A new salamander of the genus Bolitoglossa (Caudata: Plethodontidae) from the highlands of western Panama.

We describe a new species of salamander of the genus Bolitoglossa from the Cordillera de Talamanca in western Panama. The new species is distinct from its congeners by its dorsal and ventral coloration, finger and toe webbing, and a comparatively high maxillary teeth count in relation to SVL. Analysis of mitochondrial DNA sequences revealed an isolated phylogenetic position of the new species which is related to the B. robinsoni, B. subpalmata and B. epimela species groups, all four of which form a subclade within the subgenus Eladinea.

Limb regeneration in salamanders: the plethodontid tale.

Salamanders are the only vertebrates that can regenerate limbs as adults. This makes them ideal models to investigate the cellular and molecular mechanisms of tissue regeneration. Ambystoma mexicanum and Nothopthalmus viridescens have long served as primary salamander models of limb regeneration, and the recent sequencing of the axolotl genome now provides a blueprint to mine regeneration insights from other salamander species. In particular, there is a need to study South American plethodontid salamanders that present different patterns of limb development and regeneration. A broader sampling of species using next-generation sequencing approaches is needed to reveal shared and unique mechanisms of regeneration, and more generally, the evolutionary history of salamander limb regeneration.

Vastly underestimated species richness of Amazonian salamanders (Plethodontidae: Bolitoglossa) and implications about plethodontid diversification.

We present data showing that the number of salamander species in Amazonia is vastly underestimated. We used DNA sequences of up to five genes (3 mitochondrial and 2 nuclear) of 366 specimens, 189 corresponding to 89 non-Amazonian nominal species and 177 Amazonian specimens, including types or topotypes, of eight of the nine recognized species in the region. By including representatives of all known species of Amazonian Bolitoglossa, except for one, and 73% of the currently 132 recognized species of the genus, our dataset represents the broadest sample of Bolitoglossa species, specimens, and geographic localities studied to date. We performed phylogenetic analyses using parsimony with tree-alignment and maximum likelihood (ML) with similarity alignment, with indels as binary characters. Our optimal topologies were used to delimit lineages that we assigned to nominal species and candidate new species following criteria that maximize the consilience of the current species taxonomy, monophyly, gaps in branch lengths, genetic distances, and geographic distribution. We contrasted the results of our species-delimitation protocol with those of Automated Barcode Gap Discovery (ABGD) and multi-rate Poisson Tree Processes (mPTP). Finally, we inferred the historical biogeography of South American salamanders by dating the trees and using dispersal-vicariance analysis (DIVA). Our results revealed a clade including almost all Amazonian salamanders, with a topology incompatible with just the currently recognized nine species. Following our species-delimitation criteria, we identified 44 putative species in Amazonia. Both ABGD and mPTP inferred more species than currently recognized, but their numbers (23-49) and limits vary. Our biogeographic analysis suggested a stepping-stone colonization of the Amazonian lowlands from Central America through the Chocó and the Andes, with several late dispersals from Amazonia back into the Andes. These biogeographic events are temporally concordant with an early land bridge between Central and South America (~10-15 MYA) and major landscape changes in Amazonia during the late Miocene and Pliocene, such as the drainage of the Pebas system, the establishment of the Amazon River, and the major orogeny of the northern Andes.

Genetic diversity and demography of the critically endangered Roberts' false brook salamander (Pseudoeurycea robertsi) in Central Mexico.

Land use changes are threatening the maintenance of biodiversity. Genetic diversity is one of the main indicators of biological diversity and is highly important as it shapes the capability of populations to respond to environmental changes. We studied eleven populations of Pseudoeurycea robertsi, a micro-endemic and critically endangered species from the Nevado de Toluca Volcano, a mountain that is part of the Trans-Mexican Volcanic Belt, Mexico. We sequenced the mitochondrial cytochrome b gene from 71 individuals and genotyped 9 microsatellites from 150 individuals. Our results based on the cytochrome b showed two divergent lineages, with moderate levels of genetic diversity and a recently historical demographic expansion. Microsatellite-based results indicated low levels of heterozygosity for all populations and few alleles per locus, as compared with other mole salamander species. We identified two genetically differentiated subpopulations with a significant level of genetic structure. These results provide fundamental data for the development of management plans and conservation efforts for this critically endangered species.

Using transcriptomics to enable a plethodontid salamander (Bolitoglossa ramosi) for limb regeneration research.

BACKGROUND: Tissue regeneration is widely distributed across the tree of life. Among vertebrates, salamanders possess an exceptional ability to regenerate amputated limbs and other complex structures. Thus far, molecular insights about limb regeneration have come from a relatively limited number of species from two closely related salamander families. To gain a broader perspective on the molecular basis of limb regeneration and enhance the molecular toolkit of an emerging plethodontid salamander (Bolitoglossa ramosi), we used RNA-Seq to generate a de novo reference transcriptome and identify differentially expressed genes during limb regeneration. RESULTS: Using paired-end Illumina sequencing technology and Trinity assembly, a total of 433,809 transcripts were recovered and we obtained functional annotation for 142,926 non-redundant transcripts of the B. ramosi de novo reference transcriptome. Among the annotated transcripts, 602 genes were identified as differentially expressed during limb regeneration. This list was further processed to identify a core set of genes that exhibit conserved expression changes between B. ramosi and the Mexican axolotl (Ambystoma mexicanum), and presumably their common ancestor from approximately 180 million years ago. CONCLUSIONS: We identified genes from B. ramosi that are differentially expressed during limb regeneration, including multiple conserved protein-coding genes and possible putative species-specific genes. Comparative analyses reveal a subset of genes that show similar patterns of expression with ambystomatid species, which highlights the importance of developing comparative gene expression data for studies of limb regeneration among salamanders.

Phylogenomic insights into the diversification of salamanders in the Isthmura bellii group across the Mexican highlands.

Mountain formation in Mexico has played an important role in the diversification of many Mexican taxa. The Trans-Mexican Volcanic Belt in particular has served as both a cradle of diversification and conduit for dispersal. We investigated the evolutionary history of the Isthmura bellii group of salamanders, a widespread amphibian across the Mexican highlands, using sequence capture of ultraconserved elements. Results suggest that the I. bellii group probably originated in southeastern Mexico in the late Miocene and later dispersed across the Trans-Mexican Volcanic Belt and into the Sierra Madre Occidental. Pre-Pleistocene uplift of the Trans-Volcanic Belt likely promoted early diversification by serving as a mesic land-bridge across central Mexico. These findings highlight the importance of the Trans-Volcanic Belt in generating Mexico's rich biodiversity.

Molecular characterization and expression analysis of cathepsin C in Chinese giant salamander (Andrias davidianus) after Aeromonas hydrophila infection

Background: Cathepsin C (CTSC) (dipeptidyl peptidase I, DPPI), is a member of the papain superfamily of cysteine proteases and involves in a variety of host reactions. However, the information of CTST in Chinese giant salamander (Andrias davidianus), an amphibian species with important evolutionary position and economic values, remained unclear. Results: The full-length salamander CTSC cDNA contained a 96 bp of 5'-UTR, a 1392 bp of ORF encoding 463 amino acids, and a 95 bp of 3'-UTR. The salamander CTSC possessed several sequence features similar to other reported CTSCs such as a signal peptide, a propeptide and a mature peptide. The active site triad of Cys, His and Asn were also found existing in salamander CTSC. Salamander CTSC mRNA was constitutively expressed in all the examined tissues with significantly variant expression level. The highest expression of CTSC was in intestine, followed with stomach, spleen, lung and brain. Following Aeromonas hydrophila infection for 12 h, salamander CTSC was significantly up-regulated in several tissues including lung, spleen, brain, kidney, heart, stomach and skin. Conclusion: CTSC plays roles in the immune response to bacterial infection, which provided valuable information for further studying the functions of CTSC in salamander.

Taxonomic revision of the moss salamander Nototriton barbouri (Schmidt (Caudata: Plethodontidae), with description of two new species from the Cordillera Nombre de Dios, Honduras.

Moss salamanders (genus Nototriton) are represented in northern Central America by nine putative species: N. barbouri, N. brodiei, N. lignicola, N. limnospectator, N. mime, N. picucha, N. saslaya, N. stuarti, and N. tomamorum. I estimate the phylogenetic relationships for these species based on data from three mitochondrial gene fragments (16S, cytochrome b, and COI), and compare morphological variation among putative taxa. As evidenced here and in previous studies, the taxon N. barbouri is paraphyletic with respect to populations from the Cordillera Nombre de Dios in northern Honduras. I restrict this taxon to populations from the Sierra de Sulaco in central Yoro, Honduras, and describe two new species from the Cordillera Nombre de Dios.

A beautiful new yellow salamander, genus Bolitoglossa (Caudata: Plethodontidae), from the northeastern slopes of the Cordillera de Talamanca, Costa Rica.

A new yellow salamander belonging to the genus Bolitoglossa, subgenus Eladinea, is described from a premontane rainforest in the vicinity of Moravia de Chirripó, on the northeastern slopes of the Cordillera de Talamanca in Costa Rica at an elevation of ca. 1300 m. This new taxon is distinguished from its congeners by its chromatic and morphological characteristics, and by differentiation in DNA sequences of the mitochondrial 16S rRNA and cytochrome b genes.

Phylogenetic relationships of Pseudohynobius (Urodela, Hynobiidae) inferred from DNA barcoding analysis.

As a proven tool, DNA barcoding can identify species rapidly and unambiguously. In this study, we used mtDNA cyt b, COI, and 16s rRNA sequences of six species of Pseudohynobius, Protohynobius puxiongensis, Liua shihi, Ranodon sibiricus, and Pachyhynobius shangchengensis, to reconstruct the phylogenetic relationships using Bayesian inference and maximum likelihood methods. Approximate lineage divergence times were also estimated, the divergence between them was calculated to have taken place mainly in Miocene. Our results showed that: 1) Ps. guizhouensis is an independent and valid species that is a sister species to Ps. kuankuoshuiensis; 2) five Pseudohynobius species formed a monophyletic group; 3) Ps. tsinpaensis is different from L. shihi, and should be classified as belonging to the Liua genus; and 4) Pr. puxiongensis is the sister lineage to all Pseudohynobius species, and should therefore be named Pseudohynobius puxiongensis.

Population genetic structure of critically endangered salamander (Hynobius amjiensis) in China: recommendations for conservation.

Amji's salamander (Hynobius amjiensis) is a critically endangered species (IUCN Red List), which is endemic to mainland China. In the present study, five haplotypes were genotyped for the mtDNA cyt b gene in 45 specimens from three populations. Relatively low levels of haplotype diversity (h = 0.524) and nucleotide diversity (π = 0.00532) were detected. Analyses of the phylogenic structure of H. amjiensis showed no evidence of major geographic partitions or substantial barriers to historical gene flow throughout the species' range. Two major phylogenetic haplotype groups were revealed, and were estimated to have diverged about 1.262 million years ago. Mismatch distribution analysis, neutrality tests, and Bayesian skyline plots revealed no evidence of dramatic changes in the effective population size. According to the SAMOVA and STRUCTURE analyses, H. amjiensis should be regarded as two different management units.

Two new species of Chiropterotriton (Caudata: Plethodontidae) from northern Mexico.

Species of the genus Chiropterotriton are distributed through much of northern and eastern Mexico. The genus has presented taxonomic difficulties, with a number of species waiting to be described. We describe two new species, Chiropterotriton cieloensis sp. nov. and Chiropterotriton infernalis sp. nov., from the Mexican state of Tamaulipas based on both molecular and morphological data. We present a phylogenetic hypothesis for the group, with emphasis on the taxa from northern Mexico, based on mitochondrial DNA, and discuss the other species of northern Mexico, especially the wider-ranging C. multidentatus, to which the new species were formerly assigned.

Identification of conservation units of the hynobiid salamander Pachyhynobius shangchengensis.

The evolutionary significant units (ESUs) of the salamander Pachyhynobius shangchengensis (Hynobiidae) in the Dabieshan mountains, southeastern China, were identified based on mitochondrial DNA data. We used methods for detecting cryptic species, such as the minimum spanning tree, the automatic barcode gap discovery, and the generalized mixed Yule-coalescent model; geographical partitioning was also used to identify the ESUs. A total of four ESUs were identified.

Osteological Variation among Extreme Morphological Forms in the Mexican Salamander Genus Chiropterotriton (Amphibia: Plethodontidae): Morphological Evolution And Homoplasy.

Osteological variation is recorded among and within four of the most distinctive species of the Mexican salamander genus Chiropterotriton. Analysis of the data is consistent with the monophyletic status of the genus and documents previously unrecorded intraspecific and interspecific variation. Most of the recorded variation involves qualitative and quantitative proportional differences, but four fixed differences constitute autapomorphic states that affirm and diagnose some species (C. dimidiatus, C. magnipes). Osteological variation in 15 characters is analyzed with respect to predictions generated from four hypotheses: 1) phylogeny, 2) adaptation to specific habitats (the four species include cave-dwelling, terrestrial, and arboreal forms), 3) size-free shape, and 4) size. High levels of intraspecific variation suggest that the characters studied are not subject to rigid functional constraints in salamanders, regardless of size. The pattern predicted by the hypothesis based on size differences seen among these four Chiropterotriton species matches most closely the observed pattern of relative skull robustness. Since size change and heterochrony are often associated in plethodontid evolution, it is likely that changes in developmental timing play a role in the morphological transitions among these morphologically diverse taxa. Webbed feet, miniaturization, body shape, and an unusual tarsal arrangement are morphologies exhibited in species of Chiropterotrition that are shown to be homoplastic with other clades of tropical plethodontids. Although extensive homoplasy in salamanders might be seen as a roadblock to unraveling phylogenetic hypotheses, the homologous developmental systems that appear to underlie such homoplasy may reveal common and consistent evolutionary processes at work.

Bromeliad selection by two salamander species in a harsh environment.

Bromeliad phytotelmata are frequently used by several Neotropical amphibian taxa, possibly due to their high humidity, microclimatic stability, and role as a refuge from predators. Indeed, the ability of phytotelmata to buffer against adverse environmental conditions may be instrumental in allowing some amphibian species to survive during periods of environmental change or to colonize sub-optimal habitats. Association between bromeliad traits and salamanders has not been studied at a fine scale, despite the intimate association of many salamander species with bromeliads. Here, we identify microhabitat characteristics of epiphytic bromeliads used by two species of the Bolitoglossa morio group (B. morio and B. pacaya) in forest disturbed by volcanic activity in Guatemala. Specifically, we measured multiple variables for bromeliads (height and position in tree, phytotelma water temperature and pH, canopy cover, phytotelma size, leaf size, and tree diameter at breast height), as well as salamander size. We employed a DNA barcoding approach to identify salamanders. We found that B. morio and B. pacaya occurred in microsympatry in bromeliads and that phytotelmata size and temperature of bromeliad microhabitat were the most important factors associated with the presence of salamanders. Moreover, phytotelmata with higher pH contained larger salamanders, suggesting that larger salamanders or aggregated individuals might modify pH. These results show that bromeliad selection is nonrandom with respect to microhabitat characteristics, and provide insight into the relationship between salamanders and this unique arboreal environment.

New species of salamander (Caudata: Plethodontidae: Cryptotriton) from Quebrada Cataguana, Francisco Morazán, Honduras, with comments on the taxonomic status of Cryptotriton wakei.

We describe a new species of the plethodontid salamander genus Cryptotriton from Honduras after comparing morphological, molecular, and osteological data from the holotype to that of the other nominal forms of the genus. The new species differs from all of the known species of Cryptotriton in at least one character from all three datasets. We also suggest placing C. wakei in the synonymy of C. nasalis after examining the morphological and osteological characters of the single known specimen of C. wakei.

Range-wide phylogeography and conservation genetics of a narrowly endemic stream salamander, Pachyhynobius shangchengensis (Caudata, Hynobiidae): implications for conservation.

The Shangcheng stout salamander (Pachyhynobius shangchengensis) is an endangered amphibian endemic to the Dabie Mountains, southeast China, and is currently threatened by habitat loss and illegal poaching. Here we used the mitochondrial DNA control region sequence (768 bp) to conduct a comprehensive investigation of genetic diversity, phylogeographic pattern, and demographic history of the species across its geographic distribution to assist its conservation. We concluded that the levels of genetic variation are relatively low in all four populations. Analysis of molecular variance indicated that the most likely phylogeographic pattern is [JGT] [KHJ] [TM, BYM]. Two distinct clades were identified in the phylogenetic tree of 28 haplotypes, corresponding to the two southern populations (TM, BYM) and two northern populations (JGT, KHJ). Significant population differentiation (FST) was detected among all populations. Among the four populations, historical demographic analyses (e.g., the g parameter, the Tajima D test, and the Fu Fs test) did not reveal definite information on population expansion except for the BYM population, which had undergone a strong population expansion event. Based on the analysis of a Bayesian skyline plot, the total population underwent a significant population fluctuation around 20 kya. This may have been triggered by the end of the last glacial maximum. In conclusion, the existence of three evolutionarily significant units (BMY-TM, KHJ, and JGT) and four management units (BMY, TM, KHJ, and JGT) is supported by our study.

Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian.

Tracking climate change in a dispersal-limited species: reduced spatial and genetic connectivity in a montane salamander.

Tropical montane taxa are often locally adapted to very specific climatic conditions, contributing to their lower dispersal potential across complex landscapes. Climate and landscape features in montane regions affect population genetic structure in predictable ways, yet few empirical studies quantify the effects of both factors in shaping genetic structure of montane-adapted taxa. Here, we considered temporal and spatial variability in climate to explain contemporary genetic differentiation between populations of the montane salamander, Pseudoeurycea leprosa. Specifically, we used ecological niche modelling (ENM) and measured spatial connectivity and gene flow (using both mtDNA and microsatellite markers) across extant populations of P. leprosa in the Trans-Mexican Volcanic Belt (TVB). Our results indicate significant spatial and genetic isolation among populations, but we cannot distinguish between isolation by distance over time or current landscape barriers as mechanisms shaping population genetic divergences. Combining ecological niche modelling, spatial connectivity analyses, and historical and contemporary genetic signatures from different classes of genetic markers allows for inference of historical evolutionary processes and predictions of the impacts future climate change will have on the genetic diversity of montane taxa with low dispersal rates. Pseudoeurycea leprosa is one montane species among many endemic to this region and thus is a case study for the continued persistence of spatially and genetically isolated populations in the highly biodiverse TVB of central Mexico.

Early Miocene origin and cryptic diversification of South American salamanders.

BACKGROUND: The currently recognized species richness of South American salamanders is surprisingly low compared to North and Central America. In part, this low richness may be due to the salamanders being a recent arrival to South America. Additionally, the number of South American salamander species may be underestimated because of cryptic diversity. The aims of our present study were to infer evolutionary relationships, lineage diversity, and timing of divergence of the South American Bolitoglossa using mitochondrial and nuclear sequence data from specimens primarily from localities in the Andes and upper Amazon Basin. We also estimated time of colonization of South America to test whether it is consistent with arrival via the Panamanian Isthmus, or land bridge connection, at its traditionally assumed age of 3 million years. RESULTS: Divergence time estimates suggest that Bolitoglossa arrived in South America from Central America by at least the Early Miocene, ca. 23.6 MYA (95% HPD 15.9-30.3 MYA), and subsequently diversified. South American salamanders of the genus Bolitoglossa show strong phylogeographic structure at fine geographic scales and deep divergences at the mitochondrial gene cytochrome b (Cytb) and high diversity at the nuclear recombination activating gene-1 (Rag1). Species often contain multiple genetically divergent lineages that are occasionally geographically overlapping. Single specimens from two southeastern localities in Ecuador are sister to the equatoriana-peruviana clade and genetically distinct from all other species investigated to date. Another single exemplar from the Andes of northwestern Ecuador is highly divergent from all other specimens and is sister to all newly studied samples. Nevertheless, all sampled species of South American Bolitoglossa are members of a single clade that is one of several constituting the subgenus Eladinea, one of seven subgenera in this large genus. CONCLUSIONS: The ancestors of South American salamanders likely arrived at least by the Early Miocene, well before the completion of the Late Pliocene Panamanian land bridge (widely accepted as ca. 3 MYA). This date is in agreement with recent, controversial, arguments that an older, perhaps short-lived, land connection may have existed between South America and present-day Panama 23-25 MYA. Since its arrival in South America, Bolitoglossa has diversified more extensively than previously presumed and currently includes several cryptic species within a relatively small geographic area. Rather than two upper Amazonian species currently recorded for this region, we propose that at least eight should be recognized, although these additional lineages remain to be formally described.