DNA barcoding e tassonomia integrata per lo studio della biodiversità in taxa animali ad elevata diversità criptica

The study of biodiversity is still a hot research topic today. DNA barcoding, proposed by Hebert et al. (2003), has proven to be a powerful and rapid tool for sample identification and species discovery. This molecular approach finds applications in various fields including community ecology, conservation biology, public health monitoring, pharmacognosy, food industry, and forensic medicine. The success of DNA barcoding relies on two main premises: (i) the barcoding gap, which separates intra-specific genetic divergences from inter-specific genetic divergences, allowing to distinguish conspecific samples from those belonging to different species; (ii) the availability of reference sequence libraries associated with accurately identified samples by expert taxonomists. In cases where DNA barcoding analyses fails to provide certain identifications (often due to gaps in the reference library), it become necessary an integrative taxonomic investigation, incorporating independent characters sets such as morphology, ecology, and biogeography, for species validation and identification. Starting from preliminary taxonomic hypotheses, based on species delimitation methods to define molecular operational taxonomic units (MOTUs), integrative taxonomic studies can allow reaching robust taxonomic conclusions and in-depth understanding of biodiversity. The aim of the PhD thesis is to combine DNA barcoding and integrative taxonomy to assess the taxonomic diversity of two selected animal groups for which morphological diagnosis can be extremely challenging. The two animal groups selected as study model are the flea beetles (tribe Alticini Newman, 1834) and the true oysters, that belong to the families Chrysomelidae Latreille, 1802 and Ostreidae Rafinesque, 1815, respectively. Alticini are characterized by high diversity with approximately 10,000 species distributed across 601 genera. This group exhibits well-defined taxonomy; however, taxonomic experts on regional faunas are often required for correct identification at species-level. In contrast, the Ostreidae family, composed of 78 species distributed worldwide, has a poorly defined taxonomy. Taxonomic identification of most species is hampered by their phenotypic plasticity and the absence of reliable diagnostic characters. In the first part, the thesis develops studies on the biodiversity of Alticini. The DNA barcoding approach was applied by generating a reference library for 99 species belonging to 21 genera based on morphological identification and sequencing of 278 samples from 119 sites along the entire Apennine chain. The rate of correct identification of these samples based on available BOLD and GB sequence libraries was high (88%). However, 684 cases of misidentification were found in the BOLD-GB library for the studied species, which were subsequently corrected based on the reference library generated in the thesis. This study substantiates the importance of accurate reference libraries and the key role of taxonomists both in generating libraries and in their posterior validation. The solid taxonomy available for Alticini makes DNA barcoding an efficient tool for molecular species identification, with a limited number of groups requiring taxonomic reassessment. Subsequently, an integrated taxonomy approach was applied in the thesis to the Longitarsus candidulus species group. In this case, the validity of three morphologically very similar species was confirmed using a multi-locus phylogenetic approach. In particular, the species L. laureolae and L. leonardii show a peculiar disjunct Apennine-Pyrenean distribution, likely reflecting a speciation event during the Pleistocene associated with a period of severe climate change in Europe. In the second part of the thesis, the study of global oysters’ diversity was developed through the analysis of 813 samples deposited in two of the world's major collections