Prevalence of cryptic species in morphologically uniform taxa - Fast speciation and evolutionary radiation in Asian frogs.

Diversity and distributions of cryptic species have long been a vexing issue. Identification of species boundaries is made difficult by the lack of obvious morphological differences. Here, we investigate the cryptic diversity and evolutionary history of an underappreciated group of Asian frog species (Megophrys) to explore the pattern and dynamic of amphibian cryptic species. We sequenced four mitochondrial genes and five nuclear genes and delineated species using multiple approaches, combining DNA and mating-call data. A Bayesian species tree was generated to estimate divergence times and to reconstruct ancestral ranges. Macroevolutionary analyses and hybridization tests were conducted to explore the evolutionary dynamics of this cryptic group. Our phylogenies support the current subgenera. We revealed 43 cryptic species, 158% higher than previously thought. The species-delimitation results were further confirmed by mating-call data and morphological divergence. We found that these Asian frogs entered China from the Sunda Shelf 48 Mya, followed by an ancient radiation event during middle Miocene. We confirmed the efficiency of the multispecies coalescent model for delimitation of species with low morphological diversity. Species diversity of Megophrys is severely underappreciated, and species distributions have been misestimated as a result.

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

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.

Generic classification of Asian horned toads (Anura: Megophryidae: Megophryinae) and monograph of Chinese species.

The subfamily Megophryinae, as a representative batrachian group of the Oriental Realm and one of the most diverse groups of amphibians, has attracted considerable attention due to continued conjecture regarding its generic classification and failure to reach a satisfactory consensus. China boasts the richest diversity of Asian horned toads, containing some two thirds of the total species cataloged. However, most species have a complicated taxonomic history, resulting in multiple misidentifications. As such, an overall clarification of historical records and regional checklists is required. In the current investigation, we established the phylogeny of the Asian horned toads and performed detailed examinations with redefinitions of several important morphological traits. Based on the phylogenetic relationships and morphological differences, we propose a new ten-genus classification for the Asian horned toad subfamily Megophryinae: i.e., Brachytarsophrys, Atympanophrys, Grillitschia, Sarawakiphrys gen. nov., Jingophrys gen. nov., Xenophrys, Megophrys, Pelobatrachus, Ophryophryne, and Boulenophrys. Revisions on the diagnosability, distribution, and content of each genus are provided. Furthermore, we present a careful review of the taxonomic history of Asian horned toad species from China and provide a monograph of congeners, including six species of Brachytarsophrys, four species of Atympanophrys, five species of Jingophrys gen. nov., 10 species of Xenophrys, two species of Ophryophryne, and 60 species of Boulenophrys. Finally, we discuss the importance of traditional morphological traits based on multiple populations in taxonomic work as well as taxonomic inflation caused by the genetic species delimitation.

Description of a new toad of Megophrys Kuhl amp; Van Hasselt, 1822 (Amphibia: Anura: Megophryidae) from western Yunnan Province, China.

A new species of genus Megophrys from Gaoligong Mountains, Yunnan Province, China is described. Phylogenetic analyses based on mitochondrial DNA and nuclear DNA all clustered the new species as an independent clade nested into the subgenus Panophrys. The smallest genetic distance based on 16S rRNA gene between the new species and its congeners was 3.0%. The new species could be identified from its congeners by a combination of following characters: moderate body size (SVL 31.0-34.8 mm in males); vomerine ridge weak, vomerine teeth absent; dorsal skin relatively smooth; tongue slightly notched behind; tympanum rounded and relatively large, 0.54 times of eye length; a horn-like tubercle on edge of each upper eyelid small; tibio-tarsal articulation reaches middle eye when leg stretched forward; finger tips rounded, not expanded to small pad; toes with narrow fringes and rudimentary webbing; ventral hindlimbs semitransparent purplish with greyish white pigments; ventral body scattered with distinct dark patches in the middle.

The tadpoles of five Megophrys Horned frogs (Amphibia: Megophryidae) from the Hoang Lien Range, Vietnam.

Frogs in the genus Megophrys are an Asian radiation of stream-breeding frogs. The tadpoles of many Megophrys species are undescribed; those that are described are often dubiously allocated to species by association with post metamorphic specimens at collection sites and without supportive molecular data. We provide detailed descriptions of the larvae of five species of Megophrys from the Hoang Lien Range in northwest Vietnam: Megophrys fansipanensis, M. gigantica, M. hoanglienensis, M. jingdongensis and M. maosonensis. Tadpoles from different subgenera differ from each other via a combination of patternation in life, oral disc shape and tail morphology but given the small sample size, and limited number of species it is unlikely that these differences can be applied more widely to delineate subgenera. Morphological differences between tadpoles from species within the subgenus Panophrys were insufficient to clearly delineate all species. The ability to identify tadpoles is likely to advance our understanding of the frog fauna in mainland southeast Asia.

A new species of Megophrys (Amphibia: Anura: Megophryidae) from Vietnam.

A new species of Megophrys is described from Cao Bang Province, northeastern Vietnam based on morphological and molecular data. Morphologically, the new species is distinguishable from its congeners by a combination of the following characters: Size small (SVL 34.9-38.9 mm in males); tympanum visible; vomerine teeth absent; tongue not notched posteriorly; male with a single vocal sac; toes with rudimentary webbing; subarticular tubercles and lateral fringes absent on all digits; nuptial pads present on fingers I and II in males, with spicules; dorsal skin with scattered granules and tubercles; flank with tubercles; dorsum with a X-shaped dorsal ridge; dorsolateral folds prominent; a small horn-like tubercle present at the outer edge of the eyelid; dorsal surface yellowish brown with a dark brown triangle between the eyes, and a dark brown marking along the X-shaped ridge on the back. In the phylogenetic analyses, the new species is unambiguously nested within the subgenus Panophrys with interspecific uncorrected genetic p-distances (16S rRNA gene) varying from 4.26% (compared with M. rubrimera) to 10.80% (compared with M. acuta).

Description of six new species of the subgenus Panophrys within the genus Megophrys (Anura, Megophryidae) from southeastern China based on molecular and morphological data.

The diversity of the subgenus Panophrys within the genus Megophrys has been revealed to be extremely underestimated from southeastern China. Herpetological surveys coupled with extensive sampling in a longitudinal mountain belt located in southeastern China resulted in the discoveries of six new species of the subgenus Panophrys. Furthermore, the new discoveries support the findings of "micro-endemism", "sympatric phenomenon" and "sympatric but distant phylogenetically" which appear to be common among Panophrys species, and also indicates that the Asian horned toads would be good candidates for studies on speciation and biogeography, and additionally emphasizes the conservation difficulties of these toads.

A new species of the genus Megophrys (Amphibia: Anura: Megophryidae) from Hunan, China.

We describe a new species of Megophryidae frog, Megophrys shunhuangensis sp. nov., from Hunan Province, Southern China. It can be distinguished from other known species in the subgenus Panophrys by morphological characters, bioacoustic data and a molecular divergence in the mitochondrial 16S rRNA gene similar to that found among other species of Panophrys. M. shunhuangensis sp. nov. is characterized by a relatively small body size, with adult females measuring 37.6 mm and adult males measuring 30.3-33.6 mm in snout to vent length; maxillary teeth present, vomerine teeth absent; tongue smooth, not notched behind; hindlimb slender, heels overlapping, tibio-tarsal articulation reaches forward between the nasal and tip of snout. Molecular phylogenetic analyses also show that M. sp. 6 from (Mao'er Shan, China) from Chen et al. (2017) and M. sp. 24 (Anjiangping and Mao'er Shan, China) from Liu et al. (2018) may be the same species as M. shunhuangensis sp. nov., we consequently speculate that the new species also distributed in Anjiangping and Mao'er Shan, China. At present, the genus Megophrys contains 85 species of which 48 species are distributed in China, and 30 belong to the subgenus Panophrys.

A new species of Megophrys Kuhl amp; Van Hasselt (Amphibia: Megophryidae) from Borneo allied to M. nasuta (Schlegel, 1858).

Within Sundaland, the species diversity of the genus Megophrys is richest in Borneo, with some species restricted to the northern highlands of the island. On the basis of molecular, morphological and acoustic evidence, we describe a new species of large-sized Megophrys, once confused with M. nasuta. The new species Megophrys kalimantanensis sp. nov. can be differentiated from all its congeners by large genetic distance more than 6.11% in the mitochondrial 16S rRNA gene, much shorter rostral and orbital appendages, and unique advertisement call. This new species has been recorded from medium to high altitudes of mountain forest in southern and northernmost Borneo.

A New Megophrys Kuhl and Van Hasselt (Amphibia: Megophryidae) from southwestern Sumatra, Indonesia.

Megophrys lancip sp. nov., from the Bukit Barisan mountain range of southwestern Sumatra, Indonesia, is described on the basis of molecular and morphological evidence. The new species is distinguished from its congeners in Sumatra, Java, Borneo, and the Philippines by having a medium-sized body, snout with an extremely pointed rostral appendage, a medium-sized triangular eyelid appendage, a dorsolateral fold extending from just behind the eye to the groin, vomerine teeth, vocal slits, nuptial pads on the dorsomedial surface of the first and second fingers in males, and in lacking a Y, X, or H-shaped fold on the dorsum. Morphologically, the new species is most similar to M. montana, but it has a longer rostral appendage, shorter eyelid appendages, and less developed toe webbing. We also evaluate the taxonomic status of M. parallela and comment on the occurrence of M. aceras in Sumatra.

Two new and potentially highly threatened Megophrys Horned frogs (Amphibia: Megophryidae) from Indochina's highest mountains.

Megophrys are a group of morphologically conserved, primarily forest-dependent frogs known to harbour cryptic species diversity. In this study, we examined populations of small-sized Megophrys from mid- and high elevation locations in the Hoang Lien Range, northern Vietnam. On the basis of morphological, molecular and bioacoustic data, individuals of these populations differed from all species of Megophrys known from mainland Southeast Asia north of the Isthmus of Kra and from neighbouring provinces in China. Further, the newly collected specimens formed two distinct species-level groups. We herein describe two new species, Megophrys fansipanensis sp. nov. and Megophrys hoanglienensis sp. nov. Both new species are range restricted and likely to be highly threatened by habitat degradation. These discoveries highlight the importance of the Hoang Lien Range for Vietnam's amphibian diversity.

A new species of the genus Megophrys (Anura: Megophryidae) from Yunnan Province, China.

We describe a new horned toad species of the genus Megophrys from Yingjiang County of Yunnan Province, China. The new species, Megophrys feii sp. nov., can be distinguished from its congeners by a combination of the following characters: (1) body slender and small (SVL 24.3-25.1 mm in four males, 28.2-28.9 mm in two females); (2) head length about equal to head width (HDL/HDW 0.97-0.99); (3) tympanum circular and distinct; (4) maxillary teeth present; (5) vomerine ridges and vomerine teeth absent; (6) tongue round, slightly notched posteriorly; (7) tibia long (TIB/SVL 0.48-0.52 in four males, 0.54-0.55 in two females), shanks overlapping when thighs are held at right angles to the body; (8) dorsal skin distinctly granular and densely covered with small tubercles; (9) lower flanks and lateral sides of belly scattered with small but prominent, white, tubercles; (10) webbing between toes rudimentary; (11) lateral fringes on toes moderate to wide; (12) breeding males without nuptial pads and spines on fingers; (13) protruding projection posterior to cloaca present in both sexes; (14) groin and ventral thigh coloration in life not contrasting with surrounding regions; (15) the presence of an indistinct and small horn-like tubercle at the edge of the eyelid; and (16) an advertisement call with a dominant frequency of 4.74-4.91 kHz (at 18 ˚C). To date, the new species has only been found at its type locality in stream areas in montane evergreen broadleaf forests between 700-1200 m elevation.

The Vietnamese population of Megophrys kuatunensis (Amphibia: Megophryidae) represents a new species of Asian horned frog from Vietnam and southern China.

The Asian frog genus Megophrys is a diverse group of morphologically conserved, forest-dwelling frogs. The genus harbours highly localised species diversification and new species continue to be described on a regular basis. We examined the taxonomic status of a population of Megophrys frogs from the Hoang Lien Range in northern Vietnam and southern China previously identified as M. kuatunensis (subgenus Panophrys). Preliminary phylogenetic analyses using a fragment of 16S rDNA places the species in question within the Megophrys (subgenus Panophrys) species group, a primarily Chinese radiation within the genus. On the basis of morphological, molecular and bioacoustic data, we conclude that this population does not represent M. kuatunensis, or any known species in the genus. We herein describe this species of Megophrys as new. Known only from Sa Pa District, Lao Cai Province in Vietnam and Jinping County, Yunnan Province in China, the new species is likely to be threatened by ongoing deforestation in the region. We provide an updated species description of M. kuatunensis based on type specimens, and suggest that M. kuatunensis is likely to be restricted to eastern China.

The near-complete mitogenome sequence of the Omei Horned Toad Megophrys omeimontis Liu, 1950 (Anura, Megophryidae).

The nearly complete mitogenome of Megophrys omeimontis is 17,013 bp in length, including 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and a D-loop region, and it shares the identical gene arrangement with Atympanophrys shapingensis. The overall base composition of this mitogenome is biased toward AT content at 60.10%. Most PCGs begin with ATG, whereas ND1 starts with ATA, ND2 initiates from ATT, ND3 starts with ATC, and COX1 begins with GTG. Four PCGs (COX1, ATP8, ND4L and ND5) use complete stop codon TAR, and ND6 ends with AGG, but the remaining eight PCGs terminate in TA or T. The putative control region of M. omeimontis mitogenome holds two tandem repeat motifs at the 5' and 3' ends, which is different from that of A. shapingensis mtDNA. Megophrys omeimontis becomes the sister lineage to the two other species, B. carinense and A. shapingensis.