Population structure and phylogenetic analysis based on genomic
DNA
We demultiplexed raw reads and applied standard quality filters in
Ipyrad 0.7.28 (Eaton and Overcast 2020). We discarded sequences when a
single base had a Phred quality score below 10 or more than 5% of bases
had a Phred quality score below 20. Additional filtering was applied to
only retain reads that did not have enzyme cleavage sites, adaptor
sequences or index sequences and included up to 3 ambiguous sites.
Finally, we only kept loci that were present in at least 80% of the
samples. The ddRAD data is available in Dryad (doi: xxx).
We performed a principal component analysis (PCA), using the gdsfmt and
SNPRelate packages (Zheng et al. 2012) in R 3.5.1 (R Core Team, 2018),
to visualize possible clusters in the data. We used all the SNPs of the
dataset (including multiple SNPs per locus), but because the PCA is
sensitive to missing data, SNPs missing from at least one individual
were removed. We also assigned individuals to genetic clusters (K) using
Structure 2.3.4 (Pritchard et al. 2000). For this analysis we used a
single random SNP from each RAD locus. We implemented the admixture
ancestry model with correlated allele frequencies and an allele
frequency prior of λ = 1. We conducted 10 runs for each value of K =
1-4, and each run consisted in 500,000 generations following a burn-in
of 100,000. The most likely value of K was determined following the ΔK
method described by Evanno et al. (2005) and implemented in Structure
Harvester 0.6.94 (Earl and vonHoldt 2012). We averaged results across
the 10 runs using the greedy algorithm in the program CLUMPP 1.1.2
(Jakobsson and Rosenberg 2007) and visualized results using the
conStruct package (Bradburd et al. 2018) in R.
We used RAxML version 8.2.4 (Stamatakis 2014) to infer a maximum
likelihood phylogenetic tree. We implemented the ASC_GTRGAMMA model and
the Lewis correction for ascertainment bias and we conducted 200
bootstrap replicates to assess node support. We included one specimen ofT. caerulescens as outgroup (we obtained ddRADseq data for this
specimen from a previous study; Kopuchian et al. 2020; see Table S1).