The genome sequence of the Marsh Click-beetle, Actenicerus siaelandicus (Müller, O.F., 1764)
Olga Sivell, Ryan Mitchell, Michael F. Geiser, Hume B Douglas, Ruiqi Li
TL;DR
This paper reports the genome sequence of the Marsh Click-beetle, including a detailed assembly of its chromosomes and mitochondrial DNA.
Contribution
The novel contribution is the first genome assembly of Actenicerus siaelandicus, including scaffolded chromosomes and mitochondrial genome.
Findings
The genome assembly is 854.91 megabases long with 95.23% scaffolded into 10 chromosomal pseudomolecules.
The mitochondrial genome is 17.17 kilobases in length and has been fully assembled.
Abstract
We present a genome assembly from a female specimen of Actenicerus siaelandicus (Marsh Click Beetle; Arthropoda; Insecta; Coleoptera; Elateridae). The genome sequence has a total length of 854.91 megabases. Most of the assembly (95.23%) is scaffolded into 10 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 17.17 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 5| Project information | |||
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| Actenicerus siaelandicus | ||
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| PRJEB77636 | ||
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| SAMEA112964361 | ||
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| 869165 | ||
| Specimen information | |||
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| icActSiae1 | SAMEA112975526 | whole organism |
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| icActSiae1 | SAMEA112975526 | whole organism |
| Sequencing information | |||
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| ERR13498492 | 7.58e+08 | 114.51 |
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| ERR13382525 | 8.40e+06 | 83.92 |
| Genome assembly | ||
|---|---|---|
| Assembly name | icActSiae1.1 | |
| Assembly accession | GCA_964234755.1 | |
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| Assembly level for primary assembly | chromosome | |
| Span (Mb) | 854.91 | |
| Number of contigs | 560 | |
| Number of scaffolds | 373 | |
| Longest scaffold (Mb) | 130.53 | |
| Assembly metric | Measure |
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| Contig N50 length | 6.39 Mb |
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| Scaffold N50 length | 83.36 Mb |
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| Consensus quality (QV) | Primary: 59.9; alternate:
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| Primary: 82.77%; alternate:
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| BUSCO
| C:99.6%[S:96.6%,D:3.1%],
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| Percentage of assembly mapped to
| 95.2% |
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| Sex chromosomes | X |
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| Organelles | Mitochondrial genome:
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| INSDC accession | Name | Length (Mb) | GC% |
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| 1 | 130.53 | 34 | |
| 2 | 107.37 | 34.5 | |
| 3 | 97.24 | 35 | |
| 4 | 85.97 | 35 | |
| 5 | 83.36 | 35 | |
| 6 | 82.13 | 34.5 | |
| 7 | 64.33 | 35 | |
| 8 | 57.52 | 34.5 | |
| 9 | 50.24 | 34.5 | |
| X | 55.19 | 35 | |
| MT | 0.02 | 29.5 |
| Software tool | 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_windows | 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 | 0.19.8-r603 |
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| HiGlass | 44086069ee7d4d3f6f3f0012569789ec138f42b84
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| MerquryFK | d00d98157618f4e8d1a9190026b19b471055b22e |
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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 Datasets | 15.12.0 |
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| Nextflow | 23.10.0 |
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| PretextView | 0.2.5 |
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| samtools | 1.19.2 |
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| sanger-tol/ascc | - |
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| sanger-tol/blobtoolkit | 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
TopicsColeoptera Taxonomy and Distribution · Insect Resistance and Genetics · Forest Insect Ecology and Management
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Coleoptera; Polyphaga; Elateriformia; Elateroidea; Elateridae; Prosterninae; Actenicerus; Actenicerus siaelandicus (Müller, 1764) (NCBI:txid869165)
Background
Actenicerus siaelandicus (Müller, O.F., 1764) is a species from family Elateridae (Coleoptera), subfamily Dendrometrinae Gistel, 1848, tribe Prosternini Gistel, 1856. They are commonly referred to as click-beetles on account of the sound they make when they “jump” ( Evans, 1972). This leap into the air is an escape mechanism, or can be used when beetle is on its back. In Britain 73 species from that family have been recorded ( Duff, 2018), however worldwide there are about ten thousand described species making it one of the largest beetle families ( Mendel, 2024). The genus Actenicerus Kiesenwetter, 1858 contains 46 described species plus three subspecies found mostly in East Asia ( Schimmel & Tarnawski, 2015). Of the two European species, A. siaelandicus is the only species occurring in Britain ( Duff, 2018; Mendel, 2024).
Actenicerus siaelandicus is a Palaearctic species, widely distributed from Ireland to Korea ( Cate, 2013; Du Chatenet, 2000). In much of the older literature, the species name is spelt as “ sjaelandicus”, a name now considered an unjustified emendation ( Burakowski et al., 1985). The species is common and widespread in south-east England and Wales, local in rest of England, Scotland and Ireland ( Duff, 2018). It occurs in wetland habitat, e.g. raised mires, blanket bogs, wet moorland of Ireland and north and west parts of Britain; fenland, water meadow and wet heaths in the south and east England; often found at sites with sphagnum and purple moor-grass Molinia caerulea (Linnaeus). In south and east England and East Anglia the species has declined due to habitat loss ( Mendel, 2024).
Actenicerus sjaelandicus is a large beetle, measuring 11–15 mm in length ( Mendel, 2024). As in other Elateridae the body is elongated with elytra coming to a narrow point at the posterior end, while the hind angles of the pronotum are produced into sharp points. The base colour is black with metallic bronze or blue reflections. Elytra and pronotum are unevenly covered in greyish-yellowish pubescence, forming stripes or spots. It has pectinate antennae and evenly convex disc of pronotum, without the median pronotal groove. This combination of characters allows for distinguishing it from among other, similar looking British Elateridae ( Duff, 2018; Mendel, 2024).
Larvae of A. siaelandicus are soil dwelling omnivores ( Hůrka, 2005; Mendel, 2024). The larval stage lasts for at least two seasons, while pupation occurs for few weeks in late summer to early autumn ( Mendel, 2024). This species overwinters as adult in grass tussocks, sphagnum, under wood and in other sheltered locations. The beetles are most numerous from May to July and can be found on scrub, vegetation, occasionally collected from flowers, including May blossom Crataegus ( Mendel, 2024).
The high-quality genome of Actenicerus siaelandicus presented here was sequenced from a single female specimen (NHMUK015059116, SAMEA112964361) from Buxton Heath, England. The genome was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. It will aid research on genetics, biology of A. siaelandicus and phylogeny of genus Actenicerus and the family Elateridae.
Genome sequence report
Sequencing data
The genome of a specimen of Actenicerus siaelandicus ( Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating 83.92 Gb from 8.40 million reads. GenomeScope analysis of the PacBio HiFi data estimated the haploid genome size at 877.42 Mb, with a heterozygosity of 0.78% and repeat content of 45.47%. 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 93.0x coverage of the genome. Chromosome conformation Hi-C data produced 114.51 Gb from 758.32 million reads. Table 1 summarises the specimen and sequencing information, including the BioProject, study name, BioSample numbers, and sequencing data for each technology.
Photograph of the Actenicerus siaelandicus (icActSiae1) specimen used for genome sequencing.
Table 1.: Specimen and sequencing data for Actenicerus siaelandicus.
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 68 misjoins or missing joins and removed 26 haplotypic duplications. These interventions reduced the total assembly length by 1.03%, decreased the scaffold count by 4.83%, and also decreased the scaffold N50 by 1.04%. The final assembly has a total length of 854.91 Mb in 373 scaffolds, with 187 gaps, and a scaffold N50 of 83.36 Mb ( Table 2).
Table 2.: Genome assembly data for Actenicerus siaelandicus.
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 Actenicerus siaelandicus, icActSiae1.1: 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 endopterygota_odb10 set is presented at the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_964234755.1/dataset/GCA_964234755.1/snail.
Genome assembly of Actenicerus siaelandicus, icActSiae1.1: 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_964234755.1/blob.
Genome assembly of Actenicerus siaelandicus, icActSiae1.1: 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_964234755.1/dataset/GCA_964234755.1/cumulative.
Most of the assembly sequence (95.2%) was assigned to 10 chromosomal-level scaffolds, representing 9 autosomes and the X sex chromosome. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 5; Table 3). During curation, the X chromosome was identified by homology with the genome of Melanotus villosus (GCA_963082815.1) ( Sivell et al., 2024).
Genome assembly of Actenicerus siaelandicus: Hi-C contact map of the icActSiae1.1 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=V4kiNjdQRHaRpSCePff2ug.
Table 3.: Chromosomal pseudomolecules in the genome assembly of Actenicerus siaelandicus, icActSiae1.
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 59.9, and the combined primary and alternate assemblies achieve an estimated QV of 56.2. The k-mer completeness for the primary haplotype is 82.77%, and for the alternate haplotype it is 83.05%. The combined primary and alternate assemblies achieve a k-mer completeness of 99.46%. BUSCO analysis using the endopterygota_odb10 reference set ( n = 2,124) indicated a completeness score of 99.6% (single = 96.6%, duplicated = 3.1%).
Table 2 provides assembly metric benchmarks adapted from Rhie et al. (2021) and the Earth BioGenome Project Report on Assembly Standards September 2024. The achieves the EBP reference standard of 6.C.Q59.
Methods
Sample acquisition and DNA barcoding
An adult female Actenicerus siaelandicus (specimen ID NHMUK015059116, ToLID icActSiae1) was collected from Buxton Heath, England, United Kingdom (latitude 52.75, longitude 1.22) on 2022-07-04. The specimen was collected by Olga Sivell (Natural History Museum) and identified by Ryan Mitchell (National Museums Northern Ireland) and preserved by dry freezing (–80 °C).
The initial identification by morphology 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 icActSiae1 sample was prepared for DNA extraction by weighing and dissecting it on dry ice ( Jay et al., 2023). Tissue from the whole organism was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a). HMW DNA was extracted using the Automated MagAttract v2 protocol ( Oatley et al., 2023a). 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 ( Oatley et al., 2023b). 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 whole organism of the icActSiae1 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 on a Revio instrument (Pacific Biosciences, California, USA). Prepared libraries were normalised to 2 nM, and 15 μL was used for making complexes. Primers were annealed and polymerases were hybridised to create circularised complexes according to manufacturer’s instructions. The complexes were purified with the 1.2X clean up with SMRTbell beads. The purified complexes were then diluted to the Revio loading concentration (in the range 200–300 pM), and spiked with a Revio sequencing internal control. Samples were sequenced on Revio 25M SMRT cells (Pacific Biosciences, California, USA). 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. 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. Sex chromosomes were identified by synteny. 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 is 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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