The genome sequence of the wood-carving leafcutter bee, Megachile ligniseca (Kirby, 1802)

We present a genome assembly from an individual female Megachile ligniseca (the wood-carving leafcutter bee; Arthropoda; Insecta; Hymenoptera; Megachilidae). The genome sequence is 290.0 megabases in span. Most of the assembly is scaffolded into 16 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 23.71 kilobases in length. Gene annotation of this assembly on Ensembl 11,722 protein coding genes.


Background
The Wood-carving Leafcutter Bee, Megachile ligniseca (Figure 1), is a large (forewing length 8.5-12 mm), brown, solitary bee in the family Megachilidae.It occurs across northern and central Europe.In the UK is has a south-eastern distribution, extending north to Yorkshire and west into south Wales.Females have a buff-coloured scopal hairs with the hairs of the final two sternites black.Tergite six is rounded and lacks erect hairs.Males have a deep notch on the posterior margin of tergite six and the fore-tarsi are unmodified.
It can be found in a range of habitats, including woodlands, scrub and gardens, and particularly post-industrial and brownfield sites (Falk & Lewington, 2019).It is a solitary, univoltine species with a flight period from June to September, peaking in July to August.Nests are constructed in aerial cavities, typically in holes in dead wood although artificial cavities and hollow plant stems are also used (Heroldovê et al., 2021).Females cut sections of leaves and occasionally petals that are used to wrap individual cells and to partition and seal nests.Adults visit a range of flowers including thistles, bramble and Himalayan balsam (Impatiens glandulifera) (Gresty et al., 2018), and nectar rob from, occasionally destroying, deep flowers (Wildermuth & Krebs, 2010).
The complete genome sequence for this species will facilitate studies into the evolution of sociality, reproductive systems and Hymenopteran taxonomy.

Genome sequence report
The genome was sequenced from one female Megachile ligniseca collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (51.77,.A total of 80-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 one missing join. The final assembly has a total length of 290.0 Mb in 675 sequence scaffolds with a scaffold N50 of 14.1 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 (77.35%) of the assembly sequence was assigned to 16 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (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.

Sample acquisition and nucleic acid extraction
A female Megachile ligniseca (specimen ID Ox000134, ToLID iyMegLign1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2019-08-07 by netting.The specimen used for Hi-C sequencing (specimen ID Ox000489, ToLID iyMegLign2) was netted in the same location on 2020-06-15.The specimens were collected and identified by Liam Crowley (University of Oxford).A third specimen (specimen ID Ox000744, ToLID iyMegLign3), used for RNA sequencing, was collected from Wytham Woods on 2020-08-03.This specimen was collected and identified by Steven Falk (independent researcher).All specimens were snap-frozen on dry ice before processing.HMW DNA was extracted using the Automated MagAttract v1 protocol (Sheerin et al., 2023).HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (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  were also generated from head and thorax tissue of iyMegLign2 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 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.

Genome annotation
The Ensembl Genebuild system (Aken et al., 2016) was used to generate annotation for the Megachile ligniseca assembly (GCA_945859555.1)at the EBI.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).

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

Software tool Version
In this manuscript, the authors provide a chromosome-level assembly for the wood-carving leafcutter bee, Megachile ligniseca.The genome report is clearly described, with the necessary genome statistics and a table of the chromosome-level scaffolds given, allowing the reader to understand the basic size and quality of the genome.The methods for identification of the specimen and extraction and processing of HMW DNA were also concisely outlined and easy to follow.Software tools used for genome analysis are also provided in an organized manner.
Minor points: The background information has a few typos ("In the UK is has" should be "In the UK it has"; "Females have a buff-coloured scopal hairs" should be "Females have buff-coloured scopal hairs") and in general should be more clearly written to make it easier for a reader not familiar with bee biology to understand the characteristics of M. ligniseca.The methods section could use a little more detail in describing the protocol.It may be easier to follow if some of the more crucial steps are explicitly stated in this section.

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

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 The Megachilidae family is the second largest under Apoidea, distinguished by the unique pollen brush on the underside of their abdomen, which is a specialized structure for pollination.These bees are crucial pollinators for leguminous forage and fruit trees, holding significant economic value.As solitary bees, they possess strong pollination abilities and serve as excellent models for studying solitary pollinators.The Megachile rotundata, in particular, is a highly efficient pollinator of Medicago sativa and the most important insect pollinator for alfalfa.Consequently, leaf-cutting bees are vital for studying the coevolution of insects and plants.Deciphering the genome of Megachilidae will provide a valuable genomic resource for future research on the evolution and ecology of wild pollinators.
Megachile ligniseca is a leaf-cutting bee primarily found in Europe.In this study, a chromosomescale genome of Megachile ligniseca was assembled using high-throughput sequencing technology.The genome size of this species was found to be 290Mb, with a total of 16 chromosomes, consistent with most Apis and Megachile bees.The study's methodology is clearly described, complemented by detailed charts, making it a classic example of genome research.
There are three suggestions and questions: 1) The species photos of Megachile ligniseca in the article should include morphological features.
2) In Apis, male bees are haploid, and many bee genomes use males as sequencing material.Why did you not choose females for sequencing?
3) The genome size assembled by Hifiasm is 100Mb larger than expected.Why did you insist on using Hifiasm?Although using purge_dups to eliminate redundancy improved the results, was the purging process excessive?
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: Molecular phylogenetics and evolution 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 Megachile ligniseca, iyMegLign1.1: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 290,044,579 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 (20,718,490 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (14,111,993 and 139,481 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/CAMAOC01/dataset/CAMAOC01/snail.

Figure 3 .
Figure 3. Genome assembly of Megachile ligniseca, iyMegLign1.1: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/CAMAOC01/dataset/CAMAOC01/blob.

Figure 4 .
Figure 4. Genome assembly of Megachile ligniseca, iyMegLign1.1: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/CAMAOC01/dataset/CAMAOC01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Megachile ligniseca, iyMegLign1.1:Hi-C contact map of the iyMegLign1.1 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=LsvkNZzWSdmDtY48U4dLfA.