The genome sequence of the northern brown argus, Aricia artaxerxes (Fabricius, 1793)

We present a genome assembly from an individual Aricia artaxerxes (the northern brown argus; Arthropoda; Insecta; Lepidoptera; Lycaenidae). The genome sequence is 458 megabases in span. Most of the assembly (99.99%) is scaffolded into 23 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.8 kilobases in length. Gene annotation of this assembly on Ensembl has identified 12,688 protein coding genes.


Background
The northern brown argus or mountain argus, Aricia artaxerxes (Fabricius, 1793), is a small Lycaenid butterfly found throughout the Palearctic apart from North Africa and the Iberian Peninsula (Sañudo-Restrepo et al., 2013). This species is a habitat specialist that is often observed flying on alkaline slopes and grassland amongst the preferred larval host plant, the common rock-rose (Helianthemum nummularium). The northern brown argus is univoltine (Tolman & Lewington, 1997) and hibernates as a larva. Aricia artaxerxes exhibits high intraspecific morphological variation across its range, which has led to taxonomic over-splitting (Sañudo-Restrepo et al., 2013). However, two genetically and morphologically distinct subspecies are found in the British Isles (Aagaard et al., 2002): subspecies artaxerxes is found in Scotland where the species was first described and is easily distinguishable from the closely related Aricia agestis (brown argus) by a white spot on the upper forewing. Subspecies salmacis (Durham argus) occurs in northern England and lacks the distinctive white wing spot. The range of A. artaxerxes salmacis overlaps with the bivoltine A. agestis in northern England, and the two species hybridise in populations where their flight times overlap (Mallet et al., 2011).
Although A. artaxerxes is listed as a species of Least Concern on the IUCN Red List (Europe) (van Swaay et al., 2010), it is classed as vulnerable on the GB Red List (Fox et al., 2022) and is locally rare in the British Isles, exhibiting large decreases in both occurrence and abundance over the last 30 years (Fox et al., 2015). Declines in Britain can be attributed primarily to the loss and/or fragmentation of suitable grassland habitat due to intensified agricultural practices (Franco et al., 2006). Declines have also been attributed to climate change, as A. artaxerxes is shifting its range northwards and upwards in elevation in response to increased temperatures (Franco et al., 2006). At the same time, there is an increasing risk of hybridisation and/or replacement at its southern range margin, as A. agestis expands its range northwards in response to climate change. Finally, phenological shifts in response to climate change in univoltine specialists tend to come at a cost, resulting in population declines and retracting distributions as seen in A. artaxerxes (Macgregor et al., 2019).
Aricia artaxerxes has 23 chromosome pairs (Federley, 1938). The genome of spp. A. artaxerxes will be of use to researchers investigating how this habitat specialist responds to climate change.

Genome sequence report
The genome was sequenced from an individual male A. artaxerxes ( Figure 1) collected from Arthur's Seat, Edinburgh, Scotland (latitude 55.94, longitude -3.16), the type locality of this species. A total of 28-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 6 missing/misjoins and removed 3 haplotypic duplications, reducing the assembly length by 0.15% and the scaffold number by 17.24%, and increasing the scaffold N50 by 3.26%.
The final assembly has a total length of 458 Mb in 24 sequence scaffolds with a scaffold N50 of 20 Mb (Table 1).
While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
A male A. artaxerxes (ilAriArta2) was collected from Arthur's Seat, Edinburgh, Scotland (latitude 55.94, longitude -3.16) by Sam Ebdon (University of Edinburgh) and identified by Konrad Lohse (University of Edinburgh) based on wing morphology. The sample was taken from the meadow using hand netting and preserved by freezing at -80°C from live.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute. The ilAriArta2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Whole body tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts. 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. 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
A Pacific Biosciences HiFi circular consensus DNA sequencing library was constructed according to the manufacturers' instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) instrument. Hi-C data were also generated from whole body tissue ilAriArta2 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the A. artaxerxes assembly (GCA_937612035.1). 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).

Walther Traut
Universität zu Lübeck, Lübeck, Germany The Ariicia artaxerxes genome is a useful addition to the growing number of good Lepidoptera genomes. Together with the already published lycaenid genomes it opens an excellent basis for research on this family with its excessive variation of chromosome numbers. The quality of the genome assembly is good considering the BUSCO scores. When I searched for telomeres, however, only 9 of the 23 chromosomes had blocks of the insect telomere motif (TTAGG)n or its complement (CCTAA)n on both ends and in the correct orientation. This is not different though from other recently sequenced Lepidoptera genomes.
The article is clearly written and concise.
I found some minor points the authors may wish to consider: They did not mention how they identified the Z chromosome.
○ Figure 2 does not show "white scale lines".
○ "chromosomes are coloured by phylum" (Figure 3) and "chromosomes assigned to each phylum" (Figure 4) are rather cryptic remarks even to a reader familiar with Blob Tools. There is only one phylum in the in the lists.

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?

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 For the annotation of the genome, it is mentioned that transcriptomic data were used together with automated annotation but nothing is indicated about the tissue/butterfly stage used for RNAseq.

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This high quality chromosome level reference genome of A. artaxerxes will be of use to researchers investigating how habitat specialist butterflies respond to climate change.
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