Marco Casu

Small-scale morphological and genetic differentiation in the Mediterranean killifish Aphanius fasciatus (Cyprinodontidae) from a coastal brackish-water pond and an adjacent pool in northern Sardinia

Two samples of Aphanius fasciatuscollected in the Pilo pond (northern Sardinia, Italy) and in an adjacent pool of small surface area were analysed morphologically (235 individuals) and genetically (a subsample of 58 individuals). The aims of the present study were (i) to test the hypothesis that different predation pressures may be associated with morphological and/or genetic differences between samples from each habitat and (ii) to assess the level of divergence between the two populations. Morphological analysis was based on the relative size of fins because it has been shown to be associated with predation pressure. The relative caudal fin area (caudal fin area/total body surface) was smaller in specimens from the pool, in both males and females, whereas no differences were found for the dorsal and anal fin areas. Caudal fins with higher aspect ratio (fin depth/fin length) were found in fish from the pool but not in the pond, due to a higher fin depth. We hypothesised that specimens from the pool would show smaller caudal fin area, since they are subject to lower predation pressure. Random amplification of polymorphic DNA (RAPD) analysis revealed a relatively high degree of both within- and between-sample genetic heterogeneity. The pond and pool samples exhibited heterozygosities, which did not differ significantly by t-test. Between-sample genetic divergence was highlighted by the coancestry coefficient (h = 0.301 ± 0.059, P < 0.001) and analysis of molecular variance (AMOVA) (variance between sites = 41%, P < 0.001). Genetic divergence between sites with a relatively high genetic diversity within both samples suggested that the population in the pool did not originate from a single colonisation event with a small number of founders. The genetic divergence between the two populations is consistent with their differences in fin size.

Mitochondrial DNA reveals genetic structuring of Pinna nobilis across the Mediterranean Sea.

Pinna nobilis is the largest endemic Mediterranean marine bivalve. During past centuries, various human activities have promoted the regression of its populations. As a consequence of stringent standards of protection, demographic expansions are currently reported in many sites. The aim of this study was to provide the first large broad-scale insight into the genetic variability of P. nobilis in the area that encompasses the western Mediterranean, Ionian Sea, and Adriatic Sea marine ecoregions. To accomplish this objective twenty-five populations from this area were surveyed using two mitochondrial DNA markers (COI and 16S). Our dataset was then merged with those obtained in other studies for the Aegean and Tunisian populations (eastern Mediterranean), and statistical analyses (Bayesian model-based clustering, median-joining network, AMOVA, mismatch distribution, Tajima’s and Fu’s neutrality tests and Bayesian skyline plots) were performed. The results revealed genetic divergence among three distinguishable areas: (1) western Mediterranean and Ionian Sea; (2) Adriatic Sea; and (3) Aegean Sea and Tunisian coastal areas. From a conservational point of view, populations from the three genetically divergent groups found may be considered as different management units.

Population structure of the Monocelis lineata (Proseriata, Monocelididae) species complex assessed by phylogenetic analysis of the mitochondrial Cytochrome c Oxidase subunit I (COI) gene

Monocelis lineata consists of a complex of sibling species, widespread in the Mediterranean and Atlantic Ocean. Previous genetic analysis placed in evidence at least four sibling species. Nevertheless, this research was not conclusive enough to fully resolve the complex or to infer the phylogeny/phylogeography of the group. We designed specific primers aiming at obtaining partial sequences of the mtDNA gene Cytochrome c Oxidase subunit I (COI) of M. lineata, and have identified 25 different haplotypes in 32 analyzed individuals. The dendrogram generated by Neighbor-Joining analysis confirmed the differentiation between Atlantic and Mediterranean siblings, as well as the occurrence of at least two Mediterranean sibling species. Thus validated, the method here presented appears as a valuable tool in population genetics and biodiversity surveys on the Monocelis lineata complex.

Genetic characterization of Fasciola hepatica from Tunisia and Algeria based on mitochondrial and nuclear DNA sequences

Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the present study, samples identified morphologically as Fasciola hepatica from sheep and cattle from different geographical locations of Tunisia and Algeria were genetically characterised by sequences of the first (ITS-1), the 5.8S and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) and mitochondrial Cytochrome c Oxidase subunit I (COI) gene. Comparison of the ITS and COI sequences of the North African samples with sequences of Fasciola spp. from GenBank confirmed that all samples from Tunisia and Algeria samples belong to a single species, namely F. hepatica. Several specimens from Tunisia and Algeria showed a substitution C/T in position 859 in the ITS-2 sequences, previously reported from Spain, suggesting that the above mentioned variant may have a common origin and spread recently throughout the three countries because of movement of infected animals. This is the first molecular characterization of F. hepatica in North Africa which provides a foundation for further studies on Fasciola spp. in Tunisia and Algeria.

Genetic structure of Octopus vulgaris (Mollusca, Cephalopoda) from the Mediterranean Sea as revealed by a microsatellite locus

Abstract An investigation of the genetic variability of Octopus vulgaris, an intensively harvested species, was carried out using a mi crosatellite locus as genetic marker. Samples from one eastern At lantic and nine Mediterranean locations were analysed. In each population, the number of alleles at locus Ov06 varied from four to seven and was 21 overall. Observed and expected heterozy‐gosity values ranged from 0.310 to 0.655 and 0.506 to 0.841, re spectively. Permutation tests and the positive average value of FIS showed significant departures from the Hardy‐Weinberg equilibri um, due to a deficit of heterozygotes. FST showed high levels of genetic divergence among the populations. Genetic distance val ues ranged from 0.0004 to 7.1520. Isolation‐by‐distance was not evident either by the Mantel test or multidimensional scaling. Mi crosatellite results are consistent with a previous allozyme study, and suggest that the common octopus does not form a single panmictic unit in the Mediterranean. From a fishery perspective, this information leads to the conclusion that the management of O. vulgaris should be planned on a local level.

Combined analysis of four mitochondrial regions allowed the detection of several matrilineal lineages of the lessepsian fish Fistularia commersonii in the Mediterranean Sea

The bluespotted cornetfish ( Fistularia commersonii ) is an Indo-Pacific species that in the last ten years colonized a large part of the Mediterranean basin. The aim of this study was to sequence some portions of the mitochondrial DNA (D-loop II, 16S, 12S and Cyt b) of this fish from different localities of the Mediterranean Sea, in order to evaluate the level of its genetic variability in this area. The genetic analysis performed on specimens from seven localities of Sardinia, Tunisia and Libya revealed the presence of at least five mitochondrial lineages. The results obtained, compared with previous studies, indicate that the use of a sufficient number of mitochondrial regions may allow a more accurate estimate of genetic variability in lessepsian invasions.

Taxonomic distinction of Ophelia barquii and O. bicornis (Annelida: Polychaeta) in the Mediterranean as revealed by ISSR markers and the number of nephridiopores

Ophelia bicornis sensu lato is a polychaete living in intertidal sandy habitats of Mediterranean and European Atlantic coasts, whose systematics have been strongly debated in the past few decades. In the present work the count of nephridiopores was coupled with genetic analysis carried out with DNA markers (inter simple sequence repeats) for a total of 30 individuals collected at six Italian beaches. Exact test, analysis of molecular variance, non-metric multidimensional scaling and assignment tests clearly separated individuals with five nephridiopore pairs from those with six pairs. This finding validated results of a recent allozyme study in which O. bicornis sensu lato was split into O. bicornis sensu stricto (six nephridiopore pairs) and O. barquii (five nephridiopore pairs). This paper represents a further contribution to the estimation of biodiversity within marine invertebrates.

Patterns of spatial genetic structuring in the endangered limpet Patella ferruginea: implications for the conservation of a Mediterranean endemic

Patella ferruginea Gmelin, 1791 is an endangered marine gastropod endemic to the Western Mediterranean. Its range is restricted to the Sardinian-Corsican region (SCR), North Africa, a few scattered sites in Southern Spain, and Sicily. Inter-simple sequence repeat (ISSR) markers and three different mitochondrial DNA (mtDNA) regions, Cytochrome c Oxidase subunit I, 12S (small-subunit ribosomal RNA gene) and 16S (large-subunit ribosomal RNA gene), were used to investigate the presence of genetic population structuring. The mtDNA sequences showed very low levels of genetic differentiation. Conversely, ISSRs showed the presence of two main genetic groups, corresponding to Spain, North Africa and Sicily and the SCR. The SCR was further split into two subgroups. The ISSR results suggest that, on a regional scale, the genetic structure of P. ferruginea is mainly determined by the restriction of gene flow by dispersal barriers. On a more local scale human harvesting may play a crucial role in population structuring by increasing the effect of genetic drift.

PCR-RFLP: a practical method for the identification of specimens of Patella ulyssiponensis s.l. (Gastropoda: Patellidae )

Abstract Patella ulyssiponensis s.l. is widely distributed in the Mediterranean as well as in the north-eastern Atlantic, from southern Norway to north-western Africa, including Macaronesia. Throughout its range, P. ulyssiponensis s.l. shows a high degree of variability in the shape and colour pattern of the shell and in the colour pattern of the foot. In addition, the species demonstrates widely overlapping morphological parameters with the coexisting P. caerulea (in the Mediterranean), P. vulgata (in north-eastern Atlantic) and P. candei (in the Macaronesian islands). Thus P. ulyssiponensis s.l. identification proves to be difficult. So far, method to unequivocally identify uncertain specimens of P. ulyssiponensis s.l. from the coexisting species was using a molecular taxonomy approach based on the Cytochrome c Oxidase subunit I (COI) sequences analysis. The aim of the present study was to identify specific PCR-RFLP (Restriction Fragment Length Polymorphism PCR-based) markers on the COI sequences which could be used as an alternative and fast diagnostic tool for the discrimination of P. ulyssiponensis s.l. from P. caerulea, P. vulgata and P. candei, when the morphology of the coexisting individuals provided equivocal information. First, we sampled 65 Patella spp. and classified these by means of the COI sequencing. Then, we selected the TaqI endonuclease for the specific RFLP reaction, which produced a combination of RFLP electrophoretic bands, allowing an unequivocal identification of the individuals of P. ulyssiponensis s.l.

Patterns of spatial genetic variation in Patella ulyssiponensis: insights from the western Mediterranean marine ecoregion

Patterns of genetic variation in marine species reflect the interplay of species-specific traits, oceanographic features, historical processes and selection. In the Atlantic–Mediterranean regions, Patella ulyssiponensis (Mollusca: Gastropoda) was previously used as a model to investigate these patterns. Our study gained insight into the genetic patterns of P. ulyssiponensis in the western Mediterranean marine ecoregion (WME), by means of ISSRs and COI. We evaluated the genetic structure of the WME with respect to the Atlantic and eastern Mediterranean, as well as the occurrence of further structuring within this ecoregion. Both population- and individual-based analysis evidenced that WME does not appear to be sharply isolated from the adjacent regions. Within the WME, P. ulyssiponensis displays a pattern of genetic structure that may reflect a chaotic patchiness scenario: structuring is neither related to coastal distance nor to other factors that may constrain dispersal. Compared to the congener P. ferruginea, P. ulyssiponensis shows weaker spatial genetic structure, which may reflect a higher dispersal potential coupled with greater population effective size. Whilst other processes that may have influenced the genetic pattern in the WME remain to be cleared, a recent range expansion coupled with species-specific traits favouring larval dispersal may have contributed to the genetic structuring.