The genome sequence of the Blue-bordered Carpet moth Plemyria rubiginata (Denis & Schiffermüller) 1775

We present a genome assembly from an individual female Plemyria rubiginata (the Blue-bordered Carpet moth; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 356.2 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 17.64 kilobases in length.

Two subspecies have traditionally been recognised in the United Kingdom: P. rubiginata rubiginata with a whitish background and blue border, and a large brown blotch on the leading edge of the wing; and 'subspecies' P. rubiginata plumbata (Curtis, 1837), distinguished by the brown blotch extending to become a full crossband, a darker and brownish border, and a ground colour of greyish-beige (Waring et al., 2017).However, the latter is internationally treated as f.plumbata (possibly an example of increasing northwards melanism at higher latitudes and elevations in northern England and Scotland, also in Norway and possibly Japan).By contrast, P. rubiginata japonica Inoue, 1955 is treated as validly distinct from the nominotypical subspecies from eastern Siberia and Mongolia to Japan (Hausmann & Viidalepp, 2012).Randle et al. (2019) show a continuous distribution, although P. rubiginata is more scattered in Scotland and more localised in Ireland.Broadly, the species spans from 0-1600 m across almost the entire Palaearctic from north-western Portugal and Spain to China and Japan and the southern shores of the Mediterranean to northern Scandinavia and north-western Russia, with an isolated population in Turkey (GBIF Secretariat, 2024;BOLD accessed 08/04/2024;Hausmann & Viidalepp, 2012).
Despite not being a species observed in large numbers, the Blue-bordered Carpet has shown a dramatic increase in abundance of 173% since 1970, although no change in distribution (Randle et al., 2019).The moth is univoltine, flying between early June and late September in the UK with some sign of a shift to an earlier modal abundance (Randle et al., 2019); and flying from mid-July at higher elevations in Europe (Hausmann & Viidalepp, 2012).P. rubiginata overwinters as an egg on the twigs of its foodplant, and the greenish larvae with yellow lateral stripes feed among spinnings on twig from April to June, preferring alder Alnus glutinosa (L.) Gaertn.or blackthorn Prunus spinosa L., but are also found on apple Malus spp., plum Prunus spp., hawthorn Crataegus monogyna Jacq.and birch Betula spp.(Sterling et al., 2012;Waring et al., 2017) foodplants also utilised by the Nearctic sister species, P. georgii Hulst, 1896. (Choi, 1998), P. rubiginata occupies a single BIN cluster on BOLD (08/04/2024), BOLD:AAC1712, with up to 2.77% pairwise divergence within it (n = 33), with Russian and Chinese exemplars showing highest divergences (~1.43% to the nearest exemplars).There are eight exemplars DNA barcoded from the UK (08/04/2024), the most northerly being from Lancashire but none so far from Scotland and Ireland, but at present there seems to be no mitochondrial evidence for genetic distinctiveness of northern UK populations.The Nearctic P. georgii (BOLD:AAB0097) by contrast is at least 5.65% divergent from it (although a species of Chloroclystis, BOLD:AFD5638, approaches P. rubiginata more closely at 4.17%).
The morphology and taxonomy of Plemyria Hübner, 1825 was reviewed by Choi (1998) andHausmann &Viidalepp (2012), the genus comprising just the above two species.Plemyria belongs to the larentiinae tribe Cidariini.Based on a morphological analysis of adults and immatures, Choi (1997) placed the genus adjacent to Chloroclystis Hübner, 1825 and sister to Thera Stephens, 1831+ (Pennithera Viidalepp, 1980+ Heterothera Inoue, 1943).The genus was not, however, included in the molecular larentiine phylogeny of Õunap et al. (2016).The genome will be useful in investigating industrial melanism, stimulating more DNA barcoding, and clarifying the phylogeography of the species and the history of its sister species split, as well as establishing the phylogenetic placement of Plemyria.

Genome sequence report
The genome was sequenced from one female Plemyria rubiginata (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.76,.A total of 94-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 15 missing joins or mis-joins and removed 8 haplotypic duplications, reducing the assembly length by 1.07% and the scaffold number by 9.80%. The final assembly has a total length of 356.2 Mb in 45 sequence scaffolds with a scaffold N50 of 12.8 Mb (Table 1).The snail plot 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 (99.87%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 28 autosomes and the Z and W sex chromosomes.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 63.1 with k-mer completeness of 100.0%, and the assembly has a BUSCO v completeness of 98.5% (single = 97.9%,duplicated = 0.6%), using the lepidoptera_odb10 reference set (n = 5,286).

Sample acquisition and nucleic acid extraction
A female Plemyria rubiginata (specimen ID Ox000535, ToLID ilPleRubi1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.76, longitude -1.34) on 2020-06-25 using a light trap.The   HMW DNA was extracted using the Automated MagAttract v1 protocol (Sheerin et al., 2023).DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic 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.
RNA was extracted from tissue of ilPleRubi2 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).
The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the WSI 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.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 IIe (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from tissue of ilPleRubi2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Final assembly evaluation
The final assembly was post-processed and evaluated with the three Nextflow (Di Tommaso et al The sanger-tol/blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline (Challis et al., 2020).It aligns the PacBio reads with SAMtools and minimap2 (Li, 2018) and generates coverage tracks for regions of fixed size.In parallel, it queries the GoaT database (Challis et al., 2023) to identify all matching BUSCO lineages to run BUSCO (Manni et al., 2021).For the three domain-level BUSCO lineage, the pipeline aligns the BUSCO genes to the Uniprot Reference Proteomes database (Bateman et al., 2023) with DIAMOND (Buchfink et al., 2021) blastp.The genome is also split into chunks according to the density of the BUSCO genes from the closest taxonomically lineage, and each chunk is aligned to the Uniprot Reference Proteomes database with DIAMOND blastx.Genome sequences that have no hit are then chunked with seqtk and aligned to the NT database with blastn (Altschul et al., 1990).All those outputs are combined with the blobtools suite into a blobdir for visualisation.
All three pipelines were developed using the nf-core tooling (Ewels et al., 2020)  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

Varun Arya
Entomology & Agricultural Zoology, Banaras Hindu University (Ringgold ID: 30114), Varanasi, Uttar Pradesh, India This study presents a comprehensive genomic assembly of Plemyria rubiginata, commonly referred to as the 'blue-bordered carpet moth.'As present in the manuscript, the assembled sequences were successfully mapped to both autosomes and sex chromosomes.

Minor comments:
Please make the following changes in the keywords: italicize "Plemyria rubiginata."Keep "Blue-bordered Carpet moth" in lower case, also in other places in the manuscript while writing.The name of the insect orders, if used in the middle of the sentence, shall not be capitalized.Write "Lepidoptera" in lower case.

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In the background: instead of "([Denis & Schiffermüller], 1775)", write it as "(Denis & Schiffermüller, 1775)".Also correct it in the title.In the manuscript, "However, the latter is internationally treated as f.plumbata", the identity of "f.plumbata" is not clear if the authors have mentioned it as a distinct sub species or is it a typing error.The citation of "([Denis & Schiffermüller], 1775)" can be rewritten as "(Denis & Schiffermüller, 1775)" in both the background and the title.○ Instead of ".. treated as f.plumbata," do you mean "treated as P. plumbata" ?○ This might be better: "By contrast, P. rubiginata japonica Inoue, 1955, from eastern Siberia and Mongolia to Japan, is treated as validly distinct from the nominotypical subspecies (Hausmann & Viidalepp, 2012).

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From the sentence construction, is not clear which food plants are also utilized by P. georgii.All of them?Please make this more clear.

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This might be better: "The morphology and taxonomy of Plemyria Hübner, 1825, the genus comprising just the above two species, was reviewed by Choi (1998) and Hausmann & Viidalepp (2012)." ○ Is the rationale for creating the dataset(s) clearly described?

Figure 2 .
Figure 2. Genome assembly of Plemyria rubiginata, ilPleRubi1.1:metrics.The BlobToolKit snail plot 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 356,234,577 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 (15,641,424 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (12,846,343 and 8,926,186 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/Plemyria_rubiginata/dataset/GCA_963576535.1/snail.

Figure 3 .
Figure 3. Genome assembly of Plemyria rubiginata, ilPleRubi1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Plemyria_rubiginata/dataset/GCA_963576535.1/blob.

Figure 4 .
Figure 4. Genome assembly of Plemyria rubiginata, ilPleRubi1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Plemyria_rubiginata/dataset/GCA_963576535.1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Plemyria rubiginata, ilPleRubi1.1:Hi-C contact map of the ilPleRubi1.1 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=N6e0u10NSg-QOuCMZCLXhQ.

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Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Background:Change to, "…Plemyria rubiginata (Denis & Schiffermüller, 1775).

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