Evolutionary features of the mitochondrial genome of Ascidiacea (Chordata, Tunicata) at short and long phylogenetic distances

Evolutionary studies of the mitochondrial genome (mtDNA) have been widely used to solve phylogenetic controversies and to analyse the mechanisms of whole genome evolution. Indeed, the availability of several complete mtDNAs have provided, in addition to sequences of single genes, several whole-genome characters useful to solve phylogenetic questions. Moreover, the strong correlation between mitochondrial structural features and functional processes like the replication and transcription of mtDNA increases the importance to deeply investigate the mitochondrial genomic features. At present, the sequence sample of complete mtDNAs is biased toward vertebrates, and the sampling within deuterostome groups, such as hemichordates and tunicates is very poor. The mtDNA of tunicates, traditionally considered the basal group of chordates, shows several differences from the mtDNAs of remaining chordates and deuterostomes. Indeed, the tunicate mtDNA presents a different genetic code compared to that of all other deuterostomes, and encodes for two additional tRNA genes, trnG(AGR) and trnM(TAT). The trnG(AGR) gene is needed because of the different genetic code and the second trnM gene is due to the usage of different tRNA-Met as initiator and elongator. The most interesting feature of tunicate mtDNA is the high gene order variability compared to remainimg deuterostomes. This extensive gene rearrangement has been found even at intra genus level (Iannelli et al. 2007a; Iannelli et al. 2007b). However, the mtDNA has been completely sequenced only in seven tunicate species: six ascidian species (two Phallusia species and three Ciona species belonging to Phlebobranchiata order; and Halocynthia roretzi belonging to Stolidobranchiata order) and the Thaliacea Doliolum nationalis. In order to deeper analyse the structural features and the evolution of tunicate mtDNA, in this PhD project, we have sequenced the whole mtDNA of four ascidian species: the Aplousobranchiata species Clavelina lepadiformis, Clavelina phlegraea (Polycitoridae family), and Diplosoma listerianum (Didemnidae family); and the Phlebobranchiata species Ascidiella aspersa (Ascidiidae family). The two Clavelina species and D. listerianum have been selected in order to have the representatives of the unsampled Aplousobranchiata order even at intra-genus level. The mtDNA of A. aspersa has been sequenced in order to verify if the high number of gene rearrangements and the high GC content previously observed in Phallusia species, belonging to the same family Ascidiidae, are typical features of all Ascidiidae species. Thus, we have carried out comparative analyses of several mt features. also including the mtDNA of other ascidian species recently sequenced in our laboratory, and the mtDNA of other deuterostome species available in the public databases. All tunicate mtDNAs encode for the canonical mt genes but show a variable number of tRNA genes. This is in accordance with the situation observed in remaining deuterostomes, where only in few cases there is a variation of the protein gene number while changes of the tRNA gene number is more frequent. Only the mtDNA of A. aspersa shows an additional unassignable ORF of uncertain function. Tunicate mtDNAs show a great variability of gene order, indeed no conserved gene blocks are shared by all tunicates or by all ascidians. Gene rearrangements at intra-genus level have been observed in all analyzed congeneric pairs but the extent of gene rearrangements within the same genus seems to be less pronounced in the Aplousobranchiata order (the two Clavelina species show the same gene order except for the position of one tRNA gene) than in the other ascidian orders, although this observation could be the consequence of a still poor taxon sampling. The base composition is variable within tunicates and the GC content appears to follow a taxon-specific trend: the GC content is low in Aplousobranchiata, variable in Phlebobranchiata, and intermediate in Stolidobranchiata. As regard compositional asymmetry, that is the distribution of complementary bases between the two strands, the different ascidian mtDNAs show different behaviours: the AT- and GC-skews, parameters measuring the compositional asymmetry, are close to zero in most species, thus indicating an almost symmetric composition. However, in some ascidians the AT- and GC-skews show opposite signs compared to vertebrates and several cephalochordates. In vertebrates, the compositional asymmetry is related to the asymmetric mtDNA replication mechanism. Thus, the absence or the opposite orientation of the compositional asymmetry in tunicates compared to vertebrates can suggest a different replication mechanism that does not affect the base distribution between the two strands. This hypothesis is also supported by the absence, in tunicates, of a major non-coding region with size and features similar to the control region of vertebrates, which is known to be involved in mtDNA replication and transcription. All analysed mt features indicate strong differences between the mtDNA evolutionary dynamics of tunicates and those of remaining deuterostomes but at present we can not hypothesize which mechanisms are responsible of the fast mt evolutionary dynamics of tunicates.