The genome sequence of the Four-spotted Footman moth, Lithosia quadra (Linnaeus, 1758)
Finley Hutchinson, Liam M. Crowley, Arun Arumugaperumal, Sivasankaran Kuppusamy, Yu-Feng Huang, Axel Künstner
TL;DR
This paper presents the genome sequence of the Four-spotted Footman moth, including its chromosomes and mitochondrial DNA.
Contribution
The novel contribution is the high-quality genome assembly of Lithosia quadra, including chromosomal pseudomolecules and the mitochondrial genome.
Findings
The genome assembly is 456.27 megabases long with 99.91% scaffolded into 31 chromosomal pseudomolecules.
The mitochondrial genome is 15.38 kilobases in length.
Abstract
We present a genome assembly from a male Lithosia quadra (Four-spotted Footman; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence has a total length of 456.27 megabases. Most of the assembly (99.91%) is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.38 kilobases in length.
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
Figure 1
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Figure 4
Figure 5| Project information | |||
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| Lithosia quadra (four-spotting footman) | ||
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| PRJEB66748 | ||
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| SAMEA112775013 | ||
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| 987974 | ||
| Specimen information | |||
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| ilLitQuad1 | SAMEA112775073 | head and thorax |
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| ilLitQuad1 | SAMEA112775073 | head and thorax |
| Sequencing information | |||
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| ERR12102419 | 5.60e+08 | 84.6 |
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| ERR12102453 | 2.31e+06 | 27.69 |
| Genome assembly | ||
|---|---|---|
| Assembly name | ilLitQuad1.1 | |
| Assembly accession | GCA_963576445.1 | |
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| Assembly level for primary
| chromosome | |
| Span (Mb) | 456.27 | |
| Number of contigs | 110 | |
| Number of scaffolds | 43 | |
| Longest scaffold (Mb) | 22.95 | |
| Assembly metrics | Measure |
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| Contig N50 length | 8.98 Mb |
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| Scaffold N50 length | 16.41 Mb |
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| Consensus quality (QV) | Primary: 66.7; alternate:
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| Primary: 68.01%; alternate: 68.02%; combined: 99.36% |
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| BUSCO
| C:98.8%[S:98.0%,D:0.8%],
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| Percentage of assembly
| 99.92% |
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| Sex chromosomes | Z |
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| Organelles | Mitochondrial genome:
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| INSDC
| Name | Length
| GC% |
|---|---|---|---|
| 1 | 20.43 | 36.5 | |
| 2 | 19.83 | 37 | |
| 3 | 19.35 | 37 | |
| 4 | 18.95 | 37 | |
| 5 | 18.27 | 37 | |
| 6 | 18.2 | 36.5 | |
| 7 | 17.79 | 36.5 | |
| 8 | 17.38 | 36.5 | |
| 9 | 16.64 | 36.5 | |
| 10 | 16.6 | 37 | |
| 11 | 16.56 | 37 | |
| 12 | 16.41 | 37 | |
| 13 | 15.92 | 36.5 | |
| 14 | 15.38 | 37 | |
| 15 | 15.24 | 37 | |
| 16 | 15.21 | 36.5 | |
| 17 | 14.75 | 37.5 | |
| 18 | 14.73 | 37 | |
| 19 | 14.28 | 37.5 | |
| 20 | 13.38 | 37.5 | |
| 21 | 13.36 | 37 | |
| 22 | 11.35 | 37.5 | |
| 23 | 11.15 | 38 | |
| 24 | 10.54 | 40 | |
| 25 | 10.42 | 38 | |
| 26 | 10.13 | 37.5 | |
| 27 | 9.66 | 38 | |
| 28 | 7.72 | 39 | |
| 29 | 6.92 | 40.5 | |
| 30 | 6.41 | 40 | |
| Z | 22.95 | 37.5 | |
| MT | 0.02 | 20 |
| Software
| Version | Source |
|---|---|---|
| BEDTools | 2.30.0 |
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| BLAST | 2.14.0 |
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| BlobToolKit | 4.3.9 |
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| BUSCO | 5.5.0 |
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| bwa-mem2 | 2.2.1 |
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| Cooler | 0.8.11 |
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| DIAMOND | 2.1.8 |
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| fasta_
| 0.2.4 |
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| FastK | 427104ea91c78c3b8b8b49f1a7d6bbeaa869ba1c |
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| Gfastats | 1.3.6 |
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| GoaT CLI | 0.2.5 |
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| Hifiasm | 0.19.5-r587 |
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| HiGlass | 44086069ee7d4d3f6f3f0012569789ec138f42b84
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| Merqury.FK | d00d98157618f4e8d1a9190026b19b471055b2
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| Minimap2 | 2.24-r1122 |
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| MitoHiFi | 3 |
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| MultiQC | 1.14, 1.17, and 1.18 |
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| NCBI
| 15.12.0 |
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| Nextflow | 23.04.1 |
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| PretextView | 0.2 |
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| purge_dups | 1.2.5 |
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| samtools | 1.19.2 |
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| sanger-tol/
| - |
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| sanger-tol/
| 0.5.1 |
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| Seqtk | 1.3 |
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| Singularity | 3.9.0 |
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| TreeVal | 1.2.0 |
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| YaHS | 1.2a.2 |
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- —Wellcome Trust
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Taxonomy
TopicsGenomics and Phylogenetic Studies · Lepidoptera: Biology and Taxonomy · Genetic diversity and population structure
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; Noctuoidea; Erebidae; Arctiinae; Lithosiini; Lithosia; Lithosia quadra (Linnaeus, 1758) (NCBI:txid987974)
Background
Lithosia quadra, or the Four-spotted Footman, is a moth of the family Erebidae. Its range includes southern and central Europe, extending eastward across the Palaearctic to the Amur River and Japan. In the north, it occurs in southern Great Britain, Scandinavia, and Ireland ( GBIF Secretariat, 2023).
This species exhibits marked sexual dimorphism. Males have smaller wingspans (18–22 mm) with grey forewings with black and yellow blotches at the base and a blue-black stripe along the edge. Females are larger (20–26 mm) with yellow forewings marked by two distinct dark spots ( Waring et al., 2017). With a wingspan of 35–55 mm, L. quadra is a large Footman moth.
The Four-spotted Footman inhabits mature broadleaved woodlands and shows annual fluctuations in population, often reinforced by immigration. Its larvae feed on lichens and algae, overwintering in the larval stage before pupating in late spring ( Waring et al., 2017).
Here we present a chromosomally complete genome sequence for Lithosia quadra, based on a male specimen from Wytham Woods, Oxfordshire, United Kingdom ( Figure 1).
Photograph of the Lithosia quadra (ilLitQuad1) specimen used for genome sequencing.
Genome sequence report
Sequencing data
The genome of a specimen of Lithosia quadra ( Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating 27.69 Gb from 2.31 million reads. GenomeScope analysis of the PacBio HiFi data estimated the haploid genome size at 450.36 Mb, with a heterozygosity of 2.02% and repeat content of 23.25%. These values provide an initial assessment of genome complexity and the challenges anticipated during assembly. Based on this estimated genome size, the sequencing data provided approximately 58.0x coverage of the genome. Chromosome conformation Hi-C sequencing produced 84.60 Gb from 560.29 million reads. Table 1 summarises the specimen and sequencing information, including the BioProject, study name, BioSample numbers, and sequencing data for each technology.
Table 1.: Specimen and sequencing data for Lithosia quadra.
Assembly statistics
The primary haplotype was assembled, and contigs corresponding to an alternate haplotype were also deposited in INSDC databases. The assembly was improved by manual curation, which corrected 17 misjoins or missing joins and removed 2 haplotypic duplications. The final assembly has a total length of 456.27 Mb in 43 scaffolds, with 67 gaps, and a scaffold N50 of 16.41 Mb ( Table 2).
The snail plot in Figure 2 provides a summary of the assembly statistics, indicating the distribution of scaffold lengths and other assembly metrics. Figure 3 shows the distribution of scaffolds by GC proportion and coverage. Figure 4 presents a cumulative assembly plot, with separate curves representing different scaffold subsets assigned to various phyla, illustrating the completeness of the assembly.
Genome assembly of Lithosia quadra, ilLitQuad1.1: metrics.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 lepidoptera_odb10 set is presented at the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_963576445.1/dataset/GCA_963576445.1/snail.
Genome assembly of Lithosia quadra, ilLitQuad1.1: BlobToolKit GC-coverage plot.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 at https://blobtoolkit.genomehubs.org/view/GCA_963576445.1/blob.
Genome assembly of Lithosia quadra, ilLitQuad1.1: 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/GCA_963576445.1/dataset/GCA_963576445.1/cumulative.
Table 2.: Genome assembly data for Lithosia quadra.
Most of the assembly sequence (99.92%) was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 5; Table 3). During curation, chromosome Z was assigned based on synteny with the genome of Nudaria mundana (GCA_963556515.1) ( Crowley et al., 2024).
Genome assembly of Lithosia quadra: Hi-C contact map of the ilLitQuad1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom. An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=SbppNic8RpuV088Ms069EQ.
Table 3.: Chromosomal pseudomolecules in the genome assembly of Lithosia quadra, ilLitQuad1.
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 in GenBank.
Assembly quality metrics
The estimated Quality Value (QV) and k-mer completeness metrics, along with BUSCO completeness scores, were calculated for each haplotype and the combined assembly. The QV reflects the base-level accuracy of the assembly, while k-mer completeness indicates the proportion of expected k-mers identified in the assembly. BUSCO scores provide a measure of completeness based on benchmarking universal single-copy orthologues.
The primary haplotype has a QV of 66.7, and the combined primary and alternate assemblies achieve an estimated QV of 66.5. The k-mer completeness for the primary haplotype is 68.01%, and for the alternate haplotype it is 68.02%. The combined primary and alternate assemblies achieve a k-mer completeness of 99.36%. BUSCO analysis using the lepidoptera_odb10 reference set ( n = 5,286) indicated a completeness score of 98.8% (single = 98.0%, duplicated = 0.8%).
Table 2 provides assembly metric benchmarks adapted from Rhie et al. (2021) and the Earth BioGenome Project (EBP) Report on Assembly Standards September 2024. The assembly achieves the EBP reference standard of 6.C.Q66.
Methods
Sample acquisition and DNA barcoding
An adult female Lithosia quadra (specimen ID Ox003043, ToLID ilLitQuad1) was collected from Wytham Woods, Oxfordshire, United Kingdom (latitude 51.77, longitude –1.34) on 2022-07-22, using a light trap. The specimen was collected by Finley Hutchinson and Liam Crowley (University of Oxford), identified by Finley Hutchinson, and preserved on dry ice.
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) ( Pereira et al., 2022). 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 have been deposited on protocols.io ( Beasley et al., 2023).
Metadata collection for samples adhered to the Darwin Tree of Life project standards described by Lawniczak et al. (2022).
Nucleic acid extraction
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of procedures: sample preparation and homogenisation, DNA extraction, fragmentation and purification. Detailed protocols are available on protocols.io ( Denton et al., 2023b). The ilLitQuad1 sample was prepared for DNA extraction by weighing and dissecting it on dry ice ( Jay et al., 2023). Tissue from the head and thorax was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a).
HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol ( Oatley et al., 2023). The DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system ( Bates et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation, using AMPure PB beads to eliminate shorter fragments and concentrate the DNA ( Strickland et al., 2023). 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.
Hi-C sample preparation
Tissue from the head and thorax of the ilLitQuad1 sample was processed for Hi-C sequencing at the WSI Scientific Operations core, using the Arima-HiC v2 kit. In brief, 20–50 mg of frozen tissue (stored at –80 °C) was fixed, and the DNA crosslinked using a TC buffer with 22% formaldehyde concentration. After crosslinking, the tissue was homogenised using the Diagnocine Power Masher-II and BioMasher-II tubes and pestles. Following the Arima-HiC v2 kit manufacturer's instructions, crosslinked DNA was digested using a restriction enzyme master mix. The 5’-overhangs were filled in and labelled with biotinylated nucleotides and proximally ligated. An overnight incubation was carried out for enzymes to digest remaining proteins and for crosslinks to reverse. A clean up was performed with SPRIselect beads prior to library preparation. Additionally, the biotinylation percentage was estimated using the Qubit Fluorometer v4.0 (Thermo Fisher Scientific) and Qubit HS Assay Kit and Arima-HiC v2 QC beads.
Library preparation and sequencing
Library preparation and sequencing were performed at the WSI Scientific Operations core.
** PacBio HiFi **
At a minimum, samples were required to have an average fragment size exceeding 8 kb and a total mass over 400 ng to proceed to the low input SMRTbell Prep Kit 3.0 protocol (Pacific Biosciences, California, USA), depending on genome size and sequencing depth required. Libraries were prepared using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, California, USA) as per the manufacturer's instructions. The kit includes the reagents required for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead cleanup, and nuclease treatment. Following the manufacturer’s instructions, size selection and clean up was carried out using diluted AMPure PB beads (Pacific Biosciences, California, USA). DNA concentration was quantified using the Qubit Fluorometer v4.0 (Thermo Fisher Scientific) with Qubit 1X dsDNA HS assay kit and the final library fragment size analysis was carried out using the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) and gDNA 55kb BAC analysis kit.
Samples were sequenced using the Sequel IIe system (Pacific Biosciences, California, USA). The concentration of the library loaded onto the Sequel IIe was in the range 40–135 pM. The SMRT link software, a PacBio web-based end-to-end workflow manager, was used to set-up and monitor the run, as well as perform primary and secondary analysis of the data upon completion.
** Hi-C **
For Hi-C library preparation, DNA was fragmented using the Covaris E220 sonicator (Covaris) and size selected using SPRISelect beads to 400 to 600 bp. The DNA was then enriched using the Arima-HiC v2 kit Enrichment beads. Using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) for end repair, a-tailing, and adapter ligation. This uses a custom protocol which resembles the standard NEBNext Ultra II DNA Library Prep protocol but where library preparation occurs while DNA is bound to the Enrichment beads. For library amplification, 10 to 16 PCR cycles were required, determined by the sample biotinylation percentage. The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on an Illumina NovaSeq 6000 instrument.
Genome assembly, curation and evaluation
** 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 first assembled using Hifiasm ( Cheng et al., 2021) with the --primary option. Haplotypic duplications were identified and removed using purge_dups ( Guan et al., 2020). The Hi-C reads were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019). The contigs were further scaffolded using the provided Hi-C data ( Rao et al., 2014) 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 (article in preparation). Flat files and maps used in curation were generated in TreeVal ( Pointon et al., 2023). Manual curation was primarily conducted using PretextView ( Harry, 2022), with additional insights provided by JBrowse2 ( Diesh et al., 2023) and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Any identified contamination, missed joins, and mis-joins were corrected, and duplicate sequences were tagged and removed. The sex chromosome was identified based on synteny analysis. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation (article in preparation).
** Assembly quality assessment **
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 the primary and alternate haplotypes using the k-mer databases ( k = 31) that were computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.
A Hi-C contact map was produced for the final version of the assembly. The Hi-C reads were aligned using bwa-mem2 ( Vasimuddin et al., 2019) and the alignment files were combined using SAMtools ( Danecek et al., 2021). The Hi-C alignments were converted into a contact map using BEDTools ( Quinlan & Hall, 2010) and the Cooler tool suite ( Abdennur & Mirny, 2020). The contact map was visualised in HiGlass ( Kerpedjiev et al., 2018).
The blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline ( Challis et al., 2020). It aligns the PacBio reads in SAMtools and minimap2 ( Li, 2018) and generates coverage tracks for regions of fixed size. In parallel, it queries the GoaT database ( Challis et al., 2023) to identify all matching BUSCO lineages to run BUSCO ( Manni et al., 2021). For the three domain-level BUSCO lineages, the pipeline aligns the BUSCO genes to the UniProt Reference Proteomes database ( Bateman et al., 2023) with DIAMOND blastp ( Buchfink et al., 2021). The genome is also divided into chunks according to the density of the BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database using DIAMOND blastx. Genome sequences without a hit are chunked using seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). The blobtools suite combines all these 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), relying on the Conda package manager, the Bioconda initiative ( Grüning et al., 2018), the Biocontainers infrastructure ( da Veiga Leprevost et al., 2017), as well as the Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017) containerisation solutions.
Table 4 contains a list of relevant software tool versions and sources.
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 here. 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:
• Ethical review of provenance and sourcing of the material
• 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.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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