Comparative Study of the Biological Activities of the Skin Secretions from Six Common Chinese Amphibians

Water soluble skin secretions of six common Chinese amphibians were studied for their biological and enzymatic activities.The skin secretions of Tylototriton verrucosus,Bombina maxima ,and Bufo andrewsi were found toxic to mice with the intraperitoneal LD 50 of 11 5?mg/kg,18 8?mg/kg,and 264?mg/kg,respectively.No acute lethal toxicities were observed for the skin secretions of Rana nigromaculata,Rana guentheri and Rana limnocharis in a dose up to 500?mg/kg.The lethal toxicities of the skin secretions of T verrucosus and B maxima to mice are in the same grade as those of Viperidae snake venoms.The toxic components in T verrucosus and B maxima skin secretions are the proteins with molecular weights ranging from 3 to 60?kDa.All the skin secretions had both proteolytic activity and trypsin inhibitory activity.The skin secretions from T verrucosus , B maxima and B andrewsi also displayed wide spectrum antimicrobial activity.On the other hand,the skin secretions from B andrewsi and B maxima showed cytotoxicity on human cancer cells.All the six samples had not significant effects on mammalian blood coagulation system.Phospholipase A 2 activity was only found in the skin secretions of T verrucosus .None of these skin secretions showed acetylcholine esterase activity.

Amphibians and Reptiles of Luzon Island,V:The Herpetofauna of Angat Dam Watershed,Bulacan Province,Luzon Island,Philippines

We report amphibian and reptile distribution records based on recent biodiversity surveys conducted at the Angat Watershed Reservation, Bulacan Province, Luzon Island, Philippines. This watershed constitutes the principal water source for Manila, the Philippines＇ largest metropolitan area. As virtually nothing is known of the herpetological diversity of the immediate area and the surrounding Bulacan Province, all species recorded as part of our surveys constitute major geographical records and/or significant range extensions. Our data result in a total of 63 new records of amphibian （19 frogs） and reptile （22 lizards, 2 turtles, and 20 snakes） species for this protected area （and immediate vicinity） that serves as a watershed for the major metropolitan area of Manila and surrounding cities. Together with the few previous literature records, our new records bring the total number of amphibian and reptile species for Bulacan Province to 68. We discuss several strategies for future survey work （focusing on habitat type, seasonal variation, and elevational variability） that we anticipate will result in increased knowledge of diversity within the Angat Watershed Reserve. The impressive level of herpetological diversity within such a small area, so close to Metro Manila, emphasizes that the diversity and distribution patterns of amphibians and reptiles from Luzon are still poorly known and in need of further study.

Amphibians Testing Negative for Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans on the Qinghai-Tibetan Plateau,China

A disease caused by the fungi Batrachochytrium dendrobatidis（Bd） and Batrachochytrium salamandrivorans（Bsal） is responsible for recent worldwide declines and extinctions of amphibian populations.The Qinghai-Tibetan Plateau（QTP） is aglobal biodiversity hotspot,yet little is known about the prevalence of Bd and Bsal in this region.In this study,we collected 336 non-invasive skin swabs from wild amphibians（including an exotic amphibian species） on the QTP.In addition,to assess the historical prevalence of Bd and Bsal on the QTP,we collected 117 non-invasive skin swabs from museum-archived amphibian samples（from 1964-1982） originating from the QTP.Our results showed all samples to be negative for Bd and Bsal.The government should ban the potentially harmful introduction of non-native amphibian species to the QTP and educate the public about the impacts of releasing exotic amphibians from chytridinfected areas into native environments of the QTP.

Perspectives on studying molecular adaptations of amphibians in the genomic era

Understanding the genetic mechanisms underlying particular adaptations/phenotypes of organisms is one of the core issues of evolutionary biology.The use of genomic data has greatly advanced our understandings on this issue,as well as other aspects of evolutionary biology,including molecular adaptation,speciation,and even conservation of endangered species.Despite the well-recognized advantages,usages of genomic data are still limited to non-mammal vertebrate groups,partly due to the difficulties in assembling large or highly heterozygous genomes.Although this is particularly the case for amphibians,nonetheless,several comparative and population genomic analyses have shed lights into the speciation and adaptation processes of amphibians in a complex landscape,giving a promising hope for a wider application of genomics in the previously believed challenging groups of organisms.At the same time,these pioneer studies also allow us to realize numerous challenges in studying the molecular adaptations and/or phenotypic evolutionary mechanisms of amphibians.In this review,we first summarize the recent progresses in the study of adaptive evolution of amphibians based on genomic data,and then we give perspectives regarding how to effectively identify key pathways underlying the evolution of complex traits in the genomic era,as well as directions for future research.

A Revised Checklist of Amphibians and Reptiles in Camiguin Sur,Misamis Oriental,Mindaanao,Philippines

The Philippines is listed as one of the world's megadiverse countries despite its small size.Conversely,it is also on the list of global biodiversity conservation hotspots.With the threat of extinction at an all-time high,better understa nding of the archipelago's biodiversity will provide invalua ble baseline information for proper conservation efforts.Here we provide an upda ted checklist on the herpetological biodiversity of Ca miguin Sur,Misa mis Oriental,Minda nao,Philippines.Field surveys and specimen collection from the municipality of Mambajao resulted in a total of 28 species(13 anurans,11 lizards,and four snakes)-all well represented by voucher specimens.Our data provides seven additional species records,upda ting the island's total amphibian and reptile species count to 57,with about half Philippine endemic.Included in the new records are three invasive alien species of amphibians:The Cane Toad Rhinella marina,the Greenhouse Frog Eleutherodatylus planirstris,and the Asiatic Painted Narrowmouth Toad Kaloula pulchra.

Endemic Amphibians and Their Distribution in China

Amphibians are good indicators of ecosystem health. Updating the information of endemic species in time and understanding their spatial distributions are necessary for the development of integrative conservation strategies and the elucidation of geographic patterns of amphibians. We analyzed the diversity and distributions of the endemic amphibians in China based on a database of specimen records, recently published literature, and field surveys. Two hundred and sixty two endemic species of amphibians, belonging to 59 genera in 12 families and two orders, are recognized. They account for 67% of the total number of amphibian species in China, with the family Megophryidae possessing the largest number of endemics. There are also 17 genera endemic to China. Across provinces, the species richness of endemics shows five levels with a hierarchical diversification. Sichuan has the largest number of endemics, while Heilongjiang and Jilin have no endemic species. Alternatively, whether on the level of endemic genus or species, the highest diversity occurs in the Western Mountains and Plateau Subregion. The distribution ranges of most endemic species cover ≤ 4 provinces or zoogeographic subregions. Additionally, the species richness of endemics along an elevational gradient shows a bell-shaped pattern with the peak around 800 m. Most endemic species are distributed in areas of low to mid elevation(c. 700–1 500 m), while 36 species are distributed up to 3 000 m. Endemic species richness decreases with increasing of elevation range. Species at mid elevations display large range sizes, while species at lower and higher elevations exhibit smaller ranges. Our results are to be beneficial for further exploration of the underlying mechanisms of distributional patterns of amphibians in China. This study highlights a need to promote conservation programs for Chinese endemic amphibians due to their narrow distribution ranges and potential threats.

Investigating the Effectiveness of Road-related Mitigation Measures under Semi-controlled Conditions:A Case Study on Asian Amphibians

Road traffic is the main factor causing the decline in amphibian populations worldwide. The proper design of an amphibian tunnel is one of the most efficient measures to mitigate the negative impacts of road traffic on amphibians. However, no study has investigated the effectiveness of amphibian tunnels under semi-controlled conditions in Asian amphibians. Here, we selected two representative amphibian species, the Chinese brown frog, Rana chensinensis, and the Asiatic toad, Bufo gargarizans, which suffer the most severe road mortality along the roads in Northeast China. We placed experimental arrays of culverts of various sizes(diameters of 1.5, 1, and 0.5 m for circular culverts; side lengths of 1.5, 1, and 0.5 m for box culverts), and substrate type(soil, concrete, and metal) to examine the preferences of both species during the migratory season between May and September in 2016 and 2017. The results revealed that the Chinese brown frog preferred mid-and large-sized culverts as well as soil culverts. We concluded that culverts with a side length ≥ 1 m, lined with soil, and accompanied by a ≥ 0.4 m high guide drift fence and ≤ 45° gradient on the roadside ditch wall would best facilitate road crossings for both species and likely for other amphibian species in Northeast China.

Progress and Prospects for Studies on Chinese Amphibians

This work summarizes the history and progress of the studies on Chinese amphibians since they first appeared in the Chinese literature.A wide range of research has been carried out,including the history of the definition of amphibians,faunal surveys,systematic research,ecological research,biochemical research(isozyme and other proteins or peptides,chromosomes,DNA),anatomical research,embryological research,phylogenetic and zoogeographical research,and many others such as ultrastructure of organs,crossbreeding test,regeneration of organs,abnormality survey,acoustics,fossils,sperm ultrastructure and parasites.In addition,the prospects for studies on Chinese amphibians in future are proposed in this paper.

Species Diversity and Elevational Distribution of Amphibians in the Xianxialing and Wuyishan Mountain Ranges,Southeastern China

The species diversity and altitudinal distribution of amphibians along an eleva tional gradient of 200-1600 m in the Xianxialing and Wuyishan Mountain Ranges in Southeastern China were investiga ted through time-constra ined visual surveys along 32 transect lines in 9 survey areas,in which the ha bitat types were also recorded.A total of 27 amphibian species belonging to 19 genera,7 families,and 2 orders were found.The species diversity of the amphibians plateaued at low elevation,and the altitudinal boundary of their distribution was at 800 m.Their species compositions were dissimilar in the two mountain ranges probably beca use the annual average temperature and annual rainfall were different in both areas.The eleva tional Rapoport's rule demonstrated that the species range size of the amphibians expanded as the elevation increased in both mountain ranges.The results of the cross species method supported the rule only when the influence of the low-frequency occurrence proba bility of an investigated species was excluded,whereas those of the Steven's method strongly corroborated the rule rega rdless of the incidental occurrence or absence of the species.

An Integrative Taxonomy of Amphibians of Nepal:An Updated Status and Distribution

Amphibians are declining worldwide.At the same time,each year many species of amphibians are described,indicating that biological diversity on Earth remains poorly understood.This is especially true for mountainous regions,such as the Himalayas,which are also regarded as a biodiversity hotspot.However,it is most likely that the current diversity of amphibians in Nepal is highly underestimated.Amphibian inventories by the past researchers primarily used phenotypic characters as the basis for species identification.However,the application of molecular systematics methods to taxonomic studies has successfully uncovered the taxonomic status of several cryptic taxa.This study used both molecular(16 S rDNA sequences)and morphological comparisons and estimated the diversity of amphibians from Nepal.This study reports the occurrence of 38 species of amphibians based on both molecular and morphological comparisons.Among them,three species were recorded for the first time in Nepal.This study also invalids the taxonomic status of Sphaerotheca paschima.This study highlights the importa nce of integrative taxonomic approaches that help to resolve the current taxonomic ambiguities of Nepalese amphibians.

Morphological and Molecular Data Revealed One New Species of the Short-legged Toads Brachytarsophrys Tian and Hu,1983(Anura,Megophryidae)from Yunnan,China

A new species of the genus Brachytarsophrys,named Brachytarsophrys wenshanensis sp.nov.,has been identified in southeastern Yunnan,China.This new species can be readily distinguished from other known congeners by both morphological criterion and molecular analysis of three mitochondrial gene segments:16S,COI,and Cytb.This classification is based on the following morphological characters:(1)medium body size(SVL 83.8–85.1 mm in two adult males);(2)enormous head,with head width nearly 1.2 times the length;(3)tongue pyriform,feebly notched;(4)non-meeting heels;(5)male lacking nuptial pad;(6)tibiotarsal articulation reaching angle of mouth when hindlimbs are extended forward alongside the body;(7)absence of outer metatarsal tubercle,inner metatarsal tubercle elliptic and approximately equal to first toe;(8)rudimentary toe webbing,webbing formula:Ⅰ(2–)–(2^(++))Ⅱ(2^(–))–(3^(++))Ⅲ(2^(½))–(4)Ⅳ(4^(+))–(2^(⅔))V;(9)lateral fringes narrow;(10)dermal ridge or glandular fold on dorsum absent;(11)pectoral glands distinct and irregular,femoral gland small.Our work increases the number of species within the genus Brachytarsophrys to 9.

Virus-Encoded MicroRNAs Reveal How Ranavirus Interacts with Amphibian Immune Defense

Ranaviruses are harmful viruses that infect amphibians, fish, and reptiles, and have caused particularly devastating declines in amphibian populations. One particular type of ranavirus, called Frog Virus 3 (FV3), has been extensively studied due to its prevalence and impact on amphibians. Previous research has primarily focused on the virus’s genes, but little attention has been given to the non-coding regions of its genome. This article reviews recent studies that reveal the ability of ranaviruses, including FV3, to encode microRNA (miRNA), a type of regulatory RNA. These viral miRNAs play a crucial role in suppressing frog immune genes, modulating the virus-host interaction, and promoting viral infection. Understanding how ranaviruses use miRNAs to control disease progression is essential for addressing the health threat they pose to wildlife and ecosystems.

Conservation needs of amphibians in China: A review

The conservation status of all the amphibians in China is analyzed,and the country is shown to be a global priority for conservation in comparison to many other countries of the world.Three Chinese regions are particularly rich in amphibian diversity:Hengduan,Nanling,and Wuyi mountains.Sala-manders are more threatened than frogs and toads.Several smaller families show a high propensity to become seriously threatened:Bombinatoridae,Cryptobranchidae,Hynobiidae and Salamandridae.Like other parts of the world,stream-breeding,high-elevation forest amphibians have a much higher likeli-hood of being seriously threatened.Habitat loss,pollution,and over-harvesting are the most serious threats to Chinese amphibians.Over-harvesting is a less pervasive threat than habitat loss,but it is more likely to drive a species into rapid decline.Five conservation challenges are mentioned with recommendations for the highest priority research and conservation actions.

Resurvey After 20 Years:Updating the Distribution,Population,and Potential Threats of the Chinting Alpine Toad

Considering the increased anthropogenic impacts,species with a limited range and low detectability often lack fundamental information and conservation actions,placing them at a high risk of endangerment.The Chinting alpine toad Scutiger chintingensis is a rare mountain amphibian endemic to the eastern margin of the Qinghai-Xizang Plateau in China.Within its whole distribution range,only three known populations(Wolong,Emei,and Wawu)exist and no recent population status report has been documented for this species over the past two decades.From 2020 to 2023,we investigated the species distribution,and assessed the risk factors for the main populations.We recorded this species in all distribution areas,and updated a new distribution site with a lower elevation limit.The relative population density was 0.024±0.012 ind./m^(2)on Mount Emei,whereas only 0.008±0.017 ind./m^(2)on Mount Wawu.No significant difference was observed in the number of individuals between the two populations;however,the relative population density was significantly different.Sewage and waste discharge resulting from the construction of scenic areas,as well as disturbances from tourism,were the primary anthropogenic factors that influenced the survival of this species.Our results provide the updated information on the distribution and population status of the Chinting alpine toad,and suggest that unrecorded populations,as well as a wider elevation range,may exist for this species.Our findings emphasise the importance of timely updates of species distribution and population information and offer a basis for the future conservation of endangered amphibians.

When Fleeing Matters:Differences in Escape Behaviours of Three Northeast Asian Anurans

Prey species may have their own optimal escape strategy to balance predation risks and the energetic cost of fleeing.Some species have an advantage when maintaining a short fleeing distance,while others may favour an earlier escape based on microhabitat,size,or body condition.Here,we examined the escape behaviour of the three syntopic Northeast Asian anuran species:Mongolian toads(Strauchbufo raddei),Amur brown frogs(Rana amurensis),and Japanese treefrogs(Dryophytes japonicus)in Mongolia,Russia,China and DPR Korea.We examined flight initiation distance(FID;the distance from a potential predator to the point when the individual starts to flee)and distance fled(DF;distance between flight initiation and flight termination points)of each species and the effects of microhabitat,sex,and body size.Strauchbufo raddei and R.amurensis had a longer FID than D.japonicus,and S.raddei also had a longer DF than D.japonicus.These trends remained similar when dividing FID and DF by a size proxy(snout-vent length)for all individuals.This suggests that the treefrog D.japonicus used a strategy to stay immobile even when they were detected,and the toad S.raddei reacted quicker and more sensitively to predators despite the presence of toxin.Female S.raddei had a significantly longer FID than males suggesting that females are more sensitive to predation risk in this species,but body size was not significant for any of the three species.Our results indicate that the three sympatric species have different escaping strategies,likely related to differences in physiology and crypticity.

Review and synthesis of the effects of climate change on amphibians

Considerable progress has been made in understanding the responses of amphibians to climate change,with successful research carried out on climate change-associated shifts in amphibian phenology,elevational distributions and amphibian-parasite interactions.We review and synthesize the literature on this topic,emphasizing acutely lethal,sublethal,indirect and positive effects of climate change on amphibians,and major research gaps.For instance,evidence is lacking on poleward shifts in amphibian distributions and on changes in body sizes and morphologies of amphibians in response to climate change.We have limited information on amphibian thermal tolerances,thermal preferences,dehydration breaths,opportunity costs of water conserving behaviors and actual temperature and moisture ranges amphibians experience.Even when much of this information is available,there remains little evidence that climate change is acutely lethal to amphibians.This suggests that if climate change is contributing to declines,it might be through effects that are not acutely lethal,indirect,or both,but evidence in support of this suggestion is necessary.In fact,evidence that climate change is directly contributing to amphibian declines is weak,partly because researchers have not often ruled out alternative hypotheses,such as chytrid fungus or climate-fungus interactions.Consequently,we recommend that amphibian-climate research shift from primarily inductive,correlational approach as to studies that evaluate alternative hypotheses for declines.This additional rigor will require interdisciplinary collaborations,estimates of costs and benefits of climate change to amphibian fitness and populations,and the integration of correlative field studies,experiments on‘model’amphibian species,and mathematical and functional,physiological models.

Ontogenetic mechanisms underlying sexual size dimorphism in Urodele amphibians： An across-species approach

Why do two sexes of the same species differ in body size holds a long-standing question of evolutionary biology. While many across-species comparisons have focused on ultimate causes behind sexual size dimorphism （SSD）, only have a few been directed toward elucidating its ontogenetic basis. Urodeles are an amphibian group in which the direction and degree of SSD vary greatly among species. Using demographic data yielded by skeletochronology for 33 urodele species, the current study re- veals a positive across-species correlation between SSD and the sex difference in mean age of adult animals, and the latter in- creases with the corresponding difference in age at maturity; annual growth rate does not differ between the sexes. We conclude that extended longevities in one sex, which is mediated by delayed maturation, would allow it to grow for longer and get larger, with growth rate making a weak contribution to body size. The sex-specific divergence in ontogenetic trajectory might be ex- plained by potentially high growth costs of reproduction to females in association with stronger fecundity selection, and to males that are expected to experience stronger sexual selection [Current Zoology 59 （1）： 142-150, 2013].

A survey for Batrachochytrium salamandrivorans in Chinese amphibians

For the last decade, chytridiomycosis was considered to be caused by a single species of fungus, Batrachochytrium dendrobatidis （Bd）, but a second chytrid species, Batrachochytrium salamandrivorans （Bs）, was recently isolated from an in- fected Salamandra salamandra in the Netherlands. To date, Bs has only been found in the Netherlands. To assess whether Bs is present in China, we analyzed a total of 665 samples, including 425 wild samples, 41 preserved specimens, and 199 captive sam- pies, from 30 different species, including both urodeles and anurans. Our sample sites covered 15 provinces in China. All of the samples tested negative for Bs, resulting in a 95% confidence limit for a prevalence of 0.6%. The absence of Bs observed in this large-scale survey in China has significant implications for amphibian conservation and for border trade management strategies intended to control amphibian diseases. We strongly recommend the continued close monitoring of Bs to verify the status of this potentially devastating amphibian fungus in China .

Parasitic nematodes of amphibians from Lombok Island,Indonesia with description of Camallanus senaruensis sp.nov.and Meteterakis lombokensis sp.nov.

Objective:To determine and describe the nematode species of amphibians collected from Lombok Island,West Nusa Tenggara,Indonesia.Methods:The materials examined were found in the intestines of twenty-four Fejervarya cancrivora(F.cancrivora),sixteen Fejervarya verruculosa,six Duttaphyrnus melanostictus(D.melanostictus)from Senaru and Gangga District,Lombok Island on April 2015.The amphibian hosts were collected by hand.Before observing the nematodes,the hosts were anesthesized to death with chloroform.The ventral of the host was opened by longitudinal incision and the internal organs were removed,placed separately in the Petri-dish and then examined under a dissecting microscope.The nematodes found were fixed with warm 70%alcohol.The specimens for light microscope observation were cleared in glycerol and mounted in the same solution,and for the SEM Jeol scanning electron microscope 5310 low vacuum were re-fixed in caccodylate buffer and glutaraldehyde,dehydrated through a graded series of alcohol and vacuum-dried using TAITEC Vacuum-96Nitrogen,prior to attaching to stubs with double sided cello-tape,coated with gold 400?thickness in an Eico I-B2 ion coater.Drawings were made with the aid of a drawing tube attached to a Nikon compound microscope.Measurements were given in micrometers(μm)as the average,followed by the range in parentheses,unless otherwise stated.Results:Two new species of nematodes were found and described herein:Camallanus senaruensis sp.nov.,and Meteterakis lombokensis sp.nov.,parasitic in the intestine of F.cancrivora and D.melanostictus,respectively.C.senaruensis differs from other congeners in having a bluntly rounded tip of tail in the male and female,the structure of trident and having teeth in the buccal capsule.Meteterakis lombokensis differs from other previously described species in having no vulval flap,has a strongly widened proximal end of spicules,forming a cup shaped,and the number of caudal papillae.Others species found are Meteterakis singaporensis from D.melanostictus,and Chabaudus rauschi(Ch.rauschi)from sixteen F.cancrivora and eight Fejervarya verruculosa.Conclusions:The species of parasitic nematodes from Lombok Island are common in amphibians,but Camallanus,Meteterakis singaporensis,Ch.rauschi are the new record in Indonesia.The dominant species found in Lombok Island is Ch.rauschi.

Body Size but Not Food Size Determined Head Sexual Dimorphism in Rana kukunoris from the Tibetan Plateau

Sexual dimorphism between males and females is a common pattern observed in the natural world and has become one of the main focuses of evolutionary biology.Amphibian head size and shape are important factors that may influence food acquisition,but knowledge about the sexual differences between amphibian head size and shape is relatively scant.Here,intersexual differences in the head size and shape of Rana kukunoris,which is a high-elevation frog from the Qinghai-Tibet Plateau,were investigated.Significant differences were found in the head shape but not the head size between sexes.A significant correlation was also observed between prey size and head size.However,these correlations disappeared when individual body size was controlled,suggesting that body size but not food size was the main factor resulting in intersexual differences in head size/shape.The results suggested that food competition between sexes did not result in sexual dimorphism of head shape among these frogs.