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. 2025 Sep 10;10:504. [Version 1] doi: 10.12688/wellcomeopenres.24855.1

The genome sequence of the Tenthredid wasp, Eutomostethus luteiventris (Klug, 1816) (Hymenoptera: Tenthredinidae)

Andrew Halstead 1, Keith Fowler 2; Natural History Museum Genome Acquisition Lab; Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team; Wellcome Sanger Institute Scientific Operations: Sequencing Operations; Wellcome Sanger Institute Tree of Life Core Informatics team; Tree of Life Core Informatics collective; Darwin Tree of Life Consortiuma
PMCID: PMC12759282  PMID: 41488256

Abstract

We present a haploid genome assembly from an individual Eutomostethus luteiventris (Tenthredid wasp; Arthropoda; Insecta; Hymenoptera; Tenthredinidae). The genome sequence has a total length of 272.06 megabases. Most of the assembly (99.22%) is scaffolded into 6 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, with a length of 30.01 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: Eutomostethus luteiventris; Tenthredid wasp; genome sequence; chromosomal; Hymenoptera

Species taxonomy

Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Hymenoptera; Tenthredinoidea; Tenthredinidae; Blennocampinae; Eutomostethus; Eutomostethus luteiventris (Klug, 1816) (NCBI:txid1385253)

Background

Eutomostethus luteiventris is a species in which only females have been recorded in Britain and Ireland, and reproduction is believed to be parthenogenetic. It is 5–7mm long with a black head and thorax. The abdomen is mostly orange-yellow with black markings down the centre of the dorsal surface. It occurs in damp places where the larvae feed inside the foliage of rushes, Juncus spp., with the final instar feeding externally ( Benson, 1952).

Eutomostethus luteiventris a common species throughout Britain and also occurs throughout most of Europe ( GBIF Secretariat, 2024). It is an introduced species in North America. In the UK, there is a single generation with adults occurring in late April to June.

The genome of the sawfly, Eutomostethus luteiventris, 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 ( Blaxter et al., 2022). The assembly was produced using the Tree of Life pipeline from a specimen collected in Thompson Common, Norfolk, United Kingdom ( Figure 1).

Figure 1. Photograph of the Eutomostethus luteiventris (iyEutLute1) specimen used for genome sequencing.

Figure 1.

Methods

Sample acquisition and DNA barcoding

The specimen used for genome sequencing was an adult Eutomostethus luteiventris (specimen ID NHMUK015059318, ToLID iyEutLute1; Figure 1), collected from Thompson Common, Norfolk, United Kingdom (latitude 52.53, longitude 0.85) on 2022-07-06. The specimen was collected by Keith Fowler and identified by Andrew Halstead. 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 iyEutLute1 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. 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 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 14.78 ng/μL and a yield of 665.10 ng, with a fragment size of 12.7 kb. The 260/280 spectrophotometric ratio was 2.07, and the 260/230 ratio was 3.46.

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 whole organism tissue of the iyEutLute1 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 and 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 ( Cheng et al., 2021) with the --primary option. Haplotypic duplications were identified and removed using purge_dups ( Guan et al., 2020). The Hi-C reads ( 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), 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 7 breaks and 82 joins. The curation process is documented 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 Eutomostethus luteiventris specimen generated 25.44 Gb (gigabases) from 2.32 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 234.63 Mb, with repeat content of 23.03% ( Figure 2). These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 195× coverage. Hi-C sequencing produced 109.40 Gb from 724.51 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.

Figure 2.

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 PRJEB82369.

Platform PacBio HiFi Hi-C
ToLID iyEutLute1 iyEutLute1
Specimen ID NHMUK015059318 NHMUK015059318
BioSample (source
individual)
SAMEA112964468 SAMEA112964468
BioSample (tissue) SAMEA112975668 SAMEA112975668
Tissue whole organism whole organism
Instrument Sequel IIe Illumina NovaSeq 6000
Run accessions ERR13957071 ERR13947545
Read count total 2.32 million 724.51 million
Base count total 25.44 Gb 109.40 Gb

Assembly statistics

A single haplotype was assembled, with no evidence of heterozygosity. The final assembly has a total length of 272.06 Mb in 58 scaffolds, with 178 gaps, and a scaffold N50 of 44.62 Mb ( Table 2).

Table 2. Genome assembly statistics.

Assembly name iyEutLute1.1
Assembly accession GCA_964662225.1
Assembly level chromosome
Span (Mb) 272.06
Number of
chromosomes
6
Number of contigs 236
Contig N50 4.5 Mb
Number of scaffolds 58
Scaffold N50 44.62 Mb
Organelles Mitochondrion: 30.01 kb

Most of the assembly sequence (99.22%) was assigned to 6 chromosomal-level scaffolds. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 3; Table 3). The order and orientation of scaffolds between ~16.66-20.70Mb on Chromosome 5 is unsure.

Figure 3. Hi-C contact map of the Eutomostethus luteiventris genome assembly.

Figure 3.

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 Eutomostethus luteiventris iyEutLute1.

INSDC
accession
Molecule Length
(Mb)
GC%
OZ211691.1 1 51.14 38
OZ211692.1 2 45.64 39
OZ211693.1 3 44.62 38.50
OZ211694.1 4 44.43 38
OZ211695.1 5 42.07 40
OZ211696.1 6 42.05 38.50

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.

The haploid assembly achieves an estimated QV of 65.1 and the k-mer completeness is 99.41% ( Figure 4).

Figure 4. Evaluation of k-mer completeness using MerquryFK.

Figure 4.

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 hymenoptera_odb10 reference set ( n = 5 991) identified 95.9% of the expected gene set (single = 95.5%, duplicated = 0.4%). 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.

Figure 5. Assembly metrics for iyEutLute1.1.

Figure 5.

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 hymenoptera_odb10 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 iyEutLute1.1.

Figure 6.

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) the Earth BioGenome Project Report on Assembly Standards September 2024. The EBP metric, calculated for the primary assembly, is 6.C.Q65, meeting the recommended reference standard.

Table 4. Earth Biogenome Project summary metrics for the Eutomostethus luteiventris assembly.

Measure Value Benchmark
EBP summary (primary) 6.C.Q65 6.C.Q40
Contig N50 length 4.50 Mb ≥ 1 Mb
Scaffold N50 length 44.62 Mb = chromosome N50
Consensus quality (QV) Primary: 65.2 ≥ 40
k-mer completeness Primary: 99.41% ≥ 95%
BUSCO C:95.9% [S:95.5%;
D:0.4%]; F:0.9%;
M:3.2%; n:5 991
S > 90%; D < 5%
Percentage of
assembly assigned to
chromosomes
99.22% ≥ 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: 4 approved]

Data availability

European Nucleotide Archive: Eutomostethus luteiventris. Accession number PRJEB82369. The genome sequence is released openly for reuse. The Eutomostethus luteiventris 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:

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Wellcome Open Res. 2026 Mar 2. doi: 10.21956/wellcomeopenres.27379.r138244

Reviewer response for version 1

Annabel Whibley 1,2

In this data note, Halsted, Fowler and collagues report the genome assembly of the Tenthredid wasp (a type of sawfly) Eutomostethus luteiventris. The sample metadata protocols, sequencing and assembly pipelines and reporting template follow Darwin Tree of Life standards.  No gene annotation has been released at this time.

Specimen identification was verified by DNA barcoding. The sample is assumed to be female, since only females have been recorded in Britain and Ireland. The reported assembly is a primary assembly only, with the genomescope2 kmer analysis showing a single peak in the profile (albeit with a slight left shoulder).  In general, the quality metrics are excellent,with > 99% of the contigs scaffolded to one of 6 pseudochromosomes. There is some ambiguity in the ordering and orientation of chromosome 5. Busco completeness is ~95%, which seems a little low but may well be typical of the taxon.  

The recovered 30kb mitochondrial genome seems unusually large and I am a little skeptical that it is correctly assembled. E. vegetus has a more  typical 16.5kb mitogenome and the central portion of the contig shows (i) no homology to this sister taxon reference and (ii) strong self-self signal in a pairwise BLAST.  Some discussion of the validation of this organelle assembly would be welcome.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Genomics, Bioinformatics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Wellcome Open Res. 2026 Jan 21. doi: 10.21956/wellcomeopenres.27379.r142960

Reviewer response for version 1

Arun Arumugaperumal 1

The genome sequencing project of Eutomostethus luteiventris, the Tenthredid wasp, has been described. This is the first report of the genome sequence of this insect. The assembly reported here is of size 272.06 Mb, spread among 6 chromosome molecules. The sex chromosomes were not identified. The mitochondrial genome has also been assembled and reported to have a size of 30.01 kb. The authors have used PacBio long-read sequencing and Hi-C sequencing data to obtain a high-quality genome assembly. The quality is evident from the high N50 values. If the annotation details were available, it could have been used to validate the quality of the assembly.

BUSCO analysis has shown completeness of 95.9% with respect to the hymenoptera_odb10 dataset. The data note can be indexed.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Bioinformatics; Genomics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Wellcome Open Res. 2026 Jan 2. doi: 10.21956/wellcomeopenres.27379.r142964

Reviewer response for version 1

Benoit Nabholz 1

The article presents a chromosome-scale assembly of Eutomostethus luteiventris. This represents the 24th genome published for the family Tenthredinidae.

This work is part of the Darwin Tree of Life initiative, which produces high-quality genome assemblies, and the expected standards are met for this assembly. Interestingly, the introduction states that this species is believed to be parthenogenetic.

Based on the specimen photograph, it appears that a female was sequenced; however, this information is not explicitly indicated in the manuscript nor in the associated databases (ENA or the DToL portal: https://portal.darwintreeoflife.org/organism/SAMEA112964468). This is consistent with the parthenogenetic nature of the species. Interestingly, GenomeScope2 and the assembly itself did not detect any heterozygosity, suggesting that parthenogenesis in this species may be associated with a loss of heterozygosity.

Protein-coding genes have not yet been annotated, but according to the manuscript, this step will be performed in the future.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Molecular evolution

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Wellcome Open Res. 2025 Nov 12. doi: 10.21956/wellcomeopenres.27379.r133005

Reviewer response for version 1

Qing-Song Zhou 1

This paper presents a high-quality genome assembly of Eutomostethus luteiventris using PacBio HiFi and Hi-C data, which represents a significant contribution toward generating a reference genome for sawflies with high contiguity (as reflected by a high N50) and completeness (as indicated by high BUSCO scores).

However, the authors should clarify whether the sequenced individual was female or male, as this information is essential for interpreting genome structure.

Additionally, the reported length of the mitochondrial genome of E. luteiventris appears inconsistent with that of a congeneric species, E. vegetus (GenBank accession MT663219), which is 16,345 bp in length. The authors are encouraged to verify and, if necessary, revise the mitochondrial genome length accordingly.

The manuscript would also benefit from additional background information on E. luteiventris, including its ecological and economic relevance, biodiversity context, and taxonomic history. Such context would help readers better appreciate the significance of this genomic resource.

Finally, while the current assembly is already of high quality, the inclusion of Illumina short-read data for polishing and RNA-seq data for genome annotation would further enhance the accuracy and utility of this reference genome. The authors are encouraged to consider incorporating these data in future updates or supplementary analyses.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Entomology, genomics, biodiversity

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Availability Statement

    European Nucleotide Archive: Eutomostethus luteiventris. Accession number PRJEB82369. The genome sequence is released openly for reuse. The Eutomostethus luteiventris 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.


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