Heterotrophic unicellular eukaryotes feeding on the unicellular red alga <i>Cyanidiococcus</i> sp. in moderately hot geothermal sulfuric springs

Sulfuric acidic hot springs (<pH 4.0, >37°C) are found in volcanic regions worldwide, where various bacteria, archaea, and the unicellular red algae Cyanidiophyceae dominate. Regarding heterotrophic eukaryotes, the only known species was the thermophilic amoeboflagellate Tetramitus thermacidophilus (class Eutetramitea, phylum Heterolobosea), which feeds on surrounding bacteria and archaea. In this study, we investigated three sulfuric hot springs (34.7°C-50°C, ∼pH 2.0) in Japan to determine whether other heterotrophic eukaryotes inhabit these environments. As a result, we isolated and identified cultures of four species capable of surviving at pH 2.0 and 40°C: Allovahlkampfia sp. (Eutetramitea, Heterolobosea); Nuclearia sp. and Parvularia sp. (Nucleariidea, Cristidiscoidea); and Vannella sp. (Discosea, Amoebozoa). Phylogenetic analyses suggest that these four species independently evolved from mesophilic and neutrophilic ancestors, separate from each other. Additionally, Platyophrya sp. (Colpodea, Ciliophora) and two species of Neobodo (Euglenozoa, Kinetoplastea) were also found in the same environment, while their maximum survival temperatures were 35°C and 30°C, respectively. Among these, all species except Neobodo were confirmed to grow exclusively by feeding on Cyanidiococcus sp., a dominant species of Cyanidiophyceae in the environment. Thus, various lineages of heterotrophic unicellular eukaryotes have independently developed acidophilic and thermotolerant traits, allowing them to colonize sulfuric hot springs.