The genome sequence of the painted lady, Vanessa cardui Linnaeus 1758

We present a genome assembly from an individual female Vanessa cardui (the painted lady; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 425 megabases in span. The majority of the assembly is scaffolded into 32 chromosomal pseudomolecules, with the W and Z sex chromosome assembled. Gene annotation of this assembly on Ensembl has identified 12,821 protein coding genes.


Background
The painted lady, Vanessa cardui, is an extremely widespread butterfly, occurring on all continents except most of South America and Oceania (Shields, 1992). The species undertakes long-distance multi-generational migrations each year (Pollard et al., 1998;Stefanescu et al., 2013;Talavera et al., 2018;Williams, 1970). It does not overwinter and is therefore engaged in a constant movement. In the Palaearctic, migrants are known to seasonally circulate between North Africa and Europe (Pollard et al., 1998;Stefanescu, 2011;Stefanescu et al., 2013). Recent work has also revealed that autumn populations from Europe cross the Sahara Desert reaching tropical Africa (Stefanescu et al., 2016;Talavera & Vila, 2016). This journey, spanning over 4000 km, represents the longest single-leg migratory flight known in butterflies. The butterflies migrate back to Europe in spring, thus covering up to 14000 km in an annual cycle involving 8-10 generations in their Palaearctic-African range (Menchetti et al., 2019;Talavera et al., 2018). The painted lady is found throughout the British Isles but abundance varies greatly between years. Larvae are polyphagous on a large variety of plant families, but most commonly feed on thistles (Cirsium spp. and Carduus spp.) and mallows (Malva spp.). The painted lady occurs in a wide range of biomes and environments spanning semi-deserts, grasslands, meadows, and mountains to suburban areas. It is listed as Least Concern in the IUCN Red List (Walker & Coetzer, 2020). Studies of V. cardui have included thermoregulation (Tsai et al., 2020), adaptations to host plants (Celorio-Mancera et al., 2016), flight behaviour (Gamberale-Stille et al., 2019;Liu et al., 2021) and movement ecology (Suchan et al., 2018). Genes involved in the development of the distinctive eyespots on the forewings and hindwings of V. cardui have been identified (Mazo-Vargas et al., 2017;Zhang & Reed, 2016;Zhang et al., 2017). V. cardui has a karyotype of 31 chromosomes (Lorkovic, 1941).
We note the recent publication of another high-quality genome assembly for V. cardui (Zhang et al., 2021). We hope that the sequence described here, generated as part of the Darwin Tree of Life project, will further contribute to the study of V. cardui as an emerging model for the genetics of migratory behavior, ecological genomics and developmental genetics.

Genome sequence report
The genome was sequenced from a single female V. cardui (ilVanCard2; Figure 1A, B) collected from Carrifran Wildwood, Figure 1. Fore and hind wings of Vanessa cardui specimens from which the genome was sequenced. (A) Dorsal surface view of wings from specimen SC_VC_1208 (ilVanCard2) from Carrifran Wildwood, Scotland used to generate Pacific Biosciences and 10X genomics data. (B) Ventral surface view of wings from specimen SC_VC_1208 (ilVanCard2) from Carrifran Wildwood, Scotland, used to generate Pacific Biosciences and 10X genomics data. (C) Dorsal surface view of wings from specimen SC_VC_1220 (ilVanCard3) from Yellowcraig, Scotland, used to generate Hi-C data. (D) Ventral surface view of wings from specimen SC_VC_1220 (ilVanCard3) from Yellowcraig, Scotland, used to generate Hi-C data.
Scotland (latitude 55.400132, longitude -3.3352). Hi-C data were generated from a second female V. cardui (ilVanCard3; Figure 1C, D) collected from Yellowcraig, East Lothian, Scotland (latitude 56.062445, longitude -2.769836). A total of 25-fold coverage in Pacific Biosciences single-molecule long reads (N50 15 kb) and 89-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 79 missing/misjoins and removed 7 haplotypic duplications, reducing the assembly size by 0.48% and scaffold number by 61.70%, and increasing the scaffold N50 by 21.74%.
The final assembly has a total length of 425 Mb in 37 sequence scaffolds with a scaffold N50 of 15 Mb (Table 1). Of the assembly sequence, 96.0% was assigned to 32 chromosomallevel scaffolds, representing 30 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) completeness of 98.8% 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.

Gene annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Vanessa cardui assembly (GCA_905220365.1, see https://rapid.ensembl.org/Vanessa_ cardui_GCA_905220365.1/; 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
The first female V. cardui, ilVanCard2 (genome assembly), was collected from Carrifran Wildwood, Scotland (latitude 55.400132, longitude -3.3352). Two further female V. cardui specimens, ilVanCard1 (RNA-Seq) and ilVanCard3 (Hi-C), was collected from Yellowcraig, East Lothian, Scotland (latitude 56.062445, longitude -2.769836). All samples were collected and identified by Konrad Lohse, University of Edinburgh, and were snap-frozen from life in liquid nitrogen.
DNA was extracted 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 was extracted in the Tree of Life Laboratory at the WSI using TRIzol (Invitrogen), according to the manufacturer's instructions. RNA was then eluted in 50 μl RNAse-free water and its concentration RNA assessed      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;

Nicholas VanKuren
University of Chicago, Chicago, USA Lohse and colleagues assembled and annotated the genome sequence of a cosmopolitan butterfly, Vanessa cardui, and present general statistics on its quality. WSI ToL has published many short reports like this recently, and this manuscript follows a similar format as those before. Despite redundancy with an available V. cardui sequence, this genome sequence and annotation are high-quality and will be of interest to butterfly genomics, population genomics, and genetics researchers.
Very minor comments: Fig. 1: It would be nice to note that these are all females in the figure, or at least in the legend.
○ Results: BUSCO completeness in text and Fig. 2 listed as 98.8%, but Table 1 shows 98.2%. ○ Fig. 5: it would be nice to point out the W and Z here so that people can immediately visualize.

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Gene annotation: Perhaps this has been listed in previous publications, but what is the "select set of proteins from UniProt and OrthoDB"? This would be useful to know for other groups looking to annotate their genomes. In general, there should be a section on gene annotation in the Methods giving a few more details on important parameters used for predictions.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?