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Techniques of reduced-representation sequencing (RRS) have revolutionized ecological and evolutionary genomics studies. Precise establishment of orthologs is a critical challenge for RRS, especially when a reference genome is absent. The proportion of shared heterozygous sites across samples is an alternative criterion for filtering paralogs. In the prevailing pipeline for variant calling of RRS data – PYRAD/IPYRAD, maxSH is an often overlooked parameter with implications to detecting and filtering paralogs according to shared heterozygosity. Using empirical genotyping by sequencing data of two primroses (Primula alpicola Stapf and Primula florindae Ward) and their putative hybrids, and extra data sets of Californian golden cup oaks, we explore the impact of maxSH on filtering paralogs and further downstream analyses. Our study sheds light on the simultaneous validity and risk of filtering paralogs using maxSH, and its significant effects on downstream analyses of outlier detection, population assignment, and demographic modeling, emphasizing the importance of attention to detail during bioinformatic processes. The mutual confirmation between results of population assignment and demographic modeling in this study suggested maxSH = 0.10 has a potentially excessive and asymmetrical effect on the removal of truly shared heterozygous sites as paralogs. These results indicate that hybridization origin hypotheses of putative hybrids represented by results with maxSH = 0.25 and 0.50 are more credible. In conclusion, we revealed the critical hazard of paralogs filtration according to sharing heterozygosity at first, so that we propose to use specific protocols, rather than maxSH, to filter potential paralogs for closely related lineages.

Recent phylogenetic analysis encompassing multilocus nuclear-gene and matrilineal mtDNA genealogy has revealed a series of cryptic species of the subgenus Panophrys within genus Megophrys from southern and eastern China. This study demonstrates that the Panophrys specimens from the hilly areas among Guangdong, Guangxi and Hunan can be morphologically distinguished from all recognized congeners, thereby providing additional supports for the recognitions of four new species of Panophrys, namely Megophrys (Panophrys) mirabilis Lyu, Wang & Zhao, sp. nov. from northeastern Guangxi, Megophrys (Panophrys) shimentaina Lyu, Liu & Wang, sp. nov. from northern Guangdong, and Megophrys (Panophrys) xiangnanensis Lyu, Zeng & Wang, sp. nov. and Megophrys (Panophrys) yangmingensis Lyu, Zeng & Wang, sp. nov. from southern Hunan. The descriptions of these species take the number of Megophrys species to 101, 46 of which belong to the subgenus Panophrys.

The genus-level recognition of monophyletic short-legged toads (Brachytarsophrys) has been recently implicated in the taxonomic debate of Megophrys sensu lato. In the present study, Brachytarsophrys is reasonably regarded as a distinct genus based on significant morphological differentiations and recent molecular analyses. Furthermore, a comprehensive review of this genus is performed, with two species groups proposed based on morphological differences and phylogenetic relationships. Particularly, Brachytarsophrys platyparietus is removed as a synonym of Brachytarsophrys carinense and considered a valid species due to significant genetic divergence and distinct morphological differences. In addition, a new species, Brachytarsophrys orientalis sp . nov ., is described based on a series of specimens collected from southeastern China. This work takes the member species of the genus Brachytarsophrys to seven, suggesting that the diversity of Brachytarsophrys is underestimated. In addition, the genus levels of other monophyletic groups within the subfamily Megophryinae are discussed.

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.

The diversity of Panophrys horned toads is considered highly underestimated with a large number of undescribed cryptic species. In this work, we describe four Panophrys species from eastern China which were proposed as cryptic species by molecular data in previous study, additionally provide new information on the biogeography of these four species. Panophrys daiyunensis sp. nov. from southern Fujian, Panophrys daoji sp. nov. from eastern Zhejiang, Panophrys sanmingensis sp. nov. from the hilly area among Fujian, Jiangxi and Guangdong, and Panophrys tongboensis sp. nov. from northeastern Jiangxi, can be distinguished from all recognized congeners by a combination of morphological characteristics. The descriptions of these four new species take the recognized species of Panophrys to 51, which is the largest genus within the Asian horned toads subfamily Megophryinae. Considered as an appropriate arrangement for the Asian horned toads currently and applied in this study to describe the new species, the generic recognition of Panophrys is also discussed.

Recent phylogenetic analysis encompassing multilocus nuclear-gene and matrilineal mtDNA genealogy has revealed a series of cryptic species of the subgenus Panophrys within genus Megophrys from southern and eastern China. This study demonstrates that the Panophrys specimens from the hilly areas among Guangdong, Guangxi and Hunan can be morphologically distinguished from all recognized congeners, thereby providing additional supports for the recognitions of four new species of Panophrys, namely Megophrys (Panophrys) mirabilis Lyu, Wang & Zhao, sp. nov. from northeastern Guangxi, Megophrys (Panophrys) shimentaina Lyu, Liu & Wang, sp. nov. from northern Guangdong, and Megophrys (Panophrys) xiangnanensis Lyu, Zeng & Wang, sp. nov. and Megophrys (Panophrys) yangmingensis Lyu, Zeng & Wang, sp. nov. from southern Hunan. The descriptions of these species take the number of Megophrys species to 101, 46 of which belong to the subgenus Panophrys.

The taxonomy of Babina sensu lato was controversial in the past decades. In this study, the phylogeny of genus Babina sensu lato was re-constructed based on genetic analysis, morphological comparison and advertisement call analysis. We found that Babina sensu stricto and previous subgenus Nidirana should be two distinct genera in the family Ranidae. N. caldwelli is confirmed to be a synonym of N. adenopleura because of the small genetic divergence and the lack of distinct morphological differences. A new species, Nidirana nankunensis sp. nov. is described based on a series of specimens collected from Mt. Nankun, Guangdong Province, China, which can be distinguished from other known congeners by having a behavior of nest construction, distinctive advertisement calls, significant divergence in the mitochondrial genes, and a combination of morphological characters. Currently, the genus Babina contains two species and the genus Nidirana contains eight species.

Following the publication of our paper (Li et al., 2020), it has come to our attention that, due to our oversight, the contributions of some colleagues were not reflected in the "ACKNOWLEDGEMENTS" section. Hereby, we wish to add the following to the "ACKNOWLEDGEMENTS" section as a correction.

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 tree frog, G. guangdongensis sp nov., is described based on a series of specimens collected from Dawuling Forest Station, Mount Nankun and Nanling Nature Reserve of Guangdong Province, southeastern China. The new species is distinguished from all known congeners by a significant genetic divergence at the mitochondrial 16S rRNA gene fragment examined (p-distance ≥ 4.6%) and the following combination of morphological characters: relatively small body size (SVL 26.1–34.7 mm in adult males, 34.9–35.4 mm in adult females); upper eyelid and dorsum lacking spines; supratympanic fold and tympanum distinct; dorsal and lateral surface rough, sparsely scattered with tubercles; ventral skin granular; tibiotarsal projection absent; toe-webbing moderately developed, finger webbing rudimentary; heels slightly overlapping when flexed hindlimbs are held at right angles to the body axis; brown to beige above, with an inverse Y-shaped dark brown marking extendeing from the interorbital region to the centre of the dorsum; males with a single subgular vocal sac and protruding nuptial pads with minute granules on the dorsal surface of the base of first finger. The discovery and description of Gracixalus guangdongensis sp. nov. represents the 14th species known in this genus.

A new species, Amolops yunkaiensis sp. nov. is described based on a series of specimens from Ehuangzhang Nature Reserve and Yunkaishan Nature Reserve, southwestern Guangdong Province, China. The new species can be distinguished from all known congeners by molecular divergence in the mitochondrial 16S rRNA, 12S rRNA and CO1 genes, and a combination of the following characters: relatively small body size, SVL 31.8–34.1 mm in adult males, 35.2–39.0 mm in adult females; numerous raised large warts on dorsum and flanks; dorsal body olive-brown or light brown with dark brown blotches; absence of vomerine teeth; absence of tarsal glands; presence of a pair of subgular vocal sacs, nuptial spines on the first finger, and sparse translucent tubercles on the lower jaw, forechest, posterior belly and ventral thigh in male. Hence, the genus Amolops contains 52 species, 29 of which occur in China.

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.

Based on morphological and phylogenetic analyses (16S rRNA mtDNA), two new species of the genusLeptobrachellaare described from southern China, namelyL.yunkaiensisWang, Li, Lyu & Wang,sp. n.from Dawuling Forest Station of Guangdong Province andL.wuhuangmontisWang, Yang & Wang,sp. n.from Mt. Wuhuang of Guangxi Province. To date, the genusLeptobrachellacontains 68 species, among which 13 species are known from China. The descriptions of the two new species further emphasize that the species diversity of the genusLeptobrachellafrom China is still highly underestimated and requires further investigations.

The previous descriptions of Opisthotropis maxwelli Boulenger, 1914 and O. andersonii (Boulenger, 1888) were considered imperfect due to the limited number of specimens. This may in turn cause a problem for accurate species identification. In our study, the species boundaries of these two species were investigated using an integrative approach incorporating morphological characters and molecular phylogenetic analyses of the mitochondrial Cyt b gene of 26 specimens of nine known Opisthotropis species collected from south-eastern China. Our results surprisingly revealed a new cryptic species, Opisthotropis shenzhenensis sp. nov., from Shenzhen and Dongguan, Guangdong Province, southern China. Further, we re-described O. maxwelli based on several specimens from Fujian and Guangdong, and O. andersonii based on a series of specimens from Hong Kong, Shenzhen and Guangzhou, China. We provide an updated identification key to all described Opisthotropis species from China.

A new species of the Parachute Gecko, Ptychozoon bannaense sp. nov., is described on the basis of two specimens from Xishuangbanna, Yunnan Province, China. The new species can be distinguished from eight known congeners by the following combination of morphological characters: body size moderate, SVL 83.2-87.5 mm; rostral with a short dorsomedian groove; dorsal head and body covered with granular scales without enlarged tubercles, but male with several enlarged tubercles on the occipital region; two supranasals separated from each other by a large internasal; the absence of a predigital notch in the preantebrachial cutaneous flap; male possesses 17 preanofemoral pores in a continuous scale row; tail tubercle absent, 24 pairs of lateral denticulate cutaneous lobes of the tail; width of tail and caudal lobes progressively decreasing posteriorly; the terminal caudal flap short, semicircle, not expanded; lack of lobe fusion at the terminal caudal flap border; the presence of four dark dorsal bands between fore- and hind limbs insertions, the third and fourth dorsal bands fused into a X-shaped mark. The new species is also divergent from known taxa in mitochondrial gene sequences, supporting its recognition based on morphological characters. The discovery and description of Ptychozoon bannaense brings the total number of Ptychozoon to nine, and represents the first record of the genus Ptychozoon in China.

The genus Odorrana currently contains at least 56 recognized species that inhabits montane streams in subtropical and tropical Asia. Twenty new species have been described in the last decade, indicating the potential cryptic species diversity of this genus. We collected several specimens of Odorrana species from Southern China from 2007 to 2014, and on the basis of a combined morphological characters and phylogenetic analysis, we described the new species Odorrana fengkaiensis sp. nov. herein. The new species is very similar to O. hainanensis and O. bacboensis, but can be consistently separated by morphology, and allopatric distribution. It is further reciprocally monophyletic to O. hainanensis in a mitochondrial gene trees with an average genetic divergence of 2.1% (1.9%-2.4%). The new species inhabits in lowland broad streams, rivers, pools and near the riparian areas, but its general ecology remains poorly known. The new species is characterized by its body length of adult females approximately twice as long as adult males (SVL 77.8-111.9 mm in females, 37.4-51.8 mm in males); eye large in males, eye diameter 1.01-1.16 times as long as snout length; tympanum of males large and distinct, extremely close to the eye, 0.7-1.4 mm in tympanum-eye distance; dorsolateral folds absent; dorsal skin shagreened, with several large tubercles in males; flanks with tubercles and scattered larger pustules, 8-10 of which usually arranged in a dorsolateral row; ventral skin smooth, with spines in adult males during the breeding season; the tibio-tarsal articulation stretched forward beyond the tip of snout; relative finger lengths: IIlt; Ilt; IVlt; III; dorsum brown with irregularly reticulated green markings in males and young females, uniformly brown in some old adult females; males with velvety nuptial pad on thumb, paired gular pouches; mature oocytes almost purely black in life, showed dark grey animal pole and olive vegetative pole in preservative. In addition, we found O. bacboensis, a new country record from China, indicating a range extension from north-central Vietnam to southeast Yunnan and adjacent area in Guangxi.

A new species, Xenophrys insularis sp. nov., is described based on a series of specimens collected from Nan’ao Island, Shantou City, Guangdong Province, China. The new species is a separately evolving lineage that was robustly supported by the phylogenetic analysis based on the mitochondrial CO1 + 12S rRNA + 16S rRNA genes; morphologically, it can be distinguished from other known congeners by the following combination of characteristics: body size moderate, SVL 38.6–41.2 mm in adult males, 47.1 mm in a single adult female; snout obtusely rounded in dorsal view; head width slightly larger than head length; strong vomerine ridge bearing vomerine teeth; margin of tongue weakly notched behind; supratympanic fold forming a large swollen supraaxillary gland above insertion of arm; hindlimbs relatively short, heels not meeting when thighs are held at right angles to body, tibio-tarsal articulation reaches forward the posterior edge of tympanum; fingers and toes without lateral fringes, with rudiment of web at bases of toes, distinct subarticular tubercle only at the base of each finger and toe; single vocal sac in males; gravid female bears pure white oocytes. Xenophrys insularis sp. nov. is endemic to the Nan’ao Island and represents the first Xenophrys species found on and possibly endemic to a small offshore island.

Two new species, Megophrys acuta sp. nov. and Megophrys obesa sp. nov., are described based on a series of specimens collected from Heishiding Nature Reserve, Fengkai County, Guangdong Province, China. They can be distinguished from other known congeners occurred in southern and eastern China by morphological characters and molecular divergence in the mitochondrial 16S rRNA gene. M. acuta is characterized by small and slender body with adult females measuring 28.1-33.6 mm and adult males measuring 27.1-33.0 mm in snout-vent length; snout pointed, strongly protruding well beyond margin of lower jaw; canthus rostralis well developed and sharp; hindlimbs short, the heels not meeting, tibio-tarsal articulation reaching forward the pupil of eye. M. obesa is characterized by stout and slightly small body with adult females measuring 37.5-41.2 mm, adult male measuring 35.6 mm in snout-vent length; snout round in dorsal view; canthus rostralis developed; hindlimbs short, the heels not meeting, tibio-tarsal articulation reaching forward the posterior margin of eye. The discovery of these two new species further confirms that the diversity of this genus has been significantly underestimated. At present the genus Megophrys contains 56 species of which 35 species are distributed in China.

A new species of the Parachute Gecko, Ptychozoon bannaense sp. nov., is described on the basis of two specimens from Xishuangbanna, Yunnan Province, China. The new species can be distinguished from eight known congeners by the following combination of morphological characters: body size moderate, SVL 83.2-87.5 mm; rostral with a short dorsomedian groove; dorsal head and body covered with granular scales without enlarged tubercles, but male with several enlarged tubercles on the occipital region; two supranasals separated from each other by a large internasal; the absence of a predigital notch in the preantebrachial cutaneous flap; male possesses 17 preanofemoral pores in a continuous scale row; tail tubercle absent, 24 pairs of lateral denticulate cutaneous lobes of the tail; width of tail and caudal lobes progressively decreasing posteriorly; the terminal caudal flap short, semicircle, not expanded; lack of lobe fusion at the terminal caudal flap border; the presence of four dark dorsal bands between fore- and hind limbs insertions, the third and fourth dorsal bands fused into a X-shaped mark. The new species is also divergent from known taxa in mitochondrial gene sequences, supporting its recognition based on morphological characters. The discovery and description of Ptychozoon bannaense brings the total number of Ptychozoon to nine, and represents the first record of the genus Ptychozoon in China.

Two new species, Megophrys acuta sp. nov. and Megophrys obesa sp. nov., are described based on a series of specimens collected from Heishiding Nature Reserve, Fengkai County, Guangdong Province, China. They can be distinguished from other known congeners occurred in southern and eastern China by morphological characters and molecular divergence in the mitochondrial 16S rRNA gene. M. acuta is characterized by small and slender body with adult females measuring 28.1-33.6 mm and adult males measuring 27.1-33.0 mm in snout-vent length; snout pointed, strongly protruding well beyond margin of lower jaw; canthus rostralis well developed and sharp; hindlimbs short, the heels not meeting, tibio-tarsal articulation reaching forward the pupil of eye. M. obesa is characterized by stout and slightly small body with adult females measuring 37.5-41.2 mm, adult male measuring 35.6 mm in snout-vent length; snout round in dorsal view; canthus rostralis developed; hindlimbs short, the heels not meeting, tibio-tarsal articulation reaching forward the posterior margin of eye. The discovery of these two new species further confirms that the diversity of this genus has been significantly underestimated. At present the genus Megophrys contains 56 species of which 35 species are distributed in China.