Evolution and historical biogeography of the Eastern Atlantic skates

In my thesis, I tested the hypothesis that the diversification of the Eastern Atlantic skate faunas arose through vicariance rather than dispersal, using combined approach of molecular phylogeny reconstruction and zoogeography (namely historical biogeography). This analyses have been carried out independently on four Rajidae genera belonging to two different tribes: Rajini (Raja and Dipturus) and Amblyrajini (Rajella and Leucoraja). These taxa were selected because they displayed high species diversity and richness of endemic species in the Eastern Atlantic and Mediterranean. The verification of this hypothesis was carried out by reconstructing the best phylogenetic relationships among four genera and 26 species (including several endemism) based on mtDNA and nuDNA gene variation and several statistical approaches. Divergence times of taxa have been estimated based on molecular clock and fossil calibration to explain evolutionary patterns in the context of geological framework. Main issues are (i) the evidence that Eastern Atlantic skate evolution and displacement of species diversity occurred from pulsed geographical speciation (i.e. repeated series of parallel and independent speciation events) started in the Late Eocene-Early Miocene and they have occurred prevalently during Miocene; (ii) such relatively ancient origin of diversification has been allowed the sympatric displacement and evolution of several congeneric taxa likely because they have accumulated huge differences in the genomic and physiological/behavioural phenotypic traits; (iii) recently diverged sister species and taxa showed allopatric or parapatric evolution by the presence of oceanographic or hydrogeographical barriers which likely prevent large mixing between parapatric sister species.

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.

Wide-scale population genomics of Atlantic bluefin tuna (Thunnus thynnus) inferred by novel high-throughput technology

My PhD project was focused on Atlantic bluefin tuna, Thunnus thynnus, a fishery resource overexploited in the last decades. For a better management of stocks, it was necessary to improve scientific knowledge of this species and to develop novel tools to avoid collapse of this important commercial resource. To do this, we used new high throughput sequencing technologies, as Next Generation Sequencing (NGS), and markers linked to expressed genes, as SNPs (Single Nucleotide Polymorphisms). In this work we applied a combined approach: transcriptomic resources were used to build cDNA libreries from mRNA isolated by muscle, and genomic resources allowed to create a reference backbone for this species lacking of reference genome. All cDNA reads, obtained from mRNA, were mapped against this genome and, employing several bioinformatics tools and different restricted parameters, we achieved a set of contigs to detect SNPs. Once a final panel of 384 SNPs was developed, following the selection criteria, it was genotyped in 960 individuals of Atlantic bluefin tuna, including all size/age classes, from larvae to adults, collected from the entire range of the species. The analysis of obtained data was aimed to evaluate the genetic diversity and the population structure of Thunnus thynnus. We detect a low but significant signal of genetic differentiation among spawning samples, that can suggest the presence of three genetically separate reproduction areas. The adult samples resulted instead genetically undifferentiated between them and from the spawning populations, indicating a presence of panmictic population of adult bluefin tuna in the Mediterranean Sea, without different meta populations.

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.

"Tra centro e periferia" Istituzioni e processi di industrializzazione nella Sicilia del secondo dopoguerra

In Sicilia lo strumento principale che ha caratterizzato le modalità d’intervento dei poteri pubblici in economia è stato quello della partecipazione diretta o indiretta, quello cioè della Regione che si propone come ente fautore del sostegno allo sviluppo dell’industria e successivamente come soggetto imprenditore. Le formule organizzative attraverso le quali si è concretizzato l’intervento regionale in economia furono la società per azioni a partecipazione regionale, l’azienda autonoma regionale e l’ente pubblico regionale1. La storia dello sviluppo economico siciliano nel secondo dopoguerra conferma come il ricorso allo strumento dell’ente pubblico economico sia stato molto frequente nella realtà locale come, peraltro, anche in altre Regioni d’Italia. Tracce, queste, di una vicenda storica che intuiamo subito avere complesse implicazioni tali da generare la necessità di interrogarsi sul modo nel quale le istituzioni politiche hanno influito sulle dinamiche economiche siciliane nel secondo dopoguerra; per quanto noti e approfonditi siano stati infatti gli elementi caratterizzanti e i percorsi peculiari dello sviluppo economico siciliano, rimangono scarsamente approfonditi il tenore dei rapporti e i nessi politici, istituzionali ed economici tra centro e periferia, in altre parole rimane ancora parzialmente inesplorata quella parte dell’indagine inerente l’evoluzione dei processi di industrializzazione della Sicilia nel secondo dopoguerra attuata parallelamente dalle autorità regionali e dallo Stato attraverso i loro enti e strumenti. È lecito chiedersi quali siano stati i tempi, le modalità, gli ostacoli e gli eventuali risultati delle azioni di pianificazione intraprese dai poteri pubblici centrali e regionali nella prospettiva dello sviluppo economico del territorio; il coordinamento delle azioni di promozione del progresso industriale si presentava in tal senso, sin dall’inizio, come una delle sfide fondamentali per un adeguato e consistente rilancio economico delle aree più arretrate del Mezzogiorno italiano; ecco che lo studio dei provvedimenti legislativi emanati rappresenta un approfondimento indispensabile e obiettivo primario di questo lavoro.

Teoria e pratica dell'architettura solare - Morfologia, rendimento, strategia progettuale

La recente Direttiva 31/2010 dell’Unione Europea impone agli stati membri di riorganizzare il quadro legislativo nazionale in materia di prestazione energetica degli edifici, affinchè tutte le nuove costruzioni presentino dal 1° gennaio 2021 un bilancio energetico tendente allo zero; termine peraltro anticipato al 1° gennaio 2019 per gli edifici pubblici. La concezione di edifici a energia “quasi” zero (nZEB) parte dal presupposto di un involucro energeticamente di standard passivo per arrivare a compensare, attraverso la produzione preferibilmente in sito di energia da fonti rinnovabili, gli esigui consumi richiesti su base annuale. In quest’ottica la riconsiderazione delle potenzialità dell’architettura solare individua degli strumenti concreti e delle valide metodologie per supportare la progettazione di involucri sempre più performanti che sfruttino pienamente una risorsa inesauribile, diffusa e alla portata di tutti come quella solare. Tutto ciò in considerazione anche della non più procrastinabile necessità di ridurre il carico energetico imputabile agli edifici, responsabili come noto di oltre il 40% dei consumi mondiali e del 24% delle emissioni di gas climalteranti. Secondo queste premesse la ricerca pone come centrale il tema dell’integrazione dei sistemi di guadagno termico, cosiddetti passivi, e di produzione energetica, cosiddetti attivi, da fonte solare nell’involucro architettonico. Il percorso sia analitico che operativo effettuato si è posto la finalità di fornire degli strumenti metodologici e pratici al progetto dell’architettura, bisognoso di un nuovo approccio integrato mirato al raggiungimento degli obiettivi di risparmio energetico. Attraverso una ricognizione generale del concetto di architettura solare e dei presupposti teorici e terminologici che stanno alla base della stessa, la ricerca ha prefigurato tre tipologie di esito finale: una codificazione delle morfologie ricorrenti nelle realizzazioni solari, un’analisi comparata del rendimento solare nelle principali aggregazioni tipologiche edilizie e una parte importante di verifica progettuale dove sono stati applicati gli assunti delle categorie precedenti

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.