The genome sequence of a sawfly Macrophya annulata (Geoffroy, 1785)
Liam M. Crowley, Andrew Green, Arka Mukherjee, Marko Prous
TL;DR
This paper presents the genome sequence of the sawfly Macrophya annulata, including a detailed assembly of its chromosomes and mitochondrial DNA.
Contribution
The study provides the first genome assembly for Macrophya annulata, including scaffolded chromosomal pseudomolecules and the mitochondrial genome.
Findings
The genome assembly spans 236.8 megabases and is scaffolded into 8 chromosomal pseudomolecules.
The mitochondrial genome is 31.23 kilobases in length and has been fully assembled.
Abstract
We present a genome assembly from an individual male Macrophya annulata (sawfly; Arthropoda; Insecta; Hymenoptera; Tenthredinidae). The genome sequence is 236.8 megabases in span. Most of the assembly is scaffolded into 8 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 31.23 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 4
Figure 5| Project accession data | ||
|---|---|---|
| Assembly identifier | iyMacAnnu1.1 | |
| Species |
| |
| Specimen | iyMacAnnu1 | |
| NCBI taxonomy ID | 1384895 | |
| BioProject | PRJEB65725 | |
| BioSample ID | SAMEA110451611 | |
| Isolate information | iyMacAnnu1: head and thorax: DNA and Hi-C sequencing | |
| Assembly metrics
|
| |
| Consensus quality (QV) | 59.2 |
|
|
| 100.0% |
|
| BUSCO
| C:95.7%[S:95.2%,D:0.5%],F:1.4%,M:2.9%,n:5,991 |
|
| Percentage of assembly mapped to chromosomes | 99.42% |
|
| Sex chromosomes | None |
|
| Organelles | Mitochondrial genome: 31.23 kb |
|
| Raw data accessions | ||
| PacificBiosciences Sequel IIe | ERR12015770 | |
| Hi-C Illumina | ERR12035296 | |
| Genome assembly | ||
| Assembly accession | GCA_963924015.1 | |
| Span (Mb) | 236.8 | |
| Number of contigs | 273 | |
| Contig N50 length (Mb) | 2.1 | |
| Number of scaffolds | 28 | |
| Scaffold N50 length (Mb) | 38.4 | |
| Longest scaffold (Mb) | 47.94 | |
| INSDC accession | Chromosome | Length (Mb) | GC% |
|---|---|---|---|
| 1 | 47.94 | 36.0 | |
| 2 | 42.3 | 35.5 | |
| 3 | 38.43 | 34.5 | |
| 4 | 29.01 | 35.0 | |
| 5 | 26.91 | 37.0 | |
| 6 | 19.85 | 34.0 | |
| 7 | 17.31 | 34.5 | |
| 8 | 13.69 | 34.5 | |
| MT | 0.03 | 16.0 |
| Software tool | Version | Source |
|---|---|---|
| BEDTools | 2.30.0 |
|
| Blast | 2.14.0 |
|
| BlobToolKit | 4.3.7 |
|
| BUSCO | 5.4.3 |
|
| BUSCO | 5.4.3 and 5.5.0 |
|
| bwa-mem2 | 2.2.1 |
|
| Cooler | 0.8.11 |
|
| DIAMOND | 2.1.8 |
|
| fasta_windows | 0.2.4 |
|
| FastK | 427104ea91c78c3b8b8b49f1a7d6bbeaa869ba1c |
|
| GoaT CLI | 0.2.5 |
|
| Hifiasm | 0.16.1-r375 |
|
| HiGlass | 1.11.6 |
|
| HiGlass | 44086069ee7d4d3f6f3f0012569789ec138f42b84aa44357826c0b6753eb28de |
|
| MerquryFK | d00d98157618f4e8d1a9190026b19b471055b22e |
|
| MitoHiFi | 2 |
|
| MultiQC | 1.14, 1.17, and 1.18 |
|
| NCBI Datasets | 15.12.0 |
|
| Nextflow | 23.04.0-5857 |
|
| PretextView | 0.2 |
|
| purge_dups | 1.2.3 |
|
| samtools | 1.16.1, 1.17, and 1.18 |
|
| sanger-tol/genomenote | 1.1.1 |
|
| sanger-tol/readmapping | 1.2.1 |
|
| Seqtk | 1.3 |
|
| Singularity | 3.9.0 |
|
| TreeVal | 1.0.0 |
|
| YaHS | yahs-1.1.91eebc2 |
|
- —Wellcome Trust
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Taxonomy
TopicsGenomics and Phylogenetic Studies · Environmental DNA in Biodiversity Studies · Insect and Arachnid Ecology and Behavior
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Hymenoptera; Tenthredinoidea; Tenthredinidae; Tenthredininae; Macrophya; Macrophya annulata (Geoffroy, 1785) (NCBI:txid1384895).
Background
There are almost 300 Macrophya species globally, of which ten are found in Britain. The genus is characterised by elongate hind femora and coxae. Macrophya annulata (Geoffroy, 1785) is within the subgenus Macrophya. In addition to the subgenera, several species groupings have been named, and M. annulata resides in the Macrophya blanda-duodecempunctata group which consists of Macrophya blanda (Fabricius, 1775), Macrophya duodecimpunctata (Linnaeus, 1758), and M. annulata. This group is characterised by the presence of a metepimeral appendage: a small round, oval or triangular sclerite located just beneath the hind wing at the juncture of the thorax and abdomen.
The species is widely recorded in England and Wales, particularly in the south-east, but the only Scottish record derives from Benson (1952), who stated that the range extends north to Roxburghshire ( Musgrove, 2023). M. annulata is a black species with the abdomen usually girdled in red, although an all-black form is sometimes found. This species is an effective pompilid spider-hunting wasp mimic and can often be found running around at ground level in a similar manner to pompilids.
M. annulata is a comparatively large (10 to 12.5 mm), black Macrophya usually marked with red on the abdomen and often suffused with white on the front face of the fore and mid legs. Males and females are equally common and can be readily identified in the field. The similar M. blanda differs in having a white patch on the hind coxae. Most sawfly adults are predatory, or feed on pollen or nectar, though there is little published information on the feeding behaviour of M. annulata.
Benson states that in Britain the larvae of M. annulata feed on Creeping Cinquefoil ( Potentilla reptans L.) and this is supported by ovipositing results using British stock where Creeping Cinquefoil was the preferred foodplant for ovipositing and as food for early instars, with roses ( Rosa spp.) and Dewberry ( Rubus caesius L.) accepted by later instars. According to Macek (2012), larvae feed on Dog Rose ( Rosa canina L.) in the wild but will accept blackberry ( Rubus fruticosus agg.) and Creeping Cinquefoil in captivity. Larvae develop very slowly, taking several months to reach the final instar. The larvae are not considered a pest of agricultural or horticultural significance in Britain. The species is univoltine with adults on the wing between May and July. This M. annulata specimen from Wytham Woods, England was identified using Benson’s key ( Benson, 1952) and is typical of the red banded form.
Macrophya annulata specimens fall into four barcode clusters. This specimen, along with the majority of specimens recorded on BOLD, is in the AAE7614 BIN ( Boldsystem, 2023). This complete gene sequence will help our understanding of the phylogeny of this group. The comparative analysis of genomes from closely and distantly related species will benefit the knowledge of sawfly evolution.
Genome sequence report
The genome was sequenced from a male Macrophya annulata ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, –1.33). A total of 115-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 118 missing joins or mis-joins, reducing the scaffold number by 73.15%, and increasing the scaffold N50 by 5.41%.
Photograph of the Macrophya annulata (iyMacAnnu1) specimen used for genome sequencing.
The final assembly has a total length of 236.8 Mb in 28 sequence scaffolds with a scaffold N50 of 38.4 Mb ( Table 1). The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3. The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla. Most (99.42%) of the assembly sequence was assigned to 8 chromosomal-level scaffolds. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 5; Table 2). The exact order and orientation of the contigs in the centromeric repeat regions of chromosome 1 (22.1–24.9) and chromosome 2 (9.2–11.7) is unknown. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited. The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
Table 1.: Genome data for Macrophya annulata, iyMacAnnu1.1.
Genome assembly of Macrophya annulata, iyMacAnnu1.1: metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness. The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 236,804,564 bp assembly. The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (47,939,759 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (38,433,985 and 17,305,532 bp), respectively. The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude. The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot. A summary of complete, fragmented, duplicated and missing BUSCO genes in the hymenoptera_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Macrophya_annulata/dataset/GCA_963924015.1/snail.
Genome assembly of Macrophya annulata, iyMacAnnu1.1: BlobToolKit GC-coverage plot.Sequences are coloured by phylum. Circles are sized in proportion to sequence length. Histograms show the distribution of sequence length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Macrophya_annulata/dataset/GCA_963924015.1/blob.
Genome assembly of Macrophya annulata, iyMacAnnu1.1: BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences. Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Macrophya_annulata/dataset/GCA_963924015.1/cumulative.
Genome assembly of Macrophya annulata, iyMacAnnu1.1: Hi-C contact map of the iyMacAnnu1.1 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=ME0fzKJ-SAKdOmO9-jxAfA.
Table 2.: Chromosomal pseudomolecules in the genome assembly of Macrophya annulata, iyMacAnnu1.
The estimated Quality Value (QV) of the final assembly is 59.2 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.4.3 completeness of 95.7% (single = 95.2%, duplicated = 0.5%), using the hymenoptera_odb10 reference set ( n = 5,991).
Metadata for specimens, BOLD barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/1384895.
Methods
Sample acquisition and nucleic acid extraction
A male Macrophya annulata (specimen ID Ox002165, ToLID iyMacAnnu1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude –1.33) on 2022-05-19 by netting. The specimen was collected and identified by Liam Crowley (University of Oxford) and preserved on dry ice.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up. In sample preparation, the iyMacAnnu1 sample was weighed and dissected on dry ice ( Jay et al., 2023). Tissue from head and thorax was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a).
HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol ( Oatley et al., 2023). The DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system with speed setting 31 ( Bates et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation ( Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA. The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io ( Denton et al., 2023b).
Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers’ instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences Sequel IIe instrument. Hi-C data were also generated from head and thorax tissue of iyMacAnnu1 using the Arima v2 kit. The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on the Illumina NovaSeq 6000 instrument.
Genome assembly and curation
Assembly was carried out with Hifiasm ( Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using YaHS ( Zhou et al., 2023). The assembly was checked for contamination and corrected using the TreeVal pipeline ( Pointon et al., 2023). Manual curation was performed using JBrowse2 ( Diesh et al., 2023), HiGlass ( Kerpedjiev et al., 2018) and PretextView ( Harry, 2022). The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) or MITOS ( Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Final assembly evaluation
The final assembly was post-processed and evaluated with the three Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines “sanger-tol/readmapping” ( Surana et al., 2023a), “sanger-tol/genomenote” ( Surana et al., 2023b), and “sanger-tol/blobtoolkit” ( Muffato et al., 2024). The pipeline sanger-tol/readmapping aligns the Hi-C reads with bwa-mem2 ( Vasimuddin et al., 2019) and combines the alignment files with SAMtools ( Danecek et al., 2021). The sanger-tol/genomenote pipeline transforms the Hi-C alignments into a contact map with BEDTools ( Quinlan & Hall, 2010) and the Cooler tool suite ( Abdennur & Mirny, 2020), which is then visualised with HiGlass ( Kerpedjiev et al., 2018). It also provides statistics about the assembly with the NCBI datasets ( Sayers et al., 2024) report, computes k-mer completeness and QV consensus quality values with FastK and MerquryFK, and a completeness assessment with BUSCO ( Manni et al., 2021).
The sanger-tol/blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline ( Challis et al., 2020). It aligns the PacBio reads with 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 lineage, the pipeline aligns the BUSCO genes to the Uniprot Reference Proteomes database ( Bateman et al., 2023) with DIAMOND ( Buchfink et al., 2021) blastp. The genome is also split into chunks according to the density of the BUSCO genes from the closest taxonomically lineage, and each chunk is aligned to the Uniprot Reference Proteomes database with DIAMOND blastx. Genome sequences that have no hit are then chunked with seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). All those outputs are combined with the blobtools suite into a blobdir for visualisation.
All three pipelines were developed using the nf-core tooling ( Ewels et al., 2020), use MultiQC ( Ewels et al., 2016), and make extensive use of the Conda package manager, the Bioconda initiative ( Grüning et al., 2018), the Biocontainers infrastructure ( da Veiga Leprevost et al., 2017), and the Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017) containerisation solutions.
Table 3 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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