A set of 11 polymorphic markers (1 cleaved amplified polymorphic sequence (CAPS), 2 sequence-characterized amplified regions (SCARs), and 8 single-nucleotide polymorphism (SNP)-derived markers) was obtained for olive cultivar identification by comparing DNA sequences from different accessions. Marker development was more efficient, using sequences from the database rather than cloning arbitrary DNA fragments. Analyses of the sequences of 3 genes from 11 diverse cultivars revealed an SNP frequency of 1 per 190 base pairs in exons and 1 per 149 base pairs in introns. Most mutations were silent or had little perceptible effect on the polypeptide encoded. The higher incidence of transversions (55%) suggests that methylation is not the major driving force for DNA base changes. Evidence of linkage disequilibrium in 2 pairs of markers has been detected. The set of predominantly SNP-based markers was used to genotype 65 olive samples obtained from Europe and Australia, and was able clearly to discriminate 77% of the cultivars. Samples, putatively of the same cultivar but derived from different sources, were revealed as identical, demonstrating the utility of these markers as tools for resolving nomenclature issues. Genotyping data were used for constructing a dendrogram by UPGMA cluster analysis using the simple matching similarity coefficient. Relationships between cultivars are discussed in relation to the route of olive's spread.

Previous phylogenetic investigations on the mayfly Baetis rhodani Pictet from several European countries, excluding Italy, strongly suggested the presence of cryptic species. Our paper reports a DNA-taxonomy phylogenetic analysis of B. rhodani with additional populations coming from Italian and UK sites, and aims to identify potential cryptic species with a coalescent-based method (GMYC model) and to understand the mechanisms of local coexistence of cryptic species. Twenty-five haplotypes of Italian samples and five haplotypes of UK samples were identified and added to a large European dataset. A total of 11 potential cryptic species have been recognised, and three of them co-occured in one Italian area. Such cryptic species seem to be phylogenetically over-dispersed on the tree and temporally segregated, and the seasonal substitution pattern of cryptic species could explain the apparently widespread distribution of the B. rhodani complex and its ability to adapt to different temperatures and food resources, justifying some of the differences observed in the relationship between water temperature, growth rates and phenology documented from field studies.

We address the taxonomic position of the southern European individuals of pike, performing a series of tests and comparisons from morphology, DNA taxonomy and population genetics parameters, in order to support the hypothesis that two species of pike, and not only one, exist in Europe. A strong relationship emerged between a northern genotype supported by COI, Cytb, AFLP and specific fragments, and a phenotype with round spot skin colour pattern and a large number of scales in the lateral line, clearly separated from a southern genotype with other skin colour pattern and a low number of scales in the lateral line. DNA taxonomy, based on a coalescent approach (GMYC) from phylogenetic reconstructions on COI and Cytb together with AFLP admixture analysis, supported the existence of two independently evolving entities. Such differences are not simply due to geographic distances, as northern European samples are more similar to Canadian and Chinese samples than the southern Europe ones. Thus, given that the differences between the two groups of European pike are significant at the phenotypic, genotypic and geographical levels, we propose the identification of two pike species: the already known northern pike (Esox lucius) and the southern pike (E. flaviae n.sp.). The correct identification of these two lineages as independent species should give rise to a ban on the introduction of northern pikes in southern Europe for recreational fishing, due to potential problems of hybridisation.

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.

The rock partridge, Alectoris graeca, is a polytypic species declining in Italy mostly due to anthropogenic causes, including the massive releases of the closely related allochthonous chukar partridge Alectoris chukar which produced the formation of hybrids. Molecular approaches are fundamental for the identification of evolutionary units in the perspective of conservation and management, and to correctly select individuals to be used in restocking campaigns. We analyzed a Cytochrome oxidase I (COI) fragment of contemporary and historical A. graeca and A. chukar samples, using duplicated analyses to confirm results and nuclear DNA microsatellites to exclude possible sample cross-contamination. In two contemporary specimens of A. graeca, collected from an anthropogenic hybrid zone, we found evidence of the presence of mtDNA heteroplasmy possibly associated to paternal leakage and suggesting hybridization with captive-bred exotic A. chukar. These results underline significant limitations in the reliability of mtDNA barcoding-based species identification and could have relevant evolutionary and ecological implications that should be accounted for when interpreting data aimed to support conservation actions.

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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

1The Manila clam Ruditapes philippinarum -synonym Venerupis philippinarum (Adams and Reeve, 2 1850) is now one of the top 5 most commercially valuable bivalve species worldwide. Originally 3 from the Indo-Pacific region, it has been introduced in many countries for fisheries and aquaculture, 4 including estuarine environments along Atlantic and Mediterranean European coasts. Yet despite its 5 commercial value and widespread distribution, the precise origins of stocks remain speculative and 6 the genetic diversity of introduced populations is poorly known. Thus, the aim of this work was to