The genome sequence of the pale Rhogogaster, Rhogogaster chlorosoma (Benson, 1943)

We present a genome assembly from an individual female Rhogogaster chlorosoma (the pale Rhogogaster; Arthropoda; Insecta; Hymenoptera; Tenthredinidae). The genome sequence is 255 megabases in span. The whole assembly is scaffolded into 10 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 16.0 kilobases in length. Gene annotation of this assembly on Ensembl has identified 24,433 protein coding genes.


Background
Rhogogaster are green and black sawflies within the Tenthredininae subfamily.The genus is difficult to distinguish from Tenthredo.The genus Rhogogaster has around 40 species distributed across the Holarctic and Asiatic regions with the majority found in the Palaearctic region.Within this genus, numerous species clusters and subspecies have been identified.Rhogogaster chlorosoma and R. scalaris are in the scalaris cluster and, together with R. punctulata and R. viridis, form the Rhogogaster (Rhogogaster) subgenus (Liston, 1995).
Adults have been observed predating on other sawflies and flies and are not particularly associated with flowers.The larvae feed on a broad range of trees and herbs including alders, poplars, willows, rowans and meadowsweet but are not considered a pest of agricultural or horticultural crops.
The genus Rhogogaster includes taxa that are difficult to distinguish morphologically, particularly within the R. scalaris group.Intermediate forms exist between clearly identifiable R. scalaris and R. chlorosoma suggesting either they are very close sibling species or there is introgression of alleles and traits between these taxa.Indeed, a mitochondrial CO1 barcode does not consistently separate the two species (Taeger & Viitasaari, 2015).
The provision of a complete genome sequence for a member of this group will lay the foundation for future analyses, including facilitating comparison of nuclear gene variants from multiple individuals, and ultimately the genetic basis of the variant morphological traits.The genome sequence provided here derives from a female individual with several characters consistent with Benson's description of R. chlorosoma including a lack of a black stripe along the mesopleural groove, a greater extent of green in front of the ocellar triangle and larger tarsal pulvilli (Benson, 1952).The CO1 barcode groups closest to those from previously identified R. chlorosoma, although not in a monophyletic group.

Genome sequence report
The genome was sequenced from one female R. chlorosoma specimen (Figure 1) collected from Wytham Woods (51.76,.A total of 74-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 five missing or mis-joins, reducing the scaffold number by 23.08% and increasing the scaffold N50 by 23.83%. The final assembly has a total length of 255.3 Mb in 10 sequence scaffolds with a scaffold N50 of 27.7 Mb (Table 1).All of the assembly sequence was assigned to 10 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 95.8% using the hymenoptera_odb10 reference set (n = 5,911).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
A female Rhogogaster chlorosoma specimen (iyRhoChlo1) was collected by netting in Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.76, longitude -1.33) on 31 May 2021.The specimen was collected and identified by Steven Falk (independent researcher) and snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The iyRhoChlo1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Thorax tissue 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 RNA was extracted from abdomen tissue of iyRhoChlo1 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 DNA sequencing 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 was 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 iyRhoChlo1 using the Arima v2 kit and sequenced on the NovaSeq 6000 instrument.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Rhogogaster chlorosoma assembly (GCA_944452935.1) in Ensembl Rapid Release.

Ethics/compliance issues
The materials that have contributed to this genome The genome sequence is released openly for reuse.The Rhogogaster chlorosoma genome sequencing initiative is part of the Darwin Tree of Life (DToL) project.All raw sequence data and the assembly have been deposited in INSDC databases.Raw data and assembly accession identifiers are reported in Table 1.

Craig Michell
Red Sea Research Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia

Summary:
The authors here present a high-quality genome of an individual female from the species Rhogogaster chlorosoma, from the subfamily Tenthredininnae.The reported genome is 255,271,170 bases in length has a high contiguity and level of completeness.The genome will represent 1 out of the 27 reported Symphytan genomes to date and will provide an excellent resource for further studies into the evolution of Hymenopterans.

Methods:
The authors used appropriate methods to assemble the genome of this species.The technologies used for the genome assembly include 74X genome coverage PacBio HiFi sequencing and Hi-C sequencing.
RNAseq data was also prepared for this species, I presume for the annotation, but this is unclear in its current format.
The genome was annotated using BREAKER2 and then input into the Ensembl rapid release pipeline.

Datasets:
The datasets presented are clear and freely accessible online.

Comments:
I have mostly minor comments related to making the methods more reproducible especially for people interested in performing a similar project.Be explicit about the length of the genome, it is only fully listed in the Figure caption for Fig2, otherwise it is rounded.Also the genome size is different to the one listed in the Ensembl Rapid release ( https://rapid.ensembl.org/Rhogogaster_chlorosoma_GCA_944452935.1/Info/Index),not by much, but a little.I presume this is likely caused by the inclusion/exclusion of N's in the scaffolds.How many N's are filled into your scaffolds?
How was this data processed?How did you mark duplicates in this dataset?

○
In Table 3, can you add a column with the code used to run those programs?Reviewer Expertise: My areas of expertise is in genome assembly of insects (Hymenoptera; Symphyta) and other Eukaryotes.
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 Rhogogaster chlorosoma, iyRhoChlo1.1: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 255,287,193 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 (41,917,220 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 chromosome lengths (27,728,829 and 17,778,631 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 hymenoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/iyRhoChlo1_1/dataset/iyRhoChlo1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Rhogogaster chlorosoma, iyRhoChlo1.1:GC coverage.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/iyRhoChlo1_1/dataset/iyRhoChlo1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Rhogogaster chlorosoma, iyRhoChlo1.1:cumulative sequence.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/iyRhoChlo1_1/dataset/iyRhoChlo1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Rhogogaster chlorosoma, iyRhoChlo1.1:Hi-C contact map.Hi-C contact map of the iyRhoChlo1.1 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=fg3tvaqYQEGht9Fg16Ks_w.
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.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.Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.

○
What protein data was used for the annotation as BREAKER2 was run in protein mode?Did you use the BUSCO Hymenopteran protein database?○ Did you assemble the transcriptome and identify CDS?If so, how did you assemble the transcriptome and identify the CDS? ○ 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.

Table 1 . Genome data for Rhogogaster chlorosoma, iyRhoChlo1.1. Project accession data
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/iyRhoChlo1_1/dataset/iyRhoChlo1_1/busco.spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Table 3 . Software tools and versions used.
I would be useful to address comments by a previous reviewer of the manuscript, but otherwise I did not find anything to add or correct.

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.

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.