Description of two new sympatric species of the genus Leptolalax (Anura: Megophryidae) from western Yunnan of China

The Asian leaf litter toads of the genus Leptolalax represent a highly diverse species group and currently contain 53 recognized species. During herpetological surveys in Yingjiang County, western Yunnan of China, we collected series of Leptolalax specimens from an isolated small fragment of montane evergreen forest. Subsequent study based on acoustic, morphological and molecular data reveals that there were three different species among the specimens sampled: while one of them belongs to Leptolalax ventripunctataus, the other two species represent unknown taxa and are described herein: Leptolalax purpurus sp. nov. and Leptolalax yingjiangensis sp. nov. The two new species can be distinguished from other congeners by the molecular divergences, acoustic data, and by a combination of morphological characters including: body size, dorsal and ventral patterns, dorsal skin texture, sizes of pectoral and femoral glands, degree of webbing and fringing on the toes and fingers, dorsum coloration and iris coloration in life. Our results further reveal that species diversity of the genus Leptolalax still remains highly underestimated and warrants further attention.


INTRODUCTION
The Asian leaf litter toads of the genus Leptolalax Dubois, 1983 are widely distributed from southern China west to northeastern India and Myanmar, through mainland Southeast Asia to the island of Borneo. Fifty-three nominal species within the genus are recognized to date, with more than half described in the past ten years (Frost, 2017), in particular from Indochina and southern China (Yang et al., 2016;Rowley et al., 2016;Rowley, Dau & Cao, 2017;Yuan et al., 2017). Currently, two subgenera are recognized in the genus: the nominal subgenus Leptolalax is distributed south of the Isthmus of Kra, while the subgenus Lalos Dubois, Grosjean, Ohler, Adler & Zhao is distributed north of the Isthmus of Kra (Delorme et al., 2006;Dubois et al., 2010;Ohler et al., 2011). However, due to the limited mtDNA datasets of the genus, there is no firm phylogenetic support for this division (Poyarkov et al., 2015;Matsui et al., 2017). In this paper, we followed most with both forward and reverse primers using BigDye Terminator Cycle Sequencing Kit according to the guidelines of the manufacturer. The products were sequenced on an ABI Prism 3730 automated DNA sequencer in Shanghai Majorbio Bio-pharm Technology Co., Ltd. All sequences have been deposited in GenBank (Table 1). Phylogenetic analyses. In addition to the newly collected specimens, sequences of all species of the genus Leptolalax for which homologous sequences of 16S rRNA were available (38 of the 53 species) from the Genbank were included in the genetic analyses (Table 1). We used Leptobrachium chapaense (Bourret) and Xenophrys major (Boulenger) as outgroups. Sequence alignments were first conducted using Clustal X 2.0 (Thompson, Higgins & Gibson, 1994), with default parameters and the alignment being checked and manually revised, if necessary. The data were tested in jmodeltest v2.1.2 with Akaike and Bayesian information criteria, resulting the best-fitting nucleotide substitution models are GTR + I + G. Sequence data were analyzed using maximum likelihood (ML) implemented in RaxmlGUI 1.3 (Silvestro & Michalak, 2012), and Bayesian inference (BI) using MrBayes 3.12 (Ronquist & Huelsenbeck, 2003). The phylogenetic tree was constructed using ML and BI methods. For ML analysis, the bootstrap consensus tree inferred from 1,000 replicates was used to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 70% of bootstrap replicates were collapsed (Hillis & Bull, 1993). For BI analysis, two independent runs with four Markov Chain Monte Carlo simulations were performed for ten million iterations and sampled every 1,000th iteration. The first 25% of samples were discarded as burn-in. Convergence of the Markov Chain Monte Carlo simulations was assessed using Tracer v.1.4 (http://tree.bio.ed.ac.uk/software/tracer/). Pairwise distances based on 16S rRNA were calculated in MEGA 6.06 using the uncorrected p-distance model (Tamura et al., 2013). Morphological characters. Measurements followed Fei et al. (2009) and Rowley, Dau & Nguyen (2013), and were taken with digital callipers to the nearest 0.1 mm: snout-vent length (SVL); head length from tip of the snout to rear of jaws (HDL); head width at commissure of jaws (HDW); snout length from tip of the snout to anterior corner of eye (SNT); diameter of exposed portion of eyeball (EYE); interorbital distance (IOD); internasal distance (IND); upper eyelid width measured as greatest width of the upper eyelid (UEW); nostril-eyelid length (NEL); nostril-snout length (NSL); 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); length of lower arm and hand (LAHL); pes length from tip of fourth toe to proximal edge of the inner metatarsal tubercle (PL); and hindlimb length from tip of fourth toe to vent (HLL). Sex was determined by direct observation of calling in life and the presence of internal vocal sac openings, and by observation of eggs through translucent belly skin in gravid females. Comparative morphological data of Leptolalax species were obtained from the literature (see Appendix S1) and from examination of museum specimens (see Appendix S2). Due to the high likelihood of undiagnosed diversity within the genus, where available, we relied on examination of topotypic material and/or original species descriptions. Nomenclatural acts. The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:31C06247-A201-42AD-8FF8-B6510530C39D. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Description of new species
Our detailed morphological study supports the recognition of the two new species of the genus Leptolalax, which can be reliably differentiated from each other and all known congeners on the basis of body size, dorsal and ventral patterns, dorsal skin texture, sizes of pectoral and femoral glands, degree of webbing and fringing on the toes and fingers, dorsal coloration and iris coloration in life. These two new taxa are formally described below.  Description of holotype. SYS a006531, adult male, body size small (SVL 25.0 mm), head width (9.2 mm) about equal to head length (9.4 mm); snout slightly protruding, projecting slightly beyond margin of the lower jaw; nostril equidistant between snout and eye; canthus rostralis gently rounded; loreal region slightly concave; interorbital space flat, larger (IOD 2.7 mm) than upper eyelid (2.5 mm in width) and internarial distance (2.6 mm); pineal ocellus absent; pupil vertical; eye diameter smaller than snout length; tympanum distinct, round, diameter (TMP 1.8 mm) smaller than that of the eye (EYE 3.3 mm), and larger than tympanum-eye distance (TEY 1.0 mm); tympanic rim distinctly elevated relative to skin of temporal region; vomerine teeth absent; vocal sac openings slit-like, located posterolaterally on floor of mouth in close proximity to the margins of the mandible; tongue long, wide, with a small shallow notch at posterior tip; supratympanic ridge distinct, extending from eye to supra-axillary gland; a few indistinct tubercles present on supratympanic ridge. Tips of fingers rounded, slightly swollen; relative finger lengths I = II = IV < III; nuptial pad absent; subarticular tubercles absent; a large, round inner palmar tubercle distinctly separated from small, round outer palmar tubercle; finger webbing and dermal fringes absent. Tips of toes similar to that of fingers; relative toe length I <II <V <III <IV; subarticular tubercles absent; distinct dermal ridges present under the 3rd to  5th toes; large, oval inner metatarsal tubercle present, outer metatarsal tubercle absent; toe webbing rudimentary; wide lateral fringes present on all toes. Tibia 45% of snout-vent length; tibiotarsal articulation reaches to posterior corner of the eye. Skin on dorsum shagreened and scattered with irregular fine, round tubercles; longitudinal skin folds on dorsum absent; ventral skin smooth; pectoral gland large, elongated oval, 1.8 mm in length, greatly larger than tips of fingers and femoral gland; femoral gland small, round, 0.6 mm in diameter, distinctly smaller than tips of toes, situated on posteroventral surface of thigh, closer to knee than to vent; supra-axillary gland raised, 1.1 mm in diameter; ventrolateral gland distinct as small white dots forming an incomplete line.

Coloration of holotype in life.
Dorsal surface purplish brown with indistinct darker brown markings, and two indistinct light coppery orange spots on shoulder region ( Fig. 2): V-shaped interorbital marking disconnected to the W-shaped marking between axillae; dorsum scattered with indistinct irregular black spots edged with yellow pigmentation; fine, purplish red tubercles on upper eyelids, snout, head, upper lips, dorsal surfaces of body and limbs, those on flanks somewhat whitish; white bar on tip of the snout; upper lip mottled with black blotches; a distinct small coppery orange tubercle present on below posterior Temperature
corner of eye; a black spot present on loreal region; lower margin of supratympanic ridge distinctly black; tympanum nearly fully black; transverse dark brown cross-bars presents on dorsal surface of limbs; a few distinct large black spots present on flanks from groin to axilla, the one on groin largest; black markings and spots on dorsum, flank and tympanum mottled with distinct yellow pigmentation; a few coppery orange small tubercles present on flanks; elbow and upper arms in distinct coppery orange coloration; fingers and toes with transverse bars. Venter dull white with indistinct grey dusting; throat immaculate pinkish; ventral surfaces of thigh pinkish and sparsely scattered with tiny white dots; margin of lower lip scattered with tiny and small white spots. Supra-axillary gland coppery orange; femoral, pectoral and ventrolateral glands white and distinct. Iris bicolored, upper half orange yellow, lower half sliver white.

Coloration of holotype in preservative.
Dark brown markings on dorsum and flanks still visible, while transverse cross-bars on limbs become more distinct; ventral surface of body dull white with grey dusting; throat much darker; macroglands on the ventral surfaces still distinct.
Variations. The single male paratype SYS a006530 greatly matches overall characters of the holotype (for measurements of the type series see      (Dubois et al., 2010;Dehling & Matsui, 2013;Matsui, Zainudin & Nishikawa, 2014). Leptolalax purpurus sp. nov. differs from all other species in the subgenus Lalos by having purplish brown dorsum in life, pectoral gland greatly larger than femoral gland, black marking/spots on dorsum and flanks mottled with distinct yellow pigmentation, an iris of bicolored coloration, as well as a combination of male body size, presence of black spots on the flank, plus ventral coloration, degree of webbing and fringing on the toes, and dorsal skin texture (See Table 4  Leptolalax purpurus sp. nov. differs from the phylogenetically close congener, L. oshanensis (Liu), by the presence of webbing and dermal fringes on toes (vs. absent in oshanensis), purplish brown above in life (vs. reddish or greyish brown in oshanensis, see Figs. 6A-6B), a relatively shorter hindlimb (mean males HLL/SVL ratio 1.45 in purpurus sp. nov. vs. 1.56 in oshanensis), pectoral gland larger than femoral gland (vs. reversed condition in oshanensis), femoral gland closer to knee than to vent (vs. reversed condition in oshanensis), and the absence of skin folds on dorsum (vs. short skin folds present in L. oshanensis).
Leptolalax purpurus sp. nov. differs from the sympatric L. ventripunctatus by having wide dermal fringes on toes (vs. absent or narrow in ventripunctatus), absence of longitudinal skin folds on dorsum (vs. present in ventripunctatus), dorsal surface purplish brown (vs. brown in ventripunctatus, see Fig. 3), black markings and spots on dorsum and flanks mottled with yellow pigmentation (vs. black markings and spots solid black and without such yellow pigmentation in ventripunctatus), ventral surface dull white with indistinct grey dusting (vs. distinct small black spots present on venter in ventripunctatus, see Fig. 3). See below for the comparison of Leptolalax purpurus sp. nov. with the other sympatric Leptolalx species which is also described as a new species below.  Paratypes. SYS a006533, adult male, data identical to holotype; SYS a006534-a006537, adult males, same locality as holotype, collected on 10 June 2017 by JH Yang. Etymology. The specific name ''yingjiangensis'', is in reference to the type locality of the new species, Yingjiang County of Yunnan Province, China. For the common name, we suggest ''Yingjiang Leaf Litter Toad'' (English) and ''盈江掌突蟾'' (Chinese). Diagnosis. Leptolalax yingjiangensis sp. nov. can be distinguished from its congeners by a combination of the following characters: (1) small size (SVL 25.7-27.6 mm in males); (2) dorsal skin shagreened and scattered with fine, round brown tubercles; (3) tympanum distinctly discernible, upper half black; (4) fingers webbing absent, and narrow to moderate dermal fringes present on 2nd to 4th fingers; (5) toes with rudimentary webbing and wide lateral fringes; (6) pectoral gland smaller than femoral gland; (7) ventrolateral glands distinct; (8) distinct tiny white flecks present on edges of dark brown markings/blotches on dorsum; (9) flanks with distinct irregular black spots; (10) ventral surface of body creamy white and scattered with distinct small dark brown flecks on chest and lateral sides of belly; (11) iris bicolored, upper half orange yellow, lower half sliver white; (12) a call consisting of a single note and a dominant frequency of 5.7-5.9 kHz (at 19 • C). Description of holotype. SYS a006532, adult male, body size small (SVL 25.7 mm), head width (9.7 mm) slightly shorter than head length (10.3 mm); snout slightly protruding, projecting slightly beyond margin of the lower jaw; nostril equidistant between snout and eye; canthus rostralis gently rounded; loreal region slightly concave; interorbital space flat, larger (IOD 3.0 mm) than upper eyelid (2.8 mm in width) and internarial distance (2.6 mm); pineal ocellus absent; pupil vertical; eye diameter (EYE 3.9 mm) slightly smaller than snout length (SNT 4.2 mm); tympanum distinct, round, diameter (TMP 1.8 mm) smaller than that of the eye, and larger than tympanum-eye distance (TEY 1.1 mm); tympanic rim distinctly elevated relative to skin of temporal region; vomerine teeth absent; vocal sac openings slit-like, located posterolaterally on floor of mouth in close proximity to the margins of the mandible; tongue long, wide, with a small shallow notch at posterior tip; supratympanic ridge distinct, extending from eye to supra-axillary gland; a few indistinct tubercles present on supratympanic ridge. Tips of fingers rounded, slightly swollen; relative finger lengths I = II = IV < III; nuptial pad absent; subarticular tubercles absent; a large, round inner palmar tubercle distinctly separated from small, round outer palmar tubercle; narrow to moderate dermal fringes present on 2nd to 4th fingers; finger webbing absent. Tips of toes similar to that of fingers; relative toe length I <II <V <III <IV; subarticular tubercles absent; distinct dermal ridges present under the 3rd to 5th toes; large, oval inner metatarsal tubercle present, outer metatarsal tubercle absent; toe webbing rudimentary; wide lateral fringes present on all toes. Tibia 49% of snout-vent length; tibiotarsal articulation reaches to anterior corner of the eye. Skin on dorsum shagreened and scattered with fine, round tubercles; ventral skin smooth; pectoral gland tiny, round, 0.5 mm in diameter, slight smaller than tips of fingers; femoral gland small, round, 1.0 mm in diameter, distinctly larger than pectoral gland and tips of toes, situated on posteroventral surface of thigh, closer to knee than to vent; supra-axillary gland raised, 0.8 in mm diameter; ventrolateral gland distinct as small white dots forming an incomplete line.

Coloration of holotype in life.
Dorsal surface brown with indistinct dark brown markings, and two indistinct light brown spots on shoulder region (Fig. 7): V-shaped interorbital marking disconnected to the W-shaped marking between axillae; distinct tiny white flecks present on edges of dark brown markings/blotches on dorsal surfaces of head and body; fine, brown tubercles present on upper eyelids, snout, head, dorsal surfaces of body and limbs; upper lip with black large blotches and small spots; lower margin of supratympanic ridge black; upper half of tympanum dark brown; a distinct black spot present on loreal region; narrow transverse dark brown cross-bars present on dorsal surface of limbs; a few distinct irregular black spots present on flanks from groin to axilla, the one on groin largest; elbow and upper arms in light orange brown coloration; fingers and toes with transverse bars. Venter creamy white and scattered with dark brown flecks on chest and lateral sides of belly; medial belly immaculate; throat transparent pinkish; margin of lower lip scattered with dark brown spots and flecks; ventral surfaces of thigh pinkish and mottled with white and dark brown. Supra-axillary gland light brown; pectoral white and indistinct; femoral and ventrolateral glands white and distinct. Iris bicolored, upper half orange yellow, lower half sliver white. Coloration of holotype in preservative. Dorsum turned dark brown with slightly paler limbs (Fig. 4) , L. arayai, L. dringi, L. fritinniens, L. gracilis, L. hamidi, L. heteropus, L. kajangensis, L. kecil, L. marmoratus, L. maurus, L. melanoleucus, L. pictus, L. platycephalus, L. sabahmontanus and L. solus, all of which occur south of the Isthmus of Kra and lack supra-axillary and ventrolateral glands (Dubois et al., 2010;Dehling & Matsui, 2013;Matsui, Zainudin & Nishikawa, 2014). Leptolalax yingjiangensis sp. nov. differs from all other species in the subgenus Lalos by having brown dorsum in life, pectoral gland indistinct and smaller than femoral gland, narrow to moderate dermal fringes present on 2nd to 4th fingers; distinct small white flecks present on dorsum, iris bicolored, as well as a combination of male body size, presence of black spots on the flank, plus ventral coloration, degree of webbing and fringing on the toes, and dorsal skin texture (See Table 4 for a summarized comparison with all species in the subgenus Lalos). Leptolalax yingjiangensis sp. nov. differs from the phylogenetically close congener, L. khasiorum Das, Tron, Rangad & Hooroo, by having head slightly longer than wide (vs. head wider than long in khasiorum), a comparatively small tympanum (males TMP/EYE ratio 0.41-0.46 vs. 0.47-0.55 in khasiorum), supra-axillary gland and small tubercles on dorsum and hind limbs brown in life (vs. pinkish-red in khasiorum), upper parts of iris orange yellow in life (vs. bright orange in khasiorum), and pectoral gland distinctly smaller than femoral gland (vs. reversed condition in khasiorum).
Leptolalax yingjiangensis sp. nov. differs from the phylogenetically close congener, L. puhoatensis, by having ventral surface creamy white with dark brown flecks on chest and margins in males (vs. reddish brown with white dusting in puhoatensis), absence of skin folds on dorsum (vs. low skin folds present on dorsum in life in puhoatensis), wide dermal fringes on toes (vs. narrow in puhoatensis), dorsal coloration brown in life (vs. dark reddish brown in puhoatensis).
Leptolalax yingjiangensis sp. nov. differs from the phylogenetically close congener, L. petrops, by having dermal fringes present on fingers (vs. dermal fringes absent on fingers in puhoatensis), toes webbing rudimentary (vs. absent in petrops), and wide dermal fringes on toes (vs. narrow in puhoatensis), ventral surface creamy white with dark brown flecks on chest and lateral sides of belly (vs. immaculate white in petrops), and dorsal skin shagreened scattered with small tubercles (vs. dorsal skin highly tuberculate in petrops).
Leptolalax yingjiangensis sp. nov. further differs from the sympatric L. ventripunctatus by having dermal fringes present on fingers (vs. dermal fringes absent on fingers in ventripunctatus), wide dermal fringes on toes (vs. absent or narrow in ventripunctatus), absence of longitudinal skin folds on dorsum of body (vs. present in ventripunctatus), pectoral gland smaller than tips of fingers and femoral grand (vs. reversed condition in ventripunctatus), distinct white tiny flecks present on dorsum (vs. such white flecks absent in ventripunctatus), medial belly immaculate creamy white (vs. distinct small black spots present on belly in ventripunctatus, see From the rest two known Leptolalax species from Yunnan, Leptolalax yingjiangensis sp. nov. differs from L. alpinus by having dermal fringes present on fingers (vs. dermal fringes absent on fingers in alpinus), pectoral gland smaller than tips of fingers and femoral grand (vs. reversed condition in alpinus), and medial belly immaculate creamy white (vs. distinct dark brown spots/blotches present on belly in alpinus, see Figs. 6C-6D); Leptolalax yingjiangensis sp. nov. further differs from L. tengchongensis by dermal fringes present on fingers (vs. dermal fringes absent on fingers in tengchongensis), wide dermal fringes on toes (vs. narrow in tengchongensis), small tubercles on dorsum in brown coloration in life (vs. reddish in tengchongensis), distinct white tiny flecks present on dorsum (vs. such white flecks absent in tengchongensis), medial belly immaculate creamy white (vs. distinct dark brown blotches present on chest and belly in tengchongensis, see Figs. 6E-6F), and a bicolored iris (vs. uniform coloration in tengchongensis).
congeners with genetic distances between 3.2% to 20.7%, with the lowest value p = 3.2% observed in the comparison with the sequence of L. eos. This value was slightly higher than the lowest interspecific p-distance (p = 2.9%, between L. arayai and L. marmoratus) among species of Leptolalax examined and the value (3% value of p-distance in 16S rRNA) that usually represents differentiation at the species levels of anurans (Vences et al., 2005;Fouquet et al., 2007).

DISCUSSION
In the genus Leptolalax, pairs of sympatric species are commonly reported among tropical members in Indochina, e.g., L. eos and L. ventripunctatus, L. pluvialis Ohler, Marquis, Swan & Grosjean and L. bourreti, L. applebyi and L. tuberosus, L. croceus and L. applebyi, L. eos and L. puhoatensis (Rowley & Cao, 2009;Rowley et al., 2010a;Ohler et al., 2011;Rowley, Dau & Cao, 2017). Our results revealed a rare case that three species of Leptolalax co-occur in the same stream, which also represents the first record of sympatric Leptolalax species from China. According to our limited preliminary data (Table 6), the breeding season of L. purpurus starts rather early (probably in February) and ends in April; L. yingjiangensis breeds relatively later starting from May, with the peak in June; L. ventripunctatus presents the longest breeding period among the three species, with the peak in April. Thus, we assumed that the main breeding seasons of the three sympatric Leptolalax in Yingjiang County should be different but have overlapping, which needs to be verified by future study. Our survey showed that these three sympatric species overlap in time and space, but we have no data on the resources they utilize, such as food composition, trophic structure and other parameters. Future ecological studies are needed in order to understand the interspecific competition and mechanisms of niche segregation among the sympatric species of Leptolalax. The type locality of L. purpurus and L. yingjiangensis is less than one kilometer from the boundary of Tongbiguan Nature Reserve, and ca. 5 km from the international border with Myanmar's Kachin State (Fig. 1); and we can expect that these two new species and L. ventripunctatus also occur in the adjacent well-preserved natural forests of Tongbiguan Nature Reserve and Kachin State. The discovery of L. purpurus and L. yingjiangensis brings the total number of Leptolalax recorded from China up to eleven, which further reveals that the currently known species diversity of the genus Leptolalax from China and the