The complete mitogenome of an unidentified Oikopleura species

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Luscombe" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": "Appendicularians are planktonic tunicates abundant all over the world. Currently, only two complete annotated mitochondrial genome assemblies are available for appendicularians, both for cryptic species of Oikopleura dioica. This underrepresentation of available appendicularian mitochondrial genomes limits environmental DNA sequencing (eDNA) studies that rely on mitochondrial markers as a taxonomic barcode. We report the complete mitochondrial genome assembly and annotation of an unknown appendicularian species isolated from the Amami Oshima island, Kagoshima prefecture, Japan, that has significant sequence difference with other currently available assemblies and will serve as a useful resource for ecological studies and further mitochondrial studies of appendicularians." } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/13-1357/v3", "name": "The complete mitogenome of an unidentified Oikopleura species" } } ] } Home Browse The complete mitogenome of an unidentified Oikopleura species ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Wibisana JN, Plessy C, Dierckxsens N et al. The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.12688/f1000research.157311.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Genome Note Revised The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] Johannes Nicolaus Wibisana https://orcid.org/0000-0002-2452-8702 1 , Charles Plessy https://orcid.org/0000-0001-7410-6295 1 , Nicolas Dierckxsens 1 , Aki Masunaga https://orcid.org/0000-0002-6913-8417 1 , Jiashun Miao 1 , Nicholas M. Luscombe https://orcid.org/0000-0001-5293-4778 1 Johannes Nicolaus Wibisana https://orcid.org/0000-0002-2452-8702 1 , Charles Plessy https://orcid.org/0000-0001-7410-6295 1 , [...] Nicolas Dierckxsens 1 , Aki Masunaga https://orcid.org/0000-0002-6913-8417 1 , Jiashun Miao 1 , Nicholas M. Luscombe https://orcid.org/0000-0001-5293-4778 1 PUBLISHED 06 May 2025 Author details Author details 1 Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa, 9040497, Japan Johannes Nicolaus Wibisana Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Charles Plessy Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Project Administration, Supervision, Validation, Visualization, Writing – Review & Editing Nicolas Dierckxsens Roles: Conceptualization, Investigation, Methodology, Software, Writing – Review & Editing Aki Masunaga Roles: Resources Jiashun Miao Roles: Funding Acquisition, Resources Nicholas M. Luscombe Roles: Funding Acquisition, Supervision OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Genomics and Genetics gateway. Abstract Appendicularians are planktonic tunicates abundant all over the world. Currently, only two complete annotated mitochondrial genome assemblies are available for appendicularians, both for cryptic species of Oikopleura dioica. This underrepresentation of available appendicularian mitochondrial genomes limits environmental DNA sequencing (eDNA) studies that rely on mitochondrial markers as a taxonomic barcode. We report the complete mitochondrial genome assembly and annotation of an unknown appendicularian species isolated from the Amami Oshima island, Kagoshima prefecture, Japan, that has significant sequence difference with other currently available assemblies and will serve as a useful resource for ecological studies and further mitochondrial studies of appendicularians. READ ALL READ LESS Keywords tunicate, larvacean, appendicularian Corresponding Author(s) Charles Plessy ( [email protected] ) Close Corresponding author: Charles Plessy Competing interests: No competing interests were disclosed. Grant information: This study was supported by OIST core funding and JSPS KAKENHI grant number 23K14236. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Wibisana JN et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Wibisana JN, Plessy C, Dierckxsens N et al. The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.12688/f1000research.157311.3 ) First published: 12 Nov 2024, 13 :1357 ( https://doi.org/10.12688/f1000research.157311.1 ) Latest published: 06 May 2025, 13 :1357 ( https://doi.org/10.12688/f1000research.157311.3 ) Revised Amendments from Version 2 We have modified the manuscript according to the comments of the reviewers in this round of review. We have modified the manuscript according to the comments of the reviewers in this round of review. See the authors' detailed response to the review by Thomas Stach See the authors' detailed response to the review by Ayelet Voskoboynik See the authors' detailed response to the review by Carmela Gissi See the authors' detailed response to the review by Daniel M Chourrout READ REVIEWER RESPONSES 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 Sample collection and DNA extraction and sequencing We collected specimens at Tamari harbor, Amami Oshima island, Kagoshima prefecture, Japan (28.41491667 N 129.59016667 E) in July 2023. An identical specimen from the same catch was deposited at the Kagoshima University Museum ( www.museum.kagoshima-u.ac.jp ) under the voucher number KAUM-UR 1, the original specimen was consumed for sequencing. Samples were preserved in 99% ethanol at -80°C prior to DNA extraction. DNA extraction was done by firstly washing samples with 5 mL of filtered autoclaved seawater 3 times before resuspending in 200 μl of lysis buffer from the MagAttract HMW DNA Kit (Qiagen, USA #67563) with 20 μL of 10 μg/mL proteinase K and incubated for 1 h at 56°C. Next, 50 μL of 5 M NaCl was added before centrifugation of the mixture (5000 × g at 4°C) for 15 min. The supernatant was transferred into a new microtube and mixed with 400 μL of 100% EtOH and 5 μL of glycogen (20 mg/mL) and cooled at -80°C for 20 min. Further centrifugation at 6250 × g, 4°C for 5 min was performed and the supernatant removed. The obtained pellet was then washed with 1 mL of cold 70% ethanol, centrifuged, and air-dried for 5 min. The DNA was then resuspended in nuclease free water and quantified using a Qubit 3 Fluorometer (Thermo Fisher, USA). DNA was fragmented using Megaruptor 3 ® (Diagenode, USA) using the Megaruptor 3 Shearing Kit (Diagenode, USA #E07010003) at speed 32 and purified using SMRTbell cleanup beads (Pacific Biosciences, USA #102-158-300). Quality control of obtained DNA was performed using the Femto Pulse System (Agilent, USA) and the Genomic DNA 165 kb Kit (Agilent, USA #FP-1002-0275). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA) using the Sequel II sequencing kit 2.0 (Pacific Biosciences, USA #101-820-200). The DNA size profile and sequencing metrics are available on Zenodo (doi: 10.5281/zenodo.14934254 ). Assembly The sequenced reads were assembled with NOVOLoci ( https://github.com/ndierckx/NOVOLoci ) in targeted assembly mode. A partial PacBio read sequence found by a BLAST 9 search of cytochrome c oxidase subunit 1 from Okinawa O. dioica 5 to the raw whole DNA reads was used as a seed sequence. The coverage plot was generated by mapping the sequencing reads that were used to assemble the mitogenome to the assembly itself using minimap version 2.28-r1209. 10 Annotation We annotated the assembly with MITOS2 v2.1.9 11 using the ascidian mitochondrial genetic code. 7 ARWEN version 1.2.3 12 was used in addition to annotate putative tRNAs. Phylogenetic tree Protein-coding mitochondrial sequences were extracted from GenBank records with EMBOSS 13 and codon-aligned manually in SeaView 5.0.5 14 after a first alignment with Clustal Omega version 1.2.4. 15 The phylogenetic tree was computed with IQ-TREE version 2.0.7 16 using the command-line options “-T AUTO (run as many CPU threads as possible) --runs 3 (perform 3 runs to assess model convergence) --polytomy (allow unresolved branches) --ufboot 1000 (1000 boostraps with the ultrafast method) -m MFP (automatic selection of the best model for maximum likelihood inference using the ModelFinder Plus method, with one partition per gene. Sequences, accession numbers, and trees are available on Zenodo (doi: 10.5281/zenodo.14934254 ). Protein structure prediction 3D protein structure was predicted using colabfold version 1.5.5 17 and visualized with PyMOL. 18 UCSF ChimeraX 19 can be used as an alternative to PyMOL. 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. longicauda 21 (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 colabfold 17 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. longicauda 21 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. Data availability statement The mitochondrial genome sequence was deposited in GenBank under the accession number LC830956. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA1152617, SRR30429256, and SAMN43370082 respectively. The annotation and the sequences used to compute the phylogenetic tree in Fig. 3 are available in Zenodo (doi: 10.5281/zenodo.13864550 ). Underlying data Accession numbers NCBI Nucleotide database: Oikopleura sp. bigama1 mitochondrial DNA, complete genome. Accession number; LC830956. https://www.ncbi.nlm.nih.gov/nuccore/LC830956.1/ . NCBI SRA: Genome sequencing of an unknown Oikopleura species. Accession number; PRJNA1152617. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1152617/ . NCBI BioSample: Invertebrate sample from Oikopleura sp. bigama1. Accession number; SAMN43370082. https://www.ncbi.nlm.nih.gov/biosample/?term=SAMN43370082 NCBI Sequence Read Archive (SRA). WGS of Oikopleura sp. bigama1 Accession number; SRR30429256. https://www.ncbi.nlm.nih.gov/sra/?term=SRR30429256 Extended data Zenodo: Supplemental material to the journal article “The complete mitogenome of an unidentified Oikopleura species”. doi: 10.5281/zenodo.13864550 . 26 Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). Acknowledgements We thank Dr. Michael Mansfield for the invaluable suggestions and guidance in the construction of the nucleotide-based phylogenetic tree, Dr. Yongkai Tan for providing photographs of the specimens and Dr. Rade Garić for critical insights on sequence and morphological similarities to O. fusiformis. We thank the DNA Sequencing Section and the Scientific Computing and Data Analysis Section of the Research Support Division at OIST for their support. References 1. Kimura S, Ohshima C, Hirose E, et al. : Cellulose in the house of the appendicularian Oikopleura rufescens. Protoplasma. 2001 Mar; 216 (1–2): 71–74. PubMed Abstract | Publisher Full Text 2. Glover JC: Oikopleura. Curr. Biol. 2020 Oct; 30 (20): R1243–R1245. Publisher Full Text 3. Sherlock RE, Walz KR, Schlining KL, et al. : Morphology, ecology, and molecular biology of a new species of giant larvacean in the eastern North Pacific: Bathochordaeus mcnutti sp. nov. Mar. Biol. 2017 Jan; 164 (1): 20. PubMed Abstract | Publisher Full Text | Free Full Text 4. Denoeud F, Henriet S, Mungpakdee S, et al. : Plasticity of Animal Genome Architecture Unmasked by Rapid Evolution of a Pelagic Tunicate. Science. 2010 Dec 3; 330 (6009): 1381–1385. PubMed Abstract | Publisher Full Text | Free Full Text 5. 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PubMed Abstract | Publisher Full Text 15. Sievers F, Higgins DG: Clustal Omega. Curr. Protoc. Bioinformatics. 2014 Dec [cited 2024 Jul 4]; 48 (1). Publisher Full Text 16. Minh BQ, Schmidt HA, Chernomor O, et al. : IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol. Biol. Evol. 2020 May 1; 37 (5): 1530–1534. PubMed Abstract | Publisher Full Text | Free Full Text 17. Mirdita M, Schütze K, Moriwaki Y, et al. : ColabFold: making protein folding accessible to all. Nat. Methods. 2022 Jun; 19 (6): 679–682. PubMed Abstract | Publisher Full Text | Free Full Text 18. Schrödinger, LLC: The PyMOL Molecular Graphics System, Version 1.8.2015. 19. Meng EC, Goddard TD, Pettersen EF, et al. : UCSF ChimeraX: Tools for structure building and analysis. Protein Sci. 2023 Nov; 32 (11): e4792. PubMed Abstract | Publisher Full Text | Free Full Text 20. Singh TR, Tsagkogeorga G, Delsuc F, et al. : Tunicate mitogenomics and phylogenetics: peculiarities of the Herdmania momus mitochondrial genome and support for the new chordate phylogeny. BMC Genomics. 2009 Dec; 10 (1): 534. PubMed Abstract | Publisher Full Text | Free Full Text 21. Naville M, Henriet S, Warren I, et al. : Massive Changes of Genome Size Driven by Expansions of Non-autonomous Transposable Elements. Curr. Biol. 2019 Apr; 29 (7): 1161–1168.e6. PubMed Abstract | Publisher Full Text 22. Gissi C, Pesole G: Transcript Mapping and Genome Annotation of Ascidian mtDNA Using EST Data. Genome Res. 2003 Sep; 13 (9): 2203–2212. PubMed Abstract | Publisher Full Text | Free Full Text 23. Galt CP, Grober MS, Sykes PF: Taxonomic Correlates of Bioluminescence Among Appendicularians (Urochordata: Larvacea). Biol. Bull. 1985 Feb; 168 (1): 125–134. Publisher Full Text 24. Masunaga A, Mansfield MJ, Tan Y, et al. : The cosmopolitan appendicularian Oikopleura dioica reveals hidden genetic diversity around the globe. Mar. Biol. 2022 Nov 27; 169 (12): 157. Publisher Full Text 25. Plessy C, Mansfield MJ, Bliznina A, et al. : Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species. Genome Res. 2024 Apr 15; 34 : 426–440. genome;gr.278295.123v1. PubMed Abstract | Publisher Full Text | Free Full Text 26. Wibisana JN, Plessy C: Supplemental material to the journal article “The complete mitogenome of an unidentified Oikopleura species” (1.1.0). [Dataset]. Zenodo. 2024. Publisher Full Text Comments on this article Comments (0) Version 3 VERSION 3 PUBLISHED 12 Nov 2024 ADD YOUR COMMENT Comment Author details Author details 1 Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa, 9040497, Japan Johannes Nicolaus Wibisana Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Charles Plessy Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Project Administration, Supervision, Validation, Visualization, Writing – Review & Editing Nicolas Dierckxsens Roles: Conceptualization, Investigation, Methodology, Software, Writing – Review & Editing Aki Masunaga Roles: Resources Jiashun Miao Roles: Funding Acquisition, Resources Nicholas M. Luscombe Roles: Funding Acquisition, Supervision Competing interests No competing interests were disclosed. Grant information This study was supported by OIST core funding and JSPS KAKENHI grant number 23K14236. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (3) version 3 Revised Published: 06 May 2025, 13:1357 https://doi.org/10.12688/f1000research.157311.3 version 2 Revised Published: 07 Mar 2025, 13:1357 https://doi.org/10.12688/f1000research.157311.2 version 1 Published: 12 Nov 2024, 13:1357 https://doi.org/10.12688/f1000research.157311.1 Copyright © 2025 Wibisana JN et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Wibisana JN, Plessy C, Dierckxsens N et al. The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.12688/f1000research.157311.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 3 VERSION 3 PUBLISHED 06 May 2025 Revised Views 0 Cite How to cite this report: Stach T. Reviewer Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.181225.r383198 ) The direct URL for this report is: https://f1000research.com/articles/13-1357/v3#referee-response-383198 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 29 May 2025 Thomas Stach , Humboldt-Universität zu Berlin Vergleichende Elektronenmikroskopie,, Philippstraße, Germany Approved VIEWS 0 https://doi.org/10.5256/f1000research.181225.r383198 Having another mt-genome of an appendicularian species in the databases is scientific progress. So, I will tick approved this time (also, so I do not need to revise the ms again). The principal weakness of the ms still holds ... Continue reading READ ALL Having another mt-genome of an appendicularian species in the databases is scientific progress. So, I will tick approved this time (also, so I do not need to revise the ms again). The principal weakness of the ms still holds and it cannot be fixed (or only with considerably investment of resources and time). That, of course is the fact that the species the sequenced mt-genome belongs to has to remain unidentified, due to the suboptimal preservation of the specimens caught for morphological examination. Here are some minor comments: In the abstract it says "unknown appendicularian". "unknown" in this context usually means not known to science. We don't know that of the species sequenced here; in fact it is highly unlikely that it is a new species. The authors should stick to "unidentified", the adjective used - correctly - in the title. I think it should be mentioned somewhere that there are about 70 species of appendicularians described currently. Collection details (type of plankton net, mesh size, towing duration, ship speed, towing depth) should be specified. In my opinion, a few words about what is known concerning the presently described diversity of appendicularians off the coast of Japan are necessary to give the reader some context. A few considerations about the species identity beyond the sequence could also be attempted: limited as it may be, maybe some of the distinguishing features of appendicularians can still be seen? E.g., buccal glands are quite prominent and could potentially be seen, if they were present. Similarly, subchordal cells, another feature used in appendicularian taxonomy, might be visible in the recorded micrographs at higher resolution. Competing Interests: No competing interests were disclosed. Reviewer Expertise: comparative zoology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Stach T. Reviewer Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.181225.r383198 ) The direct URL for this report is: https://f1000research.com/articles/13-1357/v3#referee-response-383198 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 2 VERSION 2 PUBLISHED 07 Mar 2025 Revised Views 0 Cite How to cite this report: Stach T. Reviewer Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.178729.r374065 ) The direct URL for this report is: https://f1000research.com/articles/13-1357/v2#referee-response-374065 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 16 Apr 2025 Thomas Stach , Humboldt-Universität zu Berlin Vergleichende Elektronenmikroskopie,, Philippstraße, Germany Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.178729.r374065 The manuscript "The complete mitogenome of an unidentified Oikopleura species" submitted by J. N. Wibisana and co-authors reports the sequencing of the mitochondrial genome of an unidentified appendicularian species collected off the coast of Japan. The presented mitochondrial genome is new to ... Continue reading READ ALL The manuscript "The complete mitogenome of an unidentified Oikopleura species" submitted by J. N. Wibisana and co-authors reports the sequencing of the mitochondrial genome of an unidentified appendicularian species collected off the coast of Japan. The presented mitochondrial genome is new to the respective databases and therefore an original contribution to science. The genome has some features that are evolutionarily interesting and could potentially be used in a cladistic framework. The authors conduct a formal phylogenetic analysis using a Maximum Likelihood algorithm. I feel a bit out of my expertise in gauging the validity of the current software used, but based on my experience with other software packages, I would expect that 'ultrafast methods' to calculate bootstrap values overestimate bootstrap support of a node. The authors rationalize the study mainly by advancing the knowledge of genome sequences in an underrepresented taxon and the potential use this might have in eDNA research. My main reservation concerning this study is its surprising lack of standard taxonomy approaches. While the sequence as such might be new to science, the species it belongs to, might not be new. Or it might be. From the sequence alone, we cannot know. In order to tie the valuable data generated to the existing body of scientific knowledge, it is - in my view - imperative to at least seriously try to identify the species using the available taxonomic literature on appendicularians. To this end, specimens should have been fixed in a more suitable fixative than Ethanol, in order to preserve the taxonomically relevant characters. They probably should have also been caught more gently as judging from the Figure 1 - but the details of collecting the animals are not given. I have also a few minor comments: line 2 in the introduction: it should be 'their life cycle' instead of 'its life cycle' third paragraph of the Results and discussion: Oikopleuridae should not be italicized (only Genus and species names are). also: '... and misses some of the eight expected ...' I guess the precise number could be stated here. Eight is not so huge that the reader looses track. sixth paragraph of the Results and discussion: 'polyphylic' is not the correct term. I assume what is meant is polyphyletic (see also Carmela Gissi's review). But even this is not entirely fitting (at least in the common use of the cladistics terminology), because the depicted phylogeny would be a case of paraphyly, i.e. the genus Oikopleura is paraphyletic in this analysis. Are the rationale for sequencing the genome and the species significance clearly described? Partly Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Partly Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: comparative zoology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Stach T. Reviewer Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.178729.r374065 ) The direct URL for this report is: https://f1000research.com/articles/13-1357/v2#referee-response-374065 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 06 May 2025 Johannes Nicolaus Wibisana , Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan 06 May 2025 Author Response We agree with the reviewer’s point of view, which is why we only recommend to use the tree for guiding further data acquisition efforts.The authors rationalize the study mainly by ... Continue reading We agree with the reviewer’s point of view, which is why we only recommend to use the tree for guiding further data acquisition efforts.The authors rationalize the study mainly by advancing the knowledge of genome sequences in an underrepresented taxon and the potential use this might have in eDNA research. My main reservation concerning this study is its surprising lack of standard taxonomy approaches. While the sequence as such might be new to science, the species it belongs to, might not be new. Or it might be. From the sequence alone, we cannot know. In order to tie the valuable data generated to the existing body of scientific knowledge, it is - in my view - imperative to at least seriously try to identify the species using the available taxonomic literature on appendicularians. To this end, specimens should have been fixed in a more suitable fixative than Ethanol, in order to preserve the taxonomically relevant characters. They probably should have also been caught more gently as judging from the Figure 1 - but the details of collecting the animals are not given. Response: Thank you for the comments. The animals were obtained as a bycatch during sampling of other zooplanktons and thus the morphology was not preserved well. Unfortunately, we do not have any specimens left with better preserved morphology. However, we were able to confirm that it has characteristics pertaining to O. fusiformis owing to the shape of the gastric lobe and the slender tail. We agree that a proper taxonomic analysis is needed, but we would like to underline that situations ideal for taxonomy will not necessarily be ideal for recovering the high-molecular weight DNA needed for appendicularian mitogenome assembly. Therefore, we believe that our publication will be a steppingstone for further work, in which only the sequencing of a short PCR fragment will be needed to assess the relation between a taxonomically identified individual and our published mitochondrial genome sequence. We also underline in our conclusion that the possible existence of cryptic species, which will be difficult to resolve with only morphological information, has to be taken into account. I have also a few minor comments: line 2 in the introduction: it should be 'their life cycle' instead of 'its life cycle' third paragraph of the Results and discussion: Oikopleuridae should not be italicized (only Genus and species names are). also: '... and misses some of the eight expected ...' I guess the precise number could be stated here. Eight is not so huge that the reader looses track. sixth paragraph of the Results and discussion: 'polyphylic' is not the correct term. I assume what is meant is polyphyletic (see also Carmela Gissi's review). But even this is not entirely fitting (at least in the common use of the cladistics terminology), because the depicted phylogeny would be a case of paraphyly, i.e. the genus Oikopleura is paraphyletic in this analysis. Response: Thank you for the minor comments, we have adjusted the manuscript to reflect all these changes. We agree with the reviewer’s point of view, which is why we only recommend to use the tree for guiding further data acquisition efforts.The authors rationalize the study mainly by advancing the knowledge of genome sequences in an underrepresented taxon and the potential use this might have in eDNA research. My main reservation concerning this study is its surprising lack of standard taxonomy approaches. While the sequence as such might be new to science, the species it belongs to, might not be new. Or it might be. From the sequence alone, we cannot know. In order to tie the valuable data generated to the existing body of scientific knowledge, it is - in my view - imperative to at least seriously try to identify the species using the available taxonomic literature on appendicularians. To this end, specimens should have been fixed in a more suitable fixative than Ethanol, in order to preserve the taxonomically relevant characters. They probably should have also been caught more gently as judging from the Figure 1 - but the details of collecting the animals are not given. Response: Thank you for the comments. The animals were obtained as a bycatch during sampling of other zooplanktons and thus the morphology was not preserved well. Unfortunately, we do not have any specimens left with better preserved morphology. However, we were able to confirm that it has characteristics pertaining to O. fusiformis owing to the shape of the gastric lobe and the slender tail. We agree that a proper taxonomic analysis is needed, but we would like to underline that situations ideal for taxonomy will not necessarily be ideal for recovering the high-molecular weight DNA needed for appendicularian mitogenome assembly. Therefore, we believe that our publication will be a steppingstone for further work, in which only the sequencing of a short PCR fragment will be needed to assess the relation between a taxonomically identified individual and our published mitochondrial genome sequence. We also underline in our conclusion that the possible existence of cryptic species, which will be difficult to resolve with only morphological information, has to be taken into account. I have also a few minor comments: line 2 in the introduction: it should be 'their life cycle' instead of 'its life cycle' third paragraph of the Results and discussion: Oikopleuridae should not be italicized (only Genus and species names are). also: '... and misses some of the eight expected ...' I guess the precise number could be stated here. Eight is not so huge that the reader looses track. sixth paragraph of the Results and discussion: 'polyphylic' is not the correct term. I assume what is meant is polyphyletic (see also Carmela Gissi's review). But even this is not entirely fitting (at least in the common use of the cladistics terminology), because the depicted phylogeny would be a case of paraphyly, i.e. the genus Oikopleura is paraphyletic in this analysis. Response: Thank you for the minor comments, we have adjusted the manuscript to reflect all these changes. Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 06 May 2025 Johannes Nicolaus Wibisana , Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan 06 May 2025 Author Response We agree with the reviewer’s point of view, which is why we only recommend to use the tree for guiding further data acquisition efforts.The authors rationalize the study mainly by ... Continue reading We agree with the reviewer’s point of view, which is why we only recommend to use the tree for guiding further data acquisition efforts.The authors rationalize the study mainly by advancing the knowledge of genome sequences in an underrepresented taxon and the potential use this might have in eDNA research. My main reservation concerning this study is its surprising lack of standard taxonomy approaches. While the sequence as such might be new to science, the species it belongs to, might not be new. Or it might be. From the sequence alone, we cannot know. In order to tie the valuable data generated to the existing body of scientific knowledge, it is - in my view - imperative to at least seriously try to identify the species using the available taxonomic literature on appendicularians. To this end, specimens should have been fixed in a more suitable fixative than Ethanol, in order to preserve the taxonomically relevant characters. They probably should have also been caught more gently as judging from the Figure 1 - but the details of collecting the animals are not given. Response: Thank you for the comments. The animals were obtained as a bycatch during sampling of other zooplanktons and thus the morphology was not preserved well. Unfortunately, we do not have any specimens left with better preserved morphology. However, we were able to confirm that it has characteristics pertaining to O. fusiformis owing to the shape of the gastric lobe and the slender tail. We agree that a proper taxonomic analysis is needed, but we would like to underline that situations ideal for taxonomy will not necessarily be ideal for recovering the high-molecular weight DNA needed for appendicularian mitogenome assembly. Therefore, we believe that our publication will be a steppingstone for further work, in which only the sequencing of a short PCR fragment will be needed to assess the relation between a taxonomically identified individual and our published mitochondrial genome sequence. We also underline in our conclusion that the possible existence of cryptic species, which will be difficult to resolve with only morphological information, has to be taken into account. I have also a few minor comments: line 2 in the introduction: it should be 'their life cycle' instead of 'its life cycle' third paragraph of the Results and discussion: Oikopleuridae should not be italicized (only Genus and species names are). also: '... and misses some of the eight expected ...' I guess the precise number could be stated here. Eight is not so huge that the reader looses track. sixth paragraph of the Results and discussion: 'polyphylic' is not the correct term. I assume what is meant is polyphyletic (see also Carmela Gissi's review). But even this is not entirely fitting (at least in the common use of the cladistics terminology), because the depicted phylogeny would be a case of paraphyly, i.e. the genus Oikopleura is paraphyletic in this analysis. Response: Thank you for the minor comments, we have adjusted the manuscript to reflect all these changes. We agree with the reviewer’s point of view, which is why we only recommend to use the tree for guiding further data acquisition efforts.The authors rationalize the study mainly by advancing the knowledge of genome sequences in an underrepresented taxon and the potential use this might have in eDNA research. My main reservation concerning this study is its surprising lack of standard taxonomy approaches. While the sequence as such might be new to science, the species it belongs to, might not be new. Or it might be. From the sequence alone, we cannot know. In order to tie the valuable data generated to the existing body of scientific knowledge, it is - in my view - imperative to at least seriously try to identify the species using the available taxonomic literature on appendicularians. To this end, specimens should have been fixed in a more suitable fixative than Ethanol, in order to preserve the taxonomically relevant characters. They probably should have also been caught more gently as judging from the Figure 1 - but the details of collecting the animals are not given. Response: Thank you for the comments. The animals were obtained as a bycatch during sampling of other zooplanktons and thus the morphology was not preserved well. Unfortunately, we do not have any specimens left with better preserved morphology. However, we were able to confirm that it has characteristics pertaining to O. fusiformis owing to the shape of the gastric lobe and the slender tail. We agree that a proper taxonomic analysis is needed, but we would like to underline that situations ideal for taxonomy will not necessarily be ideal for recovering the high-molecular weight DNA needed for appendicularian mitogenome assembly. Therefore, we believe that our publication will be a steppingstone for further work, in which only the sequencing of a short PCR fragment will be needed to assess the relation between a taxonomically identified individual and our published mitochondrial genome sequence. We also underline in our conclusion that the possible existence of cryptic species, which will be difficult to resolve with only morphological information, has to be taken into account. I have also a few minor comments: line 2 in the introduction: it should be 'their life cycle' instead of 'its life cycle' third paragraph of the Results and discussion: Oikopleuridae should not be italicized (only Genus and species names are). also: '... and misses some of the eight expected ...' I guess the precise number could be stated here. Eight is not so huge that the reader looses track. sixth paragraph of the Results and discussion: 'polyphylic' is not the correct term. I assume what is meant is polyphyletic (see also Carmela Gissi's review). But even this is not entirely fitting (at least in the common use of the cladistics terminology), because the depicted phylogeny would be a case of paraphyly, i.e. the genus Oikopleura is paraphyletic in this analysis. Response: Thank you for the minor comments, we have adjusted the manuscript to reflect all these changes. Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Chourrout DM. Reviewer Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.178729.r374064 ) The direct URL for this report is: https://f1000research.com/articles/13-1357/v2#referee-response-374064 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 16 Apr 2025 Daniel M Chourrout , University of Bergen, Bergen, Norway Approved VIEWS 0 https://doi.org/10.5256/f1000research.178729.r374064 Although this appendicularian species is not named, having its mt genome sequence is useful because it contributes to increase taxonomic diversity in phylogenetic trees. The annotation is carefully performed and the conclusions make sense, with a clear position next to ... Continue reading READ ALL Although this appendicularian species is not named, having its mt genome sequence is useful because it contributes to increase taxonomic diversity in phylogenetic trees. The annotation is carefully performed and the conclusions make sense, with a clear position next to O. longicauda and two giant larvaceans. I have two comments: 1. Why not comparing the order of the coding genes with other appendicularian species ? Previous work on other species shows that synteny is not conserved with other tunicates and it could be the main contribution of the paper if this comparison was performed. 2. The picture is of extremely poor quality and it should not be index together with the main text. It may be sent to an annex as it does not bring information except that the species is an appendicularian, this is clear enough from the phylogenetic analysis. How a better picture could not be obtained is surprising, it might have helped to tentatively name the species. Are the rationale for sequencing the genome and the species significance clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: animal genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Chourrout DM. Reviewer Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.178729.r374064 ) The direct URL for this report is: https://f1000research.com/articles/13-1357/v2#referee-response-374064 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 06 May 2025 Johannes Nicolaus Wibisana , Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan 06 May 2025 Author Response 1. Why not comparing the order of the coding genes with other appendicularian species ? Previous work on other species shows that synteny is not conserved with other tunicates and ... Continue reading 1. Why not comparing the order of the coding genes with other appendicularian species ? Previous work on other species shows that synteny is not conserved with other tunicates and it could be the main contribution of the paper if this comparison was performed. Response: Thank you for this comment. We added the following to our manuscript: “Previous work on other species suggests that synteny is usually not conserved in tunicates (Singh et al., 2009). Indeed, we found that the gene order bears no resemblance to the one of O. dioica (Dierckxsens et al., 2024; Klirs et al., 2025) nor to the one of O. longicauda (Naville et al., 2019, scaffold SCLD01101138.1) nor to one in the stolidobranch ascidians Herdmania momus and Halocynthia roretzi .” 2. The picture is of extremely poor quality and it should not be index together with the main text. It may be sent to an annex as it does not bring information except that the species is an appendicularian, this is clear enough from the phylogenetic analysis. How a better picture could not be obtained is surprising, it might have helped to tentatively name the species. Response: Thank you for this comment. The specimen that we obtained was from a bycatch and thus was not preserved in the best condition. We kept the figure as it is in the main manuscript as it is the second round of review, where we would like to avoid major changes of the document layout to prevent introducing new typographical errors 1. Why not comparing the order of the coding genes with other appendicularian species ? Previous work on other species shows that synteny is not conserved with other tunicates and it could be the main contribution of the paper if this comparison was performed. Response: Thank you for this comment. We added the following to our manuscript: “Previous work on other species suggests that synteny is usually not conserved in tunicates (Singh et al., 2009). Indeed, we found that the gene order bears no resemblance to the one of O. dioica (Dierckxsens et al., 2024; Klirs et al., 2025) nor to the one of O. longicauda (Naville et al., 2019, scaffold SCLD01101138.1) nor to one in the stolidobranch ascidians Herdmania momus and Halocynthia roretzi .” 2. The picture is of extremely poor quality and it should not be index together with the main text. It may be sent to an annex as it does not bring information except that the species is an appendicularian, this is clear enough from the phylogenetic analysis. How a better picture could not be obtained is surprising, it might have helped to tentatively name the species. Response: Thank you for this comment. The specimen that we obtained was from a bycatch and thus was not preserved in the best condition. We kept the figure as it is in the main manuscript as it is the second round of review, where we would like to avoid major changes of the document layout to prevent introducing new typographical errors Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 06 May 2025 Johannes Nicolaus Wibisana , Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan 06 May 2025 Author Response 1. Why not comparing the order of the coding genes with other appendicularian species ? Previous work on other species shows that synteny is not conserved with other tunicates and ... Continue reading 1. Why not comparing the order of the coding genes with other appendicularian species ? Previous work on other species shows that synteny is not conserved with other tunicates and it could be the main contribution of the paper if this comparison was performed. Response: Thank you for this comment. We added the following to our manuscript: “Previous work on other species suggests that synteny is usually not conserved in tunicates (Singh et al., 2009). Indeed, we found that the gene order bears no resemblance to the one of O. dioica (Dierckxsens et al., 2024; Klirs et al., 2025) nor to the one of O. longicauda (Naville et al., 2019, scaffold SCLD01101138.1) nor to one in the stolidobranch ascidians Herdmania momus and Halocynthia roretzi .” 2. The picture is of extremely poor quality and it should not be index together with the main text. It may be sent to an annex as it does not bring information except that the species is an appendicularian, this is clear enough from the phylogenetic analysis. How a better picture could not be obtained is surprising, it might have helped to tentatively name the species. Response: Thank you for this comment. The specimen that we obtained was from a bycatch and thus was not preserved in the best condition. We kept the figure as it is in the main manuscript as it is the second round of review, where we would like to avoid major changes of the document layout to prevent introducing new typographical errors 1. Why not comparing the order of the coding genes with other appendicularian species ? Previous work on other species shows that synteny is not conserved with other tunicates and it could be the main contribution of the paper if this comparison was performed. Response: Thank you for this comment. We added the following to our manuscript: “Previous work on other species suggests that synteny is usually not conserved in tunicates (Singh et al., 2009). Indeed, we found that the gene order bears no resemblance to the one of O. dioica (Dierckxsens et al., 2024; Klirs et al., 2025) nor to the one of O. longicauda (Naville et al., 2019, scaffold SCLD01101138.1) nor to one in the stolidobranch ascidians Herdmania momus and Halocynthia roretzi .” 2. The picture is of extremely poor quality and it should not be index together with the main text. It may be sent to an annex as it does not bring information except that the species is an appendicularian, this is clear enough from the phylogenetic analysis. How a better picture could not be obtained is surprising, it might have helped to tentatively name the species. Response: Thank you for this comment. The specimen that we obtained was from a bycatch and thus was not preserved in the best condition. We kept the figure as it is in the main manuscript as it is the second round of review, where we would like to avoid major changes of the document layout to prevent introducing new typographical errors Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 12 Nov 2024 Views 0 Cite How to cite this report: Voskoboynik A. Reviewer Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.172738.r341076 ) The direct URL for this report is: https://f1000research.com/articles/13-1357/v1#referee-response-341076 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 06 Jan 2025 Ayelet Voskoboynik , Stanford University, Stanford, USA Approved VIEWS 0 https://doi.org/10.5256/f1000research.172738.r341076 This paper presents the mitochondrial genome assembly and annotation of an unknown appendicularian species collected from Japan. The findings contribute valuable data for future eDNA studies. The paper includes a thorough analysis of the mitochondrial genome including annotation and ... Continue reading READ ALL This paper presents the mitochondrial genome assembly and annotation of an unknown appendicularian species collected from Japan. The findings contribute valuable data for future eDNA studies. The paper includes a thorough analysis of the mitochondrial genome including annotation and phylogenetics analyses. However, the paper is missing important metrics regarding the PacBio Sequel II sequencing results. Please address the following points to make the article scientifically sound: 1. Add the following metrics regarding the PacBio Sequel II sequencing results: HiFi Reads, HiFi Yield (bp), HiFi Reads Length (Mean bp), HiFi Read Quality (median), HiFi Number of Passes (mean), HiFi Reads Length Summary, HiFi Read Length Distribution. 2. Also include the stat for the assembly itself including: Contig type (Primary vs haplotigs; polished contigs; max contigs length; median contig length, N50 Contig Length, Sum of Contig length. 3. PacBio assemblers obtain circular contigs which include the mitochondria DNA (and bacteria DNA). If circular contigs were not utilized to directly obtain mtDNA, a brief explanation of the rationale for this decision would be valuable. Are the rationale for sequencing the genome and the species significance clearly described? Yes Are the protocols appropriate and is the work technically sound? Partly Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Partly Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: genomic, evolution, comparative immunology and stem cell biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Voskoboynik A. Reviewer Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.172738.r341076 ) The direct URL for this report is: https://f1000research.com/articles/13-1357/v1#referee-response-341076 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 07 Mar 2025 Johannes Nicolaus Wibisana , Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan 07 Mar 2025 Author Response This paper presents the mitochondrial genome assembly and annotation of an unknown appendicularian species collected from Japan. The findings contribute valuable data for future eDNA studies. The paper includes a ... Continue reading This paper presents the mitochondrial genome assembly and annotation of an unknown appendicularian species collected from Japan. The findings contribute valuable data for future eDNA studies. The paper includes a thorough analysis of the mitochondrial genome including annotation and phylogenetics analyses. However, the paper is missing important metrics regarding the PacBio Sequel II sequencing results. Please address the following points to make the article scientifically sound: 1. Add the following metrics regarding the PacBio Sequel II sequencing results: HiFi Reads, HiFi Yield (bp), HiFi Reads Length (Mean bp), HiFi Read Quality (median), HiFi Number of Passes (mean), HiFi Reads Length Summary, HiFi Read Length Distribution. 2. Also include the stat for the assembly itself including: Contig type (Primary vs haplotigs; polished contigs; max contigs length; median contig length, N50 Contig Length, Sum of Contig length. 3. PacBio assemblers obtain circular contigs which include the mitochondria DNA (and bacteria DNA). If circular contigs were not utilized to directly obtain mtDNA, a brief explanation of the rationale for this decision would be valuable. Response: Thank you for the comments. We have added the PacBio sequencing metrics to the supplementary files. We have also added to the method section the method for the fragmentation step, which enables mtDNA sequencing on the PacBio platform. The mitochondrial genome presented here was produced with the NOVOLoci software in targeted assembly mode, which yielded a linear concatenate sequence which we circularised by hand. These changes have been reflected in the manuscript as follows: “The sequenced reads were assembled with NOVOLoci ( https://github.com/ndierckx/NOVOLoci ) in targeted assembly mode.” “DNA was fragmented using Megaruptor 3® (Diagenode, USA) using the Megaruptor 3 Shearing Kit (Diagenode, USA #E07010003) at speed 32 and purified using SMRTbell cleanup beads (Pacific Biosciences, USA #102-158-300). Quality control of obtained DNA was performed using the Femto Pulse System (Agilent, USA) and the Genomic DNA 165 kb Kit (Agilent, USA #FP-1002-0275). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA) using the Sequel II sequencing kit 2.0 (Pacific Biosciences, USA #101-820-200). The DNA size profile and sequencing metrics are available on Zenodo (doi: 10.5281/zenodo.14934254 ).” This paper presents the mitochondrial genome assembly and annotation of an unknown appendicularian species collected from Japan. The findings contribute valuable data for future eDNA studies. The paper includes a thorough analysis of the mitochondrial genome including annotation and phylogenetics analyses. However, the paper is missing important metrics regarding the PacBio Sequel II sequencing results. Please address the following points to make the article scientifically sound: 1. Add the following metrics regarding the PacBio Sequel II sequencing results: HiFi Reads, HiFi Yield (bp), HiFi Reads Length (Mean bp), HiFi Read Quality (median), HiFi Number of Passes (mean), HiFi Reads Length Summary, HiFi Read Length Distribution. 2. Also include the stat for the assembly itself including: Contig type (Primary vs haplotigs; polished contigs; max contigs length; median contig length, N50 Contig Length, Sum of Contig length. 3. PacBio assemblers obtain circular contigs which include the mitochondria DNA (and bacteria DNA). If circular contigs were not utilized to directly obtain mtDNA, a brief explanation of the rationale for this decision would be valuable. Response: Thank you for the comments. We have added the PacBio sequencing metrics to the supplementary files. We have also added to the method section the method for the fragmentation step, which enables mtDNA sequencing on the PacBio platform. The mitochondrial genome presented here was produced with the NOVOLoci software in targeted assembly mode, which yielded a linear concatenate sequence which we circularised by hand. These changes have been reflected in the manuscript as follows: “The sequenced reads were assembled with NOVOLoci ( https://github.com/ndierckx/NOVOLoci ) in targeted assembly mode.” “DNA was fragmented using Megaruptor 3® (Diagenode, USA) using the Megaruptor 3 Shearing Kit (Diagenode, USA #E07010003) at speed 32 and purified using SMRTbell cleanup beads (Pacific Biosciences, USA #102-158-300). Quality control of obtained DNA was performed using the Femto Pulse System (Agilent, USA) and the Genomic DNA 165 kb Kit (Agilent, USA #FP-1002-0275). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA) using the Sequel II sequencing kit 2.0 (Pacific Biosciences, USA #101-820-200). The DNA size profile and sequencing metrics are available on Zenodo (doi: 10.5281/zenodo.14934254 ).” Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 07 Mar 2025 Johannes Nicolaus Wibisana , Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan 07 Mar 2025 Author Response This paper presents the mitochondrial genome assembly and annotation of an unknown appendicularian species collected from Japan. The findings contribute valuable data for future eDNA studies. The paper includes a ... Continue reading This paper presents the mitochondrial genome assembly and annotation of an unknown appendicularian species collected from Japan. The findings contribute valuable data for future eDNA studies. The paper includes a thorough analysis of the mitochondrial genome including annotation and phylogenetics analyses. However, the paper is missing important metrics regarding the PacBio Sequel II sequencing results. Please address the following points to make the article scientifically sound: 1. Add the following metrics regarding the PacBio Sequel II sequencing results: HiFi Reads, HiFi Yield (bp), HiFi Reads Length (Mean bp), HiFi Read Quality (median), HiFi Number of Passes (mean), HiFi Reads Length Summary, HiFi Read Length Distribution. 2. Also include the stat for the assembly itself including: Contig type (Primary vs haplotigs; polished contigs; max contigs length; median contig length, N50 Contig Length, Sum of Contig length. 3. PacBio assemblers obtain circular contigs which include the mitochondria DNA (and bacteria DNA). If circular contigs were not utilized to directly obtain mtDNA, a brief explanation of the rationale for this decision would be valuable. Response: Thank you for the comments. We have added the PacBio sequencing metrics to the supplementary files. We have also added to the method section the method for the fragmentation step, which enables mtDNA sequencing on the PacBio platform. The mitochondrial genome presented here was produced with the NOVOLoci software in targeted assembly mode, which yielded a linear concatenate sequence which we circularised by hand. These changes have been reflected in the manuscript as follows: “The sequenced reads were assembled with NOVOLoci ( https://github.com/ndierckx/NOVOLoci ) in targeted assembly mode.” “DNA was fragmented using Megaruptor 3® (Diagenode, USA) using the Megaruptor 3 Shearing Kit (Diagenode, USA #E07010003) at speed 32 and purified using SMRTbell cleanup beads (Pacific Biosciences, USA #102-158-300). Quality control of obtained DNA was performed using the Femto Pulse System (Agilent, USA) and the Genomic DNA 165 kb Kit (Agilent, USA #FP-1002-0275). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA) using the Sequel II sequencing kit 2.0 (Pacific Biosciences, USA #101-820-200). The DNA size profile and sequencing metrics are available on Zenodo (doi: 10.5281/zenodo.14934254 ).” This paper presents the mitochondrial genome assembly and annotation of an unknown appendicularian species collected from Japan. The findings contribute valuable data for future eDNA studies. The paper includes a thorough analysis of the mitochondrial genome including annotation and phylogenetics analyses. However, the paper is missing important metrics regarding the PacBio Sequel II sequencing results. Please address the following points to make the article scientifically sound: 1. Add the following metrics regarding the PacBio Sequel II sequencing results: HiFi Reads, HiFi Yield (bp), HiFi Reads Length (Mean bp), HiFi Read Quality (median), HiFi Number of Passes (mean), HiFi Reads Length Summary, HiFi Read Length Distribution. 2. Also include the stat for the assembly itself including: Contig type (Primary vs haplotigs; polished contigs; max contigs length; median contig length, N50 Contig Length, Sum of Contig length. 3. PacBio assemblers obtain circular contigs which include the mitochondria DNA (and bacteria DNA). If circular contigs were not utilized to directly obtain mtDNA, a brief explanation of the rationale for this decision would be valuable. Response: Thank you for the comments. We have added the PacBio sequencing metrics to the supplementary files. We have also added to the method section the method for the fragmentation step, which enables mtDNA sequencing on the PacBio platform. The mitochondrial genome presented here was produced with the NOVOLoci software in targeted assembly mode, which yielded a linear concatenate sequence which we circularised by hand. These changes have been reflected in the manuscript as follows: “The sequenced reads were assembled with NOVOLoci ( https://github.com/ndierckx/NOVOLoci ) in targeted assembly mode.” “DNA was fragmented using Megaruptor 3® (Diagenode, USA) using the Megaruptor 3 Shearing Kit (Diagenode, USA #E07010003) at speed 32 and purified using SMRTbell cleanup beads (Pacific Biosciences, USA #102-158-300). Quality control of obtained DNA was performed using the Femto Pulse System (Agilent, USA) and the Genomic DNA 165 kb Kit (Agilent, USA #FP-1002-0275). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA) using the Sequel II sequencing kit 2.0 (Pacific Biosciences, USA #101-820-200). The DNA size profile and sequencing metrics are available on Zenodo (doi: 10.5281/zenodo.14934254 ).” Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Gissi C. Reviewer Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.172738.r341080 ) The direct URL for this report is: https://f1000research.com/articles/13-1357/v1#referee-response-341080 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 06 Jan 2025 Carmela Gissi , Department of Biosciences, Biotechnology and Environment, Università degli Studi di Bari Aldo Moro, Bari, Italy Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.172738.r341080 The paper describes the sequencing and assembly of the mitochondrial genome of a larvacean of the genus Oikopleura and also reports the mitochondrial phylogenetic analysis of Larvacea together with other chordate representatives. The manuscript is clear and well written, but ... Continue reading READ ALL The paper describes the sequencing and assembly of the mitochondrial genome of a larvacean of the genus Oikopleura and also reports the mitochondrial phylogenetic analysis of Larvacea together with other chordate representatives. The manuscript is clear and well written, but need to be improved with details on the sample and the applied protocols as well as with additional evolutionary analyses I suggest to better stress in the introduction the need to sequence other larvacean representatives, not only for eDNA studies but also for evolutionary studies aiming at understanding the origin of chordates and tunicates, and to unravel the hidden biodiversity of Larvacea, a quite neglected taxon Was the DNA extracted from a single specimen preserved in 99% ethanol or at -80 °C? In the Methods section, the Authors reports that "Quality control of obtained DNA was performed using Agilent 4200 TapeStation (Agilent, USA). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA)." Further details should be added. In particular, the Authors should report the ng and quality (i.e., absorbance values, TapeStation fragment profile) of the extracted DNA, the used PacBio library and sequencing kits, and the PacBio protocol used. Indeed, I suppose it was used the PacBio ultra-low input protocol, and, if so, how was the DNA fragmented? As for the phylogenetic tree, it seems that it was reconstructed using all 13 protein-coding genes, including those absent in larvaceans. Moreover, the tree was reconstructed from nucleotide, not from amino acid sequences, as is common when long evolutionary distances are analyzed, in order to avoid substitution saturation phenomena. I suggest reconstructing the tree also from amino acid sequences, with/without the protein-genes absent in larvaceans, so considering only positions without gaps and then to compare the obtained trees. Bayesian trees should also be reconstructed to confirm the obtained phylogeny A well formatted "full version" of the tree of Figure 3 should be provided in the Supplementary Material. Although out of the scope of the paper, a brief comment on the recovered tunicate phylogeny should be reported The Zenodo Supplementary material contains a folder with the results of IQ-TREE and RaxML analyses: could you please comment if there were differences between the trees obtained with the two methods? For not-users of the IQ-TREE software, I suggest explaining the meaning of the used command-line options and reporting that this software performs ML tree reconstructions The Authors should clarify the legend of Figure 3, including the number of genes analysed, the length of the alignment, the method and model used for the tree reconstruction. "Possible explanations for this result are heteroplasmy or that the mitogenome consists of concatemers ": please explain better why you hypothesize the existence of concatemers. "As the Oikopleura genus is polyphylic in our phylogenetic analysis and that of others,..." : the Authors should cite the references where the Oikopleura genus was already found as polyphyletic "Furthermore, these two haplotypes are supported by homopolymer variants at multiple different loci": a table with all variants between the two haplotypes should be reported in the Supplementary Material, particularly if Figure 2 shows only a portion of these variants. I suggest making available in NCBI also the second assembled haplotype Are the rationale for sequencing the genome and the species significance clearly described? Partly Are the protocols appropriate and is the work technically sound? Partly Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Partly Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: mitogenomics, tunicate evolution, long read NGS I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Gissi C. Reviewer Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.172738.r341080 ) The direct URL for this report is: https://f1000research.com/articles/13-1357/v1#referee-response-341080 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 07 Mar 2025 Johannes Nicolaus Wibisana , Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan 07 Mar 2025 Author Response I suggest to better stress in the introduction the need to sequence other larvacean representatives, not only for eDNA studies but also for evolutionary studies aiming at understanding the origin ... Continue reading I suggest to better stress in the introduction the need to sequence other larvacean representatives, not only for eDNA studies but also for evolutionary studies aiming at understanding the origin of chordates and tunicates, and to unravel the hidden biodiversity of Larvacea, a quite neglected taxon Response: Thank you for this comment. We have added the following statement in the introduction: “In addition to that, mitochondrial DNA sequence of larvaceans 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 Larvacea, which is a quite neglected taxon.” Was the DNA extracted from a single specimen preserved in 99% ethanol or at -80 °C? Response: Thank you for this comment. We preserved the specimen in 99% ethanol at -80°C. We have added the following statement to the manuscript: “Samples were preserved in 99% ethanol at -80°C prior to DNA extraction.” In the Methods section, the Authors reports that "Quality control of obtained DNA was performed using Agilent 4200 TapeStation (Agilent, USA). The sequencing was performed on a PacBio® Sequel II sequencer (Pacific Biosciences, USA)." Further details should be added. In particular, the Authors should report the ng and quality (i.e., absorbance values, TapeStation fragment profile) of the extracted DNA, the used PacBio library and sequencing kits, and the PacBio protocol used. Indeed, I suppose it was used the PacBio ultra-low input protocol, and, if so, how was the DNA fragmented? Response: Thank you for pointing this out. We have revised the quality control method, which used the Femto Pulse system after fragmentation. We also added the fragmentation protocol to the method section. As for the profile of the DNA fragments, we have attached the Femto Pulse run report to the supplementary files on Zenodo. We have adjusted the text as follows: “DNA was fragmented using Megaruptor 3 ® (Diagenode, USA) using the Megaruptor 3 Shearing Kit (Diagenode, USA #E07010003) at speed 32 and purified using SMRTbell cleanup beads (Pacific Biosciences, USA #102-158-300). Quality control of obtained DNA was performed using the Femto Pulse System (Agilent, USA) and the Genomic DNA 165 kb Kit (Agilent, USA #FP-1002-0275). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA) using the Sequel II sequencing kit 2.0 (Pacific Biosciences, USA #101-820-200). The DNA size profile and sequencing metrics are available on Zenodo (doi: 10.5281/zenodo.14934254 ).” As for the phylogenetic tree, it seems that it was reconstructed using all 13 protein-coding genes, including those absent in larvaceans. Moreover, the tree was reconstructed from nucleotide, not from amino acid sequences, as is common when long evolutionary distances are analyzed, in order to avoid substitution saturation phenomena. I suggest reconstructing the tree also from amino acid sequences, with/without the protein-genes absent in larvaceans, so considering only positions without gaps and then to compare the obtained trees. Bayesian trees should also be reconstructed to confirm the obtained phylogeny We used nucleotides instead of amino acids because we are still uncertain on how to translate the TGA codon in the Mesochordaeus and Bathochordaeus mitogenomes (Pichon et al., 2020), and worried that it may affect the results. Following the reviewer’s suggestion, we computed trees from amino acid sequences using the same IQ-TREE command line as for nucleotide sequences (as it selects the best models automatically), with or without the proteins that are not found in the appendicularian mitogenomes. In both cases the results were consistent with the nucleotide tree, by placing the new mitogenome in the same clade as O. longicauda , placing the appendicularians as a sister clade to thaliaceans except for D. nationalis , and identifying Ciona as a clade distinct from the other phlebobranchians. Unfortunately, the amino acid-based trees did not recover the expected relation between the three chordate sub-phyla nor between tunicate classes. We added a copy of these computations to our supplemental material for the reviewers and readers convenience, nevertheless we do not provide guarantee that the trees can be useful for other purposes than accountability of our work for this revision. We also did not compute Bayesian trees for this revision due to the lack of expertise in our team. A well formatted "full version" of the tree of Figure 3 should be provided in the Supplementary Material We have added the full version of the tree in the supplementary material under Figure S4. Although out of the scope of the paper, a brief comment on the recovered tunicate phylogeny should be reported We added the following: “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 orders.” The Zenodo Supplementary material contains a folder with the results of IQ-TREE and RaxML analyses: could you please comment if there were differences between the trees obtained with the two methods? The two trees have very similar topologies (identical when folded like in Figure 3), and we chose the IQ-TREE one because it provides bootstrap values that are easy to interpret. For not-users of the IQ-TREE software, I suggest explaining the meaning of the used command-line options and reporting that this software performs ML tree reconstructions We changed the method section’s text to “-T AUTO (run as many CPU threads as possible) --runs 3 (perform 3 runs to assess model convergence) --polytomy (allow unresolved branches) --ufboot 1000 (1000 boostraps with the ultrafast method) -m MFP (automatic selection of the best model for maximum likelihood inference using the ModelFinder Plus method” The Authors should clarify the legend of Figure 3, including the number of genes analysed, the length of the alignment, the method and model used for the tree reconstruction. We changed the figure legend to “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”. We did not indicate the model as we used one partition per gene and the software did not chose the same model for each partition. "Possible explanations for this result are heteroplasmy or that the mitogenome consists of concatemers ": please explain better why you hypothesize the existence of concatemers. "Furthermore, these two haplotypes are supported by homopolymer variants at multiple different loci": a table with all variants between the two haplotypes should be reported in the Supplementary Material, particularly if Figure 2 shows only a portion of these variants. I suggest making available in NCBI also the second assembled haplotype Response: Thank you for this comment. We have reanalyzed the sequences and we could not verify if the observed variation were true variants or caused by sequencing errors in homopolymers and therefore decided to remove the hypothesis of a second haplotype. The one haplotype is the one that is published online. We apologize for this error, and we confirmed that the current available sequence on NCBI is the correct one. We have also modified the text to remove the parts referencing the haplotypes. "As the Oikopleura genus is polyphylic in our phylogenetic analysis and that of others,..." : the Authors should cite the references where the Oikopleura genus was already found as polyphyletic Response: Thank you for this comment. We have added the following references to support this statement: Galt CP, Grober MS, Sykes PF. Taxonomic Correlates of Bioluminescence Among Appendicularians (Urochordata: Larvacea). The Biological Bulletin. 1985 Feb;168(1):125–34. Naville M, Henriet S, Warren I, Sumic S, Reeve M, Volff JN, et al. Massive Changes of Genome Size Driven by Expansions of Non-autonomous Transposable Elements. Current Biology. 2019 Apr;29(7):1161-1168.e6. Masunaga A, Mansfield MJ, Tan Y, Liu AW, Bliznina A, Barzaghi P, et al. The cosmopolitan appendicularian Oikopleura dioica reveals hidden genetic diversity around the globe. Mar Biol. 2022 Nov 27;169(12):157. Plessy C, Mansfield MJ, Bliznina A, Masunaga A, West C, Tan Y, et al. Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species. Genome Res. 2024 Apr 25;34(3):426–40 I suggest to better stress in the introduction the need to sequence other larvacean representatives, not only for eDNA studies but also for evolutionary studies aiming at understanding the origin of chordates and tunicates, and to unravel the hidden biodiversity of Larvacea, a quite neglected taxon Response: Thank you for this comment. We have added the following statement in the introduction: “In addition to that, mitochondrial DNA sequence of larvaceans 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 Larvacea, which is a quite neglected taxon.” Was the DNA extracted from a single specimen preserved in 99% ethanol or at -80 °C? Response: Thank you for this comment. We preserved the specimen in 99% ethanol at -80°C. We have added the following statement to the manuscript: “Samples were preserved in 99% ethanol at -80°C prior to DNA extraction.” In the Methods section, the Authors reports that "Quality control of obtained DNA was performed using Agilent 4200 TapeStation (Agilent, USA). The sequencing was performed on a PacBio® Sequel II sequencer (Pacific Biosciences, USA)." Further details should be added. In particular, the Authors should report the ng and quality (i.e., absorbance values, TapeStation fragment profile) of the extracted DNA, the used PacBio library and sequencing kits, and the PacBio protocol used. Indeed, I suppose it was used the PacBio ultra-low input protocol, and, if so, how was the DNA fragmented? Response: Thank you for pointing this out. We have revised the quality control method, which used the Femto Pulse system after fragmentation. We also added the fragmentation protocol to the method section. As for the profile of the DNA fragments, we have attached the Femto Pulse run report to the supplementary files on Zenodo. We have adjusted the text as follows: “DNA was fragmented using Megaruptor 3 ® (Diagenode, USA) using the Megaruptor 3 Shearing Kit (Diagenode, USA #E07010003) at speed 32 and purified using SMRTbell cleanup beads (Pacific Biosciences, USA #102-158-300). Quality control of obtained DNA was performed using the Femto Pulse System (Agilent, USA) and the Genomic DNA 165 kb Kit (Agilent, USA #FP-1002-0275). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA) using the Sequel II sequencing kit 2.0 (Pacific Biosciences, USA #101-820-200). The DNA size profile and sequencing metrics are available on Zenodo (doi: 10.5281/zenodo.14934254 ).” As for the phylogenetic tree, it seems that it was reconstructed using all 13 protein-coding genes, including those absent in larvaceans. Moreover, the tree was reconstructed from nucleotide, not from amino acid sequences, as is common when long evolutionary distances are analyzed, in order to avoid substitution saturation phenomena. I suggest reconstructing the tree also from amino acid sequences, with/without the protein-genes absent in larvaceans, so considering only positions without gaps and then to compare the obtained trees. Bayesian trees should also be reconstructed to confirm the obtained phylogeny We used nucleotides instead of amino acids because we are still uncertain on how to translate the TGA codon in the Mesochordaeus and Bathochordaeus mitogenomes (Pichon et al., 2020), and worried that it may affect the results. Following the reviewer’s suggestion, we computed trees from amino acid sequences using the same IQ-TREE command line as for nucleotide sequences (as it selects the best models automatically), with or without the proteins that are not found in the appendicularian mitogenomes. In both cases the results were consistent with the nucleotide tree, by placing the new mitogenome in the same clade as O. longicauda , placing the appendicularians as a sister clade to thaliaceans except for D. nationalis , and identifying Ciona as a clade distinct from the other phlebobranchians. Unfortunately, the amino acid-based trees did not recover the expected relation between the three chordate sub-phyla nor between tunicate classes. We added a copy of these computations to our supplemental material for the reviewers and readers convenience, nevertheless we do not provide guarantee that the trees can be useful for other purposes than accountability of our work for this revision. We also did not compute Bayesian trees for this revision due to the lack of expertise in our team. A well formatted "full version" of the tree of Figure 3 should be provided in the Supplementary Material We have added the full version of the tree in the supplementary material under Figure S4. Although out of the scope of the paper, a brief comment on the recovered tunicate phylogeny should be reported We added the following: “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 orders.” The Zenodo Supplementary material contains a folder with the results of IQ-TREE and RaxML analyses: could you please comment if there were differences between the trees obtained with the two methods? The two trees have very similar topologies (identical when folded like in Figure 3), and we chose the IQ-TREE one because it provides bootstrap values that are easy to interpret. For not-users of the IQ-TREE software, I suggest explaining the meaning of the used command-line options and reporting that this software performs ML tree reconstructions We changed the method section’s text to “-T AUTO (run as many CPU threads as possible) --runs 3 (perform 3 runs to assess model convergence) --polytomy (allow unresolved branches) --ufboot 1000 (1000 boostraps with the ultrafast method) -m MFP (automatic selection of the best model for maximum likelihood inference using the ModelFinder Plus method” The Authors should clarify the legend of Figure 3, including the number of genes analysed, the length of the alignment, the method and model used for the tree reconstruction. We changed the figure legend to “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”. We did not indicate the model as we used one partition per gene and the software did not chose the same model for each partition. "Possible explanations for this result are heteroplasmy or that the mitogenome consists of concatemers ": please explain better why you hypothesize the existence of concatemers. "Furthermore, these two haplotypes are supported by homopolymer variants at multiple different loci": a table with all variants between the two haplotypes should be reported in the Supplementary Material, particularly if Figure 2 shows only a portion of these variants. I suggest making available in NCBI also the second assembled haplotype Response: Thank you for this comment. We have reanalyzed the sequences and we could not verify if the observed variation were true variants or caused by sequencing errors in homopolymers and therefore decided to remove the hypothesis of a second haplotype. The one haplotype is the one that is published online. We apologize for this error, and we confirmed that the current available sequence on NCBI is the correct one. We have also modified the text to remove the parts referencing the haplotypes. "As the Oikopleura genus is polyphylic in our phylogenetic analysis and that of others,..." : the Authors should cite the references where the Oikopleura genus was already found as polyphyletic Response: Thank you for this comment. We have added the following references to support this statement: Galt CP, Grober MS, Sykes PF. Taxonomic Correlates of Bioluminescence Among Appendicularians (Urochordata: Larvacea). The Biological Bulletin. 1985 Feb;168(1):125–34. Naville M, Henriet S, Warren I, Sumic S, Reeve M, Volff JN, et al. Massive Changes of Genome Size Driven by Expansions of Non-autonomous Transposable Elements. Current Biology. 2019 Apr;29(7):1161-1168.e6. Masunaga A, Mansfield MJ, Tan Y, Liu AW, Bliznina A, Barzaghi P, et al. The cosmopolitan appendicularian Oikopleura dioica reveals hidden genetic diversity around the globe. Mar Biol. 2022 Nov 27;169(12):157. Plessy C, Mansfield MJ, Bliznina A, Masunaga A, West C, Tan Y, et al. Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species. Genome Res. 2024 Apr 25;34(3):426–40 Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 07 Mar 2025 Johannes Nicolaus Wibisana , Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan 07 Mar 2025 Author Response I suggest to better stress in the introduction the need to sequence other larvacean representatives, not only for eDNA studies but also for evolutionary studies aiming at understanding the origin ... Continue reading I suggest to better stress in the introduction the need to sequence other larvacean representatives, not only for eDNA studies but also for evolutionary studies aiming at understanding the origin of chordates and tunicates, and to unravel the hidden biodiversity of Larvacea, a quite neglected taxon Response: Thank you for this comment. We have added the following statement in the introduction: “In addition to that, mitochondrial DNA sequence of larvaceans 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 Larvacea, which is a quite neglected taxon.” Was the DNA extracted from a single specimen preserved in 99% ethanol or at -80 °C? Response: Thank you for this comment. We preserved the specimen in 99% ethanol at -80°C. We have added the following statement to the manuscript: “Samples were preserved in 99% ethanol at -80°C prior to DNA extraction.” In the Methods section, the Authors reports that "Quality control of obtained DNA was performed using Agilent 4200 TapeStation (Agilent, USA). The sequencing was performed on a PacBio® Sequel II sequencer (Pacific Biosciences, USA)." Further details should be added. In particular, the Authors should report the ng and quality (i.e., absorbance values, TapeStation fragment profile) of the extracted DNA, the used PacBio library and sequencing kits, and the PacBio protocol used. Indeed, I suppose it was used the PacBio ultra-low input protocol, and, if so, how was the DNA fragmented? Response: Thank you for pointing this out. We have revised the quality control method, which used the Femto Pulse system after fragmentation. We also added the fragmentation protocol to the method section. As for the profile of the DNA fragments, we have attached the Femto Pulse run report to the supplementary files on Zenodo. We have adjusted the text as follows: “DNA was fragmented using Megaruptor 3 ® (Diagenode, USA) using the Megaruptor 3 Shearing Kit (Diagenode, USA #E07010003) at speed 32 and purified using SMRTbell cleanup beads (Pacific Biosciences, USA #102-158-300). Quality control of obtained DNA was performed using the Femto Pulse System (Agilent, USA) and the Genomic DNA 165 kb Kit (Agilent, USA #FP-1002-0275). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA) using the Sequel II sequencing kit 2.0 (Pacific Biosciences, USA #101-820-200). The DNA size profile and sequencing metrics are available on Zenodo (doi: 10.5281/zenodo.14934254 ).” As for the phylogenetic tree, it seems that it was reconstructed using all 13 protein-coding genes, including those absent in larvaceans. Moreover, the tree was reconstructed from nucleotide, not from amino acid sequences, as is common when long evolutionary distances are analyzed, in order to avoid substitution saturation phenomena. I suggest reconstructing the tree also from amino acid sequences, with/without the protein-genes absent in larvaceans, so considering only positions without gaps and then to compare the obtained trees. Bayesian trees should also be reconstructed to confirm the obtained phylogeny We used nucleotides instead of amino acids because we are still uncertain on how to translate the TGA codon in the Mesochordaeus and Bathochordaeus mitogenomes (Pichon et al., 2020), and worried that it may affect the results. Following the reviewer’s suggestion, we computed trees from amino acid sequences using the same IQ-TREE command line as for nucleotide sequences (as it selects the best models automatically), with or without the proteins that are not found in the appendicularian mitogenomes. In both cases the results were consistent with the nucleotide tree, by placing the new mitogenome in the same clade as O. longicauda , placing the appendicularians as a sister clade to thaliaceans except for D. nationalis , and identifying Ciona as a clade distinct from the other phlebobranchians. Unfortunately, the amino acid-based trees did not recover the expected relation between the three chordate sub-phyla nor between tunicate classes. We added a copy of these computations to our supplemental material for the reviewers and readers convenience, nevertheless we do not provide guarantee that the trees can be useful for other purposes than accountability of our work for this revision. We also did not compute Bayesian trees for this revision due to the lack of expertise in our team. A well formatted "full version" of the tree of Figure 3 should be provided in the Supplementary Material We have added the full version of the tree in the supplementary material under Figure S4. Although out of the scope of the paper, a brief comment on the recovered tunicate phylogeny should be reported We added the following: “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 orders.” The Zenodo Supplementary material contains a folder with the results of IQ-TREE and RaxML analyses: could you please comment if there were differences between the trees obtained with the two methods? The two trees have very similar topologies (identical when folded like in Figure 3), and we chose the IQ-TREE one because it provides bootstrap values that are easy to interpret. For not-users of the IQ-TREE software, I suggest explaining the meaning of the used command-line options and reporting that this software performs ML tree reconstructions We changed the method section’s text to “-T AUTO (run as many CPU threads as possible) --runs 3 (perform 3 runs to assess model convergence) --polytomy (allow unresolved branches) --ufboot 1000 (1000 boostraps with the ultrafast method) -m MFP (automatic selection of the best model for maximum likelihood inference using the ModelFinder Plus method” The Authors should clarify the legend of Figure 3, including the number of genes analysed, the length of the alignment, the method and model used for the tree reconstruction. We changed the figure legend to “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”. We did not indicate the model as we used one partition per gene and the software did not chose the same model for each partition. "Possible explanations for this result are heteroplasmy or that the mitogenome consists of concatemers ": please explain better why you hypothesize the existence of concatemers. "Furthermore, these two haplotypes are supported by homopolymer variants at multiple different loci": a table with all variants between the two haplotypes should be reported in the Supplementary Material, particularly if Figure 2 shows only a portion of these variants. I suggest making available in NCBI also the second assembled haplotype Response: Thank you for this comment. We have reanalyzed the sequences and we could not verify if the observed variation were true variants or caused by sequencing errors in homopolymers and therefore decided to remove the hypothesis of a second haplotype. The one haplotype is the one that is published online. We apologize for this error, and we confirmed that the current available sequence on NCBI is the correct one. We have also modified the text to remove the parts referencing the haplotypes. "As the Oikopleura genus is polyphylic in our phylogenetic analysis and that of others,..." : the Authors should cite the references where the Oikopleura genus was already found as polyphyletic Response: Thank you for this comment. We have added the following references to support this statement: Galt CP, Grober MS, Sykes PF. Taxonomic Correlates of Bioluminescence Among Appendicularians (Urochordata: Larvacea). The Biological Bulletin. 1985 Feb;168(1):125–34. Naville M, Henriet S, Warren I, Sumic S, Reeve M, Volff JN, et al. Massive Changes of Genome Size Driven by Expansions of Non-autonomous Transposable Elements. Current Biology. 2019 Apr;29(7):1161-1168.e6. Masunaga A, Mansfield MJ, Tan Y, Liu AW, Bliznina A, Barzaghi P, et al. The cosmopolitan appendicularian Oikopleura dioica reveals hidden genetic diversity around the globe. Mar Biol. 2022 Nov 27;169(12):157. Plessy C, Mansfield MJ, Bliznina A, Masunaga A, West C, Tan Y, et al. Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species. Genome Res. 2024 Apr 25;34(3):426–40 I suggest to better stress in the introduction the need to sequence other larvacean representatives, not only for eDNA studies but also for evolutionary studies aiming at understanding the origin of chordates and tunicates, and to unravel the hidden biodiversity of Larvacea, a quite neglected taxon Response: Thank you for this comment. We have added the following statement in the introduction: “In addition to that, mitochondrial DNA sequence of larvaceans 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 Larvacea, which is a quite neglected taxon.” Was the DNA extracted from a single specimen preserved in 99% ethanol or at -80 °C? Response: Thank you for this comment. We preserved the specimen in 99% ethanol at -80°C. We have added the following statement to the manuscript: “Samples were preserved in 99% ethanol at -80°C prior to DNA extraction.” In the Methods section, the Authors reports that "Quality control of obtained DNA was performed using Agilent 4200 TapeStation (Agilent, USA). The sequencing was performed on a PacBio® Sequel II sequencer (Pacific Biosciences, USA)." Further details should be added. In particular, the Authors should report the ng and quality (i.e., absorbance values, TapeStation fragment profile) of the extracted DNA, the used PacBio library and sequencing kits, and the PacBio protocol used. Indeed, I suppose it was used the PacBio ultra-low input protocol, and, if so, how was the DNA fragmented? Response: Thank you for pointing this out. We have revised the quality control method, which used the Femto Pulse system after fragmentation. We also added the fragmentation protocol to the method section. As for the profile of the DNA fragments, we have attached the Femto Pulse run report to the supplementary files on Zenodo. We have adjusted the text as follows: “DNA was fragmented using Megaruptor 3 ® (Diagenode, USA) using the Megaruptor 3 Shearing Kit (Diagenode, USA #E07010003) at speed 32 and purified using SMRTbell cleanup beads (Pacific Biosciences, USA #102-158-300). Quality control of obtained DNA was performed using the Femto Pulse System (Agilent, USA) and the Genomic DNA 165 kb Kit (Agilent, USA #FP-1002-0275). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA) using the Sequel II sequencing kit 2.0 (Pacific Biosciences, USA #101-820-200). The DNA size profile and sequencing metrics are available on Zenodo (doi: 10.5281/zenodo.14934254 ).” As for the phylogenetic tree, it seems that it was reconstructed using all 13 protein-coding genes, including those absent in larvaceans. Moreover, the tree was reconstructed from nucleotide, not from amino acid sequences, as is common when long evolutionary distances are analyzed, in order to avoid substitution saturation phenomena. I suggest reconstructing the tree also from amino acid sequences, with/without the protein-genes absent in larvaceans, so considering only positions without gaps and then to compare the obtained trees. Bayesian trees should also be reconstructed to confirm the obtained phylogeny We used nucleotides instead of amino acids because we are still uncertain on how to translate the TGA codon in the Mesochordaeus and Bathochordaeus mitogenomes (Pichon et al., 2020), and worried that it may affect the results. Following the reviewer’s suggestion, we computed trees from amino acid sequences using the same IQ-TREE command line as for nucleotide sequences (as it selects the best models automatically), with or without the proteins that are not found in the appendicularian mitogenomes. In both cases the results were consistent with the nucleotide tree, by placing the new mitogenome in the same clade as O. longicauda , placing the appendicularians as a sister clade to thaliaceans except for D. nationalis , and identifying Ciona as a clade distinct from the other phlebobranchians. Unfortunately, the amino acid-based trees did not recover the expected relation between the three chordate sub-phyla nor between tunicate classes. We added a copy of these computations to our supplemental material for the reviewers and readers convenience, nevertheless we do not provide guarantee that the trees can be useful for other purposes than accountability of our work for this revision. We also did not compute Bayesian trees for this revision due to the lack of expertise in our team. A well formatted "full version" of the tree of Figure 3 should be provided in the Supplementary Material We have added the full version of the tree in the supplementary material under Figure S4. Although out of the scope of the paper, a brief comment on the recovered tunicate phylogeny should be reported We added the following: “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 orders.” The Zenodo Supplementary material contains a folder with the results of IQ-TREE and RaxML analyses: could you please comment if there were differences between the trees obtained with the two methods? The two trees have very similar topologies (identical when folded like in Figure 3), and we chose the IQ-TREE one because it provides bootstrap values that are easy to interpret. For not-users of the IQ-TREE software, I suggest explaining the meaning of the used command-line options and reporting that this software performs ML tree reconstructions We changed the method section’s text to “-T AUTO (run as many CPU threads as possible) --runs 3 (perform 3 runs to assess model convergence) --polytomy (allow unresolved branches) --ufboot 1000 (1000 boostraps with the ultrafast method) -m MFP (automatic selection of the best model for maximum likelihood inference using the ModelFinder Plus method” The Authors should clarify the legend of Figure 3, including the number of genes analysed, the length of the alignment, the method and model used for the tree reconstruction. We changed the figure legend to “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”. We did not indicate the model as we used one partition per gene and the software did not chose the same model for each partition. "Possible explanations for this result are heteroplasmy or that the mitogenome consists of concatemers ": please explain better why you hypothesize the existence of concatemers. "Furthermore, these two haplotypes are supported by homopolymer variants at multiple different loci": a table with all variants between the two haplotypes should be reported in the Supplementary Material, particularly if Figure 2 shows only a portion of these variants. I suggest making available in NCBI also the second assembled haplotype Response: Thank you for this comment. We have reanalyzed the sequences and we could not verify if the observed variation were true variants or caused by sequencing errors in homopolymers and therefore decided to remove the hypothesis of a second haplotype. The one haplotype is the one that is published online. We apologize for this error, and we confirmed that the current available sequence on NCBI is the correct one. We have also modified the text to remove the parts referencing the haplotypes. "As the Oikopleura genus is polyphylic in our phylogenetic analysis and that of others,..." : the Authors should cite the references where the Oikopleura genus was already found as polyphyletic Response: Thank you for this comment. We have added the following references to support this statement: Galt CP, Grober MS, Sykes PF. Taxonomic Correlates of Bioluminescence Among Appendicularians (Urochordata: Larvacea). The Biological Bulletin. 1985 Feb;168(1):125–34. Naville M, Henriet S, Warren I, Sumic S, Reeve M, Volff JN, et al. Massive Changes of Genome Size Driven by Expansions of Non-autonomous Transposable Elements. Current Biology. 2019 Apr;29(7):1161-1168.e6. Masunaga A, Mansfield MJ, Tan Y, Liu AW, Bliznina A, Barzaghi P, et al. The cosmopolitan appendicularian Oikopleura dioica reveals hidden genetic diversity around the globe. Mar Biol. 2022 Nov 27;169(12):157. Plessy C, Mansfield MJ, Bliznina A, Masunaga A, West C, Tan Y, et al. Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species. Genome Res. 2024 Apr 25;34(3):426–40 Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 3 VERSION 3 PUBLISHED 12 Nov 2024 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 4 Version 3 (revision) 06 May 25 read Version 2 (revision) 07 Mar 25 read read Version 1 12 Nov 24 read read Carmela Gissi , Università degli Studi di Bari Aldo Moro, Bari, Italy Ayelet Voskoboynik , Stanford University, Stanford, USA Daniel M Chourrout , University of Bergen, Bergen, Norway Thomas Stach , Humboldt-Universität zu Berlin Vergleichende Elektronenmikroskopie,, Philippstraße, Germany Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Stach T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 29 May 2025 | for Version 3 Thomas Stach , Humboldt-Universität zu Berlin Vergleichende Elektronenmikroskopie,, Philippstraße, Germany 0 Views copyright © 2025 Stach T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Having another mt-genome of an appendicularian species in the databases is scientific progress. So, I will tick approved this time (also, so I do not need to revise the ms again). The principal weakness of the ms still holds and it cannot be fixed (or only with considerably investment of resources and time). That, of course is the fact that the species the sequenced mt-genome belongs to has to remain unidentified, due to the suboptimal preservation of the specimens caught for morphological examination. Here are some minor comments: In the abstract it says "unknown appendicularian". "unknown" in this context usually means not known to science. We don't know that of the species sequenced here; in fact it is highly unlikely that it is a new species. The authors should stick to "unidentified", the adjective used - correctly - in the title. I think it should be mentioned somewhere that there are about 70 species of appendicularians described currently. Collection details (type of plankton net, mesh size, towing duration, ship speed, towing depth) should be specified. In my opinion, a few words about what is known concerning the presently described diversity of appendicularians off the coast of Japan are necessary to give the reader some context. A few considerations about the species identity beyond the sequence could also be attempted: limited as it may be, maybe some of the distinguishing features of appendicularians can still be seen? E.g., buccal glands are quite prominent and could potentially be seen, if they were present. Similarly, subchordal cells, another feature used in appendicularian taxonomy, might be visible in the recorded micrographs at higher resolution. Competing Interests No competing interests were disclosed. Reviewer Expertise comparative zoology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Stach T. Peer Review Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.181225.r383198) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1357/v3#referee-response-383198 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Stach T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 16 Apr 2025 | for Version 2 Thomas Stach , Humboldt-Universität zu Berlin Vergleichende Elektronenmikroskopie,, Philippstraße, Germany 0 Views copyright © 2025 Stach T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript "The complete mitogenome of an unidentified Oikopleura species" submitted by J. N. Wibisana and co-authors reports the sequencing of the mitochondrial genome of an unidentified appendicularian species collected off the coast of Japan. The presented mitochondrial genome is new to the respective databases and therefore an original contribution to science. The genome has some features that are evolutionarily interesting and could potentially be used in a cladistic framework. The authors conduct a formal phylogenetic analysis using a Maximum Likelihood algorithm. I feel a bit out of my expertise in gauging the validity of the current software used, but based on my experience with other software packages, I would expect that 'ultrafast methods' to calculate bootstrap values overestimate bootstrap support of a node. The authors rationalize the study mainly by advancing the knowledge of genome sequences in an underrepresented taxon and the potential use this might have in eDNA research. My main reservation concerning this study is its surprising lack of standard taxonomy approaches. While the sequence as such might be new to science, the species it belongs to, might not be new. Or it might be. From the sequence alone, we cannot know. In order to tie the valuable data generated to the existing body of scientific knowledge, it is - in my view - imperative to at least seriously try to identify the species using the available taxonomic literature on appendicularians. To this end, specimens should have been fixed in a more suitable fixative than Ethanol, in order to preserve the taxonomically relevant characters. They probably should have also been caught more gently as judging from the Figure 1 - but the details of collecting the animals are not given. I have also a few minor comments: line 2 in the introduction: it should be 'their life cycle' instead of 'its life cycle' third paragraph of the Results and discussion: Oikopleuridae should not be italicized (only Genus and species names are). also: '... and misses some of the eight expected ...' I guess the precise number could be stated here. Eight is not so huge that the reader looses track. sixth paragraph of the Results and discussion: 'polyphylic' is not the correct term. I assume what is meant is polyphyletic (see also Carmela Gissi's review). But even this is not entirely fitting (at least in the common use of the cladistics terminology), because the depicted phylogeny would be a case of paraphyly, i.e. the genus Oikopleura is paraphyletic in this analysis. Are the rationale for sequencing the genome and the species significance clearly described? Partly Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Partly Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise comparative zoology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 06 May 2025 Johannes Nicolaus Wibisana, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan We agree with the reviewer’s point of view, which is why we only recommend to use the tree for guiding further data acquisition efforts.The authors rationalize the study mainly by advancing the knowledge of genome sequences in an underrepresented taxon and the potential use this might have in eDNA research. My main reservation concerning this study is its surprising lack of standard taxonomy approaches. While the sequence as such might be new to science, the species it belongs to, might not be new. Or it might be. From the sequence alone, we cannot know. In order to tie the valuable data generated to the existing body of scientific knowledge, it is - in my view - imperative to at least seriously try to identify the species using the available taxonomic literature on appendicularians. To this end, specimens should have been fixed in a more suitable fixative than Ethanol, in order to preserve the taxonomically relevant characters. They probably should have also been caught more gently as judging from the Figure 1 - but the details of collecting the animals are not given. Response: Thank you for the comments. The animals were obtained as a bycatch during sampling of other zooplanktons and thus the morphology was not preserved well. Unfortunately, we do not have any specimens left with better preserved morphology. However, we were able to confirm that it has characteristics pertaining to O. fusiformis owing to the shape of the gastric lobe and the slender tail. We agree that a proper taxonomic analysis is needed, but we would like to underline that situations ideal for taxonomy will not necessarily be ideal for recovering the high-molecular weight DNA needed for appendicularian mitogenome assembly. Therefore, we believe that our publication will be a steppingstone for further work, in which only the sequencing of a short PCR fragment will be needed to assess the relation between a taxonomically identified individual and our published mitochondrial genome sequence. We also underline in our conclusion that the possible existence of cryptic species, which will be difficult to resolve with only morphological information, has to be taken into account. I have also a few minor comments: line 2 in the introduction: it should be 'their life cycle' instead of 'its life cycle' third paragraph of the Results and discussion: Oikopleuridae should not be italicized (only Genus and species names are). also: '... and misses some of the eight expected ...' I guess the precise number could be stated here. Eight is not so huge that the reader looses track. sixth paragraph of the Results and discussion: 'polyphylic' is not the correct term. I assume what is meant is polyphyletic (see also Carmela Gissi's review). But even this is not entirely fitting (at least in the common use of the cladistics terminology), because the depicted phylogeny would be a case of paraphyly, i.e. the genus Oikopleura is paraphyletic in this analysis. Response: Thank you for the minor comments, we have adjusted the manuscript to reflect all these changes. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Stach T. Peer Review Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.178729.r374065) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1357/v2#referee-response-374065 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Chourrout D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 16 Apr 2025 | for Version 2 Daniel M Chourrout , University of Bergen, Bergen, Norway 0 Views copyright © 2025 Chourrout D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Although this appendicularian species is not named, having its mt genome sequence is useful because it contributes to increase taxonomic diversity in phylogenetic trees. The annotation is carefully performed and the conclusions make sense, with a clear position next to O. longicauda and two giant larvaceans. I have two comments: 1. Why not comparing the order of the coding genes with other appendicularian species ? Previous work on other species shows that synteny is not conserved with other tunicates and it could be the main contribution of the paper if this comparison was performed. 2. The picture is of extremely poor quality and it should not be index together with the main text. It may be sent to an annex as it does not bring information except that the species is an appendicularian, this is clear enough from the phylogenetic analysis. How a better picture could not be obtained is surprising, it might have helped to tentatively name the species. Are the rationale for sequencing the genome and the species significance clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise animal genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (1) Author Response 06 May 2025 Johannes Nicolaus Wibisana, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan 1. Why not comparing the order of the coding genes with other appendicularian species ? Previous work on other species shows that synteny is not conserved with other tunicates and it could be the main contribution of the paper if this comparison was performed. Response: Thank you for this comment. We added the following to our manuscript: “Previous work on other species suggests that synteny is usually not conserved in tunicates (Singh et al., 2009). Indeed, we found that the gene order bears no resemblance to the one of O. dioica (Dierckxsens et al., 2024; Klirs et al., 2025) nor to the one of O. longicauda (Naville et al., 2019, scaffold SCLD01101138.1) nor to one in the stolidobranch ascidians Herdmania momus and Halocynthia roretzi .” 2. The picture is of extremely poor quality and it should not be index together with the main text. It may be sent to an annex as it does not bring information except that the species is an appendicularian, this is clear enough from the phylogenetic analysis. How a better picture could not be obtained is surprising, it might have helped to tentatively name the species. Response: Thank you for this comment. The specimen that we obtained was from a bycatch and thus was not preserved in the best condition. We kept the figure as it is in the main manuscript as it is the second round of review, where we would like to avoid major changes of the document layout to prevent introducing new typographical errors View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Chourrout DM. Peer Review Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.178729.r374064) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1357/v2#referee-response-374064 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Voskoboynik A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 06 Jan 2025 | for Version 1 Ayelet Voskoboynik , Stanford University, Stanford, USA 0 Views copyright © 2025 Voskoboynik A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This paper presents the mitochondrial genome assembly and annotation of an unknown appendicularian species collected from Japan. The findings contribute valuable data for future eDNA studies. The paper includes a thorough analysis of the mitochondrial genome including annotation and phylogenetics analyses. However, the paper is missing important metrics regarding the PacBio Sequel II sequencing results. Please address the following points to make the article scientifically sound: 1. Add the following metrics regarding the PacBio Sequel II sequencing results: HiFi Reads, HiFi Yield (bp), HiFi Reads Length (Mean bp), HiFi Read Quality (median), HiFi Number of Passes (mean), HiFi Reads Length Summary, HiFi Read Length Distribution. 2. Also include the stat for the assembly itself including: Contig type (Primary vs haplotigs; polished contigs; max contigs length; median contig length, N50 Contig Length, Sum of Contig length. 3. PacBio assemblers obtain circular contigs which include the mitochondria DNA (and bacteria DNA). If circular contigs were not utilized to directly obtain mtDNA, a brief explanation of the rationale for this decision would be valuable. Are the rationale for sequencing the genome and the species significance clearly described? Yes Are the protocols appropriate and is the work technically sound? Partly Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Partly Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise genomic, evolution, comparative immunology and stem cell biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (1) Author Response 07 Mar 2025 Johannes Nicolaus Wibisana, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan This paper presents the mitochondrial genome assembly and annotation of an unknown appendicularian species collected from Japan. The findings contribute valuable data for future eDNA studies. The paper includes a thorough analysis of the mitochondrial genome including annotation and phylogenetics analyses. However, the paper is missing important metrics regarding the PacBio Sequel II sequencing results. Please address the following points to make the article scientifically sound: 1. Add the following metrics regarding the PacBio Sequel II sequencing results: HiFi Reads, HiFi Yield (bp), HiFi Reads Length (Mean bp), HiFi Read Quality (median), HiFi Number of Passes (mean), HiFi Reads Length Summary, HiFi Read Length Distribution. 2. Also include the stat for the assembly itself including: Contig type (Primary vs haplotigs; polished contigs; max contigs length; median contig length, N50 Contig Length, Sum of Contig length. 3. PacBio assemblers obtain circular contigs which include the mitochondria DNA (and bacteria DNA). If circular contigs were not utilized to directly obtain mtDNA, a brief explanation of the rationale for this decision would be valuable. Response: Thank you for the comments. We have added the PacBio sequencing metrics to the supplementary files. We have also added to the method section the method for the fragmentation step, which enables mtDNA sequencing on the PacBio platform. The mitochondrial genome presented here was produced with the NOVOLoci software in targeted assembly mode, which yielded a linear concatenate sequence which we circularised by hand. These changes have been reflected in the manuscript as follows: “The sequenced reads were assembled with NOVOLoci ( https://github.com/ndierckx/NOVOLoci ) in targeted assembly mode.” “DNA was fragmented using Megaruptor 3® (Diagenode, USA) using the Megaruptor 3 Shearing Kit (Diagenode, USA #E07010003) at speed 32 and purified using SMRTbell cleanup beads (Pacific Biosciences, USA #102-158-300). Quality control of obtained DNA was performed using the Femto Pulse System (Agilent, USA) and the Genomic DNA 165 kb Kit (Agilent, USA #FP-1002-0275). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA) using the Sequel II sequencing kit 2.0 (Pacific Biosciences, USA #101-820-200). The DNA size profile and sequencing metrics are available on Zenodo (doi: 10.5281/zenodo.14934254 ).” View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Voskoboynik A. Peer Review Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . F1000Research 2025, 13 :1357 ( https://doi.org/10.5256/f1000research.172738.r341076) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1357/v1#referee-response-341076 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Gissi C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 06 Jan 2025 | for Version 1 Carmela Gissi , Department of Biosciences, Biotechnology and Environment, Università degli Studi di Bari Aldo Moro, Bari, Italy 0 Views copyright © 2025 Gissi C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The paper describes the sequencing and assembly of the mitochondrial genome of a larvacean of the genus Oikopleura and also reports the mitochondrial phylogenetic analysis of Larvacea together with other chordate representatives. The manuscript is clear and well written, but need to be improved with details on the sample and the applied protocols as well as with additional evolutionary analyses I suggest to better stress in the introduction the need to sequence other larvacean representatives, not only for eDNA studies but also for evolutionary studies aiming at understanding the origin of chordates and tunicates, and to unravel the hidden biodiversity of Larvacea, a quite neglected taxon Was the DNA extracted from a single specimen preserved in 99% ethanol or at -80 °C? In the Methods section, the Authors reports that "Quality control of obtained DNA was performed using Agilent 4200 TapeStation (Agilent, USA). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA)." Further details should be added. In particular, the Authors should report the ng and quality (i.e., absorbance values, TapeStation fragment profile) of the extracted DNA, the used PacBio library and sequencing kits, and the PacBio protocol used. Indeed, I suppose it was used the PacBio ultra-low input protocol, and, if so, how was the DNA fragmented? As for the phylogenetic tree, it seems that it was reconstructed using all 13 protein-coding genes, including those absent in larvaceans. Moreover, the tree was reconstructed from nucleotide, not from amino acid sequences, as is common when long evolutionary distances are analyzed, in order to avoid substitution saturation phenomena. I suggest reconstructing the tree also from amino acid sequences, with/without the protein-genes absent in larvaceans, so considering only positions without gaps and then to compare the obtained trees. Bayesian trees should also be reconstructed to confirm the obtained phylogeny A well formatted "full version" of the tree of Figure 3 should be provided in the Supplementary Material. Although out of the scope of the paper, a brief comment on the recovered tunicate phylogeny should be reported The Zenodo Supplementary material contains a folder with the results of IQ-TREE and RaxML analyses: could you please comment if there were differences between the trees obtained with the two methods? For not-users of the IQ-TREE software, I suggest explaining the meaning of the used command-line options and reporting that this software performs ML tree reconstructions The Authors should clarify the legend of Figure 3, including the number of genes analysed, the length of the alignment, the method and model used for the tree reconstruction. "Possible explanations for this result are heteroplasmy or that the mitogenome consists of concatemers ": please explain better why you hypothesize the existence of concatemers. "As the Oikopleura genus is polyphylic in our phylogenetic analysis and that of others,..." : the Authors should cite the references where the Oikopleura genus was already found as polyphyletic "Furthermore, these two haplotypes are supported by homopolymer variants at multiple different loci": a table with all variants between the two haplotypes should be reported in the Supplementary Material, particularly if Figure 2 shows only a portion of these variants. I suggest making available in NCBI also the second assembled haplotype Are the rationale for sequencing the genome and the species significance clearly described? Partly Are the protocols appropriate and is the work technically sound? Partly Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Partly Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise mitogenomics, tunicate evolution, long read NGS I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 07 Mar 2025 Johannes Nicolaus Wibisana, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, 9040497, Japan I suggest to better stress in the introduction the need to sequence other larvacean representatives, not only for eDNA studies but also for evolutionary studies aiming at understanding the origin of chordates and tunicates, and to unravel the hidden biodiversity of Larvacea, a quite neglected taxon Response: Thank you for this comment. We have added the following statement in the introduction: “In addition to that, mitochondrial DNA sequence of larvaceans 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 Larvacea, which is a quite neglected taxon.” Was the DNA extracted from a single specimen preserved in 99% ethanol or at -80 °C? Response: Thank you for this comment. We preserved the specimen in 99% ethanol at -80°C. We have added the following statement to the manuscript: “Samples were preserved in 99% ethanol at -80°C prior to DNA extraction.” In the Methods section, the Authors reports that "Quality control of obtained DNA was performed using Agilent 4200 TapeStation (Agilent, USA). The sequencing was performed on a PacBio® Sequel II sequencer (Pacific Biosciences, USA)." Further details should be added. In particular, the Authors should report the ng and quality (i.e., absorbance values, TapeStation fragment profile) of the extracted DNA, the used PacBio library and sequencing kits, and the PacBio protocol used. Indeed, I suppose it was used the PacBio ultra-low input protocol, and, if so, how was the DNA fragmented? Response: Thank you for pointing this out. We have revised the quality control method, which used the Femto Pulse system after fragmentation. We also added the fragmentation protocol to the method section. As for the profile of the DNA fragments, we have attached the Femto Pulse run report to the supplementary files on Zenodo. We have adjusted the text as follows: “DNA was fragmented using Megaruptor 3 ® (Diagenode, USA) using the Megaruptor 3 Shearing Kit (Diagenode, USA #E07010003) at speed 32 and purified using SMRTbell cleanup beads (Pacific Biosciences, USA #102-158-300). Quality control of obtained DNA was performed using the Femto Pulse System (Agilent, USA) and the Genomic DNA 165 kb Kit (Agilent, USA #FP-1002-0275). The sequencing was performed on a PacBio ® Sequel II sequencer (Pacific Biosciences, USA) using the Sequel II sequencing kit 2.0 (Pacific Biosciences, USA #101-820-200). The DNA size profile and sequencing metrics are available on Zenodo (doi: 10.5281/zenodo.14934254 ).” As for the phylogenetic tree, it seems that it was reconstructed using all 13 protein-coding genes, including those absent in larvaceans. Moreover, the tree was reconstructed from nucleotide, not from amino acid sequences, as is common when long evolutionary distances are analyzed, in order to avoid substitution saturation phenomena. I suggest reconstructing the tree also from amino acid sequences, with/without the protein-genes absent in larvaceans, so considering only positions without gaps and then to compare the obtained trees. Bayesian trees should also be reconstructed to confirm the obtained phylogeny We used nucleotides instead of amino acids because we are still uncertain on how to translate the TGA codon in the Mesochordaeus and Bathochordaeus mitogenomes (Pichon et al., 2020), and worried that it may affect the results. Following the reviewer’s suggestion, we computed trees from amino acid sequences using the same IQ-TREE command line as for nucleotide sequences (as it selects the best models automatically), with or without the proteins that are not found in the appendicularian mitogenomes. In both cases the results were consistent with the nucleotide tree, by placing the new mitogenome in the same clade as O. longicauda , placing the appendicularians as a sister clade to thaliaceans except for D. nationalis , and identifying Ciona as a clade distinct from the other phlebobranchians. Unfortunately, the amino acid-based trees did not recover the expected relation between the three chordate sub-phyla nor between tunicate classes. We added a copy of these computations to our supplemental material for the reviewers and readers convenience, nevertheless we do not provide guarantee that the trees can be useful for other purposes than accountability of our work for this revision. We also did not compute Bayesian trees for this revision due to the lack of expertise in our team. A well formatted "full version" of the tree of Figure 3 should be provided in the Supplementary Material We have added the full version of the tree in the supplementary material under Figure S4. Although out of the scope of the paper, a brief comment on the recovered tunicate phylogeny should be reported We added the following: “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 orders.” The Zenodo Supplementary material contains a folder with the results of IQ-TREE and RaxML analyses: could you please comment if there were differences between the trees obtained with the two methods? The two trees have very similar topologies (identical when folded like in Figure 3), and we chose the IQ-TREE one because it provides bootstrap values that are easy to interpret. For not-users of the IQ-TREE software, I suggest explaining the meaning of the used command-line options and reporting that this software performs ML tree reconstructions We changed the method section’s text to “-T AUTO (run as many CPU threads as possible) --runs 3 (perform 3 runs to assess model convergence) --polytomy (allow unresolved branches) --ufboot 1000 (1000 boostraps with the ultrafast method) -m MFP (automatic selection of the best model for maximum likelihood inference using the ModelFinder Plus method” The Authors should clarify the legend of Figure 3, including the number of genes analysed, the length of the alignment, the method and model used for the tree reconstruction. We changed the figure legend to “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”. We did not indicate the model as we used one partition per gene and the software did not chose the same model for each partition. "Possible explanations for this result are heteroplasmy or that the mitogenome consists of concatemers ": please explain better why you hypothesize the existence of concatemers. "Furthermore, these two haplotypes are supported by homopolymer variants at multiple different loci": a table with all variants between the two haplotypes should be reported in the Supplementary Material, particularly if Figure 2 shows only a portion of these variants. I suggest making available in NCBI also the second assembled haplotype Response: Thank you for this comment. We have reanalyzed the sequences and we could not verify if the observed variation were true variants or caused by sequencing errors in homopolymers and therefore decided to remove the hypothesis of a second haplotype. The one haplotype is the one that is published online. We apologize for this error, and we confirmed that the current available sequence on NCBI is the correct one. We have also modified the text to remove the parts referencing the haplotypes. "As the Oikopleura genus is polyphylic in our phylogenetic analysis and that of others,..." : the Authors should cite the references where the Oikopleura genus was already found as polyphyletic Response: Thank you for this comment. We have added the following references to support this statement: Galt CP, Grober MS, Sykes PF. Taxonomic Correlates of Bioluminescence Among Appendicularians (Urochordata: Larvacea). The Biological Bulletin. 1985 Feb;168(1):125–34. Naville M, Henriet S, Warren I, Sumic S, Reeve M, Volff JN, et al. Massive Changes of Genome Size Driven by Expansions of Non-autonomous Transposable Elements. Current Biology. 2019 Apr;29(7):1161-1168.e6. Masunaga A, Mansfield MJ, Tan Y, Liu AW, Bliznina A, Barzaghi P, et al. The cosmopolitan appendicularian Oikopleura dioica reveals hidden genetic diversity around the globe. Mar Biol. 2022 Nov 27;169(12):157. Plessy C, Mansfield MJ, Bliznina A, Masunaga A, West C, Tan Y, et al. Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species. Genome Res. 2024 Apr 25;34(3):426–40 View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Gissi C. Peer Review Report For: The complete mitogenome of an unidentified Oikopleura species [version 3; peer review: 3 approved, 1 approved with reservations] . 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