The Iberian contribution to cryptic diversity in European bats

Carlos Ibáñez, Juan L. García-Mudarra, Manuel Ruedi, Benoît Stadelmann, Javier Juste
2006 Acta Chiropterologica  
We investigate the contribution of the Iberian bat fauna to the cryptic diversity in Europe using mitochondrial (cytb and ND1) and nuclear (RAG2) DNA sequences. For each of the 28 bat species known for Iberia, samples covering a wide geographic range within Spain were compared to samples taken in Europe. In this general screening, almost 20 % of the Iberian species showed important mitochondrial discontinuities (K2P distance values > 5%) either within the Iberian or between Iberian and other
more » ... opean samples. Within Eptesicus serotinus and Myotis nattereri, levels of genetic divergence between lineages exceeded 16 %, indicating that these taxa represent a complex of several biological species. Other welldifferentiated lineages (K2P distances between 5-10 %) appeared within Hypsugo savii, Pipistrellus kuhlii and Plecotus auritus, suggesting the existence of further cryptic diversity. Most unsuspected lineages seem restricted to Iberia, although two have crossed the Pyrenees to reach, at least, Switzerland. After extraction of total DNA, samples were amplified with the primers Molcit-F (5'-AAT GACATGAAAAATCACCGTTGT-3') and MVZ-16 (Smith and Patton, 1993) or with ER-65 and ER-66 (Mayer and Helversen, 2001) designed to amplify fragments of the cytb and ND1, respectively. The PCR cocktail (20µl final reaction volume) included 2µl of DNA extract, 1µl of each primer (10µM), 0.8µl of MgCl 2 (50mM), 0.16µl of dNTP (25mM), 0.5 units of taq-polymerase. Thermocycling consisted in a 4 min initial denaturation at 94ºC 6 followed by 35 cycles of 60s at 94ºC, 30s at 45-50ºC (for the cytb), and 90s at 72ºC and a final extension of 10 min at 72ºC. The annealing temperature for the ND1 fragment was 60ºC. To amplify a fragment of the RAG2 gene, we used the primers RAG2-F1 and RAG2- R2 (Baker et al., 2000), and RAG2-R1 and RAG2-F1int (Baker et al., 2000) as internal primers. We optimized the PCR cocktails with following alterations: 0,5 µl of each primers (10µM), 1µl of MgCl 2 (50mM), and an initial denaturation of 2 min. All PCR products were sequenced in both directions using an ABI 3100 automated sequencer (PE Biosystems, Warrington, UK). Sequence and phylogenetic analyses DNA fragments were aligned and edited using Sequencher 4.1 (Gene Code Crop.). For the initial screening and for each species, Kimura 2-parameter model (K2P) was used to obtain pairwise distances among cytb sequences. We selected this model to obtain the same distance measure as previous studies on bat species (e.g. Kawai et al., 2003). Due to the inevitable heterogeneity of the cytb fragments used in the initial screening, a possible effect of fragments' length on the distance value was inspected with a Pearson's correlation coefficient. Species displaying major genetic discontinuities (i.e. distances larger than 5%, Bradley and Baker, 2001) were further investigated in more details with more individuals and markers (Appendix B). In this case, for each marker (cytb, ND1, RAG2) the best fitting substitution model was selected using hierarchical likelihood ratio tests implemented in Modeltest (Posada and Crandall, 1998). Phylogenetic reconstructions were derived from pairwise distances (NJ algorithm, Saitou and Nei, 1987) and under maximum likelihood (ML) criterion (heuristic search) using PAUP* 4.0b10 (Swofford, 2000) . For these analyses, an appropriate outgroup species was chosen according to Mayer and Helversen (2001) in order to polarize trees (Appendix B). Robustness of topologies was estimated with 5,000 bootstrap replicates (Felsenstein, 1985) for NJ and after 300,000 puzzling steps for ML reconstructions. Levels of genetic differentiation within and between groups were also calculated according to a K2P model using MEGA v. 2.1 (Kumar et al., 2001). Because of only a few mutations are present in the RAG2 sequences, relationships among haplotypes of this gene were also represented by unrooted median-joining networks (Bandelt et al., 1999) . 7 This approach combines the topology of a minimum spanning tree with a parsimony-based search of the absent nodes (median vectors) or haplotypes (Posada and Crandall, 2001). The network was obtained with the software NETWORK 4.1.1.2 (Röhl, 2005) using default parameters. RESULTS Overall genetic screening For the initial analysis, 146 aligned sequences of the mtDNA cytb gene (varying in length from 558 to 803 bp) were obtained for 28 species of bats (Table 1 , Appendix A). There was no relation across species between the length of the fragment analyzed and the maximum K2P pairwise distance found for each species (r = 0.096, P = 0.96). Maximum K2P pairwise distances were smaller than 3% in all but five species, being even less than 1% for most intra-specific comparisons (Fig. 1, Table 1 ). For the following five species, Myotis nattereri, Pipistrellus kuhlii, Hypsugo savii, Eptesicus serotinus and Plecotus auritus, comparisons reached over 5 % K2P distance values. This unusual level of intra-specific divergence is indicative of major genetic discontinuities. Genetic discontinuities The addition of many more individuals sequenced from various locations in these five species (Appendix B) confirmed the co-occurrence of major mtDNA lineages within the Iberian Peninsula (Fig. 2) , regardless of which mitochondrial marker is considered. There was always total congruence between the phylogenetic reconstructions based on NJ and ML approaches (only NJ trees are shown) and with similar bootstrap support (see Table 2 for details of the analyses). The reconstructions based on the RAG2 showed a variable level of congruence with the mitochondrial-based hypotheses, but support the existence of the most differentiated (>10% K2P distance) mtDNA lineages (Figs. 2 and 3). Within-group populations of long-eared bats, genus Plecotus (Mammalia: Chiroptera). Folia Zoologica, 53: 1-47. Benda, P., and K. A. Tsytsulina. 2000. Taxonomic revisión of Myotis mystacinus group (Mammalia: Chiroptera) in the western Palaearctic. Acta Societas Zoologicae Bohemicae, 64: 331-398.
doi:10.3161/1733-5329(2006)8[277:tictcd]2.0.co;2 fatcat:gbv23n62j5gyfmq5g7hagrc66e