The genome sequence of a drosophilid fruit fly, Hirtodrosophila cameraria (Haliday, 1833)

We present a genome assembly from an individual female Hirtodrosophila cameraria (a drosophilid fruit fly; Arthropoda; Insecta; Diptera; Drosophilidae). The genome sequence is 214.5 megabases in span. Most of the assembly is scaffolded into 4 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.94 kilobases in length.


Background
Hirtodrosophila cameraria (Haliday, 1833) is a medium sized (2.5-3.5 mm) pale greyish-brown drosophilid 'fruit fly' (Figure 1A and B), distantly related to the laboratory model Drosophila melanogaster.It is one of three British and Irish species currently classified in the genus Hirtodrosophila (Chandler, 2023).Originally placed in the genus Drosophila by Haliday, it was moved to the newly-elevated (sub-)genus Hirtodrosophila (Grimaldi, 1990) by Bächli et al. (2004).However, relationships between Drosophila and Hirtodrosophila remain unclear, with the genera being paraphyletic with respect to each other and Zygothrica and Mycodrosophila, and with no single diagnostic morphological character available to separate them (Bächli et al., 2004;Finet et al., 2021;Gautério et al., 2020;Grimaldi, 1990).Nevertheless, H. cameraria itself is easy to identify among other similar British and Irish drosophilids, having plumose aristae with a single ventral branch behind the terminal fork and lacking a pre-apical seta on the mesotibia (Bächli et al., 2004).
Like its close relatives, H. cameraria is a specialist fungus breeder and in the UK the adults are easily collected or reared from toadstools and bracket fungi, including Phallus impudicus, Lactarius quietus, Amanita rubescens, Russula cyanorantha and Paxillus (Charlesworth & Shorrocks, 1980;Gautério et al., 2020;Shorrocks & Charlesworth, 1980).Adults have also been reported in association with the violet helleborine orchid Epipactis purpurata (Roper, 2013), perhaps as a result of pheromones released by the plant to attract pollinators (Policha et al., 2019).Hirtodrosophila cameraria is broadly distributed in wooded areas across Europe, from the extreme north of Sweden, to Turkey in the east, and the Canary islands in the south west (Bächli, 2023).In the UK, breeding is likely to be focused of toadstool flushes in the late summer and early autumn (Charlesworth & Shorrocks, 1980), but adults can be caught at any time of year (Basden, 1954), and are quite commonly recorded in the months of May to November (GBIF Secretariat, 2022).It is not thought to be threatened and is in fact likely under-collected compared to its human-commensal relatives, as it rarely comes to fruit bait (Basden, 1954).
Here we present a chromosomally complete genome sequence for Hirtodrosophila cameraria, derived from the DNA of two female specimens that were collected from a bracket fungus in the Hermitage of Braid, Edinburgh, 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 female Hirtodrosophila cameraria (Figure 1C) collected from Hermitage of Braid,Edinburgh,Scotland (55.92,.A total of 45-fold  The final assembly has a total length of 214.5 Mb in 39 sequence scaffolds with a scaffold N50 of 82.8 Mb (Table 1).Most (98.38%) of the assembly sequence was assigned to 4 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).We did not identify the sex chromosome(s) as sequence data from the heterogametic sex was not available and homology is unreliable for sex chromosome identification in Diptera due to frequent sex chromosome turnover (Vicoso & Bachtrog, 2015).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/1262473.

Sample acquisition and nucleic acid extraction
The specimen used for genome sequencing was a female Hirtodrosophila cameraria (biospecimen ID SAMEA12110596, individual idHirCame2; Figure 1C).The specimen used for Hi-C scaffolding was a female Hirtodrosophila cameraria (biospecimen ID SAMEA12110595, individual idHirCame1; Figure 1C).Both specimens were collected from Hermitage of Braid, Edinburgh, Scotland, UK (latitude 55.92, longitude -3.20) on 2021-10-04.The specimens were aspirated from a bracket fungus on a deciduous tree stump in an urban woodland.The anaesthetised flies were placed directly into collection tube, and frozen from live at -80°C.The specimens were collected and identified by Darren Obbard (University of Edinburgh).
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The idHirCame2 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 whole organism tissue of idHirCame1 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    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 Tree of Life collaborator.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

Ludvik M Gomulski
University of Pavia, Pavia, Italy The manuscript reports the sequencing of the genome and mitochondrial genome from a single British female specimen of a drosophilid fly, Hirtodrosophila cameraria.The methods and analyses used are well described and appropriate and the datasets are readily accessible.
1. Hirtodrosophila cameraria should always be in italics -this is not so in the keywords and in the first sentence of the Background section.
2. It is incorrect to refer to substances released by orchids that attract pollinators as 'pheromones' -use 'semiochemicals' or 'allelochemicals'.

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 genetics and behaviour 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.
Experimental design and data analysis are comprehensively described.
Only three minor editorial issues: 1.

Hirtodrosophila cameraria
Italicize in the first sentence of the Background section.
The legend states "D: Female Hirtodrosophila cameraria photographed above a toadstool..." I can't identify a toadstool or toadstool structure in panel D.

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 1 .
Figure 1.Photographs of Hirtodrosophila cameraria specimens and locale.A: Male (above) and female (below) Hirtodrosophila cameraria presented with a 3 mm scale bar.B: Tree stump from which the sequenced individuals were collected (Hermitage of Braid, Edinburgh, Scotland; 55.92, -3.20).C: The four unrelated wild-collected females provided to the Darwin Tree of Life project.Individual idHirCame1 (biospecimen SAMEA12110595) (left) was used for Hi-C sequencing, and individual idHirCame2 (biospecimen SAMEA12110596) (second left) was used for PacBio sequencing.D: Female Hirtodrosophila cameraria photographed above a toadstool in Perth & Kinross, Scotland.

Figure 2
Figure 2 Genome assembly of Hirtodrosophila cameraria, idHirCame2.1: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 214,511,801 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 (93,358,477 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (82,766,246 and 31,806,066 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/idHirCame2.1/dataset/CATIWZ01/snail.

Figure 5 .
Figure 5. Genome assembly of Hirtodrosophila cameraria, idHirCame2.1:Hi-C contact map of the idHirCame2.1 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=WPeKseKJS1uSeK0NS7ZsOA.