The genome sequence of the Small Ranunculus, Hecatera dysodea (Denis & Schiffermüller, 1775)

We present a genome assembly from an individual female Hecatera dysodea (the Small Ranunculus; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 640.9 megabases in span. Most of the assembly is scaffolded into 32 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 15.4 kilobases in length. Gene annotation of this assembly on Ensembl has identified 12,213 protein coding genes.


Background
Hecatera dysodea, known as the Small Ranunculus, is a moth with subtly attractive markings as an adult, and an interesting history of extinction and colonisation in the British Isles.Larvae feed on the seeds or flowers of lettuces, mainly Prickly Lettuce (Lactuca serriola), but also other lettuce species, including cultivated varieties.Although H. dysodea has been reported as a pest of lettuces, as a seed and flower eater, they would only ever be eating bolted lettuces and thus a potential pest of lettuce seed crops.Adult moths visit flowers, especially of lettuces, and are readily attracted to light.Although it is sometimes reported as having one generation per year, Clancy et al., 2012 report two overlapping generations, with adults on the wing from May to October.
Found naturally across mainly Central and Southern Europe and Central Asia, H. dysodea has also been accidentally introduced to the USA, where it is now widespread in the Pacific Northwest (Landolt et al., 2010).The species name 'dysodea' is thought to originate from the larvae, 'ill-smelling' (Pratt, 1986), maybe a reference to the smell of the lettuce (they are not tasty after bolting).Chemical attractants are being used in the US to monitor and potentially control populations (Landolt et al., 2017).
The population of H. dysodea in England was always rather cyclical, with its heyday apparently around the end of the 19th century; thereafter there was a rapid decline with extinction in this country around the 1930s.Pratt (1986) summarised the history of the decline and loss of H. dysodea from Britain and suggested that more modern farming (i.e., fewer bolting lettuce plants), declines in market gardens in south-east England and a succession of wet summers could have combined to cause its local extinction.In 1997, moths were found again in Kent (Agassiz & Spice, 1998) and H. dysodea has rapidly spread since then, now being rather widespread in England and in parts of Wales and its British population is classified as being of Least Concern (Fox et al., 2019).It is a species of rough, open ground, including brownfield sites and gardens.

Genome sequence report
The genome was sequenced from one female Hecatera dysodea specimen (Figure 1) collected from Tonbridge, UK (latitude 51.186305, longitude 0.286464).A total of 47-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 60-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 70 missing or mis-joins and removed six haplotypic duplications, reducing the assembly length by 0.55%% and the scaffold number by 47.37%, and increasing the scaffold N50 by 4.54%.
The final assembly has a total length of 640.9 Mb in 40 sequence scaffolds with a scaffold N50 of 21.9 Mb (Table 1).Most (99.94%) of the assembly sequence was assigned to 32 chromosomal-level scaffolds, representing 30 autosomes and the W and Z sex chromosomes.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 99.0% (single 98.4%, duplicated 0.6%) using the lepidoptera_odb10 reference set.

Sample acquisition and nucleic acid extraction
A female Hecatera dysodea (ilHecDyso1) was collected from Tonbridge, Kent (latitude 51.186305, longitude 0.286464) on 23 June 2020.The specimen was taken from a garden by Gavin Broad (Natural History Museum) using a light trap.The specimen was identified by Gavin Broad and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilHecDyso1 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.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using 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.
RNA was extracted from head tissue of (ilHecDyso1) 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   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 H. dysodea assembly (GCA_905332915.2).Annotation was created primarily through

Ethics and compliance issues
The materials that have contributed to this genome supposed that this is a standard notation, but it is slightly confusing as the Z and W in Lepidoptera are not thought to be homologous.9) An important piece of information that is missing is how the sex chromosomes were identified.I supposed by mapping back the PacBio reads and comparing the coverage of chromosomes?Maybe for the Z by synteny analysis with a closely related species?This should be mentioned.
10) In general, the Genome annotation methods are lacking some details.For example, there should be a mention of repeat masking before annotation, and it should be precise which UniProt protein set was used.In addition, the software used for annotation should be mentioned and cited.
Is the rationale for creating the dataset(s

Zhijun Zhang
Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang, China Yunsheng Wang Bioinformatics, College of Plant Protection, Hunan Agricultural University, Changsha, Hunan, China This is the firstly high quality genome assembly report for the moth species Hecatera dysodea.The authors have used appropriate sequencing and assembly strategies to generate a chromosomelevel genome with excellent completeness and contiguity.I recommend this paper be accepted for publication after addressing the following minor comments: 1.The sample acquisition and sequencing methods are clearly described.More details on the genome assembly process would be useful -for example, the coverage cutoff used for purging haplotigs with purge_dups.
2.It would be useful to describe TE annotation process.
3.The last sentence of the Background section is unclear and seems out of place.Consider revising or removing this sentence.
The high quality genome sequences of Hecatera dysodea and will be a useful resource for further studies of this species' biology and demography.The data is openly available and clearly presented.I recommend accepting this paper pending minor revisions.

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: thrips genome analysis; Tomato spotted wilt virus\thrips \plant interaction; integrated pest management.
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Red Sea Research Center, King Abdullah University of Science and Technology, Thuwal, Makkah Province, Saudi Arabia This article is a genome assembly report for the species Hecatera dysodea, the small Ranunculus.It is a species of moth with an interesting demographic history in the British Isles and a recent introduction to North Western United States.The Authors use a combination of high coverage PacBio HiFi sequencing, 10X read cloud, Hi-C and RNA sequencing to assemble the genome and annotate the encoded genes.
The sequencing strategy and protocols have produced a high quality genome with >99% of the assembly assigned to the correct number of chromosomes, with good contiguity and completeness.The quality metrics of the genome assembly are presented in Tables 1 and 2, as well as visually in Figures 2 through 5.
The methods used to assemble the genome are briefly described; the software and versions of the programs are contained in Table 3.
All of the data used to assemble the genome is available in public databases and the accessions can be found in Table 1 and the Data availability section.
My comments are all only minor and they are found below.

Comments:
The last sentence in the background is unclear.I do not know what this is referring to."It is a species of rough, open ground, including brownfield sites and gardens."It feels like an edit that was left in during different versions. 1.
The methods are clearly described for the sample acquisition and extracts, but extremely brief for the genome assembly.It would be useful to know the settings and options used for each step, for example, purge_dups what coverage level was set for the cutoffs?The setting for this affects the removal of haplotigs.

2.
Similarly, I would be interested to know the changes after polishing as it is generally not recommended to polish HiFi assemblies as it can reduce contiguity and break phase blocks.But maybe it is different with Long Ranger.

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

Figure 2 .
Figure 2. Genome assembly of Hecatera dysodea, ilHecDyso1.2: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 640,911,623 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 (30,380,561 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (21,896,367 and 15,320,587 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 lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilHecDyso1.2/dataset/CAJOST02/snail.

Figure 5 .
Figure 5. Genome assembly of Hecatera dysodea, ilHecDyso1.2:Hi-C contact map.Hi-C contact map of the ilHecDyso1.2assembly, 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=eT_0h2ElSVOAkIDCfeUuNA.
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 used for sequencing.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.

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.