On the occurrence of Anaecypris hispanica, an extremely endangered Iberian endemism, in the Guadalquivir River basin

Journal of Fish Biology (2010) 76, 1454 1465 doi:10.1111/j.1095-8649.2010.02574.x, available online at www.interscience.wiley.com On the occurrence of Anaecypris hispanica, an extremely endangered Iberian endemism, in the Guadalquivir River basin R. De Miguel*, E. Pino*, A. Ramiro*, F. Aranda*, J. P. Peña*, I. Doadrio and C. Fernández-Delgado* *Department of Zoology, Charles Darwin Building, 3rd floor, University Campus of Rabanales, University of Córdoba, 14071 Córdoba, Spain and Museo Nacional de Ciencias Naturales, CSIC, Departamento de Biodiversidad y Biología Evolutiva, José Gutiérrez Abascal 2, 28006 Madrid, Spain (Received 5 May 2008, Accepted 5 January 2010) The jarabugo Anaecypris hispanica, considered endemic to the Guadiana River basin, has been found in the Guadalquivir River. First genetic data showed a high degree of similarity to those of the Guadiana River populations. The genetic study recovered five different groups of haplotypes, the Guadalquivir River specimens belong to the largest and most widely extended group. Journal compilation 2010 The Fisheries Society of the British Isles Key words: Cyprinidae; Iberian endemism; threatened fish. INTRODUCTION Anaecypris hispanica (Steindachner) is a small cyprinid, endemic to the Iberian Peninsula, at risk of extinction. It is catalogued as endangered in the Red Data Books of Spain and Portugal (Doadrio, 2001; Cabral et al., 2005) and in the IUCN Red List (Crivelli, 2006). It is also included in Annex III of the Berne Convention and in Annexes II and IV of the European Habitat Directive (E.U., 1992). Its conservation status is so poor that the species has been considered as the most threatened primary fish of the Iberian freshwaters (Collares-Pereira & Cowx, 2001). The species was first located in 1864 in a small stream of the Guadiana River basin near Mérida (Badajoz, Spain) (Steindachner, 1866). The first published papers after the original description considered the species to be exclusive to the Guadiana River basin (Almaça, 1976, 1978; Collares-Pereira, 1979; Doadrio & Lobón-Cerviá, 1979). In fact, when the taxonomy of the species was analysed by Collares-Pereira (1983), a monospecific genus was assigned for it: Anaecypris, meaning cyprinid of the Guadiana. Nevertheless the capture of one specimen in a small stream in the basin of Author to whom correspondence should be addressed. Tel./fax: +34 957 218605; email: carlos.fdelgado@uco.es 1454 Journal compilation 2010 The Fisheries Society of the British Isles

OCCURRENCE OF A. HISPANICA IN THE GUADALQUIVIR RIVER 1455 the River Bembézar (a tributary of the Guadalquivir River) (Barrachina et al., 1989) raised hopes that the species had a larger distribution area, but the sighting was never confirmed (Collares-Pereira et al., 1999; Collares-Pereira & Cowx, 2001). Later, exploratory trips to the same area failed to find the species, leading to the conclusion that the population of this river had completely disappeared due to pollution of its waters (Doadrio, 2001). Since this date the species has been considered endemic to the Guadiana River basin (Alves et al., 2001; Salgueiro et al., 2003; Collares-Pereira & Cowx, 2004; Crivelli, 2006; Kottelat & Freyhof, 2007). During the course of the project titled Basis for the development of a conservation programme for the freshwater fish in Andalusia (Environmental Agency of the Government of Andalusia, Spain; www.uco.es/aphanius/proyectos.php), specimens of this species were caught in 11 localities along the River Bembézar, the same tributary of the Guadalquivir River where the species had been caught 21 years before, thus confirming its presence in the Guadalquivir River basin (Fig. 1). MATERIALS AND METHODS The fish populations of the River Bembézar basin (15 514 km 2 ) were sampled by electrofishing in wadeable sections of river 100 300 m in length, depending on its width (wading upstream with one or two anodes using 240 V pulsed direct current). Collection via electrofishing began at a shallow riffle, or other physical barrier at the downstream limit of the sample reach, and terminated at a similar barrier at the upstream end of the reach. All data were standardized to river segments of 500 m 2 for comparative analysis. All fishes were treated carefully, but especially those of due to its sensitivity to handling (Collares- Pereira et al., 1999), and after being measured (fork length, L F mm) they were returned to the water. Only five specimens of were first anaesthetized (using benzocaine) before being stored in appropriate conditions in the collections of the Department of Zoology (University of Córdoba, Spain). In order to confirm the identity of the specimens and to determine their basic genetic structure, several molecular analyses were carried out. DNA samples were analysed from three of the five specimens of that had been collected from the Bembézar River (Guadalquivir basin) and preserved. DNA from another 51 individuals of, caught in different tributaries of the Guadiana River basin (Table I), were also analysed for comparison. Thirty-two of the Guadiana River basin specimens came from existing GenBank records (Table I); the remaining specimens were directly examined in this study, using previously collected specimens kept in appropriate conditions in the Museo Nacional de Ciencias Naturales (Madrid, Spain). DNA was extracted from the dorsal fin, which was preserved in 70% ethanol. The out-groups selected for phylogenetic analysis were bleak Alburnus alburnus (L.) and Iberian nase Pseudochondrostoma polylepis (Steindachner). The species is considered part of the Alburninae with A. alburnus as its sister taxon (Zardoya & Doadrio, 1999). Two overlapping fragments of the mitochondrial cyt b gene [a total of 1140 base pairs (bp)] were amplified via polymerase chain reaction (PCR) from each individual DNA sample. In all species, the primers used for cyt b and protocols were those mentioned in the study of Machordom & Doadrio (2001). The hierarchical likelihood ratio test (LRT) of the programme Modeltest 3.07 (Posada & Crandall, 1998) was used to find the best model of evolution that fitted the data under the Akaike information criterion (AIC). The aligned data were analysed by neighbour-joining inference (NJ). NJ was performed using PAUP* v. 40b10 (Swofford, 2002); confidences for this analysis were estimated by bootstrapping (1000 repetitions) (Felsenstein, 1985). A median-joining network was constructed with Network 4.502. The median-joining network (Fig. 2) reveals five groups of haplotypes. Except in the case of the Guadalmez and Ardila Rivers, haplotypes from the same locality are entirely of the same haplotype group.

1456 R. DE MIGUEL ET AL. 5 50 5 40 5 30 5 20 5 10 5 0 4 50 Guadiana River basin EXTREMADURA 8 5 1 7 SPAIN Bembézar River basin Guadalquivir River basin N Limits of Andalusia Limits of Bembézar River basin Reservoirs Rivers Sampling sites: 3 Presence of Juveniles Juveniles 4 2 Juveniles 6 0 10 20 Km 37 50 38 0 38 10 37 50 38 0 38 10 38 20 Guadalquivir River Mediterranean sea Atlantic Ocean 5 50 5 40 5 30 5 20 5 10 5 0 4 50 Fig. 1. Location of the Guadalquivir and Bembézar River basins within the borders of Andalusia, Spain. Numbers indicate localities where Anaecypris hispanica has been captured (see Table I).

OCCURRENCE OF A. HISPANICA IN THE GUADALQUIVIR RIVER 1457 Table I. Samples of Anaecypris hispanica, collection sites, number of individuals sampled in the phylogenetic analysis and haplotype network and GenBank accession numbers Species Locality, source Designation in phylogenetic tree (Fig. 3) Hydrogaphic drainage GenBank accession number Number in haplotype network Pseudochondrostoma polylepis Tiétar River, Ramacastañas (Avila, Spain) P. polylepis Tajo basin GQ228429 Alburnus alburnus Strymon River, Strimoniko (Greece) A. alburnus Strymon basin AF090745 Anaecypris hispanica Estenilla River, Afl Estena, Huertas de Sauceral (Ciudad Real, Spain) Estena River, Navas de Estena (Ciudad Real, Spain) Guadalemar River, Puebla de Don Rodrigo (Ciudad Real, Spain) Guadalemar River, Garbayuela (Badajoz, Spain) (three individuals) (three individuals) (two individuals) (two individuals) (two individuals) Guadalmez River, La Bienvenida (Ciudad Real, Spain) Guadalmez River, Guadalmez (Ciudad Real, Spain) Valdeazogues River, Almadenejos (Ciudad Real, Spain) Siruela River, Tamurejo (Badajoz, Spain) Quejigares River, Fontanosas (Ciudad Real, Spain) Gévora River, Alburquerque (Badajoz, Spain) Estenilla 1 Guadiana GQ228407 El 1 Estena 1 Guadiana GQ228408 Et 1 Guadalemar 1 Guadiana GQ228409 Gr 1 Guadalemar 2 Guadiana GQ228410 Gr 2 Guadalmez 1-3 Guadiana GQ228411-13 Gz 1-3 Guadalmez 4-6 Guadiana GQ2284114-16 Gz 4-6 Valdeazogues 1-2 Guadiana GQ228419-20 Vz 1-2 Siruela 1-2 Guadiana GQ228421-22 S 1 Quejigares 1-2 Guadiana GQ2284213-24 Q 1-2 Gevora 1 Guadiana GQ228428 Gv 1

1458 R. DE MIGUEL ET AL. Table I. Continued Species Locality, source Designation in phylogenetic tree (Fig. 3) Hydrogaphic drainage GenBank accession number Number in haplotype network (three individuals) (three individuals) (five individuals) (four individuals) (four individuals) (five individuals) (two individuals) (two individuals) (five individuals) (four individuals) Gévora River (Portugal) Gevora 2-4 Guadiana X99422 AJ427883-4 Bembezar River, Villanueva (Córdoba, Spain) Gv 2-5 Bembezar 1-3 Guadalquivir GQ228425-27 B 1-3 Vascao River (Portugal) Vascao 1-5 Guadiana AJ27828-32 AJ439881 V1-4 Odeleite River (Portugal) Odeleite 1-4 Guadiana AJ27824-7 O 1-4 Foupana River (Portugal) Foupana 1-4 Guadiana AJ427820-3 F 1-4 Degebe River (Portugal) Degebe 1-5 Guadiana AJ427815-9 D 1-5 Chanza River (Portugal) Chanza 1-2 Guadiana AJ427813-4 Cz 1-2 Carreiras River (Portugal) Carreiras 1-2 Guadiana AJ427811-2 Cr 1-2 Caia River (Portugal) Caia 1-5 Guadiana AJ427806-7 C 1-5 Ardila River (Portugal) Ardila 1-4 Guadiana AJ427801-2 AJ427804-5 A1-4

OCCURRENCE OF A. HISPANICA IN THE GUADALQUIVIR RIVER 1459 A Q 1 El 1 Gz 2 E 5 steps ET 1 C 5 5 steps 6 steps Vz 2 Guadalquivir Gz 5 Gz 4 Vz 1 C 3, S 2 Gr 1 Gv 3-5 Gz 3 B 1 Cz1 B 3 B 2 Cz 2 C 1 C 2, Gz 1, Gv 1 Gv 2 S 1 Q 2 Gr 2 C 4 Gz 6 3 steps F 1-3 O 1-2 7 Steps 4 steps D 3 D 5 D 4 A 2 D 1-2, A 1 F 4 V 1 O 3 O 4 V5 A 4 V 2 V 3 C Cr 1 V 4 Cr 2 A 3 B D Fig. 2. Median-joining network constructed from Anaecypris hispanica cyt b sequences. Size of node is proportional to haplotype (see Table I) frequency and length of branches is proportional to the number of steps. A D correspond to haplotype groups in Alves et al., (2001). E is a new group of haplotypes found in this work., new haplotypes. RESULTS The groups recovered in this study are the same as those of Alves et al. (2001) plus a group of haplotypes (E) composed of individuals from Estena and Estenilla basins on the eastern border of the current distribution area of, and one individual from the Guadalmez River. The individuals from the Guadalquivir River basin were placed in the A group of haplotypes, which is the largest and most widely extended group of the Guadiana River basin (Fig. 2). For the phylogenetic analyses, the best-fit model selected under the AIC was the GTR+I. Rate matrices for cyt b are R(a) = 407 79, R(b) = 5406 27, R(c) = 91 21, R(d) = 394 6, R(e) = 3025 83 and R(f) = 1 00. Base frequencies are: A = 0 27, C = 0 28, G = 0 16 and T = 0 28. The proportion of invariable sites is I = 0 63. These parameters were used in the subsequent phylogenetic analyses. Despite the low bootstrap support values observed in the phylogenetic tree (Fig. 3), the geographical structure is the same as observed in Fig. 2. Only the group E with individuals from the eastern border of the distribution area of obtained high support, and samples from the Guadalquivir River basin are placed together with some samples from the Guadiana River basin (Fig. 3). For cyt b sequences, the genetic uncorrected p distances between individuals from Guadiana and Guadalquivir River basins are 0 0061 ± 0 0046 (0 0009 0 193); this

1460 R. DE MIGUEL ET AL. 100 A 58 68 72 63 68 55 62 52 62 D 73 86 74 B 62 E C 65 64 86 68 81 98 Ps. polylepis A. alburnus Gevora 2 Gevora 3 Gevora 4 Bembezar 1 Bembezar 2 Bembezar 3 Guadalemar 1 Siruela 1 Quejigares 2 Guadalemar 2 Guadalmez 6 Guadalmez 5 Guadalmez 3 Chanza 1 Chanza 2 Caia 1 Caia 2 Caia 3 Guadalmez 4 Valdeazogues 2 Quejigares 1 Caia 4 Caia 5 Gevora 1 Valdeazogues 1 Guadalmez 1 Siruela 2 Vascao 1 Vascao 2 Vascao 3 Vascao 4 Carreiras 1 Carreiras 2 Ardila 3 Ardila 4 Vascao 5 Odeleite 1 Odeleite 2 Odeleite 3 Odeleite 4 Foupana 1 Foupana 2 Foupana 3 Foupana 4 Degebe 1 Degebe 2 Ardila 1 Ardila 2 Degebe 3 Degebe 4 Degebe 5 Estena 1 Estenilla 1 Guadalmez 2 Guadalquivir Fig. 3. Phylogenetic tree (50% majority-rule consensus tree) obtained by neighbour-joining analysis of the mitochondrial cyt b dataset. Numbers above nodes indicate values of the bootstrap (see Table I). is less than the distances between all the samples of 0 0074 ± 0 0049 (0 0 202). The most differentiated populations genetically are those of Estena, Estenilla and the Guadalmez River in the Guadiana River basin (Fig. 3). In conclusion, specimens from the Bembézar River show low genetic distances compared with the rest of the samples of analysed. Furthermore, their differences with respect to those of the Guadiana River basin are less than those from within the Guadiana River basin. Nevertheless, more specific analyses are necessary to determine the genetic characteristics of this new population (Salgueiro et al., 2003). The analysis of its nuclear genome will be especially interesting.

OCCURRENCE OF A. HISPANICA IN THE GUADALQUIVIR RIVER 1461 DISCUSSION The genetic data presented here suggest the existence of a new evolutionary significant unit (ESU) (Moritz, 1994) not discovered until now, which contains the populations from the eastern border, the distribution area of (haplotype group E). They also highlight the need to carry out a wider study with more specimens; for instance, when the sample size was increased in the Guadalmez River, the population showed haplotypes from two different groups (A and E haplogroups, Fig. 2). Finally, it is considered necessary to use another type of marker (e.g. microsatellites) for an adequate study of the genetic conservation of the species. Some of the variables of the population point to a viable establishment of the species: (1) specimens were found at a relatively high spatial occurrence (at 25% of sites sampled) in several streams of the Bembézar River basin; although only 36 adults were caught, all the sizes within the limits established for the species were present (Collares-Pereira et al., 1999; Ribeiro et al., 2000) (Fig. 4); 3) reproduction was confirmed, as seen by the capture of juveniles and mature adults. This natural presence of raises the question of the colonization route followed by the species in this river basin. The species is well-distributed throughout the Bembézar River basin but not so in other tributaries of the Guadalquivir River basin, where >700 sites have been analysed. Some localities are remote with very difficult access, which reduces the possibility that human introduction is responsible. A credible explanation could be that a small stream located at the sources of the Bembézar River has captured, by erosion process further up river, another stream of the Guadiana River basin where the species was present (V. Cifuentes, pers. comm.). Obviously hydrological or geological evidence is required to support this interesting hypothesis. Anaecypris hispanica was found coexisting with five autochthonous species [Andalusian barbel Luciobarbus sclateri Günther, calandino Iberocypris alburnoides 9 8 7 Number of specimens 6 5 4 3 2 1 0 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 Furcal length (mm) Fig. 4. Furcal length frequency distribution of 36 specimens of Anaecypris hispanica caught in different localities of the Bembézar River basin.

1462 R. DE MIGUEL ET AL. (Steindachner), cacho Squalius pyrenaicus (Günther), colmilleja Cobitis paludica (de Buen) and pardilla Iberochondrostoma lemmingii (Steindachner)]; and one allochthonous species (common carp Cyprinus carpio L.) which appeared in only one locality and in very low numbers (<0 5%) and probably came from the reservoir located in the centre of the river basin (Fig. 1). Table II shows the eight localities for which it was possible to calculate quantitative data, the other three localities were dominated by juveniles, whose different behaviour in response to electrofishing did not give accurate results for quantitative data collections (Cowx & Lamarque, 1990). There were large variations in the percentage contribution of to the community in different localities, comparable to the extent of variation seen between populations of within the Guadiana River (Collares-Pereira et al., 1999). None of the localities was dominated by, which in all cases accounted for a small percentage in the community except in locality 1 where the species, along with L. sclateri, dominated (33 33%). Nevertheless, the density of each species in locality 1 was very low (Table II). From this maximum value in locality 1, decreases progressively to a minimum density in locality 8, where it represents only 1 64% of the fish community (Table II). Community composition varied between localities. All the autochthonous species coexisted in only one locality (locality 6; Table II), and the rest varied; two localities were dominated by I. alburnoides (locality 4 and 8) or C. paludica (locality 3 and 7) and one by L. sclateri (locality 2) or S. pyrenaicus (locality 6) (Table II). Anaecypris hispanica showed the lowest mean ± 95% CL density (1 92 ± 2 56 specimens per 500 m 2 of river) when data of different localities were pooled, showing that has a very low mean contribution (<3%) to the fish community in the Bembézar River. Only the area of the Bembézar River basin within the borders of Andalusia was sampled. Part of the northern area of the river basin falls within the region of Extremadura (Fig. 1), which has not been examined. It is probable that the species can be found in other localities of this area not yet sampled. Regardless of this, most of the Bembézar River basin is included within the boundaries of the Natural Park of Hornachuelos (CMA, 2008), which itself is part of the Dehesas of Sierra Morena, a natural area of >400 000 ha added in 2002 to the World Network of the Biosphere Reserves by UNESCO (2007). The area where the river basin is situated is dominated by Mediterranean forest in good conservation status, which should offer more security for the future of the species, at least with respect to the conservation of riparian vegetation and low pollution of the waters of the Bembézar River. Threats are expected to come from other types of degradation of the river waters. The Bembézar River is dammed creating a reservoir (Fig. 1) that provides a habitat for C. carpio, largemouth bass Micropterus salmoides (Lacépède), pumpkinseed Lepomis gibbossus (L.) and A. alburnus. In addition to hosting these invasive species, the reservoir isolates the different subpopulations living in the river basin (Fig. 1), which could be affecting their genetic fitness. This study confirms, 21 years later, the findings of Barrachina et al. (1989) in respect of the presence of this species in the Bembézar River. It is probable that previous sampling efforts and geographic coverage were not sufficient to detect a rare species such as. According to Collares-Pereira et al. (1999), the abundance and geographic range of have progressively contracted since it was first described in Portugal

OCCURRENCE OF A. HISPANICA IN THE GUADALQUIVIR RIVER 1463 Table II. Density values (specimens per 500 m 2 of river) for the different species caught in the eight localities where adults of Anaecypris hispanica were also caught, and percentage contribution of to the fish community in each of these eight sampling sites Sampling sites (Fig. 1) Luciobarbus sclateri Iberocypris alburnoides Squalius pyrenaicus Cobitis paludica Iberochondrostoma lemmingii % contribution of 1 1 11 0 37 0 74 1 11 33 33 2 11 89 0 94 1 25 8 88 3 18 89 17 31 55 08 14 16 9 44 8 22 4 6 71 12 07 0 67 3 44 5 3 90 1 95 5 47 0 39 3 33 6 7 84 8 55 25 66 7 84 0 71 1 43 2 75 7 6 67 2 22 17 23 2 78 0 56 1 90 8 14 02 12 94 5 39 0 54 1 64 Mean 8 85 7 42 25 66 19 08 4 87 1 92 2 83 95% CL 6 30 5 43 26 02 5 26 2 56

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