The genome sequence of the early grey, Xylocampa areola (Esper, 1789)

We present a genome assembly from an individual Xylocampa areola (the early grey; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 565 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.5 kilobases in length. Gene annotation of this assembly on Ensembl identified 18,869 protein coding genes.


Background
The early grey, Xylocampa areola, is a medium sized noctuid moth, intricately grey mottled with conjoined stigmata on the forewing, that starts to emerge early in the temperate moth season (usually February in the UK).The early grey is found in deciduous woodland, hedges and a wide range of other habitats, including heathland, fens, and gardens, wherever its larval foodplant, species of Lonicera (Caprifoliaceae) and especially L. xylosteum, is found.Despite its larval preference for honeysuckle, X. areola is not generally considered to be an important garden pest.
Xylocampa areola is generally common and widespread in the western Palaearctic only, from southern Scandinavia to the northern Mediterranean; but there are relatively few records of its presence in eastern Europe (GBIF Secretariat, 2021).Populations in the UK appear to be stable or even increasing (Conrad et al., 2006;Randle et al., 2019).The adult flies from February, protracted until around the end of May, with a peak in April in the UK (Wheeler, 2022) and up to mid-June in Europe (Kettner, 2019).Adults seek nectar at night, in the Spring particularly sallow (Salicaceae) blossoms.In the Mediterranean, X. areola has been considered a very important Lepidoptera pollinator in the community there studied, especially of Arbutus unedo L., Ericaceae (Ribas-Marquès et al., 2022).
Xylocampa appears not to have been used in molecular phylogenies, and it would be interesting to determine its closest relatives.The sister group of the genus is apparently not known.
It is currently placed in the tribe Psaphidini.The genome sequence should not only be useful in phylogeny, but in studies of potentially cryptic species.There are two DNA barcode clusters (BOLD SYSTEMS, no date), i.e., the BINs BOLD: AAE4200 and BOLD:ABZ8138 (the latter recorded from Norway, Spain and France: Corsica, the first and more common of which already DNA barcoded for the UK), and these two discrete clusters are 1.6% divergent.There is only one other species classified in Xylocampa: X. mustapha (Oberthür, 1910) (BOLD:AEI5117), which is mainly from southern Italy, the Aegean Islands, north-west Africa, and the Middle East.The Italian population is only 2-3% divergent from BOLD:ABZ8138, and about 3.4-3.6%divergent from BOLD:AAE4200.

Genome sequence report
The genome was sequenced from an individual X. areola (Figure 1) collected from High Wycombe, Buckinghamshire (NHMUK014448991, latitude 51.63, longitude -0.74).A total of 58-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 8 missing/misjoins and removed 2 haplotypic duplications, reducing the scaffold number by 4.76%.
The final assembly has a total length of 565 Mb in 40 sequence scaffolds with a scaffold N50 of 20 Mb (Table 1).Most (99.96%) of the assembly sequence was assigned to 31 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 assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 99.1% (single, 98.7%, duplicated 0.3%) using the lepidoptera_odb10 reference set.

Sample acquisition and nucleic acid extraction
An unsexed individual X. areola (ilXylAreo1) was collected and identified by David Lees (Natural History Museum, London) from High Wycombe, Buckinghamshire, UK (latitude  weight DNA was removed from a 20 ng aliquot of extracted DNA using 0.8X AMpure XP purification 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. RNA was extracted from the abdomen tissue of ilXylAreo2 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 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 were performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from head tissue of ilXylAreo1 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.et al., 2021), which performed annotation using MitoFinder (Allio et al., 2020).The genome was analysed and BUSCO scores were generated within the BlobToolKit environment (Challis et al., 2020).Table 3 contains a list of all software tool versions used, where appropriate.BRAKER2 (Brůna et al., 2021) was used to generate annotation for the Xylocampa areola assembly (GCA_935421205.1).Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

Open Peer Review
Current Peer Review Status:

Lars Höök
Department of Ecology and Genetics, Uppsala University, Uppsala, Sweden In this data note, the authors present the genome assembly of Xylocampa areola, the early grey moth.The assembly consist of 31 chromosome scaffolds which is common for a Lepidoptera species.
The methods are well described and follow current standards for generating high quality genome assemblies.The genome assembly, as descibed in the note, is of high quality and is therefore a valuable resource for studies of evolution and phylogenetics.
The relevant information describing the assembly is clearly presented and the genome data is made available at ENA.

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.
Reviewer Expertise: Evolutionary genetics 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 1
Figure 1.a) Photograph of the X. areola (ilXylAreo1) specimen used for genome sequencing.b) Photograph of the X. areola (ilXylAreo2) specimen used for RNA sequencing.

Figure 2 .
Figure 2. Genome assembly of X. areola, ilXylAreo1.1: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 564,568,605 bp assembly.The distribution of chromosome lengths is shown in dark grey with the plot radius scaled to the longest chromosome present in the assembly (27,003,092 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 chromosome lengths (19,946,087 and 13,085,905 bp), respectively.The pale grey spiral shows the cumulative chromosome 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/ilXylAreo1.1/dataset/CAKXYV01/snail.

Figure 3 .
Figure 3. Genome assembly of X. areola, ilXylAreo1.1:GC coverage.BlobToolKit GC-coverage plot.Chromosomes are coloured by phylum.Circles are sized in proportion to chromosome length.Histograms show the distribution of chromosome length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilXylAreo1.1/dataset/CAKXYV01/blob.

Figure 4 .
Figure 4. Genome assembly of X. areola, ilXylAreo1.1:cumulative sequence.BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all chromosomes.Coloured lines show cumulative lengths of chromosomes assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilXylAreo1.1/dataset/ CAKXYV01/cumulative.

Figure 5 .
Figure 5. Genome assembly of X. areola, ilXylAreo1.1:Hi-C contact map.Hi-C contact map of the ilXylAreo1.1 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=ORLTYiKpR_y6hvw1MMLGGQ.
doi.org/10.21956/wellcomeopenres.20696.r68071© 2023 Seixas F. 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.Fernando SeixasDepartment of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USAThe authors assembled the genome of one Xylocampa areola individual, using PacBio and Hi-C data.This resulted in a complete (as attested by the BUSCO score) and highly contiguous (i.e.chromosome-level) assembly.I found the writing very clear, and the authors always provide complete information to understand their work.The one point that could be clarified is in the "Genome annotation" section, where the authors say "Annotation was created primarily through alignment of transcriptomic data".The primarily in that sentence makes me believe that other information was used (I could presume homology-based and/or de novo annotation) so it would be helpful if the authors could explain what other sources of evidence were used.Otherwise, I have no other remarks.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.Reviewer Expertise: Evolutonary BiologyI 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 27 November 2023 https://doi.org/10.21956/wellcomeopenres.20696.r69790© 2023 Höök L. 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.