zootaxa, a new species of leptolalax (anura: megophryidae)

ZOOTAXAISSN 1175-5326 (print edition)

ISSN 1175-5334 (online edition)Copyright © 2010 · Magnolia Press

Zootaxa 2567: 57–68 (2010) www.mapress.com/zootaxa/ Article

A new species of Leptolalax (Anura: Megophryidae)from northeastern Cambodia

JODI J. L. ROWLEY1,6, BRYAN L. STUART2,3, NEANG THY4 & DAVID A. EMMETT5

1Australian Museum, 6 College St, Sydney, NSW, 2010, Australia2North Carolina Museum of Natural Sciences, 11 West Jones Street, Raleigh, NC 27601, USA3�Museum of Vertebrate Zoology, University of California, 3101 Valley Life Sciences Building, Berkeley, CA 94720, USA

4General Department of Administration for Nature Conservation and Protection, Ministry of Environment, 48 Samdech Preah Sihanouk, Tonle Bassac, Chamkarmorn, Phnom Penh, Cambodia5Conservation International Cambodia, P.O. Box 1356, Phnom Penh, Cambodia6Corresponding author. E-mail: [email protected]

Abstract

We describe a new species of megophryid frog in the genus Leptolalax from the Kon Tum Plateau in northeastern Cambodia. Leptolalax melicus sp. nov. is distinguished from its congeners by a combination of an off-white to pale pink ventral surface with diffuse dark brown blotches and distinct white speckling, finger I < II, an absence of webbing and dermal fringes on fingers, slight basal webbing and no dermal fringes on toes, body size (19.5–22.7 mm for seven adult males), an absence of ventrolateral glandular lines, dorsum mostly smooth with no skin ridges, and a unique advertisement call consisting of a single long introductory note containing 8–50 pulses, followed by 3–11 predominantly single-pulsed notes, and with an average dominant frequency of 3560–3610 Hz. Leptolalax melicus can be further distinguished from the morphologically similar L. applebyi in having more distinct dorsal patterning, and significantly larger pectoral and femoral glands. Leptolalax melicus and L. applebyi also differ by 6.1% sequence divergence at the 16S mtDNA gene. All specimens of L. melicus were found near rocky streams in evergreen forest between 650–850 m elevation. We suggest the new species should be considered Data Deficient following IUCN’s Red List categories.

Key words: Acoustics, Anura, Leptolalax melicus sp. nov., Ratanakiri, Cambodia, Southeast Asia

Introduction

The genus Leptolalax (Dubois 1983) is a group of small, cryptic megophryid frogs associated with small to medium-sized streams in hilly evergreen forests. The genus is distributed throughout Southeast Asia, southern China and northeastern India (Frost 2010). Due to an increase in field surveys in the region, and the incorporation of acoustic and molecular data in delineating species boundaries in amphibians, there has been a rapid increase in the number of Leptolalax species described in recent decades. From only four species in 1983 (Dubois 1983), the number of known Leptolalax currently stands at 26, nine of which were described in the last decade (Frost 2010).

Many areas of Cambodia remain poorly surveyed and new species of amphibians continue to be discovered, particularly in areas of sufficient geographic relief to contain evergreen forest and swift, rocky streams (e.g. Ohler et al. 2002; Stuart et al. 2006; Grismer et al. 2007). There are three such areas in Cambodia: the Cardamom Mountains in the southwest of Cambodia, the lower slopes of the Da Lat Plateau in the extreme east, and the lower slopes of the Kon Tum Plateau in the northeast. To date, most herpetological surveys in Cambodia have focused on the Cardamom Mountains (Ohler et al. 2002; Stuart & Emmett 2006; Grismer et al. 2007).

In Cambodia, Leptolalax has only been reported from a single metamorphic individual of an undetermined species collected in the Cardamom Mountains (Ohler et al. 2002), and two undetermined

Accepted by M. Vences: 22 Jul. 2010; published: 16 Aug. 2010 57

species (Neang & Holden 2008) obtained by the authors on the western slopes of the Kon Tum Plateau in Virachey National Park. One of these species is superficially similar to the recently described L. applebyifrom the Kon Tum Plateau in central Vietnam (Rowley & Cao 2009), but is morphologically, genetically, and acoustically distinct, and is described here as new.

Material and methods

We recorded morphological data from specimens fixed in 10% formalin (MVZ 258197–258199) or 95% ethanol (MVZ 258074–258077) and then stored in 70% ethanol. Specimens were deposited at the Museum of Vertebrate Zoology, University of California, Berkeley (MVZ). Morphometric data were taken (to the nearest 0.1 mm) using digital callipers or a stereomicroscope fitted with an eyepiece micrometer. Measurements include snout-vent length (SVL); head length from tip of snout to rear of jaws (HDL); head width at the commissure of the jaws (HDW); snout length from tip of snout to the anterior corner of eye (SNT); diameter of the exposed portion of the eyeball (EYE); interorbital distance (IOD); horizontal diameter of tympanum (TMP); distance from anterior edge of tympanum to posterior corner of the eye (TEY); tibia length with the hindlimb flexed (TIB); manus length from tip of third digit to base of inner palmar tubercle (ML); pes length from tip of fourth toe to base of the inner metatarsal tubercle (PL); length of adpressed first finger from tip to distal edge of the inner palmar tubercle (F1L); length of adpressed second finger from tip to distal edge of inner palmar tubercle (F2L); and length of adpressed third finger from tip to distal edge of inner palmar tubercle (F3L). Sex was determined by direct observation of calling and the presence of internal vocal sac openings. Mass was recorded in life (to the nearest 0.1 g), using Pesola scales. Comparative morphological characters were taken from references: L. alpinis (Fei et al. 1991; Fei et al. 2009), L. applebyi (Rowley & Cao 2009), L. arayai (Matsui 1997), L. bourreti (Dubois 1983), L. dringi (Dubois 1986; Inger & Stuebing 2005), L. fuliginosus (Matsui 2006), L. gracilis (Günther 1872; Malkmus et al. 2002; Inger & Stuebing 2005), L. hamidi (Matsui 1997), L. heteropus (Boulenger 1900), L. kajangensis (Grismer et al. 2004), L. kecil (Matsui et al. 2009), L. khasiorum (Das et al. 2010), L. lateralis (Anderson 1871; Humtsoe et al. 2008), L. liui (Fei et al. 1991; Fei et al. 2009), L. maurus (Inger et al. 1997), L. melanoleucus (Matsui 2006), L. nahangensis(Lathrop et al. 1998), L. oshanensis (Lui 1950; Fei et al. 2009), L. pelodytoides (Boulenger 1893), L. pictus(Malkmus 1992; Malkmus et al. 2002), L. pluvialis (Ohler et al. 2000), L. solus (Matsui 2006), L. sungi(Lathrop et al. 1998), L. tamdil (Sengupta et al. 2010), L. tuberosus (Inger et al.1999), L. ventripunctatus (Fei et al. 1991; Fei et al. 2009). Leptolalax applebyi (AMS R 171703–171707) and L. tuberosus (AMS R 171714–171722) from Song Thanh Proposed Nature Reserve, Phouc Son district, Quang Nam Province, Vietnam, and colour photographs of the holotype of L. pluvialis (MNHN 1999.5675) in preservative were also examined. We did not examine the Leptolalax metamorph (MNHN 2001.0204) collected in the Cardamom Mountains in southwestern Cambodia (Ohler et al. 2002), but it is clearly not conspecific with the new species on the basis of reported body size and skin texture.

Advertisement calls were recorded with an Edirol R-09 24-bit WAVE/MP3 Recorder with a Røde NTG-2 condenser shotgun microphone (frequency range 20–20,000 Hz) at 44.1 kHz sampling rate and 24-bit encoding. Calls were recorded at a distance of approximately 0.2–0.5 m and ambient temperatures were taken immediately after recordings using a Kestrel 3500 hand-held weather meter. Calls were analysed with Raven Pro 1.3© software (http://www.birds.cornell.edu/raven). Audiospectrograms in figures were calculated with fast-Fourier transform (FFT) of 256 points, 50% overlap and 172 Hz grid-spacing, using Hanning windows. We examined oscillograms (waveforms) and audiospectrograms and for 40 calls per individual we measured the call duration (ms), intercall interval (ms), number of notes per call, note duration (ms), internote interval (ms), number of pulses per note, note repetition rate (notes/s) and dominant frequency (Hz). Comparisons of advertisement calls within the genus Leptolalax are complicated by a lack of clear definitions in terminology, particularly with respect to the units of a call, a note or a pulse. Here we use the definitions of Duellman (1970), except that we define a single call as vocalisations produced during a single expiration (Brown & Richards 2008), and used the term ‘call group’ to refer to temporal groups of calls, separated by intervals of

ROWLEY ET AL.58 · Zootaxa 2567 © 2010 Magnolia Press

silence distinctly greater than that between calls. Temporal and spectral parameters of calls were measured using the definitions of Cocroft & Ryan (1995). For 20 calls per individual we measured the call duration (ms), intercall interval (ms), number of notes per call, note duration (ms), internote interval (ms), number of pulses per note, note repetition rate (notes/s) and dominant frequency (Hz). Comparative advertisement call characters for Leptolalax species were taken from references (Jiang et al. 2002; Malkmus et al. 2002; Matsui 1997, 2006; Matsui et al. 2009; Xu et al. 2005; Rowley & Cao 2009), and unpublished data (Leptolalax tuberosus; Rowley and Cao).

We analyzed 537 base pairs (bp) of mitochondrial 16S ribosomal RNA from three Leptolalax melicus and compared them to two individuals of Leptolalax applebyi, the most morphologically similar and geographically proximate species. DNA was extracted using DNeasy tissue extraction kits (Qiagen, Hilden, Germany). We used the primers 16SAR and 16SBR of Palumbi et al. (1991) to amplify the 16S rRNA gene. Standard PCR protocols were used, with an initial 2 minute denaturation at 94°C; 2 cycles of 20 seconds at 94°C, 40 seconds at 52°C, and 60 seconds at 72°C; 33 cycles of 20 seconds at 94°C, 40 seconds at 50°C, and 50 seconds at 72°C; and a 5 minute final extension at 72°C. PCR products were purified using ExoSap-IT (USB Corporation, Cleveland, OH, USA). Purified templates were sequenced directly by Macrogen Inc. (Seoul, Korea). Sequences were validated using Sequencher 4.10 (Gene Codes, Ann Arbor, MI, USA), aligned using the Clustal option in MEGA 4 and refined by eye. Kimura 2 parameter mtDNA uncorrected pairwise sequence divergence was calculated using MEGA 4. DNA sequences were deposited in GenBank under the accession numbers HM133597–HM133601.

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

Zootaxa 2567 © 2010 Magnolia Press · 59A NEW LEPTOLALAX FROM CAMBODIA

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.

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).

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 V-


shaped 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.

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

*holotype

MV Z 258074

MVZ 258075 MVZ 258076 MVZ 258077 MVZ 258197 M VZ 258198*

MVZ 258199

SVL 20.7 20.2 22.8 21.8 19.8 20.1 19.5

HDL 8.2 7.9 8.6 8.2 7.6 7.7 7.7

HDW 7.5 7.4 8.2 7.6 7.3 7.4 7.0

SNT 3.3 3.2 3.2 3.2 3.1 3.0 3.1

EYE 2.8 2.6 3.1 2.8 2.3 2.7 2.6

IOD 2.9 2.5 2.9 2.8 2.5 2.4 2.4

TMP 1.5 1.1 1.3 1.2 1.3 1.2 1.0

TEY 0.7 0.7 1.0 0.7 0.7 0.6 0.7

TIB 9.9 9.2 10.4 10.1 9.1 9.6 9.4

ML 4.8 4.6 4.8 5.0 4.5 4.8 4.6

PL 9.1 8.9 9.2 9.1 8.9 9.1 8.7

F1L 1.6 1.7 1.7 1.8 1.7 1.5 1.6

F2L 1.8 1.8 2.1 2.0 2.0 1.8 1.8

F3L 4.0 3.8 3.9 4.1 3.5 3.5 3.6

TIB:SVL 0.48 0.46 0.46 0.46 0.46 0.48 0.48

HDL:SVL 0.40 0.39 0.38 0.38 0.38 0.38 0.39

HDL:HDW 1.09 1.07 1.05 1.08 1.04 1.04 1.10


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.

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 4A). 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 4B–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.

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

*holotype

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 off-white 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

Recording MVZ 258198* MVZ 258199

Temperature (ºC) 26.1 26.2

Number of notes 268 251

Call duration (ms) 292 (176–484) 278 (168–427)

Intercall interval (ms) 920 (214–3821) 1248 (226–3647)

Notes/call 6.7 (4–11) 6.2 (4–11)

Internote interval (ms) 33.7 (12–82) 31.6 (12–62)

Call repetition rate (calls/s) 0.8 0.7

Introductory note

Note duration (ms) 58 (24–106) 70 (25–132)

Pulses/note 22.7 (11–40) 26.5 (8–50)

Dominant frequency (Hz). 3561.6 (2928.5–3789.8) 3772.6 (3445.3–3789.8)

Pulse repetition rate (pulses/s) 394 (368–429) 363 (326–427)

Clicks Note duration (ms) 7.2 (2–23) 7.8 (3–24)

Pulses/note 1.2 (1–7) 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)


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).

FIGURE 4. Advertisement call of Leptolalax melicus sp. nov. (holotype MVZ 258198) recorded at ambient air temperature of 26.1º 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.

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.

Discussion

The genus Leptolalax is likely to contain a high proportion of undiagnosed diversity, with new species continuously being described from throughout their range, and further species awaiting description (J. Rowley, unpublished data). The incorporation of molecular and acoustic information has been invaluable in discovering this hitherto undiagnosed diversity given the similarity in external morphology (especially in preservative) observed among species in the genus. However, the lack of molecular and/or acoustic information for many species of Leptolalax hinders describing newly discovered species. A comprehensive phylogenetic analysis of the group is also required, but is similarly impeded by an absence of molecular material from many species. In order to resolve this dilemma, there is an urgent need to collect new specimens and associated acoustic and molecular data from type localities.

Acknowledgements

Fieldwork was funded by the Annie Alexander Endowment, Museum of Vertebrate Zoology, University of California, Berkeley and by Conservation International. This research was also supported by ADM Capital Foundation, and a grant from The John D. and Catherine T. MacArthur Foundation to the North Carolina Museum of Natural Sciences. H.E. Dr Mok Mareth, Senior Minister of Environment and H.E. Chay Samith, General Director of Administration for Nature Conservation and Protection kindly permitted us to conduct surveys and issued specimen export permits (Permit # 405 DNCP MoE [transport]). Mr Chou Sopheak and staff at Virachey National Park facilitated surveys within the park. Jimmy A. McGuire and Carol Spencer loaned specimens and tissues in their care. Rebecca Johnson, Karen Gray and Robert Mason provided assistance with obtaining DNA sequences. Annemarie Ohler shared photographs of the holotype of L. pluvialis. Tandy Rowley Photography provided photographs of specimens in preservative. Indraneil Das and Rafe Brown provided valuable comments on an earlier draft of the manuscript. For all this assistance we are most grateful.

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