidental introductions because these birds are available in pet shops in several European coun-tries (Carrete & Tella, 2008; author’s unpublished information). Here, we explore the spatial distribution and spread of mynas in Spain and Portugal as well as the success of efforts taken to mitigate this inva-sion. We used an intensive search of ornithological resources, completed with a field survey, revealing multiple introduction events, the control and eradica-tion of some populations and the spread of others, for three species of mynas (common myna, crested myna, and bank myna A. ginginianus Latham 1790) introduced in recent decades. The aim of this study was to briefly inform on the spatial distribution and fate of recent introductions in these two southern European countries.

Methods

We attempted to compile all published and unpublis-hed records of myna species in Spain and Portugal, thus covering the entire Iberian peninsula and the Canary, Balearic, Madeira and Azores archipelagos. We searched observations of non–native species in Spain and Portugal from 1912 to 2012 (see Sanz–Aguilar et al., 2014 for the same approach). Our search involved a systematic review of two national peer–reviewed journals (Ardeola from Spain and Airo from Portugal), five national and regional bird atlases, 26 regional ornithological yearbooks and monogra-phs, and 11 websites that compile bird observations or photographs of birds in Spain and Portugal (two of them devoted to exotic birds). Records were completed using our own data and unpublished observations from 45 experienced ornithologists. All localities were geo–referenced, and the data recorded included date, number of individuals observed, and any evidence of proven reproduction (e.g., active nests, fledglings accompanied by parents). As a de-monstration of the robustness of the searching effort, we compiled > 13,000 observations (involving almost 76,000 individuals) from > 370 species of non–nati-ve bird species observed in the wild in Spain and Portugal (contrasting with the just nearly 60 species recorded for the same countries by the most recently updated European catalogue; DAISIE, 2015). All this information and searching sources will be published elsewhere (Abellán et al., in prep.). Regarding records of myna species (n = 247), 77% were obtained from web sites, 15% from printed sources, and 8% from our own observations. The three myna species are easily identifiable in the field and the compiled observations were often accompanied with photographs. Moreover, 15 localities where mynas were reported were visited by the first author, allowing us to confirm the identity of the species. Therefore, we feel the data compiled here are reliable.

Based on the compiled records, we planned a field survey in the area with most of the recent observations of mynas (surroundings of Tagus Estuary, Portugal) during the 2011 breeding season. The survey was con-ducted between June 3 and June 17, visiting 11 sites with previously known presence of mynas to obtain baseline information on their distribution and rough estimates of their minimum population sizes. We looked for mynas by walking across urban and peri–urban areas where the species were previously observed. To obtain a minimum population estimate, we noted the maximum number of birds simultaneously observed at each site, thereby avoiding double–counting. Our aim was to obtain a first population assessment and to infer whether populations were increasing in an area where no control/eradication actions were undertaken.

Results

We compiled 133 observations of common mynas (in-volving at least 405 individuals) in Spain and Portugal. Records (the first obtained in 1993) were scattered in

Animal Biodiversity and Conservation 38.1 (2015) 123

space and time throughout the Iberian Peninsula, and from eight islands in the Balearic and Canary archi-pelagos and Madeira (fig. 1), suggesting they were the result of independent introduction events. The first confirmed reproduction was reported in 1993 in Tenerife, followed by reports in Gran Canaria in 2002, Fuerteventura in 2006 (islands within the Canary ar-chipelago), and Mallorca (in the Balearic archipelago) in 2001. Several control campaigns were conducted on these incipient breeding populations between 1999 and 2008, leaving only four free birds after removing 24 individuals in Fuerteventura (a further expedition to the island in 2010 verified that there were no free mynas), seven in Mallorca after removing 10 indivi-duals (the remaining seven birds were later shot by regional wildlife authorities in April 2007), and two in Tenerife after removing 10 individuals by 2009 (these two were not seen again). No further records of the species have been reported for Fuerteventura, Gran Canaria (where the three free–living individuals were removed) or Mallorca, where the species no longer exists. However, two fledglings accompanying an adult myna were observed in Tenerife in 2012 and were shot by regional wildlife authorities in 2013.

In continental Spain, observations of common my-nas seem to correspond to isolated and independent introduction events (fig. 1) and reproduction has not been confirmed. In continental Portugal, however, observations of the species were continually reported from 2001 to 2012 (n = 23 reports, groups of up to five individuals), suggesting that there is a breeding population in the estuary of the Tagus River (fig. 2). Reports correspond to urbanized areas of Lisbon, Belem, Cascais, Oeiras, Corroios and Caparica

(fig. 2). During our field survey in June 2011 (see results in appendix 1), we observed a group of about 10 individuals in a colony of crested mynas nesting in a limestone cliff in Caparica. These birds were observed in the breeding colony in three separate days (7, 15–17 VI 11), and thus their reproduction in the colony was highly probable. Moreover, a pho-tograph from an observer in Oeiras in 2008 showed an intermediate phenotype between common and crested mynas, potentially representing the natural hybridization of these species in the area.

The number of records obtained for crested mynas (n = 103) from 1997 to 2012 was similar, involving a larger number of observed individuals (n = 772). Con-trary to the widespread distribution of localities occupied by common mynas, most records of crested mynas came from the surroundings of the Tagus Estuary, Portugal (figs. 1, 2). Both the number of records and the number of individuals of crested mynas observed have increased dramatically in recent years in this area (inset in fig. 2). Our survey conducted in June 2011 (appendix 1) rendered a minimum population of 239 individuals, with about 100 individuals counted in Cor-roios, about 100 in a cliff–nesting colony in Caparica, 26 in Oeiras, nine in Belem, and four in Estoril. The true population is likely higher because time constraints impeded a thorough survey of the entire potential area occupied by the species as well as a more accurate census. We confirmed the reproduction of the species in Caparica, Oeiras, and Belem (appendix 1), mostly through the observation of adults feeding chicks in nests found in buildings and on a large limestone cliff in a periurban area. Only seven records of crested mynas were obtained outside this area (fig. 1), one

Fig. 1. Distribution of records of common mynas, crested mynas, and bank mynas in Spain and Portugal.

Fig. 1. Distribución de los registros de minás comunes, minás crestados y minás ribereños en España y Portugal.

Madeira

Canary Islands

Por

tuga

l

Spain

BalearicIslands

«p

l

Bank mynasCrested mynasCommon mynas

124 Saavedra et al.

in Evora (PO, 2006), one in Porto (PO, 2012), one in Braga (PO, 2012), three in Santander (SP, 2004, 2007–2008), and one in Mallorca (Balearic Islands, SP, 2002), all corresponding to single individuals and sug-gesting independent accidental escapes of cage birds.

Only 11 records, involving 21 observed individuals, were made of bank mynas between 2003 and 2010. These records were from three continental sites —one in Portugal and two in Spain— and and one site in the Canary Islands (La Gomera) (fig. 1). All but one record corresponded to two individuals, and the other to a single individual. The spatio–temporal distribution of records also suggests independent, accidental introductions of this species. There is no evidence of reproduction in any locality. However, three individuals captured during eradication activities in Gran Canaria in 2006 showed a phenotype that suggested the hy-bridization between bank and common mynas.

Discussion

The global compilation of introduced bird species made by Lever (2005) reported only two old records of common mynas breeding on two Spanish islands, and Saavedra (2010) summarized the eradication attempts of the species in the two Spanish archipela-gos. The Delivering Alien Invasive Species Inventories for Europe (DAISIE, 2015) lists the common myna

as unestablished in Spain and the crested myna as established in Portugal, with no additional information on their introduced populations. Here, we show a larger spatial and temporal scale for the introduction of up to three species of mynas, together with their population trends. Most introductions took place in the last decade, and several reproduction events were subsequently reported. The relative temporal synchrony and widely spaced distribution of records (fig. 1) suggest that there were at least 22 indepen-dent introduction events. Contrary to the previously reported deliberate introductions of mynas elsewhere (Lever, 2005), we did not find evidence of intentional introductions in Spain and Portugal. Wild–caught mynas of the three species were traded from their native ranges to Spanish and Portuguese cage–bird markets until 2005, and the accidental escapes of wild–caught birds may explain their successful esta-blishment in the wild (Carrete & Tella, 2008; Cabezas et al., 2013). The lack of observations of individuals banded with closed rings (typical of captive–bred birds) provides further evidence of their wild–caught origin. Moreover, three introductions could be attri-buted to escapes from owners after buying the birds in pet–shops and one from a zoological garden on four Spanish islands (Saavedra, 2010), and further records compiled here confirm the escape from a zoo on another island and the escape from a particular owner in continental Spain.

Atlantic Ocean

No

of i

ndiv

idua

ls

No of recordsNo of individuals

1995 1997 1999 2001 2003 2005 2007 2009 2011 Year

Lisbon

10 km

Fig. 2. Detailed distribution of crested and common mynas in the Tagus Estuary, Portugal. Inset: increase in the number of records and of observed individuals of crested mynas obtained through compiled records.

Fig. 2. Distribución detallada de los minás crestados y comunes en el estuario del Tajo, en Portugal. Recuadro: aumento en el número de registros y de individuos de miná crestado observados obtenidos a través de los registros recopilados.

Crested mynasCommon mynas

No

of i

ndiv

idua

ls

250

200

150

100

50

0

No

of r

ecor

ds

25

20

15

10

5

0

Animal Biodiversity and Conservation 38.1 (2015) 125

The relatively rare introductions of bank mynas did not result in established populations. The common myna, however, have established breeding popula-tions on four Spanish islands and, despite eradication efforts (see details in Saavedra, 2010), one pair was again breeding in Tenerife in 2013 and one individual appeared on another Canary island (Lanzarote) in 2013. Moreover, there is sufficient evidence to sug-gest that the species is breeding in the Tagus Estuary (Portugal) where it has been observed since 2001 and where we found up to ten individuals in a breeding colony of crested mynas. Continuous breeding of this small nucleus together with further accidental releases may increase population sizes in the future. The crested myna, however, is today of greater concern since it is now widely distributed throughout the Tagus Estuary and the number of records and of observed individuals has dramatically increased during the last decade. Given the typical lags in population growth of introduced exotic bird species (Aagaard & Lockwood, 2014), the continued growth and spread of the spe-cies throughout the Iberian Peninsula is expected in the absence of management action.

Eradication and control campaigns of common mynas occur worldwide due to their recognized ecological and economic impacts (e.g., Saavedra, 2010; Grarock et al., 2014). However, most impacts have been identified on islands (Lever, 2005) and the negative effects of mynas could be smaller in urbanized continental environments (Haythorpe et al., 2014). Introduced mynas often behave as urban exploiters and thus may have little impact on native communities (Sol et al., 2012; Orchan et al., 2013). However, recent work has shown that a typical urban dwelling exotic bird (the ring–necked parakeet Psit-tacula krameri) outcompetes most native species through aggressive interactions and may be the cause of the decline of two threatened species in a Spanish city (Hernández–Brito et al., 2014). During our short field survey, we circumstantially observed several instances of aggressions of crested mynas foraging in urban environments towards a variety of native species, from smaller–sized (barn swallow Hirundo rustica, white wagtail Motacilla alba, house sparrow Passer domesticus) to similarly–sized (black starling Sturnus unicolor, blackbird Turdus merula) and much larger–sized species (Eurasian kestrel Falco tinnuncu-lus, yellow–legged gull Larus michahellis). Moreover, mynas have the potential to spread throughout rural environments where their impact may extend to a wider array of native species (Pell & Tidemann, 1997) and agriculture. While dedicated research is needed to assess the real and potential impact of common and crested mynas in Portugal, and taking into account that the impacts of invasive species are often missed (Davidson & Hewitt, 2014), the application of the Pre-cautionary Principle calls for the eradication of these populations before it becomes unfeasible or economi-cally much more costly (Edelaar & Tella, 2012). In the case of mynas, eradication campaigns has proved to be highly effective when populations are still small (i.e., < 50 individuals), as they were on Spanish islands, but not when populations reach thousands of individuals

as was the case of St. Helena and Ascension Islands (see details on eradication efforts and their results in Saavedra, 2010). These eradication campaigns were preceded by citizen awareness campaigns to achieve a favorable public opinion (Saavedra, 2010). Moreover, eradication of mynas should consider its effects on other coexisting introduced species which may also require management. For example, Shwartz et al. (2009) suggested that common mynas may reduce the breeding success of the also invasive ring–necked parakeet, which in turn impacts on urban mammals and birds (Hernández–Brito et al., 2014a, 2014b), and thus the eradication of mynas might favor population growth of parakeets where these species may coexist (as occurs in Portugal).

Eradication campaigns may be not sufficient to avoid the establishment and spread of mynas if they are not combined with preventive measures. Since 2013, all myna species (Acridotheres sp.) have been included in the Spanish Catalogue of Invasive Alien Species, with their possession, release and commercial trade forbidden by law (Real Decreto 630/2013). This greatly helps to reduce the risk of further accidental releases in Spain, but there is no similar legislation in Portugal. Mynas are offered for sale in Portuguese and Dutch pet–shops and online through specialized websites, and are directly shipped to Spain without administrative control (J. L. Tella, unpubl. data). Therefore, a common European leg-islation against the trade of these and other invasive species is needed to successfully avoid introductions at a national scale.

Acknowledgements

We thank L. Costa (Sociedade Portuguesa para o Estudio das Aves), E. F. Vieira (Ministerio da De-fensa Nacional), Sargento–Mor V. M. de Assunçao Pereira, the staff at Forte Sao Juliao da Barra, and M. Silva (Ministerio do Ambiente e do Ordenamento do Territorio) for facilitating field work in Portugal, and Invasive Bird Management for logistic support. Two anonymous reviewers contributed greatly to improving a first version. This work was funded by Fundación Repsol (J. L. Tella) and a Fundación Do-ñana 21 grant to A. Maraver, with the support from the project Estación Biológica de Doñana–Severo Ochoa (SEV–2012–0262) and AIC–A–2011–0706.

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Carrete, M. & Tella, J. L., 2008. Wild–bird trade and exotic invasions: a new link of conservation concern? Frontiers in Ecology and Environment, 6: 207–211.

DAISIE, 2015. Delivering Alien Invasive Species In-ventories for Europe (http://www.europe–aliens.org)

Davidson, A. D. & Hewitt, C. L., 2014. How often are invasion–induced ecological impacts missed?. Biological Invasions, 16(5): 1165–1173.

Edelaar, P. & Tella, J. L., 2012. Managing non–native species: don′t wait until their impacts are proven. Ibis, 154: 635–637.

Feare, C. & Craig, A., 1998. Starlings and mynas. Cristhopher Helm, London.

Grarock, K., Tidemann, C. R., Wood, J. T. & Linden-mayer, D. B., 2014. Understanding basic species population dynamics for effective control: a case study on community–led culling of the common myna (Acridotheres tristis). Biological Invasions, 16(7): 1427–1440.

Haythorpe, K. M., Burke, D. & Sulikowski, D., 2014. The native versus alien dichotomy: relative impact of native noisy miners and introduced common mynas. Biological Invasions, 16(8): 1659–1674.

Hernández–Brito, D., Carrete, M., Popa–Lisseanu, A., Ibáñez, C. & Tella, J. L., 2014a. Crowding in the city: losing and winning competitors of an invasive

bird. PLOS ONE, 9(6): e100593.Hernández–Brito, D., Luna, A., Carrete, M. & Tella, J.

L., 2014b. Alien rose–ringed parakeets (Psittacula krameri) attack black rats (Rattus rattus) sometimes resulting in death. Hystrix, 25: 121–123.

Lever, C., 2005. Naturalised birds of the world. T & AD Poyser, London.

Lowe, S., Browne, M., Boudjelas, S. & De Poorter, M., 2000. 100 of the World’s Worst Invasive Alien Species. Available at: www.issg.org/booklet.pdf

Orchan, Y., Chiron, F., Shwartz, A. & Kark, S., 2013. The complex interaction network among multiple invasive bird species in a cavity–nesting commu-nity. Biological Invasions, 15(2): 429–445.

Pell, A. S. & Tidemann, C. R., 1997. The impact of two exotic hollow–nesting birds on two native parrots in savannah and woodland in eastern Australia. Biological Conservation, 79: 145–153.

Saavedra, S., 2010. Eradication of Invasive Mynas from islands. Is it possible? Aliens, 29: 40–47.

Sol, D., Bartomeus, I. & Griffin, A. S., 2012. The para-dox of invasion in birds: competitive superiority or ecological opportunism? Oecologia, 169: 553–564.

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Shwartz, A., Strubbe, D., Butler., C. J., Matthysen, E. & Kark, S., 2009. The effect of enemy–release and climate conditions on invasive birds: a regional test using the rose–ringed parakeet (Psittacula krameri) as a case study. Diversity and Distributions, 15: 310–318.

Animal Biodiversity and Conservation 38.1 (2015) 127

Appendix 1. Results of a baseline survey conducted in the Tagus Estuary, Portugal, in 3–17 VI 2011. The maximum number of birds observed from each species of myna in each surveyed site is shown for different dates, as well as whether breeding evidence (B) was found. Numbers for Caparica are approximated, given the difficulties to accurately census the breeding colony.

Apéndice 1. Resultados de un muestreo realizado en el estuario del Tajo, Portugal, 3–17 VI 2011. Se indica el número máximo de individuos observados de cada especie de miná en las diferentes localidades y fechas, así como si se encontraron indicios de que estuvieran reproduciéndose (B). El número de aves en Caparica es aproximado, dadas las dificultades para censar con precisión la colonia reproductora.

Date Site Locality Species Nº birds B

03 VI 2011 Sana Hotel Estoril A. cristatellus 1

05 VI 2011 San Julia Hotel Estoril A. cristatellus 3

07 VI 2011 Caparica Almada A. cristatellus + A. tristis 100 + 10 yes

09 VI 2011 Boca do inferno Oeiras 0

09 VI 2011 Train Station Carcavelos Oeiras A. cristatellus 4

10 VI 2011 Cementery of Oeiras Oeiras A. cristatellus 2 yes

10 VI 2011 Ajuda Palace Belem A. cristatellus 6 yes

10 VI 2011 Jerónimos Monastery Belem A. cristatellus 0

10 VI 2011 Empire Square Belem A. cristatellus 1

10 VI 2011 Navy Museum Belem A. cristatellus 0

10 VI 2011 Archeological Museum Belem A. cristatellus 0

11 VI 2011 Cementery of Oeiras Oeiras A. cristatellus 2 yes

12 VI 2011 Jerónimos Monastery Belem 0

12 VI 2011 Tropical garden Belem 0

12 VI 2011 Botanic garden Belem 0

12 VI 2011 Ajuda Palace Belem A. cristatellus 2

12 VI 2011 Empire Square Belem 0

13 VI 2011 Train St. Carcavelos Oeiras A. cristatellus 0

13 VI 2011 Conde Oeiras school Oeiras A. cristatellus 0

13 VI 2011 Train Station St. Oeiras Oeiras A. cristatellus 0

14 VI 2011 Benfica Seixal A. cristatellus 3

15 VI 2011 Caparica Almada A. cristatellus + A. tristis 100 + 10 yes

16 VI 2011 Molino de Mare Corroios A. cristatellus 2

16 VI 2011 Residual water facility Corroios A. cristatellus 100

17 VI 2011 Nato Headquarters 0eiras A. cristatellus 4 yes

17 VI 2011 Caparica Almada A. cristatellus + A. tristis 100 + 10 yes

128 Saavedra et al.

129Animal Biodiversity and Conservation 38.1 (2015)

© 2015 Museu de Ciències Naturals de BarcelonaISSN: 1578–665 XeISSN: 2014–928 X

Lucas, J. M., Prieto, P. & Galián, J., 205. Red squirrels from south–east Iberia: low genetic diversity at the southernmost species distribution limit. Animal Biodiversity and Conservation, 38.1: 129–138.

AbstractRed squirrels from southeast Iberia: low genetic diversity at the southernmost species distribution limit.— South–east Iberia is the southernmost limit of this species in Europe. Squirrels in the region mainly inhabit coniferous forests of Pinus. In this study, we analyzed the pattern of mitochondrial genetic variation of southern Iberian red squirrels. Fragments of two mitochondrial genes, a 350–base pair of the displacement loop (D–loop) and a 359–bp of the cytochrome b (Cytb), were sequenced using samples collected from 88 road–kill squirrels. The genetic variation was low, possibly explained by a recent bottleneck due to historical over–exploitation of forest resources. Habitat loss and fragmentation caused by deforestation and geographic isolation may explain the strong genetic subdivision between the study regions. Six new haplotypes for the D–loop and two new haplotypes for the Cytb fragments are described. A Cytb haplotype of south–east Iberia was found to be present in Albania and Japan, suggesting local extinction of this haplotype in intermediate areas. No significant clustering was found for the south–east of Spain or for the other European populations (except Calabria) in the phylogenetic analysis.

Key words: Sciurus vulgaris, Mitochondrial DNA, Genetic diversity, Population bottleneck

ResumenArdillas rojas del sureste ibérico: baja diversidad genética en el límite austral de la distribución de la especie.— El sureste ibérico es el límite más austral de la distribución de esta especie en Europa, donde las ardillas habitan principalmente en bosques de Pinus. En este estudio, se investigó el patrón de variación genética mitocondrial de las ardillas rojas del sureste ibérico. Se secuenciaron fragmentos de dos genes mitocondriales, 350 pares de bases de la región control (D–loop) y 359 pb del citocromo b (Cytb) utilizando muestras obtenidas a partir de 88 ardillas atropelladas. Se encontró una baja variación genética, lo cual podría explicarse por la existencia de un cuello de botella reciente causado por la sobreexplotación histórica de los recursos madereros de la zona. La pérdida y fragmentación del hábitat debidas a la deforestación y al aislamiento geográfico podrían explicar la fuerte subdivisión genética observada entre las regiones del estudio. Se describen seis nuevos haplotipos para el fragmento D–loop y dos para el Cytb. Un haplotipo encontrado en el sureste ibérico para el Cytb se observó también en Albania y Japón, lo que sugiere una extinción local de este haplotipo en áreas intermedias. En los análisis filogenéticos, no se detectó un agrupamiento significativo de las ardillas del sureste ibérico, ni de ninguna otra población europea (excepto en Calabria).

Palabras clave: Sciurus vulgaris, ADN mitocondrial, Diversidad genética, Cuello de botella poblacional

Received: 9 X 14; Conditional acceptance: 28 I 15; Final acceptance: 23 IV 15

J. M. Lucas, & J. Galián, Depto. de Zoología y Antropología Física, Fac. de Veterinaria, Univ. de Murcia, Campus de Espinardo, 30100 Murcia, España (Spain).– P. Prieto, Parque Natural de Cazorla, Segura y las Villas, c/ Martinez Falero 11, 23470 Cazorla, Jaén, España (Spain).

Corresponding author: José Manuel Lucas, e–mail: lucas@um.es

Red squirrels from south–east Iberia: low genetic diversity at the southernmost species distribution limit

J. M. Lucas, P. Prieto & J. Galián

130 Lucas et al.

Introduction

The red squirrel (Sciurus vulgaris Linnaeus, 1758) is widely distributed from Iberia in the west across the Palaearctic to the island of Hokkaido (Japan), and from the UK, Ireland, Scandinavia and Siberia to the Mediterranean (Corbet, 1978; Lee & Fukuda, 1999; Lurz et al., 2005). In the Iberian Peninsula, this native sciurid is continuously distributed from Girona to Galicia and the Northern Iberian Mountain Range, the Northern Plateau and the Central Mountain Range, and southwards to Valencia. It is discontinuously distributed from Cataluña to Andalucía, and widely spread in the Baetic Mountain Ranges, including Murcia, Albacete and Alicante (Valverde, 1967; PurValverde, 1967; Purroy, 2014). As the result of recent reintroductions, the species can also be found in central and north Porin central and north Portugal (Mathias & Gurnell, 1998; Ferreira et al., 2001; Ferreira & Guerreiro, 2002) (fig. 1A). Most Iberian squirrels occupy pure pine forest: Pinus halepensis in the lower altitudes, P. pinaster and P. nigra in middle levels, and P. mugo in the higher locations (Valverde, 1967). In south–east Iberia, the most common species of pine is P. halepensis (Aleppo pine), however, even at relatively medium/high altitudes. This is especially evident in the region of Murcia where red squirrels are found in urban parks and adjacent copses, in small to large villages, and even in cities where Aleppo pine can be found. In these localities, they have even been seen feeding on date palms (pers. obs.).

The species is extremely variable in color. Considerable regional variation is superimposed on a striking polymorphism and equally striking seasonal differences (Corbet, 1978). Many studies of the morphological diversity of Spanish squirrels have been made in the past century, especially in the early nineteen hundreds (Cabrera, 1905; Miller, 1907, 1909, 1912), which led to an intense taxonomical discussion. More recently, the first researcher to provide new material morphological variation was Valverde (1967). He assigned his samples to four previously described subspecies (S. v. alpinus Desmarest, 1822, S. v. numantius Miller, 1907, S. v. infuscatus Cabrera, 1905 and S. v. segurae Miller, 1912) and suggested the existence of a new subspecies, which he named S. v. hoffmanni Valverde, 1967 from Sierra Espuña (southeast Spain). However, subsequent authors considered that only two subspecies are present in Iberia: S. v. fuscoater Altum, 1876 and S. v. infuscatus (Corbet, 1978; Lurz et al., 2005; Sidorowicz, 1971) (fig. 1B).

Valverde (1967) emphasized the importance of the hoffmanni subspecies because of its ecological and morphological features. These squirrels represent the southeastern limit of the Iberian distribution of the species in the xerothermic forest–margin of the Iberian Peninsula, where it lives in pure Aleppo pine forest. Moreover, S. v. hoffmanni would be the largest of the European red squirrels, with the palest fur. Thus, this form should represent the ecological limit and the most extreme phenotype of Iberian squirrels (Valverde, 1967). According to the author, S. v. hoffmanni is restricted to the Regional Park of Sierra Espuña, but currently the hoffmanni phenotype can be

easily observed in the Regional Park of Carrascoy–El Valle further south of this region, separated from Espuña by the Guadalentín River. The Regional Park of Sierra Espuña is about 80 km east of the Natural Park of Sierra de Cazorla, Segura y Las Villas and they are connected by a northwestern green corridor (Special Protected Area of Sierra de Burete, Lavia y Cambrón, and the Northwestern Mountains of Murcia). The Natural Park of Sierra de Cazorla, Segura y Las Villas is the largest protected area in Spain, 214,300 ha, and it was designated by UNESCO as a Biosphere Reserve in 1983. There are good–sized red squirrel populations in this area, and they are still under taxonomic discussion (S. v. baeticus Cabrera, 1905 = S. v. segurae = S. v. infuscatus).

Beyond the taxonomical discussion, no recent studies of the ecological characteristics of squirrels from southeast Iberia have been published. Only one study has investigated the genetics of some Iberian populations (Lucas & Galián, 2009), and it found extremely low genetic variation in the population of the Regional Park of Sierra Espuña.

We investigated whether the low genetic diversity found in Sierra Espuña can be considered a pattern in Southeast Iberia or whether it is a peculiarity of this population. In order to study the relationships between the southeastern Iberian squirrels and the other European populations, we compared our results with those in the literature. To achieve these objectives two mitochondrial gene fragments (D–Loop and Cytb) were analyzed using samples from road–kill animals.

Material and methods

Sample collection

In southeastern Spain, most natural areas are crossed by roads and frequented by a large number of visitors. As found in other European populations (Shuttleworth, 2010), road–kill squirrels are frequent in the study area both in natural and suburban environments.

The study area was divided into five regions according to geographical and ecological barriers or distance between samples clusters (fig. 2). Samples from CSV and ESP were collected from the reported distribution of the segurae and hoffmanni subspecies. All of the samples comprised approximately 2 mm2 of muscle tissue and were preserved in absolute ethanol, then stored at –20°C until DNA purification.

DNA extraction and sequencing

Total genomic DNA was extracted from tissue samples using a Qiagen DNAeasy Tissue Kit, according to the manufacturer’s protocol. A total of 754–bp were amplified from two gene regions of the mitochondrial DNA. A 395–bp fragment of the D–loop was amplified in 12.5–µl reactions, following the protocol described by Hale et al., 2004, using 1 µl of tissue DNA and the red squirrel–specific primers, H16359 (Barratt et al., 1999) and RScont6 (Hale et al., 2004). A 359–bp region of the Cytb was amplified using the same pro

Animal Biodiversity and Conservation 38.1 (2015) 131

Fig. 1. Map of the species distribution in the Iberian peninsula (A) modified from Palomo & Gisbert (2002). Geographic distribution of red squirrel subspecies (B), obtained from Valverde (1967) and Mathias & Gurnell (1998). Square shape (£) and triangle shape (r) refer to the subspecies infuscatus and fuscoater, respectively, as synonymised in more recent studies (Sidorowicz, 1971; Corbet, 1978; Lurz et al., 2005).

Fig. 1. Mapa de distribución de la especie en la península ibérica (A), modificado de Palomo & Gisbert (2002). Distribución geográfica de las subespecies de ardilla roja (B), a partir de la información de Val-verde (1967) y Mathias & Gurnell (1998). Los cuadrados (£) y los triángulos (r) hacen referencia a las subespecies infuscatus y fuscoater respectivamente, sinonimizadas en trabajos más recientes (Sidorowicz, 1971; Corbet, 1978; Lurz et al., 2005).

tocol, except that we used the primers SV14226F and SV14647R from Grill et al. (2009). Negative (sterile water) and positive (known squirrel DNA) controls were always used and the products were visualized on 2% agarose gels alongside a 100–bp size standard to determine the success of the amplification. The PCR products were sequenced in both forward and reverse directions for each sample by Macrogen Inc., Korea.

Data analysis

Consensus sequences for each individual were obtained by aligning the forward and reverse complementary sequences of each gene (D–loop and Cytb) with Geneious 4.8.3. D–loop sequences were aligned in MUSCLE (Edgar, 2004) and Cytb sequences with ClustalW algorithm (Larkin et al., 2007). The haplotypes were identified with TCS 1.21 (Clement et al., 2000) and compared with those available in the GenBank using BLAST (Altschul et al., 1990). The relative frequencies of the Cytb and D–loop haplotypes were calculated with Arlequin 3.1.2.3 (Excoffier & Lischer, 2010). Haplotype diversity was calculated separately for each gene. Due to the low diversity found in Cytb sequences, both genes were combined to investigate the nucleotide diversity. The molecular diversity indices were determined using DnaSP 5 (Librado & Rozas, 2009).

The pairwise genetic distances between regions, which were measured as FST, were calculated from

a distance matrix of D–loop haplotypes based on the Tamura–Nei model (Tamura & Nei, 1993) in Arlequin 3.1.2.3 (Excoffier & Lischer, 2010).

The genealogical relationships between the D–loop haplotypes of southeast Iberia were assessed by constructing a median–joining network in NETWORK 4.6.1 (Bandelt et al.,1999). Haplotype networks includ. Haplotype networks including sequences from the GenBank were also calculated for both genes.

Phylogenetic analyses were conducted in MEGA6 (Tamura et al., 2013) using the maximum likelihood (ML) method with the nearest neighbour interchange algorithm. Nucleotide sequences of red squirrels from other European populations (Hale et al., 2004; Grill et al., 2009; Doziéres et al., 2012) and of the Japan squirrel Sciurus lis (Oshida & Masuda, 2000) were downloaded from GenBank and aligned with our data set. These sequences showed a 100% overlapwith the sequences we analysed. The model of nucleotide substitution that best fitted the data set was determined with MEGA6 (Tamura et al., 2013). The stability of the ML tree topologies were tested using 1,000 bootstrap replicates.

Results

A total of 88 samples from the five regions were genotyped successfully. Twenty of the samples from ESP were used in previous work (Lucas & Galián,

S. v. alpinus S. v. numanticus S. v. infuscatus S. v. segurae (Molinicos) S. v. segurae (Cazorla) S. v. hoffmanni S. v. fuscoater

A B

132 Lucas et al.

Fig. 2. Map of the study area. The black dots represent Sciurus vulgaris specimens. The green line marks the area of the Natural Park of Sierra de Cazorla, Segura y Las Villas, and the orange line delimits the area of the Natural Park of Sierra Espuña. The five regions in the study area are bounded by black lines: CSV. Natural Park of Sierra de Cazorla, Segura y Las Villas and surroundings; ESP. Regional Park of Sierra Espuña and surroundings; MUR. Copses and periurban parks near the city of Murcia; CEV. Regional Park of Carrascoy–El Valle; AAL. Albacete and Alicante.

Fig. 2. Mapa del área de estudio. Los puntos negros representan los individuos de Sciurus vulgaris. La línea verde indica el límite del Parque Natural de Sierra de Cazorla, Segura y Las Villas, y la línea naranja delimita el área del Parque Regional de Sierra Espuña. Las líneas negras definen las cinco regiones en las que se divide el área de estudio: CSV. Parque Natural de Sierra de Cazorla, Segura y Las Villas y al-rededores; ESP. Parque Regional de Sierra Espuña y alrededores; MUR. Bosquetes y parques periurbanos próximos a la ciudad de Murcia; CEV. Parque Regional de Carrasco y–El Valle; AAL. Albacete y Alicante.

2009). Fragments of the D–loop and Cytb (395–bp and 359–bp respectively) were obtained for each sample. As we found that a tRNA was present within the nucleotide spans of the D–loop fragment, they were trimmed to 350–bp to adjust the sequence length to the target gene. Aligned sequence data were submitted to the GenBank database with accession numbers KJ146734–KJ146742. We found a total of six D–loop haplotypes which have never been reported, and a total of three Cytb haplotypes, two of which were also found to be exclusive to the south east of Spain (SvCb2 and SvCb3). SvCb1 was identical to haplotypes previously found in Albania (Grill et al., 2009) and Japan (Oshida et al., 2009).

Three of the six haplotypes identified for the D–loop were found in CSV and two were present throughout the whole study area. One of the three Cytb haplotypes was exclusive to CSV but the others were present in more than one region (table 1).

The concatenated alignment was 709–bp long and contained eight variable positions. These sequences were collapsed into seven haplotypes. The nucleotide (π) diversity of the concatenated sequence was zero in CEV, low in the ESP region, intermediate in MUR

and AAL, and higher in CSV (table 2). The haplotype diversity (Hd) of the two genes varied in the same way when treated separately, although it was lower in the case of the Cytb. Genetic differentiation between regions was high in almost all cases (table 3).

In the haplotype network (fig. 3), three haplotypes were placed as external nodes, two belonging to CSV (one of them unique to this region) and one exclusive to AAL. The two most common haplotypes (SvCR1 and SvCR2) were both placed as internal nodes, as was haplotype SvCR4. This haplotype was exclusive to CSV and located in the center of the network, also being connected to SvCR3 (exclusive to AAL).

Haplotype SvCb1 was placed in the center of the Cytb network (data not shown). The SvCb2 and SvCb3 haplotypes were directly connected to this and both differed in two nucleotide positions. Haplotype networks using sequences from the GenBank (data not shown) did not show any grouping by geographic region. In the Cytb network, the only haplotype that showed a clear differentiation was that found in Calabria by Grill et al. (2009).

A phylogenetic analysis was conducted for the D–loop haplotypes, including haplotypes from Hale

AAL

MUR

CEV

ESP

CSV

Segura River

Guadalentín River

Animal Biodiversity and Conservation 38.1 (2015) 133

Fig. 3. Median–joining network of the six new D–loop haplotypes (A) and their spatial distribution (B): A. The circles (nodes) in the network represent the haplotypes and the areas of the circles are proportional to the number of samples for each haplotype. The perpendicular short black lines represent mutations; B. Each pie in the distribution map represents the proportion of haplotypes in each region and the size of the pie is proportional to the number of individuals.

Fig. 3. Red haplotípica (basada en el algoritmo de unión de medianas (median–joining) para los seis nuevos haplotipos del fragmento D–loop (A) y distribución espacial de los mismos (B): A. En la red haplotípica, los círculos (nodos) representan los haplotipos y las áreas son proporcionales al número de muestras de cada haplotipo; B. En el mapa de distribución, cada gráfica representa la proporción de haplotipos en cada región y su tamaño es proporcional al número de muestras.

AAL

MUR

CEV

ESP

CSV

A B

SvCR1 SvCR2SvCR3SvCR4SvCR5SvCR6

Table 1. Haplotype frequencies in the five regions and the overall study area. (For abbreviations see figure 2; SE Spain refers to the overall study samples.)

Tabla 1. Frecuencias haplotípicas en las cinco regiones y en toda el área de estudio. (Para las abreviaturas, véase la figura 2; SE Spain se refiere al total de muestras.)

Haplotype ESP MUR CEV AAL CSV SE Spain SvCb1 0.972 0.400 1 0.250 0.654 0.761SvCb2 0.023 0.600 0 0.750 0.192 0.193SvCb3 0 0 0 0 0.154 0.045SvCR1 0.972 0.467 1 0 0.115 0.591SvCR2 0.028 0.533 0 0.750 0.462 0.273SvCR3 0 0 0 0.250 0 0.011SvCR4 0 0 0 0 0.154 0.045SvCR5 0 0 0 0 0.231 0.068SvCR6 0 0 0 0 0.038 0.011

et al. (2004), Grill et al. (2009) and Doziéres et al. (2012). A 252–bp alignment was generated. The tree with the highest log likelihood (–762.4389) was obtained in the maximum likelihood analysis of the

D–loop haplotypes (fig. 4A). This phylogenetic tree was conducted under the Hasegawa–Kishino–Yano (HKY85) model (Hasegawa et al., 1985) with rate heterogeneity among sites (gamma distribution shape

134 Lucas et al.

Table 2. Summary of the diversity indices: N. Number of sequences/individuals; π Nucleotide diversity with standard deviation; h. Number of haplotypes; Hd. Haplotype diversity with standard deviation. (For other abbreviations see figure 2; SE Spain refers to the overall study samples.)

Tabla 2. Resumen de los índices de diversidad: N. Número de secuencias/individuos; π. Diversidad nucleotídica con desviación estándar; h. Número de haplotipos; Hd. Diversidad haplotípica con desviación estándar. (Para las otras abreviaturas, véase la figura 2; SE Spain se refiere al total de muestras.)

N π hD–loop hCytb hCombined HdD–loop HdCytb HdCombined

CSV 26 0.00339 ± 0.00030 5 3 6 0.723 ± 0.064 0.532 ± 0.092 0.831 ± 0.032

ESP 36 0.00024 ± 0.00022 2 2 2 0.056 ± 0.052 0.056 ± 0.052 0.056 ± 0.052

MUR 15 0.00226 ± 0.00022 2 2 2 0.533 ± 0.052 0.533 ± 0.052 0.533 ± 0.052

CEV 7 0.00000 1 1 1 0.000 0.000 0.000

AAL 4 0.00212 ± 0.00112 2 2 2 0.500 ± 0.265 0.500 ± 0.265 0.500 ± 0.265

SE Spain 88 0.00222 ± 0.00020 6 3 7 0.576 ± 0.044 0.385 ± 0.055 0.607 ± 0.050

parameter of 0.17). No significant clustering of the haplotypes was found for the southeast of Spain or for the rest of the European populations.

A second analysis was performed for the combined data set, that included nine haplotypes from other European populations (Grill et al., 2009). A 611–bp alignment was generated. The maximum likelihood tree of the combined mtDNA sequences (log likelihood of –1,306.7513) was inferred based on the Tamura 3–parameter model (Tamura, 1992) with invariant sites (fig. 4B). The phylogeny showed a clear differentiation for the Calabrian lineage but not for the rest of the sample. The same result was observed by analyzing the Cytb haplotypes (data not shown). Sequences of S. lis were always rooted in the phylogenetic trees.

Discussion

Capture and manipulation of living red squirrels may imply a high risk for their health, such as heart attack or dorsal spin fracture (Josep Piqué, pers. comm.).

Collecting tissue samples from road–kill squirrels avoids such risk and has been proven a suitable source of quality DNA for molecular studies (Lucas & Galián, 2009; Doziéres et al., 2012). However, this kind of sampling does not allow the development of a sampling plan where regions are equally represented. In southeast Spain, this disadvantage can be partially compensated for by the abundance of road–kill animals in rural and suburban areas.

In this study, we found a level of genetic diversity similar to that reported for Spain by Hale et al. (2004) and Grill et al. (2009). However, the extremely low genetic diversity of ESP, described by Lucas & Galián (2009), is the most striking result in this study. This contrasts sharply with the relatively high genetic variation found in CSV, despite its ecological connectivity with ESP.

Anthropogenic effects such as farming or direct human exploitation have decreased the distribution ranges and population sizes of many species in the Iberian peninsula (Gómez & Lunt, 2007). In southeastern Spain, the area occupied by ESP and CSV suffered

Table 3. FST values between pairs of regions (below diagonal) and P–values computed based on 1,000 permutations (upper diagonal): *P < 0.05, **P < 0.001. (For abbreviations see figure 2.)

Tabla 3. Valores de FST entre pares de regiones (diagonal inferior) y valores de P calculados a partir de 1.000 permutaciones (diagonal superior): * P < 0,05; ** P < 0,001. (Para las abreviaturas, véase la figura 2.)

ESP MUR CEV AAL CSVESP – – 0.99902 ± 0.0002 – –MUR 0.56208** – – 0.24805 ± 0.0161 0.19629 ± 0.0111CEV –0.07417 0.39655* – – –AAL 0.89285** 0.13125 0.82554* – 0.23828 ± 0.0161CSV 0.45766** 0.02575 0.30762* 0.05825 –

Animal Biodiversity and Conservation 38.1 (2015) 135

100

ITA VeneziaITA CalabriaITA CalabriaFRA AqUK Wb

UK EUK EUK WaFRA BrSPAITA VicoUK NSPAPRTSPAPRT

SPASPAFRA PlPRTFRA McITA VareseFRA LcITA CalabriaITA Calabria

ITA BellunoITA CalabriaSvCR1

SvCR6SvCR2SvCR3SvCR4SvCR5

Sciurus lis AB 192959Sciurus lis AB 192960

Sv66Sv67PRT 303Svh30Sv41Sv76Sv13Sv64PRT 300FRA 404ITA 209ITA 413FRA 412ITA 425ITA 414

Calabria 262

Sciurus lis AB 192923Sciurus lis AB 192922

90

98

100

100

100

98

78

69

65

51

A

B

Fig. 4. Condensed maximum–likelihood trees of the D–loop fragment (A) and the combined D–loop and Cyb sequences (B). Branches with less than 50% of bootstrap (1,000 replicates) are collapsed in both trees. The ISO 3166 code is used to designate the country of each sample taken from the literature: A. Taxon labels refer to the D–loop haplotypes from this study (SvCR#) and from other European populations (Hale et al., 2004; Grill et al., 2009); all the French sequences are obtained from Doziéres et al. (2012); B. Labels indicate the sample ID of individuals with different combined haplotypes (Sv##, Svh##) and the specimen numbers from Grill et al. (2009). GenBank accession numbers of the outgroups are indicated in the trees.

Fig. 4. Árboles condensados de máxima verosimilitud para el fragmento del D–loop (A) y para las secuencias concatenadas del D–loop y el Cytb (B). Las ramas presentes en menos del 50% de las 1.000 réplicas obtenidas por muestreo con reemplazo (bootstrap) se han condensado en ambos árboles. Se usa el código ISO 3166 para designar el país de procedencia de cada una de las muestras tomadas de la bibliografía: A. Los nombres de los taxones hacen referencia a los haplotipos del D–loop de este estudio (SvCR#) y a aquellos procedentes de otras poblaciones europeas (Hale et al., 2004; Grill et al., 2009); todas las muestras recogidas en Francia se han obtenido de Doziéres et al. (2012); B. Los nombres de los taxones indican el código de muestra de individuos con distintos haplotipos de secuencias concatenadas (Sv##, Svh##) y el número del espécimen en Grill et al. (2009). Se indican los números de acceso al GenBank de los grupos externos en ambos árboles.

136 Lucas et al.

strong deforestation caused by over–exploitation of forest resources in the 18th and 19th centuries (Valverde, 1967; Araque, 2013). As of he second half of the 19th century, reforestation works have been carried out (Codorniu, 1900; González–Pellejero & Álvarez, 2004), helping to preserve red squirrel populations in this area (Valverde, 1967) to date.

As expected given the previous scenario, the proportion of suitable habitats in the landscape decreased critically, increasing the degree of isolation with increasing habitat fragmentation. This situation may have led to a temporary decline in the local squirrel population, which reduced gene flow (Merriam & Wegner, 1992; Andrén & Delin, 1994; Wauters et al., 1994; Amos & Harwood, 1998; Wauters et al., 2010). Therefore, the low genetic variation found in southeast Iberia may be the result of a severe bottleneck, similar to that reported by Trizio et al. (2005) for Alpine squirrels. However, whereas Trizio et al. (2005) found high haplotype diversity but low nucleotide diversity, we found low genetic variation at both levels. This situation contrasts strongly with the high genetic variation found by Gallego & Galián (2008) for the other Pine–specific species Tomicus destruens in the Regional Park of Sierra Espuña.

As in other European populations (Hale et al., 2004; Finnegan et al., 2008; Doziéres et al., 2014), we found substantial genetic subdivision between regions (table 3). Habitat loss and fragmentation due to anthropogenic effects and geographical barriers may explain these results. For CEV, where SvCR1 was the only haplotype found, the high haplotype fixation may be explained by the geographical isolation caused by the Guadalentín River or by introduction of animals from other sources such as the Sierra Espuña Regional Park. The strong fixation found in CEV and AAL might also be due to low sample size, which can lead to an overestimation of the FST values.

Valverde (1967) emphasized the differentiation of the hoffmanni subspecies in Sierra Espuña and its differentiation from the populations of Sierra de Cazorla, Segura y Las Villas (S. v. segurae) and the rest of the Iberian Peninsula. This classification was achieved using morphological traits and fur colour. Nevertheless, we found no pattern of genetic variation to support this subspecific classification.

Since the internal nodes of haplotype networks are considered as ancestral and the external nodes as more recent status (Castelloe & Templeton, 1994; Templeton, 1998), and since a reduction in population size results in an accelerated increase in genetic distance in the early generations (Chakraborty & Nei, 1974; Nei, 1976; Takezaki & Nei, 1996), our results may be explained by a scenario where widely distributed ancestral haplotypes became extinct due to a bottleneck events. Thus, haplotype SvCR4 occupying the central node of the D–loop network, but in a very low frequency, is a candidate to be considered an ancestral widely distributed haplotype that became extinct in all areas but CSV, especially in ESP which is the region with the largest sample size.

The finding of a Cytb haplotype (SvCb1) that was previously described in Albania and Japan but not in other Eurasian population suggests an ancestral

wide distribution of this haplotype, followed by local extinction in intermediate areas.

Iberia and Italy have been reported as potential glacial refuges for the red squirrel (Hale et al., 2004; Finnegan et al., 2008; Grill et al., 2009; Doziéres et al., 2012) and our results confirm that Iberian samples do not show the expected high levels of genetic diversity (Hewitt, 1996; Taberlet et al., 1998). This finding would be supported by a paper by Doziéres et al. (2012) that suggested a postglacial recolonization of Europe from Asia or from the Balkans or, alternatively, a series of recent bottlenecks that reduced the genetic diversity in the Iberian and Italian populations. The finding of haplotype SvCb1 in Iberia, the Balkans and Japan favours the hypothesis of the Iberian Peninsula acting as a glacial refuge. Besides, the low genetic variation found may be explained by the recent bottleneck in these populations.

In contrast with the report by Grill et al. (2009) and Doziéres et al. (2012), we found no significant clustering for the squirrels of Calabria in the phylogenetic analysis of the D–loop haplotypes (fig. 4A). However, these individuals were clearly differentiated in the remaining the phylogenetic trees (fig. 4B). Nevertheless, the results of the phylogenetic analysis are largely dependent on the sequence length (number of informative sites) and the number of individuals analysed. Thus, this could be an explanation of the lack of clustering found in this work for the Calabrian squirrels (fig. 4A). None of the squirrels in Spain were separated in these analyses, suggesting that Iberian squirrels have not been isolated from the rest of the European populations, as found by Doziéres et al. (2012) for French squirrels. Nonetheless, Grill et al. (2009) emphasised the clear separation of the Iberian squirrels, based on the analysis of eight microsatellite loci. We noticed that the squirrels from ESP did not form a monophyletic clade in the philogenetic analyses, in contrast with what we found in previous work (Lucas & Galián, 2009). The inclusion of samples from nearby populations (CSV, CEV, ALL and MUR) shows that, in fact, the population of Sierra Espuña is very close to other Iberian populations.

A more extensive study should be carried out to understand the phylogenetic and demographic relationships between the Iberian populations, not only at a mitochondrial level, but also at a nuclear level. The recent development of next–generation sequencing methods offers a wide potential for obtaining complete genomes, allowing more accurate research into the evolutionary relationships at an intraspecific level (McCormack et al., 2013).

Acknowledgments

We wish to thank the following people who helped us by collecting road–kill squirrel samples: Antonio Ortuño, Ángel Albert, José Manuel López, Carlos González, Jorge Sánchez, Ana Miñano (C. R. F. El Valle), Cristina López, Javier García, Lidia Lorca, José Manuel Vidal, Mario León, Irene Muñoz, Carmelo Andújar, Paula Arribas, José Serrano, José Galián,

Animal Biodiversity and Conservation 38.1 (2015) 137

Rosa María Ros, and Isabel Sánchez Guiu. We also thank the environmental officers of the Natural Park of Sierra de Cazorla, Segura y Las Villas and the Regional Park of Sierra Espuña for collecting samples. And Obdulia S. Sanchez–Domingo and Ana I. Asensio for technical assistance, and thank Prof. José Serrano for useful comments on the manuscript.

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© 2015 Museu de Ciències Naturals de BarcelonaISSN: 1578–665 XeISSN: 2014–928 X

Joaquím Mateu (1921–2015), tota una vida dedicada a l'estudi dels insectes

X. Bellés

Bellés, X., 2015. Joaquím Mateu (1921–2015), tota una vida dedicada a l'estudi dels insectes. Animal Bio-diversity and Conservation, 38.1: 139–145.

Xavier Bellés, Institut de Biologia Evolutiva (CSIC–UPF), Passeig Marítim 37, 08003 Barcelona, Espanya (Spain). E-mail: xavier.belles@ibe.upf–csic.es

Joaquim Mateu Sanpere va néixer a Barcelona el 9 de gener de 1921 en el si d’una família benestant de la burgesia catalana del principi del segle XX. Els seus pares eren Cristòfol Mateu Ferrer i Teresa Sanpere Cantelis, i la família tenia un avantpassat il·lustre, l’historiador, crític i polític Salvador Sanpere i Miquel, que va ser el pare de Teresa Sanpere i va morir el 1915. Joaquim va ser el segon dels quatre fills que va tenir la parella. Eduard l’havia precedit, mentre que Josep i Elena van néixer després d’ell.

D’infant, Joaquim Mateu va estudiar al col·legi Sant Joan Baptista de La Salle, al barri barceloní de Gràcia. Tanmateix, i com relata en algunes notes autobiogràfiques inèdites, fins a l’adolescència va tenir una salut precària, afectat d’asma i de bronquitis, la qual cosa l’obligava a passar temporades llargues reclòs a casa, on ocupava la major part del temps a llegir, sovint llibres d’història natural i de viatges. Per aquí devia començar, potser, la seva vocació de naturalista i viatger. De ben jovenet va travar una amistat entranyable amb Felip Ferrer i Vert, que havia estat vicepresident de la Institució Catalana d’Història Natural i que, a la plaça Reial de Barcelona, tenia un establiment de taxidèrmia, sobretot d’ocells, tot i que també tenia col·leccions de papallones. La rebotiga d’aquest establiment singular servia per fer tertúlies improvisades sobre temes de natura, sovint centrats en els insectes, dels quals Ferrer i Vert era prou bon coneixedor. Fins a una edat molt avançada, Mateu va recordar la entranyable hospitalitat que li va brindar sempre la família Ferrer, el plaer d’aquelles tertúlies i els coneixements que li van proporcionar. Tanmateix, l’entrada formal al camp de la recerca entomològica la va fer a través del Museu de Zoologia de Barcelona.

De Barcelona al Sàhara i a Almeria (1940–1956)

L’any 1940, Mateu va entrar en contacte amb el Museu de Zoologia, concretament amb la secció d’entomologia, aleshores dirigida per Francesc Espa-ñol. Aquesta primera trobada va ser l’inici d’una gran amistat que va durar fins a la mort d’Español, l’any 1999. Dos anys després d’aquest primer contacte, però, el novembre de 1942, Mateu va començar el servei militar, que es va perllongar fins al juny de 1945. Val a dir que el servei de Mateu va ser extraor-dinàriament singular atès que, assistit pels seus co-neixements d’història natural, va poder convèncer les autoritats militars per ser destinat al nord d’Àfrica com a naturalista adjunt al govern del territori Ifni–Sàhara, llavors espanyol. Rellevat de les tasques pròpiament militars, es va poder concentrar en l’estudi de la fauna, així com de la prehistòria, d’aquests territoris. Les prospeccions es van concentrar a les regions compreses entre l’Oued Draa, al nord, i l’Agûera, al sud, als territoris del Sàhara espanyol i Río de Oro. El descobriment del majestuós paisatge del desert el va fascinar de seguida, fascinació que va perdurar al llarg de tota la seva vida. Va representar la primera trobada amb la fauna d’insectes saharians, sovint localitzada a les torturades branques de les acàcies, disperses aquí i allà, de vegades als llocs més hostils, però que concentren bona part de la biodiversitat de l’indret. Una trobada que va anar seguida d’una llarga i fructífera dedicació investigadora, com veurem després. També va representar el descobriment de la segona vocació de Mateu: l’estudi de la prehistòria africana, al qual va dedicar moltes hores de lleure i un bon nombre de publicacions.

140 Bellés

de novembre de 1948. Tres anys després, i amb el patrocini de l’Instituto de Aclimatación, un organisme estatal, va tornar al Sàhara per prospectar la zona central i nord–occidental entre març i abril de 1951, amb base al Centre National de Recherche sur les Zones Arides, a Béni–Abbés, acompanyat pel seu col·lega Franklin Pierre, i entre abril i juny, tot sol, va explorar el massís del Hoggar (fig. 2). En tornar d’aquesta expedició sahariana, el juliol de 1951, va fer prospeccions a Sierra Nevada amb el seu amic Antonio Cobos de l'Instituto de Aclimatación, i amb els col·legues francesos Albert Vandel, Jean Sermet i Guy Colas. Els mesos de febrer i maig de 1952 es va desplaçar a les illes Canàries (Gran Canària, Tenerife, La Gomera, El Hierro i Lanzarote) en companyia de Georges Pécoud i el juliol de 1952 va portar a terme prospeccions entomològiques a la Serranía de Ron-da i Benaojan, incloent–hi la visita a diverses coves acompanyat, entre d’altres, per A. Vandel, Henry Coiffait i Jacques Nègre, qui esdevindrà un amic entranyable. Vers la tardor de 1953 va fer una nova campanya a Sierra Nevada i Las Alpujarras, patrocina-da per l’Instituto de Aclimatación, acompanyat també per A. Cobos i diversos col·legues francesos. Entre juny i agost de 1954 va fer prospeccions a Tenerife, Gran Canària i La Gomera i va visitar les zones litoral i sublitoral del Rif occidental. L’any següent, entre gener i juny de 1955, va treballar a les illes de Cap Verd, Madeira, Porto Santo i Açores.

En aquestes campanyes, com hem vist, sovint anava acompanyat per col·legues francesos, amb la qual cosa va començar a establir uns lligams d’amistat i col·laboració estables amb naturalistes del país veí. Aquestes relacions es van enfortir durant els anys 1950 i 1951 en què el CSIC li va concedir una borsa de viatge per treballar al Laboratoire d’Entomologie del Muséum national d’Histoire naturelle de Paris, dirigit primerament per René Jeannel i després per Lucien Chopard. Hi va fer una estada de nou mesos, amb un parèntesi per a la campanya al Sàhara esmentada més amunt, i un altre per participar al IX Congrès International d’Entomologie, celebrat a Amsterdam l’any 1951. Va tornar a fer una estada al Laboratoire d’Entomologie del Museu de París el juliol de 1953, aprofitant el viatge per assistir al I Congrès International de Spéléologie. En aquesta etapa, i quant a congressos, va participar en les dues primeres edicions del Congreso Internacional de Estudios Pirenaicos convidat pel seu amic Enric Balcells, el primer celebrat a Sant Sebastià, el 1951, i el segon a Luchon–Pau, el 1954.

En aquests 16 anys que van des de 1940 a 1956, Mateu va publicar 42 articles d’entomologia, és a dir, una mitjana d’entre dos i tres per any, la qual cosa representa una producció notable si considerem que es tracta de l’etapa inicial de la seva carrera investi-gadora que, a més, inclou un llarg servei militar on la feina es va concentrar en tasques de camp. Producció notable, doncs, ateses les circumstàncies, i continguts ja molt madurs. El primer treball que va publicar va ser una revisió dels Steropus ibèrics, signat amb F. Español i que va aparèixer l’any 1940. Amb aquest treball va iniciar una llarga trajectòria en l’estudi de

En acabar el servei militar, Mateu va tornar a Bar-celona i, esperonat per Español, va entrar en contacte amb el món de l’espeleologia, de tal manera que l’agost de 1945 es va afegir a una campanya biospe-leològica al País Basc patrocinada per l’Instituto Espa-ñol de Entomología del CSIC, en la qual va explorar diverses coves i avencs a la serres d’Aralar i d’Hernio en companyia de F. Español, Nadal Llopis i Ramon Margalef. L’interès per la fauna cavernicola el portarà a visitar nombroses coves i avencs a Catalunya i, l’any 1946, per exemple, el trobem a l’avenc d’Olèrdola (Alt Penedès) acompanyant Español (fig. 1). Aquest mateix any es nomenat col·laborador científic del CSIC, adscrit a l’Instituto Español de Entomología, a Madrid, aleshores dirigit per Gonzalo Ceballos. Tan-mateix, aquesta adscripció va ser merament nominal ja que, de fet, va treballar en comissió de serveis al seu estimat Museu de Zoologia de Barcelona, amb el seu amic F. Español. Dos anys més tard, però, el 1948, com a col·laborador científic del CSIC, va serfou adscrit a l’Instituto de Aclimatación, a Almeria, ales-hores dirigit per Manuel Mendizábal, on finalment es va traslladar. Amb el patrocini del CSIC i de l’Instituto de Estudios Africanos, entre març i agost de 1948 va participar en una expedició a la Guinea Equatorial i Fernando Poo dirigida per Santiago Alcobé i després va continuar tot sol als mateixos territoris fins a la fi

Fig. 1. Joaquim Mateu (a la dreta) amb Francesc Español a punt d’explorar l’avenc d’Olèrdola, a l’Alt Penedès, l’any 1946. Foto: Joaquim Mateu.

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la família dels coleòpters caràbids, dels quals va arribar a ser un dels especialistes més respectats del món. Sense sortir dels caràbids, també va publicar els primers treballs sobre la subfamília Lebiinae, un dels grups favorits de Mateu, del qual va esdevenir referent indiscutible. També són d’aquesta època les primeres notes sobre coleòpters cavernícoles, els primers treballs sobre la fauna del Sàhara i de les illes atlàntiques i els resultats de les primeres campanyes a la Sierra Nevada.

La llarga etapa parisenca (1956–1987)

L’any 1956 va ser clau en la vida i la carrera de Mateu atès que va obtenir una plaça com a Attaché de Recherches del CNRS francès i es va traslladar a París, al Laboratoire d’Entomologie del Muséum national d’Histoire naturelle, llavors sota la direcció d’Eugène Séguy. Va passar més de trenta anys de la seva vida a París. En els primers temps parisencs va mantenir viu l’interès per la fauna de les illes atlànti-ques i, entre març i maig de 1957, va visitar l’illa de Madeira en una expedició dirigida per A. Vandel. Dos anys després, entre abril i maig de 1959, va prospectar a l’illa de Porto Santo i les illes Desertes, a l’arxipèlag de Madeira. Un any abans, però, s’havia traslladat al Laboratoire d’Évolution des Êtres Organisés, de la Faculté des Sciences de Paris, dirigit per Pierre–Paul Grassé. Aquest canvi va marcar profundament la ca-rrera de Mateu, sobretot per la influència de Grassé, que no solament dirigia el Laboratoire d’Évolution, sinó que també era director de la seva tesi, la qual es va orientar vers la fauna del Sàhara, en particular la que s’agrupa sota la protecció de les emblemàtiques acàcies. Inicialment, Mateu va centrar el seu interès en els aspectes taxonòmics, però Grassé també el va orientar a estudiar aspectes biològics, la qual cosa el va obligar a fer mes observacions de camp i de laboratori sobre els cicles biològics i les interac-cions entre espècies. Això va fer necessàries noves expedicions, que Mateu va assumir amb entusiasme.

Així, entre gener i abril de 1958 va portar a terme una expedició a la Mauritània meridional patrocinada pel CNRS. Va entrar per Dakar i Saint Louis al Senegal i va fer prospeccions a la zona de Kiffa, als altiplans de Tagant i a les muntanyes de l’Affolé. Entre juliol i novembre de 1958 va explorar el massís de l’Ennedi, al Tchad, i a partir de 1961 es va centrar en el Sàhara algerià, amb llargues estades al Centre National de Recherche sur les Zones Arides de Béni–Abbés. Així va desenvolupar les llargues campanyes de 1961 (de gener de 1961 a juny de 1962), de 1963 (entre febrer i abril) i de 1964 (des de gener de 1964 fins a juny de 1965). Entre 1961 i 1965, Mateu va passar un total de 48 mesos al Sàhara fent estudis sobre els insectes i també de prehistòria. I l’abril de 1968 encara va fer una escapada al desert del sud de Tunísia amb Théodore Monod. Les observacions entomològiques i el material recollit al llarg d’aquests anys li van permetre completar una tesi doctoral amb el títol La biocénose des insectes xylophages des Acacia dans les régions sahariennes que va defensar el 28 de gener de 1969

i amb la qual va obtenir el grau de doctor en Ciències Naturals per la Universitat de París amb honors i la felicitació del tribunal (fig. 3), presidit per P. P. Grassé i amb altres membres com A. Vandel i T. Monod. La tesi es va publicar el 1975, dins la sèrie dels Anais da Faculdade de Ciencias da Universidade do Porto. un contundent volum de 714 pàgines profusament il·lustrat que ha esdevingut imprescindible per entendre les biocenosis de l’acàcia i el seu paper fonamental en la biologia dels medis desèrtics.

Entre els molts assistents a la defensa de la tesi n’hi va haver un de molt especial: el seu fill Giuliano, un noiet de 14 anys que encara es trobava en procés d’adopció. Un procés que havia començat a Itàlia el 1964 i que va acabar el 1973 a París, després de gairebé 10 anys de bregar amb les administracions de tres països, Espanya, França i Itàlia, amb sistemes legals molt diferents que s’havien de posar d’acord i mai no acabaven de fer–ho. Finalment, els obstacles es van poder vèncer gràcies a la tenacitat de Mateu, de manera que Giuliano es va incorporar feliçment a la seva vida per sempre més.

Acabada la tesi, que va culminar un intens progra-ma de recerca de la fauna entomològica del desert, Mateu, amb 48 anys, no va deixar el seu interès pel continent africà, al qual va tornar el 1971 (quatre mesos al Laboratoire de Primatologie et Écologie de la Forêt

Fig. 2. Una de les fotos més emblemàtiques de Joaquim Mateu és aquesta que el mostra dalt del seu dromedari al massís del Hoggar, al Sàhara, el maig de 1951. Va portar a terme la campanya del Hoggar tot sol i la fotografia va ser feta per ell mateix amb disparador automàtic. Foto: Joaquim Mateu.

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Equatoriale de Makoku, al Gabon, i tres mesos al Marroc meridional, sobretot a les províncies d’Agadir i Tarfaia), i el 1972 (prospeccions a les regions de Tafilalet, l’Antiatles, Ifni i Tiznit, al Marroc). Aviat, però, va adreçar l’atenció al continent americà. Així, del 15 de maig a l’1 juliol de 1972 va fer campanyes a Mèxic, al desert de Chihuahua, al Sistema Transversal, a Veracruz i a la zona dels Tuxtlas. Del 8 d’agost a l’11 de novembre de 1973 va tornar a Mèxic, a Veracruz, Nuevo León, Oaxaca i Chiapas, i encara hi va tornar el 1974, del 28 d’agost a l’1 de desembre, al Sistema Transversal, San Luis Potosí, Durango, Chiapas i Yucatán, sempre ajudat pels seus amics mexicans Violeta i Gonzalo Halffter i Pedro Reyes, de l’Instituto de Ecología, a Mèxic DF. L’estiu de 1977 va explorar les zones desèrtiques litorals i els altiplans andins del Perú i després va mar-xar a Veneçuela (Maracay i els Andes veneçolans, en companyia del seu amic Carlos Bordón). Finalment, entre juliol i agost de 1981, aprofitant la participació al 4º Congreso Latinoamericano de Entomología (Mara-cay–Veneçuela), va fer una campanya als estats de Barinas i Trujillo d’aquest país, també amb C. Bordón. Un gran nombre dels treballs de coleòpters d’Amèrica del Sud es refereixen a fauna cavernícola, la qual cosa el va portar un altre cop al món de la biospeleologia i a la participació en reunions científiques d’aquest àmbit, com el Colloque sur l’évolution des coléoptères souterrains, celebrat à Moulis, al departament francès de l’Arieja, el setembre de 1979, del qual va ser un dels conferenciants convidats (fig. 4).

Durant aquesta llarga etapa parisenca, Mateu asso-leix tots els nivells de promoció al CNRS: Chargé de Recherches el 1962, Maître de Recherches el 1973, i Directeur de Recherches el 1984. També li van ser reconeguts formalment els seus mèrits científics. Per exemple, el 1969 li va ser concedit el Prix Maurice et Therèse Pic de la Société Entomologique de France, el 1973 va ser nomenat membre honorari de la Institució Catalana d’Història Natural, el 1980 va ser honorat amb el Prix Pouchard de l’Académie Française, i el 1982 va ser nomenat membre corresponent de la Reial Acadèmia de Ciències i Arts de Barcelona, a proposta del seu amic F. Español. A més de la feina al CNRS, tant al camp com al laboratori, també va dedicar temps a tasques altruistes de gestió relacionades amb la recerca. Per exemple, el 1973 va ser elegit tresorer de la Société de Biogéographie i el 1984 va assumir la direcció de la Nouvelle Revue d’Entomologie, revista que havia contribuït a fundar el 1974 amb H. Coiffait i que va dirigir fins a 1986, en què va passar a ser–ne director honorari. Aquell mateix 1986, Mateu va fer 65 anys d’edat i li va arribar la jubilació del CNRS.

Va ser una carrera llarga, de més de 31 anys, dins d’aquesta prestigiosa institució francesa, i també molt brillant i fructífera, en què als treballs de camp ja esmen-tats cal afegir la publicació de 179 treballs d’entomologia, és a dir, una mitjana d’uns sis per any, cosa que suposa una producció extraordinàriament alta i encara més tenint en compte que Mateu va signar com a únic autor gairebé tots els treballs, la quantitat ingent de feina de

Fig. 3. Joaquim Mateu va defensar la seva tesi, que havia estat dirigida per Pierre–Paul Grassé, a París, el 28 de gener de 1969. La imatge els mostra tots dos brindant amb xampany després de la defensa. Foto d'A. Devez, proporcionada per Isabelle Desportes.

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camp que va fer i que un dels treballs és la publicació de la seva tesi, de 714 pàgines. A part d’aquest volum, que el va consolidar com un dels màxims exponents en l’estudi dels insectes del desert, Mateu va publicar la major part de les seves troballes entomològiques a l’Àfrica, no sols de caràbids, sinó també d’altres grups de coleòpters, com ara cerambícids, clèrids, buprèstids, líctids i bostríquids. Va dedicar bona part de les seves publicacions a la fauna de les illes atlàntiques, de la qual va descriure dades taxonòmiques i de síntesi biogeogràfica, sobretot de Canàries, Cap Verd i Ma-deira. També va publicar una notable sèrie de notes sobre els caràbids de Madagascar i descripcions de gèneres i espècies d’aquesta família d’arreu del món, capturats per ell o comunicats per col·legues seus. Es va consolidar com a autoritat mundial de Lebiinae, amb nombrosos treballs sobre aquest grup mostrejat a tot el món, entre els quals destaca la monografia dels Microlestes de l’Àfrica, un volum de 149 pàgines publicat el 1963. Al principi d’aquest període parisenc, la major part dels treballs es basen en materials afri-cans, però el seu interès deriva progressivament cap a la fauna d’Amèrica Central i del Sud, sobretot vers els caràbids de la subfamília Trechinae, sovint cavernícoles o endogeus, de vegades recollits per ell mateix però també per altres entomòlegs. Per exemple, va estudiar les captures d’expedicions catalanes a les coves perua-nes de la dècada de 1970, cosa que va donar lloc a la descripció de nous gèneres i espècies d’aquest grup.

Retorn a Almeria i a Barcelona (1987–2015)

El 1987, l’any següent al de la jubilació, Mateu va tornar a Espanya i es va establir a Almeria, on va retrobar amics de joventut, com A. Cobos, i el seu antic Instituto de Aclimatación, ara convertit en l’Estación Experimental de Zonas Áridas, del CSIC. Tambe en va fer de nous entrant en contacte amb naturalistes i espeleòlegs joves locals que li van lliurar les seves mostres de fauna cavernícola per a estudi. Amb aquests joves i entusiastes col·legues, que el respectaven com un referent indiscutible, encara va arribar a fer exploracions espeleològiques, com la de 1989 quan, amb 68 anys, va baixar a la cova de Las Ventanas, a Piñar (Granada) a la recerca de fauna cavernícola (fig. 5). A alguns d’aquests joves col·legues els va dedicar gèneres o espècies noves trobades per ells, com el Laemostenus barrancoi Mateu, 1996, dedicat a Pablo Barranco, de la Uni-versitat d’Almeria, o el Tinautius troglophilus Mateu, 1997, dedicat a Alberto Tinaut, de la Universitat de Granada. En aquests anys a Almeria, Mateu, viatger incansable, va aprofitar la llibertat que li donava la jubilació per visitar privadament nous països en viatges que mesclaven l’interès entomològic, sempre present, amb la curiositat de viatger que mai no va perdre. Així, entre març i abril de 1990 va viatjar per Tailàndia, Malàisia i el Nepal; entre novembre de 1993 i gener de 1994 va anar a l’Argentina i el

Fig. 4. El setembre de 1979, Joaquim Mateu va ser convidat especialment al Colloque sur l’évolution des coléoptères souterrains, celebrat a Moulis. El veiem a primera fila, entre els participants (d’esquerra a dreta), Xavier Bellés, Lysiane Juberthie–Jupeau, Robert Laneyrie, René Ginet, Christian Juberthie, Joaquim Mateu i Marina Blas. Foto del Laboratoire Souterrain de Moulis.

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Paraguai, i entre abril i maig de 1995 va tornar un altre cop a Malàisia. D’aquests viatges, en van sorgir nous descobriments, especialment dels seus grups favorits, els Lebiinae i els Zuphiinae, sobre els quals va continuar publicant espècies noves i revisions. En aquesta etapa d’Almeria també va publicar nombroses espècies inèdites de trèquids cavernícoles i endogeus sud–americans de l’Equador, el Perú, Colòmbia i el Brasil. En total, 28 treballs entre 1988 i 1997, uns tres treballs per any. També va acceptar encàrrecs per fer recensions de llibres, com la que va fer el 1988 sobre la Fauna cavernícola i intersticial de la penín-sula Ibèrica i les illes Balears, de Xavier Bellés, o el 1993 sobre la Fauna Ibèrica de Coleòpters Anobiidae, de F. Español. Amb relació a F. Espanyol, també va contribuir a l’homenatge que se li va retre al seu poble natal, Valls, amb la publicació del treball Francesc Español i l’entomologia sahariana, l’any 1988. Aquest mateix any li va arribar la mala notícia de la mort a París del seu amic entranyable J. Nègre, del qual va publicar una semblança biogràfica l’any següent.

El 1997, amb 76 anys, Mateu va tornar a Barcelona on tenia la seva família i molts amics, per exemple el seu vell mentor F. Español i el carabidòleg Joan Vives. Español, però, tenia 90 anys i estava retirat de tota activitat científica; Mateu el va visitar periòdicament a casa seva fins que va morir, l’any 1999. Vives tenia

79 anys i pertanyia pràcticament a la seva generació, però ja no desenvolupava gaire activitat científica i va morir tres anys després, l’any 2000. També va retrobar altres amics de la seva època de jove espeleòleg, com els companys del GES, el Grup d’Exploracions Subterrànies, Josep Maria Thomas, Joaquim Monto-riol, Francesc Vicens i Josep Termes, amb els quals l’any 1998 va celebrar el centenari d’aquest grup. A Barcelona, Mateu es va instal·lar en un confortable pis del carrer Còrsega, on tenia allotjada la seva col·lecció de coleòpters i on va muntar un petit la-boratori d’entomologia per poder continuar treballant a casa (fig. 6). Com havia fet a París i a Almeria, a casa seva sempre hi havia un llit parat i un plat a taula per a qualsevol col·lega que volgués passar–hi una temporada, proverbial hospitalitat que aprofitaven vells i nous amics entomòlegs d’aquí i d’arreu. De seguida va reprendre el contacte amb el Museu de Zoologia, finalment integrat al gran Museu de Ciències Naturals de Barcelona, sota la direcció d’Anna Omedes. Ve recuperar el contacte directe amb antics col·legues, com ara X. Bellés, Oleguer Escolà, Jordi Ribes i Eduard Vives, i va fer noves amistats entre els entomòlegs que freqüentaven el Museu, com Lluís Auroux, Jordi Comas, Floren Fadrique, Xavier Jeremías, Josep Joaquim Pérez de Gregorio, Francesc Vallhonrat i Amador Viñolas. També va col·laborar amb la revista del Museu, l’antiga Miscel·lània Zoològica fundada per F. Español i convertida en la més internacionalitzada Animal Biodiversity and Conservation. L’any 2008 va aprofitar l’acte de recepció com a acadèmic de X. Bellés per visitar per darrera vegada la Reial Acadèmia de Ciències i Arts de Barcelona, de la qual era membre corresponent des de 1982. Va ser un dels darrers actes públics a què va assistir.

En els primers temps d’aquest retorn a Barce-lona va desplegar una important activitat d’estudis taxonòmics. Entre 1998 i 2008 va publicar 22 tre-balls científics, sobretot de caràbids cavernícoles sud–americans (sovint en col·laboració amb Mirto Etonti), ibèrics, com l’espectacular Dalyat mirabilis de les coves de la serra de Gádor, a Almeria (estudiat en col·laboració amb X. Bellés), i nord–africans, com les noves espècies d’Antoinella descobertes en les expedicions expedicions a les coves del Marroc de l’Associació Catalana de Biospeleologia (descrites amb O. Escolà i J. Comas).

L’any 2008, Mateu va publicar el seu darrer treball, en el qual proposava dues noves espècies brasileres de Negrea, un gènere que ell mateix havia descrit el 1968 i amb el qual va honorar el seu amic J. Nègre. Tenia 87 anys, les seves capacitats havien minvat significativament i va decidir deixar la recerca taxonò-mica definitivament. Durant l’etapa parisenca havia pactat que la seva col·lecció de coleòpters aniria a Torí, al Museo Regionale di Scienze Naturali, i va demanar que la vinguessin a buscar a Barcelona. Així, una tarda del final d’octubre de 2009, des del balcó del seu pis del carrer Còrsega, va veure com s’allunyava un camió amb matricula italiana que s’enduia els seus estimats coleòpters, el resultat de gairebé setanta anys d’intensa recerca. Segurament va ser un dels moments més tristos de la seva vida.

Fig. 5. Veiem Joaquim Mateu l’any 1989, amb 68 anys, baixant a la cova de Las Ventanas, a Piñar (Granada), exploració que va fer amb Alberto Tinaut abans que fos oberta al turisme. Foto: Manuel González Ríos.

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Després, les seves capacitats es van anar deteriorant fins al punt de necessitar assistència especialitzada i l’agost del 2012, als 91 anys d’edat, va ingressar en una residència d’ancians medicalitzada. Allà va viure la darrera etapa d’una vellesa tranquil·la, al final conscient tan sols dels bons vells records i ignorant les malvestats dels temps que corrien. Va morir sen-se adonar–se’n el 20 de gener de 2015 i les seves cendres reposaran aviat entre els fins i acollidors grans de sorra del desert del Sàhara.

Epíleg

Diuen que una persona no mor mentre hi ha algú que la recorda. És una bellíssima metàfora, però contradiu l’eixuta veritat biològica. La mort és consubstancial a la vida i és necessària perquè hi hagi nova vida. “Cal deixar pas als que ens seguiran” és un principi biològic i Mateu ho sabia prou bé. Allò que no morirà, però, serà la seva obra. El seu llegat importantíssim en el camp de la taxonomia dels caràbids, les contribucions a l’estudi de la biogeografia i la història del poblament entomològic del Sàhara, de les illes atlàntiques, del tròpic americà, les aportacions singulars a l’estudi de la fauna cavernícola, també les significatives contribu-cions al coneixement de la prehistòria africana. Tota aquesta obra que esdevindrà clàssica, una referència científica en cadascun dels seus camps específics.

També quedarà la seva influència en totes aquelles persones que el vam conèixer i en les quals va deixar l’empremta de la seva profunda dignitat, generositat, honestedat personal i rigor professional. Qualitats que ens agrada pensar que tots els que el vam tractar hem après en certa manera i que transmetrem “als que ens seguiran”, en la feina o en la vida. És allò que se’n diu herència cultural i que ve a constituir, també, una forma d’immortalitat.

Agraïments

Moltes de les dades esmentades aquí van ser proporcionades pel mateix Joaquim Mateu. Un cop començada aquesta biografia, em van ajudar molt, amb records, documents personals i fotografies, el seu fill Giuliano i la seva germana Elena. Isabelle Desportes i Thierry Deuve m’han enviat dades que necessitava de la seva estada a París. Vull agrair també a I. Desportes i a Terry Erwin per corregir les meves respectives versions francesa i anglesa del manuscrit original. Lluís Auroux m’ha facilitat fotografies de la darrera etapa de Barcelona i Al-berto Tinaut m’ha enviat dades de la darrera etapa d’Almeria. Anna Omedes es va brindar de seguida a publicar aquesta biografia i a considerar–la la pròpia del Museu de Ciències Naturals de Barcelona, la qual cosa m’honora especialment.

Fig. 6. Joaquim Mateu, l’any 2005, assegut davant la lupa binocular al laboratori que va instal·lar al seu pis del carrer Còrsega. Foto: Lluís Auroux.

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Joaquim Mateu (1921–2015), an entire life dedicated to the study of insects

X. Bellés

Joaquim Mateu Sanpere was born in Barcelona on January 9, 1921 within a wealthy family of the Catalan bourgeoisie of the early twentieth century. His parents were Cristòfol Mateu Ferrer and Teresa Sanpere Cantelis, and the family had an illustrious ancestor, the historian, critic and politician Salvador Sanpere i Miquel, the father of Teresa Sanpere who had died in 1915. Joaquim was the second of the couple´s four children: Eduardo had preceded him, while Josep and Elena were born after him.

As a child, Joaquim Mateu studied at the school Sant Joan Baptista de La Salle, in the Gracia district of Barcelona. However, as he describes in unpublished autobiographical notes, during his adolescence he had poor health, afflicted with asthma and bronchitis, which forced him to spend long periods at home, where he occupied most of his time reading, often books on natural history and travels. It was in this way, perhaps, that his vocation as a naturalist and traveler might have started. From a young age, he struck up a close friendship with Felip Ferrer i Vert, who had been vice president of the Institució Catalana d’Història Natural and, at the Plaça Reial of Barcelona, had a taxidermy establishment, especially centered on birds, although also showing butterfly collections. The back shop of this unique establishment served as a meeting place for young people to talk about natural history, often about insects on which Ferrer i Vert was well acquainted. Until a very old age, Mateu remembered the endearing hos-pitality that he always received from the Ferrer family, the pleasure of those gatherings and the knowledge that they provided him. However, his formal entry into the field of entomological research took place through the Museu de Zoologia of Barcelona.

From Barcelona to the Sahara and to Almería (1940–1956)

In 1940, Mateu contacted the Museu de Zoologia of Barcelona, specifically the section of entomology then headed by Francesc Español. This first meeting would be the beginning of a great friendship that would last until the death of Español in 1999. However, two years after that first contact, in November 1942, Mateu began his military service, which extended until June 1945. A military service that was exceptional, since, thanks to his knowledge of natural history, he was able to convince the military authorities to be posted to North Africa as a naturalist attached to the government of the territory Ifni–Sahara, then belonging to Spain. Relieved of military duties, he was able to concentrate on studying the natural history and prehistory in these territories. Surveys were concentrated in the regions between the Oued Dráa, in the north, and the Agûera in the south, in the Spanish territories of the Sahara and Rio de Oro. The discovery of the majestic desert landscape immediately fascinated him, a fascination that lasted

throughout his entire life. This represented his first encounter with the Saharan fauna of insects, often found in the tortured branches of acacia trees scat-tered here and there, sometimes in the most hostile places, but that concentrate much of the biodiversity of the area. This experience would be followed by a long and fruitful research dedication, as discussed be-low. It will also represent the discovery of the second vocation of Mateu: the study of African prehistory, to which he devoted much of his spare time, producing a significant number of publications.

Upon completion of his military service, Mateu re-turned to Barcelona and, stimulated by F. Español, he discovered the world of speleology, so that in August 1945 he joined a biospeleological campaign in the Basque Country sponsored by the Instituto Español de Entomología of the CSIC. Thus, accompanied by F. Español, Noel Llopis and Ramon Margalef, he explored several caves in the Sierras of Aralar and Hernio. His interest in troglobitic fauna led him to visit many caves in Catalonia, and in 1946, for example, we find him accompanying Español in the exploration of the Avenc d’Olèrdola, in the Alt Penedès, Barcelona (fig. 1). That same year, 1946, Mateu was appointed as tenured scientist in the CSIC, attached to the Instituto Español de Entomología in Madrid, then headed by Gonzalo Ceballos. It would be a nominal appointment, since in fact he really based his work in the Museu de Zoologia of Barcelona with his friend F. Español. However, two years later, in 1948, he would be attached to the Instituto de Aclimatación, in Almería, then headed by Manuel Mendizábal, where Mateu finally moved. Sponsored by the CSIC and the Instituto de Estudios Africanos, between March and August 1948, he participated in an expedition directed by Santiago Alcobé to the Spanish Guinea and Fernando Poo, and at the end of it he continued alone in the same territories until the end of November 1948. Three years later, and with the spon-sorship of the Instituto de Aclimatación, he returned to the Sahara to develop collecting campaigns in the central and northwestern areas between March and April 1951, using the Centre National de Recherche sur les Zones Arides in Béni Abbés, as a base, and accompanied by his colleague Franklin Pierre; later, between April and June, and alone again, he prospected the Hoggar Massif (fig. 2). After returning from this Saharan expedition, in July 1951, he carried out faunistic prospections in Sierra Nevada with his friend Antonio Cobos, from the Instituto de Aclimatación, and with their French colleagues Albert Vandel, Jean Sermet and Guy Colas; in February and May of 1952 he traveled to the Canary Islands (Gran Canaria, Tenerife, Gomera, Hierro and Lanzarote) with Georges Pécoud; and in July 1952 he did entomological surveys in the Serranía de Ronda and Benaojan, inclu-ding visits to several caves, accompanied by A. Vandel, Henry Coiffait and Jacques Nègre, the latter to become one of his best friends in life. By the fall of 1953, he completed a new campaign in Sierra Nevada and Las Alpujarras sponsored by the Instituto de Aclimatación, also accompanied by A. Cobos and several French colleagues; between June and August 1954 he did prospections in Tenerife, Gran Canaria and La Gomera, and in the coast and sublittoral zone of Western Rif, in

Animal Biodiversity and Conservation 38.1 (2015) 147

Northern Africa; and the following year, between January and June 1955, he worked in the islands of Cape Verde, Madeira, Porto Santo and the Azores.

As we have seen, in these campaigns Mateu is often accompanied by French colleagues, thus begin-ning to establish stable relationships of cooperation and friendship with naturalists of his neighbor country. These relationships were strengthened during 1950 and 1951 when the CSIC gave him a travel grant to work in the Laboratoire d’Entomologie of the Muséum national d’Histoire naturelle in Paris, directed by René Jeannel first and then by Lucien Chopard. He stayed there for nine months, with a break to develop the campaign in the Sahara mentioned above, and the other to participate in the IX International Congress of Entomology, held in Amsterdam in 1951. He re-turned to the Laboratoire d’Entomologie of the Paris Museum in July 1953, taking advantage of the trip to participate in the I Congrés International de Spé-léologie. During this period, and concerning scientific meetings, he contributed to the first two editions of the Congreso Internacional de Estudios Pirenaicos invited by his friend Enrique Balcells, the first held in San Sebastián (Spain) in 1951 and the second in Luchon–Pau (France) in 1954.

In the 16 years from 1940 to 1956, Mateu publis-hed 42 articles of entomology, which represents an average of two or three per year. This is a remarkable productivity if we consider that this is the initial period of his research career, and that the period includes a long military service where work was focused on field tasks. A remarkable productivity, then, under these circumstances, and a very mature content of the contributions published. His first paper was a review of the Iberian Steropus co–authored with F. Español, which appeared in 1940. This work initiated a long history in the study of carabid beetles, of which he would become one of the world’s most respected specialists. Without leaving the carabid family, he also published the first work on Lebiinae subfamily, one of Mateu’s favorite groups, of which he would become an outstanding specialist. Also from this period are the first notes on cave beetles, the earlier papers on the fauna of the Sahara and the Atlantic Islands, and the results of the first campaigns in Sierra Nevada.

The long Parisian period (1956–1987)

The year 1956 was crucial in the life and career of Mateu, as he obtained a position as Attaché de recher-ches in the French CNRS, and moved to Paris to work in the Laboratoire d’Entomologie du Museum national d’Histoire naturelle, then under the direction of Eugène Séguy. In Paris, he spent more than 30 years of his life. In the early Parisian times, he kept his original interest in the fauna of the Atlantic Islands, and between March and May 1957 he visited the island of Madeira in an expedition organized by A. Vandel. Two years later, between April and May 1959, he did prospections in the islands of Porto Santo and Desertas, also in the archipelago of Madeira. A year earlier, however, he had moved to the Laboratoire d’Évolution des Êtres Organisés, belonging to the Faculté des Sciences in

Paris, directed by Pierre–Paul Grassé. This change would profoundly affect the career of Mateu, especially because the influence of Grassé, who not only ran the Laboratoire d’Évolution, but who also became the direc-tor of his thesis, which was already centered on the insect fauna of the Sahara, in particular that grouped under the protection of the emblematic acacias. Initially, Mateu focused the project on taxonomic questions, but Grassé convinced him to extend the study to biological aspects, which led to plan new field observations and laboratory studies of biological cycles and interactions between species. This would require new expeditions, which Mateu assumed enthusiastically.

Thus, between January and April 1958 he carried out an expedition in southern Mauritania sponsored by the CNRS. He started in Dakar and Saint Louis in Senegal, and then went to undertake collecting work in the area of Kiffa, in the highlands of Tagant, and in the Affolé mountains. Between July and November 1958, he explored the Ennedi Massif in Chad, and from 1961 he focused on the Algerian Sahara, with long stays at the Centre National de Recherche sur les Zones Arides of Béni Abbès. He developed the long campaigns of 1961 (January 1961 to June 1962), 1963 (February to April) and 1964 (January 1964 to June 1965). Between 1961 and 1965, Mateu spent a total of 48 months in the Sahara, studying insects and prehistory. In April 1968, he escaped into the desert again, this time in southern Tunisia, with Théodore Monod. The entomological observations and the mate-rial collected over the years allowed him to complete a doctoral thesis entitled La biocénose des insectes xylophages des Acacia dans les régions sahariennes that he defended the January 28, 1969 and which allowed him to obtain the PhD degree in natural sci-ences from the University of Paris with honors and congratulations from the judgment committee (fig. 3) chaired by P. P. Grassé, which had A. Vandel and T. Monod as other members. The thesis was published in 1975 in the series Anais da Faculdade de Ciencias da Universidade do Porto. An overwhelming volume of 714 pages profusely illustrated, which is now es-sential to understanding the insect communities living in acacia trees and its fundamental role in the biology of desert environments.

Among the people attending the thesis defense, there was a very special person: his son Giuliano, a boy of 14 whose adoption was still underway. This process began in Italy in 1964 and ended in 1973 in Paris, after nearly 10 years of dealing with administrative issues in three countries, Spain, France and Italy, with very dif-ferent legal systems, making it very difficult to reach an agreement, and they never seemed to do so. . Finally, the obstacles were overcome thanks to the tenacity of Mateu, so Giuliano happily joined his life forever.

After the PhD thesis, which culminated in an intense research program of the entomological fauna of the desert, Mateu, then 48 years old, did not abandon his interest in the African continent, to which he returned in 1971 (four months in the Laboratoire de Primatologie et Écologie de la Forêt Equatoriale in Makoku, Gabon, and three months in southern Morocco, especially in the provinces of Agadir and Tarfaya) and in 1972

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(campaigns in regions of Tafilalet, Anti–Atlas, Ifni and Tiznit, Morocco). But he soon turned his attention to the Americas. Thus, from May 15 to July 1, 1972 he carried out collecting work in Mexico: Chihuahua desert, Transversal System, Veracruz and Tuxtlas area. From August 8 to 11 November 1973 he returned to Mexico (Veracruz, Nuevo León, Oaxaca and Chiapas), and yet again in 1974, from August 28 to December 1 (Trans-versal System, San Luis Potosí, Durango, Chiapas and Yucatán), always helped by his Mexican friends, Violeta and Gonzalo Halffter and Pedro Reyes, from the Instituto de Ecología, Mexico DF. In the summer of 1977, he explored the coastal desert and Andean highlands of Peru, and then moved to Venezuela (Maracay and Venezuelan Andes) in the company of his friend Carlos Bordón. Finally, between July and August 1981, taking advantage of his participation in the IV Congreso Latinoamericano de Entomología (Maracay, Venezuela), he campaigned in the states of Barinas and Trujillo, also with C. Bordón. Many of the works on beetles of South America refer to cave fauna, which took him back to the world of biospeleology, and to participate in scientific meetings in this area, such as the Colloque sur l’évolution des coléoptères souterrains held in Moulis, in the French department of Ariège, in September 1979, where he was one of the invited speakers (fig. 4).

During this long Parisian period, Mateu achieved all levels of promotion in the CNRS, Chargé de Re-cherches in 1962, Maître de Recherches in 1973 and Directeur de Recherches in 1984. His scientific merits also started to be formally recognized. For example, in 1969 he received the Prix Maurice et Thérèse Pic ofthe Société Entomologique de France, in 1973 he was appointed honorary member of the Institució Catalana d’Història Natural, in 1980 he was honored with the Prix Pouchard of the Académie Française, and in 1982 he was elected corresponding member of the Reial Acadèmia de Ciències i Arts of Barcelona, on a proposal of his friend F. Español. In addition to his work at the CNRS, both in the field and in the laboratory, he also devoted part of his time to covering manage-ment tasks related to research on an altruistic basis. For example, in 1973 he accepted the responsibility of treasurer of the Société de Biogéographie, and in 1984 he became director of the journal Nouvelle Revue d’Entomologie, which he had helped to found in 1974 with H. Coiffait, and that he directed until 1986, when he became honorary director. That same year, Mateu reached 65, the age of retirement in the CNRS.

He had completed a long and fruitful career of over 31 years in this prestigious French institution, in which, besides the fieldwork already mentioned, we must add the publication of 179 papers on entomology, i.e., an average of about 6 per year, which is an extraordina-rily high productivity, especially considering his sole authorship, the huge amount of fieldwork he did, and the publication of his thesis of 714 pages. Apart from this volume, which consolidates him as one of the most outstanding experts in desert entomology, Mateu published most of his entomological discoveries in Africa, not only on carabids, but also on other beetle families, such as cerambycids, clerids, buprestids,

lyctids, and bostrychids. He also devoted much of his work to the fauna of the Atlantic Islands, publishing not only taxonomic data but also biogeographical syntheses, especially from the Canary Islands, Cape Verde and Madeira. He also published a remarkable series of notes on carabids from Madagascar, and descriptions of genera and species of this family of the five continents, either collected by him, or reported by colleagues. He was indisputably recognized as a world authority on Lebiinae, with numerous papers published on this group based on samples collected around the world, for example, the monograph of Microlestes of Africa, a volume of 149 pages that appeared in 1963. At the beginning of this Parisian period, most of his work was based on African ma-terials, but gradually his interest drifted to the faunas of Central and South America, especially towards the carabids of the subfamily Trechinae, often troglobitic or endogean, sometimes collected by him, but also by other entomologists. For example, he studied the collections obtained from the Catalan expeditions to the Peruvian caves in the 1970s, which resulted in the description of new genera and species of this group.

Return to Almeria and to Barcelona (1987–2015)

In 1987, the year after his retirement, Mateu returned to Spain and settled in Almería, where he met with old friends, like A. Cobos, and with his former Instituto de Aclimatación, now converted into the modern Estación Experimental de Zonas Áridas, of the CSIC. He also made new friends when he came into contact with young local naturalists and speleologists, who reported to him the cave fauna samples that they collected. With these enthusiastic colleagues, who respected him as an indisputable senior leader, he even did speleologi-cal explorations, such as in 1989, when he explored (at 68 years old) the Cueva de las Ventanas, in Piñar (Granada), in search of cave insects (fig. 5). To some of these young colleagues, Mateu dedicated a number of genera and new species found by them, such as the Laemostenus barrancoi Mateu, 1996, dedicated to Pablo Barranco, from the University of Almería, or Tinautius troglophilus Mateu, 1997, dedicated to Alberto Tinaut, from the University of Granada. In these years in Almeria, Mateu, tireless traveler, took advantage of the freedom given by the retirement to privately visit new countries, mixing the entomological interest always present, with the curiosity of the traveler that he never lost. Thus, between March and April 1990 he traveled through Thailand, Malaysia and Nepal; between November 1993 and January 1994 through Argentina and Paraguay; and between April and May 1995 he returned to Malaysia. These trips led to new discoveries, especially of his favorite groups, Lebiinae and Zuphiinae, of which he continued publishing new species and revisions. During this stage in Almería, he also published numerous new species of troglobitic and endogean Trechinae from South America (from Ecuador, Peru, Colombia and Brazil). In all, 28 papers published between 1988 and 1997, about 3 per year. He also accepted commissions to review books of colleagues, as he did in 1988 on the Fauna cavernicola

Animal Biodiversity and Conservation 38.1 (2015) 149

i intersticial de la Península Ibèrica i les illes Balears published by Xavier Bellés, or in 1993 on the Fauna Ibérica de Coleòpters Anobiidae of F. Español. About F. Español, he also contributed to the tribute paid to him in 1988 in his hometown, in Valls, publishing the paper Francesc Español i l’entomologia sahariana. That same year he received the sad news of the death in Paris of his close friend J. Nègre, on whom he published a biographical sketch the following year.

In 1997, aged 76, Mateu returned to Barcelona where he had his family and many friends, such as his old mentor F. Español and the Catalan carabido-logist Joan Vives. Español, however, was 90 and had retired from all scientific activity; Mateu visited him regularly at his home until his friend’s death in 1999. J. Vives was 79 years old and practically belonged to Mateu’s generation, but he did not continue much scientific activity, and died three years later, in 2000. Mateu also rediscovered other friends from his time as a young speleologist, as the companions of the Grup d’Exploracions Subterrànies (GES), Josep Maria Thomas, Joaquim Montoriol, Francesc Vicens and Josep Termes, with whom in 1998 he celebrated the centenary of this group. In Barcelona, Mateu lived in a comfortable apartment on Còrsega street, which housed his collection of beetles and where he installed a small entomology laboratory to continue working at home (fig. 6). As in Paris and Almería, his home was always open to all those colleagues who wanted to spend some time studying insects, proverbial hospitality profited by old and new friends from Spain and from everywhere. Soon, he reconnected with the Museu de Zoologia, now integrated into the greater Museu de Ciències Naturals de Barcelona, under the direction of Anna Omedes. He regained direct contact with former colleagues, like X. Bellés, Oleguer Escolà, Jordi Ribes and Eduard Vives, and made new friends among the entomologists who frequented the museum, such as Lluís Auroux, Jordi Comas, Floren Fadrique, Xavier Jeremías, José Joaquín Pérez de Gregorio, Francesc Vallhonrat, and Amador Viñolas. He also collaborated with the journal of the Museum, the former Miscelánea Zoológica, founded by F. Español, now the interna-tional Animal Biodiversity and Conservation. In 2008, on the occasion of the reception of X. Bellés as new academician, he paid a visit to the Reial Acadèmia de Ciències i Arts of Barcelona, of which he had been corresponding member since 1982. This would be one of the last public events he attended.

In the early days of his return to Barcelona, he ca-rried out notable activity in taxonomic studies. Between 1998 and 2008, he published 22 scientific papers, mostly on carabid beetles from caves of South America (often in collaboration with Mirto Etonti), from the Iberian Peninsula, such as the spectacular Dalyat mirabilis from caves in the Sierra de Gádor, in Almería (studied in collaboration with X. Bellés ), and from North Africa, such as the new species of Antoinella discovered during the expeditions to the caves of Morocco organized by the Associació Catalana de Biospeleologia (described with O. Escolà and J. Comas).

In 2008, Mateu published his last paper, in which he proposed two new Brazilian species of Negrea, a

genus that he had described in 1968 and with which he paid tribute to his friend J. Nègre. He was 87 years old, his skills had diminished significantly, and he decided to leave taxonomic research. During his Paris period, he had agreed that his collection would go to Turin, to the Museo Regionale di Scienze Naturali and he asked the Museum to come to Barcelona and to collect it. So one afternoon in late October 2009, from the balcony of his home in Còrsega street, Mateu watched an Italian truck carrying away his beloved beetles, the result of nearly 70 years of intense research. Surely, this was one of the saddest moments of his life. Progressively, his capabilities deteriorated to the point of his needing specialized care, and in August 2012, when he was 91 years old, he entered a residential care facility. There he lived the last stage of a peaceful old age, in the end conscious only of the good old memories and ignorant of the turmoils of of the time. He died, without consciousness, on January 20, 2015 and his ashes will soon be laid to rest among the thin and welcoming sands of the Sahara desert.

Epilogue

It is said that a person does not die while there is someone that remembers him. It is a beautiful meta-phor, but contradicts the cold biological truth. Death is inseparable from life, and new life is necessary. “We must make way for those who will follow us” is a biological principle that Mateu knew very well. What will not die, however, is all his work. His valua-ble legacy in the field of carabid beetles taxonomy, his contributions to the study of biogeography of the Saharan insects, of the Atlantic Islands, of the Ame-rican tropics, his unique contributions to the study of cave fauna, and his significant contributions to the knowledge of African prehistory. All these works will remain and become classic as a scientific reference in each of their specific fields forever. Their influence on all those who knew him and on whom he left the imprint of his profound dignity, generosity, personal honesty and professional rigueur will also remain. Qualities that we like to think that all of us who knew him have learned to some extent, and that we will pass on to “those who will follow us” , through work or life. This is what we call cultural inheritance, and it, too, is a form of immortality.

Acknowledgements

Much of the data mentioned here were provided by Joaquim Mateu himself. His son Giuliano and his sister Elena helped me withmemories, personal documents and photographs. Isabelle Desportes and Thierry Deuve sent me information that I needed from his stay in Paris. Thanks are also due to I. Desportes and to Terry Erwin for correcting the translations of my original Catalan manuscript to French and English. Lluís Auroux provided me with photographs of the last stage in Barcelona, and Alberto Tinaut sent me data on the last stage in Almería. I also wish to thank Anna Omedes for the honor of publishing this obituary in Animal Biodiversity and Conservation.

150 Bellés

Figure legends

Fig. 1. Joaquim Mateu (right) with Francesc Español about to explore the Avenc d'Olèrdola, in the Alt Penedès, Barcelona, 1946. Photo: Joaquim Mateu.

Fig. 2. One of the most iconic photos of Joaquin Mateu on his dromedary in the Hoggar massif in the Sahara in May 1951. He embarked on the Hoggar campaign alone, and the photograph was taken by himself with an automatic shutter. Photo: Joaquim Mateu.

Fig. 3. Joaquim Mateu defended his thesis, which was directed by Pierre–Paul Grassé, in Paris on January 28, 1969. The picture shows both men celebrating the successful defense with champagne. Photo: A. Devez, provided by Isabelle Desportes.

Fig. 4. In September 1979, Joaquim Mateu was a special guest at the Colloque sur l'évolution des coléoptères souterrains held in Moulis. We see him in the front row among the participants (left to right), Xavier Bellés, Ly-siane Juberthie–Jupeau, Robert Laneyrie, René Ginet, Christian Juberthie, Joaquim Mateu and Marina Blas. Photo of the Laboratoire Souterrain of Moulis.

Fig. 5. In 1989, at 68 years old, we see Joaquim Mateu exploring the Cueva de las Ventanas, in Piñar (Granada); the exploration was carried out with Alberto Tinaut before the cave was opened to tourism. Photo: Manuel González Ríos.

Fig. 6. Joaquim Mateu in 2005 sitting at his binocular microscope in the laboratory installed in his apartment in Còrsega street. Photo: Lluís Auroux.

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Animal Biodiversity and Conservation 38.1 (2015) I

ISSN: 1578–665X eISSN: 2014–928X

© 2015 Museu de Ciències Naturals de Barcelona

Animal Biodiversity and Conservation

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Una vegada hagin estat acceptats passaran a ser propietat de la revista. Aquesta es reserva els drets d’autor, i cap part dels treballs no podrà ser reproduïda sense citar–ne la procedència.

Normes de publicació

Els treballs s'enviaran preferentment de forma electrònica (abc@bcn.cat). El format preferit és un document Rich Text Format (RTF) o DOC que inclogui les figures i les taules. es figures s'hauran d'enviar també en arxius apart en format TIFF, EPS o JPEG. Cal incloure, juntament amb l'article, una carta on es faci constar que el treball està basat en investigacions originals no publicades anteriorment i que està sotmès a Animal Biodiversity and Conservation en exclusiva. A la carta també ha de constar, per a aquells treballs en que calgui manipular animals, que els autors disposen dels permisos necessaris i que compleixen la normativa de protecció animal vigent. També es poden suggerir possibles assessors.

Quan l'article sigui acceptat, els autors hauran d'enviar a la Redacció una còpia impresa de la versió final acompanyada d'un disquet indicant el programa utilitzat (preferiblement en Word). es proves d'impremta enviades a l'autor per a la correcció, seran retornades al Consell Editor en el termini de 10 dies. Aniran a càrrec dels autors les despeses degudes a modificacions substancials introduïdes per ells en el text original acceptat.

El primer autor rebrà una còpia electrònica del treball en format PDF.

Manuscrits

Els treballs seran presentats en format DIN A –4 (30 línies de 70 espais cada una) a doble espai i amb totes les pàgines numerades. Els manus crits han de ser complets, amb taules i figures. No s'han d'enviar les figures originals fins que l'article no hagi estat acceptat.

El text es podrà redactar en anglès, castellà o català. Se suggereix als autors que enviïn els seus treballs en anglès. a revista els ofereix, sense cap càrrec, un servei de correcció per part d'una persona especialitzada en revistes científiques. En tots els casos, els textos hauran de ser redactats correctament i amb un llenguatge clar i concís. a redacció del text serà impersonal, i s'evitarà sempre la primera persona.

Els caràcters cursius s’empraran per als noms científics de gèneres i d’espècies i per als neologismes intraduïbles; les cites textuals, independentment de la llengua, seran consignades en lletra rodona i entre cometes i els noms d’autor que segueixin un tàxon aniran en rodona.

Quan se citi una espècie per primera vegada en el text, es ressenyarà, sempre que sigui possible, el seu nom comú.

Els topònims s’escriuran o bé en la forma original o bé en la llengua en què estigui escrit el treball, seguint sempre el mateix criteri.

Els nombres de l’u al nou, sempre que estiguin en el text, s’escriuran amb lletres, excepte quan precedeixin una unitat de mesura. Els nombres més grans s'escriuran amb xifres excepte quan comencin una frase.

es dates s’indicaran de la forma següent: 28 VI 99 (un únic dia); 28, 30 VI 99 (dies 28 i 30); 28–30 VI 99 (dies 28 a 30).

S’evitaran sempre les notes a peu de pàgina.

Format dels articles

Títol. Serà concís, però suficientment indicador del contingut. Els títols amb desig nacions de sèries numèriques (I, II, III, etc.) seran acceptats previ acord amb l'editor.Nom de l’autor o els autorsAbstract en anglès que no ultrapassi les 12 línies mecanografiades (860 espais) i que mostri l’essència del manuscrit (introducció, material, mètodes, resultats i discussió). S'evitaran les especulacions i les cites bibliogràfiques. Estarà encapçalat pel títol del treball en cursiva.Key words en anglès (sis com a màxim), que orientin sobre el contingut del treball en ordre d’importància.Resumen en castellà, traducció de l'Abstract. De la traducció se'n farà càrrec la revista per a aquells autors que no siguin castellano parlants. Palabras clave en castellà.Adreça postal de l’autor o autors.(Títol, Nom, Abstract, Key words, Resumen, Palabras clave i Adreça postal, conformaran la primera pàgina.)

II

Introducción. S'hi donarà una idea dels antecedents del tema tractat, així com dels objectius del treball.Material y métodos. Inclourà la informació pertinent de les espècies estudiades, aparells emprats, mètodes d’estudi i d’anàlisi de les dades i zona d’estudi.Resultados. En aquesta secció es presentaran únicament les dades obtingudes que no hagin estat publicades prèviament.Discusión. Es discutiran els resultats i es compararan amb treballs relacionats. Els sug geriments de recerques futures es podran incloure al final d’aquest apartat.Agradecimientos (optatiu).Referencias. Cada treball haurà d’anar acompanyat de les referències bibliogràfiques citades en el text.

es referències han de presentar–se segons els models següents (mètode Harvard):* Articles de revista:Conroy, M. J. & Noon, B. R., 1996. Mapping of spe

cies richness for conservation of biological diversity: conceptual and methodological issues. Ecological Applications, 6: 763–773.

* libres o altres publicacions no periòdiques:Seber, G. A. F., 1982. The estimation of animal abun-

dance. C. Griffin & Company, ondon. * Treballs de contribució en llibres:Macdonald, D. W. & Johnson, D. P., 2001. Dispersal

in theory and practice: consequences for conservation biology. In: Dispersal: 358–372 (T. J. Clober, E. Danchin, A. A. Dhondt & J. D. Nichols, Eds.). Oxford University Press, Oxford.

* Tesis doctorals:Merilä, J., 1996. Genetic and quantitative trait vari

ation in natural bird populations. Tesis doctoral, Uppsala University.

* Els treballs en premsa només han d’ésser citats si han estat acceptats per a la publicació:Ripoll, M. (in press). The relevance of population

studies to conservation biology: a review. Animal Biodiversity and Conservation.

a relació de referències bibliogràfiques d’un treball serà establerta i s’ordenarà alfabè ticament per autors i cronològicament per a un mateix autor, afegint les lletres a, b, c,... als treballs del mateix any. En el text, s’indi caran en la forma usual: "...segons Wemmer (1998)...", "...ha estat definit per Robinson & Redford (1991)...", "...les prospeccions realitzades (Begon et al., 1999)...". Taules. Es numeraran 1, 2, 3, etc. i han de ser sempre ressenyades en el text. es taules grans seran més estretes i llargues que amples i curtes ja que s'han d'encaixar en l'amplada de la caixa de la revista. Figures. Tota classe d’il·lustracions (gràfics, figures o fotografies) entraran amb el nom de figura i es numeraran 1, 2, 3, etc. i han de ser sempre ressenyades en el text. Es podran incloure fotografies si són imprescindibles. Si les fotografies són en color, el cost de la seva publicació anirà a càrrec dels autors. a mida màxima de les figures és de 15,5 cm d'amplada per 24 cm d'alçada. S'evitaran les figures tridimensionals. Tant els mapes com els dibuixos han d'incloure l'escala. Els ombreigs preferibles són blanc, negre o trama. S'evitaran els punteigs ja que no es repro dueixen bé. Peus de figura i capçaleres de taula. Seran clars, concisos i bilingües en la llengua de l’article i en anglès.

Els títols dels apartats generals de l’article (Introducción, Material y métodos, Resultados, Discusión, Conclusiones, Agradecimientos y Referencias) no aniran numerats. No es poden utilitzar més de tres nivells de títols.

Els autors procuraran que els seus treballs originals no passin de 20 pàgines (incloent–hi figures i taules).

Si a l'article es descriuen nous tàxons, caldrà que els tipus estiguin dipositats en una insti tució pública.

Es recomana als autors la consulta de fascicles recents de la revista per tenir en compte les seves normes.

Animal Biodiversity and Conservation 38.1 (2015) III

ISSN: 1578–665X eISSN: 2014–928X

© 2015 Museu de Ciències Naturals de Barcelona

Animal Biodiversity and Conservation

Animal Biodiversity and Conservation es una revista inter disciplinar, publicada desde 1958 por el Museo Ciencias Naturales de Barcelona. Incluye artículos de investigación empírica y teórica en todas las áreas de la zoología (sistemática, taxo nomía, morfología, biogeografía, ecología, etología, fisiología y genética) procedentes de todas las regiones del mundo, con especial énfasis en los estudios que permitan comprender, desde un punto de vista pluridisciplinar e integrado, los patrones de evolución de la biodiversidad en su sentido más amplio. a revista no publica compilaciones bibliográficas, catálogos, listas de especies sin más o citas puntuales. os estudios realizados con especies raras o protegidas pueden no ser aceptados a no ser que los autores dispongan de los permisos correspondientes. Cada volumen anual consta de dos fascículos.

Animal Biodiversity and Conservation está registrada en todas las bases de datos importantes y además está disponible gratuitamente en internet en www.abc.museucienciesjournals.cat, lo que permite una difusión mundial de sus artículos.

Todos los manuscritos son revisados por el editor ejecutivo, un editor y dos revisores independientes, elegidos de una lista internacional, a fin de garantizar su calidad. El proceso de revisión es rápido y constructivo, y se realiza vía correo electrónico siempre que es posible. a publicación de los trabajos aceptados se realiza con la mayor rapidez posible, normalmente dentro de los 12 meses siguientes a la recepción del trabajo.

Una vez aceptado, el trabajo pasará a ser propiedad de la revista. Ésta se reserva los derechos de autor, y ninguna parte del trabajo podrá ser reproducida sin citar su procedencia.

Normas de publicación

os trabajos se enviarán preferentemente de forma electrónica (abc@bcn.cat). El formato preferido es un documento Rich Text Format (RTF) o DOC, que incluya las figuras y las tablas. as figuras deberán enviarse también en archivos separados en formato TIFF, EPS o JPEG. Debe incluirse, con el artículo, una carta donde conste que el trabajo versa sobre inves tigaciones originales no publi cadas an te riormente y que se somete en exclusiva a Animal Bio-diversity and Conservation. En dicha carta también debe constar, para trabajos donde sea necesaria la manipulación de animales, que los autores disponen de los permisos necesarios y que han cumplido la normativa de protección animal vigente. os autores pueden enviar también sugerencias para asesores.

Cuando el trabajo sea aceptado los autores deberán enviar a la Redacción una copia impresa de la versión final junto con un disquete del manuscrito preparado con un pro cesador de textos e indicando el programa utilizado (preferiblemente Word). as pruebas de imprenta enviadas a los autores deberán

remitirse corregidas al Consejo Editor en el plazo máximo de 10 días. os gastos debidos a modificaciones sustanciales en las pruebas de im pren ta, introducidas por los autores, irán a cargo de los mismos.

El primer autor recibirá una copia electrónica del trabajo en formato PDF.

Manuscritos

os trabajos se presentarán en formato DIN A–4 (30 líneas de 70 espacios cada una) a doble espacio y con las páginas numeradas. os manuscritos deben estar completos, con tablas y figuras. No enviar las figuras originales hasta que el artículo haya sido aceptado.

El texto podrá redactarse en inglés, castellano o catalán. Se sugiere a los autores que envíen sus trabajos en inglés. a revista ofre ce, sin cargo ninguno, un servicio de corrección por parte de una persona especializada en revistas científicas. En cualquier caso debe presentarse siempre de forma correcta y con un lenguaje claro y conciso. a redacción del texto deberá ser impersonal, evitán dose siempre la primera persona.

os caracteres en cursiva se utilizarán para los nombres científicos de géneros y especies y para los neologismos que no tengan traducción; las citas textuales, independientemente de la lengua en que estén, irán en letra redonda y entre comillas; el nombre del autor que sigue a un taxón se escribirá también en redonda.

Al citar por primera vez una especie en el trabajo, deberá especificarse siempre que sea posible su nombre común.

os topónimos se escribirán bien en su forma original o bien en la lengua en que esté redactado el trabajo, siguiendo el mismo criterio a lo largo de todo el artículo.

os números del uno al nueve se escribirán con letras, a excepción de cuando precedan una unidad de medida. os números mayores de nueve se escribirán con cifras excepto al empezar una frase.

as fechas se indicarán de la siguiente forma: 28 VI 99 (un único día); 28, 30 VI 99 (días 28 y 30); 28–30 VI 99 (días 28 al 30).

Se evitarán siempre las notas a pie de página.

Formato de los artículos

Título. Será conciso pero suficientemente explicativo del contenido del trabajo. os títulos con designaciones de series numéricas (I, II, III, etc.) serán aceptados excepcionalmente previo consentimiento del editor.Nombre del autor o autoresAbstract en inglés de 12 líneas mecanografiadas (860 espacios como máximo) y que exprese la esencia del manuscrito (introducción, material, métodos, resultados y discusión). Se evitarán las especulaciones y las citas bibliográficas. Irá encabezado por el título del trabajo en cursiva.Key words en inglés (un máximo de seis) que especifiquen el contenido del trabajo por orden de importancia.

IV

Resumen en castellano, traducción del abstract. Su traducción puede ser solicitada a la revista en el caso de autores que no sean castellano hablan tes. Palabras clave en castellano.Dirección postal del autor o autores.(Título, Nombre, Abstract, Key words, Resumen, Palabras clave y Dirección postal conformarán la primera página.)

Introducción. En ella se dará una idea de los antecedentes del tema tratado, así como de los objetivos del trabajo.Material y métodos. Incluirá la información referente a las especies estudiadas, aparatos utilizados, metodología de estudio y análisis de los datos y zona de estudio.Resultados. En esta sección se presentarán únicamente los datos obtenidos que no hayan sido publicados previamente.Discusión. Se discutirán los resultados y se compararán con otros trabajos relacionados. as sugerencias sobre investigaciones futuras se podrán incluir al final de este apartado.Agradecimientos (optativo).Referencias. Cada trabajo irá acompañado de una bibliografía que incluirá únicamente las publicaciones citadas en el texto.

as referencias deben presentarse según los modelos siguientes (método Harvard):* Artículos de revista:Conroy, M. J. & Noon, B. R., 1996. Mapping of spe

cies richness for conservation of biological diversity: conceptual and methodological issues. Ecological Applications, 6: 763–773.

* ibros y otras publicaciones no periódicas:Seber, G. A. F., 1982. The estimation of animal abun-

dance. C. Griffin & Company, ondon. * Trabajos de contribución en libros:Macdonald, D. W. & Johnson, D. P., 2001. Dispersal

in theory and practice: consequences for conservation biology. In: Dispersal: 358–372 (T. J. Clober, E. Danchin, A. A. Dhondt & J. D. Nichols, Eds.). Oxford University Press, Oxford.

* Tesis doctorales:Merilä, J., 1996. Genetic and quantitative trait vari

ation in natural bird populations. Tesis doctoral,

Uppsala University. * os trabajos en prensa sólo se citarán si han sido aceptados para su publicación:Ripoll, M. (in press). The relevance of population

studies to conservation biology: a review. Animal Biodiversity and Conservation.

as referencias se ordenarán alfabética men te por autores, cronológicamen te para un mismo autor y con las letras a, b, c,... para los tra bajos de un mismo autor y año. En el texto las referencias bibliográficas se indicarán en la forma usual: "...según Wemmer (1998)...", "...ha sido definido por Robinson & Redford (1991)...", "...las prospecciones realizadas (Begon et al., 1999)...". Tablas. Se numerarán 1, 2, 3, etc. y se reseñarán todas en el texto. as tablas grandes deben ser más estrechas y largas que anchas y cortas ya que deben ajustarse a la caja de la revista.Figuras. Toda clase de ilustraciones (gráficas, figuras o fotografías) se considerarán figuras, se numerarán 1, 2, 3, etc. y se citarán todas en el texto. Pueden incluirse fotografías si son imprescindibles. Si las fotografías son en color, el coste de su publicación irá a cargo de los autores. El tamaño máximo de las figuras es de 15,5 cm de ancho y 24 cm de alto. Deben evitarse las figuras tridimen sionales. Tanto los mapas como los dibujos deben incluir la escala. os sombreados preferibles son blanco, negro o trama. Deben evitarse los punteados ya que no se reproducen bien.Pies de figura y cabeceras de tabla. Serán claros, concisos y bilingües en castellano e inglés.

os títulos de los apartados generales del artículo (Introducción, Material y métodos, Resultados, Discusión, Agradecimientos y Referencias) no se numerarán. No utilizar más de tres niveles de títulos.

os autores procurarán que sus trabajos originales no excedan las 20 páginas incluidas figuras y tablas.

Si en el artículo se describen nuevos taxones, es imprescindible que los tipos estén depositados en alguna institución pública.

Se recomienda a los autores la consulta de fascículos recientes de la revista para seguir sus directrices.

Animal Biodiversity and Conservation 38.1 (2015) V

ISSN: 1578–665X eISSN: 2014–928X

© 2015 Museu de Ciències Naturals de Barcelona

Animal Biodiversity and Conservation

Animal Biodiversity and Conservation is an interdisciplinary journal published by the Natural Science Museum of Barcelona since 1958. It includes empirical and theoretical research from around the world that examines any aspect of Zoology (Systematics, Taxonomy, Morphology, Biogeography, Ecology, Ethology, Physiology and Genetics). Special emphasis is given to integrative and multidisciplinary studies that help to understand the evolutionary patterns in biodiversity in the widest sense. The journal does not publish bibliographic compilations, listings, catalogues or collections of species, or isolated descriptions of a single specimen. Studies concerning rare or protected species will not be accepted unless the authors have been granted the relevant permits or authorisation. Each annual volume consists of two issues.

Animal Biodiversity and Conservation is registered in all principal data bases and is freely available online at www.abc.museucienciesjournals.cat assuring world–wide access to articles published therein.

All manuscripts are screened by the Executive Editor, an Editor and two independent reviewers so as to guarantee the quality of the papers. The review process aims to be rapid and constructive. Once accepted, papers are published as soon as is practicable. This is usually within 12 months of initial submission.

Upon acceptance, manuscripts become the property of the journal, which reserves copyright, and no published material may be reproduced or cited without acknowledging the source of information.

Information for authors

Electronic submission of papers is encouraged (abc@bcn.cat). The preferred format is DOC or RTF. All figures must be readable by Word, embedded at the end of the manuscript and submitted together in a separate attachment in a TIFF, EPS or JPEG file. Tables should be placed at the end of the document. A cover letter stating that the article reports original research that has not been published elsewhere and has been submitted exclusively for consideration in Animal Biodiversity and Conservation is also necessary. When animal manipulation has been necessary, the cover letter should also specify that the authors follow current norms on the protection of animal species and that they have obtained all relevant permits and authorisations. Authors may suggest referees for their papers.

Once an article has been accepted, authors should send a paper copy and an electronic copy of the final version. Please identify software (preferably Word). Proofs sent to the authors for correction should be returned to the Editorial Board within 10 days. Expenses due to any substantial alterations of the proofs will be charged to the authors.

The first author will receive electronic version of the article in PDF format.

Manuscripts

Manuscripts must be presented in DIN A–4 format, 30 lines, 70 keystrokes per page. Maintain double spacing throughout. Number all pages. Manuscripts should be complete with figures and tables. Do not send original figures until the paper has been accepted.

The text may be written in English, Spanish or Catalan, though English is preferred. The journal provides linguistic revision by an author’s editor. Care must be taken to use correct wording and the text should be written concisely and clearly. Scientific names of genera and species as well as untranslatable neologisms must be in italics. Quotations in whatever language used must be typed in ordinary print between quotation marks. The name of the author following a taxon should also be written in lower case letters.

When referring to a species for the first time in the text, both common and scientific names should be given when possible. Do not capitalize common names of species unless they are proper nouns (e.g. Iberian rock lizard). Place names may appear either in their original form or in the language of the manuscript, but care should be taken to use the same criteria throughout the text.

Numbers one to nine should be written in full within the text except when preceding a measure. Higher numbers should be written in numerals except at the beginning of a sentence.

Specify dates as follows: 28 VI 99 (for a single day); 28, 30 VI 99 (referring to two days, e.g. 28th and 30th), 28–30 VI 99 (for more than two consecutive days, e.g. 28th to 30th).

Footnotes should not be used.

Formatting of articles

Title. Must be concise but as informative as possible. Numbering of parts (I, II, III, etc.) should be avoided and will be subject to the Editor’s consent.Name of author or authorsAbstract in English, no longer than 12 typewritten lines (840 spaces), covering the contents of the article (introduction, material, methods, results and discussion). Speculation and literature citation should be avoided. The abstract should begin with the title in italics.Key words in English (no more than six) should express the precise contents of the manuscript in order of relevance. Resumen in Spanish, translation of the Abstract.Summaries of articles by non–Spanish speaking authors will be translated by the journal on request. Palabras clave in Spanish.Address of the author or authors.

(Title, Name, Abstract, Key words, Resumen, Palabras clave and Address should constitute the first page.)

Introduction. Should include the historical background of the subject as well as the aims of the paper.

VI

Material and methods. This section should provide relevant information on the species studied, materials, methods for collecting and analysing data, and the study area.Results. Report only previously unpublished results from the present study.Discussion. The results and their comparison with related studies should be discussed. Suggestions for future research may be given at the end of this section.Acknowledgements (optional).References. All manuscripts must include a bibliography of the publications cited in the text.

References should be presented as in the following examples (Harvard method):* Journal articles:Conroy, M. J. & Noon, B. R., 1996. Mapping of spe

cies richness for conservation of biological diversity: conceptual and methodological issues. Ecological Applications, 6: 763–773.

* Books or other non–periodical publications:Seber, G. A. F., 1982. The estimation of animal abun-

dance. C. Griffin & Company, ondon.* Contributions or chapters of books:Macdonald, D. W. & Johnson, D. P., 2001. Dispersal

in theory and practice: consequences for conservation biology. In: Dispersal: 358–372 (T. J. Clober, E. Danchin, A. A. Dhondt & J. D. Nichols, Eds.). Oxford University Press, Oxford.

* Ph. D. Thesis:Merilä, J., 1996. Genetic and quantitative trait variation

in natural bird populations. Ph. D. Thesis, Uppsala University.

* Works in press should only be cited if they have been accepted for publication:Ripoll, M. (in press). The relevance of population

studies to conservation biology: a review. Animal Biodiversity and Conservation.

References must be set out in alphabetical and chrono

logical order for each author, adding the letters a, b, c,... to papers of the same year. Bibliographic citations in the text must appear in the usual way: "...according to Wemmer (1998)...", "...has been defined by Robinson & Redford (1991)...", "...the prospections that have been carried out (Begon et al., 1999)..." Tables. Must be numbered in Arabic numerals with reference in the text. arge tables should be narrow (across the page) and long (down the page) rather than wide and short, so that they can be fitted into the column width of the journal.Figures. All illustrations (graphs, drawings, photographs) should be termed as figures, and numbered consecutively in Arabic numerals (1, 2, 3, etc.) with reference in the text. Glossy print photographs, if essential, may be included. The Journal will publish colour photographs but the author will be charged for the cost. Figures have a maximum size of 15.5 cm wide by 24 cm long. Figures should not be tridimensional. Any maps or drawings should include a scale. Shadings should be kept to a minimum and preferably with black, white or bold hatching. Stippling should be avoided as it may be lost in reproduction. Legends of tables and figures. egends of tables and figures should be clear, concise, and written both in English and Spanish.

Main headings (Introduction, Material and methods, Results, Discussion, Acknowledgements and References) should not be numbered. Do not use more than three levels of headings.

Manuscripts should not exceed 20 pages including figures and tables.

If the article describes new taxa, type material must be deposited in a public institution.

Authors are advised to consult recent issues of the journal and follow its conventions.

Animal Biodiversity and Conservation 38.1 (2015) VII

Welcometo the electronic version of

Animal Biodiversity and Conservation

www.abc.museucienciesjournals.cat

Animal Biodiversity and Conservation joins the worldwide Open Access Initiative

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70 Ruiz–García & Ferreras–Romero

Les cites o els abstracts dels articles d’Animal Biodiversity and Conservation es resenyen a /Las citas o los abstracts de los artículos de Animal Biodiversity and Conservation se mencionan en /Animal Biodiversity and Conservation is cited or abstracted in:

Abstracts of Entomology, Agrindex, Animal Behaviour Abstracts, Anthropos, Aquatic Sciences and Fisheries Abstracts, Behavioural Biology Abstracts, Biological Abstracts, Biological and Agricultural Abstracts, BIOSIS Previews, CiteFactor, Current Primate References, Current Contents/Agriculture, Biology & Environmental Sciences, DIALNET, DOAJ, DULCINEA, e–revist@s, Ecological Abstracts, Ecology Abstracts, Entomology Abstracts, Environmental Abstracts, Environmental Periodical Bibliography, FECYT, Genetic Abstracts, Geographical Abstracts, Índice Español de Ciencia y Tecnología, International Abstracts of Biological Sciences, International Bibliography of Periodical Literature, International Developmental Abstracts, Latindex, Marine Sciences Contents Tables, Oceanic Abstracts, RACO, Recent Ornithological Literature, Referatirnyi Zhurnal, Science Abstracts, Science Citation Index Expanded, Scientific Commons, SCImago, SCOPUS, Serials Directory, SHERPA/RoMEO, Ulrich’s International Periodical Directory, Zoological Records.

121–127Saavedra, S., Maraver, A., Anadón, J. D. & Tella, J. L.A survey of recent introduction events, spread and mitigation efforts of mynas (Acridotheres sp.) in Spain and Portugal

129–138Lucas, J. M., Prieto, P. & Galián, J.Red squirrels from south–east Iberia: low genetic diversity at the southernmost spe-cies distribution limit

Necrològica / Necrológica / Obituary

139–150 Bellés, X.Joaquím Mateu (1921–2015), tota una vida dedicada a l’estudi dels insectes

º

Índex / Índice / ContentsAnimal Biodiversity and Conservation 38.1 (2015)ISSN 1578–665 XeISSN 2014–928 X

Amb el suport de / Con el apoyo de / With the support of:

Consorci format per / Consorcio formado por / Consortium formed by:

1–10Ejigu, D., Bekele, A., Powell, L. & Lernould, J.–M.Habitat preference of the endangered Ethio-pian walia ibex (Capra walie) in the Simien Mountains National Park, Ethiopia

11–21 Rocha, G. & Quillfeldt, P.Effect of supplementary food on age ratios of European turtle doves (Streptopelia turtur L.)

23–30Prieto, M., Mederos, J. & Comas, J.A new species of Laemostenus Bonelli, 1810 (Coleoptera, Carabidae) from Els Ports Natural Park (Catalonia, northeastern Ibe-rian peninsula)

31–35García–Feria, L. M., Ureña–Aranda, C. A. & Espinosa de los Monteros, A.Minimally invasive blood sampling method for genetic studies on Gopherus tortoises

37–48Matos, M., Alves, M., Ramos Pereira, M. J., Torres, I., Marques, S. & Fonseca, C.Clear as daylight: analysis of diurnal raptor pellets for small mammal studies

49–58Vieira, B. P., Fonseca, C. & Rocha, R. G.Critical steps to ensure the successful reintroduction of the Eurasian red squirrel

59–69Ruiz–García, A. & Ferreras–Romero, M.El estado ecológico de las pequeñas cuen-cas de cabecera en las serranías béticas húmedas (parque natural Los Alcornocales, sur de España) según la Directiva Marco del Agua: ¿su aplicación garantiza la con-servación?

71–76Sánchez–Guillén R. A. & Cordero–Rivera, A.Confirmation of the presence of Ischnura senegalensis (Rambur, 1842) on the Canary Islands

77–86Jugovic, J., Praprotnik, E., Buzan, E. V. & Lužnik, M.Estimating population size of the cave shrimp Troglocaris anophthalmus (Crus-tacea, Decapoda, Caridea) using mark–re-lease–recapture data

87–100Fresneda, J., Bourdeau, C. & Faille, A.Una nueva especie troglobiomorfa de Trechus Clairville, 1806 y evidencias de coloniza-ciones múltiples del medio subterráneo de los montes cantábricos (Coleoptera, Carabidae, Trechinae)

101–119De Danieli, C. & Sarasa, M.Population estimates, density–dependence and the risk of disease outbreaks in the Alpine ibex Capra ibex