Redefinition of the Odorrana versabilis Group,with a New Species from China(Anura,Ranidae,Odorrana)

Taxonomic studies of the Odorrana versabilis group have been consistently disorganized,and they have often been incorrectly associated with an abandoned subgenus or genus Bamburana in existing literature.In this study,we conducted an integrative taxonomic analysis using molecular phylogenetic analysis of two mitochondrial gene fragments(16S rRNA and CO1)and morphological examination of 41 specimens collected during herpetological surveys in southern China.Our results confirmed the monophyly of O.versabilis group which exhibited distinct morphological traits.We also identified a cryptic lineage sister with O.exiliversabilis.Additionally,our study revealed that O.trankieni was nested within O.nasuta.Consequently,we redefined the morphological characteristics of the O.versabilis group,described a new species in this group,O.confusa sp.nov.,and proposed that O.trankieni be considered a junior synonym of O.nasuta.One of the most significant findings of this study was the observation of distinct morphological changes in adult males across all species within the O.versabilis group between the non-breeding and breeding seasons.This study contributes to research on various aspects of Asian amphibian fauna,including taxonomy,ecology,evolution,and physiology.

A New Species of Odorous Frog Genus Odorrana(Anura,Ranidae) from Southern Guizhou Province,China

We describe Odorrana liboensis sp.nov.,a new species from the Maolan National Nature Reserve,Libo County,Guizhou Province,China.Phylogenetic analyses based on DNA sequences of the mitochondrial 12 S rRNA,16 S rRNA,and ND2 genes supported the new species as an independent lineage,closely related to O.lipuensis.The uncorrected genetic distances between the 12 S rRNA and16 S rRNA in the new species and its closest congener,O.lipuensis,were 6.06%and 5.19%,respectively.The new species is distinguished from its congeners by a combination of the following morphological characters:(1)having medium body size,with the snout-vent length(SVL)of adult females approximately 1.2 times as long as males at 56.9±1.0(55.8-58.2 mm,n=9)in females and 48.7±1.2(47.1-49.9 mm,n=5)in males;(2)head length greater than width in males and females;(3)tympanum distinctly visible,greater than one-half the diameter of the eye;(4)eyes big and prominent,width of upper eyelid(UEW)approximately3/4 of eye diameter(ED);(5)dorsolateral folds absent;(6)two metacarpal tubercles;(7)relative finger lengths:Ⅱ<Ⅰ<Ⅳ<Ⅲ;(8)subarticular tubercles on fingers prominent:1,1,2,2;(9)one metatarsal tubercle;(10)tibiotarsal articulation reaching to between the eye and nostril when the leg is stretched forward;(11)toes with entire webbing to disks;(12)subarticular tubercles on toes prominent:1,1,2,3,2;(13)dorsal surfaces of limbs with distinct brownish-black bands;(14)smooth,grass-green dorsum with irregular brown mottling;(15)venter smooth,lacking black spots;and(16)lacking pectoral spinules,lacking vocal sacs,and light white nuptial pad present on finger I in males.The new species is currently only known from the type locality.

Reevaluation of the Holotype of Odorrana schmackeri Boettger,1892(Amphibia:Anura:Ranidae) and Characterization of One Cryptic Species in O.schmackeri sensu lato through Integrative Approaches

The Odorrana schmackeri sensu la to is a group of cascade frogs that are widely distributed in southern China.However,their taxonomy has long been unresolved and several cryptic species have been reported in the past decade.Previous analyses revealed the existence of three sympatrie species(i.c,O.schmackeri,O.hejiangensis and O.sp2) at the type locality of O.schmackeri(Gaojiayan Town,Yichang City,Hubei Province,China).However,the lack of an original description for the holotype specimen(SMF6241) of O.schmackeri poses a confusing and controversial problem in species nomination within this group.Here,we reevaluate the taxonomic affinities of SMF6241 and examine the distinctness of O.sp2 based on specimens from the type locality.We use integrative approaches that combine morphology with 10 microsatellite loci and bioacoustics.Canonical discriminant analysis of morphological characteristics revealed three distinct groupings(i.e.,O.schmackeri,O.hejiangensis and O.sp2) and attributed SMF6241 to O.schmackeri.Genetic clustering analysis of nuclear DNA also identified three corresponding clusters.In addition,analysis of interspecific variation in morphometrics and acoustics showed divergence between O.sp2 and its sympatric congeners.All lines of evidence from morphological,molecular and bioacoustic approaches suggest the species status of O.sp2,namely Odorrana ichangensis Chen,sp.nov.Overall,this study resolves the taxonomic assignment of the holotype of O.schmackeri and provides an example for species delimitation using integrative approaches.

Identification and characterization of two novel cathelicidins from the frog Odorrana livida

Antimicrobial peptides(AMPs) are a group of gene-encoded small peptides that play pivotal roles in the host immune system of multicellular organisms.Cathelicidins are an important family of AMPs that exclusively exist in vertebrates. Many cathelicidins have been identified from mammals, birds, reptiles and fish. To date, however, cathelicidins from amphibians are poorly understood. In the present study, two novel cathelicidins(OL-CATH1 and 2) were identified and studied from the odorous frog Odorrana livida.Firstly, the cDNAs encoding the OL-CATHs(780 and735 bp in length, respectively) were successfully cloned from a lung cDNA library constructed for the frog. Multi-sequence alignment was carried out to analyze differences between the precursors of the OL-CATHs and other representative cathelicidins.Mature peptide sequences of OL-CATH1 and 2 were predicted(33 amino acid residues) and their secondary structures were determined(OL-CATH1 showed a random-coil conformation and OL-CATH2 demonstrated α-helical conformation). Furthermore,OL-CATH1 and 2 were chemically synthesized and their in vitro functions were determined. Antimicrobial and bacterial killing kinetic analyses indicated that OL-CATH2 demonstrated relatively moderate and rapid antimicrobial potency and exhibited strong anti-inflammatory activity. At very low concentrations(10 μg/mL), OL-CATH2 significantly inhibited the lipopolysaccharide(LPS)-induced transcription and production of pro-inflammatory cytokines TNF-α, IL-1βand IL-6 in mouse peritoneal macrophages. In contrast, OL-CATH1 did not exhibit any detectableantimicrobial or anti-inflammatory activities. Overall,identification of these OL-CATHs from O. livida enriches our understanding of the functions of cathelicidins in the amphibian immune system. The potent antimicrobial and anti-inflammatory activities of OL-CATH2 highlight its potential as a novel candidate in anti-infective drug development.

无指盘臭蛙(Odorrana grahami)典型栖息地环境微生物季节变化

无指盘臭蛙(Odorrana grahami)是迄今为止发现抗菌肽种类最多的蛙类,而抗菌肽直接与环境微生物作用.为进一步研究无指盘臭蛙皮肤分泌物与环境微生物的关联性,利用平板稀释法对无指盘臭蛙典型栖息地(云南昆明、四川冕宁、四川会理)细菌、真菌、放线菌的数量随季节变化进行了研究.结果显示:1)3个地方土壤都是春季3种微生物总量菌落形成单位最多;2)放线菌都是秋季最多,春季最少;3)昆明和会理土壤春季细菌最多,冕宁夏季最多;4)昆明和冕宁土壤春季真菌最多,会理秋季最多;5)水样中,冕宁和会理微生物总数和真菌春夏秋逐渐增多,3个季节微生物变化趋势明显不同于昆明.同时对3个地方不同微生物的季节变化趋势进行了讨论.

A New Species of Odorrana Inhabiting Complete Darkness in a Karst Cave in Guangxi, China

A new species of the genusOdorrana is described from a completely dark karst cave of northeastern Guangxi, southern China. The new species,Odorranalipuensissp. nov., can be distinguished from its congeners by a combination of the following characters： medium size （SVL： 40.7–47.7 mm in males, 51.1–55.4 mm in females）;tips of all but ifrst ifnger expanded with circummarginal grooves; smooth, grass-green dorsum with irregular brown mottling; pineal body invisible; throat to upper abdomen with gray mottling; dorsal surfaces of limbs with brown bands; dorsolateral fold absent; tiny spinules on lateral body, temporal region, and anterior and posterior edge of tympanum;white nuptial pad present on ifnger I; males lacking vocal sacs; females having creamy yellow eggs, without black poles. Uncorrected sequence divergences betweenO.lipuensissp. nov. and all homologous 16S rRNA sequences ofOdorrana available on GenBank is equal to or greater than 4.9%. Currently, the new species is only known from the type locality.

Isolation and Characterization of 17 Microsatellite DNA Loci for Odorrana margaretae(Anura: Ranidae)

We isolated and characterized 17 microsatellite DNA loci for the odorous frog Odorrana margaretae from its transcriptome sequence data. These loci were screened with 24 individuals from Mt. Emei. All loci were polymorphic, with the number of alleles ranging from 2 to 8. The observed and expected heterozygosity, polymorphism information content, ranged from 0.04 to 1, 0.04 to 0.81, and 0.040 to 0.763, respectively. All loci were in linkage equilibrium and six loci were significantly deviated from Hardy-Weinberg equilibrium after sequential Bonferroni corrections. Cross- species amplification test was conducted for ten odorous frog species, and 12 loci were amplifiable in most species. With the high cross-species amplification rates, thesc markers will provide useful molecular tools for conservation genetic and phylogeographic studies on the genus Odorrana and Bamburana.

基于MHCⅡ类B基因的凹耳臭蛙(Odorrana tormota)种群遗传分化模式

利用II类MHC基因单基因座位Odto-A作为分子标记,对皖南山区凹耳臭蛙6个种群的遗传多样性和遗传分化进行研究.结果显示,皖南凹耳臭蛙总的基因多样性为0.812,核苷酸多样性为0.018.局域种群单倍型多样性变化范围为0.531-0.864,香溪种群单倍型多样性最高,最低的是漳河种群.与线粒体cyt b基因所揭示的单倍型多样性差别不大,但B基因的核苷酸多样性较之线粒体cyt b基因的高达一个数量级.暗示MHC基因丰富的核苷酸多态性可能与其病原体抗性多样性密切相关.分子变异分析结果显示,皖南山区凹耳臭蛙种群MHC II类B基因遗传变异主要来源于种群内,种群间发生了显著的遗传分化(Fst=0.05644,P=0.00391).成对种群间的遗传分化分析结果显示,直线距离最近的浮溪和香溪种群间也发生了显著的遗传分化,暗示这两个种群经历了不同的选择压力.受平衡选择的作用,MHC基因与基于中性分子标记所揭示的遗传格局不同,基于MHC基因的种群遗传分化与水系和直线地理距离均没有明显的相关性,而与种群所经历的选择压力密切相关.结果表明皖南凹耳臭蛙不同局域种群所经历的环境病原体的选择压力存在时空变异.

Sexual Dimorphism in Mass of the Hindlimb Muscles of the Piebald Odorous Frog(Odorrana schmackeri)

Male-biased sexual dimorphism in hind limb muscles is widespread in anuran species where scramble competition is common among males. Such sexual difference is thought to result from sexual selection. In this view, we tested the differences in muscle mass between the sexes and between amplectant and non-amplectant males by quantifying the mass of four hindlimb muscles （triceps femoris, sartorius, gracilis and plantaris longus） of females and males of Odorrana schmackeri. The results showed that females significantly exceeded males for muscle triceps femoris, gracilis, plantaris longus and total mass when controlled for body size. There are no significant differences between amplectant and non-amplectant males. It is probable that the maintenance of the amplectant position in O. schmackeri may depend on the strength of hindlimb muscles in females to support the pair.

Geographically Distinct Expression Profile of Host Defense Peptides in the Skin of the Chinese Odorous Frog, Odorrana margaretae

Odorrana margaretae （Anura： Ranidae） is widely distributed in the southern provinces of China. Previously, 72 antimicrobial peptides （AMPs） belonging to 21 families were identified from the skin of O. margaretae, which were captured in the Hunan province. In the present study, five O. margaretae frogs were captured from the Guizhou province and a total of 28 cDNAs encoding 17 host defense peptides （HDPs） belonging to 14 families were cloned from the skin cDNA library of O. margaretae. Among the 17 HDPs, only one （brevinin-1-Omar5） had been characterized. The distinct HDP expression profiles for O. margaretae in the previous and present study may be attributed to the environmental differences between the sampling locations and the genetic divergence among O. margaretae populations. Besides, 11 of the 17 HDPs identified in the present study were novel for ranids. In order to understand their roles in host defense reactions, three HDPs （odorranain-H-OM1, odorranain-M-OM and ranatuerin-2-OM）, which possess low sequence similarity with the known amphibian HDPs, were selected for further chemical synthesis and functional analysis. Odorranain-H-OM1 showed direct antimicrobial activity against bacteria and fungi. Odorranain-M-OM exhibited concentration-dependent anti-oxidant activity. Ranatuerin-2-OM showed lectin-like activity and could strongly hemagglu -tinate human intact erythrocytes with or without the presence of Ca2＋. The diverse activities of HDPs implied that they may play different roles in host defense reactions of O. margaretae.

Molecular Cloning, Characterization and Sequence Analysis of KCNQ4 in Large Odorous Frog, Odorrana graminea

Acoustic communication is essential for anuran survival and reproduction, and masking background noise can affect the effective acoustic communication. The larger odorous frog(Odorrana graminea) inhabits noise montane streams, and it has shown an ultrasound communication adaptation. However, the molecular mechanism underlying their ultrasonic hearing adaptation remains unknown. To characterize and investigate the molecular characteristics and evolution of the high-frequency hearing-sensitive gene(KCNQ4) in O. graminea, termed as OgKCNQ4, the rapid amplification of cDNA ends(RACE) was performed to amplify the cDNA of OgKCNQ4. Different bioinformatics analyses were used to investigate the molecular characteristics. Multiple nucleotide and amino acid sequence alignment were conducted, and phylogenies were reconstructed under the maximum likelihood and Bayesian approaches. The full-length cDNA of OgKCNQ4 was 2065 bp, and the open reading frame(ORF) was 2046 bp encoding for a putative protein with 681 amino acids. The relative molecular weight of OgKCNQ4 was 76.453 kD and the putative PI was 9.69. Secondary structure prediction analyses suggested 42.29% alpha helixes and 43.76% random coils in OgKCNQ4. Gene homology and Phylogenetic analyses revealed the closest relationship between OgKCNQ4 and KCNQ4 of Nanorana parkeri with 96.9% similarity and 95.0% identity. We first determined the full-length cDNA of OgKCNQ4 and the results here could provide foundations for further study on the evolution of KCNQ4 and its relationship to ultrasonic communication in amphibians.

Nonlinear Phenomena Conveying Body Size Information and Improving Attractiveness of the Courtship Calls in the Males of Odorrana tormota

Nonlinear phenomena are commonly shown in the vocalization of animals and exerts different adaptive functions.Although some studies have pointed out that nonlinear phenomena can enhance the individual identification of male Odorrana tormota,whether the nonlinear phenomena play a specific role in the sexual selection of O.tormota remain unclear.Here we presented evidence that there was a significant negative correlation(Pearson:n=30,r=0.65,P<0.001)between the nonlinear phenomena content and snout-vent length in the male O.tormota,and two-choice amplexus experiments showed that female O.tormota preferred male with smaller body size containing higher nonlinear phenomena content in its calls.Phonotaxis experiments also revealed that females preferred calls with higher nonlinear phenomena content.Additionally,compared to the calls with lower nonlinear phenomena content and higher fundamental frequency,there was shorter response time in phonotactic behaviour of female induced by the calls with higher nonlinear phenomena content and lower fundamental frequency.We argue that the nonlinear phenomena content in the calls of male O.tormota can convey its body size information and may provide important clues for female frogs in darkened surroundings to identify males’body size during mate choice,meanwhile,higher nonlinear phenomena content in males’calls may increase the attractiveness of males to females.The results of this study provide confirmation that,for O.tormota,nonlinear phenomena have specific function in mate choice.

Ring Clinal Variation in Morphology of the Green Odorous Frog(Odorrana margaretae)

The green odorous frog(Odorrana margaretae)has an interesting ring-shaped divergence pattern around the Sichuan Basin and documenting its morphological variations is essential in understanding its evolutionary history.Using curvilinear models,we detected significant geographical clinal variations in morphological traits,particularly sizes,of female O.margaretae.Males had significantly smaller sizes than females,and also had smaller variation ranges than females.One major trend of morphological variations was clinal:populations from the west tended to have a larger size with wider head and longer posterior limbs than populations from the east.Species history,with an early extended isolation and two subsequent secondary contacts,may explain most of the geographical clinal variations of O.margaretae.Bioclimatic factors may also contribute in explaining the variance of morphology.

Body Surface Area Prediction in Odorrana grahami

Body surface area（BSA）was regarded as a more readily quantifiable parameter relative to body mass in the normalization of comparative biochemistry and physiology.The BSA prediction has attracted unceasing research back more than a century on animals,especially on humans and rats.Few studies in this area for anurans were reported,and the equation for body surface area（S）and body mass（W）：S=9.9 W 0.56,which was concluded from toads of four species in 1969,was generally adopted to estimate the body surface areas for anurans until recent years.However,this equation was not applicable to Odorrana grahami.The relationship between body surface area and body mass for this species was established as：S=15.4 W 0.579.Our current results suggest estimation equations should be used cautiously across different species and body surface area predictions on more species need to be conducted.

安子山臭蛙(Odorrana yentuensis)的中国国家新分布纪录及其补充描述

基于采集于中国广西十万大山森林公园的标本对安子山臭蛙进行了重新描述,并基于显著的雌雄二态现象对原始文献中的鉴定特征给予补充和完善。该种的发现,是中国两栖类物种国家新纪录,并将中国臭蛙由此前的35种增加至36种。

云南产大绿臭蛙(Odorrana livida)的染色体核型研究

研究了云南勐腊县产的大绿臭蛙的核型,核型模式为2n=26(18M+8SM),5+8,NF=5;并与已报道的安徽黄山和香港的大绿臭蛙的核型进行了比较.结果表明虽然大绿臭蛙的不同地理种群之间的核型模式相同,但在SM和M的数目、顺序以及次缢痕的位置等有所不同,说明大绿臭蛙的不同地理种群的染色体具有丰富的多样性.

Embryonic development of the concave-eared torrent frog with its significance on taxonomy

We investigated the early embryonic and larval development of the concave-eared torrent frogs, Odorrana tormota （Amphibia, Anura, Ranidae）. Embryos were derived from artificial fertilization of frogs’ eggs, and the staging of development was based on morphological and physiological characteristics. Two major periods of development were designated： i） early embryonic period, from fertilization to operculum completion stage, lasted for 324 h at water temperature （WT） 18 ？23℃; ii） larval period, from operculum completion stage to tail absorbed stage, took 1207 h at WT 20 ？ 24℃. Tadpoles of the concave-eared torrent frogs showed no evidence of abdominal sucker. Absence of this key characteristic supports the view from molecular systematics that concave-eared torrent frog does not belong to the genus Amolops. Two cleavage patterns were observed in embryos at 8-cell and 16-cell stages, with Pattern I2 （latitudinal cleavage at the 8-cell stage, and meridional cleavage at the 16-cell stage with two perpendicular meridional furrows） being the predominant pattern and only 1.5% belonging to Pattern II （meridional cleavage at the 8-cell stage and latitudinal cleavage at the 16-cell stage）. The factors affecting cleavage and hatching ratios, developmental speed, and ecological adaptation were discussed.

中国四川省蛙科一新种——南江臭蛙(两栖纲:无尾目)

对分布于四川、重庆市、贵州、湖北、湖南、安徽和福建的花臭蛙[Odorrana(Odorrana)schmackeri(Boettger,1892)]标本进行了形态比较,结果发现四川省南江和万源县的花臭蛙标本与其他产地的花臭蛙标本在形态特征上存在明显区别,主要是:1)成体雄雌体长之比值较小,约为1∶1.34;2)犁骨齿列短,两内侧间距宽,距内鼻孔远;3)背面深色斑点周围无浅色边缘;4)股后部深色斑大而稀疏;5)趾间蹼较弱,第四趾蹼达远端关节下瘤;6)腹内成熟卵的动植物极均为乳白色。故将四川省南江和万源的标本订为新种--南江臭蛙[Odorrana(Odorrana)nanjiangensis,sp.nov.]。

中国蛙科一新种(两栖纲,无尾目)

对分布于广西龙胜和湖南宜章的龙胜臭蛙Odorrana(Odorrana)lungshengensis作了进一步比较研究,发现两者的形态特征有明显区别,并将湖南宜章的标本命名为新种──宜章臭蛙Odorrana(Odorrana)yizhangensis sp.nov.。

凹耳蛙的两性异形

测量和比较采自黄山的凹耳蛙成体标本(雌雄各20个),统计结果表明成年雌雄蛙的体长、头宽、吻长、鼻间距、眼间距、眼径、鼓膜宽、前臂及指长、前臂宽等两性形态学指标存在显著性差异。对与体长显著相关的形态特征,以体长为协变量的协方差分析表明凹耳蛙雌雄两性在头宽、眼间距、眼径、鼓膜宽存在显著差异。凹耳蛙两性异形比达到0.418,雌性平均体长对雄性平均体长的比达到1.716。头宽、眼间距、眼径、鼓膜宽等局部形态特征与体长的线性回归分析表明,雌性斜率大于雄性斜率,说明雌性凹耳蛙上述局部特征随体长的生长速率明显大于雄性。雌性体长的繁殖力选择能够很好地解释凹耳蛙的两性异形。