A rapid, nondestructive, reproducible and cheap DNA extraction method from body mucus and buccal cells of northern pike and brown trout is described. Buccal cells and body mucus were sampled on FTA Cards; the captured DNA was used directly for microsatellite and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analyses. A complete concordance with control DNA was found. The genotyping error rate for microsatellite ranged from 1.9% to 3.3% for the northern pike and brown trout, respectively. This methodology, using for the first time these materials as a fish DNA source, combines speed of sampling and processing, with a twofold to a threefold time and costs saving.

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Non-destructive protocols for DNA isolation from fresh or preserved specimens are fundamental to study endangered or elusive species, breeders or samples sibship and specimens derived from public or private collections (Nielsen et al. 1999;Wasko et al. 2003;Hansen and Jensen 2005;Lucentini et al. 2006). Because of the low yield and poor quality DNA, particular laboratory care and standardizations were needed to allow reproducible results. We compared six DNA extraction procedures from conservative samples: three are commercial kits [Wizard Genomic DNA Purification Kit (WGDPK) (Promega); Wizard Magnetic DNA Purification System for Food (WMDPF) (Promega); NucleoSpin Food (NSF) (Macheray-Nagel)] and three are largely employed methodologies [Trizol (Life Technologies); Chelex (Sigma-Aldrich); C-TAB]. For each method, the standard protocol (reported on data sheets or the first published one) and several variations were tested varying sample storage and pre-lysis conditions, homogenization procedures, buffer solutions and concentrations, incubations and resuspension times and temperatures. DNA was extracted from 200 northern pikes (Esox lucius L.) and 100 brown trout (Salmo trutta fario L.) (Table 1) from small (£10 mg) fin pieces and 5-10 scales stored dried or in absolute alcohol both at )20°C and at room temperature. DNA extraction was carried out on fresh materials and repeated within a period of three years (Table 1), and from 34 years old dried scales culled from a museum collection. Furthermore, DNA was extracted from liver and muscle of both species, from individuals that had naturally died and used as controls. Spectrophotometric readings (260 and 280 nm) and densitometric evaluations (Image J) of DNAs obtained through different methodologies and protocols (conservative versus control DNA) were analyzed by means of ANOVA and t-test. DNA suitability was assayed through PCR-RFLP and microsatellite analyses (Lucentini et al. (2006) (Table 2). Microsatellites were run on ABI377 DNA sequencer whereas PCR-RFLPs were assayed on 2.5% agarose electrophoresis for two mtDNA NADH coding regions (ND-1 ND5/ 6) (Cronin et al. 1993). DNA stability and amplificability was controlled every three months, for three years, in parallel with control DNA. To evaluate genotyping errors, null alleles presence and allelic dropout, all the experiments were replicated and statistically treated as suggested (Hoffman and Amos 2005; Roon et al. 2005) through MICRO-CHECKER 2.2.3 (Van oosterhout et al. 2004). Deviations from the Hardy-Weinberg equilibrium were tested by Arlequin 2000 and Genepop.Our results indicate that the quality and quantity of DNA extracted from fin clips and scales varied according to extraction methods (Table 2) and storage conditions. Alcohol preservation at )20°C is fundamental for fin specimens, although dried scales conservation at room temperature allowed good DNA extraction. One percent

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A non-invasive genetic approach has been recently employed in the population genetics of wild species, using faeces that could be easily collected, particularly of elusive or endangered species. However, faeces of pine (Martes martes) and beech marten (M. foina) can be morphologically similar and could be confused with those of polecat (Mustela putorius) and red fox (Vulpes vulpes). On this basis, a rapid, simple and inexpensive RFLP protocol using a cytochrome b (Cyt b) gene fragment of mtDNA, isolated from faecal samples, was performed to distinguish the abovementioned species.

An apparent paradox is known for crustaceans, rotifers and bryozoans living in inland small fluviatilis (Linnaeus, 1759). We expected that system genetic variability would follow enclave distributions, no clear phylogeographical patterns would be present, and nearby unconnected water bodies would show markedly different populations for this new model too. We analysed the ribosomal internal transcribed spacer regions 5.8S-ITS2-28S, the D3 domain of 28S subunit, the mitochondrial Cytochrome c Oxidase I (COI) and ten specific microsatellite markers of nine Italian and one Hungarian populations. Mitochondrial and nuclear sequences showed no or very low genetic polymorphism, whereas high levels of differ-entiation among populations and a significant polymorphism were observed using microsatellites. Microsatellite loci also showed a high proportion of private alleles for each population and an overall correlation between geographic and genetic distances among populations. All the expectations from the monopolisation hypothesis seemingly were confirmed for the analysed sponge.

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