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.

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.

First assessment on genetic structure and phylogeography of Mediterranean blue shark (prionace glauca, L. 1758) population using mitochondrial gene variation: a comparison with the Atlantic.

The blue shark, Prionace glauca, is one of the most vagile shark species worldwide distributed. The particular body shape allows blue sharks make transoceanic movements, leading to a circumglobal distribution. Due to its reproductive cycle, an extraordinarily high number of specimens is globally registered but, even if it is still a major bycatch of longline fishery rather than a commercial target, it is characterized by a high vulnerability. In this perspective it is important to increase the amount of informations regarding its population extent in the different worldwide areas, evaluating the possible phylogeographic patterns between different locations. This study, included in the "MedBlueSGen" European project, aims exactly at filling a gap in knowledges regarding the genetic population structure of the Mediterranean blue sharks, which has never been investigated before, with a comparison with the North-Eastern Atlantic blue shark population. To reach this objective, we used a dataset of samples from different Mediterranean areas implementing it with some samples from North-Eastern Atlantic. Analyzing the variability of the two mitochondrial markers control region and cytochrome b, with the design of new species-specific primer pairs, we assessed the mitochondrial genetic structure of Mediterranean and North-Eastern Atlantic samples, focusing on the analysis of their possible connectivity, and we tried to reconstruct their demographic history and population size. Data analyses highlighted the absence of a genetic structuring within the Mediterranean and among it and North-Eastern Atlantic, suggesting that the Strait of Gibraltar doesn't represent a phylogeographic barrier. These results are coherent to what has been found in similar investigations on other worldwide blue shark populations. Analysis of the historical demographic trend revealed a general stable pattern for the cytochrome-b and a slightly population expansion for the control region marker.

Modeling and simulation of a synthetic genetic circuit that implements a multicelled behavior in a growing microcolony of E. coli

Synthetic Biology is a relatively new discipline, born at the beginning of the New Millennium, that brings the typical engineering approach (abstraction, modularity and standardization) to biotechnology. These principles aim to tame the extreme complexity of the various components and aid the construction of artificial biological systems with specific functions, usually by means of synthetic genetic circuits implemented in bacteria or simple eukaryotes like yeast. The cell becomes a programmable machine and its low-level programming language is made of strings of DNA. This work was performed in collaboration with researchers of the Department of Electrical Engineering of the University of Washington in Seattle and also with a student of the Corso di Laurea Magistrale in Ingegneria Biomedica at the University of Bologna: Marilisa Cortesi. During the collaboration I contributed to a Synthetic Biology project already started in the Klavins Laboratory. In particular, I modeled and subsequently simulated a synthetic genetic circuit that was ideated for the implementation of a multicelled behavior in a growing bacterial microcolony. In the first chapter the foundations of molecular biology are introduced: structure of the nucleic acids, transcription, translation and methods to regulate gene expression. An introduction to Synthetic Biology completes the section. In the second chapter is described the synthetic genetic circuit that was conceived to make spontaneously emerge, from an isogenic microcolony of bacteria, two different groups of cells, termed leaders and followers. The circuit exploits the intrinsic stochasticity of gene expression and intercellular communication via small molecules to break the symmetry in the phenotype of the microcolony. The four modules of the circuit (coin flipper, sender, receiver and follower) and their interactions are then illustrated. In the third chapter is derived the mathematical representation of the various components of the circuit and the several simplifying assumptions are made explicit. Transcription and translation are modeled as a single step and gene expression is function of the intracellular concentration of the various transcription factors that act on the different promoters of the circuit. A list of the various parameters and a justification for their value closes the chapter. In the fourth chapter are described the main characteristics of the gro simulation environment, developed by the Self Organizing Systems Laboratory of the University of Washington. Then, a sensitivity analysis performed to pinpoint the desirable characteristics of the various genetic components is detailed. The sensitivity analysis makes use of a cost function that is based on the fraction of cells in each one of the different possible states at the end of the simulation and the wanted outcome. Thanks to a particular kind of scatter plot, the parameters are ranked. Starting from an initial condition in which all the parameters assume their nominal value, the ranking suggest which parameter to tune in order to reach the goal. Obtaining a microcolony in which almost all the cells are in the follower state and only a few in the leader state seems to be the most difficult task. A small number of leader cells struggle to produce enough signal to turn the rest of the microcolony in the follower state. It is possible to obtain a microcolony in which the majority of cells are followers by increasing as much as possible the production of signal. Reaching the goal of a microcolony that is split in half between leaders and followers is comparatively easy. The best strategy seems to be increasing slightly the production of the enzyme. To end up with a majority of leaders, instead, it is advisable to increase the basal expression of the coin flipper module. At the end of the chapter, a possible future application of the leader election circuit, the spontaneous formation of spatial patterns in a microcolony, is modeled with the finite state machine formalism. The gro simulations provide insights into the genetic components that are needed to implement the behavior. In particular, since both the examples of pattern formation rely on a local version of Leader Election, a short-range communication system is essential. Moreover, new synthetic components that allow to reliably downregulate the growth rate in specific cells without side effects need to be developed. In the appendix are listed the gro code utilized to simulate the model of the circuit, a script in the Python programming language that was used to split the simulations on a Linux cluster and the Matlab code developed to analyze the data.

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.

Genetic structuring of Eunicella singularis populations along a bathymetric gradient

Phenotypic plasticity refers to the ability of an organism to express different morphologies depending on the abiotic and biotic environment. Depth integrating many variables (e.g. temperature, light and hydrodynamics), may affect population structure and dynamics of the populations, as well as connectivity patterns and genetic diversity. Eunicella singularis is a Mediterranean arborescent gorgonian who plays an important rule as engineer species providing biomass and complexity to coralligenous habitats. It has a wide bathymetric distribution ranging from shallow rocky bottoms to deep sublittoral reefs. The species shows two depth-related morphotypes which taxonomic status is not yet clarified. The aim of the study is to analyses genetic variability and/or structuring along a vertical gradient to test the presence of the two morphotypes. Furthermore, a preliminary analyses of the phylogenetic relationship among species of the genus Eunicella has been done. Six populations of Eunicella singularis were sampled from 10 to 60 m depth in Cap de Creus and individuals belonging to Eunicella cavolinii, E. verrucosa, E. racemosa and E. stricta aphyta were collected. The genetic analyses were carried out using five microsatellite loci and ITS-1 sequence polymorphism. The results showed a reduction of genetic variability along the vertical gradient. A threshold in connectivity was observed across 30 - 40 m depth, confirming the presence of two different Eunicella singularis morphotypes. The two morphological forms could be due to phenotypic plasticity, which allowed populations to suit different environmental conditions, or to a break in gene flow that determined the isolation of the two populations and an accumulation of genetic differences. The molecular markers used were not able to clarify the phylogenetic relationship among Eunicella species and the systematic position of the two morphotypes, conversely they risen the question on the existence of single species of Mediterranean Eunicella.

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).

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.

Are there genetic breaks between Atlantic and Pacific Yellowfin tuna (Thunnus albacares) populations? A preliminary study based on microsatellites gene variation

Yellowfin tuna (Thunnus albacares, YFT, Bonnaterre 1788) is one of the most important market tuna species in the world. The high mortality of juveniles is in part caused by their bycatch. Indeed, if unregulated, it could permanently destabilize stocks health. For this reason investigating and better knowing the stock boundaries represent a crucial concern. Aim of this thesis was to preliminary investigate the YFT population structure within and between Atlantic and Pacific Oceans through the analysis of genetic variation at eight microsatellite loci and assess the occurrence of barriers to the gene flow between Oceans. For this propouse we collected 4 geographical samples coming from Atlantic and Pacific Ocean and selected a panel of 8 microsatellites loci developped by Antoni et al., (2014). Samples 71-2-Y and 77-2-Y, came from rispectively west central pacific ocean (WCPO) and east central pacific ocean (ECPO), instead samples 41-1-Y and 34-2-Y derive from west central atlantic ocean (WCAO) and east central atlantic ocean (ECAO). Total 160 specimens were analyzed (40 per sample) and were carried out several genetic information as allele frequencies, allele number, allelic richness, HWE (using He and Ho) and pairwise Fst genetic distance. Results obtained, may support the panmictic theory of this species, only one of pairwise Fst obtained is statistically significant (Fst= 0.00927; pV= 0.00218) between 41-1-Y and 71-2-Y samples. Results suggest low genetic differentiation and consequent high level of gene flow between Atlantic and Pacific populations. Furthermore, we performed an analysis of molecular taxonomy through the use of ATCO (the flaking region between ATPse6 and cytochrome oxidase subunit III genes mt DNA, to discriminate within the gener Thunnus two of the related species (Yellofin and bigeye tuna) according with their difficult recognition at certain size (<40 cm). ATCO analysis in this thesis, has provided strong discriminate evidence between the target species proving to be one of the most reliable genetic tools capable to indagate within the genus Thunnus. Thus, our study has provided useful information for possible use of this protocol for conservation plans and management of this fish stocks.

Inference of Raja miraletus population structure using mitochondrial and nuclear DNA: comparing the resolution power of molecular markers in exploring species and population boundaries

This study focused on the role of oceanographic discontinuities and the presence of transitional areas in shaping the population structure and the phylogeography of the Raja miraletus species complex, coupled with the test of the effective occurrence of past speciation events. The comparisons between the Atlantic African and the North-Eastern Atlantic-Mediterranean geographic populations were unravelled using both Cytochrome Oxidase I and eight microsatellite loci. This approach guaranteed a robust dataset for the identification of a speciation event between the Atlantic African clade, corresponding to the ex Raja ocellifera nominal species, and the NE Atlantic-Mediterranean R. miraletus clade. As a matter of fact, the origin of the Atlantic Africa and the NE Atlantic-Mediterranean deep split dated about 11.74MYA and was likely due to the synergic influence currents and two upwelling areas crossing the Western African Waters. Within the Mediterranean Sea, particular attention was also paid to the transitional area represented by Adventura and Maltese Bank, that might have contributed in sustaining the connectivity of the Western and the Eastern Mediterranean geographical populations. Furthermore, the geology of the easternmost part of Sicily and the geo-morphological depression of the Calabrian Arc could have driven the differentiation of the Eastern Mediterranean Sea. Although bathymetric and oceanographic discontinuity could represent barriers to dispersal and migration between Eastern and Western Mediterranean samples, a clear and complete genetic separation among them was not detected. Results produced by this work identified a speciation event defining Raja ocellifera and R. miraletus as two different species, and describing the R. miraletus species complex as the most ancient cryptic speciation event in the family Rajidae, representing another example of how strictly connected the environment, the behavioural habits and the evolutionary and ecologic drivers are.