2.6 Phylogeny estimation and barcoding of individual
y-larvae
Chromatograms of new forward and
reverse sequences were trimmed and assembled in Geneious Prime.
Protein-coding genes were translated (invertebrate mitochondrial code
for COX1 and universal code for H3) to proteins, aligned, and manually
checked for stop codons. Using MAFFT (–auto –maxiterate 1000),
nucleotides of each locus and specimen were then aligned with outgroup
sequences from Ascothoracida and previously sequenced material of
Facetotecta (Pérez-Losada et al., 2009). Individual locus alignments
were first inspected by eye, then subsequently curated by assessing
ambiguities with the less stringent options in GBLOCKS version 0.91b
(-t=p -b1=24 -b2=39 -b3=4 -b4=10 -b5=n -b6=y; Castresana 2000).
The nucleotide alignments were then concatenated in Geneious Prime
(6584bp and 3415bp before/after GBLOCKS), partitioned (each codon
position in protein-coding genes), and used to estimate the best-fit
substitution model with ModelFinder (Kalyaanamoorthy et al., 2017) by
allowing partitions to be merged if this increased model fit (-m
MFP+MERGE). Maximum Likelihood (ML) phylogenetic trees were built with
IQ-TREE multicore version 2.0.3 (Minh et al., 2020) and node support was
assessed with 10,000 ultrafast bootstrap (UFBoot) replicates and 10,000
replicates of the SH-like approximate likelihood ratio test (SH-aLRT;
–alrt 10,000). To alleviate issues inherent to the UFBoot
approximation method we specified the “-bnni” option. We allowed each
partition to have its own substitution rate (-spp) and performed a more
thorough nearest-neighbour interchange search (-allnni). We also
estimated a phylogeny with Bayesian inference in Mr. Bayes 3.2 (Ronquist
et al., 2012) using the concatenated dataset. We estimated posterior
probabilities through six independent runs with four Markov chains for
10,000,000 generations and with sampling every 1000 generations. We used
the ‘invgamma’ model as the lset rate and computed the potential scale
reduction factor with the ‘sump’ command. We estimated a consensus
phylogenetic tree with the first 25% of the iterations discarded as
burn-in.
The unrooted tree estimates were visualized in Geneious Prime, rooted in
Ascothoracida, and further annotated in Corel Draw© with morphological
information from live and exuvial specimens. These illustrations were
adjusted individually for brightness, contrast, and background color.