The genome sequence of the Ochreous Pearl, Anania crocealis (Hübner, 1796)

We present a genome assembly from an individual male Anania crocealis (the Ochreous Pearl; Arthropoda; Insecta; Lepidoptera; Crambidae). The genome sequence is 624.0 megabases in span. Most of the assembly is scaffolded into 29 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.33 kilobases in length. Gene annotation of this assembly on Ensembl identified 20,293 protein coding genes.


Background
The Ochreous Pearl, Anania crocealis (Hübner, 1796), is a crambid moth with a wingspan of about 22-25 mm (Goater, 1986), forewing length 11-12 mm (Sterling & Parsons, 2018), its forewings pale yellow with the very small reddish brown orbicular and reniform stigmata bounded by a convex basal transverse line and a distal transverse line which is concave around the outer stigma, and whitish grey hindwings; the terminal line in both wings is also reddish-brown.It is similar to a few other UK Pyraustinae such as Paratalanta hyalinalis (Hübner, 1796) as well and the whiter Paracorsia repandalis ([Denis & Schiffermüller], 1775] but differs in the relatively unmarked hindwings.Like many pyraustines, it has a triangular resting posture. The adult moth flies in the UK from late June to August (Goater, 1986;Parsons & Clancy, 2023), and the moth is regarded as univoltine or sometimes bivoltine flying up to September or even November in the south of the UK (Parsons & Clancy, 2023;Sterling & Parsons, 2018).The adult moth is mainly nocturnal but easily disturbed around the foodplant by day.
The Ochreous Pearl has a preference for marshy and damp habitats in the UK as well as drier slopes on chalk where its larval foodplants occur (Common Fleabane, Pulicaria dysenterica (L.) Bernh., Ploughman's Spikenard Pentanema squarrosum (L.) D.Gut.Larr., Santos-Vicente, Anderb., E.Rico & M.M.Mart.Ort.as well as, in Austria, Buphthalmum salicifolium L.; all Asteraceae; Kettner, 2023).The black headed larvae also with a black prothoracic plate and pale grey green with a darker green dorsal line, start out under a downturned leaf margin, later feeding among silk inside the shoots (Parsons & Clancy, 2023;Sterling & Parsons, 2018).
A. crocealis is more or less common and widespread in southern parts of the United Kingdom, occurring more locally in Ireland, Wales, northwest England and Scotland where scarce, as well as Isle of Man and Channel Islands (Parsons & Clancy, 2023).In Europe it occurs widely from western France to Eastern Europe, the far south of Scandinavia, as far as the eastern edge of the Black Sea, with a few records in north-west Africa (Morocco), north-eastern Spain, Italy, Greece, Croatia and western Russia (GBIF Secretariat, 2023, BOLD 2/11/2023).
A. crocealis exhibits a single mitochondrial cluster on BOLD, BOLD:AAD7537 (2023-10-30).The mitogenome of the sequenced specimen (OX438878) is identical (658 bp) to the prevalent continental haplotype and a few other DNA barcodes from UK specimens are a single nucleotide different.Anania testacealis (Zeller, 1847) from the Mediterranean regions is around 1.2% divergent but belongs to the same BIN cluster; there is an intermediate sequence identified as A. crocealis DNA barcoded from Austria.Its closest relative otherwise is unclear; the nearest cluster on BOLD is BOLD:AAQ3746 (one specimen identified as Pyrausta trimaculalis) which is morphologically dissimilar and at least 5.26% divergent, but Anania perlucidalis (Hübner, [1809]), a species treated in Yang et al. (2012) Leraut (2005); see also Trankner et al. (2009).Anania exhibit an elongated, asymmetric sclerite of the phallus apodeme and female genitalia with a digitiform sclerotization inside the antrum (Trankner et al., 2009).The genus Anania Hübner, 1823 is currently placed in the tribe Pyraustini (Mally et al., 2019).
The genome data may be useful to investigate questions such as the closest relatives of A. crocealis, and adaptations to feed on Asteraceae: Inuleae.
The genome of the Ochreous Pearl, Anania crocealis, 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 Anania crocealis, based on one male specimen from Sandwich Bay Bird Observatory, England.

Genome sequence report
The genome was sequenced from one male Anania crocealis (Figure 1) (see methods).A total of 47-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  12 missing joins or mis-joins and removed one haplotypic duplications, reducing the scaffold number by 7.69%.
The final assembly has a total length of 624.0 Mb in 35 sequence scaffolds with a scaffold N50 of 22.9 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 (99.95%) of the assembly sequence was assigned to 29 chromosomal-level scaffolds, representing 28 autosomes and the Z sex chromosome.Chromosome Z was assigned by alignment to Eudonia lacustrata (GCA_947562085.1)(Boyes et al., 2023).Chromosomescale 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 male Anania crocealis (specimen ID NHMUK013698305, ToLID ilAnaCroc1) was collected by hand in Sandwich Bay Bird Observatory, England, UK (latitude 51.27, longitude  employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.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 abdomen tissue of ilAnaCroc1 using the Automated MagMax™ mirVana protocol (do Amaral et al., 2023).The RNA concentration was 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 the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

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 remaining head and thorax tissue of ilAnaCroc1 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.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Anania crocealis assembly (GCA_949315895.1) in Ensembl Rapid Release.

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 doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material 1.
The manuscript mentions adaptations for feeding on Asteraceae: Inuleae, but it lacks specifics.Could the authors elaborate on the types of adaptations observed?Additionally, why is feeding on Inuleae noteworthy?

2.
Figure 4: Figure 4 indicates that some scaffolds lack hits.Have the authors investigated the reasons for these missing hits?Could these scaffolds be contaminations or do they represent uncharacterized sequences?Further exploration or discussion of these findings would be valuable.

3.
Is the rationale for creating the dataset(s) clearly described?

Paula Escuer
University of Neuchâtel, Neuchâtel, Switzerland The authors presented the chromosome level genome sequence of a male individual of the Ochreous Pearl (Anania crocealis).The final assembly shows a total length of 624.0 Mb condensed in 35 scaffolds with a scaffold N50 of 22.9 Mb.Mitochondrial assembly is also sequenced and available.The 99.95% of the assembly sequence is distributed in 29 chromosomal-level scaffolds, representing 28 autosomes plus the Z chromosome.The BUSCO pipeline indicated a high completeness of the genome with 98.6% of complete genes found, additional descriptive analyses are indicated in Table 1.Genome annotation identified 20,293 protein-coding genes.Standard bioinformatic pipelines were performed and software versions are indicated in Table 3.This highquality assembly is a valuable tool to understand questions related to the evolution and adaptation of this genus and about the Lepidoptera order.
While the methodological part and the data presented is robust, I would rephrase some parts of the introductory section, as sometimes can be a bit complicated to read: -In the background, sometimes it is written UK, United Kingdom, England.It is better to standarise and use one of the terms or write United Kingdom (UK) and then use UK.
I would rephrase the entire the first and second paragraph as following: Reviewer Expertise: Genomics, Biodiversity, Speciation 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 Anania crocealis, ilAnaCroc1.1: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 623,994,487 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 (51,121,332 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (22,872,268 and 14,854,060 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/Anania%20crocealis/dataset/CASGGE01/snail.

Figure 3 .
Figure 3. Genome assembly of Anania crocealis, ilAnaCroc1.1: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/Anania%20crocealis/dataset/CASGGE01/blob.

Figure 4 .
Figure 4. Genome assembly of Anania crocealis, ilAnaCroc1.1: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/Anania%20crocealis/dataset/CASGGE01/cumulative.
Legality of collection, transfer and use (national and international) 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.doi.org/10.21956/wellcomeopenres.22772.r86811© 2024 Li R.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.Ruiqi LiUniversity of Colorado Boulder, Boulder, Colorado, USA Lees et al. present a genome assembly for the Ochreous Pearl, which appears to be of high quality and holds significant value for future research.Overall, the manuscript is well-prepared, but I have a few minor suggestions :Introduction:The introduction provides a thorough overview of the natural history of the Ochreous Pearl and the divergence observed in its COI sequences.This sets a strong foundation for the study.However, the mention of the BOLD should be clarified.What is BOLD?Please include relevant citations.

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The Ochreous Pearl, Anania crocealis(Hübner, 1796), is a crambid moth, which boasts a wingspan measuring approximately 22-25 mm(Goater, 1986), with forewings spanning11-12 mm (Sterling  and Parsons, 2018).Its forewings display a pale yellow tone with minute reddish-brown orbicular and reniform stigmata, bounded by a convex basal transverse line and a distal transverse line that curves around the outer stigma.The hindwings are whitish-grey, with reddish-brown terminal lines in both wings.While resembling other Pyraustinae species found in the United Kingdom (UK), such as Paratalanta hyalinalis(Hübner, 1796)  and the whiter Paracorsia repandalis ([Denis & Schiffermüller], 1775), the Ochreous Pearl differs in its unmarked hindwings.Like many pyraustines, it adopts a triangular resting posture.Adult moths fly in the UK between late June to August (Goater, 1986; Parsons & Clancy, 2023), and It is considered as univoltine or occasionally bivoltine flying up to September or even November in the south of the UK (Parsons & Clancy, 2023; Sterling & Parsons, 2018).The adult moth is mainly nocturnal but easily spotted around the foodplant during the day.I will also correct the next sentences: ○ A. crocealis is more or less common and widespread in southern parts of the United Kingdom, occurring more locally in Ireland, Wales, northwest England and Scotland, where it is scarce, as well as the Isle of Man and the Channel Islands (Parsons & Clancy, 2023).-It was not specified if the RNA-seq reads were used for the annotation pipeline.It can clearly improve the quality of the annotation, so it is interesting to know if it was included.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.

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

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.