Figs. 3–5.
Holotype. VNMN 2016 A.22, adult male, calling in seep in evergreen forest at Pu Hoat Nature Reserve, Nghe An Province, Vietnam (19.6983° N, 104.7373° E, 1177 m a.s.l; Fig. 1). Collected on 5 June 2010 by Jodi J L Rowley, Trung Tien Cao, Vinh Quang Dau, Huong Thu Phung, Tuan Ngoc Le, Luong Thi Nguyen, Trung Danh Hoang, Thang Thanh Le.
Paratypes. VNMN 2016 A.23, VNMN 2016 A.24, NCSM 79649, three adult males, same collection locality and date as holotype. VNMN 2016 A.26 and AMS R184850, two adult males collected in evergreen forest at Pu Hoat Nature Reserve, Nghe An Province, Vietnam (19.7044° N, 104.7401° E, 1128 m a.s.l on 6 June 2010). AMS R184851, adult male, collected in evergreen forest at Pu Hoat Nature Reserve, Nghe An Province, Vietnam (19.6986° N, 104.7317° E, 1352 m a.s.l on 7 June 2010). VNMN 2016 A.21, adult female, collected in evergreen forest at Pu Hoat Nature Reserve, Nghe An Province, Vietnam (19.7001° N, 104.7381° E, 1110 m a.s.l on 5 June 2010). AMS R184853, AMS R184854, two adult females collected in evergreen forest at Pu Hoat Nature Reserve, Nghe An Province, Vietnam (21.9156° N, 104.3608° E, ~ 1300 m a.s.l in May 2010). Specimens from June 2010 were collected by Jodi J L Rowley, Trung Tien Cao, Vinh Quang Dau, Huong Thu Phung, Tuan Ngoc Le, Luong Thi Nguyen, Trung Danh Hoang, Thang Thanh Le and specimens collected in May 2010 were collected by Vinh Quang Dau, Hoang Van Bui, Trung Quang Dau and Luong Thi Nguyen.
Etymology. specific epithet is in reference to the type locality of Pu Hoat Nature Reserve.
Diagnosis. Assigned to the genus Leptolalax on the basis of the following: small size, rounded finger tips, the presence of an elevated thenar tubercle not continuous to the thumb, presence of macroglands on body, vomerine teeth absent, tubercles on eyelids, anterior tip of snout with vertical white bar (Dubois 1980; Lathrop et al. 1998; Delorme et al. 2006). Leptolalax puhoatensis sp. nov. is distinguished from its congeners by a combination of (1) a body size range of 24.2–28.1 mm in eight adult males and 27.3–31.5 mm in three adult females, (2) males with a reddish-brown venter, often with faint white dusting and females with a pale pink venter, (3) toes with webbing basal and narrow lateral fringes, (4) iris copper in upper half and golden in lower half, and (5) a call consisting of a single note and a dominant frequency of 4.9–5.6 kHz (at 22.3–25.8° C).
Description of holotype. Head wider than long; snout bluntly rounded in profile and obtusely pointed in dorsal view, projecting slightly over lower jaw; nostril closer to tip of snout than eye; canthus rostralis rounded, indistinct; lores sloping, slightly concave; vertical pupil; diameter of eye 78% length of snout; tympanum distinct, round, diameter approximately 62% that of the eye; vomerine teeth absent; pineal ocellus absent; large oval vocal sac openings present, located on either side of floor of mouth; tongue large, moderately broad, with wide notch at tip; distinct, raised supratympanic ridge running from corner of eye to axillary gland. Tips of fingers rounded, slightly wider than width of fingers; relative finger lengths I <II <IV <III; nuptial pad absent; subarticular tubercles absent; a large, inner palmar tubercle distinctly separated from slightly smaller, laterally compressed outer palmar tubercle; no finger webbing; slight lateral fringes on Finger II. Tips of toes like slightly narrow than toes; relative toe length I <II <V <III <IV; subarticular tubercles absent, replaced by dermal ridges, distinct on second, third and fourth toes; oval inner metatarsal tubercle pronounced, outer metatarsal tubercle absent; webbing basal; narrow but distinct lateral fringes. Tibia 50% of snout-vent length; tibiotarsal articulation reaches anterior edge of eye. Skin on dorsum with tiny, indistinct, low tubercles in preserve, more distinct and forming low dorsal ridges on dorsal surface in life; ventral skin smooth; pectoral gland oval, approximately 1.1 mm diameter; femoral gland elongate, 1.2 mm diameter, on posteroventral surface of thigh, closer to knee than to vent; supra-axillary gland raised, 1.1 mm diameter. Ventrolateral glandular line present as indistinct, broken lines.
Colour of holotype in life (Figs. 3 A, 4A). Dorsum dark reddish brown with faint paler brown markings between eyes and W-shaped marking on axilla; black supratympanic fold and darker brown wash over upper half of tympanum, faint barring on upper lip, and transverse barring on dorsal surface of limbs including fingers and toes; ventral surface of elbow and upper arm without dark bars; deep coppery wash on elbows. Ventral surfaces deep reddish brown, faint white speckling on chest and belly, concentrated on chest; ventral surface of arms and legs with small whitish spots concentrated on edges. Supra-axillary gland copper, pectoral gland white, femoral glands white. Iris copper in upper half and golden in lower half with minute, black reticulations.
Colour of holotype in preservative (Fig. 5 A): Dorsum medium brown with faint paler markings between eyes and on axilla, indistinct darker barring on surface of limbs including fingers and toes. Ventral surface, including throat, brown. Margins of throat and ventrolateral surfaces of arms and thighs, and entire tibiotarsus and upper arm pale brown with white speckling; macroglands cream.
Measurements. Holotype: SVL 24.8, HDL 9.2, HDW 9.4, SNT 3.8, EYE 3.2, IOD 2.9, TMP 1.7, TEY 0.9, TIB 12.2, EN 2.1, IN 2.6, NS 2.0, ML 7.3, PL 12.3, F1 2.3, F2 3.2, F3 5.6. Weight in life 1.2 g.
Measurements Males Females
Range; Mean (N=8) Range; Mean (N=3)
SVL 24.2–28.1; 25.8 27.3–31.5; 29.6 HDL 9.2–10.4; 9.9 10.9–12.3; 11.8 HDW 9.4–10.0; 9.4 10.5–12.1; 11.3 SNT 3.8–4.1; 3.9 4.1–4.5; 4.3
EYE 3.2–3.9; 3.4 3.6–4.1; 3.9
IOD 2.9–3.4; 3.1 3.1–3.3; 3.2
TMP 1.7–2.0; 1.9 2.2–2.4; 2.3
TEY 0.8–1.2; 1.0 1.0–1.7; 1.4
TIB 11.6–12.8; 12.3 12.8–15.6; 12.8 EN 1.9–2.4; 2.1 2.3–2.6; 2.5
IN 2.4–2.9; 2.6 2.7–3.1; 2.8
NS 1.6–2.0; 1.8 1.7–2.1; 2.0
ML 6.8–7.7; 7.2 6.9–9.0; 7.9
PL 11.8–13.1; 12.2 12.3–15.8; 13.9 Weight (g) 1.3–1.4; 1.3 (n=8) –
Range; Median (N=8) Range; Median (N=3)
HDL:HDW 0.97–1.05; 1.02 1.02–1.06; 1.04 HDL:SVL 0.36–0.42; 0.38 0.39–0.40; 0.40 TIB:SVL 0.45–0.52; 0.47 0.45–0.49; 0.47 TMP: SVL 0.07–0.08; 0.07 0.07–0.08; 0.07 Variation. Dorsal colour in life and in preservative is relatively uniform in males but the three females display brighter colours in life and more distinct patterning (Figs. 3, 5). In life, the ventral surfaces are deep reddish-brown in males, with varying amounts of faint white dusting in most individuals, and a pale pinkish colour in females (Fig. 4). In preservative, ventral surfaces are brown in males and very pale brownish in females. On the basis of the individuals collected, females also appear slightly larger than males (male SVL 24.2–28.1mm [N=8] and female SVL 27.3–31.5mm [N=3]). Measurements of the type series are shown in Table 3.
Advertisement call. Call descriptions are based on the calls of the holotype, recorded at 22.3°C ambient temperature. Calls were an average of 9.8 ms in duration and invariably consisted of a single note. The dominant frequency was 5.2–5.6 kHz, and harmonics were present at approximately 10.9 and 16.5 kHz (Table 4, Fig. 6). A fundamental frequency was not evident. Calls were repeated at a rate of approximately ten calls per second, and had an average intercall interval of 86 ms.
VNMN 2016 NCSM 79649 VNMN 2016 AMS AMS
A.22* A.26 R184851 R184852 Primary Call duration (ms) 9.8 (6–14) 6.2 (6–7) 8.9 (8–10) 9.9 (7–11) 10.6 (8–14) call Intercall interval (ms) 86.4 (82–93) 106.7 (102–115) 102.6 (81–128) 78.8 (74–52) 85.6 (82–99) In one individual (VNMN 2016 A.26), a secondary call type was evident (Fig. 6 Biii), this call was 299 ms in duration and consisted of 41 notes of a similar frequency and lower amplitude than other calls. In the calls of the five individuals recorded, the number of notes in primary calls was invariably one. The dominant frequency varied only slightly among individuals, from 4.9–5.6 kHz (over 3.5 °C difference in ambient temperature). To the human ear, the advertisement call of L. puhoatensis sp. nov. is a very rapid, high-pitched ticking.
Ecology. All specimens were found at night in evergreen forest between ~ 1123–1352 m a.s.l. Males were observed calling on rocks, stream banks and on vegetation within or adjacent to rocky streams (Fig. 7 A–B). The species was observed in smaller streams and seeps compared to the sympatric Leptolalax eos and Leptolalax ventripunctatus (Rowley et al. unpublished data).
Conservation status. The species is known only from Pu Hoat Nature Reserve in Nghe An Province, Vietnam. Its true extent of occurrence is unknown but the species probably extends further into adjoining areas; suitable forested regions may also include adjacent forested areas in Nghe An Province as well as parts of eastern Huaphanh Province, Laos. The predicted Extent of Occurrence (EOO) for the species is 241 km 2.Given the small number (<5) of locations and the ongoing nature of habitat loss in the region (Meyfroidt & Lambin 2008), we anticipate that the species will fulfill the criteria for being listed as Endangered according to the IUCN Red List of Threatened Species (IUCN 2012).
Comparisons. Leptolalax puhoatensis sp. nov. differs from all known congeners by a combination of (1) a body size range of 24.2–28.1 mm in eight adult males and 27.3–31.5 mm in three adult females, (2) distinct dorsolateral markings including blackish spots on the flank and dark canthal and/or temporal streaks (3) males with a reddish-brown venter, often with faint white dusting, and females with a pale pink venter, (4) skin on dorsum with tiny, indistinct, low tubercles in preservative, more distinct and forming low dorsal ridges on dorsal surface in life (5) toes with webbing basal and narrow lateral fringes, (6) iris copper in upper half and golden in lower half, and (7) a call consisting of a single note of and a dominant frequency of 4.9–5.6 kHz (at 22.3–25.8° C).
Leptolalax puhoatensis sp. nov. differs morphologically from all known Leptolalax species north of the Isthmus of Kra by having males with a reddish-brown venter, often with faint white dusting, and females with a pale pink venter (Table 5). A combination of male body size, presence of distinct blackish dorsolateral markings (canthal stripe and/or lateral blotches), and dorsal skin texture further distinguishes the new species from all other known species in the region (Table 5). In particular, the new species differs from the most closely related species, L. petrops, by having a reddish-brown venter in males and a pale pink venter in females (versus an immaculate white chest and belly), distinct blackish dorsolateral markings (versus no distinct black markings on the head) and dorsal skin with longitudinal skin ridges (versus highly tuberculate skin texture with no longitudinal ridges), having a call consisting of a single note and with a dominant frequency of 4.9–5.6 kHz at 22.3–25.8° C (versus a call with an average of four notes and a dominant frequency of 5.6–6.4 kHz at 24.5–25.3° C), and by molecular divergence (6.3% at the 16S gene fragment examined).
puhoatensis sp. nov.
Species Ventral Coloration Male SVL (mm) Distinct đorsolateral markings Dorsal skin texture In addition to morphological differences (Table 5), the advertisement call of the new species further differentiates Leptolalax puhoatensis sp. nov. from all other congeners north of the Isthmus of Kra with known calls (20 of 36 species). In having a call consisting invariably of a single note, Leptolalax puhoatensis sp. nov. differs from L. aereus, L. alpinus, L. applebyi, L. ardens, L. bidoupensis, L. botsfordi, L. croceus, L. firthi, L. fuliginosus, L. isos, L. kalonensis, L. liui, L. maculosus, L. melicus, L. oshanensis, L. pallidus, L. phyrrhops, and L. tadungensis. Of the Leptolalax species in the region with known calls, only L. tuberosus has an invariably single– note call, but this call is of a longer duration (54–78 ms at 22.4–22.5° C vs 6–14 ms at 22.3–25.8° C in the new species). In addition, the dominant frequency of 4.9–5.6 kHz (at 22.3–25.8° C) further distinguishes the call of the new species from that of the higher frequency calls of L. aereus (6.2–7.9 kHz at 22.4–25.7° C), L. alpinus (6.7 kHz at 16° C) and L. isos (5.8–6.2 kHz at 22.4–22.8° C), and the lower frequency calls of L. applebyi (4.0–4.3 kHz at 21.5° C), L. ardens (3.1–3.4 kHz at 21.4–24.7° C), L. bidoupensis (1.9–3.8 kHz at 19–21° C), L. botsfordi (2.6–3.4 kHz at 14° C), L. croceus (3.0–4.3 kHz at 21.6–25.1° C), L. fuliginosus (2.1–2.8 kHz at 19.3–19.6° C), L. kalonensis (2.8 kHz at 26.4° C), L. maculosus (2.7–2.8 kHz at 23.3–24.1° C), L. melicus (2.6–4.0 kHz at 26.1–26.2° C), L. oshanensis (4.4–4.6 kHz at 14° C), L. pallidus (2.4–2.7 kHz at 14.0–21.4° C), L. pyrrhops (1.91–2.23 kHz at 25° C), L. tadungensis (2.6–3.1 kHz at 12.9–22.3° C) and L. tuberosus (2.8–2.8 kHz at 22.5–24.5° C). Calls of the sympatric congeners L. eos and L. ventripunctatus remain unknown.