The genome sequence of the Autumnal Rustic, Eugnorisma glareosa (Esper, 1788)

We present a genome assembly from an individual male Eugnorisma glareosa (the Autumnal Rustic; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 631.0 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 15.39 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,768 protein coding genes.


Background
The Autumnal Rustic, Eugnorisma glareosa, is a medium-sized noctuid moth with a wingspan of about 32-38 mm.Its forewings usually exhibit dorsally crisp black markings on a light grey background, with two short black dashes or dots at the forewing base.The inner margins of the faintly outlined orbicular and reniform stigmata are in the form of conversely placed black arrow or axe heads.The former marking sometimes appears with adjacent black dots and often shows a diffuse rufous-grey or pinkish subterminal band.The forms in south-east England are more orange-brown and much darker forms are found in the Shetlands.As the vernacular name suggests, the adult moth emerges late in the Palaearctic Autumn, flying in the UK from August to October (Randle et al., 2019), and the moth overwinters as a small larva (Waring et al., 2017).
The Autumnal Rustic has a preference for heathland, moorland or other types of open country such as downs and shingle beaches.The larva feeds on various low growing plants such as heathers and bedstraws as well as scrub birch and sallow (Waring et al., 2017).Genera fed on include Calluna, Galium, Hieracium, Lactuca, Plantago, Poa, Rumex, and Salix (FUNET, 2023).The adult nectars on flowers such as heathers.
E. glareosa is generally fairly common and widespread in the western Palaearctic only, from northern and eastern Ireland (poorly represented in the west) to southern Scandinavia to Spain and the northern Mediterranean; avoiding Italy, but has relatively few records for eastern Europe (GBIF Secretariat, 2023).
Populations in the UK have declined severely since 1970 (Randle et al., 2019); between 1968 and 2006 reductions in Rothamsted trap numbers of at least 94% were documented with an annual change decline of about 7% (Conrad et al., 2006).
Based on mitochondrial DNA, E. glareosa shows a single cluster on BOLD, BOLD:AAE3673 (21/08/2023) with southern Mediterranean exemplars slightly (up to 1.79%) divergent from the others; south-eastern English exemplars show identity with central European populations.The genus Eugnorisma Boursin, 1946 was partly revised by Varga et al. (1990) and these authors considered a previously suggested relative Protexarnis McDunnough, 1929 as morphologically convergent, and they also considered a possible relationship based on genital characteristics with Eugraphe Hübner,1821, Paradiarsia McDunnough, 1929, andXestia Hübner, 1818.Surprisingly, the nearest neighbouring species of E. glareosa on BOLD is the dissimilar looking Double Dart Graphiphora augur, which is a mere 3.1% or more pairwise divergent in COI-5P (21/08/2023); other species of the genus Eugnorisma, including ones with similar wing patterns, are more distant.In this context the type species of the genus Eugnorisma is the Central Asian Graphiphora insignata Lederer 1853 (whereas Graphiphora Ochsenheimer, 1816 is currently considered monobasic, containing only G. augur).The genome sequence should therefore be useful in resolving the phylogenetic relationships of Eugnorisma using multiple loci.The data may also be useful to investigate genes that are potentially selected to influence forewing colouration in relation to darker resting substrates in northern Scotland and the Shetlands (see (Kettlewell & Berry, 1969), and may also be relevant to the study of the evolution of melanism.

Genome sequence report
The genome was sequenced from one male Eugnorisma glareosa (Figure 1) collected from Beinn Eighe (57.63,.A total of 42-fold coverage in Pacific Biosciences singlemolecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 12 missing joins or mis-joins and removed 3 haplotypic duplications. The final assembly has a total length of 631.0 Mb in 47 sequence scaffolds with a scaffold N50 of 22.0 Mb (Table 1).Most (99.86%) 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).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/988114.

Sample acquisition and nucleic acid extraction
A male Eugnorisma glareosa (specimen ID NHMUK014451776, individual ilEugGlar6) was collected from Beinn Eighe National Nature Reserve, Scotland, UK (latitude 57.63, longitude -5.35) on 2021-09-09, using a light trap.The specimen was collected and identified by David Lees (Natural History Museum) and dry frozen at -80°C.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilEugGlar6 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Thorax 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 solidphase 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     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 BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Eugnorisma glareosa assembly (GCA_947578425.1) in Ensembl Rapid Release.

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

Andrea Battisti
University of Padova, Padova, Italy I do not have major concerns about the manuscript, only a few comments in the attached PDF file which can be viewed here.
I congratulate the authors for the quality of the work that will contribute to the knowledge on the Lepidoptera genomic science and make it possible to compare the species with the few others for which a full genome is available.

Fahad Alqahtani
King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia In "The genome sequence of the Autumnal Rustic, Eugnorisma glareosa (Esper, 1788)," the authors have achieved a highly complete genome assembly at the chromosome level for a male Autumnal Rustic, Eugnorisma glareosa (Esper, 1788).
The genome assembly was reconstructed using two sequencing technologies: Pacific Biosciences SEQUEL II and Hi-C Illumina.The completeness of the genome assembly was assessed using BUSCO analysis, which indicated a genome size of 631 megabases, with 99% of common genes in the lepidoptera_odb10 present.
There is just one minor comment that should be addressed: 'Pretext' in the Genome Assembly section should be corrected to 'PretextView' to match the name in Table 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: 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 Eugnorisma glareosa, ilEugGlar6.1: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 631,006,646 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 (48,685,131 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (21,954,397 and 15,478,121 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/ilEugGlar6.1/dataset/CANPUN01/snail.

Figure 5 .
Figure 5. Genome assembly of Eugnorisma glareosa, ilEugGlar6.1:Hi-C contact map of the ilEugGlar6.1 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=EC1U7s0XTYaN5zpXpL2kUA.

Peer Review Current Peer Review Status: Version 1
Darwin Tree of Life (DToL) project.All raw sequence data and the assembly have been deposited in INSDC databases.Raw data and assembly accession identifiers are reported in Table 1.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.

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? No 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.