The genome sequence of the Scarce Umber, Agriopis aurantiaria (Hübner, 1799)

We present a genome assembly from an individual male Agriopis aurantiaria (the Scarce Umber; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 485.4 megabases in span. The whole assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.44 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,963 protein coding genes.


Background
Agriopis aurantiaria (Scarce Umber) is a moth from the family Geometridae with widespread distribution across Europe.It has documented increasing northward expansion across Fennoscandia, likely due to increasing temperatures (Ammunét et al., 2012;Jepsen et al., 2011).Despite its name, this species is present throughout the United Kingdom and Ireland, with broad distribution and local abundance throughout much of Britain (Randle et al., 2019).It is most abundant in broadleaved woodland with mature trees, but can also be found in scrub and gardens near well-wooded regions.The larval foodplants include a variety of deciduous trees and shrubs, including Quercus robur, Betula, Rosa, and Prunus padus (Robinson et al., 2023).This species overwinters as an egg on the foodplant, with larvae emerging between April and June, and is found on the wing from October to December (Waring et al., 2017).
Perhaps the most striking feature of this species is the strong sexual dimorphism.Similar to other closely related species in Geometridae, males have normally developed wings while females are flightless due to almost completely vestigial wings (micropterous).Females can be found by searching tree trunks in the morning, where they rest (Waring et al., 2017).Wing reduction and flightless species have evolved many times within Lepidoptera, predominantly affecting females and species which are univoltine and have flight periods in the colder winter months (Sattler, 1991), as is the case for Agriopis aurantiaria.A complete genome sequence will provide a basis for understanding how this trait has convergently evolved across Lepidoptera.
The genome of the scarce umber, Agriopis aurantiaria, 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 Agriopis aurantiaria, based on one specimen from Wytham Woods, Oxfordshire.

Genome sequence report
The genome was sequenced from one male Agriopis aurantiaria (Figure 1) collected from Wytham woods, Oxfordshire (biological vice-county Berkshire), UK (51.77,.A total of 35-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 90-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 26 missing joins or mis-joins and removed 3 haplotypic duplications, reducing the assembly length by 0.44% and the scaffold number by 41.18%, and increasing the scaffold N50 by 1.59%. The final assembly has a total length of 485.4 Mb in 30 sequence scaffolds with a scaffold N50 of 18.0 Mb (Table 1).The whole assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 29 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.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/104476.

Sample acquisition and nucleic acid extraction
A male Agriopis aurantiaria (specimen ID Ox000991, individual ilAgrAura1) was collected from Wytham Woods, Oxfordshire  (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2020-11-21, using a light trap.The specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilAgrAura1 sample was weighed and dissected on dry ice with head tissue set aside for Hi-C sequencing and abdomen tissue for RNA sequencing.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.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using the 0.8X AMpure XP RNA was extracted from abdomen tissue of ilAgrAura1 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi), Illumina HiSeq 4000 (RNA-Seq) and Illumina NovaSeq 6000 (10X) instruments.Hi-C data were also generated from head tissue of ilAgrAura1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
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 Agriopis aurantiaria assembly (GCA_914767915.1) in Ensembl Rapid Release.

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

Jan Veenstra
University of Bordeaux, Pessac, France This is yet another high quality DNA assembly from the Wellcome Sanger Institute.High quality says it all.There is really nothing here to criticize.
Feeling obliged to make a few comments, I add the following.It would have been nice to have a picture illustrating the sexual dimorphism of this species.It is very satisfying to see not only Pacific Biosciences HIFI sequences but also abundant short read illumina sequences as well at some RNAseq data.The latter in this case is exclusively from a single male and lack the brain, a tissue which as a neuroscientist I find particularly interesting.These comments should not give the impression that I am negative, that would really wrong.As stated in the first line of this review, this is high quality and as such a welcome addition to the data bank.It would be great if someone were to use this and other genome assemblies to identify genes that might be responsible for the sexual dimorphism.

Benjamin Buer
Bayer AG, Crop Science Division, Germany The Data Note describes the genome sequencing and annotation of the Scarce Umber (Agriopis aurantiaria).The genome, derived from an individual male, comprises 29 chromosomes plus the sex and the mitochondrial genome.As Scarce Umber has strong sexual dimorphism, the reference genome may be relevant to further understand the evolution of this feature in lepidopteran species in future.
The standard metrics of the genome are of high quality and the majority of methods are described well but some details of parameters may be helpful to reproduce the analysis.For the assembly, MitoHiFi was used, however the specific algorithm (MitoFinder or MITOS, or a consensus of both) was not clearly mentioned.
The genome was produced by assembling reads of an individual male collected in the UK using PacBio and Illumina sequencing data from thorax tissue and Hi-C technology for scaffolding from head tissue.In addition, the genome was annotated using BRAKER2 pipeline, however the use of the RNA sequencing data that was generated from abdomen data remains unclear in this process and may be further elaborated.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Partly
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: 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.

Figure 2 .
Figure 2. Genome assembly of Agriopis aurantiaria, ilAgrAura1.1: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 485,385,411 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 (22,923,517 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (18,049,239 and 11,825,588 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/ilAgrAura1_1.1/dataset/ilAgrAura1_1.1/snail.
Genome assembly, curation and evaluationAssembly was carried out withHifiasm (Cheng et al., 2021)   and 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 Long Ranger 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 and corrected as described previously(Howe et al., 2021).Manual curation was performed using HiGlass(Kerpedjiev et al., 2018)   andPretext (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.

Figure 5 .
Figure 5. Genome assembly of Agriopis aurantiaria, ilAgrAura1.1:Hi-C contact map of the ilAgrAura1.1 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=Of-k_IrEQt6BBHNAbX4Cqw.

Peer Review Current Peer Review Status: Version 1
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.