The genome sequence of the Notch-wing Button, Acleris emargana (Fabricius, 1775)

We present a genome assembly from an individual male Acleris emargana (the Notch-wing Button; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 691.4 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 16.34 kilobases in length. Gene annotation of this assembly on Ensembl identified 21,886 protein coding genes.


Background
The Notch-wing Button Acleris emargana (Fabricius, 1775) is a moth in the Tortricidae family.The species' vernacular name is a reference to the remarkable shape of the forewings, which show a large inward 'notch' along their leading edge.As in other members of its genus, the species is polymorphic (Bradley et al., 1973).Acleris emargana has a Palearctic distribution, being found across northern Eurasia east to at least northern China and Altai and Buryatia in Russia (Karsholt et al., 2005).The species' distribution at the eastern boundary of its range is poorly known due to confusion with its recently split sister taxon Acleris effractana, which has a Holarctic distribution (Karsholt et al., 2005).Acleris emargana is widespread across the British Isles except in the Outer Hebrides where it appears to be replaced by effractana (Elliott et al., 2018).
The species overwinters as an egg, with the larva feeding between May and July on Salix or Populus, and less commonly, Betula, Corylus or Alnus (Bradley et al., 1973;Elliott et al., 2018).The larva feeds from within a folded leaf or between spun leaves (Bradley et al., 1973;Elliott et al., 2018).Pupation occurs in the larval habitation or amongst moss.Adults occur between July and September, often found in damp areas where sallow (Salix ssp.) is established (Bradley et al., 1973;Elliott et al., 2018).
The genome of Acleris emargana 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 complete, chromosome-level genome sequence for Acleris emargana, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Acleris emargana (Figure 1) collected from Wytham Woods,Oxfordshire (51.77,.A total of 25-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 67-fold coverage in 10X Genomics read clouds was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 27 missing joins or mis-joins and removed three haplotypic duplications, reducing the assembly length by 0.99% and the scaffold number by 10.53%, and decreasing the scaffold N50 by 1.34%. The final assembly has a total length of 691.4 Mb in 33 sequence scaffolds with a scaffold N50 of 22.8 Mb (Table 1).Most (99.98%) of the 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/758706.
The resulting annotation includes 22,022 transcribed mRNAs from 21,886 protein-coding genes.

Sample acquisition and nucleic acid extraction
The specimen used for DNA sequencing was a male Acleris emargana (specimen ID Ox000806, individual ilAclEmar1), collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2020-08-01 using a light trap.The specimen used for Hi-C sequencing (specimen ID Ox000951, individual ilAclEmar2) was collected from the same location on 2020-09-08.Both   specimens were 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 ilAclEmar1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Tissue from the whole organism 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 and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (10X) instruments.Hi-C data were also generated from whole organism tissue of ilAclEmar2 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).One round of polishing was   Table 3 contains a list of relevant software tool versions and sources.

Version
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 The paper presents an outstanding genomic dataset of Acleris emargana, with detailed elaboration on the species' background and a comprehensive summary of the generated genome.Overall, this is an excellent genomic dataset, but there are a few minor comments: Q1.The authors mentioned in the research background that Acleris emargana is often confused with Acleris effractana.In this case, it is suggested to provide criteria for identification.Evidence should be given from either morphological or genetic levels to confirm that the sequenced species is indeed Acleris emargana.
Q2.The sequencing methods section should detail the sequencing strategy.

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.

Konrad Fiedler
University of Vienna, Vienna, Austria This paper presents a thorough solid technical report on the genome assembly of a tortricid moth from the UK, identified as Acleris emargana.My only question to this report is the way how the sequenced specimen has been identified in terms of taxonomy.This must be explicitly documented in the paper, since it is now well known that the taxon in question belongs to a species complex formed by 2 (if not even 3?) species.
The only reliable way to distinguish emargana from its sister taxon effractana is by anatomy of the genitalia, or by DNA barcoding.Please say clearly which way you have used to identify our focal specimen -since from the time of publication onwards the genomic information will be linked to the respective taxon name 'forever'.Hence, identification should be 100% error-free.
Because of these circumstances, it may also be noted in the paper that all information (e.g. on host plants) pre-dating the recognition of effractana as a distinct species (pre-2005) is a bit suspect, as long as no voucher specimens exist, whose taxonomic identity has been clarified in retrospect.Therefore, any paragraphs where ecological on Acleris emargana are mentioned in this paper reporting its genome should be restricted to sources in which the taxonomic identity has been rigorously checked.
Otherwise: a valid technical report that makes available important genomic data to the scientific community.Reviewer Expertise: Entomology -Insect Molecular Biology 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 Acleris emargana, ilAclEmar1.2: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 691,448,267 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 (101,224,011 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (22,775,158 and 15,148,969 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/ilAclEmar1.2/dataset/CAKMJL02/snail.

Figure 5 .
Figure 5. Genome assembly of Acleris emargana, ilAclEmar1.2:Hi-C contact map of the ilAclEmar1.2assembly, 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=CxX2ql3pQpegkujExGkbMw.
Expertise: Insect resources and their utilization; Insect systematics 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.Reviewer Report 05 June 2024 https://doi.org/10.21956/wellcomeopenres.21975.r84317© 2024 Fiedler K.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.

©
2024 Subramanian S. 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.Sabtharishi SubramanianICAR-Indian Agricultural Research Institute, Pusa, New Delhi, India This paper describes the genome assembly of a genome assembly of Notch-wing Button moth , Acleris emargana ;(Insecta; Arthropoda Lepidoptera; Tortricidae).A.emargana is widely found cross northern Eurasia east to at least northern China and Altai and Buryatia in Russia.The genome sequence is 691.4 megabases in span scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome.The mitochondrial genome has also been assembled and is 16.34 kilobases in length.Gene annotation of this assembly on Ensembl identified 21,886 protein coding genes.Queries: Q1.Since this moth's taxonomic identity has resemblance to Acleris effractana, how the authors can authentically say that the moth collected was actually Acleris emargana?Whether voucher specimen has been deposited with any repository.Q2.Whether barcoding was done for the specimen collected and accession number was obtained from Barcode of Life data base or NCBI GenBank?Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.

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.

Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? No 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, however I have significant reservations, as outlined above.
Reviewer Report 07 May 2024 https://doi.org/10.21956/wellcomeopenres.21975.r78304