Research Article |
Corresponding author: Sylvia Hofmann ( S.Hofmann@leibniz-lib.de ) Academic editor: Angelica Crottini
© 2024 Sylvia Hofmann, Daniel Jablonski, Joachim Schmidt.
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Hofmann S, Jablonski D, Schmidt J (2024) Morphological and molecular data warrant the description of a new species of the genus Scutiger (Anura, Megophryidae) from the Central Himalaya. ZooKeys 1210: 229-246. https://doi.org/10.3897/zookeys.1210.127106
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Recent phylogenetic studies in Himalayan lazy toads of the genus Scutiger Theobald, 1868 revealed the presence of genetically deeply divergent lineages. The taxonomy of S. nepalensis sensu lato was re-assessed based on museum material considering molecular and morphological data. The results strongly support the recognition of a new species, S. kanjiroba sp. nov. distributed along the Nepalese Kanjiroba massif. It is further shown that S. sikimmensis has an apparently much more restricted distribution range than previously thought. The frequent misidentification of Scutiger across large areas of the Himalaya-Tibet area highlights the significance of careful taxonomic evaluation of collection material and the need for the direct morphological comparison of closely related species when describing new species.
Genetics, Himalaya, lazy toad, montane forests, morphology, taxonomy
The Himalaya, recognised as one of the world’s biodiversity hotspots (
Lazy toads of the genus Scutiger Theobald, 1868, family Megophryidae, are endemic to the Himalaya-Tibet orogenic system and adapted to high montane and alpine areas. They occur from northern Pakistan, through Nepal, Bhutan, northern India, Myanmar, in the valleys of southern and eastern Tibet, and eastwards to southwestern China. According to
Interestingly, the assumed wide distribution range of the long-known species S. nepalensis differs considerably from the limited occurrences of other Himalayan Scutiger species. The type locality of this species is given as Khaptar [= Khaptad] area, in the Doti District of Chainpur in western Nepal. The species is assumed to occur in mountains between 3000 and 5000 m across a wide area of western Nepal (
All voucher specimens and tissue samples for newly generated molecular data were obtained from museum holdings
[Muséum national d’Histoire naturelle, Paris (
Total genomic DNA was isolated from tissues preserved in ethanol using the Qiagen DNeasy kit (Qiagen Inc.) following the manufacturer’s protocol. We amplified partial sequences of the following three mitochondrial (mt) and three nuclear (nu) loci via the polymerase chain reaction (PCR): 16S rRNA (550 bp), cytochrome oxidase subunit 1 (COI, 668 bp), and cytochrome b (Cytb, 985 bp), as well as beta-fibrinogen intron 7 (Bfib7, 508 bp), cyclin B2 gene intron 3 (Ccnb2, 777 bp), and recombination activating protein 1 gene (Rag1, 957 bp); for primers and PCR conditions see
DNA sequences from the three mitochondrial loci of each species were concatenated and used for Bayesian inference (BI) analysis, while the alignments of the three nuclear gene fragments were used separately for network analysis. Because several Scutiger species are represented in GenBank only by COI DNA sequence information, we additionally constructed a BI tree based on that single barcoding gene.
Phylogenetic trees were inferred with MrBayes using Oreolalax chuanbeiensis Tian, 1983 and O. omeimontis (Liu & Hu, 1960) (concatenated mtDNA dataset), or Oreolalax xiangchengensis Fei & Huang, 1983 (COI dataset) as outgroup. MrBayes was run for up to 5 million generations, sampling every 500th generation. We used four parallel Markov chain Monte Carlo simulations with four chains and discarded the first 25% of the samples of each run as burn-in. Chain convergence was monitored with Tracer v. 1.7.1 (
The following measurements were taken with dial callipers and recorded to the nearest 0.1 mm: Snout vent length (SVL), distance from tip of snout to posterior edge of vent; head length (HL), distance from angle of jaws to snout-tip; head width (HW), measured at posterior angle of jaws; snout length (SL), from tip of snout to anterior corner of eye; distance of naris to tip of snout (NSD); horizontal diameter of eye (ED); eyelid-naris distance (END), from naris to anterior edge of the eye; internarial distance (IND); forearm length (FAL), from flexed elbow to base of inner metacarpal tubercle; hand length (HAL), from base of inner metacarpal tubercle to tip of 3rd finger; femur (thigh) length (FEL), from vent to outer edge of flexed knee; tibia (shank) length (TIBL), distance from outer edge of flexed knee to tip of heel; length of tarsus (TaL), distance from heel to proximal edge of inner metatarsal tubercle; foot length (FOL), distance from proximal edge of inner metatarsal tubercle to tip of 4th toe. We further recorded the relative length of fingers and toes, presence/absence of subarticular tubercles, presence/absence of vocal sac, presence/absence of vomerine (v)/maxillary (m) dentation, presence/absence of tympanum, webbing of toes (developed, weak, rudimentary, absent), finger(s) with nuptial spines, presence/absence of pectoral (p)/axillary (ax) glands, gland size relation (p>>/>/= ax), presence/absence of spines on p/ax, presence/absence of spines on inner forearms, presence/absence of spines at belly, presence/absence of tubercles/warts on dorsal (and lateral) surfaces of body and/or limbs, presence/absence of black spines on dorsal tubercles. Sex was determined by the presence of nuptial spines on fingers and chest in males in breeding condition.
A Principal Component Analysis (PCA) was performed in R 4.3.3 (
The labial tooth row formula (LTRF) of the tadpole was recorded according to
Scutiger nepalensis Dubois, 1974 [1974.1096−1974.1098 and 1989.3361− 1989.3362, paratypes; one specimen of S. nepalensis (A1724/09); six specimens of S. nepalensis s. l. (A0570/99, A0574/99, A0576/99, A1248/05, A1249/05, one tadpole A1250/05) stored at the NHME]. Morphological data of other Scutiger species were extracted from original species descriptions and/or recompiled treatises, particularly (
All adult specimens were assigned to the genus Scutiger based on the following combination of features: toad-like morphology, tympanum, and tympanic ring entirely absent, pupil vertically elliptic, femoral glands indistinct, tongue rounded or slightly indented behind, fingers free, toes free or with some webbing, dorsum warty, pectoral (and axillary) glands and fingers with nuptial spines (in breeding males) (
Phylogenetic trees of the mtDNA genes (Fig.
Due to low quality and concentration of the DNA and resulting difficulties in amplification, the following specimens were only barcoded through 16S rRNA: for A0570/99 and A1254/05 we recovered > 99% identity and coverage to/of sequences of S. kanjiroba sp. nov., e.g., KY310787− KY310792 (Suppl. material
The morphological comparisons between the Scutiger specimens from the Kanjiroba range and their congeners are comprehensively outlined in Suppl. material
PCA of 15 metric variables in 17 adult specimens A with 95% confidence ellipses B with loadings. Abbreviations: SVL snout–vent length, HL head length; HW head width; SL snout length; ED horizontal diameter of eye; END eyelid-naris distance; IND internarial distance; FAL forearm length; HAL hand length; FEL femur (thigh) length; TIBL tibia (shank) length; TaL length of tarsus; FOL foot length.
The Scutiger specimens from the Kanjiroba Himal can be readily distinguished from the geographically neighbouring S. nepalensis by a narrower and smaller head shape [HW/SVL: 0.34 (0.33−0.35) vs 0.40 (0.37−0.42); HL/SVL: 0.28 (0.27−0.29) vs 0.32 (0.30−0.34), and in a shorter foot length [FOL/SVL: 0.42 (0.41−0.43) vs 0.45 (0.40−0.47)] (Suppl. material
S. adungensis − the Kanjiroba lineage is distinguished by absence of vocal sac in adult males (vs presence); smaller body size (in males; SVL 55.4 mm vs 71.0−73.0 mm); the absence of maxillary teeth (vs presence); presence of numerous small, densely arranged spines on the surface of fingers I, II, and inner surface of finger III of males in breeding condition (vs large spines on inner two fingers); two pairs of spine patches on chest of breeding male (vs one pair).
S. bangdaensis − the Kanjiroba lineage is distinguished by rudimentary webbing between toes (vs developed), and larger body size (SVL: males 55.4 mm vs 45.5−50.0 mm; females 54.2−66.7 mm vs 48.0−49.5 mm).
S. bhutanensis − the Kanjiroba lineage is distinguished by presence of numerous small, densely arranged spines on surface of fingers I, II, and inner surface of finger III of males in breeding condition (vs large nuptial spines on first two fingers of males).
S. biluoensis − the Kanjiroba lineage is distinguished by absence of vomerine and maxillary teeth (vs presence); presence of nuptial spines on dorsal surface of fingers I, II, and inner surface of finger III of males in breeding condition (vs on inner two fingers); smaller male body size (SVL 55.4 mm vs 73.0 mm).
S. boulengeri − the Kanjiroba lineage is distinguished by rudimentary webbing between toes (vs developed); absence of spine patches on belly of males in breeding condition (vs presence).
S. brevipes − the Kanjiroba lineage is distinguished by rudimentary webbing between toes (vs developed); presence of nuptial spines on dorsal surface of fingers I, II, and inner surface of finger III of males in breeding condition (vs on inner two fingers).
S. chintingensis − the Kanjiroba lineage is distinguished by larger body size (SVL: males 55.4 mm vs 42.0−42.4 mm; females 54.2−66.7 mm vs 48.0−52.8 mm); absence of maxillary teeth (vs presence); rudimentary webbing between toes (vs developed, large or reduced); absence of spines on inner surface of upper arm and forearm of males in breeding condition (vs presence).
S. feiliangi − the Kanjiroba lineage is distinguished by larger body size (SVL: males 55.4 mm vs 45.7−50.2 mm; females 54.2−66.7 mm vs 48.9−51.5 mm); absence of spines on inner surface of forearm of males in breeding condition (vs presence); the absence of maxillary teeth (vs presence); absence of pectoral/axillary glands with spines in females in breeding condition (vs presence); absence of spines on belly of females in breeding condition (vs presence).
S. ghunsa − the Kanjiroba lineage is distinguished by larger body size (male: 55.4 mm vs 42.1−47.8 mm; female: 54.2−66.7 mm vs 50.2−53.9 mm); longer head [HL/SVL: 0.28 (0.27−0.29) vs 0.25 (0.24−0.26)]; shorter hand length [HAL/SVL: 0.25 (0.24−0.26) vs 0.46 (0.43−0.54)]; longer snout (SL/SVL male: 0.15 (0.14−0.15) vs 0.11 (0.10−0.12)]; relative length of toes 4>3>5>2>1 (vs 4>5>3>2>1); absence of tympanum (vs hidden tympanum); rudimentary webbing between toes (vs absence of webbing).
S. glandulatus − the Kanjiroba lineage is distinguished by smaller body size (SVL, male: 55.4 mm vs 67.0−81.0; female: 54.2−66.7 mm vs 77.0−81.0); relative length of toes 4>3>5>2>1 (vs 4>5>3>2>1); absence of subarticular tubercles (vs presence); rudimentary webbing between toes (vs developed); presence of nuptial spines on dorsal surface of fingers I, II, and inner surface of finger III of males in breeding condition (vs on inner two fingers); presence of spines on male axillary glands (vs absence).
S. gongshanensis − the Kanjiroba lineage is distinguished by absence of vocal sac in adult males (vs presence); absence of maxillary teeth (vs presence); presence of numerous small, densely arranged spines on the surface of fingers I, II, and inner surface of finger III of males in breeding condition (vs large spines on inner two fingers); rudimentary webbing between toes (vs absence of webbing); presence of a pair of pectoral and axillary patches in breeding male (vs one pair); presence of spine on tubercles on dorsum (vs absence);
S. jiulongensis − the Kanjiroba lineage is distinguished by smaller male body size (SVL, 55.4 mm vs 67.4−81.5 mm; absence of subarticular tubercles (vs presence); presence of nuptial spines on dorsal surface of fingers I, II, and inner surface of finger III of males in breeding condition (vs on inner two fingers).
S. liupanensis − the Kanjiroba lineage is distinguished by absence of maxillary teeth (vs presence); rudimentary webbing between toes (vs developed).
S. luozhaensis − the Kanjiroba lineage is distinguished by absence of more than one and up to six separated spines on top of each dorsal tubercle of males in breeding condition (vs presence of up to six spines on tubercles); absence of yellow tubercles scattered around cloaca of males in breeding condition (vs presence).
S. maculatus − the Kanjiroba lineage is distinguished by absence of subarticular tubercles (vs presence); rudimentary webbing between toes (vs developed).
S. mammatus − the Kanjiroba lineage is distinguished by absence of subarticular tubercles (vs presence); rudimentary webbing between toes (vs developed, large or reduced); presence of nuptial spines on dorsal surface of fingers I, II, and inner surface of finger III of males in breeding condition (vs on inner two fingers); presence of spines on male axillary glands (vs absence).
S. meiliensis − the Kanjiroba lineage is distinguished by absence of maxillary teeth (vs presence); presence of nuptial spines on dorsal surface of fingers I, II, and inner surface of finger III of males in breeding condition (vs on inner two fingers).
S. muliensis − the Kanjiroba lineage is distinguished by presence of nuptial spines on dorsal surface of fingers I, II, and inner surface of finger III of males in breeding condition (vs on inner two fingers); presence of a pair of pectoral and axillary patches in breeding male (vs one pair).
S. ningshanensis − the Kanjiroba lineage di is distinguished by absence of maxillary teeth (vs presence); absence of spine patches on belly of males in breeding condition (vs presence).
S. nyingchiensis − the Kanjiroba lineage is distinguished by absence of maxillary teeth (vs presence); rudimentary webbing between toes (vs developed).
S. occidentalis − the Kanjiroba lineage is distinguished by a smaller head (HL: 14.5−19.1 mm vs 23.5 mm; HW: 18.2−23.2 mm vs 24.5 mm); absence of spines on male forearm in breeding condition (vs presence); absence of dorsal tubercles with 1−6 coloured tips (corresponding to spines in breeding condition) on top (vs presence, Suppl. material
S. pingwuensis − the Kanjiroba lineage is distinguished by absence of spines on male forearm in breeding condition (vs presence); absence of spine patches on belly of males in breeding condition (vs presence).
S. sikimmensis − the Kanjiroba lineage is distinguished by relative length of toes 4>3>5>2>1 (vs 4>5>3>2>1); presence of spines on dorsal tubercles of males (vs absence); see also Suppl. material
S. spinosus − the Kanjiroba lineage is distinguished by absence of spines on male upper arm in breeding condition (vs presence); absence of spines on dorsal tubercles of females (vs presence); wider internarial distance (5.6−6.8 mm vs 4.0−4.8 mm).
S. tengchongensis − the Kanjiroba lineage is distinguished by larger male body size (SVL: 55.4 mm vs 36.0−40.1 mm); absence of small patches of black spines near armpit of males in breeding condition (vs presence).
S. tuberculatus − the Kanjiroba lineage is distinguished by smaller male body size (SVL: 55.4 mm vs 68.0−76.0 mm); presence of nuptial spines on dorsal surface of fingers I, II, and inner surface of finger III of males in breeding condition (vs on inner two fingers).
S. wanglangensis − the Kanjiroba lineage is distinguished by absence of maxillary teeth (vs presence); absence of spine patches on belly of males in breeding condition (vs presence).
S. wuguanfui − the Kanjiroba lineage is distinguished by absence of vocal sac in adult males (vs presence); absence of small black spines on upper chest (vs presence).
Based on the synthesis of molecular phylogenetic findings and observed morphological distinctions, the Scutiger populations inhabiting the Kanjiroba Himal region in western Nepal are deemed to constitute a novel species. Consequently, we provide a detailed description of this species.
Scutiger nepalensis:
Holotype. Nepal • 1 ♂; NHME A0576/99; adult; collected at Juphal in the direction to Tripurakot, Dolpa district, Nepal (29°01'N, 82°47'E, 2600 m a.s.l.) on 31/05/1997 by Marco Fischer; deposited in the NHME. Paratypes. Nepal • 1 ♀; NHME A0570/99; adult; and 1 juvenile; NHME A1247/05; collected at Talphi, Jumla district, Nepal (29°18'N, 82°20'E, 3400 m a.s.l.) on 16/06/1997 by Marco Fischer; deposited in the NHME. 1 ♀; NHME A0574/99; adult; collected at Kaigaon, Jumla district, Nepal (29°06'N, 82°35'E, 3600 m a.s.l.) on 03/06/1997 by Marco Fischer; deposited in the NHME. 1 ♀; NHME A1248/05, adult, collected at Dhauli Lake, Jumla district, Nepal (29°21'N, 82°23'E, 4400 m a.s.l.) on 05/07/1999 by Marco Fischer, deposited in the NHME. 1 subadult; NHME A1249/05; collected at Dhauli Lake, Jumla district, Nepal (29°21'N, 82°23'E, 4400 m a.s.l.) on 05/07/1999 by Marco Fischer, deposited in the NHME. 1 tadpole; NHME A1250/05; collected at Dhauli Lake, Jumla district, Nepal (29°21'N, 82°23'E, 4400 m a.s.l.) on 05/07/1999 by Marco Fischer, deposited in the NHME. 2 juveniles; NHME A1251/05 and A1252/05; collected at Dhauli Lake, Jumla district, Nepal (29°21'N, 82°23'E, 4400 m a.s.l.) on 05/07/1999 by Marco Fischer, deposited in the NHME. 1 juvenile; NHME A1253/05; collected at Bumra [Bumramadichaur], Jumla district, Nepal (29°23'N, 82°08'E) on 22/06/1999 by Ulrich Scheidt, deposited in the NHME. 1 juvenile; NHME A1254/05; collected at Maharigaon, Jumla district, Nepal (29°20'N, 82°23'E) on 08/07/1999 by Andreas Weigel, deposited in the NHME. 1 metamorp; NHME A2014/13; collected near Khari La, Jumla district, Nepal (29°21'N, 82°09'E, 3285 m a.s.l.) in 1999 by Marco Fischer, deposited in the NHME.
Scutiger kanjiroba sp. nov. is assigned to the genus Scutiger based in the above-mentioned morphological features and by its molecular phylogenetic position relative to other Scutiger species. It can be separated from all other species of the genus Scutiger by the combination of the following characters (for character states in other species see section Morphological comparison, above): (1) moderate body size, SVL adult male 55.4 mm (n = 1), adult female 54.2−66.7 mm (n = 3); (2) vomerine and maxillary dentation absent; (3) vocal sac absent; (4) subarticular tubercles absent; (5) presence of numerous small, densely arranged spines on the surface of fingers I, II and inner surface of finger III of males in breeding condition; (6) relative length of fingers 3>4>2>1; (7) spines on (inner) (fore)arms absent; (8) toes rudimentary webbed; (9) relative length of toes 4>3>5>2>1; (10) a pair of pectoral glands and a pair of axillary glands; (11) spines on pectoral and axillary glands present in males; (12) belly without spines; (13) tubercles/warts on dorsal and lateral surfaces of body present; (14) dorsal tubercles with black spines of males in breeding condition.
(Fig.
Forelimbs strong; forearm of median length (FAL/SVL 0.25) and of similar length as the hand (HAL/SVL 0.24), without spines; fingers slender, without webbing and lateral dermal fringes; all fingertips rounded, not dilated; relative finger lengths: 3>4>2>1; subarticular tubercles absent; inner metacarpal tubercle flat and distinct, lager than the outer, indistinct metacarpal tubercle; nuptial spines on dorsal and lateral surface of first and second fingers, and on inner side of third finger.
Hindlimbs robust, moderate in length (TIBL/SVL = 0.38), thighs slightly shorter than shanks FEL/TIBL = 1.1), heels do not touch when folded at right angles to the body (see also Fig.
Dorsal and lateral skin with distinct tubercles in preservative, each tubercle with creamy coloured keratinised tip; tubercles with small, keratinised tips present below and on supratympanic fold and on shank; forehead and surfaces of lower arm relatively smooth; throat and belly surface smooth; a pair of pectoral glands and a pair of axillary glands present on chest, pectoral glands only slightly larger than axillary glands, all covered by tiny spines.
In males, a pair of pectoral glands and a pair of axillary glands present on breast (absent in females), all of them covered by spines in breeding season.
The background colouration of the dorsum fades to grey, tubercles with creamy white point, dorsal surface of finger I, II and inner surface of finger III pale brown; ventral belly pale greyish; ventral limbs, ventral chest, and ventral head greyish pale brown.
One tadpole (NHME A1250/05) of Scutiger kanjiroba sp. nov. was collected at stage 36 (Gosner, 1960) (Suppl. material
Scutiger kanjiroba is a forest-dwelling species that is currently known from high-montane areas of the Kanjiroba Himal at altitudes between ~ 3300 and 4400 m a.s.l. in west Nepal (Fig.
The specific epithet kanjiroba is a geographical proper name referring to the Kanjiroba Himal in Nepal. The southern area of the Kanjiroba Himal is so far the only known occurrence region of this new species.
Lazy toads of the genus Scutiger are a characteristic faunal element of the Himalaya-Tibet orogen. These toads possess limited dispersal capabilities, are adapted to high altitudes, and are typically found throughout the Himalaya Mountain range, exhibiting allopatric distribution with a high degree of local endemism. Previous research has revealed extensive geographically structured relationships among Himalayan Scutiger species, indicating significant physical barriers not only between species but also among populations (
The newly identified species Scutiger kanjiroba sp. nov. is phylogenetically distinct from the geographically neighbouring S. nepalensis Dubois, 1974, forming its sister lineage, as confirmed by both morphology and nuclear molecular analysis. It can be easily distinguished from S. nepalensis in the field by a much smaller and narrower head and an overall smaller body size. The two species are geographically separated by the Karnali River gorge, which probably acts as physical barrier to dispersal for terrestrial, non-flying organism groups.
The other species long known from the Central Himalaya, S. sikimmensis (Blyth, 1855), lacks a specified type locality in Sikkim. A neotype (BMNH 1887.11.2.15) had been assigned by Dubois (
The discovery of S. kanjiroba sp. nov. in the Kanjiroba massif, west Nepal, adds to the recent description of S. ghunsa in east Nepal (
We are grateful to Matthias Hartmann and Konrad Kürbis (Natural History Museum of Erfurt), and Annemarie Ohler and Christophe Dufresnes (Muséum National d’Histoire Naturelle, Paris) for the kind loan of Scutiger specimens. We also thank Morris Flecks and Pratyush P. Mohapatra for taking the photographs of specimens, Jana Polakova for her work in the DNA lab, and Jianhuan Yang and a second anonymous reviewer for their constructive comments on the manuscript.
The authors have declared that no competing interests exist.
No ethical statement was reported.
This research was financially supported by the German Research Foundation DFG (grant no. HO3792/8-1 to SH).
Conceptualization: SH. Data curation: SH, DJ. Formal analysis: SH. Funding acquisition: SH. Investigation: SH. Methodology: SH, JS. Project administration: SH. Resources: DJ, JS. Visualization: SH. Writing – original draft: SH. Writing – review and editing: DJ, JS.
Sylvia Hofmann https://orcid.org/0000-0003-0621-9049
Daniel Jablonski https://orcid.org/0000-0002-5394-0114
Joachim Schmidt https://orcid.org/0000-0002-0247-5208
All of the data that support the findings of this study are available in the main text or Supplementary Information.
Supplementary information
Data type: pdf