Leptobrachium lunatum, Stuart & Som & Neang & Hoang & Le & Dau & Potter & Rowley, 2020

Stuart, Bryan L., Som, Hannah E., Neang, Thy, Hoang, Huy Duc, Le, Duong Thi Thuy, Dau, Vinh Quang, Potter, Kathy & Rowley, Jodi J. L., 2020, Integrative taxonomic analysis reveals a new species of Leptobrachium (Anura: Megophryidae) from north-eastern Cambodia and central Vietnam, Journal of Natural History (J. Nat. Hist.) 54 (1 - 4), pp. 225-255 : 235-249

Leptobrachium lunatum sp. nov.

http://www.zoobank.org/ urn:lsid:zoobank.org:pub:CE6650AF-D9FB-40F4-8A2E-9B4E23AAC205

( Figures 7–10 )

Leptobrachium mouhoti Bain and Nguyen, 2006: 358 ; Neang and Holden, 2008: 44 (part); Nguyen, Ho and Nguyen, 2009: 78 (part); Bain and Hurley, 2011: 92 (part).

Leptobrachium pullum Nguyen, Ho and Nguyen, 2009: 80 (part); Matsui, Hamidy, Murphy, Khonsue, Yambun, Shimada, Ahmad, Belabut and Jiang, 2010: 263; Bain and Hurley 2011: 92 (part); Hamidy, Matsui, Nishikawa and Belabut, 2012: 26; Strautins, Le, Dau, Hoang and Rowley, 2014: 477; Vassilieva, Galoyan, Poyarkov and Geissler, 2016: 50 (part); Pawangkhanant, Poyarkov, Duong, Naiduangchan and Suwannapoom, 2018: Table S1.

Leptobrachium sp. Bain, Biju, Brown, Das, Diesmos, Dutta, Gower, Inger, Iskandar, Kaneko, Lau, Meegaskumbura, Ohler, Papenfuss, Pethiyagoda, Stuart, Wilkinson and Xie, 2008: 77.

Holotype. MVZ 258179 (field tag BLS 11176; Figure 7 ), adult male, Cambodia, Ratanakiri Province, Veunsai District, Virachey National Park , 14.19287°N, 106.99612°E, 650 m elev., coll. 2 October 2007 by Bryan L. Stuart, Jodi J.L. Rowley and Thy Neang.

Paratypes. Cambodia, Ratanakiri Province, Ta Veng District, Virachey National Park : MVZ 258071 (one adult male), 14.20000°N, 107.38333°E, 600 m elev ., coll. 22 June 2006 by David A. Emmett and Jodi J .L. Rowley . MVZ 258180–81 (two adult males), MVZ 258182 (one adult female), MVZ 258183 (one immature female), same data as holotype except coll . 6–13 October 2007.

Vietnam, Kon Tum Province, Dak Glei District, Ngoc Linh Nature Reserve , coll . Jodi J .L . Rowley, Huy Duc Hoang, Duong Thi Thuy Le , and Vinh Quang Dau : AMS R173756 / UNS 00159 (one adult male), AMS R173755 (one adult female), 15.23587°N, 107.70982°E, 1316 m elev ., 12 July 2009; AMS R173882 , AMS R173884 / UNS 00149 (two adult males), AMS R173881 / UNS 00150, AMS R173883 / UNS 00152, AMS R173885 (three adult females), 15.07796°N, 107.96076°E, 1992 m elev ., 17 July 2009; AMS 173617–18 (two adult males), 15.17086°N, 107.75718°E, 1104 m elev., 7 September 2009; AMS R173636 (one adult male), 15.16785°N, 107.75653°E, 1085 m elev ., 7 October 2009; NCSM 79656–57 , AMS R173433 / UNS 00153, AMS R173434 / UNS 00157 (four adult males), AMS R173429 , AMS R173432 (two adult females), 15.04069°N, 107.81649°E, 1494 m elev ., 26–30 March 2010; AMS R173430 , AMS R173431 / UNS 00163 (two adult males), 15.03227°N, 107.81529°E, 1722 m elev ., 27 March 2010; AMS R173435 , AMS R173436 / UNS 00160, AMS R173437 , AMS R173438 / UNS 00154, AMS R173439 / UNS 00155, AMS R173440 / UNS 00143, AMS R173443 / UNS 00151, AMS R173444 / UNS 00156 (eight adult males), AMS R173441 , AMS R173445 (two adult females), 15.06284°N, 107.85870°E, 1716 m elev ., 2–4 April 2010; AMS R173442 (one adult female), 15.06302°N, 107.86566°E, 2105 m elev ., 3 April 2010.

Vietnam, Gia Lai Province, Mang Yang District, Kon Ka Kinh National Park , coll . Jodi J .L . Rowley, Vinh Quang Dau, Huy Duc Hoang , Luong Thi Nguyen, Tan Thanh Le, D-jung, and Y . H’ Dun : AMS R176305–06 (two adult males), AMS R176307 (one adult female), 14.22466°N, 108.32217°E, 1097 m elev ., 21 August 2011; AMS R176310 , AMS R176313–15 (four adult males), AMS R176308 , AMS R176309 (two adult females), 14.22441°N, 108.33482°E, 1468 m elev ., 23–27 August 2011; NCSM 79658 (one adult male), 14.21964°N, 108.31979°E, 1019 m elev ., 24 August 2011; AMS R176312 (one adult male), 14.22056°N, 108.31702°E, 1003 m elev ., 24 August 2011; NCSM 79659 (one adult male), 14.22441°N, 108.33482°E, 1468 m elev ., 27 August 2011; AMS R176316 (one adult male), 14.221°N, 108.317°E, 1000 m elev ., 28 August 2011.

Referred tadpoles

MVZ 258314 , same data as holotype MVZ 258180 except 14.18778°N, 106.99573°E, 602 m elev . MVZ 258315 , same data as paratype MVZ 258183 . AMS R173631 , AMS R173633 / UNS 00372, same data as paratype AMS 173617 except 9 July 2009 . AMS R173658 / UNS 00374, same data as paratype AMS R173636 . AMS R173670 , AMS R173676 / UNS 00358, same data as paratype AMS R173756 except 15.22317°N, 107.72733°E, 1134 m elev ., 11 July 2009. AMS R173728 , same data as paratype AMS R173756 . AMS R173835 / UNS 00386, AMS R173863 , same data as paratype AMS R173882 except 15–16 July 2019 . AMS R173879 , same data as paratype AMS R173882 except 15.08°N, 107.96°E, 1905–1996 m elev . AMS R176498–99 , same data as paratype NCSM 79658 except 14.21929°N, 108.31710°E, 985 m elev .

Etymology. The specific epithet lunatum (L.) is the neuter form of the adjective lunatus for ‘shaped like a crescent moon’, in reference to the shape of the iris colour of the new species.

Diagnosis. Assigned to the genus Leptobrachium on the basis of having head width larger than shank length; skin above with a network of ridges; large axillary glands; extremities of digits rounded; upper part of iris coloured differently from lower part ( Dubois and Ohler 1998); and by its phylogenetic position ( Figure 2). A medium-sized Leptobrachium having a variably coloured scarlet, orange or yellowish scleral arc under the palpebrum, visible in the posterior corner of the eye and when the palpebrum is retracted; males with SVL 38.5–54.9 mm (mean 45.7 mm), females 44.9–71.4 mm (mean 56.7 mm); males with HDL 18.1–24.4 mm (mean 20.5 mm), SHK 13.3–20.3 mm (mean 16.7 mm), TGH 16.9–24.9 mm (mean 20.6 mm), LAL 12.2–19.8 mm (mean 14.5 mm), IML 1.7–3.6 mm (mean 2.5 mm) and IMW 0.9–2.0 mm (mean 1.4 mm); venter greyish or brownish in colouration with minute light spots on tubercles; and male advertisement calls with 9–24 pulses.

Description of holotype. Habitus moderately stocky; body slightly tapered towards groin. Head broad and depressed; head length and width subequal. Snout rounded in dorsal view, sloping in profile, projecting beyond lower jaw in profile; nostril equidistant to snout tip and eye, below canthus, internarial shorter than interorbital distance; canthus rostralis distinct; lores oblique, moderately concave; eye large, projecting from side of head, diameter 66% of snout length, upper eyelid width 78% of interorbital distance; pineal ocellus not visible; tympanum round, annulus weakly visible, horizontal diameter about 55% of eye diameter, greater than distance between tympanum and eye; tongue heart-shaped, notched posteriorly; large, slit-like vocal sac openings on floor of mouth near lateral margin of tongue; vomerine teeth absent.

Fore limb moderately slender. Fingers moderately slender, without webbing. Tip of fingers blunt, those on Finger I slightly swollen; relative finger lengths II = IV <I <III; two oval palmar tubercles in contact, inner slightly larger than outer, low callous pads on ventral surface of fingers; nuptial pad absent.

Hind limb moderately slender, relatively short. Toes moderately slender; webbing on toe I to level of distal margin of subarticular tubercle, on preaxial side of toe II to level of proximal margin of subarticular tubercle and continuing as a fringe to base of tip, on postaxial side of toe II to level of distal margin of subarticular tubercle and continuing as a fringe to base of tip, on preaxial side of toe III to level of proximal margin of proximal callous pad and continuing as a fringe to base of tip, on postaxial side of toe III to midway between proximal margin of proximal callous pad and subarticular tubercle and continuing as a fringe to base of tip, on preaxial and postaxial sides of toe IV to level of proximal margin of proximal callous pad and continuing as a fringe to base of tip, and on toe V to midway between base and tip. Tips of all toes blunt, slightly swollen; relative toe lengths I <II <V <III <IV; distinct, half-moon shaped, inner metatarsal tubercle, length about 67% distance between tip of toe I and tubercle; no outer metatarsal tubercle.

Skin above smooth with fine network of ridges, with small granules scattered posteriorly, especially near groin; no spines on upper lip; low supratympanic fold from posterior margin of eye to axilla; ventrally granular, skin smooth on ventral surfaces of limbs; large, oval axillary gland on ventrolateral surface slightly posterior to insertion of fore limb with body; round femoral gland on posteroventral surface of thigh, closer to knee than vent.

Colour of holotype in life. Dorsum dark grey-brown (Dark Neutral Gray 299), with smaller, irregular dark brown (Dusky Brown 285) markings, concentrated around lower half of dorsum; ventral side of forearm and hind legs same colour as dorsum with dark brown (Dusky Brown 285) horizontal banding, bands fainter on forearms; lateral region same colour as dorsal with minute white spots on tubercles; iris black with deep orange (Flame Scarlet 73) scleral arc under palpebrum; irregular brown patches on tympanum, lores and below eyes and supratympanic fold; ventral surface of body and limbs marbled dark grey (Vandyke Brown 281) with light grey (Pale Neutral Gray 296); minute white spots on tubercles along mouth, chin and entire ventral surface; centre of ventral surface cream coloured (Pale Buff 1); axillary and femoral glands off- white.

Colour of holotype in preservative. Colour in preservative closely resembles colour in life, except that dark grey (Dark Neutral Gray 299) on dorsal surfaces faded to purplishgrey (Vandyke Brown 282).

Measurements of holotype (mm). SVL 52.1; HDL 23.9; HDW 23.7; SNT 9.7; EYE 6.4; IOD 7.0; IND 3.8; SHK 20.2; TGH 24.8; LAL 17.7; HND 12.7; FTL 20.8; IML 2.7; IMW 1.5.

Description of tadpole. Based on largest individual in series of 11 topotypic tadpoles MVZ 258314 ( Figure 10 ). Gosner Stage 26, TL 62.9 mm; BL 23.8 mm; TAL 39.1 mm; MTH 11.9 mm; TMH 6.5 mm; TMW 6.0 mm; IOD 5.2 mm. Body oval in dorsal view, slightly compressed dorsoventrally, maximum body width midway between level of eye and anterior margin of spiracle. Nares dorsal, with slightly raised rim. Eyes dorsolateral, not visible from below. Spiracular tube single, sinistral on left side, angled slightly dorsally, aperture near midline and projecting posteriorly, approximately midway between snout and end of body. Vent tube dextral on right side of ventral fin. Tail musculature strong, level in proximal one-half, tapering in distal one-half, nearly reaching tail tip. Dorsal and ventral fin origins at end of body, widest near middle of tail, dorsal fin only slightly deeper than ventral fin. Oral disk ventral, subterminal, width subequal to TMW. Papillae moderately large, weakly conical, mostly homogeneous in length. Anterior labium bordered by single row of papillae, with short medial gap approximately two papillae in width bordering A-1. Posterior labium bordered by two rows of papillae, except single row bordering P-6. Labial tooth row formula 7(2–7)/6(1–5). A-1 and P-6 relatively short, A-1 approximately one-half length of P-6. Upper and lower jaw sheaths black with coarsely serrated margins, dark brown to black in colouration. In preservative, dorsal and lateral surfaces of body and tail muscle light brown (Cinnamon 21), belly and fins light grey (Pale Neutral Gray 296), with small, scattered, indistinct, dark brown (Verona Brown 37) spots on all surfaces except belly.

Variation. Scleral arc colouration in life varies from scarlet (Geranium 66) to yellow (Cream Colour 12) among individuals in syntopy ( Figure 8 (c,d)), but this variation is not explained by day versus night observation, sex or reproductive status ( Strautins et al. 2014). Dorsal colouration varies from brownish grey (Sepia 279) to dark grey (Dusky Brown 285), and darker markings on the dorsum, if present, are variable in size and shape. Some specimens (MVZ 258182–83, NCSM 79657 and NCSM 79659) have a uniform dark grey dorsum with few or no dark markings. Some specimens lack dark brown (Dusky Brown 285) banding on limbs. Chin and ventral colouration light brown (Greyish Horn Colour 268) or brown (Hair Brown 277) in some specimens. Some paratypes have mostly uniform ventral colouration and lack an off- white (Pale Buff 1) region in centre of belly.

Females (n = 13) are significantly larger than males (n = 34) in all 14 measurements (P <0.01 for IOD, P <0.001 for 13 other variables). Measurements are summarised in Table 5.

Some tadpoles have only six tooth rows on the anterior labium and five on the posterior labium. Tadpoles also vary in the amount of dark brown (Verona Brown 37) spots on the body and tail.

Advertisement call. Based on the calls of 10 individuals recorded at Virachey National Park ( Cambodia) and Ngoc Linh Nature Reserve ( Vietnam) at ambient temperatures of 20.3–24.0°C ( Table 6; Supplementary Appendix SIV). The call invariably contains a single, highly pulsed note lasting 0.06– 0.17 s, repeated at a variable interval (0.2– 44.0 s). Within a call, notes were relatively evenly spaced and each call contained 9–24 pulses repeated at a near-constant rate across the call. Relative amplitude varied symmetrically within each note, gradually rising over the first half of each note, reaching peak amplitude typically around 60% of the call duration and then declining gradually ( Figure 6 (b); Table 6). The dominant frequency of calls varied from 0.9 to 1.7 kHz. A low-frequency band was present at around 0.2 kHz, and a weak harmonic was present at approximately 3.0 kHz. Temporal and spectral properties of the call did not vary greatly with temperature in the 10 specimens recorded. To the human ear, the call sounds like a deep ‘waaaah’.

Distribution, natural history and conservation. Leptobrachium lunatum sp. nov. is known only from the Central Highlands (= Kon Tum Plateau) of north-eastern Cambodia and central Vietnam ( Figure 11 ). Documented locality records include Veunsai and Ta Veng Districts, Ratanakiri Province, Cambodia (this study); Dak Glei District, Kon Tum Province, Vietnam ( Strautins et al. 2014; this study); Kon Plong District, Kon Tum Province, Vietnam ( Matsui et al. 2010; Hamidy et al. 2012; Pawangkhanant et al. 2018); Mang Yang District, Gia Lai Province, Vietnam (this study); and Tra My District, Quang Nam Province, Vietnam ( Bain and Nguyen 2006; Nguyen et al. 2009). The species is found in hill and wet evergreen forest ( Figure 12 ) between 600–2105 m elevations (this study; Bain and Nguyen 2006). Males were observed calling from shallow burrows or under leaf litter near streams in Cambodia during June and October, and in Vietnam during March, April, August, September and October. MVZ 258181–82 were found in Cambodia in amplexus in shallow water in the middle of a small rocky stream on 7 October 2007. AMS R176306–07 from Gia Lai Province, Vietnam, were found in amplexus on small rocks at the base of a 2-m-wide stream pool on 21 August 2011. Tadpoles were collected at night in small stream pools with rocky or sandy substrate. The known geographic distribution of L. lunatum sp. nov. resembles that of Leptobrachella isos ( Rowley, Stuart, Neang, Hoang, Dau, Nguyen and Emmett, 2015) . Leptobrachium lunatum sp. nov. shares a type locality with Leptobrachella melica ( Rowley, Stuart, Neang and Emmett, 2010) .

Like L. pullum and L. mouhoti , the new species is dependent on forest and therefore likely to be negatively impacted by ongoing deforestation in the region (e.g. Sodhi et al. 2009). We also observed tadpoles of L. lunatum sp. nov. being harvested for human consumption at Ngoc Linh Nature Reserve. At present, the only species in the group to have had its global conservation status assessed is L. pullum , which is currently considered Least Concern ( IUCN SSC 2017). However, our findings restrict the geographic distribution of L. pullum to upper elevations of the Langbian Plateau, and this reduction in range size warrants reassessment of the conservation status of all three species.

Comparisons. Leptobrachium pullum , L. mouhoti and L. smithi are the only other Leptobrachium species in Indochina ( Vietnam, Laos and Cambodia) that have reddish colouration on the iris. Leptobrachium lunatum sp. nov. differs from L. pullum by having a variably coloured scarlet, orange or yellowish scleral arc (vs always scarlet in L. pullum ); the venter greyish or brownish in colouration with minute light spots on tubercles (vs venter white with distinct black spots in pullum ); males with longer hind limbs (SHK, TGH, P <0.001; Table 5); males with longer forearms (LAL, P <0.05; Table 5); and males with fewer (9–24) pulses per call (vs 24–44 in pullum ; Table 6; Supplementary Appendix SIV).

Leptobrachium lunatum sp. nov. differs from L. mouhoti by having smaller males, with SVL 38.5–54.9 mm, mean 45.7 mm (vs 51.3–64.7 mm, mean 56.8 mm in mouhoti ); males with shorter heads (HDL, P <0.001; Table 5); males with shorter hind limbs (SHK, TGH, P <0.001; Table 5); males with shorter forearms (LAL, P <0.001; Table 5); and males with smaller inner metatarsal tubercles (IML, IMW, P <0.001; Table 5).

Leptobrachium lunatum sp. nov. is readily distinguished from L. smithi , to which it is phylogenetically unrelated ( Figures 2 and 3 ), by having the reddish colouration of the iris restricted to a scleral arc under the palpebrum (vs orange or scarlet on the upper onethird of iris and blue on posterior margin of iris in smithi ), and by lacking large, irregular darker markings on the dorsum (present in smithi ).

Figures

Figure 3. Fifty percent majority-rule consensus phylogram resulting from partitioned Bayesian analysis of 7676 aligned characters of 11 nuclear genes from megophryid frogs in the genus Leptobrachium. Trees were rooted with the outgroup Ophryophryne synoria (not shown). Black circles at nodes indicate Bayesian posterior probabilities ≥ 0.99, open circles at nodes indicate Bayesian posterior probabilities ≥ 0.95, and numbers at nodes indicate Bayesian posterior probabilities <0.95. Numbers at terminal tips are voucher numbers. Locality data for sequenced samples are provided in Table 1.

Figure 7. (a) Lateral and (b) frontal views of the adult male holotype of Leptobrachium lunatum sp. nov. (MVZ 258179) in life. Photographs by Piotr Naskrecki.

Figure 8. (a) Dorsal, (b) ventral, and (c) ocular views of the adult male holotype of Leptobrachium lunatum sp. nov. (MVZ 258179) in life. (d) Ocular view of an adult female paratype of Leptobrachium lunatum sp. nov. (AMS R173441) in life.

Figure 9. (a) Dorsal and (b) ventral views of the adult male holotype of Leptobrachium lunatum sp.nov. (MVZ 258179) in preservative.

Figure 10. Exemplar topotypic tadpole in preservative of Leptobrachium lunatum sp. nov. (MVZ 258314) at Gosner Stage 26, TL 62.9 mm in (a) oral (insect pin visible in lower centre), (b) lateral, (c) dorsal, and (d) ventral views.

Figure 11. Map illustrating the holotype (blue star) and paratype localities (blue circles) of Leptobrachium lunatum sp. nov., the holotype (yellow star) and studied localities (yellow circles) of Leptobrachium mouhoti, and the holotype (red star) and studied localities (red circles) of Leptobrachium pullum.

Figure 12. Habitat of Leptobrachium lunatum sp. nov. at (a) the type locality in hill evergreen forest (background) as viewed from Veal Thom Grasslands (foreground) in Virachey National Park, Ratanakiri Province, Cambodia, and (b–d) paratype localities in wet evergreen forest at Ngoc Linh Nature Reserve, Kon Tum Province, Vietnam. (d) Shows the results of a destructive flood in 2010.