A Taxonomic Revision of the Kerivoula Hardwickii Complex (Chiroptera: Vespertilionidae) with the Description of a New Species

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- (Temminck, 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 phylogenetic 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 relationships within Kerivoulinae using maximum likelihood (ML) and Bayesian approaches, fo- Kerivoula species within the genus. To this end, we cusing on each gene in turn. To root the trees we used Harpiola compile molecular datasets of both mtDNA and isodon and Murina gracilis, both of which belong to the sister ncDNA sequences, to which we apply advanced subfamily Murininae (Hoofer et al., 2003; Hoofer and Van Den New species of the Kerivoula hardwickii complex 3

Bussche, 2003; Stadelmann et al., 2004; Lack et al., 2010; Lack the following named forms: hardwickii s. str. (Horsfield, 1824) and Van Den Bussche, 2010). ML analyses were performed in from Java, crypta Wroughton and Riley, 1913 from SW India, Garli 2.01 (Zwickl, 2006). To obtain node support, we per- depressa Miller, 1906a from E Myanmar, engana Miller, 1906b formed 1000 bootstrap pseudoreplicates as four independent from Engano Island near to Sumatra and malpasi Phillips, 1932 runs, each comprising 250 pseudoreplicates. These were con- from Sri Lanka; these covered most currently recognized sub- ducted with the default setting of a Garli template configuration species of K. hardwickii (Simmons, 2005). ‘garli.3diffModels.bigData’ except that less stringent searching For morphometric comparisons, the following measure- criteria with parameters ‘searchreps’ and ‘treerejectionthresh- ments were taken from adult individuals by HCK, PS or GC old’ were used (reduced from 5 to 1 and from 50 to 20 for using a digital caliper (± 0.01 mm): two external measurements respective parameters). We compensated for these relaxed set- included FA and TIB; 11 craniodental measurements included tings by examining consistency across independent runs, each greatest length of skull (GTL), condylo-basal length (CBL), of which was summarized in SumTrees 4.0.0 of the Python condylo–canine length (CCL), zygomatic breadth (ZB), greatest package DendroPy (Sukumaran and Holder, 2010; 2015). Once width of the braincase (GBB), braincase height (BH), postor- consistency among runs was confirmed, the sum mary bootstrap bital constriction (POC), maxillary toothrow length (C–M3), tree was obtained in SumTrees. posterior palatal width (M3–M3), mandible length (MDL) and

Bayesian analyses were performed in MrBayes 3.2 (Ron - mandibular toothrow length (C–M3). We followed Bates et al. quist et al., 2012) where each Metropolis coupled Markov chain (2007) for definitions of all measurements except for POC and Monte Carlo (MCMCMC) was undertaken with 11 heat chains M3–M3 which were taken at the narrowest width across the con- and a temperature parameter of 0.05. For each dataset, we per- striction posterior to the orbit and the width across the outer bor- formed two independent runs of the MCMCMC to infer chain ders of the upper third molars, respectively. convergence, with each chain run for two million iterations and Two multivariate statistical analyses were used for species sampled at each 200th iteration. In the ML and Bayesian analy- discrimination: principal component analysis (PCA) and ses of each gene, we used the software PartitionFinder to iden- Jombart et al.’s (2010) discriminant analysis of principal com- tify partitions in the sequence data and select the best fitting nu- ponents (DAPC), implemented in the R base package and the R cleotide substitution model based on the Bayesian Information add-in package adegenet (Jombart, 2008), respectively. Both Criterion (Lanfear et al., 2012). multivariate methods are based on dimension-reduction, although DAPC is expected to be more powerful than PCA in Measurements and Morphometric Analyses discriminating groups because it finds functions maximizing inter-group variation. Compared to conventional discriminant Prior to comparing morphological data across species, we anal ysis, DAPC has the advantage of using, as its input, the first tested for sexual dimorphism in the Taiwanese Kerivoula. orthogonal variables summarized by a PCA (Jombart et al., For this, we analysed unpublished physical measurements taken 2010). In both PCA and DAPC, we pooled specimens of differ- by HCK from bats captured across Taiwan during fieldwork in ent sexes due to limited material but also because the sexes 2009 and 2010 (see Kuo et al., 2014). These external measure- showed only slight differences compared to inter-species differ- ments included body mass (MASS), forearm length (FA) and ences. We used seven out of the 11 craniodental measurements length of tibia (TIB); the first one was taken with a Pesola (with GTL, CBL, CCL and ZB excluded) to achieve a greater spring scale (± 0.25 g) while the latter two were taken with dial coverage over specimens which showed missing values on callipers (± 0.1 mm). We first applied standard F-tests to evalu- some measurements. Provided that the number of groups (K) ate whether the two sexes showed unequal variances for each and their memberships needed to be predetermined in DAPC, measurement, and then applied measurement-wise Student’s these were carried out with a k-means clustering algorithm t-tests or Welch’s unequal variances t-tests for assessing dimor- which was implemented with the ‘find.clusters’ function in ade- phism between sexes, depending on the results of the F-tests genet; BIC was used in this analysis for selection of K. (see Zar, 1996). All statistical analyses were performed in R To test the performance of the discriminant functions (DFs) (R Core Team, 2015). from the DAPC, we applied these DFs to 13 other specimens To clarify the status of the Taiwanese Kerivoula, we exam- (hereafter referred to as the ‘testing set’) using the ‘predict.dapc’ ined museum specimens of this taxon together with its putative function in adegenet. These bats included one of the two indi- close relatives (based on the phylogenetic analyses, see viduals from N Myanmar that were recently collected and meas- Results). Specifically, from Taiwan we examined Kerivoula ured by PS due to their close similarity with the Taiwanese specimens held at the Endemic Species Research Institute, Nan- taxon (see Molecular Data and Phylogenetic Analyses). The re- tou (TESRI), the National Museum of Natural Science, Tai - maining 12 bats were reported in Douangboubpha et al. (2016) chung (NMNS), and Tunghai University, Taichung (THU). We as belonging to two forms of K. hardwickii s. lato based on their compared these measurements with data previously collected by groupings in a neighbor-joining phylogeny, and data ana lysed several of us from specimens held at the Institute of Ecology here were kindly provided by the authors. Douang boubpha et al. and Biological Resources (IEBR), Hanoi (catalogue numbers (2016) took a different measurement of braincase breadth (BB) starting CMR and VN), the Natural History Museum, London from ours (GBB) (see Bates et al., 2007 for the distinction); (BMNH), the Harrison Institute, Sevenoaks (HZM), the therefore, to accommodate for missing GBB values, we applied Hungarian Natural History Museum, Budapest (HNHM), the the mean value from our training data in the DAPC to each bat, Naturhistorisches Museum, Wien (NHMW) and the National such that this parameter did not contribute to any discrimination. Museum of Natural History, Smithsonian Institution, Washing - All above 13 specimens of the testing set were sequenced at ton, D.C. (USNM) (see Supplementary Table S2 for details of COI, providing opportunities to examine the concordance specimens examined). For K. hardwickii, which potentially con- between morphological-based and genetical-based clustering. tains multiple species (see Introduction), we examined either In total we compiled a dataset of craniodental measurements holotypes or specimens collected from the type localities of for 76 museum specimens (including the testing set — see 4 H.-C. Kuo, P. Soisook, Y.-Y. Ho, G. Csorba, C.-N. Wang, et al.

Supplementary Table S2) that were used in multivariate analy- showed multiple lineages. Of these, K. lenis and ses (PCA, DAPC) and species discrimination (see Results). K. papillosa together constituted a well-supported Of these individuals, 12 were also included in our phylogenetic monophyletic group, hereafter referred to as the analyses (Supplementary Tables S1 and S2). ‘K. papillosa complex’. Bats assigned to the name Echolocation Recording and Analysis K. hardwickii included three well-supported clades, with widely overlapping ranges in SE Asia, which To characterize the echolocation calls of the Taiwanese are hereafter referred to as ‘K. hardwickii A’, Kerivoula species, we recorded vocalizations from five free ‘K. hardwickii B’ and ‘K. hardwickii C’ (Fig. 1a). In flying individuals in a flight tent (3 m long × 3 m wide × 2 m addition, a fourth well-supported clade of K. hard- high) in the field. These bats, which were not included in mor- wickii was recovered by us, comprising all samples phological or genetic analyses, were captured using harp traps and released after sound recording. Calls were recorded digi- from SE China and Taiwan plus the two recently tally (500-kHz sampling rate, 16-bit resolution) using an Avisoft sampled from N Myanmar; we named this clade as CM16 condenser microphone and UltraSoundGate 416H sys- ‘K. hardwickii D’. Within the wider subfamily tem running Avisoft-Recorder USGH v4.2 software (Avisoft Kerivoulinae, K. hardwickii A, K. hardwickii D and Bioacoust ics, Berlin, Germany) and stored as .wav files. K. kachinensis formed a well-supported monophy- When analysing calls, we followed Kingston et al. (1999) in defining call groups. For each individual, we used BatSound letic group that in turn showed a sister relation- Pro (Pettersson Elektronik AB) software to analyse two call se- ship with K. hardwickii B. This above grouping as quences, each of which was selected from a different call group a whole formed a larger, well-supported monophyl - and comprised five consecutive calls with good signal-to-noise etic group with K. hard wickii C and K. krauensis; ratio. We measured duration from the time amplitude display hereafter we refer to this grouping as the ‘K. hard- and frequency information from FFT power spectrum (512- wickii complex’. All taxa of Kerivoulinae analysed point, Hanning window). Call parameters included peak frequency (frequency with most energy), maximum and minimum except K. pellucida, K. picta and Phoniscus jagorii frequency (-20 dB from peak frequency), call duration, inter- formed an even larger, well-supported monophyletic call interval, bandwidth (frequency difference between maxi- group. mum and minimum frequency), sweep rate (bandwidth/call du- The maximum likelihood (ML) tree mirrored the ration) and call repetition rate (1/call duration plus inter-call Bayesian tree in terms of recovering monophyletic interval). From four of the five individual bats analysed, we further measured parameters for the call group, including call- groups at levels of the whole subfamily, the K. pa- group size (number of calls in the group), call-group duration, pillosa complex, distinct lineages of K. hardwickii, inter-group interval, and call-group repetition rate. and most individual species. Each of these clades received good support (with a bootstrap value of ≥ 70) RESULTS with the exception of K. titania (Fig. 1a). In contrast to the Bayesian analysis, no supraspecific relation- Kerivoula hardwickii Complex Delimitation by ships were resolved with a good support in the ML Molecular Phylogenetics analysis. Levels of COI sequence divergence among members of the K. hardwickii complex were meas- The COI alignment spanned 657 base pairs (bp), ured with the Kimura 2-parameter distance (Kimura, of which 269 and 249 were variable sites and 1980) in MEGA, and are provided in Supplementary parsimony informative sites, respectively. For this Table S3. Distinct clades of K. hardwickii s. lato alignment, a best partitioning scheme selected in were divergent from each other by at least 10% at PartitionFinder was one separating each codon posi- this gene. tion into a partition. For Bayesian and ML analyses, The RAG2 alignment spanned 1054 bp, of which best nucleotide substitution models selected for the 119 and 72 were variable sites and parsimony in- first, second and third partitions were SYM + I + G, formative sites, respectively. PartitionFinder se- F81 + I and GTR + G, and TrNef + I + G, F81 + I lected a partitioning scheme separating the three and TrN + G, respectively. The Bayesian tree re - codon positions as different subsets and selected covered the Kerivoulinae as a well-supported nucleotide substitution models as HKY + I, HKY (posterior probabilities, PP ≥ 0.95) monophyletic and HKY + G for the first, second and third codon group (Fig. 1a; see also Supplementary Fig. S1 for positions, respectively (all these models were com- the same tree with voucher IDs provided). Within patible with MrBayes). Both Bayesian and ML trees this clade, samples were further clustered into well- recovered Kerivoulinae as a monophyletic group supported monophyletic groups corresponding to with good support (Fig. 1b; see also Supplementary species identities, with the exception of K. hard- Fig. S2 for the same tree with voucher IDs wickii, K. lenis and K. pa pillosa, each of which provided). Within Kerivoulinae, bats assigned to New species of the Kerivoula hardwickii complex 5 A–C are 1.00/100 D 0.69/57 K. hardwickii 1.00/100 K. hardwickii 1.00/74 0.96/75 0.72/61 1.00/88 1.00/100 Murina gracilis 0.80/61 0.005 substitutions/site K. cuprosa 1.00/86 1.00/100 bp) and (b) autosomal gene (1054 bp). For major K. titania Harpiola isodon (after slash). In (a), clades K. pellucida complex K. minuta K. intermedia K. papillosa , b. and s. lato discovered in this study named as K. hard-

complex: Kerivoula K. krauensis K. hardwickii Taiwanese K. kachinensis, wickii sensu lato probabily K. hardwickii complex K. papillosa C: A: B: D: K. minuta K. kachinensis K. hardwickii S Laos, Vietnam, Thailand, Malaya and Borneo K. hardwickii Central(C)–S Laos, C–S Vietnam and N Thailand K. krauensis K. titania K. hardwickii N–S Laos, S Vietnam and N–S Thailand K. hardwickii Taiwan, SE China and N Myanmar Phoniscus jagorii K. intermedia K. picta 1.00/100 K. pellucida 1.00/93 1.00/100 1.00/81 1.00/90 1.00/81 1.00/98 1.00/66 1.00/72 0.04 substitutions/site 1.00/96 1.00/65 1.00/99 1.00/80 Murina gracilis 1.00/100 0.94/<50 0.99/<50 0.98/<50 0.98/<50 0.92/<50 1.00/<50 Harpiola isodon 1.00/87 named following Douangboubpha et al. (2016), with an additional clade of a. . 1. Bayesian 50% majority-rule consensus trees for Kerivoulinae species reconstructed based on (a) mitochondrial COI gene (657 IG clades, node support values are shown above branches as Bayesian posterior probabilities (before slash) and ML bootstrap values clades, node support values are shown above branches as Bayesian posterior probabilities (before slash) and ML F 6 H.-C. Kuo, P. Soisook, Y.-Y. Ho, G. Csorba, C.-N. Wang, et al.

K. intermedia and K. pellucida each formed well- that 91.0% of the total variance could be explained supported monophyletic groups, while a grouping by the first two components (76.7% and 14.3% for for samples of K. minuta received virtually no sup- PC1 and PC2, respectively). For PC1, all measure- port. Among other species, membership of K. hard- ments had negative loadings, among which most wickii and K. papillosa complexes were recovered had similar loading levels (visualized in Fig. 2, with as in the COI-based analyses although the assign- values provided in Supplementary Table S4), sug- ment of K. krauensis into the former group was not gesting that this PC reflected the skull size of bats, assessed with RAG2 due to unavailability of mate- with larger-sized bats characterized by lower PC1 rial. Supraspecific relationships were better resolved scores. Thus, K. kachinensis was shown to have at this marker than at COI. Both Bayesian and ML a distinctly larger skull than all the other taxa, while trees suggested that within the Kerivoula species K. hardwickii had a smaller skull than both K. tita- studied, a close relationship exists among the nia and the Taiwanese taxon, which were compara- K. hardwickii complex along with K. titania and ble to each other (Fig. 2). On PC2, all measurements a clade comprising K. minuta plus K. intermedia. In had low loadings except for braincase height (BH) both type of analyses, strong evidence was found for and postorbital constriction (POC), which showed a sister relationship between these bats and the high positive and negative loadings, respectively K. papillosa complex to the exclusion of K. pellu- (Fig. 2). Therefore, based on PC2, the Taiwanese cida and K. cuprosa. Ke rivoula had a smaller BH and larger POC than K. titania, while specimens recognized as K. hard- Species Discrimination Revealed by Morphometric wickii showed much greater variation for these Analyses measurements, exceeding the pooled variation of the former two taxa (Fig. 2). F-tests revealed that female and male Taiwanese For our k-means clustering analysis and DAPC, Kerivoula had unequal variances for their body we did not include K. kachinensis due to this taxon mass (F67, 80 = 2.22, P < 0.001) and equal variances being clearly separated in the PCA. The DAPC con- for fore arm (F77, 81 = 1.12, P = 0.62) and tibia taining K. hardwickii s. lato, K. titania, and the Tai- (F76, 81 = 0.77, P = 0.25 — Table 1). Overall, fe- wanese taxon was able to split the former taxon into males were found to be significantly larger than two clearly distinguishable groups, but was less able males based on body mass (Welch’s t-test, t = 9.57, to separate the latter two taxa from each other (result n = 68 and 81, P < 0.001), forearm (Student’s t-test, not shown). By removing K. titania and rerunning t = 9.49, n = 78 and 82, P < 0.001) and tibia the DAPC, the split was still evident within K. hard- (Student’s t-test, t = 3.27, n = 77 and 82, P < 0.01 — wickii s. lato, and the Taiwanese taxon remained Table 1). separate (see Fig. 3a–b). Therefore we removed Given that the Taiwanese Kerivoula is phyloge- K. titania from subsequent k-means clustering netically closely related to K. hardwickii and analysis and DAPC. For k-means clustering, we K. kachinensis (which together constitute the evaluated the model fit of increasing numbers of K. hardwickii complex) we conducted multivariate clusters (K) using the Bayesian Information Cri- morphometric comparisons among these taxa, along terion (BIC). By comparing successive models, we with K. titania. Principal component analysis (PCA) recorded significantly improved fit from K = 1 to 3, based on seven craniodental measurements revealed however fit increased only marginally from K = 3 to 4, as seen in the curve of BIC values when plotted against K (Supplementary Fig. S3). We therefore chose K = 3 as the most likely number of clusters, TABLE 1. Body mass (MASS, g), forearm length (FA, mm) and length of tibia (TIB, mm) of Kerivoula furva sp. n. measured in with one cluster containing all specimens from the field (Kuo et al. 2014). Values are means and standard Taiwan plus three ‘K. hardwickii’ specimens from deviations with the range in the following row. Sample sizes are the India-Myanmar boundary, and the remaining given in parentheses two clusters both containing other ‘K. hardwickii’ Sex MASS FA TIB from across SE Asia. ♀♀ (78) 5.79 ± 0.50 35.1 ± 1.0 17.9 ± 0.6 For the discriminant analysis step of the DAPC, 4.75–7.25 (68)a 32.5–37.5 16.4–19.1 (77) we entered the first three PCs from the PCA step, ♂♂ (82) 5.10 ± 0.34 33.6 ± 1.0 17.5 ± 0.7 which together explained 91.6% of the total vari- 4.25–5.75 (81) 30.9–35.7 15.8–19.1 ance. We found two functions (DFs) for discriminat- a — excluding pregnant females ing among individuals from the three clusters from New species of the Kerivoula hardwickii complex 7

−0.75 −0.50 −0.25 0.00 0.25

? GBB, C–M3 3 3 M –M , C–M3

012 MDL PC2 (14.3%) −0.5 0.0 0.5 1.0

−1 POC

−6 −4 −2 02

PC1 (76.7%)

FIG. 2. PCA of Kerivoula spp. based on seven craniodental measurements, showing projections of individual specimens and variable loadings on the first two principal components. Scales for the projections are displayed on the bottom and left axes while those for variable loadings are displayed on the top and right axes. Specimens are recognized as K. kachinensis (black diamonds), K. titania (black triangles) and K. hardwickii as well as its allies (dots). The last group is further classified into K. furva sp. n. (green dots, with those from the India-Myanmar boundary bordered in red), K. depressa (orange dots) and K. hardwickii (blue dots) based on the DAPC. See Fig. 3 for explanation of the specimen indicated by a question mark

the k-means clustering analysis. Among the original from Kalimantan, Borneo (HZM 11.36540) (PP = variables (the seven craniodental measurements), 0.55 for most likely cluster — Fig. 3b). POC, GBB and M3–M3 (widths of the skull) had In the DAPC, bats belonging to the blue group negative higher loadings on DF1, while BH (brain- included either holotypes or specimens collect- case height) had a positive higher loading on this ed from the type localities of hardwickii, crypta, function; all original variables had low loadings on engana and malpasi (Fig. 3b and Supplementary DF2 except for BH which had a positive high load- Table S2). Given that this group was the only one of ing (visualized in Fig. 3a, with values provided in the three to contain the nominate subspecies hard- Sup plementary Table S4). Based on DF1, the Tai - wickii, we considered that all individuals in the blue wanese Ke rivoula plus the three ‘K. hardwickii’ group represent K. hardwickii s. stricto. We consid- specimens (green group in Fig. 3a) all had wider ered the bats from both the orange and green groups skulls than did the bats from the other two groups. are distinct species from each other as well as from Based on DF2, one ‘K. hardwickii’ group (blue K. hardwickii s. str. The orange group contains both group) had larger BH than the other ‘K. hardwickii’ the holotype and paratype of depressa (Fig. 3b and group (orange group) while the green group had Supplementary Table S2), and therefore we assigned intermediate BH compared to the former two (Fig. these bats to K. depressa. Other individuals from 3a). With these two DFs, unambiguous reclassifica- this group were captured in Myanmar and Vietnam tion to one of the three clusters (PP > 0.89) was ob- as well as in Cambodia, where it occurs alongside tained for all individual bats except for one collected K. hardwickii s. str. (Fig. 3b). Finally, we consider 8 H.-C. Kuo, P. Soisook, Y.-Y. Ho, G. Csorba, C.-N. Wang, et al.

a. −4 04

C–M3 M3–M3 C–M3 MDL GBB

POC 04 02

DF2 ? −4 −4 −2

−2 0 2

DF1 b.

? 0.0 1.0 Membership probability From Java From Taiwan From NE India from SW India From Sri Lanka from E Myanmar From N Myanmar from SW Sumatra crpta From Kalimantan Borneo engana depressa Type of From Cambodia and Vietnam Type of From unknown locality of Malaysia Type of c. ST NT NM 0.0 1.0 Membership probability 2011.17 (A) 2011.19 (B) 2011.52 (B) 2011.14 (A) 2012.61 (C) PSUZC-MM 2012.171 (C) 2012.163 (C) 2012.160 (C) 2012.161 (C) 2012.162 (C) 2012.170 (C) 2012.169 (C) PS160206.3 (D)

FIG. 3. DAPC of Kerivoula furva sp. n. (coloured green), K. depressa (orange) and K. hardwickii (blue) based on seven craniodental measurements. Forty seven specimens of these taxa were used to establish discriminant functions (DFs) in the DAPC. Plot (a) shows projections of these 47 specimens and variable loadings on the two DFs obtained, with scales displayed as in Fig. 2. Plot (b) shows posterior probabilities of the same 47 specimens that are reclassified by the DAPC into the three groups predetermined with a k-means clustering algorithm. Thirteen other specimens are used to assess the discriminating power of the two DFs, and the posterior probabilities of these specimens’ classification into the three groups are shown in plot (c). In (a), specimens of K. furva sp. n. from the India-Myanmar boundary are bordered in red. In (a–b), one specimen which received an ambiguous reclassification (posterior probability = 0.55 for the most likely group) is indicated by a question mark. In (c), uppercase letters above the posterior probability plots denote sampling sites (ST for South Thailand, NT for North Thailand and NM for North Myanmar), while those in the parentheses indicate mitochondrial clades recovered for corresponding specimens New species of the Kerivoula hardwickii complex 9 that bats from the green group belong to a species dry skin, skull and postcranial skeleton and TESRI that has not yet been described, and which occurs in B1401, adult ♀, dry skin, skull and postcranial Taiwan and at the India-Myanmar boundary where skeleton, the above four from TAIWAN, Taichung it is sympatric with K. depressa (Fig. 3b). We City, Heping District, Wushikeng Low Altitude name this new species as Kerivoula furva sp. n. for Experimental Station of TESRI, 24°16.42’N, which a formal description is provided in the next 120°57.059’E., 1056 m a.s.l.; HNHM 2005.66.1, section. adult ♂, in alcohol with skull extracted, collected at An analysis to assess the performance of the two TAIWAN, Tainan City, Dongshan District, Gaoyuan DFs based on a testing set of 13 bats revealed con- Village; NMNS 13946, adult ♂, dry skin, skull in siderable power. In particular, we found that bats THU (B040017), NMNS 13947, adult ♂, dry skin, belonged to distinct COI lineages (K. hardwickii A, skull in THU (B040018), NMNS 13948, adult K. hardwickii B and K. hardwickii C) were unam- ♀, dry skin, skull in THU (B030041) and NMNS bigiously separated. More specifically, the two DFs 13952, juvenile ♂, dry skin, skull in THU unambiguously classified the four specimens of (B030028), the above four from TAIWAN, K. hardwickii A and K. hardwickii B as K. depressa, Kaohsiung City, Taoyuan District, Meilan Logging and classified the eight K. hardwickii C as K. hard- Road, 23°17.24’N, 120°49.46’E, 920 m a.s.l.; wickii s. str. (PP > 0.98 in all cases — Fig. 3c). We NMNS 10806, adult ♂, dry skin, skull in THU also tested these DFs on one of the two specimens (B040007) and NMNS 14039, adult ♀, dry skin, recently captured from N Myanmar that was phylo- skull in THU (B030025), both from TAIWAN, genetically clustered into K. hardwickii D (Fig. 1a). Kaohsiung City, Taoyuan District, Meilong Logging The two DFs classified this latter specimen as K. fur - Road, 23°5.70’N, 120°44.28’E, around 800 m a.s.l.; va sp. n. with strong support (PP > 0.99 — Fig. 3c). Field number PS160206.3 (to be subsequently deposited in the Zoological Collection of the Univer- SYSTEMATIC DESCRIPTION OF NEW SPECIES sity of Mandalay), adult ♀, in alcohol with skull extracted, collected from MYANMAR, Kachin, Putao Kerivoula furva sp. n. Township, Wang Hlaing Dam Village, 27.479°N, (Figs. 1–6, Tables 1–2) 97.169°E, 863 m a.s.l.

Holotype Referred specimens NMNS 17595, adult ♀, dry skin, skull and post- BMNH 21.1.6.32, adult ♀, INDIA, Meghalaya, cranial skeleton, collected by Yi-Wen Chen, 29 Mar Jaintia Hills; BMNH 50.432, adult ♂, and BMNH 2009. 50.438, adult ♀, both from MYANMAR, Kachin, Nam Tamai; Field number PS160206.1 (to be subse- Type locality quently deposited in the Zoological Collection of the TAIWAN, Yilan County, Yuanshan Township, 3 University of Mandalay), adult ♂, from MYAN- km East of Shuanglianpi, 24°45.21’N, 121°39.63’E, MAR, Kachin, Putao Township, Wang Hlaing Dam 180 m a.s.l. Village. The two specimens from CHINA, Hainan (IBHG 08279 and IBHG 08280, both ♀) which are Paratypes consistently supported by morphological, kary- NMNS 17657, adult ♀, dry skin, skull and post- otypic and molecular evidence to have a close rela- cranial skeleton and NMNS 19136, adult ♀, dry tionship with K. furva sp. n. in Taiwan (Wu et al., skin, skull and postcranial skeleton, both from the 2012) are considered to belong to this new species. type locality; THU 07.X.25.9, adult ♀, dry skin and In addition, the following specimens from CHINA skull and THU 07.X.25.11, adult ♂, dry skin and which formed the COI lineage K. hardwickii D along skull, both from TAIWAN, Yilan County, Yuanshan with K. furva sp. n. in Taiwan (Fig. 1a) are also as- Township, Shuanglianpi, 24°45.04’N, 121°38.05’E, signed to this new species: ROM MAM 116079, ♀, around 500 m a.s.l.; HNHM 2016.8.1, adult ♂, in al- Guangxi, Jing Xin County Provincial Nature Reser - cohol with skull extracted, collected at TAIWAN, ve; ROM MAM 114918, ♂, ROM MAM 114944, Taoyuan City, Fuxing District, 2.5 km SE of Xiao ♀, both from Hunan, Shuhuangshan Nature Reser- Wulai, 24°47.01’N, 121°24.07’E, 650 m a.s.l.; ve; ROM MAM 114961, ♂, ROM MAM 114962, TESRI B1398, adult ♀, dry skin, skull and postcra- ♀, ROM MAM 114963, ♀, ROM MAM 114964, ♀, nial skeleton, TESRI B1399, adult ♀, dry skin, skull ROM MAM 115009, ♀, ROM MAM 115041, and postcranial skeleton, TESRI B1400, adult ♀, ♀, ROM MAM 115042, ♀, ROM MAM 115047, 10 H.-C. Kuo, P. Soisook, Y.-Y. Ho, G. Csorba, C.-N. Wang, et al.

♂, from Hunan, Daweishan National Park. The its width, while the anterior palatal emargination is assignments of the ROM specimens to K. furva sp. approximately the same long as its width (Fig. 5a– n. are solely informed from a mitochondrial gene, b). The sagittal crest is absent. The braincase is and thus required further verification based on mor- broadened and flattened. The basioccipital pits are phological or nuclear gene data (see Discussion). well-developed, and widen anteriorly and taper pos- teriorly beyond the front one-third of the cochleae Etymology (Fig. 5b). The C–M3 is 5.52–6.11 mm (Table 2; see The name refers to the very dark pelage of the Fig. 5c–d for qualitative characters of the upper den- new species. tition). The first upper incisor (I2) has a height approximately two-thirds that of the upper canine. The Diagnosis second upper incisor (I3) has a height half that of I2, Kerivoula furva sp. n. has a darkest dorsal pelage while the crown areas of these two are about the among the Asian Kerivoula species, with dorsal same. The upper canine has a height surpassing that hairs that are broadly uniformly coloured through- of the other teeth in the upper toothrow, and has a out their length. Its body size is comparable to that length that is approximately equal to its width. The of K. titania and K. hardwickii s. str., however, com- first upper premolar (P2) and the second upper pre- pared to the former, K. furva sp. n. has a propor- molar (P3) are both slightly shorter than their respec- tionally shorter tibia and a proportionally wider pos- tive widths; the latter tooth has a height and a crown torbital constriction, whereas compared to K. hard- area approximately 80% and 90% the respective di- wickii s. str., it has a proportionally lower braincase mensions of the former tooth. The third upper pre- height. Kerivoula furva sp. n. and K. depressa have molar (P4) has a length along its labial border longer skulls that are similar in shape while the former than that along its lingual border, making this tooth taxon has a slightly larger skull than the latter. Keri - somewhat triangular in shape. P4 has its height and voula furva sp. n. has a generally larger and more crown area 30–40% and 70% larger than respective flattened skull compared to K. krauensis. dimensions of P2; P4 has its height two-thirds that of the upper canine while having its crown area 10% Description larger than that of the latter tooth. The first and sec- A medium-sized Kerivoula, with a body mass of ond upper molars (M1 and M2, respectively) both 4–7 g and a forearm length of 30.9–37.5 mm (Tables have typical W-shaped cusp structure, with para-, 1 and 2). The ear is 14.3–15.2 mm in length (n = 4); meso- and metastyle all well developed. The third it is funnel shaped and has a fold extending from the upper molar (M3) has reduced mesostyle and meta- base of the pinna to the half of the posterior border. cone and has the metastyle absent. The edge of the pinna is darkly pigmented, contrast- The mandible has a MDL of 9.59–10.86 mm and ing strongly with the paler central part (Fig. 4a). The has a C–M3 of 5.86–6.44 mm (Table 2; see Fig. 5e– tragus is long and pointed, and curves slightly out- f for qualitative characters of the lower dentition). wards at the tip. In some individual bats examined, The first and second lower incisors (I1 and I2, re- the tip of the tragus is dark while in others the tragus spectively) are tricuspid. The third lower incisor (I3) is pale in colour throughout its length. The dorsal is unicuspid, whilst it has a large cingular cusp on pelage varies from black brown to black grey. the lingual posterior border in addition to another Individual dorsal hairs are dark brown and are smaller one on the labial posterior border. The lower broadly uniform in colour except for the tips, which canine has a large cingular cusp on its lingual ante- are even darker (Fig. 4b). The ventral pelage is grey- rior border, which is higher than I3. The first, second ish brown. Individual ventral hairs have distinct and third lower premolars (P2, P3 and P4, respec- dark brown bases; those on the chest and along the tively) are similar to each other in both height and middle line of the abdomen have medium brown tips crown area except for P3, which has a comparatively while those along the flanks of the abdomen have smaller (90%) crown area. The three lower premo- pale grey tips (Fig. 4c). Some individual bats have lars have heights of ~75% of the lower canine. The grey-tipped hairs over the entire ventral surface. The first and second lower molar (M1 and M2, respec- base of the thumb swells to form an oval, fleshy pad. tively) both have well developed talonids which The insertion point of the wing membrane is at the have widths exceeding those of the trigonids of cor- base of the first toe. responding teeth. The third lower molar (M3) has The skull has a GTL of 14.07–15.45 mm (Table a reduced talonid whose width is smaller than that of 2). The narial emargination is two-fold longer than the trigonid of the same tooth. New species of the Kerivoula hardwickii complex 11

FIG. 4. Photographs of K. furva sp. n., showing (a) a live individual (no voucher) as well as (b) dorsal and (c) ventral views of the skin specimen of holotype (NMNS 17595). Scale bars in (b–c) =10 mm. Photo (a) by Cheng-Han Chou

Comparisons the cranium, K. furva sp. n. has a proportionally While Wu et al. (2012) referred to K. furva sp. n. larger POC than K. titania (Fig. 2 and Fig. 6 bottom as K. titania, the former species is distinct in having row). a proportionally shorter tibia. In K. furva sp. n. the Among phylogenetically close relatives (i.e., ratio of the tibia to the forearm (TIB/FA) ranged K. hardwickii complex), K. kachinensis is a diagnos- 0.48–0.51 (n =13) for museum specimens and 0.48– tically larger-sized species compared to K. furva sp. 0.55 (n =159) for live individuals caught in the field. n. (Fig. 2 and Table 2). Kerivoula hardwickii s. str. In comparison, the reported TIB/FA for K. titania and K. furva sp. n. broadly overlap in both body and ranges 0.55–0.57 (n =6) for museum specimens. In skull size (Fig. 2 and Table 2), while the former 12 H.-C. Kuo, P. Soisook, Y.-Y. Ho, G. Csorba, C.-N. Wang, et al. ), et 3 –M 3 – — data from Francis d def def 3 ), posterior palatal width (M 3 5.1–5.4 (7) 7.2–8.9 (7) def — data from (1941); Tate c e MDL C–M of skull (GTL), condyle-basal length (CBL), condyle- 9.4 (1) 12.47 8.08 7.05 (7) 11.3–12.0 def def 3 –M 3 tion (POC), maxillary toothrow length (C–M M a 4.7–5.1 (7) 12.6 (6) 11.5–12.5 def 3 e a 4.9–5.9 (7) — with an ambiguous reclassification in DAPC (Fig. 3); spp. Sample sizes are given in the parentheses 13.5 13.3 (1) b ef e (2016) Kerivoula a et al. 3.2 (1) 15.4 –– – – – – 13.0–15.5 (6) def — Data from Miller (1906a); a e ); 3 — data from Struebig a c f (2016); et al. ♂ (1) 31.9 17.0 13.82 12.95♂ (1) 12.38 5.39 – 3.20 7.13 5.30 4.97 9.40 5.56 ♀♀ (9)♂♂ (17)♀♀ (4) 33.3–34.6 (6)♂♂ (4) 31.5–33.3 (7)♀♀ (4) 40.4–43.2♂♂ (4) – 40.4–43.4 (3) 15.6 (1) 32.4–34.7 33.0–34.3 (3) 22.7–23.4 (3) 21.8–23.5 13.53–15.00 (13) 17.94–18.29 18.4–19.0 (3) 18.5–19.7 (3) 12.13–13.77 (13) 14.27–14.83 (8) 17.14–18.39 15.23–15.74 15.23–15.83 12.00–13.25 16.48–17.31 13.11–13.65 (8) 16.08–16.63 13.51–14.32 (3) 12.56–13.20 14.03–14.49 8.03–8.92 (16) 16.09–16.53 13.12–13.65 (3) 15.48–16.11 7.02–7.31 (10) 8.35–8.93 (7) 13.54–13.83 (3) 8.69–9.11 10.66–10.95 10.16–10.95 6.98–7.44 7.55–7.77 (8) 8.50–9.08 8.22–8.44 8.13–8.42 7.47–7.95 ♀♀ (9)♂♂ (17)♀♀ (4)♂♂ (4) 5.39–5.81♀♀ (4) 5.44–6.02♂♂ (4) 5.11–5.86 5.48–5.88 3.02–3.29 5.17–5.67 2.95–3.42 5.29–5.58 3.49–3.70 5.47–5.80 3.51–3.70 5.21–5.78 3.17–3.58 6.64–7.15 3.22–3.29 6.91–7.16 5.23–5.60 5.98–6.21 5.04–5.67 5.80–6.24 6.35–6.47 6.39–6.59 9.83–10.31 9.28–10.47 5.34–5.68 5.05–5.60 12.15–13.01 5.79–6.18 12.60–12.95 5.73–6.09 10.34–10.81 7.14–7.58 10.26–10.63 7.44–7.56 6.25–6.51 5.98–6.41 ♀♀ (7)♂♂ (7) 31.2–34.2 (4) 30.4–33.2 (4) 16.0 (1) 15.3 (1) 13.56–14.61 (5) 13.57–14.34 12.69–13.28 (5) 12.20–12.76 (6) 12.32–13.22 (6) 8.16–8.21 (6) (2) 11.77–12.76 7.71–8.20 (5) 6.85–7.31 (5) 6.75–7.29 (5) ♀♀ (7)♂♂ (7) 4.30–5.07 4.27–5.09 3.12–3.36 2.87–3.31 5.16–5.50 4.93–5.57 4.83–5.24 4.85–5.29 9.17–9.88 9.11–9.91 5.30–5.73 5.11–5.86 ♂♂ (4) 31.7–34.7 15.8–17.7 14.07–14.96 12.60–13.50 12.47–13.34 8.45–9.25 7.15–7.40 ♀♀+♂♂ 28.0–34.0 (63) ♀♀+♂♂ 5.5 (1) Holotype ? (1) 32.0 Holotype ? (1) – – 5.54 5.22 – 5.87 b b Holotype ♀ (1) 4.86 3.07 5.38 4.91 9.50 5.80 Holotype ♀ (1) 32.8 ? ? — data from Douangboubpha e Species Sex FA TIB GTL CBL CCL ZB GBB Species Sex BH POC C–M sp. n. fromsp. n. from ♀♀ (10) ♀♀ (3) 5.01–5.38 4.75–5.11 3.35–3.74 3.32–3.41 5.66–6.11 5.64–5.77 5.15–5.69 5.38–5.59 10.00–10.86 6.02–6.44 10.24–10.51 6.05–6.17 sp. n. from Taiwansp. n. from ♀♀ (10) 33.6–36.2 (9) 16.7–18.6 (9) 14.69–15.45 12.99–13.82 12.92–13.82 9.01–9.38 (9) 7.30–7.67 sp. n. from ♀♀ (3) 34.0–36.1 18.2 (1) 14.51–14.60 (2) 13.54–13.70 (2) 12.78-13.23 8.70–8.80 (2) 7.16–7.25 2. Ranges of external and craniodental measurements (all in mm) for 2. Extended (2007); India-Myanmar ♂♂ (2) 5.02 (1) 3.42 (1) 5.52 (1) 5.52 (1) 9.59 (1) 5.86 (1) India-Myanmar ♂♂ (2) 32.2–35.3 17.0 (1) 14.27 (1) 13.28 (1) 12.49 (1) 8.62 (1) 7.34 (1) Taiwan ♂♂ (4) 4.86–5.16 3.27–3.59 5.58–5.75 5.18–5.45 9.90–10.41 5.92–6.06 ABLE ABLE K. kachinensis K. titania K. krauensis K. hardwickii K. hardwickii K. hardwickii K. hardwickii K. kachinensis K. titania K. krauensis K. depressa K. depressa K. depressa K. furva K. depressa K. depressa K. furva K. furva K. furva K. depressa T T al. External measurements include forearm length (FA) and length of tibia (TIB); craniodental measurements include greatest External measurements include forearm length (FA) canine length (CCL), zygomatic breadth (ZB), greatest width of the braincase (GBB), height (BH), postorbital constric mandible length (MDL) and mandibular toothrow (C–M New species of the Kerivoula hardwickii complex 13

Fig. 5. The skull of K. furva sp. n. (paratype, ESRI B1398), showing (a–b) dorsal and basal views of the cranium, (c) occlusal view of the left upper toothrow, (d–e) lateral views of the cranium and the mandible, and (f) occlusal view of the left lower toothrow. Scale bars = 5 mm species has a proportionally larger BH than the latter hairs of K. kachinensis have darker grey bases and one (Figs. 2–3 and Fig. 6 middle row). Kerivoula paler grey-brown upper segments (Bates et al., depressa has a smaller skull size compared to K. fur- 2004; Soisook et al., 2007), those of K. titania have va sp. n. (Fig. 2 and Table 2). Kerivoula krauensis is light grey middle segments above black bases (Bates smaller than K. furva sp. n. in general, with more ex- et al., 2007), and those of K. krauensis are dark treme differences between the two on TIB and on brown with shiny golden-brown tips (Francis et al., lengths of the skull (Table 2). In addition, the brain- 2007; Struebig et al., In press). In comparison, case of K. krauensis is domed like that of K. hard- K. furva sp. n. has a diagnostically darker dorsal wickii s. str. compared to the flat skull of K. furva sp. pelage that shows virtually no colour distinction n. The ratio of BH to C–M3 in K. furva sp. n. is between the root and the middle section of individ- smaller than that of K. hardwickii s. str. (Fig. 6 mid- ual hairs. dle right panel) and, based on a measurement reported by Douangboubpha et al. (2016), is much Echolocation smaller than that of K. krauensis (1.10). Kerivoula furva sp. n. produced echolocation The dorsal pelage of K. furva sp. n. is clearly calls in groups consisting of 4.4–9.8 pulses and distinguishable from that of all congeneric species with repetition rates among bouts of 5.0–9.3 Hz described above. Kerivoula hardwickii s. str. was (Table 3). Within bouts, individual pulses had peak described to have “dark bases of the fur on the up- frequencies at 141.8–163.3 kHz, sweeping down perparts …with the majority of the hair pale grey or from 182.4–222.1 (maximum frequency) to 105.7– brown” (Francis et al., 2007: 5–6), while K. de- 118.3 (minimum frequency) kHz, and were charac- pressa was described as having “upperparts between terized by durations of 1.4–2.0 ms and repetition buff and cream-buff” (Miller, 1906a: 65). The dorsal rates of 50–83.8 Hz. Further details of parameters 14 H.-C. Kuo, P. Soisook, Y.-Y. Ho, G. Csorba, C.-N. Wang, et al.

GBB C-M3 5.8 5.0 5.4 6.2 6.8 7.2 7.6

BH/GBB BH/C-M3 0.70 0.75 0.80 0.85 0.95 1.05 0.65

POC/GBB POC/C-M3 0.66 0.42 0.46 0.50 0.54 0.58 0.62

furT furC dep har tita furT furC dep har tita

FIG. 6. Standard boxplots for absolute values (top row) and ratios (middle and bottom rows) of selected craniodental measurements of Kerivoula spp. Abbreviations for taxa include furT for K. furva from Taiwan, furC for K. furva from the continental India- Myanmar boundary, dep for K. depressa, har for K. hardwickii and tita for K. titania New species of the Kerivoula hardwickii complex 15 for echolocation calls are presented in Table 3. In Hai nanese and Taiwanese specimens, showing general, K. furva sp. n. emitted individual calls that diploid chromosome number (2n) = 32 and funda- were typical to its genus (see Kingston et al., 1999; mental number (FN) = 51–52. The diploid chromo- Schmieder et al., 2012), characterized by very some number differed from those of K. hardwickii, broad bandwidths (69.4–103.9 kHz), extremely high K. intermedia, K. lanosa, K. lenis, K. minuta and K. frequencies and very high repetition rates, all of papillosa (reviewed in Wu et al., 2012). Phylo- which are considered to be specializations for forag- genetic analyses conducted in this study based on ing in cluttered environments such as dense vegeta- partial sequences of the mitochondrial COI gene and tion (also see Siemers and Schnitzler, 2000; Schnitz - of the nuclear RAG2 gene consistently recovered ler and Kalko, 2001; Siemers and Schnitzler, 2004). close relationships among K. furva sp. n., K. hardwickii s. lato and K. kachinensis, to the exclusion of Distribution and ecological notes other congeneric species (Fig. 1). Kerivoula krauen- Currently known from Taiwan, Meghalaya in sis may also belong to this complex based on the India, Kachin in Myanmar (this study), Hainan of COI-based phylogeny. Recently, Kuo et al. (2014) SE China (Wu et al., 2012) and probably also from investigated the phylogeography of K. furva sp. n. Hunan and Guangxi of SE China (Fig. 1). Kerivoula across its range in Taiwan. Based on genotyping in- furva sp. n. has a wide range in Taiwan where it dividuals at nine autosomal microsatellite loci, the occurs in montane and hilly areas with elevations of authors found strong genetic differentiation among <1800 m a.s.l. (Kuo et al., 2014). In such areas, populations across the island, with evidence of re- K. furva sp. n. has been in broadleaf evergreen stricted East-West gene flow, indicating that the forests with low to medium-level disturbance, in- Central Mountain Range acts as a barrier to move- cluding forests mixed with betel palm plantations, ment. No such phylogeographic structure was ob- banana orchards or bamboo stands. In Myanmar, served when studying haplotypes of the COI gene, two individuals of K. furva sp. n. were captured in although this appears to reflect this species’ recent a lowland degraded forest at a foothill. This forest colonization history on Taiwan (Kuo et al., 2014). mainly comprises bamboo and banana with some secondary growth trees, and is located between DISCUSSION a village and a primary evergreen forest. By analysing arthropod fragments in feces, Since its discovery, the taxonomy of the Chiang (2006) reported the diet of K. furva sp. n. to Kerivoula on Taiwan has been confused, with sev- be composed principally of spiders (order Araneae), eral names used. To establish its status, we applied followed by cockroaches (Blattodea). Chiang (2006) comparative genetic and morphological analyses to suggested K. furva sp. n. is a gleaner given that its this taxon and its putative relatives. As is often the diet comprised arthropods with no or poor flying case in systematic studies, archived material, meas- ability. Liao (2013) demonstrated in a behavioural urements, and genetic data were not all available experiment that K. furva sp. n. can perform aerial across all specimens. Therefore we adopted a two- hawking in addition to glean prey from the substrate step procedure. First, we used phylogenetic analyses with the interfemoral membrane. Liao’s (2013) sec- to identify all close relatives of our focal taxon ond experiment indicated that K. furva sp. n. can without making firm conclusions about boundaries hunt using prey-generated sound. It was reported between species. Second, for those taxa in our that K. furva sp. n. roosted in bamboo internodes or phylogeny that we identified as being closely re- in furled young leaves of banana plants (Chang et lated, we performed multivariate morphometric al., 2010; Cheng et al., 2010), with individuals comparisons to determine species delimitations. roosting either solitarily or in small colonies com- This approach proved to have considerable dis- posed of two to ten bats. Little is known of their re- criminating power, allowing us to conclude that productive phenology, however, females were the Kerivoula on Taiwan is a new species. Here recorded as pregnant from mid-April to mid-May we present our results and interpretations, and (n = 8) and lactating from mid-May to early July outline any caveats that should be addressed in fu- (n = 27) (H. C. Kuo, personal observation). ture studies. Our phylogenetic analyses applied separately to Genetics a mitochondrial gene (COI) and a nuclear gene Conventional, G-banded and C-banded kary- (RAG2) consistently recovered a K. hardwickii com- otypes were reported in Wu et al. (2012) for plex that comprised K. kachinensis, taxa currently 16 H.-C. Kuo, P. Soisook, Y.-Y. Ho, G. Csorba, C.-N. Wang, et al. ) Bandwidth Sweep rate Call repetition Frequency (kHz) sp. n. from Taiwan. For each individual bat, the mean value plus SD is given with the sample size in parentheses. The range of For each individual bat, the mean value plus SD is given with sample size in parentheses. Taiwan. sp. n. from K. furva size duration (ms) (ms) repetition rate (Hz) (ms) (ms) Peak Maximum Minimum (kHz) (kHz/ms) (Hz) Call-group Call-group interval Intergroup Call-group Call duration interval) Inter-call 3. Characterization of echolocation calls 3. Extended ABLE Bat ID ABLE Bat ID #1#2 1.8 ± 0.3 (10) 2.0 ± 0.1 (10) 18.7 ± 3.0 (8) 13.8 ± 1.2 (8) 141.8 ± 13.7 (10) 163.3 ± 16.2 (10) 182.4 ± 25.3 (10) 222.1 ± 10.7 (10) ± 10.2 (10) 112.9 ± 16.9 (10) 118.3 69.4 ± 20.6 (10) 103.9 ± 20.3 (10) 40.3 ± 12.4 (10) 52.0 ± 10.7 (10) 50.0 ± 7.4 (8) 63.5 ± 4.8 (8 #3#4#5All 1.6 ± 0.3 (10) 1.6 ± 0.3 (10) 1.4 ± 0.3 (10) 16.4 ± 3.1 (8) 1.4–2.0 ± 4.9 (8) 11.9 12.3 ± 1.5 (8) 147.3 ± 13.5 (10) 150.4 ± 10.3 (10) 198.4 ± 6.3 (10) (10) 142.7 ± 11.2 197.4 ± 7.8 (10) 11.9–18.7 185.2 ± 25.8 (10) 105.7 ± 6.8 (10) ± 6.1 (10) 112.4 107.1 ± 8.9 (10) (10) 92.6 ± 11.9 141.8–163.3 85.0 ± 9.2 (10) 78.0 ± 24.5 (10) (10) 60.0 ± 11.7 54.2 ± 14.0 (10) 55.7 ± 15.7 (10) 57.1 ± 8.5 (8) 182.4–222.1 83.8 ± 30.8 (8) 73.6 ± 8.5 (8) 105.7–118.3 69.4–103.9 40.3–60.0 50.0–83.8 T mean values is given in the last row #1#2#3–––– #4#5 4.4 ± 2.6 (7)All 6.0 ± 1.5 (6) 9.8 ± 12.9 (6) 72.0 ± 50.0 (7) 84.1 ± 22.7 (6) 6.7 ± 5.0 (7) 123.0 ± 141.3 (6) 76.5 ± 22.9 (6) 4.4–9.8 31.4 ± 4.0 (4) 75.3 ± 47.3 (7) 78.0 ± 55.5 (4) 6.8 ± 1.5 (6) 46.6 ± 12.5 (5) 8.5 ± 1.5 (4) 5.0 ± 2.0 (4) 72.0–123.0 9.3 ± 3.6 (5) 31.4–78.0 5.0–9.3 T New species of the Kerivoula hardwickii complex 17 recognized as K. hardwickii, and the Taiwanese and K. furva sp. n. yet continued between K. de - Kerivoula (which we name as K. furva sp. n.) (Fig. pressa populations containing divergent mito- 1). This finding complemented earlier findings that chondr ial lineages, ren dering K. depressa para - focused on mitochondrial DNA (Francis et al., 2010; phyletic in terms of mito chondrial genes. Finally, Douangboubpha et al., 2016), as well as a study that the current sympatric distribution of the two assessed K. hardwickii from the focal species com- mitochondrial lineages of K. depressa might also plex based on nuclear markers (Khan et al., 2010). result of secondary contact between previously iso- For one taxon, K. krauensis, RAG2 data were not lated populations (e.g., see Campagna et al., 2014; available, however, our COI phylogeny indicated Andriollo et al., 2015). For these reasons, species that it is a member of the K. hardwickii complex. delimitations based on mitochondrial DNA should Therefore more data from ncDNA are needed to ver- be considered equivocal without additional support- ify this relationship. ing evidence. Within the K. hardwickii complex, our COI- To determine the distinctiveness of taxa within based phylogeny revealed the presence of multiple the K. hardwickii complex, we gathered compara- divergent lineages of K. hardwickii s. lato, which we tive morphometric data from museum specimens, refer to as K. hardwickii A–D (Fig. 1a). Through including bats assigned to K. hardwickii s. lato col- cross-validations with samples included in both mo- lected across a broad geographical range. A princi- lecular and morphological analyses, we inferred that pal component analysis (PCA) supports a taxonomic K. hardwickii A–B corresponded to K. depressa, separation between K. kachinensis and K. hardwi- K. hardwickii C to K. hardwickii s. str. and K. hard- ckii s. lato based on their different skull sizes (Fig. wickii D to K. furva sp. n. (Fig. 3c). Given the topol- 2), while a discriminant analysis of principal com- ogy of our COI tree, this above classification leaves ponents (DAPC) reveals the presence of three dis- K. depressa a paraphyletic species in relation to tinct species within K. hardwickii s. lato based on K. kachinensis and K. furva sp. n., with the former clear differences in skull size and/or shape (Fig. 3). species spread across two highly divergent (13%) By relating information on the geographical ranges clades that show geographic overlap (Fig. 1a and of the specimens analysed together with the type lo- Supplementary Table S3). Such a phylogeographic calities of K. hardwickii and its relatives (detailed in pattern might reflect the presence of cryptic species Results), we consider that the three species revealed under the name K. depressa, although it could also in the DAPC correspond to K. hardwickii s. str., arise within a single species. Simulations conducted K. depressa and a new species (K. furva sp. n.). in a recent study (Dávalos and Russell, 2014) high- Consequently we argue that depressa should be re- light the ease by which distinct mitochondrial clades elevated to a species name, following its earlier syn- could form within a species as a consequence of onymization with hardwickii. Douangboubpha et al. population subdivision when females exhibit lower (2016) summarized morphometric variation of Ke - dispersal rates among subpopulations than do males. rivoula spp. in Thailand via PCAs applied to exter- This behavioural condition is widely reported in bats nal and craniodental measurements. Although these (reviewed in Nagy et al., 2013), including members authors proposed the presence of two main morpho- of the genera Murina (Kuo et al., 2015; Flanders et types in Thai K. hardwickii s. lato, this was based al., 2016) and Myotis (Kerth et al., 2002; Arnold, solely on one cranial measurement, BH. Our multi- 2007), both of which belong to subfamilies that are variate analyses provide more detailed diagnoses closely related to the Kerivoulinae (Kawai et al., between K. hardwickii s. str. and its allies on the 2002; Hoofer et al., 2003; Lack et al., 2010). Indeed basis of skull dimensions (Fig. 2–3 and Fig. 6). many studies have reported intraspecific divergent Supplementary Table S4 illustrates ways by which mitochondrial clades alongside little detectable new data points can be projected onto our Fig. 2 and genetic structure at nuclear DNA loci (e.g. Lausen Fig. 3a, so to facilitate species identifications. et al., 2008; Arnold and Wilkinson, 2015). For Moreover, by including K. titania alongside mem- example, the big brown bat (Eptesicus fuscus) bers of the K. hardwickii complex in the PCA, we shows divergence levels of up to 12% at the protein- reveal skullar differences between the former coding mitochondrial gene (ND2) (Turmelle et species and each member of the latter group, along- al., 2011). Given the potential for such contrast- side some morphological convergence between ing histories between mitochondrial and nuclear K. titania and K. furva sp. n. (Fig. 2). Further dif- genomes it is possible that nuclear gene flow could ferences between K. titania and K. furva sp. n. lie have ceased among K. depressa, K. kachinensis in their genetic divergence (Fig. 1) as well as in 18 H.-C. Kuo, P. Soisook, Y.-Y. Ho, G. Csorba, C.-N. Wang, et al. differences in the tibia and the fur colour (detailed in K. titania. Measurement values of the PSUZC specimens are Systematic description of new species). Thus com- provided by Douangboubpha et al. (2016). Specimens marked bined evidence provides strong support against with asterisks are also used in molecular analyses; Table S3. Kimura 2-parameter distances in a unit of substitutions per nu- treating these two forms as one taxon (Wu et al., cleotide site for pairwise comparisons between distinct COI 2012). clades of the K. hardwickii complex. Abbreviations for clades Although our study contains the most compre- include harA for K. hardwickii A, harB for K. hardwickii B, hensive summary to date of interspecies differences harC for K. hardwickii C, harD for K. hardwickii D, kac for among taxa affiliated to K. hardwickii s. str., there K. kachinensis and kra for K. krauensis. See Fig. 1a for phylo- are some limitations that should be addressed in fu- genetic relationships among all these clades; Table S4. Variable loadings on the principal components (PCs) and the discrimi- ture work. First, in our morphometric analyses, we nant functions (DFs) of PCA and DAPC, respectively. See the were not able to obtain and include data from a large main text for abbreviations of variables. For the PCA, means number of specimens currently recognized as and standard deviations (SD) of the analysed data are also pro- K. hardwickii s. lato, including those housed in the vided. To map a new data point onto the PCs, its values need to Royal Ontario Museum, Canada, or most of those be centered (minus means) and scaled (divided by standard de- examined by Douangboubpha et al. (2016). It would viations) before multiplied by the PC loadings. For the DAPC, means of the training data are provided. To map a new data thus be interesting to assess whether interspecies point onto the discriminant axes, its values need to be centered morphometric distinction discovered in this study is before multiplied by the DF loadings; Fig. S1. The phylogeny stable or not with the inclusion of additional mate- shown in Fig. 1a with vouchers labelled. GenBank accession rial. Second, as mentioned earlier, our morphometric numbers are provided when vouchers are unavailable; Fig. S2. analyses and phylogenetic reconstructions were The phylogeny shown in Fig. 1b with vouchers labelled. Gen - largely based on different sets of specimens due to Bank accession numbers are provided when vouchers are unavailable; Fig. S3. Model fits of the k-means clustering analysis the availability of material. Overall, while the COI applied to a dataset comprising seven craniodental measure- phylogeny and morphological analyses perfectly ments from the Taiwanese Kerivoula and from specimens re- matched each other in discriminating K. furva sp. n., ferred to as K. hardwickii. The levels of fits are assessed by K. depressa and K. hardwickii s. str. when applied to Bayesian Information Criterion (BIC) which is calculated at the a small common set of specimens (Fig. 3c), we can- number of groups (K) equal to 1–5. Supplementary Information not be certain that this will hold true in all other is available exclusively on BioOne. cases. We recommend that future genetic analyses ACKNOWLEDGEMENTS aimed at resolving problematic taxonomic groups We thank Yen-Jean Chen (NMNS), Hsi-Chi Cheng and should not rely exclusively on mitochondrial mark- Shih-Wei Chang (TESRI), Liang-Kong Lin (THU), Vu Dinh ers but should instead include nuclear markers. Thong (IEBR), Paula Jenkins, Roberto Portela Miquez and Indeed, apart from the potential effects of sex-biased Louise Tomsett (BMNH), Paul Bates, Beatrix Lanzinger and dispersal, discordant patterns from mitochondrial Malcolm Pearch (HZM), Frank Zachos and Alexander Bibl and nuclear genomes can also arise from other de- (NHMW), Kris Helgen and Darrin Lunde (USNM) for provid- mographic processes (reviewed in Toews and Brels- ing access to the specimens used in this study. In Thailand, we thank Sara Bumrungsri and Chutamas Satasook for their sup- ford, 2012) such as introgression during interspeci - port, and Sakiyah Morlor for help with genetic work. In fic hybridization (e.g., Berthier et al., 2006; Petit Myanmar, we thank U Win Naing Thaw, Director of the Nature and Excoffier, 2009; Mao et al., 2013a, 2013b; Kuo and Wildlife Conservation Division, Forest Department, Mini - et al., 2015). Finally, wherever possible, studies fo- stry of Natural Resources and Environmental Conservation, for cusing on taxonomic revisions should incorporate facilitating permissions. We also thanks U Aung Mung, U San data from common sets of specimens to promote Naing Dee, U Myint Zaw, Myint Kyaw and San Lwin Oo, and all staff of the Forest Department, as well as U Aung Zaw, reliable comparisons. Naing Lin, Zarchi Myo, all WCS Myanmar Program staff, and all UNESCO staff in Yangon and Bangkok offices, for coordi- SUPPLEMENTARY INFORMATION nating the trips. HCK was supported by a Postdoctoral Fellowship awarded to CNW and funded by ‘The Aim for the Contents: Table S1. Samples used in molecular analyses, Top University Project’ of the National Taiwan University. SJR with GenBank accession numbers for COI and RAG2 genes pro- was funded by the European Research Council, and GC was vided. Sequences newly generated in this study are shown in supported by the SYNTHESYS Project funded by European bold. Vouchers marked with asterisks are also used in morpho- Community Research Infrastructure Action under the FP7 metric analyses; Table S2. Museum specimens examined and/or “Capacities” Program, and also supported by the Hungarian used in morphometric analyses, with values of measurements Scientific Research Fund (OTKA) K112440. Finally, we are provided (all in mm; see the text for abbreviations). Abbrevia - very grateful to Chun-Chia Huang, Yin-Ping Fang, Charles tions for species include fur for K. furva sp. n., dep for K. de- Francis and three anonymous reviewers for their helpful com- pressa, har for K. hardwickii, kac for K. kachinensis and tita for ments on this research. New species of the Kerivoula hardwickii complex 19

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Received 22 June 2016, accepted 28 February 2017