Marco Arculeo

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.

Strong reproductive barriers in a narrow hybrid zone of West-Mediterranean green toads ( Bufo viridis subgroup) with Plio-Pleistocene divergence

BackgroundOne key question in evolutionary biology deals with the mode and rate at which reproductive isolation accumulates during allopatric speciation. Little is known about secondary contacts of recently diverged anuran species. Here we conduct a multi-locus field study to investigate a contact zone between two lineages of green toads with an estimated divergence time of 2.7 My, and report results from preliminary experimental crosses.ResultsThe Sicilian endemic Bufo siculus and the Italian mainland-origin B. balearicus form a narrow hybrid zone east of Mt. Etna. Despite bidirectional mtDNA introgression over a ca. 40 km North-South cline, no F1 hybrids could be found, and nuclear genomes display almost no admixture. Populations from each side of the contact zone showed depressed genetic diversity and very strong differentiation (FST = 0.52). Preliminary experimental crosses point to a slightly reduced fitness in F1 hybrids, a strong hybrid breakdown in backcrossed offspring (F1 x parental, with very few reaching metamorphosis) and a complete and early mortality in F2 (F1 x F1).ConclusionGenetic patterns at the contact zone are molded by drift and selection. Local effective sizes are reduced by the geography and history of the contact zone, B. balearicus populations being at the front wave of a recent expansion (late Pleistocene). Selection against hybrids likely results from intrinsic genomic causes (disruption of coadapted sets of genes in backcrosses and F2-hybrids), possibly reinforced by local adaptation (the ranges of the two taxa roughly coincide with the borders of semiarid and arid climates). The absence of F1 in the field might be due to premating isolation mechanisms. Our results, show that these lineages have evolved almost complete reproductive isolation after some 2.7 My of divergence, contrasting sharply with evidence from laboratory experiments that some anuran species may still produce viable F1 offspring after > 20 My of divergence.

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.

Morphological and molecular tools in identifying the Mediterranean limpets Patella caerulea, Patella aspera and Patella rustica

Allozyme electrophoresis, a partial nucleotide sequence of a mitochondrial gene for cytochrome oxidase I (COI) and discriminant analysis of shell morphometric characters were used to study the relationships among the Sicilian marine gastropods of the Patella genus. Allozyme and mtDNA markers unequivocally distinguished the species and were very useful markers in correctly classifying the different species when morphological characters overlapped each other. Several allozyme loci and many nucleotide positions were diagnostic of species. In contrast, the discriminant analysis of simple morphometric shell characters failed to adequately discriminate the species, suggesting that environmental factors influence colouration and morphological patterns in the Patella species. Our results underline the importance of a genetic approach, as compared to a morphological approach, in discriminating the Mediterranean Patella species.

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.

Where are you from, stranger? The enigmatic biogeography of North African pond turtles (Emys orbicularis)

The European pond turtle (Emys orbicularis) is a Nearctic element in the African fauna and thought to have invaded North Africa from the Iberian Peninsula. All North African populations are currently identified with the subspecies E. o. occidentalis. However, a nearly range-wide sampling in North Africa used for analyses of mitochondrial and microsatellite DNA provides evidence that only Moroccan populations belong to this taxon, while eastern Algerian and Tunisian pond turtles represent an undescribed distinct subspecies. These two taxa are most closely related to E. o. galloitalica with a native distribution along the Mediterranean coast of northern Spain through southern France to western and southern Italy. This group is sister to a clade comprising several mitochondrial lineages and subspecies of E. orbicularis from Central and Eastern Europe plus Asia, and the successive sisters are E. o. hellenica and E. trinacris. Our results suggest that E. orbicularis has been present in North Africa longer than on the Iberian Peninsula and that after an initial invasion of North Africa by pond turtles from an unknown European source region, there was a phase of diversification in North Africa, followed by a later re-invasion of Europe by one of the African lineages. The differentiation of pond turtles in North Africa parallels a general phylogeographic paradigm in amphibians and reptiles, with deeply divergent lineages in the western and eastern Maghreb. Acknowledging their genetic similarity, we propose to synonymize the previously recognized Iberian subspecies E. o. fritzjuergenobsti with E. o. occidentalis sensu stricto. The seriously imperiled Moroccan populations of E. o. occidentalis represent two Management Units different in mitochondrial haplotypes and microsatellite markers. The conservation status of eastern Algerian pond turtles is unclear, while Tunisian populations are endangered. Considering that Algerian and Tunisian pond turtles represent an endemic taxon, their situation throughout the historical range should be surveyed to establish a basis for conservation measures.

The stock genetic structure of two Sparidae species, Diplodus vulgaris and Lithognathus mormyrus, in the Mediterranean Sea

Abstract Polyacrilamide gel electrophoresis (PAGE) of allozymes was used to investigate the intraspecies genetic variation and the genetic stock structure of Diplodus vulgaris and Lithognathus mormyrus captured from eight localities in the Mediterranean Sea. Twenty-two and 20 putative enzyme-coding loci were examined, respectively, in D. vulgaris and L. mormyrus . Polymorphic loci at the 95% level were used to assess the allozyme variability in D. vulgaris ( AAT-2 ∗ , EST-1 ∗ , GLDH ∗ , PEPB-2 ∗ , PGI-2 ∗ , PGM ∗ , SDH ∗ ) and L. mormyrus ( AAT-2 ∗ , EST-1 ∗ , GLDH ∗ , MDH-2 ∗ , PGI-2 ∗ , PGM ∗ ). The proportion of polymorphic loci in both species ranged from 0.31 ( D. vulgaris ) to 0.30 ( L. mormyrus ), and the observed and expected mean heterozygosity varied between 0.082 and 0.093 ( D. vulgaris ) and between 0.069 and 0.072 ( L. mormyrus ). The mean value of observed heterozygosity in D. vulgaris showed a deficit of heterozygosites, thereby indicating a Wahlund effect in the samples examined. Significant genetic differentiation (mean value of θ =0.013, p D. vulgaris indicating an intraspecific genetic substructure among the samples examined, whereas the mean value of θ =0.001, p >0.05 found in L. mormyrus showed a high degree of genetic homogeneity. The results showed the presence of distinct subpopulations of D. vulgaris among the sampled sites, and suggested that analysis of allozymes may provide important information on the genetic stock structure of these two sparids to ensure sustainable management of these species.

Differences in gene flow in a twofold secondary contact zone of pond turtles in southern Italy (Testudines: Emydidae:Emys orbicularis galloitalica,E. o. hellenica,E. trinacris)

Using virtually range‐wide sampling for three pond turtle taxa (Emys orbicularis galloitalica, E. o. hellenica, E. trinacris), we analyse gene flow across their southern Italian contact zone. Based on population genetic analyses of 15 highly polymorphic microsatellite loci and a mitochondrial marker, we show that the general genetic pattern matches well with the current taxon delimitation. Yet, single individuals with conflicting genetic identity suggest translocation of turtles by humans. In addition, we identify in south‐western France and the vicinity of Rome populations being heavily impacted by introduced turtles. Cline analyses reveal that the major genetic break between E. o. galloitalica and E. o. hellenica corresponds well with the currently accepted intergradation zone in southern Italy. However, introgression is largely unidirectional from E. o. galloitalica into E. o. hellenica. In the distribution range of the latter subspecies, genetic footprints of E. o. galloitalica are evident along most of the Italian east coast. Our results corroborate that E. o. galloitalica was introduced long ago in Corsica and Sardinia and naturalized there. Gene flow between E. orbicularis and E. trinacris is negligible, with the Strait of Messina matching well with the narrow cline centre between the two species. This contrasts with other Mediterranean freshwater turtle species with extensive transoceanic gene flow. Compared to the two subspecies of E. orbicularis, the Sicilian E. trinacris shows an unexpectedly strong population structuring, a finding also of some relevance for conservation. The differences between the two taxon pairs E. orbicularis/E. trinacris and E. o. galloitalica/E. o. hellenica support their current taxonomic classification and make them attractive objects for follow‐up studies to elucidate the underlying mechanisms of speciation by comparing their properties.

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.