Asian Herpetological Research 2013, 4(2): 100–108 DOI: 10.3724/SP.J.1245.2013.00100

A New Species of the Genus Leptolalax (Anura: Megophryidae) from Northern Thailand

Ke JIANG1, Fang YAN1, 2, Chatmongkon SUWANNAPOOM1, 3, Siriwadee CHOMDEJ3 and Jing CHE1*

1 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China. 2 Yunnan Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China. 3 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand.

Abstract A new species of the genus Leptolalax is described from Doi Saket, Chiang Mai Province, northern Thailand. The new species is distinguished from its congeners by the following combination of characters: 1) adult size large (male SVL 45.8−52.5 mm); 2) tympanum distinct; 3) skin of dorsal surface mostly smooth, with distinct tubercles on dorsal and lateral parts of the body; 4) ventrolateral glandular ridge distinct and complete; 5) lateral body and groin covered with dark brown or black spots; 6) throat, chest, and belly nearly immaculate white; and 7) bi-colored iris, upper one- third golden, and lower two-thirds gray. Molecular phylogenetic analysis of mitochondrial 16S rRNA further distin- guishes it from its congeners for which sequences are available. Keywords Anura, new species, molecular phylogenetics, Doi Saket, Thailand

1. Introduction Following euthanasia, the voucher specimens were fixed in 10% buffered formalin, and then transferred to 70% The genus Leptolalax Dubois 1983 (Anura: Mego- ethanol. Tissue samples were taken from the liver and phryidae) currently contains 36 species, distributed preserved in 95% ethanol. Seven specimens in the best in rocky streams in hilly evergreen forest throughout condition were chosen as the type series. All specimens Southeast Asia, southern China and northeastern India were deposited in the museum of the Kunming Institute (Frost, 2013; Rowley et al., 2011; Rowley et al., 2012). of Zoology (KIZ), Chinese Academy of Sciences. Most species of Leptolalax are relatively small, with few species having snout-vent length over 40 mm in both 2.2 Morphological analyses All measurements were males and females. made with slide calipers to the nearest 0.1 mm. The During a herpetological survey in northern Thailand in measurements and their abbreviations include: SVL October 2011, a relatively large Leptolalax species was (snout-vent length); HL (head length, from posterior collected. Based on its morphological and molecular data, corner of mandible to tip of snout); HW (head width, at we conclude that it is a new species and describe it herein. the angle of jaws); IN (internarial distance); NS (distance from nostril to tip of snout); ED (horizontal eye diameter); 2. Material and Methods IO (interorbital distance, least distance between upper eyelids); UE (maximum width of upper eyelid); EN 2.1 Sampling A total of 11 individuals were collected (distance from anterior corner of eye to nostril); HTD from Doi Saket, Chiang Mai Province, northern Thailand. (horizontal tympanic diameter); FLL (forelimb length, from elbow to proximal end of outer palmar tubercle); * Corresponding author: Prof. Jing CHE, from the Kunming Institute of Zoology, Chinese Academy of Sciences, Yunnan, China, with HAL (hand length, from proximal end of outer palmar her research focusing on the systematics, molecular evolution, and tubercle to tip of the third finger); IPT (length of inner biogeography of Asian amphibians and reptiles. E-mail: [email protected] palmar tubercle, greatest length); FML [femur (thigh) Received: 9 March 2013 Accepted: 21 May 2013 length, from vent to knee]; FOL (foot length, from No. 2 Ke JIANG et al. New species of Leptolalax from Thailand 101 proximal end of inner metatarsal tubercle to tip of fourth were aligned using ClustalX 1.81 (Thompson et al., toe); IMT (length of inner metatarsal tubercle, greatest 1997) with default parameters, and then optimized by length); TFL (length of tarsus and foot, from proximal eye in MEGA 4.0 (Tamura et al., 2007). Sequences of end of tarsus to tip of the fourth toe); and TBL [tibia 16S including 18 species of the genus Leptolalax and 10 (shank) length]. species as outgroup were downloaded from GenBank to We obtained comparative morphological data from infer phylogenetic relationships (Table 1). museum specimens (Appendix) and from the literature: Phylogenetic reconstructions using Bayesian inference Leptolalax aereus (Rowley et al., 2010c), L. alpinus (Fei (BI) were executed in MrBayes 3.1.2 (Ronquist and et al., 1990, 2009), L. applebyi (Rowley and Cao, 2009), Huelsenbeck, 2003), using the model of best-fit estimated L. arayai (Matsui, 1997), L. bidoupensis (Rowley et al., with MrModeltest v 2.3 (Nylander, 2004). Bayesian 2011), L. bourreti (Dubois, 1983; Ohler et al., 2011), L. posterior probabilities (BPP) were used to estimate nodal croceus (Rowley et al., 2010b), L. dringi (Dubois, 1987), support. Two separate runs were performed with four L. eos (Ohler et al., 2011), L. firthi (Rowley et al., 2012), Markov chains. Each run was conducted for 10 million L. fuliginosus (Matsui, 2006), L. gracilis (Günther, 1872), generations and sampled every 100 generations. The first L. hamidi (Matsui, 1997), L. heteropus (Boulenger, 1900), 25% of trees were discarded, and a consensus tree was L. kajangensis (Grismer et al., 2004), L. kecil (Matsui et calculated from the remaining trees. Uncorrected pairwise al., 2009), L. khasiorum (Das et al., 2010), L. lateralis sequence divergence among species was calculated using (Anderson, 1871; Humtsoe et al., 2008), L. liui (Fei et al., MEGA 4. 1990; Fei et al., 2009), L. maurus (Inger et al., 1997), L. melanoleucus (Matsui, 2006), L. melicus (Rowley et al., 3. Results 2010a), L. minimus (Ohler et al., 2011; Taylor, 1962), L. nahangensis (Lathrop et al., 1998), L. nokrekensis (Das 3.1 Description and Deuti, 2011; Mathew and Sen, 2010), L. nyx (Ohler et Leptolalax zhangyapingi sp. nov. (Figures 2−4) al., 2011), L. oshanensis (Fei et al., 2009; Liu, 1950), L. Holotype: KIZ07258, an adult male from Phang Num pelodytoides (Boulenger, 1893, 1908; Ohler et al., 2011), Poo, Doi Saket, Chiang Mai Province, northern Thailand L. pictus (Malkmus, 1992; Malkmus et al., 2002), L. (18.98° N, 99.33° E, elevation 870 m; Figure 1), collected platycephalus (Dehling, 2012), L. pluvialis (Ohler et al., by Ke JIANG at night on 11 October 2011. 2000, 2011), L. solus (Matsui, 2006), L. sungi (Lathrop Paratypes: Three adult males, KIZ07240, KIZ07241, et al., 1998), L. tamdil (Sengupta et al., 2010), L. KIZ07259, collected at the same locality as the holotypes; tuberosus (Inger et al., 1999; Rowley et al., 2010b), and and three adult males, KIZ07456, KIZ07461, KIZ07466, L. ventripunctatus (Fei et al., 1990, 2009). The museum were collected at night on 14 October 2011, from Pang abbreviations are provided as: Chengdu Institute of Kompang Hill, Doi Saket, Chiang Mai Province, northern Biology, Chinese Academy of Sciences (CIB), and KIZ. Thailand (18.94° N, 99.34° E, elevation 1026 m). All 2.3 Molecular analyses Total DNA was extracted specimens were collected by Ke JIANG, Fang YAN, and from liver tissue using the standard phenol-chloroform Chatmongkon SUWANNAPOOM. protocols (Sambrook et al., 1989). A fragment of the 16S rRNA gene from 11 samples was PCR amplified and sequenced with the primers 16Sar 5'-CGCCTGTTTAYCAAAAACAT-3' and 16Sbr 5'-CCGGTYTGAACTCAGATCAYGT-3' (Kocher et al., 1989). Amplification was performed in a 10 μL volume reaction with the following procedures: initial denaturation step at 95°C for 5 min, 35 cycles of denaturation at 94°C for 45 sec, annealing at 55°C for 45 sec, extension at 72°C for 45 sec, and final extension at 72°C for 10 min. PCR products were enzymatically purified using a modification of the Exo-SAP method (Werle et al., 1994), and then sequenced in both Figure 1 Map showing the type locality (red dot) of Leptolalax directions with BigDye Terminator Cycle Sequencing zhangyapingi sp. nov. at Phang Num Poo, Chiang Mai Province, Kit and ABI PRISM 3730. Nucleotide sequences Thailand (18.98° N, 99.33° E). 102 Asian Herpetological Research Vol. 4

Table 1 Species, GenBank accession numbers, and localities of the individuals used in molecular analysis.

GenBank Species Country Locality Accession No. Outgroup Leptobrachium sp. 1 JN848348 India Guwahati, Assam Pradesh L. pullum 1 JN848342 Thailand Phang Nga, Phang Nga Province L. pullum 2 JN848340 Thailand Phang Nga, Phang Nga Province L.smithi 1 JN848346 Laos Ban Keng Koung, Luang Prabang Province L. smithi 2 JN848345 Laos Ban Sop Khao, Luang Prabang Province Leptobrachium. sp. 2 JN848352 Thailand Doi Chiang Dao, Chiangmai Province L. chapaense JN848353 Vietnam Ben En NP, Thanh Hoa Province Brachytarsophrys carinensis 1 JN848360 Myanmar Undisclosed locality B. carinensis 2 JN848361 Myanmar Undisclosed locality Xenophrys parva JN848362 Vietnam Sa Pa, Lao Cai Province Xenophrys sp. 1 JN848364 Laos Ban Keng Koung, Luang Prabang Province Xenophrys sp. 2 JN848365 Laos Ban Keng Koung, Luang Prabang Province Xenophrys sp. 3 JN848363 Thailand Doi Chiang Dao, Chiang Mai Province Ingroup L. aereus 1 AF285193 Vietnam Phong Nha - Ke Bang, Quang Binh Province L. aereus 2 JN848442 Vietnam Cha Noi region, Phong Nha - Ke Bang, Quang Binh Province L. aereus 3 AF285192 Vietnam Ha Thinh Province L. aereus 4 JN848440 Vietnam Cha Noi region, Phong Nha - Ke Bang, Quang Binh Province L. applebyi HM133598 Vietnam Song Thanh Proposed Nature Reserve, Quang Nam Province L. arayai DQ642119 Unknown L. bidoupensis 1 HQ902880 Vietnam Langbian Plateau, Lam Dong Province L. bidoupensis 2 HQ902881 Vietnam Langbian Plateau, Lam Dong Province L. bidoupensis 3 HQ902882 Vietnam Langbian Plateau, Lam Dong Province L. bidoupensis 4 HQ902883 Vietnam Langbian Plateau, Lam Dong Province L. bourreti JN848454 Vietnam Sa Pa, Lao Cai Province L. eos 1 JN848446 Laos Long Nai, Phongsaly Province L. eos 2 JN848447 Laos Long Nai, Phongsaly Province L. eos 3 JN848448 Laos Long Nai, Phongsaly Province L. firthi 1 JQ739208 Vietnam Ngoc Linh Nature Reserve, Kon Tum Province L. firthi 2 JQ739206 Vietnam Ngoc Linh Nature Reserve, Kon Tum Province L. firthi 3 JQ739204 Vietnam Ngoc Linh Nature Reserve, Kon Tum Province L. firthi4 JQ739205 Vietnam Ngoc Linh Nature Reserve, Kon Tum Province L. heteropus 1 JN848354 Thailand Long Nai, Long Nai Province L. heteropus 2 JN848356 Thailand Long Nai, Long Nai Province L. heteropus 3 JN848359 Thailand Long Nai, Long Nai Province L. liui EF544238 China Jinxiu County, Guangxi L. melicus 1 HM133599 Cambodia Virachey National Park, Ratanakiri Province L. melicus 2 HM133600 Cambodia Virachey National Park, Ratanakiri Province L. melicus 3 HM133601 Cambodia Virachey National Park, Ratanakiri Province L. minimus 1 JN848366 Laos Ban Nong Di, Luang Prabang Province L. minimus 2 JN848370 Thailand Mae Lao Mae Sae, Chiangmai Province L. minimus 3 JN848372 Laos Ban Nong Di, Luang Prabang Province L. minimus 4 JN848397 Laos Ban Nong Di, Luang Prabang Province L. nyx DQ283381 Vietnam Vi Xuyen, Ha Giang Province L. oshanensis 1 AY561306 China Emei Shan, Sichuan L. oshanensis 2 AY526215 China Jinfo Shan, Chongqing L. pelodytoides 1 AY236797 Vietnam Lao Cai Province L. pelodytoides 2 AY236798 Vietnam Tam Dao, Vinh Phu Province L. pelodytoides 3 EF397244 Vietnam Tam Dao, Vinh Phu Province L. pictus DQ642120 Unknown L. pluvialis 2 JN848391 Vietnam Sa Pa, Lao Cai Province Leptolalax . sp. JN848455 Thailand Doi Chiang Dao, Chiang Mai Province L. ventripunctatus 1 JN848411 Laos Phongsaly, Phongsaly Province L. ventripunctatus 2 JN848414 Laos Long Nai, Phongsaly Province L. ventripunctatus 3 JN848425 Laos Long Nai, Phongsaly Province L. ventripunctatus 4 JN848410 Laos Phongsaly, Phongsaly Province L. zhangyapingi sp. nov. JX069979 Thailand Pang Num Poo, Chiang Mai Province No. 2 Ke JIANG et al. New species of Leptolalax from Thailand 103

combination of morphological characters: 1) adult size large (male SVL 45.8−52.5 mm); 2) tympanum distinct; 3) skin of dorsal surface mostly smooth, with distinct tubercles on dorsal and lateral parts of the body; 4) ventrolateral glandular ridge distinct and complete; 5) lateral body and groin covered with dark brown or black spots; 6) throat, chest, and belly nearly immaculate white; and 7) bi-colored iris, upper one-third golden, and lower two-thirds gray. Description of the holotype (KIZ07258, adult male Figures 3−4): Measurements are provided in Table 2. Body moderately slender; head large, as wide as long; snout obtusely pointed, slightly projecting beyond jaw; canthus rostralis obtuse, loreal region oblique and concave; internarial distance approximately equal to the distance from anterior margin of eye to nostril (IN/ EN 1.02); eye large (ED/HL 0.33), diameter larger than eye to nostril distance (ED/EN 1.56); interorbital space flat; interorbital width equal to upper eyelid width (IO/ UE 1.02); pupil vertical; tympanum and tympanic

Figure 2 Male of Leptolalax zhangyapingi sp. nov. in life (KIZ07466, paratype). A: Dorsolateral view; B: Ventral view. Photo by C. SUWANNAPOOM.

Etymology: Following the Chinese style of placing the given name after the family name, the species is named after Prof. Yaping Zhang, the sponsor of the international “COLD CODE” program of DNA barcoding amphibians and reptiles. We acknowledge his continuous support and encouragement of our biodiversity survey in southeastern Asia. Its common name is suggested as Zhang’s Asian Toad. Diagnosis: Leptolalax zhangyapingi sp. nov. is assigned to the genus Leptolalax on the basis of the following characters: small size; rounded finger tips; presence of an elevated inner palmar tubercle which does not extend to the thumb; presence of macroglands on body (including supra-axillary, pectoral, femoral and ventrolateral glands); vomerine teeth absent; tubercles present on eyelids; and anterior tip of snout with pale vertical bar (Dubois, 1983; Lathrop et al., 1998; Delorme et al., 2006; Rowley et al., 2012). Leptolalax zhangyapingi sp. nov. is distinguished Figure 3 Holotype (KIZ07258) of Leptolalax zhangyapingi sp. from all other species of Leptolalax by the following nov. A: Dorsolateral view; B: Ventral view. Photo by Ke JIANG 104 Asian Herpetological Research Vol. 4

- annulus distinct; a small knob present on anterior edge 2.5 2.2 2.4 3.0 2.3 2.5 2.8 IMT of mandible; maxillary teeth present; tongue oval, very large, with small notch at posterior tip, without papillae; 2.8 2.4 2.8 3.3 2.8 2.8 3.2 IPT choanae large, located close to anterior border of palate, visible when viewed from below; vomerine teeth absent; internal single subgular vocal sac present, openings near 21.8 20.3 21.7 21.6 21.2 21.8 22.1 TBL the corners of mouth; and supratympanic fold distinct, from eye towards axillary gland. TFL 31.1 29.2 33.1 33.2 31.7 30.4 33.4 Forelimbs long; forearm almost equal to hand (FLL/ HAL 1.04); fingers slender, with no interdigital web, 20.6 19.8 21.3 21.6 20.7 20.2 21.2 FOL dermal fringes feeble; relative length of fingers: 1 = 2 < 4 < 3; finger tips rounded, slightly enlarged; subarticular 24.4 23.8 24.3 25.2 23.4 23.4 25.1

FML tubercles indistinct; inner palmar tubercle large and prominent, outer palmar tubercle small and flat, about one-third size of the inner one, the two separated by a 14.0 12.9 14.0 15.0 14.0 13.6 14.8 HAL indistinct furrow; and nuptial pads absent. Hindlimbs moderately short (TBL/SVL 0.43), tibio- FLL 14.6 14.0 15.9 14.7 14.6 14.9 14.4 tarsal articulation reaching to the middle of eye when limbs adpressed anteriorly, heels widely separated when 3.8 3.5 4.1 3.8 3.9 3.3 3.9

HTD hindlimbs flexed and held perpendicularly to body (TBL/ FML 0.89); foot much longer than shank (TFL/TBL ratio 8.7 8.3 9.2 9.3 8.5 8.2 9.1 EN 1.42); toe tips rounded, slightly enlarged, without grooves, with rudimentary webs; dermal fringes moderate; relative length of toes: 1 < 2 < 5 < 3 < 4; subarticular tubercles 5.2 4.5 5.0 5.7 4.5 3.6 4.8 UE indistinct; distinct ridges found on plantar side of toes; and inner metatarsal tubercle elliptical and prominent, IO 5.3 4.6 5.6 5.7 5.4 5.0 5.2 and outer metatarsal tubercle absent. Skin mostly smooth, with distinct tubercles on the 7.0 6.6 7.1 7.6 6.7 6.0 6.1 ED dorsal and lateral parts of the body, small but prominent

sp. nov. tubercles present on loreal and temporal regions; 4.2 4.1 4.6 5.0 4.1 4.1 4.5 NS horizontal skin ridges on the dorsal sides of the head, body and limbs, and the lateral side of the body (such ridges indistinct in preservative, rather flat); ventral IN 4.6 4.3 4.4 4.9 4.2 4.1 4.7 surface of throat, body and limbs smooth, ventrolateral glandular ridge present; oval pectoral glands present; HW 20.5 19.1 20.9 22.1 20.4 20.6 21.1 a large but flat femoral gland present on posteromedial Leptolalax zhangyapingi margin of thigh; and round glands developed and present HL 21.5 19.2 21.7 21.6 21.3 20.7 21.8 at the bases of ventral side of the thighs, and below the vent. Coloration of holotype in life: Dorsal and lateral 50.4 45.8 50.5 52.5 49.6 47.6 50.7 SVL parts of head and body yellowish brown, a dark brown subtriangular marking present between eyes, four dark Sex male male male male male male male brown markings from the shoulder region to the lower iliac region; dorsal parts of limbs yellowish brown, with dark brown transverse bands; supratympanic fold dark Status paratype paratype paratype paratype paratype paratype holotype brown; large tubercles on dorsal and lateral body reddish brown; throat, chest, and belly near immaculate white,

Measurements (in mm) of the type series the edge of chin found with small dark brown spots; pectoral glands yellowish white; ventral surface of limbs Number KIZ07258 KIZ07240 KIZ07241 KIZ07259 KIZ07456 KIZ07561 KIZ07466 SVL, snout-vent length; HL, head length, from posterior corner of mandible to tip of snout; HW, head width, at the angle of jaws; IN, internarial distance; NS, head width, at the angle of jaws; IN, internarial distance from nostril to tip snout; ED, hori from posterior corner of mandible to tip snout; HW, SVL, snout-vent length; HL, head length, zontal eye diameter; IO, interorbital distance, least distance between upper eyelids; UE, maximum width of upper eyelid; EN, distance from anterior corner meter; of FLL, eye forelimb length, to from nostril; elbow HTD, to horizontal proximal end tympanic of dia- outer palmar tubercle; HAL, hand length, from proximal end of outer palmar tubercle to tip of the vent third finger;to FML, femurknee; (thigh)FOL, length, from foot length, from proximal end of inner metatarsal tubercle to length of inner metatarsal tubercle, greatest length. tip length of inner palmar tubercle, greatest length; IMT, of length; IPT, fourth toe; TFL, length of tarsus and foot, from proximal end of tarsus to tip of the fourth toe; TBL= tibia (shank) Table 2 Table transparent and pinkish gray; and iris golden in upper No. 2 Ke JIANG et al. New species of Leptolalax from Thailand 105

Ecological notes: All specimens were collected on land at night near a slowly moving stream in montane evergreen forest, at an elevation of approximately 800−1100 m. Males of the species called continuously from beneath leaf litter and stones at night. The call is loud and rapid, sounding to the human ear like “der-der-der-der-der...”. 3.2 Molecular analyses Approximately 550 bp of 16S rRNA were sequenced for eleven individuals of L. zhangyapingi sp. nov. These eleven sequences generated only one haplotype, which was submitted to GenBank with the accession number JX069979. All aligned datasets contained 468 bp with 195 parsimony informative sites after trimming the ends. Based on the BI analysis, the genus Leptolalax formed a strongly supported monophyletic group (Figure 5). Within this genus, most relationships could not be well resolved, and L. zhangyapingi sp. nov. was, however, highly supported as an exclusive clade. Two other species of Leptolalax from Chiang Mai (L. minimus and L. sp.) highly diverged from this new species and formed distinctly separate clades (Figure 5). Three species of Leptolalax from central and southern Thailand were not included in our molecular analysis, but they are Figure 4 Holotype (KIZ07258) of Leptolalax zhangyapingi sp. distinguished from the newly collected specimens by nov. A: Right hand, palmar view; B: Right foot, plantar view. Photo morphology (see below). Uncorrected pairwise distances by Ke JIANG. of the mtDNA data were 1.11%–27.3% between the one-third, and gray in lower two-thirds, with vertical species of Leptolalax. The newly collected specimens black pupil. differed from all other species in Leptolalax for which Coloration of holotype in preservative: Dorsal and tissues are available by 10.4%–21.2%. lateral surfaces of head and body, and dorsal surface of 3.3 Comparisons Leptolalax zhangyapingi sp. nov. has limbs gray with grayish brown markings; supratympanic a distinct and complete ventrolateral glandular ridge, fold dark gray; large tubercles on dorsal and lateral body which differentiates it from L. applebyi, L. arayai, L. gray brown; ventral surfaces of head and body cream in croceus, L. dringi, L. gracilis, L. hamidi, L. heteropus, color, limbs cream colored medially with mottled grey L. kajangensis, L. kecil, L. maurus, L. melicus, L. pictus, and brown; and pectoral glands cream colored. L. platycephalus, L. solus and L. tuberosus (all of which Variation: Measurements of the type series are shown lack that character), and L. melanolecus (indistinct) and in Table 2. Individuals of the type series are relatively L. fuliginosus (incomplete). Furthermore, by having uniform in coloration, but the holotype (KIZ07258) dark brown spots on the groin and lateral body, and dark has larger makings on the dorsum of the head and body canthal and temporal streaks, L. zhangyapingi sp. nov. compared to the other individuals of the type series. differs from L. aereus, L. eos, and L. firthi, all of which KIZ07259 (one of paratypes) has more horizontal skin lack these markings. ridges on the dorsum of the body than other individuals of With the presence of an immaculate white chest and type series. belly, L. zhangyapingi sp. nov. differs from L. alpinis, Secondary sexual characters: An internal single L. bidoupensis, L. khasiorum, L. nahangensis, L. nyx, subgular vocal sac is present in males, with vocal sac L. pluvialis, and L. ventripunctatus, all of which have openings near the corners of the mouth. darker colored or spotted chests and/or bellies. Leptolalax Distribution: This species is currently known only zhangyapingi sp. nov. differs from L. bourreti, L. lateralis, from Doi Saket, Chiang Mai Province, northern L. liui, L. minimus, L. nahangensis, L. nokrekensis L. Thailand (Figure 1). oshanensis, L. pelodytoides, and L. tamdil by having a 106 Asian Herpetological Research Vol. 4 larger body size in males (45.8–52.5 mm vs. 28.0–36.2 iris in life (golden in the upper one-third and gray in the mm in L. bourreti, 26.9–28.3 mm in L. lateralis, 23.0– lower two-thirds vs. uniformly golden with minute black 28.7 mm in L. liui, 23.0–31.4 mm in L. minimus, 40.8 reticulate spots). L. zhangyapingi sp. nov. differs from L. mm in L. nahangensis, 26.0–33.0 mm in L. nokrekensis, sungi by the different color of the iris (golden in the upper 26.6–30.7 mm in L. oshanensis, 27.5–32.3 mm in L. one-third and gray in the lower two-thirds vs. almost pelodytoides, and 32.3 mm in L. tamdil), and further iridescent gold-green with only slight mottling along differs from L. nahangensis by the different color of the outer margin in L. sungi) and dorsal pattern (yellowish

L. applebyi 1.00 L. melicus 1 L. melicus 2 1.00 L. melicus 3 L. bidoupensis 1 1.00 L. bidoupensis 2 L. bidoupensis 3 L. bidoupensis 4 1.00 L. arayai L. pictus 1.00 L. hereropus 1 L. hereropus 2 L. hereropus 3 L. liui Leptolalax 1.00 L. aereus 1 L. aereus 2 L. aereus 3 L. aereus 4 L. pelodytoides 1 L. pelodytoides 2 1.00 L. pelodytoides 3 L. ventripunctatus 1 L. ventripunctatus 2 L. ventripunctatus 3 1.00 0.99 L. ventripunctatus 4 L. nyx L. minimus 1 1.00 L. minimus 2 L. minimus 3 L. minimus 4 1.00 L. pluvialis 1 L. pluvialis 2

0.90

1.00 L. zhangyapingi

0.98 1.00 L. eos 1 L. eos 2 1.00 L. eos 3 Leptolalax. sp. L. oshanensis L. bourreti L. oshanensis 1.00 L. firthi 1 L. firthi 2 L. firthi 3 0.1 L. firthi 4

Figure 5 Bayesian inference matrilineal genealogy for the species within the genus Leptolalax (Table 1). The species of the outgroup listed in Table 1 are not shown. Bayesian posterior probabilities ≥ 90% are shown near the respective branches. No. 2 Ke JIANG et al. New species of Leptolalax from Thailand 107 brown with large dark brown patches vs. uniformly light Technology of Yunnan, China (2010CI045). brown on dorsum with scattered orange tubercles on head and back of L. sungi) in life. References

4. Discussion Anderson J. 1871. A list of the reptilian accession to the Indian Museum, Calcutta, from 1865 to 1870, with a description of The genus Leptolalax has been considered to contain a some new species. J Asiatic Soc Bengal, 40: 12–39 Boulenger G. A. 1893. Concluding report on the reptiles and high proportion of cryptic diversity (Rowley et al., 2010a, batrachians obtained in Burma by Signor L. Fea, dealing with b). Our result clearly supports this view and one more the collection made in Pegu and the Karin Hills in 1887-88. new species, L. zhangyapingi is described here based on Annali del Museuo Civico di Genoa, 13: 304–347 its diagnosable morphological characters. Boulenger G. A. 1900. Descriptions of new batrachians and reptiles Presently, there are 37 described species in total in this from the Larut Hills, Perak. Ann Mag Nat Hist, 6(7): 186–194 genus, including L. zhangyapingi. However, most of them Boulenger G. A. 1908. A revision of the Oriental pelobatid batrachians (genus Megalophrys). Proc Zool Soc London, 1908: were studied based on morphology only, not combined 407–430 with acoustic data and molecular data. Recently, the Chan-ard T. 2003. A Photographic Guide to Amphibians in discovery of new species is increasing mostly aided by Thailand. Bangkok: Darnsutha Press, 1–175 (In Thai) molecular tool (Rowley et al., 2010a, 2010b; Ohler et al., Das I., Deuti K. 2011. Notes on the date of publication and generic 2011). Combined with the sequences available in this identify of Leptobrachium nokrekensis Mathew and Sen, “2009” genus (Ohler et al., 2011; Rowley et al., 2012), our 2010 (Amphibia: Anura: Megophryidae). Curr Herpetol, 30: 69–73 phylogenetic tree recovered the new described species as Das I., Tron R. K. L., Rangad D., Hooroo P. N. K. 2010. A new one clearly distinct lineage, which largely diverges from species of Leptolalax (Anura: Megophryidae) from the sacred other known species as shown in Figure 1. groves of Mawphlang, Meghalaya, north-eastern India. Zootaxa, In conclusion, at present seven described species of 2339: 44–56 Leptolalax exist in Thailand, L. fuliginosus and L. gracilis Dehling J. M. 2012. A new species of the genus Leptolalax (Anura: in the southwest, L. heteropus and L. solus in the south, L. Megophryidae) from Gunung Benom, Peninsular Malaysia. melanoleucus in the southwest and south, L. minimus and Sauria, 34(2): 9–21 Delorme M., Dubois A., Grosjean S., Ohler A. 2006. Une L. zhangyapingi sp. nov. in the north (Chan-ard, 2003; nouvelle ergotaxinomie des Megophryidae (Amphibia, Anura). Das et al., 2010; Frost, 2013; Matsui, 2006; Nabhitabhata Alytes, 24: 6–21 et al., 2000; Ohler et al., 2011). L. pelodytoides and L. Dubois A. 1981. Notes sur la systématique et la répartition des bourreti are now restricted to the Karin Hills of Myanmar Amphibiens Anoures de Chine et des régions avoisinantes. V. and Vietnam, respectively indicated by Ohler et al. (2011). Megophrys oshanensis Liu, 1950 et Leptobrachium minimum Both of them were considered to exist in Thailand by Das Taylor, 1962. Bull de la Société Linnéenne de Lyon, 50: 182–192 Dubois A. 1983. Note preliminaire sur le genre Leptolalax Dubois, et al. (2010). Recently, Ohler et al. (2011) resurrected L. 1980 (Amphibiens, Anoures), avec diagnose d’une espece minimus which was described from Chiang Mai Province, novelle du Vietnam. Altyes, 2: 147–153 Thailand by Taylor (1962), and was considered to be a Dubois A. 1987 “1986”. Miscellanea taxinomica batrachologia (I). junior synonym of L. oshanensis by Dubois (1981). Alytes, 5: 7–95 Dubois A., Grosjean S., Ohler A., Adler K., Zhao E. 2010. The Acknowledgements Field work was made possible nomenclatural status of some generic nomina of Megophryidae during the joint biological surveys at Huai Hongkhrai (Amphibia, Anura). Zootaxa, 2493: 66–68 Royal Development Study Center in Thailand. We thank Fei L., Hu S. Q., Ye C. Y., Huang Y. Z. 2009. Fauna Sinica. Amphibia, Vol. 2, Anura. Beijing: Science Press, 1–957 (In Mr. Sawing KHUNTASA for his help during collection in Chinese) Thailand. We are grateful to Dr. Theodore J. PAPENFUSS Fei L., Ye C. Y., Huang Y. Z. 1990. Key to Chinese Amphibia. (Museum of Vertebrate Zoology, University of California Chongqing: Chongqing Branch Science and Technology at Berkeley, USA) and reviewers for their comments on Literature Press, 364 (In Chinese) the manuscript. This work was supported by grants from Frost D. R. 2013. Amphibian Species of the World: an Online the Ministry of Science and Technology of China (MOST Reference. Version 5.6 (9 January 2013). 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