Metodi molecolari nello studio della variabilità genetica di Tuber borchii

Tuber borchii (Ascomycota, order Pezizales) is highly valued truffle sold in local markets in Italy. Despite its economic importance, knowledge on its distribution and population variation is scarce. The objective of this work was to investigate the evolutionary forces shaping the genetic structure of this fungus using coalescent and phylogenetic methods to reconstruct the evolutionary history of populations in Italy. To assess population structure, 61 specimens were collected from 11 different Provinces of Italy. Sampling was stratified across hosts and habitats to maximize coverage in native oak and pine stands and both mychorrizae and fruiting bodies were collected. Samples were identified considering anatomo-morphological characters. DNA was extracted and both multilocus (AFLP) and single-locus (18 loci from rDNA, nDNA, and mtDNA) approaches were used to look for polymorphisms. Screening AFLP profiles, both Jaccard and Dice coefficients of similarity were utilized to transform binary matrix into a distance matrix and then to desume Neighbour-Joining trees. Though these are only preliminary examinations, phylogenetic trees were totally concordant with those deriving from single locus analyses. Phylogenetic analyses of the nuclear loci were performed using maximum likelihood with PAUP and a combined phylogenetic inference, using Bayesian estimation with all nuclear gene regions, was carried out. To reconstruct the evolutionary history, we estimated recurrent migration, migration across the history of the sample, and estimated the mutation and approximate age of mutations in each tree using SNAP Workbench. The combined phylogenetic tree using Bayesian estimation suggests that there are two main haplotypes that are difficult to be differentiated on the basis of morphology, of ecological parameters and symbiontic tree. Between these two lineages, that occur in sympatry within T. borchii populations, there is no evidence of recurrent migration. However, migration over the history of the sample was asymmetrical suggesting that isolation was a result of interrupted gene flow followed by range expansion. Low levels of divergence between the haplotypes indicate that there are likely to be two cryptic species within the T. borchii population sampled. Our results suggest that isolation between populations of T. borchii could have led to reproductive isolation between two lineages. This isolation is likely due to sympatric speciation caused by a multiple colonization from different refugia or a recent isolation. In attempting to determinate whether these haplotypes represent separate species or a partition of the same species we applied Biological and Mechanistic species Concepts. Notwithstanding, further analyses are necessary to evaluate if selection favoured premating or post-mating isolation.

B-type cytochromes of the DOMON domain superfamily (Novel redox elements of plant plasma membrane)

The aim of the present study is understanding the properties of a new group of redox proteins having in common a DOMON-type domain with characteristics of cytochromes b. The superfamily of proteins containing a DOMON of this type includes a few protein families. With the aim of better characterizing this new protein family, the present work addresses both a CyDOM protein (a cytochrome b561) and a protein only comprised of DOMON(AIR12), both of plant origin. Apoplastic ascorbate can be regenerated from monodehydroascorbate by a trans-plasma membrane redox system which uses cytosolic ascorbate as a reductant and comprises a high potential cytochrome b. We identified the major plasma membrane (PM) ascorbate-reducible b-type cytochrome of bean (Phaseolus vulgaris) and soybean (Glycine max) hypocotyls as orthologs of Arabidopsis auxin-responsive gene air12. The protein, which is glycosylated and glycosylphosphatidylinositol-anchored to the external side of the PM in vivo, was expressed in Pichia pastoris in a recombinant form, lacking the glycosylphosphatidylinositol-modification signal, and purified from the culture medium. Recombinant AIR12 is a soluble protein predicted to fold into a β-sandwich domain and belonging to the DOMON superfamily. It is shown to be a b-type cytochrome with a symmetrical α-band at 561 nm, to be fully reduced by ascorbate and fully oxidized by monodehydroascorbate. Redox potentiometry suggests that AIR12 binds two high-potential hemes (Em,7 +135 and +236 mV). Phylogenetic analyses reveal that the auxin-responsive genes AIR12 constitute a new family of plasma membrane b-type cytochromes specific to flowering plants. Although AIR12 is one of the few redox proteins of the PM characterized to date, the role of AIR12 in trans-PM electron transfer would imply interaction with other partners which are still to be identified. Another part of the present project was aimed at understanding of a soybean protein comprised of a DOMON fused with a well-defined b561 cytochrome domain (CyDOM). Various bioinformatic approaches show this protein to be composed of an N-terminal DOMON followed by b561 domain. The latter contains five transmembrane helices featuring highly conserved histidines, which might bind haem groups. The CyDOM has been cloned and expressed in the yeast Pichia pastoris, and spectroscopic analyses have been accomplished on solubilized yeast membranes. CyDOM clearly reveal the properties of b-type cytochrome. The results highlight the fact that CyDOM is clearly able to lead an electron flux through the plasmamembrane. Voltage clamp experiments demonstrate that Xenopus laevis oocytes transformed with CyDOM of soybean exhibit negative electrical currents in presence of an external electron acceptor. Analogous investigations were carried out with SDR2, a CyDOM of Drosophila melanogaster which shows an electron transport capacity even higher than plant CyDOM. As quoted above, these data reinforce those obtained in plant CyDOM on the one hand, and on the other hand allow to attribute to SDR2-like proteins the properties assigned to CyDOM. Was expressed in Regenerated tobacco roots, transiently transformed with infected a with chimeral construct GFP: CyDOM (by A. rhizogenes infection) reveals a plasmamembrane localization of CyDOM both in epidermal cells of the elongation zone of roots and in root hairs. In conclusion. Although the data presented here await to be expanded and in part clarified, it is safe to say they open a new perspective about the role of this group of proteins. The biological relevance of the functional and physiological implications of DOMON redox domains seems noteworthy, and it can but increase with future advances in research. Beyond the very finding, however interesting in itself, of DOMON domains as extracellular cytochromes, the present study testifies to the fact that cytochrome proteins containing DOMON domains of the type of “CyDOM” can transfer electrons through membranes and may represent the most important redox component of the plasmamembrane as yet discovered.

Phylogeny and population dynamics in European Reticulitermes and Kalotermes genera (Insecta, Isoptera)

The aim of this thesis is to detect the phylogeny and the population dynamics of the European termites of the genera Reticulitermes and Kalotermes, by the use of different mitochondrial (16S, COI/tRNA/COII, CR) and nuclear (microsatellites and Inter-SINE) molecular markers. In the phylogenetic analyses, the obtained results have well defined the cladogenetic events that generated the nowadays species biodiversity of the genus Reticulitermes, while the analysis of the Kalotermes flavicollis taxon showed the presence of at least four genetic clades, defined on the basis of the geographical distance. The second part of the thesis is centred on the population dynamics of two species: Reticulitermes urbis and Kalotermes flavicollis. The first species, native of the Balkans, is known to be present in some cities of Italy and France. I’ve analyzed the colony genetic structure of the introduced population of Bagnacavallo (RA, Italy), using nine microsatellite loci. The obtained results are in accordance with those obtained from another population in France: this species in fact confirms its invasive and infestation capacities. The analysis of the natural population of K. flavicollis, performed with a combination of mitochondrial (control region) and nuclear (microsatellites and I-SINE) markers, clearly evidenced the presence of two genetic lineages that coexist in the same area. Moreover, results clearly indicate that the cross-breeding is allowed. Finally, the whole results are discussed in a comparative view to better understand the differences in ecology, evolutionary dynamics and colony social structure between these two genera.

A molecular phylogeny of bivalve mollusks: ancient radiations and divergences as revealed by mitochondrial genes

The main scope of my PhD is the reconstruction of the large-scale bivalve phylogeny on the basis of four mitochondrial genes, with samples taken from all major groups of the class. To my knowledge, it is the first attempt of such a breadth in Bivalvia. I decided to focus on both ribosomal and protein coding DNA sequences (two ribosomal encoding genes -12s and 16s -, and two protein coding ones - cytochrome c oxidase I and cytochrome b), since either bibliography and my preliminary results confirmed the importance of combined gene signals in improving evolutionary pathways of the group. Moreover, I wanted to propose a methodological pipeline that proved to be useful to obtain robust results in bivalves phylogeny. Actually, best-performing taxon sampling and alignment strategies were tested, and several data partitioning and molecular evolution models were analyzed, thus demonstrating the importance of molding and implementing non-trivial evolutionary models. In the line of a more rigorous approach to data analysis, I also proposed a new method to assess taxon sampling, by developing Clarke and Warwick statistics: taxon sampling is a major concern in phylogenetic studies, and incomplete, biased, or improper taxon assemblies can lead to misleading results in reconstructing evolutionary trees. Theoretical methods are already available to optimize taxon choice in phylogenetic analyses, but most involve some knowledge about genetic relationships of the group of interest, or even a well-established phylogeny itself; these data are not always available in general phylogenetic applications. The method I proposed measures the "phylogenetic representativeness" of a given sample or set of samples and it is based entirely on the pre-existing available taxonomy of the ingroup, which is commonly known to investigators. Moreover, it also accounts for instability and discordance in taxonomies. A Python-based script suite, called PhyRe, has been developed to implement all analyses.

Identification and genetic diversity in phytoplasmas associated with diseases of cassava and other agronomic relevant crops in south-east Asia and Latin America

Identification and genetic diversity of phytoplasmas infecting tropical plant species, selected among those most agronomically relevant in South-east Asia and Latin America were studied. Correlation between evolutionary divergence of relevant phytoplasma strains and their geographic distribution by comparison on homologous genes of phytoplasma strains detected in the same or related plant species in other geographical areas worldwide was achieved. Molecular diversity was studied on genes coding ribosomal proteins, groEL, tuf and amp besides phytoplasma 16S rRNA. Selected samples infected by phytoplasmas belonging to diverse ribosomal groups were also studied by in silico RFLP followed by phylogenetic analyses. Moreover a partial genome annotation of a ‘Ca. P. brasiliense’ strain was done towards future application for epidemiological studies. Phytoplasma presence in cassava showing frog skin (CFSD) and witches’ broom (CWB) diseases in Costa Rica - Paraguay and in Vietnam – Thailand, respectively, was evaluated. In both cases, the diseases were associated with phytoplasmas related to aster yellows, apple proliferation and “stolbur” groups, while only phytoplasma related to X-disease group in CFSD, and to hibiscus witches’ broom, elm yellows and clover proliferation groups in CWB. Variability was found among strains belonging to the same ribosomal group but having different geographic origin and associated with different disease. Additionally, a dodder transmission assay to elucidate the role of phytoplasmas in CWB disease was carried out, and resulted in typical phytoplasma symptoms in periwinkle plants associated with the presence of aster yellows-related strains. Lethal wilt disease, a severe disease of oil palm in Colombia that is spreading throughout South America was also studied. Phytoplasmas were detected in symptomatic oil palm and identified as ‘Ca. P. asteris’, ribosomal subgroup 16SrI-B, and were distinguished from other aster yellows phytoplasmas used as reference strains; in particular, from an aster yellows strain infecting corn in the same country.