Correction: Molecular species delimitation of shrub frogs of the genus Pseudophilautus (Anura, Rhacophoridae).

[This corrects the article DOI: 10.1371/journal.pone.0258594.].

Emerging threat of ranavirus: prevalence, genetic diversity, and climatic drivers of Ranavirus (Iridoviridae) in ectothermic vertebrates of Asia.

Introduction: Ranavirus disease, caused by viruses within the genus Ranavirus (Iridoviridae), is considered a globally emerging infectious disease linked to mass mortality events in both wild and cultured ectothermic vertebrates. Surveillance work is, however, limited in Asia hence prevalence and the dynamics of the disease remain poorly understood. To understand disease burden and the potential biotic and abiotic drivers in southern China region, we conducted a systematic surveillance of the ranavirus across Guangxi Zhuang Autonomous region (GAR). Methods: For this, we used a multifaceted approach involving screening of amphibians and other potential hosts, diagnostic tests, phylogenetic analyses, prevalence estimation, co-infection assessments, and climatic niche analyses. Over one thousand individuals were sampled across 25 sampling sites. Results: We found ninety-two individuals from 18 species of ectothermic vertebrates to be infected with ranavirus. Two lineages were responsible - Rana nigromaculata ranavirus and tiger frog virus were identified using phylogenetic analysis based on the major capsid protein (MCP) gene fragment. Out of these two lineages, the presence of tiger frog virus is rare as we came across only one case. We also found evidence of a co-infection with ranavirus and Batrachochytrium dendrobatidis that can be highly detrimental to host populations; possibly the first such documentation in Asia. Our niche modelling analysis suggests that precipitation seasonality plays an important role in ranavirus prevalence in GAR - southwestern, southeastern, central and northeastern regions of GAR can be considered to be optimum habitats for ranaviruses. Infection rates in wild frog species have reached 100% in some areas, even in nature reserves. Discussion: Our research also indicates that culture facilities and pet markets are frequently infected, serving as likely vectors for the regional and global spread of ranaviruses. The knowledge generated suggests the need for systematic surveillance, stringent biosecurity measures, and control of international animal trade to prevent further transmission and protection of biodiversity and aquaculture industries across Asia.

Associations of Batrachochytrium dendrobatidis with skin bacteria and fungi on Asian amphibian hosts.

Amphibian skin harbors microorganisms that are associated with the fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes chytridiomycosis, one of the most significant wildlife diseases known. This pathogen originated in Asia, where diverse Bd lineages exist; hence, native amphibian hosts have co-existed with Bd over long time periods. Determining the nuances of this co-existence is crucial for understanding the prevalence and spread of Bd from a microbial context. However, associations of Bd with the natural skin microbiome remain poorly understood for Asian hosts, especially in relation to skin-associated fungi. We used 16 S rRNA and fungal internal transcribed spacer (ITS) gene sequencing to characterize the skin microbiome of four native Asian amphibian species and examined the relationships between Bd infection and their skin bacterial and fungal communities; we also analyzed the correlates of the putative anti-Bd bacteria. We show that both skin bacterial and fungal community structure and composition had significant associations with infection status (Bd presence/absence) and infection intensity (frequency of Bd sequence reads). We also found that the putative anti-Bd bacterial richness was correlated with Bd infection status and infection intensity, and observed that the relative abundance of anti-Bd bacteria roughly correspond with changes in both Bd prevalence and mean infection intensity in populations. Additionally, the microbial co-occurrence network of infected frogs was significantly different from that of uninfected frogs that were characterized by more keystone nodes (connectors) and larger proportions in correlations between bacteria, suggesting stronger inter-module bacterial interactions. These results indicate that the mutual effects between Bd and skin-associated microbiome, including the interplay between bacteria and fungi, might vary with Bd infection in susceptible amphibian species. This knowledge will help in understanding the dynamics of Bd from a microbial perspective, potentially contributing to mitigate chytridiomycosis in other regions of the world.

Ecological Barriers for an Amphibian Pathogen: A Narrow Ecological Niche for Batrachochytrium salamandrivorans in an Asian Chytrid Hotspot.

The chytrid fungal pathogens Batrachochytrium salamandrivorans (Bsal) and B. dendrobatidis (Bd) are driving amphibian extinctions and population declines worldwide. As their origins are believed to be in East/Southeast Asia, this region is crucial for understanding their ecology. However, Bsal screening is relatively limited in this region, particularly in hotspots where Bd lineage diversity is high. To address this gap, we conducted an extensive Bsal screening involving 1101 individuals from 36 amphibian species, spanning 17 natural locations and four captive facilities in the biodiversity-rich Guangxi Zhuang Autonomous Region (GAR). Our PCR assays yielded unexpected results, revealing the complete absence of Bsal in all tested samples including 51 individuals with Bd presence. To understand the potential distribution of Bsal, we created niche models, utilizing existing occurrence records from both Asia and Europe. These models estimated potential suitable habitats for Bsal largely in the northern and southwestern parts of the GAR. Although Bsal was absent in our samples, the niche models identified 10 study sites as being potentially suitable for this pathogen. Interestingly, out of these 10 sites, Bd was detected at 8. This suggests that Bsal and Bd could possibly co-exist in these habitats, if Bsal were present. Several factors seem to influence the distribution of Bsal in Asia, including variations in temperature, local caudate species diversity, elevation, and human population density. However, it is climate-related factors that hold the greatest significance, accounting for a notable 60% contribution. The models propose that the specific climatic conditions of arid regions, primarily seen in the GAR, play a major role in the distribution of Bsal. Considering the increased pathogenicity of Bsal at stable and cooler temperatures (10-15 °C), species-dependent variations, and the potential for seasonal Bd-Bsal interactions, we emphasize the importance of periodic monitoring for Bsal within its projected range in the GAR. Our study provides deeper insights into Bsal's ecological niche and the knowledge generated will facilitate conservation efforts in amphibian populations devastated by chytrid pathogens across other regions of the world.

The two chytrid pathogens of amphibians in Eurasia-climatic niches and future expansion.

BACKGROUND: Climate affects the thermal adaptation and distribution of hosts, and drives the spread of Chytridiomycosis-a keratin-associated infectious disease of amphibians caused by the sister pathogens Batrachochytrium dendrobatidi (Bd) and B. salamandrivorans (Bsal). We focus on their climate-pathogen relationships in Eurasia, the only region where their geographical distributions overlap. Eurasia harbours invaded and native areas of both pathogens and the natural habitats where they co-exist, making it an ideal region to examine their environmental niche correlations. Our understanding of how climate change will affect their distribution is broadened by the differences in climate correlates and niche characteristics between Bd and Bsal in Asia and Europe. This knowledge has potential conservation implications, informing future spread of the disease in different regions. RESULTS: We quantified the environmental niche overlap between Bd and Bsal in Eurasia using niche analyses. Results revealed partial overlap in the niche with a unique 4% of non-overlapping values for Bsal, suggesting segregation along certain climate axes. Bd tolerates higher temperature fluctuations, while Bsal requires more stable, lower temperature and wetter conditions. Projections of their Realized Climatic Niches (RCNs) to future conditions show a larger expansion of suitable ranges (SRs) for Bd compared to Bsal in both Asia and Europe, with their centroids shifting in different directions. Notably, both pathogens' highly suitable areas in Asia are expected to shrink significantly, especially under the extreme climate scenarios. In Europe, they are expected to expand significantly. CONCLUSIONS: Climate change will impact or increase disease risk to amphibian hosts, particularly in Europe. Given the shared niche space of the two pathogens across available climate gradients, as has already been witnessed in Eurasia with an increased range expansion and niche overlap due to climate change, we expect that regions where Bsal is currently absent but salamanders are present, and where Bd is already prevalent, may be conducive for the spread of Bsal.

Pseudophilautus pardus, a junior synonym of P. viridis (Amphibia: Anura: Rhacophoridae).

Pseudophilautus pardus, was first described in 2007, based on a single specimen collected in Sri Lanka during the 19th century. Its absence in recent surveys suggested that the species was extinct. The distinctive spotted dorsal coloration, together with other morphological features, was used to distinguish it from congeners. In 2013, we discovered a specimen resembling the holotype of P. pardus in Samanala Strict Nature Reserve. A DNA analysis based on the 16S rRNA mt-DNA locus recovered a low uncorrected pairwise genetic distance of 0.77-0.96% between the recent example of 'P. pardus' and a congener, P. viridis. Molecular species delimitation methods suggest P. pardus and P. virids to be a single operational taxonomic unit. Morphological analyses using the freshly collected specimen, together with numerous photographic records show P. viridis to be a highly polychromatic species within which the coloration observed in P. pardus too occurs, though rarely. We conclude that P. pardus is a junior synonym of P. viridis.

Adapting to Novel Environments Together: Evolutionary and Ecological Correlates of the Bacterial Microbiome of the World's Largest Cavefish Diversification (Cyprinidae, Sinocyclocheilus).

The symbiosis between a host and its microbiome is essential for host fitness, and this association is a consequence of the host's physiology and habitat. Sinocyclocheilus, the largest cavefish diversification of the world, an emerging multi-species model system for evolutionary novelty, provides an excellent opportunity for examining correlates of host evolutionary history, habitat, and gut-microbial community diversity. From the diversification-scale patterns of habitat occupation, major phylogenetic clades (A-D), geographic distribution, and knowledge from captive-maintained Sinocyclocheilus populations, we hypothesize habitat to be the major determinant of microbiome diversity, with phylogeny playing a lesser role. For this, we subject environmental water samples and fecal samples (representative of gut-microbiome) from 24 Sinocyclocheilus species, both from the wild and after being in captivity for 6 months, to bacterial 16S rRNA gene profiling using Illumina sequencing. We see significant differences in the gut microbiota structure of Sinocyclocheilus, reflective of the three habitat types; gut microbiomes too, were influenced by host-related factors. There is no significant association between the gut microbiomes and host phylogeny. However, there is some microbiome related structure at the clade level, with the most geographically distant clades (A and D) being the most distinct, and the two overlapping clades (B and C) showing similarities. Microbes inhabiting water were not a cause for significant differences in fish-gut microbiota, but water quality parameters were. Transferring from wild to captivity, the fish microbiomes changed significantly and became homogenized, signifying plastic changes and highlighting the importance of environmental factors (habitat) in microbiome community assembly. The core microbiome of this group, at higher taxonomic scale, resembled that of other teleost fishes. Our results suggest that divergent natural environments giving rise to evolutionary novelties underlying host adaptations, also includes the microbiome of these fishes.

The role of climate and islands in species diversification and reproductive-mode evolution of Old World tree frogs.

Large diversifications of species are known to occur unevenly across space and evolutionary lineages, but the relative importance of their driving mechanisms, such as climate, ecological opportunity and key evolutionary innovations (KEI), remains poorly understood. Here, we explore the remarkable diversification of rhacophorid frogs, which represent six percent of global amphibian diversity, utilize four distinct reproductive modes, and span a climatically variable area across mainland Asia, associated continental islands, and Africa. Using a complete species-level phylogeny, we find near-constant diversification rates but a highly uneven distribution of species richness. Montane regions on islands and some mainland regions have higher phylogenetic diversity and unique assemblages of taxa; we identify these as cool-wet refugia. Starting from a centre of origin, rhacophorids reached these distant refugia by adapting to new climatic conditions ('niche evolution'-dominant), especially following the origin of KEIs such as terrestrial reproduction (in the Late Eocene) or by dispersal during periods of favourable climate ('niche conservatism'-dominant).

Evolving in the darkness: Phylogenomics of Sinocyclocheilus cavefishes highlights recent diversification and cryptic diversity.

Troglomorphism-any morphological adaptation enabling life to the constant darkness of caves, such as loss of pigment, reduced eyesight or blindness, over-developed tactile and olfactory organs-has long intrigued biologists. However, inferring the proximate and ultimate mechanisms driving the evolution of troglomorphism (stygomorphism) in freshwater fish requires a sound understanding of the evolutionary relationships between surface and stygomorphic lineages. We use Restriction Site Associated DNA Sequencing (RADseq) to better understand the evolution of the Sinocyclocheilus fishes of China. With a remarkable array of derived stygomorphic traits, they comprise the largest cavefish diversification in the world, emerging as a multi-species model system to study evolutionary novelty. We sequenced a total of 120 individuals throughout the Sinocyclocheilus distribution. The data comprised a total of 646,497 bp per individual, including 4378 loci and 67,983 SNPs shared across a minimum of 114 individuals at a given locus. Phylogenetic analyses using either the concatenated RAD loci (RAxML) or the SNPs under a coalescent model (SVDquartets, SNAPP) showed a high degree of congruence with similar topologies and high node support (>95 for most nodes in the phylogeny). The major clades recovered conform to a pattern previously established using Sanger-based mt-DNA sequences, with a few notable exceptions. We now recognize six major clades in this group, elevating the blind cavefish S. tianlinensis and the micro-eyed S. microphthalmus as two new distinct clades due to their deep divergence from other clades. PCA plots of the SNP data also support the recognition of six major clusters of species congruent with the identified clades in ordination space. A Bayes factor delimitation (BFD) analysis showed support for 21 species, recognizing 19 previously described species and two putative new cryptic ones. Two species whose identities were previously disputed, S. furcodorsalis and S. tianeensis, are supported here as distinct species. In addition, our multi-species calibrated tree in SNAPP suggests that the genus Sinocyclocheilus originated around 10.16 Mya, with most speciation events occurring in the last 2 Mya, likely favored by the uplift of the Qinghai-Tibetan Plateau and cave occupation induced by climate-driven aridification during this period. These results provide a firm basis for future comparative studies on the evolution of Sinocyclocheilus and its adaptations to cave life.

Patterns of infection, origins, and transmission of ranaviruses among the ectothermic vertebrates of Asia.

Ranaviral infections, a malady of ectothermic vertebrates, are becoming frequent, severe, and widespread, causing mortality among both wild and cultured species, raising odds of species extinctions and economic losses. This increase in infection is possibly due to the broad host range of ranaviruses and the transmission of these pathogens through regional and international trade in Asia, where outbreaks have been increasingly reported over the past decade. Here, we focus attention on the origins, means of transmission, and patterns of spread of this infection within the region. Infections have been recorded in both cultured and wild populations in at least nine countries/administrative regions, together with mass die-offs in some regions. Despite the imminent seriousness of the disease in Asia, surveillance efforts are still incipient. Some of the viral strains within Asia may transmit across host-taxon barriers, posing a significant risk to native species. Factors such as rising temperatures due to global climate change seem to exacerbate ranaviral activity, as most known outbreaks have been recorded during summer; however, data are still inadequate to verify this pattern for Asia. Import risk analysis, using protocols such as Pandora+, pre-border pathogen screening, and effective biosecurity measures, can be used to mitigate introduction of ranaviruses to uninfected areas and curb transmission within Asia. Comprehensive surveillance using molecular diagnostic tools for ranavirus species and variants will help in understanding the prevalence and disease burden in the region. This is an important step toward conserving native biodiversity and safeguarding the aquaculture industry.

Molecular species delimitation of shrub frogs of the genus Pseudophilautus (Anura, Rhacophoridae).

Sri Lanka is an amphibian hotspot of global significance. Its anuran fauna is dominated by the shrub frogs of the genus Pseudophilautus. Except for one small clade of four species in Peninsular India, these cool-wet adapted frogs, numbering some 59 extant species, are distributed mainly across the montane and lowland rain forests of the island. With species described primarily by morphological means, the diversification has never yet been subjected to a molecular species delimitation analysis, a procedure now routinely applied in taxonomy. Here we test the species boundaries of Pseudophilautus in the context of the phylogenetic species concept (PSC). We use all the putative species for which credible molecular data are available (nDNA-Rag-1; mt-DNA- 12S rRNA, 16S rRNA) to build a well resolved phylogeny, which is subjected to species delimitation analyses. The ABGD, bPTP, mPTP and bGMYC species delimitation methods applied to the 16S rRNA frog barcoding gene (for all species), 12S rRNA and Rag-1 nDNA grouped P. procax and P. abundus; P. hallidayi and P. fergusonianus; P. reticulatus and P. pappilosus; P. pleurotaenia and P. hoipolloi; P. hoffmani and P. asankai; P. silvaticus and P. limbus; P. dilmah and P. hankeni; P. fulvus and P. silus.. Surprisingly, all analyses recovered 14 unidentified potential new species as well. The geophylogeny affirms a distribution across the island's aseasonal 'wet zone' and its three principal hill ranges, suggestive of allopatric speciation playing a dominant role, especially between mountain masses. Among the species that are merged by the delimitation analyses, a pattern leading towards a model of parapatric speciation emerges-ongoing speciation in the presence of gene flow. This delimitation analysis reinforces the species hypotheses, paving the way to a reasonable understanding of Sri Lankan Pseudophilautus, enabling both deeper analyses and conservation efforts of this remarkable diversification. http://zoobank.org/urn:lsid:zoobank.org:pub:DA869B6B-870A-4ED3-BF5D-5AA3F69DDD27.

Evolution in Sinocyclocheilus cavefish is marked by rate shifts, reversals, and origin of novel traits.

BACKGROUND: Natural model systems are indispensable for exploring adaptations in response to environmental pressures. Sinocyclocheilus of China, the most diverse cavefish clade in the world (75 species), provide unique opportunities to understand recurrent evolution of stereotypic traits (such as eye loss and sensory expansion) in the context of a deep and diverse phylogenetic group. However, they remain poorly understood in terms of their morphological evolution. Therefore, we explore key patterns of morphological evolution, habitat utilization and geographic distribution in these fishes. RESULTS: We constructed phylogenies and categorized 49 species based on eye-related condition (Blind, Micro-eyed, and Normal-eyed), habitat types (Troglobitic-cave-restricted; Troglophilic-cave-associated; Surface-outside caves) and existence of horns. Geometric-morphometric analyses show Normal-eyed morphs with fusiform shapes segregating from Blind/Micro-eyed deeper bodied morphs along the first principal-component axis; second axis accounts for shape complexity related to horns. The body shapes showed a significant association with eye-related condition and horn, but not habitat types. Ancestral reconstructions suggest at least three independent origins of Blind morphs, each with different levels of modification in relation to their ancestral Normal-eyed morphs; Sinocyclocheilus are also pre-adapted for cave dwelling. Our geophylogeny shows an east-to-west diversification spanning Pliocene and Pleistocene, with early-diversifying Troglobitic species dominating subterranean habitats of karstic plains whereas predominantly Surface forms inhabit hills to the west. Evolutionary rates analyses suggest that lineages leading to Blind morphs were characterized by significant rate shifts, such as a slowdown in body size evolution and a 5-20 fold increase in rate of eye regression, possibly explained by limited resource availability. Body size and eye size have undergone reversals, but not horns, a trait entailing considerable time to form. CONCLUSIONS: Sinocyclocheilus occupied cave habitats in response to drying associated with aridification of China during late Miocene and the Pliocene. The prominent cave-adaptations (eye-regression, horn-evolution) occur in clades associated with the extensive subterranean cave system in Guangxi and Guizhou provinces. Integration of morphology, phylogeny, rate analyses, molecular-dating and distribution show not only several remarkable patterns of evolution, but also interesting exceptions to these patterns signifying the diversification of Sinocyclocheilus as an invaluable model system to explore evolutionary novelty.