The genome sequence of the Red-green Carpet, Chloroclysta siterata (Hufnagel, 1767)

We present a genome assembly from an individual male Chloroclysta siterata (the Red-green Carpet; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 437.9 megabases in span. Most of the assembly is scaffolded into 21 chromosomal pseudomolecules including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.7 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,814 protein coding genes.


Background
The Red-green Carpet, Chloroclysta siterata, is a delicately patterned moth in the family Geometridae.The name 'carpet moth' for this and closely related species has no relation to their diet; instead, it derives from the forewing markings which comprise a series of irregular bands, considered to resemble the ornate patterns on woven rugs and carpets.In C. siterata, these bands are alternating shades of dark and light green, suffused with ruby red streaks.The wings are held flat against the surface at rest and may provide cryptic camouflage on bark and lichen.The green colouration is vivid in freshly emerged specimens but fades with age; this may be the basis for the specific name siterata, meaning 'pertaining to corn', a plant which also changes colour (Emmet, 1991) The species is widespread across northern and eastern Europe, and in the UK is found predominantly in southern England and Wales (NBN Atlas Partnership, 2021;GBIF Secretariat, 2022).Both distribution and abundance have shown large long-term increases -over six-fold since 1970 (Randle et al., 2019).C. siterata has an unusual life-cycle, being univoltine but with two flight periods.Most records of the adult moth in Britain and Ireland are from September to November, when they will visit ivy blooms or are attracted to light.These adults mate, and the mated females overwinter as adults, emerging to give a second flight period from April to June (Newman, 1869).In Italy, Austria and Finland, overwintering in caves has been recorded (Moog et al., 2021;Soderholm, 2022;Teobladelli, 2008).After egg-laying, the larvae develop through July and August, feeding on foliage of deciduous trees -primarily oak but also apple, cherry, rose, rowan, blackthorn and birch (South, 1961;Waring et al., 2017).
A genome sequence of C. siterata will be useful in analyses of molecular adaptations to polyphagy and as part of wider comparative studies into genome evolution in the Lepidoptera.

Genome sequence report
The genome was sequenced from one male Chloroclysta siterata (Figure 1) collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.34).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 two missing joins or mis-joins.
The final assembly has a total length of 437.9 Mb in 25 sequence scaffolds with a scaffold N50 of 24.9 Mb (Table 1).Most (99.97%) of the assembly sequence was assigned to 20 chromosomal-level scaffolds, representing 19 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/934828.

Sample acquisition and nucleic acid extraction
A male Chloroclysta siterata (ilChlSite2) was collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.34) on 8 October 2020.The specimen was taken from woodland habitat by Douglas Boyes (University of Oxford) using a light trap.The specimen was identified by the collector and snap-frozen on dry ice.DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilChlSite2 sample was weighed and dissected on dry ice with head tissue set aside for Hi-C sequencing.Thorax tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.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 solid-phase 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 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 abdomen tissue of ilChlSite2 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

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 were performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina HiSeq 4000 (RNA-Seq) instruments.Hi-C data were also generated from head tissue of ilChl-Site2 using the Arima v2 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 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  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.To evaluate the assembly, MerquryFK was used to estimate consensus quality (QV) scores and k-mer completeness (Rhie et al., 2020).The genome was analysed within the BlobToolKit environment (Challis et al., 2020) and BUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated.Table 3 contains a list of software tool versions and sources.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Chloroclysta

Hana Konvičková
Institute of Entomology, Biology Centre CAS, Ceske Budejovice, Czech Republic The paper is clearly written and I appreciate its briefness with no excess information.The authors aimed to sequence a genome of widespread moth Chloroclysta siterata.This species is polyphagous and the authors hope to reveal the molecular mechanisms enabling it.They used modern method od HiFi sequencing.The method and processing the data are accurate.I have only two minor comments: 1) Figure 1 -there is only one photograph, although the title says "photographs" 2) The origin of the sample is mentioned twice (Genome sequence report and Methods), which I believe is not necessary.

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: population genetics of animals, with focus on Lepidoptera 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.
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: Insect Physiology Biochemistry and Molecular Biology, Transcriptomics 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 Chloroclysta siterata, ilChlSite2.1: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 437,887,824 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 (31,593,026 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (24,933,739 and 16,571,927 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/ilChlSite2.1/dataset/CAKOAF01/snail.

Figure 5 .
Figure 5. Genome assembly of Chloroclysta siterata, ilChlSite2.1:Hi-C contact map of the ilChlSite2.1 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=V7ANhS-_Rd6CIbouTb-IJA.

Table 3 . Software tools: sources and versions.
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 Scientific Names of the British Lepidoptera -their History and Meaning.
Colchester: Harley Books, 1991.

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