2.5 Phylogenomic analysis
We reconstructed phylogenetic trees using one representative sample from
each of the six A. cantabrica populations and all other taxa.
Some genome skimming data available online (Suppl. Table 2) were added
for reconstructing the plastid phylogeny.
Loci sequences were aligned with MAFFT (–auto; Katoh & Standley,
2013), and then the alignments were trimmed with trimAl (-automated1;
Capella-Gutiérrez et al. , 2009). For both Angiosperms353 loci and
the 125 recovered plastid fragments, we inferred nuclear and plastid
phylogenetic trees using both coalescent and concatenated maximum
likelihood (ML) approaches. In the coalescent approach, we inferred
single-locus phylogenetic trees from each trimmed alignment using
IQ-TREE (Minh et al. , 2020) with 1000 ultrafast bootstrap
replicates (-bb 1000; Hoang et al. , 2018), and branches with less
than 10% bootstrap support were collapsed with Newick utilities (Junier
& Zdobnov, 2010). We then used ASTRAL-III (Zhang et al. , 2018)
to infer the species tree (hereafter, ASTRAL tree), applying the ”-t 3”
flag to annotate local posterior probabilities (LPP) for each node.
In the concatenated ML approach, all trimmed alignments were
concatenated with FASconCAT-G (Kück & Meusemann, 2010). The best-fit
model inferred by IQ-TREE (-m MFP) was applied in RAxML-NG (Kozlovet al. , 2019) to infer the species tree using the concatenated
partitioned matrix with 1000 bootstrap replicates (–tree pars{20}
–bs-trees 1000; hereafter, RAxML tree). Additionally, we implemented
a greedy strategy (Lanfear et al. , 2012) with the relaxed
hierarchical clustering algorithm (Lanfear et al. , 2014) to
select the best partition model, which was applied in IQ-TREE to infer
the species tree with 1000 SH-like approximate likelihood ratio test
replicates (-alrt 1000; Guindon et al. , 2010) and 1000 ultrafast
bootstrap replicates (hereafter, IQ-partition tree). We visualized the
phylogenetic trees using Dendroscope (Huson & Scornavacca, 2012) and
FigTree (available at https://github.com/rambaut/figtree).
To investigate potential phylogenetic conflicts and signs of reticulate
evolution, we used SplitsTree4 (Huson & Bryant, 2006) to create a split
network based on the Neighbor-Joining algorithm with the Angiosperms353
data. In the resulting network, we masked specific samples to retain
only those within the A. cantabrica, A. adfinis and /halleri
clades for focused analysis.