The genome sequence of a kelp fly, Coelopa pilipes Haliday, 1838

We present a genome assembly from an individual male Coelopa pilipes (kelp fly; Arthropoda; Insecta; Diptera; Coelopidae). The genome sequence is 263.0 megabases in span. Most of the assembly is scaffolded into 7 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 16.86 kilobases in length.


Background
Coelopa pilipes belongs to the Coelopidae, a group of acalyptrate flies commonly named "kelp flies" or "seaweed flies" as they spend most of their life cycle on accumulations of decaying marine algae along the seashore (Dobson, 1974b;Egglishaw, 1960).By feeding on the microbial slime and marine algae at larval stage (Berdan et al., 2023;Cullen et al., 1987), Coelopidae are important ecological actors of the beach ecosystem by contributing to an efficient and rapid decomposition of stranded seaweeds (Marsh, 2008).The family is most diverse in Australia and New Zealand.The two British species, C. pilipes and C. frigida are not closely related (Meier & Wiegmann, 2002).
Coelopa pilipes are dark-coloured 'true' flies measuring between 4.5 and 7.5 mm (Egglishaw, 1960).The head is smaller than the thorax forming a typical triangle shape (Figure 1) distinguishing them from other seashore flies.Adults have small eyes, short antennae and short but strong legs.C. pilipes frequently co-occur with Coelopa frigida (Dobson, 1974a).Those two species can be distinguished by a close examination of hairs on the body and legs, with C. frigida being bristlier and C. pilipes hairier.C. pilipes is typically also darker in coloration, appearing black, while C. frigida varies from tan to dark brown.While the differences are more easily observed in males, females can be distinguished by their tibiae.The tibia of C. frigida bears a preapical bristle, which in C. pilipes is only slightly developed and is obscured by dense hairs (Egglishaw, 1960).Larvae are whitish and measure up to 15 mm but they are harder to distinguish between species (for a full description of all stages see Egglishaw (1960).
C. pilipes is very abundant throughout the year all-around Britain (Dobson, 1974b;Edward et al., 2007), although historically less common in the north and more restricted to persistent wrack beds than C. frigida.It is negatively impacted by beach grooming which consists in removing stranded seaweeds during the tourist season (Griffin et al., 2018).C. pilipes lives from the Atlantic coast of France (Bay of Biscay) up to Southern Scandinavia.Interestingly, the range of C. pilipes has been expanding towards the North over the last decades.Comparing records from 1995 to records in 2005, Edward et al. (2007) showed that C. pilipes colonised the western and northern Isles of Scotland and expanded in southern Sweden over a decade.Recent records suggest that this species is now found up to Southern Norway (https://www.gbif.org).This recent and rapid Northwards expansion is hypothesized to be associated with milder winter and warmer temperature since C. pilipes is known to prefer higher temperatures than its competitor C. frigida (Edward & Gilburn, 2007;Phillips et al., 1995).
The biology and life-cycle of Coelopa flies are well-described by Egglishaw (1960) andDobson (1974b).C. pilipes breeds all year round with shorter generations in the warmer season.It lays eggs individually or in small clutches of less than 10 eggs on deposits of different species of algae (mostly Fucaceae).The three larval instars occur in the decomposing seaweeds before the formation of a puparium in drier regions of the wrackbed.The duration of the life cycle, from egg to adult, last about 15 to 25 days at 22 °C.Adults may live a few weeks, depending on temperature, and are known to disperse across beaches.Seaweed flies are indeed distributed in meta-populations, along the coast, with patches of suitable habitat separated by stretches of shoreline without seaweed deposits.
Scientific studies focusing on C. pilipes have addressed its coexistence with C. frigida, showing some competition and an impact of density on adult size (Butlin et al., 1984;Phillips et al., 1995).The relative frequencies of each species vary across the year and across locations, possibly depending on seasonal variation in the temperature and the duration of available habitat (Dobson, 1974a;Edward et al., 2007;Egglishaw, 1960).C. pilipes has notably a longer development time than C. frigida but a higher survival at high temperatures (Phillips et al., 1995).Experiments on sexual behaviour revealed intense sexual conflict in Coelopa sp.including C. pilipes, characterized by a pre-mating struggle and important harassment behaviour by males (Dunn et al., 1999;Edward & Gilburn, 2007).More recent research has also assessed the potential of seaweed flies for animal feeding, showing that C. pilipes has a high lipid and protein content but also toxins from algae (Biancarosa et al., 2018).The genome of Coelopa pilipes was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.Here we present a chromosomally complete genome sequence for Coelopa pilipes, based on one male larva from Whitburn, Sunderland, UK.
The assembly of a chromosome-level high-quality reference for C. pilipes will offer new resources for in-depth genomic studies on this species of interest to investigate the dynamic of metapopulations and their connectivity, the architecture of sex chromosomes in a species with high sexual conflict, and genome evolution in the Coelopidae.In particular, C. frigida is known for multiple chromosomal rearrangements (Aziz, 1975;Mérot et al., 2021), while none have been reported yet in C. pilipes.On-going re-sequencing project will make use of this new reference genome and further shed light on the population genomics and evolution of C. pilipes.

Genome sequence report
The genome was sequenced from one male Coelopa pilipes (Figure 1) collected from Whitburn, UK (54.94,.A total of 76-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 109 missing joins or mis-joins, reducing the scaffold number by 68.03%, and increasing the scaffold N50 by 156.04%. The final assembly has a total length of 263.0 Mb in 38 sequence scaffolds with a scaffold N50 of 47.3 Mb (Table 1).The snail plot 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 (99.22%) of the assembly sequence was assigned to 7 chromosomal-level scaffolds, representing 5 autosomes and the X and Y sex chromosomes.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
Three Coelopa pilipes specimens were used for sequencing in this project: used for genome sequencing (specimen ID SAN00001869, ToLID idCoePili4), for Hi-C sequencing (specimen ID SAN00001866, ToLID idCoePili1) and RNA sequencing (specimen ID SAN00001874, ToLID idCoePili9).The specimens were collected from Whitburn, Sunderland, UK (latitude 54.94, longitude -1.36) on 2021-08-20 by R. Butlin.The larvae were sampled by hand with rotting seaweed and kept at ~12C in a natural day/night cycle until adult emergence (10 days to 3 weeks).Adults were sexed and identified by R. Butlin (University of Sheffield) and frozen at -80°C.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and cleanup.In sample preparation, the idCoePili4 sample was weighed and dissected on dry ice (Jay et al., 2023).Whole organism tissue was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).HMW DNA was extracted using the Automated MagAttract v1 protocol (Sheerin et al., 2023).DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB 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 the whole organism of adult idCoePili9 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' 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 whole organism tissue of idCoePili1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.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 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

Halide Nihal Açıkgöz
Ankara University, Ankara, Turkey Dear Editor, The title clearly indicates the scope of the study and presents it in an engaging manner.
The abstract summarizes the study's purpose, methods, findings, and significance.It clearly mentions the genome size, techniques used, and key results.
The introduction provides sufficient background information on Coelopa pilipes.The ecological importance of the species and the study's objectives are well highlighted.
The relevant literature is well summarized, and the importance of the study is justified.MethodsThe sampling methods and DNA/RNA extraction protocols are described in detail and clearly.
The advanced sequencing techniques used and the data analysis process are comprehensively explained, which is crucial for the reproducibility of the study.Results Details on the genome assembly and its quality (such as N50 and BUSCO scores) are thoroughly presented.The reliability and accuracy of the data are well supported.
The assembly and analysis of the mitochondrial genome are consistent with the overall findings and well integrated into the study.Discussion and Conclusions The results are compared with other studies in the literature and meaningfully interpreted.
The manuscript is well-written and logically structured.Technical details and results are presented clearly and comprehensively.
Is the rationale for creating the dataset(s) clearly described?Yes

Figure 2 .
Figure 2. Genome assembly of Coelopa pilipes, idCoePili4.1: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 262,969,196 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 (74,779,852 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (47,252,759 and 37,343,302 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 diptera_ odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANDXB01/dataset/CANDXB01/snail.
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.

Figure 3 .
Figure 3. Genome assembly of Coelopa pilipes, idCoePili4.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANDXB01/dataset/CANDXB01/blob.

Figure 4 .
Figure 4. Genome assembly of Coelopa pilipes, idCoePili4.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANDXB01/dataset/CANDXB01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Coelopa pilipes, idCoePili4.1:Hi-C contact map of the idCoePili4.1 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=bfvSTvUZRhKizeP7K5XdHg.
Are 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.Reviewer Expertise: Forensic, Biology, Forensic Entomology, DNA, mtDNA 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.Reviewer Report 31 July 2024 https://doi.org/10.21956/wellcomeopenres.23469.r90056© 2024 Cosme L. 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.Luciano CosmeYale University, New Haven, CT, USA Researchers presented a high-quality genome assembly of the kelp fly Coelopa pilipes.The genome spans 263.0 megabases, scaffolded into 7 chromosomal pseudomolecules, including X and Y sex chromosomes, and a 16.86-kilobase mitochondrial genome.This assembly, part of the Darwin Tree of Life Project, provides valuable resources for studying metapopulation dynamics, sex chromosome architecture, and genome evolution.The study also highlights the ecological importance of kelp flies in beach ecosystems and their role in decomposing marine algae.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

Table 3 . Software tools: versions and sources.
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. genome

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