Sabrina Lo Brutto

Post-Messinian evolutionary relationships across the Sicilian channel: mitochondrial and nuclear markers link a new green toad from Sicily to African relatives.

BackgroundLittle attention has been paid to the consequences of the last landbridge between Africa and Sicily on Mediterranean biogeography. Previous paleontological and scarce molecular data suggest possible faunal exchange later than the well-documented landbridge in the Messinian (5.3 My); however, a possible African origin of recent terrestrial Sicilian fauna has not been thoroughly tested with molecular methods. To gain insight into the phylogeography of the region, we examine two mitochondrial and two nuclear markers (one is a newly adapted intron marker) in green toads (Bufo viridis subgroup) across that sea barrier, the Strait of Sicily.ResultsExtensive sampling throughout the western Mediterranean and North Africa revealed a deep sister relationship between Sicilian (Bufo siculus n.sp.) and African green toads (B. boulengeri) on the mitochondrial and nuclear level. Divergence times estimated under a Bayesian-coalescence framework (mtDNA control region and 16S rRNA) range from the Middle Pliocene (3.6 My) to Pleistocene (0.16 My) with an average (1.83 to 2.0 My) around the Pliocene/Pleistocene boundary, suggesting possible land connections younger than the Messinian (5.3 My). We describe green toads from Sicily and some surrounding islands as a new endemic species (Bufo siculus). Bufo balearicus occurs on some western Mediterranean islands (Corsica, Sardinia, Mallorca, and Menorca) and the Apennine Peninsula, and is well differentiated on the mitochondrial and nuclear level from B. siculus as well as from B. viridis (Laurenti), whose haplotype group reaches northeastern Italy, north of the Po River. Detection of Calabrian B. balearicus haplotypes in northeastern Sicily suggests recent invasion. Our data agree with paleogeographic and fossil data, which suggest long Plio-Pleistocene isolation of Sicily and episodic Pleistocene faunal exchange across the Strait of Messina. It remains unknown whether both species (B. balearicus, B. siculus) occur in sympatry in northern Sicily.ConclusionOur findings on green toads give the first combined mitochondrial and nuclear sequence evidence for a phylogeographic connection across the Strait of Sicily in terrestrial vertebrates. These relationships may have implications for comparative phylogeographic research on other terrestrial animals co-occurring in North Africa and Sicily.

DNA barcoding for species assignment: the case of Mediterranean marine fishes.

Background DNA barcoding enhances the prospects for species-level identifications globally using a standardized and authenticated DNA-based approach. Reference libraries comprising validated DNA barcodes (COI) constitute robust datasets for testing query sequences, providing considerable utility to identify marine fish and other organisms. Here we test the feasibility of using DNA barcoding to assign species to tissue samples from fish collected in the central Mediterranean Sea, a major contributor to the European marine ichthyofaunal diversity. Methodology/Principal Findings A dataset of 1278 DNA barcodes, representing 218 marine fish species, was used to test the utility of DNA barcodes to assign species from query sequences. We tested query sequences against 1) a reference library of ranked DNA barcodes from the neighbouring North East Atlantic, and 2) the public databases BOLD and GenBank. In the first case, a reference library comprising DNA barcodes with reliability grades for 146 fish species was used as diagnostic dataset to screen 486 query DNA sequences from fish specimens collected in the central basin of the Mediterranean Sea. Of all query sequences suitable for comparisons 98% were unambiguously confirmed through complete match with reference DNA barcodes. In the second case, it was possible to assign species to 83% (BOLD-IDS) and 72% (GenBank) of the sequences from the Mediterranean. Relatively high intraspecific genetic distances were found in 7 species (2.2%–18.74%), most of them of high commercial relevance, suggesting possible cryptic species. Conclusion/Significance We emphasize the discriminatory power of COI barcodes and their application to cases requiring species level resolution starting from query sequences. Results highlight the value of public reference libraries of reliability grade-annotated DNA barcodes, to identify species from different geographical origins. The ability to assign species with high precision from DNA samples of disparate quality and origin has major utility in several fields, from fisheries and conservation programs to control of fish products authenticity.

Genetic architecture of the marbled goby Pomatoschistus marmoratus (Perciformes, Gobiidae) in the Mediterranean Sea.

The marbled goby Pomatoschistus marmoratus, a species inhabiting coastal Mediterranean lagoons, has been studied by measuring its mitochondrial DNA variation. This analysis revealed a Mediterranean west vs east split and, subsequently, an eastern differentiation among the Libyan-Tunisian Gulf, the Adriatic Sea and the Aegean Sea. The high cohesion between the samples collected in the vast area of western Mediterranean contrasts with the genetic mosaic of the more sub-structured eastern Mediterranean. This western homogeneity can not yet be fully explained even if a human-mediated migratory flow, due to a maritime traffic, has been posited. The pattern in the eastern basin revealed a genetic architecture possibly due to the non-migratory habit of the gobid. Within this perspective, the role of the Mediterranean lagoon habitat should be related to how much it amplifies the effects of historical (e.g. past sea-level changes) and environmental (e.g. present-day hydrographic regime) processes as regards the genetic structure of the inhabiting species.

Molecular evidence for the presence of cryptic evolutionary lineages in the freshwater copepod genus Hemidiaptomus G.O. Sars, 1903 (Calanoida, Diaptomidae)

The pattern of morphological and mtDNA cytochrome b diversity of three calanoid copepod species belonging to the diaptomid genus Hemidiaptomus has been investigated with the aim of checking the reliability of the morphological characters currently used for species identification, and the possible presence of cryptic taxa. A sharply different molecular structuring has been observed in the studied species: while Hemidiaptomus amblyodon exhibits a remarkable constancy throughout the European range of its distribution area (maximum inter-populations cytochrome b divergence of 3%), observed distances between presumed conspecific lineages of Hemidiaptomus gurneyi (maximum divergence of 21.5%) and Hemidiaptomus ingens (maximum 19.1%) suggest that under these binomens are in fact included complexes of cryptic, or currently just unrecognized, independent evolutionary lineages. The application of the “4x rule” shows that the two lineages singled out within H. ingens are in fact independent evolutionary units, while the complex molecular structure observed in H. gurneyi s.l. could not be resolved based on the currently available data. Applying standard crustacean mtDNA evolutionary rates to the observed divergence values, the separation of the main lineages within both H. ingens and H. gurneyi might dates back to the Miocene; however, it has also to be considered that the rate of mtDNA evolution might be accelerated in copepods, as already observed in other arthropod taxa. Present results gives further evidences of the high potential for copepod speciation with no or little morphological changes, and stress the need of a revision of the most controversial Palaearctic diaptomid genera.

Improving the Conservation of Mediterranean Chondrichthyans: The ELASMOMED DNA Barcode Reference Library

Cartilaginous fish are particularly vulnerable to anthropogenic stressors and environmental change because of their K-selected reproductive strategy. Accurate data from scientific surveys and landings are essential to assess conservation status and to develop robust protection and management plans. Currently available data are often incomplete or incorrect as a result of inaccurate species identifications, due to a high level of morphological stasis, especially among closely related taxa. Moreover, several diagnostic characters clearly visible in adult specimens are less evident in juveniles. Here we present results generated by the ELASMOMED Consortium, a regional network aiming to sample and DNA-barcode the Mediterranean Chondrichthyans with the ultimate goal to provide a comprehensive DNA barcode reference library. This library will support and improve the molecular taxonomy of this group and the effectiveness of management and conservation measures. We successfully barcoded 882 individuals belonging to 42 species (17 sharks, 24 batoids and one chimaera), including four endemic and several threatened ones. Morphological misidentifications were found across most orders, further confirming the need for a comprehensive DNA barcoding library as a valuable tool for the reliable identification of specimens in support of taxonomist who are reviewing current identification keys. Despite low intraspecific variation among their barcode sequences and reduced samples size, five species showed preliminary evidence of phylogeographic structure. Overall, the ELASMOMED initiative further emphasizes the key role accurate DNA barcoding libraries play in establishing reliable diagnostic species specific features in otherwise taxonomically problematic groups for biodiversity management and conservation actions.

Climate change and population genetic structure of marine species

Climate change influences populations by reducing or extirpating local populations, by disrupting patterns of migration and by shifting geographical distributions. These events can affect genetic population structure in several ways. Molecular markers have been used in numerous population genetic and phylogeographical studies of marine species and have detected population responses to climate change in the last few decades, such as range expansions, adaptative shifts and declines or increases in abundance. Little is known, however, about the molecular and physiological basis of adaptive responses to climate change in marine Mediterranean species. The Mediterranean Sea ecosystem is a ‘living laboratory’ with native species that are challenged by environmental change and by invasive species and a ‘gene-climate’ approach should be adopted as a way of focusing on the relationship between climate warming and genetic diversity.

Cryptic invasion in Southern Europe: The case of Ferrissia fragilis (Pulmonata: Ancylidae) Mediterranean populations

Four populations of the North American ancylid gastropod Ferrissia fragilis have been found in Southern Italy, thus expanding the known Eurasian distribution range of this invasive gastropod to the Mediterranean area. Both mitochondrial markers used for the molecular identification of the sampled specimens (16S and COI) showed a modest to absent haplotypic diversity in the studied Ferrissia populations from Europe and Asia, and their perfect identity with some of the haplotypes observed in North American F. fragilis populations. In the light of the scarce molecular diversity observed in the whole Palaearctic area and of the growing evidences for the ongoing spreading of the species in Europe, the occurrence of a single invasion event from North America possibly linked with aquarium plant trade followed by a rapid spreading of the species in Eurasia is suggested. The record of F. fragilis in Southern Italy caused some doubts on the presence of autochthonous Ferrissia populations in Europe and on the actual existence of Ferrissia wautieri. A molecular analysis of Ferrissia specimens from the loci typici of F. wautieri (Northern Italy) and F. clessiniana (Egypt) is definitely opportune.

Allozymic variation in Mediterranean hake Merluccius merluccius (Gadidae)

Abstract Four hundred and twenty individual hake from 10 sample sites in the Mediterranean Sea were analysed in order to study genetic variability and identify genetic stock structure. Twenty loci were identified, four of which were polymorphic at the 95% level: ADH*, PGI‐1*, PGI‐2* and SOD‐1*. Average observed and expected heterozygosity were 0.084 and 0.090, respectively. PG1–1* deviated from Hardy‐Weinberg expectations due to an excess of heterozygotes and F‐statistic analysis showed also a significant excess of heterozygosity at SOD‐1*. FST was not significant for each locus except for PGI‐2*, where a single sample from the Channel of Sicily (C5) showed a different pattern in allelic frequencies, probably caused by a stochastic event. The samples analyzed seemed to be homogeneous, but future analyses may be necessary using other complementary molecular markers.

Overlapping patterns of morphometric and genetic differentiation in the Mediterranean goby Pomatoschistus tortonesei Miller, 1968 (Perciformes, Gobiidae) in Tunisian lagoons

The genetic and morphological variations of Pomatoschistus tortonesei Miller, 1968 were studied in samples collected from three Tunisian lagoons. The morphological analysis included 18 morphometric measurements and was based on linear discriminant analysis (LDA), whereas the genetic analysis was based on the 16S-rRNA and COI mitochondrial genes. Both analyses differentiated the populations and demonstrated consistently a well-supported differentiation between the western Mediterranean samples (Bizerta and Tunis South lagoons) and the eastern Mediterranean sample (El Bibane lagoon). The observed differentiation could be explained in terms of the geographic isolation of the various populations and the influence of environmental factors, which differ greatly between the different sites. The molecular results revealed that the populations are characterised by unique haplotypes which are well defined in relation to limited gene flow and restricted dispersal abilities. Additionally, it seems that local selective pressures have modelled biometrical variation. Morphological results can reflect a differential habitat use revealed in the cephalic features and a different response to hydrodynamic constraints developed in dissimilar dorsal and pelvic fin lengths.

An additional record of Kyphosus vaigiensis (Quoy & Gaimard, 1825) (Osteichthyes, Kyphosidae) from Sicily clarifies the confused situation of the Mediterranean kyphosids

The lowfin chub, Kyphosus vaigiensis, is reported for the first time off Favignana Island, Sicily, central Mediterranean Sea. The specimen was identified on the basis of morphometric and meristic characters as well as mitochondrial DNA sequences (COI and 16S-rDNA). Two, perhaps three, Kyphosus species-K. bigibbus, K. sectatrix and K. vaigiensis-have been occasionally recorded in the Mediterranean. These species occur both in the Atlantic and Indo-Pacific regions but it is likely they entered the Mediterranean through the Strait of Gibraltar. However, it is unclear whether they have established reproductive native populations in the Mediterranean.