Megophrys (Xenophrys) flavipunctata, 2018

Mahony, Stephen, Kamei, Rachunliu G. & Teeling, Emma C., 2018, Cryptic diversity within the Megophrys major species group (Amphibia: Megophryidae) of the Asian Horned Frogs: Phylogenetic perspectives and a taxonomic revision of South Asian taxa, with descriptions of four new species, Zootaxa 4523 (1), pp. 1-96 : 46-50

Megophrys (Xenophrys) flavipunctata sp. nov.

( Figures 16 & 17 ; Table 1)

Holotype. Adult male ( BNHS 6040 [field no. SDBDU 2009.304 ]: Figures 16 & 17B ), from Mawphlang Sacred Grove , Mawphlang (25°26'37"N, 91°44'46"E, 1810 m asl.), East Khasi Hills district , Meghalaya state, Northeast India, collected by members of the Systematics Lab, University of Delhi on 0 1 June 2009.

Paratypes. One adult male ( BNHS 6044 [field no. SDBDU 2009.305 ]), and two adult females ( BNHS 6043 [field no. SDBDU 2009.303 ]: Figure 17A, C & D ; BNHS 6042 [field no. SDBDU 2009.306 ]: Figure 18A ), collected along with the holotype; one adult female ( BNHS 6041 [field no. SDBDU 2011.480 ]: Figure 17A ), collected from the type locality by SDB and RGK on 0 9 July 2011 .

Referred specimens. One adult male (SDBDU 2009.297), and one juvenile male (SDBDU 2009.298), collected from the type locality by Systematics Lab members on 30 May 2009; one adult male ( SDBDU 2007.134 : Figure 17E & F ), from near Sazolie College , Jotsoma village (25°38'23"N, 94°03'31"E, 1575 m asl.), Kohima district, Nagaland state, Northeast India, collected by RGK on 27 June 2007 .

Holotype description (measurements in mm). Mature male (SVL 68.4) ( Figures 16 & 17B ). Head moderately large, wider than long (HW 26.3, HL 25.0, IFE 10.5, IBE 18.1); snout bluntly pointed in dorsal view, obtusely protruding beyond mandible in lateral view, without rostral appendage ( Figure 16C ); loreal region acute, concave; canthus rostralis angular; dorsal surface of snout slightly concave; eye diameter 2.5 times maximum diameter of visible portion of tympanum, and shorter than snout (EL 7.6, TYD 3.1, SL 8.6); eye–tympanum distance (TYE 5.8) longer than diameter of visible portion of tympanum; tympanum oval-shaped, slightly oblique, upper border concealed by supratympanic ridge ( Figure 16C ); pupil in life vertically elliptical; nostril positioned laterally, closer to eye than to snout (EN 3.8, NS 5.4); internarial distance greater than eyelid width and narrowest point between upper eyelids (IN 7.7, UEW 6.8, IUE 5.4); pineal ocellus not visible externally; vomerine ridges medium sized, ovoid, moderately raised, orientated acutely, positioned between to slightly posterior to choanae, equidistant from each other and choanae; vomerine teeth small; maxillary teeth present; tongue moderately large, deeply notched posteriorly, medial lingual process absent.

Forelimbs moderately long, thin ( Figure 16A & B ), forearm not enlarged relative to upper forelimb, and shorter than hand (FAL 14.6; HAL 17.9); fingers moderately long, narrow, without lateral fringes ( Figure 16D ), finger length formula IV<I=II<III (FIL 8.7, FIIL 8.6, FIIIL 11.9, FIVL 7.8); interdigital webbing, subarticular, supernumerary and metacarpal tubercles all absent; thenar tubercles weak; finger tips rounded, slightly expanded relative to digit widths (FIIIW 1.3, FIIIDW 1.6), longitudinally oval-shaped pads present, terminal grooves on pads absent. Hindlimbs long, thin ( Figure 16A & B ); thighs slightly shorter than shanks, and slightly longer than feet (TL 35.5, SHL 36.7, FOL 34.7); toes long, dorsoventrally flattened, with narrow lateral fringes ( Figure 16E ), relative toe lengths I<II<V<III<IV; toe tips rounded, not dilated (TIVW 1.6, TIVDW 1.7), longitudinally ovalshaped pads distinct, terminal grooves on pads absent; webbing basal (III2.3–3.8IV3.5– 2V); inner metatarsal tubercle indistinct; subarticular, supernumerary and outer metatarsal tubercles absent; callous tissue weak on ventral surface of toes.

Skin of dorsal surfaces of body, limbs, and dorsal and lateral surfaces of head primarily smooth; tympanum smooth with borders slightly raised; outer edge of upper eyelid with a weak medial bump, remaining surface covered with small granular tubercles; supratympanic ridges narrow anteriorly gradually expanding beyond posterior edge of tympanum, becoming moderately enlarged, glandular, extending from posterior orbital border and broadly curving through upper border of tympanum, terminating above axilla; flanks with small to large scattered pustular tubercles; dorsolateral ridges thin, well defined, extending posteriorly from behind supratympanic ridges to ~75% trunk length; parietoscapular-sacral ridges indistinct; gular region, chest, abdomen and ventral surfaces of limbs smooth; pectoral glands faint, flat, level with axilla on chest; femoral glands large, flat, on posterior surface of thighs, slightly closer to knees than to cloaca; dermal asperities absent from all surfaces.

Colouration: In preservative ( Figure 16 ): Dorsal and lateral surfaces of head and body primarily mid greyishbrown mottled with lighter tones; light-edged, dark brown triangular marking present between eyes; short longitudinal brown stripe on mid-dorsal snout; small light-edged dark brown Y-shaped marking on anterior dorsum, dark brown, ragged-edged, dorsolateral stripe present on each side; tubercles on anterior flanks dark brown with light tip, on posterior flanks pale yellow with bordering small dark brown spot; front of snout greyishbrown with extensive yellowish-orange speckling, lateral canthus rostralis dark greyish-brown; light grey stripe on upper lip extends from nostril to rear of upper jaw, bordered below by thin dark brown stripe along margin of lip; dark brown blotch extends from posterior border of eye through tympanum to posterior supratympanic ridge; additional triangular blotch distinct at posterior lower orbital border, with intervening area a lighter shade of brown; outer edges of eyelids dark brown; lower half of supratympanic ridges dark brown, upper half light yellowish-cream which continues anteriorly as fine light stripe along edge of upper eyelids and canthus rostralis to tip of snout; dorsal surfaces of forelimbs and hindlimbs primarily light greyish-brown with mid brown blotches; two dark brown blotches on anterior lateral surface of forearms; dorsal surface of outer three fingers with dark brown blotches; dorsal surfaces of hindlimbs with distinct mid brown transverse crossbars; lateral surfaces of thighs and shanks with dark brown spots and blotches; throat primarily light brown with some yellow spots along its edge, light-edged wide dark brown stripe extends from posterior edge of lower jaw to ventral forearm; chest and anterior abdomen with faint large mid brown blotches on otherwise pale yellowish-grey background; ventral surfaces of forelimbs and shanks primarily pale grey, thighs pale yellowish-grey scattered with minute yellow spots; some large dark brown spots and blotches laterally along abdomen on both sides; area surrounding cloaca and posterior surfaces of thighs primarily dark brown with small yellow spots; ventral surfaces of tarsi and feet greyish-brown; hands ventrally light grey; pectoral and femoral glands creamish-white. In life ( Figure 17B ): Colouration mostly as described in preservative but richer; enlarged tubercles on posterior and ventrolateral flanks, and enlarged portion of posterior supratympanic ridge bright yellow; iris colour rich maroon. Ventral colouration in life not documented.

Variation. See Table 1 for morphometric variation within the type series and referred specimens consisting of three adult males, three adult females, and a juvenile. The paratypes and referred specimens resemble the holotype for most morphological characters with some exceptions: BNHS 6041, BNHS 6043 and SDBDU 2009.298 do not have distinct lateral fringes on toes; BNHS 6044 has finger length formula II<IV<I<III, and BNHS 6041 has the formula IV<II<I<III; vomerine teeth indistinct on BNHS 6 043; all paratypes and referred specimens bar BNHS 6044 differ from holotype by possessing dorsolateral ridges that extend almost full length of trunk (e.g., Figure 17A, C & E ); supratympanic ridges conceal up to ~20% of upper tympanum; BNHS 6044 has very small black dermal asperities forming circummarginal band on gular region, few also present on flank tubercles, but absent from other surfaces; pectoral glands on some individuals small, raised; dorsal ridges vary from a weakly raised Vshaped parietoscapular ridge only (SDBDU 2009.297, SDBDU 2009.298, BNHS 6044, BNHS 6042), Y-shaped parietoscapular ridge either without (BNHS 6043), or with an attached inverted V-shaped sacral ridge on middorsum (all remaining specimens); BNHS 6041 and BNHS 6043 have moderately well developed thenar tubercles; BNHS 6043 in life was orange-brown dorsally with dense orange spotting through upper lip stripe and throat, spotting decreased in density posteriorly on abdomen ( Figure 17C & D ); SDBDU 2009.297 in life had upper half of posterior supratympanic ridge creamish-white, blotches on central portion of throat and chest were a pale brownish-mauve; BNHS 6042 in life had chest, abdomen (excluding a large brown blotch and small speckling) and ventral surface of thighs creamish-white with distinct hint of yellow laterally on lower-most flanks; most specimens had distinct X-shaped marking on mid-dorsum.

Secondary sexual characters. Males: weakly raised nuptial pads present, sparsely covered with black microasperities, primarily covering dorsal surface of base of Finger I, narrowing distally, extending onto base of distal phalange on inner dorsal side; nuptial pad on Finger II small, oval, positioned on base of digit on inner dorsal side; external vocal sac indistinct; internal vocal slits present, one on each side on floor of mouth near rear of mandible; forearms not enlarged relative to upper forelimbs. Females: mature ova not pigmented; nuptial pads, vocal sac, vocal slits, enlarged forearms, all absent.

Morphological comparison. Megophrys flavipunctata sp. nov. (adult males, N =4, adult females, N =3) differs from M. monticola and M. zhangi by its larger adult body size, male SVL 56.9–68.4 mm, female SVL 68.0– 74.6 mm (vs. male SVL 38.2–49.5 mm, N =17, female SVL 40.5–56.1 mm, N =6; male SVL 32.5–37.2 mm, N =3, respectively); differs from M. medogensis by presence of vivid whitish-cream upper lip stripe (vs. absent), toes dorsoventrally flattened, with or without narrow lateral dermal fringes (vs. toes rounded, not flattened, lateral fringes absent); differs from M. robusta and M. major s.s. by smaller adult body size, male SVL 56.9–68.4 mm, female SVL 68.0– 74.6 mm (vs. male SVL 73.5–83.1 mm, N =6, female SVL 81.3–108.3 mm, N =4; male SVL 71.6–87.5 mm, N =12, female SVL 85.6–98.2 mm, N =3, respectively), further from M. robusta by presence of vivid whitish-cream upper lip stripe (vs. absent), further from M. major s.s. by distinct brown blotches clearly visible on chest and anterior abdomen of all specimens (vs. typically absent, if present always very faint), flank tubercles, particularly the anteriormost tubercles with dark brown blotches present (vs. absent); differs from M. mangshanensis by basal webbing between toes present (vs. absent). For comparisons with subsequent species covered in this study, refer to relevant morphological comparison sections for those species.

Systematic position. This taxon represents M. cf. major 2 in the molecular analyses of this study (discussed as OTU 6), and in Mahony et al. (2017).

The systematic position of Megophrys flavipunctata sp. nov. within the MMC clade is not fully resolved. It has consistently been found to be a member of the western clade of the MMC ( Figure 1 ), however, its relationship with other members of this clade is not clear when molecular data for the MMSG was subjected to a variety of phylogenetic techniques ( Figures 2–5 ; Appendix I, Table 3; Appendix II, Figures 1 , 2 , 5 & 6 ). Refer to Appendix I, Table 6 for uncorrected p -distances for the 16S rRNA gene between Megophrys flavipunctata sp. nov. and other MMSG species.

Etymology. The specific epithet is an adjective derived from the Latin words flavus meaning “yellow” and punctatus meaning “spotted” with reference to the bright yellow tubercles on the posterior flanks of this species.

Suggested common name: Yellow Spotted White-lipped Horned Frog.

Distribution. Megophrys flavipunctata sp. nov. is currently known with certainty from two populations, one in Meghalaya state, and a second disjunct population in Nagaland state, Northeast India ( Figure 8A ). Mawphlang Sacred Grove (25°26'37.02"N, 91°44'46.02"E, 1810 m asl.) is located next to the village of Mawphlang, on the upper limits of the Shillong Plateau, southwest from Shillong city, Meghalaya. The second known population is represented by a single specimen collected from near Sazolie College, Jotsoma village (25°38'23"N, 94°03'31"E, 1575 m asl.), Kohima district, Nagaland state, ~ 230 km east of the type locality. This species is likely to be present at other mid- to high-elevation localities in intervening areas, where suitable habitat remains. It may also extend westward in Meghalaya to Tura Peak, West Garo Hills district (see Remarks below for Sangma and Saikia 2015), however this locality requires further confirmation.

Habitat and natural history. Refer to Mahony (2008) and Mahony et al. (2013) for a general description of the habitat and forest type at Mawphlang ( Figure 9B & D ). The type series was collected on the forest floor, on or amongst dense leaf litter. Those collected at dusk were found emerging from beneath leaf litter, and one from the entrance of a rodent burrow, where presumably this species remains concealed during daylight hours. Two other individuals collected immediately after dusk, were on top of leaf litter several meters to the side of a forest trail in a part of the forest with sparse to no undergrowth. All were more than 50 meters from the nearest stream. Despite numerous visits to Mawphlang Sacred Grove between May and September 2006 to 2011, this species was only observed in 2009 and 2011, possibly because earlier surveys were primarily focused along streams and forest trails. No male advertisement calls attributable to this species were heard on any visit. This species was also not reported by others carrying out amphibian research at Mawphlang (e.g., Das et al. 2010; Purkayashta & Matsui 2010; Tron et al. 2015). The distant location of adult animals from streams in late May/early June might indicate that this species does not breed at this time of the year. Adult females had moderately well developed ova in their ovaries, and males also had moderately small (but developmentally mature) testes and very weakly developed nuptial pads indicating that they were not in full reproductive condition. Further field studies are required to determine the breeding season. The single specimen from Nagaland was collected in the month of June, on a road (at 22:30 h), adjacent to a temporary stream, nearby a degraded semi-deciduous forest patch (see Biju et al. 2010 for discussion on habitat condition at this locality). This adult male was also not in breeding condition, i.e., nuptial pads not developed, and testes not enlarged.

Remarks. A previous report of M. glandulosa from Puliebadze (near Jotsoma), Nagaland by Ao et al. (2003) might represent this species (see Remarks section of M. glandulosa ). Sangma and Saikia (2015:2410, pl. 6a) provided a figure of a specimen that superficially resembles this new species (as Xenophrys robusta ) from Tura Peak (751 m asl.), West Garo Hills, Meghalaya, however the identity of this specimen requires further confirmation pending examination.

Figures

FIGURE 1. Ultrametric guide tree topologies used for BPP analyses: A. topology based on *BEAST analyses of phased nuclear alignments, with posterior probability values for nodes; B. alternative topology guide tree based on the results of concatenated mt+nuDNA gene tree analyses (Dataset E); C. alternative topology based on *BEAST analyses of unphased nuclear alignments, with posterior probability values for nodes. Taxon names in pink represent the eastern clade, blue represents the western clade (southern subclade), and green represents the western clade (northern subclade). Refer to Appendix I, Table 2 for revised names for taxa based on the taxonomic review.

FIGURE 2. BI concatenated gene tree based on the 47 taxa concatenated mt+nuDNA alignment (Dataset C), with ML node values provided. Posterior probability/bootstrap values>95% in both analyses are represented by a black spot on the node, values <95% in one or both analyses are provided next to relevant nodes, where * is>95%, and – is given for nodes that were not resolved on the ML tree. Taxon names in pink represent the eastern clade, blue represents the western clade (southern subclade), and green represents the western clade (northern subclade). Revised names for taxa based on the taxonomic review are provided in parentheses.

FIGURE 3. Concatenated gene trees based on the 29 taxa concatenated nuDNA alignment (Dataset D): A. RAxML tree; B. MrBayes tree. Posterior probability/bootstrap values are provided next to associated nodes. Taxon names in pink represent the eastern clade, blue represents the western clade (southern subclade), and green represents the western clade (northern subclade). Refer to Appendix I, Table 2 for revised names for taxa based on the taxonomic review.

FIGURE 4. RAxML tree for the combined mt+nuDNA dataset expanded to include the Chen et al. (2017) sequences (Dataset J). Bootstrap support values are provided adjacent to the associated nodes. Refer to Appendix I, Table 2 for revised names for taxa based on the taxonomic review.

FIGURE 5. DensiTree cloudogram representations of *BEAST analyses, removing the first 10% of trees as burn-in, produced from the four nuDNA gene alignments of Dataset E: A. original unphased; B. phased. Dark blue branches represent the most frequently occurring topology, light blue branches represent the consensus trees with intensity proportional to the frequency of the topology in the dataset, and green areas represent all topologies in the dataset. Refer to Appendix I, Table 2 for revised names for taxa based on the taxonomic review.

FIGURE 6. Megophrys monticola lectotype: adult female (BMNH 1947.2.25.13) in preservation: A. dorsal view; B. ventral view; C. profile view of head; D. ventral view of hand; E. ventral view of foot.

FIGURE 8. Distribution map of the western MMSG taxa: A. All taxa (excluding M. monticola) from Northeast India and surrounding countries: diamonds represent type localities, circles represent referred localities based on molecularly sampled specimens: orange = M. zhangi; yellow = M. robusta; black = M. medogensis; dark blue = M. major; pink = Megophrys flavipunctata sp. nov.; dark green = Megophrys oreocrypta sp. nov.; light blue = Megophrys himalayana sp. nov.; light green = Megophrys periosa sp. nov.; pale blue = M. cf. periosa; purple = M. glandulosa. B. Inset rectangle in Figure A enlarged to show the distribution map for M. monticola from Darjeeling and Sikkim region, Northeast India, showing distribution of named mitochondrial haplotypes: stars represent the type localities, circles represent referred localities confirmed from sequence data: red = M. “sanu” comb. nov., light blue = M. “katabhako” comb. nov. Blue lines represent major rivers; grey lines represent international borders.

FIGURE 9. Habitat images for collection localities of MMSG species in Northeast India. A. Sessa River, near Sessa village (1110 m asl.), West Kameng dist., Arunachal Pradesh, collection locality of Megophrys robusta and Megophrys periosa sp. nov.; B. and D. Mawphlang Sacred Grove (~1810 m asl.), near Mawphlang village, East Khasi Hills dist., Meghalaya, type locality of Megophrys flavipunctata sp. nov.; C. Senchal Wildlife Sanctuary (2220 m asl.), Rambi (East Range), Darjeeling Sadar subdiv., Darjeeling dist., West Bengal, collection locality of M. monticola; E. Sessa River near Elephant village (370 m asl.), West Kameng dist., Arunachal Pradesh, off which smaller side streams were the type locality of Megophrys himalayana sp. nov.; F. stream near Pangin town (450 m asl.), East Siang dist., Arunachal Pradesh, type locality of Megophrys periosa sp. nov.

FIGURE 16. Megophrys flavipunctata sp. nov. holotype: adult male (BNHS 6040: SVL 68.4 mm) in preservation: A. dorsal view; B. ventral view; C. profile view of head; D. ventral view of hand; E. ventral view of foot.

FIGURE 17. Megophrys flavipunctata sp. nov.: A. variation of dorsal markings in adult female paratypes, left (BNHS 6043: SVL 74.6 mm), centre (BNHS 6042: SVL 68.0 mm), and right (BNHS 6041: SVL 68.0 mm) in preservation; B. dorsolateral view of holotype adult male (BNHS 6040: SVL 68.4 mm) in life; C. and D. adult female paratype (BNHS 6043: SVL 74.6 mm) in life: C. dorsolateral view; D. ventral view; E. and F. adult male referred specimen (SDBDU 2007.134: SVL 67.5 mm) from Nagaland state, in life: E. dorsolateral view; F. ventral view.

FIGURE 18. Megophrys oreocrypta sp. nov. holotype: adult female (BNHS 6045: SVL 94.9 mm) in preservation: A. dorsal view; B. ventral view; C. profile view of head; D. ventral view of hand; E. ventral view of foot.