The genome sequence of the Rock Grayling, Hipparchia semele (Linnaeus, 1758)

We present a genome assembly from an individual female Hipparchia semele (the Rock Grayling; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 403.4 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 15.22 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,540 protein coding genes.


Background
The Grayling (or Rock Grayling), Hipparchia semele (Linnaeus, 1758) (Figure 1), is a medium-sized xerothermophilous butterfly in the family Nymphalidae, occupying habitats with dry or rapidly draining soils, especially heathlands and coastal sand dunes, but also post-industrial sites (Tropek et al., 2017).It is broadly distributed across much of Europe, but has a primarily coastal distribution in northerly countries including the UK.It is declining across most of its range (Tropek et al., 2017), with habitat loss and fragmentation considered to be among the important anthropogenic drivers of change (De Ro et al., 2021).It has a fragmented distribution within its range and exhibits metapopulation dynamics (van Strien et al., 2011).Colonies can vary in size from tens of individuals to several thousand; the Darwin Tree of Life Project genome assembly presented here is from a specimen sourced from one of the largest UK colonies, on heathland in the New Forest, Hampshire.
Multiple races or subspecies of H. semele have been described within Britain alone (Eeles, 2019).Most notably, the population on the Great Orme, North Wales is distinct in both appearance and phenology (Dapporto et al., 2019;Middlebrook et al., 2019;Thompson, 1944).Variation in the size and position of wing ocelli (spots) is thought to be adaptive, and related to predator defence (Dapporto et al., 2019).Evidence also exists for local adaptation to climate (Roy et al., 2015), but the species has not advanced its phenology in recent years (Macgregor et al., 2019).The chromosome number described for a Finnish population is 29 (Federley, 1938), which is consistent with the number in the present genome assembly.

Genome sequence report
The genome was sequenced from one female Hipparchia semele collected from Beaulieu Heath,England (50.80,.A total of 58-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 assembly length by 1.23% and the scaffold number by 7.69% and decreasing the scaffold N50 by 1.04%. The final assembly has a total length of 403.4 Mb in 47 sequence scaffolds with a scaffold N50 of 14.6 Mb (Table 1).Most (99.89%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 28 autosomes and the W and Z sex chromosomes.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/111912.

Sample acquisition and nucleic acid extraction
Two female Hipparchia semele specimens were collected with permission from Beaulieu Heath, England, UK (latitude 50.80, longitude -1.50) on 2017-07-06 using a sweep net.The specimens were collected by Callum Macgregor (University of York) and formally identified by Ilik Saccheri (University of Liverpool) and stored at -80°C.One specimen (specimen ID SAN0001387, individual ilHipSeme1) was used for DNA sequencing, and a second specimen (specimen ID SAN0001388, individual ilHipSeme2) was used for Hi-C scaffolding.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilHipSeme1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Tissue from the whole organism was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.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 ud DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) et al., 2021;Simão et al., 2015) were calculated.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Hipparchia semele assembly (GCA_933228805.2) in Ensembl Rapid Release.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the

Daniel Berner
University of Basel, Basel, Switzerland Magregor and Saccheri here present a genome assembly for the Nymphalid butterfly Hipparchia semele.Based on PacBio HiFi long-read sequencing and Hi-C scaffolding, they achieve a top-quality assembly, which is then annotated using a standard pipeline.The methods are generally well described, and the results clearly presented.This new assembly certainly provides a valuable resource for future investigations.
I just have three minor suggestions: 1) 'Variation in the size and position of wing ocelli (spots)': one could be more precise by using 'eye spots' here; I guess this is what you refer to, right?
3) It is not fully clear to me which individual was used for Hi-C.First, you specify that 'One specimen (specimen ID SAN0001387, individual ilHipSeme1) was used for DNA sequencing, and a second specimen (specimen ID SAN0001388, individual ilHipSeme2) was used for Hi-C scaffolding.'But in the subsequent paragraph you state that 'The ilHipSeme1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing'.So the first individual from above was also used for Hi-C?If this is true, I would suggest writing 'One specimen (specimen ID SAN0001387, individual ilHipSeme1) was used for DNA sequencing and Hi-C scaffolding, and a second specimen (specimen ID SAN0001388, individual ilHipSeme2) was used for Hi-C scaffolding only.'

Huateng Huang
Shaanxi Normal University, Xi'an, China Delong Guan college of chemistry and biological engineering, Hechi University (Ringgold ID: 56655), Yizhou, Guangxi, China Dear authors, I have reviewed your manuscript titled "The genome sequence of the Rock Grayling, Hipparchia semele (Linnaeus, 1758)" submitted to Wellcome Open Research with great interest.This data report presents the 403.4Mb draft genome assembly of the grayling butterfly Hipparchia semele, which was generated using Pacific Biosciences long reads and scaffolded with Hi-C data.
Seventeen thousand five hundred forty protein-coding genes were annotated.This concise and well-written manuscript describes valuable genomic resources for an ecologically critical lepidopteran species.I only have several minor comments and suggestions: Page 3, third line on the right: "collected from Beaulieu Heath, England (50.80, -1.50)." are the numbers latitude and longitude?Please provide the unit.

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Details on heterozygosity and phasing of the assembly are lacking.As this is from a wild individual, heterozygosity is expected to be substantial.Please provide parameters of critical steps, like purge_dups, to ensure reproducibility of the study.Also, the parameters for Hifisam and the Hi-C scaffolding process before using yahs.
○ Tools like bwa/chromap used for reading mapping and interaction calling are not listed in Table 3.

Reviewer Expertise: phylogenomics
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 1 .
Figure 1.Image of Hipparchia semele (not the specimen used for genome sequencing).Photograph by Callum Macgregor.

Figure 2 .Figure 3 .
Figure 2. Genome assembly of Hipparchia semele, ilHipSeme1.2: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 403,420,717 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 (17,487,639 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (14,619,000 and 8,523,649 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/ilHipSeme1.2/dataset/ CAKOFV02/snail.

Figure 4 .
Figure 4. Genome assembly of Hipparchia semele, ilHipSeme1.2:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilHipSeme1.2/dataset/CAKOFV02/ cumulative.

Figure 5 .
Figure 5. Genome assembly of Hipparchia semele, ilHipSeme1.2:Hi-C contact map of the ilHipSeme1.2assembly, 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=dbfZsqrJQlqmsZnT7Knz0w.

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Page 5, under "Sequencing," "Pacific Biosciences HiFi circular consensus ud DNA" It is unclear to me what the y-axis label "ERRxxx_cov" means in Figure 3. ○ Sincerely, Huateng Huang and De-Long Guan 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 Competing Interests: No competing interests were disclosed.

Table 3 . Software tools: versions and sources. Software tool Version Open Peer Review Current Peer Review Status: Version 1
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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? Partly Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
https://doi.org/© 2024 Huang H et al.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.