A new species of Leptolalax (Anura: Megophryidae) from central Vietnam Author Rowley, Jodi J. L. Author Hoang, Huy Duc Author Dau, Vinh Quang Author Le, Duong Thi Thuy Author Cao, Trung Tien text Zootaxa 2012 3321 56 68 journal article 10.5281/zenodo.215101 b87bd712-6625-473a-8cce-db82db29dece 1175-5326 215101 Leptolalax firthi sp. nov. Holotype : AMS R 176524, adult male, calling on fern 0.1 m from slightly disturbed, 1–3 m wide, rocky stream with small cascades in montane evergreen forest in Ngoc Linh Nature Reserve, Kon Tum Province, Vietnam ( 15.0628º N , 107.8587º E , 1716 m , Figures 1 , 2 & 3 ). Collected at 20:00 h on 2 April 2010 by Jodi J. L. Rowley, Huy Duc Hoang, Duong Thi Thuy Le, and Vinh Quang Dau. Paratypes : AMS R 176525 (adult male) calling on rock at 20:05 h, and UNS 00460/ AMS R 176526 (adult male), collected at same geographic locality and date as holotype . UNS 00461/ AMS R 176527 (adult male) collected on tree root 0.3 m from 1 m wide, shallow rocky stream at 21:30 h on 4 April 2010 at same geographic locality as holotype . AMS R 176513 (adult male), calling in shallow water under 0.4 m diameter rock; UNS 00456/ AMS R 176514 (adult male) calling on gravel in stream bed; and AMS R 176515 (adult female, gravid); all collected at ~19:00 h on 27 March 2010 in a 1.5 m wide stream with medium flow and low gradient in montane evergreen forest in Ngoc Linh Nature Reserve, Kon Tum Province, Vietnam ( 15.0323º N , 107.8153º E , 1722 m , Figure 1 ). NCSM 78995, AMS R 176503–176507, UNS 00453/ AMS R 176509, UNS 00454/ AMS R 176510, and UNS 00455/ AMS R 176511 ( 9 adult males) and AMS R 176500, UNS 00452/ AMS R 176501, AMS R 176502, AMS R 176508, and AMS R 1765012 ( 5 adult females) collected at night in a large rocky stream with no riparian vegetation (due to storm damage) in montane evergreen forest in Ngoc Linh Nature Reserve, Kon Tum Province, Vietnam ( 15.0407º N , 107.8165º E , 1494 m , Figure 1 ) on 26 March 2010 . AMS R 176517, NCSM 78996–78997, AMS R 176518 ( 4 adult males) and UNS 00457/ AMS R 176516 (adult female), collected at night in large rocky stream with no riparian vegetation (due to storm damage) in montane evergreen forest in Ngoc Linh Nature Reserve, Kon Tum Province, Vietnam ( 15.0407º N , 107.8165º E , 1494 m , Figure 1 ) on 28 March 2010 . AMS R 176520, AMS R 176522 ( 2 adult males, calling) and UNS 00458/ AMS R 176519, AMS R 176521, UNS 00459/ AMS R 176523 ( 3 adult females) collected at night in large rocky stream with no riparian vegetation (due to storm damage) in montane evergreen forest in Ngoc Linh Nature Reserve, Kon Tum Province, Vietnam ( 15.0407º N , 107.8165º E , 1494 m , Figure 1 ) on 30 March 2010 . AMS R 173736 (adult female), collected at 21:05 h in 3 m wide, shallow, rocky stream in montane evergreen forest in Ngoc Linh Nature Reserve, Kon Tum Province, Vietnam ( 15.2359º N , 107.7098º E , 1316 m , Figure 1 ) on 12 July 2009 . AMS R 173774 (sub-adult female), collected in 3 m wide, shallow, rocky stream in montane evergreen forest in Ngoc Linh Nature Reserve, Kon Tum Province, Vietnam ( 15.2359º N , 107.7098º E , 1316 m , Figure 1 ) on 13 July 2009 . AMS R 171722 (adult female), collected from medium gradient, narrow, rocky stream in montane evergreen forest in Song Thanh Nature Reserve, Quang Nam Province, Vietnam ( 15.3293º N , 107.7043º E , 863 m , Figure 1 ) at 18:30 h on 26 July 2007 . AMS R 171714 (adult female), collected on leaf-litter 4 m from wide, low gradient, rocky stream in montane evergreen forest in Song Thanh Nature Reserve, Quang Nam Province, Vietnam ( 15.2681º N , 107.7546º E , 1125 m , Figure 1 ) at 19:20 h on 22 July 2007 . Specimens from 2009–2010 were collected by Jodi J. L. Rowley, Huy Duc Hoang, Duong Thi Thuy Le, Vinh Quang Dau. Specimens from 2007 were collected by Jodi J. L. Rowley, and Trung Tien Cao. Etymology : Specific epithet is a patronym honouring Denys Firth, for his support of amphibian biodiversity conservation and scientific capacity building in Asia. FIGURE 2. Adult male holotype (AMS R 176524) of L. firthi sp. nov . in life. (A) in situ , frontodorsolateral view; (B) dorsolateral view; (C) dorsal view; (D) ventral view. FIGURE 3. (A) Adult male holotype of L. firthi sp. nov . AMS R 176524 in preservative (i) dorsal view, (ii) ventral view, (iii) ventral view of left hand showing lateral dermal fringes on Finger II, and (iv) ventral view of left foot showing wide lateral dermal fringes on all toes. (B) Adult female paratype of L. firthi sp. nov . AMS R 176515 in preservative (i) dorsal view, (ii) ventral view, (iii) ventral view of left hand with Finger II lacking lateral dermal fringes, and (iv) ventral view of left foot showing poorly-developed lateral dermal fringes on all toes. Diagnosis : Assigned to the genus Leptolalax on the basis of the following: small size, rounded finger tips, the presence of an elevated inner palmar tubercle not continuous to the thumb, presence of macroglands on body (including supra-axillary, pectoral, femoral and ventrolateral glands), vomerine teeth absent, tubercles on eyelids, anterior tip of snout with pale vertical bar ( Dubois 1983 ; Lathrop et al . 1998 ; Delorme et al . 2006 ). Leptolalax firthi is distinguished from its congeners by a combination of (1) an absence of distinct dark brown/black dorsolateral markings, (2) toes with rudimentary webbing, (3) toes with wide lateral dermal fringes in males and weak or absent lateral dermal fringes in females, (4) most males with wide lateral dermal fringes on Finger II, (5) medium size for the genus ( 26.4–29.2 mm in 21 adult males, 25.7–36.9 mm in 14 females ), (6) near immaculate white chest and belly, and (7) a call consisting of 2–5 notes with a dominant frequency of 5.4–6.6 kHz (at 18.3–21.2º C). Description of holotype : Head slightly wider than long; snout rounded with slight point in dorsal view and rounded but relatively truncate in profile, projecting slightly beyond margin of the lower jaw; nostril closer to tip of snout than eye; canthus rostralis distinct, bluntly rounded; lores slightly concave; vertical pupil; eye diameter smaller than snout length; tympanum distinct, round, diameter smaller than that of the eye; tympanic rim elevated relative to skin of temporal region; vomerine teeth absent; pineal ocellus absent; vocal sac openings slit-like, located posteriolaterally on floor of mouth; tongue long, wide, with deep notch at posterior tip; supratympanic ridge distinct, running from eye towards axillary gland, with raised tubercles. Tips of fingers rounded, slightly swollen; relative finger lengths I <II = IV <III; nuptial pad absent; subarticular tubercles absent; a large, round inner palmar tubercle distinctly separated from small, laterally compressed outer palmar tubercle; no finger webbing, moderate lateral dermal fringes on Finger II only ( Figure 3 Aiii). Tips of toes like fingers; relative toe length I <II <V <III <IV; subarticular tubercles absent, replaced by dermal ridges, distinct on toes II–V; large, oval inner metatarsal tubercle pronounced, outer metatarsal tubercle absent; basal webbing; wide lateral fringes ( Figure 3 Aiv). Tibia 50% of snout-vent length; tibiotarsal articulation reaches anterior margin of eye. Skin on dorsum shagreened, with fine, round, relatively evenly scattered tubercles; ventral skin smooth; pectoral gland oval, 1.0 mm diameter, relatively indistinct in preservative; femoral gland oval, approximately 1.2 mm diameter, on posteroventral surface of thigh, closer to knee than to vent; supra-axillary gland raised, 1.1 mm diameter. Ventrolateral glands present, dorsolaterally compressed, forming an incomplete line. FIGURE 4. Paratypes of L. firthi sp. nov . in life. (A) adult male AMS R 176506, (B) adult female AMS R 176502, (C) adult female AMS R 176515, (D) adult female UNS 00458/AMS R 176519, (E) adult female AMS R 173736, and (F) sub-adult female AMS R 173774. Colour of holotype in life: Dorsal surface brown with no distinct darker markings, only faint transverse brownish grey bars on the dorsal surface of the thighs, tibia, tarsus, lower arms, fingers and toes; tiny white flecks scattered on back ( Figure 2 ). The dorsolateral surfaces of body, fingers, toes and elbow to upper arm are pale copper. Ventral surfaces of chest and belly opaque creamy white; throat transparent pink with brown dusting along anterior margin. Ventral surface of arms transparent pink with brown dusting along lateral margins; ventral surface of thighs, tibia, and tarsus brownish pink with faint cream flecking. Supra-axillary gland pale copper; pectoral glands opaque cream; femoral glands pale copper, lined with dark brown. Iris bright gold, slightly coppery orange in upper half; minute, black reticulations throughout. Iris periphery lined with black. Sclera white. Colour of holotype in preservative: Dorsum dark brown with slightly paler limbs. Banding on limbs and white flecks on dorsum are more pronounced ( Figure 3 A). Ventral surface of chest, belly, throat, interior portions of arms and thighs are creamy white. Macrogrands are creamy white. Measurements : Holotype : SVL 28.9, HDL 9.6, HDW 11.0, SNT 4.3, EYE 3.6, IOD 3.5, TMP 1.6, TEY 1.4, TIB 14.5, ML 8.4, PL 13.8, weight 1.95 g FIGURE 5. (A) Advertisement call of Leptolalax firthi sp. nov. holotype AMS R 176524 (i) 30 s waveform of relative amplitude (Rel. amp.) over time for 23 calls, (ii) waveform and (iii) corresponding spectrogram of single representative call containing two notes, expanded from section shown in i, and (iv) power spectrum (relative amplitude vs. frequency) of first note; all recorded at an ambient air temperature of 20.2º C. (B) Representative single advertisement calls of (i) paratype AMS R 176522 recorded at an ambient air temperature of 18.8º C, (ii) paratype AMS R 176520 recorded at an ambient air temperature of 19.3º C, and (iii) paratype AMS R 176513 recorded at ambient air temperature of 21.2º C. Variation : Measurements of the type series are shown in Table 1 and representative photographs of paratypes (in life) are shown in Figure 4 . Specimens vary in the extent of white speckling on the dorsum; male AMS R 176518 and to a lesser extent male AMS R 176517 and females AMS R 176515 and UNS 00459/AMS R 176523 are highly speckled; two males (AMS R 176504 and UNS 00453/AMS R 176509), and three females (AMS R 171714, AMS R 173774, and AMS R 171722) lack white specks on the dorsum entirely. In most specimens, iris colour was relatively uniformly gold or coppery gold, with minute black reticulations throughout; in some specimens (eg. UNS 00456/AMS R 176514, UNS 00458/AMS R 176519 [ Figure 4 D], AMS R 176521, AMS R 176524), the upper third to half of the iris was slightly orange or reddish orange in life. A faint silvery grey horizontal band along the middle of the iris was also indistinctly present in some specimens in life (eg. AMS R 176515, UNS 00458/AMS R 176519). Females AMS R 171722 and AMS R 173736 have more obvious limb banding and darker brown dorsal botches although patterning is still indistinct. Males vary in the extent of the lateral dermal fringes on Finger II, with AMS R 176522, NCSM 78995 and NCSM 78997 lacking distinct lateral dermal fringes on Finger II (two of which were observed calling, suggesting the degree of fringing may not reflect reproductive condition). Females are generally larger and are heavier in life (all measured males were < 2 g and females were> 2 g ). Females have, at most, weak lateral dermal fringes on the toes and all lack lateral dermal fringes on Finger II (Figure 3). TABLE 1 . Measurements (mm) of Leptolalax firthi sp. nov. Abbreviations defined in text. S. D. = standard deviation. Measurements Males Females Range; Mean ± S. D. (N=21) Range; Mean ± S. D. (N=14) SVL 26.4–29.2; 27.8 ± 0.9 25.7–36.9; 33.1 ± 2.6 HDL 9.6–12.5; 11.1 ± 0.6 11.9–14.6; 13.6 ± 1.0 HDW 10.4–11.8; 11.1 ± 0.3 10.3–14.2; 13.0 ± 1.0 SNT 3.5–4.6; 4.2 ± 0.3 4.0–5.3; 4.9 ± 0.3 EYE 3.3–4.1; 3.7 ± 0.2 3.0–4.1; 3.6 ± 0.5 IOD 2.8–3.7; 3.2 ± 0.2 3.0–4.1; 3.6 ± 0.3 TMP 1.54–2.2; 1.7 ± 0.2 1.4–2.4; 2.1 ± 0.2 TEY 0.8–1.4; 1.2 ± 0.2 1.1–3.3; 1.6 ± 0.5 TIB 12.9–14.5; 13.7 ± 0.5 13.7–17.2; 16.3 ± 0.9 ML 7.0–9.0; 7.8 ± 0.5 7.1–9.6; 8.9 ± 0.6 PL 11.0–13.8; 13.0 ± 0.7 11.7–16.0; 14.6 ± 0.6 Range; Median (N=21) Range; Median (N=14) HDL:HDW 0.87–1.11; 1.02 0.91–1.09; 1.05 HDL:SVL 0.33–0.43; 0.40 0.35–0.44; 0.41 TIB:SVL 0.47–0.51; 0.50 0.46–0.53; 0.49 Range; Mean ± S. D. (N=19) Range; Mean ± S. D. (N=12) Weight in life (g) 1.5–1.9; 1.8 ± 0.1 2.2–3.4; 2.9 ± 0.6 Advertisement call : Call descriptions are based on the calls of the holotype , recorded at 20.2º C ambient temperature. Calls were an average of 24 ms in duration and consisted of two notes ( Table 2 , Figure 5 A). Calls were highly amplitude modulated, with amplitude peaking at the start of the call and the start of each note. Notes generally contained two, rather indistinct pulses, with less distinct pulses also present ( Figure 5 Aii). The dominant frequency was 5.6–6.4 kHz, and harmonics were present at approximately 12, 18, 24 and 31 kHz. A fundamental frequency was present at approximately 0.2 kHz. Calls were repeated at a rate of approximately 0.9 calls per second, and had an average intercall interval of 1.1 s . TABLE 2. Measurements of advertisement call parameters for Leptolalax firthi sp. nov. Parameter values are given as means (and ranges). AMS R AMS R AMS R NCSM AMS R AMS R 176524* 176504 176513 78996 176520 176522 In the calls of the six individuals recorded, call repetition rate, intercall interval, the number of notes per call, the number and distinctiveness of pulses within notes and dominant frequency varied both within and among individuals ( Table 2 , Figure 5 ). Most noteworthy, while the number of notes per call was invariably two in the recording of the holotype , the number of notes per call in the recordings of paratypes ranged from 2–5, and was invariably three for two paratypes ( Table 2 ). Dominant frequency also varied within and between individuals (over <3 ºC difference in ambient temperature), from 5.4–6.6 kHz. To the human ear, the advertisement call of L. firthi is a rapid, high-pitched chirping, similar to an orthopteran.

Number of calls measured	10	10	10	10	10	10
Number of notes	20	28	30	45	30	34
Call duration (ms)	21 (18 24)	46 (40 51)	41 (39 42)	35 (32 36)	34 (31 37)	38 (35 42)
Call repetition rate (calls/s) Intercall interval (ms) Notes/call	0.86 1140 (1098 1221) 2	0.72 1346 (1075 1625) 2.8 (2 3)	0.97 920 (327 1184) 3	0.58 1674 (1261 2285) 4.5 (4 5)	0.68 1431 (1187 1716) 3	0.63 1545 (1401 2034) 3.4 (3 4)
Dominant frequency (kHz)	5.8 (5.6 6.4)	6.2 (5.8 6.6)	6.3 (6.2 6.6)	5.7 (5.4 5.8)	6.2	5.8
Temperature (°C)	20.2	20.4	21.2	18.3	19.3	18.8
*holotype

Sequence divergence : Uncorrected sequence divergences between L. firthi 16S rRNA sequences and all homologous sequences available on GenBank (sequences assigned to 16 species; Appendix II) were>10%. This degree of pairwise divergence in the 16S rRNA gene is greater than that usually representing differentiation at the species level in frogs ( Vences et al. 2005 ). Intraspecific variation in this gene fragment for L. firthi sampled from sites up to 27 km apart was <0.8%. FIGURE 6. Habitat of Leptolalax firthi sp. nov. (A) Habitat at ~900 m elevation in Song Thanh Proposed Nature Reserve, Quang Nam Province. (B) Slightly storm-damaged habitat at ~1700 m elevation, and (C–D) heavily storm-damaged habitat at ~1300–1500 m in Ngoc Linh Nature Reserve, Kon Tum Province. Ecology: All specimens of the new species were found in montane evergreen forest between ~ 860–1720 m elevation ( Figure 6 ). During wet-season surveys ( July 2007 and 2009), only females of the new species were observed, generally from within the forest, away from streams. During these surveys, females were rarely encountered (4 individuals over 19 survey nights), and no males were ever observed or heard calling. In contrast, hundreds of males were observed and heard calling at the end of the dry season in March/ April 2010 . Females were also observed at this time, but far less abundant (and/or detectable) than males. Males were observed calling on the banks of medium sized (~ 1–5 m wide), rocky streams, most of which had been highly disturbed by recent flooding (riparian vegetation was largely absent, with bare rocks for many meters at either side of most streams). The new species occurs in syntopy with at least three other species of Leptolalax : L. applebyi , L. croceus and L. tuberosus . Conservation status : Leptolalax firthi is known from a number of sites in Song Thanh Nature Reserve in Quang Nam Province, and the adjoining Ngoc Linh Nature Reserve in Kon Tum Province. The farthest distance between two collection localities is approximately 35 km . The actual distribution of the new species is unknown but probably extends to adjacent forested areas in the Kon Tum Plateau, possibly including Dong Am Pham National Biodiversity Conservation Area in Laos . Given the available information, we suggest the species should be considered Data Deficient following IUCN’s Red List categories ( IUCN 2001 ). Comparisons : In lacking distinct dorsolateral markings including blackish spots on the flank and dark canthal and/or temporal streaks, L. firthi is distinguished from all but L. aereus , L. arayai , L. croceus , L. eos and L. tuberosus ( L. alpinis , L. applebyi , L. bidoupensis , L. bourreti , L. dringi , L. fulignosus , L. gracilis , L. hamidi , L. heteropus , L. kajangensis , L. kecil , L. khasiorum , L. lateralis , L. liui , L. maurus , L. melanoleucus , L. melicus , L. minimus , L. nahangensis , L. nyx , L. oshanensis , L. pelodytoides , L. pictus , L. pluvialis , L. solus , L. sungi , L. tamdil and L. ventripunctatus all have blackish spots on the flank, and dark canthal and/or dark temporal streaks). From L. aereus , L. firthi can be distinguished molecularly, by the male advertisement call and by the degree of lateral fringing on the toes (see below); from L. arayai , L. firthi can be distinguished molecularly, by the degree of lateral fringing on toes, and by the male advertisement call (see below); from L. croceus , the new species can be distinguished molecularly, by ventral coloration, degree of lateral fringing on toes, visibility of the tympanum, and by the male advertisement call (see below); from L. eos , L. firthi can be distinguished molecularly and by size (see below); and from L. tuberosus , the new species can be distinguished molecularly, by ventral pattern, degree of lateral fringing on toes, visibility of the tympanum, and by the male advertisement call (see below) In having toes with broad lateral fringing in males, L. firthi is further distinguished from all but L. alpinus L. eos , and L. liui (all other male congeners lack fringing or have only weak fringing on their toes. In addition, topotypic adult male L. liui examined [N = 3] have considerably less developed fringing compared to L. firthi ). In having males with wide lateral dermal fringes on Finger II only, L. firthi appears to differ from all congeners. Leptolalax firthi is a medium-sized species of Leptolalax ( 26.4–29.2 mm in 21 adult males, 25.7–36.9 mm in 14 females ), and can be distinguished on the basis of size from the smaller L. applebyi (males 19.6–22.3 mm , females 21.7–25.9 mm ), L. kecil (males 19.3–20.5 mm , female 25 mm ), L. melicus (males 19.5–22.7 mm ) and L. pluvialis (males 21.3–22.3 mm ), and the larger L. bourreti (male 36.2 mm ), L. eos (males 33.1–34.7 mm ), L. gracilis (males 30–36 mm ), L. kajangensis (males 34–35 mm ), L. nahangensis (male 40.8 mm ), L. sungi (males 48.3– 52.7 mm , females 56.7–58.9), and L. tamdil (male 32.3 mm , female 31.8 mm ). In having an immaculate white chest and belly with only slight brown specking at the margins, L. firthi can be distinguished from L. alpinis , L. applebyi , L. bidoupensis , L. croceus , L. dringi , L. fulignosus , L. gracilis , L. heteropus , L. kajangensis , L. kecil , L. maurus , L. melanoleucus , L. melicus , L. nahangensis , L. pluvialis , L. solus , L. tuberosus and L. ventripunctatus , all of which have dark or otherwise maculate chests and/or bellies. In having a visible tympanum, L. firthi is differentiated from L. croceus , L. sungi and L. tuberosus . The advertisement call of L. firthi is unique among congeners with known calls. In lacking strong frequency modulation, the call of L. firthi differs from L. dringi and L. hamidi . In having a relatively high dominant frequency (5.4–6.6 kHz; 18.3–21.2 ºC) the call of L. firthi can be readily distinguished from that of L. applebyi (4.0–4.3 kHz, 21.5 ºC), L. bidoupensis (1.9–3.8 kHz, 19.9–20.0 ºC), L. croceus (2.6–3.0 kHz, 21.6–25.1 ºC), L. fuliginosus (2.3– 2.4 kHz, 19.3–19.6 ºC), L. gracilis (2.5–2.7 kHz, 20 ºC), L. heteropus (2.8 kHz, 21 ºC), L. kecil (3.2 kHz, 21.4 ºC), L. melanoleucus (3.1–3.2 kHz, 23.9 ºC), L. melicus (2.6–4.0 kHz, 26.1–26.2 ºC), L. oshanensis (4.4–4.6 kHz, 14ºC; recorded from c. 40 km from type locality of L. oshanensis ), L. solus (3.1–3.2 kHz, 24.2–24.3 ºC), and L. tuberosus (2.6– 2.8 kHz, 22.5–24.5 ºC). The call of L. firthi also appears to be lower in frequency compared to that attributed to L. dringi (7.6–8.1 kHz, 24.3 ºC). Although frequency can vary with temperature, differences among species of the scale reported here are very unlikely to be attributed to temperature differences. Congeners with known calls of a similar dominant frequency are L. aereus , L. alpinus , L. arayai , L. hamidi , L maurus and L. pictus . The advertisement call of L. firthi has a lower call repetition rate (0.58–0.97 calls/s) compared to L. aereus (2.4–8.0 calls/s), L. arayai (9.0–9.3 calls/s), L. hamidi (9.0–9.3 calls/s), and L. pictus (11–13 calls/s), has a lower number of notes per call (2–5) compared to L. alpinus (9.45 2.73), and has a lower call duration (18–51 ms) compared to L. maurus (85 ms). In addition, uncorrected sequence divergences between L. firthi 16S rRNA sequences and all homologous sequences available on GenBank (from individuals assigned to 16 species; Appendix II) were>10%.