2.3 Phylogenetic analysis
Two phylogenetic trees were generated: a non-ultrametric phylogram using
Maximum Likelihood (ML) (Felsenstein, 1981) and an ultrametric
chronogram using Bayesian inference (BI) (Drummond et al., 2002). The ML
tree was generated using Iq-Tree (Trifinopoulos et al., 2016). Node
support was assessed with 1000 ultrafast bootstrap replicates (Hoang et
al., 2018), using the GTR+G+I model, with the data partitioned according
to codon position, as recommended by PartitionFinder version 2.1.
(Lanfear et al., 2017).
The BI tree was generated using BEAST version 2.6.7 (Bouckaert et al.,
2019), using default settings for all parameters. XML files were made
with the BEAUti version 2.6.7 interface with the following settings:
GTR+G+I substitution model, empirical base frequencies, 4 gamma
categories and all codon positions partitioned with unlinked base
frequencies and substitution rates. Since there is no agreement
concerning the most appropriate clock and tree priors for reconstructing
gene trees for species delimitation (Monaghan et al., 2009; Ratnasingham
& Hebert 2013; Talavera et al., 2013; Tang et al., 2014), preliminary
analyses to compare the use of two different clock (strict and relaxed
lognormal) and two different tree priors (coalescent constant population
and Yule) were undertaken (Rodrigues et al., 2020). The results of these
exploratory analyses (data not shown) indicated the strict clock and
Yule priors as the most suitable for our data set, thus these priors
were used for the Bayesian inference analyses.
To account for mixing within chains and convergence among chains with
reversible jump MCMC (Elworth et al., 2018), a total of 10 chains were
run from different seeds for 100 million generations each. Log files
from each run were combined in LogCombiner version 2.6.7 (Drummond et
al., 2012) after removal of the first 10% of samples from each run as
burn-in. Convergence of each run and the combined data were checked for
proper mixing using effective sample size (ESS) > 200 in
Tracer version 1.7.1 (Rambaut et al., 2018). Tree files from each run
were resampled to retain only 10% of the total trees and combined using
LogCombiner after removal of the first 10% of retained trees from each
run as burn-in. A maximum clade credibility (MCC) tree was then produced
using TreeAnnotator version 2.6.6 (Drummond et al., 2012) and FigTree
version 1.4.4 (Rambaut, 2010) was used to visualize and edit the trees.
All phylogenetic analyses were conducted on the CIPRES Science Gateway
High Performance Computing platform (Miller et al., 2011).