Phylogenetic relationships
We built a multiple sequence alignment (MSA) for each 1:1 orthologs with
PRANK v.100802 (Löytynoja, 2014), using both coding sequences (CDS)
(-codon -noxml -notree -F) and amino acid sequences (-noxml -notree -F).
Individual alignments were concatenated with catfasta2phyml v1.1.0
(https://github.com/nylander/catfasta2phyml) to create a CDS supermatrix
and an amino acid supermatrix. Only locus with data for all the twelve
species have been considered. Fourfold degenerate sites (4D) for the CDS
supermatrix were extracted with MEGA X (Kumar, Stecher, Li, Knyaz, &
Tamura, 2018). We performed unpartitioned maximum likelihood (ML)
phylogenetic analyses using IQ-TREE 1.6.12 (Nguyen, Schmidt, Von
Haeseler, & Minh, 2015) (-bb 1000) both for 4D and amino acid
supermatrices. Optimal models of sequence evolution were obtained with
ModelFinder (Kalyaanamoorthy, Minh, Wong, Von Haeseler, & Jermiin,
2017) according to Bayesian information criterion (BIC), and the
resulting best-fit models were GTR+F+R2 for 4D and HIVb+F+R3 for amino
acid supermatrix. Node support was obtained with Ultrafast Bootstrap
(Hoang, Chernomor, Von Haeseler, Minh, & Vinh, 2018).
To explicitly account for incomplete lineage sorting (ILS) under the
Multispecies Coalescent Model (MSC), we inferred the species tree using
the summary coalescent approach, as implemented in ASTRAL-III 5.6.3 (C.
Zhang, Rabiee, Sayyari, & Mirarab, 2018). We first obtained all gene
trees (for each 1:1 orthologous genes) using IQ-TREE 1.6.12, and
inferred the species tree and its normalized score (from both CDS and
amino acid gene trees) using ASTRAL-III.
We generated an ultrametric tree with r8s v.1.81 (Sanderson, 2003) using
the 4D supermatrix ML tree. We used four calibration points (in myr):
root (max_age=84 min_age=73, (Braun et al., 2011), most recent common
ancestor (MRCA) of Spheniscidae (min_age=12.6, (Subramanian,
Beans-Picón, Swaminathan, Millar, & Lambert, 2013)), MRCA
Procellariiformes (min_age=49, Claramunt & Cracraft, 2015), MRCA
Procellariidae (min_age=14, (Prum et al., 2015)), retrieved from
TimeTree (Kumar, Stecher, Suleski, & Hedges, 2017), and references
therein). We used the penalized likelihood (PL) method and the Truncated
Newton (TN) algorithm, smoothing parameter was set to 100.