The genome sequence of the Beautiful Hook-tip, Laspeyria flexula (Denis & Schiffermüller, 1775)

We present a genome assembly from an individual male Laspeyria flexula (the Beautiful Hook-tip; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 450.9 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.58 kilobases in length. Gene annotation of this assembly on Ensembl identified 13,281 protein coding genes.


Background
The Darwin Tree of Life project, as part of our goals of sequencing all eukaryotic species in Ireland and Britain (Blaxter et al., 2022), is generating high quality reference genomes for many lepidoptera.Here we present a chromosomally-complete genome sequence for the Beautiful Hook-tip Laspeyria flexula ([Denis & Schiffermüller], 1775), a member of subfamily Boletobiinae within the noctuid family Erebidae (see NBN Atlas Partnership, 2023).Erebid moth species form approximately 4% of the Lepidoptera fauna of Britain and Ireland, including representatives of 63 genera (UK Species Inventory; Natural History Museum, 2023).Erebid moths are important herbivores, including invasive pests of native and agricultural ecosystems, but some species also act as sentinels for the impacts of anthropogenic impacts such as pesticide use and pollution and of the impacts of climate change (Fox, 2013).
While larvae of most moths feed on vascular plant material, some, including L. flexula, feed on lichens.The well-camouflaged caterpillars of L. flexula feed on foliose and crustose lichens that grow on the bark of both deciduous and coniferous trees.Lichenivorous moths are threatened not only through generalised habitat loss but also especially through the impacts of atmospheric pollution on their food sources.It is well recognised that atmospheric pollution, most notably SO 2 , resulting from the burning of fossil fuels, led to massive declines in many lichen species in Ireland and Britain, and more widely.Following acceptance of emission controls many lichens have rebounded, and lichenivorous species such as L. flexula are also showing recovery (albeit against a general background of declines in moth abundances (Fox et al., 2013), especially in areas that had, historically, the highest atmospheric SO 2 levels (Pescott et al., 2015).We hope that this genome sequence will assist in analysis of the population history and recovery of L. flexula, and of its adaptations to lichenivory.

Genome sequence report
The genome was sequenced from one male Laspeyria flexula (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 48-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 84-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 13 missing joins or mis-joins and removed 2 haplotypic duplications, reducing the assembly length by 0.23% and the scaffold number by 9.76%. The final assembly has a total length of 450.9 Mb in 37 sequence scaffolds with a scaffold N50 of 16.0 Mb (Table 1).Most (99.94%) 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 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/938238.

Sample acquisition and nucleic acid extraction
A male Laspeyria flexula (specimen ID Ox000046, individual ilLasFlex1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2019-06-29 using a light trap.A second specimen (ilLasFlex2, ToLID ilLasFlex2), collected from the same location on 2020-06-13, was used for RNA sequencing.Both   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 abdomen tissue of ilLasFlex2 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    Table 3. Software tools: versions and sources.

Software tool Version
The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021).Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext (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.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Laspeyria flexula assembly (GCA_905147015.1).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).

Michael Hiller
Senckenberg Nature Research Society, Frankfurt Am Main, Germany A high-quality genome of the Beautiful Hook-tip is presented.The ecological importance of the species group and factors threatening the species are clearly described.PacBio HiFi, 10X and HiC sequencing data was used to assemble the genome.The resulting assembly is highly complete, measured by BUSCO and k-mer completeness, has a high base accuracy (QV 58.3) and consists of chromosome-level scaffolds.
The assembly was annotated by the Ensembl rapid pipeline, annotating 13k coding genes.
Minor comments and suggestions 1) Which Hi-C protocol was used?Arima HiC or OmniC?
2) I would like to see the contig N50 in Table 1.This could also be mentioned in the main text, as contiguity is probably more informative than the scaffold N50, given that virtually the entire assembly consists of chromosome-level scaffolds.
3) How does the number of annotated protein-coding genes compare to related species that have a gene annotation.
4) I would suggest to run BUSCO (or better compleasm) in annotation mode on the annotated 13k proteins to estimate how complete the annotation is.

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

Figure 2 .
Figure 2. Genome assembly of Laspeyria flexula, ilLasFlex1.1: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 450,897,266 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 (24,595,128 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (16,004,308 and 10,809,102 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/Laspeyria%20flexula/dataset/CAJHUK01.1/snail.

Figure 5 .
Figure 5. Genome assembly of Laspeyria flexula, ilLasFlex1.1:Hi-C contact map of the ilLasFlex1.1 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=czjwYVChQ92r5isvbpie4g.

Table 1 . Genome data for Laspeyria flexula, ilLasFlex1.1. Project accession data
(Rhie et al., 2021)enchmarks are adapted from column VGP-2020 of "Table1: Proposed standards and metrics for defining genome assembly quality" from(Rhie et al., 2021).**BUSCO scores based on the lepidoptera_odb10 BUSCO set using v5.3.2.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/Laspeyria%20flexula/dataset/CAJHUK01.1/busco.specimenswere 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 ilLasFlex1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Tissue from the whole organism was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.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 purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing.HMW

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