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.

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.

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 phylogeny and phylogeography of the freshwater-fish genus Pethia (Teleostei: Cyprinidae) in Sri Lanka.

BACKGROUND: Sri Lanka is a continental island separated from India by the Palk Strait, a shallow-shelf sea, which was emergent during periods of lowered sea level. Its biodiversity is concentrated in its perhumid south-western 'wet zone'. The island's freshwater fishes are dominated by the Cyprinidae, characterized by small diversifications of species derived from dispersals from India. These include five diminutive, endemic species of Pethia (P. bandula, P. cumingii, P. melanomaculata, P. nigrofasciata, P. reval), whose evolutionary history remains poorly understood. Here, based on comprehensive geographic sampling, we explore the phylogeny, phylogeography and morphological diversity of the genus in Sri Lanka. RESULTS: The phylogenetic analyses, based on mitochondrial and nuclear loci, recover Sri Lankan Pethia as polyphyletic. The reciprocal monophyly of P. bandula and P. nigrofasciata, and P. cumingii and P. reval, is not supported. Pethia nigrofasciata, P. cumingii, and P. reval show strong phylogeographic structure in the wet zone, compared with P. melanomaculata, which ranges across the dry and intermediate zones. Translocated populations of P. nigrofasciata and P. reval in the Central Hills likely originate from multiple sources. Morphological analyses reveal populations of P. nigrofasciata proximal to P. bandula, a narrow-range endemic, to have a mix of characters between the two species. Similarly, populations of P. cumingii in the Kalu basin possess orange fins, a state between the red-finned P. reval from Kelani to Deduru and yellow-finned P. cumingii from Bentara to Gin basins. CONCLUSIONS: Polyphyly in Sri Lankan Pethia suggests two or three colonizations from mainland India. Strong phylogeographic structure in P. nigrofasciata, P. cumingii and P. reval, compared with P. melanomaculata, supports a model wherein the topographically complex wet zone harbors greater genetic diversity than the topographically uniform dry-zone. Mixed morphological characters between P. bandula and P. nigrofasciata, and P. cumingii and P. reval, and their unresolved phylogenies, may suggest recent speciation scenarios with incomplete lineage sorting, or hybridization.

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.

Undocumented translocations spawn taxonomic inflation in Sri Lankan fire rasboras (Actinopterygii, Cyprinidae).

A recent (2013) taxonomic review of the freshwater-fish genus Rasboroides, which is endemic to Sri Lanka, showed it to comprise four species: R. vaterifloris, R. nigromarginatus, R. pallidus and R. rohani. Here, using an integrative-taxonomic analysis of morphometry, meristics and mitochondrial DNA sequences of cytochrome b (cytb) and cytochrome oxidase subunit 1 (coi), we show that R. nigromarginatus is a synonym of R. vaterifloris, and that R. rohani is a synonym of R. pallidus. The creation and recognition of unnecessary taxa ('taxonomic inflation') was in this case a result of selective sampling confounded by a disregard of allometry. The population referred to R. rohani in the Walawe river basin represents an undocumented trans-basin translocation of R. pallidus, and a translocation into the Mahaweli river of R. vaterifloris, documented to have occurred ca 1980, in fact involves R. pallidus. A shared haplotype suggests the latter introduction was likely made from the Bentara river basin and not from the Kelani, as claimed. To stabilize the taxonomy of these fishes, the two valid species, R. vaterifloris and R. pallidus, are diagnosed and redescribed, and their distributions delineated. We draw attention to the wasteful diversion of conservation resources to populations resulting from undocumented translocations and to taxa resulting from taxonomic inflation. We argue against translocations except where mandated by a conservation emergency, and even then, only when supported by accurate documentation.