We generated DNA sequences for two Boiga sp., a specimen (ZSI-CZRC-V-7541) from Peermed, Kerala (9.602710°N, 76.937857°E, 1238 m Above Sea Level (ASL)) approximately 9 km from the type locality of B. dightoni and one more specimen (BNHS 3617) from south of Shencottah gap (Arippa, Kerala, 8.831640°N, 77.038542°E, 195 m ASL) (Fig. 1), and one specimen (BNHS 3618) of Boiga nuchalis (Yercaud, Tamil Nadu, 11.775140°N, 78.214654°E, 1300 m ASL).

Figure 1. 

Updated distribution of Boiga dightoni and B. nuchalis in the Western Ghats and B. nuchalis in peninsular India.

We extracted genomic DNA from liver samples stored in absolute ethanol at –20°C, using the DNeasy (QiagenTM) blood and tissue kit following the manufacturer’s protocol. We amplified partial sequences of two mitochondrial genes, 16S rRNA (16S) and cytochrome b (cyt b). Respective primers for these genes are as follows: 16Sar-L and 16Sbr-H (Palumbi et al. 1991) and CS1L and LTyph2R (Adalsteinsson et al. 2009). PCR conditions were as follows: Fragments of 16S were amplified using an initial denaturation at 95°C for 5 min, followed by 39 cycles of denaturation at 95°C for 45 sec, annealing at 50.4°C for 45 sec and extension at 72°C for 1 min 30 sec. Final extension was at 72°C for 10 min. Fragments of cyt b gene were amplified using an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 45 sec, annealing at 48°C for 45 sec and extension at 72°C for 55 sec. Final extension was at 72°C for 10 min. PCR reactions were carried out in 25 µl reactions containing 11µl of Takara emerald RR310B mastermix, 12 µl of PCR grade H₂O, 0.5 µl of each forward and reverse primers and 1µl (60–80 ng) of template DNA. PCR amplifications were carried out in S1000TM Thermal Cycler (Bio-Rad, USA). Amplified PCR products were run on a 2% agarose gel and viewed with an Essential V4 (UVITEC Cambridge, UK) gel documentation system to confirm the PCR amplification. PCR products were purified and Sanger sequenced in both directions at Barcode Biosciences (Bangalore, India) using the same primers that were used for amplification.

Bidirectional sequences were checked manually using CHROMAS (http://technelysium.com.au/wp/chromas) and aligned using ClustalW with default prior settings implemented in MEGA 7 (Tamura et al. 2011; Kumar et al. 2016). We checked for unexpected stop codons in the protein-coding gene cyt b by translating nucleotide alignments to amino acids in MEGA7 (Kumar et al. 2016). The new sequences generated in this study were concatenated with data for twenty-three other Boiga and three outgroups (Telescopus tripolitanus, T. variegates and Toxicodryas pulverulenta) (Appendix 1).

Maximum Likelihood (ML) analysis was performed using IQ-TREE (Nguyen et al. 2015), implemented in the web server version (http://iqtree.cibiv.univie.ac.at) (Trifinopoulos et al. 2016). The IQ-TREE server used Modelfinder (Kalyaanamoorthy et al. 2017) to find the best-fit evolutionary model for each of the four suggested partitions (16S: TIM2+F+I+G4; Cytb position 1: TIM2+F+G4; Cytb position 2: TN+F+I+G4; cyt b position3: TIM3+F+G4). Bayesian (BI) phylogenetic analysis was carried out with MrBayes 3.2 (Ronquist et al. 2012), with default prior settings and implementing the best-fit models and partitioning scheme as determined by Partition Finder V2. (Lanfear et al. 2017) with default settings. The best-fit scheme comprised three partitions, by gene and codon position (16S & cyt b position 1: GTR+I+G; cyt b position 2: TrN+I+G; cyt b position3: TVM+G). Four separate MCMC runs were initiated from random trees and allowed to run for ten million generations, sampling every 1000 generations. Analyses were terminated when the standard deviation of split frequencies was less than 0.005, the first 25% of trees were discarded as “burn-in”, and trees were constructed under the 50% majority consensus rule. Support for internal branches in ML and BI trees was quantified using Ultrafast Bootstrap (1000 pseudoreplicates) and posterior probability, respectively.

We examined 33 specimens of Boiga spp., including Boiga dightoni (n = 9), B. whitakeri (n = 2), B. nuchalis (n = 10), B. thackerayi (n = 4), B. flaviviridis (n = 1) and B. ceylonensis (n = 7) (Appendix 2). Morphological data for B. ranawanei were taken from Samarawickrama et al. (2005).

The numbers of dorsal scale rows are reported for one head length behind the head, at midbody (i.e., at the level of the ventral plate corresponding to half of the total ventral number), and at one head length anterior to the vent respectively. Dorsal scale row reduction formulae were based on Dowling (1951a). Ventral scale counts and hemipenial descriptions follow Dowling (1951b) and Dowling and Savage (1960), respectively. The terminal scute is not included in the number of subcaudals. Values for symmetric head characters are given in left/right order.

The following measurements were taken: snout-vent length (SVL); tail length (TL); head length (HL: distance between posterior edge of last supralabial and tip of the snout); head width (HW: at angle of jaws); head depth (HD: height at the occipital region); Frontal length (FL: at the longest point); frontal width (FW: at the widest point on the anterior region); eye diameter (ED: horizontal diameter); eye to nostril distance (E–N: anterior corner of eye to posterior edge of nostril); eye to snout distance (E-S: anterior corner of eye to tip of snout); frontal to snout (FrSN: anterior end of frontal to tip of snout); inter-orbital distance (IO: measured at the anterior edge of eyes); number of dorsal scale rows (DSR). All linear measurements, except SVL and TL were taken using Mitutoyo dial vernier callipers (to 0.1 mm). SVL and TL were measured using a thread and metal scale (to 1 mm).

Among the specimens checked in this study, the holotype (BNHS 3597) of Boiga whitakeri is in a poor state of preservation and the paratype (BNHS 1863) of this species is also damaged, especially its anterior ventral scales. Thus, the number of ventral scales provided here for the paratype of Boiga whitakeri (BNHS 1863) is not complete Appendix 3C). For the ventrals that are damaged, we counted the adjacent dorsal scale rows assuming that one first-row dorsal corresponds to one ventral here.

Location records for both Boiga dightoni and B. nuchalis used for the map (Fig. 1) are based on the literature and specimens examined during the study (Appendix 4). Additionally, we downloaded research-grade data for both of these species from the citizen science portals, iNaturalist (https://www.inaturalist.org) and India Biodiversity Portal (https://www.indiabiodiversity.org). All the records for both species were checked individually wherever we could count the dorsal scales on one side, especially for the records from the southern Western Ghats. Doubtful records or records with poor photographs were not considered for plotting on the mapping and distribution (see also Discussion).

To obtain counts of teeth by a non-invasive procedure, the head of the holotype of Boiga dightoni was subjected to micro-tomographic analysis at the Museum für Naturkunde Berlin, using a Phoenix nanotomX-ray|s tube. The cone-beam reconstruction was performed using the datos|x-reconstruction software (GE Sensing & Inspection Technologies GMBH phoenix|x-raydatos|x 2.0) and the data were visualised in VGStudio Max 2.2. Teeth (including empty sockets) were counted on all dentigerous bones.

BMNH: The Natural History Museum, London, UK; FMNH: Field Museum of Natural History, Chicago, USA; BNHS: Bombay Natural History Society; MCZ: Museum of Comparative Zoology, Cambridge, USA; RMNH-BBSR-R: Regional Museum of Natural History, Bhubaneswar, India; ZMB: Museum für Naturkunde (formerly Zoologisches Museum Berlin), Leibniz-Institut für Evolutions- und Biodiversitätsforschung, Berlin, Germany; ZSI-CZRC: Zoological Survey of India, Central Zone Regional Centre, Jabalpur, India; ZSI/SRS/S: Zoological Survey of India, Southern Regional Centre, Chennai, India. Museum acronyms follow Sabaj (2020).

The inferred phylogenies are broadly congruent with those presented by Ganesh et al. (2021). Boiga ceylonensisis sister to B. dightoni + B. whitakeri with strong support (ML 98, BI 1.0) and this clade is sister to B. nuchalis. Boiga nuchalis from Yercaud (BNHS 3618) is nested with other B. nuchalis from the Western Ghats (Fig. 2). Boiga cf. ranawanei (sensu Ganesh et al. 2021) is sister to B. flaviviridis (a dry zone species found in thorn forests and scrub jungle). The holotype sequence of B. whitakeri is sister to the sample from Arippa (BNHS 3617) with strong and moderate support in BI and ML, respectively (BI 0.98, ML 83) and these two samples are together sister to the B. dightoni from the type locality with strong support (ML 95, BI 1.0) (Fig. 2).

Figure 2. 

ML phylogeny showing relationships of the newly sampled Boiga (in blue) and sequences of other available congeners. ML bootstrap support and BI posterior probability support = />75 or 0.75 is shown at each internal branch. Holotype of B. whitakeri (BNHS 3597) labelled in red. Inset image: head closeups of the respective samples in life or freshly roadkill specimen (BNHS 3617). Outgroups are pruned from this tree.

The uncorrected pairwise genetic distance between the two samples of B. dightoni and the holotype of B. whitakeri, is 0.9–1.2% and 0.2–0.4% in cyt b and 16S, respectively (Table 1). These distances are almost all smaller than intraspecific distances for the other congeners B. beddomei 2.5%, B. flaviviridis 2.2%, B. nuchalis 0.4–2.3% and B. thackerayi 0.6–3.6% in cyt b and B. beddomei 0.8%, B. nuchalis 0% and B. thackerayi 0.2–0.8% in 16S.

Both specimens of Boiga sp. (ZSI-CZRC-V-7541 and BNHS 3617) collected during this study match well with the holotype of B. dightoni based on scalation data, mainly in having 23 MDSR (also see scale reduction formula for more characters). However, these two specimens differ significantly in scalation from B. whitakeri (based on data provided by Ganesh et al. 2021), despite the strong molecular similarity. MDSRs in B. dightoni are predominantly 23 from the 10^th ventral throughout most of the midbody (up to ventrals 123–144) in all of the specimens examined during the study, but the point of reduction from 23 to 21 differs slightly among the specimens examined (see scale reduction formula below). Boiga whitakeri is diagnosed from congeners mainly based on the presence of 19 MDSR as provided in the original description (Ganesh et al. 2021), but we counted 23 rows in the holotype (scale redcution formula & Table 2). Due to the poor state of preservation, we were unable to acquire the complete scale reduction formula for the paratype of B. whitakeri (BNHS 1863) but it has 21 MDSR until the level of the 145^th ventral and 19 DSR at the level of the 160^th ventral. This matches the dorsal scale reduction range of the sympatric Boiga nuchalis (see scale redcution formula). Furthermore, except for the dorsal scale rows, there is a close similarity in the arrangements of head scalation, ventrals and subcaudals between specimens identified as belonging to these “three” species (Table 2).

Dorsal scale row reduction formulae for some of the Boiga specimens examined in this study presented below. *between ventral 9 and 14 counts are not possible because of the damaged vertebral region. Additionally, there are several reductions and additions of the paravertebral scale row between the corresponding ventrals of 194 to 209. *** dorsal scales damaged and is not possible to find the area of scale reduction:

Boiga dightoni

———————————————————————————————————————————————

Boiga “whitakeri”

———————————————————————————————————————————————

Boiga dightoni (Boulenger, 1894)

Figs 3, 4, 5, 6, 7; Tables 1, 2

Dipsas dightoni Boulenger 1894, p. 528.

Dipsadomorphus dightoni – Boulenger 1896, p. 69.

Boiga dightoni – Smith 1943, p. 567; Murthy 1984, p. 84; Inger et al. 1984, p. 567; Wallach et al. 2014, p. 103; Kanagavel & Ganesh 2021, p. 67, 68, fig. 1,2; Ganesh et al. 2020, p. 314, fig. 7; Ganesh et al. 2021, p. 449–151, 453.

Boiga whitakeri Ganesh, Mallik, Achyuthan, Shanker & Vogel, 2021 p. 453, fig. 3, syn. nov.

Taxonomic comments

A detailed description of the external morphology of the holotype of Boiga dightoni (BMNH 1946.1.1.32) is presented by Ganesh et al. (2020). In this work, we provide scale reduction formula and detailed dentition based on microCT scans for the holotype of Boiga dightoni. In addition, we provide a detailed description of the hemipenis of B. dightoni based on a topotypic specimen (ZSI-CZRC-V-7541).

Based on the morphological data from the two specimens collected during this study, including the specimen from the type locality (Peermed, Kerala) of Boiga dightoni, we confidently identify these two specimens as B. dightoni. Our morphological examination of the types and non-type materials of Boiga whitakeri, B. dightoni and B. nuchalis provide evidence that led us to conclude that the holotype of B. whitakeri is conspecific with B. dightoni. This is consistent with our molecular analyses, in which the holotype of B. whitakeri is nested within the samples (including the topotype) that we identify as B. dightoni. On the other hand, the type series (BMNH 74.4.29.933–6) and two other specimens of Boiga nuchalis examined here have 21 dorsal scale rows at midbody (Scale reduction formula; Appendix 2). This further confirms that the paratype (BNHS 1863; Appendix 3) of B. whitakeri is rather B. nuchalis. Because the holotype and paratype of B. whitakeri clearly represent two already described species, we relegate Boiga whitakeri Ganesh, Mallik, Achyuthan, Shanker and Vogel, 2021 to the junior subjective synonymy of Dipsas dightoni Boulenger, 1894.

Morphology

A medium-sized Boiga (greatest TL 1000 mm (male), 935 mm (female)); 229–249 ventrals, 99–112 divided subcaudals; 13/14 teeth on maxilla and 7 on palatine; dorsal scales smooth, 23:23:19 in rows; dorsal scale reduction from 23 to 21 rows occurs between ventrals 123–144 and the reduction from 21 to 19 occurs between ventrals 148–155. Dorsum reddish dun to olive greenish with dorsal light brown to dark bands. Head with dark marking dorsally (rarely absent) and a dark laterocular stripe present.

Colouration in life and preservative

Based on the (live and museum) specimens examined and information available from the literature, we report two different colour morphs in B. dightoni.

Morph 1 (n = 5)

Reddish dun-coloured dorsum with faint reddish bands on the body (rarely absent) with or without distinct dark marking on the head, and ventral scales uniformly creamish white (Figs 3A–B, 4, 6 A–C, G–I, M–O). Holotypes of both B. dightoni and B. whitakeri are of this colour morph with no markings on the body in preservation. However, it might be noted that the recently collected specimen from Arippa (BNHS 3617) had faint markings on the body at the time of collection (3^rd February 2022) that disappeared in the preservative (Figs 2, 3C–D, 4, 6 A–C). This also applies to the holotype of B. whitakeri (Fig. 3B), which had markings on the body in life that disappeared in the preservative (Ganesh et al. 2021). A specimen from Aanapara, Kerala reported by Kanagavel and Ganesh (2021) also belongs to this morph, with very faint bands.

Figure 3. 

Representative images of B. dightoni in life. Morph 1: A Uncollected individual from Arippa, Kerala (female), B BNHS 3597 (male); Morph 2: C ZSI-CZRC-V-7541 (male), D uncollected individual from Arippa, Kerala (male).

Figure 4. 

Representative image of B. dightoni Morph 1. A–B BMNH 1946.1.1.32 (female), C–D BNHS 3617 (female), E–F FMNH 217699 (male). Scale bar = 5 cm.

Morph 2 (n = 5)

Olive greenish dorsum with black bands (76–80) on the body, with distinct marks on the head and a postocular stripe that ends shortly behind the fissure of the mouth, and irregular small dark blotches along the paraventral scales (Figs 3C–D, 5, 6D–F, J–L, P–R). The topotypic specimen (ZSI-CZRC-V-7541) of B. dightoni collected during this study is of this morph (Fig. 3C) and we observed several specimens from museum collections of this morph including a specimen (ZSI/SRS/S-73) collected from the Anamalais in Southern India.

Figure 5. 

Representative image of B. dightoni Morph 2. A–B ZSI-CZRC-V-7541 (male), C–D BNHS 1842 (unknown), E–F ZSI-CZRC-V-7542 (male). Scale bar = 5 cm.

Figure 6. 

Head closeup showing colour and pattern in B. dightoni Morph 1: A–C BNHS 3617, G–I BNHS 3597, M–O BMNH 1946.1.1.32; Morph 2: D–F ZSI-CZRC-V-7542, J–L BNHS 1842 and P–R ZSI-CZRC-V-7541. Scale bar = 10 mm.

Based on the specimens examined here, it is also clear that these two colour morphs are not explained by sexual dichromatism because both male and female specimens are known for both morphs. For example, the male specimens BNHS 3597 and ZSI-CZRC-V-7541 and the female specimens BMNH 1946.1.1.32 and BMNH 1940.10.13.19 belong to Morph 1 and 2, respectively. Both the morphs are found in sympatry in at least one location (Arippa, Kerala), so they additionally cannot be explained as purely geographic variation. Furthermore, these colour morphs cannot be currently explained as simple ontogenetic variation, because all the specimens examined here are adults.

Description of hemipenis of ZSI-CZRC-V-7541 (Fig. 7)

The right hemipenis is fully everted and removed in situ for further analysis. The hemipenis is sub-cylindrical and moderately elongate (length: 17.0 mm, maximum width: 5.7 mm), extending to the 7^th subcaudal. The sulcus is undivided, bounded by thick walls on both sides, and terminates at the centre of the lobe. It can be differentiated into three zones; the proximal zone is covered with 4–6 rows of spines (~40% of the total length), the middle zone with 5 or 6 rows of spinulate flounces arranged transversely (~35% of the total length), and the distal calyculate area (~25%) with 4 or 5 rows of irregular calyces with papilate edges. The sulcus spermaticus is exposed before entering the calyculate area. There is not much variation in the arrangements of spines and body calyces on sulcate and asulcate sides. The overall structure of the hemipenis of ZSI-CZRC-V-7542 is similar to that described for ZSI-CZRC-V-7541.

Figure 7. 

Hemipenis of Boiga dightoni (Right organ of ZSI-CZRC-V-7541). A sulcate view; B asulcate view; C apex view. Scale bar = 10 mm.

Dentition based on the holotype of B. dightoni (BMNH 1946.1.1.32) (left/right order)

Maxillary bone with 13/14 prediastemal teeth, followed by a distinct diastema that is as long as the socket of the last prediastemal tooth and followed by two distinctly enlarged, grooved and posteriorly bent postdiastemal teeth. Prediastemal teeth increase in size posteriorly, the anterior three distinctly posteriorly hooked, the following with less pronounced curvature. On the left side, prediastemal teeth 1, 4, 5, 7, 9, 11, and 13 missing, maxilla broken behind the diastema. On the right side, prediastemal teeth 2–4, 6, 8, 10, 12, 13 and anterior postdiastemal tooth are missing.

Palatine bone with 7/7 posteriorly curved teeth, anterior ones as long as the middle prediastemal teeth, slightly decreasing in size posteriorly. Teeth 1, 5 and 7 are loose, and tooth 3 missing on left side. Teeth 1, 3 and 5 are loose on the right side. Lateral to each palatine tooth is a single replacement tooth at different growth stages. Pterygoid bone with 18/16 posteriorly curved teeth, first one half as long as last palatine tooth, gradually decreasing in size posteriorly, last one minute. Teeth 2, 4, 6, 8, 10, 12, and 14–16 missing on left side, teeth 2, 4, 6, 8, and 10 loose, and 11, 13, and 15 missing on right side. The posterior 45% of the pterygoid bone is without teeth.

Mandibular bone with 20/20 posteriorly curved teeth, shorter than maxillary and palatine teeth, gradually decreasing in size posteriorly. Medial to each mandibular tooth is a single replacement tooth in different growth stages. Teeth 1, 3–7, 9, 11, 13, 15–17, and 19 missing, tooth 2 loose on left side, teeth 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 missing, and tooth 2 loose on right side. Mandibular bone broken behind tooth 13 on left side.

Distribution

Based on currently available data, Boiga dightoni is widely distributed in the southern Western Ghats (south of the Palghat Gap), at elevations of 9–1258 m (Appendix 4). Murthy (1984) extended the northern range of this species to Topslip in Anamalais. Murthy (1984) reported 23 dorsal scale rows at midbody for the specimen he collected, which is known only for B. dightoni among Western Ghats’ Boiga. The identity of this specimen (ZSI/SRS/S-73) is confirmed by photographs presented by Murthy (1984). With an additional specimen from the same locality (ZSI-CZRC-V-7541), we reconfirm the distribution of B. dightoni in Topslip in the Anamalai hills. The northernmost known distribution of B. dightoni is based on a specimen (BNHS 1842) from Palagapandy in the Nelliyampathy Hills, Kerala, a specimen that was previously (Ganesh et al. 2020) misidentified as B. nuchalis. The southernmost known occurrence of this species is Ponmudi in Kerala (Fig. 1). Thus, B. dightoni is found only south of the Palghat Gap in the Western Ghats. Boiga dightoni in parts of its range is probably sympatric with B. nuchalis and B. thackerayi immediately south of the Palghat Gap, based on distribution data (Fig. 1) and the sequences reported by Ganesh et al. (2021).