The genome sequence of the Hoary Footman, Eilema caniola (Hübner, 1808)

We present a genome assembly from one female Eilema caniola (the Hoary Footman; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 781.7 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 15.42 kilobases in length. Gene annotation of this assembly on Ensembl identified 22,953 protein coding genes.


Background
The Hoary Footman, Eilema caniola, is a moth in the arctiine tribe Lithosiini, with a wingspan of 26-36 mm (male) and 25-32 mm (female) (Macià et al., 2022), forewing 15-17 mm (Waring et al., 2017).Its forewings are silky pale grey with an ochreous costa and the head is yellow-orange.The forewings lack underside androconia (Macià et al., 2022).The hindwings are also whiter than the very similar E. complana (Hübner, 1808); like it, the moth rolls its wings at rest.Although difficult to identify superficially, the male and female genitalia are distinctive (Macià et al., 2022: 12-13, Figs. 49-50).The adult moth flies in the UK from late June to early August (Randle et al., 2019).However, Macià et al. (2022) characterise the species in Europe as bivoltine or even trivoltine, with a continuous flight period from May to October, sometimes November in Europe.
The Hoary Footman has a preference for cliffs, quarries and shingle in the UK (Waring et al., 2017), preferring xerothermic environments up to 1800 m elevation in Europe (Macià et al., 2022).The larva feeds from September to late June on lichens and algae on rocks and artificial substrates as well as on the leaves of legumes such as trefoils and Kidney Vetch Anthyllis vulneraria (Henwood et al., 2020).The full-grown larva is over 20 mm long (see Macià et al., 2022: Figs. 75-76, Fig. 94).Pupation is on the ground in a loose cocoon.
Eilema caniola occurs in England, historically occurring in its southwest, western Wales, Anglesey, Scilly Isles and in the Channel Isles (Waring et al., 2017).It also occurs in southeast England as a migrant and since 2000 has spread rapidly inland; it is locally common in England and Wales and near threatened in Ireland, occurring on its southeastern coast (Randle et al., 2019).In Western Europe it is widely distributed, ranging from the shores of the Mediterranean as far north as Denmark, and also eastwards to the borders of the Black Sea (GBIF Secretariat, 2023).Macià et al. (2022) describe the species as holomediterranean, occurring also in North Africa, Asia Minor and large Mediterranean islands.Macià et al. (2022) propose the genus Eilema Hübner, [1808] as monotypic, including only E. caniola (Bombyx caniola Hübner, 1808 is the type species).The species exhibits a single mitochondrial cluster on BOLD, the BIN BOLD: AAF6264 (30/10/2023).The DNA barcode of the individual from south-eastern Kent on which the genome is based does not differ from the most prevalent continental haplotype.Macià et al. (2022) recognise an additional subspecies, E. c. torstenii Mentzer, 1980, as endemic to the Balearics, although thus identified individuals are (30/10/2023) part of two subclusters (one of which includes individuals currently identified on BOLD as E. complana from Corsica; a second subcluster from there are mostly (mis-) identified as E. complana).The correct BIN cluster for E. complana is BOLD:AAB6846 (which is shared also with E. pseudocomplana Daniel, 1939 andE. iberica Mentzer, 1980;Ortiz et al., 2017), which considering UK sequences on BOLD, is about 5.2% divergent to E. caniola (BOLD:AAF6264).In a tree based on COX1, ArgK and DDX23, the sister clade of E. caniola was supported as the clade E. palliatella (Scopoli, 1763) + E. costalis (Zeller, 1847) + E. pseudocomplana + (E.complana + E. iberica), all of which were placed in the genus Manulea Wallengren, 1863(Macià et al., 2022).The genome will be useful in helping to resolve or confirm such phylogenetic relationships, if not overall classification of Eilema (s.l.), which is highly polyphyletic in the latest scheme (Macià et al., 2022).
The genome of the Hoary Footman, Eilema caniola, 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 Eilema caniola, based on one female specimen from Sandwich Bay, SE Kent.

Genome sequence report
The genome was sequenced from one female Eilema caniola (Figure 1) (see Methods).A total of 39-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 21 missing joins or mis-joins and removed 4 haplotypic duplications, reducing the scaffold number by 7.14%.
The final assembly has a total length of 781.7 Mb in 51 sequence scaffolds with a scaffold N50 of 25.9 Mb (Table 1).
The snailplot 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 (99.71%)

Genome annotation
Number of the assembly sequence was assigned to 31 chromosomallevel scaffolds, representing 29 autosomes and the W and Z sex chromosomes.The order and orientation of the W chromosome is or uncertain order and orientation.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.
The estimated Quality Value (QV) of the final assembly is 65.1 with k-mer completeness of 100%, and the assembly has a BUSCO Metadata for specimens, barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/ 987929.

Genome annotation report
The Eilema caniola genome assembly (GCA_949126895.1)was annotated using the Ensembl rapid annotation pipeline (Table 1; https://rapid.ensembl.org/Eilema_caniola_GCA_949126895.1/Info/Index).The resulting annotation includes 23,146 transcribed mRNAs from 22,953 protein-coding genes.3 system with speed setting 31 (Bates 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.

Sample acquisition and nucleic acid extraction
Protocols developed in the Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

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 instrument.Hi-C data were also generated from remaining head and thorax tissue of ilEilCani1 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 corrected 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.
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

Chiara Bortoluzzi
Department of Environmental Systems Science, ETH Zurich (Ringgold ID: 27219), Zürich, Zürich, Switzerland In this genome note, the Darwin Tree of Life consortium report the sequencing of the Hoary Footman (Eilema caniola) genome, a species of moth endemic to the United Kingdom.This genome is a high quality one and will play a significant role in resolving or confirming the evolutionary relationships of Eilema caniola.
The report is very well written and datasets are presented in a clear manner.The data is readily accessible to the public through both ENA and NCBI, with all provided links functioning properly.The values presented in tables are accurate and align with the information available in public repositories.
I suggest the following changes to the main text: Page 3/11: Replace "Kidney Vetch" with "kidney vetch" 1.
Replace "Although thus identified individuals" with "Although these identified individuals" 2.
Replace "if not overall classification" with "if not the overall classification" 3.
Page 5/11: Replace "The order and orientation of the W chromosome is or uncertain order and orientation" with "The order and orientation of the W chromosome is of uncertain order and orientation" Page 7/11: Replace "ilAnaCroc1" with "ilEilCani1" Change the orientation of the label on the y-axis of Figure 3  Reviewer Expertise: Comparative genomics, population genomics, conservation genomics 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 Eilema caniola, ilEilCani1.1: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 781,750,262 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 (51,636,824 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (25,911,362 and 17,159,098 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/Eilema%20caniola/dataset/CASBPR01/snail.
A female Eilema caniola (specimen ID NHMUK013698301, ToLID ilEilCani1) was collected by hand from Restharrow Dunes National Nature Reserve, Sandwich Bay, England, UK (latitude 51.27, longitude 1.38) on 2021-09-24.The specimen was collected and identified by David Lees (Natural History Museum) and preserved by dry freezing at -80°C.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 ilAnaCroc1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing (Jayet al., 2023).Tissue from the whole organism was homogenised using a PowerMasher II tissue disruptor(Denton et al., 2023a).HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol(Oatley et al., 2023).The DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor

Figure 5 .
Figure 5. Genome assembly of Eilema caniola, ilEilCani1.1:Hi-C contact map of the ilEilCani1.1 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=cu-2fCbuQ1yiZr4aQHR1pQ.
and Figure 4 to improve readability.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.

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:
The data generated in this article were deposited in public databases.The link to access the datasets and annotations were provided in the manuscript.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.