On the importance of correct DNA sequence alignment in plastid phylogenomics – an example from revisiting the relationships of the water lilies

The water lily clade represents the second earliest diverging branch of angiosperms. Most of its species belong to Nymphaeaceae, whose dominant clade (the “core Nymphaeaceae”) comprises the genera Barclaya, Euryale, Nymphaea, and Victora. Despite previous molecular phylogenetic studies on the core Nymphaeaceae, various aspects of their evolutionary relationships have remained unresolved. Among these unresolved questions are (i) the potential sister relationship of Euryale and Victoria to the subgenera Hydrocallis and Lotos of Nymphaea, which would render Nymphaea paraphyletic, (ii) the potential sister relationship of the type subgenus of Nymphaea to all other species of the genus, and (iii) the unclear relationships within the subgenera Brachyceras, Hydrocallis, and Nymphaea of Nymphaea. New molecular phylogenetic studies on the core Nymphaeaceae need to accommodate the idiosyncrasies of the non-coding plastome regions, as most of the DNA sequence variability and, by extension, phylogenetic information between the water lily genomes is located in the length-variable introns and intergenic spacers. For example, new studies must emphasize correct, motif-based DNA sequence alignments of intron and intergenic spacer regions before phylogenetic tree reconstruction. In this study, we revisit the phylogenetic history of the core Nymphaeaceae by employing such an approach. Specifically, we use a plastid phylogenomics analysis strategy in which the coding and non-coding partitions of the plastome are separated by region and undergo an automatic as well as a manual DNA sequence alignment. Our approach allows us to increase the reliability of the character base compared to the default practice of aligning complete plastomes through automated algorithms. It produces significantly different phylogenetic tree reconstructions for several of the regions under study. The results of these reconstructions indicate that Nymphaea is paraphyletic in its current circumscription, that each of the five subgenera of Nymphaea is monophyletic, and that the type subgenus is sister to all other subgenera of Nymphaea. Our results also clarify many relationships within the Nymphaea subgenera Brachyceras, Hydrocallis, and Nymphaea. Finally, we discuss whether the phylogenetic reconstructions received through our approach are in line with morphological evidence.