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.
Highlights
The debate regarding multiple sequence alignment (MSA) construction [1,2,3,4,5,6,7,8] and molecular marker selection [9] for use in phylogenetic estimates has been well established in the literature
Sequences obtained for phylogenetic analyses and phylogenetic informativeness (PI) profiles are labelled in table 1, and all alignments have been deposited in TreeBASE
Our findings suggested that MSA methodology affected phylogenetic estimates of the Doridina, especially regarding how we decided to align highly variable rDNA regions
Summary
The debate regarding multiple sequence alignment (MSA) construction [1,2,3,4,5,6,7,8] and molecular marker selection [9] for use in phylogenetic estimates has been well established in the literature. There is little consensus, on the most accurate and replicable approach when considering MSA assemblies [10]. In most instances manual curation is loosely defined— which makes subsequent studies difficult to replicate—and even though mathematical algorithms allow for studies to be replicated, they do not take into account evolutionary history and consider homology exclusively as similarity [3]. They often fail to accurately align variable regions [11]
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