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1.  Phylogenomic analyses support the position of turtles as the sister group of birds and crocodiles (Archosauria) 
BMC Biology  2012;10:65.
Background
The morphological peculiarities of turtles have, for a long time, impeded their accurate placement in the phylogeny of amniotes. Molecular data used to address this major evolutionary question have so far been limited to a handful of markers and/or taxa. These studies have supported conflicting topologies, positioning turtles as either the sister group to all other reptiles, to lepidosaurs (tuatara, lizards and snakes), to archosaurs (birds and crocodiles), or to crocodilians. Genome-scale data have been shown to be useful in resolving other debated phylogenies, but no such adequate dataset is yet available for amniotes.
Results
In this study, we used next-generation sequencing to obtain seven new transcriptomes from the blood, liver, or jaws of four turtles, a caiman, a lizard, and a lungfish. We used a phylogenomic dataset based on 248 nuclear genes (187,026 nucleotide sites) for 16 vertebrate taxa to resolve the origins of turtles. Maximum likelihood and Bayesian concatenation analyses and species tree approaches performed under the most realistic models of the nucleotide and amino acid substitution processes unambiguously support turtles as a sister group to birds and crocodiles. The use of more simplistic models of nucleotide substitution for both concatenation and species tree reconstruction methods leads to the artefactual grouping of turtles and crocodiles, most likely because of substitution saturation at third codon positions. Relaxed molecular clock methods estimate the divergence between turtles and archosaurs around 255 million years ago. The most recent common ancestor of living turtles, corresponding to the split between Pleurodira and Cryptodira, is estimated to have occurred around 157 million years ago, in the Upper Jurassic period. This is a more recent estimate than previously reported, and questions the interpretation of controversial Lower Jurassic fossils as being part of the extant turtles radiation.
Conclusions
These results provide a phylogenetic framework and timescale with which to interpret the evolution of the peculiar morphological, developmental, and molecular features of turtles within the amniotes.
doi:10.1186/1741-7007-10-65
PMCID: PMC3473239  PMID: 22839781
2.  Phylogeography of Sardinian Cave Salamanders (Genus Hydromantes) Is Mainly Determined by Geomorphology 
PLoS ONE  2012;7(3):e32332.
Detecting the factors that determine the interruption of gene flow between populations is key to understanding how speciation occurs. In this context, caves are an excellent system for studying processes of colonization, differentiation and speciation, since they represent discrete geographical units often with known geological histories. Here, we asked whether discontinuous calcareous areas and cave systems represent major barriers to gene flow within and among the five species of Sardinian cave salamanders (genus Hydromantes) and whether intraspecific genetic structure parallels geographic distance within and among caves. We generated mitochondrial cytochrome b gene sequences from 184 individuals representing 48 populations, and used a Bayesian phylogeographic approach to infer possible areas of cladogenesis for these species and reconstruct historical and current dispersal routes among distinct populations. Our results show deep genetic divergence within and among all Sardinian cave salamander species, which can mostly be attributed to the effects of mountains and discontinuities in major calcareous areas and cave systems acting as barriers to gene flow. While these salamander species can also occur outside caves, our results indicate that there is a very poor dispersal of these species between separate cave systems.
doi:10.1371/journal.pone.0032332
PMCID: PMC3299655  PMID: 22427830
3.  Base composition, selection, and phylogenetic significance of indels in the recombination activating gene-1 in vertebrates 
Frontiers in Zoology  2009;6:32.
Background
The Recombination Activating Proteins, RAG1 and RAG2, play a crucial role in the immune response in vertebrates. Among the nuclear markers currently used for phylogenetic purposes, Rag1 has especially enjoyed enormous popularity, since it successfully contributed to elucidating the relationships among and within a large variety of vertebrate lineages. We here report on a comparative investigation of the genetic variation, base composition, presence of indels, and selection in Rag1 in different vertebrate lineages (Actinopterygii, Amphibia, Aves, Chondrichthyes, Crocodylia, Lepidosauria, Mammalia, and Testudines) through the analysis of 582 sequences obtained from Genbank. We also analyze possible differences between distinct parts of the gene with different type of protein functions.
Results
In the vertebrate lineages studied, Rag1 is over 3 kb long. We observed a high level of heterogeneity in base composition at the 3rd codon position in some of the studied vertebrate lineages and in some specific taxa. This result is also paralleled by taxonomic differences in the GC content at the same codon position. Moreover, positive selection occurs at some sites in Aves, Lepidosauria and Testudines. Indels, which are often used as phylogenetic characters, are more informative across vertebrates in the 5' than in the 3'-end of the gene. When the entire gene is considered, the use of indels as phylogenetic character only recovers one major vertebrate clade, the Actinopterygii. However, in numerous cases insertions or deletions are specific to a monophyletic group.
Conclusions
Rag1 is a phylogenetic marker of undoubted quality. Our study points to the need of carrying out a preliminary investigation on the base composition and the possible existence of sites under selection of this gene within the groups studied to avoid misleading resolution. The gene shows highly heterogeneous base composition, which affects some taxa in particular and contains sites under positive selection in some vertebrate lineages in the 5'-end. The first part of the gene (5'-end) is more variable than the second (3'-end), and less affected by a heterogeneous base composition. However, in some vertebrate lineages the 5'-end of the gene is not yet widely used for phylogenetic studies.
doi:10.1186/1742-9994-6-32
PMCID: PMC2803162  PMID: 20015384
4.  Morphometrics Parallel Genetics in a Newly Discovered and Endangered Taxon of Galápagos Tortoise 
PLoS ONE  2009;4(7):e6272.
Galápagos tortoises represent the only surviving lineage of giant tortoises that exhibit two different types of shell morphology. The taxonomy of Galápagos tortoises was initially based mainly on diagnostic morphological characters of the shell, but has been clarified by molecular studies indicating that most islands harbor monophyletic lineages, with the exception of Isabela and Santa Cruz. On Santa Cruz there is strong genetic differentiation between the two tortoise populations (Cerro Fatal and La Reserva) exhibiting domed shell morphology. Here we integrate nuclear microsatellite and mitochondrial data with statistical analyses of shell shape morphology to evaluate whether the genetic distinction and variability of the two domed tortoise populations is paralleled by differences in shell shape. Based on our results, morphometric analyses support the genetic distinction of the two populations and also reveal that the level of genetic variation is associated with morphological shell shape variation in both populations. The Cerro Fatal population possesses lower levels of morphological and genetic variation compared to the La Reserva population. Because the turtle shell is a complex heritable trait, our results suggest that, for the Cerro Fatal population, non-neutral loci have probably experienced a parallel decrease in variability as that observed for the genetic data.
doi:10.1371/journal.pone.0006272
PMCID: PMC2707613  PMID: 19609441
5.  Evidence for recent gene flow between north-eastern and south-eastern Madagascan poison frogs from a phylogeography of the Mantella cowani group 
Background
The genus Mantella, endemic poison frogs of Madagascar with 16 described species, are known in the field of international pet trade and entered under the CITES control for the last four years. The phylogeny and phylogeography of this genus have been recently subject of study for conservation purposes. Here we report on the studies of the phylogeography of the Mantella cowani group using a fragment of 453 bp of the mitochondrial cytochrome b gene from 195 individuals from 21 localities. This group is represented by five forms: M. cowani, a critically endangered species, a vulnerable species, M. haraldmeieri, and the non-threatened M. baroni, M. aff. baroni, and M. nigricans.
Results
The Bayesian phylogenetic and haplotype network analyses revealed the presence of three separated haplotype clades: (1) M. baroni, M. aff. baroni, M. nigricans, and putative hybrids of M. cowani and M. baroni, (2) M. cowani and putative hybrids of M. cowani and M. baroni, and (3) M. haraldmeieri. The putative hybrids were collected from sites where M. cowani and M. baroni live in sympatry.
Conclusion
These results suggest (a) a probable hybridization between M. cowani and M. baroni, (b) a lack of genetic differentiation between M. baroni/M. aff. baroni and M. nigricans, (c) evidence of recent gene-flow between the northern (M. nigricans), eastern (M. baroni), and south-eastern (M. aff. baroni) forms of distinct coloration, and (d) the existence of at least three units for conservation in the Mantella cowani group.
doi:10.1186/1742-9994-4-1
PMCID: PMC1785372  PMID: 17207286
6.  Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians 
Background
Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI) has been proposed as universal marker for this purpose among animals.
Results
Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%), with low degrees of pairwise haplotype divergence within populations (0–1%).
Conclusion
We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.
doi:10.1186/1742-9994-2-5
PMCID: PMC555853  PMID: 15771783

Results 1-6 (6)