Ascidians are a fascinating group of filter-feeding marine chordates characterized by rapid evolution of both sequences and structure of their nuclear and mitochondrial genomes. Moreover, they include several model organisms used to investigate complex biological processes in chordates. To study the evolutionary dynamics of ascidians at short phylogenetic distances, we sequenced 13 new mitogenomes and analyzed them, together with 15 other available mitogenomes, using a novel approach involving detailed whole-mitogenome comparisons of conspecific and congeneric pairs. The evolutionary rate was quite homogeneous at both intra-specific and congeneric level, and the lowest congeneric rates were found in cryptic (morphologically undistinguishable) and in morphologically very similar species pairs. Moreover, congeneric nonsynonymous rates (dN) were up to two orders of magnitude higher than in intra-species pairs. Overall, a clear-cut gap sets apart conspecific from congeneric pairs. These evolutionary peculiarities allowed easily identifying an extraordinary intra-specific variability in the model ascidian Botryllus schlosseri, where most pairs show a dN value between those observed at intra-species and congeneric level, yet consistently lower than that of the C. intestinalis cryptic species pair. These data suggest ongoing speciation events producing genetically distinct B. schlosseri entities. Remarkably, these ongoing speciation events were undetectable by the cox1 barcode fragment, demonstrating that, at low phylogenetic distances, the whole mitogenome has a higher resolving power than cox1. Our study shows that whole-mitogenome comparative analyses, performed on a suitable sample of congeneric and intra-species pairs, may allow detecting not only cryptic species but also ongoing speciation events.

Background: Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results: As a starting step in this direction, in this work we performed a large scale human-mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions: We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts) we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species-specific, suggests that, still maintaining the conventional definition of gene orthology, a new concept of "splicing orthology" can be defined at transcript level.

Ascidians or sea squirts form a diverse group within chordates, which includes a few thousand members of marine sessile filter-feeding animals. Their mitochondrial genomes are characterized by particularly high evolutionary rates and rampant gene rearrangements. This extreme variability complicates standard PCR-based techniques for molecular characterization studies, and consequently only a few complete ascidian mitochondrial genome sequences are available. Using the standard PCR and Sanger sequencing approach, we produced the mitochondrial genome of Ascidiella aspersa only after a great effort. In contrast, we produced five additional mitogenomes (Botrylloides aff. leachii, Halocynthia spinosa, Polycarpa mytiligera, Pyura gangelion, and Rhodosoma turcicum) with a novel strategy, consisting in sequencing the pooled total DNA samples of these five species using one Illumina HiSeq 2000 flow cell lane. Each mitogenome was efficiently assembled in a single contig using de novo transcriptome assembly, as de novo genome assembly generally performed poorly for this task. Each of the new six mitogenomes presents a different and novel gene order, showing that no syntenic block has been conserved at the ordinal level (in Stolidobranchia and in Phlebobranchia). Phylogenetic analyses support the paraphyly of both Ascidiacea and Phlebobranchia, with Thaliacea nested inside Phlebobranchia, although the deepest nodes of the Phlebobranchia-Thaliacea clade are not well resolved. The strategy described here thus provides a cost-effective approach to obtain complete mitogenomes characterized by a highly plastic gene order and a fast nucleotide/amino-acid substitution rate.

The few sequenced mitochondrial (mt) genomes of the class Ascidiacea (Chordata, Tunicata), mostly belonging to congeneric species of the Phlebobranchia order, show extraordinary gene order rearrangements. In order to assess if this hypervariability in gene order is a general feature of Ascidiacea, we report here the gene arrangement of five ascidians belonging to the Aplousobranchia and Stolidobranchia orders. Our data show that Ascidiacea are characterized by: 1) extensive gene order rearrangements both within and between the three major lineages; 2) lack of significant similarities to the gene order of other deuterostomes; and 3) an extent of rearrangements comparable with that of Mollusca (especially the Gastropoda, Bivalvia, and Scaphopoda classes), a phylum with highly rearranged mtDNAs. The only conserved feature is the location of all genes on the same strand, which suggests that selective constraints are related to the mt transcription. Finally, a higher mobility of the tRNA genes is undetectable because of saturation effect, and only the partially conserved cox2-cob gene block seems to retain some phylogenetic signals.

The MITOchondrial genome database of metaZOAns (MitoZoa) is a public resource for comparative analyses of metazoan mitochondrial genomes (mtDNA) at both the sequence and genomic organizational levels. The main characteristics of the MitoZoa database are the careful revision of mtDNA entry annotations and the possibility of retrieving gene order and non-coding region (NCR) data in appropriate formats. The MitoZoa retrieval system enables basic and complex queries at various taxonomic levels using different search menus. MitoZoa 2.0 has been enhanced in several aspects, including: a re-annotation pipeline to check the correctness of protein-coding gene predictions; a standardized annotation of introns and of precursor ORFs whose functionality is post-transcriptionally recovered by RNA editing or programmed translational frameshifting; updates of taxon-related fields and a BLAST sequence similarity search tool. Database novelties and the definition of standard mtDNA annotation rules, together with the user-friendly retrieval system and the BLAST service, make MitoZoa a valuable resource for comparative and evolutionary analyses as well as a reference database to assist in the annotation of novel mtDNA sequences. MitoZoa is freely accessible at http://www.caspur.it/mitozoa.

MitoZoa is a relational database collecting curated metazoan entries of complete or nearly complete mitochondrial genomes (mtDNA), specifically designed to assist comparative studies of mitochondrial genome-level features in a given taxon or in congeneric species of Metazoa. The principal novelties of MitoZoa are extensive corrections/improvements of the mtDNA annotations and the possibility of easily searching for data on: (1) gene order, a genomic feature useful as phylogenetic marker; (2) sequence, size and location of non-coding regions, likely containing the regulatory signals for mtDNA replication and transcription; (3) mt features/sequences of congeneric species, where saturation phenomena in nucleotide substitutions and gene order changes are expected to be absent or at least minimal. In addition, MitoZoa allows the exploration of basic mt features such as molecule topology, genetic code, gene content, and compositional parameters of the entire genome. Finally, in order to facilitate downstream analyses of retrieved data, MitoZoa entry lists can be visualized and downloaded in a tabular format, while sequences and gene order data are provided in FASTA and FASTA-like formats, respectively. The MitoZoa database is available at http://www.caspur.it/mitozoa. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confdently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration

According to the tRNA punctuation model, the mitochondrial genome (mtDNA) of mammals and arthropods is transcribed as large polycistronic precursors that are maturated by endonucleolytic cleavage at tRNA borders and RNA polyadenylation. Starting from the newly sequenced mtDNA of Ixodes ricinus and using a combination of mitogenomics and transcriptional analyses, we found that in all currently-sequenced tick lineages (Prostriata, Metastriata and Argasidae) the 3′-end of the polyadenylated nad1 and rrnL transcripts does not follow the tRNA punctuation model and is located upstream of a degenerate 17-bp DNA motif. A slightly different motif is also present downstream the 3′-end of nad1 transcripts in the primitive chelicerate Limulus polyphemus and in Drosophila species, indicating the ancient origin and the evolutionary conservation of this motif in arthropods. The transcriptional analyses suggest that this motif directs the 3′-end formation of the nad1/rrnL mature RNAs, likely working as a transcription termination signal or a processing signal of precursor transcripts. Moreover, as most regulatory elements, this motif is characterized by a taxon-specific evolution. Although this signal is not exclusive of ticks, making a play on words it has been named "Tick-Box", since it is a check mark that has to be verified for the 3′-end formation of some mt transcripts, and its consensus sequence has been here carefully characterized in ticks. Indeed, in the whole mtDNA of all ticks, the Tick-Box is always present downstream of nad1 and rrnL, mainly in non-coding regions (NCRs) and occasionally within trnL(CUN). However, some metastriates present a third Tick-Box at an intriguing site - inside the small NCR located at one end of a 3.4 kb translocated region, the other end of which exhibits the nad1 Tick-Box - hinting that this motif could have been involved in metastriate gene order rearrangements