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 bats 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, Russia, 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, Plecotus auritus/ognevi and Miniopterus schreibersii/ fuliginosus) and suggested previously unreported splits within Eptesicus 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 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, Vespertilionidae, alpha-taxonomy, bat 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 China 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 Fur thermore, 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, Korea (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, Vespertilio 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 Mongolia. Exclusion of Japan 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. brand tii, V. murinus, and E. nils sonii 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