The complete mitogenome of an unidentified Oikopleura species

Introduction

Appendicularians (synonym: larvaceans) are tunicates distributed all over the world’s ocean that do not have a sessile stage, remaining free-swimming throughout their life cycle, and construct a cellulose “house” which is used for feeding and protection.¹ The best studied appendicularian is the ~4 mm length O. dioica, ² but there are also large species with a body size ranging between 3–10 cm.³ Appendicularian mitochondrial genomes use the ascidian mitochondrial genetic code,^4–8 which differs from the invertebrate one by the reassignment of AGR codons from serine to glycine. In one clade within appendicularians containing O. dioica, homopolymers interrupt coding sequences and are resolved to hexamers by an unknown editing process.^4–6 In this study, we sequenced the mitochondrial genome of an unknown appendicularian species sampled from the Amami Oshima island, Japan (Figure 1), in order to increase the taxonomic power of eDNA studies based on the sequence of mitochondrial genes. In addition to that, mitochondrial DNA sequence of appendicularians can be useful in evolutionary studies to the origin of tunicates, as well as chordates, and as a means to understand the hidden biodiversity of Appendicularia, which is a quite neglected taxon.

Figure 1. Photographs of a specimen.

Photographs of a specimen of the unidentified Oikopleura species preserved in 70% EtOH of (A) the whole body and (B) the trunk. Photos were taken by Dr. Yongkai Tan.

Methods

Results and discussion

We noticed large appendicularians during a sampling trip targeting O. dioica in the Amami Oshima island, Kagoshima, Japan. We took the opportunity to collect several of these large appendicularians and sequenced a single individual, from which we assembled a circular mitogenome of 13,058 bp length (Figure 2). We mapped the fraction of sequencing reads that were used for the assembly to the assembled sequence and obtained a sequencing depth between 36–176 × and an average depth of 122.4 × (Fig. S1).

Figure 2. Circular plot of the mitogenome.

Circular plot generated by Geneious Prime version 2024.0.5. Protein coding genes and tRNAs are displayed on a yellow and pink background respectively. The circle in the middle illustrates the homopolymers; 6 or more successive Ts (forward) or As (reverse) in red and Cs (forward) or Gs (reverse) in blue. Abbreviations as follows, cox2: cytochrome c oxidase subunit 2, nad5: NADH dehydrogenase subunit 5, cob: cytochrome b, nad4: NADH dehydrogenase subunit 4, nad1: NADH dehydrogenase subunit 1, putative nad2: putative NADH dehydrogenase subunit 2, atp6: ATP synthase Fo subunit 6, cox1: cytochrome c oxidase subunit 1, cox3: cytochrome c oxidase subunit 3.

The mitogenome does not possess stretches of homopolymers like the ones observed in O. dioica. There is also no evidence of mitochondrial introns. Thus, we could translate its open reading frames (ORFs) with no interruptions. We found a total of 9 protein coding genes, two pseudogenes (fragments of cox3) and 2 tRNA genes, all on the same strand (Table S1), but could not annotate ribosomal RNA genes, although the length of the remaining unannotated regions suggest that they may be present. We were only able to detect tRNA genes for Leucine and Valine.

Eight out of the nine protein-coding genes match known mitochondrial proteins without ambiguity. Previous work on other species suggests that synteny is usually not conserved in tunicates.²⁰ Indeed, we found that the gene order bears no resemblance to the one of O. dioica^5,6 nor to the one of O. longicauda²¹ (scaffold SCLD01101138.1) nor to one in the stolidobranch ascidians Herdmania momus and Halocynthia roretzi.

We also found an ORF with homology to the putative NADH dehydrogenase 2 (nad2) reported by Klirs et al.,⁶ and we also found matches in other appendicularian species, confirming its presence across Oikopleuridae (Fig. S2). Searches using BLASTp on the non-redundant protein sequences database did not yield hits, and a tBLASTx search on whole-genome shotgun contigs database of tunicates (taxid:7712) matched a predicted Oikopleura longicauda mitochondrial contig (SCLD01139119.1) which is different from the one we used for the phylogenetic analysis and misses four of the eight expected mitochondrial proteins. The predicted structure of the putative nad2 using colabfold¹⁷ consists of alpha helices (Fig. S3), similar to reported nad2 protein from human (PDB IDs: 5XTC chain Q). This observation might have been caused by tunicates having fast evolving mitochondria,²⁰ and the coverage gap in the database that currently is available.

We extended the automatic gene annotation to the longest ORF which has stop codons (TAA, TAG) and start codons (TTG, ATA, ATG, GTG) accepted by the ascidian mitochondrial code.⁷ Nevertheless, due to the variability of initiation tRNA, we cannot rule out the possibility of the translation start codon being different, for example Halocynthia roretzi uses ATT as a start codon.²²

The codon usage (table S2) shows that, while TGA codes for tryptophan in tunicates, it is used in less than 5% of the tryptophan positions. Furthermore, these TGA codons were only found in the most N-terminal region of cox2, which is not well supported by alignment to other appendicularians and has a possible alternative start site downstream of these codons. Thus, depending on the real position of cox2’s translation start site, it is possible that the TGA codon is not used in this genome, similar to what was reported for O. longicauda on the cox1 and cob genes.⁷ Other than that, there are several other codon biases, such as towards TTG (39.7% and TTA (30.4%) for leucine. Another bias is present towards GTG that is coding for valine (56.7%).

The phylogenetic tree using protein-coding mitochondrial sequences (Figure 3, Figure S4) shows that this unknown species belongs to the clade of appendicularians that includes Bathochordaeus, Mesochordaeus and Oikopleura longicauda but not O. dioica. This clade was also found in a phylogenetic analysis of ribosomal protein sequences.²¹ The split between O. dioica and the other appendicularians in our tree corresponds to the bioluminescent/non-bioluminescent classification of Galt et al., 1985.²³ This is also reflected in the situation of homopolymers which are not abundant in this mitogenome, similar to O. longicauda²¹ and not O. dioica.⁵ As the Oikopleura genus is paraphyletic in our phylogenetic analysis and that of others, further work not in the scope of this manuscript will be needed to resolve which genus has to be corrected. Considering the tunicates phylogeny, the tree recovered clades for the free-living Appendicularia, Thaliacea, and for the sessile Stolidobranchia, Aplousobranchia and Phlebobranchia, from which it detached the Ciona genus as a separate clade. A single thaliacean species, Doliolum nationalis, grouped with the sessile tunicates, however this is not fully supported by bootstrap values. This computed tree suggests that targeted sampling and sequencing of additional doliolids and Ciona species may be useful to further clarify the phylogeny of tunicates classes and order.

Figure 3. Phylogenetic tree of mitochondrial genomes.

Phylogenetic tree computed by maximum likelihood inference on a ~13 kbp codon alignment of 13 mitochondrial genome protein-coding genes collected from publicly available aquatic chordate genomes.

As a final attempt to identify the species of this appendicularian, we extracted the sequence of the nuclear ribosomal RNA gene from one sequence read (see supplemental material), which we used to screen the GenBank database. The best hit (MK621860) has 1757 identical nucleotides over a length of 1773 (99%), and is from an O. fusiformis individual sampled in Croatia.

Conclusion

We present here the complete mitogenome of an unidentified Oikopleura species. Our phylogenetic analysis and the lack of homopolymer insertions show that it is closer to the lineage of O. longicauda than to the one of O. dioica. Morphological similarity and a preliminary analysis using nuclear genome rRNA sequences suggest that this unknown appendicularian is most closely related to O. fusiformis, however as our recent studies of O. dioica^24,25 uncovered cryptic speciation in appendicularians, and in the absence of specimen preservation allowing for confident taxonomic identification, we refrain from naming the species at this current stage. We project that the data produced in this study will be useful in future eDNA studies.

Ethical approval

Ethical approval and consent were not required.

Author contributions

JNW, CP, and NML conceived the study. AM and JM collected samples and AM performed sequencing. JNW, ND, and CP performed bioinformatics analysis. JNW drafted the manuscript. CP, ND, and NML critically revised the manuscript. All authors approved the final manuscript and agreed to be accountable for all aspects of this work.