Elevational Distribution of Amphibians: Resolving Distributions, Patterns, and Species Communities in the Republic of Korea.

Species biodiversity organises along elevational trends and is generally expected to decrease with increasing elevation. This pattern is regulated by numerous factors, although principally overridden by temperature in ectotherms such as amphibians. Here, we collated elevation data (n = 55,182) collected between 1909 and 2020. We then determined elevation distribution patterns and species communities for all amphibians in the Republic of Korea. Species were found to range from sea level up to 1,393 m a.s.l. The average elevational distribution was significantly different between species but also between anura and caudata. On average, anura were found at lower elevations with a peak in species richness and abundance matching with the lowlands. In opposition, the peak in species richness and abundance for caudata matched with low hilly landscapes. The altitudinal distributions of species were strongly skewed, with all 23 species found within the 0-199 m range, and steadily decreasing with only five species within the last elevational range (1,200-1,399 m). Finally, only a few species were found below 30 m, reflecting a likely risk of salinisation in this environment. Our results help understand the altitudinal distribution of amphibians in the Republic of Korea.

Latitude-Induced and Behaviorally Thermoregulated Variations in Upper Thermal Tolerance of Two Anuran Species.

Although thermal tolerance along geographical gradients gives an insight into species' response to climate change, current studies on thermal tolerance are strongly skewed towards global-scale patterns. As a result, intraspecific variations are often assumed to be constant, despite a lack of evidence. To understand population-specific responses to thermal stress, we investigated the presence of intraspecific variations in the critical thermal maximum (CTmax) of tadpoles in two anuran species, Rana uenoi and Bufo sachalinensis. The study was conducted across a five-degree latitudinal gradient in the Republic of Korea. We exposed the tadpoles to increasing temperatures and recorded the CTmax for 270 R. uenoi individuals from 11 sites, collected in rice paddies, and for 240 B. sachalinensis individuals from ten sites, collected in reservoirs. We also recorded the swimming performance and behavior of the tadpoles when placed in an experimental apparatus during CTmax measurements. We then used linear regressions to determine the relationship between abiotic factors and CTmax. In R. uenoi, we found a positive relationship between latitude and CTmax, but the tadpoles did not display specific thermoregulatory behaviors. In B. sachalinensis, none of the abiotic factors such as climate and geographic coordinates were related to CTmax, but we detected a tendency to swim close to the water surface when water temperature was increasing. For R. uenoi, we tentatively relate the CTmax variability across the latitudinal gradient to a physiological adaptive response associated with habitat characteristics that are assumed to be fluctuating, as the species inhabits small water bodies prone to drying out. In the case of B. sachalinensis, the behavior observed may be linked to oxygen depletion and thermoregulation, as it may buffer temperature changes in the absence of physiological adjustment. These findings suggest that intra-specific variations in CTmax are greater than generally accounted for, and thermal conditions of natural environments are important for understanding thermal tolerance in ectothermic species. Our results highlight that species' specific responses to climate warmings need to be studied to better protect species against climate change.

Increasing salinity stress decreases the thermal tolerance of amphibian tadpoles in coastal areas of Taiwan.

Global warming is the main cause for the rise of both global temperatures and sea-level, both major variables threatening biodiversity. Rising temperatures threaten to breach the thermal limits of organisms while rising sea-level threatens the osmotic balance of coastal animals through habitat salinization. However, variations in thermal tolerance under different salinity stresses have not yet been thoroughly studied. In this study, we assessed the critical thermal maxima (CTmax) of amphibian tadpoles in different salinity conditions. We collected tadpoles of Duttaphrynus melanostictus, Fejervarya limnocharis and Microhyla fissipes from coastal areas and housed them in freshwater, low, and high salinity treatments for 7 days of acclimation. The CTmax, survival rate, and development rate of tadpoles in high salinity treatments were significantly lower than that of the two other treatments. Our results indicate that physiological performances and heat tolerances of tadpoles are negatively affected by salinization. Maximum entropy models showed that CTmax and sea-level rise are predicted to negatively affect the distribution of the three focal species. The present results suggest that global warming can lead to negative dual-impacts on coastal animals because of reduced thermal tolerances at elevated salinity. The impacts of global warming on anurans in coastal areas and other habitats impacted by salinization may be more severe than predicted and it is likely to cause similar dual-impacts on other ectotherms.

From Gondwana to the Yellow Sea, evolutionary diversifications of true toads Bufo sp. in the Eastern Palearctic and a revisit of species boundaries for Asian lineages.

Taxa with vast distribution ranges often display unresolved phylogeographic structures and unclear taxonomic boundaries resulting in hidden diversity. This hypothesis-driven study reveals the evolutionary history of Bufonidae, covering the phylogeographic patterns found in Holarctic bufonids from the West Gondwana to the phylogenetic taxonomy of Asiatic true toads in the Eastern Palearctic. We used an integrative approach relying on fossilized birth-death calibrations, population dynamics, gene-flow, species distribution, and species delimitation modeling to resolve the biogeography of the clade and highlight cryptic lineages. We verified the near-simultaneous Miocene radiations within Western and Eastern Palearctic Bufo, c. 14.49-10.00 Mya, temporally matching with the maximum dust outflows in Central Asian deserts. Contrary to earlier studies, we demonstrated that the combined impacts of long dispersal and ice-age refugia equally contributed to the current genetic structure of Bufo in East Asia. Our findings reveal a climate-driven adaptation in septentrional Eastern Asian Bufo, explaining its range shifts toward northern latitudes. We resolve species boundaries within the Eastern Palearctic Bufo, and redefine the taxonomic and conservation units of the northeastern species: B. sachalinensis and its subspecies.

The east Asian Asiatic toad (also known by the latin name Bufo gargarizans) lives in a wide range of habitats across East Asia including forests, meadows and cultivated land. However, it remains unclear how these toads evolved and became so widespread ­ partly because it has proved difficult for researchers to clearly define the species and what distinguishes it from other closely-related species of toads (collectively known as Bufo toads). Othman et al. combined several bioinformatics techniques to study Asiatic toads and 38 other species of bufonid toads from across the globe. This approach found that Bufo toads first emerged in eastern Asia between 14 and 10 million years ago. This coincides with a point in time when large swathes of land in central Asia turned from adequate to sustain toad populations into desert, suggesting the change in climate prompted the toads to migrate eastwards from central Asia. The Bufo toads then divided into two groups of species: one in mainland East Asia and the other in Japan. Furthermore, the study revealed there is more genetic diversity ­ that is, more variety in the DNA of individuals ­ in Asiatic toads than previously thought. The findings also help to define several other species of Bufo toads more clearly and describe a new toad species restricted to the Korean Peninsula, northeastern China and the Russian Primorye region: the Sakhalin toad (Bufo sachalinensis). This work demonstrates that a large-scale study of many species across the globe can be used to understand how the species evolved and more clearly distinguish one species from another. The findings of Othman et al. will be of interest to both professional and citizen scientists interested in the natural history of Asia. Furthermore, as several species of Bufo toads are in decline in the wild, they provide evidence that may aid future efforts to conserve them.

Coexisting good neighbours: acoustic and calling microhabitat niche partitioning in two elusive syntopic species of balloon frogs, Uperodon systoma and U. globulosus (Anura: Microhylidae) and potential of individual vocal signatures.

BACKGROUND: Most amphibians use a repertoire of acoustic signals to propagate signals in social contexts. The description of these repertoires provides a key towards the understanding of the behaviour of individuals and the evolutionary functions of calls. Here, we assessed the variations in advertisement calls within and between two fossorial sympatric species, Uperodon systoma and Uperodon globulosus, that share their breeding season and breeding sites. For each species, we applied Beecher's index of total information capacity (HS) for the individual vocal signature, determined the difference in call properties and demonstrated the segregation in the calling microhabitat niche between the two species. RESULTS: Our results demonstrated that the advertisement calls of U. systoma are pulsatile with a call rate of 3.00 ± 0.97 calls per second while those of U. globulosus are not pulsatile with a lower call rate of 0.53 ± 0.22 calls per second. For both species, the variations in call properties among individuals was higher than that within individual, a pattern consistent with that of other fossorial anurans. The body condition and air temperature did not significantly impact the call properties of either species. The outcome of the Beecher's index (HS) showed that the calls of U. systoma can be used to identify 14 different individuals and the calls of U. globulosus can be used to identify 26 different individuals. The statistical analyses on the advertisement call of the two species showed a significant difference in the temporal properties as the call duration, and fall time and rise time were significantly different between the two species. Lastly, we successfully demonstrated that there is a clear segregation in calling site microhabitat between the two species, where U. globulosus calls floating close to the bank of the waterbody while U. systoma calls floating further away from the bank. CONCLUSION: This study highlights the potential for pre-mating isolation, character displacement and assortative mating in two syntopic fossorial anurans, leading to association between acoustic, calling microhabitat niche and body index divergence as important behavioural and ecological traits.

Endurance rivalry and female choice jointly influence male mating success in the emerald treefrog (Zhangixalus prasinatus), a lek-chorusing anuran.

BACKGROUND: Endurance rivalry and female choice are two important mechanisms of sexual selection in lek-breeding species. Endurance rivalry is when males compete for opportunities to mate by spending more time in leks than others (interaction-independent male-male competition). Because high-quality males can afford to have high lek attendance, females have a higher chance of mating with good-quality males even when they mate randomly. The good gene hypothesis proposes that females can pass good genes on to their offspring by choosing males that display elaborate morphological and/or behavioral traits that reflect the males' genetic quality. The relative importance of lek attendance and female choice to males' mating success in anurans is rarely evaluated. In this study, we investigated how these two mechanisms might jointly shape males' morphological traits in the lek-chorusing emerald treefrog Zhangixalus prasinatus. RESULTS: Our results show that (1) male lek attendance is positively correlated with body size and condition, and males with higher lek attendance have higher mating success, (2) the dominant frequency of males' advertisement calls are negatively correlated with body size and males producing lower frequency calls have higher mating success, (3) male body size, but not body condition, has a non-significant positive relationship with mating success and (4) females show preference for calls with lower dominant frequencies in two-choice playback. CONCLUSIONS: Overall, both endurance rivalry and female choice play an important role in the mating success of male emerald treefrogs in the field and both are influenced by male body size/condition. By mating with males that have higher lek attendance and produce lower frequency calls, selection may indirectly favor larger males.

Impact of the Miocene orogenesis on Kaloula spp. radiation and implication of local refugia on genetic diversification.

The phylogeography of the Kaloula genus in East Asia is still poorly understood. One of the difficulties is the absence of fossils to corroborate molecular dating estimates. Here, we examined the mitochondrial structure of Kaloula spp. in East Asia and focused on the impact of glaciations on the northernmost species: Kaloula borealis. We determined the phylogenetic relationships, molecular dating, and genetic connectivity assessments within the genus from 1211 bp of concatenated mitochondrial 12S and 16S. The relaxed clock analyses reveal the emergence of Kaloula spp. common ancestor in East and Southeast Asia between the Eocene and Oligocene, c. 38.47 Ma (24.69-53.65). The genetic diversification of lineages then increased on the East Asian Mainland during the Lower Miocene, c. 20.10 (8.73-30.65), most likely originating from the vicariance and radiation triggered by the orogeny of the Qinghai-Tibetan Plateau. Later, the dispersal towards the North East Asian Mainland during the Upper Miocene drove the population diversification of K. borealis c. 9.01 Ma (3.66-15.29). Finally, the central mainland population became isolated following orogenesis events and diverged into K. rugifera during the Pliocene, c. 3.06 Ma (0.02-10.90). The combination of population genetic and barrier analyses revealed a significant genetic isolation between populations of Kaloula spp. matching with the massive Qinling-Daba Mountain chain located in south-central China. Finally, we highlight a young divergence within the Eastern Mainland population of K. borealis, possibly attributed to refugia in south eastern China from which populations later expanded.

Description of a new Kurixalus species (Rhacophoridae, Anura) and a northwards range extension of the genus.

Knowledge of biodiversity before species become extinct is paramount to conservation, especially when the relevant species are far from their expected distribution and, thus, likely overlooked. Here, we describe a new Kurixalus species corresponding to a range extension of Kurixalus on the Asian mainland, with the closest population in Taiwan. The species diverged from its closest relative during the Late Pliocene to Pleistocene, ca. 3.06 Mya (HPD 95%: 5.82-0.01), based on calibrations with a relaxed clock species tree of unlinked mtDNA 12S rRNA and nuclear DNA TYR. The status of the newly-described species is also supported by a divergence in call properties and morphometrics. We named the species described here as Kurixalusinexpectatus sp. nov. due to the nature of the discovery, as well as the adjunct distribution of the species relative to its closest congeners. The species was found in Zhejiang Province and it represents a range extension of 663 km for the Kurixalus genus.

Using the 2020 global pandemic as a springboard to highlight the need for amphibian conservation in eastern Asia.

Emerging infectious diseases are on the rise in many different taxa, including, among others, the amphibian batrachochytrids, the snake fungal disease and the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) virus, responsible for Coronavirus disease 2019 (COVID-19) in mammals. Following the onset of the pandemic linked to COVID-19, eastern Asia has shown strong leadership, taking actions to regulate the trade of potential vector species in several regions. These actions were taken in response to an increase in public awareness, and the need for a quick reaction to mitigate against further pandemics. However, trade restrictions rarely affect amphibians, despite the risk of pathogen transmission, directly, or indirectly through habitat destruction and the loss of vector consumption. Thus, species that help alleviate the risk of zoonoses or provide biological control are not protected. Hence, in view of the global amphibian decline and the risk of zoonoses, we support the current wildlife trade regulations and support measures to safeguard wildlife from overexploitation. The current period of regulation overhaul should be used as a springboard for amphibian conservation. To mitigate risks, we suggest the following stipulations specifically for amphibians. I) Restrictions to amphibian farming in eastern Asia, in relation to pathogen transmission and the establishment of invasive species. II) Regulation of the amphibian pet trade, with a focus on potential vector species. III) Expansion of the wildlife trade ban, to limit the wildlife-human-pet interface. The resulting actions will benefit both human and wildlife populations, as they will lead to a decrease in the risk of zoonoses and better protection of the environment. SIGNIFICANCE STATEMENT: There is an increasing number of emerging infectious diseases impacting all species, including amphibians, reptiles and mammals. The latest threat to humans is the virus responsible for COVID-19, and the resulting pandemic. Countries in eastern Asia have taken steps to regulate wildlife trade and prevent further zoonoses thereby decreasing the risk of pathogens arising from wild species. However, as amphibians are generally excluded from regulations we support specific trade restrictions: I) Restrictions to amphibian farming; II) regulation of the amphibian pet trade; III) expansion of the wildlife trade ban. These restrictions will benefit both human and wildlife populations by decreasing the risks of zoonoses and better protecting the environment.

Impact of the Mid-Pleistocene Revolution and Anthropogenic Factors on the Dispersion of Asian Black-Spined Toads (Duttaphrynus melanostictus).

Divergence-time estimation critically improves the understanding of biogeography processes underlying the distribution of species, especially when fossil data is not available. We hypothesise that the Asian black-spined toad, Duttaphrynus melanostictus, expanded into the Eastern Indomalaya following the Quaternary glaciations with the subsequent colonisation of new landscapes during the Last Glacial Maximum. Divergence dating inferred from 364 sequences of mitochondrial tRNAGly ND3 supported the emergence of a common ancestor to the three D. melanostictus clades around 1.85 (±0.77) Ma, matching with the Lower to Mid-Pleistocene transition. Duttaphrynus melanostictus then dispersed into Southeast Asia from the central Indo-Pacific and became isolated in the Southern Sundaic and Wallacea regions 1.43 (±0.10) Ma through vicariance as a result of sea level oscillations. The clade on the Southeast Asian mainland then colonised the peninsula from Myanmar to Vietnam and expanded towards Southeastern China at the end of the Mid-Pleistocene Revolution 0.84 (±0.32) Ma. Population dynamics further highlight an expansion of the Southeast Asian mainland population towards Taiwan, the Northeastern edge of the species' range after the last interglacial, and during the emergence of the Holocene human settlements around 7000 BP. Thus, the current divergence of D. melanostictus into three segregated clades was mostly shaped by Quaternary glaciations, followed by natural dispersion events over land bridges and accelerated by anthropogenic activities.

Yellow sea mediated segregation between North East Asian Dryophytes species.

While comparatively few amphibian species have been described on the North East Asian mainland in the last decades, several species have been the subject of taxonomical debates in relation to the Yellow sea. Here, we sampled Dryophytes sp. treefrogs from the Republic of Korea, the Democratic People's Republic of Korea and the People's Republic of China to clarify the status of this clade around the Yellow sea and determine the impact of sea level change on treefrogs' phylogenetic relationships. Based on genetics, call properties, adult morphology, tadpole morphology and niche modelling, we determined the segregated status species of D. suweonensis and D. immaculatus. We then proceeded to describe a new treefrog species, D. flaviventris sp. nov., from the central lowlands of the Republic of Korea. The new species is geographically segregated from D. suweonensis by the Chilgap mountain range and known to occur only in the area of Buyeo, Nonsan and Iksan in the Republic of Korea. While the Yellow sea is the principal element to the current isolation of the three clades, the paleorivers of the Yellow sea basin are likely to have been the major factor for the divergences within this clade. We recommend conducting rapid conservation assessments as these species are present on very narrow and declining ranges.

Parentage of overlapping offspring of an arboreal-breeding frog with no nest defense: implications for nest site selection and reproductive strategy.

Overlapping offspring occurs when eggs are laid in a nest containing offspring from earlier reproduction. Earlier studies showed that the parentage is not always obvious due to difficulties in field observation and/or alternative breeding tactics. To unveil the parentage between overlapping offspring and parents is critical in understanding oviposition site selection and the reproductive strategies of parents. Amplectant pairs of an arboreal-breeding frog, Kurixalus eiffingeri, lay eggs in tadpole-occupied nests where offspring of different life stages (embryos and tadpoles) coexist. We used five microsatellite DNA markers to assess the parentage between parents and overlapping offspring. We also tested the hypothesis that the male or female frog would breed in the same breeding site because of the scarcity of nest sites. Results showed varied parentage patterns, which may differ from the phenomenon of overlapping egg clutches reported earlier. Parentage analyses showed that only 58 and 25% of the tadpole-occupied stumps were reused by the same male and female respectively, partially confirming our prediction. Re-nesting by the same individual was more common in males than females, which is most likely related to the cost of tadpole feeding and/or feeding schemes of females. On the other hand, results of parentage analyses showed that about 42 and 75% of male and female respectively bred in tadpole-occupied stumps where tadpoles were genetically unrelated. Results of a nest-choice experiment revealed that 40% of frogs chose tadpole-occupied bamboo cups when we presented identical stumps, without or with tadpoles, suggesting that the habitat saturation hypothesis does not fully explain why frogs used the tadpole-occupied stumps. Several possible benefits of overlapping offspring with different life stages were proposed. Our study highlights the importance of integrating molecular data with field observations to better understand the reproductive biology and nest site selection of anuran amphibians.

Short amplexus duration in a territorial anuran: a possible adaptation in response to male-male competition.

Mating duration is a reproductive behaviour that can impact fertilization efficiency and offspring number. Previous studies of factors influencing the evolution of mating duration have focused on the potential role of internal sperm competition as an underlying source of selection; most of these studies have been on invertebrates. For vertebrates with external fertilization, such as fishes and frogs, the sources of selection acting on mating duration remain largely unknown due, in part, to the difficulty of observing complete mating behaviours in natural conditions. In this field study, we monitored breeding activity in a population of the territorial olive frog, Rana adenopleura, to identify factors that affect the duration of amplexus. Compared with most other frogs, amplexus was short, lasting less than 11 min on average, which included about 8 min of pre-oviposition activity followed by 3 min of oviposition. We evaluated the relationship between amplexus duration and seven variables: male body size, male condition, operational sex ratio (OSR), population size, clutch size, territory size, and the coverage of submerged vegetation in a male's territory. We also investigated the influence of these same variables, along with amplexus duration, on fertilization rate. Amplexus duration was positively related with clutch size and the degree of male-bias in the nightly OSR. Fertilization rate was directly related to male body size and inversely related to amplexus duration. Agonistic interactions between males in amplexus and intruding, unpaired males were frequent. These interactions often resulted in mating failure, prolonged amplexus duration, and reduced fertilization rates. Together, the pattern of our findings indicates short amplexus duration in this species may be an adaptive reproductive strategy whereby males attempt to reduce the risks of mating and fertilization failures and territory loss resulting from male-male competition.