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Research Article
The grey zone of taxonomy—The case of the Sikkim Myotis (Chiroptera: Vespertilionidae: Myotis sicarius), first recorded from Southeast Asia
expand article infoDorottya Győrössy§, Vuong Tan Tu|, Gábor Csorba, Sanjan Thapa#¤, Péter Estók«, Gábor Földvári»˄, Tamás Görföl˅»¦
‡ Hungarian Natural History Museum, Budapest, Hungary
§ Hungarian University of Agriculture and Life Sciences, Gödöllő, Hungary
| Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hanoi, Vietnam
¶ Graduate University of Science and Technology, Hanoi, Vietnam
# Guangzhou University, Guangzhou, China
¤ Small Mammals Conservation and Research Foundation, Kathmandu, Nepal
« Eszterházy Károly Catholic University, Eger, Hungary
» Institute of Evolution, HUN-REN Centre for Ecological Research, Budapest, Hungary
˄ Centre for Eco-Epidemiology, National Laboratory for Health Security, Budapest, Hungary
˅ Department of Zoology, Hungarian Natural History Museum, Budapest, Hungary
¦ National Laboratory of Virology, Szentágothai Research Centre, University of Pécs, Pécs, Hungary
Open Access

Abstract

In taxonomic works, the weight to be given to morphological, mitochondrial, or nuclear signals, and the assessment of differences as species or subspecies distinctions has also varied considerably over the past decades and is largely a subjective research decision. This apparent example of the “grey zone of taxonomy” underpins the need of critical studies of as many specimens as possible and of using both mitochondrial and nuclear genes in taxonomic-systematic studies, as phylogeny based on uniparentally inherited genes alone may not represent true evolutionary scenarios. Myotis sicarius, a species occurring thorough the Himalayan foothills was found for the first time out of South Asia, in North Vietnam. Analysis of topotypical and Vietnamese specimens revealed high mitochondrial heterogeneity – at the upper limit of the usual threshold of intraspecific difference – but only minute nuclear sequence and negligible morphological differences. Albeit the large geographic distance between the two records might suggest the existence of two putative reproductively isolated taxonomic units, based on the incongruent results we concluded that the split of geographic populations of M. sicarius into different taxa is unsupported. As a morphologically closely resembling species, we also reviewed the taxonomic status of the two morphological forms of M. annectans and synonymizing M. primula with M. annectans was also corroborated by our phylogenetic analyses.

Keywords

Area expansion, bats, mito-nuclear discordance, multilocus phylogeny, Vietnam

Introduction

In the era of genetics, molecular data are more commonly used in studies on taxonomy and phylogeny, particularly for interpreting relationships among closely related taxa. In most of these studies, mitochondrial genes have usually been chosen because of their suitable characteristics i.e., maternal inheritance, lack of recombination, haploid status, high mutational rate, and easier recoverability from degraded samples (Hebert et al. 2003; Antil et al. 2023; Elyasigorji et al. 2023). However, owning to their maternal inheritance, phylogenetic reconstructions based on mitochondrial signals may be inconsistent with the true evolutionary scenarios due to biological processes e.g., mitochondrial introgression, sex-biased dispersal (Castella et al. 2008; Flanders et al. 2009; Vallo et al. 2013; Andriollo et al. 2015; Platt et al. 2017) or misleading due to NUMTs (Nuclear mitochondrial DNA segments; Shaw 2002; Song et al. 2008). Hence, taxonomic inference based on mtDNA markers of certain taxa of interest need to be confirmed by additional phenetic analyses of morphological traits and/or nuclear gene (nuDNA) sequences (e.g., Tu et al. 2017, 2018; Hassanin et al. 2018; Petzold and Hassanin 2020). As the coalescence time of nuclear markers on average are several times longer than that of the uniparental markers (mtDNA), the use of nuDNA genes for delimitating species boundaries among recently diverged taxa (such as incipient species) and/or within-species lineages (subspecies) is also confounded with low genetic variation and/or incomplete lineage sorting of ancestrally polymorphic alleles (e.g., Hassanin et al. 2018). Nevertheless, the weight to be given to morphological, mitochondrial, or nuclear signals, and the assessment of differences as species or subspecies distinctions (or other operational taxonomic units) has also varied considerably over the past decades and is largely a subjective research decision (Zachos 2018). This “grey zone of taxonomy” underpins the need of critical studies of as many specimens as possible and of using integrated taxonomic approaches.

During the systematic exploration of Southeast Asian bats, a specimen of Myotis was captured in Xuan Lien Nature Reserve (Vietnam), near the Lao border which was morphologically identifiable as M. sicarius, a species that was previously known only from a handful of records in montane forests on hill sides and in valleys from Nepal and India, South Asia (Bates and Harrison 1997; Srinivasulu and Srinivasulu 2019) (Fig. 1). Given the biogeographic barriers to dispersal and the possibly of limited gene flow between bats from the two known sampling sites in a distance of over 1700 km, further investigation is required to confirm the validity of the morphology-based species identification of the newly collected Vietnamese specimen and to explore its phylogenetic relationship with Indian and Nepalese congeners (Slatkin 1985; Avise 2000; Castella et al. 2008).

Figure 1. 

Known occurrences of Myotis sicarius. Yellow dots: former occurrences, blue star: new Nepalese records (CDZTU B4–B6), red square: new Vietnamese record (IEBR VN14-0252). The basemap from Natural Earth is used for better visualization only and does not indicate any territorial claims.

Morphologically, M. sicarius closely resembles species of the montivagus-complex, especially M. indochi­nensis and M. annectans (Son et al. 2013). By contrast, a previous phylogenetic study based on cyt b and Rag2 genes found that this species forms a clade with M. frater, M. bechsteinii and M. daubentonii, and is only distantly related to the montivagus-complex (Ruedi et al. 2013). Such incongruences between morphology and molecular phylogeny of Myotis species are relatively common, suggesting that this genus contains potential high cryptic (i.e., different species subsumed in one) and pseudocryptic (i.e., individuals of a single species were misidentified into different sister ones) diversity and that further comprehensive studies using integrated analyses of multiple datasets (morphology, molecular and/or acoustic data) are required to confirm the taxonomic status of certain formerly recognized morphological species (Ruedi et al. 2015, 2021; Novaes et al. 2022; Kruskop et al. 2023).

An example of the above taxonomic issues can be found in a recent study of Chakravarty et al. (2020) who identified three Myotis bats – released on the spot – from Uttarakhand, India as M. cf. annectans on the bases of their forearm length (46.1–46.4 mm) following the keys published in previous studies (Görföl et al. 2013; Son et al. 2013). Nevertheless, one of these individuals’ COI sequence appeared to be only distantly related to all other bats assigned to M. annectans collected from Southeast Asia suggesting that it may represent another species (Chakravarty et al. 2020). This external morphological confusion warranted the further investigation of the taxon annectans described originally in the genus Pipistrellus due to its missing middle upper premolar. Myotis annectans now includes M. primula as a junior synonym, the type specimen of which morphologically differs only by the presence of a middle upper premolar (Topál 1970). To elucidate the systematic position of the Vietnamese M. sicarius, the relationship between this species and M. annectans, and the intraspecific variations in the dental formula of the latter, we studied new material from Nepal and Vietnam using both mitochondrial and nuclear markers completed with morphological comparisons.

Methods

Collection acronyms

BNHSBombay Natural History Society, Mumbai, India; CDZTUCentral Department of Zoology, Tribhuvan University, Kathmandu, Nepal; EBDEstación Biológica de Doñana, Sevilla, Spain; FMNHField Museum of Natural History, Chicago, USA; GJ – Gareth Jones field number; GZHU – Guangzhou University, Guangzhou, China; HNHMHungarian Natural History Museum, Budapest, Hungary; HZM – Harrison Institute, Sevenoaks, UK; IEBR – Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hanoi, Vietnam; KK – Kuniko Kawai personal collection; KM – Kishio Maeda personal collection; MHNGMuséum d’histoire naturelle de Genève, Geneva, Switzerland; M or MR – Manuel Ruedi field collection; NHMUKNatural History Museum, London, UK; NMPNational Museum Prague, Prague, Czech Republic; ROMRoyal Ontario Museum, Toronto, Canada; SMFSenckenberg Museum of Frankfurt, Frankfurt, Germany; TiK – Tigga Kingston field number; UKMUniversiti Kebangsaan Malaysia, Bangi, Malaysia; ZMMUZoological Museum of Moscow State University, Moscow, Russia.

Sample collection

Bats were captured and handled in the field with methods conforming to the guidelines approved by the American Society of Mammalogists for the use of wild mammals in research and education (Sikes 2016). Mist-nets were erected before dusk and were regularly checked at least for four hours. Individuals representing different morphological forms that could not be identified to species level in the field were taken as vouchers. Tissue samples (pectoral muscles of voucher specimens or wing punches of released individuals) were also collected and preserved in 99% ethanol.

Three M. sicarius were caught in Nepal, Kathmandu, Bajrabarahi Religious Forest, 1485 m a.s.l., 27°36.0’N, 85°19.2’E, on 30 August 2016 by Sanjan Thapa and Gábor Csorba. All individuals were released after careful examination and taking a wing punch sample (samples registered under the numbers CDZTU B4–B6).

In Vietnam, a single specimen of the same species was collected in Vin village, Xuan Lien National Reserve, Thanh Hoa Province (19°59.28’N, 104°58.62’E, 717 m a.s.l.), near the border with Laos, on 17 October 2014 by Tamás Görföl, Vuong Tan Tu and Péter Estók. The specimen was taken as a voucher for further examination (IEBR VN14-0252).

Comparative material

Myotis annectans: CAMBODIA: HNHM 2005.82.8., sex unknown, Keo Seima Wildlife Sanctuary; HZM 1.32758, ♂, Cardamom Mts.; INDIA: NHMUK 1816.3.25.30 (primula holotype), ♂, Pashok, Darjeeling; NHMUK 1920.7.27.2, ♂, Teesta Valley; NHMUK 1920.7.27.3, ♀, Teesta Valley; THAILAND: NHMUK 1978.2355, ♀, Chiang Ma; VIETNAM: HNHM 24987, ♂, Xuan Lien Nature Reserve; HNHM 26056, ♂, Mu Cang Chai Nature Reserve; HNHM 2008.23.10., ♂, Pu Huong Nature Reserve.

Myotis sicarius: INDIA: NHMUK 1891.10.7.56 (holotype), sex unknown, Sikkim; FMNH 35419, ♂, Sikkim; FMNH 35424, sex unknown, Sikkim; NEPAL: BNHS 3783, ♀, Bans Bahari; BNHS 3784, ♂, Bans Bahari; CDZTU B4, ♀, Lalitpur,; CDZTU B5, ♀, Lalitpur,; CDZTU B6, ♀, Lalitpur,; CDZTU BAT24, ♀, Lalitpur,; CDZTU BAT25, ♀, Lalitpur,; HZM 1.16284, ♂, Godawari; NHMUK 1923.1.9.4, ♀, Bans Bahari; NHMUK 1923.1.9.5, ♀, Bans Bahari; ZMMU 164493, ♀, Sudame; ZMMU 164494, ♀, Sudame; ZMMU 164495, ♂, Sudame; ZSI 17429, ♀, Bans Bahari; VIETNAM: IEBR VN14-0252, ♂, Xuan Lien Nature Reserve.

Myotis indochinensis: Vietnam: IEBR M-839-2 (holotype) ♀, A Luoi; HNHM 24210 (paratype) ♀, A Luoi.

Measurements and morphometrics

External measurements were taken from live animals (forearm of released bats) or fluid-preserved vouchers to the nearest 0.1 mm, and craniodental measurements to 0.01 mm using digital callipers. Measurements include only those taken from fully-grown individuals, as indicated by the presence of fully ossified metacarpal-phalangeal joints. Means and standard deviations were calculated with R v. 4.2.1 (R Core Team 2018).

Abbreviations and definitions for external and craniodental measurements include FA: forearm length – from the extremity of the elbow to the extremity of the carpus with the wings folded; TAIL: tail length – from the base to the tip of the tail; EAR: ear length – from the lower border of the external auditory meatus where it joins with the body to the tip of the pinna; TIB: tibia length – from the knee joint to the ankle; HF: hind foot – from the tip of the longest digit, excluding the claw, to the extremity of the heel, behind the os calcis; GTL: greatest length of skull – from the front of the 1st upper incisor to the most projecting point of the occipital region; CCL: condylo-canine length – from the exoccipital condyle to the most anterior part of the canine; C1C1W: width across the upper canines – greatest width across the outer borders of the upper canines; M3M3W: width across the upper molars – greatest width across the outer crowns of the last upper molars; IOW: interorbital width – least width of the interorbital constriction; ZYW: zygomatic width – greatest width of the skull across the zygomatic arches; MAW: mastoid width – greatest distance across the mastoid region; BCW: braincase width – greatest width of the braincase; BCH: braincase height – from the basisphenoid at the level of the hamular processes to the highest part of the skull, including the sagittal crest (if present); AOB: anteorbital width – the distance by which the anteorbital foramen is separated from orbit, measured from the foramen infraorbitale to the foramen lachrymale; CM3L: maxillary toothrow length – from the front of the upper canine to the back of the crown of the third molar; CP4L: upper canine–premolar length – from the front of the upper canine to the back of the crown of the last premolar; MANL: mandible length – from the anterior rim of the alveolus of the 1st lower incisor to the most posterior part of the condyle; CM3L: mandibular toothrow length – from the front of the lower canine to the back of the crown of the 3rd lower molar; CP4L: lower canine–premolar length – from the front of the lower canine to the back of the crown of the last premolar; and CPH: least height of the coronoid process – from the tip of the coronoid process to the apex of the indentation on the inferior surface of the ramus adjacent to the angular process. Absolute crown height was used in all height comparisons for individual teeth (e.g., C1 versus P4).

Molecular phylogenetics

Total genomic DNA was extracted with DNeasy Blood & Tissue Kit (QIAGEN, Germany) according to the instructions of the manufacturer. Considering the discrepancy in the available genetic sequences of Asiatic Myotis spp. deposited in GenBank by previous studies, in the first phase, we sequenced three commonly used genes from both mitochondrial (cyt b, 1140 bp and COI, 657 bp) and nuclear (Rag2, 1148 bp) genomes of selected specimens to reconstruct their phylogenetic relationships and to explore the congruences between gene trees. The primers used for PCR amplification of these three genes were Molcit-F/Cytb-H (Ibáñez et al. 2006; Weyeneth et al. 2008), VF1d/VR1d (Ivanova et al. 2006), and 179F/1458R (Stadelmann et al. 2007), respectively. Then, in the second phase, three additional nuDNA genes were used to ascertain the genetic signals obtained from the mtDNA and Rag2 phylogenies of our specimens of interest: Thyrotropin (THY), Protein kinase C iota (PRKC1), and Abhydrolase domain containing 11 (ABHD11). The respective primers used for PCR amplification of these three genes were THY_F/THY_R, BatPKa_F/BatPKa_R (Eick et al. 2005), and ABHD11-F1/ABHD11-R1 (Salicini et al. 2011).

PCR reactions were performed in 50 µl using 1 µl (ca. 20 ng) of genomic DNA, 1-1 µl of the primers (10 mM), 40.25 µl of nuclease-free water, 1.5 µl of dNTP, 5 µl of DreamTaq Green Buffer (Thermo Scientific, USA) and 0.25 µl of DreamTaq DNA Polymerase (Thermo Scientific, USA). The PCR conditions are summarized in the Table 1.

Table 1.

The PCR conditions for amplifying target genes.

Gene Initial denaturation Cycles (denaturation / annealing / extension) Final extension Reference
cyt b 3 min at 94°C 40 cycles (45 sec at 94°C / 45 sec at 50->45°C (touchdown) / 1.5 min at 72°C) 5 min at 72°C based on Weyeneth et al. (2008)
COI 1 min at 94°C 5 cycles (30 sec at 94°C / 40 sec at 50°C / 1 min at 72°C), followed by 35 cycles (30 sec at 94°C / 40 sec at 55°C / 1 min at 72°C) 10 min at 72°C De Pasquale and Galimberti (2014)
Rag2 3 min at 94°C 39 cycles (45 sec at 94°C, 45 sec at 60°C and 1.5 min at 72°C) 5 min at 72°C Stadelmann et al. (2007)
ABHD11 & THY 2 min at 94°C 2-2 cycles (15 sec at 95°C / 30 sec at 65, 63, 61, 59, and 57°C (touchdown) / 1 min at 72°C), followed by 30 cycles (15 sec at 95°C / 30 sec at 55°C / 1 min at 72°C) 5 min at 72°C Dool et al. (2016)
PRKC1 3 min at 94°C 39 cycles (30 sec at 94°C / 1.5 min at 53°C / 1 min at 72°C) 10 min at 72°C Eick et al. (2005)

Sequencings were done with the PCR primers in the Molecular Taxonomy Laboratory of the Hungarian Natural History Museum, Budapest, Hungary; at Macrogen Europe, Maastricht, The Netherlands; and, in case of the Nepalese sample, in the Center for Molecular Dynamics Nepal, Kathmandu, Nepal. The newly generated sequences were deposited in GenBank under accession numbers, OR413179OR413180 and OR413539OR413554; additional sequences of related species used in the phylogenetic reconstructions were downloaded from GenBank (File S1 and Table S1).

The cyt b, Rag2, and COI sequences were aligned with related taxa of Myotis as well as with Kerivoula, Murina, and Harpiocephalus as outgroups (Ruedi and Mayer 2001; Kawai et al. 2003; Stadelmann et al. 2004a, 2004b, 2007; Jones et al. 2006; Lack et al. 2010; Ruedi et al. 2012, 2013; Wang et al. 2017; and see File S1 and Table S1) with MAFFT v. 7.505 (Katoh and Standley 2013). Separate trees (cyt b, Rag2 and COI) were generated using Bayesian inference method (BI) with MrBayes v. 3.2.7a (Ronquist and Huelsenbeck 2003). It was run for 10 million generations and sampled every 1000th generation. Model parameters (HKY+I+G and GTR+I+G in case of cyt b and Rag2, COI dataset, respectively) was determined with MrModeltest2 v. 2.4 (Nylander 2004). Ten percent of the generations were treated as burn-in and discarded. Posterior probabilities were calculated from the consensus of the remaining trees. Trees were visualized with iToL v. 3 (Letunic and Bork 2016). Pairwise distances were calculated using Kimura 2-parameter model (Kimura 1980) in MEGA X v. 10.2.6 (Kumar et al. 2018).

Results

Morphology

The combination of the external traits mentioned below are typical of the species, and are essentially similar in the three Nepalese (CDZTU B4–B6), the Vietnamese (IEBR VN14-0252) specimens, and the fluid-preserved holotype of M. sicarius (NHMUK 1891.10.7.56): ears reach the tip of the nostrils when laid forward; tragus with a well-developed basal lobe, and reaches almost half the length of the pinna; wing attaches at the base of the first toe; calcar extends to the half of the free edge of uropatagium, with a small, but visible “notch” at the end; the fur on the dorsum is uniform dark brown; crown hairs are with whitish tips; ventral surface of the fur is dark brown (as on the dorsum) basally, but tips lighter whitish brown; and conspicuous reddish-chestnut brown coloration can be observed on the belly. The only notable exception is that the Vietnamese specimen had a developed whitish “pad” at the thumb (Fig. 2).

Figure 2. 

External traits of M. sicarius. a portrait of female CDZTU B4 bat from Nepal; b portrait; c ventral aspect; d genital region (note the reddish-chestnut fur on the belly); e thumb of the male IEBR VN14-0252 specimen from Vietnam; f thumb of holotype (NHMUK 1891.10.7.56).

Compared to other closely related Myotis species, the skull of M. sicarius is massive with a long rostrum; cranial profile flattened, the depression between the rostrum and braincase is shallow. Zygomas are strong, the sagittal crest is pronounced; anteorbital bridge is moderate to relatively wide (Fig. 3). Upper incisors are bicuspid. The inner upper incisor (I2) has the same height as the outer upper incisor (I3) which exceeds the secondary cusp of I2. Upper canine (C1) heavy and is higher than the third upper premolar (P4). There is a definite gap between I3 and C1. Middle upper premolar (P3) minute, not visible when viewed laterally, totally intruded from the toothrow. Basal dimension of the anterior upper premolar (P2) is one-fifth of P4 and is approximately fourth the size of P3. First and second lower incisors (I1 and I2) with four lobes. Lower canine (C1) about as high as third lower premolar (P4). First lower premolar (P2) about half the height of the corresponding canine and last premolar. Middle lower premolar (P3) small, only slightly intruded from the toothrow, visible laterally; P2 and P4 are not in contact.

The (damaged) skull and dentition of the holotype (not figured) and a Nepalese specimen (NHMUK 1923.1.9.4) agrees well in all important details with the skull of the specimen collected in Vietnam (Fig. 3).

Figure 3. 

Lateral view of skulls of M. sicarius. a IEBR VN14-0252 from Vietnam; b NHMUK 1923.1.9.4 from Nepal; c occlusal view upper toothrow; d lower toothrow of M. sicarius IEBR VN14-0252 from Vietnam. Upper scale 5 mm, lower scale 3 mm.

The external and craniodental dimensions of the bats from the Himalayas and the new specimen from Vietnam are concordant, with no substantial differences found except the narrower AOB of the latter (Table 2).

Table 2.

Selected external and craniodental measurements (in mm) of Myotis sicarius specimens. Values are given as mean±SD; min-max (n).

M. sicarius (Himalayas) M. sicarius (Vietnam)
FA 49.36±2.07; 46.1–54.5 (18) 49.7
TAIL 42.9
EAR 17.9
TIB 21.07±0.81; 20.6–22 (3) 21.7
HF 9.63±0.99; 8.5–10.8 (4) 9.7
GTL 18.84±0.18; 18.68–19.02 (4) 18.92
CCL 16.59±0.38; 16.19–17 (5) 16.37
C1C1W 5.12±0.18; 4.89–5.50 (10) 5.21
M3M3W 7.9±0.21; 7.59–8.20 (10) 7.97
IOW 4.47±0.22; 4.10–4.70 (9) 4.24
ZYW 12.03±0.27; 11.70–12.30 (5) 11.95
MAW 9.13±0.24; 8.80–9.50 (7) 9.00
BCW 8.19±0.16; 8.01–8.34 (4) 8.18
BCH 6.37±0.19; 6.14–6.60 (4) 6.16
AOB 1.05±0.13; 0.90–1.23 (7) 0.74
CM3L 7.38±0.16; 7.18–7.66 (11) 7.54
CP4L 3.59±0.12; 3.45–3.76 (7) 3.53
MANL 14.1±0.32; 13.60–14.69 (10) 14.61
CM3L 7.91±0.31; 7.20–8.40 (11) 8.16
CP4L 3.15±0.16; 3.05–3.33 (3) 3.32
CPH 4.73±0.17; 4.52–5.01 (6) 4.60

Molecular phylogenetics

Consistent with the morphology-based species identification, our phylogenetic analyses using cyt b and COI sequences (Figs 4 and S1) indicated that the new material of M. sicarius collected from Nepal and Vietnam, and the Nepalese and Indian conspecifics found by previous authors (e.g., Stadelmann et al. 2004b; Chakravarty et al. 2020) group into a well-supported monophyletic lineage (PP=1), which mostly clusters with M. longicaudatus kaguyae, M. bechsteinii, M. frater, and M. daubentonii, and is only distantly related to M. annectans. Within M. sicarius, both analyses showed that Nepalese bats have low intraspecific variation (Kimura’s two-parameter, K2P distance ≤0.53%) but they differ from the Vietnamese specimen by 8.30–9.78% K2P distances (Tables 3 and S1). Within M. annectans, three examined specimens including two from Vietnam (HNHM 26056 and HNHM 24987, with and without the middle upper premolar, respectively), constituted a clade with very low genetic divergences (K2P≤0.6%) (Fig. 4; Table 3).

Figure 4. 

Bayesian inference tree based on cyt b sequences of selected species of Myotis (Kerivoula, Murina and Harpiocephalus sequences were included as outgroups). Numbers at splits indicate posterior probabilities. Red background colour indicates M. sicarius, whereas blue colour designates M. annectans samples.

Table 3.

Estimates of evolutionary divergence between cyt b sequences (in %).

N Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1 Myotis sicarius IEBR VN14-0252 Vietnam
2 Myotis sicarius CDZTU B4 Nepal 9.78
3 AJ841951 Myotis sicarius Nepal 9.59 0.53
4 AF376847 Myotis daubentonii 19.28 20.33 19.47
5 AF376843 Myotis bechsteinii 18.02 18.78 17.95 13.84
6 KF312534 Myotis frater 20.53 19.64 18.77 15.66 16.91
7 AB106593 Myotis longicaudatus kaguyae 18.22 16.05 15.55 13.91 15.19 15.47
8 KF312533 Myotis secundus 19.42 20.77 19.99 19.91 22.20 20.23 16.89
9 Myotis indochinensis GZHU15186 20.30 20.66 19.90 20.14 20.55 20.04 19.35 18.10
10 KF312522 Myotis cf. montivagus 21.88 20.14 19.71 20.05 21.53 22.51 19.42 17.53 12.33
11 AB106609 Myotis gracilis 21.85 23.47 23.18 21.23 22.20 21.68 21.28 22.51 23.42 21.43
12 Myotis annectans HNHM 24987 Vietnam 26.01 25.13 24.72 24.43 24.07 26.73 25.24 25.28 21.33 21.81 25.13
13 Myotis annectans HNHM 26056 Vietnam 26.40 25.50 25.10 24.80 24.44 27.13 25.62 25.66 21.67 22.16 25.50 0.18
14 AJ841956 Myotis annectans 26.40 25.50 25.10 24.61 24.44 27.13 25.62 25.66 22.02 21.81 25.50 0.44 0.62
15 AM261886 Myotis brandtii 20.73 20.20 20.15 21.30 22.02 20.98 19.28 22.76 20.23 21.67 14.58 24.47 24.84 24.84

In relation to results obtained from mtDNA markers, our Rag2 sequence analysis revealed a similarly distant interrelationship between M. sicarius and M. annectans (Fig. 5). Bats of these two taxa differed from each other by a K2P distance of ca. 1% which is regarded as a potential minimum interspecific distance between accepted species of the genus (Ruedi et al. 2013; Table 4). Within M. annectans low genetic divergence was observed (K2P distances ≤0.44%, Table 4), whereas within M. sicarius, Nepalese and Vietnamese bats exhibited very low genetic divergence (0.088% K2P distance, Table 4) with only a few heterozygous or transitional positions. Such low genetic divergence was also recovered in the pairwise comparisons of the nucleotide sequences in three other nuclear genes (ABHD11, PRKC1 and THY) of the two newly sequenced M. sicarius specimens.

Figure 5. 

Bayesian inference tree based on Rag2 sequences of selected species of Myotis (Kerivoula, Murina and Harpiocephalus sequences were included as outgroups). Numbers at splits indicate posterior probabilities. Red background colour indicates M. sicarius, whereas blue colour designates M. annectans samples.

Table 4.

Estimates of evolutionary divergence between Rag2 sequences (in %).

N Sample 1 2 3 4 5 6 7 8 9 10 11 12 13
1 Myotis sicarius IEBR VN14-0252 Vietnam
2 Myotis sicarius CDZTU B4 Nepal 0.088
3 Myotis annectans HNHM 24987 Vietnam 1.074 1.248
4 Myotis annectans HNHM 26056 Vietnam 0.984 1.159 0.439
5 AM265663 Myotis annectans 0.985 1.160 0.000 0.175
6 KF312580 Myotis frater 0.991 1.166 0.800 0.711 0.712
7 AM265653 Myotis daubentonii 1.260 1.435 1.067 0.797 0.888 0.984
8 AM265643 Myotis bechsteinii 1.539 1.714 1.711 1.435 1.529 1.352 1.161
9 AM265647 Myotis brandtii 1.257 1.432 1.155 1.066 1.067 1.073 1.158 1.804
10 KF312561 Myotis longicaudatus kaguyae 1.072 1.245 1.243 1.153 1.156 1.071 1.428 1.705 1.517
11 KF312579 Myotis secundus 1.381 1.559 1.557 1.277 1.373 1.475 1.372 1.656 1.841 1.463
12 Myotis indochinensis GZHU15186 1.254 1.428 1.062 0.973 0.974 0.799 1.155 1.706 1.334 1.424 1.743
13 KF312565 Myotis cf. montivagus 1.351 1.526 1.157 1.067 1.069 0.892 1.251 1.713 1.431 1.520 1.845 0.440
14 AM265660 Myotis gracilis 1.075 1.250 1.066 0.977 0.978 0.983 1.069 1.713 0.885 1.427 1.748 1.244 1.341

Discussion

Taxonomic decisions in grey areas: The case of Myotis sicarius

Comprehensive studies suggested that the levels of mitochondrial sequence divergence below 2% may reflect intraspecific variation, whereas over 10% is indicating the presence of separate species (Bradley and Baker 2001). Between these (soft) limits are the grey-area cases where careful examinations of additional data (nuclear gene sequences, morphology, morphometrics, sound analysis, ecological information) are required to validate taxonomic decisions to split groups of organisms into separate species or to lump them together into a single taxon (Roux et al. 2016; Zachos 2018).

In the case of M. sicarius, the relatively high (8–10%) divergences in mtDNA sequences amongst Nepalese and Vietnamese samples are comparable with the variations found between several traditionally accepted and phylogenetically sister species of Myotis, i.e., Myotis fimbriatus vs. M. pilosus, M. hasseltii vs. M. macrotarsus, M. emarginatus vs. M. formosus, M. alticraniatus vs. M. annamiticus, and M. punicus vs. M. myotis (Ruedi et al. 2013; our own data).

The large geographic distance (which limit their gene flow, even if bats can be strong dispersers) between these samples suggests the existence of two putative reproductively isolated taxonomic units; however, the nuclear gene sequence data and the morphological results are incongruent with this taxonomic inference. In fact, the lack of or low genetic variation in nuDNA markers are attributable to the slower rate of evolution of the nuclear genome (Allio et al. 2017), and speciation is not always accompanied by morphological change, as it was confirmed in the Myotis ater complex (Kruskop et al. 2023), Aselliscus (Tu et al. 2015), Tylonycteris (Tu et al. 2017), or Kerivoula hardwickii-complex (Kuo et al. 2017; Tu et al. 2018). Nevertheless, the observed genetic structure of M. sicarius might be explained by sex-biased gene flow with female philopatry and male dispersal (Funk and Omland 2003). This inference is consistent with previous ecological studies of females of many bat species, especially in case of Myotis which are philopatric to their nursery roosts and to their swarming sites (Rivers et al. 2005). Hence, although further examination is needed to confirm the shallow genetic variation of nuDNA genes and only one Vietnamese specimen was studied morphologically, the available data suggest that the split of geographic populations of M. sicarius into different taxonomic units is unsupported.

Myotis sicarius was previously thought to be a montane forest dwelling bat species, endemic to the southern slopes of the Himalayas between 950–1600 m a.s.l. (Srinivasulu and Srinivasulu 2019) (Fig. 1). With the new record, the occurrence of this species was extended by 1700 km south-eastwards to the border between Vietnam and Laos; nevertheless, the Vietnamese locality is connected to the southern part of the Himalayas through continuous mountains in North Vietnam, North Laos, North Myanmar, and South China. The Vietnamese specimen was caught in a mist-net set at the edge of a mixed evergreen secondary forest adjacent to a mosaic of terraced paddy fields, small streams, gardens and houses of Vin village at 717 m a.s.l. However, the area above the sampling site is extending up to the mountain peak (approximately 1500 m a.s.l.) and is covered by less disturbed evergreen forests.

Based on our data, it is postulated that M. sicarius can adapt to a wider range of habitats than previously known and that the dispersal of individuals (e.g., at least males to find counterparts during mating season) among its geographic populations might be less influenced by physical and ecological barriers (Tu et al. 2021). As such, geographic populations of M. sicarius are expected to maintain a long-range gene flow even under the impacts of extreme habitat changes during the Pleistocene glacial and interglacial periods and this allowed them to evolve a generalist phenotype to suit a variety of environments throughout its distribution range (Uhrin et al. 2010; Hollander et al. 2014; Tu et al. 2021).

Given that M. sicarius is a widespread species, its apparent rarity might be due to gaps in survey coverage in Myanmar, Laos, and Northeast India. The increasing intensity of bat research in these regions will therefore undoubtedly document additional bats that so far remain hidden from the eyes of researchers. Based on these and possible further results, the reassessment of the IUCN Red List status (Vulnerable) of the species will be needed, but at this point we suggest retaining it as it is.

The identification of M. sicarius by external characters may be challenging, as it was indicated by individuals caught in Uttarakhand, India (Chakravarty et al. 2020). These three individuals have the shortest forearm length recorded for the species to date and were initially identified as M. cf. annectans on the bases of these FA values. Although fur colour can be helpful in the identification of certain Myotis species, the reddish coloration around the genital region can also be observed in both M. annectans and M. sicarius, and currently no mutually exclusive external character is known to tell apart these taxa. Chakravarty et al. (2020) provided information about the call parameters of M. sicarius (under the name Myotis cf. annectans) which can be useful to identify the species with acoustic methods. Nevertheless, the following craniodental features can be used to differentiate the two species: the interorbital bridge is narrow (0.4–0.7 mm) in annectans (vs. above this value in sicarius), the palatal emargination is wider than long and widening posteriorly in annectans (vs. quadrate or antero-posteriorly elongated in sicarius), P2 and P4 are in contact and P3 is completely internal to the toothrow in annectans (vs. lower premolars are more or less in line in sicarius).

The dental anomalies in bats can be categorized into two groups: oligodontia and polyodontia. They are uncommon phenomena and may have a significant phylogenetic signal (Esquivel et al. 2021). In case of vespertilionid bats, the most common anomaly is the lack of premolars. There are several Myotis species with such anomalies (Esquivel et al. 2021); however, amongst the Asian species M. annectans seems to be one of the most common species showing this anomaly resulted in taxonomic complications (Topál 1970). Both morphological variants of M. annectans (with and without second upper premolar) were sequenced for mitochondrial and nuclear genes and the subsequent analyses showed low genetic divergences amongst them (0.18–0.62% for cyt b and 0.439% for Rag2). These values are comparable to intraspecific variations of the genus Myotis (Ruedi et al. 2013) which provides robust evidence that M. primula is a junior objective synonym of M. annectans. The localities of individuals with or without second upper premolar show no signs of geographical structuring suggesting that even subspecific separation does not seem justified.

Our article presents a typical example of the “grey zone of taxonomy”, and highlights the need to use different analytical methods and approaches to clarify taxonomic actions based on dense geographical sampling and voucher specimens.

Acknowledgements

In Vietnam, we would like to thank the directorate and staff of the Xuan Lien Nature Reserve and the IEBR for their support during the field survey. The field research was done under the permissions of the People’s Committees of Thanh Hoa provinces and the Vietnamese Ministry of Agriculture and Rural Development (Vietnam Administration of Forestry). In Nepal, we would like to express our grateful thanks to the Department of Forests and Soil Conservation for tissue sample collection permission to ST. This research received support from the National Research, Development, and Innovation Fund of Hungary (NKFIH FK137778, RRF-2.3.1-21-2022-00010 and RRF-2.3.1-21-2022-00006) to GC, TG and GF, the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00825/21) to TG, the Hungarian-Vietnamese bilateral mobility grant (NKM-2021-39) to GF, TG and VTT, and the Vietnam Academy of Science and Technology (Project No: QTHU01.01/22–23) to VTT.

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Supplementary materials

Supplementary material 1 

Figure S1

Győrössy D, Tu VT, Csorba G, Thapa S, Estók P, Földvári G, Görföl T (2024)

Data type: .jpg

Explanation notes: Bayesian inference tree based on COI sequences of selected species of Myotis (Kerivoula, Murina and Harpiocephalus sequences were included as outgroups). Numbers at splits indicate posterior probabilities.

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.
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Supplementary material 2 

Tables S1, S2

Győrössy D, Tu VT, Csorba G, Thapa S, Estók P, Földvári G, Görföl T (2024)

Data type: .zip

Explanation notes: Table S1. Origin of the specimens analysed for the COI genes — Table S2. Estimates of evolutionary divergence between COI sequences.

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.
Download file (21.31 kb)
Supplementary material 3 

File S1

Győrössy D, Tu VT, Csorba G, Thapa S, Estók P, Földvári G, Görföl T (2024)

Data type: .docx

Explanation notes: Origin of the specimens analysed for the cyt b and Rag2 genes. NA denotes that Rag2 sequence is not available from that specimen.

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.
Download file (43.54 kb)
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