Evolutionary Insights from the Mitochondrial Genome of Oikopleura dioica: Sequencing Challenges, RNA Editing, Gene Transfers to the Nucleus, and tRNA Loss

Abstract

Sequencing the mitochondrial genome of the tunicate Oikopleura dioica is a challenging task due to the presence of long poly-A/T homopolymer stretches, which impair sequencing and assembly. Here, we report on the sequencing and annotation of the majority of the mitochondrial genome of O. dioica by means of combining several DNA and amplicon reads obtained by Illumina and MinIon Oxford Nanopore Technologies with public RNA sequences. We document extensive RNA editing, since all homopolymer stretches present in the mitochondrial DNA correspond to 6U-regions in the mitochondrial RNA. Out of the 13 canonical protein-coding genes, we were able to detect eight, plus an unassigned open reading frame that lacked sequence similarity to canonical mitochondrial protein-coding genes. We show that the nad3 gene has been transferred to the nucleus and acquired a mitochondria-targeting signal. In addition to two very short rRNAs, we could only identify a single tRNA (tRNA-Met), suggesting multiple losses of tRNA genes, supported by a corresponding loss of mitochondrial aminoacyl-tRNA synthetases in the nuclear genome. Based on the eight canonical protein-coding genes identified, we reconstructed maximum likelihood and Bayesian phylogenetic trees and inferred an extreme evolutionary rate of this mitochondrial genome. The phylogenetic position of appendicularians among tunicates, however, could not be accurately determined.

Keywords: Appendicularia; Oxford Nanopore Technologies; Tunicata; mtDNA; phylogenomics; poly-A/T homopolymers.

Fig. 1.

Oikopleura dioica mitochondrial genome map. Protein-coding genes are in green, rRNA genes (rns, rnl) in dark blue, tRNA-Met (UAU) in pink, CDSs in yellow, and poly-A/T stretches in light blue. The genes are pointing in the direction of their transcriptional orientation. A nonsequenced region, indicated as “gap,” is in gray.

Fig. 2.

A typical poly-A/T region in the O. doica cox1 gene. The reference sequence represents the assembled mitochondrial genome. The sequence of part of the cox1 gene is indicated in dark green. Above it are the nucleotide and amino acid sequences of the reference sequence. The poly-A/T region is in light blue. Reads from five different sequencing sources mapped to this region are shown below the reference sequence: our ONT data, our Illumina reads, public EST data from Norway, public RNA reads from Japan, and public RNA reads from the North Sea (see supplementary table S3a, Supplementary Material online for accession numbers). Each line represents a different read. Note that the different ONT reads do not have the same length, exemplifying the fact that the length of the poly-T region could not be accurately determined. The EST and RNA reads lack a long poly-U region. Rather, the poly-T region corresponds to 6U-bases. This difference (long poly-T stretch in DNA vs. 6U in the EST and RNA reads) provides evidence of editing at the RNA level. Illumina reads can map to each side of the poly-T stretch, but because poly-T stretches can be longer than the read length, we were unable to assemble the entire region using Illumina only or to determine the length of the poly-T region.

Fig. 3.

Presence–absence of aaRS orthologs in different species. Single eukaryotic (Euk): single-copy aaRSs that function in both the cytosol and mitochondria. Other aaRSs are expressed either only in the cytosol (cytosolic) or in the mitochondrion (mitochondrial). aaRSs presence is shown in pink, aaRSs absence in white, and aaRS-Gly in blue. Protein accession numbers are provided in supplementary table S5, Supplementary Material online.

Fig. 4.

The position of O. dioica among chordates. The phylogenetic tree was reconstructed based on mitochondrial protein sequences under the MtZoa+EHO+F+I+G4. Branch supports are only indicated for those cases in which the bootstrap support values were lower than 100%. The tunicate classes: Stolidobranchia in blue, Thaliacea in red, Phlebobranchia in green, Aplousobranchia in yellow, and Appendicularia in purple.