The genome sequence of the slender grass hoverfly, Melanostoma scalare (Fabricius, 1794)

We present a genome assembly from an individual male Melanostoma scalare (the slender grass hoverfly; Arthropoda; Insecta; Diptera; Syrphidae). The genome sequence is 738.2 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 16.08 kilobases in length.


Background
The slender grass hoverfly, Melanostoma scalare, is a medium to small hoverfly.This is one of the commonest in the UK, and along with other members of the genus, is one of the most abundant hoverflies of the Palaeartic (Haarto & Ståhls, 2014).These hoverflies are black and yellow in colouration and are sexually dimorphic.Males of this species are narrowly built and elongate, making them rather distinctive (Ball & Morris, 2015).Females have triangular abdominal markings and resemble female Melanostoma mellinum but are more elongate and have larger dust spots on their frons (Stubbs & Falk, 2002).
Melanostoma scalare larvae are thought to be generalist predators on aphids and other small arthropods found in leaf litter or tussocky grass (Wilkinson & Rotheray, 2017).While many other aphidophagous hoverfly lay only on previously infested plants, M. scalare females will lay on plants even in the absence of aphids, perhaps as because of their generalist food preferences (Chandler, 1968).This behaviour lends the species to pest management options in agricultural settings (Chandler, 1968).Adults have a long flight period spanning from March to November (Stubbs & Falk, 2002).The adults feed on a wide variety of flowers making them important generalist pollinators (Doyle et al., 2020).The mitochondrial genome of this species has been previously sequenced (Liu et al., 2020).The production of a high quality Melanostoma scalare genome sequence described here, generated as part of the Darwin Tree of Life project, will further aid understanding of the biology and ecology of this hoverfly.

Genome sequence report
The genome was sequenced from one male Melanostoma scalare (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 31-fold coverage in Pacific Biosciences single-molecule HiFi long was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 157 missing joins or mis-joins and removed 34 haplotypic duplications, reducing the assembly length by 0.84% and the scaffold number by 48.2%, and increasing the scaffold N50 by 120.8%. The final assembly has a total length of 738.2 Mb in 143 sequence scaffolds with a scaffold N50 of 241.7 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 (99.22%) of the assembly sequence was assigned to 5 chromosomal-level scaffolds, representing 3 autosomes and the X and Y sex chromosomes.Sex chromosomes were identified using coverage information.Chromosome Y scaffolds determined by mapping Hi-C data from a female sample to the male assembly.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.
Metadata for specimens, barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/ species/92598.

Sample acquisition and nucleic acid extraction
Specimens of Melanostoma scalare were netted in Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2019-07-31.The specimens were collected and identified by Will Hawkes (University of Exeter) and preserved on dry ice.A male specimen (specimen ID Ox000110, individual idMelScal2) was used for DNA sequencing and Hi-C data and a female specimen was used for RNA sequencing (specimen ID Ox000111, ToLID idMelScal1).

Number of scaffolds 143
Scaffold N50 length (Mb) 241.7 Longest scaffold (Mb) 281.9DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The idMelScal2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Head and 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.
RNA was extracted from head and thorax tissue of idMelScal1 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed 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   et al., 2020) andBUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated.
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 align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material • Legality of collection, transfer and use (national and international) Table 3. Software tools: versions and sources.

Software tool Version
It is modern, the raw data is satisfactory, and the methodology is appropriate and understandable.Additionally, the authors have made the raw data and genome publicly available.
Based on my own investigation of the data, the resulting genome is high quality and has excellent coverage.
It is difficult to determine the identity of the specimen from the provided photograph.While I have no reason to doubt the identification, I urge caution with this group as it can be tricky.There are four species of Melanostoma in Northern Europe: M. scalare, M. mellinum, M. mellarium and M. certum.M. scalare is most easily distinguished from these by longer pruinosity on the arista as compared to the other species.
It would be helpful if the literary resource used to determine the identity of this species was provided in the text.

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

Claus Vogl
Vetmeduni Vienna, Veterinärplatz, Wien, Austria I am not a fly specialist, but rather a biostatistician/bioinformatician.Reading the note, I was mainly concerned about the identification of the species.However, upon reading further material it seems that both sexes of the species can be identified confidently.It should be made clear in the text that identification of both the male and female sequenced specimens was made with confidence.
It has turned out (eg in Anopheles mosquitos) by the use of modern genetic methods (such as those employed in the note) that what was once thought to be a single wide-spread species constitutes in fact a species complex.Since both specimens derive from the same location it is unlikely that they actually are different cryptic species (should M scalare be a cryptic species complex).All methods and tools seem state of the art and the description in the text is confident and without fuzz.The DNA data (from the male) are clearly documented with some figures and corresponding text in the results section.On the other hand, the RNAseq data of the female are not documented at all in the results section.I wonder if this intentional.

Andrés López Rubio
Tecnológico de Antioquia -University Institute, Antioquia, Colombia The article shows the publishing for the complete genome of Melanostooma scalare with 99.2 % coverage.
The methods on the paper are well described.It would be good to show the results on RNA sequencing.Any replicate was deposited in a Biological collection?
I found this paper useful since sequencing and produce genomic data from non model species is having great impact in the understanding of the biology of every species

Darren J Obbard
The University of Edinburgh, Edinburgh, Scotland, UK This data note reports the sequencing and assembly of the genome of Melanostoma scalare for the "Darwin Tree of Life" programme.In common with other data notes from this research effort, the reporting is standardised and quite brief.As such, I have very few comments to make.The approach is state-of-the-art, the raw data appear to be of a suitably high quality, and the assembly methods are appropriate.The public availability of raw data and genome assembly are appropriate.The resulting genome is likely to be of very high quality, and I have no doubt that it will be of great value to any researchers working on the comparative or evolutionary genomics of insects.
I have a couple of very minor suggestions In addition to the photograph of the sequenced male specimen, it would be very nice to have images of both the male and female in life.

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The introduction is well written, and not excessively brief.However, it would be nice to have a little more detail on the distribution, ecology, or behaviour (if papers exist)

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How does the sequenced mtDNA compare to the previously published one?Are they the same length with similar gene content?

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The assembly of X and Y is great -but both appear to be very small.Is that expected?Or, is it more likely that one or both are mostly unassembled due to repeats?It would be great to have a comparison with (say) expected genome size based on DNA content, and/or previously published cytology if any exists ○ 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

Figure 2 .
Figure 2. Genome assembly of Melanostoma scalare, idMelScal2.1: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 738,179,629 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 (281,865,375 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (241,717,587 and 185,741,843 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/Melanostoma%20scalare/dataset/CATKHO01/snail.

Figure 5 .
Figure 5. Genome assembly of Melanostoma scalare, idMelScal2.1:Hi-C contact map of the idMelScal2.1 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=WFlEul_nRvWxkL2eXYGk3A.
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.

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.

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
Reviewer Expertise: Phylogeny, Entomology, Evolutionary 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.
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.