genetic diversity of northeastern palaearctic bats as...

INTRODUCTION

The incorporation of molecular methods intosystematic research has provided valuable insightsinto chiropteran species diversity and led to the tax-onomic reevaluation of some complex speciesgroups. While it is not surprising that molecular datahighlighted potentially new taxonomic discoveriesin species rich and understudied tropical areas (e.g.,Clare et al., 2007; Francis et al., 2010), it has alsoled to interesting taxonomic findings even in rela-tively species-depauperate temperate faunas (Mayerand von Helversen, 2001; Mayer et al., 2007). ThePalaearctic bat fauna has undergone comprehensivetaxonomic revisions in the last three decades with a dramatic boost in the number of recognizedspecies around the turn of the last century (Horáčeket al., 2000). Several bat groups have been identifiedas taxonomic ‘hot spots’, in which unexpected levels of cryptic diversity were revealed (Mayer andvon Helversen, 2001).

The combination of molecular, acoustic and refined morphological approaches has led bat researchers to revisit the systematics of several

common species with broad geographic distribution.Among them were the reevaluation of the status of two ‘phonic types’ in the Pipistrellus pipistrellus/pygmaeus species complex (Hulva et al., 2004), the morphologically based revision of geographicmorphs within Myotis mystacinus (Benda andTsytsulina, 2000), and a complete revamping of spe -cies content within Plecotus (Spitzenberger et al.,2001, 2003; Kiefer et al., 2002).

These efforts, although targeting the Palaearcticbat fauna as a whole, were heavily biased towardsspecies from Western Europe whereas EasternEurope and Asia remained underrepresented. Fewstudies using broader geographic sampling suggest-ed the existence of phylogeographic splits indicativeof past speciation events. For example, Myotis petaxfrom Siberia and China has been proposed as a geo-graphic vicariate of the European Myotis dauben-tonii (Kawai et al., 2003; Kruskop, 2004; Matveevet al., 2005). Similarly, molecular studies of Minio -pterus schreibersii (Appleton et al., 2004; Tian etal., 2004) demonstrated that it is restricted to Europeand North Africa, while Miniopterus fuliginosus re-places it further east. However, these studies used

Acta Chiropterologica, 14(1): 1–14, 2012PL ISSN 1508-1109 © Museum and Institute of Zoology PAS

doi: 10.3161/150811012X654222

Genetic diversity of northeastern Palaearctic bats as revealed by DNA barcodes

SERGEI V. KRUSKOP1, ALEX V. BORISENKO2, NATALIA V. IVANOVA2, BURTON K. LIM3, and JUDITH L. EGER3

1Zoological Museum of Moscow University, Ul Bol’shata Nikitskaya, 6, Moscow, Russia, 1250092Canadian Centre for DNA Barcoding, Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road E, Guelph,

Ontario, Canada, N1G 2W13Department of Natural History, Royal Ontario Museum, 100 Queen’s Park, Toronto, Ontario, Canada, M5S 2C6

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

Sequences of the DNA barcode region of the cytochrome oxidase subunit I gene were obtained from 38 species of northeasternPalaearctic bats to assess patterns of genetic diversity. These results confirmed earlier findings of deep phylogeographic splits in fourpairs of vicariant species (Myotis daubentonii/petax, M. nattereri/bombinus, Plecotus auritus/ognevi and Miniopterus schreibersii/fuliginosus) and suggested previously unreported splits within Eptesicus nilssoni and Myotis aurascens. DNA barcodes support alltaxa raised to species rank in the past 25 years and suggest that an additional species — Myotis sibiricus — should be separated fromMyotis brandtii. Major phylogeographic splits occur between European and Asian populations of Myotis aurascens, Rhinolophusferrumequinum and Myotis frater; smaller scale splits are observed between insular and mainland populations in the Far East (M. frater, Myotis ikonnikovi and Murina ussuriensis) and also between southeastern Europe and Ciscaucasia (Myotis daubentonii,Plecotus auritus, and Pipistrellus pipistrellus). One confirmed case of sequence sharing was observed in our dataset — Eptesicusnilssoni/serotinus. This study corroborates the utility of DNA barcodes as a taxonomic assessment tool for bats.

Key words: cytochrome oxidase, phylogeography, Vespertilionidae, alpha-taxonomy, bat fauna, Russia

different morphological and molecular charactersets, thus preventing broad comparisons across taxa.Fur thermore, the poor representation of collectionspecimens from Siberia and the Far East has hin-dered the recovery of the geographic patterns of genetic divergence among widely distributed spe -cies, such as Myot is brandtii, Vespertilio murinus,and Eptesicus nilssoni.

Our paper aims to fill these knowledge gaps andprovide baseline information on the genetic diversi-ty of northeastern Palaearctic bats using the DNAbarcoding approach (Hebert et al., 2003) which hasbeen proposed as a standard molecular tool for pro-visional taxonomic assessment. Its utility in evaluat-ing chiropteran taxonomic diversity has been previ-ously demonstrated for other geographic areas inSouth America and southeastern Asia (Clare et al.,2007; Francis et al., 2010).

We focus on the continental part of northeasternPalaearctic, defined as the boreal and temperate re-gions of Russia and adjacent territories in China,Korea, and Mongolia. Exclusion of Japan was due tolack of material and the high level of species en-demicity in the bat fauna of these islands (Yoshi -yuki, 1989; Ohdachi et al., 2009). The number of batspecies occurring within this continental region isestimated at 56, including 44 species occurring inRussia (Pavlinov and Rossolimo, 1987; Pavlinov etal., 1995, 2002; Simmons, 2005). The boreal andtemperate bat assemblages in this area are heavilydominated by Vespertilionidae. Several members ofthis family have extensive distributional rangesacross the northeastern Palaearctic and exhibit distinct morphological variation that led earlier au-thors to describe several geographic forms. The tax-onomic composition of some species, particularly M. brand tii, V. murinus, and E. nils sonii remainscontroversial, calling for a reappraisal of their statususing an independent molecular dataset.

MATERIAL AND METHODS

Sampling

Most tissue samples used in this study were obtained frommuseum preserved specimens fixed in 70–75% ethanol. Piecesof pectoral muscle were the preferred source, due to relativelyquick ethanol penetration during fixation and good preservationof mitochondrial DNA. Some tissues were sampled from fresh-ly collected bats during field surveys. Tissue samples from theseanimals (muscle, heart, kidney, spleen, and liver) were taken immediately following euthanasia and preserved in 95–99%ethanol or frozen in liquid nitrogen (Engstrom et al., 1999).Approximately 5 mg or 2 mm3 of tissue was subsampled fromeach specimen for analysis. Voucher specimens were deposited

in the following collections: Zoological Museum of MoscowState University (ZMMU); Royal Ontario Museum (ROMMAM); Paleontological Institute, Russian Academy of Sciences(PIN RAS); Kirov City Zoological Museum (KCZM); NationalInstitute of Biological Resources, Korea (NIBR); and Instituteof Biology and Soil Science, Russian Academy of Sciences, FarEastern Branch (IBSS RAS). Five specimens were obtainedfrom the Museum of Natural History, Geneva (MHNG) as anexchange with ZMMU.

Molecular Protocols

Tissues were arrayed into 96-well microplates (Borisenko etal., 2008, 2009) and submitted for molecular analysis to the coreanalytical facility at the Canadian Centre for DNA Barcoding(CCDB), Biodiversity Institute of Ontario, University ofGuelph. Prior to DNA extraction, each plate well was filled with50 μL of lysis buffer with Proteinase K and the plates were in-cubated overnight (12–18 h) at 56°C, followed by a roboticDNA extraction protocol (Ivanova et al., 2006, In press).

Standard mammalian barcoding protocols for PCR amplifi-cation and sequencing were employed (Ivanova et al., In press):12.5 μl of PCR master mix was added to the wells; vertebrateM13-tailed primer cocktail [C_VF1LFt1 + C_VR1LRt1] wasused to recover the full length DNA barcode region (657 basepairs) and a shorter fragment (421 base pairs) was recovered using the M13-tailed modification of the internal primer RonM(Pfunder et al. 2004) with the reverse cocktail [RonM_t1 +C_VR1LRt1] (Borisenko et al., 2008; Ivanova et al., In press).PCR products were visualized on a 2% agarose gel using an E-Gel96 Pre-cast Agarose Electrophoresis System (Invitrogen)(Ivanova et al., In press).

The standard CCDB protocol with 1/24 BigDye dilution(Ivanova and Grainger, 2007) was used for sequencing.Products were labelled using the BigDye© Terminator v.3.1Cycle Sequencing Kit, Applied Biosystems, Inc. (Hajibabaei etal., 2005) and sequenced bidirectionally using an ABI 3730XLcapillary sequencer following manufacturer’s instructions.Sequences were assembled from raw sequencer trace files usingSeqScape v 2.1.1 (Ap plied Biosystems) and CodonCode align-er v. 3.5.2 (CodonCode Corporation) and verified by eye.

Sequence data were stored and initially analyzed using theBarcode of Life Data System — BOLD (Ratnasingham andHebert, 2007) using its online analytical tools. Pairwise nearestneighbour distances were calculated using the built-in toolsavailable in BOLD. Sequence data were then exported fromBOLD for further analysis in Molecular Evolutionary GeneticsAnalysis (MEGA) software (Tamura et al., 2007) using themaximum composite likelihood substitution model and pairwisedeletion of missing data. Neighbour-joining trees (NJ) wereboot strapped at 500 replicates. Transition saturation patterns ofCOI nucleotide sequences were calculated for the completedataset using the DAMBE software package for molecular dataanalysis (Xia and Xie, 2001).

The results of this study (sequences, trace files, and associ-ated detailed specimen information) are available online athttp://www.barcodinglife.org in a published BOLD projeccalled “Bats of North eastern Palaearctic” [SKBPA]. Sequencedata were also submitted to NCBI GenBank (accession nos.JF442793–JF443154 and JX008034–JX008092). In order to addcomparative zoogeographic context to our data on NortheasternPalaearctic, we used data from another published BOLD project:“Bats of Southeast Asia” [BM] (Francis et al., 2010); GenBank

2 S. V. Kruskop, A. V. Borisenko, N. V. Ivanova, B. K. Lim, and J. L. Eger

accession nos. HM540109–HM542004. Detailed lists of speci-mens with provenance information can be retrieved from theseBOLD projects online.

RESULTS

DNA barcodes were obtained from 388 speci-mens representing 38 recognized species — 68% ofthe 56 species (sensu Simmons, 2005) presentlyrecorded from the study area. Most of them wererepresented by multiple individuals sampled fromacross their range and including both ‘western’ and‘eastern’ representatives of widely distributedspecies and/or species complexes (Fig. 1). Of the se-quences generated, 355 were over 500 bp and 302were over 650 bp in length.

Genetic Distances

A calculation of average pairwise distances forthe entire dataset (Table 1) was skewed by the pres-ence of two species, one of which (M. brandtii) displayed a deep (13%) genetic split and another (E. serotinus) displayed a case of ‘barcode sharing’with a congeneric species (E. nilssonii). With thesetwo species removed (Table 1), there was almost no overlap between intraspecific and interspecificdistances.

Transition saturation (Fig. 2) is evident in our dataset at pairwise genetic distances > 12%,

indicating the lack of phylogenetic signal at the intrageneric level (Table 2). This is in general agree-ment with bootstrap support values for branches onthe NJ tree (Fig. 3).

DISCUSSION

High Concordance between Molecular andTaxonomic Hypotheses

Our results show high concordance between ge-netic divergence in COI and taxonomic identifica-tions, suggesting DNA barcoding to be an instru-mental diagnostic tool for northeastern Palaearcticbats. Patterns of COI sequence divergence stronglysupport all taxa raised to species status in the pastthree decades (Table 3), including taxa whose statusremains controversial. For example, the validity ofMyotis aurascens has been contested by Mayer andHelversen (2001), although this was not corrobo-rated by subsequent molecular data (Mayer et al.,2007). DNA barcode data show that M. aurascensis more divergent from M. mystacinus (9.8%), itspurported senior synonym, than from its nearestneighbour M. ikonnikovi (8.7%). DNA barcodes fur-ther added clarity to the distributional status ofspecies with obscure morphological differences,such as the P. pipistrellus/pygmaeus complex. Bothspecies occur in the Russian fauna, but, based on ourmolecular data, P. pygmaeus is more abundant and

Genetic diversity of northeastern Palaearctic bats as revealed by DNA barcodes 3

Map Legend:

Siberia Transbaikalia

East Europe Caucasus and C. Asia

Mainland Far East

Insular Far East

extralimital localities

FIG. 1. Collecting localities of bat specimens used in this study

widespread across the European part, while P. pipi -strellus is restricted to the Caucasus. This corrobo-rates an earlier suggestion on the distribution ofthese species based on morphometrics (Kruskop,2007). In addition, comparison of our results withrecently published DNA barcode data on SoutheastAsian bats (Francis et al., 2010) has allowed us toadd broader taxonomic context to some of the re-cently adopted names for Far Eastern ‘counterparts’of European species.

Several bat taxa previously considered to bespecies with broad Palaearctic distribution (e.g.,Corbet, 1978) have been split in the past threedecades. Many of them are now represented by an‘eastern’ and ‘western’ species (Table 3); such are

Plecotus ognevi and P. auritus (Spitzenberger et al.,2006; Bulkina and Kruskop, 2009), or Myotis bom -binus and M. nattereri (Horáček and Hanak, 1984;Kawai et al., 2003). Other similar cases deserve a more in-depth discussion and will be consideredbelow.

Although the reconstruction of phylogenetic re-lationships was not the aim of this study, it can bespeculated that branches with high bootstrap sup-port representing closely related species, (Fig. 3)will prove to be valid monophyletic clades. For example, our NJ trees demonstrate strong bootstrapsupport for branches containing Myotis bombinus/blythii/nattereri, and M. daubentonii/bechsteinii/frater (Figs. 3–4). This agrees with the recent results


FIG. 2. Patterns of transition saturation in northeastern Palaearctic bats

Comparison between N specimens N taxa N comparisons Min P-dist (%) Max P-dist (%) 0 ± SE (%)

Entire datasetConspecifics 372 32 3739 0.000 13.397 2.504 ± 0.071Congenerics 377 10 16649 0.622 20.190 14.47 ± 0.018

Reduced dataset (M. brandtii and E. serotinus removed)Conspecifics 295 30 2187 0.000 5.477 0.442 ± 0.017Congenerics 300 10 9052 5.463 18.721 13.408 ± 0.020

TABLE 1. Average pairwise distance within species and genera of northeast Palaearctic bats

y = -1.8984x2 + 1.0039xR2 = 0.7604

× Transitions (Ti)

¡ Transversions (Tv)

y = 1.0858x2 + 0.0081xR2 = 0.7401

Ti/Tv distance

Cu

mu

lative

ge

ne

tic d

ista

nce

0.20

0.16

0.12

0.08

0.04

0.00

0 0.10 0.20 0.30 0.40

SpeciesIntraspecific distances

Nearest neighbour (NN) species Distances to NNMean Maximum

Rhinolophus ferrumequinum 3.63 5.44 Rhinolophus mehelyi 10.41R. hipposideros 0.08 0.15 R. ferrumequinum 12.95R. mehelyi N/A N/A R. ferrumequinum 10.41Barbastella barbastellus 0.00 0.00 Barbastella darjelingensis 17.19B. darjelingensis N/A N/A B. barbastellus 17.19Barbastella sp. TMP1 N/A N/A Myotis brandtii 18.41Eptesicus gobiensis N/A N/A Eptesicus serotinus 6.53E. nilssonii 0.88 2.83 E. serotinus 0.63E. serotinus 4.61 9.12 E. nilssonii 0.63Hypsugo alashanicus N/A N/A Nyctalus leisleri 14.24Miniopterus fuliginosus 0.00 0.00 Miniopterus schreibersii 17.70M. schreibersii 0.10 0.15 M. fuliginosus 17.70Murina hilgendorfi 0.31 1.01 Murina ussuriensis 17.99M. ussuriensis 0.61 1.54 Myotis mystacinus 16.03Myotis aurascens 1.59 3.18 M. ikonnikovi 8.74M. bechsteinii N/A N/A M. daubentonii 10.63M. blythii 1.91 4.27 M. nattereri 11.17M. bombinus 0.43 0.92 M. nattereri 11.36M. brandtii 6.76 15.35 M. aurascens 13.63M. dasycneme 0.27 0.70 M. daubentonii 11.07M. daubentonii 0.33 2.33 M. macrodactylus 10.53M. emarginatus 0.00 0.00 M. macrodactylus 12.47M. frater 0.81 2.43 M. daubentonii 11.43M. ikonnikovi 0.93 1.56 M. aurascens 8.74M. macrodactylus 1.99 2.99 M. petax 8.51M. mystacinus 0.34 0.91 M. aurascens 9.88M. nattereri 0.00 0.00 M. blythii 11.17M. petax 0.28 1.16 M. macrodactylus 8.51Nyctalus leisleri 0.23 0.31 Nyctalus noctula 13.57N. noctula 0.12 0.31 N. leisleri 13.57Pipistrellus kuhlii 1.10 5.77 Pipistrellus pipistrellus 14.06P. nathusii 0.21 0.61 Nyctalus leisleri 15.43P. pipistrellus 0.72 1.44 Pipistrellus pygmaeus 5.79P. pygmaeus 0.25 0.92 P. pipistrellus 5.79Plecotus auritus 1.42 4.06 Plecotus ognevi 14.22P. ognevi 0.24 1.45 P. auritus 14.22Vespertilio murinus 0.30 0.72 Vespertilio sinensis 9.85V. sinensis 0.33 0.72 V. murinus 9.85


TABLE 2. Intraspecific and nearest neighbour distances for northeast Palaearctic bats. Values of intraspecific distances over 3% andnearest neighbour (NN) distances below 5% marked in bold italics

of a phylogenetic study based on complete Cytbgene sequences (Zhang et al., 2009). The concor-dance of results obtained using different geneticmarkers is important, because the use of molec-ular data in reconstructing phylogenetic affinities in Myotis resulted in the complete revamping of its intrageneric taxonomy (Ruedi and Mayer, 2001).

Murina hilgendorfi has been synonymized withMurina leucogaster for much of the 20th century(e.g., Ellerman and Morrison-Scott, 1966; Corbet,1978). It was first separated from M. leucogaster byYoshiyuki (1989) who applied the name hilgendorfionly to the Japanese specimens. Later on, Simmons(2005) redefined the distribution for the two species,

extrapolating the range of M. hilgendorfi to borealand temperate Asia and that of M. leucogaster totropical Asia. While the distinction between thesetwo species was later adopted (Kruskop, 2005;Ohdachi et al., 2009), it has not been formally sup-ported by genetics or morphology. The pattern ofnucleotide divergence in COI for these named formsrevealed in our study (Fig. 5) provides the first ge-netic support for the distinction of M. hilgendorfiand M. leucogaster.

Similarly, Murina ussuriensis and Murina aura-ta were also traditionally synonymized (e.g., Eller -man and Morrison-Scott, 1966; Corbet, 1978) until the former was raised to full species (Maeda,1980). The taxonomic composition and geographic


TABLE 3. Northeast Palaearctic bat species raised to species rank since 1980

Species Previously synonymized with Raised to species rank by Mean COI P-distance (%)

Myotis aurascens Myotis mystacinus Benda and Tsytsulina (2000) 8.6M. bombinus M. nattereri Horáček and Hanák (1984) 10.3M. petax M. daubentonii Matveev et al. (2005) 12.0Plecotus ognevi Plecotus auritus Spitzenberger et al. (2006) 12.1Murina ussuriensis Murina aurata Maeda (1980) unknownM. hilgendorfi M. leucogaster Yoshiyuki (1989) 14.1Miniopterus fuliginosus Miniopterus schreibersii Tian et al. (2004) 15.1Pipistrellus pygmaeus Pipistrellus pipistrellus Häussler et al. (2000) 3.6Eptesicus gobiensis Eptesicus nilssonii Strelkov (1986) 6.3Hypsugo alaschanicus Hypsugo savii Horáček et al. (2000) unknownBarbastella darjelingensis Barbastella leucomelas Benda et al. (2008) unknown

East Europe, n=33

Caucasus, n=3

Myotis daubentonii

Myotis bechsteinii Far East, n=2

Transbaikalia, n=10 Myotis frater

Myotis dasycneme, n=8 n=2

Myotis macrodactylus

Myotis petax, n=23

Asia, n=6 Crimea+Caucasus, n=2

Myotis blythii

Myotis nattereri, n=4 Myotis bombinus, n=6

Myotis emarginatus, n=3 Mainland Far East, n=6

Insular Far East, n=4

Myotis ikonnikovi

Asia, n=7 Europe, n=3

Myotis aurascens

Myotis mystacinus, n=11

Europe, n=25

Asia, n=26

Myotis brandtii

Murina hilgendorfi, n=13

Insular Far East, n=4 Mainland Far East, n=6

Murina ussuriensis

Korea, n=2Caucasus

Rhinolophus ferrumequinum100

100

100

70100

94

98

100

100

100

100

100

100

100

100

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100

97

87

96

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100100

100

80

81

99

92

99

90

71

77

87

79 98

100

89

distribution of M. aurata is pending further scrutiny,following a recent description of a new species —Murina eleryi (Furey et al., 2009) from NorthVietnam; see further discussion by Eger and Lim

(2011). No material is available from Moupin(Sichuan, China), which is the type locality for M. aurata (Corbet and Hill, 1992). Of the samplesavailable at our disposal, the DNA barcode of

M. ussuriensis displays greater similarity with thatof M. huttoni than M. eleryi and ‘M. aurata’ (sensuFrancis et al., 2010), while being genetically distantfrom all nearest neighbour species (Fig 5).

Major Trans-Palaearctic Splits

Myotis petax was formally raised to species rankfrom M. daubentonii by Matveev et al. (2005). COIdata (Fig. 4) suggest that these species are not only

divergent, but represent two separate speciesgroups: the ‘daubentonii’ group which also includesM. bechsteini, and M. frater; and an East Asiangroup containing M. petax, M. macrodactylus andM. pilosus. These provisional conclusions are pend-ing validation by a targeted phylogenetic study.

Similarly, the genetic split of the ‘Palaearctic’and ‘Oriental’ lineages within the Miniopterusschreibersii species complex was first proposed byAppleton et al. (2004) and Tian et al. (2004). The


FIG. 3. Cumulative NJ tree for northeastern Palaearctic bats. Marked in grey are branches where the genetic diversity within currentlyrecognized species exceeds 3%. Markers next to selected branches correspond to markers on map (Fig. 1). Open square marker nextto Myotis frater indicates combined mainland and insular Far East. Bootstrap values < 70% not shown; basal branches with < 70%

bootstrap support are depicted as dotted lines

Insular Far East, n=4 Mainland Far East, n=6

Murina ussuriensis

Korea, n=2 Caucasus

Rhinolophus ferrumequinum

Rhinolophus mehelyi Rhinolopus hipposideros, n=4

Miniopterus fuliginosus, n=3 Miniopterus schreibersii, n=3

Barbastella sp. TMP1 Barbastella darjelingensis

Barbastella barbastellus, n=2

Plecotus ognevi, n=28

Cis-Caucasia East Europe, n=6 Plecotus auritus

Eptesicus serotinus (East Europe), n=11

Eptesicus serotinus (Korea), n=4 Eptesicus gobiensis Eptesicus nilssoni (East-Central Europe), n=4

Eptesicus serotinus (Central Europe), n=3 Eptesicus nilssoni (East Europe+Siberia), n=9

Vespertilio murinus, n=16

Vespertilio sinensis, n=4 Hypsugo alashanicus

Pipistrellus nathusii, n=13

Nyctalus noctula, n=5 Nyctalus leisleri, n=4

East Europe, n=16

West EuropePipistrellus kuhlii

Caucasus, n=4 Central Europe, n=2

Pipistrellus pipistrellus

Pipistrellus pygmaeus, n=16

100

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0.02í

î

authors lacked material from the type locality of M. fuliginosus Hodgson, 1835 from Nepal, but pro-visionally applied this name to the ‘Oriental’ line-age. Based on our COI data (Figs. 3, 6), Europeanand Asian specimens are separated by a genetic dis-tance of 16%, and the specimen from the RussianFar East is similar to the specimen from Nepal.While M. fuliginosus has already been treated as a valid species by Sano (2009), our data provide independent confirmation of its species status. A combined COI tree (Fig. 6) including northeast-ern Palaearctic and Southeast Asian Miniopterus(data from Francis et al., 2010) shows much higher

genetic similarity (5%) and bootstrap support be-tween specimens of M. fuliginosus and M. magna -ter, compared to M. schreibersii, which is in agree-ment with the conclusions of Appleton et al. (2004)and Tian et al. (2004).

The deep COI divergence between Asian andEuropean haplogroups of Myotis brandtii (Figs.3–4) corroborates earlier findings made by Sta -delmann et al. (2007) using Cyt b data on samplesfrom Europe and the Far East. In our dataset, easternand western haplogroups are separated by a pairwisegenetic distance of 13% which exceeds the level ofinterspecific divergence between most northeastern


East Europe, n=33

Caucasus, n=3

Myotis daubentonii

Myotis bechsteinii Far East, n=2

Transbaikalia, n=10 Myotis frater

Myotis dasycneme, n=8 Myotis macrodactylus, n=3

n=6

n=4Myotis pilosus

Myotis petax, n=23

Asia, n=6

Caucasus, n=2Myotis blythii

Myotis chinensis, n=2 Myotis nattereri, n=4

Myotis bombinus, n=6 Myotis emarginatus, n=3

Mainland Far East, n=6

Insular Far East, n=4

Myotis ikonnikovi

Asia, n=7

Europe, n=3Myotis aurascens

Myotis mystacinus, n=11

Europe, n=25

Asia, n=26

Myotis brandtii

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FIG. 4. NJ tree for selected species of Myotis. Markers next to selected branches correspond to markers on map (Fig. 1). Open squaremarker next to M. frater indicates combined mainland and insular Far East. Extralimital species names are underlined. Bootstrap

values < 70% not shown; basal branches with < 70% bootstrap support are depicted as dotted lines

Palaearctic Myotis (Table 2). Geographically, thesplit is confined to the Urals, which contradicts thecurrently accepted view that M. brandtii is repre-sented by a single nominotypical subspecies acrossmost of its Palaearctic range (e.g., Corbet, 1978;Pavlinov and Rossolimo, 1987; Pavlinov et al.,1995; Simmons, 2005). The area around the Sea ofJapan was traditionally thought to be inhabited by a distinct subspecies — M. brandtii gracilis Ognev,1928 described from Vladivostok (Ognev, 1928;Tiunov, 1997; Sim mons, 2005); this named form istreated as a full species by some Japanese authors(Yoshiyuki, 1989; Ka wai et al., 2003). In our data -set, no significant differences were found among thespecimens collected east of the Yenisei River, in-cluding the Russian Far East. COI data suggest thatall eastern mainland populations formerly referredto as sibiricus and gracilis should be regarded as a single species, distinct from European M. brandtii.Fur ther more, the name sibiricus proposed for a sub-species of ‘Vespertilio mystacinus’ from the vicinityof Tomsk, Russia (Kastshenko, 1905), would havepriority over gracilis Ognev, 1928. Although thisname was suggested as conjectural, it should be regarded as valid, according to ICZN Art. 11.4.2.,11.4.3. and 11.5.1., because it is consistent with thePrinciple of Binominal Nomen clature, contains anexplicit description and reference to specimens examined and is proposed conditionally for a taxonbefore 1961. Unfortunately, no collection material is available to us from Western and Central Siberia;thus further sampling is required to clarify the dis-tributional boundary of the two putative species. As well, the relationships between sibiricus, gracilisand insular forms need further clarification usingseveral independent character sets.

Barbastella is another case of genetic separationof eastern and western forms. The sole specimen

available from the Far East, provisionally referred tohere as Barbastella sp. TMP1 is clearly divergent(Fig. 3) from its congeners (B. barbastellus fromEurope and B. darjelingensis from Nepal) and mayrepresent an undescribed species. This is consistentwith the recent suggestion that the taxonomic com-plexity of Barbastella is underestimated (Zhang etal., 2007).

In three other bat species the genetic divergencebetween eastern and western populations is shallow-er. The deepest split (5%) was between Korean andCaucasian specimens of Rhinolophus ferrumequ i -num — R. f. nippon and R. f. colchicus, respectively(see Wallin, 1969). Although only three specimensof this species were available to us for molecularanalysis, our results are in general agreement withearlier molecular studies invoking much larger sam-ple sizes (Rossiter et al., 2007; Flanders et al., 2009)that also show distinct western and oriental lineages.

Myotis mystacinus aurascens Kuzyakin, 1935was first elevated to species status on morphologicalgrounds (Benda and Tsytsulina, 2000), then syn-onymized with M. mystacinus in later revisions(Mayer and von Helversen, 2001; Simmons, 2005)and then resurrected again by Mayer et al. (2007).The latter study compared patterns of divergence inthe ND1 gene and found two specimens the authorscaptured in Bulgaria whose sequences differed fromthose of M. mystacinus and were similar to ND1 se-quences of M. aurascens deposited in NCBI Gen -Bank by Tsytsulina et al. (AY699856, AY699858and AY699860 — K. A. Tsytsulina, M. H. Dick, K.Maeda, and R. Masuda, unpublished data). Thisstudy corroborates the view that M. aurascens isspecifically distinct. The morphologically similarform mongolicus Borissenko and Kruskop, 1996,described as a subspecies of M. mystacinus, wassynonimized with M. nipalensis with no explanation


FIG. 5. NJ tree for selected species of Murina. Extralimital species names are underlined. Bootstrap values < 70% not shown; basal branches with < 70% bootstrap support are depicted as dotted lines

0.02

(Simmons, 2005). COI data demonstrate strong sim-ilarity between aurascens from southeastern Europeand mongolicus (3%) from southern Siberia andMongolia, and their distinctiveness from mystacinus(8.6% — Table 3). Unfortunately, no material isavailable from Central Asia to clarify the status ofthe named forms nipalensis, transcaspicus, sogdi-anus and przewalskii described from there. It is pos-sible that ‘Myotis new species’ from Mongolia list-ed by Dolch et al. (2007) also represents M. a. mon-golicus, because the authors provide no evidence ofits distinctiveness from the latter.

Eptesicus nilssonii has a shallow genetic split between eastern and western populations; however,in contrast to all other species, its border between‘east ern’ and ‘western’ haplogroups lies within Cen -tral Russia and does not coincide with any ap parentgeographic barrier (Fig. 3). Eptesicus nilssonii hasthe northernmost distributional limits of all Palae -arctic bats, and its unique phylogeographic pattern isconsistent with the possible existence of a glacialrefugium in the Ural Mountains (e.g., see Markovaet al., 2008), where E. nilssonii was common at leastin the late Pleistocene–early Holocene (Fadeeva andKruskop, 2008). From this area, the species couldhave recolonized Siberia and easternmost Europe.Genetic distance between haplogroups suggests that they became isolated prior to the latest glacialcycle (Artyushin et al., 2009). No morphological

differences have been found between these two ge-netic clusters.

Vespertilio murinus is the only species with a broad Palaearctic distribution that shows geneticuniformity across its entire range — below 1% COI divergence, similar to that found in its eastern con-gener V. sinensis (Fig. 3). Colonies of V. murinus inthe Asian part of its range live in buildings and itsdistribution seems to be confined to human settle-ments. We speculate that its eastward expansionmay have been recent and linked with the spread ofhuman-altered habitats.

Local Phylogeographic Splits

Several species with restricted geographic distri-bution show notable phylogeographic divergence.Myotis frater shows a split of 2% between popula-tions from Siberia and the Far East, correspond-ing to M. f. yeniseensis and M. f. longicaudatus, respectively (Tsytsulina and Strelkov, 2001 — Figs.4–5).

Myotis ikonnikovi and Murina ussuriensis haveshallow splits between mainland and insular pop-ulations within the Far East. The insular form of M. ussuriensis is also morphologically divergentand was recently described as a separate sub spe-cies M. u. katerinae (Kruskop, 2005). The isola-tion of Sakhalin, Hokkaido, and the Kurile Islands


FIG. 6. NJ tree for selected species of Miniopterus. Extralimital species names are underlined. Bootstrap values < 70% not shown

0.02

supposedly commenced in late Pleistocene as a re-sult of ocean transgression, and their full separationprobably took place around the Pleisto cene–Holo -cene transition (Velizhanin, 1976). Tim ing the diver-gence of the insular forms to the last ocean trans-gression would imply a much higher mitochondrialsubstitution rate that the average calculated for bats (Nabholz, et al. 2007); therefore it is possi-ble that the insular lineages of these bat species be-came geographically isolated long before the oceantransgression.

Myotis daubentonii, Plecotus auritus, and Pipi -strellus pipistrellus have similar levels of genetic divergence (2 –3% — Fig. 3) between populationsinhabiting the lowlands of southeastern Europe(lower Volga and Don River basins) and Ciscaucasia(North Caucasus and adjacent plains); however, oursampling from those areas is limited. Despite the ex-istence of drastic climatic differences and a promi-nent zoogeographic barrier (Volga Basin), no phylo-geographic splits could be detected between theEuropean and Central Asian populations of E. se ro -ti nus and Nyctalus noctula.

Odd Cases of Sequence Divergence or Sharing

The COI divergence observed among specimensof Pipistrellus kuhlii from West Europe (Fig. 3) isconsistent with Mayer et al. (2007) who found twodistinct haplogroups with about 5% divergence co-existing within western European populations. Todate, there is no evidence that these haplogroups arereproductively isolated. This conflicts with the no-tion that mtDNA is prone to selective sweeps(Ballard and Whitlock, 2004) that would help main-tain genetic uniformity in panmictic populationsacross large time scales. It is therefore highly unlike-ly that the two haplogroups have diverged within a panmictic population. All specimens availablefrom southwestern Europe and the Caucasus com-pose a single haplogroup which also includes thesingle available specimen from Iran and is distinctfrom both West European mitochondrial lineages.This corresponds to the recent morphology-basedassessment of the distribution of the subspecies P. kuhlii lepidus across south-central Europe (Barti,2010). Interest ing ly, specimens from different partsof Euro pe are morphologically indistinguishablefrom each other and all contrast with the distinctivemorphological features of Iranian specimens of P. kuhlii (Kruskop and Lavrenchenko, 2006). It ispossible that this is the result of recent mitochon-drial introgression events, but in-depth analysis of

nuclear markers is required to clarify the picture (M.Ruedi, personal communication).

Our data show a genetic split between popula-tions of M. blythii from southeastern Europe andCentral Asia (Figs. 3–4). While our Caucasus speci-mens may represent the form oxygnathus, it is un-known whether their genetic distinctiveness is a re-sult of mitochondrial divergence from M. blythiiproper or due to past introgression with M. myotisdocumented for western Europe (Ruedi and Mayer,2001; Berthier et al., 2006).

Earlier studies found a 6% occurrence of mito-chondrial introgression among European mammals(Mallet, 2005), although it has been speculated thatcases of mitochondrial sequence sharing do not seri-ously undermine the discriminatory power of DNAbarcoding (Hebert and Gregory, 2005). Our datasetcomprising of the 38 currently recognized spe-cies confirms one case of ‘barcode sharing’ amongbats within the northeastern Palaearctic (E. seroti-nus/nilssonii — Fig. 3) that has been documentedearlier, based on other markers (Berthier et al.,2006; Artyu shin et al., 2009). It is interesting to notethat individuals with traces of introgression are pres-ent only in the western part of the range of E. serot-inus and all of them represent haplotypes distinctfrom those of the available E. nilssonii.

General Conclusions

The diverse patterns of COI variation amongnortheastern Palaearctic bats highlight the diversityof speciation and recolonization events that tookplace in the history of this faunal assemblage. Oneof the most striking patterns is the profound geneticsplit between morphologically similar but allopatri-cally distributed species occupying the Euro-pean and Asian parts of this area. This break is like-ly a result of either ancient speciation (e.g., Myotisbrandtii/sibiricus, M. nattereri/bombinus, Plecotusauritus/ognevi) or independent faunal origins of thecounterpart species (e.g., M. daubentonii/petax, Mi -nio pterus schreibersii/fuliginosus).

On a more refined level, several species (e.g.,My otis aurascens, Rhinolophus ferrumequinumand M. frater) appear to demonstrate speciation inprogress. It is reasonable to speculate that the shal-low genetic splits observed in our dataset amongpopulations of these species can be linked to morerecent glaciation events in the northeastern Palae -arctic. The current distribution of geographicallyisolated populations of these species is consistentwith the existence of isolated glacial refugia, where


populations accumulated not only genetic, but alsomorphological differences. As a result, the descen-dants of these populations represent currently accepted subspecies (see Wallin, 1969; Kruskop andBo rissenko, 1996; Benda and Tsytsulina, 2000; Tsy -tsulina and Strelkov, 2001).

The results of this study are generally congruentwith recent taxonomic findings, offer a more com-prehensive picture of the alpha-taxonomic structureof Northeast Palaearctic bats and suggest future avenues for in-depth taxonomic enquiry. Due to thehigh levels of transition saturation, the DNA bar-code region cannot be recommended as a reliablemarker in phylogenetic reconstructions. Nonethe -less, these data corroborate an earlier suggestion(Kruskop et al., 2007) that DNA barcodes will behelpful in highlighting phylogeographic splitsamong Palaearctic bats.

ACKNOWLEDGEMENTS

Tissue collection and processing of the museum materialwas carried out with financial support from the RussianFoundation for Basic Research (RFBR grants No 09-04-00283-a and 10-04-00683-a). Processing specimens at the ZMMU wasdone with administrative support from Igor Pavlinov and OlgaRossolimo. Exchange of samples between Moscow zoologicalmuseum and Geneva natural history museum was made possi-ble due to a travel grant from GNHM and personal support fromManuel Ruedi. Fieldwork in China by the Royal Ontario Muse -um was supported by the ROM Governors Fund, Department ofNatural History, and United States National Sci ence Foundation(DEB-0344430). Sequencing was done at the Canadian Centrefor DNA Barcoding with administrative support from PaulHebert; funding for molecular analyses was provided by theNatural Sciences and Engineering Research Council, GenomeCanada through the Ontario Genomics Institute (2008-OGI-ICI-03) and Gordon and Betty Moore Foundation. Molecular analy-ses were performed by the staff of the CCDB, particularly AgataPavlowski. Ilija Artyushin, Tatiana Bulkina, Elena Sitnikova,Andrey Lissovsky and Vladimir Lebedev helped during differ-ent stages of this research. Manuel Ruedi made insightful com-ments on the draft manuscript. We also thank two anonymousreferees for their insightful comments.

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Received 16 January 2012, accepted 30 May 2012

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