﻿Tadpoles of four sympatric megophryinid frogs (Anura, Megophryidae, Megophryinae) from Mangshan in southern China

﻿Abstract Sympatric distribution and potentially long larval development time make the assignment of tadpoles confusing in Asian-horned frogs of the subfamily Megophryinae. In this study, we used molecular data to identify four syntopic megophryinid tadpoles from Mangshan on the border between Hunan and Guangdong provinces in southern China: Brachytarsophryspopei, Boulenophrysshimentaina, Bo.cf.ombrophila, and Bo.nanlingensis. A detailed re-description of the Br.popei tadpoles is provided as well as the first descriptions of three Boulenophrys tadpoles based on external morphology and coloration. An effort is attempted to distinguish these tadpoles by coloration patterns: the dorsal pattern, ventral pattern, and pattern on tail are useful for field identification of these tadpoles. However, the variation of color pattern could sometimes make species delineation difficult. Researchers are encouraged to document coloration in life with photographs and the collection of tadpoles of different development stages and sizes advocated in order to better understand how color may change during larval development.


Introduction
The Asian Horned Frogs in the subfamily Megophryinae Bonaparte, 1850 belong to seven genera and 129 recognized species with wide distributed in Asia, ranging from India and Bhutan to China and south to the Sundas and the Philippines (Frost 2022). Tadpoles of Megophryinae are characterized by a funnel-like oral disc, which allows them to feed beneath the water surface or anchor to a substrate for keeping safe during floods or other threats; this mouthpart specialization had intrigued scientists for almost a century (Hora 1928;Pope 1931;Liu 1950;Huang et al. 1991;Zeng 2021).
Seven monophyletic clades were found within Megophryinae based on molecular analysis , previously regarded as seven subgenera of Megophrys sensu lato: Atympanophrys Tian & Hu, 1983;Boulenophrys Fei, Ye andJiang in Fei &Ye, 2016 (under the name Panophrys Rao &Yang, 1997); Brachytarsophrys Tian & Hu, 1983;Megophrys Kuhl & Van Hasselt, 1822;Ophryophryne Boulenger, 1903;Pelobatrachus Beddard, 1908;and Xenophrys Günther, 1864. Recent taxonomic studies have elevated the seven subgenera to the level of genera Lyu et al. 2021;Qi et al. 2021). However, the morphological differences remain insufficient for clear delineation of these genera. A recent review of the genus Brachytarsophrys by  regarded the tadpole ventral pattern as a diagnostic character for species identification. Subsequently, Tapley et al. (2020a) tentatively suggested the presence of "white longitudinal stripe on the ventrolateral surface of the head and body" in tadpoles as a diagnostic character for Brachytarsophrys. However, there are no known larval characters that differentiate the remaining megophryinid genera from one another. The external morphology, including oral disc, as well as internal buccal features are highly conserved in all species in this subfamily (Huang et al. 1991;Grosjean 2003). The coloration in life, although rarely described, is considered useful for species identification since Leong and Chou (1998) and has attracted more and more attention (Oberhummer et al. 2014;Poyarkov et al. 2017;Tapley et al. 2017Tapley et al. , 2020aTapley et al. , 2020bLi et al. 2018;Munir et al. 2019;Liu et al. 2020;Shi et al. 2020aShi et al. , 2020bShi et al. , 2021Wu et al. 2020). However, compared to the rate of new megophryinid frog species descriptions, tadpole descriptions remain scarce or are very brief, resulting in insufficient diagnostic characters for comparison and/or field recognition.
Before molecular analysis was widely used in megophryinid taxonomic studies, tadpoles were assigned to species based on their association with post-metamorphic/adult specimens collected from the same site, e.g., Boulenophrys jinggangensis , Xenophrys mangshanensis (Fei & Ye in Fei et al. 1990), tadpoles described by Fei and Ye (2016) and Xenophrys longipes (Boulenger, 1886), tadpoles described by Leong and Chou (1998). Notably, many megophryinid species are reported to be in sympatry, including sister species (e.g., Wang et al. 2014Wang et al. , 2019Chen et al. 2017;Poyarkov et al. 2017;Liu et al. 2018;Mahony et al. 2018Mahony et al. , 2020Shi et al. 2020a;Tapley et al. 2018Tapley et al. , 2020bTapley et al. , 2021. Some megophryinid tadpoles are suspected of having long larval development periods of over one year (Grosjean 2003;Tapley et al. 2020a). Consequently, cf. tadpoles collected from the same site with adult frogs may not necessarily belong to the same species. Thus, the description of tadpoles without molecular data has become suspicious if there are any other megophyinid species discovered in sympatry.
Mangshan, a part of Nanling Mountains, is located on the border between Hunan and Guangdong provinces in southern China. The first megophryinid record from this area was a tadpole with a funnel-like oral disc collected at the mountain top of Guangdong/Hunan border (Mell 1922), which was identified as "Megalophrys boettgeri" (currently Boulenophrys boettgeri). This record has been shown to be erroneous as no adult B. boettgeri frogs were found in this area (Shen 1983;Shen et al. 2014), and the horned frogs collected in Mangshan were identified as "Megophrys kuatunensis" (currently Bo. kuatunensis), "Megophrys brachykolos" (currently Bo. brachykolos), and Brachytarsophrys carinense in "Fauna Hunan, Amphibia" (Shen et al. 2014). Subsequently, this "Brachytarsophrys carinense" population was described as a new species Brachytarsophrys popei , while the identity of the two Boulenophrys frogs, that were both previously regarded as widespread (e.g., Fei et al. 2009;Fei and Ye 2016) was questioned after molecular analysis based on large-scale sampling (Tapley et al. 2017;Liu et al. 2018;Gao et al. 2022).
In this study, we collected tadpoles bearing funnel-like oral disc from Mangshan. Using DNA barcoding, we confirmed that this collection of syntopic tadpoles was composed of four species: Brachytarsophrys popei, Boulenophrys nanlingensis (Lyu, Wang, Liu & Wang in Wang et al. 2019), Bo. shimentaina , and Bo. cf. ombrophila (Messenger & Dahn in Messenger et al. 2019). Based on the examination of external morphology and coloration in life, we re-described the tadpoles of Br. popei and provided the first description of the three Boulenophrys tadpoles.

Samples
All tadpoles were collected from Mangshan, Yizhang, Hunan Province, China during field surveys in June, July, and November 2021. Specimens were photographed in life in a transparent acrylic box before being euthanized with buffered tricaine methanesulfonate (MS-222) and then fixed with 10% formalin for storage. Tissue samples (tail fin/muscle) were preserved in 95% ethanol for molecular analysis. Specimens were deposited at the Institute of Wildlife Conservation, Central South University of Forestry and Technology (CSUFT), Changsha, China.

Molecular analysis
Segments of the 16S ribosomal RNA gene (16S) were used for species identification. Primer sequences (L3975 and H4551) from Simon et al. (1994) were used for PCR amplification in 50 µl reaction volumes under the following conditions: 98 °C for 2 min; followed by 30 cycles of 98 °C for 10 sec, 55 °C for 10 sec, and 72 °C for 10 sec, with a final extension step at 72 °C for 5 min. PCR purification and sequencing were performed by Biomarker Technologies Co. (Beijing, China). The new sequences were then searched on BLAST (NCBI) to verify their approximate identity. The identification of GenBank accession numbers were retrieved from the BLAST result and manually verified by checking the original references. Uncorrected p-distance between the new sequences and sequences from the BLAST result was calculated using MEGA 6 (Tamura et al. 2013). Before calculating uncorrected p-distances, sequences were aligned using the MUSCLE algorithm with default parameters (Edgar 2004) and trimmed with gaps partially deleted in MEGA 6.

Morphology
Image J 1.53k software (Schneider et al. 2012) was used to measure the tadpoles from photographs of preserved specimens taken next to a scale (10 mm length). The staging follows Gosner (1960), and the terminology for external morphology follows Altig and McDiarmid (1999). Measurements and morphometric abbreviations follow Oberhummer et al. (2014) For the measurement of oral disc width (ODW) in preserved specimens, we expanded the oral disc by anchoring it to a glass (as shown in Fig. 3D).

Tadpole identification
Two tadpoles were identified as Brachytarsophrys popei based on an uncorrected pdistance of 0.0-0.7% from the samples in the type series from Hunan, Guangdong, and Jiangxi Provinces (GenBank accession numbers: KM504251-KM504258). Three tadpoles from the same collection site and bearing the same characteristics as the above two tadpoles but without molecular data were also assigned to Br. popei. The collection site of these tadpoles is only ~ 5 km from the collection site of paratype SYS a00589 (GenBank accession number: KM504051) in the adjacent Nanling Nature Reserve, Guangdong Province.
A total of 14 tadpoles was identified as Boulenophrys nanlingensis, which exhibited an uncorrected p-distance of 0.0-0.4% from the holotype SYS a001964 (GenBank accession number: MH406646) collected ~ 10 km in the adjacent Nanling Nature Reserve, Guangdong Province.
Five tadpoles were identified as Boulenophrys shimentaina, which exhibited an uncorrected p-distance of 1.2% from the type series (GenBank accession numbers: MH406655-MH406656, and 787-788) collected from Shimentai Nature Reserve, Guangdong Province, China ~ 70 km to the south of Mangshan. This genetic distance is smaller than the interspecies p-distance in the subfamily Megophryinae used to identify tadpoles (1.4% in Br. intermedia, Tapley et al. 2020a).
Four tadpoles showed an uncorrected p-distance of 1.8-2.1% from the type series of Bo. ombrophila (GenBank accession numbers: KX856397-KX856404) collected from Wuyishan, Fujian Province, China ~ 500 km to the northeast of Mangshan. This genetic distance almost equals to the threshold (2.0% in 16S gene, proposed by Chen et al. 2017) for a potential new species in this subfamily. However, the other two populations of Bo. ombrophila suspected by Messenger et al. (2019) from Jiulianshan, Jiangxi Province (Megophrys sp 8 in Liu et al. 2018) and Renhua County, Guangdong Province (M. sp 9 in Liu et al. 2018) were closer to our samples both in the geographical distance of collection sites and genetic distances based on 16S gene. The Jiulianshan population of Bo. ombrophila (GenBank accession numbers: MH406836-MH406840) collected from ~ 160 km to the east of Mangshan showed an uncorrected p-distance of 1.6-1.8% from our samples. The Renhua population of Bo. ombrophila (GenBank accession numbers: MH406650-MH406653, and MH406834) collected from ~ 70 km to the east of Mangshan showed an uncorrected p-distance of 1.4-1.6% from our samples. Further examination of adult frogs revealed morphometric differences between the Mangshan population and the type series from Wuyishan, Fujian. However, the morphometric and morphological data were unavailable for the Jiulianshan population and Renhua Population. Thus, we currently identified the Mangshan population as Bo. cf. ombrophila.

Morphological description
All examined specimens exhibited a funnel-like oral disc corresponding to the tadpole description of "Megophrys minor" by Liu (1950): "The mouth is terminal, with a large funnel that has two long lateral wings, a short ventral wing and a comparatively narrow convex flap above. The tips of the lateral and ventral wings are bluntly pointed." Detailed tadpole descriptions are given below. Fig. 1 Remark. The following description is based on five tadpoles at Stages 26-27 (N = 2) and 36-37 (N = 3). Body ratio ranges represent all specimens. Raw measurements are given in Table 1. External morphology. The body is oval, robust, and flattened above (BW/BL 53.3-55.7% at Stages 26-27, N = 2; and 53.6-55.2% at Stages 36-37, N = 3); the head is wider than the trunk; the eyes are located dorsolaterally, the pupils are round; the nares are oval, opening laterally, closer to the eye than to the tip of the snout (NE/SN 68.8-73.3% at Stages 26-27, N = 2; and 73.7-83.3% at Stages 36-37, N = 3); the internarial distance  Table 1. Morphometric data of the tadpole specimens used in this study. For abbreviations, see Materials and Methods. "*" indicates specimens with broken tails, and "\" indicates "no data". Coloration. In life, the background color of the head and trunk is dark brown; the dorsal pattern is pale brown interspersed with dark brown chromocytes, extending to above the horizontal level of the spiracle on the trunk from a lateral perspective; the dorsal surface of the anterior part of the tail is pale brown marbled with dark brown speckles; neuromasts are distinctly visible on the head, trunk and tail; the region between the anterior edges of the eyes and the median point of the upper lip is pigmented with a dark brown V-shaped pattern; the narial rims are pale brown; the oral disc is golden-pigmented, with a translucent edge; the submarginal papillae on lips are dark brown-pigmented. Laterally, the tail is pale brown-pigmented; dense goldish spots are located at the anterior part of the lateral surface of tail muscle, becoming smaller and at the middle, then disappearing posteriorly; three distinct dark brown stripes extended from the body-tail junction, and horizontally positioned at the anterior part of the tail; the upper and lower stripes end before reaching the maximum tail height, while the middle stripe is about half the length of the others; the upper and middle stripes are incomplete; the anterior part of the upper fin is opaque, marbled with goldish pigmentation and brown speckles; the anterior part of the ventral fin, as well as the anal tube are semi-translucent with dense large golden spots; the rest of the fins are semi-translucent, and exhibit sparse dark brown speckles interspersed with small goldish dots. The ventral surface of the body is rather dark; the belly is dark purplish covered with dense white spots; two longitudinal stripes, positioned ventrolaterally, extending from the snout to the vertical edge of the eyes posteriorly, and sometimes appear to broken; a transverse bar is positioned at the head-trunk junction of the vertical edge of the anterior spiracle and is always interrupted at the middle; the spiracle region and the corresponding region on the other side of the body, are covered with a short white stripe, that starts from the head-body connection, and terminated before reaching the region of the spiracle tube opening; regions without white pigmentation have less melanocytes; the gills and gut coils are indistinctly visible through the ventral skin. The eye sclera is silver with black dots; the iris periphery is wide and black; the iris is golden sprinkled with black dots; and the spiracle is translucent without pigmentation. In tadpoles at Stages 36-37, the hindlimbs are semi-transparent, and the outer aspect of the legs exhibits brown pigmentation interspersed with goldish chromocytes.

Brachytarsophrys popei
In preserved specimens, the tail stripes are still prominent; an incomplete V-shaped pigmentation pattern is still visible; the ventral pattern is translucent milky white; the golden pigmentation wanes on the oral disc; and the hindlimb bones are visible in ventral view in Stage 36-37 tadpoles.
Comparisons. Tadpoles of Br. popei differ significantly from the three syntopic Boulenophrys tadpoles described below by the unique pattern of two longitudinal white ventrolateal stripes on head, a transverse white bar on chest, and distinct large spots on belly (vs. absence of stripes and bars, and smaller spots/speckles on belly).
The differences in ventral pattern between four Brachytarsophrys tadpoles were compared by  and summarized in Table 2. The tadpole of Br. popei (Stage 29, N = 1) illustrated in their paper (also in Zhao et al. 2014, but marked as Table 2. Comparison of color pattern among tadpoles of the subfamily Megophryinae which were identified based on molecular data. "*" indicates characteristics not mentioned in the text but were illustrated in the figure, and "\" indicates "no data". . We believe this pattern should be confirmed using more specimens at an earlier developmental stage in case this is a transitional form during metamorphosis. Further comparisons between Br. popei tadpoles and all megophryinid tadpoles that were identified using molecular data are shown in Tables 2 and 3. Ecology notes. All tadpoles were collected from an artificial roadside drainage ditch (Fig. 5C) at night while feeding beneath the water surface. Upstream of the ditch is a narrow, slow-moving stream with many rocks covered by moss. The ditch was rocky with a sandy substrate. The maximum depth of this ditch was ~ 0.5 m. Branches of plants from the mountain side of the road extended over this ditch, however, sunlight did reach the water surface at certain times of the day. Tadpoles were observed in a still water stretch behind big rocks, or a small dam formed by submerged leaf litter. Three tadpoles at Stages 36-37 were collected on 30 May 2021 at 22:30 h, together with tadpoles of Bo. shimentaina and Bo. nanlingensis with an ambient air temperature of ~ 20 °C. Two tadpoles at Stages 2627 were collected on 16 November 2021 at 19:30 h with an ambient temperature of ~ 13 °C. Tadpoles were considered nocturnal because we did not encounter any tadpoles during the day. Male Br. popei frogs began their calling activities under rock crevices in this ditch in late July. Zhao et al. (2014) reported that the breeding season of Br. popei is July to September, and that their tadpoles (Stages 26-29) were collected in April and December. This indicates that the development of these tadpoles may be very prolonged, and it is likely that they can over winter. Interestingly, it is unknown why no tadpoles were collected during the breeding season both in this study and in Zhao et al. (2014).  Table 3. Comparison of morphological characteristics among tadpoles of the subfamily Megophryinae, which was identified based on molecular data. "*" indicates characteristics not mentioned in the text but were illustrated in the figure, and "\" indicates "no data".  Fig. 2 Remark. The following description is based on five tadpoles at Stages 25-28 (N = 5). Body ratio ranges represent all specimens. Raw measurements are given in Table 1. External morphology. The body is oval and flattened above (BW/BL 51.3-55.0%, N = 5); the eyes are located dorsolaterally, and the pupils are round; the nares are oval, open laterally, closer to the eye than to the tip of the snout (NE/SN 62.5-71.4%, IND/IOD 67.6-71.9%, N = 5); the rims of nares are serrated, slightly raised from the body wall; the spiracle is sinistral, low on the left flank; the spiracle tube is short, free from the body at the tip and opens laterally (SS/BL 53.4-58.0%, N = 5); the anal tube opens medially, unattached to the ventral fin; the dorsal fin arises behind the body-tail junction while the ventral fin is connected to the trunk; the tail muscle is massive, taller than tail fins before reaching the 2/3 part of the tail length (TMH/MTH 50.0-55.6%, N = 5); the tail tip is bluntly pointed, the tail length accounts for 69.5-76.1% (N = 4) of the total length; the mouth is terminal and the oral disc is funnel-like (BW/ODW 65.2-77.2%, N = 5); four rows of oval submarginal papillae are visible on the upper lip, and five rows of oval submarginal papillae on the lower lip; keratodonts are absent; the upper jaw sheath is comb-like, exhibiting a small median notch; the lower jaw sheath is thin and sickle-shaped, weakly keratinized, and finely serrated.

Boulenophrys shimentaina
Coloration. The following description is based on a tadpole at Stage 27 (CSUFT T10285). In life, the background color of the body and tail is semi-transparent dark brown; the dorsum is pigmented pale brown which extends to the dorsal surface of anterior tail and gradually becomes golden; a distinct circled marking is present at the center of dorsum, forming a saddle with the background dark brownish coloration; the middle of the saddle is pigmented pale brown; and the neuromasts are distinctly visible. Laterally, the dorsal pattern extends to the region above the horizontal level of the spiracle on the trunk, and covers the whole lateral surface of head; the lateral surface of tail is pigmented brown; the tail and fins are covered with irregularly shaped pale golden spots, interspersed with dense dark brown speckles; the fins are semi-transparent; the anterior part of the dorsal fin is marbled with golden and dark brown speckles; the junction of the anterior half of the dorsal fin and the caudal muscle is pigmented dark brown, forming an incomplete line; the anterior part of the ventral fin and the anal tube exhibit minimal dark brown pigmentation; the posterior part of tail and fins are pigmented with dense dark brown markings. The ventral body is semitranslucent grey, pigmented with dark brown chromocytes, and is covered with dense small, indistinct milky-white speckles; the gills and gut coils are visible through the ventral skin; two large, milky-white spots are present on each side of the ventrolateral surface of head-body connection and are followed by a cluster of smaller spots. The oral disc is translucent milky white; the lateral and middle wings are covered with orangish pigmentation; the tips of the wings and the middle of the upper lip exhibit dark brown pigmentation; the submarginal papillae on lips are dark brown, and the narial rims are pigmented beige. The eye sclera is silver with black dots; the iris is orange sprinkled with black dots; and the spiracle is translucent without pigmentation.

Variation of coloration in life.
The other four tadpole specimens match most of the descriptions above. However, the dorsum pattern of a saddle is not clearly visible in CSUFT T10156 and the dorsum is almost uniform pale brown in CSUFT T10177. The ventrolateral spots on head-body connection are very large in CSUFT T10283 (Stage 28, Fig. 2F), but smaller in CSUFT T10277 (Stage 26, Fig. 2E). In preserved specimens, the pale brown pigmentation on the dorsal surfaces of the body and tail are still visible; the golden and orangish pigmentation fade to milky white; the white spots on each side of the ventrolateral surface of head-body connection become translucent; there is no orange pigmentation on the mouthparts, and prominent black pigmentation can be observed on the tail.
Comparisons. The two distinct, conspicuous ventrolateral spots on ventrolateral surface of head-body connection could distinguish the tadpoles of Bo. shimentaina from most Boulenophrys tadpoles, including Bo. fansipanensis, which have a single spot visible on each side, and Bo. rubrimera, Bo. hoanglienensis, Bo. jingdongensis, Bo. leishanensis, Bo. jiangi, and Bo. lushuiensis with no ventrolateral spots; the ventral pattern of indistinct, small speckles on belly could distinguish Bo. shimentaina tadpoles from Bo. lini, which have dense large speckles (see Wang et al. 2014: fig. 5F). Furthermore, the tadpoles of Bo. shimentaina differs from Bo. lushuiensis by having a silver sclera with black dots (vs. black with golden pigments); and from Bo. baishanzuensis by having a pale brown pattern on dorsum (vs. uniformly brownish black).
Tadpoles of Bo. shimentaina could be distinguished from the syntopic Boulenophrys tadpoles in Mangshan (see below for the descriptions) by having a dark brown background coloration of body and tail (vs. pale brown in Bo. cf. ombrophila and Bo. nanlingensis), and a tail pattern of dense dark brown markings posteriorly (vs. several large brown spots along tail muscle in Bo. cf. ombrophila; and many brown speckles in Bo. nanlingensis). Further comparisons between Bo. shimentaina tadpoles and all megophryinid tadpoles identified based on molecular data are shown in Tables 2, 3.
Ecology notes. A single tadpole at Stage 25 was collected on 30 May 2021, together with the tadpoles of Bo. nanlingensis and Br. popei from the road ditch (Fig. 5C) that was mentioned above in the Br. popei section. Four tadpoles at Stages 25-28 were collected together with tadpoles of Bo. nanlingensis and Bo. cf. ombrophila from a rocky, slow-flowing narrow stream (Fig. 5B) on 14 July 2021 at 23:20 h while nearby adult males were calling. As this stream is located near the mountain top, it is narrow and slow. There were low trees and bamboo on both sides of the stream, and many fallen logs lay across the stream with a rocky stream bed. This site was used by many species as a breeding site including Bo. nanlingensis, Bo. shimentaina, Br. popei, Leptobrachella mangshanensis (Hou, Zhang, Hu, Li, Shi, Chen, Mo & Wang, 2018), and Quasipaa exilispinosa (Liu & Hu, 1975). The tadpoles of Bo. shimentaina found in this stream were observed at night in an area with sandy substrate near the stream bank or in still water behind a small dam formed by submerged leaf litter. Sunlight could reach the surface of these areas at certain times during the day. While feeding beneath the water surface, the tadpoles rely on submerged leaf litter or rocks (Fig. 5A). Once disturbed, they hid quickly under the submerged leaf litter and emerged from the leaf litter after several seconds. In the still water area where these tadpoles were found, we also encountered many Q. exilispinosa tadpoles on the stream substrate, and a subadult newts, Pachytriton xanthospilos Wu, Wang & Hanken, 2012, hiding under submerged leaf litter. Male Bo. shimentaina frogs were observed calling from late June to August in Mangshan, and it was suggested that the breeding season of Bo. shimentaina is from April to August in Shimentai Nature Reserve, Guangdong Province . It is not clear if tadpoles complete metamorphosis within a single year, and we didn't collect any tadpoles of more advanced developmental stages. Fig. 3 Remark. The following description is based on four tadpoles at Stages 25-27 (N = 3) and Stage 36 (N = 1). Body ratio ranges represent all specimens except where specified. Raw measurements are given in Table 1. External morphology. The body is flattened and oval (BW/BL 52.9-54.8% at Stages 25-27, N = 3; and 51.0% at Stage 36, N = 1); the eyes are located dorsolaterally, the pupils are round; the nares are oval, opening laterally, closer to the eye than to the tip of the snout (NE/SN 60.0-73.3% at Stages 25-27, N = 3; and 82.4% at Stage 36, N = 1); the internarial distance is smaller than interorbital distance (IND/IOD 65.8-68.2% at Stages 25-27, N = 3; and 66.7% at Stage 36, N = 1); the rims of nares are serrated, slightly raised from the body wall; the spiracle is sinistral, low on the left flank, and opens posterolaterally; the spiracle tube protrudes posteriorly, free from the body at the tip (SS/BL 54.9-59.6% at Stages 25-27, N = 3; and 56.0% at Stage 36, N = 1); the anal tube opens medially, unattached to the ventral fin; the dorsal fin arises behind the body-tail junction while the ventral fin is connected to the trunk; the tail muscle is massive, taller than tail fins until reaching 2/3 of the tail length (TMH/MTH 45.8-53.3% at Stages 25-27, N = 3; and 48.3% at Stage 36, N = 1); the tail tip is usually sharply pointed (bluntly rounded in one small-sized specimen CSUFT T10992, Stage 25, TTL 20.9 mm); the tail length accounts for 72.4-75.6% of the total length at Stages 25-27 (N = 3), and 70.3% at Stage 36 (N = 1); the mouth is terminal and the oral disc is funnel-like (BW/ODW 61.5-69.2% at Stages 25-27, N = 3; and 61.4% at Stage 36, N = 1); three and four rows of short oval submarginal papillae are present on the upper and lower lips, respectively; keratodonts are absent; the upper jaw sheath is comb-like, exhibiting a small median notch, whereas the lower jaw sheath is thin and sickle-shaped, weakly keratinized, and finely serrated.

Boulenophrys cf. ombrophila
Coloration. The following description is based on a tadpole at Stage 27 (CSUFT T10272, Fig. 3A-C). In life, the background color of the body and tail is semi-transparent beige; the dorsal surface of the body is covered by a pale brown pattern that extends to the dorsal surface of the anterior part of the tail; a dark spot is present between the eyes and followed by a short beige vertical line on the anterior dorsum; the neuromasts are distinctly visible; and sparse dark brown markings alongside the vertical line and the dorsolateral neuromasts. Laterally, the dorsal pattern extends to above the horizontal level of the spiracle; three large, whitish, and golden pigmented spots are present behind the eyes on each side of the lower part of head-body connection, two of them are visible from the ventral view; the lateral surface of the tail and fins is covered by irregularly shaped pale golden spots, interspersed with whitish chromocytes which form short lines, and brown chromocytes which gather into large spots along the tail muscle at the posterior part of the tail; the fins are semi-transparent; the anterior part of the dorsal fin is marbled with golden and dark brown speckles; the anterior part of the ventral fin and the anal tube exhibit minimal brown pigmentation whereas the rest of fins that exhibits sparse dark brown speckles. The ventral body skin is translucent beige, covered by dense milky white speckles; the gills and gut coils are indistinctly visible through the ventral skin. The oral disc is translucent beige; the lateral and middle wings are covered by orange pigmentation; the submarginal papillae on lips are dark brown; the narial rims are yellow; the eye sclera is silver with black dots; the iris is bright orange sprinkled with black dots; the spiracle is translucent, with scattered golden pigmentation.

Variation of coloration in life.
Among the remaining three specimens examined, two tadpoles at Stages 26 and 36 match most of the coloration pattern of the description for CSUFT T10272 above (see Fig. 3E, F for ventral patterns). However, the dark spot between the eyes is not present in both; the vertical line is more distinct in CSUFT T10281, which extends from the middle of the eyes to the body-tail connection; and the vertical line in CSUFT T10270 is a bit longer than CSUFT T10272, which extend to the posterior dorsum. In the Stage 36 tadpole (CSUFT T10270), the hindlimbs are semi-transparent, and the legs are covered externally by brown chromocytes. A smallsized tadpole at Stage 25 (CSUFT T10992, TTL 20.9 mm) exhibited a significantly different coloration from other three tadpoles of larger body size (Stages 26-27, and 36; TTL 30.4-33.7 mm): the dorsal pattern is pale brown, with scattered dense dark melanocytes, especially at the vertebral line region; an inconspicuous orange V-shaped pattern between the anterior edges of the eyes and the median point of the upper lip; two orangish spots at the posterior edge of the eyes; the orangish pigmentation is also diffuse on the dorsal aspect of the body and tail; the ventral skin is almost clear, translucent milky, with sparse goldish speckles on the edge of the belly; the belly is covered with dense melanocytes, however, gut coils clearly visible through these melanocytes.
In preserved specimens, a fading of the dorsal pattern is observed; the golden spots on the lateral surfaces of the tail are not visible; the large spots on the anterior corner of the spiracle and gills become translucent, and the orange pigmentation on the lips disappears.
Comparisons. The tadpoles of Bo. cf. ombrophila differ from the syntopic tadpoles of Bo. shimentaina by the semi-translucent beige background coloration of body and tail (vs. dark brown), a ventral pattern of dense milky white speckles on belly (vs. indistinct small speckles), and the pattern on tail of large spots along tail muscle (vs. dense markings posteriorly); from Bo. nanlingensis (see below for tadpole description) by the ventral pattern of dense whitish speckles (vs. sparse speckles), and the pattern on tail of several large spots (vs. many speckles).
Compared to other described Boulenophrys tadpoles where species identification is supported with molecular data, the tadpoles of Bo. cf. ombrophila differs by the ventral pattern of dense whitish speckles (vs. relatively sparse metallic blue speckles in Bo. fansipanensis; sparse whitish speckles in Bo. jingdongensis; sparse metallic grey blue speckles in Bo. hoanglieneneis; and scattered with silver tiny patches in Bo. lushuiensis), and the tail pattern of several large spots along tail muscle (vs. few dark brown speckles in Bo. fansipanensis; absence of large spots in Bo. jingdongensis; many dark brown speckles in Bo. hoanglienensis; small black spots in Bo. baishanzuensis; few dark spots on posterior tail muscle in Bo. jiangi; small black spots on tail muscle in Bo. leishanensis; and dozens of small dark brown patches in Bo. lushuiensis). Further comparisons between Bo. cf. ombrophila tadpoles and all megophryinid tadpoles that were identified based on molecular data are shown in Tables 2, 3.
Ecology notes. One observed breeding site of Bo. cf. ombrophila was a relatively broad wetland crossed by a small shallow creek. Several water sources from the gentle slope of the bamboo forest fed this creek and made the entire area very wet. This breeding site was muddy, covered with leaf litter and fallen logs. The creek was narrow and slow flowing with maximum depth of 0.2 m. Some fallen logs blocked the creek and created still water areas. Only male Bo. cf. ombrophila and Q. exilispinosa were observed calling in this site during our visits from May to August, and in November. The potential predator of these frogs, an aquatic snake Opisthotropis cheni Zhao, 1999 which was observed once, in July, in this creek. A single small-sized tadpole specimen (CSUFT T10992, TTL 20.9 mm) at Stage 25 was collected from this site while the male frogs were heard calling before a heavy rainstorm on 3 June 2021 at 19:30 h at dusk. Three tadpoles were collected from the rocky area (Fig. 5B, mentioned above in the Bo. shimentaina section) 20 m downstream of this creek together with tadpoles of Bo. shimentaina and Bo. nanlingensis. Interestingly, male Bo. cf. ombrophila frogs were not observed calling in the rocky area, and the other two species did not breed in this wetland. This indicates a different microhabitat preference between these congeneric species. The breeding season of Bo. cf. ombrophila ends in mid-July in Mangshan. It is not clear if tadpoles will complete metamorphosis during the year. Fig. 4 Remark. The following description is based on 14 tadpoles at Stages 25-29 (N = 12), and 34-35 (N = 2). Body ratio ranges represent all specimens except where specified. Raw measurements are given in Table 1. External morphology. The body is elongated, oval, and flattened above N = 11;N = 2); the eyes are located dorsolaterally, and the pupils are round; the nares are oval, closer to the eye than to the tip of the snout N = 12;.5% at Stages 34-35, N = 2); the internarial distance is smaller than interorbital distance (IND/IOD 61.5-71.0% at Stages 25-29, N = 12; and 68.1-69.2% at Stages 34-35, N = 2); the nares open laterally; the rims of nares are serrated, slightly raised from the body wall; the spiracle is sinistral, low on the left flank, and opens posteriorly; the spiracle tube is short and slightly protrudes posteriorly (SS/BL 54.8-62.7% at Stages 25-29, N = 12; and 59.3-63.6% at Stages 34-35, N = 2). The anal tube opens medially and is unattached to the ventral fin; the dorsal fin arises behind the body-tail junction, and the ventral fin is connected to the trunk. The tail muscle is massive, deeper than tail fins before reaching the maximum tail height (TMH/MTH 43.4-63.0% at Stages 25-29, N = 11; and 50.7-54.5% at Stages 34-35, N = 2); the tail tip is pointed, the tail length accounts for 69.5-76.1% (at Stages 25-29, N = 11) and 73.1-74.4% (at Stages 34-35, N = 2) of the total length; the mouth is terminal and the oral disc is funnel-like (BW/ODW 63.3-79.6% at Stages 25-29, N = 12; and 73.1-85.7% at Stages 34-35, N = 2); four and five rows of short oval submarginal papillae can be observed on the upper and lower lips, respectively; keratodonts are absent; the upper jaw sheath is comb-like, exhibiting a weak median notch; the lower jaw sheath is thin and sickle-shaped, weakly keratinized, and finely serrated.

Boulenophrys nanlingensis
Coloration. The following description is based on a tadpole at Stage 25 (CSUFT T10303, Fig. 4A-C). In life, the background color of the body and tail are semitransparent grey; the dorsal surface of the body is covered by a pale brown pattern that extends to the dorsal surface of the anterior part of the tail; roughly symmetrical dark brown pigmentation can be observed on the dorsal body; and the neuromasts are distinctly visible. Laterally, the dorsal pattern extends to above the horizontal level of the spiracle; the lateral surface of the tail is pigmented brown, interspersed with pale golden spots and irregular dark brown speckles; the fins are semi-transparent and scattered with pale golden spots; the anterior part of the dorsal fin is marbled with golden and dark brown speckles, with the dark brown speckles forming an incomplete line; the anterior part of the ventral fin and the anal tube, lacks brown pigmentation but with sparse golden speckles. The ventral surface of the body is semi-transparent grey; the gills appear pink through the ventral skin; two large gold-pigmented white spots are present at ventrolateral head-body connection; the gut coils are distinctly visible through the ventral skin, the belly is scattered with small whitish speckles; the oral disc is translucent beige; the lateral and middle wings are covered by orangish pigmentation; the submarginal papillae on lips are dark brown; the narial rims are beige; the eye sclera is silver with black dots; the iris is copper-colored sprinkled with black dots, comparable to the iris coloration in adults; and the spiracle is translucent.
Variation of coloration in life. The dorsal pattern coloration in tadpoles of Bo. nanlingensis is subject to significant variation both in same stages and between stages. At Stage 25, a small-sized tadpole (CSUFT T10144, TTL 18.7 mm) exhibits a yellowish dorsum with pale orange blotches, and dark brown pigmentation present posteriorly; another small tadpole (CSUFT T10284, TTL 18.9 mm) displays a brown dorsum with whitish patterns on the dorsolateral surfaces of the trunk and these extend to the tip of the tail. The coloration of the medium-sized tadpoles at Stage 25 (CSUFT T10261, TTL 25.1 mm; and CSUFT T10262, TTL 27.2 mm) and a broken-tailed individual at Stage 25 (CSUFT T10302) correspond to the dorsal pattern of CSUFT T10303 described above. However, the shape and coverage of dark brown markings varies between individuals. At later Stages 27-29, three mediumsized tadpoles (CSUFT 10377, Stage 27, TTL 28.1 mm; CSUFT T10376, Stage 27, TTL 24.8 mm; and CSUFT 10378, Stage 28, TTL 26.9 mm) exhibit a bi-colored   ) showed a ventral pattern of large spots on belly that was different with other specimens. For tadpoles at Stages 34-35 (CSUFT T10969, and CSUFT T10986), the hindlimbs are semi-transparent, the outer aspect of the legs is pigmented yellow and interspersed with brown chromocytes on top.
In preserved specimens, a fading of the dorsal pattern is observed; the tail is translucent with sparse dark-brown pigmentation; the orange pigmentation on lips is no longer visible; the whitish speckle on the ventral surface and the nares are translucent.
Comparisons. The variation of dorsum pattern makes the tadpoles of Bo. nanlingensis are sometimes confused with the syntopic tadpoles of Bo. cf. ombrophila. Usually, the ventral pattern of sparse speckles (vs. dense speckles) and the tail pattern of many small speckles (vs. large spots) could distinguish them. An exception is the smallsized tadpole CSUFT 10144 (Stage 25, TTL 18.7 mm), which bears almost the same pattern as a small-sized Bo. cf. ombrophila tadpole CSUFT T10992 (Stage 25, TTL 20.9 mm). The tadpoles of Bo. nanlingensis differ from the syntopic Bo. shimentaina tadpoles by the pale brownish background coloration of the body and tail (vs. dark brown), the ventral pattern of sparse speckles (vs. dense small speckles), and the tail pattern of small dots (vs. large speckles).
Compared to other described Boulenophrys tadpoles where species identification is supported by molecular data, the tadpoles of Bo. nanlingensis differs by the presence of ventrolateral spots on each side of head-body connection (vs. absent in Bo. jingdongensis, Bo. hoanglienensis, Bo. leishanensis, and Bo. lushuiensis); the tail pattern of many brown speckles (vs. small spots on tail muscle in Bo. leishanensis; few dark spots on posterior tail muscle in Bo. jiangi; and small white and black dots in Bo. baishanzuensis). Further comparisons between Bo. nanlingensis tadpoles and all megophryinid tadpoles that were identified based on molecular data are shown in Tables 2, 3.
Ecology notes. Tadpoles of Bo. nanlingensis were discovered in all collection sites during our field surveys in 2021, which perhaps implies that this species has larger population size, or it might exhibit less microhabitat specificity. Besides the three sites mentioned above, four Bo. nanlingensis tadpoles were collected from a relatively wide stream (3-5 m wide), with a maximum depth of 0.5 m. An adult male was observed calling under rocks near the stream bank with its feet standing in shallow water on 28 July 2021.
The male calling activities of Bo. nanlingensis, which began in late July, had increased during our visit in November in Mangshan. It seems the newborn larva would have to over-winter. Thus, we suspected the tadpoles of early Stages 25-29 collected in May and July were born in the previous year. Two tadpoles at advanced Stages 34-35 were collected on the 19 th of November. Considering tadpoles in late stages develop relatively fast (Grosjean, 2003;TQ, personal observation). It was likely these advanced tadpoles would finish metamorphosis in cold season at the beginning of the next year. However, this assumption needs further confirmation because the cold weather and scarce food in winter may not be suitable for the survival of froglets.

Discussion
In this study, we attempted to identify sympatric megophryinid tadpoles using external morphology and color patterns, especially ventral patterns. However, our sample size was small. Tadpoles of Bo. shimentaina and Bo. cf. ombrophila bearing stable and distinct ventral pattern were collected from a single site. It is not clear if the color pattern may differ between sites. In Bo. nanlingensis, the color pattern varied between different body size ranges rather than stages or collection sites. This provides new insight into that the coloration pattern perhaps should be classified by body size ranges in megophyinid tadpoles, and not only development stages. Diagnostic larval characters for delineating megophryid genera are still insufficient except for the ventrolateral pattern in Brachytarsophrys. However, there are characters shared within genera, such as the rim of nares is "tubular" in Ophryophryne elfina , and O. microstoma Boulenger, 1903(tadpoles described by Grosjean, 2003 without molecular data), but this rim is "serrated" in Boulenophrys tadpoles, described both in this study and in Tapley et al. (2020b); or the oral disc is "hastate shaped" in Atympanophrys gigantica (Tapley et al. 2020b), which has not been reported in other genera.
We failed to find any Xenophrys mangshanensis adult or larva during our field surveys, despite Mangshan being its type locality. However, it was reported to be in sympatry with Bo. nanlingensis and Br. popei in Guangdong Province . As earlier mentioned under the ecology notes for Boulenophrys cf. ombrophila, sometimes larvae maybe washed downstream where adult frogs are not thought to be present. Therefore, if megophryinid species occur in sympatry with others, the tadpole identification without molecular data should be re-considered. Perhaps, the tadpole specimens previously described as X. mangshanensis, for example, by Fei and Ye (2016) should be re-examined after molecular identification.
The presence of Bo. kuatunensis and Bo. brachykolos in Mangshan was not confirmed in this study, which agrees with the conjecture proposed by Liu et al. (2018) and Tapley et al. (2017) that both species are restricted to their type localities. However, taxonomic revisions need adequate field surveys and detailed examination of museum specimens (Qi et al. 2021). This study provides an example of using tadpole identification to document the presence of species where adults may not always be visible.
This proved particularly useful here as the tadpoles of megophryinid frogs at this site appear to be relatively slow to develop, and they could always be found outside the breeding season.