The genome sequence of the Ruby Tiger, Phragmatobia fuliginosa (Linnaeus, 1758)

We present a genome assembly from an individual male Phragmatobia fuliginosa (the Ruby Tiger; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 629.4 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.4 kilobases in length. Gene annotation of this assembly on Ensembl identified 13,338 protein coding genes.


Background
The ruby tiger Phragmatobia fuliginosa is a distinctive moth in the subfamily Arctiiinae, the only representative of its genus recorded in the UK.In southern Britain, adult moths have pinkish-red or pinkish-brown forewings and mostly bright pink hindwings that are usually hidden when the moth is settled.Moths from northern Britain are generally darker and have been placed in the subspecies borealis (Staudinger) (Waring et al., 2017).
Phragmatobia fuliginosa has a range that extends across much of Europe and Asia, as well as parts of northern North America (GBIF Secretariat, 2022).It has a wide distribution in Great Britain and Ireland, occurring mostly in in open habitats, and is absent only from Shetland.Adults are occasionally active during the day but are more likely to be recorded at light (South, 1961).In northern Britain there is typically a single annual generation (Waring et al., 2017), but in southern Britain there are usually two generations, with adult moths recorded in small numbers from April until June, and in much higher numbers during July and August (Randle et al., 2019).The apparent high abundance of the second generation relative to the first may in part result from the late summer generation being more attracted to light traps (Waring et al., 2017).
The spherical white eggs of P. fuliginosa are deposited in batches, and the larvae are polyphagous, consuming a wide variety of mostly herbaceous plants, with a particular fondness for ragworts (Senecio spp.) (Henwood et al., 2020).The hairy larvae overwinter fully-grown.South (1961) comments that "the vitality of caterpillars is extraordinary", reporting an observation of a larva that was embedded in ice for at least 14 days without apparent harm.In the spring, the dark-coloured larvae bask in sunshine to raise their body temperature well above ambient, and the speedy larvae are often observed crossing roads and paths.
Male pheromones used in P. fuliginosa courtship are derived from pyrrolizidine alkaloids (PAs) obtained during larval feeding (Krasnoff & Roelofs, 1990).A genome sequence for Phragmatobia fuliginosa will facilitate studies into molecular adaptations to polyphagy, the evolution of pheromone-based courtship, and contribute to a growing data set of resources for understanding lepidopteran biology more widely.
The genome of Phragmatobia fuliginosa 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 Phragmatobia fuliginosa, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Phragmatobia fuliginosa specimen (Figure 1) collected from Wytham Woods, Oxfordshire, UK (latitude 51.77, longitude -1.34).A total of 35-fold coverage in Pacific Biosciences single-molecule HiFi long was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 29 missing joins or mis-joins and removed seven haplotypic duplications, reducing the assembly length by 2.82% and the scaffold number by 15.79%, and decreasing the scaffold N50 by 2.33%.
The final assembly has a total length of 629.4 Mb in 32 sequence scaffolds with a scaffold N50 of 22.9 Mb (Table 1).Most (99.97%) of the assembly sequence was assigned to 28 chromosomal-level scaffolds, representing 27 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).The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 98.7% (single 97.9%, duplicated 0.8%) using the lepidoptera_odb10 reference set.While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
A male P. fuliginosa specimen (ilPhrFuli1) was collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire) (latitude 51.77, longitude -1.34) on 13 June 2020.The specimen was taken from woodland habitat by Douglas Boyes (University of Oxford) using a light trap.The specimen was identified by Douglas Boyes using field ID and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute.The ilPhrFuli1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Abdomen tissue was cryogenically disrupted to a

Andrew Mongue
Entomology and Nematology, University of Florida, Gainesville, Florida, USA A concise and well-written example of a genome report.The manuscript is perfectly intelligible and suitable for indexing as is.In particular, I appreciate the table format for datasets, accessions, tools, and version numbers.I have only a couple of minor suggestions, ordered by importance: Chromosome number.The authors report n = 28 chromosomal scaffolds.The ancestral lepidopteran karyotype is thought to be n = 31, so this suggests a number of fusion events in the lineage leading to ruby tigers.I recognize it as beyond the scope of this paper to identify which syntenic units fused, but I believe it is worth mentioning as a point of future study, given the research interest in comparative lepidopteran genomics.

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The background on life-history includes the phrase "the hairy larvae overwinter fullygrown".The phrasing is slightly ambiguous, as "fully-grown" often colloquially means "mature" or "adult", but I take the authors to mean that ruby tigers overwinter as last-instar caterpillars, as becomes apparent in the next sentence, but I still feel that "fully-grown" is a somewhat ambiguous term to use.

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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?

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Genome assembly, Lepidoptera, Hemiptera, evolutionary genetics, population 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.
Boyes and Lewis present a chromosome scale assembly of a a single male moth (Phragmatobia fulginosa) native to Britain, which was sequenced as part of the Darwin Tree of Life Project.The primary reads were generated using PacBio HiFI and HiC was performed for chromosomal scaffolding.They were able to isolate the putative Z sex chromosome and the mitochondrial genome.
Although the methods are sound and the assembly seems pretty typical given the species/technologies, I have three comments to aid in clarity for future readers and to put this paper in context.As such I have reservations as I would like these relatively small things to be addressed but also admit they are minor clarifications (#1 and #2) and addition of some more citations/context (#3), in large part since a big result I get out of this is you can use HiFi and HiC to assemble Lepidopteran genomes pretty well.
First, there are no details in the manuscript how they determined which of the scaffolds was the Z scaffold.If this is because it is established in Leps that the largest scaffold is the sex chromosome they should add a citation, or if there are genetic markers that would help to assign the scaffolds to previously published linkage groups.I believe heterochromatic sex chromosomes was published in the early 1900s using this species 1 but not sure what types of more traditional genetics has been done since.
Second, it is unclear from the text how the authors computed k-mer completeness presented in Table 1 since it isn't defined as a footnote and doesn't appear in other legends where BlobToolkit is referred to for the "traditional" assembly quality measures (e.g., Figure 2).Please make it clear how this was computed either as a footnote or in the methods.
Finally, I don't expect any new biology in an open research note but the authors should put their work in context.For example, a PubMed search of "molecular adaptations to polyphagy" returns over 180 results, including crop pests that are probably better studied like fall army worm 2 and other Leps.Are there unique life history or other aspects that could be explored in the future, other than adding another Lep to (future) comparative genome analysis?Also, I know a lot of work on pheromones has been done in Heliconius (e.g., Byers et al., 2021 3 to cite a more recent one).Some citations and context would help clarify why this could be an important resource other than being one of the many genomes sequenced by the Darwin Tree of Life project.

Figure 2 .
Figure 2. Genome assembly of Phragmatobia fuliginosa, ilPhrFuli1.1: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 629,457,366 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 (81,383,725 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (22,865,098 and 15,039,256 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/ilPhrFuli1.1/dataset/CAKOBC01/snail.

Figure 5 .
Figure 5. Genome assembly of Phragmatobia fuliginosa, ilPhrFuli1.1:Hi-C contact map.Hi-C contact map of the ilPhrFuli1.1 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=Cc6lMkieRfalxi0HQfR6Xw.

Table 2 . Chromosomal pseudomolecules in the genome assembly of Phragmatobia fuliginosa, ilPhrFuli1. INSDC accession Chromosome Size (Mb) GC%
(Kerpedjiev et al., 2018)and Pretext (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2022), which performed annotation using MitoFinder (Allio et al., 2020).The genome was analysed, and BUSCO scores were generated within the BlobToolKit environment (Challis et al., 2020).Table3contains a list of all software tool versions used, where appropriate.Ethics and compliance issuesThe 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.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.All efforts are undertaken to minimise the suffering of animals used for sequencing.Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.The genome sequence is released openly for reuse.The Phragmatobia fuliginosa genome sequencing initiative is part of the 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

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.

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.