Leptolalax melicus, Rowley, Jodi J. L., Stuart, Bryan L., Thy, Neang & Emmett, David A., 2010

Rowley, Jodi J. L., Stuart, Bryan L., Thy, Neang & Emmett, David A., 2010, A new species of Leptolalax (Anura: Megophryidae) from northeastern Cambodia, Zootaxa 2567, pp. 57-68 : 59-66

Leptolalax melicus sp. nov.

Holotype: MVZ 258198, an adult male, calling on 2 cm diameter tree root, 0.5 m from 2 m wide, swift, rocky stream in Virachey National Park, Ratanakiri Province, Cambodia (14.19287º N, 106.99612º E, 650 m, Figures 1–2 ). Collected by Jodi J. L. Rowley, Bryan L. Stuart and Neang Thy at 19:35 h on 13 October 2007.

Paratypes: MVZ 258199, an adult male, same data as holotype except collected at 19:40 h on 13 October, 2007. MVZ 258197, adult male, same data as holotype except collected at 19:40 h on 10 October 2007. MVZ 258074, adult male, calling from boulder, collected by Jodi J. L. Rowley on 22 June 2006 from Virachey National Park, Ratanakiri Province, Cambodia (ca. 14.3º N, 107.37º E, 600 m, Figure 1 ). MVZ 258075– 258077, adult males, all calling on rocks, collected by David A. Emmett on 22 June 2006 from Virachey National Park, Ratanakiri Province, Cambodia (ca. 14.2º N, 107.38º E, 850 m, Figure 1 ). All specimens were found <10 m from rocky streams in evergreen forest.

Etymology: Specific name from melicus L., meaning musical or lyrical, in reference to the complex call structure of the new species.

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, and femoral 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 melicus is distinguished from its congeners by a combination of (1) a white to pale pink ventral surface with diffuse dark brown blotches and distinct white speckling, (2) finger I <II, (3) an absence of webbing and dermal fringes on fingers, (4) slight basal webbing and no dermal fringes on toes, (5) body size (19.5–22.7 mm for seven adult males), (6) absence of ventrolateral glandular lines, (7) mostly smooth dorsum with no skin ridges, and (8) unique advertisement call consisting of a single long introductory note containing 8–50 pulses, followed by 3–11 predominantly single-pulse notes, and with an average dominant frequency of 3560–3610 Hz.

Description of holotype: Head longer than wide; snout rounded in profile, projecting slightly over lower jaw; nostril closer to tip of snout than eye; canthus rostralis rounded; lores sloping, concave; vertical pupil; eye diameter equal to snout length; tympanum distinct, round, diameter smaller than that of the eye; tympanic annulus elevated relative to skin of temporal region; vomerine teeth absent; pineal ocellus absent; vocal sac openings large, oval; tongue large, broad, with small notch at tip; raised supratympanic ridge running from eye towards axillary gland. Tips of fingers rounded, slightly enlarged; 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; small, oval inner metatarsal tubercle pronounced, outer metatarsal tubercle absent; webbing basal, confined to very base of toes; no lateral fringes. Tibia relatively short and stout, width approximately one-third of length; tibiotarsal articulation reaches snout. Skin on dorsum mostly smooth, with fine, scattered tubercles concentrated on eyelids and ventrolateral surfaces; ventral skin smooth; pectoral gland circular, 0.8 mm diameter; femoral gland oval, 0.7 mm diameter, on posteroventral surface of thigh, closer to knee than to vent; supra-axillary gland raised, 0.7 mm diameter. Ventrolateral glandular line absent.

Colour of holotype in life: Dorsal surface brown; distinct dark brown markings lined with diffuse pale copper colour on dorsum, V-shaped interorbital marking, W-shaped marking between axillae and inverted Vshaped marking above sacrum; dorsal surface of head anterior to eyes pale copper; fine tubercles on dorsum reddish; vertical blackish brown bars on background of pale copper on upper lip; black line along canthus rostralis, through eye, and continuing along supratympanic ridge, encompassing most of tympanum, terminating above axilla; pale copper coloured area under supratympanic ridge, fine reddish line above supratympanic ridge; transverse blackish brown bars on dorsal surface of limbs; large, black blotch on flank and numerous, smaller black spots on sides from groin to axilla; ventral surface of elbow and upper arms without dark bars but with distinct copper colouration; fingers and toes with transverse barring; pale pink ventral surface with diffuse dark blotches and distinct white speckling concentrated on belly, but also scattered on throat, and lateral surfaces of body and upper arms, nearly absent from ventral surface of legs; ventral margin of throat bearing broken row of slightly larger white spots. At night, ventral surface much paler, off-white. Supra-axillary gland copper; femoral and pectoral glands white, lined with dark brown. Iris dark gold with minute, black reticulations.

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

Measurements: Holotype: SVL 20.1, HDL 7.7, HDW 7.4, SNT 3.0, EYE 2.7, IOD 2.4, TMP 1.2, TEY 0.6, TIB 9.6, ML 4.8, PL 9.1, F1L 1.5, F2L 1.8, F3L 3.5, weight 0.7g

Variation. Size and extent of white speckling on ventral and ventrolateral surfaces may be such that anterior half of belly is mostly white with pinkish brown mottling (MVZ 258075, 258197), rather than mostly off-white or pale pink. Specimens vary in the number and size of black blotches ventrolaterally. MVZ 258197 has large white blotches ventrolaterally, in addition to black blotches. In some specimens, dorsal markings join along midline to form a continuous dark brown marking (MVZ 258077, 258199). A single specimen (MVZ 258199) has small non-pigmented patches of skin on knee and groin. Relative finger lengths may be I <II ≤ IV <III. Measurements of the type series are shown in Table 1 . Females of the new species are unknown.

M V Z MVZ 258075 MVZ 258076 MVZ 258077 MVZ 258197 M V Z MVZ 258199

258074 258198*

Advertisement call: Advertisement call descriptions are based on the calls of the holotype MVZ 258198 and paratype MVZ 258199, taken at ambient temperatures of 26.1ºC and 26.2ºC respectively. In both recorded calls, call intensity and call rate were highly variable ( Figure 4 A). Although both the amplitude and rate of calling increased and decreased during recordings, calls did not form distinct call groups (in up to 120 s of calling). Individual calls were consistent in overall structure, consisting of a single introductory note of a variable length (24–132 ms), containing 8–50 pulses, followed by 4–11 short (2–24 ms) notes, or clicks, of 1– 2 pulses repeated at a rate of approximately 15–26 notes per second ( Table 2 , Figure 4 B–C). Pulses in the introductory notes were of less than half the amplitude of clicks, and peak amplitude was usually towards the middle of the click series. Both note types had similar dominant frequencies of 2584–3962.1 Hz ( Figure 4 ), with faint harmonics at approximately 7752 Hz. To the human ear, the call of L. melicus contains a barely discernable ‘squeak’ (introductory note), followed by a faint clicking, and resembles the call of an orthopteran.

means (and ranges).

Sequence divergence: Uncorrected sequence divergences between L. melicus (MVZ 258197–258199) and L. applebyi (holotype AMS R 171703, paratype AMS R171704) collected from approximately 100 km away were 6.1% 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). There was no intraspecific variation in this gene fragment.

Ecology: All specimens were found in evergreen forest between 600–850 m elevation. Males were observed calling on and between rocks and on tree roots, or under leaf litter, less than 1 m from small (<5 m wide) rocky streams. Leptolalax melicus was heard calling in both June and October, during and shortly after rainfall, suggesting that breeding in L. melicus occurs throughout the monsoon season (approximately April– November).

Conservation status: The seven type specimens are the only known representatives of the new species. Given the available information, we suggest the species should be considered Data Deficient following IUCN’s Red List categories (IUCN 2001). Being known only from Virachey National Park, its extent of occurrence is unknown but probably extends further into adjoining areas of the Kon Tum Plateau. Virachey National Park is bordered by two other protected areas; Dong Amphan National Protected Area in Laos and Mom Ray Nature Reserve in Vietnam, both of which contain evergreen forest at similar elevations that may represent suitable habitat for L. melicus .

Comparisons: Leptolalax melicus is distinguished from all other species in the genus by having an offwhite to pale pink ventral surface with diffuse dark brown blotches and distinct white speckling on the chest, belly and throat ( 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 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. 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).

In having finger I <II, L. melicus differs from L. arayai , L. gracilis , L. heteropus , L. kajangensis , L. kecil , L. pelodytoides , L. pictus , and L. sungi , which have fingers I and II equal, and from L. solus , in which Finger I> II. In lacking webbing and dermal fringes on fingers and having only slight basal webbing and no dermal fringes on toes, L. melicus differs from L. alpinis , L. bourreti , L. fuliginosus , L. heteropus , L. kecil , L. liui , L. pelodytoides , and L. sungi , all of which have lateral fringes or more extensive webbing on toes.

The small size of male Leptolalax melicus (19.5–22.7 mm) further distinguishes it from all but L. alpinis (24.0– 26.4 mm), L. applebyi (19.6–20.8 mm), L. kecil (19.3–20.5 mm), L. khasiorum (24.5–27.3 mm), L. liui (23.0– 28.7 mm), and L. pluvialis (21.3–22.3 mm). All other congeners have distinctly larger male body sizes ( L. arayai 29.6 mm; L. bourreti 36.2 mm; L. dringi 28.7–30.3 mm; L. fuliginosus 28.2–30.0 mm; L. gracilis 30–36 mm; L. hamidi 28.7–31.3 mm; L. heteropus 33 mm; L. kajangensis 34–35 mm; L. lateralis 26.9–28.3 mm; L. maurus 26.1 mm; L. melanoleucus 26.6–28.8 mm; L. nahangensis 40.8 mm; L. oshanensis 26.6–30.7 mm; L. pelodytoides 37 mm; L. pictus 31–34 mm; L. solus 27.6 mm; L. sungi 48.3–52.7 mm; L. tamdil 32.3 mm; L. tuberosus 24.4–29.5 mm; L. ventripunctatus 25.5–28.0 mm).

Leptolalax melicus is also differentiated from L. alpinis , L. fuliginosus , L. khasiorum , L. liui , L. oshanensis , L. pelodytoides , L. pluvialis and L. tamdil by the absence of ventrolateral glandular lines and from L. arayai , 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.

Leptolalax melicus can be further distinguished from the most morphologically similar species, L. applebyi , in having a more distinct dorsal patterning, significantly larger pectoral glands ( L. melicus mean diameter 0.9 mm ± 0.07 S. E., L. applebyi mean diameter 0.4 mm ± 0.03 S. E.; Mann-Whitney U -test, Z= - 2.847, p = 0.004; N=12), and significantly larger femoral glands ( L. melicus mean diameter 0.9 mm ± 0.08 S. E., L. applebyi mean diameter 0.5 mm ± 0.05 S. E.; Mann-Whitney U -test, Z= -2.847, p = 0.004; N=12).

The advertisement call of L. melicus differs structurally from all fourteen Leptolalax species with described calls; L. alpinis , L. applebyi , L. arayai , L. dringi , L. fuliginous , L. gracilis , L. hamidi , L. heteropus , L. kecil , L. melanoleucus , L. pelodytoides , L. pictus , L. oshanensis and L. solus , and also from L. tuberosus . Leptolalax melicus is the only species of Leptolalax with a call containing a single long introductory note containing 8–50 pulses, followed by a series of predominantly single-pulse notes or clicks. All other described calls of Leptolalax species contain notes that are similar in structure and duration. The only species with noticeably different first notes in the advertisement call are L. hamidi and L. heteropus , which have introductory notes of a slightly longer duration but similar amplitude to successive notes, and L. arayai , which has introductory notes of a slightly shorter duration but similar amplitude to successive notes.

Figures

FIGURE 1. Collection sites of Leptolalax melicus sp. nov. from Virachey National Park, Cambodia (black circles), Leptolalax applebyi from Song Thanh Proposed Nature Reserve, Vietnam (dark grey square; Rowley & Cao 2009) and Leptolalax sp. from the Cardamom Mountains, Cambodia (dark grey triangle; Ohler et al. 2002). Areas over 600 m elevation are pale grey. Boundaries of Virachey National Park (hatched area) were obtained from the World Database on Protected Areas 2009.

FIGURE 2. Male holotype of Leptolalax melicus sp. nov. (MVZ 258198) in life. (A) Lateral view in the field, in calling posture (nocturnal ventral colouration), (B) dorsal and (C) ventral views (diurnal ventral colouration).

FIGURE 3. (A) Dorsal view, (B) ventral view and (C) lateral view of head of preserved holotype (MVZ 258198) of Leptolalax melicus sp. nov. Scale bars = 2 mm.

FIGURE 4. Advertisement call of Leptolalax melicus sp. nov. (holotype MVZ 258198) recorded at ambient air temperature of 26.1 º C. (A) 60 s waveform of relative amplitude over time, (B) waveform and (C) corresponding spectrogram of single representative call expanded from section shown in A, and (D) power spectrum (relative amplitude vs. frequency) of introductory note and (E) click.

Tables

TABLE 1. Measurements (mm) of adult male Leptolalax melicus, sp. nov. Abbreviations defined in text.

SVL HDL	20.7 20.2 8.2 7.9	22.8 8.6	21.8 8.2	19.8 7.6	20.1 7.7	19.5 7.7
HDW	7.5 7.4	8.2	7.6	7.3	7.4	7.0
SNT EYE	3.3 3.2 2.8 2.6	3.2 3.1	3.2 2.8	3.1 2.3	3.0 2.7	3.1 2.6
IOD	2.9 2.5	2.9	2.8	2.5	2.4	2.4
TMP TEY	1.5 1.1 0.7 0.7	1.3 1.0	1.2 0.7	1.3 0.7	1.2 0.6	1.0 0.7
TIB	9.9 9.2	10.4	10.1	9.1	9.6	9.4
ML PL	4.8 4.6 9.1 8.9	4.8 9.2	5.0 9.1	4.5 8.9	4.8 9.1	4.6 8.7
F1L	1.6 1.7	1.7	1.8	1.7	1.5	1.6
F2L F3L	1.8 1.8 4.0 3.8	2.1 3.9	2.0 4.1	2.0 3.5	1.8 3.5	1.8 3.6

TABLE 2. Measurements of advertisement call parameters for Leptolalax melicus sp. nov. Parameter values are given as

Recording Temperature (ºC)	MVZ 258198* 26.1	MVZ 258199 26.2
Number of notes	268	251
Call duration (ms) Intercall interval (ms)	292 (176–484) 920 (214–3821)	278 (168–427) 1248 (226–3647)
Notes/call	6.7 (4–11)	6.2 (4–11)
Internote interval (ms) Call repetition rate (calls/s)	33.7 (12–82) 0.8	31.6 (12–62) 0.7
Introductory Note duration (ms) note Pulses/note Dominant frequency (Hz).	58 (24–106) 22.7 (11–40) 3561.6 (2928.5–3789.8)	70 (25–132) 26.5 (8–50) 3772.6 (3445.3–3789.8)
Pulse repetition rate (pulses/s)	394 (368–429)	363 (326–427)
Clicks Note duration (ms) Pulses/note	7.2 (2–23) 1.2 (1–7)	7.8 (3–24) 1.2 (1–7)
Dominant frequency (Hz)	3560.4 (2584–3798.8)	3602.9 (2756.2–3962.1)
Note repetition rate (notes/s)	24 (16–26)	23 (15–26)
*holotype