Molecular phylogeny reveals distinct evolutionary lineages of the banded krait, Bungarusfasciatus (Squamata, Elapidae) in Asia

Abstract

The banded krait, Bungarusfasciatus is a widespread elapid snake, likely to comprise several distinct species in different geographic regions of Asia. Therefore, based on molecular phylogenetics and comparative morphology data, we present an overview of the systematic composition of the species to delimit potential biogeographic boundaries. Our phylogenetic analyses, based on four mitochondrial genes, reveal the existence of at least three evolutionary lineages within B.fasciatus, corresponding to Indo-Myanmar, Sundaic and eastern Asian lineages. We are convinced that there are at least three taxonomic entities within the nomen B.fasciatus and restrict the distribution of B.fasciatus sensu stricto to the Indo-Myanmar region. We also provide additional natural history data of the taxon from eastern India. Finally, we advocate further studies to establish the degree of reproductive isolation among these diverging evolutionary lineages and to reassess the systematic status of this species complex especially the Sundaic and eastern Asian lineages.

Full text

1
Vol.:(0123456789)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports
Molecular phylogeny reveals
distinct evolutionary lineages
of the banded krait, Bungarus
fasciatus (Squamata, Elapidae)
in Asia
Lal Biakzuala
1, Hmar T. Lalremsanga
1*, Vishal Santra
2,3, Arindam Dhara
2, Molla T. Ahmed
2,
Ziniya B. Mallick
2, Sourish Kuttalam
2,4, A. A. Thasun Amarasinghe
5* & Anita Malhotra
4*
The banded krait, Bungarus fasciatus is a widespread elapid snake, likely to comprise several
distinct species in dierent geographic regions of Asia. Therefore, based on molecular phylogenetics
and comparative morphology data, we present an overview of the systematic composition of the
species to delimit potential biogeographic boundaries. Our phylogenetic analyses, based on four
mitochondrial genes, reveal the existence of at least three evolutionary lineages within B. fasciatus,
corresponding to Indo-Myanmar, Sundaic and eastern Asian lineages. We are convinced that there
are at least three taxonomic entities within the nomen B. fasciatus and restrict the distribution of
B. fasciatus sensu stricto to the Indo-Myanmar region. We also provide additional natural history
data of the taxon from eastern India. Finally, we advocate further studies to establish the degree of
reproductive isolation among these diverging evolutionary lineages and to reassess the systematic
status of this species complex especially the Sundaic and eastern Asian lineages.
Aside from its taxonomical importance, recognition and ascertainment of independently evolving lineages is
crucial for understanding the evolutionary processes aecting the origin of population structure and species
diversication1. Because of the growing availability of genetic methods for species delineation2, numerous studies
have uncovered cryptic diversity within the widespread vertebrate species including in tropical and sub-tropical
Asia; for instance, among shes3–5, amphibians6–8, birds9–11, and mammals12–14. Moreover, recent phylogeographi-
cal and molecular studies have rened our understanding of cryptic speciation across biogeographic boundaries
or within biogeographic regions15,16, and even propounded the suitability of reptiles in particular as biogeographic
indicators17,18. Recent studies focussing on widespread reptilian species have also established the existence of
previously unnoticed cryptic diversity, including in lizards19–22 and snakes23–30.
Bungarus Daudin, 1803, collectively known as kraits, are venomous elapid snakes which inhabit the Asian
subcontinent31. Most of the nominal Bungarus species are poorly understood. However, recent study on the
diversication and evolution of elapid snakes have highlighted that the diversication of kraits occurred around
30–25 million years ago, and are close relatives of other Australasian elapid genera and sea snakes32. Bungarus
fasciatus (Schneider, 1801), commonly known as the banded krait, is a nocturnal and conspicuous krait that
grows up to 2,250mm in total length and is morphologically characterized by its yellow (or cream) and black
banded body33. It occurs in various habitat types such as primary forests, agricultural lands as well as domestic
gardens up to 2,300m above sea level33,34. So far, B. fasciatus has been reported from eastern India, Nepal, Bhutan,
Bangladesh, and Myanmar, extending southwards through ailand, Malaysia and Singapore into the Indone-
sian archipelago, and eastwards through Laos, Vietnam and China35,36. e species is currently listed as a Least
OPEN
1Developmental Biology and Herpetology Laboratory, Department of Zoology, Mizoram University, Aizawl,
Mizoram 796004, India. 2Society for Nature Conservation, Research and Community Engagement, Nalikul,
Hooghly, West Bengal 712407, India. 3Captive and Field Herpetology, 13 Hirfron, Anglesey LL65 1YU,Wales,
UK. 4Molecular Ecology and Evolution at Bangor, School of Natural Sciences, College of Environmental Sciences
andEngineering,EnvironmentCentreWales,BangorUniversity,BangorLL572UW,UK.5Department of Biology,
FacultyofMathematicsandNaturalSciences,UniversitasIndonesia,KampusUI,Depok16424,Indonesia. *email:
htlrsa@yahoo.co.in; thasun.amarasinghe@ui.ac.id; a.malhotra@bangor.ac.uk
Content courtesy of Springer Nature, terms of use apply. Rights reserved

2
Vol:.(1234567890)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
Concern (LC) species in the IUCN Red List35. Despite its wide distribution, studies have so far been conducted
mainly on its potential medical signicance37, ecological importance38,39, or characterization of venom40–45.
Although there are no studies specically on the molecular systematics of this species, several previous
studies have highlighted intra-specic or geographical variability based on genetic barcoding46–48. Accurate
species delimitation is crucial in view of the variability in snake venom composition49 and its potential eects
on antivenom ecacy50. Most of the existing taxonomic and systematic literature on Bungarus have apparently
overlooked the intraspecic diversity of B. fasciatus51–58. erefore, in this study we ll in the inherent knowledge
gaps by providing comparative morphological evidence and molecular phylogeny based on four mitochondrial
genes (COI, CYTB, ND4 and 16S rRNA) based on sequences from east and northeast India, Indochina, and the
Greater Sunda islands. Moreover, given the minimal knowledge on the natural history, reproductive behaviour,
and ecology, which are important for assessing the population status of the species34,59, we also provide natural
history data for the populations of B. fasciatus from India.
Materials and methods
Sampling. For this study, we collected both morphological and genetic data for Bungarus fasciatus, which we
compared to publicly available or unpublished data. We collected morphological data for the B. fasciatus popula-
tion represented by 15 specimens from northeastern India between the years 2007–2022. We surveyed during
the day and night, collected individuals by hand, and euthanized them with MS-222 following the standard
procedure60 in compliance with the American Veterinary Medical Association (AMVA) guidelines and approved
by the Institutional Animal Ethics Committee (IAEC) (Permission No. MZU-IAEC/2018/12). We then xed
the specimens in 10% buered formalin solution overnight, prior to their storage in 70% ethanol. We preserved
liver tissue samples for DNA analysis in 95% ethanol, which were stored at −20°C. Vouchered specimens were
deposited at the Departmental Museum of Zoology, Mizoram University (MZMU). Additional blood samples
from the caudal sinus were collected from the West Bengal (WB) populations and preserved in EDTA-Tris
buer; these specimens were subsequently released aer taking necessary scale counts. Our study is reported in
accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of InVivo Experiments)61. e dis-
tribution map was prepared using QGIS v3.16.2 and the digital elevation model (DEM) was downloaded from
Open Topography (https:// opent opogr aphy. org/).
DNA extraction, amplication and molecular analyses. Liver tissue or blood was used to extract
genomic DNA using DNeasy (Qiagen™) blood and tissue kits following the manufacturer’s instructions. Frag-
ments of four mitochondrial (mt) markers (16S, COI, ND4 and CYTB) were amplied in a 20 μL reaction
volume, containing 1X DreamTaq PCR Buer, 2.5mM MgCl2, 0.25mM dNTPs, 0.2pM of each gene primer
pair, approximately 3.0ng of extracted DNA, and 1 U of Taq polymerase. A negative control with reagent grade
water instead of DNA template was always included. Target mt gene sequences were amplied using the thermal
proles and primers given in Supplementary TableS1. PCR products were checked using gel electrophoresis on
a 1.5% agarose gel containing ethidium bromide. e PCR products were cleaned using ermoFisher ExoSAP-
IT PCR product cleanup reagent and subsequently sequenced using the Sanger dideoxy method using the ABI
3730xl DNA Analyzer at Barcode BioSciences, Bangalore, India. e generated partial gene sequences were
deposited on the NCBI repository (GenBank accession numbers are given in Supplementary TableS2). In this
study, a total of one COI, six 16S, six ND4, and nine CYTB sequences were generated and were combined with
published sequences of B. fasciatus obtained from the NCBI database; database sequences of B. caeruleus, B.
candidus, B. ceylonicus, B. sindanus, and B. multicinctus were used as outgroups. e four mt gene alignments
were concatenated in SequenceMatrix62. Using the CYTB dataset, the uncorrected p-distance was estimated
in MEGA X using the complete deletion option for the treatment of gaps/missing data63. Prior to the Bayesian
analysis, PartitionFinder v2.164 was utilized to search the best partitioning schemes and the best tting model
through Bayesian Information Criterion (BIC) (Supplementary TableS3). Bayesian phylogeny (BI) was recon-
structed using the selected models in Mr.Bayes v3.2.565. e MCMC was run with four chains (one cold and
three hot chains) for 20 million generations and sampled every 5000 generations. Tracer v1.766 was used to check
the convergence of likelihood and the burn-in cut-o. e diagnosis of topological convergence and MCMC
and mixing of chains was done in R-Studio67 using the package, R We ere Yet (RWTY)68. e BI tree was
further illustrated using web-based tree annotator iTOL soware v569. e Maximum Likelihood (ML) tree
was reconstructed in IQ-TREE70 using 10,000 Ultrafast Bootstrap (UFB)71 based on the dataset partitioned by
codon positions with the most appropriate model selected for each partition using ModelFinder72 integrated
in IQ-TREE70. e CYTB dataset, partitioned by codon, was utilized for performing BI and ML based Poisson
Tree Processes (PTP) species delineation analyses73 implemented in iTaxoTools v0.174. For the input le of PTP,
a non-ultrametric tree was produced in IQ-TREE70 with 10,000 UFB replicates71 using the models selected for
CYTB partitions. Only the CYTB dataset was selected for the species delimitation analysis as it contains more
samples from dierent geographical regions compared to the other three genes.
Morphology. We obtained morphometric (mensural and meristic) data for species comparisons, and distri-
bution data from examined specimens (Java (JV), Mizoram (MZ) and WB) and published literature54,75–77. We
measured the following characters to the nearest millimetre with a Mitutoyo digital caliper and Leica M50 (Leica
Microsystems Inc.) dissecting microscopes: eye diameter (ED, horizontal diameter of orbit); eye–nostril length
(EN, distance between anteriormost point of eye and middle of nostril); snout length (ES, distance between
anteriormost point of eye and snout); head length (HL, distance between posterior edge of mandible and tip
of snout); head width (HW, maximum width of head); snout–vent length (SVL, measured from tip of snout to
anterior margin of vent); tail length (TaL, measured from anterior margin of vent to tail tip). Meristic characters
Content courtesy of Springer Nature, terms of use apply. Rights reserved

3
Vol.:(0123456789)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
were taken as follows: supralabials (SL) and infralabials (IL) (rst labial scale to last labial scale bordering gape);
dorsal scale rows (DSR, counted around the body from one side of ventrals to the other in three positions, on one
head length behind neck, at midbody and at one head length prior to cloacal plate); when counting the number
of ventral scales (Ve), we scored values according to the method described by Dowling78. We counted subcaudal
scales (Sc) from the rst subcaudal scale meeting its opposite to the scale before the tip of the tail, the terminal
scute is excluded when counting. Sex of the specimens was identied by examining everted hemipenes or by
ventral tail dissection. We evaluated the relative size of the nuchal band, the number of the black cross bands
of each individual. e number of cross bands on the body (BB) were counted from the rst band posterior to
the nuchal band on the nape up to the level of cloaca, the count on the tail from the level of cloaca to the tip of
tail (BT), and number of vertebral scales covering the nuchal band (NBW). In addition, the number of vertebral
scales covering the rst cross band is also considered a reliable character for adult individuals. Values for bilateral
head characters are given in le/right order. We followed Keogh79 for hemipenial terminology, and the extent of
inverted hemipenis in terms of percentage of subcaudal scales (HpR).
Statistical analyses. e morphological information was obtained from three dierent populations exam-
ined by us: recent and long-term preserved specimens from JV in Indonesia (n = 15), live specimens from WB
(n = 8) and live, recent and long-term preserved specimens from MZ (n = 15) states in India. Before performing
any further analyses, the meristic data were standardized to zero mean and unit standard deviation to avoid
potential bias due to dierence in the range of measurement among variables; for mensural data, the combina-
tion of characters with the highest R-squared score obtained through linear regression was selected as the best
log transformation model to make linear relationship with body size. Since we do not have gender information
from the WB population, the meristics of the remaining populations (JV and MZ) were rst tested using sepa-
rate one-way analysis of variance (ANOVA) using sex and locality as factors along with Levene’s test80 to test the
homogeneity of variances; if the assumption of homoscedascity was violated, Brown-Forsythe test81 was utilised
as an alternative approach. For mensurals (TaL, HL, and HW), a two-way analysis of covariance (ANCOVA)
was carried out with snout-vent length (SVL) as a covariate. e meristic variables identied with no sexual
dimorphism were utilised for multiple comparison among the three populations by pooling sexes using one-
way ANOVA using locality as a factor, and post-hoc was performed with applying Bonferroni correction. In
addition, a potential observer dierence was screened by repeating measurements on the same specimens and
then tested using one-way ANCOVA. e variable characters among lineages identied through the univariate
analyses were utilized further for Principal Component Analysis (PCA) to visualize the clustering of the dierent
populations. e correlation matrices between all pairs of the morphological variables, variance explained by
each eigenvalue as well as the correlations of each variable to the rst two components are explored. Specimens
with missing characters were excluded in the multivariate analysis. Statistical analyses were performed using the
SPSS v.25.0 statistical package (Armonk, NY: IBM Corp.).
Results
Phylogenetic relationship. e rst 25% of trees from the BI analysis were discarded as burn-in, and
the standard deviation of split frequencies were < 0.005 when analyses terminated. e graphs created using
RWTY in R-Studio also indicated satisfactory topological mixing. e inferred concatenated trees from BI and
ML analyses were congruent with each other. e BI tree, created using Mr.Bayes v3.2.565 and further illustrated
using iTOL soware v569, is show in Fig.1, with Bayesian posterior probabilities from the BI analysis and UFB
values from the ML analysis. e CYTB dataset consisted of a total of 1047 aligned characters, with 97 variable
sites (excluding outgroups).
Molecular phylogenetic based on the concatenated aligned matrix for four mitochondrial genes (16S, COI,
ND4, and CYTB; 2850bp in length), recovered a monophyletic clade consisting of three lineages within Asia.
Both the phylogenetic analyses and the single-locus-based PTP species delineation approach signicantly support
these three distinct clades which we describe as, (i) B. fasciatus from the Sundaic region, especially from Great
Sunda islands which we describe as the Sundaic lineage (Clade I; Fig.1); (ii) B. fasciatus from Indo-Myanmar
(Clade II; Fig.1), and (iii) B. fasciatus from mainland Sundaland including southern China, here described as
east Asian lineage (Clade III; Fig.1).
e overall mean intra-specic divergence across all lineages of B. fasciatus (uncorrected p-distance) was
3.5%. Furthermore, 0.4% intra-clade genetic divergence was observed within Clade I (between two locations in
JV), 0.0%–1.3% within Clade II (between India and Myanmar), and 0.0%–6.5% within Clade III (among China,
Vietnam, ailand, and an unknown locality). e mean inter-clade genetic divergence is 5.0% between Clade
I (Sundaic) and Clade II (Indo-Myanmar), 5.3% between Clade II (Indo-Myanmar) and III (east Asia); 5.7%
between Clade I (Sundaic) and III (east Asia). Combined B. fasciatus (Clades I + II + III) shows the least inter-
specic genetic divergence (19.5%–19.8%) with B. candidus, while inter-specic distances among other species
(B. sindanus, B. caeruleus, B. candidus, B. ceylonicus, and B. multicinctus) range from 3.0% (between B. candidus
and B. multicinctus) to 19.0% (between these two species and B. ceylonicus) (also see Supplementary TableS4).
Morphometric analysis. In this study, despite limited sampling, morphometric analyses were performed
to identify taxonomically informative characters among the examined populations (WB, MZ and JV). Only the
mensurals such as TaL (p < 0.001), HW (p < 0.05) and HL (p < 0.05) showed signicantly dimorphic characters
between males and females within JV and MZ populations. For meristic characters, inter-population dier-
ences were statistically signicant (p < 0.001) for Ve (MZ vs. JV), BB, BT, and NBW (the latter three characters
are tested among three populations), all of which showed a higher number in the MZ population; for mensural
characters, inter-population dierences were also statistically signicant for TaL (p < 0.05) and HL (p < 0.001)
Content courtesy of Springer Nature, terms of use apply. Rights reserved

4
Vol:.(1234567890)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
Figure1. Bayesian inference (BI) phylogenetic tree based on concatenated mitochondrial 16S, COI, ND4 and
CYTB genes; lineage partitions recovered from CYTB-based PTP analyses are presented besides the BI tree
(only the CYTB dataset was utilized for PTP analyses because it contains more representative samples from the
three clades compared to the other genes). Values at each node represent Bayesian posterior probabilities (PP)
and Ultrafast Bootstrap (UFB) values from the Maximum Likelihood (ML) analysis (PP/UFB). Abbreviations of
country and state/province names are: ID Indonesia, JW/J Java, MM Myanmar, AY Ayeyarwady, IN India, WB
West Bengal, MZ Mizoram, AS Assam, VN Vietnam, VC Vinh Phuc, CN China, GZ Guizhou, GX Guangxi, GD
Guangdong, YN Yunnan, TH ailand.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

5
Vol.:(0123456789)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
(Table1). Post-hoc tests conducted among the three populations for BB, BT, and NBW showed that, except for
BT between MZ and WB populations (p > 0.05), signicant dierences are seen for all characters: BB (p < 0.001
across all the populations), NBW (p < 0.001 in MZ vs. WB, and JV vs. WB; p < 0.05 in MZ vs. JV), and BT
(p < 0.001 in MZ vs. JV; p < 0.01 in JV vs. WB). Comparison was also made based on the identied variable
meristic characters among the three populations using a PCA. e correlation matrix showed weak correlations
between pairs of variables (r < 0.7); thus, all variables were retained for this analysis. e rst two components
accounted for 84% of the total variation of the data, with PC1, PC2 and PC3 representing 64%, 20% and 11%,
respectively. e loadings of all variables are high on the rst axis, while only Ve loads considerably highly on
the second axis, with Ve having less eect on PC1 than PC2 (Supplementary TableS5). e representation of the
rst two components depicts substantial separation of the Javanese and the Indian populations on the rst axis
(PC1), and marginal separation of the WB and MZ populations on the second axis (PC2) (Fig.2). Given that the
samples from the three populations (WB, MZ and JV) were examined by dierent recorders, we also tested for
potential recorder bias between the East Indian and northeast Indian specimens; however, no signicant dier-
ences were seen aer re-examination of the same specimens (p > 0.05).
Systematics. We present diagnostic morphological, morphometric, and meristic data taken for Bungarus
fasciatus Clade II from east and northeast India (Supplementary TableS6). e examined specimens of B. fas-
ciatus from India are morphologically distinguishable from the Sundaic population (see Table2). Based on the
present study, we postulate the existence of at least three dierent taxonomic entities within the nomen B. fascia-
tus, and also conrm that populations in eastern India (e.g. Odisha, WB, etc.) and northeastern India (e.g. MZ,
Assam, etc.) are conspecic. Based on the original description of Pseudoboa fasciata, minimum three specimens
were available or referable to Schneider82; hence syntypes. Among these syntypes two specimens (ZMB 2771,
2772) have been deposited at ZMB from the collection of Marcus Bloch (de Bauer83). In addition, one of syn-
types was depicted in Russell84 (page 3, plate 3) as the “Bungarum Pamah”, an adult from “Mansoor Cottah” (now
Gobalpur, Odisha (Orissa), India), specimen is now lost (de Bauer85). So far, the only existing name-bearing
type specimens are the two syntypes in the collection of Berlin Zoological Museum (ZMB 2771–72) originat-
ing from “Indien” (= India) de ZMB catalogue36 a detailed taxonomic revision will be published elsewhere
(Amarasinghe etal. in preparation). We arm that the specimen used by Russell84 for his illustration is the same
specimen (syntype) housed in the ZMB, thus we adhere with the type locality given by Russell84. erefore, here
we postulate the Indo-Myanmar populations (Clade II) as B. fasciatus sensu stricto, while considering the popu-
Table 1. Evaluation on the meristic and mensural characters measured for 38 Bungarus fasciatus individuals
from Java (JV), Mizoram (MZ), and West Bengal (WB), including mean, standard deviation, minimum and
maximum values. Standardized meristic data were utilised for the following tests: Ve of JV and MZ was tested
for inter-population dierence and sexual dimorphism using separate one-way ANOVA with locality and
sex as the factors, respectively; Sc of JV and MZ was tested using two-way ANCOVA using sex and locality
as factors; BB and NBW were tested for inter-population dierence (among the three populations) and
sexual dimorphism (within JV and MZ) using separate one-way ANOVA with locality and sex as the factors,
respectively; since BT violated the assumption of homoscedascity, it was tested using the alternative Brown-
Forsythe test and was indicated by octothorp (#). For mensurals, two-way ANCOVA was performed for the log
transformed TaL, HL, and HW values from JV and MZ by using the log transformed SVL as a covariate, with
locality and sex as the factors. e characters with statistically signicant variations at the alpha level of 0.05
are shown in boldface. e characters tested for inter-population dierence across the three populations are
indicated by asterisk (*). Signicant values are in bold.
Characters Sex
Java (n = 15) Mizoram (n = 15) West Bengal (n = 8)
unsexed
Sexual dimorphism Inter-population dierenceMean ± SD Range Mean ± SD Range Mean ± SD Range
Ve Male 205.44 ± 3.43 199–210 226 ± 2.10 222–228 217.63 ± 3.12 212–222 F1,28 = 1.35 p = 0.256 F1,28 = 469.80 p < 0.001
Female 206.83 ± 1.94 205–210 229.11 ± 2.15 224–231
Sc Male 34.43 ± 0.98 33–36 35.83 ± 0.75 35–37 34.63 ± 1.49 31–36 F1,25 = 2.44 p = 0.131 F1,25 = 1.30 p = 0.266
Female 31.17 ± 1.60 30–34 33.75 ± 1.28 32–36
BB Male 22.67 ± 1.12 21–25 24.33 ± 1.97 22–27 28.38 ± 1.73 26–31 F1,28 = 0.44 p = 0.511 F2,35 = 39.78* p < 0.001*
Female 21.83 ± 1.17 20–23 25.00 ± 1.58 23–27
BT Male 3.22 ± 0.67 2–4 5.00 ± 0.00 55.25 ± 1.09 4–7 F1,21 = 0.12#p = 0.728#F2,12 = 17.86*#p < 0.001*#
Female 3.17 ± 0.41 3–4 4.22 ± 0.44 4–5
NBW Male 19.00 ± 1.00 18–20 18.20 ± 0.45 18–19 15.63 ± 1.11 14–17 F1,27 = 0.40 p = 0.533 F2,34 = 22.16* p < 0.001*
Female 19.00 ± 0.63 18–20 17.67 ± 1.73 15–20
TaL Male 120.74 ± 20.01 90–145 101 ± 38.92 47–133 – – F1,24 = 18.96 p < 0.001 F1,24 = 6.01 p = 0.022
Female 107.86 ± 23.43 85–145 97.88 ± 15.56 76–119
HL Male 35.06 ± 4.97 27.10–40.90 21.60 ± 5.71 12.80–26.60 – – F1,24 = 4.37 p = 0.047 F1,24 = 79.38 p < 0.001
Female 34.81 ± 6.19 25.90–44.50 21.03 ± 5.03 15.74–29.68
HW Male 20.88 ± 4.03 13.80–25.70 17.79 ± 5.10 12.18–22.46 – – F1,25 = 4.33 p = 0.048 F1,25 = 0.97 p = 0.334
Female 20.70 ± 3.13 16.40–26.20 16.12 ± 4.30 10.40–22.76
Content courtesy of Springer Nature, terms of use apply. Rights reserved

6
Vol:.(1234567890)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
lations from Sundaic region, especially from Greater Sunda Islands (Clade I) and mainland Sundaland including
southern China (Clade III) as B. fasciatus sensu lato. Consequently, we redescribe the B. fasciatus sensu stricto,
including hemipenis morphology, based on MZ population, from where a large number of samples are available.
Bungarus fasciatus (Schneider, 1801) sensu stricto. (Tables1, 2; Figs.3A–E, 4A,B, 5).
[English: Banded krait; Bengali: Sankhamuti/Sankhini/Chamorkasa; Mizo: Chawnglei/Tiangsir].
Pseudoboa fasciata Schneider, 1801.
Bungarus annularis Daudin, 1803.
Bungarus fasciatus bifasciatus Mell, 1929.
Bungarus fasciatus insularis Mell, 1930.
Examined materials. Males (n = 7; MZMU 933, 1314, 1320, 1417, 1421, 1883, 2935) and Females (n = 8;
MZMU 1319, 1321, 1550, 1562, 1561, 1548, 1572, 2481) collected from MZ, northeast India.
Species redescription. Based on the overall examined MZ materials with combined sexes, adults SVL
444.0–1220.0mm, tail length 47.0–133.0mm; head elongate (HL 2.0–3.5% of SVL), wide (HW 71.8–92.1%
of HL), slightly attened, indistinct from neck; snout elongate (ES 22.8–40.1% of HL), moderate, at in dorsal
view, rounded in lateral prole, rather depressed. Rostral shield large, at, slightly visible from above, pointed
posteriorly; interorbital width broad; internasals subtriangular; nostrils rather large, nasals large, divided, and
elongated, in anterior contact with rostral, and internasal and prefrontal dorsally, 1st and 2nd supralabial ven-
trally, preocular posteriorly; no loreal; prefrontal rather large, broader than long, and pentagonal; frontal large,
hexagonal, short, slightly longer than width; supraoculars narrow, elongate, subrectangular, posteriorly wider;
parietals large, elongate, buttery wing-like in shape, bordered by supraoculars, frontal, upper postocular ante-
riorly, anterior and upper posterior temporals, and ve or six nuchal scales posteriorly; one preocular, vertically
slightly elongated, hexagonal, in contact with prefrontal and posterior nasal anteriorly, supraocular dorsally, and
Figure2. Ordination of Bungarus fasciatus populations from Mizoram (MZ), West Bengal (WB) and Java (JV)
along the rst two principal components based on a PCA of the characters Ve, BB, BT, and NBW. Total variance
associated with PC1 and PC2 are 64% and 20%, respectively.
Table 2. Some comparative morphological data of Bungarus fasciatus sensu lato in each biogeographic region,
based on this study and published data.
Character
Population/clade
Indo-Myanmar (n = 23) East Asia (n = 11) Greater Sunda (n = 15)
Ventrals 200–234 217–237 199–210
Subcaudals 23–39 33–41 30–36
Number of dorsal bands on body 22–31 19–21 20–25
Number of dorsal bands on tail 4–7 ? 2–4
Nuchal band covered by vertebral scales 14–20 ? 18–20
Background body color Yellow/cream Yellow Yellow/cream
Source Smith75
is study Yang & Rao76;
Chen etal.54; Leviton etal.77 is study
Content courtesy of Springer Nature, terms of use apply. Rights reserved

7
Vol.:(0123456789)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
2nd and 3rd supralabials ventrally; eye moderate (ED 10.7–21.7% of HL), round, about half of the size of snout
length (ED 41.7–69.9% of ES), pupil rounded; two postoculars, subequal or upper one larger, pentagonal, upper
postocular in broad contact with supraocular, parietal and anterior temporal, lower postocular in contact with
anterior temporal and 5th supralabials; temporals 1 + 2, large, slightly elongated, subrectangular or pentagonal;
anterior temporal larger than posterior temporal, in contact with parietal and both postoculars dorsally, and
5th and 6th supralabial ventrally; lower posterior temporal in contact with 6th and 7th supralabials ventrally.
Supralabials seven (on both sides), 5th–7th largest in size; 1st supralabial in contact with rostral anteriorly, nasals
dorsally, 2nd with posterior nasal and preocular dorsally, 3rd with preocular and orbit dorsally, 4th with orbit;
Figure3. Bungarus fasciatus sensu stricto (MZMU1883) from Northeast India: (a) dorsal view of full body, (b)
ventral view of full body, (c) dorsal view of head, (d) lateral view of the le side of head, and (e) ventral view of
head.
Figure4. Live individuals of Bungarus fasciatus sensu stricto (a) from Keitum village, Mizoram, India
(MZMU1421), and (b) a juvenile with creamish dorsum coloration from Saikhawthlir village, Mizoram, India.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

8
Vol:.(1234567890)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
5th with orbit, lower postocular, and anterior temporal dorsally, and 6th with anterior and lower posterior tem-
porals dorsally, 7th with lower posterior temporal dorsally and scales of the neck posteriorly.
Mental large, triangular, blunt posteriorly; rst infralabial pair larger than mental plate and in broad contact
with each other, in contact with anterior chin shields posteriorly; seven infralabials, 1st–4th in contact with ante-
rior chin shields, 4th infralabial largest in size in contact with both anterior and posterior chin shields; 4th–7th
infralabials in contact with gular scales; two larger anterior chin shields, and two slightly smaller posterior chin
shields; anterior chin shields in broad contact between them; posterior chin shields bordered posteriorly by
seven gular scales.
Body robust, elongate and subcylindrical; dorsal scales in 15 midbody rows, all smooth and pointed poste-
riorly; 222–228 ventrals in males and 224–231 in females; cloacal plate divided. Tail comparatively short, TaL
8.9–10.4% of total length in males and 13.5–17.1% of total length in males, robust and thick; subcaudals 35–37
in males and 32–36 in females, divided.
Coloration. In preservative, dorsum and venter white or yellow; 22–27 black cross bands along the body and
4 or 6 on the tail; cross bands complete laterally, and reaching the ventrals except the nuchal band; the bands
on the tail distinct; the nuchal band on the nape anteriorly inverted V-shaped covering 15–20 vertebral scales;
nuchal band starts from mid frontal; snout, anterior head, and lateral head black making remaining the white
dorsal color an inverted V-shaped marking; rst black band on the body covering 6 or 7 vertebral scales; inter-
band width covers with 3–5 vertebral scales; lower parts of the supralabials white; ventral head white until the
rst black band; tail tip black dorsally, white ventrally.
In life (Fig.4A), same color as in preservative, but the white body color may vary from white, cream, pale
yellow to bright yellow. One juvenile with cream and black body bands was encountered in Saikhawthlir, MZ
(Fig.4B), but the snake escaped before recording morphological data.
Variation. Except the anomalous specimen (MZMU1321) which had three postoculars on le and two on
right, and temporals 1 + 2 on the le and 2 + 2 on the right, all other meristic and morphometric characters
obtained so far did not show any signicant variation between the examined populations, and also correspond
to the conventional taxonomical characters provided in previously published literature77,86,87.
Hemipenis. Based on MZMU2935, the organ is single and subcylindrical, relatively short, robust, and capi-
tate; inverted hemipenis extends to 4th–7th subcaudal level (i.e. 11.1–20% from the total number of Sc); sulcus
spermaticus bifurcate below the crotch, shallow and centripetal; apical lobe less evident with only slight apical
aring; calyculate organ with a complex ornamentation of retiform ridges, papillate ounces, and spines; spines
on the upper basal areas enlarged and decreasing the size towards the proximal portion; apical region sharply
separated from the basal portion by a well-dened demarcation, so the apex is free and the apical part of the
hemipenis is richly capitate (Fig.5).
Distribution. Within India, B. fasciatus has been reported from Uttar Pradesh (Gorakhpur, de Masson88;
also see Anwar89 and Das etal.90) in the north and central Maharashtra in the west91–93, extending across Telan-
gana (Hyderabad, de Kinnear94, Andhra Pradesh95, Chhattisgarh96,97, Jharkhand (Koderma, de Smith86; also
see Husain98), Bihar99, Odisha (Mahanadi valley, de Wall99; also see Boruah etal.100), and northern part of WB101
to northeastern India, including Arunachal Pradesh102,103, Assam99,104,105, Meghalaya106, MZ107,108, Tripura109,
Figure5. Sulcal (le) and asulcal (right) views of the right hemipenis of Bungarus fasciatus sensu stricto
(MZMU2935) from Mizoram, India.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

9
Vol.:(0123456789)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
Manipur110 and Nagaland111. A few unveried records are available from Madhya Pradesh36, Uttarakhand35,and
southern peninsular India in Tamil Nadu, Karnataka and Kerala98.
Here we provide additional distributional records for B. fasciatus sensu stricto based on 44 new localities from
MZ, and two from WB, India (Supplementary TableS7). e lowest elevation among these new records is 4m
a.s.l. at Chitrasali in Hooghly District, WB and the highest is 1426m a.s.l. at Champhai Jailveng in Champhai
District, MZ. Based on the previous distribution of the species, the elevation range was between 40 and 2300m
a.s.l.33,34. Moreover, an estimated distribution range of the species was plotted (Fig.6) following WHO’s range
estimation for B. fasciatus112.
Natural history. Although B. fasciatus is a common species, details on the ecology, habitat, population, and
breeding are still sparse and further studies are needed. erefore, here we provide some natural history data
based on two clutches of eggs encountered from two localities in WB State, India:
(i) On 16th May 2020, at ca. 20:00h, from Chitrasali village, Hooghly, the snake was encountered on the bank
of a pond adjacent to a house in the middle of a village. e female was found coiling around a clutch of 19 eggs.
e breeding site was located inside a naturally occurring burrow at the base of a dead tree with decayed roots.
e burrow was on the bank ca. 6 feet from the pond. e pond had a gentle slope and was surrounded by plenti-
ful vegetation. On the day of the egg collection, the recorded ambient temperature at the natural breeding site was
28–38°C with average humidity of 78%. e eggs were relocated and incubated in a dedicated herpetoculture
room at 27.6°C using 3cm thick vermiculite bedding in a perforated box. On 10th June at 20:18h, the rst egg
slitswere observed, and hatching was completed on 18th June at 05:45h. e uctuating room temperature and
Figure6. Map showing the distribution range of Bungarus fasciatus sensu lato, based on the latest species map
provided by the World Health Organization (2022); the coloration corresponds to the three distinct evolutionary
lineages recovered in the phylogenetic analyses. e type locality of Bungarus fasciatus sensu stricto is indicated
by a black star. Localities of specimens used in the morphological analyses are indicated by black lled diamonds
(WB), circles (MZ), and triangles (JV). Abbreviations for countries are: IN India, NP Nepal, BT Bhutan, BD
Bangladesh, LK Sri Lanka, CN China, MM Myanmar, LA Laos, TH ailand, VN Vietnam, KH Cambodia, MY
Malaysia, BN Brunei Darussalam, ID Indonesia (KA Kalimantan, SM Sumatra, JW Java).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

10
Vol:.(1234567890)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
average humidity from the start of hatching until hatching was completed were 26–35°C and 81.1%, respectively.
Notably, hatchlings crawled out from the pipped eggs on the 12th, 13th, and 14th June. Upon investigation, we
found that a total of six eggs failed to hatch, out of which three eggs were unfertilized, two contained partially
developed embryos showing deformities, and one egg had a fully developed embryo, possibly unable to cut
through the eggshell. On 18th June, we recorded the biometric data of the 13 hatchlings (5 females with aver-
age SVL 322.2mm, TaL 32.4mm, and body weight 21.2g; 8 males with average SVL 318.6mm, TaL 36.5, body
weight 19.9g), and were subsequently released close to where the eggs were collected.
(ii) On 05th May 2021 at 12:30pm, from a construction site at Ankuni village, Hooghly. A clutch of eight
eggs were uncovered under a pile of old bricks at the base of a dead tree with lots of burrows. e breeding site
was located on the bank of a pond, and the entire rubble pile was covered in vegetation. However, in this case,
the female snake was not found near the eggs, and it is possible that the excavation work might have scared the
female away. e eggs were relocated and incubated in the same herpetoculture room using 3cm thick ver-
miculite bedding in a perforated box. e room temperature recorded on 5th May uctuated between 24 and
33°C, with a relative humidity of 65%. Egg slits were seen on 6th June at ca. 22:00h. On 8th June at ca. 08:00h,
hatching was completed and all of the juveniles had emerged from the eggs. From the egg relocation until the
completed hatching (6th–8th June), the temperature and humidity uctuated between 24 and 39°C and 65–75%,
respectively. On 8th June, the biometric data of the eight hatchlings were taken (3 females with average SVL
333.3mm, TaL 38.7mm, and body weight 21.3g; 5 males with average SVL 351.0mm, TaL 43.2, body weight
21.4g), and they were also released close to the site from which the eggs had been collected.
Discussion
Bungarus fasciatus sensu stricto. Evidence from this study, based on morphology and molecular data,
denes three distinct clades of B. fasciatus with non-overlapping distribution clusters. e high genetic diver-
gence among lineages also suggests distinct species-level groups within B. fasciatus as currently conceived. Our
morphometric data analysis also provides evidence of their morphological distinctiveness between Clade I and
II. Moreover, the lineage from east Asia is basal to the other two lineages but, if these clades were to be accepted
as full species, the name-bearing lineage is Clade II. us, according to our newly presented evidence, and partly
according to Russell84, the distribution range of Bungarus fasciatus sensu stricto (Indo-Myanmar clade) com-
prises east and northeast India extending towards Myanmar. (Figs.1, 6).
Systematic challenges. In this study, we elucidate the presence of three independent lineages within B.
fasciatus, which is crucial for future nomenclatural revision. In the CYTB gene, while negligible intra-clade
genetic divergence was observed within Clade I (0.4%; between two locations in JV) and Clade II (0.0–1.3%;
Myanmar, east and northeast India), a wide range of intra-clade genetic divergence (00.0–6.5%) was evident
within Clade III (China, Vietnam, ailand). Consequently, we speculate that there might still be cryptic diver-
sity within the east Asian lineage (Clade III). Moreover, for robust delimitation of the B. fasciatus complex, it is
necessary to establish whether these lineages have undergone some degree of extrinsic or intrinsic reproductive
isolation to be evolving separately113. For instance, due to the high evolutionary rate of hemipenial traits com-
pared to the other morphological traits114,115, the organ has commonly been used to provide a picture of sexual
barrier even among cryptic species116–118.
Although it has been previously stressed that delimiting the taxonomic status of geographically diversied
populations of venomous snakes alone cannot necessarily predict patterns of venom variation, it can play a
pivotal role in overcoming the consequential variability of venoms119–121. Fry etal.120 further indicated that the
medical importance of B. fasciatus has been overestimated. Moreover, the possible existence of undiscovered
cryptic species accompanied by more venom diversity with uncharacterized components had been pointed out122.
Siqueira-Silva etal.123 observed that more productive environments favour more complex venom, with more
toxins in similar proportions. Based on the verbal autopsy we have conducted so far within MZ, there are three
cases of fatal envenomation potentially from the bite of banded krait. erefore, here we highlight the importance
of analyzing the venom compositions in dierent populations in each biogeographically isolated clade.
Further work. e combination of multivariate morphometric analysis and mitochondrial gene-based
phylogeography has been applied successfully for species delineation24,124,125 as well as for testing species
boundaries126. However, nuclear genes provide an independent test of species boundaries127 as they are capable
of measuring the extent of gene ow, and for this reason, recent work has increasingly used a combination of
nuclear and mitochondrial genes for phylogeographic analyses and species delineation128. Consequently, we
believe that the potentially species-level diversity across dierent B. fasciatus populations depicted in this study
cannot be overlooked, and a thorough comprehension of B. fasciatus systematics is still a fundamental challenge.
Data availability
e generated partial gene sequences were deposited on the NCBI repository (GenBank accession numbers are
given in Supplementary TableS2).
Received: 18 August 2022; Accepted: 16 January 2023
References
1. Jirsová, D. et al. From taxonomic deation to newly detected cryptic species: Hidden diversity in a widespread African squeaker
catsh. Sci. Rep. 9, 1–13. https:// doi. org/ 10. 1038/ s41598- 019- 52306-2 (2019).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

11
Vol.:(0123456789)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
2. Luo, A., Ling, C., Ho, S. Y. & Zhu, C. D. Comparison of methods for molecular species delimitation across a range of speciation
scenarios. Syst. Biol. 67, 830–846. https:// doi. org/ 10. 1093/ sysbio/ syy011 (2018).
3. Dwivedi, A. K. et al. Cryptic diversity in the Indian clade of the catsh family Pangasiidae resolved by the description of a new
species. Hydrobiologia 797, 351–370 (2017).
4. Halasan, L. C., Geraldino, P. J. L. & Lin, H. C. First evidence of cryptic species diversity and population structuring of Selaroides
leptolepis in the tropical western Pacic. Front. Mar. Sci. 8, 756163. https:// doi. org/ 10. 3389/ fmars. 2021. 756163 (2021).
5. Matsumoto, S. et al. Cryptic diversication of the swamp eel Monopterus albus in East and Southeast Asia, with special reference
to the Ryukyuan populations. Ichthyol. Res. 57, 71–77. https:// doi. org/ 10. 1007/ s10228- 009- 0125-y (2010).
6. Nishikawa, K. et al. Molecular phylogeny and biogeography of caecilians from Southeast Asia (Amphibia, Gymnophiona, Ich-
thyophiidae), with special reference to high cryptic species diversity in Sundaland. Mol. Phylogenet. Evol. 63, 714–723. https://
doi. org/ 10. 1016/j. ympev. 2012. 02. 017 (2012).
7. R amesh, V., Vijayakumar, S. P., Gopalakrishna, T., Jayarajan, A. & Shanker, K. Determining levels of cryptic diversity within the
endemic frog genera, Indirana and Walkerana, of the Western Ghats, India. PLoS ONE 15, e0237431. https:// doi. org/ 10. 1371/
journ al. pone. 02374 31 (2020).
8. Stuart, B. L., Inger, R. F. & Voris, H. K. High level of cryptic species diversity revealed by sympatric lineages of Southeast Asian
forest frogs. Biol. Lett. 2, 470–474. https:// doi. org/ 10. 1098/ rsbl. 2006. 0505 (2006).
9. Lohman, D. J. et al. Cryptic genetic diversity in “widespread” Southeast Asian bird species suggests that Philippine avian end-
emism is gravely underestimated. Biol. Conserv. 143, 1885–1890. https:// doi. org/ 10. 1016/j. biocon. 2010. 04. 042 (2010).
10 . Outlaw, D. C. & Voelker, G. Pliocene climatic change in insular Southeast Asia as an engine of diversication in Ficedula ycatch-
ers. J. Biogeogr. 35, 739–752. https:// doi. org/ 10. 1111/j. 1365- 2699. 2007. 01821.x (2008).
11 . Rheindt, F. E., Wu, M. Y., Movin, N. & Jønsson, K. A. Cryptic species-level diversity in Dark-throated Oriole Oriolus xanthonotus.
Bull. Br. Ornithol. Club. 142, 254–267. https:// doi. org/ 10. 25226/ bboc. v142i2. 2022. a10 (2022).
12. Chattopadhyay, B. et al. Cryptic diversity of Rhinolophus lepidus in South Asia and dierentiation across a biogeographic barrier.
Front. Biogeogr. https:// doi. org/ 10. 21425/ F5FBG 49625 (2021).
13. Chen, S. et al. Multilocus phylogeny and cryptic diversity of white-toothed shrews (Mammalia, Eulipotyphla, Crocidura) in
China. BMC Evol. Biol. 20, 1–14. https:// doi. org/ 10. 1186/ s12862- 020- 1588-8 (2020).
14. Nater, A. et al. Morphometric, behavioral, and genomic evidence for a new orangutan species. Curr. Biol. 27, 3487–3498. https://
doi. org/ 10. 1016/j. cub. 2017. 09. 047 (2017).
15. Pfenninger, M. & Schwenk, K. Cryptic animal species are homogeneously distributed among taxa and biogeographical regions.
BMC Evol. Biol. 7, 1–6. https:// doi. org/ 10. 1186/ 1471- 2148-7- 121 (2007).
16 . Vodă, R., Dapporto, L., Dincă, V. & Vila, R. Cryptic matters: Overlooked species generate most buttery beta-diversity. Ecography
38, 405–409. https:// doi. org/ 10. 1111/ ecog. 00762 (2014).
17. Bauer, A. M. Reptiles and the biogeographic interpretation of New Caledonia. Tuatara 30, 39–50 (1989).
18. Camargo, A., Sinervo, B. & Sites, J. W. Lizards as model organisms for linking phylogeographic and speciation studies. Mol. Ecol.
19, 3243–3488. https:// doi. org/ 10. 1111/j. 1365- 294x. 2010. 04722.x (2010).
19. Gowande, G. et al. Molecular phylogenetics and taxonomic reassessment of the widespread agamid lizard Calotes versicolor
(Daudin, 1802) (Squamata, Agamidae) across South Asia. Vertebr. Zool. 71, 669–696. https:// doi. org/ 10. 3897/ vz. 71. e62787
(2021).
20. Guo, P. et al. Cryptic diversity of green pitvipers in Yunnan, South-west China (Squamata, Viperidae). Amphib. Reptil. 36,
265–276 (2015).
21. Wagner, P. et al. Integrative approach to resolve Calotes mystaceus Duméril & Bibron, 1837 species complex (Squamata: Agami-
dae). Bonn Zool. Bull. 70, 141–171. https:// doi. org/ 10. 20363/ BZB- 2021. 70.1. 141 (2021).
22. Zug, G., Brown, H., Schulte, J. & Vindum, J. Systematics of the Garden Lizards, Calotes versicolor Group (Reptilia, Squamata,
Agamidae), in Myanmar: Central Dry Zone Populations. Proc. Calif. Acad. Sci. 57, 35–68 (2007).
23. Alfaro, M. E., Karns, D. R., Voris, H. K., Abernathy, E. & Sellins, S. L. Phylogeny of Cerberus (Serpentes: Homalopsinae) and
phylogeography of Cerberus rynchops: Diversication of a coastal marine snake in Southeast Asia. J. Biogeogr. 31, 1277–1292.
https:// doi. org/ 10. 1111/j. 1365- 2699. 2004. 01114.x (2004).
24. Malhotra, A., Dawson, K., Guo, P. & orpe, R. S. Phylogenetic structure and species boundaries in the mountain pitviper
Ovophis monticola (Serpentes: Viperidae: Crotalinae) in Asia. Mol. Phylogenet. Evol. 59, 444–457. https:// doi. org/ 10. 1016/j.
ympev. 2011. 02. 010 (2011).
25. Mallik, A. K. et al. Disentangling vines: A study of morphological crypsis and genetic divergence in vine snakes (Squamata:
Colubridae: Ahaetulla) with the description of ve new species from Peninsular India. Zootaxa 4874, 1–62. https:// doi. org/ 10.
11646/ zoota xa. 4874.1.1 (2020).
26. Shankar, P. G. et al. King or royal family? Testing for species boundaries in the King Cobra, Ophiophagus hannah (Cantor, 1836),
using morphology and multilocus DNA analyses. Mol. Phylogenet. Evol. 165, 107300. https:// doi. org/ 10. 1016/j. ympev. 2021.
107300 (2021).
27. orpe, R. S., Pook, C. E. & Malhotra, A. Phylogeography of Russell’s viper (Daboia russelii) complex in relation to variation in
the colour pattern and symptoms of envenoming. Herpetol. J. 10, 209–218 (2007).
28. Wüster, W. Taxonomic changes and toxinology: Systematic revisions of the Asiatic cobras (Naja naja) species complex. Tox i con
34, 399–406. https:// doi. org/ 10. 1016/ 0041- 0101(95) 00139-5 (1996).
29. Wüster, W. & orpe, R. S. Naja siamensis, a cryptic species of venomous snake revealed by mtDNA sequencing. Experientia
50, 75–79. https:// doi. org/ 10. 1007/ BF019 92054 (1994).
30. Wüster, W., Otsuka, S., Malhotra, A. & orpe, R. S. Population systematics of Russell’s viper: A multivariate study. Biol. J. Linn.
Soc. 47, 97–113. https:// doi. org/ 10. 1111/j. 1095- 8312. 1992. tb006 58.x (1992).
31. Midtgaard, R. Repfocus, a Survey of the Reptiles of the World. http:// repfo cus. dk/ Bunga rus. html (2022).
32. Lee, M. S., Sanders, K. L., King, B. & Palci, A. Diversication rates and phenotypic evolution in venomous snakes (Elapidae). R.
Soc. Open Sci. 3, 150277. https:// doi. org/ 10. 1098/ rsos. 150277 (2016).
33. Ahmed, M. F., Das, A. & Dutta, S. K. Amphibians and Reptiles of Northeast India. A Photographic Guide (Aaranyak, 2009).
34. Knierim, T. K., Strine, C. T., Suwanwaree, P. & Hill III, J. G. Spatial ecology study reveals nest attendance and habitat preference
of banded kraits (Bungarus fasciatus). Herpetol. Bull. 150, 6–13 (2019).
35. Stuart, B. etal. Bungarus fasciatus. e IUCN Red List of reatened Species 2013. https:// www. iucnr edlist. org/ speci es/ 192063/
20349 56 (2013).
36. Wallach, V., Williams, K. L. & Boundy, J. Snakes of the World : A Catalogue of Living and Extinct Species (CRC Press, 2014).
37. Pe, T. et al. Envenoming by Chinese krait (Bungarus multicinctus) and banded krait (B. fasciatus) in Myanmar. Trans. R. Soc.
Trop. Med. Hyg. 91, 686–688. https:// doi. org/ 10. 1016/ S0035- 9203(97) 90524-1 (1997).
38. Ahsan, M. F. & Rahman, M. M. Status, distribution and threats of kraits (Squamata: Elapidae: Bungarus) in Bangladesh. J. re at.
Taxa. 9, 9903–9910. https:// doi. org/ 10. 11609/ jott. 2929.9. 3. 9903- 9910 (2017).
39. Tongpoo, A. et al. Krait envenomation in ailand. er. Clin. Risk Manag. 14, 1711–1717. h t tps:// doi. org/ 10. 2147/ TCRM. S1695
81 (2018).
40. Lo, T. B. & Lu, H. S. Studies on Bungarus fasciatus Venom . (Toxins, 1978).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

12
Vol:.(1234567890)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
41. Lu, J. et al. A novel serine protease inhibitor from Bungarus fasciatus venom. Peptides 29, 369–374. https:// doi. org/ 10. 1016/j.
pepti des. 2007. 11. 013 (2008).
42. Rusmili, M. R. A., Yee, T. T., Mustafa, M. R., Hodgson, W. C. & Othman, I. Proteomic characterization and comparison of
Malaysian Bungarus candidus and Bungarus fasciatus venoms. J. Proteom. 110, 129–144. https:// doi. org/ 10. 1016/j. jprot. 2014.
08. 001 (2014).
43. Tan, N. H. & Ponnudurai, G. A comparative study of the biological properties of krait (genus Bungarus) venoms. Comp. Biochem.
Physiol. C. Toxicol. Pharmacol. 95, 105–109. https:// doi. org/ 10. 1016/ 0742- 8413(90) 90089-r (1990).
44 . Tsai, I. H., Tsai, H. Y., Saha, A. & Gomes, A. Sequences, geographic variations and molecular phylogeny of venom phospholipases
and threenger toxins of eastern India Bungarus fasciatus and kinetic analyses of its Pro31 phospholipases A2. FEBS J. 274,
512–525. https:// doi. org/ 10. 1111/j. 1742- 4658. 2006. 05598.x (2007).
45. Ziganshin, R. H. et al. Quantitative proteomic analysis of Vietnamese krait venoms: Neurotoxins are the major components in
Bungarus multicinctus and phospholipases A2 in Bungarus fasciatus. Tox i con 107, 197–209. https:// doi. org/ 10. 1016/j. toxic on.
2015. 08. 026 (2015).
46. Kundu, S. et al. Mitochondrial DNA discriminates distinct population of two deadly snakes (Reptilia: Elapidae) in Northeast
India. Mitochondrial DNA B Resour. 5, 1530–1534. https:// doi. org/ 10. 1080/ 23802 359. 2020. 17422 10 (2020).
47. Laopichienpong, N. et al. Assessment of snake DNA barcodes based on mitochondrial COI and Cytb genes revealed multiple
putative cryptic species in ailand. Gene 594, 238–247. https:// doi. org/ 10. 1016/j. gene. 2016. 09. 017 (2016).
48. Supikamolseni, A. et al. Molecular barcoding of venomous snakes and species-specic multiplex PCR assay to identify snake
groups for which antivenom is available in ailand. Genet. Mol. Res. 14, 13981–13997. https:// doi. org/ 10. 4238/ 2015. octob er.
29. 18 (2015).
49. Chippaux, J. P., Williams, V. & White, J. Snake venom variability: Methods of study, results and interpretation. Tox i c on 29,
1279–1303. https:// doi. org/ 10. 1016/ 0041- 0101(91) 90116-9 (1991).
50. Harrison, R. A., Wüster, W. & eakston, R. D. G. e conserved structure of snake venom toxins confers extensive immuno-
logical cross-reactivity to toxin-specic antibody. Toxicon 41, 441–449. https:// doi. or g/ 10. 1016/ s004 1- 0101 (02) 00360-4 (2003).
51. Abtin, E., Nilson, G., Mobaraki, A., Hosseini, A. A. & Dehgannejhad, M. A new species of krait, Bungarus (Reptilia, Elapidae,
Bungarinae) and the rst record of that genus in Iran. Russ. J. Herpetol. 21, 243–250. https:// doi. o rg/ 10. 30906/ 1026- 2296- 2014-
21-4- 243- 250 (2014).
52. Ashraf, M. R. et al. Phylogenetic analysis of the Common Krait (Bungarus caeruleus) in Pakistan based on mitochondrial and
nuclear protein coding genes. Amphib. Reptile Conserv. 13, 203–211 (2019).
53. Biakzuala, L., Purkayastha, J., Rathee, Y. S. & Lalremsanga, H. T. New data on the distribution, morphology, and molecular
systematics of two venomous snakes, Bungarus niger and Bungarus lividus (Serpentes: Elapidae), from north-east India. Sala-
mandra. 57, 219–228 (2021).
54. Chen, Z. N., Shi, S. C., Vogel, G., Ding, L. & Shi, J. S. Multiple lines of evidence reveal a new species of Krait (Squamata, Elapidae,
Bungarus) from Southwestern China and Northern Myanmar. Zookeys. 1025, 35–71. https:// doi. org/ 10. 3897/ zooke ys. 1025.
62305 (2021).
55. Keogh, J. S. Molecular phylogeny of elapid snakes and a consideration of their biogeographic history. Biol. J. Linn. Soc. 63,
177–203. https:// doi. org/ 10. 1111/J. 1095- 8312. 1998. TB015 13.X (1998).
56. Kuch, U. et al. A new species of krait (Squamata: Elapidae) from the Red River system of northern Vietnam. Copeia 818–833,
2005. https:// doi. org/ 10. 1643/ 0045- 8511% 282005% 29005% 5B0818% 3AANS OKS% 5D2.0. CO% 3B2 (2005).
57. Slowinski, J. B. A phylogenetic analysis of Bungarus (Elapidae) based on morphological characters. J. Herpetol. 28, 440–446.
https:// doi. org/ 10. 2307/ 15649 56 (1994).
58. Slowinski, J. B. & Keogh, J. S. Phylogenetic relationships of elapid snakes based on cytochrome b mtDNA sequences. Mol.
Phylogenet. Evol. 15, 157–164. https:// doi. org/ 10. 1006/ mpev. 1999. 0725 (2000).
59. Maritz, B. et al. Identifying global priorities for the conservation of vipers. Biol. Conserv. 204, 94–102. https:// doi. org/ 10. 1016/j.
biocon. 2016. 05. 004 (2016).
60. Conroy, C. J., Papenfuss, T., Parker, J. & Hahn, N. E. Use of tricainemethanesulfonate (MS222) for euthanasia of reptiles. J. Am.
Assoc. Lab. Anim. Sci. 48, 28–32 (2009).
61. Percie du Sert, N. et al. e ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. J. Cereb. Blood Flow
Metab. 40, 1769–1777 (2020).
62. Vaidya, G., Lohman, D. J. & Meier, R. SequenceMatrix: Concatenation soware for the fast assembly of multi-gene datasets with
character set and codon information. Cladistics 27, 1716–2180. https:// doi. org/ 10. 1111/j. 1096- 0031. 2010. 00329.x (2011).
63. Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing
platforms. Mol. Biol. Evol. 35, 1547–1549. https:// doi. org/ 10. 1093/ molbev/ msy096 (2018).
64. Lanfear, R., Frandsen, P. B., Wright, A. M., Senfeld, T. & Calcott, B. PartitionFinder 2: New methods for selecting partitioned
models of evolution for molecular and morphological phylogenetic analyses. Mol. Biol. Evol. 34, 772–773. https:// doi. org/ 10.
1093/ molbev/ msw260 (2017).
65. Ronquist, F. et al. MrBayes 3.2: Ecient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol.
61, 539–542. https:// doi. org/ 10. 1093/ sysbio/ sys029 (2012).
66. Rambaut, A., Drummond, A. J., Xie, D., Baele, G. & Suchard, M. A. Posterior summarization in Bayesian phylogenetics using
Tracer 1.7. Syst. Biol. 67, 901–904. https:// doi. org/ 10. 1093/ sysbio/ syy032 (2018).
67. R Core Team. R: A Language and Environment for Statistical Computing https:// www.R- proje ct. org/ (R Foundation for Statistical
Computing, 2020).
68. Warren, D. L., Geneva, A. J. & Lanfear, R. RWTY (R We ere Yet): An R package for examining convergence of Bayesian
phylogenetic analyses. Mol. Biol. Evol. 34, 1016–1020. https:// doi. org/ 10. 1093/ molbev/ msw279 (2017).
69. Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic
Acids Res. 49, W293–W296. https:// doi. org/ 10. 1093/ nar/ gkab3 01 (2021).
70. Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: A fast and eective stochastic algorithm for estimating
maximum likelihood phylogenies. Mol. Biol. Evol. 32, 268–274. https:// doi. org/ 10. 1093/ molbev/ msu300 (2015).
71. Minh, B. Q., Nguyen, M. A. T. & von Haeseler, A. Ultrafast approximation for phylogenetic bootstrap. Mol. Biol. Evol. 30,
1188–1195. https:// doi. org/ 10. 1093/ molbev/ mst024 (2013).
72. Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K., Von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate
phylogenetic estimates. Nat. Methods. 14, 587–589. https:// doi. org/ 10. 1038/ nmeth. 4285 (2017).
73. Zhang, J., Kapli, P., Pavlidis, P. & Stamatakis, A. A general species delimitation method with applications to phylogenetic place-
ments. Bioinform. 29, 2869–2876. https:// doi. org/ 10. 1093/ bioin forma tics/ btt499 (2013).
74. Vences, M. et al. iTaxoTools 0.1: Kickstarting a specimen-based soware toolkit for taxonomists. Megataxa. 6, 77–92. https://
doi. org/ 10. 11646/ megat axa.6. 2.1 (2021).
75. Smith, M. A. e Fauna of British India, Ceylon and Burma. Reptilia and Amphibia. Vol. 3. Serpentes (Taylor & Francis, 1943)
76. Yang, D. T. & Rao, D. Q. Amphibia and Reptilia of Yunnan (Yunnan Science and Technology Press, 2008).
77. Leviton, A. E. et al. e dangerously venomous snakes of Myanmar illustrated checklist with keys. Proc. Cal. Acad. Sci. 54,
407–462 (2003).
78. Dowling, H. G. A proposed standard system of counting ventrals in snakes. Br. J. Herpetol. 1, 97–99 (1951).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

13
Vol.:(0123456789)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
79. Keogh, J. S. Evolutionary implications of hemipenial morphology in the terrestrial Australian elapid snakes. Zool. J. Linn. Soc.
125, 239–278. https:// doi. org/ 10. 1111/j. 1096- 3642. 1999. tb005 92.x (1999).
80. Levene, H. Robust Tests for Equality of Variances. Contributions to Probability and Statistics. Essays in Honor of Harold Hotelling
(eds. Olkin, I. etal.). 279–292 (Stanford University Press, 1961).
81. Brown, M. B. & Forsythe, A. B. Robust tests for the equality of variances. J. Am. Stat. Assoc. 69, 364–367 (1974).
82. Schneider, J. G. Historiae Amphibiorum Naturalis et Literariae. Fasciculus Secundus Continens Crocodilos, Scincos, Chamaesauras,
Boas. Pseudoboas, Elapes, Angues. Amphisbaenas et Caecilias. (Frommanni, 1801).
83. Bauer, A. M. & Lavilla, E. O. (eds.). J. G. Schneider’s Historiae Amphibiorum: Herpetology at the Dawn of the 19th Century. (SSAR,
2021).
84. Russell, P. An Account of Indian Serpents, Collected on the Coast of Coromandel: Containing Descriptions and Drawings of Each
Species; Together with Experiments and Remarks on eir Several Poisons (W. Bulmer and Co., 1796).
85. Bauer, A. M. Patrick Russell’s snakes and their role as type specimens. Hamadryad. 37, 18–65 (2015).
86. Smith, O. A. Large common and banded krait. J. Bombay Nat. Hist. Soc. 21, 283–284 (1911).
87. Whitaker, R. & Captain, A. Snakes of India: e Field Guide (Draco Books, 2008).
88. Masson, J. e distribution of the Banded Krait (Bungarus fasciatus). J. Bombay Nat. Hist. Soc. 34, 256–257 (1930).
89. Anwar, M. First record of banded krait (Bungarus fasciatus) from Pilibhit District, Uttar Pradesh-India. Taprobanica. 3, 102–103.
https:// doi. org/ 10. 4038/ tapro. v3i2. 3967 (2012).
90. Das, A., Basu, D., Converse, L. & Suresh, C. C. Herpetofauna of Katerniaghat Wildlife Sanctuary, Uttar Pradesh, India. J. reat.
Taxa. 4, 2553–2568. https:// doi. org/ 10. 11609/ JoTT. o2587. 2553- 68 (2012).
91 . Bhandarkar, W. R., Paliwal, G. T., Bhandarkar, S. V. & Kali, A. A. Herpetofaunal diversity at navegaon national park, Distt. Gondia
Maharashtra. Int. J. Environ. Rehabil. Conserv. 3, 42–49 (2012).
92. Deshmukh, R. V., Deshmukh, S. A., Badhekar, S. A. & Naitame, R. Y. Snakes of Bhandara District, Maharashtra, Central India
with notes on natural history. Reptil. Amphib. 27, 10–17 (2020).
93. Joshi, P. S., Charjan, A. P. & Tantarpale, V. T. A herpetofaunal inventory of Vidarbha region, Maharashtra. India. Bio. Disc. 8,
582–587 (2017).
94. Kinnear, N. B. Banded Krait (Bungarus fasciatus) in Hyderabad State. J. Bombay Nat. Hist. Soc. 22, 635–636 (1913).
95. Srinivasulu, C., Venkateshwarlu, D. & Seetharamaraju, M. Rediscovery of the Banded Krait Bungarus fasciatus (Schneider 1801)
(Serpentes: Elapidae) from Warangal District, Andhra Pradesh, India. J. reat. Taxa. 1, 353–354. https:// doi. org/ 10. 11609/ JoTT.
o1986. 353-4 (2009).
96. Chandra, K., Raha, A., Majumder, A., Parida, A. & Sarsavan, A. First Record of Banded Krait, Bungarus fasciatus (Schneider,
1801), (Reptilia: Elapidae), from Guru Ghasidas National Park, Koriya District, Chhattisgarh. India. Rec. Zool. Surv. India. 113,
77–80 (2013).
97. Ingle, M. Herpetofauna of Naglok Region, Jashpur District. Chhattisgarh. Rec. Zool. Surv. India. 111, 99–109 (2011).
98. Hussain, A. New record of banded krait Bungarus fasciatus (Schneider, 1801) from Ranchi (Jharkhand) with its preying on
checkered keel-back snake. Biol. Forum. 12, 29–32 (2020).
99. Wall, F. A popular treatise on the common Indian snakes. Part 15. Bungarus fasciatus and Lycodon striatus. J. Bombay Nat. Hist.
Soc. 20, 933–953 (1912).
100. Boruah, B. et al. Diversity of herpetofauna and their conservation in and around North Orissa University Campus, Odisha,
India. NeBIO. 7, 138–145 (2016).
101. Sharma, R. C. e Fauna of India and the Adjacent Countries. Vol. 3. Reptilia (Serpentes). (Zoological Survey of India, 2007).
102. Borang, A., Bhatt, B. B., Chaudhury, S. B., Borkotoki, A. & Bhutia, P. T. Checklist of the snakes of Arunachal Pradesh, northeast
India. J. Bombay Nat. Hist. Soc. 102, 19–26 (2005).
103. Das, A. Notes on Snakes of the Genus Bungarus (Serpentes: Elapidae) from Northeast India. Indian Hotspots (Springer, 2018).
104. Mathew, R. On a collection of snakes from North-east India (Reptilia: Serpentes). Rec. Zool. Surv. India. 80, 449–458 (1983).
105. Purkayastha, J., Das, M. & Sengupta, S. Urban herpetofauna: A case study in Guwahati City of Assam. India. Herpetol. Notes. 4,
195–202 (2011).
106. Mathew, R. State Fauna Series 4: Fauna of Meghalaya, Part I; Reptilia (ed. Director). 379–454 (Zoological Survey of India, 1995).
107. Lalremsanga, H. T., Sailo, S. & Chinliansiama, H. Diversity of snakes (Reptilia: Squamata) and role of environmental factors in
their distribution in Mizoram, Northeast India. Proc. Adv. Environ. Chem. 64, 265–269 (2011).
108. Pawar, S. & Birand, A. A Survey of Amphibians, Reptiles, and Birds in Northeast India (Centre for Ecological Research and
Conservation, 2001).
109. Majumder, J., Bhattacharjee, P. P., Majumdar, K., Debnath, C. & Agarwala, B. K. Documentation of herpetofaunal species rich-
ness in Tripura, northeast India. NeBio 3, 60–70 (2012).
110. Singh, S. On a collection of reptiles and amphibians of Manipur. Geobios New Rep. 14, 135–145 (1995).
111. Dasgupta, G. & Raha, S. Fauna of Nagaland, State Fauna Series 12; Reptilia (ed. Director). 433–460 (Zoological Survey of India,
2006).
112. World Health Organization. Snakebite Information and Data Platform. https:// www. who. int/ teams/ contr ol- of- negle cted- tropi
cal- disea ses/ snake bitee nveno ming/ snake bite- infor mation- and- data- platf orm/ overv iew# tab= tab_1 (2022).
113. Hillis, D. M. Species delimitation in herpetology. J. Herpetol. 53, 3–12. https:// doi. org/ 10. 1670/ 18- 123 (2019).
114. Gilman, C. A., Corl, A., Sinervo, B. & Irschick, D. J. Genital morphology associated with mating strategy in the polymorphic
lizard, Uta stansburiana. J. Morphol. 280, 184–192. https:// doi. org/ 10. 1002/ jmor. 20930 (2019).
115. Klaczko, J., Ingram, T. & Losos, J. Genitals evolve faster than other traits in Anolis lizards. J. Zool. 295, 44–48. https:// doi. org/
10. 1111/ jzo. 12178 (2015).
116. Arnold, E. N. Why copulatory organs provide so many useful taxonomic characters: the origin and maintenance of hemipenial
dierences in lacertid lizards (Reptilia: Lacertidae). Biol. J. Linn. Soc. 29, 263–281. https:// doi. org/ 10. 1111/j. 1095- 8312. 1986.
tb002 79.x (1986).
117. Myers, C. W. & McDowell, S. B. New taxa and cryptic species of neotropical snakes (Xenodontinae), with commentary on
hemipenes as generic and specic characters. Bull. Am. Museum Nat. Hist. 385, 1–112. https:// doi. org/ 10. 1206/ 862.1 (2014).
118. Nunes, P. M. S., Fouquet, A., Curcio, F. F., Kok, P. J. R. & Rodrigues, M. T. Cryptic species in Iphisa elegans Gray, 1851 (Squamata:
Gymnophthalmidae) revealed by hemipenial morphology and molecular data. Zool. J. Linn. Soc. 166, 361–376. https:// doi. org/
10. 1111/j. 1096- 3642. 2012. 00846.x (2012).
119. Daltry, J. C., Wüster, W. & orpe, R. S. Diet and snake venom evolution. Nature 379, 537–540 (1996).
120. Fry, B. G., Winkel, K. D., Wickramaratna, J. C., Hodgson, W. C. & Wüster, W. Eectiveness of snake antivenom: Species and
regional venom variation and its clinical impact. J. Toxicol. Toxin Rev. 22, 23–34. https:// doi. org/ 10. 1081/ TXR- 12001 9018 (2003).
121. Williams, H. F. et al. e urgent need to develop novel strategies for the diagnosis and treatment of snakebites. Toxins 11, 363.
https:// doi. org/ 10. 3390/ toxin s1106 0363 (2019).
122. Chatrath, S. T. et al. Identication of novel proteins from the venom of a cryptic snake Drysdalia coronoides by a combined
transcriptomics and proteomics approach. J. Proteome Res. 10, 739–750. https:// doi. org/ 10. 1021/ pr100 8916 (2011).
123. Siqueira-Silva, T. et al. Ecological and biogeographic processes drive the proteome evolution of snake venom. Glob. Ecol. Biogeogr.
30, 1978–1989. https:// doi. org/ 10. 1111/ geb. 13359 (2021).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

14
Vol:.(1234567890)
Scientic Reports | (2023) 13:2061 | https://doi.org/10.1038/s41598-023-28241-8
www.nature.com/scientificreports/
124. Wüster, W. & Broadley, D. G. A new species of spitting cobra from northeastern Africa (Serpentes: Elapidae: Naja). J. Zool. 259,
345–359. https:// doi. org/ 10. 1017/ S0952 83690 20033 33 (2003).
125. Wüster, W. & Broadley, D. G. Get an eyeful of this: a new species of giant spitting cobra from eastern and north-eastern Africa
(Squamata: Serpentes: Elapidae: Naja). Zootaxa 1532, 51–68. https:// doi. org/ 10. 11646/ zoota xa. 1532.1.4 (2007).
126. Puorto, G. et al. Combining mitochondrial DNA sequences and morphological data to infer species boundaries: phylogeography
of lanceheaded pitvipers in the Brazilian Atlantic forest, and the status of Bothrops pradoi (Squamata: Serpentes: Viperidae). J.
Evol. Biol. 14, 527–538. https:// doi. org/ 10. 1046/j. 1420- 9101. 2001. 00313.x (2001).
127. Hare, M. P. Prospects for nuclear gene phylogeography. Trends Ecol. Evol. 16, 700–706. https:// doi. org/ 10. 1016/ S0169- 5347(01)
02326-6 (2001).
128. Wüster, W. et al. Integration of nuclear and mitochondrial gene sequences and morphology reveals unexpected diversity in the
forest cobra (Naja melanoleuca) species complex in Central and West Africa (Serpentes: Elapidae). Zootaxa 4455, 68–98. https://
doi. org/ 10. 11646/ zoota xa. 4455.1.3 (2018).
Acknowledgements
We thank the Chief Wildlife Warden of Environment, Forests and Climate change Department, Government of
Mizoram for providing collection permits (No.A.33011/2/99-CWLW/22 and No.B.19060/5/2020-CWLW/20-26).
We also thank the Directorate of Forests, Wildlife Wing, Government of West Bengal for continued support
to carry out our research and conservation activities. is work is supported by DST-SERB, New Delhi (DST
No: EMR/2016/002391), DBT, New Delhi (DBT-NER/AAB/64/2017), National Mission for Himalayan Stud-
ies (NMHS), Uttarakhand (GBPNI/NMHS-2017/MG-22/566), DRDO, New Delhi (DGTM/DFTM/GIA/19-
20/0422), and DST-SERB, New Delhi (DST No: EEQ/2021/000243). AM would like to acknowledge EU Marie
Curie Action PIRSES-GA-2013-612131 (the BITES project); the Bangor University ESRC Impact Acceleration
Account, for funding; and Ashok Mallik for his assistance in this work. HTL is grateful to the International Her-
petological Symposium (IHS), USA for the award grant. LB is also thankful for the small grant he received from
e Ruord Foundation, UK (grant number 36737-1). LB and HTL would like to thank Mathipi Vabeiryureilai,
Fanai Malsawmdawngliana, Lal Muansanga, Lalengzuala Tochhawng, Ht. Decemson, Gospel Zothanmawia
Hmar, Vanlal Siammawii, H. Laltlanchhuaha; VS would like to thank Biswajit Das, Lakshmi Santra, Aritra
Dhara, Pallab Das, Ayan Koley, Rakesh Koley, Rajkumar Chakraborty, Amal Kr Santra, Ananta Katwal, Ishan
Santra, Shrabani Santra for their assistance in this study. AATA thanks the Ministry of Environment and Forestry
(KLHK) and e Directorate General of Conservation of Natural Resources and Ecosystems (KSDAE) of the
Republic of Indonesia for granting research permits; C. Rahmadi, A. Riyanto, A. Hamidy, Syaripudin, and W.
Trilaksano (MZB) for their support and facilitating the in-house study of specimens under their care. We are
deeply thankful to Patrick David for the insightful taxonomical comments in the dra version of the manuscript.
Author contributions
L.B.: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Soware, Visualization,
Writing—original dra. H.T.L.: Conceptualization, Data curation, Funding acquisition, Investigation, Method-
ology, Project administration, Validation, Visualization, Writing—original dra, Resources, Supervision. V.S.:
Investigation, Resources, Writing—review & editing. A.A.T.A.: Data curation, Formal analysis, Soware, Valida-
tion, Visualization, Investigation, Writing—review & editing. A.D.: Investigation, Resources. M.T.A.: Investiga-
tion, Resources. Z.B.M.: Investigation, Resources. S.K.: Investigation, Soware, Writing—review & editing. A.M.:
Formal analysis, Validation, Writing—review & editing.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 023- 28241-8.
Correspondence and requests for materials should be addressed to H.T.L., A.A.T.A.orA.M.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
Open Access is article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. e images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
© e Author(s) 2023
Content courtesy of Springer Nature, terms of use apply. Rights reserved

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com