Patterns of endemism and species richness in Malagasy cophyline frogs support a key role of mountainous areas for speciation

Cophyline narrow-mouthed frogs (Anura: Microhylidae) are a diverse endemic radiation of Madagascar. Cophylines contain a high proportion of range restricted species and constitute a good model system to understand patterns of evolutionary diversification in tropical ecosystems. We combine spatial and phylogenetic analyses for a near-complete taxon sample to test competing explanations for patterns of species richness (SR) and endemism. Our reconstruction of the phylogeny of cophylines indicates the presence of 22 new species and several instances of nonmonophyly. We found a strong historical signal in current cophyline ranges indicating a high degree of spatial niche conservatism in clade diversification, with clades occurring in the North of Madagascar constituting the most derived in the phylogeny. We identified six positively correlated centers of SR and endemism that can neither be explained by stochastic models such as elevational or latitudinal mid-domain effect, nor by low-elevation river catchments. Instead, the locations of these centers in areas spanning a high altitudinal range in combination with specific climatic parameters support a key role of mountainous areas for speciation of these anurans, although we cannot exclude an influence of habitat loss due to human impact. High conservation priority is ascribed to these areas.     

Molecular Detection of <Emphasis Type="Italic">Xenostrobus securis</Emphasis> and <Emphasis Type="Italic">Mytillus Galloprovincialis</Emphasis> Larvae in Galician Coast (Spain)

The mussel species Xenostrobus securis from New Zealand was detected in the Spanish coast recently, in the mouth of the Verdugo River into the Vigo Ria. In view of the great importance of the farm mussel sector in this region, the presence of this alien species greatly concerned producers and administration authorities, because of its potential medium- or long-term effects on the autochthonous species, Mytilus galloprovincialis, an important marine resource widely exploited in this location. The goal of this study was to develop a DNA-based technique to identify X. securis and M. galloprovincialis larvae in plankton samples, which would allow monitoring for the presence of X. securis in different points of the Vigo Ria. The techniques used were simplex and multiplex polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and fragment analysis. The application of this system to planktonic samples could be an effective means to assess the presence of the alien species, allowing monitoring if its dispersion is increasing, or on the contrary, if its distribution is restricted to the mouth of the Verdugo River, where X. securis was first detected. In addition, the application of this system at different times could be useful to assess the presence of larvae of these two species in the plankton.     

DNA conformational changes and cleavage by ruthenium(II) nitrofurylsemicarbazone complexes

Metal complexes that establish interactions with DNA are being studied not only because of their potential use as therapeutic agents but also as tools for biochemistry and molecular biology. Searching for drugs with anti-trypanosome activity, we previously synthesized a series of ruthenium mixed ligand dimethyl sulfoxide complexes of the type [Ru(II)Cl(2)(DMSO)(2)L], where L is 5-nitrofurylsemicarbazone derivatives and DMSO is dimethyl sulfoxide. Though they present the ability to bind DNA, no activity against parasites in cell culture was observed. Considering their potential application as molecular tools we further analyzed the interactions with DNA through an electrophoretic approach. Non covalent withdrawal of superhelicity and a rapid nicking activity upon covalent interaction was observed. Inhibition of both effects was observed in the presence of distamycin suggesting the involvement of the DNA minor groove in the interaction with the nitrofurylsemicarbazone ruthenium complexes. In addition cleavage inhibition by dimethyl sulfoxide suggests an oxidative mechanism of action.     

Multiple overseas dispersal in amphibians

Amphibians are thought to be unable to disperse over ocean barriers because they do not tolerate the osmotic stress of salt water. Their distribution patterns have therefore generally been explained by vicariance biogeography. Here, we present compelling evidence for overseas dispersal of frogs in the Indian Ocean region based on the discovery of two endemic species on Mayotte. This island belongs to the Comoro archipelago, which is entirely volcanic and surrounded by sea depths of more than 3500 m. This constitutes the first observation of endemic amphibians on oceanic islands that did not have any past physical contact to other land masses. The two species of frogs had previously been thought to be nonendemic and introduced from Madagascar, but clearly represent new species based on their morphological and genetic differentiation. They belong to the genera Mantidactylus and Boophis in the family Mantellidae that is otherwise restricted to Madagascar, and are distinguished by morphology and mitochondrial and nuclear DNA sequences from mantellid species occurring in Madagascar. This discovery permits us to update and test molecular clocks for frogs distributed in this region. The new calibrations are in agreement with previous rate estimates and indicate two further Cenozoic transmarine dispersal events that had previously been interpreted as vicariance: hyperoliid frogs from Africa to Madagascar (Heterixalus) and from Madagascar to the Seychelles islands (Tachycnemis). Our results provide the strongest evidence so far that overseas dispersal of amphibians exists and is no rare exception, although vicariance certainly retains much of its importance in explaining amphibian biogeography.     

Improving anti-trypanosomal activity of 3-aminoquinoxaline-2-carbonitrile N1,N4-dioxide derivatives by complexation with vanadium

New vanadium complexes of the type [V(IV)O(L)(2)], where L are 3-aminoquinoxaline-2-carbonitrile N(1),N(4)-dioxide derivatives, were prepared as an effort to obtain new anti-trypanosomal agents improving the bioactivity of the free ligands. Complexation to vanadium of the quinoxaline ligands leads to excellent antiprotozoal activity, similar to that of the reference drugs nifurtimox and benznidazole and in all cases higher than that of the corresponding free ligands. In addition, it is for the first time that the V((IV))O-quinoxaline complexes are reported as a family of anti-Trypanosoma cruzi agents. Finally, the anti-trypanosomal activity of these vanadium complexes could be explained on the basis of their lipophilicity and the electronic characteristics of the quinoxaline substituents.     

Deciphering amphibian diversity through DNA barcoding: chances and challenges

Amphibians globally are in decline, yet there is still a tremendous amount of unrecognized diversity, calling for an acceleration of taxonomic exploration. This process will be greatly facilitated by a DNA barcoding system; however, the mitochondrial population structure of many amphibian species presents numerous challenges to such a standardized, single locus, approach. Here we analyse intra- and interspecific patterns of mitochondrial variation in two distantly related groups of amphibians, mantellid frogs and salamanders, to determine the promise of DNA barcoding with cytochrome oxidase subunit I (cox1) sequences in this taxon. High intraspecific cox1 divergences of 7-14% were observed (18% in one case) within the whole set of amphibian sequences analysed. These high values are not caused by particularly high substitution rates of this gene but by generally deep mitochondrial divergences within and among amphibian species. Despite these high divergences, cox1 sequences were able to correctly identify species including disparate geographic variants. The main problems with cox1 barcoding of amphibians are (i) the high variability of priming sites that hinder the application of universal primers to all species and (ii) the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species. Common discordances between geographical signatures of mitochondrial and nuclear markers in amphibians indicate that a single-locus approach can be problematic when high accuracy of DNA barcoding is required. We suggest that a number of mitochondrial and nuclear genes may be used as DNA barcoding markers to complement cox1.     

Recurrent ecological adaptations revealed through a molecular analysis of the secretive cophyline frogs of Madagascar

The cophyline microhylid frogs of Madagascar show a wide range of habitat specialization, ranging from terrestrial/burrowing and semi-arboreal to entirely arboreal species. The classification of these frogs is thus far mainly based upon morphological, largely osteological, characters that might be homoplastic. Using 1173 bp of DNA sequences from the mitochondrial 12S and 16S rRNA genes, we here present a molecular phylogeny for 28 species of all known genera, except for the genus Madecassophryne. The resulting maximum likelihood tree contained four major clades: one represented by the genus Anodonthyla, the second by Cophyla and Platypelis, the third by several terrestrial and semi-arboreal species of the genus Plethodontohyla, and the fourth by species of the genera Stumpffia, Plethodontohyla, and Rhombophryne. The results confirm that several cophyline lineages adapted independently to similar habitats, with multiple shifts among terrestriality and arboreality. The direction of these shifts cannot be ascertained due to unclarified relationships among the most basal lineages, but for one terrestrial species (Anodonthyla montana), it is most parsimonious to assume that it evolved from arboreal ancestors. Our results suggest that the genus Plethodontohyla is probably paraphyletic, and that the classification of this and of the genus Rhombophryne needs to be re-assessed.     

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.