The genome sequence of the Ingrailed Clay, Diarsia mendica (Fabricius, 1775)

We present a genome assembly from an individual male Diarsia mendica (the Ingrailed Clay; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 727.9 megabases in span. Most of the assembly is scaffolded into 32 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.37 kilobases in length. Gene annotation of this assembly on Ensembl identified 14,077 protein coding genes.


Background
Diarsia mendica (Ingrailed Clay) is a macro-moth in the family Noctuidae.The species has a southerly distribution in Britain and is mainly found throughout northern and central Europe; however there are a few records as far east as Siberia (GBIF Secretariat, 2023).In the UK, the species has undergone a significant decline in abundance since the 1970s (Randle et al., 2019).
The moth is highly variable both within local populations, and across its range.Northern populations are generally darker and smaller, with recognised sub-species in Shetland and Orkney.D. mendica can usually be identified by the outline of the kidney mark and a black dot between the oval mark and the trailing edge of the forewing.The forewing length is 13-17 mm (Waring et al., 2017).The common name of this moth includes an heraldic term, 'ingrailed', indicating a decorative border (Marren, 2019).Close examination reveals arrowhead-shaped marks along the outer edge of the forewing.
D. mendica has one generation a year, flying between late May and July in the southern part of its range and up to a month later in the north.It regularly comes to light; can be attracted by sugaring; and also feeds on flowers.The moth is found in a variety of habitats including woodland and gardens in the south of its range; but favouring heathlands and moorlands in the north.D. mendica overwinters as a small larva.It feeds at night on a range of herbaceous plants before pupating underground (Waring et al., 2017).
The genome sequence from D. mendica will be useful for research into colour variation in moths, and more generally for comparative studies across the Lepidoptera.The genome of D. mendica 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 D. mendica based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Diarsia mendica (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 40-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 58 missing joins or mis-joins and removed 33 haplotypic duplications, reducing the assembly length by 6.04% and the scaffold number by 33.96%, and decreasing the scaffold N50 by 3.29%. The final assembly has a total length of 727.9 Mb in 34 sequence scaffolds with a scaffold N50 of 25.4 Mb (Table 1).A summary of the assembly statistics is shown in Figure 2, 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 (99.99%) of the assembly sequence was assigned to 32 chromosomal-level scaffolds, representing 31 autosomes and the Z sex chromosome.The Z chromosome was identified based on synteny with Diarsia rubi (GCA_932274075.1)(Boyes et al., 2023).Chromosome 31/B1 is a putative supernumerary B chromosome.Alignments show lack of homology of B 1 with assembled chromosomes of Diarsia rubi.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, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/987924.

Sample acquisition and nucleic acid extraction
A male Diarsia mendica (specimen ID Ox001878, individual ilDiaMeni1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.32) on 2021-05-28 using a light trap.The specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.
The sample was prepared for DNA extraction at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilDiaMeni1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Thorax tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.DNA was extracted at the WSI Scientific Operations core using the Qiagen MagAttract HMW DNA kit, according to the manufacturer's instructions.
RNA was extracted from abdomen tissue of ilDiaMeni1 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 using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was 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 head tissue of ilDiaMeni1 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 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.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Diarsia mendica assembly (GCA_949316265.1).Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

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 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

Kay Lucek
Department for Environmental Sciences, University of Basel, Basel, Switzerland This is the data note for the genome assembly of Diarsia mendica, which provides a great resource for future comparative studies, especially on the evolution of B chromosomes if this holds up.
Overall the genome sequencing and assembly follow the standard approaches of the Darwin Tree of Life project.However, for a full replication of the assembly methods, one would need the specific settings of all the programs that were used for assembly and annotation.
Concerning the annotation, it would be great if the authors could elaborate what fraction of the genes of the genome were actually annotated.Interestingly, they used an abdomen for RNAseq, which also contains the gut and therefore many exogenous transcripts.Similarly, transcripts in other body parts such as the brain, may have been missing.

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? Partly
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Speciation, Genomics, Evolutionary Biology, Lepidoptera 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 Diarsia mendica, ilDiaMeni1.1: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 727,952,529 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 (34,520,335 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (25,354,251 and 16,374,863 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/Diarsia%20mendica/dataset/CASGGF01/snail.

Figure 5 .
Figure 5. Genome assembly of Diarsia mendica, ilDiaMeni1.1:Hi-C contact map of the ilDiaMeni1.1 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=WNkhkJMKSum9Dm1JKAnxRg.

Table 3 . Software tools: versions and sources. Software tool Version 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.

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.