Abstract
We present a genome assembly from an individual male Melanostoma mellinum (the dumpy grass hoverfly; Arthropoda; Insecta; Diptera; Syriphidae). The genome sequence is 731 megabases in span. The majority of the assembly (99.67%) is scaffolded into five chromosomal pseudomolecules, with the X and Y sex chromosomes assembled. The complete mitochondrial genome was also assembled and is 16.1 kilobases in length.
Keywords: Melanostoma mellinum, dumpy grass hoverfly, genome sequence, chromosomal, Diptera
Species taxonomy
Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Diptera; Brachycera; Muscomorpha; Syrphoidea; Syrphidae; Syrphinae; Melanostomini; Melanostoma; Melanostoma mellinum (Linnaeus, 1758) (NCBI:txid653684).
Background
Melanostoma mellinum is a small hoverfly with yellow and black markings, perhaps using this aposematic colourway to loosely mimic wasps and so gain protection from predators ( Ball & Morris, 2015; van Veen, 2010). These hoverflies have dark faces and dark scutellums. Female Melanostoma species have distinctive inverted yellow triangle abdominal markings and can be separated from the other common species M. scalare by the narrow dust spots on the otherwise shining frons ( Ball & Morris, 2015). Male M. mellinum ( Figure 1) have square yellow abdominal markings with tergite 2 and 3 being as long as wide, while M. scalare males have longer tergites ( Ball & Morris, 2015; van Veen, 2010). Colour and morphological variation is high and M. mellinum may comprise a species complex ( Haarto & Ståhls, 2014). M. mellinum is an abundant grassland species in the UK and can be found in greatest numbers during summer, feeding on the pollen of wind pollinated flowers such as grasses, plantain and sedges ( Ball & Morris, 2015). The larvae of this species feed on aphids in the leaf litter and ground layer and are incredibly generalist predators, preying on at least 32 different aphid species ( Dziock, 2005) and gall forming Psyllidae ( Hodkinson & Flint, 1971). Like some other generalist hoverflies, M. mellinum adults display seasonal migratory behaviour ( Aubert et al., 1976; Gatter & Schmid, 1990). This species seems to be heavily affected by the Entomophora fungal pathogen ( Jensen & Eilenberg, 2001) and dead M. mellinum individuals can be often found hanging beneath flower heads with fungal spores exuding (WH, personal observation; Figure 1). Migration in this species may function to reduce the negative effects of this fungus on the population by allowing escape from contaminated habitats and by culling infected animals, both phenomena seen in other migrants (see Satterfield et al. (2018)). This is the first production of a high quality M. mellinum genome and we believe that the sequence described here, generated as part of the Darwin Tree of Life project, will further aid understanding of the biology and ecology of this hoverfly.
Figure 1. Example images of Melanostoma mellinum.
Top: female (left) and male (right) examples of M. mellinum; photographs provided by WH. Bottom left: examples of dead M. mellinum individuals that have succumbed to the Entomophora fungal pathogen; photograph provided by WH. Bottom right: the male M. mellinum specimen used for genome sequencing; photograph provided by Liam Crowley, University of Oxford.
Genome sequence report
The genome was sequenced from a single male M. mellinum collected from Wytham Great Wood, Oxfordshire (Biological vice-county: Berkshire), UK (latitude 51.769, longitude -1.330) ( Figure 1). A total of 26-fold coverage in Pacific Biosciences single-molecule long reads and 55-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 332 missing/misjoins and removed 35 haplotypic duplications, reducing the assembly size by 2.84% and the scaffold number by 74.75%, and increasing the scaffold N50 by 395.90%.
The final assembly has a total length of 731 Mb in 76 sequence scaffolds with a scaffold N50 of 235 Mb ( Table 1). The majority, 99.67%, of the assembly sequence was assigned to five chromosomal-level scaffolds, representing three autosomes (numbered by sequence length), and the X and Y sex chromosomes ( Figure 2– Figure 5; Table 2). The assembly has a BUSCO v5.1.2 ( Simão et al., 2015) completeness of 96.3% (single 94.8%, duplicated 1.5%) using the diptera_odb10 reference set (n=3285). While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.
Figure 2. Genome assembly of Melanostoma mellinum, idMelMell2.1: metrics.
The BlobToolKit Snailplot 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 731,040,377 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 (265,601,162 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (235,131,548 and 204,347,527 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 diptera_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idMelMell2.1/dataset/CAJZBU01/snail.
Figure 5. Genome assembly of Melanostoma mellinum, idMelMell2.1: Hi-C contact map.
Hi-C contact map of the idMelMell2.1 assembly, visualised in HiGlass. Chromosomes are arranged in size order from left to right and top to bottom.
Table 1. Genome data for Melanostoma mellinum, idMelMell2.1.
| Project accession data | |
|---|---|
| Assembly identifier | idMelMell2.1 |
| Species | Melanostoma mellinum |
| Specimen | idMelMell2 |
| NCBI taxonomy ID | 653684 |
| BioProject | PRJEB46300 |
| BioSample ID | SAMEA7520051 |
| Isolate information | Male, head/thorax (genome assembly), abdomen (Hi-C) |
| Raw data accessions | |
| PacificBiosciences SEQUEL II | ERR6636092, ERR6636093 |
| 10X Genomics Illumina | ERR6688425-ERR6688428 |
| Hi-C Illumina | ERR6688429 |
| Genome assembly | |
| Assembly accession | GCA_914767635.1 |
| Accession of alternate haplotype | GCA_914767615.1 |
| Span (Mb) | 731 |
| Number of contigs | 479 |
| Contig N50 length (Mb) | 4.6 |
| Number of scaffolds | 76 |
| Scaffold N50 length (Mb) | 235 |
| Longest scaffold (Mb) | 266 |
| BUSCO * genome score | C:96.3%[S:94.8%,D:1.5%],F:0.9%,M:2.8%,n:3285 |
*BUSCO scores based on the diptera_odb10 BUSCO set using v5.1.2. C= complete [S= single copy, D=duplicated], F=fragmented, M=missing, n=number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/idMelMell2.1/dataset/CAJZBU01/busco.
Figure 3. Genome assembly of Melanostoma mellinum, idMelMell2.1: GC coverage.
BlobToolKit GC-coverage plot. Scaffolds are coloured by phylum. Circles are sized in proportion to scaffold length. Histograms show the distribution of scaffold length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idMelMell2.1/dataset/CAJZBU01/blob.
Figure 4. Genome assembly of Melanostoma mellinum, idMelMell2.1: cumulative sequence.
BlobToolKit cumulative sequence plot. The grey line shows cumulative length for all scaffolds. Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idMelMell2.1/dataset/CAJZBU01/cumulative.
Table 2. Chromosomal pseudomolecules in the genome assembly of Melanostoma mellinum, idMelMell2.1.
| INSDC accession | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| OU612058.1 | 1 | 265.60 | 36.7 |
| OU612059.1 | 2 | 235.13 | 37.0 |
| OU612060.1 | 3 | 204.35 | 37.4 |
| OU612061.1 | X | 20.89 | 36.7 |
| OU612062.1 | Y | 1.11 | 36.8 |
| OU612063.1 | MT | 0.02 | 18.7 |
| - | Unplaced | 3.94 | 37.0 |
Methods
Sample acquisition, DNA extraction and sequencing
One male M. mellinum sample, idMelMell2, was collected from Wytham Great Wood, Oxfordshire, (Biological vice-county: Berkshire), UK (latitude 51.769, longitude -1.330) by Will Hawkes, University of Exeter on 7 August 2019. The specimen was caught with a net, identified by the same individual, snap-frozen on dry ice and stored using a CoolRack.
DNA was extracted from the head/thorax of idMelMell2 at the Wellcome Sanger Institute (WSI) Scientific Operations core from the whole organism using the Qiagen MagAttract HMW DNA kit, according to the manufacturer’s instructions. Pacific Biosciences HiFi circular consensus and 10X Genomics Chromium read cloud sequencing libraries were constructed according to the manufacturers’ instructions. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II (HiFi) and Illumina HiSeq X (10X) instruments. Hi-C data were generated from abdomen tissue of idMelMell2 using the Arima v2 Hi-C kit in the Tree of Life laboratory and sequenced at the Scientific Operations core on an Illumina NovaSeq 6000 instrument.
Genome assembly
Assembly was carried out with Hifiasm ( Cheng et al., 2021); haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). One round of polishing was performed by aligning 10X Genomics read data to the assembly with longranger align, calling variants with freebayes ( Garrison & Marth, 2012). The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using SALSA2 ( Ghurye et al., 2019). The assembly was checked for contamination as described previously ( Howe et al., 2021). Manual curation was performed using HiGlass ( Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2021), which performs annotation using MitoFinder ( Allio et al., 2020). The genome was analysed and BUSCO scores generated within the BlobToolKit environment ( Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.
Table 3. Software tools used.
| Software tool | Version | Source |
|---|---|---|
| Hifiasm | 0.15.2 | Cheng et al., 2021 |
| purge_dups | 1.2.3 | Guan et al., 2020 |
| SALSA2 | 2.2 | Ghurye et al., 2019 |
| longranger align | 2.2.2 |
https://support.10xgenomics.com/genome-exome/
software/pipelines/latest/advanced/other-pipelines |
| freebayes | 1.3.1-17-gaa2ace8 | Garrison & Marth, 2012 |
| MitoHiFi | 2.0 | Uliano-Silva et al., 2021 |
| HiGlass | 1.11.6 | Kerpedjiev et al., 2018 |
| PretextView | 0.2.x | https://github.com/wtsi-hpag/PretextView |
| BlobToolKit | 2.6.4 | Challis et al., 2020 |
Ethics/compliance issues
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. 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. 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.
Data availability
European Nucleotide Archive: Melanostoma mellinum (dumpy grass hoverfly). Accession number PRJEB46300; https://identifiers.org/ena.embl/PRJEB46300.
The genome sequence is released openly for reuse. The M. mellinum genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. The genome will be annotated and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (206194) and the Darwin Tree of Life Discretionary Award (218328).
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]
Author information
Members of the University of Oxford and Wytham Woods Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.5746938.
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.5744972.
Members of the Wellcome Sanger Institute Tree of Life programme are listed here: https://doi.org/10.5281/zenodo.5744840.
Members of Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective are listed here: https://doi.org/10.5281/zenodo.5746904.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5743293.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.5638618.
References
- Allio R, Schomaker-Bastos A, Romiguier J, et al. : MitoFinder: Efficient Automated Large-Scale Extraction of Mitogenomic Data in Target Enrichment Phylogenomics. Mol Ecol Resour. 2020;20(4):892–905. 10.1111/1755-0998.13160 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Aubert J, Aubert JJ, Others: Douze Ans de Captures Systematiques de Syrphides (Diptères) Au Col de Bretolet (Alpes Valaisannes).1976. Reference Source [Google Scholar]
- Ball S, Morris R: Britain’s Hoverflies: A Field Guide - Revised and Updated Second Edition.Princeton University Press.2015. Reference Source [Google Scholar]
- Challis R, Richards E, Rajan J, et al. : BlobToolKit - Interactive Quality Assessment of Genome Assemblies. G3 (Bethesda). 2020;10(4):1361–74. 10.1534/g3.119.400908 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheng H, Concepcion GT, Feng X, et al. : Haplotype-Resolved de Novo Assembly Using Phased Assembly Graphs with Hifiasm. Nat Methods. 2021;18(2):170–75. 10.1038/s41592-020-01056-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dziock F: Evolution of Prey Specialization in Aphidophagous Syrphids of the Genera Melanostoma and Platycheirus (Diptera: Syrphidae) 1. Body Size, Development and Prey Traits. Eur J Entomol. 2005;102(3):413– 421. 10.14411/eje.2005.059 [DOI] [Google Scholar]
- Garrison E, Marth G: Haplotype-Based Variant Detection from Short-Read Sequencing. arXiv: 1207.3907. 2012. Reference Source [Google Scholar]
- Gatter W, Schmid U: Hoverfly Migration (Diptera Syrphidae) at Randecker Maar Southwest Germany. Spixiana. 1990;15:1–100. [Google Scholar]
- Ghurye J, Rhie A, Walenz BP, et al. : Integrating Hi-C Links with Assembly Graphs for Chromosome-Scale Assembly. PLoS Comput Biol. 2019;15(8):e1007273. 10.1371/journal.pcbi.1007273 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guan D, McCarthy SA, Wood J, et al. : Identifying and Removing Haplotypic Duplication in Primary Genome Assemblies. Bioinformatics. 2020;36(9):2896–98. 10.1093/bioinformatics/btaa025 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haarto A, Ståhls G: When mtDNA COI Is Misleading: Congruent Signal of ITS2 Molecular Marker and Morphology for North European Melanostoma Schiner, 1860 (Diptera, Syrphidae). Zookeys. 2014; (431):93–134. 10.3897/zookeys.431.7207 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hodkinson ID, Flint PWH: Some Predators from the Galls of Psyllopsis Fraxini L.(Hem., Psyllidae). Entomologist’s Monthly Magazine. 1971;107:11–12. Reference Source [Google Scholar]
- Howe K, Chow W, Collins J, et al. : Significantly Improving the Quality of Genome Assemblies through Curation. GigaScience. 2021;10(1):giaa153. 10.1093/gigascience/giaa153 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jensen AB, Eilenberg J: Genetic Variation within the Insect-Pathogenic Genus Entomophthora, Focusing on the E. Muscae Complex, Using PCR–RFLP of the ITS II and the LSU rDNA. Mycol Res. 2001;105(3):307–12. 10.1017/S0953756201003434 [DOI] [Google Scholar]
- Kerpedjiev P, Abdennur N, Lekschas F, et al. : HiGlass: Web-Based Visual Exploration and Analysis of Genome Interaction Maps. Genome Biol. 2018;19(1):125. 10.1186/s13059-018-1486-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rao SS, Huntley MH, Durand NC, et al. : A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping. Cell. 2014;159(7):1665–80. 10.1016/j.cell.2014.11.021 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Satterfield DA, Marra PP, Sillett TS, et al. : Responses of Migratory Species and Their Pathogens to Supplemental Feeding. Philos Trans R Soc Lond B Biol Sci. 2018;373(1745):20170094. 10.1098/rstb.2017.0094 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simão FA, Waterhouse RM, Ioannidis P, et al. : BUSCO: Assessing Genome Assembly and Annotation Completeness with Single-Copy Orthologs. Bioinformatics. 2015;31(19):3210–12. 10.1093/bioinformatics/btv351 [DOI] [PubMed] [Google Scholar]
- Uliano-Silva M, Nunes JGF, Krasheninnikova K, et al. : marcelauliano/MitoHiFi: mitohifi_v2.0.2021. 10.5281/zenodo.5205678 [DOI] [Google Scholar]
- Van Veen MP: Hoverflies of Northwest Europe: Identification Keys to the Syrphidae.BRILL.2010. Reference Source [Google Scholar]