Version Changes
Revised. Amendments from Version 1
In version 2 of this data note we have added information about karyotyping of the Hesperiidae family to the Background section. Information about genome annotation by Ensembl has been added. We have used an additional table to clarify which specimens were used for different types of sequence data. We have replaced the chromosome map in Figure 5 with a labelled version.
Abstract
We present a genome assembly from an individual male Thymelicus sylvestris (the Small Skipper; Arthropoda; Insecta; Lepidoptera; Hesperiidae). The genome sequence is 471 megabases in span. Most of the assembly (99.97%) is scaffolded into 28 chromosomal pseudomolecules, including the Z sex chromosome. Gene annotation of this assembly on Ensembl identified 13,941 protein-coding genes. The mitochondrial genome was also assembled and is 17.1 kilobases in length.
Keywords: Thymelicus sylvestris, small skipper, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Hesperioidea; Hesperiidae; Hesperiinae; Hesperiini; Thymelicus; Thymelicus sylvestris (Poda, 1761) (NCBI:txid272628).
Background
The Small Skipper ( Thymelicus sylvestris) is a butterfly within the skipper family Hesperiidae. The skippers are named for their characteristically quick, darting flight. The common name of T. sylvestris is a clear reference to its small size: the adult wingspan ranges from 27–34 mm ( Tomlinson & Still, 2002). However, it is not the smallest of the skippers, with four other British species being an equivalent size or smaller. Similar to other skippers, T. sylvestris has golden-orange wings with clear sex brands on males, but it can be distinguished by a lack of coloured patches on its wings and a dull brown or orange colouration to its antennae ( Tomlinson & Still, 2002).
Thymelicus sylvestris is widespread across the European continent with a habitat range paralleling that of other skipper species. This range encompasses the northernmost reaches of Morocco and Algeria all the way to the bordering regions between the Baltic states and Russia ( Tolman & Lewington, 2008). However, it is noticeably absent from northern Scandinavia, Corsica and Sardinia ( Tolman & Lewington, 2008). In the British Isles the small skipper is found across most of Wales and England with recent trends showing a northward expansion in range, beyond the England-Scotland border. Recently, T. sylvestris individuals have also been observed in Ireland, where they had not been reported previously ( Harding & Jacob, 2013). Thymelicus sylvestris populations appear stable and it is listed as a species of least concern by the IUCN ( van Swaay et al., 2010).
The small skipper is a habitat generalist ( Louy et al., 2007) and can be found in open areas with long grass, such as rough grasslands and roadside verges ( Tomlinson & Still, 2002). It is most associated with Yorkshire fog ( Holcus lanatus), its main food plant, on which it often basks and lays eggs from June to July. Females are known to be meticulous with their choice of oviposition sites, spending up to 15 minutes inspecting potential host plants prior to laying eggs ( Tolman & Lewington, 2008). After approximately a month, eggs hatch into caterpillars which develop through 5 instar stages. Come winter, caterpillars spin cocoons within which they undergo diapause. The caterpillars re-emerge in spring, constructing a ‘leaf tube’ by joining together the ends of a leaf, where they live and feed, moving to new leaves as necessary. Small skipper caterpillars usually pupate by June, with adult butterflies emerging in July, to spend their remaining days in tall grassland until the summer’s end in September.
Most skippers retain the ancestral lepidopteran haploid number n = 31, which is also inferred to be ancestral for Hesperiidae overall ( Lukhtanov, 2014). Within Hesperiinae, the tribe Thymelicini most commonly shows n = 29, with a report of n = 27 for Thymelicus sylvestris based on cytology ( Lukhtanov, 2014). We present the first chromosome-level genome sequence for Thymelicus sylvestris, the Small Skipper. The karyotype reported here is n = 28.
The assembly was generated as part of the Darwin Tree of Life Project, which aims to generate high-quality reference genomes for all named eukaryotic species in Britain and Ireland to support research, conservation, and the sustainable use of biodiversity.
Genome sequence report
The genome was sequenced from a single male T. sylvestris collected from Ruan Minor, Cornwall, UK (latitude 49.9942295, longitude –5.1974720) ( Figure 1). Two other specimens were used for Hi-C scaffolding and RNA sequencing. Table 1 summarises the specimen and sequencing details. PacBio sequencing generated 20.16 Gb (gigabases) from 1.99 million reads, which were used to assemble the genome. Based on the estimated genome size, the sequencing data provided approximately 40× coverage. Hi-C sequencing produced 122.17 Gb from 809.08 million reads, which were used to scaffold the assembly. RNA sequencing data were also generated and are available in public sequence repositories.
Figure 1. Fore and hind wings of the Thymelicus sylvestris specimen from which the genome was sequenced.
Dorsal (left) and ventral (right) surface view of wings from specimen EN_RM_1378 (ilThySylv1) from Ruan Minor, Cornwall, UK, used to generate Pacific Biosciences and 10X genomics data.
Table 1. Specimen and sequencing data for BioProject PRJEB45673.
| Platform | PacBio HiFi | Hi-C | RNA-seq |
|---|---|---|---|
| ToLID | ilThySylv1 | ilThySylv3 | ilThySylv2 |
| Specimen ID | SAN0000837 | SAN0000839 | SAN0000838 |
| BioSample (source individual) | SAMEA7523279 | SAMEA7523281 | SAMEA7523280 |
| BioSample (tissue) | SAMEA7523375 | SAMEA7523377 | SAMEA7523376 |
| Tissue | whole organism | whole organism | whole organism |
| Instrument | Sequel II | Illumina NovaSeq 6000 | Illumina HiSeq 4000 |
| Run accessions | ERR6608659 | ERR6363315 | ERR6363314 |
| Read count total | 1.99 million | 809.08 million | 38.45 million |
| Base count total | 20.16 Gb | 122.17 Gb | 5.81 Gb |
Manual assembly curation corrected 9 missing/misjoins and removed 3 haplotypic duplications, reducing the assembly size by 0.06% and the scaffold number by 20.00%.
The final assembly has a total length of 471 Mb in 32 sequence scaffolds with a scaffold N50 of 16.61 Mb ( Table 2). Of the assembly sequence, 99.97% was assigned to 28 chromosomal-level scaffolds, representing 27 autosomes (numbered by sequence length), and the Z sex chromosome ( Figure 2– Figure 5; Table 3). The assembly curator noted that this differs from the published karyotype for this species ( Lukhtanov, 2014), but the available data did not support making additional joins. One chromosome is very small scaffold, at about 8 Mb, which could plausibly have been overlooked in earlier cytological work; alternatively, differences between cytological and genome-assembly interpretations could explain the discrepancy.
Figure 2. Genome assembly of Thymelicus sylvestris, ilThySylv1.1: metrics.
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 470,727,450 bp assembly. The distribution of chromosome lengths is shown in dark grey with the plot radius scaled to the longest chromosome present in the assembly (37,236,842 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 chromosome lengths (17,253,319 and 11,732,099 bp), respectively. The pale grey spiral shows the cumulative chromosome 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/ilThySylv1.1/dataset/CAJVQR01/snail.
Figure 5. Genome assembly of Thymelicus sylvestris, ilThySylv1.1: Hi-C contact map.
Hi-C contact map of the ilThySylv1.1 assembly, visualised in PretextView and PretextSnapshot. Chromosomes are arranged in size order from left to right and top to bottom.
Table 2. Genome data for Thymelicus sylvestris, ilThySylv1.1.
| Assembly name | ilThySylv1.1 |
| Assembly accession | GCA_911387775.1 |
| Alternate haplotype accession | GCA_911387695.1 |
| Assembly level | chromosome |
| Span (Mb) | 470.71 |
| Number of chromosomes | 28 |
| Number of contigs | 46 |
| Contig N50 | 16.61 Mb |
| Number of scaffolds | 32 |
| Scaffold N50 | 17.25 Mb |
| Sex chromosomes | Z |
| Organelles | Mitochondrion: 17.11 kb |
Figure 3. Genome assembly of Thymelicus sylvestris, ilThySylv1.1: GC coverage.
BlobToolKit GC-coverage plot. Scaffolds are coloured by phylum. Circles are sized in proportion to scaffold length. Histograms show the distribution of scaffold length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilThySylv1.1/dataset/CAJVQR01/blob.
Figure 4. Genome assembly of Thymelicus sylvestris, ilThySylv1.1: cumulative sequence.
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/ilThySylv1.1/dataset/CAJVQR01/cumulative.
Table 3. Chromosomal pseudomolecules in the genome assembly of Thymelicus sylvestris, ilThySylv1.1.
| INSDC accession | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| OU426886.1 | 1 | 20.99 | 36.0 |
| OU426887.1 | 2 | 20.74 | 36.9 |
| OU426888.1 | 3 | 20.31 | 36.3 |
| OU426889.1 | 4 | 19.76 | 36.3 |
| OU426890.1 | 5 | 19.60 | 36.5 |
| OU426891.1 | 6 | 19.09 | 35.7 |
| OU426892.1 | 7 | 18.50 | 35.9 |
| OU426893.1 | 8 | 18.16 | 35.9 |
| OU426894.1 | 9 | 17.98 | 35.8 |
| OU426895.1 | 10 | 17.90 | 36.0 |
| OU426896.1 | 11 | 17.25 | 35.6 |
| OU426897.1 | 12 | 17.04 | 35.8 |
| OU426898.1 | 13 | 16.99 | 36.2 |
| OU426899.1 | 14 | 16.61 | 36.0 |
| OU426900.1 | 15 | 16.11 | 36.2 |
| OU426901.1 | 16 | 16.10 | 35.9 |
| OU426902.1 | 17 | 15.35 | 35.9 |
| OU426903.1 | 18 | 15.05 | 36.0 |
| OU426904.1 | 19 | 14.87 | 36.2 |
| OU426905.1 | 20 | 14.87 | 36.6 |
| OU426906.1 | 21 | 14.38 | 36.2 |
| OU426907.1 | 22 | 13.89 | 36.3 |
| OU426908.1 | 23 | 11.73 | 36.0 |
| OU426909.1 | 24 | 11.38 | 36.7 |
| OU426910.1 | 25 | 10.65 | 36.1 |
| OU426911.1 | 26 | 9.84 | 36.9 |
| OU426912.1 | 27 | 8.15 | 36.4 |
| OU426885.1 | Z | 37.24 | 35.7 |
| OU426913.1 | MT | 0.02 | 16.7 |
The assembly has a BUSCO ( Simão et al., 2015) v5.1.2 completeness of 98.5% (single 98.1%, duplicated 0.5%) 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 report
The Thymelicus sylvestris genome assembly (GCA_911387775.1) was annotated by Ensembl at the European Bioinformatics Institute (EBI). This annotation includes 38,357 transcribed mRNAs from 13,941 protein-coding and 6 462 non-coding genes. The average transcript length is 18,220.25 bp, with an average of 1.88 coding transcripts per gene and 7.23 exons per transcript. For further information about the annotation, please refer to the annotation page on Ensembl.
Methods
Specimen acquisition and nucleic acid extraction
Three male T. sylvestris (ilThySylv1, ilThySylv2 and ilThySylv3) specimens were collected from Ruan Minor, Cornwall, UK (latitude 49.9942295, longitude –5.1974720) using a net by Alex Hayward in May 2019. The samples were identified by the same person, and then snap-frozen on dry ice.
DNA was extracted from the whole organism of ilThySylv1 (specimen ID: EN_RM_1378) 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 from whole organism tissue of ilThySylv2 was extracted in the Tree of Life Laboratory at the WSI using TRIzol, 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 the 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) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II (HiFi), Illumina HiSeq X (10X) and Illumina HiSeq 4000 (RNA-Seq) instruments. Hi-C data were generated from head tissue of ilThySylv3 in the Tree of Life Laboratory using the Arima Hi-C+ kit and sequenced on an Illumina NovaSeq 6000 instrument.
Genome assembly
Assembly was carried out with Hifiasm ( Cheng et al., 2021). Haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). One round of polishing was performed by aligning 10X Genomics read data to the assembly with longranger align, calling variants with freebayes ( Garrison & Marth, 2012). The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using SALSA2 ( Ghurye et al., 2019). The assembly was checked for contamination and corrected using the gEVAL system ( Chow et al., 2016) as described previously ( Howe et al., 2021). Manual curation was performed using gEVAL, HiGlass ( Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2021). The genome was analysed and BUSCO scores generated within the BlobToolKit environment ( Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.
Table 3. Software tools used.
| Software tool | Version | Source |
|---|---|---|
| Hifiasm | 0.15 | Cheng et al., 2021 |
| purge_dups | 1.2.3 | Guan et al., 2020 |
| SALSA2 | 2.2 | Ghurye et al., 2019 |
| longranger align | 2.2.2 | https://support.10xgenomics.com/genome-exome/software/pipelines/latest/advanced/other-pipelines |
| freebayes | 1.3.1-17-gaa2ace8 | Garrison & Marth, 2012 |
| MitoHiFi | 2.0 | Uliano-Silva et al., 2021 |
| gEVAL | N/A | Chow et al., 2016 |
| HiGlass | 1.11.6 | Kerpedjiev et al., 2018 |
| PretextSnapshot | N/A | https://github.com/sanger-tol/PretextSnapshot |
| PretextView | 0.2.x | https://github.com/sanger-tol/PretextView |
| BlobToolKit | 2.6.4 | Challis et al., 2020 |
Data availability
European Nucleotide Archive: Thymelicus sylvestris (small skipper). Accession number PRJEB45673; https://identifiers.org/ena.embl/PRJEB45673. The genome sequence is released openly for reuse. The Thymelicus sylvestris genome sequencing initiative is part of the Darwin Tree of Life Project (PRJEB40665), the Sanger Institute Tree of Life Programme (PRJEB43745) and Project Psyche (PRJEB71705).
All raw sequence data and the assembly have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1 and Table 2.
Production code used in genome assembly at the WSI Tree of Life is available at https://github.com/sanger-tol.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (206194) and the Darwin Tree of Life Discretionary Award (218328). AH is supported by a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Fellowship (BB/N020146/1).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 2; peer review: 4 approved]
Author information
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.5744972.
Members of the Wellcome Sanger Institute Tree of Life programme are listed here: https://doi.org/10.5281/zenodo.5744840.
Members of Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective are listed here: https://doi.org/10.5281/zenodo.5746904.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org10.5281/zenodo.5743293.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.5638618.
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