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Genetic analyses reveal further cryptic lineages within the Myotis nattereri species complex
Mammalian Biology 77 (2012) 224–228
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Mammalian Biology journal homepage: www.elsevier.de/mambio
Short Communication
Genetic analyses reveal further cryptic lineages within the Myotis nattereri species complex Sébastien J. Puechmaille a,b,c,∗ , Benjamin Allegrini d , Emma S.M. Boston a , Marie-Jo Dubourg-Savage c , Allowen Evin e,f , Alexandre Knochel g , Yann Le Bris h , Vincent Lecoq i , Michèle Lemaire j , Delphine Rist h , Emma C. Teeling a a
School of Biology and Environmental Sciences, University College Dublin, Dublin, Ireland Sensory Ecology group, Max Planck Institute for Ornithology, Seewiesen, Germany c Groupe Chiroptères de Midi-Pyrénées (CREN-GCMP), Toulouse, France d Naturalia, Avignon, France e Département Systématique et Evolution, Origine Structure et Evolution de la Biodiversité, Muséum National d’Histoire Naturelle, Paris, France f Department of Archaeology, University of Aberdeen, Aberdeen, Scotland, United Kingdom g Conservatoire des Sites Lorrains, Fénétrange, France h Groupe Chiroptères Corse, Corte, France i Association Myotis, France j Muséum d’Histoire naturelle de la ville de Bourges, Bourges, France b
a r t i c l e
i n f o
Article history: Received 2 July 2011 Accepted 3 November 2011 Keywords: Myotis nattereri Chiroptera Cryptic diversity Phylogeny
a b s t r a c t In recent years, new cryptic mammalian species have been discovered in Europe, many of which belong to the order Chiroptera. Within this order, some species such as Myotis nattereri contain several cryptic lineages/species, especially in the Mediterranean region. Here we present genetic, phylogenetic and morphological analysis on the Myotis nattereri species complex, focusing on France which is thought to be a contact zone for the different lineages/species. We sequenced the full Cytochrome b gene from individuals from 23 localities and investigated diagnostic morphological characteristics. Our results reveal new phylogenetic relationships within the Myotis nattereri species complex and among closely related species. We discuss morphological characters which are synapomorphic to each lineage and can be used to differentiate them. Our results also demonstrate the presence of a new lineage within the Myotis nattereri species complex. This lineage, endemic to Corsica, possibly represents a new cryptic species for which we present preliminary ecological data. We further identify the presence of three lineages/species in France and detail their distribution with potential contact zones. © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved.
In the last decade, genetic screening has been successfully carried out to assess the presence of undiscovered or unrecognized species, especially cryptic species that are morphologically difficult to tell apart. Assigning individuals to biological species is essential for understanding the ecology, behavioural strategies, distribution and conservation status of species. Morphologically, Myotis nattereri Kuhl (1817) is easily differentiated from its congenerics by having an S-shape spur and the presence of a hair fringe at the hind margin of the uropatagium (Horácek and Hanák 1984; Dietz and Von Helversen 2004). This species has traditionally been reported to range from Ireland, throughout Europe (except North Scandinavia), Morocco, Northern Algeria, Lebanon, Israel, parts of
∗ Corresponding author at: School of Biology and Environmental Sciences, University College Dublin, Dublin, Ireland. Tel.: +353 1 716 2262. E-mail address: [email protected] (S.J. Puechmaille).
Turkey and Western Russia, Crimea and Caucasus to Turkmenistan (Simmons 2005). Recent genetic studies on the Western Palaearctic region have reported the presence of four divergent lineages within ˜ et al. 2006; Mayer et al. 2007; Garcia-Mudarra M. nattereri (Ibanez et al. 2009). Nevertheless, the phylogenetic relationships between these lineages and other lineages/species previously belonging to M. nattereri (i.e. M. bombinus, M. nattereri tschuliensis) are yet to be determined, as previous studies only included a subset of these in ˜ et al. their phylogenetic reconstruction (Kawai et al. 2003; Ibanez 2006; Jones et al. 2006; Mayer et al. 2007; Garcia-Mudarra et al. 2009). In the Western Palearctic, the lineage occurring from Greece, Hungary, Switzerland and Germany retains the name M. nattereri as the species was originally described from Hanau in Germany (Kuhl 1817). A second lineage, known from the Iberian Peninsula and the Pyrénées Orientales in Southern France (Evin et al. 2009; GarciaMudarra et al. 2009), has been attributed the name M. escalerai based on a description of specimens from Foyos, near Valencia and
1616-5047/$ – see front matter © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.mambio.2011.11.004
S.J. Puechmaille et al. / Mammalian Biology 77 (2012) 224–228
Bellver, near Lérida (Spain) by Cabrera (1904, 1912, 1914). The two remaining lineages are likely different species, yet to be included in a detailed taxonomic assessment (Garcia-Mudarra et al. 2009). The first of these is known from Northern Iberia, the Pyrénées Mountains, Northern Italy and Southern Austria and is provisionally referred to as Myotis sp. A, and the second lineage is present in Morocco and Northern Algeria and is referred to as Myotis sp. B (Mayer et al. 2007; Evin et al. 2009; Garcia-Mudarra et al. 2009; Flaquer et al. 2010). Due to a limited sampling across Europe, our knowledge on the distribution of these lineages/species remains poorly known. Similarly, areas of sympatry or contact zones between these lineages/species are not known apart from Northern Iberia where M. escalerai and M. sp. A are found in sympatry. Based on our current knowledge of these lineages/species distributions (Garcia-Mudarra et al. 2009), France is expected to be a contact zone between M. nattereri, M. escalerai and M. sp. A (Fig. 1). This study aimed to (i) test whether these lineages/species have overlapping distribution in France and whether there are other new lineages present in the area, especially in the island of Corsica which has a particular fauna and flora with endemic species, and (ii) reconstruct the phylogenetic relationships between the studied species/lineages and closely related species and discuss the morphological characters synapomorphic to each lineage which can be used to differentiate them. A total of 37 samples were obtained from dead individuals as well as live individuals identified as Myotis nattereri sensu lato using currently available morphological characters (Dietz and Von Helversen 2004). Attention was paid to the attachment of the plagiopatagium which was the main criteria given by Cabrera to differentiate M. escalerai from M. nattereri (Cabrera 1904). The samples originated from 20 localities mainly in France and Corsica as well as one locality in each of the following countries, Germany, Ireland and Greece (Fig. 1). Total genomic DNA was extracted from ethanol, dry or frozen preserved material using the Qiagen Tissue kit and following the manufacturer protocol. The complete mitochondrial Cytochrome b gene (Cytb) was amplified using the forward primer mtDNAR3-F (5 -TGGCATGAAAAATCACCGTTGT-3 , Puechmaille et al. in press) and the reverse CytB-H (5 -CTTTTCTGGTTTACAAGACCAG-3 , Weyeneth et al. 2008). For some samples that did not amplify or sequence well using the reverse primer CytB-H, either BSVES268H (Stadelmann et al. 2004) or MVZ16 (Irwin et al. 1991) was used in combination with mtDNA-R3-F. The PCR amplifications were completed as in Puechmaille et al. (2010) except for the annealing time that was set to 30 s. PCR products were sent for sequencing in both directions to Macrogen Inc. (Korea). Sequences belonging to the M. nattereri species complex (M. nattereri, M. sp. A, M. escalerai, M. sp. B, M. schaubi, M. n. tschuliensis, M. bombinus, M. b. amurensis), closely related species (M. pequinius, M. cf. pequinius, M. myotis, M. blythii blythii, M. b. ancilla, M. punicus, M. chinensis) and distant species (M. velifer, M. lucifugus, M. brandtii, M. gracilis) from the New World Clade (sensu Stadelmann et al. 2007) were retrieved from GenBank and used in phylogenetic reconstructions undertaken using the Bayesian inference in BEAST v1.6.1 (Drummond and Rambaut 2007). The GTR + + I substitution model was used as determined by ModelTest version 3.7 (Posada and Crandall 1998). A strict molecular clock model was applied with a fixed mean substitution rate of 1.30 × 10−8 subs/site/year (calculated after Nabholz et al. 2008; Nabholz, pers. com.; n = 223 Chiropteran species). No outgroup was specified and the Yule process was used as a tree prior. The UPGMA method was used to construct the starting tree. The program was run for 10,000,000 generations (10% discarded as burn-in) and sampled every 500. Two replicate analyses were performed to ensure convergence and the results were then pooled. Mean pairwise distances in Cytb between species/lineages were
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calculated using the Kimura two parameters (K2P) model in MEGA4 (Tamura et al. 2007). The data set consisted in 79 sequences (37 from this study submitted to GenBank with accession numbers JF412390-JF412415; 42 retreived from GenBank) and a total length of 1140 bp, shorter sequences were considered as containing missing data. For at least one individual per clade of M. nattereri sensu lato investigated in this study, PCR and sequencing were completed using different primer sets which recovered identical sequences. Furthermore, all our sequences showed a single band on a gel after DNA amplification and none of the sequences presented stop codons in the Cytb. These data suggest that the fragments amplified in this study are of mitochondrial DNA origin as opposed to nuclear DNA (Numts) (Puechmaille et al. in press). Phylogenetic reconstructions based on mitochondrial DNA support the paraphyly of M. nattereri sensu lato as identified based on morphological characters. Two clades are well differentiated in the phylogeny, which correspond to the M. escalerai and M. nattereri clades, both supported with a posterior probability of 1. The M. escalerai clade can be further divided into a Western Paleartic (M. schaubi, M. n. tschuliensis, M. sp. B and M. escalerai) and an Eastern Paleartic clade (M. bombinus, M. pequinius) whereas the M. nattereri clade contains only Western Paleartic lineages/species (M. nattereri, M. sp. A and M. sp. C). The topology and position in the tree of the large Myotis clade (Western Paleartic species: M. myotis, M. blythii, M. punicus, Eastern Paleartic species: M. chinensis) are weakly supported and although the clade appears monophyletic and more closely related to the M. nattereri clade, more data and analysis are clearly needed to resolve these relationships. The reconstruction confirmed the existence of multiple lineages/species within M. nattereri sensu lato from the Western ˜ et al. 2006; Mayer Palearctic (Fig. 2) as previously reported (Ibanez et al. 2007; Garcia-Mudarra et al. 2009) and further identified a new lineage referred to M. sp. C (Fig. 2). This new lineage from Corsica (M. sp. C) is closely related to M. sp. A, from which it is estimated to have diverged from around 2.8 MYA (CI95%: 2.3–3.7). Intra- and interspecific K2P distances calculated for species/lineages belonging to the M. nattereri, M. escalerai and M. myotis clades are presented in Table 1. All six individuals from the Pyrénées Orientales department in Southern France grouped with the M. escalerai from Iberia. These individuals are known to roost in caves during the breeding season, which corresponds well with the ecology of M. escalerai from ˜ et al. 2006). Other underground sites monitored by Iberia (Ibanez the Association Myotis in the Conflent region (Pyrénées Orientales) are known to contain important summer maternity colonies of M. nattereri sensu lato but given their ecology, they are thought to be ˜ M. escalerai (Ibanez et al. 2006). Interestingly, individuals group in the tree according to geography with one clade for Southern Iberian individuals, one for Central and Northern Iberia and the last one for the individuals from Southern France, at the border with Spain (Fig. 2). Further samples from Southern France (n = 12) formed a monophyletic clade with samples from Northern Iberia which likely correspond to a new cryptic species yet to be described ˜ et al. 2006; Garcia-Mudarra et al. 2009). The (Myotis sp. A; Ibanez three individuals from Corsica form a well supported monophyletic group sister to M. sp. A. Finally, all sequences from Central and eastern France (n = 11), Ireland (n = 1), Germany (n = 2) and Greece (n = 1) formed a well supported monophyletic clade which we refer to as M. nattereri. The type locality of M. nattereri is Hanau (Germany) (Kuhl 1817), which is just 40 km south of the locality of samples Mnat 23–24 (Fig. 2). We therefore assume that this clade represents M. nattereri sensu stricto. The actual range of M. escalerai and M. sp. A are overlapping in some areas of Northern Spain and in Southern France (this study, ˜ et al. 2006) (Fig. 2). In his description of M. escalerai, Cabrera Ibanez
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Fig. 1. Localisation of samples collected of Myotis nattereri sensu stricto (squares), M. sp. A (triangles), M. sp. C (crosses) and M. escalerai (circles). Asterisks represent the type localities for Myotis escalerai Cabrera (1904), near Foyos (Spain) and Myotis nattereri Kuhl (1817), near Hanau (Germany). The distribution of Myotis nattereri sensu stricto (grey dots), M. sp. A (vertical lines), M. sp. C (black dots), M. escalerai (horizontal lines) and M. sp. B (grid) were drawn according to data presented in Garcia-Mudarra et al. (2009) and this study. By default, regions where no individuals have been genetically investigated are attributed to M. nattereri (grey dots).
(1904; plate V) showed that the plagiopatagium was inserted at the mid-metatarsus (according to terminology described in Findley 1972) a character that allowed the discrimination with M. nattereri. Ten years later, the same author did not mention M. escalerai as a valid species and only mentioned M. nattereri but reported that the insertion of the plagiopatagium varied in this species, sometimes at the mid-metatarsus and sometimes at the base of the toe (Cabrera 1914). In the individuals genetically confirmed as M. escalerai from Southern France (n = 6, this study), the plagiopatagium inserted at the mid-metatarsus whereas in the individuals genetically
confirmed as M. sp. A (n = 12, this study), it inserted at the base of the toe (Fig. 3). Interestingly, in the sister taxa to M. escalerai, M. sp. B from Morocco and Algeria, the plagiopatagium also inserted at the midmetatarsus (B. Allegrini, unpublished data), character also found in M. pequinius (Thomas 1908). Our observations in the field show that in individuals genetically confirmed as M. nattereri sensu stricto, M. sp. A and M. sp. C, the plagiopatagium inserted at the base of the toe. The specimens observed by Cabrera in 1914 were probably a mix of M. escalerai and M. sp. A. In the subspecies M. nattereri
Table 1 Taxa pairwise sequence divergence (Kimura 2-parameter) estimates across Cytochrome b (1140 bp.) sequence pairs between and within taxa. The number of base substitutions per site was estimated by averaging over all sequence pairs between taxa (average: lower triangular matrix; sd: upper triangular matrix) and within each taxa (diagonal). Positions containing missing data were eliminated in pairwise sequence comparisons only.
M. nattereri M. sp. A M. sp. C M. myotis M. blythii M. punicus M. chinensis M. escalerai M. sp. B M. tschuliensis M. schaubi M. cf. pequinius M. pequinius M. bombinus
M. nat
M. sp. A
M. sp. C
M. myo
M. bly
M. pun
M. chi
M. esc
M. sp. B
0.7 10.0 9.2 13.4 13.0 14.7 13.4 15.2 17.8 16.5 18.2 14.2 14.2 14.8
1.1 0.8 7.5 13.9 14.0 14.6 16.4 17.2 17.4 18.9 17.7 15.1 15.2 15.2
1.1 0.9 0.1 13.7 14.4 14.1 15.1 15.6 16.5 16.3 16.5 13.4 14.0 15.0
1.1 1.2 1.1 0.7 5.4 11.4 12.9 16.1 16.8 16.3 16.5 13.5 14.1 15.2
1.1 1.2 1.1 0.7 1.7 10.9 13.9 15.5 16.2 14.3 15.0 12.4 13.5 14.9
1.4 1.3 1.3 1.2 1.1 1.6 13.3 13.7 15.9 16.1 13.9 13.9 13.0 15.2
1.1 1.3 1.3 1.1 1.1 1.2 1.1 16.2 16.4 15.1 15.6 12.7 13.3 13.7
1.2 1.4 1.4 1.4 1.4 1.3 1.4 0.6 9.1 13.9 10.0 13.9 13.8 14.4
1.6 1.6 1.5 1.7 1.6 1.7 1.6 1.0 1.0 14.0 13.6 14.3 14.3 14.1
M. tsc 1.8 1.8 1.8 1.5 1.4 2.1 1.6 1.7 2.2 N/A 19.2 14.4 15.1 15.4
M. sch 1.7 1.6 1.6 1.7 1.6 1.5 1.5 1.2 1.6 12.1 1.8 13.6 13.7 14.7
M. cf. peq 1.3 1.4 1.2 1.2 1.1 1.4 1.2 1.3 1.6 1.7 1.5 N/A 6.7 8.8
M. peq 1.3 1.3 1.3 1.2 1.1 1.2 1.1 1.2 1.5 1.6 1.4 0.9 N/A 9.3
M. bom 1.2 1.3 1.3 1.3 1.2 1.3 1.2 1.3 1.5 1.5 1.6 1.0 0.9 4.5
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Fig. 2. Bayesian consensus tree (GTR + + I substitution model) showing the phylogenetic relationship between individuals of different taxa based on 1140 bp of Cytochrome b. Posterior probability support is shown above each branch. For clarity reasons, posterior probability supports are not shown within clades represented by only one taxon. Tip labels are composed of species names followed in brackets by GenBank accession number (for published sequences) or sequence reference (this study) and country of origin (when available). The insertion of the plagiopatagium is shown for specific clades (see text and Fig. 3 for details).
hoveli described from Israel (Harrison 1964) the plagiopatagium also inserted at the base of the toe (P. Bates, pers. com.), suggesting it is more closely related to M. nattereri, M. sp. A or M. sp. C than M. escalerai or M. sp. B. Further investigations are necessary however to confirm that the insertion of the plagiopatagium is a criterion to discriminate species between the M. escalerai clade and the M. nattereri clade (Fig. 2). So far, there are no known morphological or acoustic characters to differentiate M. sp. A and M. nattereri sensu stricto and only genetic analyses can tell them apart.
Fig. 3. Drawing of the insertion of the plagiopatagium (A) at the mid-metatarsus for M. escalerai (individual 110910.1) and (B) directly at the base of the toe for M. sp. A (individual 160910.1).
It is also unknown whether their ranges do overlap despite our investigation. Sampling individuals from more geographic locations in France will enable us to answer this question. As M. nattereri sensu lato is widespread in France and has a ‘continuous’ distribution range (Arthur and Lemaire 2009), we expect a range overlap between M. nattereri sensu stricto and M. sp. A or at least a narrow contact zone. Identifying this zone is important for conservation purposes but could also be of particular interest in investigating whether the two potential species are completely genetically isolated or if they occasionally form hybrids as it is the case between M. myotis and M. blythii (Berthier et al. 2006). The taxonomic status of individuals referred to as M. nattereri in Corsica is uncertain. The important genetic divergence (7.4% K2P divergence at Cytb; Table 1) strongly suggests they represent an undescribed species that would be endemic to the island. M. nattereri has not been reported to occur in Sardinia (Courtois et al. 1997) and is rare in Corsica with less than 20 records (mainly between 1000 and 1500 m a.s.l.) and only one known summer roost with 75 individuals occupying an underground tunnel. This colony has been observed from May until early July but without evidence for reproduction. These data suggest that the species might have a cave roosting ecology but further research is needed to confirm this. The use of nuclear markers combined with the collection of further samples is necessary before the taxonomic status of the M.
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nattereri sensu lato lineages herein mentioned can be confirmed. Similarly, more markers are needed to unambiguously resolve whether the large Myotis species (M. myotis, M. blythii, M. punicus, M. chinensis) form a monophyletic clade and where they branch in the tree in relation to the M. nattereri and M. escalerai clades. Acknowledgements This study was funded by a grant from The President of Ireland Young Researcher Award Science foundation Ireland to E.C.T, an IRCSET-Marie Curie International Mobility Fellowship in Science, Engineering and Technology to S.J.P and supported by the ‘Direction de l’Environement et du Développement Durable’ of the French Forest Office (ONF). Thanks to two anonymous reviewers for their comments on the manuscript. We would like to thank Paul Bates, Laurent Arthur and Lisa Ferrand for their help with this project and the Bibliothèque mondiale des chauves-souris (Genève) and Jean-Marc Pons for providing us with references. Thanks to Mathieu Faure, Yannis Kazoglou, Elena Papadatou, Xavier Grémillet and other volunteers as well as the Society for the Protection of Prespa (SPP) for assisting us during samples’ collection. Data collected in the Pyrénées-Orientales has been possible through the collaboration between the Conseil Général 66 (Operator of the Natura 2000 “Sites à Chiroptères des PyrénéesOrientales”), the Association Myotis and the Groupe Chiroptères Languedoc-Roussillon. Thanks to the Groupe Chiroptères Corse, the “direction régionale de l’environnement Corse” and the “office de l’environnement de la Corse” for enabling the work in Corsica. Samples collection by the authors was done under permits from the National Parks and Wildlife Service (Licence No. 74C/2008), Ireland; the Greek Ministry of Agricultural Development and Food (Permit No. 104694/2439) and the French Direction Régionale de l’Environnement, de l’Aménagement et du Logement (Arrêté n◦ 2009-11). Thanks to Manuel Ruedi, Javier Juste and Irene Salinici for their comments on this manuscript. References Arthur, L., Lemaire, M., 2009. Les Chauves-souris de France, Belgique, Luxembourg et Suisse, Mèze: Biotope. Muséum national d’Histoire naturelle, Paris. Berthier, P., Excoffier, L., Ruedi, M., 2006. Recurrent replacement of mtDNA and cryptic hybridization between two sibling bat species Myotis myotis and Myotis blythii. Proc. R. Soc. Lond. B 273, 3101–3109. ˜ Mem. Soc. Cabrera, A., 1904. Ensayo monográfico sobre los quirópteros de Espana. ˜ Hist. Nat. 2, 249–292. Espan. Cabrera, A., 1912. El concepto de tipo en zoología y los tipos de Mamíferos del Museo de ciencias naturales. Trab. del Mus. de C. Nat. 3, 3–32. Cabrera, A., 1914. Fauna Ibérica: Mamíferos. Museo Nacional de Ciencias Naturales, Madrid. Courtois, J.-Y., Mucedda, M., Salotti, M., Casale, A., 1997. Deux îles, deux peuplements: comparaison des populations de chiroptères troglophiles de Corse et de Sardaigne. Arvicola 9, 15–18.
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Mammalian Biology journal homepage: www.elsevier.de/mambio
Short Communication
Genetic analyses reveal further cryptic lineages within the Myotis nattereri species complex Sébastien J. Puechmaille a,b,c,∗ , Benjamin Allegrini d , Emma S.M. Boston a , Marie-Jo Dubourg-Savage c , Allowen Evin e,f , Alexandre Knochel g , Yann Le Bris h , Vincent Lecoq i , Michèle Lemaire j , Delphine Rist h , Emma C. Teeling a a
School of Biology and Environmental Sciences, University College Dublin, Dublin, Ireland Sensory Ecology group, Max Planck Institute for Ornithology, Seewiesen, Germany c Groupe Chiroptères de Midi-Pyrénées (CREN-GCMP), Toulouse, France d Naturalia, Avignon, France e Département Systématique et Evolution, Origine Structure et Evolution de la Biodiversité, Muséum National d’Histoire Naturelle, Paris, France f Department of Archaeology, University of Aberdeen, Aberdeen, Scotland, United Kingdom g Conservatoire des Sites Lorrains, Fénétrange, France h Groupe Chiroptères Corse, Corte, France i Association Myotis, France j Muséum d’Histoire naturelle de la ville de Bourges, Bourges, France b
a r t i c l e
i n f o
Article history: Received 2 July 2011 Accepted 3 November 2011 Keywords: Myotis nattereri Chiroptera Cryptic diversity Phylogeny
a b s t r a c t In recent years, new cryptic mammalian species have been discovered in Europe, many of which belong to the order Chiroptera. Within this order, some species such as Myotis nattereri contain several cryptic lineages/species, especially in the Mediterranean region. Here we present genetic, phylogenetic and morphological analysis on the Myotis nattereri species complex, focusing on France which is thought to be a contact zone for the different lineages/species. We sequenced the full Cytochrome b gene from individuals from 23 localities and investigated diagnostic morphological characteristics. Our results reveal new phylogenetic relationships within the Myotis nattereri species complex and among closely related species. We discuss morphological characters which are synapomorphic to each lineage and can be used to differentiate them. Our results also demonstrate the presence of a new lineage within the Myotis nattereri species complex. This lineage, endemic to Corsica, possibly represents a new cryptic species for which we present preliminary ecological data. We further identify the presence of three lineages/species in France and detail their distribution with potential contact zones. © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved.
In the last decade, genetic screening has been successfully carried out to assess the presence of undiscovered or unrecognized species, especially cryptic species that are morphologically difficult to tell apart. Assigning individuals to biological species is essential for understanding the ecology, behavioural strategies, distribution and conservation status of species. Morphologically, Myotis nattereri Kuhl (1817) is easily differentiated from its congenerics by having an S-shape spur and the presence of a hair fringe at the hind margin of the uropatagium (Horácek and Hanák 1984; Dietz and Von Helversen 2004). This species has traditionally been reported to range from Ireland, throughout Europe (except North Scandinavia), Morocco, Northern Algeria, Lebanon, Israel, parts of
∗ Corresponding author at: School of Biology and Environmental Sciences, University College Dublin, Dublin, Ireland. Tel.: +353 1 716 2262. E-mail address: [email protected] (S.J. Puechmaille).
Turkey and Western Russia, Crimea and Caucasus to Turkmenistan (Simmons 2005). Recent genetic studies on the Western Palaearctic region have reported the presence of four divergent lineages within ˜ et al. 2006; Mayer et al. 2007; Garcia-Mudarra M. nattereri (Ibanez et al. 2009). Nevertheless, the phylogenetic relationships between these lineages and other lineages/species previously belonging to M. nattereri (i.e. M. bombinus, M. nattereri tschuliensis) are yet to be determined, as previous studies only included a subset of these in ˜ et al. their phylogenetic reconstruction (Kawai et al. 2003; Ibanez 2006; Jones et al. 2006; Mayer et al. 2007; Garcia-Mudarra et al. 2009). In the Western Palearctic, the lineage occurring from Greece, Hungary, Switzerland and Germany retains the name M. nattereri as the species was originally described from Hanau in Germany (Kuhl 1817). A second lineage, known from the Iberian Peninsula and the Pyrénées Orientales in Southern France (Evin et al. 2009; GarciaMudarra et al. 2009), has been attributed the name M. escalerai based on a description of specimens from Foyos, near Valencia and
1616-5047/$ – see front matter © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.mambio.2011.11.004
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Bellver, near Lérida (Spain) by Cabrera (1904, 1912, 1914). The two remaining lineages are likely different species, yet to be included in a detailed taxonomic assessment (Garcia-Mudarra et al. 2009). The first of these is known from Northern Iberia, the Pyrénées Mountains, Northern Italy and Southern Austria and is provisionally referred to as Myotis sp. A, and the second lineage is present in Morocco and Northern Algeria and is referred to as Myotis sp. B (Mayer et al. 2007; Evin et al. 2009; Garcia-Mudarra et al. 2009; Flaquer et al. 2010). Due to a limited sampling across Europe, our knowledge on the distribution of these lineages/species remains poorly known. Similarly, areas of sympatry or contact zones between these lineages/species are not known apart from Northern Iberia where M. escalerai and M. sp. A are found in sympatry. Based on our current knowledge of these lineages/species distributions (Garcia-Mudarra et al. 2009), France is expected to be a contact zone between M. nattereri, M. escalerai and M. sp. A (Fig. 1). This study aimed to (i) test whether these lineages/species have overlapping distribution in France and whether there are other new lineages present in the area, especially in the island of Corsica which has a particular fauna and flora with endemic species, and (ii) reconstruct the phylogenetic relationships between the studied species/lineages and closely related species and discuss the morphological characters synapomorphic to each lineage which can be used to differentiate them. A total of 37 samples were obtained from dead individuals as well as live individuals identified as Myotis nattereri sensu lato using currently available morphological characters (Dietz and Von Helversen 2004). Attention was paid to the attachment of the plagiopatagium which was the main criteria given by Cabrera to differentiate M. escalerai from M. nattereri (Cabrera 1904). The samples originated from 20 localities mainly in France and Corsica as well as one locality in each of the following countries, Germany, Ireland and Greece (Fig. 1). Total genomic DNA was extracted from ethanol, dry or frozen preserved material using the Qiagen Tissue kit and following the manufacturer protocol. The complete mitochondrial Cytochrome b gene (Cytb) was amplified using the forward primer mtDNAR3-F (5 -TGGCATGAAAAATCACCGTTGT-3 , Puechmaille et al. in press) and the reverse CytB-H (5 -CTTTTCTGGTTTACAAGACCAG-3 , Weyeneth et al. 2008). For some samples that did not amplify or sequence well using the reverse primer CytB-H, either BSVES268H (Stadelmann et al. 2004) or MVZ16 (Irwin et al. 1991) was used in combination with mtDNA-R3-F. The PCR amplifications were completed as in Puechmaille et al. (2010) except for the annealing time that was set to 30 s. PCR products were sent for sequencing in both directions to Macrogen Inc. (Korea). Sequences belonging to the M. nattereri species complex (M. nattereri, M. sp. A, M. escalerai, M. sp. B, M. schaubi, M. n. tschuliensis, M. bombinus, M. b. amurensis), closely related species (M. pequinius, M. cf. pequinius, M. myotis, M. blythii blythii, M. b. ancilla, M. punicus, M. chinensis) and distant species (M. velifer, M. lucifugus, M. brandtii, M. gracilis) from the New World Clade (sensu Stadelmann et al. 2007) were retrieved from GenBank and used in phylogenetic reconstructions undertaken using the Bayesian inference in BEAST v1.6.1 (Drummond and Rambaut 2007). The GTR + + I substitution model was used as determined by ModelTest version 3.7 (Posada and Crandall 1998). A strict molecular clock model was applied with a fixed mean substitution rate of 1.30 × 10−8 subs/site/year (calculated after Nabholz et al. 2008; Nabholz, pers. com.; n = 223 Chiropteran species). No outgroup was specified and the Yule process was used as a tree prior. The UPGMA method was used to construct the starting tree. The program was run for 10,000,000 generations (10% discarded as burn-in) and sampled every 500. Two replicate analyses were performed to ensure convergence and the results were then pooled. Mean pairwise distances in Cytb between species/lineages were
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calculated using the Kimura two parameters (K2P) model in MEGA4 (Tamura et al. 2007). The data set consisted in 79 sequences (37 from this study submitted to GenBank with accession numbers JF412390-JF412415; 42 retreived from GenBank) and a total length of 1140 bp, shorter sequences were considered as containing missing data. For at least one individual per clade of M. nattereri sensu lato investigated in this study, PCR and sequencing were completed using different primer sets which recovered identical sequences. Furthermore, all our sequences showed a single band on a gel after DNA amplification and none of the sequences presented stop codons in the Cytb. These data suggest that the fragments amplified in this study are of mitochondrial DNA origin as opposed to nuclear DNA (Numts) (Puechmaille et al. in press). Phylogenetic reconstructions based on mitochondrial DNA support the paraphyly of M. nattereri sensu lato as identified based on morphological characters. Two clades are well differentiated in the phylogeny, which correspond to the M. escalerai and M. nattereri clades, both supported with a posterior probability of 1. The M. escalerai clade can be further divided into a Western Paleartic (M. schaubi, M. n. tschuliensis, M. sp. B and M. escalerai) and an Eastern Paleartic clade (M. bombinus, M. pequinius) whereas the M. nattereri clade contains only Western Paleartic lineages/species (M. nattereri, M. sp. A and M. sp. C). The topology and position in the tree of the large Myotis clade (Western Paleartic species: M. myotis, M. blythii, M. punicus, Eastern Paleartic species: M. chinensis) are weakly supported and although the clade appears monophyletic and more closely related to the M. nattereri clade, more data and analysis are clearly needed to resolve these relationships. The reconstruction confirmed the existence of multiple lineages/species within M. nattereri sensu lato from the Western ˜ et al. 2006; Mayer Palearctic (Fig. 2) as previously reported (Ibanez et al. 2007; Garcia-Mudarra et al. 2009) and further identified a new lineage referred to M. sp. C (Fig. 2). This new lineage from Corsica (M. sp. C) is closely related to M. sp. A, from which it is estimated to have diverged from around 2.8 MYA (CI95%: 2.3–3.7). Intra- and interspecific K2P distances calculated for species/lineages belonging to the M. nattereri, M. escalerai and M. myotis clades are presented in Table 1. All six individuals from the Pyrénées Orientales department in Southern France grouped with the M. escalerai from Iberia. These individuals are known to roost in caves during the breeding season, which corresponds well with the ecology of M. escalerai from ˜ et al. 2006). Other underground sites monitored by Iberia (Ibanez the Association Myotis in the Conflent region (Pyrénées Orientales) are known to contain important summer maternity colonies of M. nattereri sensu lato but given their ecology, they are thought to be ˜ M. escalerai (Ibanez et al. 2006). Interestingly, individuals group in the tree according to geography with one clade for Southern Iberian individuals, one for Central and Northern Iberia and the last one for the individuals from Southern France, at the border with Spain (Fig. 2). Further samples from Southern France (n = 12) formed a monophyletic clade with samples from Northern Iberia which likely correspond to a new cryptic species yet to be described ˜ et al. 2006; Garcia-Mudarra et al. 2009). The (Myotis sp. A; Ibanez three individuals from Corsica form a well supported monophyletic group sister to M. sp. A. Finally, all sequences from Central and eastern France (n = 11), Ireland (n = 1), Germany (n = 2) and Greece (n = 1) formed a well supported monophyletic clade which we refer to as M. nattereri. The type locality of M. nattereri is Hanau (Germany) (Kuhl 1817), which is just 40 km south of the locality of samples Mnat 23–24 (Fig. 2). We therefore assume that this clade represents M. nattereri sensu stricto. The actual range of M. escalerai and M. sp. A are overlapping in some areas of Northern Spain and in Southern France (this study, ˜ et al. 2006) (Fig. 2). In his description of M. escalerai, Cabrera Ibanez
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Fig. 1. Localisation of samples collected of Myotis nattereri sensu stricto (squares), M. sp. A (triangles), M. sp. C (crosses) and M. escalerai (circles). Asterisks represent the type localities for Myotis escalerai Cabrera (1904), near Foyos (Spain) and Myotis nattereri Kuhl (1817), near Hanau (Germany). The distribution of Myotis nattereri sensu stricto (grey dots), M. sp. A (vertical lines), M. sp. C (black dots), M. escalerai (horizontal lines) and M. sp. B (grid) were drawn according to data presented in Garcia-Mudarra et al. (2009) and this study. By default, regions where no individuals have been genetically investigated are attributed to M. nattereri (grey dots).
(1904; plate V) showed that the plagiopatagium was inserted at the mid-metatarsus (according to terminology described in Findley 1972) a character that allowed the discrimination with M. nattereri. Ten years later, the same author did not mention M. escalerai as a valid species and only mentioned M. nattereri but reported that the insertion of the plagiopatagium varied in this species, sometimes at the mid-metatarsus and sometimes at the base of the toe (Cabrera 1914). In the individuals genetically confirmed as M. escalerai from Southern France (n = 6, this study), the plagiopatagium inserted at the mid-metatarsus whereas in the individuals genetically
confirmed as M. sp. A (n = 12, this study), it inserted at the base of the toe (Fig. 3). Interestingly, in the sister taxa to M. escalerai, M. sp. B from Morocco and Algeria, the plagiopatagium also inserted at the midmetatarsus (B. Allegrini, unpublished data), character also found in M. pequinius (Thomas 1908). Our observations in the field show that in individuals genetically confirmed as M. nattereri sensu stricto, M. sp. A and M. sp. C, the plagiopatagium inserted at the base of the toe. The specimens observed by Cabrera in 1914 were probably a mix of M. escalerai and M. sp. A. In the subspecies M. nattereri
Table 1 Taxa pairwise sequence divergence (Kimura 2-parameter) estimates across Cytochrome b (1140 bp.) sequence pairs between and within taxa. The number of base substitutions per site was estimated by averaging over all sequence pairs between taxa (average: lower triangular matrix; sd: upper triangular matrix) and within each taxa (diagonal). Positions containing missing data were eliminated in pairwise sequence comparisons only.
M. nattereri M. sp. A M. sp. C M. myotis M. blythii M. punicus M. chinensis M. escalerai M. sp. B M. tschuliensis M. schaubi M. cf. pequinius M. pequinius M. bombinus
M. nat
M. sp. A
M. sp. C
M. myo
M. bly
M. pun
M. chi
M. esc
M. sp. B
0.7 10.0 9.2 13.4 13.0 14.7 13.4 15.2 17.8 16.5 18.2 14.2 14.2 14.8
1.1 0.8 7.5 13.9 14.0 14.6 16.4 17.2 17.4 18.9 17.7 15.1 15.2 15.2
1.1 0.9 0.1 13.7 14.4 14.1 15.1 15.6 16.5 16.3 16.5 13.4 14.0 15.0
1.1 1.2 1.1 0.7 5.4 11.4 12.9 16.1 16.8 16.3 16.5 13.5 14.1 15.2
1.1 1.2 1.1 0.7 1.7 10.9 13.9 15.5 16.2 14.3 15.0 12.4 13.5 14.9
1.4 1.3 1.3 1.2 1.1 1.6 13.3 13.7 15.9 16.1 13.9 13.9 13.0 15.2
1.1 1.3 1.3 1.1 1.1 1.2 1.1 16.2 16.4 15.1 15.6 12.7 13.3 13.7
1.2 1.4 1.4 1.4 1.4 1.3 1.4 0.6 9.1 13.9 10.0 13.9 13.8 14.4
1.6 1.6 1.5 1.7 1.6 1.7 1.6 1.0 1.0 14.0 13.6 14.3 14.3 14.1
M. tsc 1.8 1.8 1.8 1.5 1.4 2.1 1.6 1.7 2.2 N/A 19.2 14.4 15.1 15.4
M. sch 1.7 1.6 1.6 1.7 1.6 1.5 1.5 1.2 1.6 12.1 1.8 13.6 13.7 14.7
M. cf. peq 1.3 1.4 1.2 1.2 1.1 1.4 1.2 1.3 1.6 1.7 1.5 N/A 6.7 8.8
M. peq 1.3 1.3 1.3 1.2 1.1 1.2 1.1 1.2 1.5 1.6 1.4 0.9 N/A 9.3
M. bom 1.2 1.3 1.3 1.3 1.2 1.3 1.2 1.3 1.5 1.5 1.6 1.0 0.9 4.5
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Fig. 2. Bayesian consensus tree (GTR + + I substitution model) showing the phylogenetic relationship between individuals of different taxa based on 1140 bp of Cytochrome b. Posterior probability support is shown above each branch. For clarity reasons, posterior probability supports are not shown within clades represented by only one taxon. Tip labels are composed of species names followed in brackets by GenBank accession number (for published sequences) or sequence reference (this study) and country of origin (when available). The insertion of the plagiopatagium is shown for specific clades (see text and Fig. 3 for details).
hoveli described from Israel (Harrison 1964) the plagiopatagium also inserted at the base of the toe (P. Bates, pers. com.), suggesting it is more closely related to M. nattereri, M. sp. A or M. sp. C than M. escalerai or M. sp. B. Further investigations are necessary however to confirm that the insertion of the plagiopatagium is a criterion to discriminate species between the M. escalerai clade and the M. nattereri clade (Fig. 2). So far, there are no known morphological or acoustic characters to differentiate M. sp. A and M. nattereri sensu stricto and only genetic analyses can tell them apart.
Fig. 3. Drawing of the insertion of the plagiopatagium (A) at the mid-metatarsus for M. escalerai (individual 110910.1) and (B) directly at the base of the toe for M. sp. A (individual 160910.1).
It is also unknown whether their ranges do overlap despite our investigation. Sampling individuals from more geographic locations in France will enable us to answer this question. As M. nattereri sensu lato is widespread in France and has a ‘continuous’ distribution range (Arthur and Lemaire 2009), we expect a range overlap between M. nattereri sensu stricto and M. sp. A or at least a narrow contact zone. Identifying this zone is important for conservation purposes but could also be of particular interest in investigating whether the two potential species are completely genetically isolated or if they occasionally form hybrids as it is the case between M. myotis and M. blythii (Berthier et al. 2006). The taxonomic status of individuals referred to as M. nattereri in Corsica is uncertain. The important genetic divergence (7.4% K2P divergence at Cytb; Table 1) strongly suggests they represent an undescribed species that would be endemic to the island. M. nattereri has not been reported to occur in Sardinia (Courtois et al. 1997) and is rare in Corsica with less than 20 records (mainly between 1000 and 1500 m a.s.l.) and only one known summer roost with 75 individuals occupying an underground tunnel. This colony has been observed from May until early July but without evidence for reproduction. These data suggest that the species might have a cave roosting ecology but further research is needed to confirm this. The use of nuclear markers combined with the collection of further samples is necessary before the taxonomic status of the M.
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nattereri sensu lato lineages herein mentioned can be confirmed. Similarly, more markers are needed to unambiguously resolve whether the large Myotis species (M. myotis, M. blythii, M. punicus, M. chinensis) form a monophyletic clade and where they branch in the tree in relation to the M. nattereri and M. escalerai clades. Acknowledgements This study was funded by a grant from The President of Ireland Young Researcher Award Science foundation Ireland to E.C.T, an IRCSET-Marie Curie International Mobility Fellowship in Science, Engineering and Technology to S.J.P and supported by the ‘Direction de l’Environement et du Développement Durable’ of the French Forest Office (ONF). Thanks to two anonymous reviewers for their comments on the manuscript. We would like to thank Paul Bates, Laurent Arthur and Lisa Ferrand for their help with this project and the Bibliothèque mondiale des chauves-souris (Genève) and Jean-Marc Pons for providing us with references. Thanks to Mathieu Faure, Yannis Kazoglou, Elena Papadatou, Xavier Grémillet and other volunteers as well as the Society for the Protection of Prespa (SPP) for assisting us during samples’ collection. Data collected in the Pyrénées-Orientales has been possible through the collaboration between the Conseil Général 66 (Operator of the Natura 2000 “Sites à Chiroptères des PyrénéesOrientales”), the Association Myotis and the Groupe Chiroptères Languedoc-Roussillon. Thanks to the Groupe Chiroptères Corse, the “direction régionale de l’environnement Corse” and the “office de l’environnement de la Corse” for enabling the work in Corsica. Samples collection by the authors was done under permits from the National Parks and Wildlife Service (Licence No. 74C/2008), Ireland; the Greek Ministry of Agricultural Development and Food (Permit No. 104694/2439) and the French Direction Régionale de l’Environnement, de l’Aménagement et du Logement (Arrêté n◦ 2009-11). Thanks to Manuel Ruedi, Javier Juste and Irene Salinici for their comments on this manuscript. References Arthur, L., Lemaire, M., 2009. Les Chauves-souris de France, Belgique, Luxembourg et Suisse, Mèze: Biotope. Muséum national d’Histoire naturelle, Paris. Berthier, P., Excoffier, L., Ruedi, M., 2006. Recurrent replacement of mtDNA and cryptic hybridization between two sibling bat species Myotis myotis and Myotis blythii. Proc. R. Soc. Lond. B 273, 3101–3109. ˜ Mem. Soc. Cabrera, A., 1904. Ensayo monográfico sobre los quirópteros de Espana. ˜ Hist. Nat. 2, 249–292. Espan. Cabrera, A., 1912. El concepto de tipo en zoología y los tipos de Mamíferos del Museo de ciencias naturales. Trab. del Mus. de C. Nat. 3, 3–32. Cabrera, A., 1914. Fauna Ibérica: Mamíferos. Museo Nacional de Ciencias Naturales, Madrid. Courtois, J.-Y., Mucedda, M., Salotti, M., Casale, A., 1997. Deux îles, deux peuplements: comparaison des populations de chiroptères troglophiles de Corse et de Sardaigne. Arvicola 9, 15–18.
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