The genome sequence of the speckled wood butterfly, Pararge aegeria (Linnaeus, 1758) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual female Pararge aegeria (the speckled wood butterfly; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 517 megabases in span. The majority of the assembly (99.68%) is scaffolded into 29 chromosomal pseudomolecules, with the W and Z sex chromosome assembled. Gene annotation of this assembly on Ensembl has identified 12,288 protein coding genes.


Introduction
The speckled wood butterfly, Pararge aegeria (Linnaeus, 1758), is a common woodland species that occurs across the Palearctic, from north Africa to Europe and western Asia, reflecting wide environmental tolerances. Pararge aegeria is a multivoltine species, producing a variable number of generations each year and can over-winter in two life stages; as a larva or pupa, using a variety of grasses as the larval host plant (Shreeve, 1986). Two main lineages are recognised: subspecies aegeria in north Africa (Linnaeus, 1758) and tircis in Eurasia (Godart, 1821;Livraghi et al., 2018). Two additional subspecies, oblita (western Scotland) (Harrison, 1949) and insula (Isles of Scilly) (Howarth, 1971), have been described based on wing pattern variation. The distribution of P. aegeria in the UK has been dynamic in recent history: the range contracted from a widespread distribution, due to cooler climates in the 1950s, to south-west England and Wales, as well as a refugial population in western Scotland (Hill et al., 1999). Since the 1970s, a warming climate has enabled P. aegeria to expand its range and to recolonise northern England and the whole of Scotland. Morphological differences in wing size, shape and colour have been described across the UK distribution (Taylor-Cox et al., 2020) and life-history evolution and plasticity have been studied extensively in this species (Nylin et al., 1995). The standard haploid karyotype of P. aegeria consists of 27 autosomes and one sex chromosome (Federley, 2010), and the female is heterogametic (WZ).

Genome sequence report
The genome was sequenced from a single female P. aegeria (ilParAegt1) collected from West Saltoun Forest, Scotland (latitude 55.884404, longitude -2.857514). Hi-C data were generated from a male P. aegeria (ilParAegt2) collected from the same location (Figure 1). A total of 42-fold coverage in Pacific Biosciences single-molecule long reads and 66-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 12 missing/misjoins, reducing the scaffold number by 2.78%.
The final assembly has a total length of 517 Mb in 70 sequence scaffolds with a scaffold N50 of 19 Mb (Table 1). Of the assembly sequence, 99.68% was assigned to 29 chromosomal-level scaffolds, representing 27 autosomes (numbered by sequence length), and the W and Z sex chromosome (Figure 2- Figure 5; Table 2). The assembly has a BUSCO (Simão et al., 2015) v5.1.2 completeness of 98.6% 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.

Gene annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Pararge aegeria assembly (GCA_905163445.1, see https://rapid.ensembl.org/Pararge_ aegeria_GCA_905163445.1/; Table 1). The annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019) and OrthoDB (Kriventseva et al., 2008). Prediction tools, CPC2 (Kang et al., 2017) and RNAsamba (Camargo et al., 2020), were used to aid determination of protein coding genes.

Sample acquisition and nucleic acid extraction
One female (ilParAegt1, genome assembly) and one male (ilParAegt2, Hi-C) P. aegeria specimen were collected from West Saltoun Forest, Scotland (latitude 55.884404, longitude -2.857514) by Konrad Lohse, University of Edinburgh. A further female specimen, ilParAegt3 (RNA-Seq), was collected from Yellowcraig, Scotland (latitude 56.062447, longitude 2.769836) by the same individual, who also identified both specimens. All samples were snap-frozen in liquid nitrogen.
DNA was extracted from the whole organism of ilParAegt1 at the Wellcome Sanger Institute (WSI) Scientific Operations core from the whole organism using the Qiagen MagAttract HMW DNA kit, according to the manufacturer's instructions. RNA (also from the whole organism) was extracted from ilParAegt3 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 RNA 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 and 10X Genomics read cloud 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 II (HiFi), Illumina HiSeq X (10X) and Illumina HiSeq 4000 (RNA-Seq) instruments. Hi-C data were generated from the whole organism of ilParAegt2 using the Qiagen EpiTect Hi-C kit and sequenced on HiSeq X.

Genome assembly
Assembly was carried out with Hifiasm (Cheng et al., 2021). Haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020). One round of polishing was performed by aligning 10X Genomics read data to the assembly with longranger align, calling variants with freebayes (Garrison & Marth, 2012). The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using SALSA2 (Ghurye et al., 2019).
The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021). Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext.  The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2021). The genome was analysed and BUSCO scores generated within the BlobToolKit environment (Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.

Ethical/compliance issues
The materials that have contributed to this genome note were 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,  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 genome sequence is released openly for reuse. The P. aegeria 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 Table 1.