Leptolalax bidoupensis, Rowley, Jodi J. L., Le, Duong Thi Thuy, Tran, Dao Thi Anh & Hoang, Huy Duc, 2011

Rowley, Jodi J. L., Le, Duong Thi Thuy, Tran, Dao Thi Anh & Hoang, Huy Duc, 2011, A new species of Leptolalax (Anura: Megophryidae) from southern Vietnam, Zootaxa 2796, pp. 15-28 : 17-26

Leptolalax bidoupensis sp. nov.

Holotype: AMS R 173133, adult male, calling on clay bank 0.2 m from 1–4 m wide, medium-high gradient, rocky stream in montane evergreen forest in Bidoup-Nui Ba National Park, Lam Dong Province, Vietnam (12.19225º N, 108.71494º E, 1730 m, Figure 1 ). Collected at 23:55 h on 19 May 2008 by J. J. L. Rowley, Hoang D. H., Le T. T. D., and Tran T. A. D.

Paratypes: UNS 00101/AMS R 173135, adult male, calling on tree root 0.2 m above 2–5 m wide, mediumhigh gradient, rocky stream in montane evergreen forest in Bidoup-Nui Ba National Park, Lam Dong Province, Vietnam (12.19106º N, 108.71703º E, 1641 m), collected at 20:25 h on 20 May 2008. UNS 00102/AMS R 173137, metamorph, in water of swampy area adjacent to a swift, rocky stream in cloud forest in Bidoup-Nui Ba National Park, Lam Dong Province, Vietnam (12.18644º N, 108.71486º E, 1627 m), collected at 19:45 h on 18 May 2008. AMS R 173134, adult female, on clay bank 0.2 m from 1–4 m wide, medium-high gradient, rocky stream in montane evergreen forest in Bidoup-Nui Ba National Park, Lam Dong Province, Vietnam (12.19225º N, 108.71494º E, 1730 m), collected at 23:50 h on 19 May 2008, in close proximity to holotype. AMS R 173136, adult male, calling on leaf litter 0.2 m from 2–5 m wide, medium-high gradient, rocky stream in montane evergreen forest in Bidoup- Nui Ba National Park, Lam Dong Province, Vietnam (12.19106º N, 108.71703º E, 1641 m), collected at 21:40 h on 20 May 2008. NCSM 77320, adult female, in water of swampy area off swift, rocky stream in montane evergreen forest in Bidoup-Nui Ba National Park, Lam Dong Province, Vietnam (12.18644º N, 108.71486º E, 1627 m), collected at 19:50 h on 18 May 2008. NCSM 77321, adult male, sitting in upright posture (previously calling?) in leaf litter, 0.1 m from swift, rocky stream in montane evergreen forest in Bidoup-Nui Ba National Park, Lam Dong Province, Vietnam (12.18644º N, 108.71486º E, 1627 m), collected at 21:45 h on 18 May 2008. NCSM 77322, metamorph, on clay bank 0.5 m from 2–5 m wide, medium-high gradient, rocky stream in montane evergreen forest in Bidoup-Nui Ba National Park, Lam Dong Province, Vietnam (12.19106º N, 108.71703º E, 1641 m), collected at 21:30 h on 20 May 2008. All specimens were collected by J. J. L. Rowley, Hoang D. H., Le T. T. D., and Tran T. A. D.

Etymology. specific epithet is in reference to the type locality of Bidoup-Nui Ba National Park.

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 vertical white bar ( Dubois 1983; Lathrop et al. 1998; Delorme et al. 2006). Leptolalax bidoupensis is distinguished from its congeners by a combination of (1) a dark brownish red ventral surface with white speckling on entire ventral surface including throat, arms and legs, often forming distinct marbling on chest and belly, (2) small size (23.6–24.6 mm in four adult males and 29.2–29.4 mm in two adult females), (3) bicoloured iris (coppery red upper half, fading to pale silver ventrally), (4) a mostly smooth skin texture with no skin ridges, and (5) relatively short tibia (male TIB:SVL 0.44–0.46). The male advertisement call of the new species, consisting of 6–9 single-pulsed notes with a dominant frequency of 1.9–3.8 kHz, is also unique among Leptolala x species for which calls are known.

Description of holotype. Head slightly longer than wide; snout bluntly rounded in dorsal view and in profile, projecting slightly beyond margin of the lower jaw; nostril closer to tip of snout than eye; canthus rostralis distinct, gently rounded; lores sloping; 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 oval, located posteriolaterally on floor of mouth; tongue long, moderate width, with slight notch at posterior tip; raised supratympanic ridge running from eye towards axillary gland. Tips of fingers rounded, not 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 or lateral fringes. Tips of toes like fingers; relative toe length I <II <V <III <IV; subarticular tubercles absent, replaced by dermal ridges, distinct on second, third, fourth and fifth toes; large, oval inner metatarsal tubercle pronounced, outer metatarsal tubercle absent; basal webbing; weak lateral fringes. Tibia relatively short and stout, width approximately one third of length, tibia 46% of snout-vent length; tibiotarsal articulation reaches eye. Skin on dorsum mostly smooth, with fine, scattered tubercles concentrated on eyelids, ventrolateral surfaces and on upper surfaces on tibiotarsus; ventral skin smooth; pectoral gland oval, 1.2 mm diameter; femoral gland oval, 0.8 mm diameter, on posteroventral surface of thigh, closer to knee than to vent; supraaxillary gland raised, 1.2 mm diameter. Ventrolateral glands present, dorsolaterally compressed, forming an incomplete line that is difficult to distinguish from white marbling.

Males Females Metamorphs

Colour of holotype in life. Dorsal surface brown with distinct darker brown markings; V-shaped interorbital marking, W-shaped marking between axillae and inverted V-shaped, irregular marking above sacrum; fine, pale greyish-blue tubercles on upper eyelids, snout, lateral margins of dorsum and upper surfaces of legs; blackish brown patch on upper lip under eye; blackish brown line along canthus rostralis through eye, and continuing below supratympanic ridge, terminating above axilla, encompassing nare and most of tympanum; transverse dark brown bars on dorsal surface of limbs; large, black blotch on posterior flank joining with inverted V-shaped marking above sacrum and numerous, smaller black spots on sides from groin to axilla; elbow and upper arms without dark bars but with distinct copper colouration; fingers and toes with transverse barring. Dark brownish red ventral surface with white speckling on entire ventral surface including throat, arms and legs, forming distinct marbling on chest and belly; ventral margin of throat bearing broken row of slightly larger white spots. Supra-axillary gland copper; femoral glands white; pectoral glands white, lined with dark brown. Iris bicoloured, with upper half coppery red, fading to pale silver in lower half; minute, black reticulations throughout.

Colour of holotype in preservative. Dorsum dark brown with slightly paler limbs. Ventral surface brown, with white speckling and marbling. Macroglands white. Patterns on dorsal surface less distinct ( Figure 3 ).

Measurements. Holotype: SVL 23.6, HDL 9.2, HDW 8.9, SNT 3.6, EYE 2.5, IOD 2.9, TMP 1.4, TEY 1.3, TIB 10.4, ML 5.2, PL 9.9, F1L 2.0, F2L 2.3, F3L 3.8, weight 1.1g

Variation. Specimens vary slightly in colour in life. AMS R 173135 and NCSM 77320 have a paler brown dorsum compared to the holotype, and dorsal patterns lined with diffuse, paler brown. AMS R 173136 and NCSM 77321 also have paler brown dorsal surfaces, but with only indistinct, diffuse dorsal markings; in NCSM 77321 the only distinct dark markings are over the tympanum, below the supratympanic ridge and as a single patch on the flank; AMS R 173136 has larger and more numerous brownish black patches on the flanks. AMS R 173134 and UNS 00101/AMS R 173135 have more mottled dorsal patterns. The ventral surfaces of specimens in the type series vary in their degree of white speckling and in the presence or absence of white marbling on parts of the chest and belly; UNS 00101/AMS R 173135 and NCSM 77320–77321 have less intense white speckling on ventral surfaces and only slight white marbling on parts of their chest; AMS R 173133 has slightly less intense white speckling and no white marbling. Pectoral glands are more distinct in specimens with less marbling. Ventrolateral glands are most easily detected in the holotype AMS R 173133, and more widely spaced and difficult to distinguish from white ventral patterning on all other specimens in the type series. UNS 00102/AMS R 173137 and NCSM 77322 are metamorphs with incompletely reabsorbed tails (tail length 2 mm in UNS 00101/AMS R 173137 and 8 mm in NCSM 77322), almost uniformly dark brown dorsal surfaces, dark brown ventral surfaces with white speckling, and indistinct tympana. Iris colouration varies slightly in the balance of coppery red versus pale silver ( Figure 5 A– C).

Advertisement call. Call descriptions are based on the calls of the holotype, taken at 19.0 ºC ambient temperature. Calls were an average of 479 ms in duration and consisted of 6–9 (usually 7) singly pulsed notes, or clicks, of variable (1–13 ms) duration, repeated at a rate of 18–21 notes per second ( Table 2 , Figures 4 A, C). Calls were highly amplitude modulated, with amplitude peaking towards the middle of the call. The dominant frequency was 2.3–2.6 kHz, and harmonics were weak or absent. Slight frequency modulation was present within each call, with lower amplitude notes, particularly the last note, often slightly higher or lower frequency. Calls were repeated at a rate of approximately 0.2 calls per second, and had an average intercall interval of 4 s. Call repetition rate, intercall interval, and the number of notes per call varied slightly within calling bouts and among individuals ( Table 2 , Figure 4). To the human ear, the advertisement call of L. bidoupensis is a slow rasping, similar to an orthopteran, with each note distinctly discernable. The call is extremely faint and difficult to locate, even when compared to other species within the genus.

Sequence divergence. Uncorrected sequence divergences between L. bidoupensis (NCSM 77320–77321, AMS R 173133–173134) and L. applebyi (holotype AMS R 171703, paratype AMS R 171704) collected from approximately 350 km away were 9.3% at the 16S rRNA gene. Uncorrected sequence divergences between L. bidoupensis and L. melicus (MVZ 258197–258199) collected from approximately 300 km away were 9.6% at the 16S rRNA gene. This degree of pairwise divergence in the 16S rRNA gene in frogs usually represents differentiation at the species level (Vences et al. 2005), and is greater than that between L. applebyi and L. melicus (6.6%). There was no intraspecific variation in this gene fragment for L. bidoupensis , L. applebyi , and L. melicus .

Ecology. All specimens of the new species were found in montane evergreen forest between 1620–1730 m elevation. Males were observed calling on stream banks, less than 0.5 m from small (<5 m wide) rocky streams in May and July. The new species was not heard or observed during surveys in the month of March, when conditions were cooler and drier.

Conservation status. Leptolalax bidoupensis is known only from an area of approximately 1 km 2 in Bidoup Nui Ba National Park. The actual distribution of the new species is unknown but probably extends to adjacent forested areas in the Langbian Plateau, including Chu Yang Sin National Park in Dak Lak Province, and Phuoc Binh National Park in Ninh Thuan Province, both of which contain montane evergreen forest at similar elevations and continuous with Bidoup-Nui Ba National Park. Given the available information, we suggest the species should be considered Data Deficient following IUCN’s Red List categories ( IUCN 2001).

Comparisons. Leptolalax bidoupensis is distinguished from all other species in the genus except for L. applebyi by having a dark brownish red ventral surface with white speckling on entire ventral surface including throat, arms and legs, forming distinct marbling on chest and belly ( L. aereus , L. alpinis , L. arayai , L. bourreti , L. dringi , L. fuliginosus , L. gracilis , L. hamidi , L. khasiorum , L. lateralis , L. liui , L. nahangensis , L. oshanensis , L. pelodytoides , L. pictus , L. solus , L. sungi , L. tamdil and L. tuberosus have mostly white or pale grey/brown venters, with or without dark spots or mottling; L. croceus has a bright orange belly; L. melicus has an off-white to pale pink ventral surface with diffuse dark brown blotches and distinct white speckling on the chest, belly and throat; L. pluvialis has a dirty white/grey venter with dark brown/grey marbling, and uniform pale dirty white/grey throat with pale speckling only around the margins; L. melanoleucus and L. ventripunctatus display large patches of distinct black and white marbling, L. heteropus has a grey venter, speckled with black; L. maurus has a black or dark grey brown venter, with indistinct small light areas, and L. kecil has a uniformly dark venter with large, dark orange pectoral glands).

The small size of Leptolalax bidoupensis (23.6–24.6 mm in four adult males and 29.2–29.4 mm in two adult females) further distinguishes it from the larger L. arayai (male 29.6 mm), L. bourreti (male 36.2 mm, females 42– 45 mm). L. dringi (males 28.7–30.3 mm, female 37.5 mm), L. fuliginosus (males 28.2–30.0 mm), L. gracilis (males 30–36 mm), L. hamidi (males 28.7–31.3 mm, females 36.1–42.8 mm), L. heteropus (male 33 mm), L. kajangensis (males 34–35 mm), L. nahangensis (male 40.8 mm), L. pelodytoides (male ~ 30 mm, female ~ 37 mm), L. pictus (males 31–34 mm, female 47 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). The remaining, small-sized congeners may have overlapping body-sizes for either or both sexes ( L. aereus males 25.1–28.9 mm, females 27.1–38.6 mm; L. alpinis males 24.0– 26.4 mm, females 31.7–32.5 mm; L. applebyi males 19.6–20.8 mm, females 21.7 mm; L. croceus males 22.2–27.3 mm; L. kecil males 19.3–20.5 mm, female 20.5 mm; L. khasiorum males 24.5–27.3 mm, females 31.2–33.5 mm; L. lateralis males 26.9–28.3 mm, female 36 mm; L. liui males 23.0– 28.7 mm, females 23.1–28.1 mm; L. maurus male 26.1 mm, female 31.8 mm; L. melanoleucus males 26.6–28.8 mm, female 32.7 mm; L. melicus males 19.5–22.7 mm; L. oshanensis males 26.6–30.7 mm, female 31.6 mm; L. pluvialis males 21.3–22.3 mm; L. solus male 27.6 mm; L. tuberosus males 24.4–29.5 mm, female 30.2 mm; and L. ventripunctatus males 25.5–28.0 mm).

In having a bicoloured iris, with the upper half coppery red and the lower half fading to pale silver, L. bidoupensis differs from at least L. aereus , L. applebyi , L. croceus , L. kajangensis , L. kecil , L. maurus , L. melicus , L. nahangensis , L. sungi and L. tuberosus , all of which have uniform iris colouration under black reticulations ( Table 3 ; Figure 5 ). Leptolalax bidoupensis is also differentiated from L. arayai , L. croceus , L. khasiorum , L. lateralis , L. maurus , L. solus , L. tamdil , L. tuberosus and L. ventripunctatus in having mostly smooth (versus tuberculate) skin texture with no skin ridges, and from L. pictus and L. pluvialis in having relatively short tibia (male TIB:SVL 0.44– 0.46, versus 0.55–0.61 in L. pictus and 0.52–0.56 in L. pluvialis ).

Leptolalax bidoupensis can be further differentiated from the two most morphologically similar species, L. applebyi and L. melicus , by having slight lateral fringing on feet, and by being approximately 19% larger than L.

applebyi (Wilcoxin rank-sum test, W= 16, p = 0.0284; N=8) and 14% larger than L. melicus (Wilcoxin rank-sum test, W= 28, p = 0.0106; N=11). In life, L. bidoupensis also weighs more than either species ( L. bidoupensis males 1.1–1.4 g, N=4; L. applebyi males 0.8–0.9 g, N=7; L. melicus males 0.7–0.75 g, N=2).

L. arayai Yes Upper 100–120° bright red-brown, rest whitish grey to light Malkmus et al. 2002

greyish green, with black reticulations.

L. bidoupensis sp. nov. * Yes Upper half coppery red, lower half fading to pale silver. Exten- Present paper sive black reticulations.

L. bourreti ?? –

L. croceus* No Pale gold with distinct, black reticulations encircling the pupil. Rowley et al. 2010a

L. dringi Yes Upper section reddish, rest pale golden grey. Black reticula- Inger & Stuebing tions. 2005 (from Fig. 19)

L. fuliginosus* Yes Upper half reddish orange, lower half gold. Black reticulations. Matsui 2006

L. gracilis Yes Upper section reddish orange, rest pale golden grey. Black Inger & Stuebing reticulations. 2005 (from Fig. 20)

L. hamidi Yes Upper half reddish, rest pale golden grey, with black reticula- Inger & Stuebing tions. 2005 (from Fig. 21) L. khasiorum* Yes Upper third bright orange, rest yellowish cream. Black reticula- Das et al. 2010

tions.

L. tamdil* Yes Upper third bright orange, rest greyish cream, edged with black Sengupta et al. 2010

reticulations.

Species Notes/ call Call Call Variable note Pulses/note Note duration Dominant Frequency Temp ºC Source

duration repetition structure (ms) frequency modulation

(ms) rate (calls/s) (kHz)

aereus * 2–16 13 –130 2.4–8.0 Slight 1–2 3–30 6.2–7.9 No 22.4–25.7 Rowley et al. 2010c alpinis 9.45±2.73 270±69? No? 1? 3 6.7±0.1 No? 16 Xu et al. 2005 applebyi * 4–5 280–441 1.7 No 1–5 4–32 4.0–4.3 No 21.5 Rowley & Cao 2009 arayai * 2 29 9.0–9.3 Note 1 shorter 1? 5.4–5.8 Slight decrease 17.4 Matsui et al. 1997 bidoupensis * 6–9 277–543 0.1–0.2 No 1 1–13 1.9–3.8 Very slight 19–21 Present paper croceus * 1–31 (variable

4–6 164–249 0.1–0.7 Yes note type) Variable 4.6–3.0 No 21.6–25.1 Rowley et al. 2010a dringi 2–4 47 11.0–11.5 No?? 7.6–8.1 Strongdecrease 24.3 Matsui et al. 1997 fuliginosus * 6–9 51 –80 2.9 No??? 2.3–2.4 No 19.3–19.6 Matsui2006 gracilis 3–4 47 6.9–7.2 No 1?? 2.5–2.7 Slight 20.0 Matsui et al. 1997 hamidi 2–3 33 9 –9.3 Note 1 longer??? 6.7–7.3 Strong decrease 22.9 Matsui et al. 1997 heteropus 3–6 207–513? Yes Note 1: c. 20 Note 1: 80 2.8 Slight 21.0 Matsui et al. 1997

Notes 2–3: 5–6 Notes 2–3: 28

kecil * 4 110–130 c. 0.2 No? 1–2? 3.2 Very weak 18.5–21.4 Matsui et al. 2009 maurus 4 85 No 1 8 5.2 No? Sukumaran et al.

2010

melanoleucus * 3–4 40 –63 6.1 No??? 3.1–3.3 No 23.9 Matsui2006 melicus * 4–11 168–484 0.7–0.8 Yes Note 1: 8–50 Note 1: 24–132 2.6–4.0 No 26.1–26.2 Rowley et al. 2010b

Clicks: 1–7 Clicks: 2–24

oshanensis 3? c.2–3 No?? 29–67 4.4–4.6 No? 14 Jiang et al. 2002 pictus 1 30– 35 11–13 No? na 6.8–7.2 Strongdecrease 19–22 Malkmus et al. 2002 solus * 4–8 130–260 2.1–2.3 No?? 3.1–3.2 No 24.2–24.3 Matsui2006 tuberosus Indistinctly

1 54–78 0.1–0.5 No pulsed na 2.6–2.8 No 22.5–24.5 Rowley et al. 2010a * documented from type specimens.

The advertisement call of L. bidoupensis differs in terms of structure and/or frequency from all other Leptolalax with described calls ( Table 4). In addition, uncorrected sequence divergences between L. bidoupensis and all homologous 16S rRNA sequences available on GenBank (from individuals assigned to L. applebyi , L. arayai , L. bourreti , L. liui , L. melicus , L. oshanensis , L. pelodytoides , and L. pictus ) were>9%.

Figures

FIGURE 1. Collection site of Leptolalax bidoupensis sp. nov. from Bidoup-Nui Ba National Park (black star) and type localities of Leptolalax applebyi (white circle) and Leptolalax melicus (black circles).

FIGURE 3. (A) Dorsal, (B) ventral and (C) lateral view of preserved holotype (AMS R 173133) of L. bidoupensis sp. nov. Scale bars = 5 mm.

FIGURE 4. Advertisement call of Leptolalax bidoupensis sp. nov. (holotype AMS R 173133) recorded at ambient air temperature of 19.0 º C. (A) 48 s waveform of relative amplitude over time for 11 calls, (B) waveform and (C) corresponding spectrogram of single representative call containing eight notes, expanded from section shown in A, and (D) power spectrum (relative amplitude vs. frequency) of fourth note shown in B – C.

FIGURE 5. Iris colouration in life of (A) Leptolalax bidoupensis sp. nov. NCSM 77320, (B) Leptolalax bidoupensis sp. nov. AMS R 173134, (C) Leptolalax bidoupensis sp. nov. AMS R 173133, (D – E) Leptolalax applebyi, and (F) Leptolalax melicus MVZ 258199.

Tables

TABLE 2. Measurements of advertisement call parameters for Leptolalax bidoupensis sp. nov. Parameter values are given as means (and ranges).

	AMS R 173133* UNS 00101/ AMS R 173135	AMS R 173136	Non-vouchered 1 Non-vouchered 2
Number of calls	10 10	10	8 10
Number of notes	85 53	67	50 72
Call duration (ms)	479 (326 543) 320 (278 343)	334 (234 391)	394 (380 411) 401 (277 463)
Call repetition rate (calls/s)	0.22 0.17	0.13	0.14 0.1
Intercall interval (ms)	4014 (3590 5537 (4393 4521) 7430)	7071 (4984 13106)	6689 9511 (5153 8492) (7560 12284)
Notes/call	8.2 (6 9) 6.7 (6 7)	6.7 (5 8)	7 (7) 7.2 (5 8)
Note duration (ms)	8.4 (1 13) 6.3 (2 9)	8.3 (4 11)	7.9 (4 12) 7.4 (4 11)
Internote interval (ms)	55 (18 70) 49 (42 59)	49 (9 76)	56 (46 76) 55 (22 66)
Note repetition rate (notes/s)	18 21 21	14 21	18 18
Dominant frequency (kHz)	2.1(2.3 2.6) 2.1 (2.1 3.6)	2.5 (1.9 3.4)	2.0 (1.9 2.3) 2.5 (2.3 3.8)
Temperature (°C)	19.0 20.4	21.0	20.2 20.4
*holotype

TABLE 3. Iris colouration in life for species within the genus Leptolalax.

Species	Iris bi- Iris colouration coloured	Source
L. aereus *	No Bronze with minute, black reticulations.	Rowley et al. 2010c
L. alpinis	No? Copper/gold with black reticulations.	Fei et al. 2010 (from figures on p. 169)
L. applebyi *	No Coppery gold to gold with black reticulations.	Rowley & Cao 2009