Deep sequencing of mixed total DNA without barcodes allows efficient assembly of highly plastic ascidian mitochondrial genomes

Abstract

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 polymerase chain reaction (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.

Keywords: Ascidians; Illumina; Tunicates; gene order; genome assembly; mitochondrial genome; mitogenomics; mixture models; next-generation sequencing; phylogeny; rearrangements.

Fig. 1.—

Ascidian species sequenced in this work. (A) Rhodosoma turcicum (Corellidae), (B) Ascidiella aspersa (Ascidiidae), (C) Botrylloides aff. leachii (Styelidae), (D) Polycarpa mytiligera (Styelidae), (E) Halocynthia spinosa (Pyuridae), and (F) Pyura gangelion (Pyuridae).

Fig. 2.—

Organization of the assembled tunicate mt genomes. Red background highlights extra and lost (in brackets) genes. tRNA genes are marked in black by their one-letter code, except for G(a), Gly(AGR); G(g), Gly(GGN); L(u), Leu(UUR); L(c), Leu(CUN); M(c), Met(CAU); M(u), Met(UAU); S(a), Ser(AGY); and S(u), Ser(UCN). P2 and Q2 indicate the extra trnP-2 and trnQ-2 genes of A. aspersa. rRNAs are marked in red. ATP synthase genes are marked in orange. NADH dehydrogenase genes (Complex I) are marked in blue. The cytochrome b (complex III) is marked in purple. Cytochrome c oxidase genes (Complex VI) are marked in green. Noncoding regions are marked in white. Ticks are set every 400 bp. All genes are transcribed clockwise.

Fig. 3.—

Comparison of mt gene order between closely related species. Syntenic regions within each pair of species are marked by the same color and indicated by connected rectangles. Noncoding (NC) regions > 20 bp are marked by a black background, with numbers corresponding to the NC size (in bp). Gene abbreviations: 8, atp6: subunits 8 and 6 of the F0 ATPase; cox1-3: cytochrome c oxidase subunits 1-3; cob: cytochrome b; nad1-6 and nad4L: NADH dehydrogenase subunits 1-6 and 4L; rrnS and rrnL: small and large subunit rRNAs. tRNA genes are indicated by the one-letter code of the transported amino acid, except for: F2: duplicated trnF gene; Ga, Gly(AGR); Gg, Gly(GGN); Lu, Leu(UUR); Lc, Leu(CUN); Mc, Met(CAU); Mu, Met(UAU); Sa, Ser(AGY); Su, Ser(UCN).

Fig. 4.—

Phylogeny of Deuterosomes inferred from the concatenation of the 13 mitochondrial proteins. Bayesian consensus tree of 4 independent MCMC runs obtained using the CAT + GTR + Γ mixture model (38 taxa and 3,038 amino acid sites). Values at nodes correspond to Bayesian PP. Circles indicate strongly supported nodes with PP ≥ 0.99.