Yingyong Wang; Jian Zhao; Jianhuan Yang; Zhixin Zhou; Guoling Chen; Yang Liu

“ Live holotype coloration: Olive-brown above with distinct dark brown markings bordered with light edge and obscure markings; a distinct dark triangular marking between the eyes, apex of triangle over occiput; a distinct X-shaped dark marking on the back; a small longitudinal dark stripe on the dorsum of the snout; dorsal surface of the limbs and digits with dark brown transverse bands; side of head with dark brown vertical bars, one from the tip of the snout to behind the nares; one under the eye, one along the supratympanic fold, coving tympanum; supratympanic fold light colored ”

“ We recorded the male calls of 13 individuals from three sympatric Xenophrys species in Mt. Jinggang, X. jinggangensis, X. chenisp. nov., and X. linisp. nov. (four, three, and six individuals, respectively) . We also recorded the vocalizations of X. kuatunensis (N = 2) , X. huangshanensis (N = 3) , and X. boettgeri (N = 3) permitting additional comparisons (the typical calls of six analyzed Xenophrys species are available in Appendix S1) .
The male call of X. chenisp. nov. has a much slower pace than X. jinggangensis and X. linisp. nov. ”

“ Both of these species are small in body length (<45 mm) , they can be assigned to the genus Xenophrys based on the following characteristics: head broad and depressed, tympanum distinct, tubercles on the outer edge of the upper eyelids short, tubercles on the snout absent, no mid-dorsal fold, no black horny spines on dorsum, hind limbs long, and heels overlap [12] . Furthermore, we also noticed that the vocalizations and phylogenetic relationships of these two unknown Xenophrys toads seemed to be distinct from that of X. jinggangensis. ”

“ To implement unbiased species delineation in cryptic amphibians, integrating evidence from morphology, DNA sequence data, and behavior may be critically necessary [16] .
In particular, molecular genetic approaches enable us to decipher phylogenetic relationships and thus the evolutionary history of a large number of species, thereby solving taxonomic uncertainties [17] . In addition, bioacoustic analysis is a very useful approach in species diagnosis because many male frogs and toads have species-specific advertising vocalization during breeding seasons [18] . ”

“ The male paratype SYS a001421 has mature spermaries in the abdominal cavity, measuring 4.9×2.1 mm in the major and minor axes, respectively. All female paratypes bear pure yellowish mature eggs and atrophic ovary fat. According tadpole stages defined by Gosner [38] , the individuals in 28–34th stages were found under rocks in the stream on the 5th December, 2011. Juveniles were collected on 21th, May, 2013. Thus, we assume the breeding season of this species likely begins September–October.
Xenophrys cheniWang and Liu sp. ”

“ Genomic DNA was extracted from the muscle tissue using a standard phenol-chloroform extraction protocol [22] . We amplified a fragment of the mitochondrial 16s rRNA gene from Xenophrys species using the primer pairs L3975 and H4551 [23] , and mitochondrial 12s rRNA gene using the primer pair Fphe40L and 12S600H [24] . PCR amplifications were performed in a reaction volume of 25 μl containing 100 ng of template DNA, 0.3 mM of each PCR primer and 10 μl Premix EX TaqTM (Takara, Dalian, China) . The PCR condition for 16s rRNA was, an initial denaturation step at 94°C for 1.5 min ”

“ Dorsal surface sallow with darker brown reticular markings; triangular and X-shaped markings on dorsum and transverse bands on limbs and digits distinct; ventral surface yellowish with grey spots; black zigzag lines distinct; creamy-white substitutes the pinkish in the anterior surface of the thighs and lateral surface of the trunk.
Variation: Measurements and body proportions of type series are given in Table 4.
Color patterns in 14 male paratypes are more similar to the holotype. In the male paratypes SYS a002124, 002126, 002140, 002141 and 002145, tympanum or its edge is indistinct ”

“ Furthermore, in consistent with the phylogenetic gene trees, the inferred multi-locus network based on concatenated sequences of 16s and 12s rRNA (910 bp) strongly supported the genetic distinctness of X. chenisp. nov. and X. linisp. nov. (Figure 3) .
Bayesian inference and maximum-likelihood phylogenies.The species Paramegophrys oshanensis and Megophrys nasuta were included as outgroup. Numbers above or below branches are bootstrap values based on 1000 replicates for maximum-likelihood analyses (left, >50 retained) and Bayesian posterior probabilities (right, >0.5 retained) . ”

“ Hind limbs relatively elongated and moderately robust; heels overlapping when the flexed legs are held at right angles to the body axis; tibio-tarsal articulation reaching the anterior corner of the eye, when leg stretched along the side of the body; tibia length 49% of SVL; foot length 66% of SVL; relative toe lengths I ”

“ Jinggang, Jiangxi Province, China (Figure 6: A, B, C) .
Xenophrys cheni sp. nov.A: Dorsolateral view of the live adult male Xenophrys chenisp. nov. holotype SYS a001873. B: Ventral view of the live holotype. C: Foot with wide lateral fringes and rudimentary webbing on the toes in the live holotype. D and E: Dorsolateral and ventral views of the live adult female paratype SYS r001429. F and G: Hand and foot of the live paratype SYS r001429. ”

“ Xie, F & Jiang, JP, “Endemic amphibians and their distribution in China”, Asian Herpetol Res 3: 163–171 HuJHLiCXieFJiangJP ↑ Jiang, J & Zhou, K, “Phylogenetic relationships among Chinese ranids inferred from sequence data set of 12S and 16S rDNA”, Herpetol J 15: 1–8 JiangJZhouK ↑ ↑ ↑ Karraker, NE; Arrigoni, J & Dudgeon, D, “Effects of increased salinity and an introduced predator on lowland amphibians in Southern China: Species identity matters”, Biol Conserv 143: 1079–1086 KarrakerNEArrigoniJDudgeonD ↑ Wake, D & Vredenburg, V, “Are we in the midst of the sixth mass extinction? ”