The genome sequence of a drosophilid fruit fly, Chymomyza fuscimana (Drosophilidae) (Zetterstedt, 1838)

We present a genome assembly from an individual male Chymomyza fuscimana (drosophilid fruit fly; Arthropoda; Insecta; Diptera; Drosophilidae). The genome sequence is 338.0 megabases in span. Most of the assembly is scaffolded into 5 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 16.47 kilobases in length.


Background
Chymomyza fuscimana (Zetterstedt, 1838) is a medium sized (ca.3.0-3.5 mm) drosophilid 'fruit fly' with striking red eyes and contrasting yellowish-brown and black body colouration (Figure 1A).It is very distantly related to the laboratory model Drosophila melanogaster within the subfamily Drosophilinae (Finet et al., 2021), and is one of six species of Chymomyza recorded from Britain and Ireland (Chandler, 2023).It is morphologically close to Chymomyza distincta, but without dissection can still be distinguished by the absence (in male fuscimana) of long white setulae on the inner side of the procoxa (Bächli et al., 2004).
As in many other species of Chymomyza, the larvae of C. fuscimana feed in fermenting sap under the bark of fallen and damaged trees, including both broad-leaved and coniferous species (Burla, 1995;Burla, 1997;Rotheray & Robertson, 1998).The adults can often be seen on freshly exposed timber surfaces, including newly fallen trees, broken branches, and sawn logs and stumps (Figure 1B and C) -where males display and compete by wing-waving and raising their front legs (Burla, 1995;Burla, 1997;Chandler, 1978;Perry, 2019).
Chymomyza fuscimana is broadly distributed across the northern Palearctic, from Ireland in the west to the far east of Russia, and from Greece in the south to the north of Finland (Bächli, 2023).Although relatively few records are available in the UK, adults appear to be most active between May and October (GBIF Secretariat, 2023).The species is not reported to be threatened, and it seems likely that the scarcity of UK records reflects the challenge of identification (e.g.Basden, 1961), the failure of these flies to come to baits, and/or the transience and accessibility of freshly exposed timber (Burla, 1995;Chandler, 1978).
Here we present a chromosomally complete genome sequence for Chymomyza fuscimana, derived from the DNA and RNA of three adult male flies collected from a fallen oak tree on the Penns in the Rocks estate, East Sussex, as part of the Darwin Tree of Life Project.This genome sequence will help to resolve relationships among the Drosophilidae and will further build on the value of this family as a model clade for comparative genomics and molecular evolution.This project is a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.

Genome sequence report
The genome was sequenced from one male Chymomyza fuscimana (Figure 1) collected from Penns in the Rocks Estate, East Sussex, UK (51.09, 0.17).A total of 65-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 69 missing joins or mis-joins and removed 5 haplotypic duplications, reducing the assembly length by 0.55 % and the scaffold number by 76.81%, and increasing the scaffold N50 by 60.81%.
The final assembly has a total length of 338.0 Mb in 15 sequence scaffolds with a scaffold N50 of 104.9 Mb (Table 1).A summary of the assembly statistics is shown in Figure 2, 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 (99.78%) of the assembly sequence was assigned to 5 chromosomal-level scaffolds, representing 3 autosomes and the X and Y sex chromosomes.Chromosomescale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).The X and Y chromosome were identified by half read coverage, and chromosome 3 might be the equivalent of a Drosophila dot chromosome.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.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/1692350.

Sample acquisition and nucleic acid extraction
Chymomyza fuscimana specimens were collected from the broken surface of a fallen oak in Penns in the Rocks Estate, East Sussex, UK (latitude 51.09, longitude 0.17) on 2021-09-07.The specimens were collected and identified by Darren Obbard (University of Edinburgh) and frozen from live (-80°C).
The specimen used for genome sequencing was specimen ID SAN00002034, ToLID idChyFusc2), while the specimen used for Hi-C scaffolding was specimen ID SAN00002033, ToLID idChyFusc1.A third specimen ID SAN00002035, ToLID idChyFusc3, was used to generate poly-A selected RNAseq data.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The idChyFusc2 sample was weighed and dissected on dry ice.Tissue from the whole organism was disrupted using a Nippi Powermasher fitted with a BioMasher pestle.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30.Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample.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.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from the whole organism tissue of idChyFusc1 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 as described previously (Howe et al., 2021).Manual curation was performed using 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.
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This work was done using Nextflow (Di Tommaso et al., 2017)   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

Bernard Moussian
University of Tübingen, Tübingen, Germany The genome of a male of the drosophila fly Chymomyza fuscimana presented.The sequence is of high quality.For instance the Busco value is 98,8%.Only little unclear sequence information persists.For more clarity for the reader, I have two points: -in the abstract, the author writes "most of the assembly...".Information on the portion not being assembled should be provided.
-in the genome sequence report, the author states that <"5 haplotypic duplications" were removed: was this a technical or a biological problem?
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? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Insect 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.

Figure 1 .
Figure 1.A: Male (above) and female (below) Chymomyza fuscimana presented with a 3 mm scale bar.B: The fallen oak from which the sequenced individuals were collected (Penns in the Rocks estate, East Sussex, England; 51.093N,0.1698E).C: Male fly on a freshly broken surface of the oak.

Figure 2 .
Figure 2. Genome assembly of Chymomyza fuscimana, idChyFusc2.1: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 337,977,858 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 (146,831,255 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (104,850,594 and 65,373,579 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/Chymomyza%20fuscimana/dataset/CATLJI01/snail.

Figure 5 .
Figure 5. Genome assembly of Chymomyza fuscimana, idChyFusc2.1:Hi-C contact map of the idChyFusc2.1 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=cxKt_xm7TFqYAi236-3nRg.

Table 3
contains a list of relevant software tool versions and sources.

Table 3 . Software tools: versions and sources. Software tool Version Open Peer Review Current Peer Review Status: Version 1
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.
Tyler Scott AliotoFundacion Centro Nacional de Analisi Genomico, Barcelona, Catalonia, SpainThe genome note submitted by Obbard et al. reports the genome sequence of the drosophilid fruit fly, Chymomyza fuscimana.The assembly is chromosome-scale and of high quality, meeting the minimum standards recommended by the Earth Biogenome Project.All protocols are appropriate and well-documented.All data conforms with FAIR principles, with read data and assemblies being available in the ENA.Blobtoolkit figures are interactive.Additional QC data are provided at https://tolqc.cog.sanger.ac.uk/, supplementing the figures published in the data note.

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? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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.