The mitochondrial genome of Phallusia mammillata and Phallusia fumigata (Tunicata, Ascidiacea): high genome plasticity at intra-genus level

Abstract

Background: Within Chordata, the subphyla Vertebrata and Cephalochordata (lancelets) are characterized by a remarkable stability of the mitochondrial (mt) genome, with constancy of gene content and almost invariant gene order, whereas the limited mitochondrial data on the subphylum Tunicata suggest frequent and extensive gene rearrangements, observed also within ascidians of the same genus.

Results: To confirm this evolutionary trend and to better understand the evolutionary dynamics of the mitochondrial genome in Tunicata Ascidiacea, we have sequenced and characterized the complete mt genome of two congeneric ascidian species, Phallusia mammillata and Phallusia fumigata (Phlebobranchiata, Ascidiidae). The two mtDNAs are surprisingly rearranged, both with respect to one another and relative to those of other tunicates and chordates, with gene rearrangements affecting both protein-coding and tRNA genes. The new data highlight the extraordinary variability of ascidian mt genome in base composition, tRNA secondary structure, tRNA gene content, and non-coding regions (number, size, sequence and location). Indeed, both Phallusia genomes lack the trnD gene, show loss/acquisition of DHU-arm in two tRNAs, and have a G+C content two-fold higher than other ascidians. Moreover, the mt genome of P. fumigata presents two identical copies of trnI, an extra tRNA gene with uncertain amino acid specificity, and four almost identical sequence regions. In addition, a truncated cytochrome b, lacking a C-terminal tail that commonly protrudes into the mt matrix, has been identified as a new mt feature probably shared by all tunicates.

Conclusion: The frequent occurrence of major gene order rearrangements in ascidians both at high taxonomic level and within the same genus makes this taxon an excellent model to study the mechanisms of gene rearrangement, and renders the mt genome an invaluable phylogenetic marker to investigate molecular biodiversity and speciation events in this largely unexplored group of basal chordates.

Figure 1

Gene organization of tunicate mitochondrial genomes, and taxonomic classification of the analysed species. Gene blocks conserved between P. mammillata and P. fumigata are reported in different colours, with yellow indicating the gene pair conserved in all analysed tunicates. Sequences almost identical in the same mtDNA are underlined and linked by a red line. Genes transposed among the three Ciona species are underlined and linked by a black line. Non-coding (NC) regions equal or longer that 40 bp are indicated by black background, with numbers corresponding to their size (in bp). Abbreviations for protein-coding and rRNA genes are as in the main text, except for atp8 gene (abbreviation: 8). Transfer RNA genes are indicated according to the transported amino acid, except for: G1: Gly(AGR); G2: Gly(GGN); L1: Leu(UUR); L2: Leu(CUN); M1: Met(AUG); M2: Met(AUA); S1: Ser(AGY); S2: Ser(UCN). Diamond: trnX gene of P. fumigata. Star: intra-genome duplicated genes, that is trnI genes in P. fumigata (named I1 and I2), and trnF in Halocynthia roretzi. Feature table of Halocynthia roretzi mtDNA is as reported in [38].

Figure 2

Similarity between the two trnI sequences of P. fumigata, including flanking non-coding (NC) regions. A gray background highlights the 208 bp sequence with 95% identity between the two regions, with trnI-1 and trnI-2 sequences boxed. Gene abbreviations are as in the main text. Non-coding regions are named according to their length (in bp). Underlined sequences indicate inverted repeats.

Figure 3

Similarity between the non-coding region 134 bp-long (NC-134) and the trnP gene of P. fumigata, together with the tRNA-like structure assumed by NC-134. A gray background indicates the 29-bp identical sequences, and boxed sequences identify tRNA and tRNA-like structures. In the tRNA-like structure, canonical and G-U base pairing are differently indicated. Stop codons are in black background.

Figure 4

Alignment of the C-terminal region of cytochrome b. All available tunicate species and representatives of remaining deuterostomes are shown. Identical and conserved residues are shown by reverse contrast with black and dark gray background, respectively. Similar residues are indicated by light gray background. The "ENK" consensus sequence of non-ascidian proteins is highlighted by asterisks. Numbers refer to amino acid position. Species classification and AC number of the analysed sequences are listed in Additional file 2.

Figure 5

tRNA genes of P. fumigata sharing a long and almost identical sequence (length ≥ 30 bp; identity ≥ 85%). (A) Similarity between trnX and trnY, and alternative secondary structures of the tRNA encoded by trnX (tRNA-Tyr2: position 13667–13730; tRNA-Thr2: position 13665–13729). (B) Similarity between trnR and trnQ. A gray background indicates almost identical sequences. Anticodon sequences are in bold. Canonical and G-U base pairing are differently indicated in the tRNA structure.