Coprophilic amoebae and flagellates, including Guttulinopsis, Rosculus and Helkesimastix, characterise a divergent and diverse rhizarian radiation and contribute to a large diversity of faecal-associated protists.

A wide diversity of organisms utilize faecal habitats as a rich nutrient source or a mechanism to traverse through animal hosts. We sequenced the 18S rRNA genes of the coprophilic, fruiting body-forming amoeba Guttulinopsis vulgaris and its non-fruiting relatives Rosculus 'ithacus' CCAP 1571/3, R. terrestris n. sp. and R. elongata n. sp. and demonstrate that they are related to the coprophilic flagellate Helkesimastix in a strongly supported, but highly divergent 18S sister clade. PCR primers specific to both clades were used to generate 18S amplicons from a range of environmental and faecal DNA samples. Phylogenetic analysis of the cloned sequences demonstrated a high diversity of uncharacterised sequence types within this clade, likely representing previously described members of the genera Guttulinopsis, Rosculus and Helkesimastix, as well as so-far unobserved organisms. Further, an Illumina MiSeq sequenced set of 18S V4-region amplicons generated from faecal DNAs using universal eukaryote primers showed that core-cercozoan assemblages in faecal samples are as diverse as those found in more conventionally examined habitats. These results reveal many novel lineages, some of which appear to occur preferentially in faecal material, in particular cercomonads and glissomonads. More broadly, we show that faecal habitats are likely untapped reservoirs of microbial eukaryotic diversity.

Phylogeny and Classification of Novel Diversity in Sainouroidea (Cercozoa, Rhizaria) Sheds Light on a Highly Diverse and Divergent Clade.

Sainouroidea is a molecularly diverse clade of cercozoan flagellates and amoebae in the eukaryotic supergroup Rhizaria. Previous 18S rDNA environmental sequencing of globally collected fecal and soil samples revealed great diversity and high sequence divergence in the Sainouroidea. However, a very limited amount of this diversity has been observed or described. The two described genera of amoebae in this clade are Guttulinopsis, which displays aggregative multicellularity, and Rosculus, which does not. Although the identity of Guttulinopsis is straightforward due to the multicellular fruiting bodies they form, the same is not true for Rosculus, and the actual identity of the original isolate is unclear. Here we isolated amoebae with morphologies like that of Guttulinopsis and Rosculus from many environments and analyzed them using 18S rDNA sequencing, light microscopy, and transmission electron microscopy. We define a molecular species concept for Sainouroidea that resulted in the description of 4 novel genera and 12 novel species of naked amoebae. Aggregative fruiting is restricted to the genus Guttulinopsis, but other than this there is little morphological variation amongst these taxa. Taken together, simple identification of these amoebae is problematic and potentially unresolvable without the 18S rDNA sequence.