2.4 | Statistical Methods
Sequence fragments were assembled, edited, and aligned with MUSCLE
(Edgar 2004) within Geneious Prime. The COI alignment was
translated with the Mold, Protozoan, and Coelenterate translation
mitochondrial code table to ensure that the sequences were aligned in
the correct reading frame and that no stop codons were present.
We used jModelTest (Posada & Crandall 1998) within Geneious to select
the best evolutionary model. We estimated phylogenies with MrBayes
(v3.2, Ronquist et al. 2012) and IQ-Tree 2 with 1000 bootstrap
replicates (Minh et al. 2020) for all ctenophores available based
on the most appropriate models selected by the AIC (Akaike 1974).
Bayesian phylogenies estimated with MrBayes included multiple runs of
5x106 generations with a 10% burn-in, with six
chains, that were sampled and printed every 1000 generations.
Convergence was determined with TRACER v1.7 (Rambaut et al. 2018)
and by comparing topologies of multiple runs.
Phylogenies were visualized with FigTree (v1.4.4,
tree.bio.ed.ac.uk/software/figtree/).
To illuminate within-order diversity, alignments of lobate species for
both COI and 18S fragments were analyzed separately with
the same parameters that had been used for the full alignment, and both
Bayesian and likelihood support values were reported on the phylogeny.
The lobate phylogenies were rooted with the cydippid Pleurobrachia
bachei as the outgroup.
To assess diversity within sequenced ctenophores for COI we
calculated percent general time reversible (GTR) distance within and
between molecular operational taxonomic units (MOTUs). Base composition
was calculated with MegaX (v10.0.5, Kumar et al. 2018) and
pairwise distances were calculated in Geneious Prime. We calculated the
number of parsimony-informative sites within the Lobata for bothCOI and 18S with DnaSP (v6.12.01, Rozas et al.2017). We tested for saturation of observed proportions of transitions
and transversions versus GTR distance among all MOTUs with the software
DAMBE7 (v7, Xia 2018). We also tested five other mitochondrial loci for
saturation from the published mitochondrial genomes of eight species of
ctenophores with DAMBE7.
To illustrate how a more complete reference library can affect
metabarcoding sequence assignments, we used data for the COIfragment for ctenophores from Pitz et al. (2020) and queried our
ctenophore-specific library with the same methods as the authors of the
study. Data for stations where no ctenophore sequences were recovered
were not reported. We plotted species assignments based on the presence
or absence of reads in each sample in Rstudio (Team 2015) with ggplot2
(Wickham 2016).