A new species of Leptolalax (Anura: Megophryidae) from Vietnam and further information on Leptolalax tuberosus Author Rowley, Jodi J. L. Author Huy, Hoang Duc Author Duong, Le Thi Thuy Author Vinh, Dau Quang Author Trung, Cao Tien text Zootaxa 2010 2660 33 45 journal article 10.5281/zenodo.199018 85b1ce1c-89e6-450c-9e54-b4d051b74a84 1175-5326 199018 Leptolalax croceus sp. nov. Holotype : AMS R 173740, adult male, calling on a leaf at the edge of a 3 m wide, shallow, rocky stream in evergreen forest in Ngoc Linh Nature Reserve, Dak Glei District, Kon Tum Province, Vietnam ( 15.23587º N , 107.70982º E , 1316 m ). Collected at 21:50 h on 12 July 2009 by J. J. L. Rowley, Hoang D. H., Le T. T. D., and Dau Q. V. Paratypes : AMS R 173738, adult male, calling on a rock in the stream, collected at 19:55 h on 12 July 2009 ; AMS R 173739, adult male, collected on a fern leaf, 1 m from a 5 m wide, shallow, rocky stream on 12 July 2009 ; AMS R 173741, adult male, collected from beneath leaf litter at the edge of a stream on 12 July 2009 ; UNS 00106/AMS R 173742, adult male, collected from leaf litter on a rock on 12 July 2009 ; UNS 00107/AMS R 173743, adult male, collected from a plant stem on 12 July 2009 ; AMS R 173744, adult male, calling on leaf litter 6 m from stream, collected at 20:25 h on 12 July 2009 ; AMS R 173745, adult male, calling on leaf of plant, 0.3 m above ground, collected at 20:52 h on 12 July 2009 ; AMS R 173746, adult male, collected on 12 July 2009 ; AMS R 173747, adult male, calling under leaf litter 1 m from stream, collected at 20:50 h on 12 July 2009 ; AMS R 173748, adult male, collected on leaf of plant on 12 July 2009 ; UNS 00108/ AMS R 173750, adult male, calling on leaf litter 2 m from stream, collected at 19:35 h on 12 July 2009 ; AMS R 173751, adult male, calling on leaf litter 0.5 m from shallow pool near stream, collected at 21:55 h on 12 July 2009 ; UNS 00109/AMS R 173752, adult male, collected on 12 July 2009 ; UNS 00110/AMS R 173775, adult male, collected on 13 July 2009 ; UNS 00111/AMS R 173779, adult male, collected on 13 July 2009 . All specimens were collected at night at type locality by J. J. L. Rowley, Hoang D. H., Le T. T. D., and Dau Q. V. Etymology: specific epithet from croceus L., meaning saffron-coloured or golden, and is in reference to the bright orange colour evident in life on the ventral surface of the new species. Suggested common name: Orange-bellied leaf-litter toad (English), Cóc mày bụng cam (Vietnamese). Diagnosis: Assigned to the genus Leptolalax on the basis of the following: small size, rounded finger tips; presence of an elevated inner palmar tubercle not continuous with thumb; presence of macroglands on body (including supra-axillary, pectoral and femoral glands); vomerine teeth absent; tubercles on eyelids; anterior tip of snout with pale, vertical bar (Dubois 1980; Lathrop et al . 1998 ; Delorme et al . 2006). Leptolalax croceus is distinguished from all other Leptolalax by its bright orange belly in life and by its unique, complex advertisement call consisting of 4–6 variably pulsed notes with a dominant frequency of 2635–3000 Hz. Leptolalax croceus can be further distinguished from its congeners by a combination of (1) an indistinct tympanum, (2) highly tuberculate dorsum, (3) medium body size ( 22.2–27.3 mm in 16 adult males), (4) head slightly wider than long, (5) rudimentary webbing between toes I–III, no webbing between toes III– V, and no lateral fringing on toes, and (6) no ventrolateral glands. Description of holotype : Head length 91% of head width; snout gently truncate in profile, projecting only slightly over 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 indistinct, obscured by skin, rim not visible; vomerine teeth absent; pineal ocellus absent; vocal sac openings slit-like, located laterally on floor of mouth; tongue large, broad, with small notch at posterior tip; supratympanic ridge indistinctly visible as a series of large, connected tubercles running from eye towards axillary gland. Tips of fingers blunt, swollen, slightly broader than phalanges; finger I almost as long as finger II, relative finger lengths I <II = IV <III; nuptial pads absent; subarticular tubercles absent; a large, laterally compressed inner palmar tubercle distinctly separated from smaller, laterally compressed outer palmar tubercle; no finger webbing or lateral fringes. Tips of toes like tips of fingers; relative toe length I <II <V <III <IV; subarticular tubercles absent, replaced by dermal ridges, distinct on second, third, fourth and fifth toes; oval inner metatarsal tubercle pronounced, outer metatarsal tubercle absent; webbing rudimentary between toes I–III, absent between toes III–V; lateral fringing absent. Tibia 57% of snout-vent length; tibiotarsal articulation reaches beyond tip of snout. Skin on entire dorsum covered in tubercles of varying sizes, smaller dorsolateraly; eyelids and snout with raised, rounded tubercles; ventral skin smooth; pectoral gland distinct in life, but indistinct in preserved specimens; femoral gland oval, 0.9 mm diameter, on posteroventral surface of thigh, closer to knee than to vent; supra-axillary gland present as large tubercle, 0.5 mm diameter; ventrolateral glands absent. FIGURE 1. Collection sites of Leptolalax croceus sp. nov. (black circle) and L. tuberosus (white circle). Boundaries of Ngoc Linh Nature Reserve (hatched area) were obtained from the World Database on Protected Areas 2009. Colour of holotype in life: Dorsal surface brown with slight pale orange wash; darker, weakly defined brown interorbital bar; weakly defined, W-shaped marking between axillae; inverted V-shaped marking between sacrum; transverse darker greyish-brown bars on dorsal surface of limbs; elbow and upper arm paler brown without dark bars or distinct patterning; fingers and toes with faint transverse barring; upper two-thirds of skin over tympanum darker brown. Belly bright orange, most vivid posteriorly, fading to pale orange anteriorly and laterally, pale orange on chest; two symmetrical, paler orange longitudinal lines running along chest and belly; throat whitish grey with pale orange speckling; dark grey and pale orange speckling along margins of throat, chest and belly. Ventral surface of thighs and upper arms with a network of small pale orange spots on a background of pale pinkish grey; lateral surfaces of thighs marbled with pale orange and pinkish-grey. Ventral surface of lower arms and tibiotarsus pinkish-grey with pale pinkish-orange speckling. Supra-axillary gland orange; pectoral gland bright orange on a background of pale orange and pinkish grey marbling; femoral gland orange with a background of pinkish grey. Iris pale gold with distinct, black reticulations encircling the pupil. Colour of holotype in preservative: Dorsal surface dark brown, with less distinct markings compared to in life. Ventral surface creamy white, ventrolateral margins of chest and belly with brown speckling. Entire throat speckled with pale brown, concentrated along margins. Symmetrical, longitudinal lines running along chest and belly no longer visible. Macroglands are creamy white; pectoral glands have become indistinct in preservative. Measurements: Holotype : SVL 25.3, HDL 9.6, HDW 10.5, SNT 3.5, EYE 2.8, IOD 2.4, TIB 14.3, ML 6.3, PL 12.7, F1L 2.5, F2L 2.9, F3L 5.0. Variation: Paratypes closely resemble the holotype in appearance. Measurements of the type series are shown in Table 1 . Females of the new species are unknown. FIGURE 2. (A) Dorsolateral and (B) ventral view of male holotype of Leptolalax croceus sp. nov. (AMS R 173740) in life, and (C) dorsolateral and (B) ventral view of male L. tuberosus (AMS R 171721) in life. Advertisement call: Call descriptions are based on the calls of the holotype (AMS R 173740), recorded at 23.2ºC ambient temperature ( Table 2 , Figure 7 ). Calls were an average of 208 ms in duration. Each call consisted of 4–5 (usually 4) pulsed notes, with an approximate pulse duration of 2–3 ms. The first note of each call typically had the greatest number of pulses, the lowest amplitude, and the least amplitude modulation ( Table 2 ). Within each call, successive notes tended to increase in amplitude relative to each other, with each note approximately double the amplitude of the previous note. Within each note, there was typically a decrease in amplitude over time. The last note in each call typically displayed both the highest amplitude and the highest degree of amplitude modulation. The dominant frequency was 2625–3000 Hz, a weak fundamental frequency was present at 375 Hz, and harmonics were detected at 5625 Hz, 8250 Hz, 11625 Hz, and 14250Hz. Frequency modulation was absent. Calls were repeated at a rate of approximately 0.2 calls per second, and had an average intercall interval of 4.1 s . Call repetition rate, intercall interval, number of notes per call, and number of pulses per note varied within calling bouts and among individuals, and calls did not form distinct call groups ( Table 2 , Figure 7 ). The dominant frequency was relatively constant among individuals recorded from 21.6 ºC to 25.1 ºC. To the human ear, the advertisement call of L. croceus sounds similar to an orthopteran, with each note distinctly discernable. TABLE 1. Measurements (mm) of adult Leptolalax croceus sp. nov . Abbreviations defined in text. Measurements Range; Mean ± S. D. (N=16) SVL 22.2–27.3; 24.6 ± 1.6 HDL 8.5–10.8; 9.6 ± 0.7 HDW 9.2–11.0; 10.1 ± 0.6 SNT 3.2–3.9; 3.5 ± 0.2 EYE 2.7–3.3; 2.9 ± 0.2 IOD 2.0–2.8; 2.5 ± 0.2 TIB 12.8–15.0; 13.9 ± 0.6 ML 5.7–7.0; 6.5 ± 0.4 PL 11.5–14.8; 12.6 ± 0.8 F1L 2.4–2.8; 2.6 ± 0.2 F2L 2.6–3.1; 2.9 ± 0.2 F3L 4.5–5.3; 4.9 ± 0.3 Weight 1.2–2.1; 1.5 ± 0.3 Range; Median (N=16) TIB:SVL 0.52–0.59; 0.57 HDL:SVL 0.37–0.41; 0.40 HDL:HDW 0.90–0.99; 0.96 Ecology: Leptolalax croceus was abundant at the type locality, at approximately 1300 m elevation. Habitat at the site consisted of hilly evergreen forest ( Figure 3 A). The stream at the site was rocky, shallow and low gradient. Leptolalax croceus were only observed at night, and located almost entirely by their call. Males were observed calling up to 10 m from the stream, on and under leaf litter ( Figure 3 B) and on low (< 1 m high) vegetation ( Figure 3 C). Females were not observed. Leptolalax croceus was sympatric with, and calling at the same time as the recently described L. applebyi , however the two species appeared to differ in terms of microhabitat, with L. applebyi always found in small seeps and wet rocky areas away from the main stream channel or in stream headwaters, and often in steep terrain. At the type locality, L. croceus was found in relatively flat terrain, within 10 m from the main stream. Leptolalax croceus may also be sympatric with L. tuberosus , as this species has been recorded from only 11 km away, however the two species may be partitioned in terms of elevation, with L. tuberosus known mainly from lower elevations ( 900–1200 m , Inger et al . 1999 ; c. 850–900 m , Bain et al. 2007; 836–1401 m , Rowley & Cao 2009 ). Both L. croceus and L. tuberosus have been documented calling in the month of July, while L. applebyi has been heard calling in March and July (Rowley et al . unpublished data). The eggs and larvae of L. croceus (along with L. applebyi and L. tuberosus ) remain unknown. TABLE 2. Measurements of advertisement call parameters for Leptolalax croceus sp. nov. Parameter values are given as means (and ranges).

Number of calls recorded	AMS R AMS R 173738 173740* 7 10	AMS R 173744 10	AMS R 173745 10	AMS R 173747 10	Non- vouchered 10
Number of notes recorded	28 41	41	44	48	44
Temperature (°C)	21.6 23.2	23.4	22.2	25.1	22.5
Call duration (ms)	171 (164–178) 208 (181–248)	190 (173–214)	209 (178–229)	211 (169–249)	190 (164–239)
Call repetition rate (calls/s)	0.1 0.2	0.3	0.7	0.4	0.3
Intercall interval (s)	10.7 (6.9–14.1) 4.1 (1.1–6.6)	3.4 (0.9–7.8)	1.3 (0.4–3.7)	2.2 (0.5–6.3)	3.6 (0.7–11.1)
Notes/call	4 4.1 (4–5)	4.1 (4–5)	4.4 (4–5)	4.8 (4–6)	4.4 (4–6)
Pulses/note
Note 1	11.3 (9–14) 14.4 (6–23)	16.6 (6–31)	12.9 (4–24)	11.7 (6–19)	9.2 (2–17)
Note 2	6.3 (5–7) 4.2 (2–6)	3.1 (2–5)	3 (1–5)	4 (2–5)	4.1 (3–5)
Note 3	4.3 (4–5) 3.2 (2–4)	3 (2–4)	2.9 (2–4)	3.1 (2–4)	3.3 (3–4)
Note 4	7.7 (7–8) 9.4 (3–11)	7 (3–12)	5.6 (3–8)	3.4 (2–6)	8.4 (3–13)
Note 5	– 8	8	6 (5–8)	3.9 (2–5)	7 (3–10)
Note 6	– –	–	–	4	7
Dominant frequency (Hz)	2625 2958 (2625– 3000)	3000	3000	3000	3000
* holotype

Conservation status: Given the difficulty in detecting Leptolalax , particularly when not calling, the discovery of L. croceus from a single locality does not necessarily indicate that the species is range-restricted. To date, the new species is only known from Ngoc Linh Nature Reserve, Kon Tum Province, but it probably occurs in adjoining areas of Laos and Vietnam . The habitat of L. croceus is contiguous with similar areas of habitat in the central Truong Son range. Considering that L. croceus was found sympatric with L. applebyi , it is highly likely that the new species also occurs in the adjoining Song Thanh Proposed Nature Reserve in Quang Nam Province, the type locality of L. applebyi . Until the distribution and habitat requirements of this species are more adequately documented, we suggest the species should be considered Data Deficient following IUCN’s Red List categories ( IUCN 2001 ). Comparisons: Leptolalax croceus is unique in the genus in having a bright orange belly in life. Leptolalax 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 venters, without white speckling and with or without dark spots or mottling; L. applebyi has a dark brownish pink ventral surface with white speckling over the entire ventral surface, including tibiotarsus and both lower and upper arms; 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 grey venter with dark grey marbling, uniform pale grey throat with speckling around the border; 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 distinct orange belly of L. croceus fades to cream in preservative, making specimens less obviously distinct from L. tuberosus . In preserved specimens, the ventral surface of L. tuberosus (AMS R 171715– 171721) is more heavily speckled/streaked with dark grey and the throat, thigh and tibiotarsus have a dark grey background bearing white spots, as compared to the cream background bearing pale grey speckling in L. croceus ( Figures 4 , 6 ). FIGURE 3. (A) Habitat at type locality of L. croceus sp. nov. , and in situ calling postures of male L. croceus sp. nov. (B) AMS R 173738 and (C) AMS R 173751. In having an indistinct tympanum, L. croceus differs from all species of Leptolalax except L. sungi and L. tuberosus . In having a heavily tubercled dorsum, L. croceus differs from all species except L. arayai , L. lateralis , L. maurus , and L. tuberosus ( L. alpinis , L. applebyi , L. bourreti , L. fulignosus , L. gracilis , L. hamidi , L. heteropus , L. kajangensis , L. liui , L. melanoleucus , L. melicus , L. nahangensis , L. oshanensis , L. pelodytoides , L. pictus , and L. pluvialis have mostly smooth skin with or without skin ridges, and L. dringi , L. kecil , L. khasiorum , L. solus , L. sungi , L. tamdil , and L. ventripunctatus are more weakly tuberculated). Leptolalax croceus is a medium-sized species of Leptolalax ( 22.2–27.3 mm in 16 adult males), and can be distinguished on the basis of size from the smaller L. applebyi (males 19.6–20.8 mm ), and L. kecil (males 19.3–20.5 mm ), and the larger L. bourreti (male 36.2 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 ), and L. tamdil (male 32.3 mm ). Based upon the type series of L. croceus and seven male L. tuberosus (AMS R 171715–171721), L. croceus is also significantly smaller than L. tuberosus (male L. croceus mean SVL 24.6 mm ± 1.6 S . D., male L. tuberosus mean SVL 29.0 mm ± 1.3 S . D.; Mann-Whitney U -test, Z =3.74, p <0.001; N=23). FIGURE 4. (A) Dorsal, (B) ventral and (C) dorsolateral view of preserved holotype of L. croceus sp. nov. (AMS R 173740). Scale bar = 5 mm. Leptolalax croceus , with a head slightly wider than long, can be further distinguished from L. applebyi , L. arayai , L. fuliginosus , L. gracilis , L hamidi , L. kajangensis , L. kecil , L. liui , L. maurus , L. melanoleucus , L. melicus , L. pictus , L. pluvialis , and L. solus , all of which have heads distinctly longer than wide. In having rudimentary webbing between toes I–III, no webbing between toes III–V and no lateral fringing, L. croceus can be differentiated from L. alpinis L. kecil , L. khasiorum , L. liui , L. sungi , and L. tamdil which have some degree of lateral fringing on toes, and from L. pelodytoides , which has more extensive webbing and wide lateral fringes between toes. In lacking ventrolateral glands, L. croceus is further distinguished from L. alpinis , L. bourreti , L. fuliginosus , L. khasiorum , L. lateralis , L. liui , L. nahangensis , L. oshanensis , L. pelodytoides , L. pluvialis , L. tamdil and L. ventripunctatus . FIGURE 5. Ventral surfaces of the right hand and right foot of preserved holotype of Leptolalax croceus sp. nov. (AMS R 173740) Scale bars = 2 mm. FIGURE 6. (A) Dorsal and (B) ventral view of preserved of Leptolalax tuberosus (AMS R 171720), Quang Nam Province, Vietnam. Scale bar = 5 mm. The advertisement call of L. croceus appears structurally unique and unusually complex among members of the genus with described calls. Most species of Leptolalax with described calls have simple notes containing few pulses. Leptolalax croceus is the only species of Leptolalax with 4–6 pulsed, structurally different notes per call. In having a dominant frequency of 2625–3000 Hz (at 21.6–25.1ºC), the advertisement call of L. croceus also differs markedly from the higher frequency calls of L. alpinis (6600–6800 Hz; 16ºC), L. applebyi (3962–4306.6 Hz; 21.5ºC), L. arayai (5400–5800 Hz; 17.4ºC), L. dringi (7600–8100 Hz; 24.3ºC), L. hamidi (6715–7295 Hz; 22.9ºC) and L. pictus (6800–7200 Hz; 19–22ºC). Differences in dominant frequencies among species of the scale reported here are unlikely to be attributed to temperature. The advertisement call of L. croceus is also significantly different from the morphologically similar L. tuberosus . The call of L. tuberosus is described here based upon the calls of three L. tuberosus collected approximately 11 km from the type locality of L. croceus and recorded at 22.5–24.5 ºC ambient temperature. Each call consisted of a single, highly, but weakly pulsed note lasting 54–78 ms ( Table 3 , Figure 8 ). The dominant frequency of calls was 2584–2756.2 Hz, similar to that recorded in L. croceus . Call repetition rate and intercall frequency were variable, and calls did not form distinct call groups.