The genome sequence of the Glanville fritillary, Melitaea cinxia (Linnaeus, 1758)

We present a genome assembly from an individual male Melitaea cinxia (the Glanville fritillary; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 499 megabases in span. The complete assembly is scaffolded into 31 chromosomal pseudomolecules, with the Z sex chromosome assembled. Gene annotation of this assembly on Ensembl has identified 13,666 protein coding genes.


Species taxonomy Introduction
The Glanville fritillary (Melitaea cinxia) is a non-migratory butterfly named after the naturalist, Lady Eleanor Glanville, and the distinctive chequered orange and brown markings on the underside of its wings. This species forms discrete colonies and inhabits dry meadows containing its host plants Plantago and Veronica, across North Africa, Europe and temperate Asia (Wahlberg & Saccheri, 2007). M. cinxia shows strong phylogeographic structure in the mitochondrial DNA, consisting of four major clades across its range; Mococco, Western, Central and Eastern (Wahlberg & Saccheri, 2007). In the British Isles, colonies are virtually restricted to coastal regions on the southern half of the Isle of Wight and the Channel Islands, in addition to a few mainland coastal locations. Over the past 50 years, this species has faced a sharp decline in the UK (Fox et al., 2015). However, it is listed as Least Concern in the IUCN Red List (Europe) (Van Swaay et al., 2010). This species is univoltine, except for a few bivoltine populations; adults can be seen in flight from April to July and occasionally in August, and larvae diapause over winter. A large metapopulation of M. cinxia in the Åland archipelago of Finland, covering 4,000 dry meadows, is an established model system for studies focusing on the effects of habitat fragmentation on ecology, genetics and evolution (Hanski, 1999;Hanski, 2011). The first reference genome for M. cinxia (N50=331 kb) was used to demonstrate remarkable conservation of chromosome synteny across distantly-related lepidopteran species (Ahola et al., 2014). M. cinxia has a karyotype of 31 chromosomes (Federley, 1938).

Genome sequence report
The genome was sequenced from a single male M. cinxia collected from El Brull, Catalunya, Spain (latitude 41.8103, longitude 2.3054) (Figure 1). A total of 28-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 69 missing/ misjoins and removed 10 haplotypic duplications, reducing the assembly size by 0.97% and scaffold number by 56.94%, and increasing the scaffold N50 by 11.21%.
The final assembly has a total length of 499 Mb in 32 sequence scaffolds with a scaffold N50 of 12 Mb (Table 1). Of the assembly sequence, 100% was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes (numbered by sequence length), and the Z sex chromosome (Figure 2- Figure 5; Table 2). The assembly has a BUSCO (Simão et al., 2015) v5.1.2 completeness of 98.4% 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.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Melitaea cinxia assembly (GCA_905220565.1, see https://rapid.ensembl.org/Meli-taea_cinxia_GCA_905220565.1/; 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.

Methods
Sample acquisition, nucleic acid extraction and sequencing A single male M. cinxia was collected from El Brull, Catalunya, Spain (latitude 41.8103, longitude 2.3054) by Roger Vila (Institut de Biologia Evolutiva, CSIC-UPF), Alex Hayward (University of Exeter) and Konrad Lohse (University of Edinburgh). The specimen was collected using a net, identified by Roger Vila and flash-frozen in liquid nitrogen.
DNA was extracted from the whole organism of ilMelAtha1 using the Qiagen MagAttract HMW DNA kit in the Scientific Operations core at the Wellcome Sanger Institute (WSI), according to the manufacturer's instructions. Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud sequencing libraries were constructed according to the manufacturers' instructions. Sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II and Illumina HiSeq X instruments. Hi-C data were generated using the Arima v2.0 kit and sequenced on an Illumina NovaSeq 6000 instrument.   Pretext. Regions of concern were identified and resolved using 10X longranger and genetic mapping data. The genome was analysed 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, 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; • Legality of collection, transfer and use (national and international).
Each transfer of samples is undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Tree of Life collaborator, Genome Research Limited (operating as the Wellcome Sanger Institute) and in some circumstances other Tree of Life collaborators.
The genome sequence is released openly for reuse. The A. urticae 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.
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