Abstract
We present a genome assembly from a female specimen of Erebia triaria (de Prunner’s Ringlet; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The assembly contains two haplotypes with total lengths of 521.30 megabases and 412.03 megabases. Most of haplotype 1 (99.7%) is scaffolded into 17 chromosomal pseudomolecules, including the Z 1, Z 2, and W sex chromosomes. Haplotype 2 was assembled to scaffold level. The mitochondrial genome has also been assembled, with a length of 15.26 kilobases.
Keywords: Erebia triaria, de Prunner's Ringlet, genome sequence, chromosomal, Lepidoptera
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; Papilionoidea; Nymphalidae; Satyrinae; Erebiini; Erebia; Erebia triaria von Prunner, 1798 (NCBI:txid242256)
Background
De Prunner’s Ringlet, Erebia triaria, is a European montane species, distributed from the Iberian Peninsula through the western and central Alps to the Balkans, where it is extremely localised ( Micevski, 2014; Tolman & Lewington, 2008). The scientific name frequently appears as Erebia triarius in the literature ( Wiemers et al., 2018) as, for instance, in the phylogenomic study of the genus Erebia that confirmed that de Prunner’s Ringlet was a member of the medusa clade and revealed that it is the sister species of the monophyletic group formed by E. polaris, E. medusa and E. epipsodea ( Augustijnen et al., 2024).
Erebia triaria inhabits roadside verges, clearings, and edges of open pine forests and oak woodlands, favouring warm, dry, steep, and rocky locations with generally southern exposures. Its typical habitat includes arid stony slopes or limestone crags, often exposed to full midday sun. Typical habitats are regions such as the Valais, a characteristic inner-Alpine dry valley, with rocky heaths with xerophilous and steppe vegetation ( Nardelli, 1988; Sonderegger, 2005; Tolman & Lewington, 2008). It has a very wide elevation range, from 200 m to 2 500 m, with most of the records occurring between 1 000 and 1 500 m in Spain and between 600 and 1 600 m in Switzerland.
This medium-sized butterfly ( Figure 1) has a forewing length of 21–25 mm and exhibits the typical earthy and muted colouration of the genus Erebia. The dorsal side of the wing is dark brown with a postdiscal band that is reddish-orange in males or orange in females. The forewings display four or five ocelli with pupils. On the hindwings, three or four dorsal ocelli are visible, situated on a discontinuous orange band. The ventral surface is dark. The ventral ocelli of the hindwings are barely visible in males, whereas in females the underside is lighter and more contrasting. Final instar larvae are light green, with a brown head and dorsal and lateral black bands, the dorsal band being flanked by two white lines. The substantial geographical variability in the wing pattern of de Prunner’s Ringlet has led to the description of at least 12 subspecies (reviewed by Vila, 2004). However, the only available phylogeographic study on this species focused on the genetic differentiation of the western-most one, Erebia triaria pargapondalense, whose allopatric isolation likely predates the last glaciation ( Vila et al., 2005).
Figure 1. Photograph of Erebia triaria by Miguel Carballa (not the specimen used for genome sequencing).
Erebia triaria is a univoltine species, with adults mostly flying from May to the first half of July, depending on altitude. It may occur in sympatry with E. meolans, which typically flies during the summer season. This coexistence may lead to misidentification during late spring/early summer. Males of E. triaria are particularly active during their flight period, patrolling their territories in search of mates. The female lays eggs on various grasses, such as Festuca spp. and Stipa pennata L. ( Clarke, 2024).
The species is classified as Least Concern (LC) at the European level ( van Swaay et al., 2010). However, it receives a higher level of protection in certain countries, being listed as Vulnerable (VU) in Switzerland, for example. Furthermore, specific lineages or populations may warrant particular attention. For instance, the aforementioned westernmost subspecies has been identified as a distinct conservation unit based on genetic analyses, as well as morphological and phenological observations ( Vila et al., 2006). Predictive models for the period 2061–2080 revealed that E. triaria will be one of the three species of Erebia likely to remain in Spain (almost entirely restricted to the Pyrenees and the Cantabrian Mountains), where the suitable areas for E. triaria will decrease by up to 77% ( Romo et al., 2023).
The first karyotypic analysis suggested E. triaria has an approximate haploid number of 16 chromosomes ( de Lesse, 1961). As the chromosome numbers of the medusa clade range between 11 and 18, the chromosome-level reference genome of E. triaria provides an invaluable resource to unravel the chromosome evolution in Erebia, which has likely driven diversification in this highly speciose genus ( Augustijnen et al., 2024). Moreover, this reference genome provides the opportunity to resolve the cryptic diversity within E. triaria.
Methods
Sample acquisition
The specimen used for genome sequencing was an adult female Erebia triaria (specimen ID SAN20001744, ToLID ilEreTria2), collected from Hohtenn, Valaid, Switzerland (latitude 46.33, longitude 7.763; elevation 1 443 m) on 06/06/2022. The specimen was collected and identified by Yannick Chittaro (Info Fauna, Neuchâtel, Switzerland).
Nucleic acid extraction
Protocols for high molecular weight (HMW) DNA extraction developed at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory are available on protocols.io ( Howard et al., 2025). The ilEreTria2 sample was weighed and triaged to determine the appropriate extraction protocol. Tissue from the whole organism was homogenised by powermashing using a PowerMasher II tissue disruptor.
HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol. DNA was sheared into an average fragment size of 12–20 kb following the Megaruptor®3 for LI PacBio protocol. Sheared DNA was purified by automated SPRI (solid-phase reversible immobilisation). The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system. For this sample, the final post-shearing DNA had a Qubit concentration of 25.8 ng/μL and a yield of 1 496.40 ng, with a fragment size of 13.7 kb. The 260/280 spectrophotometric ratio was 1.92, and the 260/230 ratio was 2.21.
PacBio HiFi library preparation and sequencing
Library preparation and sequencing were performed at the WSI Scientific Operations core. Libraries were prepared using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, California, USA), following the manufacturer’s instructions. The kit includes reagents for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead clean-up, and nuclease treatment. Size selection and clean-up were performed using diluted AMPure PB beads (Pacific Biosciences). DNA concentration was quantified using a Qubit Fluorometer v4.0 (ThermoFisher Scientific) and the Qubit 1X dsDNA HS assay kit. Final library fragment size was assessed with the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) using the gDNA 55 kb BAC analysis kit.
The sample was sequenced on a Revio instrument (Pacific Biosciences). The prepared library was normalised to 2 nM, and 15 μL was used for making complexes. Primers were annealed and polymerases bound to generate circularised complexes, following the manufacturer’s instructions. Complexes were purified using 1.2X SMRTbell beads, then diluted to the Revio loading concentration (200–300 pM) and spiked with a Revio sequencing internal control. The sample was sequenced on a Revio 25M SMRT cell. The SMRT Link software (Pacific Biosciences), a web-based workflow manager, was used to configure and monitor the run and to carry out primary and secondary data analysis.
Specimen details, sequencing platforms, and data yields are summarised in Table 1.
Table 1. Specimen and sequencing data for BioProject.
| Platform | PacBio HiFi | Hi-C |
|---|---|---|
| ToLID | ilEreTria2 | ilEreTria2 |
| Specimen ID | SAN20001744 | SAN20001744 |
|
BioSample (source
individual) |
SAMEA114286221 | SAMEA114286221 |
| BioSample (tissue) | SAMEA114286282 | SAMEA114286282 |
| Tissue | whole organism | whole organism |
|
Sequencing
platform and model |
Revio | Illumina NovaSeq X |
| Run accessions | ERR12954116 | ERR12982554 |
| Read count total | 1.75 million | 667.67 million |
| Base count total | 19.49 Gb | 100.82 Gb |
Hi-C
Sample preparation and crosslinking
The Hi-C sample was prepared from 20–50 mg of frozen whole organism tissue of the ilEreTria2 sample using the Arima-HiC v2 kit (Arima Genomics). Following the manufacturer’s instructions, tissue was fixed and DNA crosslinked using TC buffer to a final formaldehyde concentration of 2%. The tissue was homogenised using the Diagnocine Power Masher-II. Crosslinked DNA was digested with a restriction enzyme master mix, biotinylated, and ligated. Clean-up was performed with SPRISelect beads before library preparation. DNA concentration was measured with the Qubit Fluorometer (Thermo Fisher Scientific) and Qubit HS Assay Kit. The biotinylation percentage was estimated using the Arima-HiC v2 QC beads.
Hi-C library preparation and sequencing
Biotinylated DNA constructs were fragmented using a Covaris E220 sonicator and size selected to 400–600 bp using SPRISelect beads. DNA was enriched with Arima-HiC v2 kit Enrichment beads. End repair, A-tailing, and adapter ligation were carried out with the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs), following a modified protocol where library preparation occurs while DNA remains bound to the Enrichment beads. Library amplification was performed using KAPA HiFi HotStart mix and a custom Unique Dual Index (UDI) barcode set (Integrated DNA Technologies). Depending on sample concentration and biotinylation percentage determined at the crosslinking stage, libraries were amplified with 10–16 PCR cycles. Post-PCR clean-up was performed with SPRISelect beads. Libraries were quantified using the AccuClear Ultra High Sensitivity dsDNA Standards Assay Kit (Biotium) and a FLUOstar Omega plate reader (BMG Labtech).
Prior to sequencing, libraries were normalised to 10 ng/μL. Normalised libraries were quantified again and equimolar and/or weighted 2.8 nM pools. Pool concentrations were checked using the Agilent 4200 TapeStation (Agilent) with High Sensitivity D500 reagents before sequencing. Sequencing was performed using paired-end 150 bp reads on the Illumina NovaSeq X.
Specimen details, sequencing platforms, and data yields are summarised in Table 1.
Genome assembly
Prior to assembly of the PacBio HiFi reads, a database of k-mer counts ( k = 31) was generated from the filtered reads using FastK. GenomeScope2 ( Ranallo-Benavidez et al., 2020) was used to analyse the k-mer frequency distributions, providing estimates of genome size, heterozygosity, and repeat content.
The HiFi reads were assembled using Hifiasm in Hi-C phasing mode ( Cheng et al., 2021; Cheng et al., 2022), producing two haplotypes. Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019). Contigs were further scaffolded with Hi-C data in YaHS ( Zhou et al., 2023), using the --break option for handling potential misassemblies. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al., 2022), BUSCO ( Manni et al., 2021) and MERQURY.FK ( Rhie et al., 2020).
The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Assembly curation
The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants ( ASCC) pipeline. TreeVal was used to generate the flat files and maps for use in curation. Manual curation was conducted primarily in PretextView and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Manual corrections included 4 breaks and 205 joins. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation. PretextSnapshot was used to generate a Hi-C contact map of the final assembly.
Assembly quality assessment
Chromosomal painting was performed using lep_busco_painter using Merian elements, which represent the 32 ancestral linkage groups in Lepidoptera ( Wright et al., 2024). Painting was based on gene locations from the lepidoptera_odb10 BUSCO analysis and chromosome lengths from the genome index produced using SAMtools faidx ( Danecek et al., 2021). Each complete BUSCO (including both single-copy and duplicated BUSCOs) was assigned to a Merian element using a reference database, and coloured positions were plotted along chromosomes drawn to scale.
The Merqury.FK tool ( Rhie et al., 2020), run in a Singularity container ( Kurtzer et al., 2017), was used to evaluate k-mer completeness and assembly quality for both haplotypes using the k-mer databases ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.
The genome was analysed using the BlobToolKit pipeline, a Nextflow implementation of the earlier Snakemake BlobToolKit pipeline ( Challis et al., 2020). The pipeline aligns PacBio reads using minimap2 ( Li, 2018) and SAMtools ( Danecek et al., 2021) to generate coverage tracks. Simultaneously, it queries the GoaT database ( Challis et al., 2023) to identify relevant BUSCO lineages and runs BUSCO ( Manni et al., 2021). For the three domain-level BUSCO lineages, BUSCO genes are aligned to the UniProt Reference Proteomes database ( Bateman et al., 2023) using DIAMOND blastp ( Buchfink et al., 2021). The genome is divided into chunks based on the density of BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database with DIAMOND blastx. Sequences without hits are chunked using seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). The BlobToolKit suite consolidates all outputs into a blobdir for visualisation. The BlobToolKit pipeline was developed using nf-core tooling ( Ewels et al., 2020) and MultiQC ( Ewels et al., 2016), with package management via Conda and Bioconda ( Grüning et al., 2018), and containerisation through Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017).
Genome sequence report
Sequence data
The genome of a specimen of Erebia triaria was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating 19.49 Gb (gigabases) from 1.75 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 455.82 Mb, with a heterozygosity of 0.95% and repeat content of 28.80%. These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 42× coverage. Hi-C sequencing produced 100.82 Gb from 667.67 million reads, which were used to scaffold the assembly. Table 1 summarises the specimen and sequencing details.
Assembly statistics
The genome was assembled into two haplotypes using Hi-C phasing. Haplotype 1 was curated to chromosome level, while haplotype 2 was assembled to scaffold level. The final assembly has a total length of 521.30 Mb in 57 scaffolds, with 317 gaps, and a scaffold N50 of 33.97 Mb ( Table 2).
Table 2. Genome assembly statistics.
| Assembly name | ilEreTria2.hap1.1 | ilEreTria2.hap2.1 |
| Assembly accession | GCA_964166005.1 | GCA_964166405.1 |
| Assembly level | chromosome | scaffold |
| Span (Mb) | 521.30 | 412.03 |
|
Number of
chromosomes |
17 | N/A |
|
Number of
contigs |
374 | 149 |
| Contig N50 | 5.93 Mb | 5.95 Mb |
| Number of scaffolds | 57 | 49 |
| Scaffold N50 | 33.97 Mb | 33.89 Mb |
|
Longest scaffold
length (Mb) |
52.36 | N/A |
| Sex chromosomes | W; Z1 and Z2 | N/A |
| Organelles | Mitochondrial
genome: 15.26 kb |
N/A |
Most of the assembly sequence (99.7%) was assigned to 17 chromosomal-level scaffolds, representing 14 autosomes and the Z 1, Z 2, and W sex chromosomes. Chromosomes Z and W were assigned based on read coverage and Hi-C data. Chromosome W is comprised of contigs of an uncertain order and orientation. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 2; Table 3). Chromosome painting with Merian elements illustrates the distribution of orthologues along chromosomes and highlights patterns of chromosomal evolution relative to Lepidopteran ancestral linkage groups ( Figure 3).
Figure 2. Hi-C contact map of the Erebia triaria genome assembly.
Assembled chromosomes are shown in order of size and labelled along the axes. The plot was generated using PretextSnapshot.
Figure 3. Merian elements painted across chromosomes in the ilEreTria2.hap1.1 assembly of Erebia triaria.
Chromosomes are drawn to scale, with the positions of orthologues shown as coloured bars. Each orthologue is coloured by the Merian element that it belongs to. All orthologues which could be assigned to Merian elements are shown.
Table 3. Chromosomal pseudomolecules in the haplotype 1 genome assembly of Erebia triaria ilEreTria2.
| INSDC
accession |
Molecule | Length
(Mb) |
GC% | Assigned
Merian elements |
|---|---|---|---|---|
| OZ073195.1 | 1 | 43.41 | 37 | M11;M13;M30 |
| OZ073197.1 | 2 | 38.09 | 37 | M14;M29;M8 |
| OZ073198.1 | 3 | 35.54 | 37 | M22;M3 |
| OZ073199.1 | 4 | 33.99 | 37 | M12;M4 |
| OZ073200.1 | 5 | 33.97 | 37 | M23;M9 |
| OZ073201.1 | 6 | 33.39 | 37 | M15;M5 |
| OZ073202.1 | 7 | 31.18 | 37.50 | M10;M27;M31 |
| OZ073203.1 | 8 | 28.09 | 37 | M28;M7 |
| OZ073204.1 | 9 | 27.22 | 37 | M21;M24 |
| OZ073205.1 | 10 | 24.10 | 37 | M25;M6 |
| OZ073206.1 | 11 | 21.20 | 37.50 | M19;M26 |
| OZ073207.1 | 12 | 20.24 | 37 | M1 |
| OZ073208.1 | 13 | 20.17 | 37 | M2 |
| OZ073209.1 | 14 | 19.82 | 37 | M17;M20 |
| OZ073194.1 | W | 52.36 | 36 | N/A |
| OZ073196.1 | Z1 | 39.69 | 36.50 | M18;MZ |
| OZ073210.1 | Z2 | 17.29 | 37 | M16 |
The mitochondrial genome was also assembled. This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.
Assembly quality metrics
For haplotype 1, the estimated QV is 65.1, and for haplotype 2, 67.1. When the two haplotypes are combined, the assembly achieves an estimated QV of 65.8. The k-mer completeness is 84.89% for haplotype 1, 74.12% for haplotype 2, and 99.33% for the combined haplotypes ( Figure 4). BUSCO analysis using the lepidoptera_odb10 reference set ( n = 5 286) ( Kriventseva et al., 2019) identified 98.4% of the expected gene set (single = 97.8%, duplicated = 0.5%) for haplotype 1. The snail plot in Figure 5 summarises the scaffold length distribution and other assembly statistics for haplotype 1. The blob plot in Figure 6 shows the distribution of scaffolds by GC proportion and coverage for haplotype 1.
Figure 4. Evaluation of k-mer completeness using MerquryFK.
This plot illustrates the recovery of k-mers from the original read data in the final assemblies. The horizontal axis represents k-mer multiplicity, and the vertical axis shows the number of k-mers. The black curve represents k-mers that appear in the reads but are not assembled. The green curve (the homozygous peak) corresponds to k-mers shared by both haplotypes and the red and blue curves (the heterozygous peaks) show k-mers found only in one of the haplotypes.
Figure 5. Assembly metrics for ilEreTria2.hap1.1.
The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1,000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer.
Figure 6. BlobToolKit GC-coverage plot for ilEreTria2.hap1.1.
Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available on the BlobToolKit viewer.
Table 4 lists the assembly metric benchmarks adapted from Rhie et al. (2021) the Earth BioGenome Project Report on Assembly Standards September 2024. The EBP metric, calculated for the haplotype 1, is 6.C.Q65, meeting the recommended reference standard.
Table 4. Earth Biogenome Project summary metrics for the Erebia triaria assembly.
| Measure | Value | Benchmark |
|---|---|---|
| EBP summary (haplotype 1) | 6.7.Q65 | 6.C.Q40 |
| Contig N50 length | 5.93 Mb | ≥ 1 Mb |
| Scaffold N50 length | 33.97 Mb | = chromosome N50 |
| Consensus quality (QV) | Haplotype 1: 65.1; haplotype 2: 67.1; combined: 65.8 | ≥ 40 |
| k-mer completeness | Haplotype 1: 84.89%; Haplotype 2: 74.12%; combined:
99.33% |
≥ 95% |
| BUSCO | C:98.4%[S:97.8%‚D:0.5%]‚ F:0.3%‚M:1.3%‚n:5 286 | S > 90%; D < 5% |
| Percentage of assembly
assigned to chromosomes |
0% | ≥ 90% |
Wellcome Sanger Institute – Legal and Governance
The materials that have contributed to this genome note have been 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.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (220540). KL was supported by the Swiss National Science Foundation Grant PCEFP3_202869. MV was supported by Xunta de Galicia (ED431B 2024/23) and the University of A Coruña.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 1; peer review: 3 approved]
Data availability
European Nucleotide Archive: Erebia triaria (de Prunner’s ringlet). Accession number PRJEB75277. The genome sequence is released openly for reuse. The Erebia triaria genome sequencing initiative is part of 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. The genome will be annotated using available RNA-Seq data and presented through Ensembl at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1 and Table 2.
Pipelines used for genome assembly at the WSI Tree of Life are available at https://pipelines.tol.sanger.ac.uk/pipelines. Table 5 lists software versions used in this study.
Table 5. Software versions and sources.
Author information
Contributors are listed at the following links:
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