Abstract
We present a genome assembly from an individual male Erebia ligea (Arran brown; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 506 megabases in span. The majority (99.92%) of the assembly is scaffolded into 29 chromosomal pseudomolecules, with the Z sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.2 kilobases in length.
Keywords: Erebia ligea, Arran brown, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Papilionoidea; Nymphalidae; Satyrinae; Satyrini; Erebiina; Erebia; Erebia ligea (Linnaeus, 1758) (NCBI:txid111903).
Background
The Arran brown, Erebia ligea, is one of the most widespread species of the genus Erebia, occurring from the Russian Kamchatka Peninsula and Japan in eastern Asia ( Dubatolov et al., 1998) to central and northern Europe ( Kudrna et al., 2015). Although the species takes its common name from the Isle of Arran in Scotland, where it was first recorded in 1803, the current and historic presence of this butterfly in the British Isles remains disputed ( Salmon, 1995). The intraspecific phenotypic diversity present throughout the distribution of E. ligea has triggered the description of several subspecies ( Dubatolov et al., 1998; Warren, 1937; Zakharova & Tatarinov, 2016), however, a formal biogeographic assessment remains lacking.
E. ligea is characterised as a woodland species associated with clearings and meadows, and occurs at relatively low altitudes compared to most other Erebia butterflies ( Kleckova et al., 2014). Recorded host plants include a variety of grasses (Poaceae) and sedges ( Carex, Cyperaceae). It is univoltine and in some northern localities it is recorded only every second year ( Tolman & Lewington, 2008). Although E. ligea is considered a species of Least Concern according to the IUCN Red List (Europe) ( van Swaay et al., 2010), the species can be locally endangered ( Fichefet et al., 2008).
While the first karyotypic analysis suggested that male Erebia ligea from Finland have 29 chromosomes ( Federley, 1938), Japanese individuals from Hokkaido were found to have only 28 chromosomes ( Saitoh & Abe, 1997). These values are close to the most common and putatively ancestral chromosomal state for Lepidoptera (n=31; Robinson, 1971), although Erebia is one of the most karyologically diverse known genera of butterflies ( Robinson, 1971; de Vos et al., 2020).
Genome sequence report
The genome was sequenced from a single male E. ligea ( Figure 1) collected from Borzont, Joseni, Harghita, Romania (latitude 46.664, longitude 25.317). A total of 34-fold coverage of Pacific Biosciences single-molecule circular consensus (HiFi) long reads and 63-fold coverage of 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 47 missing/misjoins and removed 10 haplotypic duplications, reducing the assembly length by 3.59% and the scaffold number by 39.39%, and increased the scaffold N50 by 4.29%.
Figure 1. Forewings and hindwings of the male Erebia ligea specimen from which the genome was sequenced.
Dorsal (left) and ventral (right) surface view of wings from specimen RO_EE_997 (ilEreLige1) from Borzont, Joseni, Harghita, Romania, used to generate Pacific Biosciences, 10X genomics and Hi-C data.
The final assembly has a total length of 506 Mb in 40 sequence scaffolds, with a scaffold N50 of 19.1 Mb ( Table 1). The majority, 99.92%, of assembly sequence was assigned to 40 chromosomal-level scaffolds, representing 28 autosomes (numbered by sequence length), and the Z sex chromosome ( Figure 2– Figure 5; Table 2). The assembly has a BUSCO v5.2.2 ( Manni et al., 2021) completeness of 97.9% (single 97.4%, duplicated 0.5%) using the lepidoptera_odb10 reference set (n=5,286). While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.
Figure 2. Genome assembly of Erebia ligea, ilEreLige1.2: 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 506,397,422 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 (22,722,498 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 chromosome lengths (19,149,538 and 12,368,103 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/ilEreLige1.2/dataset/CAKAVA02/snail.
Figure 5. Genome assembly of Erebia ligea, ilEreLige1.2: Hi-C contact map.
Hi-C contact map of the ilEreLige1.2 assembly, visualised in HiGlass. Chromosomes are shown in size order from left to right and top to bottom. The interactive Hi-C map can be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=L3267sJjSyakmh-bayAgPg.
Table 1. Genome data for Erebia ligea, ilEreLige1.2.
| Project accession data | |
|---|---|
| Assembly identifier | ilEreLige1.2 |
| Species | Erebia ligea |
| Specimen | ilEreLige1 (genome assembly,
Hi-C) |
| NCBI taxonomy ID | NCBI:txid111903 |
| BioProject | PRJEB42125 |
| BioSample ID | SAMEA7523313 |
| Isolate information | Male, whole organism |
| Raw data accessions | |
| PacificBiosciences SEQUEL II | ERR7141799 |
| 10X Genomics Illumina | ERR6745725-ERR6745728 |
| Hi-C Illumina | ERR6745729-ERR6745732 |
| Genome assembly | |
| Assembly accession | GCA_917051295.2 |
| Span (Mb) | 506 |
| Number of contigs | 78 |
| Contig N50 length (Mb) | 14.9 |
| Number of scaffolds | 40 |
| Scaffold N50 length (Mb) | 19.1 |
| Longest scaffold (Mb) | 22.7 |
| BUSCO * genome score | C:97.9%[S:97.4%,D:0.5%],F:0.2%,
M:1.9%,n:5,286 |
*BUSCO scores based on the lepidoptera_odb10 BUSCO set using v5.2.2. C= complete [S= single copy, D=duplicated], F=fragmented, M=missing, n=number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/ilEreLige1.2/dataset/CAKAVA02/busco.
Figure 3. Genome assembly of Erebia ligea, ilEreLige1.2: 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/ilEreLige1.2/dataset/CAKAVA02/blob.
Figure 4. Genome assembly of Erebia ligea, ilEreLige1.2: 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/ilEreLige1.2/dataset/CAKAVA02/cumulative.
Table 2. Chromosomal pseudomolecules in the genome assembly of Erebia ligea, ilEreLige1.2.
| INSDC accession | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| OU785219.1 | 1 | 22.72 | 37.2 |
| OU785220.1 | 2 | 22.11 | 37.3 |
| OU785221.1 | 3 | 22.01 | 37.4 |
| OU785223.1 | 4 | 21.42 | 37.3 |
| OU785224.1 | 5 | 20.97 | 37.3 |
| OU785225.1 | 6 | 20.77 | 37.1 |
| OU785226.1 | 7 | 20.52 | 37.1 |
| OU785227.1 | 8 | 20.11 | 37.4 |
| OU785228.1 | 9 | 20.06 | 37.3 |
| OU785229.1 | 10 | 19.31 | 37.4 |
| OU785230.1 | 11 | 19.22 | 37.3 |
| OU785231.1 | 12 | 19.15 | 37.2 |
| OU785232.1 | 13 | 18.89 | 37.3 |
| OU785233.1 | 14 | 18.5 | 37.4 |
| OU785234.1 | 15 | 18.36 | 37.3 |
| OU785235.1 | 16 | 17.54 | 37.4 |
| OU785236.1 | 17 | 17.22 | 37.7 |
| OU785237.1 | 18 | 16.82 | 37.2 |
| OU785238.1 | 19 | 16.82 | 37.4 |
| OU785239.1 | 20 | 16.64 | 37.5 |
| OU785240.1 | 21 | 15.51 | 37.3 |
| OU785241.1 | 22 | 15.26 | 37.6 |
| OU785242.1 | 23 | 12.47 | 37.7 |
| OU785243.1 | 24 | 12.37 | 38.2 |
| OU785244.1 | 25 | 11.94 | 37.5 |
| OU785245.1 | 26 | 10.28 | 37.7 |
| OU785246.1 | 27 | 9.22 | 37.8 |
| OU785247.1 | 28 | 8.14 | 38.1 |
| OU785222.1 | Z | 21.64 | 37.3 |
| OU785248.1 | MT | 0.02 | 20.2 |
| - | Unplaced | 0.39 | 41.4 |
Methods
Sample acquisition and nucleic acid extraction
A single male E. ligea specimen (ilEreLige1, genome assembly, HiC) was collected from Borzont, Joseni, Harghita, Romania (latitude 46.664, longitude 25.317) using a handnet by Konrad Lohse, Dominik Laetsch (both University of Edinburgh) and Alex Hayward (University of Exeter). The sample was identified by Roger Vila (Institut de Biologia Evolutiva, Barcelona) and snap-frozen from live in a dry shipper.
DNA was extracted at the Scientific Operations Core, Wellcome Sanger Institute. The ilEreLige1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Whole organism tissue was disrupted by manual grinding with a disposable pestle. Fragment size analysis of 0.01–0.5 ng of DNA was then performed using an Agilent FemtoPulse. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. Low molecular weight DNA was removed from a 200-ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing. HMW DNA was sheared into an average fragment size between 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 and 10X Genomics read cloud DNA 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 (HiFi) and Illumina HiSeq X (10X) instruments. Hi-C data were also generated from remaining whole organism tissue of ilEreLige1 using the Arima v1 Hi-C kit and sequenced on an Illumina HiSeq X (10X) 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 ( Howe et al., 2021) was performed using gEVAL, HiGlass ( Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2021), which performed annotation using MitoFinder ( Allio et al., 2020). 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 | 0.12 | 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 | 1 | Uliano-Silva et al., 2021 |
| HiGlass | 1.11.6 | Kerpedjiev et al., 2018 |
| PretextView | 0.1.x |
https://github.com/wtsi-hpag/
PretextView |
| BlobToolKit | 3.0.5 | Challis et al., 2020 |
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). KL and DL are supported by an ERC grant (ModelGenomLand 757648). KL was also supported by a NERC fellowship (NE/L011522/1). AH is supported by a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Fellowship (BB/N020146/1). RV was supported by the Spanish government through grant PID2019-107078GB-I00/ MCIN/AEI/ 10.13039/501100011033.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 1; peer review: 2 approved]
Data availability
European Nucleotide Archive: Erebia ligea (Arran brown). Accession number PRJEB42125; https://identifiers.org/ena.embl/PRJEB42125.
The genome sequence is released openly for reuse. The E. ligea 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.
Author information
Members of the Wellcome Sanger Institute Tree of Life programme are listed here: https://doi.org/10.5281/zenodo.6866293.
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.org/10.5281/zenodo.6125046.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.6418363.
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