Combining molecular data with classical morphology for uncultured phagotrophic euglenids (Excavata): a single-cell...

Area of Science:

• Protistology
• Molecular Evolution
• Microbial Ecology

Background:

• Phagotrophic euglenids are crucial heterotrophic flagellates in sediments, yet their biodiversity and evolutionary links to photosynthetic forms remain poorly understood.
• Existing knowledge relies heavily on light microscopy, with limited integration of molecular data for phylogenetic analysis.

Purpose of the Study:

• To develop and validate a protocol for obtaining both light microscopy and molecular data from single, isolated phagotrophic euglenid cells.
• To investigate the biodiversity and phylogenetic relationships of phagotrophic euglenids using this integrated approach.

Main Methods:

• Isolation and single-cell rinsing of euglenids from freshwater and marine benthic samples via micropipetting.
• High-resolution photomicroscopy for morphological documentation.
• Single-cell nested PCR amplification of SSU rDNA using taxon-specific and universal eukaryotic primers.

Main Results:

• Successful generation of >75% or full-length SSU rDNA sequences from individual cells.
• Phylogenetic placement of identified Anisonema, Dinema, and Heteronema c.f. exaratum, revealing a misidentified 'Peranema sp.' and the first sequence data for a 'skidding heteronemid'.
• Demonstration of distinct phylogenetic positions for Heteronema c.f. exaratum (sister to primary osmotrophs) and a Ploeotia-like cell (unrelated to P. costata).

Conclusions:

• The established protocol is effective for analyzing phagotrophic euglenid biodiversity and evolution.
• This method provides a foundation for future environmental sequencing surveys and systematic studies of euglenids.