A New Species of Genus Microhyla (Amphibia: Anura:Microhylidae) from Zhejiang Province, China

Meihua ZHANG, Liang FEI, Changyuan YE, Yufan WANG, Bin Wang* and Jianping JIANG*

1 CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization and Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China

2 University of Chinese Academy of Sciences, Beijing 100049, China

3 Zhejiang Forest Resource Monitoring Center, Hangzhou 310020, China

Abstract We described a new species, Microhyla beilunensis sp. nov., from Zhejiang Province of China. Phylogenetic analyses based on the mitochondrial 12S, 16S and CO1 gene sequences suggested that the new taxon was distinctly separated from its congeners and closed to M. mixtura and M. okinavensis. Morphologically, the new species could be identified from its congeners except M. mixtura by several characters： (1) rudimentary webs on toe base; (2) absence of disks and dorsal median longitudinal grooves on finger tips; (3) presence of disks and dorsal median longitudinal grooves on toe tips. As well, the new species could be identified from topotype M. mixtura by the combination of characters： (1) apart from the stripes, bar-shaped and oval-shaped patterns, the rounded spots present on the dorsum of body and legs; (2) the outer metacarpal tubercles prominently larger than the inner one; (3) of males, the ratios of HW,IND, UEW and LAW to SVL of the new species were significantly larger than those of M. mixtura (P ＜ 0.01), and the ratios of SL, IOD, LAHL, HLL, TL, TFL and FL to SVL of the new species were significantly less than those of M.mixtura (P ＜ 0.05).

Keywords Microhyla, morphological comparison, new species, phylogenetic analysis, taxonomy, Zhejiang

1. Introduction

The genusMicrohylaTschudi, 1838 (Amphibia：Anura： Microhylidae) is widely distributed in Eastern,Southeastern and Southern Asia. It currently contains 41 species (Frost, 2018). As noted, 13Microhylaspecies have been described in the past decade, of which, five species,M. arboricola,M. darevskii, M.minuta,M. pineticolaandM. pulchellafrom Vietnam(Poyarkovet al., 2014), three species,M. mukhlesuri,M.mymensinghensis,M. nilphamariensisfrom Bangladesh(Hasanet al., 2014; Howladeret al., 2015), and the remaining five species,M. malangfrom Malaysia(Matsui, 2011),M. orientalisfrom Indonesia (Matsuiet al., 2013),M. lateritefrom India (Seshadriet al., 2016),M. mihintaleifrom Sri Lanka (Wijayathilakaet al., 2016),M. taraiensisfrom Nepal (Khatiwadaet al., 2017). Even so, molecular studies still indicated that several cryptic species in the genus remained to be described, especially in the species with a wide geographical range inhabiting varied habitats (Howladeret al., 2015; Khatiwadaet al.,2017; Matsuiet al., 2011).

Microhyla mixturaLiu and Hu, 1966 is an endemic species to China, and was widely distributed in Shaanxi,Henan, Chongqing, Sichuan, Hubei, Guizhou, Anhui and Zhejiang provinces of China (Fei and Ye, 1982; Chen,1991; Feiet al., 2012). Nevertheless, many previous studies proposed that there were significant differences on morphology (e.g. skin smoothness, colour spots,supratympanic fold and canthus rostralis) between the population from the type locality, Hua’e mountain of Sichuan Province of China, and other populations,such as, populations from Yangxian County of Shaanxi Province (Huet al., 1966), Shangcheng County of Henan Province (Chenet al., 2003), and Beilun District of Ningbo City of Zhejiang Province (Feiet al., 1999, 2012).These work indicated that there may be cryptic species inM. mixturacomplex. However, to now, there has been no molecular phylogenetic examination in this group, which was proved to be effective in evaluating species diversity or species boundary in Microhylidae (e.g. Howladeret al., 2015; Khatiwadaet al., 2017; Matsui, 2011; Matsuiet al., 2011).

From 1997 to 2017, we collected 41 adult specimens of the genusMicrohylafrom Beilun District, Ningbo City, Zhejiang Province of China, which were initially identified asM. mixtura(Huanget al., 1990; Feiet al., 1999, 2012). Our morphological comparisons and phylogenetic analyses consistently suggested that these specimens were distinctly different from its congeners.Therefore, we described it as a new species in this study.

2. Materials and Methods

2.1. SamplingDuring April 1997, March to April 1998,and April 2017, a total of 41 adult specimens (list in Table S1) initially classified intoM. mixturawere collected from Chaiqiao Town (29.86667° N, 121.55000° E,120 m a.s.l.), Beilun District, Ningbo City, Zhejiang Province of China (Figure 1; here after we called Beilun population). For comparisons, ten adult specimens ofM.mixturafrom Hua’e mountain of Sichuan Province, the type locality of the species (here after we called Hua’e mountain population), and eight adult specimens of it from Yangxian County of Shaanxi Province (here after we called Yangxian population; list in Table S1; Figure 1). All specimens were fixed in 10% buffered formalin for one day, and then transferred to 70% ethanol. Tissues samples were preserved separately in 95% ethanol prior to fixation. Specimens collected in this work were deposited in Chengdu Institute of Biology (CIB), Chinese Academy of Sciences (CAS), Chengdu, China.

Figure 1 Sampling localities in this study. 1, the type locality of Microhyla beilunensis sp. nov., Chaiqiao Town, Beilun District, Ningbo City, Zhejiang Province, China; 2, the type locality of M. mixtura, Hua’e Mountain, Wanyuan City, Sichuan Province, China; 3, another locality of M. mixtura, Huayang Town, Yangxian County, Shaanxi Province, China.

2.2. Molecular phylogeneticsFor molecular work, five samples from Beilun population, eight samples from Hua’e mountain population and eight samples from Yangxian population ofM. mixturawere used (list see Table 1).

Total genomic DNA was extracted from the muscles using a DNA extraction kit (Tiangen Biotech, Beijing).For molecular analyses, DNA fragments of mitochondrial12S rRNA, 16S rRNA, and CO1 genes were amplified and sequenced for 11 samples, 11 samples and 21 samples, respectively (see Table 1). Primers were presented in Table 2. PCR amplifications were performed as following conditions： an initial denaturing step at 95 °C for 4 min; followed by 35 cycles of denaturing at 94 °C for 35 s, annealing at 46-52 °C for 30s, then extending at 72 °C for 1 min, and the final extending step of 72 °C for 10 min. PCR products were purified using a Qiagen PCR purification kit, and sequenced on an ABI3500 automated sequencer using the front/back primers same to PCR. The newly sequences were deposited in GenBank (GenBank accession numbers were shown in Table 1).

(Continued Table 1)

Sequences were aligned using the clustal walgorithm in MEGA v. 6.0 (Tamuraet al., 2013) with default parameters, and then checked by eyes for accuracy. For phylogenetic analyses, we downloaded the compiled available sequence data for the related species from GenBank (GenBank accession numbers see Table 1).Concatenated sequence data of 12S (387 bp) and 16S (540 bp) of 29Microhylaspecies were used for phylogenetic reconstructions. According to Matsuiet al. (2011), oneChaperina fuscasample was chosen as outgroup.

Phylogenetic relationships were reconstructed by Maximum likelihood (ML) and Bayesian Inference(BI) methods, implemented in RAxMLHPC v. 8.1.20(Stamatakiset al., 2006) and MrBayes v. 3.2 (Ronquistet al., 2012), respectively. Before that, the bestfit substitution model was selected by jModeltest v.2.1.4 (Darribaet al., 2012) under the corrected Akaike Information Criterion (AICc; Hurvich and Tsai, 1989),and as results, GTR+I+G model and GTR+I+G+X model were selected for the BI and ML phylogenetic analyses,respectively. Non-parametric bootstrapping with heuristic searches of 1000 replicates was used to assess confidences of branches in ML trees. In BI, we initiated two dependent runs each with four simultaneous Monte Carlo Markov chains (MCMC) for 20 million generations with sampling every 1000 generations and discarded the first 25% of generations as burn-in after the convergence of chains was confirmed. The final majority tree and posterior probabilities (pp) were achieved from the remaining trees.In addition, to further visualize the degree of genetic splits among the new taxon and its closely-related species,we reconstructed a phylogenetic network based on the CO1 gene sequences dataset using maximum parsimony method in SplitsTree v. 4.11.3 (Huson and Bryant, 2006).The supports of Splittree lineages were evaluated by 1000 bootstrap replicates. Finally, we calculated pairwise uncorrectedp-distance between the newly-found taxon and its congeners by the combination of 12S rRNA and 16S rRNA gene sequences in MEGA.

2.3. Morphological comparisonsWe compared morphological characters of the Beilun population with otherMicrohylaspecies. Morphological data for comparisons were obtained from the available literatures(Andersson, 1942; Bain and Nguyen, 2004; Blyth, 1856;Boulenger, 1884, 1897, 1990; Daset al., 2007; Duméril and Bibron, 1841; Dutta and Ray, 2000; Fernando and Siriwardhane, 1996; Hallowell, 1861; Hasanet al.,2014; Howladeret al., 2015; Inger, 1989; Inger and Frogner, 1979; Jerdon, 1853; Khatiwadaet al., 2017; Huet al., 1966; Matsui, 2011; Matsuiet al., 2013; Parker,1928, 1934; Parker and Osman-Hill, 1948; Pillai, 1977;Poyarkovet al., 2014; Schenkel, 1901; Seshadriet al.,2016; Smith, 1923; Stejneger, 1901; Tschudi, 1838; Vogt,1911; Wijayathilakaet al., 2016).

For morphometric analyses, 41 specimens of Beilunpopulation, and ten adult specimens of Hua’e mountain population were measured. Morphological measurements were taken with a digital caliper to the nearest 0.01 mm.The terminology and methods follows Feiet al. (2005)and Watterset al. (2016). Totally 16 characters were measured： snout-vent length (SVL, distance from the tip of the snout to the posterior edge of the vent), head length(HL, distance from the tip of the snout to the articulation of jaw), head width (HW, greatest width between the left and right articulations of jaw), snout length (SL, distance from the tip of the snout to the anterior corner of the eye), eye diameter (ED, distance from the anterior corner to the posterior corner of the eye), internasal distance(IND, minimum distance between the inner margins of the external nares), interorbital distance (IOD, minimum distance between the inner edges of the upper eyelids),upper eyelid width (UEW, greatest width of the upper eyelid margins measured perpendicular to the anteriorposterior axis), lower arm and hand length (LAHL,distance from the elbow to the distal end of the Finger IV), lower arm width (LAW, maximum width of the lower arm), hand length (HAL, distance from the posterior end of the inner metacarpal tubercle to the distal tip of Finger IV), hindlimb length (HLL, maximum length from the vent to the distal tip of the Toe IV), tibia length (TL,distance from the outer surface of the flexed knee to the tibiotarsal articulation), tibia width (TW, maximum width of the tibia), length of tarsus and foot (TFL, distance from the tibiotarsal articulation to the distal end of the Toe IV),length of foot (FL, distance from the posterior end of the inner metatarsal tubercle to the distal tip of the Toe IV).

Table 2 Primers used in this study.

Principal component analysis (PCA) of size-corrected valuables and simple bivariate scatterplots were used to explore and reflect the morphometric differences between the Beilun population and Hua’e mountain population.Independent-SampleTtest was applied to further explore the morphometric differences between these two populations. Independent-SampleTtest was also conducted to test the morphometric differences between the males and the females of the new species. The statistical analyses were performed using SPSS v. 21.0(SPSS, Inc., Chicago, IL, USA), and differences were considered significant atP＜ 0.05.

3. Results

3.1. Phylogenetic analysesThe aligned dataset of the mitochondrial 12S and 16S genes contained 927 bp, in which 608 sites were variable, and 304 were parsimony sites. BI and ML analyses resulted in a consistent topology (Figure 2A). Specimens of Beilun population were clustered into one clade, which was sister to theM.mixturaandM. okinavansisclades. SplitTree analyses based on CO1 gene sequences also showed that the Beilun population formed an independent lineage, which was distinctly separated from its closely-related species(Figure 2B). In addition, the estimated genetic distance based on 12S and 16S gene dataset (Table 3) between the Beilun population andM. mixturapopulations was 2.6%,being higher than that betweenM. mukhlesuriandM.fissipes(2.4%) and equal to that betweenM. malangandM. borneensis(2.6%).

3.2. Morphological analysesAccording to the available morphological data concerningMicrohylataxa, the Beilun population differs fromM. annamensis,M.annectens,M. berdmorei,M. darevskii,M.laterite,M.malang,M. mantheyi,M. marmorata, M. nanapollexa,M. perparva,M. petrigena,M. pulchella,M. pulverata,M.sholigariby presence of basal rudimentary webbing(versus well developed toe webbing reaching disks or reaching distal subarticular tubercles); differentiated fromM. arboricola,M. achatina,M. butleri,M. fusca,M. heymonsi,M. minuta,M. orientalis,M. pineticola,M. pulchella,andM. karunaratneiby having disks only on the toes (versus both fingers and toes disks present); distinguish fromM. fissipes,M. mihintalei,M.mukhlesuri,M. mymensinghensis,M. niphamariensis,M.pulchra,M. picta,M. rubra,M.ornata,M. inornata,M.taraiensisby presenting toe disks (versus both finger and toe disks entirely absent); distinct fromM. zeylanica,M.chakrapanii, M. maculifera,M. palmipes,M. superciliarisby presence of dorsal median longitudinal grooves on the toe disks (versus absent of longitudinal grooves on toe disks).

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The Beilun population could be identified from Hua’e mountain population in the following characters： 1) apart from the stripes, bar-shaped and oval-shaped patterns,the rounded spots present on the dorsum of body and legs (versus absent in the latter); 2) the outer metacarpal tubercles prominently larger than the inner one (versus the outer metacarpal tubercle slightly larger than the inner one in the latter).

Meanwhile, the results of morphometric analyses indicated that the Beilun population could be well separated from Hua’e mountain population. PCA extracted four principal component axes with eigenvalues greater than one, and of these, the first two component axes accounted for 53.34% of the variation (Table 4).The first two principal component axes could separate the Beilunpopulationfrom Hua’e mountain population(Figure 3) mainly based on limb and head characteristics,namely, TFL, FL, TL, HLL, LAHL, SL, IOD, HL, UEW and LAW (Table 4). Independent-SampleTtest showed that in male, there are significant differences on 11 morphometric characters between Beilun population and Hua’e mountain population： the ratios of HW, IND, UEW and LAW to SVL of Beilun population were significantly larger than those of Hua’e mountain population(P＜0.01), and the ratios of SL, IOD, LAHL, HLL, TL, TFL and FL to SVL of Beilun population were significantly less than those of Hua’e mountain population (P＜ 0.05,Table S2).

3.3. Taxonomic conclusionThe results of morphological and molecular phylogenetic analyses supported that Beilun population was distinctly differed from its congeners. Thus, we described it as a new species here：

Microhyla beilunensis sp. nov.

Microhylamixtura： Fei, Ye, Xie and Cai. 1999. Chin Zool Res, Beijing (Beilun, Zhejiang)： 239-240.

Holotype:CIBA980059, adult male (Figure 4A, C, E, G,I, K), SVL 22.09 mm, from Chaiqiao Town (29.86667 °N,121.55000 °E, 120 m a.s.l), Beilun District, Ningbo City,Zhejiang Province, China (Figure 1), collected by Liang Fei on 25 April 1998.

Allotype:CIBA980062, adult female (Figure 5), SVL 28.02 mm, collected on the same day and the same location by Liang Fei.

Figure 2 Phylogenetic relationships within the genus Microhyla. A, the Bayesian inference tree for 29 Microhyla species reconstructed based on 12S rRNA and 16S rRNA gene sequences. Bayesian posterior probabilities/maximum likelihood bootstrap supports were noted near each node, the symbol“-”indicates value below 50. B, phylogenetic network constructed by SplitTree for seven Microhyla species based on CO1 gene sequences.

Figure 3 Plots of the first principal component (PC1) versus the second (PC2) for the males of Microhyla beilunensis sp. nov. and topotype M.mixtura.

Figure 4 Morphological comparisons between Microhyla beilunensis sp. nov. and M. mixtura. Microhyla beilunensis sp. nov. (holotype,male)： dorsal view (A), ventral view (C), lateral view (E), ventral view of hand (G), ventral view of foot (I), dordal median longitudinal grooves of Toe IV (K). M. mixtura (topotype, male)： dorsal view (B), ventral view (D), lateral view (F), ventral view of hand (H), ventral view of foot (J), dorsal median longitudinal grooves of Toe IV (L).

Paratype:36 adult males (CIBA97001-CIB A97023,CIBA98060, CIBA98063-CIBA98067) and three adult females (CIBA97024, CIBA97025, CIBA980061) were collected from the type locality in April 1997, March to April in 1998 by Liang Fei, and seven adult males(CIBBL001-CIBBL007) were collected in April in 2017 by Yufan Wang.

These specimens are all deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS),Chengdu, China.

Etymology: This specific namebeilunensisis a Latinize toponymic adjective that refers to Beilun District, Ningbo City, Zhejiang Province of China, where the new species was collected. For the common name, we suggest Beilun Pygmy frog (English) and Bei lun ji wa (Chinese).

Diagnosis:The new species is allocated to the genusMicrohyla, because it is consistent with the generic diagnostic characters： relatively small body size;maxillary and vomerine teeth absent; vomer divided into two parts and disappearing at the posterior edge of the choana; tongue posteriorly rounded; skin smooth or with tubercles; tympanum hidden or indistinct; 1-2 row of horizontal skin ridges on the palate; fingers without webbing; toes webbed or free of webbing; 2 or 3 metacarpal tubercles; the absence of supernumerary tubercles below the base of fingers and toes; the absence of skin ridge or skin projection between the subarticular tubercles of toes III and IV (Parker, 1934; Feiet al., 2005,2009).

Table 4 Variable loadings for principal components with Eigenvalue greater than 1, from morphometric characters corrected by SVL. All measurements were given in millimeter (mm).

The new species could be identified from its congeners exceptM. mixturaby the several characters： (1)rudimentary webs on toe base; (2) absence of disks and dorsal median longitudinal grooves on finger tips; (3)presence of disks and dorsal median longitudinal grooves on toe tips. As well, the new species could be identified from Hua’e mountain population in the following characters： 1) apart from the stripes, bar-shaped and ovalshaped patterns, the rounded spots present on the dorsum of body and legs; 2) the outer metacarpal tubercles prominently larger than the inner one.

In male, between the new species andM. mixtura,there is significant differences on eleven morphometric characters： 1) the ratios of HW, IND, UEW and LAW to SVL of the new species were significantly larger than those ofM. mixtura(P＜ 0.01); 2) the ratios of SL, IOD,LAHL, HLL, TL, TFL and FL to SVL of the new species were significantly less than those ofM. mixtura(P＜0.05).

Figure 5 Allotype of Microhyla beilunensis sp. nov. (CIBA980062, adult female, SVL 28.02 mm). A, dorsal view; B, ventral view; C, lateral view; D, ventral view of hand; E, ventral view of foot; F, dorsal median longitudinal groove of Toe IV.

Figure 6 Variation in Microhyla beilunensis sp. nov. from Chaiqiao Town, Beilun District, Ningbo City, Zhejiang Province, China. A and B,dorsal and ventral views of the holotype in life, respectively; C and D, dorsal and ventral views of the allotype in life, respectively; E, mating couples; F, breeding site.

Description of holotype:Small body size (SVL 22.09 mm). Head length 75% head width. Snout short, rounded,and projecting beyond the lower jaw. Nostril rounded,distinct, and obviously closer to the tip of the snout than the eye. Interorbital distance (2.46 mm) greater than internasal distance (2.10 mm) and upper eyelid width (1.60 mm), but smaller than eye diameter (3.09 mm). Canthus rostralis and supratympanic fold distinct,tympanum hidden. Tongue posteriorly oval. Maxillary and vomerine edentate.

Forelimbs relatively short and thin. Lower arm (14%of SVL) shorter than hand (24% of SVL). Fingers short, distally rounded, not webbed, disks and dorsal longitudinal grooves absent, the relative finger lengths： III＞ IV ＞ II ＞ I. Subarticular tubercles roughly circle-shaped and distinct. Supernumerary tubercles below the base of finger absent. Inner and outer metacarpal tubercles prominent, oval-shaped, the inner one distinctly larger than the outer one, which close to each other (Figure 4 G). Nupital pads and spines absent.

Hind limb length 170% of SVL, tibia length 55% of SVL, length of tarsal and foot 76% of SVL, and foot length 53% of SVL. Tibiotarsal articulation reaching the eyes or the posterior margin of the eyes when hindlimb extend along the body side. Heels overlapped when hind limbs folded at right angles. Toes distally rounded, and equipped with disks and longitudinal grooves except for the toe I, webbing poorly developed at base of totes,relative toe lengths： IV ＞ III ＞ V ＞ II ＞ I. Inner metatarsal tubercle long and thin, while outer metatarsal tubercle relatively small and rounded, they widely separated from each other (Figure 4I). Subarticular tubercles approximately circle-like and markedly prominent.Supernumerary tubercles below the base of toes absent.

Dorsal skin surface roughish with dense tiny tubercles.Ventral surface of posterior part of body, cloacal region,thigh granules. Throat, chest, and ventral part of thigh and tibia smooth.

Color in life:Dorsal body brownish or gray-brownish,the presence of dark brown markings with light brown margins (Figure 6A, C, E). A thick dark brown butterfly marking between the eyes and on the dorsal body with three pairs of symmetrical figures or streaks or projections, the anterior pair of figures shorter and extending laterally to the middle of upper eyelid; the middle pair relatively long and extending laterally;posterior pair of streaks running obliquely from above the shoulder to the hip, these streaks in connection with each another or not. Dorsum of posterior part of body and legs brownish scattered with dense dark brown circle-like spots; rounded, bar-shaped pattern and streaks with light margins. Supratympanic fold dark brown. Spots on body sides contiguous or not. Limbs indefinitely barred with dark brown. One back brown patches on anterior surface of thigh. Vent light brown with a large black patch. No black and yellowish streaks on the flanks, throat dark purple, chest and belly white mottled with purple (Figure 6B, D).

Variation:The morphometric variation of 37 adult males and 4 adult females (listed in Table S1). Basic statistics for morphological measurements was presentedin Table 5. The butterfly dorsal marking interrupted behind head in some individuals. Behind the butterfly marking, bar-shaped and rounded patterns presented in most individuals; but in some specimens, there one or two stripes parallel with the posterior part of the butterfly marking. Also, the number of spots on body variation,such as, spots on ventral of forelimbs 0-6, those on posterior part of thigh 0-4, those on anterior surface of shank 1-9, and those on posterior surface of shank 2-8.Two patterns near the vent continuous or discontinuous.

Table 5 Measurements of Microhyla beilunensis sp. nov. and M. mixtura. All measurements were given in millimeter (mm).

Sexual dimorphism:The snout-vent length of females significantly larger than that of males (t39= -9.58,P＜0.001). Besides, the ratios of HL, HW, SL, IND, ED,LAHL, LAW, HLL, TL, TW, TFL and FL to SVL of females were significantly greater than those of males(P＜ 0.05; Table S3). Adult males with a single external subgular vocal sac, dark purple throat, occipital furrow remarkably prominent, pink linea musculina present on dorsum and belly of body. Nupital pads and spines absent. The gravid females containing brownish oocytes in oviduct which clearly visible through the belly skin.

Distribution and habits:Microhyla beilunensissp. nov.is now known only from the type locality, Beilun District,Ningbo City, Zhejiang Province of China. This species lives in the water pits or ponds about 3 m diameter or adjacent grass thickets, subterranean holes and mud pits in the mountainous regions at elevation 1400 m a.s.l.The male frogs make vibrating calls on the last 10 days of March. Small tadpoles seen in the early April would dive to the bottom when disturbed. On the 9th April 1998, four gravid females, egg groups, and small tadpoles were found. Mating of females and males took place in the pond of imitated natural eco-breeding in Chengdu(Feiet al., 1999). Small tadpoles were found in the last 10 days April. It took 60 days for the fertilized eggs to finish metamorphosis. Newly metamorphic frogs were 8-10 mm. Sympatric species of amphibians in the Beilun District during the surveys includesHynobius yiwuensis,Echinotriton chinhaiensis,Bufo gargarizans,Rana zhenhaiensis,Pelophylax nigromaculatus,Sylvirana latouchii,Fejervarya multistriata.

Eggs:Animal pole brown, vegetative beige, egg diameter on average 1.0 mm, clutch size 400-657 eggs found in Beilun District, Ningbo City, Zhejiang province of China.

Larvae:At Gosner’s stages 31-35 (early May; Gosner,1960), tadpole’s dorsum dark brown, front part lightly colored on both sides, upper and lower fins darkly colored on the margins, tail end thin and pointed, mouth dorsoterminal, lower labial fold relatively wide, concaved in the middle with 4-5 papillae on the margin of the fold on each side of the middle, no labial teeth or horny beak, the snout-vent length of newly metamorphosed about 10.0 mm (Feiet al., 1999).

4. Discussion

IdentifyingMicrohylaspecies only based on morphology is difficult and/or possibly problematic, because of their relatively high morphological similarity (Howladeret al.,2015; Seshadriet al., 2016). The molecular technology has made the taxonomic research, especially diagnosing cryptic taxa, more efficient and reliable (Khatiwadaet al., 2017; Matsuiet al., 2011). The specimens ofMicrohyla beilunensissp. nov. has been ever recognized asM. mixtura(Huanget al., 1990; Feiet al., 1999, 2009,2012). In this study, the phylogenetic data showed that the Beilun population formed a clade, and was sister toM. mixtura.Besides, the genetic distance between the new species andM. mixturapopulations is 2.6% on 12S and 16S genes, equal to that betweenM. malangandM.borneensis(2.6%), and even greater than that betweenM. mukhlesuriandM. fissipes(2.4%), which conforms to the general species-level in amphibian sister taxa (Venceset al., 2005). Additionally, both the unrooted tree and morphometric analyses could distinctly separate the new species fromM. mixtura.Indeed, there are some subtle morphological characters between these two species after careful comparisons. All the evidences support thatMicrohyla beilunensissp. nov. is a valid species.

The straight-line distance between the type locality ofMicrohyla beilunensissp. nov. and the type locality ofM.mixturais approximately 1342 km. There are some high mountains (e.g. Daba Mountain and Dabie Mountain) and great river (e.g. Yangtze River) between the distributional ranges of the two species, which are often considered as the geographic barriers to block gene flow, facilitating genetic divergence and speciation (Chaveset al., 2011;Crawet al., 2008).Microhyla beilunensissp. nov. prefers to the environment of standing water, and inhabits and breeds in hilly paddy fields, pools and grasslands (Feiet al., 1999, 2009, 2012), possibly, indicating their limited dispersal ability. Therefore, the external barriers and inherent features have probably facilitated the genetic differentiation and speciation of this species from its sister species.

AcknowledgementsWe are grateful to editors and reviewers for their working on the manuscript. Thanks give to Yuxiao HE and Xiong GONG for their help on data analyses. Collections in field were permitted by Zhejiang Forest Resource Monitoring Center (No.ZJFRMC1998034). This study was approved by the animal ethical committee of Chengdu Institute of Biology,Chinese Academy of Sciences, and animal experiments were carried out following the institutional guidelines(No. 2016CIBAEC0622). This work was supported by National Key Programme of Research and Development,Ministry of Science and Technology (2017YFC0505202),National Natural Sciences Foundation of China (NSFC-31471964 and 31201702), Important Research Project of Chinese Academy of Sciences (KJZG-EW-L13) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA19050201).