Research Article |
Corresponding author: Gábor Csorba ( csorba.gabor@nhmus.hu ) Academic editor: Clara Stefen
© 2024 Dorottya Győrössy, Vuong Tan Tu, Gábor Csorba, Sanjan Thapa, Péter Estók, Gábor Földvári, Tamás Görföl.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Győrössy D, Tu VT, Csorba G, Thapa S, Estók P, Földvári G, Görföl T (2024) The grey zone of taxonomy—The case of the Sikkim Myotis (Chiroptera: Vespertilionidae: Myotis sicarius), first recorded from Southeast Asia. Vertebrate Zoology 74: 737-749. https://doi.org/10.3897/vz.74.e127269
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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.
Area expansion, bats, mito-nuclear discordance, multilocus phylogeny, Vietnam
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 (
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 (
Morphologically, M. sicarius closely resembles species of the montivagus-complex, especially M. indochinensis and M. annectans (
An example of the above taxonomic issues can be found in a recent study of
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 (
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
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).
Myotis annectans: CAMBODIA:
Myotis sicarius: INDIA:
Myotis indochinensis: Vietnam: IEBR M-839-2 (holotype) ♀, A Luoi;
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).
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 (
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
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 |
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 |
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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 |
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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 |
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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 |
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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, OR413179–OR413180 and OR413539–OR413554; 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 (
The combination of the external traits mentioned below are typical of the species, and are essentially similar in the three Nepalese (
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.
The (damaged) skull and dentition of the holotype (not figured) and a Nepalese specimen (
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
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 |
Consistent with the morphology-based species identification, our phylogenetic analyses using cyt b and COI sequences (Figs
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.
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 |
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 |
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 |
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.
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.
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 |
0.088 | ||||||||||||
3 |
Myotis annectans |
1.074 | 1.248 | |||||||||||
4 |
Myotis annectans |
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 |
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 (
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 (
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 (
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. (
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 (
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 (
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 (
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.
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.
Figure S1
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.
Tables S1, S2
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.
File S1
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.