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.

Feeding behaviour of the Corydoradinae

The evolution of a sub-family of south-american freshwater catfishes has created a great diversification of its representants into a huge number of species, usually members of characteristic mimicry communities. After the work of Alexandrou (Alexandrou, 2011) on the phylogeny of the group that discovered the existence of more deep divisions, from the taxonomic point of view, of the sub-family, even from the ecological point of view, I tried to reconfirm these statements from the ethological point of view, settling watching and recording sessions and an experiment that could show their interactions on the environment by observing their substrate mixing action.

Morphological-molecular taxonomy and population genetic structure of common and Egyptian soles of the Adriatic Sea

A total of 352 specimens were analyzed to achieve the different aims of this thesis. 255 central-northern Adriatic specimens of S. solea and S. aegyptiaca were molecularly analysed using microsatellite locus Sos(AC)40 and 205 also morphologically due to evaluate the abundance and the distribution of the cryptic species S. aegyptiaca and to confirm morphologic analyses. Morphological and molecular analyses comparated show a correspondence of 96%. A combined morphologic approach could be proposed to apply multiple criteria on the analyzed external morphological keys. The Adriatic Egyptian soles may lives in shallow waters (up 30 m) and in brackish lagoon. 127 samples of Adriatic common sole added to 326 samples of previous studies showed, using mitochondrial marker (CytB), that the Adriatic Sea as contact zone between Tyrrhenian and Aegean Sea, the divergence within the Adriatic Sea is low but significant between central-north and south, with a longitudinal strong gene flow in central-northern side. It’s also showed as in the Adriatic Sea two near-panmictic populations of common sole exist.

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.

Molecular phylogeny and historical zoogeography of four genera of Eastern Atlantic skates (Rajiformes, Rajidae)

The aim of this study was to reconstruct a solid phylogeny of four genera of the Rajidae family (Chondrichthyans: Batoidea) using a concatenated alignment of mtDNA genes. Then use the resultant tree to estimate divergence time between taxa based on molecular clock and fossil calibration and conduct biogeographic analysis. The intent was to prove that the actual distribution of species of Eastern Atlantic and Mediterranean skates is due to a series of vicariant events. The species considered belongs to two different tribe: Rajini (Raja and Dipturus) and Amblyrajini (Leucoraja and Rajella). The choice of this genera is due to their high presence in the area of interest and to the richness of endemic species. The results show that despite the ancient origin of Rajidae (97 MYA), the Eastern Atlantic and Mediterranean faunas originated more recently, during Middle Miocene-Late Pliocene, after the closure of connection between these areas and the Indo-Pacific ocean (15 MYA). The endemic species of the Mediterranean (Raja asterias, R. radula, R. polystigma and Leucoraja melitensis) originated after the Messinian salinity crisis (7-5 MYA), when the recolonization of the basin occurred, and are still maintained in allopatric distribution by the presence of biogeographic barriers. Moreover from 4 to 2.6 MYA we can observe the formation of sister species for Raja, Leucoraja and Rajella, one of which has a Northern distribution, and the other has a Southern distribution (R. clavata vs R. straeleni, L. wallacei vs L. naevus, R. fyllae vs R. caudaspinosa and R. kukujevi vs R. leopardus + R. barnardi). The Quaternary and present oceanographic discontinuities that occur along the western African continental shelf (e.g., Cape Blanc and the Angola–Benguela Front) might contribute to the maintenance of low or null levels of gene flow between these closely related siblings species. Also sympatric speciation must be invoked to explain the evolution of skates, for example for the division between R. leopardus and R. barnardi. The speciation processes followed a south-to-north pathways for Dipturus and a north-to-south pathways for Raja, Leucoraja and Rajella underling that the evolution of the genera occurred independently. In the end, it is conceivable that the evolutionary pathways of the tribes followed the costal line during the gondwana fragmentation. The results demonstrate that the evolution of this family is characterized by a series of parallel and independent speciation events, strictly correlated to the tectonic movement of continental masses and paleogeographic and paleoclimatic events and so can be explained by a panbiogeographical (vicariance) model.

Analisi della diversità e della variazione delle comunità ittiche demersali dell'Alto e Medio Adriatico

Il Mar Adriatico presenta una grande ricchezza di diversità di specie ittiche, molte delle quali sono rilevanti dal punto di vista commerciale, altre rappresentano un contributo alla complessità biologica dell’ambiente. In questo lavoro di tesi tecniche di analisi multivariata sono state utilizzate per analizzare la composizione in specie ittiche demersali dell’Alto e Medio Adriatico e la diversità, per arrivare a delineare un quadro generale di tali comunità. I dati utilizzati sono stati raccolti nelle campagne GRUND effettuate in Adriatico dal 1982 al 2007 nell’area delle acque nazionali italiane ed internazionali, al limite delle acque croate e slovene. La Cluster Analysis effettuata sui dati di abbondanza (kg/h) delle specie ha permesso di definire quattro assemblaggi principali di specie (40% di similarità) associati all’area di costa, all’area costiera fuori Venezia, a un area detritica e a un area più profonda. All’interno di questi assemblaggi, stabili per tutti gli anni, sono stati ritrovate delle associazioni più ristrette (similarità del 50%). Profondità e tipologia di fondale sembrano essere i fattori determinanti la divisione di questi assemblaggi. Tali risultati sono stati confermati anche dall’analisi di ordinamento non metrico MDS. Con l’analisi ANOSIM si è cercato di vedere se ci sono differenze significative tra gli assemblaggi annuali delle aree identificate, e se gli assemblaggi di specie variano significativamente nel corso degli anni all’interno di ciascuna area. Con l’analisi SIMPER si sono identificate quelle specie caratterizzanti gli assemblaggi e le specie che sono responsabili della diversità tra aree. Sono stati calcolati gli indici di diversità per indagare la diversità e la variabilità temporale delle comunità demersali che caratterizzano le quattro aree principali. E’ stata fatta un’analisi temporale delle abbondanze medie delle specie commerciali maggiormente rappresentative dei quattro assemblaggi principali ritrovati, e un’analisi su come variano le taglie nel corso degli anni.

Genetic and morphological variation and differentiation of Raja clavata populations in the European seas: marker development and preliminary data

In this study the population structure and connectivity of the Mediterranean and Atlantic Raja clavata (L., 1758) were investigated by analyzing the genetic variation of six population samples (N = 144) at seven nuclear microsatellite loci. The genetic dataset was generated by selecting population samples available in the tissue databases of the GenoDREAM laboratory (University of Bologna) and of the Department of Life Sciences and Environment (University of Cagliari), all collected during past scientific surveys (MEDITS, GRUND) from different geographical locations in the Mediterranean basin and North-east Atlantic sea, as North Sea, Sardinian coasts, Tuscany coasts and Cyprus Island. This thesis deals with to estimate the genetic diversity and differentiation among 6 geographical samples, in particular, to assess the presence of any barrier (geographic, hydrogeological or biological) to gene flow evaluating both the genetic diversity (nucleotide diversity, observed and expected heterozygosity, Hardy- Weinberg equilibrium analysis) and population differentiation (Fst estimates, population structure analysis). In addition to molecular analysis, quantitative representation and statistical analysis of morphological individuals shape are performed using geometric morphometrics methods and statistical tests. Geometric coordinates call landmarks are fixed in 158 individuals belonging to two population samples of Raja clavata and in population samples of closely related species, Raja straeleni (cryptic sibling) and Raja asterias, to assess significant morphological differences at multiple taxonomic levels. The results obtained from the analysis of the microsatellite dataset suggested a geographic and genetic separation between populations from Central-Western and Eastern Mediterranean basins. Furthermore, the analysis also showed that there was no separation between geographic samples from North Atlantic Ocean and central-Western Mediterranean, grouping them to a panmictic population. The Landmark-based geometric morphometry method results showed significant differences of body shape able to discriminate taxa at tested levels (from species to populations).

Identification of stock units of Mullus barbatus (Linnaeus, 1758) in the Italian seas: development of genomic 2b-RAD markers and biological variation analysis.

This thesis is settled within the STOCKMAPPING project, which represents one of the studies that were developed in the framework of RITMARE Flagship project. The main goals of STOCKMAPPING were the creation of a genomic mapping for stocks of demersal target species and the assembling of a database of population genomic, in order to identify stocks and stocks boundaries. The thesis focuses on three main objectives representing the core for the initial assessment of the methodologies and structure that would be applied to the entire STOCKMAPPING project: individuation of an analytical design to identify and locate stocks and stocks boundaries of Mullus barbatus, application of a multidisciplinary approach to validate biological methods and an initial assessment and improvement for the genotyping by sequencing technique utilized (2b-RAD). The first step is the individuation of an analytical design that has to take in to account the biological characteristics of red mullet and being representative for STOCKMAPPING commitments. In this framework a reduction and selection steps was needed due to budget reduction. Sampling areas were ranked according the individuation of four priorities. To guarantee a multidisciplinary approach the biological data associated to the collected samples were used to investigate differences between sampling areas and GSAs. Genomic techniques were applied to red mullet for the first time so an initial assessment of molecular protocols for DNA extraction and 2b-RAD processing were needed. At the end 192 good quality DNAs have been extracted and eight samples have been processed with 2b-RAD. Utilizing the software Stacks for sequences analyses a great number of SNPs markers among the eight samples have been identified. Several tests have been performed changing the main parameter of the Stacks pipeline in order to identify the most explicative and functional sets of parameters.

Stock boundaries of Parapenaeus longirostris (Lucas, 1846) populations in the Italian seas: Development of genomic 2b-RAD markers and biological variation analysis.

This thesis is developed in the contest of Ritmare project WP1, which main objective is the development of a sustainable fishery through the identification of populations boundaries in commercially important species in Italian Seas. Three main objectives are discussed in order to help reach the main purpose of identification of stock boundaries in Parapenaeus longirostris: 1 -Development of a representative sampling design for Italian seas; 2 -Evaluation of 2b-RAD protocol; 3 -Investigation of populations through biological data analysis. First of all we defined and accomplished a sampling design which properly represents all Italian seas. Then we used information and data about nursery areas distribution, abundance of populations and importance of P. longirostris in local fishery, to develop an experimental design that prioritize the most important areas to maximize the results with actual project funds. We introduced for the first time the use of 2b-RAD on this species, a genotyping method based on sequencing the uniform fragments produced by type IIB restriction endonucleases. Thanks to this method we were able to move from genetics to the more complex genomics. In order to proceed with 2b-RAD we performed several tests to identify the best DNA extraction kit and protocol and finally we were able to extract 192 high quality DNA extracts ready to be processed. We tested 2b-RAD with five samples and after high-throughput sequencing of libraries we used the software “Stacks” to analyze the sequences. We obtained positive results identifying a great number of SNP markers among the five samples. To guarantee a multidisciplinary approach we used the biological data associated to the collected samples to investigate differences between geographical samples. Such approach assures continuity with other project, for instance STOCKMED, which utilize a combination of molecular and biological analysis as well.

Identificazione di un metodo statistico per la descrizione della popolazione di residui dello sparo da inneschi innovativi

La tesi, in collaborazione con il Maggiore Donghi del reparto di Balistica del RIS di Parma, individua un metodo statistico di descrizione della popolazione di particelle residue dello sparo, attraverso numerose sperimentazioni effettuate con inneschi innovativi. Lo studio propone la comparazione degli inneschi utilizzati e i successivi confronti tra i GSR rinvenuti sulle mani dello sparatore e le famiglie di inneschi utilizzati.

Osservazioni in ambiente controllato del ciclo riproduttivo e delle prime fasi di accrescimento di Chiloscyllium punctatum (Muller & Henle 1838)

La presente tesi tratta delle attività riproduttive e delle prime fasi di crescita dello squalo Chiloscyllium punctatum. Da Dicembre 2013 a Giugno 2014, all’Acquario di Cattolica, sono state effettuate osservazioni su una coppia di adulti riproduttori e sui relativi neonati mantenuti in ambiente controllato. Il Chiloscyllium punctatum è una specie ovipara che rilascia uova di forma rettangolare, ha un ciclo riproduttivo relativamente rapido ed è molto sfruttata come specie ornamentale dagli acquari di tutto il mondo. Per ottenere una corretta gestione in ambiente controllato, la riproduzione ed estrapolare il maggior numero d’informazioni certe su gli stadi di vita, sulla dieta di divezzamento dei neonati dal sacco vitellino, sull’illuminazione più consona da adottare, sulle dimensioni medie di uova, nascituri e adulti, sulla crescita dei nascituri, sui tempi di sviluppo embrionale, sulla frequenza di deposizione delle uova e sui tempi di schiusa è stato necessario sviluppare un corretta procedura di gestione ordinaria. In 7 mesi, gli adulti di C. punctatum, in condizioni ambientali ottimali e costanti, hanno dato vita a 33 neonati. Questi sono stati sottoposti a misurazioni di lunghezza (LT, cm) e peso corporeo (PC, g) per valutare l’influenza dell’illuminamento e della dieta sulla crescita. Inoltre, sono stati confrontati dati sperimentali (e.g. tempistiche di sviluppo embrionale) con dati bibliografici (Haraush et al., 2007) che hanno evidenziato alcune similitudini e differenze.

Metodi matematici per lo sviluppo di una nuova distanza genetica per inferire eventi demografici da dati di sequenziamento di popolazioni umane

La tesi individua un metodo matematico per inferire alcuni eventi demografici relativi a popolazioni umane, attraverso l’analisi di dati, reali e simulati, e con strumenti di statistica e analisi numerica (Cluster Analysis, Analisi Discriminate, Analisi della varianza, Interpolazione).

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.

Struttura di età di nasello (Merluccius merluccius, Linnaeus, 1758) in alto-medio Adriatico mediante analisi degli otoliti e frequenza di taglia

The aim of present study is to define the general framework of Merluccius merluccius population structure, to estimate the growth rate and to assess the recruitment dynamics of juveniles from Northern and Central Adriatic, through otoliths analysis. The otoliths of hake specimens collected during the MedITS trawl survey in the 2012 in GSA 17, were cleaned and 102 otoliths out of 506 were embedded, sectioned, grindined and polished to obtain frontal and sagittal sections. The whole sample were analysed under stereomicroscope and optical microscope, with camera and connected to PC provided of an image analyses program. The frequency analysis of size classes and age revealed that the species is dominated by hake with >200mm TL and > one year old. The fish average size of M. merluccius at the end of the first year of life is about 199 mm TL. Allometrics analyses between fish TL and Feret (major axis), MiniFeret (minor axis), Area, Perimeter, showed a direct proportionality among lengths. Among the 88 otoliths sections analysed, the number of daily increments read ranged from 86 to 206, within 55 and 175mm TL range. The age estimate ranged from about 2-3 to 9 months and the growth rate from 20.99 to 27.15mm TL. The hatch-date distribution, obtained by back calculation, showed that the hatching occurs in November-March. In conclusion, strong preventive measures are needed for hake adults because the success of this species seems to be linked to deep water ecosystem protection where big spawners dwell.