Abstract
We examined selected external characteristics and measurements of Pipistrellus k. kuhlii and P. k. lepidus representatives from the Balkans and Central Europe, whose ranges have rapidly expanded over the past few decades. We also sequenced and analysed two mitochondrial (16S and COI genes) and one nuclear (RAG2) markers of these two bat morphotypes to determine haplotype diversity and distribution patterns with a wider geographic perspective. We found that bats of the two taxa differed markedly with regard to the overall body coloration, size (P. k. lepidus is larger than P. k. kuhlii), extent and shape of the pale wing margin, and penis coloration, a finding which seems to be of diagnostic value, similarly to other Pipistrellus species. No polymorphism in RAG2 marker was found, but in both mtDNA markers we detected different haplotypes characteristic for both taxa, corresponding to morphological and morphometric patterns established in this study. Our genetic analysis results confirmed a clear division into two phylogenetic lineages and may indicate their allopatric speciation and a very recent simultaneous expansion to the Balkans and Central Europe from the Mediterranean region (P. kuhlii/deserti) and south-west Asia across eastern Europe (P. k. lepidus). We also show that P. k. lepidus distribution is wider than previously reported, and that the ranges of P. k. lepidus and P. k. kuhlii have already contacted in Central Europe.
References
Andriollo, T., Naciri, Y. & Ruedi, M. (2015) Two mitochondrial barcodes for one biological species: the case of European Kuhl's pipistrelles (Chiroptera). PLoS ONE, 10 (8), e0134881.
https://doi.org/10.1371/journal.pone.0134881
Barti, L. (2010) First record of Pipistrellus kuhlii (Chiroptera: Vespertlionidae) from Transylvania and a morphological approach to the Lepidus taxon. Acta Siculica, 2010, 155–168.
Baker, R.J., Porter, C.A., Patton, J.C. & Van Den Bussche, R.A. (2000) Systematics of bats of the family Phyllostomidae based on RAG2 DNA sequences. Occasional Papers of the Museum of Texas Tech University, 202, 1–16.
Bauer, K. (1996) Ausbreitung der Weißrandfledermaus Pipistrellus kuhlii (Kuhl, 1819) in Österreich. Mitteilungen der Abteilung für Zoologie am Landesmuseum Joanneum, 50, 17–24.
Benda, P., Andriollo, T. & Ruedi, M. (2015) Systematic position and taxonomy of Pipistrellus deserti (Chiroptera: Vespertilionidae). Mammalia, 79, 419–438.
https://doi.org/10.1515/mammalia-2014-0024
Benson, D. A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J. & Wheeler, D.L. (2008) GenBank. Nucleic Acids Research, 36 (Database Issue), D25–30.
Çoraman, E., Furman, A., Karataş, A. & Bilgin, R. (2013) Phylogeographic analysis of Anatolian bats highlights the importance of the region for preserving the Chiropteran mitochondrial genetic diversity in the Western Palearctic. Conservation Genetics, 14, 1205–1216.
https://doi.org/10.1007/s10592-013-0509-4
Cheesman, R.E. & Hinton, A.C. (1924) On the mammals collected in the Desert of Central Arabia by Major R. E. Cheesman. Annals and Magazine of Natural History, Series 9, 14, 548–557.
https://doi.org/10.1080/00222932408633158Danko, Š. (2007) Reproduction of Hypsugo savii and Pipistrellus kuhlii in eastern Slovakia: further evidence of their spreading northwards. Vespertilio, 11, 13–24. [in Slovak with an abstract in English]
Dietz, C., von Helversen, O. & Nill, D. (2009) Bats of Britain, Europe and Northeastern Africa. A&C Black, London, 398 pp.
Dietz, C. & Kiefer, A. (2014) Die Fledermäuse Europas: kennen, bestimmen, schützen. Franckh-Kosmos Verlags-GmbH and Co. KG, Stuttgart, 394 pp.
Gaisler, J. (1970) The bats (Chiroptera) collected in Afghanistan by the Czechoslovak expeditions of 1965–1967. Acta Scientarum Naturalium Academiae Scientiarum Bohemicae Brno, 4, 1–56.
Galimberti, A., Spada, M., Russo, D., Mucedda, M., Agnelli, P., Crottini, A., Ferri, E., Martinoli, A. & Casiraghi, M. (2012) Integrated operational taxonomic units (IOTUs) in echolocating bats: a bridge between molecular and traditional taxonomy. PLoS ONE, 7 (6), e40122.
https://doi.org/10.1371/journal.pone.0040122
Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.
Hammer, Ř., Harper, D.A.T. & Ryan, P.D. (2001) PAST: Paleontological Statistics Software Package for Education and Data Analysis. Palaeontologia Electronica, 4, 1–9.
Harrison, D.L. (1964) The mammals of Arabia. Vol. 1. Insectivora, Chiroptera, Primates. Ernest Benn Limited, London, 192 pp.
Hasegawa, M., Kishino, H. & Yano, T. (1985) Dating the human-ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution, 22, 160–174.
https://doi.org/10.1007/BF02101694
Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogeny. Bioinformatics, 17, 754–755.
https://doi.org/10.1093/bioinformatics/17.8.754Hutton, T. (1845) Rough notes on the zoology of Candahar and the neighbouring districts. With notes by Ed. Blyth, Curator of the Asiatic Society’s Museum. Journal of the Asiatic Society of Bengal, 14, 340–354.
Kruskop, S.V., Borisenko, A.V., Ivanova, N.V., Lim, B.K. & Eger, J.L. (2012) Genetic diversity of northeastern Palearctic bats as revealed by DNA barcodes. Acta Chiropterologica, 14, 1–14.
https://doi.org/10.3161/150811012X654222Mayer, F., Dietz, C. & Kiefer, A. (2007) Molecular species identification boosts bat diversity. Frontiers in Zoology, 4, 4.
https://doi.org/10.1186/1742-9994-4-4Paunović, M. & Marinković, S. (1998) Kuhl’s pipistrelle Pipistrellus kuhlii Kuhl, 1817 (Chiroptera, Vespertilionidae)—a new species in the mammal fauna of Serbia, with data on its Balkan distribution range, status and ecology. Proceedings of the Fauna of Serbia, Natural and Mathematical Sciences, 5, 167–180.
Pestano, J., Brown, R.P., Suárez, N.M. & Fajardo, S. (2003) Phylogeography of pipistrelle-like bats within the Canary Islands, based on mtDNA sequences. Molecular Phylogenetics and Evolution, 26, 56–63.
https://doi.org/10.1016/S1055-7903(02)00307-XRahmatulina, I.K. (2005) Рукокрылые Азербайджана (фауна, экология, зоогеография) [Bats of Azerbaijan (Fauna, Ecology, Zoogeography)]. Nacional’naâ Akademiâ Nauk Azerbajdžana, Baku, 480 pp. [in Russian, with summaries in English and Azeri]
Sachanowicz, K., Wower, A. & Bashta, A.-T. (2006) Further range extension of Pipistrellus kuhlii (Kuhl, 1817) in central and eastern Europe. Acta Chiropterologica, 8, 543–548.
https://doi.org/10.3161/1733-5329(2006)8[543:FREOPK]2.0.CO;2
Steiner, H.M. & Gaisler, J. (1994) On a collection of bats (Chiroptera) from NE Turkey and N Iran. Acta Scientiarum Naturalium, Brno 28, 1–37.
Strelkov, P.P., Sosnovtzeva, V.P. & Babaev, H.B. (1978) The bats of Turkmenia. Proceedings of Zoological Institute Academy of Sciences CCCP [Trudy Zool. Inst. AN SSSR], 79, 3–71. [in Russian]
Strelkov, P.P., Unkurova, V.I. & Medvedeva, G.A. (1985) New data on Pipistrellus kuhlii and dynamics of its range in the USSR. Zoologichesky Zhurnal, 64, 87–97. [in Russian with English abstract]
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution, 30, 2725–2729.
https://doi.org/10.1093/molbev/mst197Tilova, E.K., Stoycheva, S.B. & Georgiev, D.G. (2005) New information on the distribution of some bat species (Mammalia: Chiroptera) from Bulgaria. Animalia, 41, 135–144.
Wawrocka, K., Bartonička, T. & Reiter, A. (2012) Pipistrellus kuhlii, a bat species breeding and hibernating in the Czech Republic. Vespertilio, 16, 351–356.
Veith, M., Mucedda, M., Kiefer, A. & Pidinchedda, E. (2011) On the presence of pipistrelle bats (Pipistrellus and Hypsugo; Chiroptera: Vespertilionidae) in Sardinia. Acta Chiropterologica, 13, 89–99.
https://doi.org/10.3161/150811011X578642Vierhaus, H. (2004) Pipistrellus nathusii (Keyserling & Blasius, 1839) – Rauhhautfledermaus. In: Krapp, F. (Eds.), Handbuch der Säugetiere Europas. Band 4. Fledertiere, Teil II. Chiroptera II. Vespertilionidae 2, Molossidae, Nycteridae. Aula Verlag, Wiebelsheim, pp. 825–873.
Yang, Z. (1996) Among-site rate variation and its impact on phylogenetic analyses. Trends in Ecology & Evolution, 11, 367–372.
https://doi.org/10.1016/0169-5347(96)10041-0