Eur J Wildl Res (2011) 57:313–321 DOI 10.1007/s10344-010-0430-9 ORIGINAL PAPER Hares in Corsica: high prevalence of Lepus corsicanus and hybridization with introduced L. europaeus and L. granatensis Christian Pietri & Paulo Célio Alves & José Melo-Ferreira Received: 26 May 2010 /Revised: 31 August 2010 /Accepted: 1 September 2010 /Published online: 23 September 2010 # Springer-Verlag 2010 Abstract The Italian hare, Lepus corsicanus, was first showed an inconsistent molecular assignment between described in Corsica more than 100 years ago, but the markers suggesting a hybrid origin: L. corsicanus×L. knowledge on the status of the species in this island europaeus, L. corsicanus×L. granatensis, and L. euro- remains scarce. Moreover, frequent introductions of paeus×L. granatensis. The first two cases of hybridization thousands of individuals from other hare species, namely had never been described in nature, even in studies focusing Lepus europaeus and Lepus granatensis, into Corsica are on hares from Italy where L. corsicanus and L. europaeus known to have occurred and an updated assessment of the are often sympatric. These results stress the real risk of prevalence of L. corsicanus in Corsica is therefore of corrosion of the native gene pool of L. corsicanus via utmost importance. Here, to estimate the relative prevalence hybridization with introduced species. We highlight the of the hare species present in Corsica, we conducted a need of urgently rethinking the management plan of hare molecular analysis on 67 samples collected by hunters populations in Corsica. between 2002 and 2007 in 36 Corsican communes. Sequencing of portions of the nuclear gene transferrin and Keywords Conservation . Corsica . Italian hare . of the control region of the mitochondrial DNA allowed Hybridization . Lepus . Introduced species classifying most of the collected samples as belonging to L. corsicanus (70.1%). Of the sampled Corsican communes, 86.1% contained this species, while only in 11.1%, L. Introduction europaeus was present. Three of the analyzed specimens The taxonomic status of the Italian hare, Lepus corsicanus, Communicated by C. Gortázar has historically been a source of major controversy. This species was first described in 1898 in Corsica, a French C. Pietri Fédération Départementale des Chasseurs de Haute-Corse island close to the Italian coast, by De Winton who (FDCHC), suggested that the species could also be found in Central Résidence Nouvelle-Corniche, St Joseph, and Southern Italy. Later, it was instead considered to be 20600 Bastia, France asubspeciesofLepus europaeus, the European brown P. C. Alves : J. Melo-Ferreira (*) hare (Miller 1912; Ellerman and Morrison-Scott 1951; CIBIO, Centro de Investigação em Biodiversidade de Recursos Toschi 1965). However, recent studies on morphometrics Genéticos, Universidade do Porto, (Palacios 1996) and genetics (Pierpaoli et al. 1999) Campus Agrário de Vairão, identified the Italian hare as a distinct entity, re-establishing 4495-661 Vairão, Portugal e-mail: [email protected] its specific status despite extensive similarity to the Iberian broom hare, Lepus castroviejoi (Palacios 1996; Alves et al. P. C. Alves 2008a). Departamento de Biologia da Faculdade de Ciências, Lepus is thought to have been originally brought into Universidade do Porto, R. Campo Alegre s/n, Corsica from mainland Italy for hunting purposes before 4169-007 Porto, Portugal the fifteenth century (Vigne 1999), but the current status of 314 Eur J Wildl Res (2011) 57:313–321 the genus in the island is largely unknown (Dubray et al. European Brown Hare Syndrome virus (EBHS) or road kill. 1984; Pietri 2007). In the beginning of the twenty-first These samples were preserved and stored using the same century, Angelici and Luiselli (2001) assessed the distribu- procedure. Specimens recently introduced in Corsica are tion of L. corsicanus in peninsular Italy and Sicily, tagged, and we did not include these marked individuals in concluding that the species was still present in these our sample, therefore restricting our analyses to resident regions but stressing that, in Italy, the densities of the specimens occurring in the Island. species have been strongly declining over the years. No data were however collected from Corsica in this work. Some studies have later identified L. corsicanus as the sole Table 1 Origin of the samples used in this study (codes are those in Fig. 1) species present in Corsica, but were essentially focused in limited areas of the island (Scalera and Angelici 2003; Riga Commune Code Sample size Origin et al. 2003; Suchentrunk et al. 2006). Pierpaoli and Randi (2005) have also identified this species in specimens Haute-Corse (2B) collected in Corsica between 1999 and 2001, but no details Aghione A 1 Hunting on the location or the number of specimens studied were Albertacce B 2 Hunting provided. Although these studies suggested that L. corsi- Aleria C 3 Hunting canus was the sole hare species present in Corsica, it is Antisanti D 1 Hunting known that hunting associations introduced thousands of Bigorno E 1 Hunting hares from different species in the region up to this day: L. Castello di Rostino F 1 Road kill europaeus all over the island, and the Iberian hare, Lepus Castifao G 1 EBHS virus granatensis, in its southern part (Corse-du-Sud). While in Chiatra H 1 Hunting northern Corsica, restocking with L. europaeus stopped in Corte I 1 Hunting 1995, these procedures continue in the southern region of Erbajolo J 1 Hunting the island. Also, the release of several hundreds of L. Gavignano K 2 EBHS virus granatensis individuals of Spanish origin in Corse-du-Sud Gavignano K 1 Hunting was done over the course of many years, from 1984–2000 Ghisonaccia L 3 Hunting (Pietri 2007). Although these practices may be a threat to Giuncaggio M 1 Hunting local populations of L. corsicanus, no assessment of the Lento N 1 Hunting prevalence of each of the hare species occurring in Corsica Linguizzetta O 2 Hunting has been done so far. Linguizzetta O 1 EBHS virus In this work, we intend to assess which hare species are Lozzi P 1 Hunting present in Corsica and which of these prevail in the island, Lumio Q 4 Hunting given the long history of multi-species restocking. We use a Moltifao R 1 Hunting genetic approach to identify the species present in hunting Morosaglia S 1 Hunting bags across Corsica. Our results indicate that L. corsicanus Murato T 2 Hunting may predominate in the island, but that L. europaeus and Olmeta di Tuda U 1 Hunting perhaps L. granatensis are also present. We furthermore Omessa V 4 Hunting show strong evidences that these species are hybridizing in Pietralba W 1 Hunting the region and suggest the urgent development of effective Pieve X 1 Hunting management plans that allow preserving the gene pool of L. Pioggiola Y 1 Hunting corsicanus. Santo Pietro di Tenda Z 1 Hunting Sermano Aa 1 Hunting Tallone Ab 1 EBHS virus Methods Corse-du-Sud (2A) Calcatoggio 2 1 Hunting Between 2002 and 2007, hare hunters in Corsica were Cargese A 1 Hunting contacted by hunting federations of Haute-Corse and Casaglione B 2 Hunting Corse-du-Sud. For each hare harvested, the hunters kept a Figari C 15 Hunting piece of muscle along with information about its capture, Sarrola Carcopino D 1 Hunting such as geographic area (commune) and date (Table 1). Valle-di-Mezzana E 1 Hunting After collection, tissues were placed in 90% ethanol for Vico F 1 Hunting storing. In addition, some tissue samples were collected Zicavo G 1 Hunting from hare carcasses for which the cause of death was the Eur J Wildl Res (2011) 57:313–321 315 Total genomic DNA was extracted from the collected used as controls for identifying specific clades (see Table 2). tissues using standard high-salt methods similar to those For the TF, the allelic phase was determined using the described by Sambrook et al. (1989). Amplification of program PHASE 2.1.1 (Stephens et al. 2001; Stephens and fragments of the nuclear gene transferrin (TF) and of the Scheet 2005). Three replicate runs were performed, mitochondrial DNA control region (CR) was performed consisting of a burn-in of 100 iterations followed by using the polymerase chain reaction (PCR) conditions and 1,000 main iterations, each consisting of ten steps through primers as described by Alves et al. (2003) and Melo- the Markov chain. Given the low level of genetic diversity Ferreira et al. (2007), respectively. For each specimen, found in this marker, the relationships among the detected sequences were obtained following the ABI PRISM BigDye alleles were reconstructed using a median-joining network Terminator Cycle Sequencing 3.1 (Applied Biosystems) (Bandelt et al. 1999) as implemented in the computer standard protocol using the forward and/or the reverse PCR program Network v4.51 (www.fluxus-technology.com). primers. Again, sequences with known species provenance available Sequences were visually inspected and then aligned in GenBank were included (see Table 2). Basic genetic using CLUSTAL W (Thompson et al. 1994). For the CR, diversity indices were estimated using Arlequin 3.11 assignment of haplotypes to the correspondent specific (Excoffier et al. 2005). lineage was done by determining a neighbor-joining In order to calculate the 95% confidence intervals (CI) phylogeny using Mega v4.0 (Tamura et al. 2007) and the for the frequencies and proportions of each species found in Tamura–Nei model of sequence evolution, which is our sample, we assumed binomial and Poisson distributions appropriate for describing the pattern of evolution of for proportional data on hares and communes as suggested mtDNA control region sequences (Tamura and Nei 1993). by Scherrer (1984). The 95% CI estimated with the “binom. Sequences previously published in GenBank (Pierpaoli et exact” and “pois.exact” functions of the “epitools” library al.