The genome sequence of the Mazarine Blue, Cyaniris semiargus (Rottemburg, 1775)

We present a genome assembly from an individual male Cyaniris semiargus (the Mazarine Blue; Arthropoda; Insecta; Lepidoptera; Lycaenidae). The genome sequence is 441.5 megabases in span. Most of the assembly is scaffolded into 24 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.4 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,408 protein coding genes.


Background
The Mazarine Blue, Cyaniris semiargus (Rottemburg, 1775), is a butterfly within the Lycaenidae family (the Blues, Coppers and Hairstreaks).Its English name comes from the blue-purple colour of the upper side of the male's wings, 'mazarine' being derived either from Cardinal Mazarin (1602-1661, sometime first minister of France), who popularised a brilliant cut of diamonds that became known as 'mazarines' (Bretherton, 1989) or alternatively his niece Hortense Mancini, Duchess Mazarin (1645-99) (Marren, 2019).Like many 'Blues', the female's wings are mostly brown, with blue basal scales that may extend notably in some specimens.In some populations, especially of the taxon hellenus Staudinger, 1862, from southern Greece, females display orange submarginal spots.The species has a Palearctic distribution, ranging from Morocco through much of Europe and Central Asia to China and Korea (Tolman & Lewington, 1997).
The Mazarine Blue is listed as a species of Least Concern, with stable populations on a European assessment (van Swaay et al., 2010), but with a decreasing trend on a Mediterranean assessment (van Swaay et al., 2014) for the IUCN Red List.In Britain, the Mazarine Blue has been known as a rare indigenous species since 1710, but was last seen as a native around 1905 (Andrews, 2015).It was formerly distributed widely in England and Wales, mainly in the south.The cause of extinction is suggested to be a change in agricultural practices (Thomas & Lewington, 2010).
Cyaniris semiargus can be found in dry to damp meadows, grasslands, cultivations, forest clearings and margins, and mountain slopes up to 2,800 m, where the adults fly, usually as a single generation, between May and early August (Haahtela, 2019).The larvae feed on various species of Fabaceae, including the genera Trifolium, Melilotus, Coronilla, Vicia and Anthyllis, and also on Armeria spp.(Plumbaginaceae).In the southern part of its Palearctic distribution, this butterfly is restricted to somewhat isolated populations in mid to high altitude areas, except for restricted populations in south-western Iberia, where it inhabits coastal dunes and feeds exclusively on Armeria velutina.Again, like many other Blues, the larva and pupa have strong associations with ants of the genera Lasius and Camponotus, which obtain a sweet secretion from the larva and provide protection (Thomas & Lewington, 2010).
The genus Cyaniris seems to be closely related to Rimisia and Agriades, with a divergence of about 4.4 Myr (Talavera et al., 2013).The Mazarine Blue has a haploid chromosome number of 24 (de Lesse, 1960;Federley, 1938;Lorković, 1941).The genome sequence now provides the basis for understanding the genetic relationships of the Mazarine Blue to other Lycaenidae and for studies on sexual dimorphism, myrmecophily and ecological adaptation.It will also help make sense of the genetic diversity and phylogeography of this species and its multiple recorded subspecies.

Genome sequence report
The genome was sequenced from one male Cyaniris semiargus specimen (Figure 1) collected from Apuseni Mountains, Lupșa, Alba, Romania (latitude 46.42, longitude 23.19).A total of 42-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 77-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected five missing joins or mis-joins, reducing the scaffold number by 10.71%.
The final assembly has a total length of 441.5 Mb in 25 sequence scaffolds with a scaffold N50 of 19.4 Mb (Table 1).Most (99.99%) of the assembly sequence was assigned to 24 chromosomal-level scaffolds, representing 23 autosomes, and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-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.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found here.

Sample acquisition and nucleic acid extraction
A male Cyaniris semiargus specimen (specimen number  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 and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and HiSeq X Ten (10X) instruments.Hi-C data were also generated from tissue of ilCyaSemi1 using the Arima v1 kit and sequenced on the HiSeq X Ten 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).One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes (Garrison & Marth, 2012).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using SALSA2 (Ghurye et al., 2019).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., 2022), 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.To evaluate the assembly, MerquryFK was used to estimate consensus quality (QV) scores and k-mer completeness (Rhie et al., 2020).The genome was analysed within the BlobToolKit environment (Challis et al., 2020) and BUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated.Table 3 contains a list of software tool versions and sources.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Cyaniris semiargus assembly (GCA_905187585.1) in Ensembl Rapid Release.

Ethics and compliance issues
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The   The current work is relevant and of general interest to researchers interested in genomic assembly, butterfly evolution, and Lepidoptera.It provides relevant information about Palearctic Lycaenidae.
The studied butterfly, C. semiargus, is declining in the Mediterranean region and is extinct locally in Britain.An annotated genome of this species would be useful for its conservation.The manuscript clearly describes the methodology, including the butterfly collection, laboratory procedures, genome assembly, and annotation.Unfortunately, the butterfly wings were deteriorated during the collection process (Figure 1).For further genomic studies, I encourage the researchers to manipulate the specimens more carefully.Additionally, it is suggested that these wings be mounted on a plate and included in a natural history collection.
The manuscript retains some mistakes mentioned by the previous reviewer, such as the author's affiliation, Tyler-Smith, and the link to the interactive version of Figure 5.

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: Butterfly's systematic and ecology chromosomes.(This is mentioned because in another similar Data Note in this journal, there is an a similar Hi-C contact plot with an online interactive version, in which the chromosome accessions are clearly shown for each box).Furthermore, the link to the interactive version of this Figure, found in the figure legend, does not seem to work.The HiGlass website opens up, but the figure never shows up and seems to be "Loading" indefinitely.It was attempted to access the link with different web-browsers, with no luck, and there were no issues accessing the other interactive online figures.
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?Partly Competing Interests: No competing interests were disclosed.
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 1 .
Figure 1.Forewings and hindwings of the male Cyaniris semiargus (ilCyaSemi1) specimen from which the genome was sequenced.Dorsal (a) and ventral (b) surface view of wings from specimen RO_CS_951 from Lupşa, Alba, Romania, used to generate Pacific Biosciences, 10X genomics and Hi-C data.

Figure 2 .
Figure 2. Genome assembly of Cyaniris semiargus, ilCyaSemi1.1: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 441,519,306 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 (28,312,907 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (19,361,588 and 14,815,647 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/ilCyaSemi1.1/dataset/CAJJIN01/snail#Filters.

Figure 5 .
Figure 5. Genome assembly of Cyaniris semiargus, ilCyaSemi1.1:Hi-C contact map of the ilCyaSemi1.1 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=RaIZ0Z3sQmercnKuDiEVdQ.

Table 3 . Software tools: versions and sources. Software tool Version 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.