Species identification, distributioin and reproductive interactions of common smoot-hound and blackspotted smooth-hound shark (Genus mustelus) in the Adriatic Sea

Longstanding taxonomic ambiguity and uncertainty exist in the identification of the common (M. mustelus) and blackspotted (M. punctulatus) smooth-hound in the Adriatic Sea. The lack of a clear and accurate method of morphological identification, leading to frequent misidentification, prevents the collation of species-specific landings and survey data for these fishes and hampers the delineation of the distribution ranges and stock boundaries of the species. In this context, adequate species-specific conservation and management strategies can not be applied without risks of population declining and local extinction. In this thesis work I investigated the molecular ecology of the two smooth-hound sharks which are abundant in the demersal trawl surveys carried out in the NC Adriatic Sea to monitor and assess the fishery resources. Ecological and evolutionary relationships were assessed by two molecular tests: a DNA barcoding analysis to improve species identification (and consequently the knowledge of their spatial ecology and taxonomy) and a hybridization assay based on the nuclear codominant marker ITS2 to evaluate reproductive interactions (hybridization or gene introgression). The smooth-hound sharks (N=208) were collected during the MEDITS 2008 and 2010 campaigns along the Italian and Croatian coasts of the Adriatic Sea, in the Sicilian Channel and in the Algerian fisheries. Since the identification based on morphological characters is not strongly reliable, I performed a molecular identification of the specimens producing for each one the cytochrome oxidase subunit 1 (COI) gene sequence (ca. 640 bp long) and compared them with reference sequences from different databases (GenBank and BOLD). From these molecular ID data I inferred the distribution of the two target species in the NC Adriatic Sea. In almost the totality of the MEDITS hauls I found no evidence of species sympatry. The data collected during the MEDITS survey showed an almost different distribution of M. mustelus (confined along the Italian coasts) and M. punctulatus (confined along the Croatian coasts); just one sample (Gulf of Venice, where probably the ranges of the species overlap) was found to have catches of both the species. Despite these data results suggested no interaction occurred between my two target species at least during the summertime (the period in which MEDITS survey is carried out), I still wanted to know if there were inter-species reproductive interactions so I developed a simple molecular genetic method to detect hybridization. This method is based on DNA sequence polymorphism among species in the nuclear ribosomal Internal Transcribed Spacer 2 locus (ITS2). Its application to the 208 specimens collected raised important questions regarding the ecology of this two species in the Adriatic Sea. In fact results showed signs of hybridization and/or gene introgression in two sharks collected during the trawl survey of 2008 and one collected during the 2010 one along the Italian and Croatian coasts. In the case that it will be confirmed the hybrid nature of these individuals, a spatiotemporal overlapping of the mating behaviour and ecology must occur. At the spatial level, the northern part of the Adriatic Sea (an area where the two species occur with high frequency of immature individuals) could likely play the role of a common nursery area for both species.

Statistical methods for the analysis of DNA sequences: application to dinucleotide distribution in the human genome

Questa tesi si inserisce nell'ambito delle analisi statistiche e dei metodi stocastici applicati all'analisi delle sequenze di DNA. Nello specifico il nostro lavoro è incentrato sullo studio del dinucleotide CG (CpG) all'interno del genoma umano, che si trova raggruppato in zone specifiche denominate CpG islands. Queste sono legate alla metilazione del DNA, un processo che riveste un ruolo fondamentale nella regolazione genica. La prima parte dello studio è dedicata a una caratterizzazione globale del contenuto e della distribuzione dei 16 diversi dinucleotidi all'interno del genoma umano: in particolare viene studiata la distribuzione delle distanze tra occorrenze successive dello stesso dinucleotide lungo la sequenza. I risultati vengono confrontati con diversi modelli nulli: sequenze random generate con catene di Markov di ordine zero (basate sulle frequenze relative dei nucleotidi) e uno (basate sulle probabilità di transizione tra diversi nucleotidi) e la distribuzione geometrica per le distanze. Da questa analisi le proprietà caratteristiche del dinucleotide CpG emergono chiaramente, sia dal confronto con gli altri dinucleotidi che con i modelli random. A seguito di questa prima parte abbiamo scelto di concentrare le successive analisi in zone di interesse biologico, studiando l’abbondanza e la distribuzione di CpG al loro interno (CpG islands, promotori e Lamina Associated Domains). Nei primi due casi si osserva un forte arricchimento nel contenuto di CpG, e la distribuzione delle distanze è spostata verso valori inferiori, indicando che questo dinucleotide è clusterizzato. All’interno delle LADs si trovano mediamente meno CpG e questi presentano distanze maggiori. Infine abbiamo adottato una rappresentazione a random walk del DNA, costruita in base al posizionamento dei dinucleotidi: il walk ottenuto presenta caratteristiche drasticamente diverse all’interno e all’esterno di zone annotate come CpG island. Riteniamo pertanto che metodi basati su questo approccio potrebbero essere sfruttati per migliorare l’individuazione di queste aree di interesse nel genoma umano e di altri organismi.