The genome sequence of the Beautiful China-mark moth Nymphula nitidulata (Hufnagel, 1767)

We present a genome assembly from an individual female Nymphula nitidulata (the Beautiful China-mark moth; Arthropoda; Insecta; Lepidoptera; Crambidae). The genome sequence is 635.8 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 15.36 kilobases in length. Gene annotation of this assembly on Ensembl identified 20,031 protein coding genes.


Background
Nymphula nitidulata, aptly named the Beautiful China-mark moth, is one of the more distinctive and charismatic species of the subfamily Acentropinae.The forewings are a shining white with brown, rounded markings.Like other species in the subfamily, N. nitidulata is associated with freshwater environments, where the larvae live, while the adults are terrestrial (De-Freitas et al., 2019;Pabis, 2018).It is a small moth with a wingspan of 20-25 mm.Fairly widespread throughout Britain and Ireland, this species is classified as local by the Butterfly Conservations' Microlepidoptera report.This species comes to light and is easily disturbed by day from vegetation near the waterside.
The larvae are a bright yellow with a dark brown dorsal line and pale brown head.They live in streams, lakes, as well as fens and marshes and feed on bur-reed (Sparganium spp.) and yellow water-lily (Nuphar lutea).The genome of this species is a key addition to the underrepresented aquatic insects (Hotaling et al., 2020), and will provide insights into how this subfamily adapted to live in freshwater habitats.
The genome of the Beautiful China-mark, Nymphula nitidulata, 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 Nymphula nitidulata, based on one female specimen from Wytham Woods.

Genome sequence report
The genome was sequenced from one female Nymphula nitidulata (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 41-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 6 missing joins or mis-joins and removed one haplotypic duplications, reducing the assembly length by 0.30% and the scaffold number by 4.65%, and increasing the scaffold N50 by 3.51%. The final assembly has a total length of 635.8 Mb in 40 sequence scaffolds with a scaffold N50 of 22.2 Mb (Table 1).The snail plot 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 (99.89%) of the assembly sequence was assigned to 31 chromosomallevel scaffolds, representing 30 autosomes and the Z and W sex chromosomes.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 specimens of Nymphula nitidulata used for genome sequencing (specimen ID Ox000517, ToLID ilNymNiti1) and   In sample preparation, the ilNymNiti1 sample was weighed and dissected on dry ice (Jay et al., 2023).Whole organism tissue was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).HMW DNA was extracted using the Automated MagAttract v1 protocol (Sheerin et al., 2023).HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.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) instruments.Hi-C data were also generated from whole organism tissue of ilNymNiti2 using the Arima2 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 Nymphula nitidulata assembly (GCA_947347705.1) in Ensembl Rapid Release at the EBI.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome   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 The DNA for the genome sequence was extracted using standard Darwin Tree of Life protocols and also the sequencing and sequencing pipeline were also standard for the Darwin Tree of Life.Hence, the assembly is of very high quality, with a BUSCO completeness score of 98.9%.The sequences are scaffolded into 31 chromosomal pseudomolecules, including the sex chromosomes.This will be a very useful resource for future studies in a variety of different areas: evolution, ecological adaptation, sex chromosomes and entomology.

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: Genomics, transcriptomics, ecological adaptation 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.

Axel Künstner
University of Lübeck, Lübeck, Germany The authors present the genome assembly from an individual Beautiful China-mark moth ( Nymphula nitidulata).
The manuscript is clearly written, methods well explained and I did not found any technical flaws in the assembly strategy.The raw and the assembled data is available via ENA.
I do not have any comments to improve the presented manuscript.

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: My area of research is mainly Bioinformatics.
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 Nymphula nitidulata, ilNymNiti1.1:metrics.The BlobToolKit snail plot 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 635,785,502 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 (35,422,410 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (22,210,000 and 14,630,000 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/CANAFG01/dataset/CANAFG01/snail.

Figure 3 .
Figure 3. Genome assembly of Nymphula nitidulata, ilNymNiti1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANAFG01/dataset/CANAFG01/blob.
Assembly was carried out withHifiasm (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 PretextView(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.

Figure 4 .
Figure 4. Genome assembly of Nymphula nitidulata, ilNymNiti1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANAFG01/dataset/CANAFG01/cumulative.
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

Figure 5 .
Figure 5. Genome assembly of Nymphula nitidulata, ilNymNiti1.1:Hi-C contact map of the ilNymNiti1.1 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=LBpzVD6nQHKGvY3czdx4Sg.

Reviewer Report 09
July 2024 https://doi.org/10.21956/wellcomeopenres.23342.r88073© 2024 Künstner A. 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.

Table 1 . Genome data for Nymphula nitidulata, ilNymNiti1.1. Project accession data
(Denton et al., 2023b)hmarks are adapted from column VGP-2020 of "Table1: Proposed standards and metrics for defining genome assembly quality" from(Rhie et al., 2021).Hi-C sequencing (specimen ID Ox000518, ToLID ilNym-Niti2) were collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.33) on 2020-06-25 using a light trap.The specimens were collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.Protocols developed by the Wellcome Sanger Institute (WSI) Tree of Life core laboratory have been deposited on protocols.io(Dentonetal., 2023b).The workflow for high molecular weight (HMW) DNA extraction at the WSI includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.

Table 3
contains a list of relevant software tool versions and sources.