L. Gigliarelli

Geographical and seasonal evidence of cryptic diversity in the Baetis rhodani complex (Ephemeroptera, Baetidae) revealed by means of DNA taxonomy

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.

Molecular and Phenotypic Evidence of a New Species of Genus Esox (Esocidae, Esociformes, Actinopterygii): The Southern Pike, Esox flaviae

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.

Genetic characterization of a putative indigenous brown trout (Salmo trutta fario) population in a secondary stream of the Nera River Basin (Central Italy) assessed by means of three molecular markers

Genetic diversity was analysed in a putative autochthonous brown trout (Salmo trutta fario L.) population (Monterivoso Stream, Tyrrhenian Apennine Slope) by means of seven microsatellite loci and PCR‐RFLP of two mitochondrial (ND1 and ND5/6) and one nuclear (LDH‐C1*) locus. Monterivoso data were compared to those obtained analysing populations of the same basin (Nera River) and of the Po basin; Irish populations were used as a source of Atlantic strain brown trout. Haplotypes distributions, heterozygosity, F‐statistic and UPGMA analyses indicated a genetic diversification between these populations, suggesting a widespread alteration of the genetic structure due to repeated stocking with allochthonous material, mainly of Atlantic origin, that has partially polluted the Monterivoso population. This population appeared to be constituted of Mediterranean strain samples and might represent a residue of an indigenous pool: it shows high specificity characteristics and it is genetically separate from the others observed and from populations of the Adriatic slope of the Apennines. This population should be employed in managing and breeding programmes finalised to an eco‐sustainable restocking of brown trout in the Nera River Basin, with periodic monitoring by genetic analyses.

PCR-RFLP approaches to easily identify Pleuronectes platessa from other flatfishes: a rapid and efficient tool to control label information

European plaice (Pleuronectes platessa) is quite frequently substituted with other flatfish, especially with Yellow fin sole (Limanda aspera), which differs not only in meat quality but also particularly in its origin and manipulation chain. We propose an integrated approach of laboratory and in silico mtDNA PCR-RFLP procedures generating a set of restriction patterns easily resolvable in agarose gel, able to discriminate with certainty P. platessa from other 20 flatfish species. The herein proposed procedure is an economical and valid tool in detecting mislabelled seafoods.

Isolation of microsatellite markers in Squalius lucumonis (Bianco, 1983) and cross-species amplification within the family Cyprinidae and other freshwater fish species

Abstract The Etruscan chub Squalius lucumonis (Bianco, 1983) is an endemic endangered fish inhabiting the lakes and rivers of Central Italy. A microsatellite-enriched genomic library was constructed in order to develop microsatellite loci and eight polymorphic loci were isolated from this species to assess the level of genetic diversity in fifty individuals of S. lucumonis collected in the Umbria Region. The number of alleles ranged from four (Sluc12) to 18 (Sluc4). Observed and expected heterozygosity per locus ranged from 0.05 (Sluc12) to 0.80 (Sluc4; Sluc7 and Sluc11) and from 0.22 (Sluc12) to 0.90 (Sluc4), respectively. Furthermore, these loci were tested on ten other Cyprinidae species and four freshwater fish species belonging to other families. Successful cross-priming amplifications were obtained for several species of Cyprinidae, whereas no positive results were obtained for other species. The availability of the reported microsatellite loci will facilitate population structure investigations of these species aiming to phylogeographical approaches and conservation strategies.

Nuclear PCR-RFLP detects the brook chub, Squalius lucumonis (Leuciscinae: Cyprinidae), and related hybrids with other cyprinid species

Abstract The brook chub, Squalius lucumonis (Bianco, 1983), is an endemic endangered species inhabiting the rivers of Central Italy. Being subject to hybridizations with other cyprinids, particularly the congeneric S. squalus (Bonaparte, 1837), genetic overestimation can emerge when molecular markers are applied and mtDNA barcoding is not sufficient to ensure a correct attribution, due to its maternal inheritance. In this perspective, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach on rDNA is proposed to rapidly discriminate S. lucumonis from nine other cyprinids. For its capacity to identify hybrids of these species with S. lucumonis, this method should be considered as a fundamental tool to be applied prior to the application of other molecular markers in extensive genetic characterizations for phylogenetic, phylogeographic, population and management analyses.