The genome sequence of the Arctic Skipper, Carterocephalus palaemon (Pallas, 1771)

We present a genome assembly from an individual male Carterocephalus palaemon (the Arctic Skipper; Arthropoda; Insecta; Lepidoptera; Hesperiidae). The genome sequence is 394.5 megabases in span. The whole assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.78 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,032 protein coding genes.


Background
The Chequered Skipper (also known as the Arctic Skipper), Carterocephalus palaemon (Pallas, 1771), is a small butterfly that flies in rapid zigzagging dashes, inhabiting woodland glades or edges and wet meadows.It has a broadly Holarctic distribution that is often patchy, with relatively low local abundances.Phylogenomic analysis (Zhang et al., 2021) supports the distinction of three subspecies (or species): C. palaemon palaemon (Eurasia); C. palaemon skada (northern North America); and C. palaemon magnus (western North America).The Chequered Skipper is listed as a species of Least Concern both on the IUCN Red List (Europe) (Van Swaay et al., 2010) and the GB Red List (Fox et al., 2022).
In Britain, until its reintroduction into England starting in 2018, the species was restricted to an isolated population in western Scotland (source for the DToL genome assembly), having gone extinct from England in 1976(Asher et al., 2001), owing to the decline in coppicing and other changes to forestry practice.
Although mitochondrial DNA evidence suggests that the historical English population is closely related to the Scottish one (Joyce & Pullin, 2004), the source material used for the reintroduction into Rockingham Forest (Northamptonshire) came from Belgian sites with similar ecological conditions (Maes et al., 2019).In Scotland, the main host plant is Purple Moor-grass (Molinia caerulea), whereas in England it is Wood Small-reed (Calamagrostis epigejos) and False-brome (Brachypodium sylvaticum).
The karyotype of the Chequered Skipper is unknown.The Darwin Tree of Life reference genome we present here will facilitate non-invasive monitoring of genetic variability and ancestry in the reintroduced population.

Genome sequence report
The genome was sequenced from one male Carterocephalus palaemon (Figure 1) collected from Glasdrum National Nature Reserve,Scotland (56.57,.A total of 45-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 16 missing joins or mis-joins and removed one haplotypic duplication, reducing the scaffold number by 20%. The final assembly has a total length of 394.5 Mb in 44 sequence scaffolds with a scaffold N50 of 13.9 Mb (Table 1).The whole 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/218720.

Sample acquisition and nucleic acid extraction
The specimen used for genome sequencing was a male Carterocephalus palaemon (ilCarPala2), hand netted in Glasdrum National Nature Reserve, Scotland, UK (latitude 56.57, longitude -5.23) on 2021-06-01.The collectors were   Konrad Lohse, Sam Ebdon, Alex Mackintosh and Simon Martin (all University of Edinburgh).The specimen was identified by Konrad Lohse and then snap-frozen from live in a dry shipper.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilCarPala2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Whole organism tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle.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.

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 the Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from tissue of ilCarPala2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.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, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Carterocephalus palaemon assembly (GCA_944567765.1) in Ensembl Rapid Release.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Tree of Life collaborator.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

Jerome H L Hui
The Chinese University of Hong Kong, Hong Kong, Hong Kong In this Data Note, Lohse and colleagues reported the genome assembly of a male butterfly Carterocephalus palaemon (Pallas, 1771), which is also commonly known as the chequered skipper or arctic skipper.According to the UK BAP, it is protected under Schedule 5 of the Wildlife and Countryside Act 1981, with respect to sale only.Molecular data of this species are limited to few marker genes deposited to the NCBI database.Therefore, this new genome resource will be useful for further studies into many areas, including but not limited to understanding its population structure, ecology, insect-plant interaction, and evolution with other insects more widely.In addition, given it is a priority species listed in the UK BAP, the high-quality genomic resource provided in this study will also be useful for designing and carrying out its different conservation measures.
This genome resource is excellent from the summary statistics, with high BUSCO number scores, high sequence continuity (scaffold N50), and majority of sequences contained on the 30 pseudochromosomes (plus sex chromosome and mitochondrion).All in all, this is a valuable contribution by the Darwin Tree of Life.

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

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: phylogenetics, evolutionary biology, conservation biology, bioinformatics 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 Carterocephalus palaemon, ilCarPala2.1: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 394,538,208 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 (18,628,966 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (13,897,380 and 9,164,508 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/ilCarPala2.1/dataset/CALYMH01/snail.

Figure 5 .
Figure 5. Genome assembly of Carterocephalus palaemon, ilCarPala2.1:Hi-C contact map of the ilCarPala2.1 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=GepxHsyRR8-FnB77oidXAw.

Table 3
contains a list of relevant software tool versions and sources.

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