Genetic and Distribution Data of the Bramble Shark Echinorhinus brucus (Bonnaterre, 1788) and the Prickly Shark Echinorhinus cookei Pietschmann, 1928 to Better Reconstruct Their Conservation Status.

Elasmobranch species show low resilience in relation to anthropogenic stressors such as fishing efforts, loss of habitats, and climate change. In this sense, the elasmobranch populations appear to be at risk of extinction in many cases. Despite conservation researchers making efforts to implement knowledge, the information on the biology, reproduction, distribution, or genetic structure of some species is still scattered, often caused by the occurrence of species in inaccessible habitats. Echinorhinus brucus is a deep benthic shark evaluated as "Endangered" on which little information is available, particularly about its geographical range and genetic structure, while E. cookei is listed as "Data Deficient". Echinorhinus brucus belongs to the Echinorhinidae family, and its unique congeneric species is E. cookei. The main morphological diagnostic characteristic of both species is the presence of denticles with different shapes and patterns on the derma. In the present paper, mitochondrial COI and NADH2 sequences were retrieved from both E. brucus and E. cookei species, and analyses were conducted by applying different models of phylogenetic inference. Sequences of E. brucus captured in the Indian Ocean (IOS) did not cluster with the Atlantic E. brucus counterparts (AOS) but instead with E. cookei sequences; the different models showed an overlapping tree topology. Concurrently, a review of the historical and recent captures of the two species was carried out. The worldwide distribution of E. brucus excludes the Pacific Ocean area, where E. cookei occurs, and is characterised by presumably current local extinctions in the North Sea and the western Mediterranean Sea. The dataset describes two definite areas of significantly high abundance of E. brucus located in the Atlantic Ocean (Brazil) and the Indian Ocean (India). These areas suggest zones for conservation plans, especially considering the two lineages identified through molecular approaches.

The extinct Sicilian wolf shows a complex history of isolation and admixture with ancient dogs.

The Sicilian wolf remained isolated in Sicily from the end of the Pleistocene until its extermination in the 1930s-1960s. Given its long-term isolation on the island and distinctive morphology, the genetic origin of the Sicilian wolf remains debated. We sequenced four nuclear genomes and five mitogenomes from the seven existing museum specimens to investigate the Sicilian wolf ancestry, relationships with extant and extinct wolves and dogs, and diversity. Our results show that the Sicilian wolf is most closely related to the Italian wolf but carries ancestry from a lineage related to European Eneolithic and Bronze Age dogs. The average nucleotide diversity of the Sicilian wolf was half of the Italian wolf, with 37-50% of its genome contained in runs of homozygosity. Overall, we show that, by the time it went extinct, the Sicilian wolf had high inbreeding and low-genetic diversity, consistent with a population in an insular environment.

Effect of different packaging methods on the free amino acid profiles of the deep-water rose shrimp (Parapenaeus longirostris) during frozen storage.

The composition of free amino acids (FAAs) in seafood products contributes to characterizing their flavor, as well as freshness and quality during storage. Deep-water rose shrimps (Parapenaues longirostris, Lucas, 1846) (DWRS) are being increasingly harvested in the Mediterranean Sea, and the captured specimens are quickly frozen onboard fishing trawlers to preserve freshness and post-harvest quality. Here, we quantified the FAA profiles of DWRS packaged using five methods: (1) 100% N2; (2) vacuum; (3) 50% N2 + 50% CO2; (4) commercial anhydrous sodium sulfite; and (5) air (control). All samples were quickly frozen at -35°C and stored for 12 months at -18°C. Arginine (661 mg/100 g), proline (538 mg/100 g), and glycine (424 mg/100 g) were the most abundant FAAs, whereas the least abundant were tyrosine (67 mg/100 g), histidine (58 mg/100 g), and aspartic acid (34 mg/100 g). FAAs in all samples gradually (and significantly) increased in the first 6 to 8 months of storage, and then significantly decreased. The sodium sulfite treatment (Method 4) kept the initial FAA contents lower than the other treatments, due to the strong antioxidant action of sulfite agents. Interestingly, similar results were obtained for vacuum packaging (Method 2). Thus, combining frozen storage with vacuum packaging represents an alternative approach to chemical additives in shrimp/prawn processing to meet the increasing demand for high-quality seafood products with long shelf-life.

The invasive seaweed Asparagopsis taxiformis erodes the habitat structure and biodiversity of native algal forests in the Mediterranean Sea.

Invasive seaweeds are listed among the most relevant threats to marine ecosystems worldwide. Biodiversity hotspots, such as the Mediterranean Sea, are facing multiple invasions and are expected to be severely affected by the introduction of new non-native seaweeds in the near future. In this study, we evaluated the consequences of the shift from the native Ericaria brachycarpa to the invasive Asparagopsis taxiformis habitat on the shallow rocky shores of Favignana Island (Egadi Islands, MPA, Sicily, Italy). We compared algal biomass and species composition and structure of the associated epifaunal assemblages in homogenous and mixed stands of E. brachycarpa and A. taxiformis. The results showed that the biomass of primary producers is reduced by 90% in the A. taxiformis invaded habitat compared to the E. brachycarpa native habitat. The structure of the epifaunal assemblages displayed significant variations among homogenous and mixed stands. The abundance, species richness and Shannon-Wiener diversity index of the epifaunal assemblages decreased by 89%, 78% and 40%, respectively, from homogenous stands of the native E. brachycarpa to the invasive A. taxiformis. Seaweed biomass was the structural attribute better explaining the variation in epifaunal abundance, species richness and diversity. Overall, our results suggest that the shift from E. brachycarpa to A. taxiformis habitat would drastically erode the biomass of primary producers and the associated biodiversity. We hypothesize that a complete shift from native to invasive seaweeds could ultimately lead to bottom-up effects on rocky shore habitats, with negative consequences for the ecosystem structure, functioning, and the services provided.

Small-scale patches of detritus as habitat for invertebrates within a Zostera noltei meadow.

Seagrass detritus can attract numerous invertebrates as it provides food and substrate within the meadow or in adjacent environments. Nonetheless, several factors could modify the invertebrate response to this habitat. In this study, we tested if epifaunal colonisation of Zostera noltei detritus was related to substrate availability rather than food and whether colonising assemblages were similar according to the meadow structural complexity. Litterbags filled with natural or artificial detritus were deployed within an eelgrass meadow in a Mediterranean coastal lagoon (Thau lagoon, France). Colonisation appeared to be driven by the presence of detritus, with similar assemblages in natural and artificial substrate, but with more individuals than the empty bags, used as controls. There were also no differences according to habitat complexity. These findings show that detritus, acting as a faunal magnet, plays an important role in maintaining biodiversity, as epifauna is a critical trophic link between primary producers and consumers.

First Assessment of Plasticizers in Marine Coastal Litter-Feeder Fauna in the Mediterranean Sea.

Micro and nanoplastics are harmful to marine life due to their high level of fragmentation and resistance to degradation. Over the past two decades, marine coastal sediment has shown an increasing amount of microplastics being a sort of trap for debris wastes or chemicals. In such an environment some species may be successful candidates to be used as monitors of environmental and health hazards and can be considered a mirror of threats of natural habitats. Such species play a key role in the food web of littoral systems since they are litter-feeders, and are prey for fishes or higher trophic level species. A preliminary investigation was conducted on five species of small-sized amphipod crustaceans, with the aim to understand if such an animal group may reflect the risk to ecosystems health in the central Mediterranean area, recently investigated for seawater and fish contamination. This study intended to gather data related to the accumulation of plasticizers in such coast dwelling fauna. In order to detect the possible presence of xenobiotics in amphipods, six analytes were scored (phthalic acid esters and non-phthalate plasticizers), identified and quantified by the gas chromatography mass spectrometry (GC-MS) method. The results showed that among all the monitored contaminants, DEP and DiBP represented the most abundant compounds in the selected amphipods. The amphipod crustaceans analyzed were a good tool to detect and monitor plasticizers, and further studies of these invertebrates will help in developing a more comprehensive knowledge of chemicals spreading over a geographical area. The results are herein presented as a starting point to develop baseline data of plasticizer pollution in the Mediterranean Sea.

New records of amphipod crustaceans along the Israeli Mediterranean coast, including a rare Mediterranean endemic species, Maera schieckei Karaman & Ruffo, 1971.

BACKGROUND: A survey has been carried out at four Israeli rocky sites to evaluate the diversity of the amphipod fauna on various hard substrates, still scarcely monitored, as potential pabulum for amphipod crustacean species. NEW INFORMATION: A survey of shallow rocky reefs along the Mediterranean coast of Israel recovered 28 species and integrated the Amphipoda checklist for the country ofIsrael with 12 newly-recorded species. Such renewed national list includes Maera schieckei Karaman & Ruffo, 1971, a rare species endemic to the Mediterranean Sea, recorded here for the first time from the southern Levant Basin. The species, described from specimens collected in the Tyrrhenian Sea in 1970, has been only recorded eight times within the whole Mediterranean Sea. A revision of the bibliography on the distribution and ecology of M. schieckei showed that, although mentioned only for the western Mediterranean basin by some authors, it is listed in the checklist of amphipods of the Aegean Sea and neighbouring seas and has been found in the eastern Mediterranean basin since 1978. Maera schieckei was rarely found in the Mediterranean, one of the most studied marine biogeographic region as concerns the amphipod fauna; and the species seems to prefer bays or gulf areas. The role of updating and monitoring faunal composition should be re-evaluated.

Reducing the data-deficiency of threatened European habitats: Spatial variation of sabellariid worm reefs and associated fauna in the Sicily Channel, Mediterranean Sea.

Biogenic reefs, such as those produced by tube-dwelling polychaetes of the genus Sabellaria, are valuable marine habitats which are a focus of protection according to European legislation. The achievement of this goal is potentially hindered by the lack of essential empirical data, especially in the Mediterranean Sea. This study addresses some of the current knowledge gaps by quantifying and comparing multi-scale patterns of abundance and distribution of two habitat-forming species (Sabellaria alveolata and S. spinulosa) and their associated fauna along 190 km of coast on the Italian side of the Sicily Channel. While the abundance of the two sabellariids and the total number of associated taxa did not differ at any of the examined scales (from tens of centimetres to tens-100 of kilometres), the structure (composition in terms of both the identity and the relative abundance of constituting taxa) of the associated fauna and the abundance of several taxa (the polychaetes Eulalia ornata, Syllis pulvinata, S. garciai, Nereis splendida and Arabella iricolor, and the amphipods Apolochus neapolitanus, Tethylembos viguieri and Caprella acanthifera) varied among locations established ∼50-100 km apart. Syllis pulvinata also showed significant variation between sites (hundreds of metres apart), analogously to the other syllid polychaetes S. armillaris and S. gracilis, the nereidid polychaete Nereis rava, and the amphipod Gammaropsis ulrici. The largest variance of S. spinulosa, of the structure of the whole associated fauna and of 56% of taxa analysed individually occurred at the scale of replicates (metres apart), while that of the dominant bio-constructor S. alveolata and of 25% of taxa occurred at the scale of sites. The remaining 19% and the total richness of taxa showed the largest variance at the scale of locations. Present findings contribute to meet a crucial requirement of any future effective protection strategy, i.e., identifying relevant scales of variation to be included in protection schemes aiming at preserving representative samples not only of target habitats and organisms, but also of the processes driving such variability.

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.

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.

Overlooked cryptic endemism in copepods: systematics and natural history of the calanoid subgenus Occidodiaptomus Borutzky 1991 (Copepoda, Calanoida, Diaptomidae).

Our comprehension of the phylogeny and diversity of most inland-water crustaceans is currently hampered by their pronounced morphological bradytely, which contributed to the affirmation of the "Cosmopolitanism Paradigm" of freshwater taxa. However, growing evidence of the existence of cryptic diversity and molecular regionalism is available for calanoid copepods, thus stressing the need for careful morphological and molecular studies in order to soundly investigate the systematics, diversity and distribution patterns of the group. Diaptomid copepods were here chosen as model taxa, and the morphological and molecular diversity of the species belonging to the west-Mediterranean diaptomid subgenus Occidodiaptomus were investigated with the aim of comparing the patterns of morphological and molecular evolution in freshwater copepods. Three species currently lumped under the binomen Hemidiaptomus (Occidodiaptomus) ingens and two highly divergent clades within H. (O.) roubaui were distinguished, thus showing an apparent discordance between the molecular distances recorded and Occidodiaptomus morphological homogeneity, and highlighting a noteworthy decoupling between the morphological and molecular diversity in the subgenus. Current Occidodiaptomus diversity pattern is ascribed to a combined effect of ancient vicariance and recent dispersal events. It is stressed that the lack of sound calibration points for the molecular clock makes it difficult to soundly temporally frame the diversification events of interest in the taxon studied, and thus to asses the role of morphological bradytely and of accelerated molecular evolutionary rates in shaping the current diversity of the group.

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.

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

BACKGROUND: Little 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. RESULTS: Extensive 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. CONCLUSION: Our 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.