The genome sequence of the light-bulb sea squirt, Clavelina lepadiformis (Müller, 1776)

We present a genome assembly from an individual Clavelina lepadiformis (the light-bulb sea squirt; Chordata; Ascidiacea; Aplousobranchia; Clavelinidae). The genome sequence is 210.1 megabases in span. Most of the assembly is scaffolded into 9 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 14.48 kilobases in length.


Background
Clavelina lepadiformis is a colonial (budding) ascidian (sea squirt) of the Order Aplousobranchia.The colony is formed by the repeated budding of zooids ('individual' sea squirt bodies) starting with the founding zooid formed by the metamorphosis of a swimming larva.The zooids of a colony remain linked basally; they are up to 45 mm tall, elongate in shape with the inhalant and exhalant siphons at the upper end, and are largely transparent, although the branchial basket and siphon rims are marked with white, yellow or pink lines and circles.This pigmentation accounts for the common name 'Light-bulb sea squirt', referring to the fancied resemblance of colonies to clusters of incandescent light bulbs with glowing filaments.As is typical of colonial ascidians, embryos are brooded until released as larvae.
C. lepadiformis grows on solid surfaces from the low shore down to c. 100 m, and is frequent in harbours.It ranges from southern Scandinavia to the Mediterranean, and is present as an introduced species in the Azores, South Africa, South Korea, Japan and on the US east coast (Fofonoff et al., 2018;Nishikawa & Namikawa, 2018).Molecular (COI sequence) evidence identified two distinct clades in the western Mediterranean, one in natural rocky habitats on the open coast and one in harbours and marinas (Turon et al., 2003); the form in harbours and marinas is very closely related to the clade found in both habitats in the European Atlantic, and seems to represent a recent anthropogenic introduction from the Atlantic.The clades could not be distinguished morphologically.However, de Caralt et al. (2002) found clear differences in the phenology of harbour and open rocky littoral populations of C. lepadiformis in the western Mediterranean.The harbour population brooded larvae from November to June with several gonadal cycles over that time.In contrast, the open-coast colonies brooded larvae for only 2 to 3 months in winter-spring, with a single gonadal cycle per year.The zooids of open coast colonies regressed over the summer, such summer inactivity (aestivation) being a pattern seen in a range of shallow-water marine invertebrates in the Mediterranean.
The zooids of C. lepadiformis appear particularly clean and transparent, lacking fouling by other sessile species.This may in large part reflect chemical defence against colonisation of their surface.Crude aqueous extracts of C. intestinalis tissue showed strong toxicity to a range of invertebrate larvae, greater than the effect of extracts from three other tunicate species (Teo & Ryland, 1995).C. intestinalis produces a range of cytotoxic alkaloids: lepadins (Casertano et al., 2022;Kubanek et al., 1995;Steffan, 1991); lepadiformine 1 (Biard et al., 1994);and villatamines (Kubanek et al., 1995).Several of these substances were also found in the flatworm Prostheceraeus villatus, which feeds on C. lepadiformis (Kubanek et al., 1995).

Genome sequence report
The genome was sequenced from Clavelina lepadiformis (Figure 1) collected from Queen Anne's Battery Marina visitors ' pontoon,Plymouth,UK (50.36,.A total of 117-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 60 missing joins or mis-joins and removed 9 haplotypic duplications, reducing the assembly length by 5.78% and the scaffold number by 25.9%, and increasing the scaffold N50 by 35.45%. The final assembly has a total length of 210.1 Mb in 102 sequence scaffolds with a scaffold N50 of 25.1 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (98.62%) of the assembly sequence was assigned to 9 chromosomal-level scaffolds.Karyotyping information was used from Colombera (1971).Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
Specimens of Clavelina lepadiformis were collected by hand from submerged rope from the Queen Anne's Battery Marina visitors' pontoon, Plymouth, Devon, UK (latitude 50.36, longi- High molecular weight (HMW) DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI),    beads to sample to eliminate shorter fragments and concentrate the DNA.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.
Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from kaClaLepa9 using the Automated MagMax™ mirVana protocol (https://dx.doi.org/10.17504/protocols.io.6qpvr36n3vmk/v1).The RNA concentration was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
All wet lab protocols developed by the Tree of Life laboratory are publicly available on protocols.io:https://dx.doi.org/10.17504/protocols.io.8epv5xxy6g1b/v1.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' Table 3. Software tools: versions and sources.

Software tool Version
instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from tissue of kaClaLepa3 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021).Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Table 3 contains a list of relevant software tool versions and sources.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material
The reported assembly appears to be of high quality and the assembly and analysis methods used are adequate.This article reports the first high-quality genome of an aplousobranch ascidian.It is thus an important milestone.
The authors are encouraged to indicate in the methods section the parameters used when running the various pieces of assembly and analysis software.They should also cite previous mitochondrial genome sequencing efforts [1], [2].
Our main concern relates to the level of confidence that can be given to the species assignment of the sequenced individuals.A photograph is not sufficient, and the authors should describe in greater detail how they convinced themselves that they are reporting a Clavelina lepadiformis genome.There is at least one case of innacurate assignment of a tunicate genome to a species [3], a study to which J. Bishop participated.This manuscript reports the sequencing of the genome of a colonial sea squirt.Given the relatively small number of high-quality tunicate genome this is a nice resource.Overall, the manuscript is clearly written, and procedures appropriately explained.I have two main comments: The authors do not comment on the expected chromosome number in comparison with other tunicates and potentially also in line with previous cytogenetic studies of tunicates.Moreover, tunicates appear to exhibit a particular type of chromosomal architecture (type I according to Hoencamp et al. 2021) in which centromeres and telomeres contact each other with relatively limited contacts elsewhere (as seen with the cross pattern in the HiC map) ○ which could be a problem for HiC scaffolding.I feel as previously commented for other DToL reports, that the procedure for the HiC scaffolding is insufficiently detailed compared to the ones for DNA extraction and sequencing.Which tissue was used, how was it fixed and/or pulverized?Similarly, YAHS is mentioned but not the details of processing (YAHS takes a BAM file, which should be preprocessed using a tool like pair tools).I think it would really be crucial to release some protocols as the DToL did for the DNA extraction and library making.Reviewer Expertise: genomics, phylogeny, transposable elements 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.

Figure 2 .
Figure 2. Genome assembly of Clavelina lepadiformis, kaClaLepa1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 210,089,204 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (28,789,166 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (25,070,715 and 19,509,797 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the metazoa_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Clavelina%20lepadiformis/dataset/CANQJE01/snail.

Figure 5 .
Figure 5. Genome assembly of Clavelina lepadiformis, kaClaLepa1.1:Hi-C contact map of the kaClaLepa1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=X_VH7rrISNydJ-6yEBmyFw.

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information necessary to reproduce the work.Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.

Table 2 . Chromosomal pseudomolecules in the genome assembly of Clavelina lepadiformis, kaClaLepa1. INSDC accession Chromosome Length (Mb) GC%
Clavelina lepadiformis (lightbulb sea squirt).Accession number PRJEB57668; https://identifiers.org/ena.embl/PRJEB57668(WellcomeSangerInstitute,2022).The genome sequence is released openly for reuse.The Clavelina lepadiformis genome sequencing initiative is part of the Darwin Tree of Life (DToL) project.All raw sequence data and the assembly have been deposited in INSDC databases.The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute.Raw data and assembly accession identifiers are reported in Table1.Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.4893703.Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory Team are listed here: https://doi.org/10.5281/zenodo.10066175.

Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Partly Competing Interests
Comparison of the mitochondrial genome of the sequenced individual to two independently sequenced C. lepadiformis mitochondrial genomes (see above) could strengthen the authors' claim.genomes on different levels of analysis.Mol Phylogenet Evol.2010; 55 (3): 860-70 PubMed Abstract | Publisher Full Text 3. Viard F, Roby C, Turon X, Bouchemousse S, et al.: Cryptic Diversity and Database Errors Challenge Non-indigenous Species Surveys: An Illustration With Botrylloides spp. in the English Channel and Mediterranean Sea.Frontiers in Marine Science.2019; 6. Publisher Full Text : No competing interests were disclosed.Reviewer Expertise: Tunicate biology; Aniseed database 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.
https://doi.org/10.21956/wellcomeopenres.22606.r71068© 2024 Marlétaz F. 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.Ferdinand Marlétaz Centre for Life's Origin & Evolution, Department of Ecology, Evolution & Environment, University College London, London, UK