Mitochondrial phylogenetic tree and divergence dating
Complete mitochondrial genomes were annotated with MitoFinder (48) and
aligned with MAFFT l-ins-i. We first investigated phylogenetic
relationships among Glaucopsyche individuals by analyzing the
entire mitochondrial genome. We used IQ-TREE2 (49) to select the best
fitting substitution model for each partition and merge similar
partitions and build a maximum likelihood tree and assessed support with
1000 ultrafast bootstrap replicates.
To infer a time-calibrated phylogeny, we selected one individual each of
Xerces Blue and Silvery Blue in addition to 12 related Polyommatinae. We
extracted the sequences for protein-coding genes, aligned each with the
codon-aware aligner MACSE, and concatenated all. We used BEAST2 (50)
with the bModelTest package to perform phylogenetic site model averaging
for each of the merged partitions. Because there is no accepted
molecular clock rate for butterflies and no fossils to apply in this
part of the phylogeny, we used two strategies to apply time constraints
to the analysis. First, we used two published molecular clock rates for
the mitochondrial COX1 gene (1.5% divergence/Ma estimated for various
invertebrates (51), and the ‘standard’ insect mitochondrial clock 2.3%
divergence/Ma (51). We applied a strict clock with a normal prior set up
to span the 1.5-2.3% range with the 95% HPD interval (mean=1.9%,
sigma=0.00119). Second, we borrowed the age of the most recent common
ancestor of our sampled taxa from fossil-calibrated analyses across
butterflies, which has been estimated to ~33 Ma(7, 52).
We fixed the root age to 33 Ma and allowed the remaining node ages to be
estimated using a strict clock. Analyses were run twice from different
starting seeds for 10 million MCMC generations and trees were sampled
every 1000 generations. Runs were checked for convergence with Tracer
and all effective sample size (ESS) values were >200. Runs
were combined with LogCombiner, after removing the first 30% of
topologies as burn-in, and a maximum credibility tree was generated with
TreeAnnotator (50). Phylogenetic analyses were performed on the National
Life Science Supercomputing Center - Computerome 2.0
(www.computerome.dk).
Xerces Blue and
Silvery Blue population histories
We used the Pairwise Sequentially Markovian Coalescent (PSMC) model to
explore the demographic history of both species (Xerces Blue and Silvery
Blue). We obtained a consensus fastq sequence of the mappable fraction
of the genome for each autosomal chromosome (total of 22 chromosomes ofG. alexis assembly). Only positions with a depth of coverage
above 4X and below 15X were kept. Posteriorly a PSMC was built using the
following parameters: -N25 -t15 -r5 -p ”28*2+3+5”. We used 1 year for
the generation time and a mutation rate of 1.9x10-9,
estimated in Heliconius melpomene (53). Considering that calling
consensus sequences from low coverage samples (< 10x) can
underestimate heterozygous sites (54), and given the different coverage
between samples, we corrected by False Negative Rate the samples with
coverage lower than the coverage of L005 (for Xerces Blue) and L013 (for
Silvery Blue), as recommended by the developers of the software, so that
all samples are comparable with each other. However, since in our
dataset we do not reach a coverage > 20x, we acknowledge
that we are not capturing the whole diversity and thus our PSMC might
infer lower historical effective population sizes.