A Taxonomic Revision of the Kerivoula Hardwickii Complex (Chiroptera: Vespertilionidae) with the Description of a New Species
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Acta Chiropterologica, 19(1): xxx–xxx, 2017 PL ISSN 1508-1109 © Museum and Institute of Zoology PAS doi: 10.3161/15081109ACC2017.19.1.0xx A taxonomic revision of the Kerivoula hardwickii complex (Chiroptera: Vespertilionidae) with the description of a new species HAO-CHIH KUO1, 6, PIPAT SOISOOK2, YING-YI HO3, GABOR CSORBA4, CHUN-NENG WANG1, and STEPHEN J. ROSSITER6 1Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei 10617, Taiwan 2Princess Maha Chakri Sirindhorn Natural History Museum, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand 3Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 80424, Taiwan 4Department of Zoology, Hungarian Natural History Museum, Baross u. 13, H-1088 Budapest, Hungary 5School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, United Kingdom 6Corresponding author: E-mail: [email protected] Since its discovery, the taxonomic status of the only species of Kerivoula (Chiroptera: Vespertilionidae: Kerivoulinae) to be found on Taiwan has been confused. Previous studies have assigned this species to either Kerivoula hardwickii or K. titania, both of which occur on continental SE Asia. This uncertainty supports repeated suggestions in the literature that specimens of K. hardwickii collected and/or sampled across SE Asia are likely to represent multiple cryptic taxa. To address these issues, we combined new and existing data from the genus Kerivoula on Taiwan and continental Asia, and performed diagnostic analyses in steps. First, phylogenetic reconstructions based on mitochondrial and nuclear DNA revealed a well-supported group comprising all taxa currently recognized as K. hardwickii, together with the Taiwanese Kerivoula and Kerivoula kachinensis to the exclusion of all other congeneric species. Second, focusing on all members of this monophyletic clade (i.e., K. hardwickii complex) together with K. titania, we used multivariate statistical methods to separate taxa based on morphometric data. Our results provide strong evidence that among these bats, the Taiwanese Kerivoula is a new species that also occurs on continental Asia, for which we provide a formal description and name. In addition, we show that the subspecies K. hardwickii depressa should be elevated to species status. We discuss our findings and the caveats of this and similar studies. Key words: Kerivoula, new species, systematics, taxonomic revision, woolly bats INTRODUCTION inventories until the introduction and establishment of the harp trap as the preferred method for survey- The genus Kerivoula contains species that are ing forest-interior bat species in the Old World commonly referred to as woolly bats. This genus, to- (Fran cis, 1989). gether with its sister genus Phoniscus, is classified Although the taxonomy and systematics of under the subfamily Kerivoulinae. Woolly bats Kerivoula species have remained poorly known for occur in both the Indomalaya-Australasia and Afro - many years, several recent studies have gathered tropic ecozones, where, respectively, 12 and seven morphological and/or molecular evidence from mul- distinct species were recognized by Simmons tiple Kerivoula specimens from Indo-Malaya, pro- (2005), although three additional species have been viding some valuable insights into species delimita- described from the former region since then (Bates tion. For example, the systematics and taxonomy of et al., 2004, 2007; Francis et al., 2007). All Keri - Kerivoula from Peninsula Malaysia and Borneo voula species appear to forage in dense vegetation have been explored by combining evidence from using broadband calls (Kingston et al., 1999, 2003; mitochondrial DNA, nuclear DNA, karyotypes and Schmieder et al., 2012) and roost in foliage or tree morphological characters (Khan et al., 2010) as well cavities (e.g., Rossiter et al., 2012). The apparent as by multivariate morphometric analyses (Hasan ability of Kerivoula species to detect and avoid mist and Abdullah, 2011). Similarly, Douangboubpha nets explains their poor representation in species et al. (2016) combined mitochondrial DNA and 2 H.-C. Kuo, P. Soisook, Y.-Y. Ho, G. Csorba, C.-N. Wang, et al. morphometric data to study species delimitation phyl ogenetic analyses. We also collect morphomet- among Thai species, while Francis et al. (2010) gen- ric data from putative related taxa and use multivari- erated mitochondrial cytochrome c oxidase subunit 1 ate statistical analyses. Our phylogenies consistently (COI) barcoding sequences for 110 individual bats point to a monophyletic clade that contains the Tai - covering ten Kerivoula spp. from several SE Asian wan ese Kerivoula, multiple lineages of K. hard- countries. Although such studies have clarified sev- wickii and an additional Kerivoula species from SE eral species’ relationship, they have also highlighted Asia, all to the exclusion of other congeneric species paraphyletic groupings. For example, bats identified including K. titania. Our morphometric analyses in- as K. hardwickii (Horsfield, 1824), K. papillosa dicate specimens currently recognized as K. hard- (Tem minck, 1840) and K. lenis (Thomas, 1916) wickii represents more than one taxon, including the were all shown to comprise multiple divergent line- Taiwanese form, each of which is morphologically ages (Francis et al., 2010), consistent with earlier distinguishable from K. titania. Finally, we provide studies that also suggested ambiguous delimitation a systematic description for the Taiwanese species. or the likely presence of cryptic species (Corbet and Hill, 1992; Bates and Harrison, 1997; Bates et al., MATERIALS AND METHODS 2007). The Taiwanese Kerivoula was first discovered in Molecular Data and Phylogenetic Analyses 1998 by Dr. Ling-Ling Lee (Cheng et al., 2010), however, it has not been formally described and its To determine the phylogenetic position of the Taiwanese taxonomic status has remained confused. Yoshiyuki taxon within the genus Kerivoula we performed separate phylo- genetic tree reconstructions based on the barcoding fragment et al. (2010) considered that this taxon was K. hard- COI (cytochrome c oxidase 1) and on the autosomal nuclear wickii. Later, Wu et al. (2012) examined specimens gene RAG2 (recombination activating gene 2). from Taiwan and Hainan island using morphological For COI, we included published sequences generated by and karyotypic characters as well as a mitochondrial Kuo et al. (2014) and Francis et al. (2010) that were available phylogeny (cytochrome b). The authors concluded from the DRYAD (DOI:10.5061/dryad.f5th5) and BOLD data- bases (Ratnasingham and Hebert, 2007), respectively. In addi- that these bats represented island populations of the tion, we included 16 unpublished COI sequences from Thai continental taxon K. titania (Bates et al., 2007), al- Kerivoula spp. that were newly reported by Douangboubpha et though karyotype or genetic data from K. titania al. (2016), which we obtained from the authors. Finally, we were unavailable and thus did not contribute to this generated COI sequences from two individuals from North assessment. In a more recent study of phylogeogra- Myanmar that showed resemblance to the Taiwanese taxon, collected by P. Soisook, S. S. Lin Oo and U. Pimsai on 6 Febru - phy, Kuo et al. (2014) generated COI barcoding ary 2016. For details of primers and PCR conditions used for sequences from 143 individual bats sampled across these new sequences see Soisook et al. (2016). Sequencing was the whole of Taiwan and found that the 15 unique performed in both directions on a 3500xL Genetic Analyzer haplotypes were similar to those from specimens (Life Technologies). The resultant set of COI sequences sampled from Hunan and Guangxi of China; these (n = 347 — Supplementary Table S1) was aligned using the MUSCLE algorithm (Edgar, 2004) in MEGA 6 (Tamura et al., taxa together represented one of several divergent 2013) and then reduced to sets of unique haplotypes for each lineages referred to as K. hardwickii by Francis et al. taxon (n = 151). (2010). Kuo et al. (2014) emphasized that each of For RAG2, we generated new sequences from one individ- these divergent lineages of ‘K. hardwickii’ was dis- ual each from five taxa; these were K. cuprosa and K. titania, tantly related to the single lineage of K. titania re- both held at the Hungarian Natural History Museum, and the Taiwanese taxon together with the outgroups Harpiola iso- covered in Francis et al. (2010) and, importantly, don and Murina gracilis (see Supplementary Table S1 for de- that specimens referred to as K. titania by Francis tails). For PCR we used the primer pair 179F and 1458R et al. (2010) included multiple vouchers of this (Stadelmann et al., 2007) fol lowing the protocol described in species reported in Bates et al. (2007). Therefore, Kuo et al. (2014). Sequencing was performed in both directions while Kuo et al.’s (2014) finding appeared to dis- on an ABI 3730 DNA Analyzer (Applied Biosystems). These new RAG2 sequences were used as queries in standard nucleo - agree with Wu et al.’s (2012) classification, it did tide BLAST searches (http://www.ncbi.nlm.nih.gov/) to retrieve not clarify the status of the Taiwanese and Hai - RAG2 from other members of the Kerivoulinae. Resulting nanese Kerivoula. RAG2 sequences were aligned by MUSCLE and revealed 25 In this current paper we aim to resolve compre- unique haplotypes (Supplementary Table S1). hensively the phylogenetic position of Taiwanese We inferred phylogenetic