Differentiation in neutral genes and a candidate gene in the pied flycatcher: using biological archives to track global climate change.

Global climate change is one of the major driving forces for adaptive shifts in migration and breeding phenology and possibly impacts demographic changes if a species fails to adapt sufficiently. In Western Europe, pied flycatchers (Ficedula hypoleuca) have insufficiently adapted their breeding phenology to the ongoing advance of food peaks within their breeding area and consequently suffered local population declines. We address the question whether this population decline led to a loss of genetic variation, using two neutral marker sets (mitochondrial control region and microsatellites), and one potentially selectively non-neutral marker (avian Clock gene). We report temporal changes in genetic diversity in extant populations and biological archives over more than a century, using samples from sites differing in the extent of climate change. Comparing genetic differentiation over this period revealed that only the recent Dutch population, which underwent population declines, showed slightly lower genetic variation than the historic Dutch population. As that loss of variation was only moderate and not observed in all markers, current gene flow across Western and Central European populations might have compensated local loss of variation over the last decades. A comparison of genetic differentiation in neutral loci versus the Clock gene locus provided evidence for stabilizing selection. Furthermore, in all genetic markers, we found a greater genetic differentiation in space than in time. This pattern suggests that local adaptation or historic processes might have a stronger effect on the population structure and genetic variation in the pied flycatcher than recent global climate changes.

Conservation of structural elements in the mitochondrial control region of Daphnia.

Mitochondrial DNA is well characterized in vertebrates and insects and represents a prime target for phylogenetic and population genetic analyses. In particular the highly variable control region, responsible for regulation of replication and transcription, has been studied by molecular biologists and geneticists alike. However, we lack basic information on the structure and molecular evolution of the control region in major animal groups, such as the crustaceans. Here we present a genetic analysis of the mitochondrial DNA control regions of three microcrustacean species of the genus Daphnia. Their control regions are located between the tRNA-Ile and the 12S rRNA and exhibit conserved structural elements previously described only for insects. We identified the (TA(A))n-block, a putative hairpin structure with a 5' conserved flanking region and the GA-block. Moreover, not only the presence of these elements, but also their relative location on the mtDNA is conserved among D. longispina and insects. A comparative analysis of Malacostraca and Branchiopoda, which form a sister clade of insects revealed that the presence of structural elements per se is conserved between insects and crustaceans, but the arrangement of the elements along the control region as well as the number of each element is variable. Finally we discuss the usefulness of the mitochondrial control region for population genetic studies in Daphnia, with respect to sequence divergence and heteroplasmy.

Determination of polycyclic aromatic hydrocarbons in smoked pork by effect-directed bioassay with confirmation by chemical analysis.

Polycyclic aromatic hydrocarbons (PAHs) are generated during smoke curing and other heating treatments of food and represent a large class of chemical pollutants including a number of carcinogens. At present, PAHs are frequently detected by costly and time-consuming chemical analysis. Effect-directed in vitro cell-based bioassays of contaminants can offer a rapid, sensitive, and relatively inexpensive alternative for screening of contaminants in comparison to instrumental analysis. They enable estimation of total biological activity of all compounds acting through the same mode of binding. The aryl hydrocarbon receptor as a binding site plays an important role in PAH-induced carcinogenesis. The in vitro chemical-activated luciferase expression assay (using conditions to detect PAH) was investigated for its applicability for effect-directed analysis of PAH levels in smoked meat. There was an intra-assay variability of 0 to 15% and a mean coefficient of variation of 25% (3 to 50%) for the cleanup and bioassay analysis of the smoked pork samples. There was a correlation between the total responses of the bioassay and the individual amounts of the PAHs with a high molecular weight. The comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[k]fluoranthene used as standard in the in vitro chemical-activated luciferase expression assay resulted in benzo[k]fluoranthene being able to be used as an alternative, nontoxic standard in the bioassay. This bioassay is an applicable effect-directed functional prescreening method for the analysis of PAHs in smoked meat and appears to have potential in being used for food control in the future.

From sea to land and beyond--new insights into the evolution of euthyneuran Gastropoda (Mollusca).

BACKGROUND: The Euthyneura are considered to be the most successful and diverse group of Gastropoda. Phylogenetically, they are riven with controversy. Previous morphology-based phylogenetic studies have been greatly hampered by rampant parallelism in morphological characters or by incomplete taxon sampling. Based on sequences of nuclear 18S rRNA and 28S rRNA as well as mitochondrial 16S rRNA and COI DNA from 56 taxa, we reconstructed the phylogeny of Euthyneura utilising Maximum Likelihood and Bayesian inference methods. The evolution of colonization of freshwater and terrestrial habitats by pulmonate Euthyneura, considered crucial in the evolution of this group of Gastropoda, is reconstructed with Bayesian approaches. RESULTS: We found several well supported clades within Euthyneura, however, we could not confirm the traditional classification, since Pulmonata are paraphyletic and Opistobranchia are either polyphyletic or paraphyletic with several clades clearly distinguishable. Sacoglossa appear separately from the rest of the Opisthobranchia as sister taxon to basal Pulmonata. Within Pulmonata, Basommatophora are paraphyletic and Hygrophila and Eupulmonata form monophyletic clades. Pyramidelloidea are placed within Euthyneura rendering the Euthyneura paraphyletic. CONCLUSION: Based on the current phylogeny, it can be proposed for the first time that invasion of freshwater by Pulmonata is a unique evolutionary event and has taken place directly from the marine environment via an aquatic pathway. The origin of colonisation of terrestrial habitats is seeded in marginal zones and has probably occurred via estuaries or semi-terrestrial habitats such as mangroves.

The Trox-2 Hox/ParaHox gene of Trichoplax (Placozoa) marks an epithelial boundary.

Hox and ParaHox genes are implicated in axial patterning of cnidarians and bilaterians, and are thought to have originated by tandem duplication of a single "ProtoHox" gene followed by duplication of the resultant gene cluster. It is unclear what the ancestral role of Hox/ParaHox genes was before the divergence of Cnidaria and Bilateria, or what roles the postulated ProtoHox gene(s) played. Here we describe the full coding region, spatial expression and function of Trox-2, the single Hox/ParaHox-type gene identified in Trichoplax adhaerens (phylum Placozoa) and either a candidate ProtoHox or a ParaHox gene. Trox-2 is expressed in a ring around the periphery of Trichoplax, in small cells located between the outer margins of the upper and lower epithelial cell layers. Inhibition of Trox-2 function, either by uptake of morpholino antisense oligonucleotides or by RNA interference, causes complete cessation of growth and binary fission. We speculate that Trox-2 functions within a hitherto unrecognized population of possibly multipotential peripheral stem cells that contribute to differentiated cells at the epithelial boundary of Trichoplax.