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.