Aligning evidence: concerns regarding multiple sequence alignments in estimating the phylogeny of the Nudibranchia suborder Doridina

Abstract

Molecular estimates of phylogenetic relationships rely heavily on multiple sequence alignment construction. There has been little consensus, however, on how to properly address issues pertaining to the alignment of variable regions. Here, we construct alignments from four commonly sequenced molecular markers (16S, 18S, 28S and cytochrome c oxidase subunit I) for the Nudibranchia using three different methodologies: (i) strict mathematical algorithm; (ii) exclusion of variable or divergent regions and (iii) manually curated, and examine how different alignment construction methods can affect phylogenetic signal and phylogenetic estimates for the suborder Doridina. Phylogenetic informativeness (PI) profiles suggest that the molecular markers tested lack the power to resolve relationships at the base of the Doridina, while being more robust at family-level classifications. This supports the lack of consistent resolution between the 19 families within the Doridina across all three alignments. Most of the 19 families were recovered as monophyletic, and instances of non-monophyletic families were consistently recovered between analyses. We conclude that the alignment of variable regions has some effect on phylogenetic estimates of the Doridina, but these effects can vary depending on the size and scope of the phylogenetic query and PI of molecular markers.

Keywords: Cryptobranchia; Dorid systematics; Phanerobranchia; multiple sequence alignment; sequence homology.

Figure 1.

Phylogenetic informativeness profiles estimated by PhyDesign. (a) MAFFT alignment. (b) Gblocks alignment. (c) Curated alignment.

Figure 2.

Phylograms of Doridina phylogenetic estimates from all three different alignment constructions. Topologies represent Bayesian estimates. (a) MAFFT alignment. (b) Gblocks alignment. (c) Curated alignment.

Figure 3.

Cladogram of Doridina phylogenetic estimates. Topology represents Bayesian estimate. Branches are coloured based on superfamily designations, which are pictured adjacent to the phylogenies. Only four of the five families are depicted due to the inability to obtain an image of Bathydoridoidea. Circles represent posterior probabilities (top) and non-parametric bootstrap support values (bottom). Closed circles indicate high Bayesian and ML support (pp ≥ 0.95; bs ≥ 75). Red circles indicate moderate support values (pp: 0.95–0.90; bs: 75–70). Open circles indicate no support (pp < 0.90; bs < 70). Relationships that were not recovered by ML analysis are represented by dashes. (a) MAFFT alignment. (b) Gblocks alignment. (c) Curated alignment.

Figure 4.

Phylogenetic estimate from MAFFT Alignment. Topology represents Bayesian estimate, with posterior probabilities (pp) and non-parametric bootstrap (bs) support values depicted above and below each branch, respectively. Relationships that were not recovered by ML analysis are represented by dashes. Branches are coloured based on family designations and represented on the exterior of the phylogeny.

Figure 5.

Phylogenetic estimate from Gblocks alignment. Topology represents Bayesian estimate, with posterior probabilities (pp) and non-parametric bootstrap (bs) support values depicted above and below each branch, respectively. Relationships that were not recovered by ML analysis are represented by dashes. Branches are coloured based on family designations and represented on the exterior of the phylogeny.

Figure 6.

Phylogenetic estimate from Curated alignment. Topology represents Bayesian estimate, with posterior probabilities (pp) and non-parametric bootstrap (bs) support values depicted above and below each branch, respectively. Relationships that were not recovered by ML analysis are represented by dashes. Branches are coloured based on family designations and represented on the exterior of the phylogeny.