Fine-scale genetic structure, phylogeny and systematics of threatened crayfish species complex.

Systematic uncertainties in the crayfish Austropotamobius pallipes are well grounded by the number of species and subspecies described using different approaches, causing scientists to define this taxon as "complex". However, a key task that conservation programmes are facing regarding the recent and drastic decline of European populations, is the coherent systematic classification of this threatened species. Here we present results obtained by coupling mtDNA and genome analysis suggestive of a novel evolutionary framework to explain the relationships among phylogenetic lineages of A. pallipes. The direct sequencing of mtDNA COI gene fragment revealed a strong geographic structure with four distinct haplogroups separated by a range of 5-25 mutations. However, mitochondrial data were not supported by genomic fingerprinting based on 535 AFLP polymorphisms. Nuclear markers showed an unexpected moderate level of genetic differentiation and the absence of any geographic structure. Consequently, this study proposes that the taxonomic hypothesis of a single species of A. pallipes settling the Italian continental waters, is affected by complex evolutionary events. To solve the paradox, we hypothesized an evolutive scenario in which the separation of ancient mtDNA lineages likely occurred before the latest glacial periods. However, the speciation process remained incomplete due to secondary intensive postglacial contacts that forced the mingling of the genomes, and confounds the phylogeographic signature still detectable within mtDNA. Postglacial dispersion and the following demographic events, such as founder effects, drift and bottlenecks, abruptly depleted the local mtDNA variation, and shaped the current genetic population structure of white-clawed crayfish.

An improved protocol for the production of AFLP markers in complex genomes by means of capillary electrophoresis.

The amplified fragment-length polymorphism (AFLP) technology is a recently introduced method to investigate genomes of different complexity, from microbial to higher organisms. It is applied to purposes as diverse as identification of species, strain and varieties, investigation of genetic diversity within and between populations, simple and complex trait mapping, and construction of linkage and physical maps. This technology has been designed on the use of primers labelled with radioactivity and on AFLP fragment separation on sequencing gel. We show that the original EcoRI/TaqI AFLP protocol does not perform appropriately when transferred to fluorescent labelling and capillary electrophoresis (CE), and propose an improved protocol for the production of high-quality AFLP markers in fish, rodents and artiodactyles by means of the Beckman-Coulter CEQ2000 automatic DNA sequencer. In addition, we describe the procedure routinely used in our laboratory to obtain binary matrices from AFLP profiles with the aid of Genographer free-share software (vers. 1.6.0, J.J. Benham, Montana State University), able to elaborate original fragment data and convert them to standard graphical formats for phylogenetic analyses. Comparison with radioactive AFLPs in goats confirmed the reliability of the protocol developed for CE. In fact, 107 fragments generated by two primer combinations and identified by both techniques were attributed the same scoring. Compared with traditional methods, the use of capillary systems and automated analysis increases data throughput and scoring reliability, decreasing the overall experimental error.

AFLP to assess genetic variation in laboratory gerbils (Meriones unguiculatus).

The amplified fragment length polymorphism (AFLP) technique has been increasingly employed for characterizing inbred breeds of animals and detecting strain-specific polymorphisms. The majority of animals studies conducted in biomedical research are performed on rodent species, among which laboratory-reared Mongolian gerbils can be included. Despite the wide use of gerbils in scientific studies, their genetics has rarely been studied. Therefore we investigated the genetic differentiation of laboratory bred gerbils by means of AFLP markers. Six EcoRI/TaqI primer combinations were selected among 13 different combinations to assess the genetic polymorphisms in four stocks of animals: Charles River (CR), Harlan (Ha), Parma (Pr), and Crossbred (Cb). CR and Ha gerbils were purchased from commercial vendors, while Pr and Cb were derived from animals bred in our animal colony. A total of 228 fragments ranging between 70 and 650 bp were obtained. The mean percentage of polymorphic loci across primer combinations was 7.5%. Calculation of genetic distances through application of different algorithms (Nei's, BSI, and Jaccard's indexes) confirmed the poor genetic diversity between stocks. Nevertheless, a differentiation of the Pr and Cb stocks from the more homogeneous CR and Ha was revealed, in agreement with the different breeding derivation and management of the stocks.

Plutonium, 241Am, 90Sr, and 137Cs concentrations in some Antarctic matrices.

A radioecological survey in Antarctica shows that the 239 + 240Pu, 238Pu, 241Am, 90Sr, and 137Cs activities were detectable in nearly all the samples. The activity level of 239 + 240Pu, 241Am, and 137Cs in antarctic sediments was about 5-20 times lower than in the northern Adriatic Sea sediments, but the 238Pu activities were relatively high. It was interesting to note that the 90Sr concentrations in all the sediments tended to be low, which could be the result of the easier exchangeable behavior of 90Sr in water. High concentrations were detected in mosses and lichens and their activity levels were comparable to those in central Italy. The radionuclide ratio analyses show that the major part of 239 + 240Pu, 241Am, 90Sr, and 137Cs was a result of nuclear weapon tests. The higher 241Am/239 + 240Pu ratio was observed and it could perhaps be the result of fallout of nuclear weapon tests prior to 1962. The 238Pu/239 + 240Pu ratio in the antarctic matrices was about seven times higher than in the Northern hemisphere and it could be inferred that the major part of 238Pu was originating from the SNAP-9A satellite accident.