The genome sequence of the fork-jawed nomad bee, Nomada ruficornis (Linnaeus, 1758)

We present a genome assembly from an individual male Nomada ruficornis (the fork-jawed nomad bee; Arthropoda; Insecta; Hymenoptera; Apidae). The genome sequence is 273.0 megabases in span. Most of the assembly is scaffolded into 16 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 20.66 kilobases in length.


Background
Bees from the Nomada genus are commonly called Nomad Bees since they frequently roam close to the ground in search of their host's nests.All Nomada species practise kleptoparasitism, depositing their eggs in other bees' nests and utilizing the pollen gathered by those host bees.The host species are often from the Andrena genus.
Nomada ruficornis Linnaeus is a medium-sized kleptoparasitic bee (females 8-11mm, males 7-11 mm).The species can be distinguished from all other UK Nomada species, excluding N. fabricana (Linnaeus), due to the bidentate mandibles, which has given it the common name of 'fork-jawed nomad bee'.
Nomada ruficornis is univoltine, active from March to July (Smit, 2018).The host is recorded as Andrena haemorrhoa (Fabricius), a ground nesting polylectic species.The host is common throughout the UK, nesting in a wide range of habitat types.In turn N. ruficornis is found throughout the UK but becomes more scarce further north.Outside of the UK, the distribution extends across the Palearctic to Japan (Ascher & Pickering, 2016).
The genome of the fork-jawed nomad bee, Nomada ruficornis, was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.Here we present a chromosomally complete genome sequence for Nomada ruficornis, based on one male specimen from Wytham Woods.

Genome sequence report
The genome was sequenced from one male Nomada ruficornis (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.76, -1.34).A total of 92-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 17 missing joins or mis-joins, reducing the scaffold number by 8.6% and increasing the scaffold N50 by 1.69%.
The final assembly has a total length of 273.0 Mb in 169 sequence scaffolds with a scaffold N50 of 15.0 Mb (Table 1).Most (92.79%) 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 2-Figure 5; Table 2).The specimen is a haploid male.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/601849.

Sample acquisition and nucleic acid extraction
Two male Nomada ruficornis specimens were netted in Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.76, longitude -1.34) on 2021-04-23.The specimens were collected and identified by Steven Falk (University of Oxford) and snap-frozen on dry ice.The specimen with ID Ox001289 (ToLID iyNomRufi1) was used for DNA sequencing, while specimen with ID Ox001295 (ToLID iyNomRufi2) was used for Hi-C scaffolding.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The iyNomRufi1 sample was weighed and dissected on dry ice.Tissue from the head and thorax 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.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using the 0.8X 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.Sequencing Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from whole organism tissue of iyNomRufi2 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 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.
A Hi-C map for 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

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The materials that have contributed to this genome  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.
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Trevor Sless
Biology, York University, Toronto, Ontario, Canada The submitted article reports on the complete genome of the fork-jawed nomad bee Nomada ruficornis, the type species of its genus.This is a significant contribution to the genomic study of cuckoo bees such as Nomada and also serves to further the Darwin Tree of Life Project's broader goal.The genome assembly was generated using modern standard methods including the PacBio HiFi and Hi-C sequencing platforms, and all assembly metrics support the high quality of this new reference genome.The sequence dataset is openly accessible and will be useful for a variety of future studies.

Suggestions:
Background: "Nomada ruficornis Linnaeus is a medium-sized…" Linnaeus author citation should be in parentheses (due to basionym Apis ruficornis)

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Although not strictly necessary, I feel that it is worthwhile to mention the particular taxonomic significance of N. ruficornis as the type species of the genus Nomada.
○ "The species can be distinguished from all other UK Nomada species, excluding N. fabricana…" "species" is unnecessarily italicized Here are some other minor suggestions: One aspect that I think could be expanded on a bit more is the rationale for sequencing this bee.I agree that including it for the Darwin Tree of Life project is important but I think there could be something added to the background that could tie into pollinator diversity and/or conservation.

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Maybe add another sentence to the second paragraph of how this species differs from N. fabriciana.

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: Nomada 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 Nomada ruficornis, iyNomRufi1.1: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 273,031,491 bp assembly.The distribution of sequence lengths is shown in dark grey with the plot radius scaled to the longest sequence present in the assembly (27,766,539 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 sequence lengths (14,982,175 and 9,633,086 bp), respectively.The pale grey spiral shows the cumulative sequence 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/iyNomRufi1.1/dataset/CATOQH01/snail.
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

Figure 5 .
Figure 5. Genome assembly of Nomada ruficornis, iyNomRufi1.1:Hi-C contact map of the iyNomRufi1.1 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=TxoFhDkFSK6cHDFb9syBjw.

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

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.
○"N. fabricana" is misspelled, it should be N. fabriciana ○ Genome Sequence Report: "Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size…"The chromosomes listed in Table2are slightly out of order, with the chromosome designated as #5 confusingly being the eighth largest.Please either rename or explain the reasoning for this.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Reviewer Report 21 September 2023 https://doi.org/10.21956/wellcomeopenres.22067.r66796