The genome sequence of the flutter-wing fly, Palloptera scutellata (Macquart, 1835)

We present a genome assembly from an individual female Palloptera scutellata (the flutter-wing fly; Arthropoda; Insecta; Diptera; Pallopteridae). The genome sequence is 415.6 megabases in span. Most of the assembly is scaffolded into 5 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.93 kilobases in length.


Background
The Pallopteridae is a family of small to medium sized (2.5-7 mm), acalypterate flies sometimes known as the flutter-wing or trembling-wing flies, because of the rapid wing movements made by many species.The family is identified by the following characters: subcosta complete and curving smoothly to end in the costa, a costal break at the junction of the subcosta, first radial vein (R 1 ) bare on the dorsal surface, head with one pair of reclinate orbital setae, postvertical bristles parallel to divergent, vibrissae absent, and wings usually with markings consisting of clouded areas and spots.On account of their patterns of wing spots, in the British fauna, the Pallopteridae -along with the families Tephritidae, Opomyzidae, Platystomatidae and Ulidiidae -are often known collectively as the picture-winged flies.There are around 83 described species of the Pallopteridae in 15 genera which are found in the temperate regions of the globe (GBIF Secretariat, 2023).
With 46 species, the genus Palloptera contains the majority of the Pallopteridae.Palloptera scutellata (Macquart, 1835) (Diptera: Pallopteridae) has orange-brown abdomen and legs, a blue-grey thorax and an orange frons.It is readily identified by the wing pattern with four spots, comprising the wing stigma in the subcostal cell, two small patches covering the anterior and posterior crossveins, and a shaded area towards the wing tip but not including the wing apex, which is clear.P. scutellata is found in northern Europe with most records in Great Britain and Northern Ireland, Germany, the Netherlands and Belgium.Females have a long piercing ovipositor and lay eggs in the stems of the Soft Rush, Juncus effusus, where the larvae are phytophagous (Rotheray & Hewitt, 2015).The life cycle is unusual within the genus with females emerging in the autumn and overwintering in the mated state (Bland & Horsfield, 2016).This life cycle is distinct from that of other European species of Palloptera, in which larvae are the overwintering stage (Rotheray, 2014).
In the United Kingdom, Palloptera scutellata was first recorded in 1950 at Common, Surrey (Parmenter, 1950).Subsequent records show a widespread but scattered distribution across England and Wales.It was first recorded in Scotland as recently as 2015 but has been found to be widespread (Bland & Horsfield, 2016).The presence of the species in Ireland was noted by Speight (1979), andSmit et al. (2009) reported the species as new to Belgium.This flurry of new announcements is indicative of a fly whose populations are thriving and increasing in range.Nevertheless, it is found much less frequently than its host plant implying that it may have special requirements, as yet unknown, but probably including warm, sheltered locations.
A female Palloptera scutellata was taken on 15 May 2021 at Stover Country Park, South Devon in the south-west of England during a meeting of the Devon Fly Group, a local group of the Dipterists Forum, the UK's national society for the study of flies.The specimen was sent live to the Natural History Museum, London.Stover Country Park is a Local Nature Reserve and designated as a Site of Special Scientific Interest.The site comprises 46 hectares of mixed habitats including woodland, lake, marsh, heathland, and grassland.
The generation of a high-quality genome sequence for Palloptera scutellata is an important step in advancing the understanding of these fascinating flies and in determining relationships between species within the genus Palloptera and within the Pallopteridae.

Genome sequence report
The genome was sequenced from one female Palloptera scutellata (Figure 1) collected from Stover Country Park, England (50.57,.A total of 46-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 50 missing joins or mis-joins, reducing the scaffold number by 24.14%, and increasing the scaffold N50 by 0.58%. The final assembly has a total length of 415.6 Mb in 65 sequence scaffolds with a scaffold N50 of 98.6 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 (99.57%) of the assembly sequence was assigned to 5 chromosomal-level scaffolds.We did not identify the sex chromosome 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).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 genome sequencing was a female Palloptera scutellata (specimen ID NHMUK014036893, ToLID idPalScut3) collected using an aerial net from Stover Country Park, England (latitude 50.57, longitude -3.65) on 2021-05-15.
The specimen was collected and identified by Michael Ashworth (independent researcher) and then dry frozen (-80°C).The specimen used for Hi-C sequencing (specimen ID NHMUK014449035, ToLID idPalScut1) was collected using an aerial net from Bookham Common on 2021-04-20.
The specimen was collected and identified by Duncan Sivell (Natural History Museum) and preserved in ethanol.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation; DNA extraction; HMW DNA fragmentation;

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 idPalScut1 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.
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 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

Software tool
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.
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 This genome resource is excellent from the summary statistics, with high BUSCO numbers, high sequence continuity (scaffold N50), and majority of sequences contained on the 5 pseudochromosomes (plus mitochondrion).To sum up, this is a valuable contribution setting up a nice foundation for further studies.

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: I have published with Peter W.H. Holland more than three years ago, and confirm that this potential conflict of interest did not affect my ability to write an objective and unbiased review of the article.
Reviewer Expertise: Genomics, evolution, invertebrates 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.Reviewer Expertise: Population genetics, genomics, and pest control.
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 Palloptera scutellata, idPalScut3.1: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 415,657,720 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 (158,007,244 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (98,550,696 and 68,594,659 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/Palloptera%20scutellata/dataset/idPalScut3_1/snail.

Figure 5 .
Figure 5. Genome assembly of Palloptera scutellata, idPalScut3.1:Hi-C contact map of the idPalScut3.1 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=LehCkdUsR3u6lpCbO5ESsA.

Reviewer Report 02
February 2024 https://doi.org/10.21956/wellcomeopenres.22568.r71309© 2024 Wei S. 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.Shu-Jun Wei Beijing Academy of Agriculture and Forestry Sciences, Beijing, China Michael Ashworth and Duncan Sivell have assembled the nuclear and mitochondrial genomes of the flutter-wing fly, Palloptera scutellata (Diptera; Pallopteridae) using Pacific Biosciences singlemolecule HiFi long reads and chromosome conformation Hi-C data.The final assembly has a total length of 415.6 Mb and a BUSCO completeness of 98.6%.This high-quality genome is a valuable resource for understanding the genetics and evolution of this fly and other dipterans.However, it should be noted that this genome assembly has not yet been annotated.Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.