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

Area of Science:

• Molecular Evolution
• Phylogenetics
• Microbial Ecology

Background:

• Hypervariable regions (V4, V9) of small subunit ribosomal DNA (SSU-rDNA) are challenging to align and often excluded from phylogenetic analyses.
• Next-generation sequencing frequently yields only these variable regions, limiting phylogenetic inferences for ecological and evolutionary questions.
• Ciliates, a diverse group of protists, require robust phylogenetic methods for understanding their evolutionary history and ecological roles.

Purpose of the Study:

• To investigate the impact of including V4 or V9 SSU-rDNA regions on ciliate phylogenetic tree topologies.
• To evaluate different phylogenetic methods, including a novel PairDist approach, for inferring evolutionary relationships using variable rDNA regions.
• To determine the optimal strategy for incorporating V4 amplicons into phylogenetic analyses of ciliates.

Main Methods:

• Phylogenetic analyses were performed using distinct methods, including RAxML and the newly introduced PairDist, on SSU-rDNA sequences.
• The study compared the effects of including V4 and V9 regions, irrespective of alignment quality, on tree reconstruction.
• A sliding window algorithm within RAxML was employed to assess the utility of individual nucleotide positions within the V4 region.

Main Results:

• Integrating V4 amplicons with full-length Sanger SSU-rDNA sequences and inferring trees with RAxML yielded the most robust phylogenetic results.
• Not all nucleotide positions within the V4 region consistently outperformed the V9 region in phylogenetic inference.
• Ancestral-state reconstruction using V4 amplicons suggested repeated transitions of colpodean ciliates from terrestrial/freshwater to marine environments.

Conclusions:

• The optimal approach for ciliate phylogenetics involves combining V4 amplicons with full-length SSU-rDNA data using RAxML.
• The V4 region, despite alignment challenges, offers valuable phylogenetic signal for ciliate evolutionary studies.
• Colpodean ciliates exhibit greater habitat plasticity than previously assumed, with evidence of marine incursions.