
==== Front
Genome Biol Evol
Genome Biol Evol
gbe
Genome Biology and Evolution
1759-6653
Oxford University Press UK

39162185
10.1093/gbe/evae182
evae182
Letter
AcademicSubjects/SCI01130
AcademicSubjects/SCI01140
Tracing Homopolymers in Oikopleura dioica's Mitogenome
https://orcid.org/0000-0001-8051-6602
Dierckxsens Nicolas Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

Watanabe Kosei Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan
Keio University, School of Medicine, Tokyo, Japan

https://orcid.org/0000-0002-1413-3424
Tan Yongkai Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

https://orcid.org/0000-0002-6913-8417
Masunaga Aki Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

https://orcid.org/0000-0003-4717-4721
Mansfield Michael J Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

https://orcid.org/0000-0002-5227-5431
Miao Jiashun Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

https://orcid.org/0000-0001-5293-4778
Luscombe Nicholas M Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

https://orcid.org/0000-0001-7410-6295
Plessy Charles Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

Pisani Davide Associate Editor
Corresponding author: E-mail: charles.plessy@oist.jp.
9 2024
20 8 2024
20 8 2024
16 9 evae18213 8 2024
10 9 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.
2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Oikopleura dioica is a planktonic tunicate (Appendicularia class) found extensively across the marine waters of the globe. The genome of a single male individual collected from Okinawa, Japan was sequenced using the single-molecule PacBio Hi-Fi method and assembled with NOVOLoci. The mitogenome is 39,268 bp long, featuring a large control region of around 22,000 bp. We annotated the proteins atp6, cob, cox1, cox2, cox3, nad1, nad4, and nad5, and found one more open reading frame that did not match any known gene. This study marks the first complete mitogenome assembly for an appendicularian, and reveals that A and T homopolymers cumulatively account for nearly half of its length. This reference sequence will be an asset for environmental DNA and phylogenetic studies.

tunicate
Oikopleura dioica
homopolymers
larvacean
Appendicularia
OIST 10.13039/501100004199 OIST x KEIO Long-term Research Internship Program Japan Society for the Promotion of Science 10.13039/501100001691 JSPS International Research Fellow Luscombe Unit
==== Body
pmcSignificance

Appendicularians, such as Oikopleura dioica, are planktonic tunicates which play an important role in the carbon cycle and the food web. Until this year, no annotated mitochondrial genome assembly was available for any appendicularian species. This lack of reference data hampers studies of their geographical ecology using environmental DNA sequencing (eDNA), which relies on the mitochondrial cytochrome oxidase 1 sequence as a taxonomic barcode, resulting in appendicularians often being categorized as “uncultured eukaryotes” by default. Moreover, the mitochondrial transcriptome of O. dioica is subjected to post-transcriptional editing, which further complicates the prediction of DNA sequences from RNA and vice versa. Therefore, curated reference annotation of appendicularian mitogenomes are essential not only for eDNA surveys, but also for supporting phylogenomic studies of the emergence of vertebrates, to which tunicates are the sister group, and for the enabling studies of the mechanism of homopolymer post-transcriptional edition. The complete mitochondrial genome assembly and its annotation published in this manuscript reveal the principles of Oikopleura mitogenome organization and pave the way for the important studies summarized above.

Introduction

Oikopleura dioica (Fol 1872), a marine tunicate within the Appendicularia class (larvacean), is notable for its widespread distribution across oceans. Throughout its lifecycle, O. dioica remains adrift, carried by ocean currents, and constructs a distinctive cellulose apparatus called the “house”, which it uses for protection and food collection. O. dioica frequently replaces its house when it becomes clogged, contributing to marine snow as discarded houses descend to the ocean floor. This process is important for Earth's carbon cycling, emphasizing the species' ecological significance (Glover 2020).

We reported earlier the possibility of cryptic speciation in O. dioica (Masunaga et al. 2022), and that cox1 nucleotide sequence similarity could be as low as ∼85% similar when comparing O. dioica isolated from Europe (Denoeud et al. 2010; Danks et al. 2013), Okinawa (Bliznina et al. 2021), or the main Japanese islands (Wang et al. 2015, 2020). Appropriate adjustment of the taxonomy is currently being discussed in the tunicate community. Nevertheless the evolutionary distance between these O. dioica cryptic species is large enough that separate reference sequences are needed to support applications of molecular biology to experimental research and taxonomic surveys using mitochondrial sequences as barcodes. This evolutionary distance also provides useful data to support phylogenetic studies of appendicularians and tunicates, which are needed to understand the evolution of early vertebrates.

The mitogenome of O. dioica was first characterized in the Supplementary material in Denoeud et al. (2010), revealing eight genes (cox1, cox2, cox3, nad1, nad4, nad5, cob, and atp6). The existence of nad2 remained under question. The ascidian mitochondrial genetic code was used by Denoeud et al. (2010) to translate these coding genes. Furthermore, Pichon et al. (2019) used multiple sequence alignments of Cox1 and Cob to demonstrate that this genetic code arose early in the tunicate history and confirm its appropriateness for O. dioica sequences (Pichon et al. 2019) despite they are not ascidians.

Denoeud et al. (2010) also noted the presence of poly-T insertions in coding regions, and stated the hypothesis that they are reduced to 6-mers in the transcriptome with an RNA editing mechanism. Bliznina et al. (2021) produced a partial assembly of 9,225 kbp containing the previously reported genes except nad5 (Bliznina et al. 2021). Unfortunately the length of the poly-T insertions could not be assessed with confidence because of limitations in the Nanopore basecalling technology, and the use of post-assembly polishing methods. Neither of the two studies could confirm whether the O. dioica mitochondrial genome was a single circle, linear, or a collection of minicircles like in Salpa thompsoni (Goodall-Copestake 2017). Sequencing complete O. dioica mitochondrial genomes has remained a challenge until now because of the lack of technologies accurate over long homopolymers, and the lack of software capable to assemble these regions.

Materials and Methods

Sample Collection and DNA Extraction

We collected O. dioica specimens at Ishikawa harbor, Okinawa, Japan (26.114N, 127.665E) in June 2018. The samples were washed with 5 mL of filtered autoclaved seawater three times, and then resuspended in 200 µL lysis buffer from the MagAttract HMW DNA Kit (Qiagen, 67563) with 20 µL of 10 µg/mL proteinase K and incubated for 1 h at 56 °C. After adding 50 µL of 5 M NaCl, the mixture was centrifuged at 5,000 × g at 4 °C for 15 min. The supernatant was transferred into a new microtube with 400 µL of 100% EtOH and 5 µL of glycogen (20 mg/mL) and cooled at −80 °C for 20 min. After centrifuging at 6,250 × g, 4 °C for 5 min, the supernatant was removed. The pellet was washed with 1 mL of cold 70% ethanol, centrifuged, and air-dried 5 min. Finally, the DNA was resuspended in molecular biology grade for quantitation using a Qubit 3 Fluorometer (Thermo Fisher Scientific, Q32850), and quality controlled using an Agilent 4200 TapeStation (Agilent, 5067-5365).

Sequencing and Assembly

The genome of a single individual (“I25”) was sequenced on PacBio Sequel II using a low-input HiFi library kit. Genomic DNA was sheared with Megarupter3 to an average size of 10 kbp. Library size distribution and concentration were assessed using the FEMTO Pulse system.

The sequence reads were assembled with NOVOLoci (Dierckxsens 2024), a newly developed targeted haplotype-aware assembler based on the same principle as the organelle assembler NOVOPlasty (Dierckxsens et al. 2017), using a partial nad1 gene sequence of 350 bp from the O. dioica OKI2018_I69_1.0 sequence (Bliznina 2021) as a seed. The seed sequence is only used to recruit reads for the first iteration of the assembly and will therefore not affect the final assembly. Nevertheless, it is important not to select a repetitive or duplicated region to initiate the assembly. Hence, we choose a region that was deprived of long homopolymer sequences.

Annotation

We attempted to annotate the assembly with MITOS (Bernt et al. 2013) using the ascidian mitochondrial genetic code (Pichon et al. 2019). However, the long length of the homopolymers (Figs. 1 and 3, and Table 1) made it problematic for MITOS to detect entire genes with enough precision and lead to a large number of false positives. We hence compared the genome to the Trinity transcriptome assembly of OKI2018_I69 (Bliznina 2021) to detect each gene. We queried the transcript models using amino acid sequences from MITOS or from related tunicates using tblastn (Altschul et al. 1997). The matched models, which were polycistronic, are shown in Table 1 and provided in the Supplementary material. We determined the extent of each gene's coding sequence using the getorf command from EMBOSS (Rice et al. 2000) with -table 13. The region encoding the gene was then aligned to the genome to discover the locations of poly-A or poly-T insertions, by Smith–Waterman alignments with the water command. The Circular plot was drawn by Circos version 0.69-9 (Krzywinski et al. 2009).

Fig. 1. Pie charts showing the proportion of the homopolymers of six successive bases or longer. C repeats do not exist and G repeats are slight in proportion, thus excluded from the charts.

Fig. 2. Relation between cox1 sequenced this work and the other publicly available appendicularian sequences. Bootstrap values are displayed on each node.

Fig. 3. Known protein-coding gene symbols are displayed on a green background for plus-strand genes and yellow for minus strand. Unknown proteins and transcript models are displayed in gray. The inner circle illustrates the repetitive regions; six or more successive A or T's are colored, respectively, red and blue.

Table 1 Coordinates of the annotated genes, IDs of transcripts used for the annotation, number of edited homopolymers

Gene	Start	End	Number of n-mers	Strand	Transcript ID	
nd1	98	820	4	+	TRINITY_DN9989_c0_g1_i1	
cox1	859	2625	5	+	TRINITY_DN9989_c0_g1_i1	
cob	5787	7021	7	+	TRINITY_DN19786_c0_g1_i4	
cox2	7963	8857	4	+	TRINITY_DN19786_c0_g1_i4	
nd5	8914	10959	12	+	TRINITY_DN19786_c0_g1_i4	
cox3	37885	39219	3	+	TRINITY_DN9989_c0_g1_i1	
atp6	7110	7738	4	−	TRINITY_DN19786_c0_g1_i4	
nd4	36676	37828	5	−	TRINITY_DN9989_c0_g1_i1	

Phylogenetic Tree

We collected chordate cox1 nucleotide sequences from publicly available complete mitogenomes assemblies. In addition, we also extracted cox1 from genomic scaffolds for Mesochordaeus erythrocephalus (SCLF01725989), Bathochordaeus stygius (SCLE01415711), and Oikopleura longicauda (SCLD01101138) (Naville et al. 2019). O. dioica sequences are from this work (I25) or were extracted from transcriptomes (Danks et al. 2013; Wang et al. 2015; Bliznina et al. 2021). A copy of these sequences are available as supplementary data, Supplementary Material online. We aligned the codons within sequences with Clustal Omega (Sievers et al. 2011) and Seaview (Gouy et al. 2021), and computed a tree with IQTREE (Kalyaanamoorthy et al. 2017; Hoang et al. 2018; Minh et al. 2020) with parameters –polytomy –ufboot 1000 -m MFP, with the –polytomy flag to collapse near-zero branches into polytomies.

Results

We looked for mitochondrial sequences in 58 samples sequenced with Nanopore and 3 samples sequenced with PacBio. From the 58 Nanopore sequencing runs, 26 did not contain any mitochondrial sequences and none resulted in a circular assembly. The high error rate in the control region, caused by the abundance of homopolymers, made it impossible to assemble the entire genome. Assembly lengths ranged from 800 to 26,000 bp. Assembly lengths of the 3 PacBio runs were generally longer and one resulted in a complete circular genome, which we selected for this study. The circular mitogenome has a length of 39,283 bp, containing 34.8% A-homopolymers of length six or more and 13.6% T-homopolymers of length six or more (Fig. 1). While existing algorithms were incapable of assembling the complete mitochondrial genome, NOVOLoci succeeded by step-wise extending the seed into a complete circular genome. We confirmed the existence of each gene reported earlier (Denoeud et al. 2010) (Fig. 2, Table 1). All coding genes ended with TAA juxtaposing A-homopolymers of length greater than six. No A-homopolymer was found within open reading frames (ORFs) (a feature which in retrospect makes the genome very easy to annotate), with the possible exception of cob, for which we lack phylogenomic or proteomic evidence to determine if the translation starts before or after an A-homopolymer present at the beginning of the ORF. Every T-homopolymer longer than 6 in the coding genes was reduced to 6-mers in the transcriptome. We note that some poly-T regions had non-T insertions which were also removed. Visual inspection of sequence read alignments to the genome confirmed the accuracy of the sequence at these insertions.

The region between cox1 and cob does not encode known proteins, but we found a long ORF at the same position where Denoeud et al. (2010) hypothesized nad2. Unfortunately, the identity of this gene could not be deciphered as its sequence did not have matches in the NCBI BLAST databases. The other ORFs of the region were all shorter and also without known protein or nucleotide matches. The cox1 sequence reported here is 99.4% similar to the transcript model we used as a seed, and encodes for the same protein sequence.

To place our reference genome into context and to illustrate appendicularian relationship to other taxa, we computed a phylogenetic tree on 87 codon-aligned cox1 chordate sequences (Fig. 2). The appendicularian branch placement is different from our previous analysis based on nuclear gene orthogroups (Plessy et al. 2024), but the discrepancy may be caused by long branch attraction. We also note that the Ciona genus grouped with Aplousobranchia instead of the Phlebobranchia where current taxonomy places it, but this discrepancy has been observed in numerous phylogenomic analysis before. Finally, the division between O. dioica and other appendicularians corresponds to the Coecaria (O. longicauda group) and Vexillaria (O. dioica group) and is well supported by taxonomy (Galt et al. 1985) and genomics (Naville et al. 2019). Sequence identity between O. dioica and O. longicauda is below 60%; no full-length cox1 sequence is available for closer relatives of O. dioica such as O. albicans or O. vanhoeffeni (Naville et al. 2019).

Discussion and Conclusion

We present here the first complete mitogenome of O. dioica. The assembly is circular, but we cannot exclude the possibility that the mitochondrial genome is actually a linear concatenate or present in multiple copies in a circle, due to the length of the control region. Our findings confirm the presence of genes previously reported and identify an unusual high amount of poly-T insertions in the control region. Our annotation reveals a general principle that will facilitate the study of other mitogenomes containing homopolymer insertions in the Oikopleura genus. First, genes are separated by poly-A regions directly encoded in the genome. Second, poly-T insertions containing a few non-T bases may also be edited. The discovery of this principle enables the annotation of homopolymer-containing Oikopleura mitogenome in the absence of transcriptome annotation.

Our protein-coding gene annotation does not resolve the possible loss of the ATP synthase subunit atp8 and the dehydrogenase subunits nad2, nad3, nad4L, and nad6 since the last common ancestor with ascidians. Investigation of the ORFs between cox1 and cob is currently hampered by the lack of sequence homology with other tunicates.

During the preparation of this manuscript, we became aware of the publication of a mitogenome assembly for an European isolate of O. dioica (Klirs et al. 2024). In order to avoid confusion caused by the high nucleotide divergence, we assigned our assembly to NCBI's taxon ID 3071372 as “unclassified Oikopleura”, pending the needed taxonomic adjustments. These two assemblies will be an asset for detecting O. dioica in environmental DNA (eDNA) studies of diverse geographical regions, and open the way for comparative approaches to elucidate noncoding regions and small ORFs. Future production of mitogenomes from the Oikopleura genus will be needed, which will increase the power of these methods.

We confirmed that the gene order in our assembly is identical to that of Denoeud et al. (2010), extracted from a Norwegian laboratory line, and that of Bliznina et al. (2021), extracted from our laboratory line from Okinawa, Japan. Plessy et al. (2024) showed that in the nuclear genome, the order of protein-coding genes is “scrambled” when comparing these two O. dioica lines (Plessy et al. 2024). It is therefore noticeable that although the order of genes is said to be less conserved in mitogenomes (Singh et al. 2009), no change took place in O. dioica at the time scale separating these two populations.

The mechanism of poly-T editing in the mitochondrial mRNAs is not understood. Despite the existence of this editing is common knowledge in the scientific community working with appendicularian mitochondrial sequences, our publication and that of Klirs et al. (2024) are only the second ones after Denoeud et al. (2010) to provide evidence matching genome and transcriptome data. Because of (1) this reason, (2) the uncertainty on the coding gene count, and (3) the absence of ncRNA annotation, we were only allowed to deposit the genome's sequence in GenBank as “UNVERIFIED”, with no annotation, which we provide as Supplementary material. This is unfortunate because it hides essential taxonomic information to metagenomics and eDNA studies that rely on the existence of annotated hits with strong sequence similarity in public databases. This publication aims as build up the evidence available to curators regarding the existence of homopolymer editing in the Oikopleura genus, and the need to adjust database infrastructure so that this biological phenomenon can be properly represented in annotations distributed by public databases.

Acknowledgments

We thank the DNA Sequencing Section and the Scientific Computing and Data Analysis Section of the Research Support Division at OIST for their support.

Author Contributions

A.M. collected samples, Y.T. cultured and sequenced them. N.D., K.W., M.J.M., J.M., and C.P. performed bioinformatics analysis. K.W. and N.D. drafted the manuscript. C.P. and N.M.L. critically revised the manuscript. All authors approved the final manuscript and agreed to be accountable for all aspects of this work.

Funding

This work was supported by OIST core funding. K.W. was supported by the OIST x KEIO Long-term Research Internship Program. M.J.M. acknowledges funding from the Japan Society for the Promotion of Science as a JSPS International Research Fellow (Luscombe Unit, Okinawa Institute of Science and Technology Graduate University).

Conflict of Interest

No potential conflict of interest was reported by the authors.

Ethical Approval

No ethical issues were involved in this study.

Data Availability

The mitochondrial genome sequence was deposited in GenBank under the accession number PP146516. The annotation and the sequences used to compute Fig. 2’s tree are available in Zenodo under DOI: https://doi.org/10.5281/zenodo.11142978.
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Literature Cited

Altschul  SF, Madden  TL, Schäffer  AA, Zhang  J, Zhang  Z, Miller  W, Lipman  DJ. Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res. 1997:25 (17 ):3389–3402. 10.1093/nar/25.17.3389.9254694
Bernt  M, Donath  A, Jühling  F, Externbrink  F, Florentz  C, Fritzsch  G, Pütz  J, Middendorf  M, Stadler  PF. Mitos: improved de novo metazoan mito chondrial genome annotation. Mol Phylogenet Evol. 2013:69 (2 ):313–319. 10.1016/j.ympev.2012.08.023.22982435
Bliznina  A, Masunaga  A, Mansfield  MJ, Tan  Y, Liu  AW, West  C, Rustagi  T, Chien  HC, Kumar  S, Pichon  J, et al  Telomere-to-telomere assembly of the genome of an individual Oikopleura dioica from Okinawa using nanopore-based sequencing. BMC Genomics. 2021:22 (1 ):222. 10.1186/s12864-021-07512-6.33781200
Danks  G, Campsteijn  C, Parida  M, Butcher  S, Doddapaneni  H, Fu  B, Petrin  R, Metpally  R, Lenhard  B, Wincker  P, et al  Oikobase: a genomics and developmental transcriptomics resource for the urochordate Oikopleura dioica. Nucleic Acids Res. 2013:41 (D1 ):D845–D853. 10.1093/nar/gks1159.23185044
Denoeud  F, Henriet  S, Mungpakdee  S, Aury  JM, Da Silva  C, Brinkmann  H, Mikhaleva  J, Olsen  LC, Jubin  C, Cañestro  C, et al  Plasticity of animal genome architecture unmasked by rapid evolution of a pelagic tunicate. Science. 2010:330 (6009 ):1381–1385. 10.1126/science.1194167.21097902
Dierckxsens  N . 2024. NOVOLoci. [accessed 2024 May]. https://github.com/ndierckx/NOVOLoci.
Dierckxsens  N, Mardulyn  P, Smits  G. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 2017:45 (4 ):e18. 10.1093/nar/gkw955.28204566
Fol  H . Études sur les appendiculaires du détroit de messine. Memoires de la Société de physique et d’histoire naturelle de. Genève. 1872:21 :445–499.
Galt  C, Grober  M, Sykes  P. Taxonomic correlates of bioluminescence among appendicularians (urochordata: larvacea). Biol Bull.  1985:168 (1 ):125–134. 10.2307/1541178.
Glover  J . Oikopleura. Curr Biol.  2020:30 (20 ):R1243–R1245. 10.1016/j.cub.2020.07.075.33080189
Goodall-Copestake  W . One tunic but more than one barcode: evolutionary insights from dynamic mitochondrial DNA in salpa thompsoni (tunicata: salpida). Biol J Linn Soc Lond.  2017:120 (3 ):637–648. 10.1111/bij.12915.
Gouy  M, Tannier  E, Comte  N, Parsons  DP. Seaview version 5: a multiplatform software for multiple sequence alignment, molecular phylogenetic analyses, and tree reconciliation. Methods Mol Biol. 2021:2231 :241–260. 10.1007/978-1-0716-1036-7_15.33289897
Hoang  DT, Chernomor  O, von Haesele  A, Minh  BQ, Vinh  LS. UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol. 2018:35 (2 ):518–522. 10.1093/molbev/msx281.29077904
Bliznina  A (2021) Oki2018_i69 assembly and annotation of the genome of an individual oikopleura dioica from okinawa (1.1). Data set. 10.5281/zenodo.4604144.
Kalyaanamoorthy  S, Minh  BQ, Wong  TKF, von Haeseler  A, Jermiin  LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods.  2017:14 (6 ):587–589. 10.1038/nmeth.4285.28481363
Klirs  Y, Novosolov  M, Gissi  C, Garic  R, Pupko  T, Stach  T, Huchon  D. Evolutionary insights from the mitochondrial genome of Oikopleura dioica: sequencing challenges, RNA editing, gene transfers to the nucleus, and tRNA loss. Genome Biol Evol.  2024:evae181 . 10.1093/gbe/evae181.
Krzywinski  M, Schein  J, Birol  I, Connors  J, Gascoyne  R, Horsman  D, Jones  SJ, Marra  MA. Circos: an information aesthetic for comparative genomics. Genome Res. 2009:19 (9 ):1639–1645. 10.1101/gr.092759.109.19541911
Masunaga  A, Mansfield  MJ, Tan  Y, Liu  AW, Bliznina  A, Barzaghi  P, Hodgetts  TL, Ferrández-Roldán  A, Cañestro  C, Onuma  TA, et al  The cosmopolitan appendicularian Oikopleura dioica reveals hidden genetic diversity around the globe. Mar Biol. 2022:169 (12 ):157. 10.1007/s00227-022-04145-5.
Minh  BQ, Schmidt  HA, Chernomor  O, Schrempf  D, Woodhams  MD, von Haeseler  A, Lanfear  R. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020:37 (8 ):2461. 10.1093/molbev/msaa131.32556291
Naville  M, Henriet  S, Warren  I, Sumic  S, Reeve  M, Volff  JN, Chourrout  D. Massive changes of genome size driven by expansions of non-autonomous transposable elements. Curr Biol. 2019:29 (7 ):1161–1168.e6. 10.1016/j.cub.2019.01.080.30880010
Pichon  J, Luscombe  NM, Plessy  C. Widespread use of the “ascidian” mitochondrial genetic code in tunicates. F1000Res. 2019:8 :2072. 10.12688/f1000research.21551.2.32148763
Plessy  C, Mansfield  MJ, Bliznina  A, Masunaga  A, West  C, Tan  Y, Liu  AW, Grašič  J, Del Río Pisula  MS, Sánchez-Serna  G, et al  Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species. Genome Res. 2024:34 (3 ):426–440. 10.1101/gr.278295.123.38621828
Rice  P, Longden  I, Bleasby  A. Emboss: the European molecular biology open software suite. Trends Genet. 2000:16 (6 ):276–277. 10.1016/s0168-9525(00)02024-2.10827456
Sievers  F, Wilm  A, Dineen  D, Gibson  TJ, Karplus  K, Li  W, Lopez  R, McWilliam  H, Remmert  M, Söding  J, et al  Fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega. Mol Syst Biol. 2011:7 (1 ):539. 10.1038/msb.2011.75.21988835
Singh  TR, Tsagkogeorga  G, Delsuc  F, Blanquart  S, Shenkar  N, Loya  Y, Douzery  EJ, Huchon  D. Tunicate mitogenomics and phylogenetics: peculiarities of the herdmania momus mitochondrial genome and support for the new chordate phylogeny. BMC Genomics. 2009:10 (1 ):534. 10.1186/1471-2164-10-534.19922605
Wang  K, Omotezako  T, Kishi  K, Nishida  H, Onuma  TA. Maternal and zygotic transcriptomes in the appendicularian, Oikopleura dioica: novel protein-encoding genes, intra-species sequence variations, and trans-spliced RNA leader. Dev Genes Evol. 2015:225 (3 ):149–159. 10.1007/s00427-015-0502-7.26032664
Wang  K, Tomura  R, Chen  W, Kiyooka  M, Ishizaki  H, Aizu  T, Minakuchi  Y, Seki  M, Suzuki  Y, Omotezako  T, et al  A genome database for a Japanese population of the Larvacean Oikopleura dioica. Dev Growth Differ. 2020:62 (6 ):450–461. 10.1111/dgd.12689.32677034
