An appropriate management of fisheries resources can only be achieved with the continuous supply of information on the structure and biology of populations, in order to predict the temporal fluctuations. This study supports the importance of investigating the bio-ecology of increasingly exploited and poorly known species, such as gurnards (Osteichthyes, Triglidae) from Adriatic Sea (Mediterranean), to quantify their ecological role into marine community. It also focuses on investigate inter and intra-specific structuring factor of Adriatic population. These objectives were achieved by: 1) investigating aspects of the population dynamics; 2) studying the feeding biology through the examination of stomach contents; 3) using sagittal otoliths as potential marker of species life cycle; 4) getting preliminary data on mDNA phylogeny. Gurnards showed a specie-specific “critical size” coinciding with the start of sexual maturity, the tendency to migrate to greater depths, a change of diet from crustaceans to fish and an increase of variety of food items eaten. Distribution of prey items, predator size range and depth distribution were the main dimensions that influence the breadth of trophic niche and the relative difference amongst Adriatic gurnards. Several feeding preferences were individuated and a possible impact among bigger-size gurnards and other commercial fishes (anchovy, gadoids) and Crustacea (such as mantis prawn and shrimps) were to be necessary considered. Otolith studies showed that gurnard species have a very fast growth despite other results in other areas; intra-specific differences and the increase in the variability of otolith shape, sulcus acusticus shape, S:O ratios, sulcus acusticus external crystals arrangement were shown between juveniles and adults and were linked to growth (individual genetic factors) and to environmental conditions (e.g. depth and trophic niche distribution). In order to facilitate correct biological interpretation of data, molecular data were obtained for comparing morphological distance to genetic ones.

L’anguilla europea, è una specie eurialina catadroma con un complesso ciclo biologico: l’area di riproduzione, unica, si trova molto distante da quella di distribuzione. La specie necessita di una gestione dello stock a fini conservazionistici. Il problema è europeo: lo stock è unico, distribuito in Europa e nell’Africa settentrionale, si riproduce in Atlantico ed è panmittico. C’è preoccupazione per il declino del reclutamento e delle catture di adulti. Lo scopo del progetto è di individuare possibili unità di stock nella penisola italiana. La ricerca è basata sullo studio degli otoliti mediante analisi morfometrica e microchimica. I contorni degli otoliti sono sottoposti ad analisi ellittica di Fourier per individuare eventuali gruppi. Gli otoliti sono stati levigati per effettuare: letture d’età, indagini microstrutturali al SEM delle fasi larvali, analisi microchimiche LA-ICP-MS del nucleo, studiarne l’origine e valutare l’ambiente di sviluppo. Le indagini morfometriche mostrano evidenti pattern ontogenetici, ma non legati ocorrelati alla località, sesso o anno di nascita. Le indagini microstrutturali hanno evidenziano l’alto contenuto organico nucleare, un pattern comune di crescita ed eventi chiave delle fasi larvali, con una media di 212 anelli giornalieri. La microchimica rivela che le larve si sviluppano in acque salate fino alla metamorfosi, poi migrano verso acque meno salate. Le analisi su campioni nati nello stesso anno, evidenziano due gruppi: individui di rimonta naturale e individui di ripopolamento. I profili nucleo bordo evidenziano la permanenza a salinità intermedie degli adulti. L’attività di ricerca si è dimostrata proficua dal punto di vista tecnico con la messa a punto di protocolli innovativi e con forti ricadute sulla riduzione dei tempi e costi d’analisi. Il debole segnale di possibili unità di stock andrà verificato in futuro mediante analisi più dettagliate discriminando meglio la storia di ogni singolo individuo. / The European eel is euryhaline catadromous with a complex life cycle: spawning area, unique, is far from that of distribution. The species needs a stock management and its conservation. The problem is European: the stock is unique, distributed in Europe and North Africa, spawning in Atlantic Sea and is panmictic. There is concern about the decline of recruitment and catches of adults. The purpose of the project is to identify possible units of stock in the Italian peninsula. The research is based on the study of otoliths by morphometric and microchemical analyses . The otoliths contours are analyzed by elliptic Fourier function to discover any groups. Otoliths were polished to make: ageing, SEM microstructural investigations of larval stages, LA-ICP-MS microchemical analysis of the core to study the origin and along a transect core to edge to evaluate the development environment. Morphometric investigations show obvious ontogenetic pattern, but no location, gender or year of birth correlation. The microstructural investigations showed high organic content in the core, a common pattern of growth and key events of the larval stages, with an average of 212 daily rings. The microchemical reveals that the larvae develop into salt water until metamorphosis, then migrate to waters less salty. Analyses of samples born in the same year, showed two groups: wild and restocked. The profiles confirm that the adult stay in brackishwater. The development of innovative protocols and improved technics have guaranteed a strong reduction of time and cost of analysis. The weak signal of possible stock units will be further tested through more detailed analysis discriminating about the life history of each individual.

In recent years the advances in genomics allowed to understand the importance of Transposable Elements (TE) in the evolution of eukaryotic genomes. In this thesis I face two aspects of the TE impact on the in the animal kingdom. The first part is a comparison of the dynamics of the TE dynamics in three species of stick-insects of the Genus Bacillus. I produced three random genomic libraries of 200 Kbps for the three parental species of the taxon: a gonochoric population of Bacillus rossius (facultative parthenogenetic), Bacillus grandii (gonochoric) and Bacillus atticus (obligate parthenogenetic). The unisexual taxon Bacillus atticus does not shows dramatic differences in TE total content and activity with respect to Bacillus grandii and Bacillus rossius. This datum does not confirm the trend observed in other animal models in which unisexual taxa tend to repress the activity of TE to escape the extinction by accumulation of harmful mutations. In the second part I tried to add a contribute to the debate initiated in recent years about the possibility that a high TE content is linked to a high rate of speciation. I designed an evolutionary framework to establish the different rate of speciation among two or more taxa, then I compared TE dynamics considering the different rates of speciation. The species dataset comprises: 29 mammals, four birds, two fish and two insects. On the whole the majority of comparisons confirms the expected trend. In particular the amount of species analyzed in Mammalia allowed me to get a statistical support (p<0,05) of the fact that the TE activity of recently mobilized elements is positively related with the rate of speciation.

Many bivalve species possess two distinct mtDNA lineages, called F and M, respectively inherited maternally and paternally: this system is called doubly uniparental inheritance (DUI). The main experimental project of my PhD was the quantification of the two mtDNAs during the development of the DUI species Ruditapes philippinarum, from early embryos to sub-adults, using Real-Time qPCR. I identified the time interval in which M mtDNA is lost from female individuals, while it is retained in males (which are heteroplasmic through all of their life cycle). The results also suggested absence of mtDNA replication during early embryogenesis, a process constituting a bottleneck that highly reduces the copy number of mtDNA molecules in cells of developing larvae. In males this bottleneck may produce cells homoplasmic for M mtDNA, and could be considered as a first step of the segregation of M in the male germ line. Another finding was the characterization, in young clams approaching the first reproductive season, of a significant boost in copy number of F mtDNA in females and of M in males. Given the age of animals in which this mtDNA-specific growth was observed, the finding could probably be the outcome of the first round of gonads and gametes production. Other lines of research included the characterization of the unassigned regions in mt genomes of DUI bivalves. These regions can harbor signals involved in the control of replication and/or transcription of the mtDNA molecule, as well as additional open reading frames (ORFs) not related to oxidative phosphorylation. These features in DUI species could be associated to the maintenance of separate inheritance routes for the two mtDNAs. Additional ORFs are also found in other animal mt genomes: I summarized the presence of gene duplications as a co-author in a review focusing on animal mt genomes with unusual gene content.

In this PhD thesis I will describe different aspects of conservation genetics and genomics of two wild Canidae species, the wolf (Canis lupus) and the golden jackal (Canis aureus), through the study of two of the most variable gene families: the Major Histocompatibility Complex genes (MHC), and Olfactory Receptors genes (OR). In order to perform these studies both Sanger and next generation sequencing (NGS) DNA techniques have been used. The background of the thesis is described in the “General introduction” with phylogeny, classification and evolutionary ecology of the Canidae, with a focus on the species Canis lupus and its main conservation concerns in Italy. Moreover, I will introduce the importance to perform genetic studies as tools for wild-life conservation and management, with a description of the framework of the principal historical and currently used molecular markers that had driven to develop MHC and OR sequencing projects. The thesis is divided into two parts, “PART I – The MHC typing project” and “PART II – The OR genes typing project”. A total of four scientific papers (already published or under revision) will be introduced and illustrated as result of three years of PhD activities at ISPRA’s (Istituto Superiore per la Protezione e la Ricerca Ambientale), Laboratory of conservation genetics, in Ozzano dell’Emilia, and thankfully to a PhD fellowship granted by the Università di Bologna.

In the marine realm, two of the main stressors causing significant changes are ocean warming and acidification. Of particular concern are organisms reliant on the generation of calcium carbonate, such as corals. Reproduction and population structure are strongly related to environmental parameters and may be indicators for stability or decline of coral populations. Part of my research focused on the reproductive cycle of the Mediterranean non-zooxanthellate coral Caryophyllia inornata in relation to seasonal variations of seawater temperature and photoperiod. Moreover, an unusual embryogenesis was found in females, males and inactive individuals during the entire year, indicating a possible agamic origin of embryos. This was a pilot study for a broader investigation along a wide latitudinal gradient of temperature and solar radiation. Reproductive traits of this coral do not vary along the gradient as observed for another non-zooxanthellate species, Leptopsammia pruvoti, indicating that these species could be quite tolerant to environmental changes, probably due to the lack of symbiosis with the zooxanthellae. A study on the spermatogenesis of the zooxanthellate coral Balanophyllia europaea living along a natural pCO2 gradient showed no significant variations with low pH. We hypothesized that high CO2 levels probably enhance zooxanthellae photosynthesis, leading to an increase of the available energy for gonadal development. The ability to reproduce in particular conditions is related to population structure, providing information on coral responses to the environment. I investigated the population structure of the tropical coral Montastraea cavernosa along a depth gradient in Bermuda. Deeper populations were characterized by smaller but more numerous colonies compared to shallow ones, with no variation in the percent cover among depths. Thus, mesophotic populations of M. cavernosa in Bermuda seem quite stable, indicating that these reefs may serve as a source of propagules to maintain shallower reefs and help guide future management and conservation strategies.

Gli elementi trasponibili (TEs) sono componenti universali dei genomi di tutti gli esseri viventi. L'attività ed il movimento degli TEs hanno importanti effetti sul genoma ospite e per questo sono considerati dei fattori importanti nell'evoluzione del genoma. Le relazioni che intercorrono tra TEs e il loro genoma ospite sono ancora ampiamente dibattute. Hickey già nel 1982 suggerì che queste relazioni potevano essere influenzate dalla modalità di riproduzione del genoma ospite. Organismi bisessuali, attraverso i meccanismi propri della meiosi, riuscirebbero a contrastare la proliferazione degli TEs, al contrario negli organismi unisessuali, proprio a causa della mancanza di questi meccanismi, gli TEs tendono a proliferare ed accumulare all'interno del genoma. Al fine di valutare queste ipotesi, ho isolato e caratterizzato gli TEs in organismi con strategie riproduttive non canoniche. Ho condotto le mie analisi in due organismi: gli insetti stecco del genere Bacillus e nel fossile vivente Triops cancriformis. Entrambi presentano strategie riproduttive che vanno dal gonocorismo alla partenogenesi, rendendoli un eccellente modello per lo studio e la caratterizzazione degli TEs. Nel genere Bacillus mi sono focalizzata sullo studio del retrotransposone non-LTR R2. Ho isolato e caratterizzato sette elementi R2 completi; sia in specie gonocoriche che partenogenetiche. Ho trovato un elemento R2 degenerato presente nel genoma di B. rossius da almeno 5 milioni di anni. In oltre, le mie analisi suggeriscono per la prima volta che anche gli elementi R2 possono trasferirsi attraverso eventi di trasferimento orizzontale. I miei dati mostrano una panoramica della composizione di TEs nel genoma di una popolazione partenogenetica di Triops cancriformis. Dalle mie analisi si evince che il 20% della library di triops è composta da TEs. / Transposable elements (TEs) are universal components of all living organisms. TEs activity and movement have profound effects on host genome and today many researchers agree to consider them as important actors in genome evolution. The relationships between TEs and their host genomes are still under debate. Different hypotheses were proposed to explain TEs dynamics in host genome; one of these propose that host reproductive strategies can influence TEs evolutionary dynamics (Hickey, 1982). In fact, bisexual organisms, through homologous chromosomes recombination and reassortment during meiosis and amphimixis, can control the spread and proliferation of mobile elements, while unisexual organisms would experience an increase of these elements density due to the inability to eliminate them through exclusive mechanisms of sexual reproduction. In order to evaluate these hypothesis, I isolated and characterized TEs in organisms with non-canonical reproductive strategies. I performed my analyses in two organisms: the stick insects of the Bacillus genus and in the tadpole shrimps T. cancriformis. In both instances reproductive strategies range from bisexual gonochoric reproduction, to unisexual parthenogenesis, making them an excellent model for the study and characterization of TEs. In the Bacillus genus I focused on the R2 non-LTR retrotranposon. I isolated and sequenced by primer walking seven R2 complete elements; both gonochoric and parthenogenetic Bacillus species. I found a R2 degenerate element present in the B. rossius genome since 5 Myr ago. In addition, my analyses for the first time suggest that also R2 retrotransposons can use horizontal transfer as a strategy to colonize a new genome. My data showed a TEs overview in a T. cancriformis parthenogenetic population. In contrast with the theoretical, my analyses highlighted that the 20% of the library is composed by TEs, in which both TEs classes are widely represented (class I, 11.4% and class II, 8.9%).

Yellowfin tuna (Thunnus albacares; YFT) represents one of the most important seafood commodities in the world. The rationale of this Ph.D. project was identified by prioritizing key issues as objectives for contributing to the conservation of YFT and helping to develop a more realistic stock assessment and sustainable management of this species. Specifically, in this work the current mismatch between the biology and/or ecology and the realized management strategies was highlighted, putting particular emphasis on YFT population structure, which is currently characterized by a high degree of uncertainty at both local and global scale. This general pattern was confirmed by the results obtained using a panel of microsatellite loci, which cannot reject the null hypothesis of the existence of only one panmictic population at the global scale. On the contrary, the access to more powerful and cost effective genetic tools would represent the first step for resolving YFT population structure at both global and local scale. After having evaluated the efficiency and usefulness of 2b-RAD genotyping technique for investigating population genetic structure in highly migratory fish species, a panel of 972 SNPs (Single Nucleotide Polymorphisms) was generated. Using this panel, three distinct populations were identified in the Atlantic, Indian and Pacific Oceans. Additionally, it was possible to define a subset of 33 outlier loci putatively under selection to delineate and separate sub-populations within both the Atlantic and the Pacific Oceans (following an east-west division). Finally, it was emphasized for the first time that in the Atlantic Ocean larger YFT females allocate a greater fraction of surplus energy to egg production than smaller ones, improving noticeably the spawning quality. This result sheds light on the important contribution that larger and most experienced spawners have for the YFT productivity.

Global climate changes, driven by increasing temperature and pCO2, negatively affect marine ecosystem and mainly calcifying organisms. The reproduction of the zooxanthellate coral, Balanophyllia europaea, varied significantly along a latitudinal gradient of temperature and solar radiation. The warmer populations showed a lower reproductive efficiency due to the inhibition of the zooxanthellae photosynthesis, leading less available energy for reproduction. On the contrary, the B. europaea population, naturally living along a pCO2 gradient, did not show differences in oocytes development, production and morphology, probably by reallocating additional energy due to increasing of photosynthetic efficiency of zooxanthellae under pCO2 conditions. Increasing pCO2 negatively influenced spermaries production and development in L. pruvoti, causing a delayed in fertilization process, probably due to the lack of zooxanthellae involving in a lack of additional energy available for coral. The effects of high temperature, pCO2 and their interaction were tested on two symbiotic tropical corals, Fungia granulosa (solitary) and Pocillopora verrucosa (colonial). F. granulosa showed negative effects only in interaction treatment, while P. verrucosa showed decreased photosynthetic efficiency, increased bleaching tissue and mortality in all treatments. The more sensitivity of P. verrucosa could be explained by lower tissue thickness, making it more exposed and vulnerable to external environmental conditions. To investigate the role of intra-skeletal organic matrix in coral biomineralization, four Mediterranean species with different trophic strategy and growth form were selected. B. europaea organic matrix presented stronger control and higher independence from the crystallization environment than the other corals. Shell features of clam Chamelea gallina varied along a latitudinal gradient of temperature and solar radiation. Shells from the warmest and the most irradiated population were ~30% lighter due to thinner and more porous shells. No variation was observed in shell polymorphism at the nanoscale level, indicating no effects of environmental parameters on its composition and crystallography.

As a large and long-lived species with high economic value, restricted spawning areas and short spawning periods, the Atlantic bluefin tuna (BFT; Thunnus thynnus) is particularly susceptible to over-exploitation. Although BFT have been targeted by fisheries in the Mediterranean Sea for thousands of years, it has only been in these last decades that the exploitation rate has reached far beyond sustainable levels. An understanding of the population structure, spatial dynamics, exploitation rates and the environmental variables that affect BFT is crucial for the conservation of the species. The aims of this PhD project were 1) to assess the accuracy of larval identification methods, 2) determine the genetic structure of modern BFT populations, 3) assess the self-recruitment rate in the Gulf of Mexico and Mediterranean spawning areas, 4) estimate the immigration rate of BFT to feeding aggregations from the various spawning areas, and 5) develop tools capable of investigating the temporal stability of population structuring in the Mediterranean Sea. Several weaknesses in modern morphology-based taxonomy including demographic decline of expert taxonomists, flawed identification keys, reluctance of the taxonomic community to embrace advances in digital communications and a general scarcity of modern user-friendly materials are reviewed. Barcoding of scombrid larvae revealed important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology-based methods. Using a Genotyping-by-Sequencing a panel of 95 SNPs was developed and used to characterize the population structuring of BFT and composition of adult feeding aggregations. Using novel molecular techniques, DNA was extracted from bluefin tuna vertebrae excavated from late iron age, ancient roman settlements Byzantine-era Constantinople and a 20th century collection. A second panel of 96 SNPs was developed to genotype historical and modern samples in order to elucidate changes in population structuring and allele frequencies of loci associated with selective traits.