The genome sequence of the Downland Villa bee-fly, Villa cingulata (Meigen, 1804)

We present a genome assembly from an individual male Villa cingulata (the Downland Villa bee-fly; Arthropoda; Insecta; Diptera; Bombyliidae). The genome sequence is 412.6 megabases in span. Most of the assembly is scaffolded into 10 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 22.43 kilobases in length.

A photographic identification guide to the UK Villa spp.has been compiled for the Soldierflies and Allies Recording Scheme (Harvey, 2022), and a detailed description and key is provided in Stubbs and Drake (2014).All three UK species are medium-to-large flies with a characteristic overall appearance of a fairly broad, flat abdomen, clear wings with dark bases, and varying degrees and arrangements of golden hairs and dark scales (Harvey, 2022;Stubbs & Drake, 2014).Differences in abdominal banding and extent of darkened wings are particularly useful in separating the species, with all three exhibiting varying degrees of sexual dimorphism.
While the three UK Villa species are very similar in appearance, clear differences in the associated habitats and current distributions of each are currently observed, with V. cingulata showing a preference for inland grasslands.Until quite recently V. cingulata was very predominantly an insect of calcareous meadows and chalk downland with short turf and/or minimal vegetation cover (Harvey, 2022;Stubbs & Drake, 2014).However, since 2013 it has also been found in neutral hay meadow habitats (e.g.Chandler, 2015) and in the last few years has also colonised some acid grasslands, e.g. in the New Forest (Soldierflies and Allies Recording Scheme, 2023).Some of these recent sites have tall grassland swards, but paths and patches of bare ground are usually present as well.
Historically recorded, albeit in very low numbers, from several southern UK counties, where it was described as occurring on shrubby, chalky slopes with tall vegetation (Drake, 2017;Stubbs & Drake, 2014;Verrall, 1909).A period of about 60 years with no records of V. cingulata led to concerns that the species might be extinct in the UK; however, in 2000, the species was rediscovered in the Cotswolds, with additional records from nearby areas subsequently reported (Stubbs & Drake (2014) and references therein).The threat status for V. cingulata was assessed as "Least Concern" by Drake (2017), and at that time it was given a rarity status of Nationally Rare.However, that assessment was based largely on data prior to 2016, and since then the number of records per year has greatly increased, as has the number of occupied ten-kilometre squares, and the species would no longer qualify as Nationally Rare based on current data.Since 2000 it has been recorded from 21 vice-counties in southern England and south-east Wales (Soldierflies and Allies Recording Scheme, 2023).Adults of V. cingulata are found in summer, from June to August (Harvey, 2022;Stubbs & Drake, 2014).Adult flies can be observed visiting umbellifers (Fam.Apiaceae), such as Wild Parsnip and Hogweed, while females will often rest on warm ground, gathering dust on their abdomens, which subsequently adheres to their eggs to enable effective dispersal via flicking (Falck, 2009).The host of V. cingulata in the UK remains unknown (e.g.Stubbs & Drake, 2014).Males have been observed to be territorial/defensive to conspecific males (Falck, 2009).
The specimen was collected in July 2021.On the continent V. cingulata is found throughout Europe and the Middle East (Evenhuis & Greathead, 2015).
The genome of V. cingulata 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 Villa cingulata, based on one male specimen from Holtspur Bottom Nature Reserve, UK.

Genome sequence report
The genome was sequenced from one male Villa cingulata (Figure 1) collected from Holtspur Bottom Nature reserve, England, UK (51.61, -0.67).A total of 54-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 removed contaminating scaffolds and corrected 5 missing joins or mis-joins and removed one haplotypic duplication, reducing the assembly length by 47.54% and the scaffold number by 99.35%, and increasing the scaffold N50 by 221.12%.
The final assembly has a total length of 412.6 Mb in 45 sequence scaffolds with a scaffold N50 of 45.3 Mb (Table 1).
The snailplot in Figure 2 represents 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 (99.29%) of the assembly sequence was assigned to 10 chromosomal-level scaffolds, representing 8 autosomes and the X and Y sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5, Table 2).The Y chromosome is not scaffolded due  to lack of Hi-C signal, as the Hi-C data appears to be from a female sample whereas the PacBio data used for de novo assembly is from a male.(Figure 2-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.The estimated Quality Value (QV) of the final assembly is 66.9 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 completeness of 94.5% (single = 93.5%,duplicated = 1.0%), using the diptera_odb10 reference set (n = 3,285).
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/2753613.DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The idVilCing2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Thorax tissue 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 head and thorax tissue of idVilCing1 using the Arima2 kit and sequenced on the HiSeq X Ten 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) andPretext (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.

Software tool Version
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

Luciano Cosme
Yale University, New Haven, CT, USA This manuscript presents the genome assembly of the Villa cingulata, commonly known as the Downland Villa bee-fly.The genome sequence is 412.6 megabases, scaffolded into 10 chromosomal pseudomolecules, including the X and Y sex chromosomes.The mitochondrial genome has also been assembled, with a length of 22.43 kilobases.

Replication Concerns:
The manuscript lacks sufficient detail on how to replicate the findings using the raw data.Although the authors mention the sequencing methods and tools used, they do not provide a detailed workflow or pipeline for processing the raw data to achieve the same genome assembly results.
Recommendation for Authors: Include a supplementary section or a dedicated repository that outlines a step-by-step protocol for processing the raw data, including all software parameters, commands, and scripts used.This will enable other researchers to replicate the analysis and verify the results.A detailed methods section will enhance transparency and reproducibility, which are crucial for scientific research.Does the project cover genome annotation?
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? No
Are the datasets clearly presented in a useable and accessible format?Partly

INRAE, Castanet Tolosan, France
The article presents read sets and a principal genome assembly for Villa cingulata species.The assembly contigs were built with hifiasm which is the most commonly used assembler for PacBio HiFi reads.The contigs were scaffolded using Hi-C reads with YaHS.The assembly is of high quality.The authors should check the presence of telomers at both ends of their chromosomes.
Is the rationale for creating the dataset(s) clearly described?

Yiyuan Li
Ningbo University, Ningbo, Zhejiang, China In this paper, the authors assembled the genome of a male Downland Villa bee-fly, Villa cingulata.They assembled the 10 chromosomes including the X and Y chromosomes.The genome has been manually curated with high quality.The authors have also included the picture of the specimen they sequenced with detailed information on the collecting time and location.The manuscript is well written with very useful details of species and its genome.I only have two minor comments: 1. "the three UK Villa species are very similar in appearance" Would it be possible to distinguish the three species using the COI gene?If so, as the authors already assembled and annotated its mitochondrial genome, it would be helpful to search for the COI gene on the Barcode of Life Database (https://www.boldsystems.org/)or NCBI NT database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm the taxonomy of the sequenced specimen.
2. Hifiasm has a few parameters during assembly.It would be helpful to also include the parameters they use for assembling.For example, did the authors use --primary and HiC options (--h1 and --h2) for their assembly?
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: Comparative 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.

Figure 2 .
Figure 2. Genome assembly of Villa cingulata, idVilCing2.1: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 412,610,433 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 (65,149,795 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (45,252,737 and 35,763,846 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/idVilCing2.1/ dataset/CATOAS01/snail.

Figure 3 .
Figure 3. Genome assembly of Villa cingulata, idVilCing2.1: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/idVilCing2.1/dataset/CATOAS01/blob.

Figure 4 .
Figure 4. Genome assembly of Villa cingulata, idVilCing2.1: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/idVilCing2.1/dataset/CATOAS01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Villa cingulata, idVilCing2.1:Hi-C contact map of the idVilCing2.1 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=SXAgkaluToGo3fqjAOJUjw.

Table 3
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

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.