The genome sequence of the meadow brown, Maniola jurtina (Linnaeus, 1758)

We present a genome assembly from an individual female Maniola jurtina (the meadow brown; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 402 megabases in span. The complete assembly is scaffolded into 30 chromosomal pseudomolecules, with the W and Z sex chromosome assembled. Gene annotation of this assembly on Ensembl has identified 12,502 protein coding genes.


Introduction
The meadow brown Maniola jurtina is a common, Palearctic butterfly occurring throughout Europe, the Middle East, and North Africa (gbif.org, 2021). Both widespread and often abundant, the species is associated with almost any grassy habitats (South, 1906), reaching highest densities in areas where grazing or other pressures keep the sward at an intermediate height (Maitland Emmet & Heath, 1989). Although M. jurtina is consistently univoltine, emergence occurs over a prolonged period in summer, which varies in length geographically and with habitat types (Brakefield, 1987). In many Mediterranean populations, females aestivate during the hottest months of the year (Scali, 1971). Eggs are laid singly or in small clusters, both on individual blades of grass or loose into a suitable tuft (Maitland Emmet & Heath, 1989), preferentially on Poa, Agrostis and Lolium. Larvae overwinter, but do not undergo true diapause, and feed intermittently in warm spells. Pupae show considerable variation in colouration which is affected by light and temperature (Brakefield, 1979). The species exhibits a great deal of phenotypic variation both within and between populations (Thomson, 1969). Four sub-species are known from the British Isles -ssp. splendida, insularis, iernes, and cassiteridum -although the validity of these taxa is questionable (Weir & Others, 2016), since they seem to be phenotypic extremes at opposing ends of clines (Maitland Emmet & Heath, 1989). In their pioneering work in ecological genetics, Ford and Dowdeswell considered the evolutionary factors shaping variation in the spot patterning of the underside of the hindwings in M. jurtina, initially on the Isles of Scilly, then the British mainland (reviewed in (Ford, 1964) and (Dowdeswell, 1981). Several early studies (Bigger, 1960;Federley, 1938;Lorković, 1941), summarised in (Robinson, 1971, report a karyotype of 29 chromosomes. The genome size has been estimated as 367.3 Mb (Mackintosh et al., 2019). We note the publication of a de novo genome assembly of M. jurtina by (Singh et al., 2020) and believe that the sequence described here, generated as part of the Darwin Tree of Life project, will further aid understanding of the biology of this butterfly.

Genome sequence report
The genome was sequenced from a female M. jurtina (ilManJurt1; Figure 1A, B) collected from Aberlady Bay, East Lothian, Scotland, UK (latitude 56.019964, longitude -2.85808). Hi-C data were generated from another individual (ilManJurt3; Figure 1E, F) collected from East Linton, East Lothian, Scotland, UK (latitude 55.977161, longitude -2.667545). A total of 76-fold coverage in Pacific Biosciences single-molecule long reads (N50 14 kb) and 88-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 24 missing/misjoins and removed two haplotypic duplications, reducing the assembly size by 1.67% and scaffold number by 24.39%.
The final assembly has a total length of 402 Mb in 30 sequence scaffolds with a scaffold N50 of 15 Mb (Table 1). Of the assembly sequence, 100% was assigned to 30 chromosomal-level scaffolds, representing 28 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 98.3% using the lepidoptera_odb9 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 Maniola jurtina assembly (GCA_905333055.1, see https://rapid.ensembl.org/Maniola_jur-tina_GCA_905333055.1/; 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.
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 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 Chromium read cloud sequencing libraries were constructed according to the manufacturers' instructions. Poly(A)    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.   This article reports the genome assembly of a Maniola jurtina female, allowing access to both the autosomes and the W and Z sexual chromosomes. This butterfly observed in Europe in particular, has raised interest for its ecology. The article clearly written and particularly pleasant to read, It shows convincing evidences for a high-quality assembly. The methods for genome assembly, quality test and Hi-C scaffolding are relevant and up-to-date. Maybe the authors could also add the default parameters used during the hifiasm run and particularly if any purging option was used. They may also provide the version of the softwares used, as well as the percentage of BUSCO proteome complete/fragmented.
The article also describes mitogenome assembly with mito-hifi, therefore providing a complete view about genomic content for this species. Given our own interest about butterfly genomes, we would be keen on knowing the genome-wide level of heterozygosity, as well as the TE content per chromosomes and throughout the whole genome.
Overall, we think the release of this well-assembled and annotated genome is a useful contribution and we recommend the publication of this article. Violaine Llaurens & Manuela Lopez-Villavicencio.

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