Spatio-temporal analysis of toxigenic genes expression in the growing Bufo gargarizans based on RNA sequencing data.

BACKGROUND: Bufo gargarizans Cantor, a widely distributed amphibian species in Asia, produces and releases toxins through its retroauricular and granular glands. Although various tissues have been sequenced, the molecular mechanisms underlying the toxin production remain unclear. To elucidate these mechanisms, abdominal skin (non-toxic secretory glands) and retroauricular gland (toxic secreting glands) samples were collected at different time points (3, 6, 12, 24, and 36 months) for RNA sequencing (RNA-seq) and analysis. RESULTS: In comparison to the S group during the same period, a total of 3053, 3026, 1516, 1028, and 2061 differentially expressed genes (DEGs) were identified across five developmental stages. Gene Ontology (GO) analysis revealed that DEGs were primarily enriched in biological processes including cellular processes, single-organism processes, metabolic processes, and biological regulation. In terms of cellular components, the DEGs were predominantly localized in the cell and cell parts, whereas molecular function indicated significant enrichment in binding and catalytic activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the metabolism and synthesis of various substances, such as lipid metabolism, cofactor and vitamin metabolism, tryptophan metabolism, steroid biosynthesis, and primary bile acid biosynthesis, were accompanied by the development of toads. Additionally, using trend analysis, we discovered candidate genes that were upregulated in the retroauricular glands during development, and the abundance of these genes in the abdominal skin was extremely low. Finally, we identified 26 genes that are likely to be involved in toxin production and that are likely to be involved in toxin anabolism. CONCLUSION: Overall, these results provide new insights into the genes involved in toxin production in B. gargarizans, which will improve our understanding of the molecular mechanisms underlying toxigenic gene expression.

Field-based molecular detection of Batrachochytrium dendrobatidis in critically endangered Atelopus toads and aquatic habitats in Ecuador.

Batrachochytrium dendrobatidis (Bd) is a lethal fungal species that parasitizes vertebrates and is associated with the worldwide decline of amphibian populations. The development of sensitive, rapid detection methods, particularly DNA-based techniques, is critical for effective management strategies. This study evaluates the efficacy of DNA extraction and a portable PCR device in a mountable field laboratory setup for detecting Bd near the habitats of three critically endangered Atelopus toad species in Ecuador. We collected skin swabs from Atelopus balios, A. nanay, and A. bomolochos, and environmental DNA (eDNA) samples from streams in Andean and coastal regions of Ecuador. For eDNA, a comparison was made with duplicates of the samples that were processed in the field and in a standard university laboratory. Our findings revealed Bd detection in eDNA and swabs from 6 of 12 water samples and 10 of 12 amphibian swab samples. The eDNA results obtained in the field laboratory were concordant with those obtained under campus laboratory conditions. These findings highlight the potential of field DNA-based monitoring techniques for detecting Bd in amphibian populations and their aquatic habitats, particularly in remote areas. Furthermore, this research aligns with the National Action Plan for the Conservation of Ecuadorian Amphibians and contributes to the global effort to control this invasive and deadly fungus.

Using genome-wide data to ascertain taxonomic status and assess population genetic structure for Houston toads (Bufo [= Anaxyrus] houstonensis).

The Houston toad (Bufo [= Anaxyrus] houstonensis) is an endangered amphibian with a small geographic range. Land-use changes have primarily driven decline in B. houstonensis with population supplementation predominant among efforts to reduce its current extinction risk. However, there has been historic uncertainty regarding the evolutionary and conservation significance of B. houstonensis. To this end, we used 1170 genome-wide nuclear DNA markers to examine phylogenetic relationships between our focal taxon, representatives of the Nearctic B. americanus group, and B. nebulifer, a sympatric Middle American species. Phylogenetic analyses indicate B. houstonensis is a taxon that is distinct from B. americanus. We corroborated such genetic distinctiveness with an admixture analysis that provided support for recent reproductive isolation between B. americanus and B. houstonensis. However, ABBA-BABA tests for ancient admixture indicated historic gene flow between Nearctic species while no signal of historic gene flow was detected between Nearctic and Middle-American species. We used an admixture analysis to recognize four Management Units (MU) based on observed genetic differentiation within B. houstonensis and recommend captive propagation, population supplementation, and habitat restoration efforts specific to each MU. Our results re-affirm the evolutionary novelty of an endangered relict.

[Molecular identification of animal species of Bufonis Venenum from secretions].

The animal species is one of the key factors affecting the quality of Bufonis Venenum. The quality of Bufonis Venenum derived from Bufo bufo gargarizans is significantly higher than that from B. melanostictus. Since Bufonis Venenum is from secretions, the conventional identification methods are difficult to identify the animal species due to the lack of the appearance and morphology of the animals. The rapid development of molecular identification technology has provided new methods for the identification of Bufonis Venenum. However, because of the low content and serve degradation of residual DNA in secretions, the research on the molecular identification of Chinese medicinal materials from secretions remains to be carried out. To understand the animal species of Bufonis Venenum, this study collected 83 samples of Bufonis Venenum, including 7 commercially available samples, 5 reference medicinal materials, and 71 animal samples from which Bufonis Venenum was prepared according to the method in the 2020 edition of the Chinese Pharmacopoeia. Different DNA extraction methods were used and compared, and the mitochondrial 16S rRNA gene fragments were amplified, on the basis of which the phylogenetic trees were built. Finally, molecular identification of the animal species of the samples was performed. The results showed that the DNA extracted from Bufonis Venenum by the reagent kit had good quality, and 16S rRNA sequences were successfully amplified from 80 out of the 83 samples. In addition, 71 16S rRNA sequences of the animal species of Bufonis Venenum were downloaded from GenBank. The phylogenetic trees constructed based on the neighbor-joining(NJ) method and the Bayesian inference(BI) method showed that the samples derived from B. bufo gargarizans and B. melanostictus were clustered into separate monophyletic clades, with the support of 100%(NJ) and 1.00(BI), respectively. The animal species of both commercially available samples and reference medicinal materials were B. bufo gargarizans. In conclusion, DNA can be extracted from Bufonis Venenum derived from secretions, and the 16S rRNA gene sequences can be amplified, which can be used for molecular identification of the animal species of Bufonis Venenum. The findings provide a reference for the quality control of Bufonis Venenum and the identification of animal species of medicinal materials derived from secretions.

Batrachochytrium dendrobatidis strain affects transcriptomic response in liver but not skin in latitudinal populations of the common toad (Bufo bufo).

Batrachochytrium dendrobatidis (Bd) is a fungal pathogen that has decimated amphibian populations worldwide for several decades. We examined the changes in gene expression in response to Bd infection in two populations of the common toad, Bufo bufo, in a laboratory experiment. We collected B. bufo eggs in southern and northern Sweden, and infected the laboratory-raised metamorphs with two strains of the global panzoonotic lineage Bd-GPL. Differential expression analysis showed significant differences between infected and control individuals in both liver and skin. The skin samples showed no discernible differences in gene expression between the two strains used, while liver samples were differentiated by strain, with one of the strains eliciting no immune response from infected toads. Immune system genes were overexpressed in skin samples from surviving infected individuals, while in liver samples the pattern was more diffuse. Splitting samples by population revealed a stronger immune response in northern individuals. Differences in transcriptional regulation between populations are particularly relevant to study in Swedish amphibians, which may have experienced varying exposure to Bd. Earlier exposure to this pathogen and subsequent adaptation or selection pressure may contribute to the survival of some populations over others, while standing genetic diversity in different populations may also affect the infection outcome.

Larval Body Shape and Eye Size Variation in Japanese Toads (Anura: Bufonidae: Bufo): An Ecological Implication for Lentic and Lotic Habitats.

Variation in morphological traits of anurans has evolved due to the pressures imposed by inhabiting different environments. The Japanese stream toad, Bufo torrenticola, breeds in running water, such as mountain streams, where the larvae grow. This lotic-breeding habit of B. torrenticola was suggested to have evolved from a lentic-breeding ancestor. Evolutionary shift of breeding habit from lentic- to lotic-breeding caused larval morphological changes to adapt to the stream habitat. However, morphological adaptation associated with the larval habitat of these three types of Japanese toads has not been explored well. In this study, we applied geometric morphometrics and distance measurements to compare body and eye characters among the tadpoles of three Japanese toads. The results showed that B. torrenticola has more dorsally and posteriorly positioned eyes, and a longer and wider rostrum because of having a larger mouth and more-developed oral muscles than its close relatives. These characters might be related to lotic lifestyle. Meanwhile, tadpoles of B. torrenticola and its sister taxon B. japonicus japonicus showed similar eye growth pattern, supporting their close phylogenetic relationship. Some of the lotic-adapted characters have also been reported in other lotic tadpoles, which is indicative of convergent evolution among stream-adapted tadpoles.

Population genetic structure and hybrid zone analyses for species delimitation in the Japanese toad (Bufo japonicus).

Hybridization following secondary contact may produce different outcomes depending on the extent to which genetic diversity and reproductive barriers have accumulated during isolation. The Japanese toad, Bufo japonicus, is distributed on the main islands of Japan. In the present study, we applied multiplexed inter-simple sequence repeat genotyping by sequencing to achieve the fine-scale resolution of the genetic cluster in B. j. japonicus and B. j. formosus. We also elucidated hybridization patterns and gene flow degrees across contact zones between the clusters identified. Using SNP data, we found four genetic clusters in B. j. japonicus and B. j. formosus and three contact zones of the cluster pairs among these four clusters. The two oldest diverged lineages, B. j. japonicus and B. j. formosus, formed a narrow contact zone consistent with species distinctiveness. Therefore, we recommend that these two subspecies be elevated to the species level. In contrast, the less diverged pairs of two clusters in B. j. japonicus and B. j. formosus, respectively, admixed over a hundred kilometers, suggesting that they have not yet developed strong reproductive isolation and need to be treated as conspecifics. These results will contribute to resolving taxonomic confusion in Japanese toads.

Conservation genomics of an endangered montane amphibian reveals low population structure, low genomic diversity and selection pressure from disease.

Wildlife diseases are a major global threat to biodiversity. Boreal toads (Anaxyrus [Bufo] boreas) are a state-endangered species in the southern Rocky Mountains of Colorado and New Mexico, and a species of concern in Wyoming, largely due to lethal skin infections caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). We performed conservation and landscape genomic analyses using single nucleotide polymorphisms from double-digest, restriction site-associated DNA sequencing in combination with the development of the first boreal toad (and first North American toad) reference genome to investigate population structure, genomic diversity, landscape connectivity and adaptive divergence. Genomic diversity (π = 0.00034-0.00040) and effective population sizes (Ne = 8.9-38.4) were low, likely due to post-Pleistocene founder effects and Bd-related population crashes over the last three decades. Population structure was also low, likely due to formerly high connectivity among a higher density of geographically proximate populations. Boreal toad gene flow was facilitated by low precipitation, cold minimum temperatures, less tree canopy, low heat load and less urbanization. We found >8X more putatively adaptive loci related to Bd intensity than to all other environmental factors combined, and evidence for genes under selection related to immune response, heart development and regulation and skin function. These data suggest boreal toads in habitats with Bd have experienced stronger selection pressure from disease than from other, broad-scale environmental variations. These findings can be used by managers to conserve and recover the species through actions including reintroduction and supplementation of populations that have declined due to Bd.

Regulation mechanisms underlying tail resorption in Bufo gargarizans metamorphosis.

Anurans have been excellent organisms for studying amphibian metamorphosis. Tail resorption is a remarkable event that occurs during amphibian metamorphosis. Although tail resorption has been previously studied in other anurans like Xenopus laevis and Rana chensinensis, there is no report on Bufo gargarizans. This paper thus explored the mechanism of tail resorption during metamorphosis in Bufo gargarizans tadpoles through some biological research methods. Histological results showed that the tail tissues of tadpoles gradually degraded as metamorphosis progressed. RNA sequencing analysis was performed to examine the expression level and functional enrichment of differentially expressed genes in the tail. In addition, we analyzed the mRNA expression levels of genes related to tail resorption by quantitative real-time polymerase chain reaction. We also speculated on three pathways that participate in the regulation of tail resorption based on the above results. The present study might provide a theoretical basis and novel insights for further research of complex molecular mechanisms of tail resorption in amphibians.

Mining and Characterization of Indolethylamine N-Methyltransferases in Amphibian Toad Bufo gargarizans.

Strong, psychedelic indolethylamines (IAAs) are typically present in trace amounts in the majority of species, but they build up significantly in the skin of amphibian toads, especially N-methylated 5-hydroxytryptamine (5-HT) analogues. However, there is no pertinent research on the investigation of indoleamine N-methyltransferase (INMT) in amphibians, nor is there any adequate information on the key amino acids that influence the activity of known INMTs from other species. Herein, we focused on Bufo toad INMT (BINMT) for the first time and preliminarily identified BINMT 1 from the transcriptomes of Bufo gargarizans active on tryptamine, 5-HT, and N-methyl-5-HT. We established the enzyme kinetic characteristics of BINMT 1 and identified the essential amino acids influencing its activity via molecular docking and site-directed mutagenesis. Subsequently, we carried out sequence alignment and phylogenetic tree analysis on 43 homologous proteins found in the genome of B. gargarizans with BINMT 1 as the probe and selected seven of them for protein expression and activity assays. It was found that only three proteins possessing the highest similarity to BINMT 1 had INMT activity. Our research unveils the binding residues of BINMT for 5-HT analogues for the first time and initiates the study of INMTs in amphibian toads, serving as a tentative reference for further study of BINMT and providing insight into the comprehension of BINMT's catalytic mechanism and its role in the biosynthesis of 5-HT analogues in Bufo toads. It also contributes to the expansion of the INMT library to help explore and explain interspecies evolution in the future.

Generic classification of Asian horned toads (Anura: Megophryidae: Megophryinae) and monograph of Chinese species.

The subfamily Megophryinae, as a representative batrachian group of the Oriental Realm and one of the most diverse groups of amphibians, has attracted considerable attention due to continued conjecture regarding its generic classification and failure to reach a satisfactory consensus. China boasts the richest diversity of Asian horned toads, containing some two thirds of the total species cataloged. However, most species have a complicated taxonomic history, resulting in multiple misidentifications. As such, an overall clarification of historical records and regional checklists is required. In the current investigation, we established the phylogeny of the Asian horned toads and performed detailed examinations with redefinitions of several important morphological traits. Based on the phylogenetic relationships and morphological differences, we propose a new ten-genus classification for the Asian horned toad subfamily Megophryinae: i.e., Brachytarsophrys, Atympanophrys, Grillitschia, Sarawakiphrys gen. nov., Jingophrys gen. nov., Xenophrys, Megophrys, Pelobatrachus, Ophryophryne, and Boulenophrys. Revisions on the diagnosability, distribution, and content of each genus are provided. Furthermore, we present a careful review of the taxonomic history of Asian horned toad species from China and provide a monograph of congeners, including six species of Brachytarsophrys, four species of Atympanophrys, five species of Jingophrys gen. nov., 10 species of Xenophrys, two species of Ophryophryne, and 60 species of Boulenophrys. Finally, we discuss the importance of traditional morphological traits based on multiple populations in taxonomic work as well as taxonomic inflation caused by the genetic species delimitation.

First Discovery of Phenuiviruses within Diverse RNA Viromes of Asiatic Toad (Bufo gargarizans) by Metagenomics Sequencing.

Most zoonotic pathogens originate from mammals and avians, but viral diversity and related biosafety risk assessment in lower vertebrates also need to be explored. Amphibians are an important group of lower vertebrates that played a momentous role in animal evolution. To elucidate the diversity of RNA viruses in one important species of amphibians, the Asiatic toad (Bufo gargarizans), we obtained 44 samples including lung, gut, liver, and kidney tissues from Asiatic toads in Sichuan and Jilin provinces, China, for viral metagenomics sequencing. More than 20 novel RNA viruses derived from the order Bunyavirales and 7 families of Astroviridae, Dicistroviridae, Leviviridae, Partitiviridae, Picornaviridae, Rhabdoviridae, and Virgaviridae were discovered, which were distinct from previously described viruses and formed new clusters, as revealed by phylogenetic analyses. Notably, a novel bastrovirus, AtBastV/GCCDC11/2022, of the family Astroviridae was identified from the gut library, the genome of which contains three open reading frames, with the RNA-dependent RNA polymerase (RdRp) coded by ORF1 closely related to that of hepeviruses, and ORF2 encoding an astrovirus-related capsid protein. Notably, phenuiviruses were discovered for the first time in amphibians. AtPhenV1/GCCDC12/2022 and AtPhenV2/GCCDC13/2022 clustered together and formed a clade with the group of phenuiviruses identified from rodents. Picornaviruses and several invertebrate RNA viruses were also detected. These findings improve our understanding of the high RNA viral diversity in the Asiatic toad and provide new insights in the evolution of RNA viruses in amphibians.

The structural discrepancy between the small and large gut microbiota of Asiatic toad (Bufo gargarizans) during hibernation.

Hibernating amphibians are suitable for the research on the adaptation of gut microbiota to long-term fasting and cold stresses. However, the previous studies mainly focus on the large or whole gut microbiota but not the small gut microbiota. To test the structural discrepancy between the small and large gut microbiota during hibernation, we performed two independent batches of 16S rRNA gene amplicon sequencing to profile the small and large gut microbiota of hibernating Asiatic toad (Bufo gargarizans) from two wild populations. Both batches of data revealed that Proteobacteria, Bacteroidetes, and Firmicutes were the three most dominant phyla in the small and large gut microbiota. Three core OTUs with 100% occurrence in all gut microbiotas were annotated as Pseudomonas. A significant structural discrepancy was detected between the small and large gut microbiota. For instance, Proteobacteria assembled in the small intestine with a higher proportion than it did in the large intestine, but Bacteroidetes and Firmicutes assembled in the large intestine with a higher proportion than they did in the small intestine. The large gut microbiota exhibited higher diversity than the small gut microbiota. Nevertheless, a severe batch effect existed in the structural analysis of the gut microbiotas. The large gut microbiota showed a better resistance to the batch effect than the small gut microbiota did. This study provides preliminary evidence that microbes assemble in the small and large intestines of amphibians with discrepant patterns during hibernation.

Easternmost distribution of Bufo bufo (Linnaeus, 1758) in Türkiye: implications for the putative contact zone between B. bufo and B. verrucosissimus.

The geographic range of a species is crucial for obtaining information on the exact distribution of the species. The geographic data are important for delimiting distinct species or exploring the degree of differentiation among different populations of a species. The local details of species boundaries facilitate the study of the importance of phylogeographic background, secondary contacts, and hybrid zones, along with the relations between the species and its extrinsic environmental factors. In the present study, the range boundaries of Bufo bufo and Bufo verrucosissimus in the north-eastern region of Türkiye were delineated using an integrative taxonomic approach that utilized a combination of molecular and morphological data. According to the mtDNA results of the present study, B. bufo inhabits a single distribution from Iyidere town to Çayeli town in Rize, while B. verrucosissimus is distributed from Savsat town of Artvin to Ardesen town in Rize. In addition, the two species coexist in Pazar, Hemsin, and Çamlihemsin towns in Rize. The demographic analyses indicated a distinct population expansion for the B. verrucosissimus species after the Last Glacial Maximum, while the same did not occur for B. bufo. The univariate and multivariate statistical analyses conducted for the morphological data of the two species corroborated the presence of a putative contact zone between B. bufo and B. verrucosissimus. In summary, the present study resolved the non-distinct geographic boundaries between B. bufo and B. verrucosissimus species and also revealed the easternmost distribution of B. bufo in Türkiye. In addition, important evidence on the putative contact zone between the two species was indicated using an integrative taxonomic approach.

Landscape genetics of a sub-alpine toad: climate change predicted to induce upward range shifts via asymmetrical migration corridors.

Climate change is expected to have a major hydrological impact on the core breeding habitat and migration corridors of many amphibians in the twenty-first century. The Yosemite toad (Anaxyrus canorus) is a species of meadow-specializing amphibian endemic to the high-elevation Sierra Nevada Mountains of California. Despite living entirely on federal lands, it has recently faced severe extirpations, yet our understanding of climatic influences on population connectivity is limited. In this study, we used a previously published double-digest RADseq dataset along with numerous remotely sensed habitat features in a landscape genetics framework to answer two primary questions in Yosemite National Park: (1) Which fine-scale climate, topographic, soil, and vegetation features most facilitate meadow connectivity? (2) How is climate change predicted to influence both the magnitude and net asymmetry of genetic migration? We developed an approach for simultaneously modeling multiple toad migration paths, akin to circuit theory, except raw environmental features can be separately considered. Our workflow identified the most likely migration corridors between meadows and used the unique cubist machine learning approach to fit and forecast environmental models of connectivity. We identified the permuted modeling importance of numerous snowpack-related features, such as runoff and groundwater recharge. Our results highlight the importance of considering phylogeographic structure, and asymmetrical migration in landscape genetics. We predict an upward elevational shift for this already high-elevation species, as measured by the net vector of anticipated genetic movement, and a north-eastward shift in species distribution via the network of genetic migration corridors across the park.

Identification and characterization of a new family of long satellite DNA, specific of true toads (Anura, Amphibia, Bufonidae).

Amphibians have some of the most variable genome sizes among vertebrates. Genome size variation has been attributed to repetitive and noncoding DNA, including satellite repeats, transposable elements, introns, and nuclear insertions of viral and organelle DNA. In vertebrates, satellite DNAs have been widely described in mammals, but few molecular studies have been carried out in amphibians. Here, we provide a detailed characterization of a new family of satellite DNA, present in all 15 examined species of the family Bufonidae. Southern-blot analysis and PCR reveal that this satellite is formed by monomers of 807 bp, is organized in tandem arrays, and has an AT-content of 57.4%. Phylogenetic analyses show that most clades exhibit species-specific variances, indicating that this satellite DNA has evolved by concerted evolution. The homogenization/fixation process is heterogeneous in Bufonidae, where the genera Bufo and Bufotes do not show species-specific differences, while populations from Rhinella marina exhibit population-specific changes. Additionally, variants of this satellite DNA have been identified in Duttaphrynus melanostictus and R. marina, supporting the 'library hypothesis' (a set, 'library', of satellite DNAs is shared by a species group). Physical mapping in Bufo bufo, Bufo spinosus, Epidalea calamita and Bufotes viridis provides evidence that this repetitive DNA is not dispersed in the karyotype, but accumulated in pericentromeric regions of some chromosomal pairs. This location, together with its presence in the transcriptomes of bufonids, could indicate a role in centromere function or heterochromatin formation and maintenance.

Modified Metabolism and Response to UV Radiation: Gene Expression Variations Along an Elevational Gradient in the Asiatic Toad (Bufo gargarizans).

High-elevation adaptation provides an excellent system for examining adaptive evolution, and adaptive variations may manifest at gene expression or any other phenotypic levels. We examined gene expression profiles of Asiatic toads (Bufo gargarizans) along an elevational gradient from both wild and common-garden acclimated populations. Asiatic toads originated from high altitudes have distinctive gene expression patterns. We identified 18 fixed differentially expressed genes (DEGs), which are different in both wild and acclimated samples, and 1217 plastic DEGs, which are different among wild samples. The expression levels of most genes were linearly correlated with altitude gradient and down-regulated in high-altitude populations. Expression variations of several genes associated with metabolic process are fixed, and we also identified a co-expression module that is significantly different between acclimated populations and has functions related to DNA repair. The differential expression of the vast majority genes, however, are due to phenotypic plasticity, revealing the highly plastic nature of gene expression variations. Expression modification of some specific genes related to metabolism and response to UV radiation play crucial role in adaptation to high altitude for Asiatic toads. Common-garden experiments are essential for evaluating adaptive evolution of natural populations.

Cytogenetic Analysis in the Toad Species Bufo spinosus, Bufotes viridis and Epidalea calamita (Anura, Bufonidae) from the Mediterranean Area.

Taxonomy in Bufonidae witnessed notable transformations. Bufotes viridis and Epidalea calamita, previously included in genus Bufo, were relocated in other genera, while the genus Bufo was restricted to members of the earlier Bufo bufo group. On the other hand, Bufo bufo sensu lato now includes four species: Bufo bufo, Bufo spinosus, Bufo verrucosissimus and Bufo eichwaldi. In this study, we examined three species of three Bufonidae genera (B. spinosus, B. viridis and E. calamita) by conventional (C-banding and Ag-NOR staining) and molecular (in situ hybridization with probes for telomeric repeats and rDNA loci, and genomic in situ hybridization (GISH)) cytogenetic methods. C-banding patterns are reported for the first time for B. spinosus and E. calamita populations from Iberian Peninsula and for B. viridis from Greece, and reveal several differences with the reported C-banded karyotypes described for other European populations of these species. Silver staining shows size heteromorphisms of the signals at the Nucleolar Organizing Region (NOR). By contrast, FISH with ribosomal probes only reveal size heteromorphism of rDNA sequences in E. calamita, suggesting that the differences observed after silver staining in B. spinosus and B. viridis should be attributed to differences in chromosomal condensation and/or gene activity rather than to differences in the copy number for ribosomal genes. Regarding telomeric repeats, E. calamita is the only species with interstitial telomeric sequences (ITS) located on centromeric regions, probably originated by accumulation of telomeric sequences in the centromeric heterochromatin. Finally, we analyzed the composition and distribution of repetitive sequences by genome in situ hybridization. These experiments reveal the accumulation of repetitive sequences in centromeric regions of the three species, although these sequences are not conserved when species from different genera are compared.

Body condition, skin bacterial communities and disease status: insights from the first release trial of the limosa harlequin frog, Atelopus limosus.

Many endangered amphibian species survive in captive breeding facilities, but there have been few attempts to reintroduce captive-born individuals to rebuild wild populations. We conducted a soft-release trial of limosa harlequin frogs, Atelopus limosus, which are highly susceptible to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), to understand changes associated with the transition from captivity to the wild. Specifically, we assessed changes in body condition, skin-associated bacterial communities and disease status after release. Frogs were housed individually in field mesocosms and monitored for 27 days. Body condition did not significantly change in the mesocosms, and was similar to, or higher than, that of wild conspecifics at day 27. The skin bacteria of captive-born frogs, based on 16S rRNA gene amplicons, became similar to that of wild conspecifics after 27 days in mesocosms. Prevalence of Bd in wild conspecifics was 13-27%, and 15% of the A. limosus in mesocosms became infected with Bd, but no mortality of infected frogs was observed. We conclude that mesocosms are suitable for systematically and repeatedly monitoring amphibians during release trials, and that body condition, the skin microbiome, and Bd status can all change within one month of placement of captive-born individuals back into the wild.

Genetic diversity and demography of Bufo japonicus and B. torrenticola (Amphibia: Anura: Bufonidae) influenced by the Quaternary climate.

The Quaternary climate affected the present species richness and geographic distribution patterns of amphibians by limiting their activities during the glacial period. The present study examined the phylogenetic relationships of Japanese toads (Bufo japonicus and B. torrenticola) and the demography of each lineage from the past to the present based on mitochondrial sequences and ecological niche models. Japanese toads are a monophyletic group with two main clades (clades A and B). Clade A represents B. j. formosus, including three clades (clades A1, A2, and A3). Clade B contains three clades, two of which corresponded to B. j. japonicus (clades B1 and B2) and the other to B. torrenticola. Clade B2 and B. torrenticola made a sister group, and, thus, B. j. japonicus is paraphyletic. Clades A and B diverged in the late Miocene 5.7 million years ago (Mya) during the period when the Japanese archipelago was constructed. The earliest divergence between the three clades of clade A was estimated at 1.8 Mya. Clades A1 and A2 may have diverged at 0.8 Mya, resulting from the isolation in the multiple different refugia; however, the effects of the glacial climate on the divergence events of clade A3 are unclear. Divergences within clade B occurred from the late Pliocene to the early Pleistocene (3.2-2.2 Mya). Niche similarity between the parapatric clade in clade B (clades B1 and B2) indicated their allopatric divergence. It was suggested that niche segregation between B. japonicus and B. torrenticola contributed to a rapid adaptation of B. torrenticola for lotic breeding. All clade of Japanese toads retreated to each refugium at a low elevation in the glacial period, and effective population sizes increased to construct the current populations after the Last Glacial Maximum. Furthermore, we highlight the areas of climate stability from the last glacial maximum to the present that have served as the refugia of Japanese toads and, thus, affected their present distribution patterns.