Acta Chiropterologica, 14(1): 1–14, 2012 PL ISSN 1508-1109 © Museum and Institute of Zoology PAS doi: 10.3161/150811012X654222

Genetic diversity of northeastern Palaearctic as revealed by DNA barcodes

SERGEI V. K RUSKOP1, ALEX V. B ORISENKO2, NATALIA V. I VANOVA2, BURTON K. LIM3, and JUDITH L. EGER3

1Zoological Museum of Moscow University, Ul Bol’shata Nikitskaya, 6, Moscow, , 125009 2Canadian Centre for DNA Barcoding, Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road E, Guelph, Ontario, Canada, N1G 2W1 3Department 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 northeastern Palaearctic bats to assess patterns of genetic diversity. These results confirmed earlier findings of deep phylogeographic splits in four pairs of vicariant species (Myotis daubentonii/petax, M. nattereri/bombinus, auritus/ognevi and Miniopterus schreibersii/ fuliginosus) and suggested previously unreported splits within nilssoni and Myotis aurascens. DNA barcodes support all taxa raised to species rank in the past 25 years and suggest that an additional species — Myotis sibiricus — should be separated from Myotis brandtii. Major phylogeographic splits occur between European and Asian populations of Myotis aurascens, Rhinolophus ferrumequinum 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). One confirmed case of sequence sharing was observed in our dataset — Eptesicus nilssoni/serotinus. This study corroborates the utility of DNA barcodes as a taxonomic assessment tool for bats.

Key words: cytochrome oxidase, phylogeography, , alpha-, fauna, Russia

INTRODUCTION common species with broad geographic distribution. Among them were the reevaluation of the status The incorporation of molecular methods into of two ‘phonic types’ in the Pipistrellus pipistrellus/ systematic research has provided valuable insights pygmaeus species complex (Hulva et al., 2004), into chiropteran species diversity and led to the tax- the morphologically based revision of geographic onomic reevaluation of some complex species morphs within Myotis mystacinus (Benda and groups. While it is not surprising that molecular data Tsytsulina, 2000), and a complete revamping of spe- highlighted potentially new taxonomic discoveries cies content within Plecotus (Spitzenberger et al., in species rich and understudied tropical areas (e.g., 2001, 2003; Kiefer et al., 2002). Clare et al., 2007; Francis et al., 2010), it has also These efforts, although targeting the Palaearctic led to interesting taxonomic findings even in rela- bat fauna as a whole, were heavily biased towards tively species-depauperate temperate faunas (Mayer species from Western Europe whereas Eastern and von Helversen, 2001; Mayer et al., 2007). The Europe and Asia remained underrepresented. Few Palaearctic bat fauna has undergone comprehensive studies using broader geographic sampling suggest- taxonomic revisions in the last three decades with ed the existence of phylogeographic splits indicative a dramatic boost in the number of recognized of past speciation events. For example, Myotis petax species around the turn of the last century (Horáček from Siberia and has been proposed as a geo- et al., 2000). Several bat groups have been identified graphic vicariate of the European Myotis dauben- as taxonomic ‘hot spots’, in which unexpected tonii (Kawai et al., 2003; Kruskop, 2004; Matveev levels of cryptic diversity were revealed (Mayer and et al., 2005). Similarly, molecular studies of Minio - von Helversen, 2001). pterus schreibersii (Appleton et al., 2004; Tian et The combination of molecular, acoustic and al., 2004) demonstrated that it is restricted to Europe refined morphological approaches has led bat and North Africa, while Miniopterus fuliginosus re- researchers to revisit the systematics of several places it further east. However, these studies used 2 S. V. Kruskop, A. V. Borisenko, N. V. Ivanova, B. K. Lim, and J. L. Eger different morphological and molecular character in the following collections: Zoological Museum of Moscow sets, thus preventing broad comparisons across taxa. State University (ZMMU); Royal Ontario Museum (ROM Furthermore, the poor representation of collection MAM); Paleontological Institute, Russian Academy of Sciences (PIN RAS); Kirov City Zoological Museum (KCZM); National specimens from Siberia and the Far East has hin- Institute of Biological Resources, (NIBR); and Institute dered the recovery of the geographic patterns of of Biology and Soil Science, Russian Academy of Sciences, Far genetic divergence among widely distributed spe - Eastern Branch (IBSS RAS). Five specimens were obtained cies, such as Myot is brandtii, murinus, from the Museum of Natural History, Geneva (MHNG) as an and Eptesicus nilssoni. exchange with ZMMU. Our paper aims to fill these knowledge gaps and provide baseline information on the genetic diversi- Molecular Protocols ty of northeastern Palaearctic bats using the DNA Tissues were arrayed into 96-well microplates (Borisenko et barcoding approach (Hebert et al., 2003) which has al., 2008, 2009) and submitted for molecular analysis to the core been proposed as a standard molecular tool for pro- analytical facility at the Canadian Centre for DNA Barcoding visional taxonomic assessment. Its utility in evaluat- (CCDB), Biodiversity Institute of Ontario, University of ing chiropteran taxonomic diversity has been previ- Guelph. Prior to DNA extraction, each plate well was filled with 50 μL of lysis buffer with Proteinase K and the plates were in- ously demonstrated for other geographic areas in cubated overnight (12–18 h) at 56°C, followed by a robotic South America and southeastern Asia (Clare et al., DNA extraction protocol (Ivanova et al., 2006, In press). 2007; Francis et al., 2010). Standard mammalian barcoding protocols for PCR amplifi- We focus on the continental part of northeastern cation and sequencing were employed (Ivanova et al., In press): Palaearctic, defined as the boreal and temperate re- 12.5 μl of PCR master mix was added to the wells; vertebrate M13-tailed primer cocktail [C_VF1LFt1 + C_VR1LRt1] was gions of Russia and adjacent territories in China, used to recover the full length DNA barcode region (657 base Korea, and . Exclusion of was due to pairs) and a shorter fragment (421 base pairs) was recovered lack of material and the high level of species en- using the M13-tailed modification of the internal primer RonM demicity in the bat fauna of these islands (Yoshi - (Pfunder et al. 2004) with the reverse cocktail [RonM_t1 + yuki, 1989; Ohdachi et al., 2009). The number of bat C_VR1LRt1] (Borisenko et al., 2008; Ivanova et al., In press). species occurring within this continental region is PCR products were visualized on a 2% agarose gel using an E-Gel96 Pre-cast Agarose Electrophoresis System (Invitrogen) estimated at 56, including 44 species occurring in (Ivanova et al., In press). Russia (Pavlinov and Rossolimo, 1987; Pavlinov et The standard CCDB protocol with 1/24 BigDye dilution al., 1995, 2002; Simmons, 2005). The boreal and (Ivanova and Grainger, 2007) was used for sequencing. temperate bat assemblages in this area are heavily Products were labelled using the BigDye© Terminator v.3.1 dominated by Vespertilionidae. Several members of Cycle Sequencing Kit, Applied Biosystems, Inc. (Hajibabaei et al., 2005) and sequenced bidirectionally using an ABI 3730XL this family have extensive distributional ranges capillary sequencer following manufacturer’s instructions. across the northeastern Palaearctic and exhibit Sequences were assembled from raw sequencer trace files using distinct morphological variation that led earlier au- SeqScape v 2.1.1 (Ap plied Biosystems) and CodonCode align- thors to describe several geographic forms. The tax- er v. 3.5.2 (CodonCode Corporation) and verified by eye. onomic composition of some species, particularly Sequence data were stored and initially analyzed using the Barcode of Life Data System — BOLD (Ratnasingham and M. brandtii , V. murinus, and E. nilssonii remains Hebert, 2007) using its online analytical tools. Pairwise nearest controversial, calling for a reappraisal of their status neighbour distances were calculated using the built-in tools using an independent molecular dataset. available in BOLD. Sequence data were then exported from BOLD for further analysis in Molecular Evolutionary Genetics Analysis (MEGA) software (Tamura et al., 2007) using the MATERIAL AND METHODS maximum composite likelihood substitution model and pairwise deletion of missing data. Neighbour-joining trees (NJ) were Sampling boot strappedat 500 replicates. Transition saturation patterns of COI nucleotide sequences were calculated for the complete Most tissue samples used in this study were obtained from dataset using the DAMBE software package for molecular data museum preserved specimens fixed in 70–75% ethanol. Pieces analysis (Xia and Xie, 2001). of pectoral muscle were the preferred source, due to relatively The results of this study (sequences, trace files, and associ- quick ethanol penetration during fixation and good preservation ated detailed specimen information) are available online at of mitochondrial DNA. Some tissues were sampled from fresh- http://www.barcodinglife.org in a published BOLD projec ly collected bats during field surveys. Tissue samples from these called “Bats of Northeastern Palaearctic” [SKBPA]. Sequence (muscle, heart, kidney, spleen, and liver) were taken data were also submitted to NCBI GenBank (accession nos. immediately following euthanasia and preserved in 95–99% JF442793–JF443154 and JX008034–JX008092). In order to add ethanol or frozen in liquid nitrogen (Engstrom et al., 1999). comparative zoogeographic context to our data on Northeastern Approximately 5 mg or 2 mm3 of tissue was subsampled from Palaearctic, we used data from another published BOLD project: each specimen for analysis. Voucher specimens were deposited “Bats of Southeast Asia” [BM] (Francis et al., 2010); GenBank Genetic diversity of northeastern Palaearctic bats as revealed by DNA barcodes 3 accession nos. HM540109–HM542004. Detailed lists of speci- indicating the lack of phylogenetic signal at the mens with provenance information can be retrieved from these intrageneric level (Table 2). This is in general agree- BOLD projects online. ment with bootstrap support values for branches on the NJ tree (Fig. 3). RESULTS DISCUSSION DNA barcodes were obtained from 388 speci- mens representing 38 recognized species — 68% of High Concordance between Molecular and the 56 species (sensu Simmons, 2005) presently Taxonomic Hypotheses recorded from the study area. Most of them were represented by multiple individuals sampled from Our results show high concordance between ge- across their range and including both ‘western’ and netic divergence in COI and taxonomic identifica- ‘eastern’ representatives of widely distributed tions, suggesting DNA barcoding to be an instru- species and/or species complexes (Fig. 1). Of the se- mental diagnostic tool for northeastern Palaearctic quences generated, 355 were over 500 bp and 302 bats. Patterns of COI sequence divergence strongly were over 650 bp in length. support all taxa raised to species status in the past three decades (Table 3), including taxa whose status Genetic Distances remains controversial. For example, the validity of Myotis aurascens has been contested by Mayer and A calculation of average pairwise distances for Helversen (2001), although this was not corrobo- the entire dataset (Table 1) was skewed by the pres- rated by subsequent molecular data (Mayer et al., ence of two species, one of which (M. brandtii) 2007). DNA barcode data show that M. aurascens displayed a deep (13%) genetic split and another is more divergent from M. mystacinus (9.8%), its (E. serotinus) displayed a case of ‘barcode sharing’ purported senior synonym, than from its nearest with a congeneric species (E. nilssonii). With these neighbour M. ikonnikovi (8.7%). DNA barcodes fur- two species removed (Table 1), there was almost ther added clarity to the distributional status of no overlap between intraspecific and interspecific species with obscure morphological differences, distances. such as the P. pipistrellus/pygmaeus complex. Both Transition saturation (Fig. 2) is evident in species occur in the Russian fauna, but, based on our our dataset at pairwise genetic distances > 12%, molecular data, P. pygmaeus is more abundant and

Map Legend: extralimital localities East Europe Caucasus and C. Asia Siberia Transbaikalia Mainland Far East Insular Far East

FIG. 1. Collecting localities of bat specimens used in this study 4 S. V. Kruskop, A. V. Borisenko, N. V. Ivanova, B. K. Lim, and J. L. Eger

0.20

× Transitions (Ti) ¡ Transversions (Tv) 0.16 y = -1.8984x2 + 1.0039x R2 = 0.7604

0.12

0.08 Cumulative genetic distance

y = 1.0858x2 + 0.0081x 0.04 R2 = 0.7401

0.00 0 0.10 0.20 0.30 0.40 Ti/Tv distance

FIG. 2. Patterns of transition saturation in northeastern Palaearctic bats widespread across the European part, while P. pipi - Plecotus ognevi and P. auritus (Spitzenberger et al., strellus is restricted to the Caucasus. This corrobo- 2006; Bulkina and Kruskop, 2009), or Myotis bom- rates an earlier suggestion on the distribution of binus and M. nattereri (Horáček and Hanak, 1984; these species based on morphometrics (Kruskop, Kawai et al., 2003). Other similar cases deserve 2007). In addition, comparison of our results with a more in-depth discussion and will be considered recently published DNA barcode data on Southeast below. Asian bats (Francis et al., 2010) has allowed us to Although the reconstruction of phylogenetic re- add broader taxonomic context to some of the re- lationships was not the aim of this study, it can be cently adopted names for Far Eastern ‘counterparts’ speculated that branches with high bootstrap sup- of European species. port representing closely related species, (Fig. 3) Several bat taxa previously considered to be will prove to be valid monophyletic clades. For species with broad Palaearctic distribution (e.g., example, our NJ trees demonstrate strong bootstrap Corbet, 1978) have been split in the past three support for branches containing Myotis bombinus/ decades. Many of them are now represented by an blythii/nattereri, and M. daubentonii/bechsteinii/ ‘eastern’ and ‘western’ species (Table 3); such are frater (Figs. 3–4). This agrees with the recent results

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

Comparison between N specimens N taxa N comparisons Min P-dist (%) Max P-dist (%) 0 ± SE (%) Entire dataset Conspecifics 372 32 3739 0.000 13.397 2.504 ± 0.071 Congenerics 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 ± 0.017 Congenerics 300 10 9052 5.463 18.721 13.408 ± 0.020 Genetic diversity of northeastern Palaearctic bats as revealed by DNA barcodes 5

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

Intraspecific distances Species Nearest neighbour (NN) species Distances to NN Mean Maximum Rhinolophus ferrumequinum 3.63 5.44 Rhinolophus mehelyi 10.41 R. hipposideros 0.08 0.15 R. ferrumequinum 12.95 R. mehelyi N/A N/A R. ferrumequinum 10.41 barbastellus 0.00 0.00 Barbastella darjelingensis 17.19 B. darjelingensis N/A N/A B. barbastellus 17.19 Barbastella sp. TMP1 N/A N/A Myotis brandtii 18.41 Eptesicus gobiensis N/A N/A Eptesicus serotinus 6.53 E. nilssonii 0.88 2.83 E. serotinus 0.63 E. serotinus 4.61 9.12 E. nilssonii 0.63 alashanicus N/A N/A leisleri 14.24 Miniopterus fuliginosus 0.00 0.00 Miniopterus schreibersii 17.70 M. schreibersii 0.10 0.15 M. fuliginosus 17.70 Murina hilgendorfi 0.31 1.01 Murina ussuriensis 17.99 M. ussuriensis 0.61 1.54 Myotis mystacinus 16.03 Myotis aurascens 1.59 3.18 M. ikonnikovi 8.74 M. bechsteinii N/A N/A M. daubentonii 10.63 M. blythii 1.91 4.27 M. nattereri 11.17 M. bombinus 0.43 0.92 M. nattereri 11.36 M. brandtii 6.76 15.35 M. aurascens 13.63 M. dasycneme 0.27 0.70 M. daubentonii 11.07 M. daubentonii 0.33 2.33 M. macrodactylus 10.53 M. emarginatus 0.00 0.00 M. macrodactylus 12.47 M. frater 0.81 2.43 M. daubentonii 11.43 M. ikonnikovi 0.93 1.56 M. aurascens 8.74 M. macrodactylus 1.99 2.99 M. petax 8.51 M. mystacinus 0.34 0.91 M. aurascens 9.88 M. nattereri 0.00 0.00 M. blythii 11.17 M. petax 0.28 1.16 M. macrodactylus 8.51 Nyctalus leisleri 0.23 0.31 Nyctalus noctula 13.57 N. noctula 0.12 0.31 N. leisleri 13.57 Pipistrellus kuhlii 1.10 5.77 Pipistrellus pipistrellus 14.06 P. nathusii 0.21 0.61 Nyctalus leisleri 15.43 P. pipistrellus 0.72 1.44 Pipistrellus pygmaeus 5.79 P. pygmaeus 0.25 0.92 P. pipistrellus 5.79 Plecotus auritus 1.42 4.06 Plecotus ognevi 14.22 P. ognevi 0.24 1.45 P. auritus 14.22 Vespertilio murinus 0.30 0.72 Vespertilio sinensis 9.85 V. sinensis 0.33 0.72 V. murinus 9.85 of a phylogenetic study based on complete Cytb extrapolating the range of M. hilgendorfi to boreal gene sequences (Zhang et al., 2009). The concor- and temperate Asia and that of M. leucogaster to dance of results obtained using different genetic tropical Asia. While the distinction between these markers is important, because the use of molec- two species was later adopted (Kruskop, 2005; ular data in reconstructing phylogenetic affinities Ohdachi et al., 2009), it has not been formally sup- in Myotis resulted in the complete revamping of ported by genetics or morphology. The pattern of its intrageneric taxonomy (Ruedi and Mayer, nucleotide divergence in COI for these named forms 2001). revealed in our study (Fig. 5) provides the first ge- Murina hilgendorfi has been synonymized with netic support for the distinction of M. hilgendorfi Murina leucogaster for much of the 20th century and M. leucogaster. (e.g., Ellerman and Morrison-Scott, 1966; Corbet, Similarly, Murina ussuriensis and Murina aura- 1978). It was first separated from M. leucogaster by ta were also traditionally synonymized (e.g., Eller - Yoshiyuki (1989) who applied the name hilgendorfi man and Morrison-Scott, 1966; Corbet, 1978) only to the Japanese specimens. Later on, Simmons until the former was raised to full species (Maeda, (2005) redefined the distribution for the two species, 1980). The taxonomic composition and geographic 6 S. V. Kruskop, A. V. Borisenko, N. V. Ivanova, B. K. Lim, and J. L. Eger

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) 8.6 M. bombinus M. nattereri Horáček and Hanák (1984) 10.3 M. petax M. daubentonii Matveev et al. (2005) 12.0 Plecotus ognevi Plecotus auritus Spitzenberger et al. (2006) 12.1 Murina ussuriensis Murina aurata Maeda (1980) unknown M. hilgendorfi M. leucogaster Yoshiyuki (1989) 14.1 Miniopterus fuliginosus Miniopterus schreibersii Tian et al. (2004) 15.1 Pipistrellus pygmaeus Pipistrellus pipistrellus Häussler et al. (2000) 3.6 Eptesicus gobiensis Eptesicus nilssonii Strelkov (1986) 6.3 Hypsugo alaschanicus Hypsugo savii Horáček et al. (2000) unknown Barbastella darjelingensis Barbastella leucomelas Benda et al. (2008) unknown distribution of M. aurata is pending further scrutiny, (2011). No material is available from Moupin following a recent description of a new species — (, China), which is the type locality for Murina eleryi (Furey et al., 2009) from North M. aurata (Corbet and Hill, 1992). Of the samples Vietnam; see further discussion by Eger and Lim available at our disposal, the DNA barcode of

89 East Europe, n=33 100 Myotis daubentonii 98 79 Caucasus, n=3 Myotis bechsteinii 87 98 100 Far East, n=2 Myotis frater Transbaikalia, n=10 100 Myotis dasycneme, n=8 80 99 n=2 Myotis macrodactylus 77 99 Myotis petax, n=23 100 100 Asia, n=6 100 Myotis blythii 90 Crimea+Caucasus, n=2 100 92 Myotis nattereri, n=4 100 Myotis bombinus, n=6 100 Myotis emarginatus, n=3 97 Mainland Far East, n=6 87 100 Insular Far East, n= 4 Myotis ikonnikovi

71 96 100 Asia, n=7 100 Myotis aurascens Europe, n=3 100 Myotis mystacinus, n=11

100 Europe, n=25 81 Myotis brandtii 100 Asia, n=26

100 Murina hilgendorfi, n=13 100 Insular Far East, n=4 70 Murina ussuriensis Mainland Far East, n=6 100 100 Korea, n=2 Rhinolophus ferrumequinum 94 Caucasus Genetic diversity of northeastern Palaearctic bats as revealed by DNA barcodes 7

M. ussuriensis displays greater similarity with that divergent, but represent two separate species of M. huttoni than M. eleryi and ‘M. aurata’ (sensu groups: the ‘daubentonii’ group which also includes Francis et al., 2010), while being genetically distant M. bechsteini, and M. frater; and an East Asian from all nearest neighbour species (Fig 5). group containing M. petax, M. macrodactylus and M. pilosus. These provisional conclusions are pend- Major Trans-Palaearctic Splits ing validation by a targeted phylogenetic study. Similarly, the genetic split of the ‘Palaearctic’ Myotis petax was formally raised to species rank and ‘Oriental’ lineages within the Miniopterus from M. daubentonii by Matveev et al. (2005). COI schreibersii species complex was first proposed by data (Fig. 4) suggest that these species are not only Appleton et al. (2004) and Tian et al. (2004). The

100 Insular Far East, n=4 70 Murina ussuriensis Mainland Far East, n=6 100 100 Korea, n=2 Rhinolophus ferrumequinum 94 Caucasus 100 Rhinolophus mehelyi 100 72 Rhinolopus hipposideros, n=4 100 99 Miniopterus fuliginosus, n=3 100 Miniopterus schreibersii, n=3 Barbastella sp. TMP1 80 Barbastella darjelingensis 100 Barbastella barbastellus, n=2

100 Plecotus ognevi, n=28

100 Cis-Caucasia 100 Plecotus auritus East Europe, n=6 100 Eptesicus serotinus (East Europe), n=11 100 100 Eptesicus serotinus (Korea), n=4 Eptesicus gobiensis 88 80 Eptesicus nilssoni (East-Central Europe), n=4 99 Eptesicus serotinus (Central Europe), n=3 Eptesicus nilssoni (East Europe+Siberia), n=9 100 99 Vespertilio murinus, n=16 100 Vespertilio sinensis, n=4 Hypsugo alashanicus 100 Pipistrellus nathusii, n=13 100 Nyctalus noctula, n=5 100 Nyctalus leisleri, n=4 99 East Europe, n=16 100 Pipistrellus kuhlii 88 West Europe

90 99 Caucasus, n=4 88 Pipistrellus pipistrellus 100 Central Europe, n=2 100

Pipistrellus pygmaeus, n=16 í 0.02 î

FIG. 3. Cumulative NJ tree for northeastern Palaearctic bats. Marked in grey are branches where the genetic diversity within currently recognized species exceeds 3%. Markers next to selected branches correspond to markers on map (Fig. 1). Open square marker next to 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 8 S. V. Kruskop, A. V. Borisenko, N. V. Ivanova, B. K. Lim, and J. L. Eger authors lacked material from the type locality of genetic similarity (5%) and bootstrap support be- M. fuliginosus Hodgson, 1835 from Nepal, but pro- tween specimens of M. fuliginosus and M. magna- visionally applied this name to the ‘Oriental’ line- ter, compared to M. schreibersii, which is in agree- age. Based on our COI data (Figs. 3, 6), European ment with the conclusions of Appleton et al. (2004) and Asian specimens are separated by a genetic dis- and Tian et al. (2004). tance of 16%, and the specimen from the Russian The deep COI divergence between Asian and Far East is similar to the specimen from Nepal. European haplogroups of Myotis brandtii (Figs. While M. fuliginosus has already been treated as 3–4) corroborates earlier findings made by Sta- a valid species by Sano (2009), our data provide delmann et al. (2007) using Cyt b data on samples independent confirmation of its species status. from Europe and the Far East. In our dataset, eastern A combined COI tree (Fig. 6) including northeast- and western haplogroups are separated by a pairwise ern Palaearctic and Southeast Asian Miniopterus genetic distance of 13% which exceeds the level of (data from Francis et al., 2010) shows much higher interspecific divergence between most northeastern

84 East Europe, n=33 100 Myotis daubentonii 92 85 Caucasus, n=3 Myotis bechsteinii 90 95 100 Far East, n=2 Transbaikalia, n=10 Myotis frater 100 Myotis dasycneme, n=8 99 Myotis macrodactylus, n=3 88 96 100 n=6 93 Myotis pilosus 89 n=4 99 Myotis petax, n=23 99 100 Asia, n=6 100 Myotis blythii 95 Caucasus, n=2 100 Myotis chinensis, n=2 100 80 Myotis nattereri, n=4 100 Myotis bombinus, n=6 100 Myotis emarginatus, n=3 97 83 Mainland Far East, n=6 73 100 Insular Far East, n=4 Myotis ikonnikovi

84 100 Asia, n=7 97 Myotis aurascens Europe, n=3 100 Myotis mystacinus, n=11 100 Europe, n=25 91 Myotis brandtii 100 Asia, n=26

0.02

FIG. 4. NJ tree for selected species of Myotis. Markers next to selected branches correspond to markers on map (Fig. 1). Open square marker 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 Genetic diversity of northeastern Palaearctic bats as revealed by DNA barcodes 9

0.02 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

Palaearctic Myotis (Table 2). Geographically, the available from the Far East, provisionally referred to split is confined to the Urals, which contradicts the here as Barbastella sp. TMP1 is clearly divergent currently accepted view that M. brandtii is repre- (Fig. 3) from its congeners (B. barbastellus from sented by a single nominotypical subspecies across Europe and B. darjelingensis from Nepal) and may most of its Palaearctic range (e.g., Corbet, 1978; represent an undescribed species. This is consistent Pavlinov and Rossolimo, 1987; Pavlinov et al., with the recent suggestion that the taxonomic com- 1995; Simmons, 2005). The area around the Sea of plexity of Barbastella is underestimated (Zhang et Japan was traditionally thought to be inhabited by al., 2007). a distinct subspecies — M. brandtii gracilis Ognev, In three other bat species the genetic divergence 1928 described from Vladivostok (Ognev, 1928; between eastern and western populations is shallow- Tiunov, 1997; Simmons, 2005); this named form is er. The deepest split (5%) was between Korean and treated as a full species by some Japanese authors Caucasian specimens of Rhinolophus ferrumequ i - (Yoshiyuki, 1989; Ka wai et al., 2003). In our data - num — R. f. nippon and R. f. colchicus, respectively set, no significant differences were found among the (see Wallin, 1969). Although only three specimens specimens collected east of the Yenisei River, in- of this species were available to us for molecular cluding the Russian Far East. COI data suggest that analysis, our results are in general agreement with all eastern mainland populations formerly referred earlier molecular studies invoking much larger sam- to as sibiricus and gracilis should be regarded as ple sizes (Rossiter et al., 2007; Flanders et al., 2009) a single species, distinct from European M. brandtii. that also show distinct western and oriental lineages. Fur ther more, the name sibiricus proposed for a sub- Myotis mystacinus aurascens Kuzyakin, 1935 species of ‘Vespertilio mystacinus’ from the vicinity was first elevated to species status on morphological of Tomsk, Russia (Kastshenko, 1905), would have grounds (Benda and Tsytsulina, 2000), then syn- priority over gracilis Ognev, 1928. Although this onymized with M. mystacinus in later revisions name was suggested as conjectural, it should be (Mayer and von Helversen, 2001; Simmons, 2005) regarded as valid, according to ICZN Art. 11.4.2., and then resurrected again by Mayer et al. (2007). 11.4.3. and 11.5.1., because it is consistent with the The latter study compared patterns of divergence in Principle of Binominal Nomen clature, contains an the ND1 gene and found two specimens the authors explicit description and reference to specimens captured in Bulgaria whose sequences differed from examined and is proposed conditionally for a taxon those of M. mystacinus and were similar to ND1 se- before 1961. Unfortunately, no collection material quences of M. aurascens deposited in NCBI Gen - is available to us from Western and Central Siberia; Bank by Tsytsulina et al. (AY699856, AY699858 thus further sampling is required to clarify the dis- and AY699860 — K. A. Tsytsulina, M. H. Dick, K. tributional boundary of the two putative species. Maeda, and R. Masuda, unpublished data). This As well, the relationships between sibiricus, gracilis study corroborates the view that M. aurascens is and insular forms need further clarification using specifically distinct. The morphologically similar several independent character sets. form mongolicus Borissenko and Kruskop, 1996, Barbastella is another case of genetic separation described as a subspecies of M. mystacinus, was of eastern and western forms. The sole specimen synonimized with M. nipalensis with no explanation 10 S. V. Kruskop, A. V. Borisenko, N. V. Ivanova, B. K. Lim, and J. L. Eger

0.02

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

(Simmons, 2005). COI data demonstrate strong sim- differences have been found between these two ge- ilarity between aurascens from southeastern Europe netic clusters. and mongolicus (3%) from southern Siberia and Vespertilio murinus is the only species with Mongolia, and their distinctiveness from mystacinus a broad Palaearctic distribution that shows genetic (8.6% — Table 3). Unfortunately, no material is uniformity across its entire range — below 1% COI available from to clarify the status of divergence, similar to that found in its eastern con- the named forms nipalensis, transcaspicus, sogdi- gener V. sinensis (Fig. 3). Colonies of V. murinus in anus and przewalskii described from there. It is pos- the Asian part of its range live in buildings and its sible that ‘Myotis new species’ from Mongolia list- distribution seems to be confined to human settle- ed by Dolch et al. (2007) also represents M. a. mon- ments. We speculate that its eastward expansion golicus, because the authors provide no evidence of may have been recent and linked with the spread of its distinctiveness from the latter. human-altered habitats. Eptesicus nilssonii has a shallow genetic split between eastern and western populations; however, Local Phylogeographic Splits in contrast to all other species, its border between ‘east ern’ and ‘western’ haplogroups lies within Cen - Several species with restricted geographic distri- tral Russia and does not coincide with any ap parent bution show notable phylogeographic divergence. geographic barrier (Fig. 3). Eptesicus nilssonii has Myotis frater shows a split of 2% between popula- the northernmost distributional limits of all Palae - tions from Siberia and the Far East, correspond- arctic bats, and its unique phylogeographic pattern is ing to M. f. yeniseensis and M. f. longicaudatus, consistent with the possible existence of a glacial respectively (Tsytsulina and Strelkov, 2001 — Figs. refugium in the Ural Mountains (e.g., see Markova 4–5). et al., 2008), where E. nilssonii was common at least Myotis ikonnikovi and Murina ussuriensis have in the late Pleistocene–early Holocene (Fadeeva and shallow splits between mainland and insular pop- Kruskop, 2008). From this area, the species could ulations within the Far East. The insular form of have recolonized Siberia and easternmost Europe. M. ussuriensis is also morphologically divergent Genetic distance between haplogroups suggests and was recently described as a separate subspe- that they became isolated prior to the latest glacial cies M. u. katerinae (Kruskop, 2005). The isola- cycle (Artyushin et al., 2009). No morphological tion of Sakhalin, , and the Kurile Islands Genetic diversity of northeastern Palaearctic bats as revealed by DNA barcodes 11 supposedly commenced in late Pleistocene as a re- nuclear markers is required to clarify the picture (M. sult of ocean transgression, and their full separation Ruedi, personal communication). probably took place around the Pleistocene–Holo - Our data show a genetic split between popula- cene transition (Velizhanin, 1976). Tim ing the diver- tions of M. blythii from southeastern Europe and gence of the insular forms to the last ocean trans- Central Asia (Figs. 3–4). While our Caucasus speci- gression would imply a much higher mitochondrial mens may represent the form oxygnathus, it is un- substitution rate that the average calculated for known whether their genetic distinctiveness is a re- bats (Nabholz, et al. 2007); therefore it is possi- sult of mitochondrial divergence from M. blythii ble that the insular lineages of these bat species be- proper or due to past introgression with M. myotis came geographically isolated long before the ocean documented for western Europe (Ruedi and Mayer, transgression. 2001; Berthier et al., 2006). Myotis daubentonii, Plecotus auritus, and Pipi - Earlier studies found a 6% occurrence of mito- strellus pipistrellus have similar levels of genetic chondrial introgression among European divergence (2–3% — Fig. 3) between populations (Mallet, 2005), although it has been speculated that inhabiting the lowlands of southeastern Europe cases of mitochondrial sequence sharing do not seri- (lower Volga and Don River basins) and Ciscaucasia ously undermine the discriminatory power of DNA (North Caucasus and adjacent plains); however, our barcoding (Hebert and Gregory, 2005). Our dataset sampling from those areas is limited. Despite the ex- comprising of the 38 currently recognized spe- istence of drastic climatic differences and a promi- cies confirms one case of ‘barcode sharing’ among nent zoogeographic barrier (Volga Basin), no phylo- bats within the northeastern Palaearctic (E. seroti- geographic splits could be detected between the nus/nilssonii — Fig. 3) that has been documented European and Central Asian populations of E. se ro - earlier, based on other markers (Berthier et al., ti nus and Nyctalus noctula. 2006; Artyu shin et al., 2009). It is interesting to note that individuals with traces of introgression are pres- Odd Cases of Sequence Divergence or Sharing ent only in the western part of the range of E. serot- inus and all of them represent haplotypes distinct The COI divergence observed among specimens from those of the available E. nilssonii. of Pipistrellus kuhlii from West Europe (Fig. 3) is consistent with Mayer et al. (2007) who found two General Conclusions distinct haplogroups with about 5% divergence co- existing within western European populations. To The diverse patterns of COI variation among date, there is no evidence that these haplogroups are northeastern Palaearctic bats highlight the diversity reproductively isolated. This conflicts with the no- of speciation and recolonization events that took tion that mtDNA is prone to selective sweeps place in the history of this faunal assemblage. One (Ballard and Whitlock, 2004) that would help main- of the most striking patterns is the profound genetic tain genetic uniformity in panmictic populations split between morphologically similar but allopatri- across large time scales. It is therefore highly unlike- cally distributed species occupying the Euro- ly that the two haplogroups have diverged within pean and Asian parts of this area. This break is like- a panmictic population. All specimens available ly a result of either ancient speciation (e.g., Myotis from southwestern Europe and the Caucasus com- brandtii/sibiricus, M. nattereri/bombinus, Plecotus pose a single haplogroup which also includes the auritus/ognevi) or independent faunal origins of the single available specimen from Iran and is distinct counterpart species (e.g., M. daubentonii/petax, Mi - from both West European mitochondrial lineages. nio pterus schreibersii/fuliginosus). This corresponds to the recent morphology-based On a more refined level, several species (e.g., assessment of the distribution of the subspecies My otis aurascens, Rhinolophus ferrumequinum P. kuhlii lepidus across south-central Europe (Barti, and M. frater) appear to demonstrate speciation in 2010). Interesting ly, specimens from different parts progress. It is reasonable to speculate that the shal- of Euro pe are morphologically indistinguishable low genetic splits observed in our dataset among from each other and all contrast with the distinctive populations of these species can be linked to more morphological features of Iranian specimens of recent glaciation events in the northeastern Palae - P. kuhlii (Kruskop and Lavrenchenko, 2006). It is arctic. The current distribution of geographically possible that this is the result of recent mitochon- isolated populations of these species is consistent drial introgression events, but in-depth analysis of with the existence of isolated glacial refugia, where 12 S. V. Kruskop, A. V. Borisenko, N. V. Ivanova, B. K. Lim, and J. L. Eger populations accumulated not only genetic, but also BARTI, L. 2010. First record of Pipistrellus kuhlii (Chiroptera: morphological differences. As a result, the descen- Vespertilionidae) from Transylvania and a morphological dants of these populations represent currently approach to the lepidus taxon. Acta Siculica, 2010: 155–168. BENDA, P., and K. A. TSYTSULINA. 2000. Taxonomic revision of accepted subspecies (see Wallin, 1969; Kruskop and Myotis mystacinus group (Mammalia: Chiroptera) in the Bo rissenko, 1996; Benda and Tsytsulina, 2000; Tsy - western Palaearctic. Acta Societatis Zoologicae Bohemicae, tsulina and Strelkov, 2001). 64: 331–398. The results of this study are generally congruent BENDA, P., C. DIETZ, M. ANDREAS, J. HOTOVY, R. K. LUCAN, with recent taxonomic findings, offer a more com- A. MALTBY, K. MEAKIN, J. TRUSCOTT, and P. VALLO. 2008. prehensive picture of the alpha-taxonomic structure Bats (Mammalia: Chiroptera) of the Eastern Mediterranean and Middle East. Part 6. Bats of Sinai (Egypt) with some of Northeast Palaearctic bats and suggest future taxonomic, ecological and echolocation data on that fauna. avenues for in-depth taxonomic enquiry. Due to the Acta Societatis Zoologicae Bohemicae, 72: 1–103. high levels of transition saturation, the DNA bar- BERTHIER, P., L. EXCOFFIER, and M. RUEDI. 2006. Recurrent code region cannot be recommended as a reliable replacement of mtDNA and cryptic hybridization between marker in phylogenetic reconstructions. Nonethe - two sibling bat species Myotis myotis and Myotis bly- thii. Proceedings of the Royal Society of London, 273B: less, these data corroborate an earlier suggestion 3101–3123. (Kruskop et al., 2007) that DNA barcodes will be BORISENKO, A. V., B. K. LIM, N. V. IVANOVA, R. H. HANNER, and helpful in highlighting phylogeographic splits P. D. N. HEBERT. 2008. DNA barcoding in surveys of small among Palaearctic bats. communities: a field study in Suriname. Molecular Ecology Resources, 8: 471–479. BORISENKO, A. V., J. E. SONES, and P. D. N. HEBERT. 2009. The ACKNOWLEDGEMENTS front-end logistics of DNA barcoding: challenges and pros- Tissue collection and processing of the museum material pects. Molecular Ecology Resources, 9: 27–34. was carried out with financial support from the Russian BULKINA, T. M., and S. V. KRUSKOP. 2009. Search for mor- Foundation for Basic Research (RFBR grants No 09-04-00283- phological difference between genetically distinct brown a and 10-04-00683-a). Processing specimens at the ZMMU was long-eared bats (Plecotus auritus s. lato, Vespertilionidae). done with administrative support from Igor Pavlinov and Olga Ple cotus et al., 11–12: 3–13. Rossolimo. Exchange of samples between Moscow zoological CLARE, E. L., B. K. LIM, M. D. ENGSTROM, J. L. EGER, and P. D. museum and Geneva natural history museum was made possi- N. HEBERT. 2007. DNA barcoding of Neotropical bats: spe- ble due to a travel grant from GNHM and personal support from cies identification and discovery within Guyana. Molecular Manuel Ruedi. Fieldwork in China by the Royal Ontario Muse- Ecology Notes, 7: 184–190. um was supported by the ROM Governors Fund, Department of CORBET, G. B. 1978. The mammals of the Palaearctic Re- Natural History, and United States National Sci ence Foundation gion: a taxonomic review. Cornell University Press, Lon don, (DEB-0344430). Sequencing was done at the Canadian Centre 314 pp. for DNA Barcoding with administrative support from Paul CORBET, G. B., and J. E. HILL. 1992. The mammals of the Hebert; funding for molecular analyses was provided by the Indomalayan Region. Oxford University Press, Oxford, Natural Sciences and Engineering Research Council, Genome 488 pp. Canada through the Ontario Genomics Institute (2008-OGI-ICI- DOLCH, D., N. BATSAIKHAN, K. THIELE, F. BURGER, I. 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