The genome sequence of the Feathered Bright, Incurvaria masculella (Denis & Schiffermüller, 1775)

We present a genome assembly from an individual male Incurvaria masculella (the Feathered Bright; Arthropoda; Insecta; Lepidoptera; Incurvariidae). The genome sequence is 552 megabases in span. Most of the assembly is scaffolded into 26 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.3 kilobases in length.


Background
The Incurvariidae is a small taxonomic family of moths containing only 50 to 100 species worldwide and five species in the UK.Phylogenetically, Incurvariidae lie outside the Ditrysia within the paraphyletic 'Monotrysia' (Dugdale, 1974;Regier et al., 2013); they therefore occupy an important evolutionary position for studies investigating the evolution of lepidopteran genetic and phenotypic characters.Incurvaria masculella is one of the more frequently encountered UK species of Incurvariidae, commonest in central and southern counties of England, and it is also found across northern Europe and coastal regions of Scandinavia (GBIF Secretariat, 2022).Despite having a wingspan of just 12-16 mm, I. masculella is a striking moth with a yellow head, glossy purplish wings held tent-like over the body, and two sharply defined lemon-yellow diamond-shaped patches along the dorsal midline where the forewings meet.The antennae of the male are particularly notable, being large and deeply pectinate.In contrast, the antennae of the female are simple and thread-like.This sex-specific difference suggests that the unusual comb-like shape of the male antenna is likely an adaptation for increased olfactory reception in mate-finding.
The larvae of I. masculella are initially leaf-miners, forming circular blotch-shaped mines on leaves of the food plant, typically hawthorn Crataegus monogyna.The larvae then cut around the blotch and, sandwiched by leaf tissue, they drop to the ground to feed on dead leaves (Heath & Pelham-Clinton, 1983).The moth has one generation per year in the UK with the day-flying adult on the wing in May (NBN Gateway, 2022;Sterling & Parsons, 2018).
The complete genome sequence of I. masculella was sequenced as part of the Darwin Tree of Life Project based on long-read sequence data from the ilIncMasc1 male specimen from Wytham Woods, UK, scaffolded using Hi-C data from the ilIncMasc2 male specimen from Wallingford, UK.Due to the phylogenetic position of Incurvariidae, the genome sequence will be particularly useful in comparative studies investigating the evolution of lepidopteran genetic and phenotypic characters.

Genome sequence report
The genome was sequenced from one male Incurvaria masculella specimen (Figure 1) collected from Wytham Woods, Oxfordshire, UK (latitude 51.772, longitude -1.338).A total of 32-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 16 missing joins or mis-joins and removed five haplotypic duplications], reducing the assembly length by 0.77%, and decreasing the scaffold N50 by 0.75%.
The final assembly has a total length of 552.0 Mb in 31 sequence scaffolds with a scaffold N50 of 21.9 Mb (Table 1).Most (99.97%) of the assembly sequence was assigned to 26 chromosomal-level scaffolds, representing 25 autosomes, and the Z sex chromosome.The Z chromosome was identified by similarity.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 91.9% (single 91.1%, duplicated 0.8%) using the lepidoptera_odb10 reference set.While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
A male I. masculella (Figure 1) was collected from Wytham Woods, Oxfordshire (biological vice-country: Berkshire), UK (latitude 51.772, longitude -1.338) by William Langdon (University of Oxford) on 12 May 2021; this specimen (ToLID ilIncMasc1, specimen Ox001334) was used for acquisition of the genome sequence.A male I. masculella was found on a windowpane during daytime in Wallingford, Oxfordshire, UK (latitude 51.604, longitude -1.139) by Peter Holland  (University of Oxford) on 9 May 2021; this specimen (ToLID ilIncMasc2, specimen Ox001335) was used for Hi-C scaffolding.Each specimen was identified by the collector and frozen at -80°C.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilIncMasc1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Whole organism 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 Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from whole organism tissue of ilIncMasc2 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.The genome was analysed and BUSCO scores generated within the BlobToolKit environment (Challis et al., 2020).Table 3 contains a list of all software tool versions used, where appropriate.

Ethics and compliance issues
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    I am very approving of the release of the genome for open re=use.
I would have thought the authors could comment on the synteny of the 26 chromosomes identified at least in a somewhat superficial way.A number of studies have shown that Lepidoptera chromosomes are strongly conserved with a basal number of 31 across the whole order.Thus, unless Incurvaria is exceptional (like some Pieridae), one would expect around 5 simple fusions from the ancestral karyotype to explain the Incurvaria n = 26.Is this the case?
The only indication of synteny interest I could find in the draft paper was "The Z chromosome was identified by similarity."--but similarity to what, exactly?"BUSCO v5.3.2 completeness of 91.9% (single 91.1%, duplicated 0.8%) using the lepidoptera_odb10 reference set."This seems good, but unexpectedly low for a well-sequenced genome.However, I assume that the BUSCO orthologs are largely based on Ditrysians, and maybe the reduced BUSCO score achieved here is due to the standard not really applying so well to this more "basal" lineage.
Overall, I applaud this effort to gain excellent genome assemblies of obscure taxa and hope my comments are not discouraging.Maybe a few more quick pointers to the interest in the particular species and data may help generate even more interest.

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: Lepidoptera evolution and 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.
strong statement, do you have a reference to back this up?
"The assembly was scaffolded with Hi-C data (Rao et al., 2014)" this is ambiguous as it would suggest that the Hi-C data came from Rao et al 2014.This is not the case.I am not sure what it could be referring to.The library was produced using the Armina kit, and the scaffolding was done using YaHS.If it is a citation for Hi-C in general then the sentence needs to be reworded.

2.
In terms of the tools used, all are cited, however BUSCO which is used through blobtools should also be cited.

3.
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: Genome assembly of non-model organisms 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 Incurvaria masculella, ilIncMasc1.2: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 552,037,208 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 (36,252,389 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (21,889,303 and 17,498,119 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 lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilIncMasc1.2/dataset/CAMPPI02/snail.

Figure 5 .
Figure 5. Genome assembly of Incurvaria masculella, ilIncMasc1.2:Hi-C contact map.Hi-C contact map of the ilIncMasc1.2assembly, 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=H2Dc25S1RqW8TtUHTYp_Xg.

Table 3 . Software tools and versions used.
Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.