The genome sequence of the Forest Cuckoo Bee, Bombus sylvestris (Lepeletier, 1832)

We present a genome assembly from an individual male Bombus sylvestris (the Forest Cuckoo Bee; Arthropoda; Insecta; Hymenoptera; Apidae). The genome sequence is 303 megabases in span. Most of the assembly is scaffolded into 24 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 23.6 kilobases in length. Gene annotation of this assembly on Ensembl has identified 13,025 protein coding genes.


Background
The Forest Cuckoo Bee, Bombus sylvestris, is one of six 'cuckoo bumblebee' species in the UK.It is a social parasite of Bombus pratorum, and possibly also B. jonellus and B. monticola, usurping colonies of these species and using the workers to raise its own offspring.This species does not produce workers.Females search out and enter a host nest, cohabiting in the nest before dominating the host queen and preventing oviposition of host eggs (Küpper & Schwammberger, 1995).The B. sylvestris female then lays her own eggs in the host nest, that are reared by the host workers.Cuckoo bumblebees were formerly placed in their own genus Psithyrus, which has subsequently dropped to the rank of subgenus (Pedersen, 1996).
Bombus sylvestris is widespread throughout much of Europe and can be found across the same habitats as its host(s), although it is rarely common.It is a small species -around 15 mm in length -and is covered in black hairs with a band of yellow hairs across the pronotum and white hairs on the apical segments of the abdomen.The white hairs are variably buff/darker in some individuals.The apex of the abdomen is strongly curled under in the female.Males have a tuft of orange hairs on the very tip of the abdomen and may have some buff/yellow hairs on tergite one (Edwards & Jenner, 2005).Males produce a distinctive 'mousy' scent that is detectable by humans, derived from cephalic secretions related to territory marking (Kullenberg et al., 1970).
The phenology of this species matches the host species, with overwintered females emerging from late march and new males and females produced from July to September.It is likely to be bivoltine in areas where the host is also bivoltine, although this remains unclear.A wide range of flowers are visited for nectar.Pollen is not collected, although females may feed on pollen to facilitate ovary development.
A complete genome sequence for B. sylvestris will facilitate studies into the evolution of social parasitism and reproductive systems, as well as conservation of pollinator species.

Genome sequence report
The genome was sequenced from one male Bombus sylvestris (Figure 1) collected from Wytham Woods, Oxfordshire (latitude 51.78, longitude -1.34).A total of 79-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 74-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 48 missing joins or mis-joins, increasing the assembly length by 1.72%, reducing the scaffold number by 5.41%, and increasing the scaffold N50 by 80.9%.
The final assembly has a total length of 302.6 Mb in 140 sequence scaffolds with a scaffold N50 of 12.4 Mb (Table 1).Most (97.15%) of the assembly sequence was assigned to 24 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 97.5% (single 97.2%, duplicated 0.3%) using the hymenoptera_odb10 reference set (n = 5,911).

Genome annotation report
Annotation of the B. sylvestris GCA_911622165.1 assembly was generated using the Ensembl genome annotation pipeline (Table 1; https://rapid.ensembl.org/Bombus_sylvestris_GCA_911622165.1/).The resulting annotation includes 13,025 protein coding genes with an average length of 12,696.31and an average coding length of 1,413.16,and 5,798 non-protein coding genes.There is an average of 6.12 exons and 5.12 introns per canonical protein coding transcript, with an average intron length of 1,644.29.A total of 7,657 gene loci have more than one associated transcript.

Sample acquisition and nucleic acid extraction
A single male Bombus sylvestris (iyBomSyle1) was collected by netting in Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.78, longitude -1.34) on 1 June 2020.The specimen was collected and identified by Liam Crowley (University of Oxford) and then snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The iyBomSyle1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Head and thorax tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.High molecular  RNA was extracted from abdomen tissue of iyBomSyle1 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 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), Illumina HiSeq 4000 (RNA-Seq) and HiSeq X Ten (10X) instruments.Hi-C data were also generated from tissue of iyBomSyle1 using the Arima v2 kit and sequenced on the HiSeq X Ten instrument.

Genome assembly
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with 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.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the B. sylvestris assembly (GCA_911622165.1).Annotation was created primarily through alignment of transcriptomic data to the genome, with

Shaoying Wu
Hainan University, Haikou, China The introduction briefly describes the remarkable biology of the species.The genome was produced using an excellent method, the manuscript provides the details of the analysis, the data is excellent, and the resulting genome is of high quality.This paper describes the genome sequencing of a social parasitic bumblebee, Bombus sylvestris.It can provide a rich database for entomologists around the world, especially a specialist in bees.

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.
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.

Peter K Dearden
University of Otago, Dunedin, Aotearoa,, New Zealand This manuscript describes the genome sequencing of Bombus sylvestris, a socially parasitic bumblebee species.The genome has been produced using an excellent methodology and the manuscript provides details of the analysis, which are standard, and have produced a high-quality genome.
The introduction provides a brief description of the remarkable biology of this species.The figures are excellent, and the resulting genome is high quality and scores highly with BUSCO analysis.
My only comment would be the HiC plot, figure 5, is usual, and it would be nice (though not crucial) to know what is causing that.

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: Insect genomics 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 Bombus sylvestris, iyBomSyle1.2: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 302,565,711 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 (18,982,394 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (12,445,291 and 8,163,122 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 hymenoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/iyBomSyle1.1/dataset/ CAJVRE01/snail.

Figure 3 .
Figure 3. Genome assembly of Bombus sylvestris, iyBomSyle1.2: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/iyBomSyle1.1/dataset/CAJVRE01/blob.

Figure 4 .
Figure 4. Genome assembly of Bombus sylvestris, iyBomSyle1.2: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/iyBomSyle1.1/dataset/ CAJVRE01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Bombus sylvestris, iyBomSyle1.2:Hi-C contact map.Hi-C contact map of the iyBomSyle1.2assembly, 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=DBoJSZEqSRaXzTmtjCWZ4A.

Table 1 . Genome data for Bombus sylvestris, iyBomSyle1.2. Project accession data
8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing.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.

Table 3 . Software tools and versions used. Software tool Version Source
Ethics and compliance issuesThe 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.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.All efforts are undertaken to minimise the suffering of animals.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.

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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.
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.