The genome sequence of the soldier beetle, Malthodes minimus (Linnaeus, 1758) (Coleoptera: Cantharidae)
Liam M. Crowley, James McCulloch, Maria Antonia Madrid Restrepo, Hume Douglas
TL;DR
This paper presents the genome sequence of the soldier beetle Malthodes minimus, including its chromosomes and mitochondrial DNA, as part of a larger project to sequence species in Britain and Ireland.
Contribution
The paper provides a high-quality reference genome for Malthodes minimus, including sex chromosomes and mitochondrial DNA.
Findings
The genome assembly is 583.60 megabases long, with 97.75% scaffolded into 7 chromosomal pseudomolecules.
The mitochondrial genome is 19.48 kilobases in length and has been fully assembled.
The work is part of the Darwin Tree of Life project, which aims to sequence eukaryotic species in Britain and Ireland.
Abstract
We present a genome assembly from an individual male Malthodes minimus (soldier beetle; Arthropoda; Insecta; Coleoptera; Cantharidae). The genome sequence has a total length of 583.60 megabases. Most of the assembly (97.75%) is scaffolded into 7 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled, with a length of 19.48 kilobases. This assembly was generated as part of the Darwin Tree of Life project, which produces reference genomes for eukaryotic species found in Britain and Ireland.
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
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6| Platform | PacBio HiFi | Hi-C |
|---|---|---|
|
| icMalMini1 | icMalMini3 |
|
| ||
|
| SAMEA112232515 | SAMEA114644949 |
|
| SAMEA112232957 | SAMEA114645632 |
|
| whole organism | whole organism |
|
| Sequel IIe | Illumina NovaSeq X |
|
| ERR13420776 | ERR13389760 |
|
| 3.07 million | 715.27 million |
|
| 29.87 Gb | 108.01 Gb |
|
| icMalMini1.1 |
|
| GCA_964263285.1 |
|
| GCA_964263265.1 |
|
| chromosome |
|
| 583.60 |
|
| 7 |
|
| 691 |
|
| 1.84 Mb |
|
| 190 |
|
| 89.91 Mb |
|
| X and Y |
|
| Mitochondrion: 19.48 kb |
| INSDC accession | Molecule | Length (Mb) | GC% |
|---|---|---|---|
| 1 | 192.32 | 34.50 | |
| 2 | 93.95 | 34.50 | |
| 3 | 89.91 | 34.50 | |
| 4 | 86.73 | 35 | |
| 5 | 72.51 | 35.50 | |
| X | 33.39 | 34.50 | |
| Y | 1.67 | 39.50 |
| Measure | Value | Benchmark |
|---|---|---|
| EBP summary (primary) | 6.C.Q63 | 6.C.Q40 |
| Contig N50 length | 1.84 Mb | ≥ 1 Mb |
| Scaffold N50 length | 89.91 Mb | = chromosome N50 |
| Consensus quality (QV) | Primary: 63.1; alternate: 60.5; combined: 61.7 | ≥ 40 |
|
| Primary: 73.25%; alternate: 60.86%; combined:
| ≥ 95% |
| BUSCO | C:99.1% [S:97.6%; D:1.5%]; F:0.3%; M:0.6%; n:2 124 | S > 90%; D < 5% |
| Percentage of assembly assigned to
| 97.75% | ≥ 90% |
- —Wellcome Trust
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Taxonomy
TopicsGenomics and Phylogenetic Studies · Beetle Biology and Toxicology Studies · Environmental DNA in Biodiversity Studies
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Coleoptera; Polyphaga; Elateriformia; Elateroidea; Cantharidae; Malthininae; Malthodes; Malthodes minimus (Linnaeus, 1758) (NCBI:txid878135)
Background
There are 14 British species in the genus Malthodes Kiesenwetter, 1852, a group of very small soldier beetles (family Cantharidae). Malthodes minimus (Linnaeus, 1758) is one of the more frequently recorded species in southern Britain ( Fitton & Eversham, 2008). Adults are only 3–4 mm long, with dark elytra usually tipped yellow so that part of the hind wings and abdomen remain exposed, and males can only be identified with confidence by microscopic examination of the terminal abdominal appendages, while females are seldom identifiable to species level ( Fitton & Eversham, 2008; NatureSpot, 2025).
Malthodes minimus occurs in well-wooded areas, long grass and water meadows, where adults prey mainly on other insects. In Britain adults are recorded from early May to late August and become scarcer towards the north ( Fitton & Eversham, 2008; NatureSpot, 2025). GBIF lists 3 948 georeferenced occurrence records for M. minimus, all from Europe, with most records from the United Kingdom and Sweden ( GBIF Secretariat, 2025).
We present a chromosome-level genome sequence for Malthodes minimus, produced using the Tree of Life pipeline from a specimen collected from Wytham Woods, Oxfordshire, UK ( Figure 1).
Photograph of the Malthodes minimus (icMalMini1) specimen used for genome sequencing.
Methods
Sample acquisition and DNA barcoding
The specimen used for genome sequencing was an adult male Malthodes minimus (specimen ID Ox002280, ToLID icMalMini1; Figure 1), collected from Wytham Woods, Oxfordshire, UK (latitude 51.772, longitude -1.338) on 2022-07-06. The specimen was collected by James McCulloch and Liam Crowley and identified by James McCulloch. A second specimen collected from the same location on 2023-06-17 was used for Hi-C sequencing (specimen ID Ox004022, ToLID icMalMini3).
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
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 icMalMini1 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. We used centrifuge-mediated fragmentation to produce DNA fragments in the 8–10 kb range, following the Covaris g-TUBE protocol for ultra-low input (ULI). 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.
PacBio HiFi library preparation and sequencing
Library preparation and sequencing were performed at the WSI Scientific Operations core. Prior to library preparation, the DNA was fragmented to ~10 kb. Ultra-low-input (ULI) libraries were prepared using the PacBio SMRTbell® Express Template Prep Kit 2.0 and gDNA Sample Amplification Kit. Samples were normalised to 20 ng DNA. Single-strand overhang removal, DNA damage repair, and end-repair/A-tailing were performed according to the manufacturer’s instructions, followed by adapter ligation. A 0.85× pre-PCR clean-up was carried out with Promega ProNex beads.
The DNA was evenly divided into two aliquots for dual PCR (reactions A and B), both following the manufacturer’s protocol. A 0.85× post-PCR clean-up was performed with ProNex beads. DNA concentration was measured using a Qubit Fluorometer v4.0 (Thermo Fisher Scientific) with the Qubit HS Assay Kit, and fragment size was assessed on an Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) using the gDNA 55 kb BAC analysis kit. PCR reactions A and B were then pooled, ensuring a total mass of =500 ng in 47.4 μl.
The pooled sample underwent another round of DNA damage repair, end-repair/A-tailing, and hairpin adapter ligation. A 1× clean-up was performed with ProNex beads, followed by DNA quantification using the Qubit and fragment size analysis using the Agilent Femto Pulse. Size selection was performed on the Sage Sciences PippinHT system, with target fragment size determined by Femto Pulse analysis (typically 4–9 kb). Size-selected libraries were cleaned with 1.0× ProNex beads and normalised to 2 nM before sequencing.
The sample was 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, and to perform primary and secondary analysis of the data upon completion.
Hi-C
** Sample preparation and crosslinking **
The Hi-C sample was prepared from 20–50 mg of frozen tissue from the icMalMini3 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 ( Cheng et al., 2021) with the --primary option. The Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019), and the contigs were scaffolded in YaHS ( Zhou et al., 2023) with 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).
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 119 breaks and 155 joins. This reduced the scaffold count by 11.2% and reduced the total assembly length by 1.6%. 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 Merqury.FK tool ( Rhie et al., 2020) was run in a Singularity container ( Kurtzer et al., 2017) to evaluate k-mer completeness and assembly quality for the primary and alternate 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 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 Malthodes minimus specimen generated 29.87 Gb (gigabases) from 3.07 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 529.70 Mb, with a heterozygosity of 1.81% and repeat content of 39.72% ( Figure 2). These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 53× coverage. Hi-C sequencing produced 108.01 Gb from 715.27 million reads, which were used to scaffold the assembly. Table 1 summarises the specimen and sequencing details.
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.
Assembly statistics
The primary haplotype was assembled, and contigs corresponding to an alternate haplotype were also deposited in INSDC databases. The final assembly has a total length of 583.60 Mb in 190 scaffolds, with 501 gaps, and a scaffold N50 of 89.91 Mb ( Table 2).
Most of the assembly sequence (97.75%) was assigned to 7 chromosomal-level scaffolds, representing 5 autosomes and the X and Y sex chromosomes. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 3; Table 3). Chromosomes X and Y assigned by read coverage and HiC signal.
Hi-C contact map of the Malthodes minimus 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 primary genome assembly of Malthodes minimus icMalMini1.
The mitochondrial genome was also assembled (length 19.48 kb, OZ181730.1). This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.
The combined primary and alternate assemblies achieve an estimated QV of 61.7. The k-mer completeness is 73.25% for the primary assembly, 60.86% for the alternate haplotype, and 96.59% for the combined assemblies ( 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 v.5.5.0 analysis using the endopterygota_odb10 reference set ( n = 2 124) identified 99.1% of the expected gene set (single = 97.6%, duplicated = 1.5%). The snail plot in Figure 5 summarises the scaffold length distribution and other assembly statistics for the primary assembly. The blob plot in Figure 6 shows the distribution of scaffolds by GC proportion and coverage.
Assembly metrics for icMalMini1.1.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 endopterygota_odb10 set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer.
BlobToolKit GC-coverage plot for icMalMini1.1.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) and the Earth BioGenome Project Report on Assembly Standards September 2024. The EBP metric, calculated for the primary assembly, is 6.C.Q63, meeting the recommended reference standard.
Table 4.: Earth Biogenome Project summary metrics for the Malthodes minimus assembly.
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:
Ethical review of provenance and sourcing of the materialLegality 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.
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