Mapping Statistics of the analysed historical specimens.

Mapping statistics of the 4 historical G. xerces (L003, L005, L007 and L009) and the 7 historical G. lydagmus (L002, L004, L006, L008, L011, L012 and L013) specimens mapped against the G. alexis reference genome. Average depth is displayed for the covered regions of each individual.

Phylogenetic placement of the Xerces Blue.

A: Maximum likelihood tree from whole mitochondrial genomes of Xerces Blue, Silvery Blue and Green-Underside Blue. Node labels are bootstrap support values. Asterisk indicates the position of a previously published mitochondrial DNA genome (MW677564.1)4. B: Time-calibrated phylogeny from Bayesian inference using mitochondrial protein-coding genes of Xerces Blue and related butterflies. Node values show median age estimates from dating analysis with a molecular clock (above nodes) or from fixing the age of the root (below nodes). Bars are 95% HPD intervals for node ages. All posterior probabilities were 1, except for one node annotated in black. Images show upper sides (left) and undersides (right) of male specimens for a selection of species. We used MN974526 as a proxy for P. argus; however, after detecting a high sequence identity (>99%) with a previously published P. melissa mitogenome, we have decided to label it as Plebejus sp. in the tree.

PSMC plot of one Xerces Blue (Glaucopsyche xerces) (L05) specimen and one Silvery Blue specimen (Glaucopsyche lygdamus).

The two historical samples are those with higher average coverage. Individual PSMC plots were bootstrapped 100 times each (lighter lines). One year of generation time and a mutation rate of μ=1.9×10-9 were used. The peak of the Marine Isotopic Stage 7 interglacial is marked in yellow.

Runs of Homozygosity (RoH) in the genomes of Xerces Blue and Silvery Blue (modern and historical).

a: Percentage of the autosomal genome in RoH by size bins: very short RoH (<100 Kb), short RoH (100-500 Kb), intermediate RoH (500Kb-1Mb) and long (1-5Mb). Short RoH reflect LD patterns, intermediate size RoH describe background inbreeding due to genetic drift and long RoH appear in the case of very recent inbreeding due to consanguinity. Error bars show the standard deviation. b: Distribution of RoH in the autosomal genome of a Xerces specimen, L05 c) Distribution of RoH in the autosomal genome of a Silvery specimen L13.

Functional effect prediction on the fixed amino acid-changing alleles observed in Xerces Blue and Silvery Blue.

a: Wide genome Ka/Ks ratio comparison. b: High-to-moderate effect variant comparison in homozygous sites. c: High-to-moderate effect variant comparison in heterozygous sites. d: Presence of high-to-moderate variants in regions of the genome in RoH. Error bars show the standard deviation.