DNA barcoding taxonomy and diversity of the Mediterranean sharks and rays

Elasmobranchs are an important by-catch of commercial fisheries targeting bony fishes. Fisheries targeting sharks are rare, but usually almost all specimen bycatched are marketed. They risk extinction if current fishing pressure continues (Ferretti et al., 2008). Accurate species identification is critical for the design of sustainable fisheries and appropriate management plans, especially since not all species are equally sensitive to fishing pressure (Walker & Hislop 1998). The identification of species constitutes the first basic step for biodiversity monitoring and conservation (Dayrat B et al., 2005). More recently, mtDNA sequencing has also been used for species identification and its use has become widespread under the DNA Barcode initiative (e.g. Hebert et al. 2003a, 2003b; Ward et al. 2005, 2008a; Moura et al 2008; Steinke et al. 2009). The aims of this work were: 1) identify sharks and skates species using DNA barcode; 2) compare species of different provenance; 3) use DNA barcode for misidentified species. Using DNA barcode 15 species of sharks (Alopias vulpinus, Centrophorus granulosus, Cetorhinus maximus, Dalatias licha, Etmopterus spinax, Galeorhinus galeus, Galeus melastomus, Heptranchias perlo, Hexanchus griseus, Mustelus mustelus, Mustelus punctulatus, Oxynotus centrina, Scyliorhinus canicula Squalus acanthias, Squalus blainville), 1 species of chimaera (Chimaera monstrosa) and 21 species of rays/skayes (Dasyatis centroura, Dasyatis pastinaca, Dasyatis sp., Dipturus nidarosiensis, Dipturus oxyrinchus, Leucoraja circularis, Leucoraja melitensis, Myliobatis aquila, Pteromylaeus bovinus, Pteroplatytrygon violacea, Raja asterias, Raja brachyura, Raja clavata, Raja miraletus, Raja montagui, Raja radula, Raja polystigma, Raja undulata, Rostroraja alba, Torpedo marmorata, Torpedo nobiliana, Torpedo torpedo) was identified.

Genetic structure and connectivity between populations of two common Mediterranean sessile invertebrates

Population genetic and phylogeography of two common mediterranean species were studied in 10 localities located on the coasts of Toscana, Puglia and Calabria. The aim of the study was to verify the extent of genetic breaks, in areas recognized as boundaries between Mediterranean biogeographic sectors. From about 100 sequences obtained from the mitochondrial Cytochrome Oxidase subunit I (COI) gene of Halocynthia papillosa and Hexaplex trunculus genetic diversity, genetic structure at small and large distances and demographic history of both specieswere analyzed. No evidences of genetic breaks were found for the two species in Toscana and Puglia. The genetic structure of H. trunculus evidences the extent of a barrier to gene flow localized in Calabria, which could be represented by the Siculo-Tunisian Strait and the Strait of Messina. The observed patterns showed similar level of gene flow at small distances in both species, although the two species have different larval ecology. These results suggest that other factors, such as currents, local dynamics and seasonal temperatures, influence the connectivity along the Italian peninsula. The geographic distribution of the haplotypes shows that H. papillosacould represent a single genetic pool in expansion, whereas H. trunculus has two distinct genetic pools in expansion. The demographic pattern of the two species suggests that Pleistocene sea level oscillations, in particular of the LGM, may have played a key role in shaping genetic structure of the two species. This knowledge provides basic information, useful for the definition of management plans, or for the design of a network of marine protected areas along the Italian peninsula.

Molecular phylogenesis of Mediterranean Octocorals

In order to support the conservation of the Mediterranean octocorals improvements on information regarding their taxonomic units and phylogenetic relationships are strongly needed. In the present thesis work, phylogenetic analyses based on the mitochondrial mtMSH and 16S genes were performed including 15 Mediterranean octocorals species on the 56 recognized to date. Moreover, an extended datasets with Atlanto/Pacific congeners Octocorallia species was implemented to clarify their phylogenetic relationships and estimate the divergence times of the Mediterranean species. Results indicated that: 1) there are similarity and differences among molecular and morphological traits depending on the taxonomical level considered; 2) the molecular phylogeny of the Mediterranean octocorals retrace the previous relationships based on wide octocorals analyses; and 3) the divergence time among Mediterranean and Atlanto/Pacific species varies depending on analysed taxa. At higher taxonomic level, the Mediterranean trees supported the division of the Mediterranean Octocorallia into one major clade (Alcyoniina-Holaxonia) plus two unresolved branch including the single species available of Scleraxonia and Stolonifera respectively. This topology was better supported including the Atlanto/Pacific congeners species. The molecular evidence suggested that Alcyonium palmatum and Corallium rubrum species are the youngest with a divergence time estimated around 4 MYA. Particularly, C. rubrum results were in agreement with the hypothesis that recent orogenesis process of the Mediterranean Sea promoted the allopatric speciation of this specie. Increasing the sample design and implementing the emerging next-generation genomic-sequencing technologies, further studies would be able to improve the understanding of the Mediterranean octocorals phylogenetic relationships and evolution.

Genetic characterisation and global comparison of ancient Mediterranean Great White Sharks population (Carcharodon carcharias, L.1758)

This study poses as its objective the genetic characterization of the ancient population of the Great White shark, Carcharodon carcharias, L.1758, present in the Mediterranean Sea. Using historical evidence, for the most part buccal arches but also whole, stuffed examples from various national museums, research institutes and private collections, a dataset of 18 examples coming from the Mediterranean Sea has been created, in order to increase the informations regarding this species in the Mediterranean. The importance of the Mediterranean provenance derives from the fact that a genetic characterization of this species' population does not exist, and this creates gaps in the knowledge of this species in the Mediterranean. The genetic characterization of the individuals will initially take place by the extraction of the ancient DNA and the analysis of the variations in the sequence markers of the mitochondrial DNA. This approach has allowed the genetic comparison between ancient populations of the Mediterranean and contemporary populations of the same geographical area. In addition, the genetic characterization of the population of white sharks of the Mediterranean, has allowed a genetic comparison with populations from global "hot spots", using published sequences in online databases (NCBI, GenBank). Analyzing the variability of the dataset, both in terms space and time, I assessed the evolutionary relationships of the Mediterranean population of Great Whites with the global populations (Australia/New Zealand, South Africa, Pacific USA, West Atlantic), and the temporal trend of the Mediterranean population variability. This method based on the sequencing of two portions of mitochondrial DNA genes, markers showed us how the population of Great White Sharks in the Mediterranean, is genetically more similar to the populations of the Australia Pacific ocean, American Pacific Ocean, rather than the population of South Africa, and showing also how the population of South Africa is abnormally distant from all other clusters. Interestingly, these results are inconsistent with the results from tagging of this species. In addition, there is evidence of differences between the ancient population of the Mediterranean with the modern one. This differentiation between the ancient and modern population of white shark can be the result of events impacting on this species occurred over the last two centuries.

Genetic variability and differentiation in the Mediterranean endemic starry ray Raja asterias (Delaroche, 1809)

The present study deal with the population structure and connectivity of the Mediterranean endemic starry ray Raja asterias (Delaroche, 1809) in the Western and Eastern Mediterranean basin. A panel of eight microsatellite loci which cross-amplify in Rajidae (El Nagar, 2010) was used to assess population connectivity and structure. Those aims were investigated by analyzing the genetic variation of 9 population sample for a total of 185 individuals collected during past scientific surveys (MEDITS, GRUND), commercial trawling and also directly at fish markets. The purpose of this thesis is to estimate the genetic divergence occurring between the Mediterranean populations and, in particular, to assess the presence of any barrier (geographic, hydrogeological and biological) to gene flow for this species. Different statistical approaches were performed to reach this aim evaluating both the genetic diversity (nucleotide diversity, allelic richness, observed and expected heterozygosity and Hardy-Weinberg equilibrium test) and the population differentiation patterns (pairwise Fst estimated and population structure analysis). The results obtained from the analysis of the microsatellite dataset suggest a geographic and genetic separation between the starry ray populations of the Mediterranean basin into three or four distinct groups: Western and Eastern Mediterranean basins and Sicilian coast always clustering as an independent group and Algeria which could be or not considered another separate group. The data were discussed from both an evolutionary and a conservation point of view and in relation to previous results obtained by the analysis of mitochondrial marker. A comparison with other Mediterranean demersal skate species was performed in order to better contextualise our results. Finally, our results could offer useful information to protect vulnerable species as R. asterias and developing effective conservation plans in the Mediterranean.

Climatological analysis of temperature and salinity fields in the Mediterranean sea

A climatological field is a mean gridded field that represents the monthly or seasonal trend of an ocean parameter. This instrument allows to understand the physical conditions and physical processes of the ocean water and their impact on the world climate. To construct a climatological field, it is necessary to perform a climatological analysis on an historical dataset. In this dissertation, we have constructed the temperature and salinity fields on the Mediterranean Sea using the SeaDataNet 2 dataset. The dataset contains about 140000 CTD, bottles, XBT and MBT profiles, covering the period from 1900 to 2013. The temperature and salinity climatological fields are produced by the DIVA software using a Variational Inverse Method and a Finite Element numerical technique to interpolate data on a regular grid. Our results are also compared with a previous version of climatological fields and the goodness of our climatologies is assessed, according to the goodness criteria suggested by Murphy (1993). Finally the temperature and salinity seasonal cycle for the Mediterranean Sea is described.

Thermal structure and dynamical modeling of a Mediterranean tropical-like cyclone

In the present work, a detailed analysis of a Mediterranean TLC occurred in January 2014 has been conducted. The author is not aware of other studies regarding this particular event at the publication of this thesis. In order to outline the cyclone evolution, observational data, including weather-stations data, satellite data, radar data and photographic evidence, were collected at first. After having identified the cyclone path and its general features, the GLOBO, BOLAM and MOLOCH NWP models, developed at ISAC-CNR (Bologna), were used to simulate the phenomenon. Particular attention was paid on the Mediterranean phase as well as on the Atlantic phase, since the cyclone showed a well defined precursor up to 3 days before the minimum formation in the Alboran Sea. The Mediterranean phase has been studied using different combinations of GLOBO, BOLAM and MOLOCH models, so as to evaluate the best model chain to simulate this kind of phenomena. The BOLAM and MOLOCH models showed the best performance, by adjusting the path erroneously deviated in the National Centre for Environmental Prediction (NCEP) and ECMWF operational models. The analysis of the cyclone thermal phase shown the presence of a deep-warm core structure in many cases, thus confirming the tropical-like nature of the system. Furthermore, the results showed high sensitivity to initial conditions in the whole lifetime of the cyclone, while the Sea Surface Temperature (SST) modification leads only to small changes in the Adriatic phase. The Atlantic phase has been studied using GLOBO and BOLAM model and with the aid of the same methodology already developed. After tracing the precursor, in the form of a low-pressure system, from the American East Coast to Spain, the thermal phase analysis was conducted. The parameters obtained showed evidence of a deep-cold core asymmetric structure during the whole Atlantic phase, while the first contact with the Mediterranean Sea caused a sudden transition to a shallow-warm core structure. The examination of Potential Vorticity (PV) 3-dimensional structure revealed the presence of a PV streamer that individually formed over Greenland and eventually interacted with the low-pressure system over the Spanish coast, favouring the first phase of the cyclone baroclinic intensification. Finally, the development of an automated system that tracks and studies the thermal phase of Mediterranean cyclones has been encouraged. This could lead to the forecast of potential tropical transition, against with a minimum computational investment.

Temporal variation of genetic diversity and differentiation of albacore, Thunnus alalunga, in the Mediterranean Sea

This study is on albacore (Thunnus alalunga, Bonnaterre 1788), an epi- and mesopelagic oceanic tuna species cosmopolitan in the tropical and temperate waters of all oceans including the Mediterranean Sea, extending in a broad band between 40°N and 40°S. What it’s known about albacore population structure is based on different studies that used fisheries data, RFLP, mtDNA control region and nuDNA markers, blood lectins analysis, individual tags and microsatellite. At the moment, for T. alalunga six management units are recognized: the North Pacific, South Pacific, Indian, North Atlantic, South Atlantic and Mediterranean stocks. In this study I have done a temporal and spatial comparison of genetic variability between different Mediterranean populations of Thunnus alalunga matching an historical dataset ca. from 1920s composed of 43 individuals divided in 3 populations (NADR, SPAIN and CMED) with a modern dataset composed of 254 individuals and 7 populations (BAL, CYP, LIG, TYR, TUR, ADR, ALB). The investigation was possible using a panel of 94 nuclear SNPs, built specifically for the target species at the University of Basque Country UPV/EHU. First analysis done was the Hardy-Weinberg, then the number of clusters (K) was determined using STRUCTURE and to assess the genetic variability, allele frequencies, the average number of alleles per locus, expected (He) and observed (Ho) heterozygosis, and the index of polymorphism (P) was used the software Genetix. Historical and modern samples gives different results, showing a clear loss of genetic diversity over time leading to a single cluster in modern albacore instead of the two found in historical samples. What this study reveals is very important for conservation concerns, and additional research endeavours are needed.

Species identification of archived fish bones collected from the Mediterranean Sea 100 years ago using molecular techniques

With the discovery that DNA can be successfully recovered from museum collections, a new source of genetic information has been provided to extend our comprehension of the evolutionary history of species. However, historical specimens are often mislabeled or report incorrect information of origin, thus accurate identification of specimens is essential. Due to the highly damaged nature of ancient DNA many pitfalls exist and particular precautions need to be considered in order to perform genetic analysis. In this study we analyze 208 historical remains of pelagic fishes collected in the beginning of the 20th century. Through the adaptation of existing protocols, usually applied to human remains, we manage to successfully retrieve valuable genetic material from almost all of the examined samples using a guanidine and silica column-based approach. The combined use of two mitochondrial markers cytochrome-oxidase-1(mtDNA COI) and Control Region (mtDNA CR), and the nuclear marker first internal transcriber space (ITS1) allowed us to identify the majority of the examined specimens using traditional PCR and Sanger sequencing techniques. The creation of primers capable of amplifying heavily degraded DNA have great potential for future uses, both in ancient and in modern investigation. The methodologies developed in this study can in fact be applied for other ancient fish specimens as well as cooked or canned samples.

A study of air-sea interaction processes on water mass formation and upwelling in the Mediterranean sea

Air-sea interactions are a key process in the forcing of the ocean circulation and the climate. Water Mass Formation is a phenomenon related to extreme air-sea exchanges and heavy heat losses by the water column, being capable to transfer water properties from the surface to great depth and constituting a fundamental component of the thermohaline circulation of the ocean. Wind-driven Coastal Upwelling, on the other hand, is capable to induce intense heat gain in the water column, making this phenomenon important for climate change; further, it can have a noticeable influence on many biological pelagic ecosystems mechanisms. To study some of the fundamental characteristics of Water Mass Formation and Coastal Upwelling phenomena in the Mediterranean Sea, physical reanalysis obtained from the Mediterranean Forecating System model have been used for the period ranging from 1987 to 2012. The first chapter of this dissertation gives the basic description of the Mediterranean Sea circulation, the MFS model implementation, and the air-sea interaction physics. In the second chapter, the problem of Water Mass Formation in the Mediterranean Sea is approached, also performing ad-hoc numerical simulations to study heat balance components. The third chapter considers the study of Mediterranean Coastal Upwelling in some particular areas (Sicily, Gulf of Lion, Aegean Sea) of the Mediterranean Basin, together with the introduction of a new Upwelling Index to characterize and predict upwelling features using only surface estimates of air-sea fluxes. Our conclusions are that latent heat flux is the driving air-sea heat balance component in the Water Mass Formation phenomenon, while sensible heat exchanges are fundamental in Coastal Upwelling process. It is shown that our upwelling index is capable to reproduce the vertical velocity patterns in Coastal Upwelling areas. Nondimensional Marshall numbers evaluations for the open-ocean convection process in the Gulf of Lion show that it is a fully turbulent, three-dimensional phenomenon.

Genetic patterns in recovered specimens of Caretta caretta from the Adriatic Sea

The Adriatic Sea is considered a feeding and developmental area for Mediterranean loggerhead turtles, but this area is severely threatened by human impacts. In the Adriatic Sea loggerhead turtles are often found stranded or floating, but they are also recovered as by-catch from fishing activities. Nevertheless, information about population structuring and origin of individuals found in the Adriatic Sea are still limited. Cooperation with fishermen and a good network of voluntary collaborators are essential for understanding their distribution, ecology and for developing conservation strategies in the Adriatic Sea. In this study, a comparative analysis of biometric data and DNA sequence polymorphism of the long fragment of the mitochondrial control region was carried out on ninety-three loggerheads recovered from three feeding areas in the Adriatic Sea: North-western, North-eastern and South Adriatic. Differences in turtles body sizes (e.g. Straight Carapace Length) among the three recovery areas and relationship between SCL and the type of recovery were investigated. The origin of turtles from Mediterranean rookeries and the use of the Adriatic feeding habitats by loggerheads in different life-stages were assessed to understand the migratory pathway of the species. The analysis of biometric data revealed a significant difference in turtle sizes between the Southern and the Northern Adriatic. Moreover, size of captured turtles resulted significantly different from the size of stranded and floating individuals. Actually, neritic sub-adults and adults are more affected by incidental captures than juveniles because of their feeding behavior. The Bayesian mixed-stock analysis showed a strong genetic relationship between the Adriatic aggregates and Mediterranean rookeries, while a low pro¬portion of individuals of Atlantic origin were detected in the Adriatic feeding grounds. The presence of migratory pathways towards the Adriatic Sea due to the surface current system was reinforced by the finding of individuals bearing haplotypes endemic to the nesting populations of Libya, Greece and Israel. A relatively high contribution from Turkey and Cyprus to the Northwest and South Adriatic populations was identified when the three sampled areas were analyzed independently. These results have to be taken in account in a conservative perspective, since coastal hazards, affecting the population of turtles feeding in the Adriatic Sea may also affect the nesting populations of the Eastern Mediterranean with a unique genetic pattern.