The genome sequence of the Lulworth Skipper, Thymelicus acteon (Rottemburg, 1775)
Konrad Lohse, Roger Vila, Alex Hayward, Xiaoling Fan, Li-Wei Wu, Elena Pazhenkova
TL;DR
This paper provides the genome sequence of the Lulworth Skipper butterfly, including detailed gene annotations and chromosomal scaffolding.
Contribution
The study presents a high-quality genome assembly and gene annotation for Thymelicus acteon, including chromosomal pseudomolecules and mitochondrial DNA.
Findings
The genome assembly spans 537.0 megabases and includes 28 chromosomal pseudomolecules.
Gene annotation identified 12,813 protein-coding genes using Ensembl.
The mitochondrial genome is 17.08 kilobases long and was successfully assembled.
Abstract
We present a genome assembly from an individual male Thymelicus acteon (the Lulworth Skipper; Arthropoda; Insecta; Lepidoptera; Hesperiidae). The genome sequence is 537.0 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 17.08 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,813 protein-coding genes.
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
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Figure 1
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Figure 4
Figure 5| Project accession data | ||
|---|---|---|
| Assembly identifier | ilThyActe1.1 | |
| Species |
| |
| Specimen | ilThyActe1 | |
| NCBI taxonomy ID | 876078 | |
| BioProject | PRJEB59958 | |
| BioSample ID | SAMEA110069525 | |
| Isolate information | ilThyActe1, male: head (PacBio sequencing)
| |
| Assembly metrics
|
| |
| Consensus quality (QV) | 65.0 |
|
|
| 100.0% |
|
| BUSCO
| C:98.4%[S:98.0%,D:0.5%],F:0.4%,M:1.2%,n:5,286 |
|
| Percentage of assembly mapped to chromosomes | 98.98% |
|
| Sex chromosomes | Z |
|
| Organelles | Mitochondrial genome: 17.08 kb |
|
| Raw data accessions | ||
| PacificBiosciences Sequel IIe | ERR10906098 | |
| Hi-C Illumina | ERR10908636 | |
| Genome assembly | ||
| Assembly accession | GCA_951805285.1 | |
|
| GCA_951805295.1 | |
| Span (Mb) | 537.0 | |
| Number of contigs | 120 | |
| Contig N50 length (Mb) | 10.7 | |
| Number of scaffolds | 66 | |
| Scaffold N50 length (Mb) | 19.7 | |
| Longest scaffold (Mb) | 40.67 | |
| Genome annotation | ||
| Number of protein-coding genes | 12,813 | |
| Number of non-coding genes | 2,647 | |
| Number of gene transcripts | 25,933 | |
| INSDC accession | Chromosome | Length (Mb) | GC% |
|---|---|---|---|
| 1 | 23.4 | 36.5 | |
| 2 | 24.07 | 37.0 | |
| 3 | 22.12 | 36.5 | |
| 4 | 22.48 | 36.5 | |
| 5 | 21.31 | 36.5 | |
| 6 | 21.33 | 36.0 | |
| 7 | 20.81 | 36.0 | |
| 8 | 20.0 | 36.0 | |
| 9 | 20.19 | 36.0 | |
| 10 | 20.0 | 36.5 | |
| 11 | 19.68 | 36.0 | |
| 12 | 18.87 | 36.0 | |
| 13 | 18.82 | 36.0 | |
| 14 | 18.75 | 36.0 | |
| 15 | 18.52 | 36.5 | |
| 16 | 18.31 | 36.0 | |
| 17 | 17.7 | 36.0 | |
| 18 | 17.2 | 36.0 | |
| 19 | 17.38 | 36.5 | |
| 20 | 17.12 | 36.5 | |
| 21 | 16.51 | 36.5 | |
| 22 | 15.74 | 36.5 | |
| 23 | 13.45 | 36.0 | |
| 24 | 13.2 | 36.5 | |
| 25 | 12.18 | 36.5 | |
| 26 | 12.01 | 37.0 | |
| 27 | 9.73 | 36.5 | |
| Z | 40.67 | 35.5 | |
| MT | 0.02 | 16.5 |
| Software tool | Version | Source |
|---|---|---|
| BlobToolKit | 4.2.1 |
|
| BUSCO | 5.3.2 |
|
| Hifiasm | 0.16.1-r375 |
|
| HiGlass | 1.11.6 |
|
| Merqury | MerquryFK |
|
| MitoHiFi | 2 |
|
| PretextView | 0.2 |
|
| purge_dups | 1.2.3 |
|
| sanger-tol/genomenote | v1.0 |
|
| sanger-tol/readmapping | 1.1.0 |
|
| YaHS | 1.1a.2 |
|
- —Biotechnology and Biological Sciences Research Council
- —ERC
- —Wellcome Trust
- —NERC
- —Ministerio de Ciencia e Innovación
- —Agencia Estatal de Investigación
- —European Regional Development Fund
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Taxonomy
TopicsGenomics and Phylogenetic Studies · Genetic diversity and population structure · Aquaculture disease management and microbiota
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysia; Obtectomera; Hesperioidea; Hesperiidae; Hesperiinae; Hesperiini; Thymelicus; Thymelicus acteon (Rottemburg, 1775) (NCBI:txid876078).
Background
The Lulworth Skipper ( Thymelicus acteon) is a habitat specialist of warm grasslands in North Africa, Southern and Central Europe, the Middle East and Western Asia. Thymelicus acteon reaches its Northern range limit in the South of England where it occurs in a number of localities on south-facing limestone and chalk grasslands in Dorset, centred around the village of Lulworth where it was first recorded in the UK in 1832. It is absent from Wales, Scotland and Ireland.
The Lulworth Skipper is listed as Near Threatened on the IUCN and UK Red lists ( Fox et al., 2015; Van Swaay et al., 2010), but in the UK it has increased in abundance since 2014, possibly as a result of climate change. The species is univoltine with adults occurring from the end of April to August depending on locality. Eggs are laid in a row in flower-sheaths of Tor-grass ( Brachypodium pinnatum), the principal larval host plant in the UK, although it feeds on several other grasses across its range.
The Lulworth skipper has lower overall genetic diversity compared to its congeners T. sylvestris and T. lineola ( Mackintosh et al., 2019). At the local scale in Central Europe it shows no isolation by distance, but relatively high genetic structure ( Louy et al., 2007). Population connectivity has been shown to be strongly influenced by land use patterns ( Engler et al., 2014). In the Western Palearctic, this species shows several geographically-structured mitochondrial lineages ( Dapporto et al., 2022) as well as phenotypic variation: darker forms occur in north-west Africa, Iberia, Elba, Crete, and other eastern Mediterranean islands. The taxon christi, previously considered a subspecies of T. acteon, is now generally treated as a species endemic to the Canary Islands.
The Lulworth skipper has 28 chromosomes ( de Lesse, 1970); the genome size has been estimated as 517.4 Mb ( Mackintosh et al., 2019).
Genome sequence report
The genome was sequenced from a male Thymelicus acteon ( Figure 1) collected from El Serrat, Catalunya, Spain (41.69, 2.25). A total of 42-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 14 missing joins or mis-joins and removed 6 haplotypic duplications, reducing the assembly length by 0.77% and the scaffold number by 9.46%.
Photographs of forewings and hindwings of the Thymelicus acteon specimen ES_TS_344 (ilThyActe1) used for genome sequencing. a) Dorsal and b) ventral surface views of wings from the specimen.
The final assembly has a total length of 537.0 Mb in 66 sequence scaffolds with a scaffold N50 of 19.7 Mb ( Table 1). The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3. The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla. Most (98.98%) of the assembly sequence was assigned to 28 chromosomal-level scaffolds, representing 27 autosomes and the Z sex chromosome. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( 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.
Table 1.: Genome data for Thymelicus acteon, ilThyActe1.1.
Genome assembly of Thymelicus acteon, ilThyActe1.1: metrics.The BlobToolKit snail plot 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 536,997,220 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 (40,667,834 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (19,676,428 and 13,448,605 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/ilThyActe1_1/dataset/ilThyActe1_1/snail.
Genome assembly of Thymelicus acteon, ilThyActe1.1: BlobToolKit GC-coverage plot.Sequences are coloured by phylum. Circles are sized in proportion to sequence length. Histograms show the distribution of sequence length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilThyActe1_1/dataset/ilThyActe1_1/blob.
Genome assembly of Thymelicus acteon, ilThyActe1.1: BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences. Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilThyActe1_1/dataset/ilThyActe1_1/cumulative.
Genome assembly of Thymelicus acteon, ilThyActe1.1: Hi-C contact map of the ilThyActe1.1 assembly, 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=DwelZModSOCAr3bDC4wEig.
Table 2.: Chromosomal pseudomolecules in the genome assembly of Thymelicus acteon, ilThyActe1.
The estimated Quality Value (QV) of the final assembly is 65.0 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.3.2 completeness of 98.4% (single = 98.0%, duplicated = 0.5%), using the lepidoptera_odb10 reference set ( n = 5,286).
Metadata for specimens, barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/876078.
Genome annotation report
The Thymelicus acteon genome assembly (GCA_951805285.1) was annotated at the European Bioinformatics Institute (EBI) on Ensembl Rapid Release. The resulting annotation includes 25,933 transcribed mRNAs from 12,813 protein-coding and 2,647 non-coding genes ( Table 1; https://rapid.ensembl.org/Thymelicus_acteon_GCA_951805285.1/Info/Index).
Methods
Sample acquisition and nucleic acid extraction
Two male Thymelicus acteon specimens were collected from El Serrat, Catalunya, Spain (latitude 41.69, longitude 2.25) on 2017-05-28 using a sweep net. The specimen used for genome sequencing had ID SAN00002425 (ToLID ilThyActe1), while the specimen used for Hi-C sequencing had ID SAN00002426 (ToLID ilThyActe2). Both specimens were collected by Konrad Lohse (University of Edinburgh) and Alex Hayward (University of Exeter) and identified by Roger Vila (Universitat Pompeu Fabra). Samples were snap frozen at –80 °C in a dry shipper.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up. In sample preparation, the ilThyActe1 sample was weighed and dissected on dry ice ( Jay et al., 2023). Tissue from the head was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a).
HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol ( Oatley et al., 2023). The DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system with speed setting 31 ( Bates et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation ( Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA. 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.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io ( Denton et al., 2023b).
Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers’ instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences Sequel IIe instrument. Hi-C data were also generated from head tissue of ilThyActe2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm ( Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using YaHS ( Zhou et al., 2023). The assembly was checked for contamination and corrected as described previously ( Howe et al., 2021). Manual curation was performed using HiGlass ( Kerpedjiev et al., 2018) and PretextView ( Harry, 2022). The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) or MITOS ( Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
A Hi-C map for the final assembly was produced using bwa-mem2 ( Vasimuddin et al., 2019) in the Cooler file format ( Abdennur & Mirny, 2020). To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury ( Rhie et al., 2020). This work was done using Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines “sanger-tol/readmapping” ( Surana et al., 2023a) and “sanger-tol/genomenote” ( Surana et al., 2023b). The genome was analysed within the BlobToolKit environment ( Challis et al., 2020) and BUSCO scores ( Manni et al., 2021; Simão et al., 2015) were calculated.
Table 3 contains a list of relevant software tool versions and sources.
Genome annotation
The Ensembl Genebuild annotation system ( Aken et al., 2016) was used to generate annotation for the Thymelicus acteon assembly (GCA_951805285.1) in Ensembl Rapid Release at the EBI. 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).
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 Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, 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 further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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