The genome sequence of the parsley Cheilosia, Cheilosia pagana (Meigen, 1822)

We present a genome assembly from an individual female Cheilosia pagana (the parsley Cheilosia; Arthropoda; Insecta; Diptera; Syrphidae). The genome sequence is 354.1 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 16.76 kilobases in length.


Background
The parsley Cheilosia, Cheilosia pagana, is a small (wing length 4.75-8.5 mm) Holarctic hoverfly.Individuals of C. pagana are a distinct shiny black colour and possess orange antennae above their hairless eyes.Variation between spring and summer broods of this species is often expressed in body hair and should be noted when attempting identification (Stubbs, 2021).Female antennae are significantly larger than those of males, providing a useful identification feature for this cryptic species (Ball & Morris, 2013).C. pagana is both common and widespread, partly driven by its multiple broods occurring from Spring through to late Summer (Ball & Morris, 2013).
The preferred environment of C. pagana is broadly forest with open ground; however, they also show an affinity to anthropogenic landscapes including unimproved grassland, hedgerows, and roadsides (Speight, 2017).As adults they frequent yellow composites, white umbellifers, and willow during their flight periods (May/June and July/September) and can be seen as early as March in southern Europe (Speight, 2017).
The larval morphology of C. pagana is described by Rotheray (1990) but complete larval developmental stages are yet undescribed.Rotheray and Gilbert (1999) conclude that the larvae are most likely mycophagous, due to Stubbs (1980) providing an account of larvae feeding on the rotting roots of Cow Parsley, Anthriscus sylvestris.With Cheilosia being one of the most diverse and species-rich hoverfly genera, molecular investigation has been used to clarify phylogeny and taxonomy (Ståhls & Nyblom, 2000).We hope this high-quality chromosomally complete genome sequence of Cheilosia pagana, generated as part of the Darwin Tree of Life project, will contribute to the growing database of Cheilosia genomes and used to progress in understanding the biology and ecology of this species.

Genome sequence report
The genome was sequenced from one female Cheilosia pagana (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.34).A total of 52-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 10 missing joins or mis-joins, reducing the scaffold number by 9.09%.
The final assembly has a total length of 354.1 Mb in 10 sequence scaffolds with a scaffold N50 of 63.9 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 (99.95%) of the assembly sequence was assigned to 6 chromosomal-level scaffolds, representing 5 autosomes and the X sex chromosome.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
The specimen used for DNA sequencing, a female Cheilosia pagana (specimen ID Ox000866, ToLID idChePaga1), was netted in Wytham Woods, Oxfordshire, UK (latitude 51.77, longitude -1.34) on 2020-08-20.The specimen used for Hi-C sequencing, also a female, (specimen ID Ox001734, ToLID idChePaga2) was netted in the same location on 2021-07-17.Both specimens were collected and identified by Steven Falk (independent researcher) and preserved on dry ice.
The workflow for high molecular weight (HMW) DNA extraction at the WSI includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the idChePaga1  sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue from the whole organism was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).HMW DNA was extracted using the Automated MagAttract v1 protocol (Oatley et al., 2023).The 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

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 instrument.Hi-C data were also generated from whole organism tissue of idChePaga2 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 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 In this assembly and some other Cheilosia assemblies as well, chromosome 1 is noticeably longer than the others.Hi-C data shows a repeated region near its center.Can the authors comment on whether they think it is a centromere, and if the one of chr1 is much larger than the others, or if centromeres are just missing from the other assembled chromosomes?
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: Pairwise genome alignment 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.

Background
It might be adequate to add the sex determination system of this species explicitly (in one sentence) within the first paragraph.I understood this species follows an XY sex-determination system, like other congeneric species (Crowley et al., 2023).
I would add some examples of pollinated plants.It might be interesting to link with The Database of Pollinator Interactions (DoPI;https://www.sussex.ac.uk/lifesci/ebe/dopi/), which contains some plants pollinated by this species.
I would emphasize a bit more that this species participates in pollinator webs.As it is commented on the Center for Plant Conservation website (see https://saveplants.org/pollinator-search/) we cannot conserve plants without knowledge about (and conservation of) their pollinators.

Methods
To increase reproducibility, I highly recommend creating a Supplementary Material or a GitHub page containing all the commands with the selected parameters/options.This will be very useful for the whole scientific community.
I strongly recommend integrating the following sentence better within the main text: "Table 3 contains a list of relevant software tool versions and sources."It could be at the beginning of the "Genome assembly, curation and evaluation" subsection.

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?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Population genomics, Conservation genomics, Bioinformatics, Aquaculture 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 Cheilosia pagana, idChePaga1.1: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 354,125,056 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 (118,147,124 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (63,862,789 and 52,569,716 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/CAKZFA01/dataset/CAKZFA01/snail.

Figure 3 .
Figure 3. Genome assembly of Cheilosia pagana, idChePaga1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAKZFA01/dataset/CAKZFA01/blob.

Figure 4 .
Figure 4. Genome assembly of Cheilosia pagana, idChePaga1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAKZFA01/dataset/CAKZFA01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Cheilosia pagana, idChePaga1.1:Hi-C contact map of the idChePaga1.1 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=E2sLf6zXS7mwEXMoSQEXQw.

Table 1 :
Proposed standards and metrics for defining genome assembly quality" from(Rhie et al., 2021).