Discussion
We have used a modern reference genome and ancient DNA genome sequence data from museum specimens to explore the relationships and historical population genetic history of an extinct butterfly, the Xerces Blue. Based upon a near-complete mtDNA genome from a Xerces Blue specimen, Grewe et al. (2021) (3) proposed that the Xerces Blue and the Silvery Blue were distinct species. We confirm this finding using full mitochondrial genomes and extensive nuclear genomic data from multiple specimens. Given the lack of evidence for Wolbachia infection, a detailed analysis of genomic architectures could help identify barriers to introgression between these species.
We provide evidence for low population size in Xerces Blue, correlated with low genetic variation, a higher proportion of runs of homozygosity and increased frequency of deleterious, amino acid-changing alleles (14–16). However, there was no genetic evidence of recent inbreeding, which sometimes occurs in critically endangered species (17,18). Our analyses indicate that the Xerces Blue had experienced a severe demographic decline for tens of thousands of years, likely associated with changing climatic factors. Thus, the destruction of the Xerces Blue habitat by humans was likely the final blow in the extinction process. We suggest that endangered insects with demographic traits indicative of long-term low effective population size should be considered to be especially vulnerable to extinction.
The generation of these paleogenomes is the first step towards understanding the specific adaptations of Xerces Blue butterfly. Maybe the most conspicuous of these was a pattern of white spots, usually without black ocelli, on their ventral wing surfaces. Genes such asWnt , optix and cortex are associated with pigmentation wing patterns in butterflies (19–23). It may be informative to identify amino acid differences in these and other genes and to undertake functional studies, such as those based on CRIPSR-Cas 9 knock-outs, in extant relatives to elucidate relevant phenotypic and genomic characteristics of this extinct butterfly. However, in our preliminary screening in these pigmentation genes, no amino acid changes as compared to the Silvery Blue genomes have been discovered, raising the question if potential differences could be in fact in regulatory regions. However, it is likely that this question would need to further increase the coverage of the paleogenomes generated. Irrespective of this, our study further demonstrates the value of ancient DNA in museum specimens for evolutionary studies at a population scale.
In addition to, Glaucopsyche alexis  is a good reference genome to study the transition from mutualism with ants to parasitism on ants within subtribe Glaucopsychina; the latter group includes the remarkable Phengaris  butterflies, which are ant-parasites in the last instars and are currently endangered in Europe (mainly due to their highly specialised strategy) (24). The G. alexis reference genome (coupled with those of the Xerces Blue and Silvery Blue) will help to understand the evolution from an exclusively herbivorous diet in later instars to a aphytophagous one and the biochemical synthesis of molecules used in the ant-parasitic relationship.