Abstract
We present a genome assembly from an individual male Aethes tesserana (Downland Conch; Arthropoda; Insecta; Lepidoptera; Tortricidae). The assembly contains two haplotypes with total lengths of 486.12 megabases and 482.92 megabases. Most of haplotype 1 (99.81%) is scaffolded into 30 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 15.54 kilobases. This assembly was generated as part of the Darwin Tree of Life project, which produces genomes for eukaryotic species found in Britain and Ireland.
Keywords: Aethes tesserana, Downland Conch, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Altocrustacea; Allotriocarida; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Eumetabola; Endopterygota; Aparaglossata; Panorpida; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysia; Apoditrysia; Tortricoidea; Tortricidae; Tortricinae; Cochylini; Aethes; Aethes tesserana (Denis & Schiffermüller), 1775 (NCBI:txid1660574).
Background
Aethes tesserana (Downland Conch) ( Figure 1) is a tortricid moth (family Tortricidae), a group of small moths in which many species have patterned forewings. In A. tesserana, the forewings are variable in ground colour, ranging from yellowish to greenish yellow, with orangey-brown or brown markings that often form four squarish blotches; in some individuals these appear instead as cross-lines or cross-bands, giving the moth a distinctive chequered appearance ( Kimber, 2025; Waring et al., 2017). The larvae feed in the roots of a range of Asteraceae, including hawk’s-beards, hawkweeds, bristly oxtongue, hawkweed oxtongue, and ploughman’s-spikenard ( Waring et al., 2017). UKMoths similarly notes larval feeding in the roots of oxtongues ( Picris) and hawkweeds ( Hieracium), among other plants ( Kimber, 2025).
Figure 1. Photograph of Aethes tesserana (This image is Image Number 5429023 at Insect Images).
GBIF records indicate that A. tesserana is a western Palaearctic species with a broad distribution across much of Europe, extending eastwards into western Asia ( GBIF Secretariat, 2025). In Britain, it is a local species ( Davis, 2012). UKMoths states that its British distribution is centred in southern England, extending north to Yorkshire, with occasional records from Wales ( Kimber, 2025).
We present a chromosome-level genome sequence for Aethes tesserana, the Downland Conch. The assembly was produced using the Tree of Life pipeline from a specimen collected from Bradenham, England, UK. This 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 ( Darwin Tree of Life Project Consortium, 2022).
Methods
Sample acquisition and DNA barcoding
The specimen used for genome sequencing was an adult male Aethes tesserana (specimen ID NHMUK013697087, ToLID ilAetTess1; Figure 1), collected from Bradenham, England, UK (latitude 51.68, longitude −0.81) on 2022-05-22. The specimen was collected and identified by David Lees.
The initial identification was verified by an additional DNA barcoding process according to the framework developed by Twyford et al. (2024). A small sample was dissected from the specimen and stored in ethanol, while the remaining parts were shipped on dry ice to the Wellcome Sanger Institute (WSI) (see the protocol). The tissue was lysed, the COI marker region was amplified by PCR, and amplicons were sequenced and compared to the BOLD database, confirming the species identification ( Crowley et al., 2023). Following whole genome sequence generation, the relevant DNA barcode region was also used alongside the initial barcoding data for sample tracking at the WSI ( Twyford et al., 2024). The standard operating procedures for Darwin Tree of Life barcoding are available on protocols.io.
Nucleic acid extraction
Detailed protocols for nucleic acid extraction developed at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory are available on protocols.io ( Howard et al., 2025). The ilAetTess1 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. High molecular weight (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 12.16 ng/μL and a yield of 571.52 ng, with a fragment size of 16.8 kb. The Genomic Quality Number (GQN) was 8.1.
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 25 M 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.
Hi-C
Sample preparation and crosslinking
The Hi-C sample was prepared from 20–50 mg of frozen tissue from the ilAetTess1 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 to create 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.
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, 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 MerquryFK ( Rhie et al., 2020).
The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023).
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 13 breaks and 67 joins. This reduced the scaffold count by 34.0%. The curation process is described 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
The MerquryFK tool ( Rhie et al., 2020) was run in a Singularity container ( Kurtzer et al., 2017) to evaluate k-mer completeness and assembly quality for both haplotypes using the k-mer database ( 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 version ( Challis et al., 2020). The pipeline aligns PacBio reads using minimap2 ( Li, 2018) and SAMtools ( Danecek et al., 2021) to generate coverage tracks. It runs BUSCO ( Manni et al., 2021) using lineages identified from the NCBI Taxonomy ( Schoch et al., 2020). For the three domain-level 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 containerisation through Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017).
Genome sequence report
Sequence data
PacBio sequencing of the Aethes tesserana specimen generated 42.44 Gb (gigabases) from 3.68 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 482.81 Mb, with a heterozygosity of 1.09% and repeat content of 41.79% ( Figure 2). These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 85× coverage. Hi-C sequencing produced 116.40 Gb from 385.44 million reads, which were used to scaffold the assembly. Table 1 summarises the specimen and sequencing details.
Figure 2. Frequency distribution of k-mers generated using GenomeScope2.
The plot shows observed and modelled k-mer spectra, providing estimates of genome size, heterozygosity, and repeat content based on unassembled sequencing reads.
Table 1. Specimen and sequencing data for Aethes tesserana (BioProject PRJEB79700).
| Platform | PacBio HiFi | Hi-C |
|---|---|---|
| ToLID | ilAetTess1 | ilAetTess1 |
| Specimen ID | NHMUK013697087 | NHMUK013697087 |
| BioSample (source individual) | SAMEA114805586 | SAMEA114805586 |
| BioSample (tissue) | SAMEA114805705 | SAMEA114805705 |
| Tissue | whole organism | whole organism |
| Instrument | Revio | Illumina NovaSeq X |
| Run accessions | ERR13650037 | ERR13654234 |
| Read count total | 3.68 million | 385.44 million |
| Base count total | 42.44 Gb | 116.40 Gb |
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 486.12 Mb in 69 scaffolds, with 86 gaps, and a scaffold N50 of 17.54 Mb ( Table 2).
Table 2. Genome assembly statistics for Aethes tesserana.
| Genome assembly | Haplotype 1 | Haplotype 2 |
|---|---|---|
| Assembly name | ilAetTess1.hap1.1 | ilAetTess1.hap2.1 |
| Assembly accession | GCA_964270645.1 | GCA_964270635.1 |
| Assembly level | chromosome | scaffold |
| Span (Mb) | 486.12 | 482.92 |
| Number of chromosomes | 30 | scaffold-level |
| Number of contigs | 155 | 138 |
| Contig N50 | 10.34 Mb | 8.28 Mb |
| Number of scaffolds | 69 | 53 |
| Scaffold N50 | 17.54 Mb | 17.15 Mb |
| Longest scaffold length (Mb) | 38.41 | - |
| Sex chromosomes | Z | - |
| Organelles | Mitochondrial genome: 15.54 kb | - |
Most of the haplotype 1 assembly sequence (99.81%) was assigned to 30 chromosomal-level scaffolds, representing 29 autosomes and the Z sex chromosome. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 3 and Table 3). During curation, we noted that contigs in the following regions have uncertain order and orientation: chromosome 26 (4.9–5.9 Mbp) and chromosome 18 (4.5–5.4 Mbp). The Z chromosome was identified through synteny analysis with the genome of Aethes cnicana (GCA_963971205.1).
Figure 3. Hi-C contact map of the Aethes tesserana genome assembly.
Assembled chromosomes are shown in order of size and labelled along the axes, with a megabase scale shown below. The plot was generated using PretextSnapshot.
Table 3. Chromosomal pseudomolecules in the haplotype 1 genome assembly of Aethes tesserana ilAetTess1.
| INSDC accession | Molecule | Length (Mb) | GC% |
|---|---|---|---|
| OZ185030.1 | 1 | 22.33 | 39 |
| OZ185031.1 | 2 | 22.17 | 39.50 |
| OZ185032.1 | 3 | 20.32 | 39 |
| OZ185033.1 | 4 | 20.06 | 39.50 |
| OZ185034.1 | 5 | 19.38 | 39 |
| OZ185035.1 | 6 | 18.89 | 39 |
| OZ185036.1 | 7 | 18.71 | 39 |
| OZ185037.1 | 8 | 18.04 | 39.50 |
| OZ185038.1 | 9 | 17.84 | 39 |
| OZ185039.1 | 10 | 17.81 | 39.50 |
| OZ185040.1 | 11 | 17.54 | 39 |
| OZ185041.1 | 12 | 17.12 | 39.50 |
| OZ185042.1 | 13 | 16.39 | 39.50 |
| OZ185043.1 | 14 | 16.39 | 39.50 |
| OZ185044.1 | 15 | 16.34 | 39 |
| OZ185045.1 | 16 | 15.69 | 39.50 |
| OZ185046.1 | 17 | 15.56 | 39.50 |
| OZ185047.1 | 18 | 14.69 | 40 |
| OZ185048.1 | 19 | 14.06 | 39 |
| OZ185049.1 | 20 | 13.79 | 39.50 |
| OZ185050.1 | 21 | 13.76 | 39.50 |
| OZ185051.1 | 22 | 12.56 | 40 |
| OZ185052.1 | 23 | 11.50 | 39 |
| OZ185053.1 | 24 | 10.92 | 39.50 |
| OZ185054.1 | 25 | 10.89 | 39.50 |
| OZ185055.1 | 26 | 9.26 | 41.50 |
| OZ185056.1 | 27 | 8.30 | 39.50 |
| OZ185057.1 | 28 | 8.28 | 39.50 |
| OZ185058.1 | 29 | 8.19 | 40.50 |
| OZ185029.1 | Z | 38.41 | 39 |
The mitochondrial genome was also assembled (length 15.54 kb, OZ185059.1). 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 65.6, and for haplotype 2, 66.3. When the two haplotypes are combined, the assembly achieves an estimated QV of 65.9. The k-mer completeness is 77.87% for haplotype 1, 77.86% for haplotype 2, and 99.16% for the combined haplotypes ( Figure 4).
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 corresponds to k-mers shared by both haplotypes, and the red and blue curves show k-mers found only in one of the haplotypes.
BUSCO analysis using the lepidoptera_odb10 reference set ( n = 5 286) identified 98.5% of the expected gene set (single = 97.9%, duplicated = 0.6%) in haplotype 1. For haplotype 2, BUSCO v.5.5.0 analysis identified 98.5% of the expected gene set (single = 97.9%, duplicated = 0.6%). 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 5. Assembly metrics for ilAetTess1.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 blob plot for ilAetTess1.hap1.1.
The plot shows base 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) and the Earth BioGenome Project Report on Assembly Standards January 2026. The EBP metric, calculated for the haplotype 1, is 7.C.Q65, meeting the recommended reference standard.
Table 4. Earth Biogenome Project summary metrics for the Aethes tesserana assembly.
| Measure | Value | Benchmark |
|---|---|---|
| EBP summary (haplotype 1) | 7.C.Q65 | 6.C.Q40 |
| Contig N50 length | 10.34 Mb | ≥ 1 Mb |
| Scaffold N50 length | 17.54 Mb | = chromosome N50 |
| Consensus quality (QV) | Haplotype 1: 65.6; haplotype 2: 66.3; combined: 65.9 | ≥ 40 |
| k-mer completeness | Haplotype 1: 77.87%; Haplotype 2: 77.86%; combined: 99.16% | ≥ 95% |
| BUSCO | C:98.5% [S:97.9%, D:0.6%], F:0.3%, M:1.2%, n:5 286 | S > 90%; D < 5% |
| Percentage of assembly assigned to chromosomes | 99.81% | ≥ 90% |
Note: The EBP summary uses log10(Contig N50); chromosome-level (C) or log10(Scaffold N50); Q (Merqury QV). BUSCO: C = complete; S = single-copy; D = duplicated; F = fragmented; M = missing; n = orthologues.
Author information
Contributors are listed at the following links:
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Members of the Natural History Museum Genome Acquisition Lab
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Members of the Darwin Tree of Life Barcoding collective
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Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team
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Members of Wellcome Sanger Institute Scientific Operations – Sequencing Operations
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Members of the Wellcome Sanger Institute Tree of Life Core Informatics team
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Members of the Tree of Life Core Informatics collective
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Members of the Darwin Tree of Life Consortium
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. 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:
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Ethical review of provenance and sourcing of the material
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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.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (220540) and the Darwin Tree of Life Discretionary Award [218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328 </a>].
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, 1 approved with reservations]
Data availability
European Nucleotide Archive: Aethes tesserana. Accession number PRJEB79700. The genome sequence is released openly for reuse. The Aethes tesserana genome sequencing initiative is part of the Darwin Tree of Life Project (PRJEB40665), 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 the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Tables 1 and 2.
Production code used in genome assembly at the WSI Tree of Life is available at https://github.com/sanger-tol . Table 5 lists software versions used in this study.
Table 5. Software versions and sources used for Aethes tesserana.
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