The genome sequence of the Chevron, Eulithis testata (Linnaeus, 1761)

TL;DR

This paper provides the genome sequence of the Chevron moth, including 308.1 megabases of DNA and annotations for over 16,000 genes.

Contribution

The study presents a high-quality genome assembly and gene annotation for the Chevron moth, Eulithis testata.

Findings

The genome assembly spans 308.1 megabases and is scaffolded into 30 chromosomal pseudomolecules.

Gene annotation identified 16,167 protein coding genes using Ensembl.

The mitochondrial genome is 15.9 kilobases in length.

Abstract

We present a genome assembly from an individual male
Eulithis testata (the Chevron; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 308.1 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.9 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,167 protein coding genes.

Figures

Photograph of Eulithis testata. https://www.inaturalist.org/photos/97801019 (c) Michel Langeveld, some rights reserved (CC BY-SA).

Genome assembly of Eulithis testata, ilEulTest2.1: 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 308,098,708 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 (13,628,168 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (10,981,015 and 7,954,720 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 lepidoptera_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Eulithis%20testata/dataset/CANNPX01/snail.

Genome assembly of Eulithis testata, ilEulTest2.1: 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/Eulithis%20testata/dataset/CANNPX01/blob.

Genome assembly of Eulithis testata, ilEulTest2.1: 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/Eulithis%20testata/dataset/CANNPX01/cumulative.

Genome assembly of Eulithis testata, ilEulTest2.1: Hi-C contact map of the ilEulTest2.1 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=dYxE9IdaTNu__uKX_RLc4g.

Tables

Table 1.. Genome data for Eulithis testata, ilEulTest2.1.

Project accession data
Assembly identifier	ilEulTest2.1
Species	Eulithis testata
Specimen	ilEulTest2
NCBI taxonomy ID	326959
BioProject	PRJEB55881
BioSample ID	SAMEA14448148
Isolate information	ilEulTest2, male; head and thorax (genome sequencing) ilEulTest3, head and thorax (Hi-C scaffolding)
Assembly metrics *		Benchmark
Consensus quality (QV)	67.7	≥ 50
k-mer completeness	100%	≥ 95%
BUSCO **	C:98.1%[S:97.8%,D:0.4%], F:0.5%,M:1.4%,n:5,286	C ≥ 95%
Percentage of assembly mapped to chromosomes	99.92%	≥ 95%
Sex chromosomes	Z chromosome	localised homologous pairs
Organelles	Mitochondrial genome assembled	complete single alleles
Raw data accessions
PacificBiosciences SEQUEL II	ERR10224850
Hi-C Illumina	ERR10177756
Genome assembly
Assembly accession	GCA_947507515.1
Accession of alternate haplotype	GCA_947507505.1
Span (Mb)	308.1
Number of contigs	69
Contig N50 length (Mb)	9.3
Number of scaffolds	36
Scaffold N50 length (Mb)	11.0
Longest scaffold (Mb)	13.6
Genome annotation
Number of protein-coding genes	16,167
Number of gene transcripts	16,359

Table 2.. Chromosomal pseudomolecules in the genome assembly of Eulithis testata, ilEulTest2.

INSDC accession	Chromosome	Size (Mb)	GC%
OX382194.1	1	13.13	37.7
OX382195.1	2	12.79	37.4
OX382196.1	3	12.65	37.5
OX382197.1	4	12.36	37.6
OX382198.1	5	11.88	36.9
OX382199.1	6	11.76	37.1
OX382200.1	7	11.68	37
OX382201.1	8	11.67	37.1
OX382202.1	9	11.41	36.9
OX382203.1	10	11.33	37.5
OX382204.1	11	11.16	36.9
OX382205.1	12	10.98	37.3
OX382206.1	13	10.9	37.4
OX382207.1	14	10.66	37
OX382208.1	15	10.41	37.5
OX382209.1	16	10.39	37.5
OX382210.1	17	10.37	37.3
OX382211.1	18	10.31	37.4
OX382212.1	19	10.23	37.6
OX382213.1	20	9.97	37.1
OX382214.1	21	9.57	37.8
OX382215.1	22	8.19	37.1
OX382216.1	23	8.17	38.7
OX382217.1	24	7.96	37
OX382218.1	25	7.95	37.9
OX382219.1	26	7.73	37.2
OX382220.1	27	7.18	37.2
OX382221.1	28	5.84	37.3
OX382222.1	29	5.58	37.5
OX382193.1	Z	13.63	37.5
OX382223.1	MT	0.02	19.4
-	unplaced	0.23	42.2

Table 3.. Software tools: versions and sources.

Software tool	Version	Source
BlobToolKit	4.0.7	https://github.com/blobtoolkit/blobtoolkit
BUSCO	5.3.2	https://gitlab.com/ezlab/busco
Hifiasm	0.16.1-r375	https://github.com/chhylp123/hifiasm
HiGlass	1.11.6	https://github.com/higlass/higlass
Merqury	MerquryFK	https://github.com/thegenemyers/MERQURY.FK
MitoHiFi	2	https://github.com/marcelauliano/MitoHiFi
PretextView	0.2	https://github.com/wtsi-hpag/PretextView
purge_dups	1.2.3	https://github.com/dfguan/purge_dups
sanger-tol/genomenote	v1.0	https://github.com/sanger-tol/genomenote
sanger-tol/readmapping	1.1.0	https://github.com/sanger-tol/readmapping/tree/1.1.0
YaHS	yahs-1.1.91eebc2	https://github.com/c-zhou/yahs

Full text

Species taxonomy

Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Geometroidea; Geometridae; Larentiinae; Eulithis; Eulithis testata (Linnaeus, 1761) (NCBI:txid326959).

Background

The Chevron, Eulithis testata, is a medium sized geometrid moth, quite variable in ground colour, often with a beautiful mixture of yellowish scales frosted with grey on the forewing, distinguished by a series of six strongly angled chevron markings following one another, and a somewhat semicircular brown blotch at the termen ( Figure 1). The Chevron emerges in late June or July and flies (at dusk) until mid-September in the UK ( Randle et al., 2019), overwintering as an egg.

Photograph of Eulithis testata. https://www.inaturalist.org/photos/97801019 (c) Michel Langeveld, some rights reserved (CC BY-SA).

The Chevron is found in a range of habitats especially moorland and heathland, also marshes and sand dunes ( Randle et al., 2019). Its larval foodplants comprise mainly different shrubs particularly Salix species, as well as birch and heather further north ( Waring et al., 2017).

E. testata is one of a few geometrid moths that span the Holarctic ( GBIF Secretariat, 2022), and it is widespread throughout the United Kingdom ( NBN Atlas Partnership, 2021). Populations in the UK show a significant decrease since 1970 in both abundance and distribution ( Conrad et al., 2006; Randle et al., 2019).

The genus Eulithis is currently placed in the larentiine tribe Cidariini ( Choi, 1997). It is sister to the genus Ecliptopera in the study of Õunap, et al. ( 2016; Figure 2). Evolution of its wing patterns, including those of its many congeners have been studied, and one species Eulithis convergenata (Bremer, 1864) appears to be mimetic of the aposematic geometrid genus Abraxas ( Choi, 2001).

The genome sequence should not only be useful in phylogeny, but also in studies of potentially cryptic species. There are two DNA barcode clusters on BOLD (17 April 2023), the BINs BOLD:AAB0154 and BOLD:ABZ0682 (the former in the USA and Canada, the latter in the Palaearctic including the UK and Europe), and these two discrete clusters are only about 1.47% pairwise divergent from each other.

The genome of Eulithis testata 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. Here we present a chromosomally complete genome sequence for Eulithis testata, based on one male specimen from Beinn Eighe National Nature Reserve, Scotland, UK.

Genome sequence report

The genome was sequenced from one male Eulithis testata. A total of 60-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 12 missing joins or mis-joins, reducing the scaffold number by 12.2%.

The final assembly has a total length of 308.1 Mb in 36 sequence scaffolds with a scaffold N50 of 11.0 Mb ( Table 1). Most (99.92%) of the assembly sequence was assigned to 28 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 2– Figure 5; Table 2). 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 Eulithis testata, ilEulTest2.1.

Genome assembly of Eulithis testata, ilEulTest2.1: 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 308,098,708 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 (13,628,168 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (10,981,015 and 7,954,720 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 lepidoptera_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Eulithis%20testata/dataset/CANNPX01/snail.

Genome assembly of Eulithis testata, ilEulTest2.1: 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/Eulithis%20testata/dataset/CANNPX01/blob.

Genome assembly of Eulithis testata, ilEulTest2.1: 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/Eulithis%20testata/dataset/CANNPX01/cumulative.

Genome assembly of Eulithis testata, ilEulTest2.1: Hi-C contact map of the ilEulTest2.1 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=dYxE9IdaTNu__uKX_RLc4g.

Table 2.: Chromosomal pseudomolecules in the genome assembly of Eulithis testata, ilEulTest2.

The estimated Quality Value (QV) of the final assembly is 67.7 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 completeness of 98.1% (single = 97.8%, duplicated = 0.4%), using the lepidoptera_odb10 reference set ( n = 5,286).

Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/326959.

Genome annotation report

The E. testata genome assembly (GCA_947507515.1) was annotated using the Ensembl rapid annotation pipeline ( Table 1; https://rapid.ensembl.org/Eulithis_testata_GCA_947507515.1/Info/Index). The resulting annotation includes 16,359 transcribed mRNAs from 16,167 protein-coding genes.

Methods

Sample acquisition and nucleic acid extraction

Two Eulithis testata specimens (ilEulTest2 and ilEulTest3) were collected from Beinn Eighe National Nature Reserve, Scotland, UK (latitude 57.63, longitude –5.35) on 10 September 2021 using a light trap. The specimens were collected and identified by David Lees (Natural History Museum) and dry frozen at –80°C. Individual ilEulTest2 (specimen no. NHMUK014543798) was used for genome sequencing, while individual ilEulTest3 (specimen no. NHMUK014543792) was used for Hi-C scaffolding.

DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilEulTest2 sample was weighed and dissected on dry. Head and thorax tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. HMW DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system with speed setting 30. Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample. 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.

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 the Pacific Biosciences SEQUEL II instrument. Hi-C data were also generated from head and thorax tissue of ilEulTest3 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation

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 as described previously ( Howe et al., 2021). Manual curation was performed using HiGlass ( Kerpedjiev et al., 2018) and Pretext ( Harry, 2022). The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2022), 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.

A Hi-C map for the final assembly was produced using bwa-mem2 ( Vasimuddin et al., 2019) in the Cooler file format ( Abdennur & Mirny, 2020). To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury ( Rhie et al., 2020). This work was done using Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines “sanger-tol/readmapping” ( Surana et al., 2023a) and “sanger-tol/genomenote” ( Surana et al., 2023b). The genome was analysed within the BlobToolKit environment ( Challis et al., 2020) and BUSCO scores ( Manni et al., 2021; Simão et al., 2015) were calculated.

Table 3 contains a list of relevant software tool versions and sources.

Genome annotation

The BRAKER2 pipeline ( Brůna et al., 2021) was used in the default protein mode to generate annotation for the Eulithis testata assembly (GCA_947507515.1) in Ensembl Rapid Release.

Ethics and 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.