The genome sequence of the Twin-spot Plume, Stenoptilia bipunctidactyla (Scopoli, 1763)

We present a genome assembly from an individual male Stenoptilia bipunctidactyla (the Twin-spot Plume; Arthropoda; Insecta; Lepidoptera; Pterophoridae). The genome sequence is 822.9 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 17.8 kilobases in length. Gene annotation of this assembly on Ensembl has identified 22,137 protein coding genes.


Background
Stenoptilia bipunctidactyla (Scopoli, 1763), known as the 'Twin-Spot Plume' (Hart, 2011), is a micro moth of the family Pterophoridae, commonly known as 'Plume moths' for their thin, elongated wings which are often held rolled-up when at rest, at right angles to the body.
It is a relatively widespread member of the group, known from all mainland European countries (Gielis, no date), but precise understanding of its range and biology have been clouded by the relatively recent description of a number of other species formerly included under S. bipunctidactyla (Arenberger, 2005;Gielis, 1996;Gielis, 2003).Adults of this 'S.bipunctidactyla complex' are extremely similar externally, but species can be separated on the basis of their genital morphology, and to some extent their foodplants.Five of the species described in this complex occur in Europe alongside the original S. bipunctidactyla (Rennwald, no date), although S. succisae (Gibeaux & Nel, 1991) has recently been reduced back to a synonym of S. bipunctidactyla by (Huemer et al., 2021).In the UK, three members of the complex are known, of which S. bipunctidactyla appears to be by far the most widespread.S. annadactyla ('Small Scabious Plume') is currently known only from the Brecklands of East Anglia, while S. scabiodactyla ('Gregson's Plume') appears to be a more northern species, but its distribution is poorly known (Emmet et al., no date).
Confusion over the taxonomy of S. bipunctidactyla has also fuelled confusion over its biology (Rennwald, no date), but in the UK it has two main foodplants -Field Scabious (Knautia arvensis L.) and Devil's Bit Scabious (Succisa pratensis Moench) (Hart, 2011).In the south of England there are two broods a year, with larvae of the first brood emerging from hibernation (probably at the base of the plant) in early spring (Hart, 2011).On K. arvensis they then mine a growing stem, causing blackening near the tips of the newest leaves, while on S .pratensislarvae initially mine the midrib of an old leaf, before feeding under the new leaves, spinning them together as they grow older (Hart, 2011).Adults then fly in late May and June, and second brood larvae can be found from late June to mid-August, feeding concealed inside the flowers of the foodplants, with another brood of adults in late July and August (Hart, 2011).
In the UK, both foodplants of S. bipunctidactyla are species of un-improved, low-nutrient grasslands, and though the moth is widespread it has probably declined as a result of the improvement of such habitats (Hart, 2011).K. arvensis occurs mostly on drier, often calcareous sites, and S. pratensis in similar habitats, but also on damper neutral and acid grasslands.S. bipunctidactyla occurs in all these habitats.
The genome of Stenoptilia bipunctidactyla 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 Stenoptilia bipunctidactlya, based on one male specimen from Wytham Woods, Oxfordshire, UK.This specimen was captured in the area of the woods known as 'Upper Seeds' where S. bipunctidactyla larvae have been found feeding on Field Scabious.

Genome sequence report
The genome was sequenced from one male S. bipunctidactyla (Figure 1) collected from Wytham Woods, UK (latitude 51.77, longitude -1.33).A total of 24-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 76 missing or mis-joins and removed 41 haplotypic duplications, reducing the assembly length by 2.8% and the scaffold number by 7.4%, reducing the scaffold N50 by 0.51%.
The final assembly has a total length of 822.9 Mb in 313 sequence scaffolds with a scaffold N50 of 27.0 Mb (Table 1).Most (96.41%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 29 autosomes and the Z sex chromosome.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 97.6%% (single 96.7%, duplicated 0.9%) 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
Three Stenoptilia bipunctidactyla specimens (ilSteBipu1, ilSteBipu2 and ilSteBipu3) were collected in Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.33) on 10 August 2021.The specimens were taken from a grassland habitat by netting and were snap-frozen on dry ice.William Langdon (University of Oxford) collected and identified the specimens.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilSteBipu1 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.
RNA was extracted from ilSteBipu3 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 was 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 using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing were performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from ilSteBipu2 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the S. bipunctidactyla assembly (GCA_944452665.1) in Ensembl Rapid Release.The researchers present a chromosome-level genome assembly of the "Twin-Spot Plume" moth, Stenoptilia bipunctidactyla.The authors employed state-of-the-art methods for sequencing, assembling, and annotation of insect genomes.The paper is clearly written, including information about other moth species in the same complex along with observations on their natural history.There are just some minor comments: Some references are currently missing the date.For instance: (Gielis, no date) and (Rennwald, no date). 1.
This sentence might not be clear to the general public: "In the UK, both foodplants of S. bipunctidactyla are species of un-improved, low-nutrient grasslands, and though the moth is widespread it has probably declined as a result of the improvement of such habitats (Hart, 2011)."Maybe explaining what "un-improved" and "improvement" refer to in this context would help.

2.
It would be useful to cite the link for the lepidoptera_odb10 database.3. Do the authors also use transcriptomic data for annotation as part of their annotation pipeline?If this is the case, it would be important to include this information.

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: 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.Reviewer Expertise: Cytogenomics 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 Stenoptilia bipunctidactyla, ilSteBipu1.1: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 822,897,450 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 (51,424,521 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (27,025,175 and 20,980,724 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/ilSteBipu1.1/dataset/CALYBW01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Stenoptilia bipunctidactyla, ilSteBipu1.1:Hi-C contact map.Hi-C contact map of the ilSteBipu1.1 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=Dv3cV6lwQC-nrfcURO3Aew.

Reviewer
Report 05 June 2023 https://doi.org/10.21956/wellcomeopenres.21156.r55892© 2023 Lourenço L. 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.Luciana Bolsoni Lourenço Laboratório de Estudos Cromossômicos (LabEsC), Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, State of São Paulo, Brazil The text is clear and the data are very useful.The figures are informative and the datasets can be easily accessed.I would only suggest that the authors indicate how they identified the sex chromosome.Is there any previous genetic or cytogenetic paper that might support the identification of the Z chromosome?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.

Table 3 . Software tools and versions used. Software tool Version Source
Ethics and compliance issuesThe 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.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.All efforts are undertaken to minimise the suffering of animals used for sequencing.Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material 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.