The genome sequence of the Summer Chafer, Amphimallon solstitiale (Linnaeus, 1758)

We present a genome assembly from an individual male Amphimallon solstitiale (the Summer Chafer; Arthropoda; Insecta; Coleoptera; Scarabaeidae). The genome sequence is 1,584.1 megabases in span. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 19.29 kilobases in length.


Background
The tilt of the Earth's axis of rotation means that once each year the northern hemisphere experiences the day with the longest period of daylight, which may fall on either 20 or 21 June.Before the summer solstice the hours of daylight are increasing each day; after the summer solstice the hours of daylight are decreasing.Many species of insect have life-history traits attuned to day length changes, a trait known as photoperiodism (Denlinger et al., 2017).In many cases, there is interplay between photoperiodism and temperature sensitivity, with each contributing to when an insect enters or emerges from diapause or changes behaviour.The cellular and molecular basis of these responses is incompletely understood although progress is being made in studies using Lepidoptera (Denlinger et al., 2017;Kozak et al., 2019); case studies from other insect orders are needed to further an understanding of this important biological phenomenon.
Amphimallon solstitiale (order Coleoptera, family Scarabaeidae) is a species of beetle with a life history that seems exquisitely linked to day length in the northern hemisphere.The larvae feed underground on roots and tubers, spending two or three years as a larva before pupating underground in the final spring (UK Beetles, no date).In midsummer, notably around the summer solstice, adults emerge to mate.Males gather at the tops of trees and tall shrubs in late evening, or even above chimney stacks on houses, swarming in numbers ranging from a few individuals to over a hundred.Not every tree is chosen with apparent preference for the tallest (PWHH, personal observation).These impressive aggregations are often seen on the evening of the summer solstice itself and may continue until mid-July.After mating, females descend to the ground to lay eggs in the soil.A female sex pheromone has been identified (Tolasch et al., 2003).The behavioural link to midsummer has given rise to the common name Summer Chafer, European June beetle or the Solstice beetle, and the specific name solstitiale.
A. solstitiale is found across central and northern Europe including southern regions of England and Wales and southern Scandinavia.Further east, there are abundant records from Russia and scattered records from Georgia, Armenia, Tajikistan, Kyrgyzstan and Uzbekistan (GBIF Secretariat, 2023).
The underground larva can occasionally reach pest proportions in garden lawns and commercial horticulture (Royal Horticultural Society, 2023).Proposed control measures include the use of entomopathogenic nematodes; however, microbiota screening suggests that bacteria in the gut of A. solstitiale include species antagonistic to symbiotic bacteria of nematodes (Skowronek et al., 2021).
Here we report a complete genome sequence for the Summer Chafer Amphimallon solstitiale determined as part of the Darwin Tree of Life project.The genome sequence of A. solstitiale will facilitate research into the molecular control of photoperiodism and contribute to the rapidly growing resources for studying genome evolution in insects.

Genome sequence report
The genome was sequenced from one male Amphimallon solstitiale (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 47-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 122 missing joins or mis-joins and removed 25 haplotypic duplications, reducing the assembly length by 1.00% and the scaffold number by 6.10%. The final assembly has a total length of 1,584.1 Mb in 461 sequence scaffolds with a scaffold N50 of 150.9 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (97.69%) of the assembly sequence was assigned to 11 chromosomal-level scaffolds, representing 9 autosomes and the X and Y sex chromosomes.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).Chromosomes X and Y were assigned by read coverage statistics and synteny to the genome assembly of Melolontha melolontha (GCA_935421215.2) (Ashworth et al., 2023).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.The estimated Quality Value (QV) of the final assembly is 63.1 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.3.2 completeness of 99.2% (single = 96.9%,duplicated = 2.3%), using the endopterygota_odb10 reference set (n = 2,124).

Sample acquisition and nucleic acid extraction
A male Amphimallon solstitiale (specimen ID Ox001707, ToLID icAmpSols1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2021-07-17 in a light trap.The specimen was collected by Douglas Boyes (University of Oxford) and identified by Liam Crowley (University of Oxford), and then snap-frozen on dry ice.remaining abdomen tissue of icAmpSols1 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).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).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) and PretextView (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.
A Hi-C map of the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file   format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This work was done using Nextflow (Di Tommaso et al.Table 3 contains a list of relevant software tool versions and sources.

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 However, the data provided in Figure 2 is too complex.It is recommended to draw statistical charts according to the data category, so that other peers, especially those who are not specialized in bioinformatics research, can quickly and clearly read the information of interest.

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: Resource insects and microbiome 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, however I have significant reservations, as outlined above.

Phillip Davidson
Department of Biology, Indiana University Bloomington, Bloomington, Indiana, USA In their genome report, Boyes, Crowley, and Holland present a complete and high quality reference assembly for the Summer Chafer Amphimallon solstitiale.Overall, the assembly and report are of superb quality and represent a much needed addition for Melolonthinae genomic resources.The rationale as a model for photoperiodism is well described and the methods are sound.Further, the data are accessible online.I have only a handful of minor revision suggestions that I think will clarify the results and significance of this data set, as listed below.
Genome Sequence Report: I see the PacBio coverage is stated at 47X coverage, however, please also state the coverage for the HiC data. 1.
Figure 1: Please add a scale bar or if not possible, consider substituting a separate image that includes one.

2.
The genome of Amphimallon solstitiale is remarkably large for a scarab (nearly 1.6Gb), which is very interesting.I think a nice addition to the results would describe what genomic elements compose this enormous expansion (repetitive elements I may guess) and their biological implication.I do not think a formal repetitive element analysis (annotation, classification) is necessary for a data report, but an estimation of what % of the genome is composed of repeats would improve the description of the results.

3.
Figure 2: The BUSCO scores mini-circle plot states the middle shade of green is 99.2% and represents the completion score.However, I think this label should be changed to "single copy (96.9%)" score as the middle shade green does not appear to compose 99.2% of the plot.Further, listing the SC, Duplication, Frag, and Missing is more standard as it distinguishes single copy and duplication composition.Then, the complete score of 99.2% can be listed separately or perhaps in the legend.

Methods:
For the clause "snap-frozen on dry ice".Should this instead read "snap-frozen in liquid nitrogen"?Typically dry ice is used for storage and transportation, so I wanted to check. 1.
Data Availability: When the gene annotations are complete and if possible, it may be nice to update this report with a link to those files. 1.

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 have expertise in analyzing genomic data from beetle species.Hence, I was able to assess the majority of the manuscript.
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 Amphimallon solstitiale, icAmpSols1.1: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 1,584,161,395 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 (260,577,686 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (150,921,111 and 118,082,474 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 endopterygota_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAUJHA01/dataset/CAUJHA01/snail.

Figure 3 .
Figure 3. Genome assembly of Amphimallon solstitiale, icAmpSols1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAUJHA01/dataset/CAUJHA01/blob.

Figure 4 .
Figure 4. Genome assembly of Amphimallon solstitiale, icAmpSols1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAUJHA01/dataset/CAUJHA01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Amphimallon solstitiale, icAmpSols1.1:Hi-C contact map of the icAmpSols1.1 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=bmH40SVZR06IKUSWBLb7PA.

Table 1 . Genome data for Amphimallon solstitiale, icAmpSols1.1. Project accession data
(Strickland et al., 2023)rks are adapted from column VGP-2020 of "Table1: Proposed standards and metrics for defining genome assembly quality" fromRhie et al. (2021).**BUSCOscoresbasedon the endopterygota_odb10 BUSCO set using version 5.3.2.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/CAUJHA01/dataset/CAUJHA01/busco.preparation, the icAmpSols1 sample was weighed and dissected on dry ice(Jay et al., 2023).Tissue from the abdomen was homogenised using a PowerMasher II tissue disruptor(Denton et al., 2023a).HMW DNA was extracted using the(Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.The concentration of the sheared and purified DNA was assessed using a Nanodrop

Table 2 . Chromosomal pseudomolecules in the genome assembly of Amphimallon solstitiale, icAmpSols1. INSDC accession Chromosome Length (Mb) GC%
Reviewer Report 22 May 2024 https://doi.org/10.21956/wellcomeopenres.23340.r83352This 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.