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1 Zootaxa 4039 (3): Copyright 2015 Magnolia Press Article A new species of Leptolalax (Anura: Megophryidae) from Vietnam and Cambodia ISSN (print edition) ZOOTAXA ISSN (online edition) JODI J. L. ROWLEY 1,2,10, BRYAN L. STUART 3, THY NEANG 4,5, HUY D. HOANG 6, VINH Q. DAU 7, TAO T. NGUYEN 8 & DAVID A. EMMETT 9 1 Australian Museum Research Institute, Australian Museum, 1 William St, Sydney NSW 2010, Australia 2 College of Marine and Environmental Science, Centre for Tropical Biodiversity and Climate Change, James Cook University, Townsville, Qld 4811 Australia 3 North Carolina Museum of Natural Sciences, 11 West Jones Street, Raleigh, NC 27601, USA 4 Fauna & Flora International (FFI), Cambodia Programme #19, St. 360, Boeng Keng Kang I, Phnom Penh, Cambodia 5 Department of National Parks, Ministry of Environment, 48 Samdech Preah Sihanouk, Tonle Bassac, Chamkarmorn, Phnom Penh, Cambodia 6 Faculty of Biology, Vietnam National University Ho Chi Minh City, University of Science, 227 Nguyen Van Cu, District 5, Ho Chi Minh City, Vietnam 7 Institute of Ecology and Biological Resources, 18 Hoang Quoc Viet, Nghia Do, Tu Liem, Hanoi, Vietnam 8 Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam 9 Conservation International, Asia-Pacific Field Division, 318 Tanglin Road #01-30, Block B, Singapore Corresponding author. Jodi.Rowley@austmus.gov.au; Phone: Abstract We describe a new, medium-sized Leptolalax species from the Kon Tum Plateau of Vietnam and adjacent Cambodia. Leptolalax isos sp. nov. is distinguished from its congeners by a combination of an absence of distinct dark brown/black dorsolateral markings; toes with rudimentary webbing, wide lateral dermal fringes in males and weak or absent lateral dermal fringes in females; most males with wide lateral dermal fringes on Finger II, a body size of mm in 38 adult males and mm in 9 adult females, near immaculate white chest and belly; absence of white speckling on the dorsum; and a call consisting of 2 3 notes with a dominant frequency of khz (at º C). Uncorrected sequence divergences between L. isos sp. nov. and all homologous 16S rrna sequences available are >10%. At present, the new species is known from montane evergreen forest between ~ m elevation in northeastern Cambodia and central Vietnam. Habitat within the range of the new species is threatened by deforestation and upstream hydroelectric dams. Key words: Acoustics, Anura, Leptolalax isos sp. nov., Cambodia, Southeast Asia, Vietnam Introduction The genus Leptolalax (Dubois 1983) is an assemblage of small frogs associated with the forest floor and rocky streams in hilly evergreen forest in Southeast Asia, southern China and northeastern India (Frost 2015). Due to their small size, cryptic coloration and often faint, insect-like calls, frogs in the genus are often difficult to detect in the field, particularly outside of their breeding seasons. They are also highly morphologically conserved and are subsequently difficult to identify to species, which has resulted in an underestimation of true diversity in the genus. Recently, the incorporation of molecular and acoustic data in delineating species boundaries in the group, along with increased survey efforts, has resulted in a rapid increase in the number of known species in the genus. There are currently 43 species of Leptolalax known, 42% of which have been described in the last five years (Frost 2015). Herein we describe a new, medium-sized species of Leptolalax from the Kon Tum Plateau of Vietnam and adjacent Cambodia. The new species is morphologically, molecularly and acoustically most similar to Leptolalax firthi. Accepted by M. Vences: 7 Oct. 2015; published: 5 Nov

2 Material and methods We recorded morphological data from specimens fixed in 10% formalin and then stored in 70% ethanol. Specimens were deposited at the Australian Museum (AMS), Museum of Vertebrate Zoology, Berkeley (MVZ), and North Carolina Museum of Natural Sciences (NCSM). Some specimens currently at the AMS will be deposited at the Vietnam National Museum of Nature (VNMN) and have been cross-catalogued at both institutions. In these instances, voucher numbers are reported as VNMN/AMS. Morphometric data were taken (to the nearest 0.1 mm) with digital callipers. Measurements include snout-vent length (SVL); head length from tip of snout to rear of jaws (HDL); head width at commissure of jaws (HDW); snout length from tip of snout to anterior corner of eye (SNT); diameter of exposed portion of eyeball (EYE); interorbital distance (IOD); horizontal diameter of tympanum (TMP); distance from anterior edge of tympanum to posterior corner of eye (TEY); tibia length with hindlimb flexed (TIB); manus length from tip of third digit to proximal edge of inner palmar tubercle (ML); and pes length from tip of fourth toe to proximal edge of the inner metatarsal tubercle (PL). Sex was determined by direct observation of calling in life, and the presence of internal vocal sac openings and/or gonadal inspection. Mass was recorded in life (to the nearest 0.1 g), using Pesola scales. Geographic coordinates were obtained using a Garmin GPSMAP 60CSx GPS receiver and recorded in datum WGS 84. We obtained comparative morphological data from museum specimens of Leptolalax and photographs of these specimens in life (Appendix I) and from the literature: L. aereus (Rowley et al. 2010a), L. alpinis (Fei et al. 1991, 2009, 2010), L. applebyi (Rowley & Cao 2009), L. arayai (Matsui 1997), L. bidoupensis (Rowley et al. 2011), L. botsfordi (Rowley et al. 2013), L. bourreti (Dubois 1983; Ohler et al. 2011), L. croceus (Rowley et al. 2010b), L. dringi (Dubois 1987; Inger et al. 1995), L. eos (Ohler et al. 2011), L. firthi (Rowley et al. 2012), L. fuliginosus (Matsui 2006), L. fritinniens (Dehling & Matsui 2013), L. gracilis (Günther 1872; Inger & Stuebing 2005; Dehling 2012a), L. hamidi (Matsui 1997), L. heteropus (Boulenger 1900), L. kecil (Matsui et al. 2009), L. khasiorum (Das et al. 2010), L. kajangensis (Grismer et al. 2004), L. lateralis (Anderson 1871; Humtsoe et al. 2008), L. laui (Sung et. al. 2014), L. liui (Fei et al. 1991, 2009, 2010), L. marmoratus (Matsui et al. 2014a), L. maurus (Inger et al. 1997), L. melanoleucus (Matsui 2006), L. melicus (Rowley et al. 2010c), L. minimus (Ohler et al. 2011), L. nahangensis (Lathrop et al. 1998), L. nokrekensis (Mathew & Sen 2010), L. nyx (Ohler et al. 2011), L. oshanensis (Liu 1950; Fei et al. 2009, 2010), L. pelodytoides (Boulenger 1893, 1908; Ohler et al. 2011), L. pyrrhops (Poyarkov et al. 2015) L. pictus (Malkmus 1992; Malkmus et al. 2002), L. platycephalus (Dehling 2012b), L. pluvialis (Ohler et al. 2000, 2011), L. sabahmontanus (Matsui et al. 2014b), L. solus (Matsui 2006), L. sungi (Lathrop et al. 1998), L. tamdil (Sengupta et al. 2010), L. tuberosus (Inger et al.1999; Rowley et al. 2010b), L. ventripunctatus (Fei et al. 1991, 2009, 2010), and L. zhangyangpingi (Jiang et al. 2013). Due to the degree of undiagnosed diversity within the genus, where available, we relied on examination of topotypic material and/or original species descriptions. Advertisement calls were recorded with an Edirol R-09HR WAVE/MP3 Recorder (96 khz sampling rate and 24-bit encoding) with a Røde NTG-2 condenser shotgun microphone. Calls were recorded at a distance of approximately m and ambient air temperatures at calling sites were taken immediately after recordings using a Kestrel 3500 hand-held weather meter. Calls were analysed with Raven Pro 1.3 software ( Audiospectrograms for analysis were calculated with fast-fourier transform (FFT) of 1024 points, 50% overlap and 172 Hz grid-spacing, using Hanning windows. We used 512 points to generate figures. In describing the advertisement calls, we used the definitions of Duellman (1970), except that we defined a single call as vocalisations produced during a single expiration. Temporal and spectral parameters of calls were measured using the definitions of Cocroft & Ryan (1995), except for fundamental frequency, where the definition of Duellman (1970) was used. For each call recording, we measured the call duration (ms), call repetition rate (calls/s), intercall interval (ms), number of notes per call, note duration (ms) and dominant frequency (khz). Comparative advertisement call characters for Leptolalax species were taken from references, with advertisement calls known for 25 of the 43 known species of Leptolalax (Matsui 1997, 2006; Jiang et al. 2002; Malkmus et al. 2002; Matsui et al. 2009, 2014a, 2014b; Xu et al. 2005; Rowley & Cao 2009; Rowley et al. 2010a, 2010b, 2010c, 2011, 2012, 2013; Sukumaran et al. 2010; Dehling & Matsui 2013; Poyarkov et al. 2015). To maintain consistency and facilitate meaningful comparisons, we have used the terminology defined above to compare calls, regardless of terms used in these references. Total genomic DNA was extracted from tissues using DNeasy tissue extraction kits (Qiagen). We used the 402 Zootaxa 4039 (3) 2015 Magnolia Press ROWLEY ET AL.

3 primers 16SAR and 16SBR of Palumbi et al. (1991) to amplify ~550 base pairs of the 16S rrna gene for the new species. Standard PCR protocols were used and PCR products were purified using ExoSap-IT (USB Corporation, OH, USA). Purified templates were sequenced directly by Macrogen (Seoul, Korea). Sequences were validated using Sequencher 4.10 (Gene Codes, Ann Arbor, MI), aligned using the Clustal option in MEGA 5 and refined by eye. We used Akaike Information Criterion as implemented in jmodeltest (Darriba et al. 2012) to select the best-fit model of nucleotide substitutions, which was then used in model-based phylogenetic inference. In addition to the newly collected specimens, all species belonging to the genus Leptolalax for which homologous sequences were available (25 of the 43 species) were trimmed to match the length of the newly generated sequences and included in the phylogenetic analysis. Leptobrachium cf. chapaense was included in the analysis as an outgroup. Locality information and accession numbers for all sequences included in the analysis can be found in Table 1. TABLE 1. Sequences used in this study. *generated as part of this study. Species Locality Voucher no. GenBank no. Leptolalax aereus Vietnam, Quang Binh Province RH60165 JN Leptolalax applebyi Vietnam, Quang Nam Province AMS R HM Leptolalax arayai Malaysia, Borneo BORNEENSIS AB Leptolalax bidoupensis Vietnam, Lam Dong Province AMS R HQ Leptolalax bourreti Lao Cai Province, Vietnam AMS R KR Leptolalax dringi Malaysia, Borneo KUHE:55610 AB Leptolalax eos Laos, Phongsaly Province MNHN: JN Leptolalax firthi Vietnam, Quang Nam Province AMS R , , JQ Leptolalax firthi Vietnam, Kon Tum Province AMS R , , JQ Leptolalax fritinniens Malaysia, Borneo KUHE AB Leptolalax gracilus Malaysia, Borneo KUHE AB Leptolalax hamidi Malaysia, Borneo KUHE AB Leptolalax heteropus Malaysia, Peninsula KUHE AB Leptolalax isos sp. nov. Vietnam, Gia Lai Province AMS R , AMS R , KT * VNMN A /AMS R Leptolalax isos sp. nov. Cambodia, Ratanakiri Province MVZ KT * Leptolalax laui China, Shenzhen SYS A KM Leptolalax liui China, Jiangxi Province SYS A KM Leptolalax marmoratus Malaysia, Borneo KUHE AB Leptolalax maurus Malaysia, Borneo SP AB Leptolalax melicus Cambodia, Ratanakiri Province MVZ HM Leptolalax minimus Thailand, Chiangmai Province - JN Leptolalax nyx Vietnam, Ha Giang Province AMNH A DQ Leptolalax oshanensis China, Sichuan Province SYS A KM Leptolalax pictus Malaysia, Borneo UNIMAS 8705 KJ Leptolalax pluvialis Vietnam, Lao Cai Province MNHN: JN Leptolalax pyrrhops Lam Dong Province, Vietnam ZMMU A-5208 KP Leptolalax sabahmontanus Malaysia, Borneo BORNEENSIS AB Leptolalax ventripunctatus Laos, Phongsaly Province MNHN JN Leptobrachium cf. chapaense Vietnam, Lao Cai Province AMS R KR Bayesian phylogenetic analyses were performed in MrBayes 3.2 (Ronquist et al. 2012) under a GTR+I +Γ (general time reversible model with Gamma distributed substitution rates and a proportion of the sites invariable; A NEW MEGOPHRYID FROG SPECIES FROM INDOCHINA Zootaxa 4039 (2) 2015 Magnolia Press 403

4 model parameters estimated during the search). Four independent Markov Chain Monte Carlo searches were run for 2 million generations, sampled every 2000 generations, each with four chains, and default priors. Output files were examined visually in Tracer v1.5 (Rambaut & Drummond 2007) to assess stationarity and determine the number of generations to remove as burn-in. To be conservative, we considered 200,000 generations from each run as burn-in, and removed 100 trees before summarizing topology and posterior probabilities. Trees were visualized using the FigTree v1.3.1 program, available at We consider branches receiving 0.95 posterior probabilities to be well-supported (Wilcox et al. 2002). Uncorrected pairwise sequence divergence was calculated using MEGA 5. FIGURE 1. Type locality (yellow star) and collection localities (yellow circles) of Leptolalax isos sp. nov. and type locality (orange star) and collection localities (orange circles) of L. firthi. Leptolalax isos sp. nov. Figs. 2 4, 7. Holotype. VNMN A /AMS R (Fig. 2A, Fig. 3), adult male, on wet rock at edge of 3 4 m wide stream in evergreen forest at Kon Ka Kinh National Park, Gia Lai Province, Vietnam ( º N, º E, 985 m; Fig. 1). Collected on 20 August 2011 by Jodi J. L. Rowley, Nguyen Thien Tao, Dau Quang Vinh, Nguyen Thi Luong and Hoang Duc Huy. Paratypes. The following specimens were collected in evergreen forest in Kon Ka Kinh National Park, Gia Lai Province, Vietnam by Jodi J. L. Rowley, Nguyen Thien Tao, Dau Quang Vinh, Nguyen Thi Luong and Hoang Duc Huy. AMS R , , and NCSM 79652, adult males, and AMS R , adult female, same collection data as holotype. AMS R , , NCSM 79651, adult males and AMS R , , adult females, collected on 19 August 2011 near 4 m wide rocky stream ( º N, º E, 1003 m). AMS R , and AMS R , adult males, collected on 22 August and 23 August 2011, respectively, near 4 m wide rocky stream ( º N, º E, 1003 m). AMS R , adult male, collected near 1 2m wide rocky on 21 August ( º N, º E, 1097 m). 404 Zootaxa 4039 (3) 2015 Magnolia Press ROWLEY ET AL.

5 AMS R , adult male, collected on 24 August 2011 at edge of 4 m wide, rocky, low gradient stream ( º N, º E, 1058 m). VNMN A /AMS R , adult female and VNMN A /AMS R , adult male, collected on 24 August 2011 in a swampy area with small rocky stream ( º N, º E, 1019 m). AMS R , adult male, collected on 25 August 2011 in 2 3 m wide, rocky stream ( º N, º E, 1072 m). AMS R , adult female, and AMS R , adult males, collected on 28 August 2011 in 4 m wide stream (14.221º N, º E, 1000 m). FIGURE 2. Leptolalax isos sp. nov. in life. (A) Male holotype VNMN A /AMS R , (B) male paratype AMS R , (C) male paratype AMS R in lateral view (D) male paratype AMS R in ventral view. (E) female paratype MVZ (F) male paratype MVZ The following specimens were collected in hill evergreen forest in Virachey National Park, Ratanakiri Province, Cambodia ( º N, º E, 650 m) by Jodi J. L. Rowley, Bryan L. Stuart and Neang Thy. MVZ , adult males, collected near 2 m wide, swift, rocky stream on 7 October 2007; MVZ , adult male, calling on fallen branch over seep on 7 October 2007; MVZ , adult male, on ground under herbaceous A NEW MEGOPHRYID FROG SPECIES FROM INDOCHINA Zootaxa 4039 (2) 2015 Magnolia Press 405

6 plants, 3 m from 1.5 m wide, swift, rocky stream on 7 October 2007; MVZ , adult male, on gravel bank at edge of 3 m wide, swift, low-gradient, rocky stream on 7 October 2007; MVZ , adult male, on bank of swift, rocky stream on 7 October 2007; MVZ , adult male, on rock in 3 m wide, low-gradient, swift, rocky stream, collected on 2 October 2007; MVZ , MVZ adult females, and MVZ , MVZ adult males, collected near swift, rocky stream, on 2 October MVZ , adult male, collected in an almost dry, 1 m wide stream on 22 June 2006 by Jodi J. L. Rowley in hill evergreen forest in Virachey National Park (approximately 14.3º N, º E, 600 m), and MVZ , adult male, collected near a rocky stream in Virachey National Park (approximately 14.2º N, º E, 850 m) on 22 June, 2006 by David A. Emmett. Etymology. Specific epithet is from the Greek isos, meaning equal or like, in reference to the similarity of the new species to Leptolalax firthi. Diagnosis. Assigned to the genus Leptolalax on the basis of the following: small size, rounded finger tips, the presence of an elevated thenar tubercle not continuous to the thumb, presence of macroglands on body, vomerine teeth absent, tubercles on eyelids, and anterior tip of snout with vertical white bar (Dubois 1980; Lathrop et al. 1998; Delorme et al. 2006). Leptolalax isos sp. nov. 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) males with wide lateral dermal fringes on Finger II, (5) a body size of mm in 38 adult males, mm in 9 adult females, (6) near immaculate white chest and belly, (7) absence of white speckling on the dorsum and (8) a call consisting of 2 5 notes with a dominant frequency of khz (at º C). FIGURE 3. (A) ventral, (B) dorsal and (C) lateral views of male holotype of Leptolalax isos sp. nov. (VNMN A /AMS R ) in preserve. Scale bar = 5 mm. 406 Zootaxa 4039 (3) 2015 Magnolia Press ROWLEY ET AL.

7 FIGURE 4. Right hand and left foot of preserved paratype of Leptolalax isos sp. nov. (MVZ ) Scale bar = 2 mm. Description of holotype. Head longer than wide; snout rounded in profile and obtusely pointed in dorsal view, projecting over lower jaw; nostril closer to tip of snout than eye; canthi rostralis oval, constricted; lores sloping, slightly concave; vertical pupil; diameter of eye slightly less or equal to length of snout; tympanum distinct, round, diameter approximately one-half that of the eye; vomerine teeth absent; pineal ocellus absent; large oval vocal sac openings present, located on either side of floor of mouth; paired internal subgular vocal sac, loose skin on either side of gular region just anterior to insertion of arms; tongue large, moderately broad, with slight notch at tip; distinct, raised supratympanic ridge running from corner of eye to axillary gland. Tips of fingers rounded, narrower than width of fingers; relative finger lengths I < II = IV < III; nuptial pad absent; subarticular tubercles absent; a large, inner palmar tubercle distinctly separated from slightly smaller, laterally compressed outer palmar tubercle; no finger webbing; slight lateral fringes on Finger III and IV, Finger II with distinct lateral fringes (Fig 4A). Tips of toes like fingers; relative toe length I < II < V < III < IV; subarticular tubercles absent, replaced by dermal ridges, distinct on second, third and fourth toes; oval inner metatarsal tubercle pronounced, outer metatarsal tubercle absent; webbing basal; wide lateral fringes (Fig. 4B). Tibia 48% of snout-vent length; tibiotarsal articulation reaches to nostril. Skin on dorsum mostly smooth, with indistinct, low tubercles in preserve, more obvious in life; ventral skin smooth; dorsal ridges or dorsolateral folds absent; pectoral gland approximately 1.4 mm diameter; femoral gland oval, 0.8 mm diameter, on posteroventral surface of thigh, closer to knee than to vent; supra-axillary gland raised, 1.0 mm diameter; pair of small glands above vent, ~0.5 mm. Ventrolateral glandular line present as indistinct, broken lines, on anterior half of body only. Colour of holotype in life. Dorsum medium brown with slightly darker brown markings, the most distinct of these being an interorbital bar, a W-shaped mark on axilla, indistinct wash over most of tympanum, barring on upper lip, and transverse barring on dorsal surface of limbs including fingers and toes; ventral surface of elbow and upper arm without dark bars; pale orange wash on elbows. Throat and ventral surface of arms pale pink and slightly transparent, particularly at either side of throat; edges of throat pale brown with white speckling, concentrated towards snout; ventral surface of chest and abdomen immaculate white; ventral surface of legs pinkish grey with A NEW MEGOPHRYID FROG SPECIES FROM INDOCHINA Zootaxa 4039 (2) 2015 Magnolia Press 407

8 small whitish spots concentrated on edges. Supra-axillary gland and glands above vent orange, pectoral gland white, edged with pale pink, femoral glands white, edged with brown. Iris gold, slightly more coppery in upper half, with minute, black reticulations. Colour of holotype in preservative. Nearly uniform dark brownish grey dorsal surface, with indistinct darker barring on surface of limbs including fingers and toes (Fig. 3). Ventral surface including throat pale brown to creamy white. Margins of ventral surface with pale brown speckling, margins of throat and ventrolateral surfaces of arms and thighs, and entire tibiotarsus and upper arm pale brown with white speckling; macroglands white. Measurements. Holotype: SVL 25.7, HDL 9.9, HDW 9.5, SNT 4.1, EYE 3.3, IOD 3.2, TMP 1.8, TEY 1.1, TIB 12.3, EN 2.3, IN 2.4, NS 1.6, ML 7.1, PL 11.1, F1 2.4, F2 2.9, F3 5.3, PEC 1.4. Weight in life 1.6 g. Variation. Like L. firthi, L. isos sp. nov. is sexually dimorphic in regards to body size, skin texture and the extent of lateral fringes and webbing on feet and fringes on finger II. Male L. isos sp. nov. are significantly smaller than females (Wilcoxon test, Z= , p < ), and generally have only low, flattened tubercles spaced relatively widely on the dorsum, in contrast to the highly tuberculate dorsum of females (although there is much variation in skin texture among individuals). Fringes on finger II are present only in males and are most pronounced in the holotype VNMN A /AMS R and paratypes AMS R , AMS R , AMS R , AMS R , AMS R , MVZ , MVZ , MVZ , MVZ , and MVZ Webbing and fringes on the feet are pronounced in males. Dorsal colour in life varies from pale to medium brown. In both sexes, skin texture more tuberculate in life compared to in preserve. Measurements of the type series are shown in Table 2. TABLE 2. Measurements (mm) of adult Leptolalax isos sp. nov. Abbreviations defined in text. * Not recorded in all specimens from Cambodia (13 male, 2 female). Measurements Males Females Range; Mean ± S. D. (N=38) Range; Mean ± S. D. (N=9) SVL ; 25.1 ± ; 29.4 ± 0.9 HDL ; 9.7 ± ; 11.6 ± 0.4 HDW ; 9.0 ± ; 10.7 ± 0.5 SNT ; 3.8 ± ; 4.3 ± 0.1 EYE ; 3.2 ± ; 3.8 ± 0.2 IOD ; 2.8 ± ; 3.2 ± 0.4 TMP ; 1.7 ± ; 2.1 ± 0.1 TEY ; 1.1 ± ; 1.3 ± 0.2 TIB ; 12.1 ± ; 14.1 ± 0.4 EN ; 2.1 ± 0.2* ; 2.4 ± 0.2* IN ; 2.6 ± 0.2* ; 2.8 ± 0.2* NS ; 1.7 ± 0.1* ; 1.8 ± 0.1* ML ; 6.9 ± ; 7.9 ± 0.3 PL ; 11.2 ± ; 12.5 ± 0.8 F ; 2.6 ± ; 3.1 ± 0.2 F ; 3.1 ± ; 3.8 ± 0.8 F ; 5.3 ± ; 5.9 ± 0.3 PEC ; 1.0 ± ; 1.3 ± 0.3 Range; Mean ± S. D. (N=31) Range; Mean ± S. D. (N=9) Weight (g) ; 1.4 ± ; 2.1 ± 0.2 Range; Median (N=38) Range; Median (N=9) HDL:HDW ; ; 1.09 HDL:SVL ; ; 0.39 TIB:SVL ; ; 0.48 TMP:SVL ; ; Zootaxa 4039 (3) 2015 Magnolia Press ROWLEY ET AL.

9 FIGURE 5. Advertisement call of Leptolalax isos sp. nov. (A) 30 s waveform of relative amplitude (Rel. amp.) over time for (i) paratype AMS R , (ii) holotype VNMN A /AMS R , (iii) non-vouchered male, (iv) JR8. (B) (i)waveform and (ii) corresponding spectrogram of single representative call containing two notes, and (iii) power spectrum (relative amplitude vs. frequency) of first note. (C) Representative single advertisement calls of (i) paratype AMS R , (ii) holotype VNMN A /AMS R , (iii v) non-vouchered individual 1, (vi) non-vouchered individual 2. All recorded at C. A NEW MEGOPHRYID FROG SPECIES FROM INDOCHINA Zootaxa 4039 (2) 2015 Magnolia Press 409

10 Advertisement call. Call descriptions are based on the calls of the holotype, recorded at 22.8ºC ambient temperature. Calls were an average of 34 ms in duration and consisted of three notes 92% of the time (92/100 notes recorded; Table 3). Within each call, amplitude and note duration decreased with each successive note. Notes contained a single pulse. The dominant frequency was khz, and harmonics were present at approximately 15.4, and 18.7 khz (Fig. 5). A fundamental frequency was not evident. Calls were repeated at a rate of approximately one call per second, and had an average intercall interval of one second. In the calls of the four individuals recorded, the number of notes with two notes per call varied from 1 8%. Dominant frequency varied only slightly among individuals, from khz (over 0.4 ºC difference in ambient temperature). To the human ear, the advertisement call of L. isos sp. nov. is a rapid, high-pitched chirping, similar to an orthopteran. TABLE 3. Measurements of advertisement call parameters for Leptolalax isos sp. nov. Parameter values are given as means (and ranges). * holotype. AMS R VNMN A /AMS Non-vouchered Non-vouchered R * Number of calls measured Number of notes measured Call duration (ms) 31 (30 34) 34 (31 38) 29.7 (28 32) 33 (31 34) Call repetition rate (calls/s) Intercall interval (ms) 968 ( ) 349 ( ) 380 ( ) 565 ( ) Notes/call Dominant frequency (khz) ( ) 6.1 ( ) 6.2 ( ) Note 1 duration 10 (8 11) 5 (4 7) 6 (4 7) 9 (7 12) Note 2 duration 8 (6 10) 5 (4 7) 6 (5 6) 8 (6 9) Note 3 duration 6 (4 7) 8 (5 10) 7 (6 9) 12 (10 13) Temperature ( C) Molecules. Leptolalax isos sp. nov. was recovered within the clade of northern Leptolalax, and sister to L. firthi (Fig. 6). Uncorrected 16S rrna sequence divergences between L. isos sp. nov. and all homologous sequences available on GenBank (sequences assigned to 25 species; Table 1) 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). The uncorrected pairwise divergence between L. isos sp. nov. and L. firthi collected from ~ km apart was %. In contrast, intraspecific variation in this gene fragment for L. isos sp. nov. sampled up to ~140 km apart was only %. Ecology. All specimens were found at night in semi-evergreen and hill evergreen forest between ~ m asl. Males were observed calling from on rocks, stream banks and on vegetation within or adjacent to rocky streams and were heard calling in June and August (Fig. 7). Leptolalax isos sp. nov. was sympatric with L. melicus at Virachey National Park, and with L. cf. melicus at Kon Ka Kinh National Park. Conservation status. The species is known only from Virachey National Park in Cambodia and Kon Ka Kinh National Park in Vietnam. Its true extent of occurrence is unknown but the species 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 Chu Mom Ray Nature Reserve in Vietnam, both of which contain evergreen forest at similar elevations that may represent suitable habitat for L. isos sp. nov. Suitable forested regions may also include Kon Chu Rang Nature Reserve and An Toan Nature Reserve in Vietnam. Comparisons. In lacking distinct dorsolateral markings including blackish spots on the flank and dark canthal and/or temporal streaks, L. isos sp. nov. is distinguished from all but L. aereus, L. arayai, L. croceus, L. eos, L. firthi, L. laui, L. tuberosus and L. zhangyangpingi (L. alpinis, L. applebyi, L. bidoupensis, L. botsfordi, L. bourreti, L. dringi, L. fulignosus, L. fritinniens, L. gracilis, L. hamidi, L. heteropus, L. kajangensis, L. kecil, L. khasiorum, L. lateralis, L. liui, L. marmoratus, L. maurus, L. melanoleucus, L. melicus, L. minimus, L. nahangensis, L. nokrekensis, L. nyx, L. oshanensis, L. pelodytoides, L. pictus, L. platycephalus, L. pluvialis, L. pyrrhops, L. 410 Zootaxa 4039 (3) 2015 Magnolia Press ROWLEY ET AL.

11 sabahmontanus, L. solus, L. sungi, L. tamdil and L. ventripunctatus all have blackish spots on the flank, and dark canthal and/or dark temporal streaks). FIGURE 6. Bayesian inference (BI) trees on ~561 bp fragment of the 16S gene for Leptolalax firthi and L. isos sp. nov., along with representatives of all Leptolalax species for which comparable sequences are available. Node support is indicated by circles; those with Bayesian posterior probabilities of are grey and 1.00 are black. Distance between sample localities and between species are indicated via arrows to the right. From L. aereus, L. isos sp. nov. can be distinguished by the degree of lateral fringes on the toes, the male advertisement call and molecular data (see below); from L. arayai, L. isos sp. nov. can be distinguished by the degree of lateral fringes on the toes, the male advertisement call, and molecular data (see below); from L. croceus, the new species can be distinguished by the degree of lateral fringes on toes, ventral coloration, visibility of the tympanum, the male advertisement call and molecular data (see below); from L. eos, L. isos sp. nov. can be distinguished molecularly and by size (see below); from L. firthi, L. isos sp. nov. can be distinguished by body size and weight, relative tympanum size in females, absence of white speckling on the dorsum, male advertisement call and molecular data (see below); from L. laui, the new species can be distinguished by the degree of lateral fringes on toes and molecular data (see below); from L. tuberosus, the new species can be distinguished by the ventral pattern, degree of lateral fringes on toes, visibility of the tympanum, male advertisement call and molecular data (see below); and from L. zhangyangpingi the new species can be distinguished by body size, degree of lateral fringes on the toes and molecular data (see below). In having toes with broad lateral fringes in males, L. isos sp. nov. is further distinguished from all but L. alpinus L. eos, L. firthi, L. liui, L. zhangyangpingi (all other male congeners lack fringes or have only weak fringes on their toes). L. firthi appears to be the only species with similarly broad fringes. Similarly, in having most males with wide lateral dermal fringes on Finger II, L. isos sp. nov. appears to differ from all congeners except for L. firthi. A NEW MEGOPHRYID FROG SPECIES FROM INDOCHINA Zootaxa 4039 (2) 2015 Magnolia Press 411

12 FIGURE 7. (A) Female paratype AMS R , (B) male paratype VNMN A /AMS R , (C) unvouchered individual and (D) male paratype AMS R of Leptolalax isos sp. nov. in situ. (E F) Representative breeding habitat for Leptolalax isos sp. nov. in Kon Ka Kinh National Park, Vietnam. Leptolalax isos sp. nov. is a medium-sized species of Leptolalax ( mm in 38 adult males, mm in 9 adult females), and can be distinguished on the basis of at least male body size from the smaller L. applebyi (males mm, females mm), L. kecil (males mm, female 25 mm), L. melicus (males mm) and L. pluvialis (males mm), and the larger L. bourreti (male 36.2 mm), L. botsfordi (males , females mm), L. dringi (males ), L. eos (males ), L. 412 Zootaxa 4039 (3) 2015 Magnolia Press ROWLEY ET AL.

13 fritinniens (males , females mm), L. fulignosus (males mm), L. gracilis (males mm, females mm), L. hamidi (males mm), L. kajangensis (males mm), L. lateralis ( mm), L. marmoratus (males , females ), L. nahangensis (male 40.8 mm), L. pictus (males mm), L. platycephalus (male 35.1, female 46.0 mm), L. pyrrhops (males mm, females mm), L. sungi (males mm, females ), L. tamdil (male 32.3 mm, female 31.8 mm), and L. zhangyangpingi (males ). In having an immaculate white chest and belly with only slight brown specking at the margins, L. isos sp. nov. can be distinguished from L. alpinis, L. applebyi, L. bidoupensis, L. botsfordi, L. croceus, L. dringi, L. fritinniens, L. fulignosus, L. gracilis, L. heteropus, L. kajangensis, L. kecil, L. marmoratus, L. maurus, L. melanoleucus, L. melicus, L. nahangensis, L. pluvialis, L. pyrrhops, 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. isos sp. nov. is differentiated from L. croceus, L. sungi and L. tuberosus. In having a relatively uniformly coloured gold iris, often only slightly more coppery in the upper half, L. isos sp. nov. is differentiated from species with distinctly bicolored irises (L. arayai [bright red brown upper, white/grey below], L. bidoupensis [coppery red upper, silver/gold below], L. gracilis [bright red upper and around pupil, dull greyish red below], L. frittiniens [red upper and around pupil, grey below], L. fuliginosus [red upper, copper/gold below], L. khasiorum [bright orange upper, cream below], L. marmoratus [reddish upper and around pupil, white below], L. melanoleucus [orange upper, silver below], L. nokrekensis [bright red upper], L. pictus [upper and around pupil golden brown, pale golden/grey below], L. sabahmontanus [red upper and around pupil, silver below], L. solus [upper dark red, brown below], L. tamdil [upper bright orange, greyish cream below], L. zhangyangpingi [golden upper, grey below]), and from species with relatively uniform but very different iris colours (L. kecil [dark red], L. maurus [red brown], L. sungi [greenish gold]). The new species is most morphologically similar to L. firthi, but differs in a number of characteristics. Although L. isos sp. nov. overlaps slightly in body size with L. firthi, L. isos sp. nov. has significantly smaller males ( mm [x = 25.1 mm] mm versus mm [x = 27.8 mm] in L. firthi; Wilcoxon test, Z= , p < ; Fig. 8A) and smaller females ( mm [x = 29.4 mm] versus mm [x = 33.1 mm] in L. firthi; Wilcoxon test, Z= , p = , Fig. 8B). L. isos sp. nov. also weighs significantly less in life than L. firthi in both males ( g [x = 1.4g] versus g [x = 1.8g] in L. firthi; Wilcoxon test, Z= , p < , Fig 8C), and females ( g [x = 2.1g] versus g [x = 2.9g] in L. firthi; Wilcoxon test, Z= , p = , Fig 8D). Female L. isos sp. nov. also have larger relative tympana compared to female L. firthi (TMP/SVL Wilcoxon test, Z= , p < 0.001; Fig. 8E). In addition, no L. isos sp. nov. in the type series (n=47) display white speckling on the dorsum, while 90% of male L. firthi and 79% of female L. firthi in the type series (n=35) have white speckling on the dorsum. The advertisement call of L. isos sp. nov. is unique among congeners with known calls. In lacking strong frequency modulation, the call of L. isos sp. nov. differs from L. dringi, L. hamidi and L. sabahmontanus. In having a relatively high dominant frequency ( khz; ºC) the call of L. isos sp. nov. can be readily distinguished from that of L. applebyi ( khz, 21.5ºC), L. bidoupensis ( khz, ºC ), L. croceus ( khz, ºC), L. fuliginosus ( khz, ºC), L. gracilis ( khz, 20ºC), L. heteropus (2.8 khz, 21ºC), L. kecil (3.2 khz, 21.4ºC), L. melanoleucus ( khz, 23.9ºC), L. melicus ( khz, ºC), L. oshanensis ( khz, 14ºC; recorded from c. 40 km from type locality of L. oshanensis), L. pyrrhops ( khz at 25 C), L. solus ( khz, ºC), and L. tuberosus ( khz, ºC). The call of L. isos sp. nov. also appears to be lower in frequency compared to that attributed to L. dringi ( khz, 24.3 ºC) and L. sabahmontanus ( khz, ºC). Although frequency can vary with temperature, differences among species of the scale reported here are 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. firthi, L. maurus and L. pictus. The advertisement call of L. isos sp. nov. has a lower call repetition rate ( calls/s) compared to L. arayai ( calls/s), L. hamidi ( calls/s), and L. pictus (11 13 calls/s), has a lower number of notes per call (2 3) compared to L. alpinus (9.45 ± 2.73), and has a lower call duration (18 51 ms) compared to L. maurus (85 ms). The call of L. isos sp. nov. is most similar to that of L. firthi, but based upon the recordings available the two species differ in call repetition rate ( calls/s compared to in L. firthi at similar temperatures) and variability in the number of notes per call (2 3 notes/call [92 100% 3-note calls] in L. isos sp. nov. versus 2 5 notes/call [0 98% 3 note calls] in L. firthi). A NEW MEGOPHRYID FROG SPECIES FROM INDOCHINA Zootaxa 4039 (2) 2015 Magnolia Press 413

14 FIGURE 8. Statistically significant morphometric differences between L. firthi and L. isos sp. nov. (A) Male and (B) female SVL (mm), (C) male and (D) female weight in life (g), and (E) female relative tympanum diameter (TMP/SVL). Abbreviations defined in text. In addition, uncorrected sequence divergences between L. isos sp. nov. 16S rrna sequences and all homologous sequences available on GenBank (Table 1) were >10%. Discussion Leptolalax isos sp. nov. is known from mid-high elevations (~ m) in the western and southern slopes of the Kon Tum Plateau. Its sister species, L. firthi, occurs on the northern edge of the same plateau. Similar distribution patterns have been documented in the smaller-bodied Leptolalax, L. applebyi and L. melicus. Leptolalax applebyi is only known from Quang Nam and Kon Tum Provinces in Vietnam, at the same two sites at which L. firthi is known, while L. melicus is only known from Virachey National Park in Cambodia (Rowley et al. 2010c). Leptolalax isos sp. nov. is only the second confirmed species in the genus from Cambodia after L. melicus, but the 16th species of Leptolalax confirmed from Vietnam (Frost 2015). At present, Vietnam is the country with the highest known number of Leptolalax species recorded. The historical distribution of L. isos sp. nov. is likely to have been considerably reduced due to forest loss, and illegal logging and agricultural expansion are ongoing threats, even within Virachey and Kon Ka Kinh National Parks (Ha et al. 2011; Grimsditch 2012; Birdlife International 2015). Hydroelectric projects are likely to pose an additional threat within Virachey National Park (Bottomley 2000). Acknowledgements Research was supported by grants from the ADM Capital Foundation, The John D. and Catherine T. MacArthur Foundation, the Annie Alexander Endowment, Museum of Vertebrate Zoology, University of California, Berkeley, Conservation International and a STMVQG 06G14-16 project. 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]). 414 Zootaxa 4039 (3) 2015 Magnolia Press ROWLEY ET AL.

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