Hybridization of rRNAs to microarrays is a promising approach for prokaryotic and eukaryotic species identification. Typically, the amount of bound target is measured by fluorescent intensity and it is assumed that the signal intensity is directly related to the target concentration. Using thirteen different eukaryotic LSU rRNA target sequences and 7693 short perfect match oligonucleotide probes, we have assessed current approaches for predicting signal intensities by comparing Gibbs free energy (ΔG°) calculations to experimental results. Our evaluation revealed a poor statistical relationship between predicted and actual intensities. Although signal intensities for a given target varied up to 70-fold, none of the predictors were able to fully explain this variation. Also, no combination of different free energy terms, as assessed by principal component and neural network analyses, provided a reliable predictor of hybridization efficiency. We also examined the effects of single-base pair mismatch (MM) (all possible types and positions) on signal intensities of duplexes. We found that the MM effects differ from those that were predicted from solution-based hybridizations. These results recommend against the application of probe design software tools that use thermodynamic parameters to assess probe quality for species identification. Our results imply that the thermodynamic properties of oligonucleotide hybridization are by far not yet understood.

Background: Identification of species via DNA sequences is the basis for DNA taxonomy and DNA barcoding. Currently there is a strong focus on using a mitochondrial marker for this purpose, in particular a fragment from the cytochrome oxidase I gene (COI). While there is ample evidence that this marker is indeed suitable across a broad taxonomic range to delineate species, it has also become clear that a complementation by a nuclear marker system could be advantageous. Ribosomal RNA genes could be suitable for this purpose, because of their global occurrence and the possibility to design universal primers. However, it has so far been assumed that these genes are too highly conserved to allow resolution at, or even beyond the species level. On the other hand, it is known that ribosomal gene regions harbour also highly divergent parts. We explore here the information content of two adjacent divergence regions of the large subunit ribosomal gene, the D1-D2 region.

Aim The biogeographical origins of the extant vertebrates endemic to Madagascar are largely unsolved, but have often been related to vicariance in the context of fragmentation of the supercontinent Gondwana in the Mesozoic. Such hypotheses are especially appealing in the case of cichlid ®shes, which show phylogenetic relationships re¯ecting the temporal successions of the breakup of Gondwana. We used molecular clock data to test this assumption.Location Fragments of the 16S rRNA gene and of the nuclear Tmo-4C4 locus, partly obtained from Genbank from South American, African, Malagasy and Indian cichlids were analysed.Methods Based on monophyletic cichlid radiations in African lakes, we calibrated a molecular clock. The obtained rates were used to estimate the age of divergence of the major cichlid clades. ResultsThe results agreed better with a Cenozoic than with a Mesozoic divergence, and were in accordance with the fossil record. Sequence divergences of the 16S and 12S rRNA genes of most lineages of Malagasy terrestrial and freshwater vertebrates from their non-Malagasy sister groups were below saturation and many were relatively similar to those of cichlids.Main conclusions A Cenozoic dispersal from continental landmasses may explain the origin of most extant Malagasy vertebrate groups better than a Jurassic/Cretaceous vicariance.

African killifishes of the genus Chromaphyosemion show a high degree of phenotypic and karyotypic diversity. The latter is especially pronounced in C. riggenbachi, a morphologically defined species restricted to a small distribution area in Cameroon. This study presents a detailed reconstruction of karyotype differentiation within C. riggenbachi using conventional Giemsa staining and sequential chromosome banding as well as a phylogenetic analysis based on part of the mitochondrial (mt) cytochrome b gene from eleven populations. The cytogenetic analysis revealed differences in chromosome morphology, banding patterns and/or diploid chromosome number (2n) among all populations examined. Diploid number ranged from 2n = 20 to 2n = 36 and varied mainly among populations, while C-banding patterns and NOR phenotypes showed fixed differences among populations as well as some variability within populations. The mtDNA analysis disclosed five clearly differentiated haplotype groups. Mapping the karyotype data onto the mtDNA dendrogram revealed a decrease in 2n from the most basal to the most derived groups, thus demonstrating a reduction of 2n during their evolutionary history. Our results indicate that karyotype differentiation involved Robertsonian fusions as well as non-Robertsonian processes. Causes of the high karyotypic variability may include an elevated chromosomal mutation rate as well as certain features of the ecology and mating system that could facilitate the fixation of chromosomal rearrangements. The pattern of karyotype and haplotype differentiation and the results of previous crossing experiments suggest incipient speciation in C. riggenbachi.

The phylogeny of the West African genus Archiaphyosemion was studied with mitochondrial and nuclear DNA sequences. The results of the combined dataset presented here did not support a monophyletic group. After the exclusion of the type species of the genus, A. guineense, the remaining species form a well-supported monophyletic group. Based on these molecular results and supported by morphological data, we suggest a new name for this group, Nimbapanchax, new genus. Additionally, based on a recent collection in Guinea, two new Nimbapanchax species were described. The taxon Nimbapanchax leucopterygius, new species, is described for a nothobranchiid fish formerly misidentified as Archiaphyosemion maeseni (Poll, 1941). Nimbapanchax melanopterygius, new species, is described from the Mount Nimba region in southeastern Guinea. Both new Nimbapanchax species are clearly distinguished from their congeners by the coloration pattern of adult males. The results of the DNA data support the assumption based on color pattern and morphological characters that the new described species are sister taxa. The type of Aphyosemion maeseni Poll, 1941 was reexamined and transferred to the genus Epiplatys, a decision based on diagnostic morphological characters. RésuméLa phylogénie du genre Ouest Africain Archiaphyosemion été étudié en utilisant de l'ADN mitochondrial et nucléaire. Le résultat de la combinaison de ces deux jeux de données, présenté ici, ne supporte pas un seul groupe monophylétique. Cependant, après l'exclusion d'espèce-type du genre, A. guineense, les individus restant forment un groupe monophylétique bien supporté. En se basant sur les résultats moléculaires et morphologiques, nous proposons un nom pour ce group: Nimbapanchax, nouveau genre. De plus, deux nouvelles espèces de Nimbapanchax ont été décrites grâce à un récent échantillonnage en Guinée. Le taxon Nimbapanchax leucopterygius, nouvelle espèce, a été décrit chez le poisson nothobranchiid qui avait été précédemment identifié à tort comme étant Archiaphyosemion maeseni (Poll, 1941). Nimbapanchax melanopterygius, nouvelle espèce, quant à elle, a été décrite dans la région du mont Nimba au sud-est de la Guinée. Ces deux nouvelles espèces de Nimbapanchax sont clairement identifiables grâce aux motifs colorés des mâles adultes. L'ADN confirme l'hypothèse basée sur la coloration et les caractères morphologiques prédisant que les deux nouvelles espèces décrites sont des taxa frères. Le type d'Aphyosemion maeseni Poll, 1941 a été réétudié et transféré au genre Epiplatys, cette décision étant basée sur des caractères morphologiques.