Placing environmental next generation sequencing amplicons from microbial eukaryotes into a phylogenetic context

Nucleotide positions in the hyper-variable V4 and V9 regions of the SSU-rDNA locus are normally difficult to align and are removed prior to standard phylogenetic analyses. But with next generation sequencing data, amplicons of these regions are all there is to answer ecological and evolutionary questions that depend on phylogenetic inferences. With ciliates, we asked how inclusion of the V4 or V9 regions, regardless of alignment quality, affects tree topologies using distinct phylogenetic methods (including PairDist that is introduced here). Results show that the best approach is to place V4 amplicons into an alignment of full-length Sanger SSU-rDNA sequences and to infer the phylogenetic tree with RAxML. A sliding window analysis as implemented in RAxML shows, though, that not all nucleotide positions in the V4 region are better than V9 at inferring the ciliate tree. With this approach and an ancestral-state reconstruction, we use V4 amplicons from European near-shore sampling sites to infer that rather than being primarily terrestrial and freshwater, colpodean ciliates may have repeatedly transitioned from terrestrial/freshwater to marine environments.