A new species of rain-pool frog (Dicroglossidae: Fejervarya) from western Thailand

We describe a new species, Fejervarya muangkanensis sp. nov., based on a series of specimens collected from Ban Tha Khanun, Thong Pha Phum District, Kanchanaburi Province, Thailand. The new species is easily distinguished from its congeners by morphological and molecular data, and can be diagnosed by the following characters: (1) small size (adult male snout-vent length (SVL) 33.5 mm; female SVL 40.0-40.9 mm); (2) tympanum small, discernible but unclear; (3) poorly developed toe webbing; (4) no lateral line system in adults; (5) characteristic "Fejervaryan" lines present in females; and (6) femoral glands absent. Molecular phylogenetic analysis of mitochondrial 16S rRNA further supports it as a distinct lineage and distinguishes it from its congeners for which sequences are available.

Recent morphological and genetic comparisons have revealed several new species of Thai anurans, including one new species of Fejervarya from northern Thailand (Suwannapoom et al., 2016). During herpetological surveys in 2013 in the Kanchanaburi Province of Thailand, we found a morphologically distinct population of Fejervarya. We compared the morphology of this species with its congeners as well as levels of genetic divergence with species having comparable data in GenBank. These analyses supported the recognition of a new species. 1

Sampling
Five individuals (KIZ 024627, KIZ 024675-78) were captured during fieldwork in the village of Tha Khanun, Thong Pha Phum District, Kanchanaburi Province, Thailand (Figure 1), from June to September 2013. After euthanization using a chlorobutanol solution, muscle and liver tissues were taken from the frogs and preserved in 95% ethanol for genetic analysis. Specimens were later fixed in 10% buffered formalin and then transferred to 70% ethanol. All specimens were deposited at the Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS).

Molecular analysis
Total genomic DNA was extracted from a tissue sample of specimen KIZ 024627 using the standard phenol-chloroform protocol (Sambrook et al., 1989). A fragment of 16S rRNA was amplified for one individual using the primers 16Sar (5′-CGCCTGTTTAYCAAAAACAT-3′) and 16Sbr (5′-CCGGTYTG AACTCAGATCAYGT-3′) from Kocher et al. (1989). Amplification involved an initial cycle of denaturation at 95 °C for 5 min, and 35 subsequent cycles of 95 °C for 1 min; the annealing temperature was 55 °C for 1 min and 72 °C for 1 min, followed by a final extension step of 72 °C for 7 min. The resulting PCR products were directly cycle-sequenced with the same primers as those used for PCR. Sequence analysis was performed on an ABI PRISM ® 3730xl DNA Analyzer (Applied Biosystems, UK) at KIZ, CAS.

Phylogenetic analysis
New sequences were examined for signal quality and confirmed for completeness using DNASTAR 5.0. Nineteen sequences were downloaded from GenBank for analysis (Table 1). Occidozyga lima was chosen as an outgroup taxon (Pyron & Wiens, 2011). After trimming the ends, sequences were aligned with gaps using MUSCLE (Edgar, 2004) with default settings. Genetic distances among the taxa were calculated using the pdistance model in MEGA 6.0 (Tamura et al., 2013). Phylogenetic reconstructions were executed using Bayesian inference (BI) and maximum likelihood (ML). The best-fit model of DNA sequence evolution was chosen using MrModeltest v2.3 (Nylander, 2004) under the Akaike information criterion. The GTR+I+G model was selected as the best model. A Bayesian tree was generated using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). For BI analyses, two independent searches with random starting trees were run for 5 million generations while sampling over 1 000 generations and compared using four Markov Chain Monte Carlo (MCMC) chains (temp=0.2). Convergence was assessed by plotting the log-likelihood scores in Tracer v.1.5 (Rambaut et al., 2013), and data from the first 25% were discarded as burn-in before building a consensus tree. Maximum likelihood analyses were performed using RAxML 7.0.4 (Stamatakis et al., 2008). The same model of nucleotide substitution as for the BI analyses was used for ML tree-searching and nodal stability was estimated with 1 000 bootstrap pseudoreplicates.

Phylogenetic analyses
The unique de novo sequence was deposited in GenBank under accession No. MF166918 (Table 1). A total of 721 base pairs (bp) of 16S rRNA data were generated, among which 549 positions were potentially parsimony-informative. Similar topologies were produced by ML and BI analyses. Major clades I and II were identified within Fejervarya (Figure 2), which corresponded to the groups of Fejervarya identified by Dinesh et al. (2015).  Table 2). These results revealed a substantial genetic divergence between the specimens from Thong Pha Phum and the other species, suggesting that this population represents an undescribed species. Furthermore, distinct morphological differences were also found. For example, the "Fejervaryan" lines on legs characteristic, present in females, occurred in the Thong Pha Phum population. Considering its independent evolutionary history, level of genetic divergence, and distinct morphological characters, a new species is described. Paratypes: Four females (KIZ 024675-78), collected by Chatmongkon Suwannapoom, Jing Che, Fang Yan, and Wei Gao at the same locality as the holotype.
Snout smooth, with rare indistinct dermal tubercles; nares with low dermal flaps; small tubercles on upper eyelid. Dorsal and lateral surfaces of head and body, including body flanks, shagreened; posterior part of dorsum with distinct, round glandular warts, continuing on dorsal surfaces of legs and arms; small tubercles on the anterodorsal part of thigh, cloacal region, dorsal surface of tibia, and tarsus; lateral sides of body, ventral surfaces of body, and limbs smooth. Dorsal skin showing small, rare, and longitudinal dermal ridges arranged in series.

Coloration of holotype in life:
Male dorsal ground color varies from brown to dark green, and transverse black bands are present on the dorsal surface of the thigh, tibia, and tarsus region (Figure 3). In females, the mid-dorsal stripe is orange, and bands run from the anterior side between the eyes to the vent and from the posterior side of the thigh to the tarsus; forearm has prominent orange spots, nearly touching the subtympanic orange streak.
Coloration of holotype in preservative: Dorsum grayish brown with many large black spots ( Figure 4). Thin, creamcolored mid-dorsal stripe runs from between the eyes to the vent and from the posterior side of the thigh to the tarsus (females). Lateral side with many small black dots; ventral side immaculate, except for white with black bands across the throat. Transverse black bands on upper surface of the thigh, tibia, and tarsus to outer edge of the foot.

Etymology:
The specific epithet muangkanensis is derived from the common name of the Kanchanaburi province, Thailand.

Suggested common names:
We suggest the following common names: Kanchanaburi Rain-Pool Frog (English).

Ecology:
The species is found in small swamps in secondary forests at elevations between 700-900 m a.s.l. Advertisement calls of the males can be heard in small ponds from July to September in Thong Pha Phum, Kanchanaburi Province. Calling males are usually observed within or beside the swamp ( Figure 6).
Distribution: This species is currently known only from Thong Pha Phum, Kanchanaburi Province, western Thailand ( Figure 1).
The new species can be distinguished from F. pierrei by relative finger lengths, with the second finger being shorter than the fourth finger (II<IV<I<III vs. II=IV<I<III;Howlader, 2011). F. muangkanensis sp. nov. can be differentiated from F. syhadrensis by its head width being less than head length (HW/HL=0.7; Kuramoto et al., 2007) and by relative finger lengths (II<IV<I<III vs. I=II<IV<III;Howlader, 2011).
Although comparative data are limited, the SVL of F. muangkanensis sp. nov. overlaps with the SVL of male F. sengupti (23.0-37.8 mm, Meghalaya, India;Purkayastha & Matsui, 2012). However, the new species clearly differs from it in relative finger lengths II<IV<I<III and shagreened dorsum, vs. II<I<IV<III and warty dorsum.
F. nilagirica, F. caperata, and F. mudduraja from the Western Ghats differ from the new species based on their body sizes and proportions and by the presence of warts and dermal ridges on the dorsum (Kuramoto et al., 2007). F. nilagirica is a large-bodied species and can be easily distinguished from the small-bodied F. muangkanensis sp. nov. (SVL=33.5 mm in males); it can be further distinguished from the new species by numerous warts and dermal ridges on the dorsum (vs. smooth to shagreened dorsum with glandular warts on the posterior part) and by relatively smaller eyes, EHD/SVL=0.1 (vs. EHD/SVL=0.1). F. caperata is smaller than the new species, with a mean SVL of 29 mm in males and 33 mm in females (vs. SVL 33.5 mm in males and 40.3 mm in females). It can also be differentiated from F. muangkanensis sp. nov. by relative finger lengths (IV<II<I<III vs. II<IV<I<III). F. mudduraja can be distinguished from the new species by its larger female body size with a mean SVL of 45 mm (vs. 40.0-40.9 mm); no information on male SVL exists for F. mudduraja. The species also differs from F. muangkanensis sp. nov. by having a head width greater than head length, HW/HL=1.1 (vs. head width less than head length, HW/HL=0.7).
The new species can be easily distinguished from F. teraiensis of Nepal and northeast India by its smaller body size (SVL 33.5 mm vs. 37.8-44.1 in males;Howlader, 2011), relative finger lengths (II<IV<I<III vs. II=IV<I<III;Howlader, 2011), and having a head width less than head length (HW/HL=0.7 vs. HW/HL=1.0, i.e., head width almost equal to head length).
The two species from Sri Lanka, F. kirtisinghei and F. greenii, can be easily differentiated from F. muangkanensis sp. nov. by their dorsums being covered with well-developed, long, continuous dermal ridges (vs. shagreened dorsum with rare low dorsal ridges, never forming continuous rows).
Further comparisons of F. muangkanensis sp. nov. with other species in the region is complicated due to their unclear taxonomic status. The locality of F. brevipalmata, originally designated as Bago, Myanmar, appears to be uncertain, and might also include the Western Ghats of southern India; thus, the status of this taxon is unclear (AmphibiaWeb, 2017;Boulenger, 1920). Furthermore, F. sauriceps and F. parambikulamana are endemic to Kerala and Karnataka in southern India. Both are known only from holotypes that appear to have been lost. Regardless, the numerous differences in morphology, coloration, and mtDNA gene sequences support the recognition of the specimens collected from Thong Pha Phum as a new species.

DISCUSSION
Although our discovery of F. muangkanensis increases the total number of Fejervarya species of Thailand to nine (Suwannapoom et al., 2016), the diversity of this group may still be underestimated. For example, previous molecular studies have identified several distinct lineages diverged from closely related, recognized species, including Fejervarya sp. hp3 from Pilok, Thailand, and F. sp. hp2 from Bangkok, Thailand (Kotaki et al., 2010). Future studies should examine morphological characteristics of specimens from these regions in detail to confirm their taxonomic identities. Moreover, as many areas of Thailand are still poorly or never surveyed for amphibian diversity, especially in southern Thailand, unrecognized diversity of the genus could still exist. Closer inspections of previously collected congeners from these regions are necessary to better understand amphibian diversity in Thailand, which could help to manage and conserve this unique diversity effectively.