Patterns of genetic diversity within and between Myotis d. daubentonii and M. d. nathalinae derived...

INTRODUCTION

In Europe, the southern Mediterraneanarea has the greatest diversity among plants,insects, small mammals and other groups(Mitchell-Jones et al., 1999). This diversityincludes many endemic species (Myers etal., 2000) and there is also substantial in-traspecific genetic variation within manyspecies (Hewitt, 1999, 2000). The Mediter-ranean peninsulas (Iberia, Italy and Bal-kans) may have acted as refugia wheremany species have survived the glacia-tions in small favourable areas (Taberlet andCheddadi, 2002). After climate warming,

some of these surviving species expandedtheir distribution whereas others remaintrapped in the southern parts of Europe. Asa consequence of prolonged isolation, theseisolated populations should have divergedfrom each other, especially if there was lim-ited gene flow among them (Petit et al.,2003). In this type of evolutionary history a high level of cryptic diversity is expected.Even within glacial refugia, complex pat-terns of ‘refugia within refugia’ are knownto have occurred, leading to even greater di-versity that may frequently only be detectedthrough extensive sampling (Gomez andLunt, 2007).

Acta Chiropterologica, 9(2): 379–389, 2007PL ISSN 1508-1109 © Museum and Institute of Zoology PAS

Patterns of genetic diversity within and between Myotis d. daubentoniiand M. d. nathalinae derived from cytochrome b mtDNA sequence data

BRUNO F. SIMsES1, 2, 4, HUGO REBELO1, 3, RICARDO J. LOPES1, PAULO C. ALVES1, 2, and D. JAMES HARRIS1

1CIBIO, Centro de Investigaçno em Biodiversidade e Recursos Genéticos, Rua Padre Armando Quintas-Crasto, 4485-601 Vairno, Portugal

2Faculdade de CiLncias da Universidade do Porto, Praça Gomes Teixeira, 4099-002 Porto, Portugal3School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, United Kingdom

4Corresponding author: E-mail: [email protected]

We analysed the phylogenetic relationships between M. d. daubentonii and M. d. nathalinae based on 1,010 bpof the cytochrome b mtDNA gene. The inference based on molecular phylogenetics methods shows that thesetwo morphotypes correspond to two mitochondrial groups within the Iberian Peninsula. Our results also supportthe model of ‘refugia within refugia’, where M. d. daubentonii has spread north and M. d. nathalinae has becamean Iberian endemism. The haplotype network indicates haplotype sharing between Monfurado and S. Mamede(Portugal) and Bavaria (Germany), demonstrating current or recent dispersal and gene flow between thesepopulations. Myotis d. nathalinae displays a substructure showing that populations under the same climate type are more related. As a distinct Iberian endemism, the conservation status of M. d. nathalinae should bereviewed.

Key words: phylogeography, phylogeny, M. d. daubentonii, M. d. nathalinae, Iberian Peninsula, cytochrome b

The bats are a group in which the degreeof specific diversity is still uncertain, sincecryptic species are common (e.g., IbáZez etal., 2006). Prior to the emergence of DNAsequencing as a taxonomic aid, 37 morpho-logical defined verpertilionid bat specieswere accepted for the Western Palaearcticrealm. Now a total of 51 species can be dis-tinguished according to mitochondrial DNAsequences (Mayer et al., 2007). One of themost common bats of Europe, Pipistrelluspygmaeus, was first discovered by its dis-tinct echolocation call frequency (Jones andParijs, 1993) and then its specific status wasconfirmed by molecular markers (Barrat etal., 1997). Myotis alcathoe was also discov-ered by molecular methods (Helversen etal., 2001), and now, it appears that the ge-nus Plecotus is represented by at least sixdifferent species in Europe: P. auritus,P. austriacus, P. kolombatovici, P. macro-bullaris (Kiefer et al., 2002; Spitzenbergeret al., 2003), P. sardus (Mucedda et al.,2002) and P. begognae (IbaZez et al., 2006).Molecular methods have also been used todistinguish the cryptic species M. ikonni-kovi and M. brandtii gracilis (Kawai et al.,2006).

Another species in which cryptic diver-sity is a distinct possibility is Daubenton’sbat, M. daubentonii. One species of theDaubenton’s complex, M. petax, was re-cently revalidated using DNA sequence data(Matveev et al., 2005). Another form with-in this complex, M. nathalinae, was first de-scribed in Spain by Tupinier (1977). Thisproposed species was described mainly bythe absence of a protoconus in the last upperlarge premolar (P4). Further, according tothis author, M. nathalinae could be distin-guished from M. daubentonii by its slight-ly smaller dimensions, by the darker coatand by the shape of its penis. However, the systematic status of the form nathalinae(Tupinier, 1977) is still unclear, since themorphological diagnosis fails in some of the

southern lineages (Bogdanowicz, 1990).For this reason, the species has not beenuniversally recognized and consequentlytreated as subspecies of M. daubentonii oreven as a synonym to the nominate species(Bogdanowicz and Wójcik, 1986; Bogdano-wicz, 1990; Simmons, 2005) The mtDNAanalysis of the Ruedi and Mayer study(2001) in the genus Myotis shows that na-thalinae is genetically closely related withdaubentonii. This conclusion was alsoachieved by Mayer et al. (2001) based onmtDNA. However, these studies were basedon very reduced number of specimens,namely from the Iberian Peninsula.

The aim of this study was to examine genetic variation within populations of M. d. daubentonii and M. d. nathalinaefrom known glacial refugia, the IberianPeninsula, using cytochrome b sequencedata. Additional samples from northernEurope and Japan were included to assesshow the species spread from this refugium.The data should also be useful in determin-ing the distinction between M. daubentonii and M. d. nathalinae, and thus make a con-tribution towards a better understanding ofEuropean bat diversity.

MATERIAL AND METHODS

Sampling

This study included 24 specimens of M. d. dau-bentonii and M. d. nathalinae, collected under licenseduring the summers of 2005/2006. To achieve thecaptures, an initial prospecting of suitable habitatswas made with an ultra-sound detector and, after-wards, mist-nets were mounted above the surface ofsmall rivers and extended to bankside vegetation(Warren et al., 2000). In addition, potential andknown roosts were visited and the specimens werecaught by hand whenever found (Fig. 1). A 3 mm diameter wing-membrane biopsy punch was taken fortissue sampling from the plagiopatagium of each in-dividual following Worthington-Wilmer and Barratt(1996). Samples were stored in 70% ethanol untilDNA extraction. The specimens were classified asdaubentonii or nathalinae based on external morpho-

380 B. F. Simtes, H. Rebelo, R. J. Lopes, P. C. Alves, and D. J. Harris

logical characteristics following Tupinier (1977) andPalmeirim (1990). The smaller and darker specimenswere classified as nathalinae, whereas individualswith bigger size and with grey or brownish colourwere classified as daubentonii. Sometimes specimensmorphologically intermediate between daubentoniiand nathalinae were present in the samples, as for ex-ample in PN Montesinho, and consequently it wasdifficult to separate consistently the two forms. Allthe sampled individuals were adults as determined byinspection of the epiphysis calcification. The forearmwas measured in all specimens using a calliper andthey were subsequently released.

Furthermore, one additional samples from a Na-tural History Museum and 26 sequences were obtain-ed from collaborators. Eight sequences of M. dauben-tonii from previous studies were downloaded fromGenBank, as were three sequences of M. bechsteinii,M. sicarus and M. latirostris, that were designated asoutgroups (Appendix).

DNA Extraction and Sequencing

Genomic DNA was extracted from half wingpunches, following a high-salt procedure (Miller etal., 1988). The final extraction product was dilutedinto 50 µl of water and stored at -20°C. Fragments ofthe cytochrome b gene were obtained by two overlap-ping PCR products with the following primer pairs:L14724 — MVZ16 (Kocher et al., 1989, Smith andPatton, 1993) and L15162 — H15915 (Irwin et al.,1991). The PCR cocktail (25 µl final reaction vol-umes) consists of 2.5 µl of DNA extract, 0.2 µM eachprimer, 2.5 mM MgCl2, 1 mM dNTP, and 1 unit ofTaq polymerase with appropriate buffer (Ecotaq) andwater. Thermal profiles of amplifications started withthree minutes of denaturation at 94°C, followed by 39 cycles at 94°C (45 s), 45–50°C (45 s) and 72°C(1.5 min.), with a final extension at 72°C (5 min.). AllPCR products were visualised in agarose gels (2%)with ethidium bromide and sequenced using an auto-mated DNA sequencer (Applied Biosystems Hitachi3130 xl).

Alignment and Data Analysis

The two overlapping PCR fragments were assem-bled manually, edited and aligned with the softwareBioEdit (Hall, 1999). For all samples (except theUK), 1010 bp were sequenced for this study (position19 – 1,029 of human cytochrome b). The UK sampleswere approximately 200bp shorter. Alignment wasfacile as no indels were found. A total of 61 speci-mens were analysed. Maximum likelihood (ML),

maximum parsimony (MP), and Bayesian analysiswere performed. To select the model of nucleotidesubstitution that better fits our data, the AIC test wascarried out using Modeltest 3.7 (Posada and Crandall,1998). Sequences were imported into PAUP*4.0b10(Swofford, 2002) to determine the phylogenetic rela-tionships among the taxa. MP trees were generat-ed with unweighted parameters using heuristicsearches started with random stepwise addition treesand 100 replicates. For ML and MP, stability of nodeswas estimated using the bootstrap technique(Felsentein, 1985) with 1,000 pseudoreplicates. TheBayesian analysis was performed using MrBayes(Ronquist and Huelsenbeck, 2003) using default heating values run for 2 x 106 generations. Afterreaching stationarity, trees were saved every 100 gen-erations and a 50% majority rule consensus tree was constructed to determine posterior probabilitiesof nodes.

To further assess variation within species, haplo-type networks from M. d. daubentonii and M. d. na-thalinae were also produced, using the statistical par-simony algorithm with the program TCS (Clement etal., 2000). These two groups were too divergent to beunambiguously connected, so two separate networkswere produced. The daubenton’s network was basedin 1,010 bp and the sequences from the UK were ex-cluded due to the difference in length. The networkfrom M. d. nathalinae was based on only 780 bp, sothat shorter sequences for this species from GenBankcould also be incorporated.

To investigate for significant differences in theforearm length between the two subspecies we usedthe Mann-Whitney test (Zar, 1996). All tests were cal-culated using the statistics software R 2.4 (R Devel-opment Core Team, 2006).

RESULTS

The forearm length of Iberian samplesshowed significant differences between thetwo forms. Over all, M. d. daubentonii indi-viduals were bigger than M. d. nathalinae(W = 112, P < 0.001 — Fig. 1).

For the phylogenetic analysis, we used36 sequences from cytochrome b (1,010bp). Of the 1,010 nucleotide positions, 303were variable and 196 were parsimony in-formative. All variable positions involvedsingle base substitutions. The sequences ex-hibited a low proportion of guanine residues(A: 0.30; C: 0.26; G: 0.13; T: 0.31).

Genetic distinction between M. d. daubentonii and M. d. nathalinae 381


FIG. 1. Maximum parsimony (MP) estimates of relationships between M. d. daubentonii and M. d. nathalinaebased on the cytochrome b sequence data. Other equally parsimonious trees differed only in short internalbranches. M. sicarus, M. latirostris and M. bechsteinii are used as outgroups. Haplotypes are coded by samplinglocation and by specimen number (see Appendix). Bootstraps values for MP, ML and Bayesian posterior probabilities (PP) are shown in percentage (ML/MP/PP). An asterisk (*) identifies nodes with less than 50%

support with a particular method. Also the forearm length is shown to each sample

All phylogenetic methods produced sim-ilar estimates of phylogenetic relationships(Fig. 1). Clade II consists of an Iberian lin-eage corresponding to specimens identifiedin the field as M. d. nathalinae, with dif-ferent sub-clades: one that can be found in thermo-mediterranean climates including

the specimens of Vale do Guadiana, DouroInternacional, that group with the south-ern Iberian population and one divergenthaplotype of north Iberia and another atlan-tic subclade, with specimens of Monte-sinho and Peneda-GerLs. Group I (Fig. 1) corresponding to samples morphologically

identified as M. d. daubentonii includesspecimens found in Atlantic climates or inhumid climates with Atlantic vegetation in Portugal (Montesinho, S. Mamede andMonfurado, respectably) and similar haplo-types from specimens from Bavaria (Ger-many) and Sweden. In these group one sub-clades includes specimens of Monfuradoand Montesinho. Although well supportedas a clade using MP, its monophyly is notsupported by ML or Bayesian analyses, butno alternative relationships are supported,rather the subclades arise from an unresolvedpolytomy. Two different haplotypes wererecovered in the UK. Of the 26 British spec-imens, 16 belong to the haplotype UK001and the other 10 belong to the haplotypeUK002 both of which integrated in group I.The two major divisions (I and II) are sepa-rated by 2.5–3% of sequence divergence.

The parsimony network of haplotypes(Fig. 2) revealed a star-like topology of theDaubenton’s samples/sequences. The hap-lotype in the centre of the star was the mostabundant and can be found on Monfurado,S. Mamede and Bavaria (Germany). Whilehaplotypes observed on the lateral brancheswere usually specific to one place. Thenathalinae network showed that these hap-lotypes tend to be more similar to thosesampled from the same or nearby regionsthan to those from more geographically dis-tant populations. However, close affinitieswere also found between haplotypes sam-pled in distant areas such Vale do Guadianaand Douro Internacional.

DISCUSSION

In this phylogenetic analysis, daubento-nii and nathalinae correspond to distinctgroups within the Iberian Peninsula (Fig. 1).Cytochrome b divergence among sister taxain bats varies between 3.3–14.7% (Bakerand Bradley, 2006) and but may vary fromas low as 2.3–3.8% in Myotis (Rodriguez

and Ammerman, 2004). Although the genet-ic distance between the two groups is low,several species show a shallow sequence di-vergence. Examples of this are Eptesicus se-rotinus and E. nilssonii (< 2%), the siblingspecies M. myotis and M. oxygnathus (2.5%— Ruedi and Mayer, 2001) and Pipistrelluskuhlii/P. deserti. More surprising is the se-quence divergence of 1.2% between Ny-ctalus leisleri and N. azoreum, two specieswith marked phenotypic difference (smallersize, darker pelage, higher echolocation fre-quency and day time activity) (Salgueiro etal., 2007). This lower divergences can becaused by recent speciation or introgressionof mitochondrial haplotypes. Also, diver-gent phenotypic traits are adaptive featuresthat can evolved fast in response to selec-tion while neutral mitochondrial markersare not expected to evolved like genes underselection (Polly, 2001).

Interestingly, the specimens collectedfrom a single roost in Montesinho NaturalPark, were darker then usual and the fore-arm length were very close to the specimensdiagnosed genetically as M. d. daubentonii.These individuals were confirmed geneti-cally as M. d. nathalinae and consequential-ly indicate that mixed roosts can occur.These two genetic units, M. d. daubentoniiand M. d. nathalinae have some phylogeo-graphic structure within them, further sup-porting the hypothesis of “refugia withinrefugia” within the Iberian Peninsula. Theorientation of the mountains allowed thepresence of microclimates during glacialperiods that allowed the survival and isola-tion of populations within the Iberian refugia (Goméz and Lunt, 2007). Similarpatterns are seen in a variety of Iberian ver-tebrates, such as roe deer (Royo et al.,2002), Podarcis wall lizards (Harris and Sa-Sousa, 2001) and the newt Lissotriton bo-scai (Martínez-Solano et al., 2006). With the end of the last Ice Age some of these M. d. daubentonii populations might have



FIG. 2. A) Haplotype networks (statistical parsimony) for the two genetic units M. d. daubentonii and M. d. nathalinae, based on 1,010 bp and 738 bp of the cytochrome b, respectively, produced by TCS. All theindividuals analysed are identified by their code (see Appendix). B) Location of the sampling places with

patterns used for geographical discrimination in the haplotype networks


spread northwards and colonized at least thewestern parts of Europe. On the other hand,some populations might have been repeat-edly trapped in southern regions during theseveral glacial events. This complex repeat-ed pattern might have lead to the M. d. na-thalinae type as an Iberian endemism. TheJapanese and Russian specimens reported asM. daubentonii on Genbank are not relatedwith European lineages with a sequence di-vergence that varies between 11.1–13% asalready show by Kawai et al. (2003). Fromtheir location these specimens may belongto the revalidated M. petax (Matveev et al.,2005).

The statistical test used in the forearmlength shows the presence of two groupsand demonstrate that sometimes it’s possi-ble to recognize the two subspecies in thefield. These data differ from previous stud-ies where no significant differences werefound between the subspecies. Neverthe-less, in previous studies (Palmeirim, 1990)it was found that M. daubentonii present inPortugal are unusually large and the diver-gence of values can be consequence of this.Moreover, our field observation reveals thatsometimes intermediate morphotypes canbe found at least for the morphologicalcharacters analysed here.

The network of the M. d. daubentoniilineage, which indicates haplotype sharingbetween Monfurado, S. Mamede NaturalPark and Bavaria (Germany), does not showany apparent large-scale phylogeographypattern (Fig. 2). This lack of an evident ge-ographic structure can be explained by cur-rent or historical extensive dispersal andgene flow. This hypothesis is supported byecological studies, since M. daubentonii hasbeen reported to migrate distances of up to260 km under natural conditions (Urbañ-czyk, 1991). On the other hand, M. d. natha-linae displays a substructure (Fig. 2), wherehaplotypes situated under the same climatetype (Atlantic versus Mediterranean) are

more closely related to each other (DouroInternacional/Vale do Guadiana; PenLda-GerLs/North of Spain). The closely inter-spersed haplotypes from the Vale do Gua-diana Natural Park and Douro InternacionalNatural Park, points to recurrent gene flowbetween these areas, which are under thesame climate term. However, the presenceof homoplasy makes more detailed infer-ences of evolutionary history hard to make(Avise, 2000). Since homoplasy in the net-work probably arose from the high rate ofmutation of the mitochondrial DNA coupledwith a long population history, resolutionmight be improved by examining longerDNA sequences, particularly of polymor-phic sites in less variable portions of the mi-tochondrial genome. Further geographicsampling is also needed to full determinethe ranges of the lineages within the IberianPeninsula.

The aim of this paper was to attempt tounderstand the phylogenetic relationshipbetween these two groups. Our data suggestthat the two forms founded in the field cor-respond to distinct genetic lineages. How-ever some intermediate specimens can befound and a genetically identification isneeded. The use of mtDNA has proved to bea powerful tool in phylogeographic studies,and especially mitochondrial protein-cod-ing genes, like cytochrome b, that evolvequickly are powerful markers for inferringevolution history in lower categorical levelssuch genera and species (Wan et al., 2004).This feature of mtDNA in phylogeny is suit-able for resolving taxonomic uncertainties.However, analyses based only in mtDNAsometimes provide an incomplete represen-tation of the population histories (Ballardand Whitlock, 2004) because it’s matrilinealmode of inheritance and consequently mayfail to validate species.

Thus, although our results appear to in-dicate that M. d. daubentonii and M. d. na-thalinae represent distinct evolutionary


units, more studies including nuclear mark-ers are needed to fully address this issue.Also, the presence of a sympatric popu-lation in Montesinho Natural Park offers a good opportunity to study sympatric spe-ciation and gene flow between morphologi-cal forms. By this reason, deeper studies inthis mixed roost, using nuclear genes and/ormicrosatellites are needed to confirm repro-ductive isolation or hybridization betweenthe two forms. Clearly an integrative ap-proach with phenotypic and genetic data is necessary to detect all species. Only withexternal morphological characters is a fastidentification in the field possible which is essential for ecological, behavioural orconservation programs (Mayer et al., 2007).However the present and future mol-ecular studies are fundamental to supportconservation strategies to protect cryptic batfauna.

ACKNOWLEDGEMENTS

We especially thank to Lars Lundqvist, curator ofthe Museum of Zoology of Lund University (Swe-den), Joël Clary, Jean Louis Martelli and Yves Tu-pinier of the Natural History Museum of Lyon(France) for providing samples. We want to thankProf. Jorge Palmeirim, Prof. Gareth Jones and SarahHarris for samples and sequences. We thank our col-leagues of CIBIO, Albano Beja-Pereira, Pedro Tar-roso, Joana Silva, Tiago Antno, Catarina Rato, MiguelFonseca and Cláudia Almendra for valuable help andsuggestions on the manuscript. HR, RJL and DJH aresupported by grants from Portuguese Science andTechnology Foundation (FCT) (SFRH/BD/17755/2004, SFRH/BPD/14953/2004 and SFRH/BPD/26738/2006 respectively).

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Received 14 March 2007, accepted 27 July 2007

APPENDIX

Specimens of Myotis used in this study and their respective locations

Species/ Specimen Locality GenBank ReferenceMorphotype NumberM. daubentonii Mt001 PN Montesinho, Portugal EU153103 This paperM. daubentonii Mt002 PN Montesinho, Portugal EU153104 This paperM. daubentonii Mt003 PN Montesinho, Portugal EU153107 This paperM. daubentonii Mf001 Monfurado, Portugal EU153105 This paperM. daubentonii Mf002 Monfurado, Portugal EU153106 This paperM. daubentonii SM001 PN Serra S. Mamede, Portugal EU153124 This paperM. daubentonii SM002 PN Serra S. Mamede, Portugal EU153125 This paperM. daubentonii UK001 United Kingdom – S. Harris et al. (unpubl. data)M. daubentonii UK002 United Kingdom – S. Harris et al. (unpubl. data)M. daubentonii Sw001 Blentarp, Sweden EU153102 This paper

(NHMUL – L970/3496)M. daubentonii Md853 Northern Iberia DQ120896 IbaZez et al. (2006)M. daubentonii Md854 Southern Iberia DQ120897 IbaZez et al. (2006)M. daubentonii Md855 Southern Iberia DQ120898 IbaZez et al. (2006)M. daubentonii Rs001 Russia AY665137 K. Tsytsulina et al. (unpubl.

data)M. daubentonii Rs002 Novosibirsk, Russia AB106589 Kawai et al. (2003)M. daubentonii Jp001 Hokkaido, Japan AB106590 Kawai et al. (2003)M. daubentonii Gm001 Bavaria, Germany AF376847 Ruedi and Mayer (2001)M. d. nathalinae Sp001 Ciudad Real, Spain AF376862 Ruedi and Mayer (2001)M. d. nathalinae VG001 PN Vale do Guadiana, Portugal EU153112 This paperM. d. nathalinae VG002 PN Vale do Guadiana, Portugal EU153113 This paperM. d. nathalinae VG003 PN Vale do Guadiana, Portugal EU153114 This paper


APPENDIX. Continued

Species/ Specimen Locality GenBank ReferenceMorphotype NumberM. d. nathalinae VG004 PN Vale do Guadiana, Portugal EU153115 This paperM. d. nathalinae VG005 PN Vale do Guadiana, Portugal EU153116 This paperM. d. nathalinae VG006 PN Vale do Guadiana, Portugal EU153117 This paperM. d. nathalinae VG007 PN Vale do Guadiana, Portugal EU153118 This paperM. d. nathalinae VG008 PN Vale do Guadiana, Portugal EU153119 This paperM. d. nathalinae DI001 PN Douro Internacional, Portugal EU153120 This paperM. d. nathalinae DI002 PN Douro Internacional, Portugal EU153121 This paperM. d. nathalinae DI003 PN Douro Internacional, Portugal EU153122 This paperM. d. nathalinae DI004 PN Douro Internacional, Portugal EU153123 This paperM. d. nathalinae Mt004 PN Montesinho, Portugal EU153108 This paperM. d. nathalinae Mt005 PN Montesinho, Portugal EU153109 This paperM. d. nathalinae Mt006 PN Montesinho, Portugal EU153110 This paperM. d. nathalinae PG001 PN Peneda-Geres, Portugal EU153111 This paperM. bechsteinii Mb001 Jura, Switzerland AF376843 Ruedi and Mayer (2001)M. sicarus Ms001 Annapurna, Nepal AJ841959 Stadelmann et al. (2004)M. latirostris Ml001 Moi-Li Co, Taiwan AM262329 Stadelmann et al. (2007)

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