The genome sequence of the Mottled Pug, Eupithecia exiguata (Hübner, 1813)

We present a genome assembly from an individual male Eupithecia exiguata (the Mottled Pug; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 372.9 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.39 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,194 protein coding genes.


Background
The Mottled Pug (Eupithecia exiguata) is a small geometrid moth.Its larvae feed on Hawthorn, Blackthorn and other shrubs (Henwood et al., 2020;Waring et al., 2017).It occurs in woodland, hedgerow and garden habitats and is common and widespread across much of England and Wales.It is also widespread in Ireland but there are fewer records from Scotland, where it is spreading; its distribution overall has increased markedly since 1970 (Randle et al., 2019).Globally, The Mottled Pug occurs across Europe and Asia to the Pacific coast of Russia and China (GBIF Secretariat, 2023).
The genome of the mottled pug, Eupithecia exiguata, 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 Eupithecia exiguata, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Eupithecia exiguata (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.32).A total of 56-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 20 missing joins or mis-joins and removed one haplotypic duplication, reducing the scaffold number by 15.38%.
The final assembly has a total length of 372.9 Mb in 32 sequence scaffolds with a scaffold N50 of 13.1 Mb (Table 1).Most (99.99%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 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/934847.
The resulting annotation includes 19,529 transcribed mRNAs from 11,194 protein-coding and 1,243 non-coding genes.

Sample acquisition and nucleic acid extraction
A male Eupithecia exiguata (specimen ID Ox001895, individual ilEupExig1) was collected from Wytham Woods, Oxfordshire, 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 workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation; DNA extraction; HMW DNA fragmentation; and fragmented DNA clean-up.The sample was prepared for DNA extraction at the WSI Tree of Life laboratory: the ilEupExig1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing (https://dx.doi.org/10.17504/protocols.io.x54v9prmqg3e/v1).Tissue from the whole organism was disrupted using a Nippi Powermasher fitted with a BioMasher pestle (https://dx.doi.org/10.17504/protocols.io.5qpvo3r19v4o/v1).DNA was extracted at the WSI Scientific Operations core using the Qiagen MagAttract HMW DNA kit, according to the manufacturer's instructions.A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, 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 Eupithecia exiguata assembly (GCA_947086465.1).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 This data note reports the high-quality genome of the geometrid moth Eupithecia exiguata (Mottled Pug).Genome sequencing, assembly and annotation is based PacBio-HiFi and Hi-C data.
Overall, the dataset is presented clearly and concisely.The methods are state-of-the-art and follow the usual DToL procedures.Nevertheless, as with other DToL genome reports, I would wish for more details in the method section, particularly concerning the software settings and the protein set used for genome annotation.Likewise, the background could be more elaborate with some explanation about its relevance.It seems like the background section of DToL genome reports gets shorter and shorter.Still, the genome provides a valuable resource for evolutionary and comparative genomic research.
Is the rationale for creating the dataset(s) clearly described?

Jaakko Pohjoismäki
University of Eastern Finland, Joensuu, Finland The manuscript by Boyes, Lewis & the DToL consortium presents the reference-level genome assembly for the geometrid moth Eupithecia exiguata.The genome quality and assembly corresponds to the standards of previous reference genome releases and has been obtained using the standard DToL pipeline.
Unfortunately the genome is again from a male and thus lacks the W sex chromosome.I think that more effort should be put into obtaining the heterogametic sex for the reference genomes, as sequencing another reference genome from the same species is unlikely or at least low priority.Also this species is very common and can be also found resting on walls etc. during days, so obtaining a female is not very difficult.Otherwise I have only minor suggestions: The background is quite sparingly written.Eupithecia is a very species-rich genus, with diverse host plant associations and habitat requirements.Some more information about the diversity in the UK would be useful.Also as some (or most) species can be difficult to determine, it would be important to explain how exiguata can be differentiated from other similar species.Despite these minor shortcomings, this high quality genome assembly is a nice addition to the growing number of available lepidopteran reference genomes.

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.

Figure 2 .
Figure 2. Genome assembly of Eupithecia exiguata, ilEupExig1.1: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 372,887,712 bp assembly.The distribution of sequence lengths is shown in dark grey with the plot radius scaled to the longest sequence present in the assembly (19,876,806 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 sequence lengths (13,143,396 and 8,794,744 bp), respectively.The pale grey spiral shows the cumulative sequence 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/Eupithecia%20exiguata/dataset/CAMTYU01/snail.

Figure 3 .
Figure 3. Genome assembly of Eupithecia exiguata, ilEupExig1.1: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/Eupithecia%20exiguata/dataset/CAMTYU01/blob.

Figure 4 .
Figure 4. Genome assembly of Eupithecia exiguata, ilEupExig1.1: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/Eupithecia%20exiguata/dataset/CAMTYU01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Eupithecia exiguata, ilEupExig1.1:Hi-C contact map of the ilEupExig1.1 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=PURr_tyLQ6Obof4vztyYVA.

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
Reviewer Expertise: Genome sequencing, genomics, phylogenomics, Hymenoptera 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.
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.