Abstract
We present a genome assembly from an individual male Kyklioacalles roboris (cryptorhynchine weevil; Arthropoda; Insecta; Coleoptera; Curculionidae). The assembly contains two haplotypes with total lengths of 698.93 megabases and 609.02 megabases. Most of haplotype 1 (87.44%) is scaffolded into 20 chromosomal pseudomolecules, including the X and Y sex chromosomes. Haplotype 2 was assembled to scaffold level. The mitochondrial genome has also been assembled, with a length of 20.21 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.
Keywords: Kyklioacalles roboris; cryptorhynchine weevil; genome sequence; chromosomal; Coleoptera
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Coleoptera; Polyphaga; Cucujiformia; Curculionoidea; Curculionidae; Cryptorhynchinae; Kyklioacalles; Kyklioacalles roboris (Stüben, 2003) (NCBI:txid501664)
Background
Kyklioacalles roboris (Curtis, 1834) is a small cryptorhynchine weevil found in broadleaf woodland litter, especially among dead leaves and twig accumulations around oak growths such as witches’ brooms and epicormic shoots ( Gurney, 2018). Adults have dense tufted scales on the pronotum and elytra, and the pronotum shows pronounced lateral bumps ( Gurney, 2018). In Britain it is the least often encountered of the three similar species in this group ( Gurney, 2018).
The genus Kyklioacalles was separated from Acalles by Stüben in 1999 based on male endophallus characters, with later work refining generic limits and intrageneric groupings ( Stüben & Astrin, 2010). The species belongs to the hidden snout weevils (Curculionidae: Cryptorhynchinae), a hyperdiverse lineage with many thousands of species worldwide. Adults commonly feign death by folding the legs and tucking the rostrum into a ventral groove. Phylogenetic and biogeographical analyses indicate a Late Cretaceous origin for Cryptorhynchinae with later dispersal into the Western Palaearctic ( Letsch et al., 2020).
We present a chromosome-level genome sequence for Kyklioacalles roboris, based on a specimen collected from Wytham Woods, Oxfordshire, UK ( Figure 1), as part of the Darwin Tree of Life Project. This genome sequence will enable molecular investigations of cryptorhynchine evolution across this hyperdiverse clade.
Figure 1. Photograph of the Kyklioacalles roboris (icKykRobo1) specimen used for genome sequencing.
Methods
Sample acquisition and DNA barcoding
The specimen used for genome sequencing was an adult male Kyklioacalles roboris (specimen ID Ox003277, ToLID icKykRobo1; Figure 1), collected from Wytham Woods, Oxfordshire, UK (latitude 51.772, longitude –1.338) on 2022-10-31. The specimen was collected and identified by Liam Crowley (University of Oxford). Sample metadata were collected in line with the Darwin Tree of Life project standards described by Lawniczak et al. (2022).
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 icKykRobo1 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. Sheared DNA was purified by manual 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 6.51 ng/μL and a yield of 299.46 ng, with a fragment size of 13.2 kb.
PacBio HiFi library preparation and sequencing
Library preparation and sequencing were performed at the WSI Scientific Operations core. Libraries were prepared using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, California, USA), following the manufacturer’s instructions. The kit includes reagents for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead clean-up, and nuclease treatment. Size selection and clean-up were performed using diluted AMPure PB beads (Pacific Biosciences). DNA concentration was quantified using a Qubit Fluorometer v4.0 (ThermoFisher Scientific) and the Qubit 1X dsDNA HS assay kit. Final library fragment size was assessed with the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) using the gDNA 55 kb BAC analysis kit.
The sample was sequenced on a Revio instrument (Pacific Biosciences). The prepared library was normalised to 2 nM, and 15 μL was used for making complexes. Primers were annealed and polymerases bound to generate circularised complexes, following the manufacturer’s instructions. Complexes were purified using 1.2X SMRTbell beads, then diluted to the Revio loading concentration (200–300 pM) and spiked with a Revio sequencing internal control. The sample was sequenced on a Revio 25M SMRT cell. The SMRT Link software (Pacific Biosciences), a web-based workflow manager, was used to configure and monitor the run and to carry out primary and secondary data analysis.
Hi-C
Sample preparation and crosslinking
The Hi-C sample was prepared from 20–50 mg of frozen tissue from the whole organism of the icKykRobo1 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.
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 organelle genomes were 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 41 breaks and 48 joins. This reduced the scaffold count by 0.7% and increased the scaffold N50 by 2.1%. 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 Kyklioacalles roboris specimen generated 40.45 Gb (gigabases) from 3.57 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 616.98 Mb, with a heterozygosity of 0.61% and repeat content of 46.12% ( Figure 2). These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 63× coverage. Hi-C sequencing produced 293.49 Gb from 1 943.62 million reads, which were used to scaffold the assembly. Table 1 summarises the specimen and sequencing details.
Figure 2. Frequency distribution of k-mers generated using GenomeScope2.
The plot shows observed and modelled k-mer spectra, providing estimates of genome size, heterozygosity, and repeat content based on unassembled sequencing reads.
Table 1. Specimen and sequencing data for BioProject PRJEB76839.
| Platform | PacBio HiFi | Hi-C |
|---|---|---|
| ToLID | icKykRobo1 | icKykRobo1 |
| Specimen ID | Ox003277 | Ox003277 |
| BioSample (source individual) | SAMEA113425821 | SAMEA113425821 |
| BioSample (tissue) | SAMEA113426007 | SAMEA113426007 |
| Tissue | whole organism | whole organism |
| Instrument | Revio | Illumina NovaSeq 6000 |
| Run accessions | ERR13304160 | ERR13317840 |
| Read count total | 3.57 million | 1 943.62 million |
| Base count total | 40.45 Gb | 293.49 Gb |
Assembly statistics
The genome was assembled into two haplotypes using Hi-C phasing. Haplotype 1 was curated to chromosome level, while haplotype 2 was assembled to scaffold level. The final assembly has a total length of 698.93 Mb in 964 scaffolds, with 467 gaps, and a scaffold N50 of 30.38 Mb ( Table 2).
Table 2. Genome assembly statistics.
| Assembly name | icKykRobo1.hap1.1 | icKykRobo1.hap2.1 |
| Assembly accession | GCA_964211925.1 | GCA_964211975.1 |
| Assembly level | chromosome | scaffold |
| Span (Mb) | 698.93 | 609.02 |
| Number of chromosomes | 20 | scaffold-level |
| Number of contigs | 1 431 | 689 |
| Contig N50 | 2.97 Mb | 4.11 Mb |
| Number of scaffolds | 964 | 424 |
| Scaffold N50 | 30.38 Mb | 30.51 Mb |
| Longest scaffold length (Mb) | 56.91- | |
| Sex chromosomes | X and Y | - |
| Organelles | Mitochondrion: 20.21 kb | - |
Most of the haplotype 1 assembly sequence (87.44%) was assigned to 20 chromosomal-level scaffolds, representing 18 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 were assigned based on read coverage statistics and Hi-C signal.
Figure 3. Hi-C contact map of the Kyklioacalles roboris 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 Kyklioacalles roboris icKykRobo1.
| INSDC accession | Molecule | Length (Mb) | GC% |
|---|---|---|---|
| OZ125620.1 | 1 | 56.91 | 34.50 |
| OZ125621.1 | 2 | 48.63 | 34.50 |
| OZ125622.1 | 3 | 41.22 | 35 |
| OZ125623.1 | 4 | 37.52 | 34.50 |
| OZ125624.1 | 5 | 35.12 | 35 |
| OZ125625.1 | 6 | 34.58 | 35 |
| OZ125626.1 | 7 | 34.19 | 34.50 |
| OZ125627.1 | 8 | 30.92 | 34 |
| OZ125628.1 | 9 | 30.38 | 34.50 |
| OZ125629.1 | 10 | 30.07 | 34.50 |
| OZ125630.1 | 11 | 30.03 | 35 |
| OZ125632.1 | 12 | 27.70 | 35.50 |
| OZ125633.1 | 13 | 27.49 | 35 |
| OZ125634.1 | 14 | 26.69 | 35 |
| OZ125635.1 | 15 | 23.83 | 35.50 |
| OZ125636.1 | 16 | 23.10 | 35 |
| OZ125637.1 | 17 | 20.75 | 35 |
| OZ125638.1 | 18 | 12.72 | 36 |
| OZ125631.1 | X | 27.86 | 34.50 |
| OZ125639.1 | Y | 11.46 | 35.50 |
The mitochondrial genome was also assembled (length 20.21 kb, OZ125640.1). 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 61.6, and for haplotype 2, 63.0. When the two haplotypes are combined, the assembly achieves an estimated QV of 62.2. The k-mer completeness is 89.18% for haplotype 1, 83.76% for haplotype 2, and 98.94% for the combined haplotypes ( Figure 4).
Figure 4. Evaluation of k-mer completeness using MerquryFK.
This plot illustrates the recovery of k-mers from the original read data in the final assemblies. The horizontal axis represents k-mer multiplicity, and the vertical axis shows the number of k-mers. The black curve represents k-mers that appear in the reads but are not assembled. The green curve corresponds to k-mers shared by both haplotypes, and the red and blue curves show k-mers found only in one of the haplotypes.
BUSCO analysis using the endopterygota_odb10 reference set ( n = 2 124) identified 98.7% of the expected gene set (single = 97.8%, duplicated = 0.9%) 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.
Figure 5. Assembly metrics for icKykRobo1.hap1.1.
The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1 000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer.
Figure 6. BlobToolKit GC-coverage plot for icKykRobo1.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 6.7.Q61.
Table 4. Earth Biogenome Project summary metrics for the Kyklioacalles roboris assembly.
| Measure | Value | Benchmark |
|---|---|---|
| EBP summary (haplotype 1) | 6.7.Q61 | 6.C.Q40 |
| Contig N50 length | 2.97 Mb | ≥ 1 Mb |
| Scaffold N50 length | 30.38 Mb | = chromosome N50 |
| Consensus quality (QV) | Haplotype 1: 61.6; haplotype 2: 63.0; combined: 62.2 | ≥ 40 |
| k-mer completeness | Haplotype 1: 89.18%; Haplotype 2: 83.76%; combined: 98.94% | ≥ 95% |
| BUSCO | C:98.7% [S:97.8%; D:0.9%]; F:0.3%; M:1.0%; n:2 124 | S > 90%; D < 5% |
| Percentage of assembly
assigned to chromosomes |
87.44% | ≥ 90% |
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 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.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (220540) and the Darwin Tree of Life Discretionary Award [218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328 </a>].
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 1; peer review: 2 approved]
Data availability
European Nucleotide Archive: Kyklioacalles roboris. Accession number PRJEB76839. The genome sequence is released openly for reuse. The Kyklioacalles roboris genome sequencing initiative is part of the Darwin Tree of Life Project (PRJEB40665) and the Sanger Institute Tree of Life Programme (PRJEB43745). All raw sequence data and the assembly have been deposited in INSDC databases. The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1 and Table 2.
Production code used in genome assembly at the WSI Tree of Life is available at https://github.com/sanger-tol. Table 5 lists software versions used in this study.
Table 5. Software versions and sources.
Author information
Contributors are listed at the following links:
Members of the University of Oxford and Wytham Woods Genome Acquisition Lab
Members of the Darwin Tree of Life Barcoding collective
Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team
Members of Wellcome Sanger Institute Scientific Operations – Sequencing Operations
Members of the Wellcome Sanger Institute Tree of Life Core Informatics team
Members of the Tree of Life Core Informatics collective
Members of the Darwin Tree of Life Consortium
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