The genome sequence of a longhorn beetle, Stictoleptura scutellata (Fabricius, 1781) (Coleoptera: Cerambycidae)

TL;DR

This paper presents the genome sequence of the longhorn beetle Stictoleptura scutellata, including two haplotypes and a mitochondrial genome, as part of a larger project to sequence species in Britain and Ireland.

Contribution

The study provides a high-quality reference genome for Stictoleptura scutellata, including chromosomal pseudomolecules and a mitochondrial genome.

Findings

The genome assembly includes two haplotypes with lengths of 1,471.09 and 1,480.10 megabases.

Haplotype 1 is scaffolded into 10 chromosomal pseudomolecules, including the X chromosome.

The mitochondrial genome is 17.97 kilobases long.

Abstract

We present a genome assembly from an individual female
Stictoleptura scutellata (longhorn beetle; Arthropoda; Insecta; Coleoptera; Cerambycidae). The assembly contains two haplotypes with total lengths of 1 471.09 megabases and 1 480.10 megabases. Most of haplotype 1 (99.33%) is scaffolded into 10 chromosomal pseudomolecules, including the X sex chromosome, while haplotype 2 was assembled to scaffold level. The mitochondrial genome has also been assembled, with a length of 17.97 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.

Figures

Photograph of the Stictoleptura scutellata (icStiScut1) specimen used for genome sequencing.

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.

Hi-C contact map of the Stictoleptura scutellata 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.

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.

Assembly metrics for icStiScut1.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.

BlobToolKit GC-coverage plot for icStiScut1.hap1.1.Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available on the BlobToolKit viewer.

Tables

Table 1.. Specimen and sequencing data for BioProject PRJEB77316.

Platform	PacBio HiFi	Hi-C	RNA-seq
ToLID	icStiScut1	icStiScut1	icStiScut1
Specimen ID	NHMUK015059141	NHMUK015059141	NHMUK015059141
BioSample (source individual)	SAMEA112964455	SAMEA112964455	SAMEA112964455
BioSample (tissue)	SAMEA112975689	SAMEA112975689	SAMEA112975653
Tissue	head and thorax	head and thorax	abdomen
Instrument	Sequel IIe	Illumina NovaSeq 6000	Illumina NovaSeq X
Run accessions	ERR13362602; ERR13362603	ERR13350325	ERR14792835
Read count total	10.95 million	719.46 million	91.56 million
Base count total	116.52 Gb	108.64 Gb	13.82 Gb

Table 2.. Genome assembly statistics.

Assembly name	icStiScut1.hap1.1	icStiScut1.hap2.1
Assembly accession	GCA_964212005.1	GCA_964212015.1
Assembly level	chromosome	scaffold
Span (Mb)	1 471.09	1 480.10
Number of chromosomes	10	Scaffold-level
Number of contigs	502	438
Contig N50	10.87 Mb	10.13 Mb
Number of scaffolds	230	201
Scaffold N50	172.53 Mb	170.6 Mb
Longest scaffold length (Mb)	286.49	-
Sex chromosomes	X	-
Organelles	Mitochondrion: 17.97 kb	-

Table 3.. Chromosomal pseudomolecules in the haplotype 1 genome assembly of Stictoleptura scutellata icStiScut1.

INSDC accession	Molecule	Length (Mb)	GC%
OZ125664.1	1	286.49	33.50
OZ125665.1	2	225.97	33.50
OZ125666.1	3	175.87	33.50
OZ125667.1	4	172.53	33.50
OZ125668.1	5	146.22	33.50
OZ125669.1	6	131.20	33.50
OZ125670.1	7	98.91	33.50
OZ125671.1	8	83.23	33.50
OZ125672.1	9	83.17	34
OZ125673.1	X	57.67	34

Table 4.. Earth Biogenome Project summary metrics for the Stictoleptura scutellata assembly.

Measure	Value	Benchmark
EBP summary (haplotype 1)	7.C.Q64	6.C.Q40
Contig N50 length	10.87 Mb	≥ 1 Mb
Scaffold N50 length	172.53 Mb	= chromosome N50
Consensus quality (QV)	Haplotype 1: 64.6; haplotype 2: 64.5; combined: 64.6	≥ 40
k-mer completeness	Haplotype 1: 79.54%; Haplotype 2: 79.68%; combined: 99.60%	≥ 95%
BUSCO	C:99.2% [S:96.6%; D:2.7%]; F:0.3%; M:0.4%; n:2 124	S > 90%; D < 5%
Percentage of assembly assigned to chromosomes	99.33%	≥ 90%

Full text

Species taxonomy

Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Coleoptera; Polyphaga; Cucujiformia; Chrysomeloidea; Cerambycidae; Lepturinae; Lepturini; Stictoleptura; Stictoleptura scutellata (Fabricius, 1781) (NCBI:txid879004)

Background

We present a chromosome-level genome sequence for Stictoleptura scutellata. This longhorn beetle is considered scarce in Britain and depends on ancient broadleaved woodland and wood pasture, with records concentrated in the New Forest, North Downs and the Epping Forest area ( Falk, 2025). Across its wider range it is a western Palaearctic, thermophilic species associated with beech forests and other deciduous woodland ( Hoskovec et al., 2025). In Britain and Ireland, records are largely from south-east England ( NBN Atlas Partnership, 2025).

Stictoleptura scutellata is ecologically linked to deciduous trees, especially beech ( Fagus sylvatica), but it is polyphagous, developing in the dead wood of a range of broadleaved hosts including Fagus, Carpinus, Quercus, Alnus, Betula, Corylus avellana, Fraxinus, Castanea, Populus and others ( Ellis, 2025; Hoskovec et al., 2025). Larvae feed in dead wood of moderate to large diameter in standing trunks, fallen timber, trunk cavities and dead branches; pupation occurs in the wood. It has a prolonged life cycle (typically lasting 2 to 3 years, up to 4 years in some observations), and adults are active from late spring to late summer (May to September), often visiting flowers such as hawthorn and bramble ( Falk, 2025; Hoskovec et al., 2025).

Adult S. scutellata beetles are medium-sized (12–21 mm), blackish, strongly punctate, and show a partially yellow scutellum, aiding field recognition ( Falk, 2025; Hoskovec et al., 2025).

A total of 5 495 GBIF occurrence records with coordinates are available for this species, with observations primarily from Europe and smaller numbers from Asia and North Africa; records are most frequent from France, Sweden, the United Kingdom, Germany, Austria and the Netherlands ( GBIF Secretariat, 2025). This assembly was generated as part of the Darwin Tree of Life Project, which aims to deliver high-quality reference genomes for all named eukaryotic species in Britain and Ireland to support research, conservation and sustainable use of biodiversity ( Blaxter et al., 2022). This assembly is the first genome for the genus Stictoleptura as of September 2025 (NCBI datasets, O’Leary et al., 2024).

Methods

Sample acquisition and DNA barcoding

The specimen used for genome sequencing was an adult female Stictoleptura scutellata (specimen ID NHMUK015059141, ToLID icStiScut1; Figure 1), collected from Catfield Fen, England, United Kingdom (latitude 52.73, longitude –1.51) on 2022-07-04. The specimen was collected by Steve Crellin and identified by Chris Spilling. The same specimen was used for RNA sequencing.

Photograph of the Stictoleptura scutellata (icStiScut1) specimen used for genome sequencing.

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.

Sample metadata were collected in line with the Darwin Tree of Life project standards described by Lawniczak et al. (2022).

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 icStiScut1 sample was weighed and triaged to determine the appropriate extraction protocol. Tissue from the head and thorax was homogenised by powermashing using a PowerMasher II tissue disruptor. HMW DNA was extracted 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 43 ng/μL and a yield of 2 021.00 ng, with a fragment size of 11.7 kb. The 260/280 spectrophotometric ratio was 1.94, and the 260/230 ratio was 2.03.

RNA was also extracted from abdomen tissue of icStiScut1 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol. The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit. Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

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 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 head and thorax tissue of the icStiScut1 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 6000.

RNA library preparation and sequencing *

Libraries were prepared using the NEBNext ^®^ Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer’s instructions. Poly(A) mRNA in the total RNA solution was isolated using oligo(dT) beads, converted to cDNA, and uniquely indexed; 14 PCR cycles were performed. Libraries were size-selected to produce fragments between 100–300 bp. Libraries were quantified, normalised, pooled to a final concentration of 2.8 nM, and diluted to 150 pM for loading. Sequencing was carried out on the Illumina NovaSeq X to generate 150-bp paired-end reads.### Genome assembly

Prior to assembly of the PacBio HiFi reads, a database of k-mer counts ( k = 31) was generated from the filtered reads using FastK. GenomeScope2 ( Ranallo-Benavidez et al., 2020) was used to analyse the k-mer frequency distributions, providing estimates of genome size, heterozygosity, and repeat content.

The HiFi reads were assembled using Hifiasm in Hi-C phasing mode ( Cheng et al., 2021; Cheng et al., 2022), producing two haplotypes. Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019). Contigs were further scaffolded with Hi-C data in YaHS ( Zhou et al., 2023), using the --break option for handling potential misassemblies. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al., 2022), BUSCO ( Manni et al., 2021) and MERQURY.FK ( Rhie et al., 2020).

The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Assembly curation

The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants ( ASCC) pipeline. TreeVal was used to generate the flat files and maps for use in curation. Manual curation was conducted primarily in PretextView and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Manual corrections included 22 breaks, 46 joins, and removal of 9 haplotypic duplications. 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 both haplotypes using the k-mer databases ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.

The genome was analysed using the BlobToolKit pipeline, a Nextflow implementation of the earlier Snakemake 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 Stictoleptura scutellata specimen generated 116.52 Gb (gigabases) from 10.95 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 1 476.23 Mb, with a heterozygosity of 0.98% and repeat content of 39.25% ( Figure 2). These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 77× coverage. Hi-C sequencing produced 108.64 Gb from 719.46 million reads, which were used to scaffold the assembly. RNA sequencing data were also generated and are available in public sequence repositories. 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 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 1 471.09 Mb in 230 scaffolds, with 272 gaps, and a scaffold N50 of 172.53 Mb ( Table 2).

Most of the assembly sequence (99.33%) 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 3; Table 3). Chromosome X was assigned based on aligment to that of Leptura quadrifasciata (GCA_963675555.1). During curation, we observed a haplotypic inversion on Chromosome 5 in the region ~85.47–88.39 Mb.

Hi-C contact map of the Stictoleptura scutellata 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 Stictoleptura scutellata icStiScut1.

The mitochondrial genome was also assembled (length 17.97 kb, OZ125674.1). This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.

For haplotype 1, the estimated QV is 64.6, and for haplotype 2, 64.5. When the two haplotypes are combined, the assembly achieves an estimated QV of 64.6. The k-mer completeness is 79.54% for haplotype 1, 79.68% for haplotype 2, and 99.60% for the combined haplotypes ( 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 endopterygota_odb10 reference set ( n = 2 124) identified 99.2% of the expected gene set (single = 96.6%, duplicated = 2.7%) for haplotype 1. The snail plot in Figure 5 summarises the scaffold length distribution and other assembly statistics for haplotype 1. The blob plot in Figure 6 shows the distribution of scaffolds by GC proportion and coverage for haplotype 1.

Assembly metrics for icStiScut1.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.

BlobToolKit GC-coverage plot for icStiScut1.hap1.1.Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available on the BlobToolKit viewer.

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 haplotype 1, is 7.C.Q64, meeting the recommended reference standard.

Table 4.: Earth Biogenome Project summary metrics for the Stictoleptura scutellata 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.