Initial diversification of living amphibians predated the breakup of Pangaea

The origin and divergence of the three living orders of amphibians (Anura, Caudata, Gymnophiona) and their main lineages are one of the most hotly debated topics in vertebrate evolution. Here, we present a robust molecular phylogeny based on the nuclear RAG1 gene as well as results from a variety of alternative independent molecular clock calibrations. Our analyses suggest that the origin and early divergence of the three living amphibian orders dates back to the Palaeozoic or early Mesozoic, before the breakup of Pangaea, and soon after the divergence from lobe-finned fishes. The resulting new biogeographic scenario, age stimate, and the inferred rapid divergence of the three lissamphibian orders may account for the lack of fossils that represent plausible ancestors or immediate sister taxa of all three orders and the heretofore paradoxical distribution of some amphibian fossil taxa. Furthermore, the ancient and rapid radiation of the three lissamphibian orders likely explains why branch lengths connecting their early nodes are particularly short, thus rendering phylogenetic inference of implicated relationships especially difficult

Next-generation mitogenomics: A comparison of approaches applied to caecilian amphibian phylogeny

Mitochondrial genome (mitogenome) sequences are being generated with increasing speed due to the advances of next-generation sequencing (NGS) technology and associated analytical tools. However, detailed comparisons to explore the utility of alternative NGS approaches applied to the same taxa have not been undertaken. We compared a 'traditional' Sanger sequencing method with two NGS approaches (shotgun sequencing and non-indexed, multiplex amplicon sequencing) on four different sequencing platforms (Illumina's HiSeq and MiSeq, Roche's 454 GS FLX, and Life Technologies' Ion Torrent) to produce seven (near-) complete mitogenomes from six species that form a small radiation of caecilian amphibians from the Seychelles. The fastest, most accurate method of obtaining mitogenome sequences that we tested was direct sequencing of genomic DNA (shotgun sequencing) using the MiSeq platform. Bayesian inference and maximum likelihood analyses using seven different partitioning strategies were unable to resolve compellingly all phylogenetic relationships among the Seychelles caecilian species, indicating the need for additional data in this case

Limited Lifespan of Fragile Regions in Mammalian Evolution

An important question in genome evolution is whether there exist fragile regions (rearrangement hotspots) where chromosomal rearrangements are happening over and over again. Although nearly all recent studies supported the existence of fragile regions in mammalian genomes, the most comprehensive phylogenomic study of mammals (Ma et al. (2006) Genome Research 16, 1557-1565) raised some doubts about their existence. We demonstrate that fragile regions are subject to a "birth and death" process, implying that fragility has limited evolutionary lifespan. This finding implies that fragile regions migrate to different locations in different mammals, explaining why there exist only a few chromosomal breakpoints shared between different lineages. The birth and death of fragile regions phenomenon reinforces the hypothesis that rearrangements are promoted by matching segmental duplications and suggests putative locations of the currently active fragile regions in the human genome

Reversal to air-driven sound production revealed by a molecular phylogeny of tongueless frogs, family Pipidae

<p>Abstract</p> <p>Background</p> <p>Evolutionary novelties often appear by conferring completely new functions to pre-existing structures or by innovating the mechanism through which a particular function is performed. Sound production plays a central role in the behavior of frogs, which use their calls to delimit territories and attract mates. Therefore, frogs have evolved complex vocal structures capable of producing a wide variety of advertising sounds. It is generally acknowledged that most frogs call by moving an air column from the lungs through the glottis with the remarkable exception of the family Pipidae, whose members share a highly specialized sound production mechanism independent of air movement.</p> <p>Results</p> <p>Here, we performed behavioral observations in the poorly known African pipid genus <it>Pseudhymenochirus </it>and document that the sound production in this aquatic frog is almost certainly air-driven. However, morphological comparisons revealed an indisputable pipid nature of <it>Pseudhymenochirus </it>larynx. To place this paradoxical pattern into an evolutionary framework, we reconstructed robust molecular phylogenies of pipids based on complete mitochondrial genomes and nine nuclear protein-coding genes that coincided in placing <it>Pseudhymenochirus </it>nested among other pipids.</p> <p>Conclusions</p> <p>We conclude that although <it>Pseudhymenochirus </it>probably has evolved a reversal to the ancestral non-pipid condition of air-driven sound production, the mechanism through which it occurs is an evolutionary innovation based on the derived larynx of pipids. This strengthens the idea that evolutionary solutions to functional problems often emerge based on previous structures, and for this reason, innovations largely depend on possibilities and constraints predefined by the particular history of each lineage.</p

Molecular evolution of HoxA13 and the multiple origins of limbless morphologies in amphibians and reptiles

Developmental processes and their results, morphological characters, are inherited through transmission of genes regulating development. While there is ample evidence that cis-regulatory elements tend to be modular, with sequence segments dedicated to different roles, the situation for proteins is less clear, being particularly complex for transcription factors with multiple functions. Some motifs mediating protein-protein interactions may be exclusive to particular developmental roles, but it is also possible that motifs are mostly shared among different processes. Here we focus on HoxA13, a protein essential for limb development. We asked whether the HoxA13 amino acid sequence evolved similarly in three limbless clades: Gymnophiona, Amphisbaenia and Serpentes. We explored variation in ω (dN/dS) using a maximum-likelihood framework and HoxA13sequences from 47 species. Comparisons of evolutionary models provided low ω global values and no evidence that HoxA13 experienced relaxed selection in limbless clades. Branch-site models failed to detect evidence for positive selection acting on any site along branches of Amphisbaena and Gymnophiona, while three sites were identified in Serpentes. Examination of alignments did not reveal consistent sequence differences between limbed and limbless species. We conclude that HoxA13 has no modules exclusive to limb development, which may be explained by its involvement in multiple developmental processes

The Development of Three Long Universal Nuclear Protein-Coding Locus Markers and Their Application to Osteichthyan Phylogenetics with Nested PCR

BACKGROUND: Universal nuclear protein-coding locus (NPCL) markers that are applicable across diverse taxa and show good phylogenetic discrimination have broad applications in molecular phylogenetic studies. For example, RAG1, a representative NPCL marker, has been successfully used to make phylogenetic inferences within all major osteichthyan groups. However, such markers with broad working range and high phylogenetic performance are still scarce. It is necessary to develop more universal NPCL markers comparable to RAG1 for osteichthyan phylogenetics. METHODOLOGY/PRINCIPAL FINDINGS: We developed three long universal NPCL markers (>1.6 kb each) based on single-copy nuclear genes (KIAA1239, SACS and TTN) that possess large exons and exhibit the appropriate evolutionary rates. We then compared their phylogenetic utilities with that of the reference marker RAG1 in 47 jawed vertebrate species. In comparison with RAG1, each of the three long universal markers yielded similar topologies and branch supports, all in congruence with the currently accepted osteichthyan phylogeny. To compare their phylogenetic performance visually, we also estimated the phylogenetic informativeness (PI) profile for each of the four long universal NPCL markers. The PI curves indicated that SACS performed best over the whole timescale, while RAG1, KIAA1239 and TTN exhibited similar phylogenetic performances. In addition, we compared the success of nested PCR and standard PCR when amplifying NPCL marker fragments. The amplification success rate and efficiency of the nested PCR were overwhelmingly higher than those of standard PCR. CONCLUSIONS/SIGNIFICANCE: Our work clearly demonstrates the superiority of nested PCR over the conventional PCR in phylogenetic studies and develops three long universal NPCL markers (KIAA1239, SACS and TTN) with the nested PCR strategy. The three markers exhibit high phylogenetic utilities in osteichthyan phylogenetics and can be widely used as pilot genes for phylogenetic questions of osteichthyans at different taxonomic levels

What are the consequences of combining nuclear and mitochondrial data for phylogenetic analysis? Lessons from Plethodon salamanders and 13 other vertebrate clades

<p>Abstract</p> <p>Background</p> <p>The use of mitochondrial DNA data in phylogenetics is controversial, yet studies that combine mitochondrial and nuclear DNA data (mtDNA and nucDNA) to estimate phylogeny are common, especially in vertebrates. Surprisingly, the consequences of combining these data types are largely unexplored, and many fundamental questions remain unaddressed in the literature. For example, how much do trees from mtDNA and nucDNA differ? How are topological conflicts between these data types typically resolved in the combined-data tree? What determines whether a node will be resolved in favor of mtDNA or nucDNA, and are there any generalities that can be made regarding resolution of mtDNA-nucDNA conflicts in combined-data trees? Here, we address these and related questions using new and published nucDNA and mtDNA data for <it>Plethodon </it>salamanders and published data from 13 other vertebrate clades (including fish, frogs, lizards, birds, turtles, and mammals).</p> <p>Results</p> <p>We find widespread discordance between trees from mtDNA and nucDNA (30-70% of nodes disagree per clade), but this discordance is typically not strongly supported. Despite often having larger numbers of variable characters, mtDNA data do not typically dominate combined-data analyses, and combined-data trees often share more nodes with trees from nucDNA alone. There is no relationship between the proportion of nodes shared between combined-data and mtDNA trees and relative numbers of variable characters or levels of homoplasy in the mtDNA and nucDNA data sets. Congruence between trees from mtDNA and nucDNA is higher on branches that are longer and deeper in the combined-data tree, but whether a conflicting node will be resolved in favor mtDNA or nucDNA is unrelated to branch length. Conflicts that are resolved in favor of nucDNA tend to occur at deeper nodes in the combined-data tree. In contrast to these overall trends, we find that <it>Plethodon </it>have an unusually large number of strongly supported conflicts between data types, which are generally resolved in favor of mtDNA in the combined-data tree (despite the large number of nuclear loci sampled).</p> <p>Conclusions</p> <p>Overall, our results from 14 vertebrate clades show that combined-data analyses are not necessarily dominated by the more variable mtDNA data sets. However, given cases like <it>Plethodon</it>, there is also the need for routine checking of incongruence between mtDNA and nucDNA data and its impacts on combined-data analyses.</p

Late Cretaceous Vicariance in Gondwanan Amphibians

Overseas dispersals are often invoked when Southern Hemisphere terrestrial and freshwater organism phylogenies do not fit the sequence or timing of Gondwana fragmentation. We used dispersal-vicariance analyses and molecular timetrees to show that two species-rich frog groups, Microhylidae and Natatanura, display congruent patterns of spatial and temporal diversification among Gondwanan plates in the Late Cretaceous, long after the presumed major tectonic break-up events. Because amphibians are notoriously salt-intolerant, these analogies are best explained by simultaneous vicariance, rather than by oceanic dispersal. Hence our results imply Late Cretaceous connections between most adjacent Gondwanan landmasses, an essential concept for biogeographic and palaeomap reconstructions

Parental investment by skin feeding in a caecilian amphibian

Although the initial growth and development of most multicellular animals depends on the provision of yolk, there are many varied contrivances by which animals provide additional or alternative investment in their offspring(1). Providing offspring with additional nutrition should be favoured by natural selection when the consequent increased fitness of the young offsets any corresponding reduction in fecundity(2). Alternative forms of nutrition may allow parents to delay and potentially redirect their investment. Here we report a remarkable form of parental care and mechanism of parent-offspring nutrient transfer in a caecilian amphibian. Boulengerula taitanus is a direct-developing, oviparous caecilian(3), the skin of which is transformed in brooding females to provide a rich supply of nutrients for the developing offspring. Young animals are equipped with a specialized dentition, which they use to peel and eat the outer layer of their mother's modified skin. This new form of parental care provides a plausible intermediate stage in the evolution of viviparity in caecilians. At independence, offspring of viviparous and of oviparous dermatotrophic caecilians are relatively large despite being provided with relatively little yolk. The specialized dentition of skin-feeding (dermatophagous) caecilians may constitute a pre-adaptation to the fetal feeding on the oviduct lining of viviparous caecilians.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62957/1/nature04403.pd

Microsatellite discovery in an insular amphibian (Grandisonia alternans) with comments on cross-species utility and the accuracy of locus identification from unassembled Illumina data

The Seychelles archipelago is unique among isolated oceanic islands because it features an endemic radiation of caecilian amphibians (Gymnophiona). In order to develop population genetics resources for this system, we identified microsatellite loci using unassembled Illumina MiSeq data generated from a genomic library of Grandisonia alternans, a species that occurs on multiple islands in the archipelago. Applying a recently described method (PALFINDER) we identified 8001 microsatellite loci that were potentially informative for population genetics analyses. Of these markers, we screened 60 loci using five individuals, directly sequenced several amplicons to confirm their identity, and then used eight loci to score allele sizes in 64 G. alternans individuals originating from five islands. A number of these individuals were sampled using non-lethal methods, demonstrating the efficacy of non-destructive molecular sampling in amphibian research. Although two loci satisfied our criteria as diploid, neutrally evolving loci with the statistical power to detect population structure, our success in identifying reliable loci was very low. Additionally, we discovered some issues with primer redundancy and differences between Illumina and Sanger sequences that suggest some Illumina-inferred loci are invalid. We investigated cross-species utility for eight loci and found most could be successfully amplified, sequenced and aligned across other species and genera of caecilians from the Seychelles. Thus, our study in part supported the validity of using PALFINDER with unassembled reads for microsatellite discovery within and across species, but importantly identified major limitations to applying this approach to small datasets (ca. 1 million reads) and loci with small tandem repeat sizes