A new species of Myotis from China with notes on the siligorensis species group (Chiroptera, Vespertilionidae)

333 A new species of Myotis from China with notes on the siligorensis species group (Chiroptera, Vespertilionidae) Mikhail Petrovich Tiunov1, Sen Liu2, Jiang Feng3, Pipat Soisook4, Tinglei Jiang3 1 Federal Science Center of East Asian Terrestrial Biodiversity, Far East Branch, Russian Academy of Sciences, Pr-t 100-let Vladivostoka 159, Vladivostok 690022, Russia 2 College of Life Sciences, Henan Normal University, 46 Jianshe East Road, Xinxiang 453007, China 3 Jilin Provincial Key Laboratory of Animal Resource Conservation and Utilization, Northeast Normal University, Changchun 130117, China 4 Princess Maha Chakri Sirindhorn Natural History Museum, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Corresponding authors: Mikhail Petrovich Tiunov ([email protected]); Tinglei Jiang ([email protected]) Copyright: © Mikhail Petrovich Tiunov et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract On the basis of molecular and morphological studies of samples collected in China, a new Myotis species belonging to the siligorensis group is described, Myotis kalkoae Tiunov, Jiang, & Liu, sp. nov. The species rank of M. sowerbyi and M. alticraniatus was confirmed. All three taxa under consideration belong to different genetic lines and can be distinguished from each other and from other morphologically similar species based on the shape of their baculum and tragus. The difficulties of taxonomy within the M. siligorensis species group are discussed. Key words: Baculum, bats, morphology, new species, taxonomy, tragus Introduction Myotis is one of the most diverse chiropteran genera (Koopman 1994; Simmons 2005). The content, worldwide distribution, and separate taxonomic position (as a member of a separate subfamily; refer to Hoofer and van den Busche 2003) of this genus highlight the importance of taxonomic studies within this group. Molecular genetics methods have been applied for the taxonomic studies of Myotis since the early 2000s (Ruedi and Mayer 2001), resulting in several rearrangements at the taxonomic levels (e.g., Stadelmann et al. 2004, 2007; Lack et al. 2010; Larsen et al. 2012; Novaes et al. 2021; Saikia et al. 2025), which contributed to increased interest in taxonomic studies of bats in general. However, some of the recent successful taxonomic publications (not particularly concerning Myotis) are not based on molecular data (Csorba et al. 2011; Reeder et al. 2013). Some species groups of tropical Myotis have been studied insufficiently, and their taxonomic status and species delimitation require further investigations. Previous studies have revealed cryptic diversity within the Myotis siligorensis species group, and two new species were described, namely, M. phanluongi Borisenko, Kruskop & Ivanova, 2008 and M. badius Tiunov, Kruskop & Feng, Academic editor: Wieslaw Bogdanowicz Received: 25 December 2024 Accepted: 1 September 2025 Published: 7 November 2025 ZooBank: https://zoobank.org/ C432C924-474E-4932-8AB9B07E56709243 Citation: Tiunov MP, Liu S, Feng J, Soisook P, Jiang T (2025) A new species of Myotis from China with notes on the siligorensis species group (Chiroptera, Vespertilionidae). ZooKeys 1258: 333–356. https://doi. org/10.3897/zookeys.1258.145290 ZooKeys 1258: 333–356 (2025) DOI: 10.3897/zookeys.1258.145290 334 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China 2011. However, the taxonomy of this group remains unclear to date, although new undescribed forms have been identified (Francis et al. 2010; Ruedi et al. 2013). For example, two subspecies of M. siligorensis (alticraniatus and sowerbyi) have been elevated to the species level, and M. alticraniatus Osgood, 1932 may include three subspecies (alticraniatus, thaianus, and badius) (Ruedi et al. 2015, 2021). However, the status of the subspecies of M. alticraniatus is debatable, especially for M. alticraniatus badius. Therefore, M. badius is considered a separate species and here raised to specific status (Fig. 1). Kawai et al. (2003) published the molecular taxonomic data on the rare bat species Myotis davidii (Peters, 1869), a species endemic to East Asia. Since these data were placed in GenBank, the number of fresh records of this species in China has increased markedly. Further study was conducted on the genetic diversity of 126 individuals, which were originally identified as Myotis davidii, obtained from seventeen Chinese localities (You et al. 2010). This study, which was based on nuclear and mitochondrial DNA markers, demonstrated that individuals can be subdivided into three groups based on their geographical origin. Moreover, P. Benda investigated the holotype of Vespertilio davidii (Peters, 1869) and allocated it into a synonymy of the ‘mystacinus’ species Figure 1. Distribution map of the forms within the M. siligorensis species group and the sampling locations of the three species, namely, M. badius, M. sowerbyi, and M. kalkoae sp. nov., used in this study. The ranges for M. badius and M. siligorensis are based on the IUCN Red List of Threatened Species (https://www.iucnredlist.org/), whereas the ranges for M. phanluongi, M. sowerbyi, and M. alticraniatus are based on the Mammal Diversity Database (2005) (https://www. mammaldiversity.org/). Note that the ranges of these species are currently controversial. 335 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China group, considering that “this name represents the senior synonym of the whole group of morphotypes included … into species rank of Myotis nipalensis...” (Benda and Karatas 2005). Later, P. Benda accepted M. davidii as a valid and senior name for the M. nipalensis / aurascens complex (Benda et al. 2012), placing it apart from the siligorensis species group. Ruedi et al. (2021) also considered M. davidii to be a well-defined species but distinguished it from M. nipalensis, which inhabits the southern slopes of the Himalayan region, as a separate species. S. Kruskop also examined three specimens of M. davidii from Central China (including the holotype MNHN1987-296), and confirmed (pers. comm., oral) that all three correspond to the M. mystacinus species group in skull shape and tooth structure (although they represent the extreme variants of the P3 displacement). According to their morphological characteristics, none of the samples (identified genetically as Myotis davidii sensu Kawai et al. 2003) fit the original description of this species. According to Tate (1941), “A specimen, U.S.N.M. 219175 from Chi-li, has a forearm length of 33 mm, thumbs and wing attachment as in true daubentonii, the skull with full braincase, large anteorbital foramen of Leuconoe, but with the muzzle and palate so much shortened that p3 is wholly excluded and contact is established between p4 and p2.” All the individuals that were identified previously as M. davidii based on the molecular analysis (You et al. 2010) exhibited a different dental structure. A single specimen in the collection of the Museum of Natural History (London), originally labeled M. davidii NHMUK 9.1.1.1, does not present skull and teeth proportions typical of the M. siligorensis species group, whereas Kawai’s ‘M. davidii’ are definitely within the siligorensis group (clade IX sensu Ruedi et al. 2013; refer also to Zhang et al. 2009). It was assumed that all the questionable Chinese samples studied belonged to the Myotis siligorensis species group, although their affiliations with a particular species remained uncertain. All of these specimens possess lower molars of the nyctalodont or seminyctalodont type, with a postcristid connected chiefly to a hypoconulid (Menu 1987), a feature that is typical of the M. siligorensis group (although not for all its members), in contrast to the vast majority of the other myotines, which are myotodonts. The individuals also possess skulls of recognizable shape, with a high and rounded braincase and a low rostral portion, which divides them from other nyctalodont myotines, such as Submyotodon latirostris and S. caliginosus (Ruedi et al. 2015). Most species in the M. siligorensis species group are quite similar in overall size and skull proportions. However, our study shows that they differ in the shapes of their tragus and penial bone (baculum). Despite a certain level of individual variation, the latter structure is known to be a useful species diagnostic feature, at least in some Vespertilionid genera, separating even the morphologically similar species (e.g., Thomas 1915; Topal 1958; Brown 1967; Hill and Harrison 1987; Benda and Tsytsulina 2000; Matveev et al. 2005). Based on these findings, the bacular morphologies of all taxa belonging to the group M. siligorensis and the material belonging to the different genetic lineages previously assigned to ‘M. davidii’ were studied. Thus, using an integrative combination of molecular and morphological analyses, a new species was identified in the M. siligorensis group, Myotis kalkoae Tiunov, Jiang, & Liu, sp. nov. 336 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China Materials and methods Morphological and morphometric studies Three specimens of the Myotis kalkoae sp. nov. were captured during the MPT’s field work and fixed in 75% ethanol, and eighty-four samples were used for qualitative and quantitative morphological comparisons (adult individuals of both sexes; dryor alcohol-preserved skins with extracted skulls; see Suppl. material 1) (Fig. 1). The abbreviations of the collections are as follows: FMNH Field Museum of Natural History, Chicago, USA HNHM Hungarian Natural History Museum, Budapest, Hungary HZM Harrison Institute, formerly the Harrison Zoological Museum, Sevenoaks, Kent, Great Britain IBSS Institute of Biology and Soil Science, Far East Branch of the Russian Academy of Sciences, Vladivostok, Russia MNH Museum of Natural History, London, Great Britain MNHN National Museum of Natural History, Paris, France NHMUK Museum of Natural History, London, Great Britain NNU Northeast Normal University, Changchun, China ROM Royal Ontario Museum, Toronto, Canada SMF Senckenberg Museum of Natural History, Frankfurt am Main, Germany ZMMU Zoological Museum of Moscow State University, Moscow, Russia External measurements were taken to the nearest 0.1 mm using a dial caliper. In the laboratory, a set of 19 cranial measurements was taken to the nearest 0.01 mm using an electronic caliper in combination with a binocular microscope. The following external measurements were taken: HB head and body length, T tail length, TT length of the free tail tip, E ear length, Tr tragus length, Tib tibia length, F foot length (including claws, measured to the most remote part of the claw), FA forearm length, Mc length of the first digit, including the claw, length of the metacarpal of the second digit, and lengths of the metacarpals, Ph phalanxes of the third through fifth digits. All measurements for the wings were taken using the right wing. The following cranial characteristics were measured (appropriate abbreviations are provided in parentheses): CBL condylobasal length, CCL condylocanine length, W width of the skull at the level of the auditory bullae, 337 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China BCW width of the braincase, BCH height of the braincase posterior to the auditory bullae, IOW least interorbital width, ZW zygomatic width, WR rostral width at the level of the preorbital foramina, LR rostral length from the preorbital foramen to the anterior edges of alveolus of the inner incisor, CM3 C–M3 crown length, ‘pseudodiastema’ PD length of the interval between the cingulum of the upper canine and large premolar, P4 M3 molariform tooth row length, CC crown width between the outer margins of the upper canines, M3 M3 crown width between the outer margins of M3, lmd lower jaw length from the alveolus of I 1 to the articulated process, hmd lower jaw height from the level of the tip of the coronoid process, MCM3 crown length of the maxillary tooth row. This list of measurements was limited to 16 items for statistical purposes. Only intact skulls with a complete set of measurements were used in the analysis. In order to assess the pattern of variation in the quantitative characteristics, principal component (PC) analysis and discriminant factor (DF) analysis were performed for cranial measurements using the appropriate modules of STATISTICA for Windows (Stat-Soft Inc. 1999). In the PC analysis, the measurements were standardized [(raw score - mean)/SD] to decrease the influence of the overall size. The shapes of the tragus and baculum were examined in three samples of M. kalkoae sp. nov., 11 of M. badius, nine of M. sowerbyi, three of M. siligorensis, two of M. alticraniatus, and two of M. phanluongi. The drawings of the baculum M. alticraniatus and M. phanluongi were taken from the literature (Borisenko et al. 2008; Kruskop 2013a). Molecular studies All samples in which the cytochrome b (Cytb) and 12S ribosomal DNA (12S rDNA) genes were amplified and were obtained from specimens deposited at the Museum of Natural History of Northeast Normal University, Jilin Province, China. Total genomic DNA was extracted from the muscle tissues using a UNIQ-10 column animal genomic DNA isolation kit (Sangon, Shanghai, China). The Cytb gene of all samples was amplified using the universal primers L14724/ H15915 (Kocher et al. 1989; Irwin et al. 1991), whereas the 12S rDNA gene was amplified with the primer pair 12c/12g (Springer et al. 1995). Each PCR mixture contained 50 ng of genomic DNA, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, 0.5 μM of each primer, and 2.5 U of Taq DNA polymerase (TaKaRa, Dalian, China) in a total volume of 25 μL. The PCRs were carried out in a thermocycler for 5 min at 94 °C, followed by 35 cycles of 45 s at 94 °C, 45 s at 44 °C, and 90 s at 72 °C, followed by one final extension at 72 °C for 5 min. 338 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China The PCR products were purified using an EZ-10 Spin column DNA Gel extraction kit (BBI, Shanghai, China) and then sequenced using an ABI PRISM 3730 sequencer (Applied Biosystems, Foster City, USA). The GenBank accession numbers are given in Table 1. In order to investigate the phylogenetic position of these bats, the available Cytb sequences and 12S rDNA genes of the other bats were obtained from GenBank (Table 1). Molecular analyses of the Cytb and 12S rDNA data were performed based on the maximum likelihood (ML) criteria, using the MEGA11 molecular genetic analysis software (Tamura et al. 2021). The maximum likelihood search used the maximum composite likelihood method and pairwise deletion of the missing data. The tree was rooted with a composite outgroup containing Kerivoula titania and K. hardwickii (basal Vespertilionids, which were also used as an outgroup in Borisenko et al. 2008). The reliability of the nodes of ML phylogenetic trees was assessed by performing 1000 non-parametric bootstraps (Felsenstein 1985), and nodes with over 70% bootstrap support were considered strongly supported (Hillis and Bull 1993). Table 1. Origin of the samples analyzed for Cytb and 12S rDNA. Species Cytb 12S rDNA Reference/voucher (Cytb; 12S rDNA) M. badius GD-08-38 GD-08-38 KF894921; KF894928 GX-07-10 GX-07-10 KF894922; KF894929 MW054891.1 Ruedi et al. 2021 M. kalkoae sp. nov. HUN-08c-16 GZ-07-74 KF894923; KF894930 CQ-08c-16 KF894920 M. sowerbyi AH-08-9 AH-08-9 KF894919; KF894927 ZJ-08-47 JS-08-31 KF894926; KF894931 JS-08-31 JX-09-89 KF894924; KF894932 JS-08-37 KF894925 M. alticraniatus OR096759.1 AY495508.1 Liu et al. 2023; Hoofer et al. 2003 FJ755898.1 Borisenko et al. 2008 M. davidii EF570884.1 hn1161 M. muricola AJ841957.1 FJ755896.1 Stadelmann et al. 2004 Borisenko et al. 2008 M. capaccinii AF376845.1 AY495494.1 Ruedi et al. 2001 Hoofer and Van Den Bussche 2003 M. ikonnikovi AY665162.1 Tsytsulina et al. 2012 M. altarium FJ215677.1 Zhang et al. 2009 M. mystacinus AF376861.1 Ruedi and Mayer 2001 M. brandtii AF376844.1 Ruedi and Mayer 2001 M. longipes EF555231.1 Zhang et al. 2009 M. petax EF555237.1 (Tan and Feng, unpublished) M. frater FJ215682.1 Zhang et al. 2009 M. phanluongi FJ755897.1 Borisenko et al. 2008 M. annamiticus FJ755901.1 Borisenko et al. 2008 M. csorbai FJ755892.1 Borisenko et al. 2008 M. alcathoe AJ841955.1 Stadelmann et al. 2004 Kerivoula hardwickii GU585657 FJ755904.1 Khan et al. 2010 Borisenko et al. 2008 Kerivoula titania JN112246 FJ755902.1 Wu et al. 2012 Borisenko et al. 2008 339 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China Results Molecular results The genetic differences between the specimens of Myotis kalkoae sp. nov., M. sowerbyi, and M. badius were minimal (Suppl. materials 2, 3). The divergence in the Cytb gene between each species pair did not exceed 2.0% (Fig. 2). Nevertheless, each of the three forms generated a monophyletic clade with high bootstrap support. The 12S rDNA gene was significantly different between at least M. kalkoae sp. nov. and M. badius at ~2.2% (Fig. 3). All three Chinese species formed a highly supported monophyletic clade, with M. alticraniatus as a sister group. The three Chinese forms, as well as other studied members of the ‘siligorensis’ species group, possess certain qualitative morphological differences in tragus and baculum shapes. These characteristics are described in the Systematics section below. Different bacular shapes were associated with particular genetic lineages. The distribution of bats with different bacular morphologies essentially corresponds to the distribution of the different Figure 2. Maximum likelihood tree constructed using complete cytochrome b gene sequences. The nodes were considered supported if the indicated bootstrap exceeded 70%. 340 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China genetic races of ‘M. davidii’ and the accepted subspecies of M. siligorensis. Animals from one of the genetically differentiated forms assigned previously to ‘M. davidii’ from the Chinese provinces of Yunnan were recently described as a separate species, Myotis badius Tiunov, Kruskop & Feng, 2011 (Tiunov et al. 2011). Moreover, the degree of skull variation between the forms is low. A PC analysis, conducted with different datasets to include or exclude the partly damaged samples, clearly divided at least a portion of the studied taxa. A bivariate scatter plot, shown in Fig. 4, was generated based on the two ‘first principal components’, which were calculated from the set of all seventeen craniodental measurements taken for the 74 samples of only the nyctalodont Myotis species. These two principal components cumulatively accounted for ~70.8% of the total variance (Table 2). PC I was positively correlated with the overall skull size and length of the upper and lower tooth rows; PC II was positively correlated with the length and width of the rostrum. Myotis annamiticus and M. phanluongi were plotted, and no overlap with other species was noted; for M. badius, this overlap was minimal. Moreover, in this particular analysis, M. alticraniatus and M. sowerbyi could not be fully separated from M. siligorensis. In another analysis with a partially reduced dataset, M. alticraniatus was well separated from at least M. sowerbyi based on the first Figure 3. Maximum likelihood tree constructed from the 12S rDNA gene sequences. The nodes were considered supported if the indicated bootstrap exceeded 70%. 341 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China Figure 4. Bivariate scatter plot for the first and second principal components, which was generated through calculations related to the 17 cranial and dental measurements of the 74 specimens of Asiatic mouse-eared bats from the M. siligorensis species group. Factor loadings and eigenvalues are presented in Table 2. The filled symbols indicate centroids. and third PCs (the third one exhibited a high positive correlation with the width and height of the braincase). Only the animals from North Vietnam and adjacent areas of China were included in the analysis as M. alticraniatus individuals, while the other samples from Central Vietnam were included as ‘M. cf. siligorensis’ because their species affiliation was unclear and could not be determined from the geographical data. Myotis kalkoae sp. nov. tended to group with M. badius. However, only two samples of M. kalkoae sp. nov. were included in the PC analysis, and this data may, therefore, be inadequate. In the DF analysis, based on the same dataset, no significant difference was found between M. alticraniatus and M. siligorensis (Table 3). The specimens of M. kalkoae sp. nov. were not distant from those of M. sowerbyi and M. siligorensis thaianus, but they were separated from the centroids of any group by greater distances than some groups were from each other. Despite the craniometric results, the presence of qualitative morphological features in combination with the level of genetic difference allowed us to suggest a specific rank for the unnamed form from China. 348 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China at the base was thicker than those of M. kalkoae sp. nov. and of M. sowerbyi. The lobe’s external angle was a bluntly rounded shape, with the apex directed downwards (Fig. 5E). The margin of the plagiopatagium was attached to the metatarsus of the first toe. The length of the foot with the claw was ~50% (42%–53%, n = 10) of the tibia length. The frontal part of the skull was distinctly elevated above the rostrum (Fig. 6C). The brain sample was inflated, and its height was ~81.3% (78.4%–84.0%, n = 13) of the skull width. The interorbital constriction was narrow; the interorbital width was ~47%–51% of the skull width. The posterior border of the nasal emargination extended to a level comparable to the middle of the upper canine. Sagittal and occipital crests were almost absent, and the lambdoid crests were reduced but visible laterally. The outer upper incisor (I3) was equal in size to or slightly longer than the internal incisor (I2). The upper canine was small, only slightly exceeded or was equal in height, and it was smaller in crown area than the corresponding larger premolar (P4). Both small upper premolars (P2 and P3) were essentially in the toothrow and clearly visible from a lateral view. The first lower molar was a nyctalodont type, and the second was a nyctalodont or seminyctalodont. Baculum. The penial bone was very small, ~0.35 mm in length. The structure was simple in shape and proportionally wider than that in M. sowerbyi and M. kalkoae sp. nov. The baculum was wider basally, evenly narrowing to the distal end without any abrupt constrictions. The urethral groove was reduced but was present as a depression on the lower surface of the bone and projects forward, almost to its tip (Fig. 8D). Remarks Traditionally, all siligorensis-like Myotis individuals from Vietnam and adjacent parts of China and Laos have been referred to as M. s. alticraniatus (Kruskop 2013b). However, the genetic diversity of the COI gene in Indochinese and southern Chinese M. siligorensis sensu lato (Francis et al. 2010) exceeded the intraspecific level of variation common for other Myotis species. This suggests the existence of more than one species within that region, with an uncertain taxonomic position of animals from central Indochina. Thus, it is suggested to tentatively restrict the distribution of M. alticraniatus sensu stricto to the northern provinces of Vietnam and Laos and the adjacent territories of China (Fig. 1). Additional studies with different gene markers are, however, needed to conclude this topic. The new species was slightly longer than M. siligorensis: the forearm length was 34.9 and 35.3, the condylobasal length was 11.9 and 12.1 vs. 29.1–29.5 and 10.2–10.8 mm in M. s. thaianus (data of this study) and 30.8 and 11.2 mm in M. s. siligorensis (Bates and Harrison 1997), respectively. Myotis kalkoae sp. nov. clearly differed from M. siligorensis in terms of baculum shape. The baculum in M. siligorensis was wedge-shaped with small lateral ‘wings’ (Fig. 8C). The structure was shorter but wider at the base than that of M. kalkoae sp. nov. (Fig. 7). The maximum baculum length was 0.45 mm; the maximum baculum width was 0.225 mm. The maximum width/length ratio of the baculum of M. siligorensis was 0.5, that of M. alticraniatus was 0.4, of M. sowerbyi was 0.36, and of M. kalkoae sp. nov. was 0.32. Myotis kalkoae sp. nov. clearly differs in the tragus shape from those of M. s. siligorensis and M. s. thaianus (these two species have the same tra- 349 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China Figure 8. Bacula of the selected Myotis siligorensis species group. A. Myotis sowerbyi (dorsal, ventral, and lateral views); B. Myotis badius (dorsal, ventral, and lateral); C. Myotis siligorensis (dorsal, ventral, and lateral); D. Myotis alticraniatus (ventral and lateral); E. Myotis phanluongi (dorsal and lateral). Scale bars: 0.2 mm. gus shape). In contrast to M. kalkoae sp. nov., the tragus of M. siligorensis was lancet-shaped and wide at the base, tapering evenly to the top (Fig. 5D). Myotis kalkoae sp. nov. differs from M. siligorensis in terms of the maximum width/length ratio of the tragus. In M. kalkoae sp. nov., the ratio was 0.29, whereas in M. siligorensis, M. badius, and M. sowerbyi, the ratios were 0.38, 350 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China 0.27, and 0.26, respectively. The lobe at the base of the tragus of M. kalkoae sp. nov. was thinner than that of M. sowerbyi and M. badius. The lobe’s exterior angle was directed upward, which is in contrast to M. sowerbyi, in which it was directed horizontally, and to M. badius, in which it was angled downward. Most members of the Myotis siligorensis species group were very similar morphologically and could barely be distinguished based on traditional features such as skull proportions. Therefore, a taxonomic investigation is required to further research, with the evaluation of other morphological structures such as the baculum and shape of the tragi, and thorough attention to the peculiarities of the observed morphologies. According to the results of a study by Ruedi (Ruedi et al. 2021), M. badius belongs to the subspecies M. alticraniatus. However, the structure of the baculum and tragus in the studied topotypes remains unknown to date. In addition, the distribution of M. alticraniatus in China remains unclear; therefore, this decision from Ruedi may be premature. The results of the PC and DF analyses indicate very low skull variation within the M. siligorensis complex, which may explain why M. alticraniatus, M. siligorensis, and cf. M. sowerbyi were thought to be conspecific for so long. Most likely, a different set of measurements is required to discriminate the species. Unequal sample sizes could also have affected the results of the morphometric analyses. Finally, not all the samples were identified by bacular/ear morphology or by genetics and were assigned to a specific sample based on geographical distribution. Therefore, some misidentifications could have occurred if more than one species were present in the same region. One of the main problems encountered in the clarification of the taxonomy of the M. siligorensis species group is the lack of genetic data, as few genes have been sequenced for the members of this group to date (Borisenko et al. 2008; Zhang et al. 2009; Francis et al. 2010; Ruedi et al. 2013), and only a few specimens have been sampled (Ruedi et al. 2021). The identification of new cryptic forms expands the current knowledge of the taxonomic diversity of the M. siligorensis species group and highlights the intriguingly high species diversity within a morphologically uniform species complex. Most likely, the high diversity may be explained by the low mobility of the group members. The post-glacial periods of rapidly changing natural zones can easily lead to the appearance of isolated populations (e.g., those in separate mountainous ranges). The same scenario is most likely true for some other bats with the low mobility, such as Murina or Rhinolophus (Francis et al. 2010), for which many cryptic species have been recently identified (Csorba et al. 2011; Francis and Eger 2012; Patrick et al. 2013). There are still a number of issues regarding the taxonomy of this group, which remain to be investigated in detail. The absence of suitable genetic material from a typical M. siligorensis from India and Myanmar, as well as that from M. s. thaianus, prevented the investigation of their genetic differences from each other and East Asian relatives. This difference, in light of the aforementioned results, can be considerable despite the morphological similarities. The relationships between the nyctalodont and myotodont members of the species group remain unclear. The authors are fairly confident that M. laniger is a member of this complex (see Ruedi et al. 2013), whereas the relationships be- 351 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China tween the morphologically similar species, such as M. longipes and M. csorbai, remain unknown. Therefore, further studies aimed at evaluating the phylogenetic history of the M. siligorensis complex require including novel material from all parts of the group distribution. Nuclear genetic markers should also be incorporated in future analyses because the similarities demonstrated only by the mitochondrial DNA may represent an artifact caused by a former gene flow, as reported for some Palaearctic bat species (Berthier et al. 2006; Artyushin et al. 2009, 2012). Acknowledgements Authors are very thankful to Dr. Paul Bates for providing image of the penial bone of typical M. siligorensis and for substantial help with different stages of the manuscript preparation. We thank S. Kruskop for providing drawings of the tragus of Myotis alticraniatus, a photograph of the skull of M. alticraniatus, and valuable comments during the writing of the manuscript, and A. M. Omelko for assistance in statistical processing of the material. The language of this study was professionally edited by ExEditing.com. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of AI No use of AI was reported. Funding The research was conducted within the state assignment of Ministry of Science and Higher Education of the Russian Federation (theme No. 124012200182-1), was supported by the Special Foundation for National Science and Technology Basic Research Program of China (2021FY100301), and the National Natural Science Foundation of China (Grant No. 32371562). Author contributions Mikhail Petrovich Tiunov, Jiang Feng, and Tinglei Jiang designed the study; Mikhail Petrovich Tiunov, Sen Liu, Pipat Soisook and Tinglei Jiang collected and provided materials for the study; Mikhail Petrovich Tiunov, Sen Liu, and Tinglei Jiang performed morphometric and phylogenetic analyses; Mikhail Petrovich Tiunov and Tinglei Jiang interpreted the results and prepared the manuscript, photographs, and figures for the study. All authors read and approved the final version of the manuscript. Author ORCIDs Mikhail Petrovich Tiunov https://orcid.org/0000-0002-4276-4266 Sen Liu https://orcid.org/0000-0003-2258-3676 Jiang Feng https://orcid.org/0000-0002-7503-1069 352 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China Pipat Soisook https://orcid.org/0000-0002-1404-3568 Tinglei Jiang https://orcid.org/0000-0003-3858-9458 Data availability In this study, all the gene sequences have been deposited in GenBank. The other data are available from the corresponding author (Mikhail Petrovich Tiunov: [email protected]) upon reasonable request. References Artyushin IV, Bannikova AA, Lebedev VS, Kruskop SV (2009) Mitochondrial DNA relationships among North Palaearctic Eptesicus (Vespertilionidae, Chiroptera) and past hybridization between Common Serotine and Northern Bat. 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Link: https://doi.org/10.3897/zookeys.1258.145290.suppl1 356 ZooKeys 1258: 333–356 (2025), DOI: 10.3897/zookeys.1258.145290 Mikhail Petrovich Tiunov et al.: A new species of Myotis from China Supplementary material 2 Genetic distances based on Cytb sequences Authors: Mikhail Petrovich Tiunov, Sen Liu, Jiang Feng, Pipat Soisook, Tinglei Jiang Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1258.145290.suppl2 Supplementary material 3 Genetic distances based on 12S rDNA sequences Authors: Mikhail Petrovich Tiunov, Sen Liu, Jiang Feng, Pipat Soisook, Tinglei Jiang Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1258.145290.suppl3