Differentiating Migration and Dispersal Processes for Pond-Breeding Amphibians

Abstract Understanding the movement of animals is critical to many aspects of conservation such as spread of emerging disease, proliferation of invasive species, changes in land-use patterns, and responses to global climate change. Movement processes are especially important for amphibian management and conservation as species declines and extinctions worldwide become ever more apparent. To better integrate behavioral and ecological data on amphibian movements with our use of spatially explicit demographic models and guide effective conservation solutions, I present 1) a synopsis of the literature regarding behavior, ecology, and evolution of movement in pond-breeding amphibians possessing biphasic life cycles to distinguish between migration and dispersal processes, 2) a working hypothesis of juvenile-based dispersal, and 3) a discussion of conservation issues that follow from distinguishing the spatial and temporal movements of amphibians at different scales. I define amphibian migration as intrapopulational, round-trip movements toward and away from aquatic breeding sites. Population-level management, in general, can be focused on spatial scales of <1.0 km with attention focused on adult population and juveniles that remain near the natal wetland. I define amphibian dispersal as interpopulational, unidirectional movements from natal sites to other breeding sites. Metapopulation- or landscape-level management can be focused on movements among populations at spatial scales >1.0–10.0 km and on importance of terrestrial connectivity. The ultimate goal of conservation for amphibians should be long-term regional persistence by addressing management issues at both local and metapopulation scales.     

Microsatellite variation and population structure of the moor frog (Rana arvalis) in Scandinavia

Several recent studies have found amphibian populations to be genetically highly structured over rather short geographical distances, and that the rate of genetically effective dispersal may differ between the sexes. However, apart from the common frog ( Rana temporaria ) little is known about the genetic structuring and sex-biased dispersal in northern European amphibians. We investigated the patterns of genetic diversity and differentiation within and among Scandinavian populations of the moor frog ( Rana arvalis ) using microsatellite markers. The genetic diversity within local R. arvalis populations was not a simple linear negative function of latitude but a convex one: genetic diversity peaked in mid-latitude populations, and declined thereafter dramatically towards the north. The average degree of genetic differentiation among populations ( F _ST = 0.14) was lower than that observed for the common frog ( F _ST = 0.21), though the pattern of isolation by distance was similar for both species. Contrary to common frogs, no evidence for female-biased dispersal was found. The results reinforce the view that amphibian populations are—in general—highly structured over relatively small geographical distances, even in comparatively recently colonized areas.     

Dispersal and the metapopulation paradigm in amphibian ecology and conservation: are all amphibian populations metapopulations?

Amphibians are frequently characterized as having limited dispersal abilities, strong site fidelity and spatially disjunct breeding habitat. As such, pond‐breeding species are often alleged to form metapopulations. Amphibian species worldwide appear to be suffering population level declines caused, at least in part, by the degradation and fragmentation of habitat and the intervening areas between habitat patches. If the simplification of amphibians occupying metapopulations is accurate, then a regionally based conservation strategy, informed by metapopulation theory, is a powerful tool to estimate the isolation and extinction risk of ponds or populations. However, to date no attempt to assess the class‐wide generalization of amphibian populations as metapopulations has been made. We reviewed the literature on amphibians as metapopulations (53 journal articles or theses) and amphibian dispersal (166 journal articles or theses for 53 anuran species and 37 salamander species) to evaluate whether the conditions for metapopulation structure had been tested, whether pond isolation was based only on the assumption of limited dispersal, and whether amphibian dispersal was uniformly limited. We found that in the majority of cases (74%) the assumptions of the metapopulation paradigm were not tested. Breeding patch isolation via limited dispersal and/or strong site fidelity was the most frequently implicated or tested metapopulation condition, however we found strong evidence that amphibian dispersal is not as uniformly limited as is often thought. The frequency distribution of maximum movements for anurans and salamanders was well described by an inverse power law. This relationship predicts that distances beneath 11–13 and 8–9 km, respectively, are in a range that they may receive one emigrating individual. Populations isolated by distances approaching this range are perhaps more likely to exhibit metapopulation structure than less isolated populations. Those studies that covered larger areas also tended to report longer maximum movement distances – a pattern with implications for the design of mark‐recapture studies. Caution should be exercised in the application of the metapopulation approach to amphibian population conservation. Some amphibian populations are structured as metapopulations – but not all.     

Outbreeding depression in the common frog, <Emphasis Type="Italic">Rana temporaria</Emphasis>

Theory suggests that parental relatedness is a continuous variable with a fitness optimum that we heretoforth will refer to as optimal outbreeding. In the present paper, we test this proposition from a conservation (translocation) perspective. Amphibians are facing a global decline and many amphibian populations are today small and threatened by extinction. Because genetic differentiation is often high between amphibian populations, they could be particularly sensitive to outbreeding depression, e.g. due to breakdown of locally adapted gene complexes. We tested if outbreeding would reduce fitness in common frogs, Rana temporaria, crossed from a large and an isolated, small population, separated by 130km, using artificial fertilization. For females from the large population, tadpoles were significantly smaller and more malformed in crosses with males from the small population, than with males from the large population. For the small population, however, no significant paternal genetic effects could be found. The difference in response to outbreeding between populations was accompanied with significant differences in the importance of maternal effects. We conclude that care should be taken when translocating frogs between distantly related populations to avoid outbreeding depression.     

Small body size increases the regional differentiation of populations of tropical mantellid frogs (Anura: Mantellidae)

The processes affecting species diversification may also exert an influence on patterns of genetic variability within species. We evaluated the contributions of five variables potentially influencing clade diversification (body size, reproductive mode, range size, microhabitat and skin texture) on mtDNA divergence and polymorphism among populations of 40 species of frogs (Mantellidae) from two rainforest communities in Madagascar. We report an inverse association between body size and nucleotide divergence between populations but find no influence of other variables on genetic variation. Body size explained ca. 11% of the variation in nucleotide divergence between populations and was coupled with high F_ST levels and an absence of haplotype sharing in small‐bodied and medium‐sized frogs. Low dispersal ability is likely the proximate mechanism producing higher population differentiation in small mantellids. The lack of genetic cohesion among populations establishes regional genetic fragmentation which in turn has the potential to accelerate rates of allopatric speciation in small frogs relative to large species. However, there is little evidence of increased speciation rates in these or other small‐bodied organisms. We reconcile these contradictory observations by suggesting that lower dispersal ability also curbs colonization of new areas, decelerating diversification in weak dispersers. Our results imply that the intermediate dispersal model also applies to amphibians and may explain inconsistent previous results on the correlation of body size and speciation rate.     

Density dependence in the terrestrial life history stage of two anurans

Populations of species with complex life cycles have the potential to be regulated at multiple life history stages. However, research tends to focus on single stage density-dependence, which can lead to inaccurate conclusions about population regulation and subsequently hinder conservation efforts. In amphibians, many studies have demonstrated strong effects of larval density and have often assumed that populations are regulated at this life history stage. However, studies examining density regulation in the terrestrial stages are rare, and the functional relationships between terrestrial density and vital rates in amphibians are unknown. We determined the effects of population density on survival, growth and reproductive development in the terrestrial stage of two amphibians by raising juvenile wood frogs (Rana sylvatica) and American toads (Bufo americanus) at six densities in terrestrial enclosures. Density had strong negative effects on survival, growth and reproductive development in both species. We fitted a priori recruitment functions to describe the relationship between initial density and the density of survivors after one year, and determined the functional relationship between initial density and mass after one year. Animals raised at the lowest densities experienced growth and survival rates that were over twice as great as those raised at the highest density. All female wood frogs in the lowest density treatment showed signs of reproductive development, compared to only 6% in the highest density treatment. Female American toads reached minimum reproductive size only at low densities, and male wood frogs and American toads reached maturity only in the three lowest density treatments. Our results demonstrate that in the complex life cycle of amphibians, density in the terrestrial stage can reduce growth, survival and reproductive development and may play an important role in amphibian population regulation. We discuss the implications of these results for population regulation in complex life cycles and for amphibian conservation.     

A sex-specific marker reveals male heterogamety in European tree frogs

Most amphibians examined so far show undifferentiated sex chromosomes. The heterogametic sex's identity, usually revealed through indirect means, often varies among closely related species or even populations (as do sex-linkage groups), suggesting great evolutionary instability of the sex-determining genes. Here we take advantage of a sex-specific marker that amplifies in several related species of European tree frogs (Hyla arborea group) to disclose a homogeneous pattern of male heterogamety. Besides relevance for evolutionary studies of sex determination in amphibians, our results have potential for addressing practical issues in conservation biology because sex reversal by anthropogenic endocrine disruptors is considered one possible cause of amphibian decline.     

Gene flow in a direct-developing,leaf litter frog between isolated mountains in the Taita Hills,Kenya

Amphibians are in decline worldwide, and high altitude tropical areas appear to be the worst affected. This is in stark contrast with current information we have on gene flow in amphibian populations which focus on temperate pond breeding species. Using AFLP markers, we show that a small, direct-developing, leaf litter frog from the Taita Hills in south–west Kenya (Schoutedenella xenodactyloides) has extended populations covering large areas (>3.5 km) of fragmented, forest habitat, uncharacteristic of typical amphibian models. Further, we demonstrate high levels of gene flow (F _ST < 0.065) through unsuitable dry savannah habitat which might otherwise be considered a barrier to dispersal. Landscape genetic analysis demonstrates a strong link between hydrologic features, and further highlights links between sites through specific catchments. We propose a model of passive-active dispersal for the Dwarf Squeaker, S. xenodactyloides, which features passive downhill and active uphill movements over large areas, contrasting with limited cross slope movements. Our study highlights the importance of the diverse reproductive strategies of the Amphibia when considering dispersal and gene flow, and hence conservation management.     

Amphibian decline,pond loss and reduced population connectivity under agricultural intensification over a 38 year period

Habitat loss, together with less obvious land-use changes such as intensified farming practice, can have significant adverse impacts on biodiversity. An important factor in determining the ability of species to cope with such changes is their potential to sustain a populations network by dispersal across the landscape. Habitat quality and structure are particularly important for surface-dwelling species with low dispersal abilities, such as amphibians. To assess this ecological function, ponds in a coastal and typically rural area of northern France were surveyed for amphibians in 1974, 1992 and 2011. These repeated surveys yielded different outcomes for different species groups. Three rare species persisted in more or less specialized habitat types. Two moderately common species declined, but kept strongholds in coastal dunes and associated marshes. Five common species with broad ecological niches remained equally widespread. The Northern crested newt declined markedly and the Midwife toad declined dramatically, as did their breeding habitats in vegetated ponds and cattle drinking troughs. One species, the Moor frog, may have gone locally extinct. A model of relative resistance to amphibian dispersal was created for different landscape types, on a scale from 0 (low resistance) to 1 (high resistance). This generated values of 0.23 for pasture, 0.72 for arable and 0.98 for urban and transport. As pasture declined in the study area, while arable and urban/transport infrastructure increased, amphibian dispersal became more difficult. However, dispersal paths proved difficult to evaluate in a patchy landscape like the one surveyed, due to a paucity of spatial signal. Pond loss is a more tractable reason for the observed amphibian species decline than is the quality of intervening terrestrial habitat matrix. In 2011, 22 newly created ponds had species richness in line with pre-existing ponds and this will have counteracted a dwindling metapopulation structure, indicating that habitat creation/restoration can play a valuable role in conservation. The colonization of new ponds may also prove more informative for gauging the potential for amphibian dispersal in the landscape than the preceding decline.     

Multiple overseas dispersal in amphibians

Amphibians are thought to be unable to disperse over ocean barriers because they do not tolerate the osmotic stress of salt water. Their distribution patterns have therefore generally been explained by vicariance biogeography. Here, we present compelling evidence for overseas dispersal of frogs in the Indian Ocean region based on the discovery of two endemic species on Mayotte. This island belongs to the Comoro archipelago, which is entirely volcanic and surrounded by sea depths of more than 3500 m. This constitutes the first observation of endemic amphibians on oceanic islands that did not have any past physical contact to other land masses. The two species of frogs had previously been thought to be nonendemic and introduced from Madagascar, but clearly represent new species based on their morphological and genetic differentiation. They belong to the genera Mantidactylus and Boophis in the family Mantellidae that is otherwise restricted to Madagascar, and are distinguished by morphology and mitochondrial and nuclear DNA sequences from mantellid species occurring in Madagascar. This discovery permits us to update and test molecular clocks for frogs distributed in this region. The new calibrations are in agreement with previous rate estimates and indicate two further Cenozoic transmarine dispersal events that had previously been interpreted as vicariance: hyperoliid frogs from Africa to Madagascar (Heterixalus) and from Madagascar to the Seychelles islands (Tachycnemis). Our results provide the strongest evidence so far that overseas dispersal of amphibians exists and is no rare exception, although vicariance certainly retains much of its importance in explaining amphibian biogeography.     

Fine-scale population differentiation and gene flow in a terrestrial salamander (Plethodon cinereus) living in continuous habitat

Several recent studies have shown that amphibian populations may exhibit high genetic subdivision in areas with recent fragmentation and urban development. Less is known about the potential for genetic differentiation in continuous habitats. We studied genetic differentiation of red-backed salamanders (Plethodon cinereus) across a 2-km transect through continuous forest in Virginia, USA. Mark-recapture studies suggest very little dispersal for this species, whereas homing experiments and post-Pleistocene range expansion both suggest greater dispersal abilities. We used six microsatellite loci to examine genetic population structure and differentiation between eight subpopulations of red-backed salamanders at distances from 200 m to 2 km. We also used several methods to extrapolate dispersal frequencies and test for sex-biased dispersal. We found small, but detectable differentiation among populations, even at distances as small as 200 m. Differentiation was closely correlated with distance and both Mantel tests and assignment tests were consistent with an isolation-by-distance model for the population. Extrapolations of intergenerational variance in spatial position (sigma(2)<15 m(2)) and pair-wise dispersal frequencies (4 Nm < 25 for plots separated by 300 m) both suggest limited gene flow. Additionally, tests for sex-biased dispersal imply that dispersal frequency is similarly low for both sexes. We suggest that these low levels of gene flow and the infrequent dispersal observed in mark-recapture studies may be reconciled with homing ability and range expansion if dispersing animals rarely succeed in breeding in saturated habitats, if dispersal is flexible depending on the availability of habitat, or if dispersal frequency varies across the geographic range of red-backed salamanders.     

Population genetic structure of orchid bees (Euglossini) in anthropogenically altered landscapes

Habitat degradation and fragmentation are widespread phenomena in tropical regions. Negative effects on the biota are numerous, ranging from interruption of gene flow among populations, to the loss of genetic diversity within populations, to a decline in species richness over time. Orchid bees (Hymenoptera: Apidae: Euglossini) are of major conservation interest due to their function as pollinators of numerous orchid species and other tropical plants. Here, we used microsatellite markers to investigate the effects of geographic distance and habitat fragmentation on gene flow among populations. Populations of Euglossa dilemma in three geographic regions??the Yucat??n peninsula (Mexico), Veracruz (Mexico), and Florida (USA)??were genetically structured predominantly across the regions, with the strength of differentiation among populations being positively correlated with geographic distance. Within geographic regions only little substructure was found, suggesting that dispersal is substantial in the absence of geographic or ecological barriers. In a second study, patterns of genetic differentiation among eight species of Euglossa were not related to habitat fragmentation following deforestation in southern Mexico (Veracruz). Specifically, most bee populations in the 9,800?ha forest remnant of Los Tuxtlas (Volcano San Martin) were neither differentiated from, nor had less genetic diversity than, populations in near-continuous forest separated from Los Tuxtlas by 130?km of agricultural land. Either occasional long distance dispersal across open areas has buffered the expected genetic effects of fragmentation, or the history of fragmentation in southern Mexico is too recent to have caused measurable shifts in allelic composition.     

Dispersal and gene flow in the rare, parasitic Large Blue butterfly Maculinea arion

Dispersal is crucial for gene flow and often determines the long‐term stability of meta‐populations, particularly in rare species with specialized life cycles. Such species are often foci of conservation efforts because they suffer disproportionally from degradation and fragmentation of their habitat. However, detailed knowledge of effective gene flow through dispersal is often missing, so that conservation strategies have to be based on mark–recapture observations that are suspected to be poor predictors of long‐distance dispersal. These constraints have been especially severe in the study of butterfly populations, where microsatellite markers have been difficult to develop. We used eight microsatellite markers to analyse genetic population structure of the Large Blue butterfly Maculinea arion in Sweden. During recent decades, this species has become an icon of insect conservation after massive decline throughout Europe and extinction in Britain followed by reintroduction of a seed population from the Swedish island of Öland. We find that populations are highly structured genetically, but that gene flow occurs over distances 15 times longer than the maximum distance recorded from mark–recapture studies, which can only be explained by maximum dispersal distances at least twice as large as previously accepted. However, we also find evidence that gaps between sites with suitable habitat exceeding ～20 km induce genetic erosion that can be detected from bottleneck analyses. Although further work is needed, our results suggest that M. arion can maintain fully functional metapopulations when they consist of optimal habitat patches that are no further apart than ～10 km.     

Treatment of chytridiomycosis with reduced-dose itraconazole

Effective treatment methods to eliminate infection with Batrachochytrium dendrobatidis (Bd) are required for development of sustainable captive survival assurance populations of amphibians and to reduce the risk of introducing Bd to new locations as part of amphibian trade or reintroduction programs. Treatment with itraconazole baths at 100 mg l-1 is commonly used in captive amphibians, but side effects are observed in some amphibian species and life stages. Naturally occurring outbreaks of chytridiomycosis in Wyoming toads Anaxyrus baxteri and White's tree frogs Litoria caerulea were treated with lower-dose itraconazole baths (e.g. 50 mg l-1 for White's tree frogs) and followed post-treatment with serial Taqman PCR testing to confirm elimination of Bd infection. Post-treatment PCR tests were consistently negative for the presence of Bd and treatment was deemed successful. Although this was not a controlled clinical trial, results suggest that lower doses of itraconazole may be effective for treatment of chytridiomycosis with resulting cost savings to amphibian conservation programs and a potential for a reduction in dose-related side effects from itraconazole treatment. Prospective clinical trials of alternative itraconazole treatment protocols are encouraged.     

Pathophysiology in mountain yellow-legged frogs (Rana muscosa) during a chytridiomycosis outbreak

The disease chytridiomycosis is responsible for declines and extirpations of amphibians worldwide. Chytridiomycosis is caused by a fungal pathogen (Batrachochytrium dendrobatidis) that infects amphibian skin. Although we have a basic understanding of the pathophysiology from laboratory experiments, many mechanistic details remain unresolved and it is unknown if disease development is similar in wild amphibian populations. To gain a better understanding of chytridiomycosis pathophysiology in wild amphibian populations, we collected blood biochemistry measurements during an outbreak in mountain yellow-legged frogs (Rana muscosa) in the Sierra Nevada Mountains of California. We found that pathogen load is associated with disruptions in fluid and electrolyte balance, yet is not associated with fluctuations acid-base balance. These findings enhance our knowledge of the pathophysiology of this disease and indicate that disease development is consistent across multiple species and in both laboratory and natural conditions. We recommend integrating an understanding of chytridiomycosis pathophysiology with mitigation practices to improve amphibian conservation.     

Amphibian declines: future directions

Abstract. The amphibian decline problem is complex, and there is no easy solution. I highlight four major areas of future research that should increase our ability to detect declines, elucidate their underlying mechanisms, and advance our capacity to manage and conserve amphibian populations. First, a statistically sensitive monitoring approach is necessary to determine the distribution and abundance of amphibian populations, to assess whether they are declining, and to quantify the extent of declines. Most amphibian populations characteristically fluctuate, detection probabilities may be low for many species and populations tend to decline in numbers between years more often than they increase. These traits make establishing monitoring programmes difficult and distinguishing declines from natural fluctuations challenging. It is thus necessary to determine the best monitoring techniques based on their statistical power and to use appropriate statistical methods for detecting population trends. Secondly, although amphibian population studies occur most commonly at single or few breeding sites, research should occur often at the landscape level, and conservation efforts should focus on suitable habitat (whether or not it is occupied) and dispersal capabilities of species. Metapopulation dynamics are probably important for many species, but we must be cautious how we define metapopulations. That is, the term ‘metapopulation’ is currently used to define a wide range of demographic situations in amphibian populations, each with different management implications. Thirdly, recent advances in molecular genetic techniques make it possible to infer demographic events such as effects of recent fragmentation, bottlenecks or hybridization. Molecular techniques can be used in conjunction with census surveys to bolster knowledge about demographic processes such as declines. Alternatively, in the absence of long‐term census data, molecular data can be used to infer population trends. New genomic approaches may make estimating adaptive genetic variation more feasible. Fourthly, multi‐factorial studies are needed to disentangle the complexity of the several putative causes that probably interact to cause amphibian declines. Recent studies demonstrate the value of a multi‐factorial approach, and more work is needed to elucidate the synergistic effects of multiple environmental factors affecting amphibian populations simultaneously worldwide.     

Population differentiation of temperate amphibians in unpredictable environments

Amphibians are a globally distributed and diverse lineage, but much of our current understanding of their population genetic structure comes from studies in mesic temperate habitats. We characterize the population genetic structure of two sympatric explosive breeding amphibians in the southwestern deserts of the United States: the Great Plains toad ( Anaxyrus cognatus ) and Couch's spadefoot toad ( Scaphiopus couchii ). For both species, we find limited genetic differentiation even between populations in adjacent valleys separated by dispersal barriers such as mountainous habitats. To understand how population genetic patterns in these two arid-adapted species compare to taxa in more mesic environments, we computed a standardized measure of population differentiation for A. cognatus , S. couchii , and for pond-breeding amphibians that inhabit mesic temperate environments. Our results indicate that the arid-adapted species have lower population genetic structure at fine and moderate scales than most other amphibian species we surveyed. We hypothesize that stochasticity in the availability of appropriate breeding sites as well as landscape homogeneity may result in increased population connectivity in desert-adapted frogs. Future work examining fine-scale population structure in amphibians from a diversity of habitats will test the generality of our findings. Intraspecific comparisons among localities with varied seasonality and habitats will be particularly useful for investigating the interaction between species-typical population dynamics and environmental characteristics as determinants of population connectivity in pond-breeding amphibians.     

Batrachochytrium dendrobatidis in the live frog trade of Telmatobius (Anura: Ceratophryidae) in the tropical Andes

Species of frogs in the genus Telmatobius are traded and sold for human consumption in the Andes and in coastal cities of Peru and Bolivia. These frogs are harvested from wild populations. We report high prevalence of infection by the pathogenic fungus Batrachochytrium dendrobatidis in live frogs purchased at the main market in Cusco, Peru, from January 2008 to January 2010. We suggest that the transport of native anurans through the live frog trade could facilitate the spread of this fungus among Andean frogs. The tropical Andes are the most important biodiversity hotspot for amphibians. Because many neotropical taxa are known to be susceptible to chytridiomycosis, the presence of a large reservoir of infection in the frog trade poses a significant threat to amphibian conservation.     

Behaviour of Australian rainforest stream frogs may affect the transmission of chytridiomycosis

The amphibian disease chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis, has been implicated in mass mortalities, population declines and extinctions of amphibians around the world. In almost all cases, amphibian species that have disappeared or declined due to chytridiomycosis coexist with non-declining species. One reason why some species decline from chytridiomycosis and others do not may be interspecific differences in behaviour. Host behaviour could either facilitate or hinder pathogen transmission, and transmission rates in the field are likely to vary among species according the frequency of factors such as physical contact between frogs, contact with infected water and contact with environmental substrates containing B. dendrobatidis. We tracked 117 frogs (28 Litoria nannotis, 27 L. genimaculata and 62 L. lesueuri) at 5 sites where B. dendrobatidis is endemic in the rainforest of tropical northern Queensland and recorded the frequency of frog-to-frog contact and the frequency of contact with stream water and environmental substrates. Frequency of contact with other frogs and with water were highest in L. nannotis, intermediate in L. genimaculata and lowest in L. lesueueri. Environmental substrate use also differed among species. These species-specific opportunities for disease transmission were correlated with conservation status: L. nannotis is the species most susceptible to chytridiomycosis-related declines and L. lesueuri is the least susceptible. Interspecific variation in transmission probability may, therefore, play a large role in determining why chytridiomycosis drives some populations to extinction and not others.     

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.