# The genome sequence of the Alpine Heath, Coenonympha gardetta Hübner, 1819 (Lepidoptera: Nymphalidae)

Camille Cornet, Mathieu Joron, Kay Lucek, Charlotte J Wright, Joana I Meier, Mark L Blaxter

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## To cite this version:

Camille Cornet, Mathieu Joron, Kay Lucek, Charlotte J Wright, Joana I Meier, et al.. The genome sequence of the Alpine Heath, Coenonympha gardetta Hübner, 1819 (Lepidoptera: Nymphalidae). Wellcome Open Research, 2025, 10, pp.403. 10.12688/wellcomeopenres.24645.1. hal-05345624

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DATA NOTE

# The genome sequence of the Alpine Heath, *Coenonympha gardetta* Hübner, 1819 (Lepidoptera: Nymphalidae)

[version 1; peer review: awaiting peer review]

Camille Cornet ID<fnref n="1" />, Mathieu Joron ID<fnref n="2" />, Kay Lucek ID<fnref n="1" />, Charlotte J. Wright ID<fnref n="3" />,
Joana I. Meier<fnref n="3" />, Mark L. Blaxter ID<fnref n="3" />,
Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory
team,
Wellcome Sanger Institute Scientific Operations: Sequencing Operations,
Wellcome Sanger Institute Tree of Life Core Informatics team,
Tree of Life Core Informatics collective, Project Psyche Community

**V1**
First published: 31 Jul 2025, 10:403
https://doi.org/10.12688/wellcomeopenres.24645.1

Latest published: 31 Jul 2025, 10:403
https://doi.org/10.12688/wellcomeopenres.24645.1

Open Peer Review

**Approval Status** AWAITING PEER REVIEW

Any reports and responses or comments on the
article can be found at the end of the article.

Abstract

We present a genome assembly from a female specimen of
*Coenonympha gardetta* (Alpine Heath; Arthropoda; Insecta;
Lepidoptera; Nymphalidae). The assembly contains two haplotypes
with total lengths of 500.33 megabases and 474.74 megabases. Most
of haplotype 1 (99.65%) is scaffolded into 29 chromosomal
pseudomolecules, including the Z sex chromosome. Haplotype 2 was
assembled to scaffold level. The mitochondrial genome has also been
assembled, with a length of 15.29 kilobases.

Keywords

Coenonympha gardetta, Alpine Heath, genome sequence,
chromosomal, Lepidoptera

<figure><img src="image_4.png" /></figure>

This article is included in the Tree of Life
gateway.

<footnote n="1">¹University of Neuchâtel, Neuchâtel, Switzerland</footnote>

<footnote n="2">²Centre for Evolutionary and Functional Ecology (CEFE CNRS), Montpellier, France</footnote>

<footnote n="3">³Tree of Life, Wellcome Sanger Institute, Hinxton, England, UK</footnote>
**Corresponding author:** Charlotte J. Wright (cw22@sanger.ac.uk)

**Author roles:** Cornet C: Investigation, Resources; Joron M: Writing - Original Draft Preparation; Lucek K: Resources, Supervision;

Wright CJ: Funding Acquisition, Project Administration, Supervision; Meier JI: Funding Acquisition, Project Administration, Supervision;

Blaxter ML: Funding Acquisition, Project Administration, Supervision;

**Competing interests:** No competing interests were disclosed.

**Grant information:** This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (220540). CC and KL were supported by Swiss National Science Foundation (SNSF) Grant ID 202869.

*The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.*

**Copyright:** © 2025 Cornet C et al. This is an open access article distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**How to cite this article:** Cornet C, Joron M, Lucek K et al. *The genome sequence of the Alpine Heath, Coenonympha gardetta Hübner, 1819 (Lepidoptera: Nymphalidae) [version 1; peer review: awaiting peer review]* Wellcome Open Research 2025, 10:403
https://doi.org/10.12688/wellcomeopenres.24645.1

**First published:** 31 Jul 2025, 10:403 https://doi.org/10.12688/wellcomeopenres.24645.1
## Species taxonomy

Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysis; Obtectomera; Papilionoidea; Nymphalidae; Satyrinae; Satyrini; Coenonymphina; Coenonympha; Coenonympha gardetta Hübner, 1819 (NCBI: txid554481)

## Background

Coenonympha gardetta (de Prunner, 1798), known as the Alpine Heath, is a small nymphalid butterfly from a diverse genus with a Holarctic distribution (Bozano, 2002). *C. gardetta* is part of a clade that has diversified in the Western Palaearctic (Kodandaramaiah *et al.*, 2009). *C. gardetta* is a high-altitude specialist restricted to the European Alps, and the Massif Central of France (Bozano, 2002). This species is listed as Least Concern in the IUCN Red List (Europe) with a decreasing trend (Van Swaay *et al.*, 2010). Subspecies *C. g. lecerfi* de Lesse 1949, the only non-Alpine population with a disjunct distribution on the hilltops of the Forez massif of France, is listed as Vulnerable (UICN-France, 2014).

Coenonympha gardetta inhabits open grasslands around the treeline and above into alpine meadows from 1 400 to 2 800 metres above sea level. It depends on grasses for larval development, laying eggs singly in the shade of herbs or shrubs. Adults fly from late June to late July, depending on geography, and may be found perching or sun basking on plants and rocks. The wing pattern includes a dark brown upper side and a grey and buff colour on the underside, featuring a regular series of black eyespots with a white centre, which contrast prominently against a crescent of cream-coloured scales. It resembles *C. orientalis*, a closely related high-altitude species in the Balkan Peninsula (Bozano, 2002).

Coenonympha gardetta is part of a clade that displays significant signatures of interspecific gene flow (Greenwood *et al.*, 2025), and is therefore worthy of further investigation to reveal the role of gene flow in local adaptation and speciation. *C. gardetta* diverged from its lowland relative *C. arcania* approximately 1.7 million years ago, replacing it on the altitudinal gradient and suggesting specific altitudinal adaptations. For instance, genomic signals of selection have been found in genes related to anoxia (*Capblancq* *et al.*, 2024), and experiments have uncovered thermal adaptation to adult flight, larval cold hardiness, and egg heat tolerance (*Doniol-Valcroze* *et al.*, 2024; Nève & Després, 2020; Vrba *et al.*, 2022). *C. gardetta* is the subject of ongoing research into hybrid speciation, with population resequencing of genomes from diverse Alpine locations and from the contact zones with its close allies (*Capblancq* *et al.*, 2024; Doniol-Valcroze, 2024). Although *C. gardetta* exhibits strong premating isolation with *C. arcania*, involving chemical divergence and genetic incompatibilities (*Capblancq* *et al.*, 2019; Doniol-Valcroze *et al.*, 2025), hybridisation around 300 thousand years ago has formed two local hybrid lineages in the Alps, to which *C. gardetta* contributes about 23% of its genome (*Capblancq* *et al.*, 2015; *Capblancq* *et al.*, 2024). Understanding how this genome fraction contributes

to adaptation and to barriers to gene flow with related species is crucial to our understanding of the evolutionary history of this clade. More generally, this case of hybrid speciation is an excellent model for investigating the modalities of the emergence of new species (*Capblancq* *et al.*, 2015; *Capblancq* *et al.*, 2024).

A reference genome for *C. gardetta* is an invaluable resource for advancing our understanding of clade diversification, hybrid speciation, and altitudinal adaptation. The sequence data were derived from a female specimen (Figure 1) collected from Brochwald, Meiringen, Switzerland.

## Methods

### Sample acquisition

The specimen used for genome sequencing was an adult female *Coenonympha gardetta* (specimen ID SAN28000081, ToLID ilCoeGard1; Figure 1), collected from Brochwald, Meiringen, Switzerland (latitude 46.6743, longitude 8.1334; elevation 1470 m) on 09/07/2023. The specimen was collected and identified by Camille Cornet (University of Neuchâtel).

### 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 ilCoeGard1 sample was weighed and triaged to determine the appropriate extraction protocol. Tissue from the thorax 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

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Voucher photograph of the *Coenonympha gardetta* (ilCoeGard1) specimen used for genome sequencing.</figcaption></figure>
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 32.23 ng/µL and a yield of 1,514.81 ng, with a fragment size of 13.6 kb. The 260/280 spectrophotometric ratio was 1.87, and the 260/230 ratio was 2.72.

## 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.

### Hi-C

#### Sample preparation and crosslinking

The Hi-C sample was prepared from 20–50 mg of frozen head tissue of the ilCoeGard1 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

<table><caption>**Table 1. Specimen and sequencing data for BioProject PRJEB78768.**</caption><thead><tr><th>Platform</th><th>PacBio HiFi</th><th>Hi-C</th></tr></thead><tbody><tr><td>ToLID</td><td>ilCoeGard1</td><td>ilCoeGard1</td></tr><tr><td>Specimen ID</td><td>SAN28000081</td><td>SAN28000081</td></tr><tr><td>BioSample (source individual)</td><td>SAMEA115109803</td><td>SAMEA115109803</td></tr><tr><td>BioSample (tissue)</td><td>SAMEA115109828</td><td>SAMEA115109827</td></tr><tr><td>Tissue</td><td>thorax</td><td>head</td></tr><tr><td>Sequencing platform and model</td><td>Revio</td><td>Illumina NovaSeq X</td></tr><tr><td>Run accessions</td><td>ERR13485730</td><td>ERR13493987</td></tr><tr><td>Read count total</td><td>1.82 million</td><td>774.20 million</td></tr><tr><td>Base count total</td><td>20.32 Gb</td><td>116.90 Gb</td></tr></tbody></table>

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 8 breaks and 17 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_buscoPainter 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 *Coenonympha gardetta* was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating 20.32 Gb (gigabases) from 1.82 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 480.04 Mb, with a heterozygosity of 1.81% and repeat content of 38.66%. These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 41× coverage. Hi-C sequencing produced 116.90 Gb from 774.20 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 500.33 Mb in 74 scaffolds, with 178 gaps, and a scaffold N50 of 18.14 Mb (Table 2).

Most of the assembly sequence (99.65%) was assigned to 29 chromosomal-level scaffolds, representing 28 autosomes and the Z sex chromosome. The specimen appears to be a ZO female. 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).

<table><caption>**Table 2. Genome assembly statistics.**</caption><thead><tr><th></th><th>ilCoeGard1.hap1.1</th><th>ilCoeGard1.hap2.1</th></tr></thead><tbody><tr><td>Assembly name</td><td>ilCoeGard1.hap1.1</td><td>ilCoeGard1.hap2.1</td></tr><tr><td>Assembly accession</td><td>GCA_964258855.1</td><td>GCA_964258745.1</td></tr><tr><td>Assembly level</td><td>chromosome</td><td>scaffold</td></tr><tr><td>Span (Mb)</td><td>500.33</td><td>474.74</td></tr><tr><td>Number of chromosomes</td><td>29</td><td>N/A</td></tr><tr><td>Number of contigs</td><td>252</td><td>264</td></tr><tr><td>Contig N50</td><td>4.46 Mb</td><td>4.91 Mb</td></tr><tr><td>Number of scaffolds</td><td>74</td><td>122</td></tr><tr><td>Scaffold N50</td><td>18.14 Mb</td><td>17.71 Mb</td></tr><tr><td>Longest scaffold length (Mb)</td><td>26.01</td><td>N/A</td></tr><tr><td>Sex chromosomes</td><td>Z</td><td>N/A</td></tr><tr><td>Organelles</td><td>Mitochondrial genome: 15.29 kb</td><td>N/A</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Hi-C contact map of the *Coenonympha gardetta* genome assembly. Assembled chromosomes are shown in order of size and labelled along the axes. The plot was generated using PretextSnapshot.</figcaption></figure>

<table><caption>**Table 3.** Chromosomal pseudomolecules in the haplotype 1 genome assembly of *Coenonympha gardetta* ilCoeGard1.</caption><thead><tr><th>INSDC accession</th><th>Molecule</th><th>Length (Mb)</th><th>GC%</th><th>Assigned Merian elements</th></tr></thead><tbody><tr><td>OZ177574.1</td><td>1</td><td>21.17</td><td>38.50</td><td>M17;M20</td></tr><tr><td>OZ177575.1</td><td>2</td><td>21.08</td><td>38</td><td>M2</td></tr><tr><td>OZ177576.1</td><td>3</td><td>21.05</td><td>38</td><td>M1</td></tr><tr><td>OZ177577.1</td><td>4</td><td>20.85</td><td>38</td><td>M3</td></tr><tr><td>OZ177578.1</td><td>5</td><td>20.64</td><td>38</td><td>M19;M26</td></tr><tr><td>OZ177579.1</td><td>6</td><td>19.82</td><td>37.50</td><td>M9</td></tr><tr><td>OZ177580.1</td><td>7</td><td>19.73</td><td>39</td><td>M14;M29</td></tr><tr><td>OZ177581.1</td><td>8</td><td>19.58</td><td>38</td><td>M8</td></tr><tr><td>OZ177582.1</td><td>9</td><td>19.17</td><td>38</td><td>M5</td></tr><tr><td>OZ177583.1</td><td>10</td><td>18.71</td><td>38</td><td>M18</td></tr><tr><td>OZ177584.1</td><td>11</td><td>18.43</td><td>38.50</td><td>M7</td></tr><tr><td>OZ177585.1</td><td>12</td><td>18.14</td><td>38</td><td>M12</td></tr><tr><td>OZ177586.1</td><td>13</td><td>17.86</td><td>38.50</td><td>M6</td></tr></tbody></table>

<table><thead><tr><th>INSDC accession</th><th>Molecule</th><th>Length (Mb)</th><th>GC%</th><th>Assigned Merian elements</th></tr></thead><tbody><tr><td>OZ177587.1</td><td>14</td><td>17.67</td><td>38</td><td>M4</td></tr><tr><td>OZ177588.1</td><td>15</td><td>17.46</td><td>37.50</td><td>M16</td></tr><tr><td>OZ177589.1</td><td>16</td><td>16.76</td><td>37.50</td><td>M21</td></tr><tr><td>OZ177590.1</td><td>17</td><td>16.66</td><td>38.50</td><td>M15</td></tr><tr><td>OZ177591.1</td><td>18</td><td>16.58</td><td>38</td><td>M22</td></tr><tr><td>OZ177592.1</td><td>19</td><td>16.49</td><td>39</td><td>M11</td></tr><tr><td>OZ177593.1</td><td>20</td><td>16.47</td><td>38.50</td><td>M10</td></tr><tr><td>OZ177594.1</td><td>21</td><td>15.73</td><td>39</td><td>M23</td></tr><tr><td>OZ177595.1</td><td>22</td><td>14.60</td><td>38</td><td>M13</td></tr><tr><td>OZ177596.1</td><td>23</td><td>13.05</td><td>41</td><td>M30</td></tr><tr><td>OZ177597.1</td><td>24</td><td>12.28</td><td>39</td><td>M24</td></tr><tr><td>OZ177598.1</td><td>25</td><td>11.86</td><td>39</td><td>M28</td></tr><tr><td>OZ177599.1</td><td>26</td><td>10.63</td><td>38.50</td><td>M27</td></tr><tr><td>OZ177600.1</td><td>27</td><td>10.56</td><td>40</td><td>M25</td></tr><tr><td>OZ177601.1</td><td>28</td><td>9.55</td><td>41.50</td><td>M31</td></tr><tr><td>OZ177573.1</td><td>Z</td><td>26.01</td><td>37</td><td>MZ</td></tr></tbody></table>
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Merian elements painted across chromosomes in the *ilCoeGard1.hap1.1* assembly of *Coenonympha gardetta*. 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.</figcaption></figure>

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 63.7, and for haplotype 2, 64.2. When the two haplotypes are combined, the assembly achieves an estimated QV of 63.9. The k-mer completeness is 72.16% for haplotype 1, 68.67% for haplotype 2, and 99.33% for the combined haplotypes (Figure 4). BUSCO analysis using the lepidoptera_odb10 reference set (n = 5286) (Kriventseva et al., 2019) identified 98.2% of the expected gene set (single = 97.5%, duplicated = 0.7%) 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.

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.Q63**, meeting the recommended reference standard.

## 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
<figure><img src="image_8.png" /><figcaption>**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.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 5.** Assembly metrics for ilCoeGard1.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.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Figure 6.** BlobToolKit GC-coverage plot for ilCoeGard1.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.</figcaption></figure>

<table><caption>**Table 4.** Earth Biogenome Project summary metrics for the Coenonympha gardetta assembly.</caption><thead><tr><td>Measure</td><td>Value</td><td>Benchmark</td></tr></thead><tbody><tr><td>EBP summary (haplotype 1)</td><td>6.C.Q63</td><td>6.C.Q40</td></tr><tr><td>Contig N50 length</td><td>4.46 Mb</td><td>≥ 1 Mb</td></tr><tr><td>Scaffold N50 length</td><td>18.14 Mb</td><td>= chromosome N50</td></tr><tr><td>Consensus quality (QV)</td><td>Haplotype 1: 63.7; haplotype 2: 64.2; combined: 63.9</td><td>≥ 40</td></tr><tr><td>k-mer completeness</td><td>Haplotype 1: 72.16%; Haplotype 2: 68.67%; combined: 99.33%</td><td>≥ 95%</td></tr><tr><td>BUSCO</td><td>C:98.2%[S:97.5%,D:0.7%], F:0.3%,M:1.5%,n:5286</td><td>S > 90%; D < 5%</td></tr><tr><td>Percentage of assembly assigned to chromosomes</td><td>99.65%</td><td>≥ 90%</td></tr></tbody></table>
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.

## Data availability

European Nucleotide Archive: Coenonympha gardetta (alpine heath). Accession number PRJEB78768. The genome sequence is released openly for reuse. The Coenonympha gardetta 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.

## Author information

Contributors are listed at the following links:

- Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team

- Wellcome Sanger Institute Scientific Operations – Sequencing Operations

- Wellcome Sanger Institute Tree of Life Core Informatics team

- Tree of Life Core Informatics collective

- Project Psyche Community.

<table><caption>Table 5. Software versions and sources.</caption><thead><tr><td>Software</td><td>Version</td><td>Source</td></tr></thead><tbody><tr><td>BEDTools</td><td>2.30.0</td><td>https://github.com/arq5x/bedtools2</td></tr><tr><td>BLAST</td><td>2.14.0</td><td>ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/</td></tr><tr><td>BlobToolKit</td><td>4.3.9</td><td>https://github.com/blobtoolkit/blobtoolkit</td></tr><tr><td>BUSCO</td><td>5.5.0</td><td>https://gitlab.com/ezlab/busco</td></tr><tr><td>bwa-mem2</td><td>2.2.1</td><td>https://github.com/bwa-mem2/bwa-mem2</td></tr><tr><td>Cooler</td><td>0.8.11</td><td>https://github.com/open2c/cooler</td></tr><tr><td>DIAMOND</td><td>2.1.8</td><td>https://github.com/bbuchfink/diamond</td></tr><tr><td>fasta_windows</td><td>0.2.4</td><td>https://github.com/tolkit/fasta_windows</td></tr><tr><td>FastK</td><td>1.1</td><td>https://github.com/thegenemyers/FASTK</td></tr><tr><td>GenomeScope2.0</td><td>2.0.1</td><td>https://github.com/tbenavi1/genomescope2.0</td></tr><tr><td>Gfastats</td><td>1.3.6</td><td>https://github.com/vgl-hub/gfastats</td></tr><tr><td>GoaT CLI</td><td>0.2.5</td><td>https://github.com/genomehubs/goat-cli</td></tr><tr><td>Hifiasm</td><td>0.19.8-r603</td><td>https://github.com/chhylp123/hifiasm</td></tr><tr><td>HiGlass</td><td>1.13.4</td><td>https://github.com/higlass/higlass</td></tr><tr><td>lep_buscoPainter</td><td>1.0.0</td><td>https://github.com/charlottewright/lep_busco_painter</td></tr><tr><td>MerquryFK</td><td>1.1.1</td><td>https://github.com/thegenemyers/MERQURY.FK</td></tr><tr><td>Minimap2</td><td>2.24-r1122</td><td>https://github.com/lh3/minimap2</td></tr></tbody></table>
<table><thead><tr><td>Software</td><td>Version</td><td>Source</td></tr></thead><tbody><tr><td>MitoHiFi</td><td>3</td><td>https://github.com/marcelauliano/MitoHiFi</td></tr><tr><td>MultiQC</td><td>1.14; 1.17 and 1.18</td><td>https://github.com/MultiQC/MultiQC</td></tr><tr><td>Nextflow</td><td>23.10.0</td><td>https://github.com/nextflow-io/nextflow</td></tr><tr><td>PretextSnapshot</td><td>N/A</td><td>https://github.com/sanger-tol/PretextSnapshot</td></tr><tr><td>PretextView</td><td>0.2.5</td><td>https://github.com/sanger-tol/PretextView</td></tr><tr><td>samtools</td><td>1.19.2</td><td>https://github.com/samtools/samtools</td></tr><tr><td>sanger-tol/asc</td><td>0.1.0</td><td>https://github.com/sanger-tol/asc</td></tr><tr><td>sanger-tol/ blobtoolkit</td><td>0.6.0</td><td>https://github.com/sanger-tol/blobtoolkit</td></tr><tr><td>Seqtk</td><td>1.3</td><td>https://github.com/lh3/seqtk</td></tr><tr><td>Singularity</td><td>3.9.0</td><td>https://github.com/sylabs/singularity</td></tr><tr><td>TreeVal</td><td>1.2.0</td><td>https://github.com/sanger-tol/treeval</td></tr><tr><td>YaHS</td><td>1.2a.2</td><td>https://github.com/c-zhou/yahs</td></tr></tbody></table>

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