The genome sequence of the Pine Hawkmoth, Sphinx pinastri (Linneaus 1758)

We present a genome assembly from an individual male Sphinx pinastri (the Pine Hawkmoth; Arthropoda; Insecta; Lepidoptera; Sphingidae). The genome sequence is 509.2 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.3 kilobases in length.


Background
The Pine Hawkmoth (Sphinx pinastri) is a moth in the family Sphingidae found throughout Europe eastwards to the Balkans.The moth was accidentally introduced to North America and, in parts of its range, is a serious forest pest (Leraut, 2006).The distribution of this moth increased in southern England during the 20th century, with a major spread occurring over the last 40 years (Randle et al., 2019).The increase in conifer plantations has aided the expansion of its range (Heath & Emmet, 1963).
The Pine Hawkmoth is a large (forewing length 35-41 mm), rather plain, brownish-grey moth with longitudinal black streaks on its wings, which have chequered fringes.The thorax is bordered in black.In the UK, the moth is single-brooded, flying from May until early August (Waring et al., 2017).The moth lays its eggs in small groups on the needles of Scots pine and sometimes Norway spruce.The pupa is found either on, or just under, the ground, often under pine needles.The moth overwinters as a pupa for up to two years (Heath & Emmet, 1963).
The Pine Hawkmoth sometimes comes to light, and feeds at flowers including the lesser butterfly orchid (Platanthera bifolia) (Steen, 2013).Research in Norway demonstrated that the timing of flower visits by the hawkmoth coincided with the release of terpenoids by the orchids.This strongly suggests that these chemicals play an important role in guiding the moth to the flowers (Steen et al., 2019).
A genome sequence from S. pinastri will be useful for comparative studies across the Lepidoptera.The genome of S. pinastri was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all the named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.Here we present a chromosomally complete genome sequence for S. pinastri based on a male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Sphinx pinastri (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.34).A total of 46-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 9 missing joins or mis-joins and removed 5 haplotypic duplications, reducing the assembly length by 0.46% and the scaffold number by 6.38%.
The final assembly has a total length of 509.2 Mb in 43 sequence scaffolds with a scaffold N50 of 18.7 Mb (Table 1).Most (99.92%) of the assembly sequence was assigned to 28 chromosomal-level scaffolds, representing 27 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/987436.

Sample acquisition and nucleic acid extraction
A male Sphinx pinastri (specimen ID Ox000585, individual ilSphPina1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2020-07-05 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 ilSphPina1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Abdomen tissue 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.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 thorax tissue of ilSphPina1 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

Software tool Version
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

Ljiljana Šašić Zorić
BioSense Institute, University of Novi Sad, Novi Sad, Serbia "The genome sequence of the Pine Hawkmoth, Sphinx pinastri (Linneaus 1758)" is a technical report on the genome assembly of the forest pest species, Pine Hawkmoth.The sequencing approach included Pacific Biosciences HiFi sequencing and scaffolding with chromosome conformation using Hi-C data.Additionally, RNA sequencing was also done.
Methodology is well described, and genome assembly data is clearly presented.There is no thorough analysis on RNA-Seq data, only raw data accession.However, authors stated that RNA-Seq data will be used for genome annotation and provided through Ensembl pipeline (European Bioinformatics Institute).
A genome sequence of the Pine Hawkmoth will be useful for new marker development as well as comparative studies across Lepidoptera.

Suggestions for correction:
In the section "Genome sequence report" number of sequence scaffolds is 43, while in Table1 is 44.
Is the rationale for creating the dataset(s) clearly described?

Marko Mutanen
University of Oulu, Oulu, Finland This study publishes a reference genome for a moth of the family Sphingidae, Sphinx pinasti (Lepidoptera).The species is widespread and common in Europe, and it represents a species of the type genus of the family.The article does not summarize how many members of the family are with reference genomes generated before, and what is the closest relatives with genome available.The manuscript would benefit from such a short overview.
The genome is chromosomal-level, and the quality measures all indicate that it is of high quality and reliability.There is very little missing data and for example very large proportion of Lepidoptera BUSCO markers were mapped.The work is therefore technically sound.
While there is no doubt about the correct identification in this case, I find that it would be a good practise to validate the identification through DNA barcode region comparison as the assembly includes also the full mitochondrial genome.In some cases this might reveal that the sequenced genome does not represent the species it was believed to represent.
This work is an important contribution which helps us to understand e.g.Lepidoptera phylogenomics, but is useful in various other comparative settings.

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: I am an expert of Lepidoptera systematic and taxonomy, including molecular tools.I have a plenty of experience of genomic methods, but little about genome assembly.I do not have personal experience of several of the used software.
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 Sphinx pinastri, ilSphPina1.1: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 509,238,608 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 (27,562,872 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (18,699,573 and 14,095,973 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/ilSphPina1.1/dataset/CANOQI01/snail.

Figure 3 .
Figure 3. Genome assembly of Sphinx pinastri, ilSphPina1.1: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/ilSphPina1.1/dataset/CANOQI01/blob.

Figure 4 .
Figure 4. Genome assembly of Sphinx pinastri, ilSphPina1.1: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/ilSphPina1.1/dataset/CANOQI01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Sphinx pinastri, ilSphPina1.1:Hi-C contact map of the ilSphPina1.1 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=X0FKgs1LRFu4Xc1NtTgp0Q.

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

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.

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.