Lower termites are classified as termites that require a symbiotic relationship with their hindgut community of single-celled protozoa in order to gather nutrients to survive. The class Spirotrichonymphea is one of the six classes of protists that make up the Phylum Parabasalia. Within the class Spirotrichonymphea, there are 3 families and 11 genera. In this study, the Spirotrichonympha, Spironympha, and Microjoenia genera (family Spirotrichonymphidae), Holomastigotes genus (family Holomastigotidae), along with a new genus Brugerollea were targeted for molecular analysis. Protist cells were collected from Reticulitermes tibialis (Rhinotermitidae), Hodotermopsis sjostedti (Archotermopsidae), and Paraneotermes simplicornis (Kalotermidiae). Most molecular phylogenetic studies of termite-associated protists have used the 18S rRNA gene, however, there have been some ambiguities in the phylogeny of this gene. EF1-α, also known as EF1A, is a protein whose sequence can additionally be used to study the evolution of protists. EF1-α gene sequences were obtained from isolated protist cells by reverse transcription PCR (RT-PCR). Additionally, the 18S rRNA gene was amplified to confirm the isolated cells’ identity and compare the two phylogenetic methods, to see which would better resolve phylogenetic ambiguities. Sequences were compiled into an alignment for each target gene, and then a maximum likelihood tree was created for each using RAxML. Results from both trees supports the monophyly of Spirotrichonymphea and the polyphyly of genus Spirotrichonympha. However, neither gene fully resolves the phylogeny of Spirotrichonymphea.

The termite Coptotermes gestroi is a small subterranean termite originating from Southeast Asia. The hindgut of C. gestroi contains five distinct species of parabasalid: Pseudotrichonympha leei, two undescribed species from the genus Holomastigotoides, and two undescribed species from the genus Cononympha. This study investigates the protist symbionts in C. gestroi and the relationship between their relative abundance as inferred by Illumina sequence reads and the directly observed abundances for each protist genus. Illumina amplicon sequencing as a means of DNA analysis is a proven method for identification and diversity analysis, although the specific ratios of sequence reads to cell abundance in protists is not well known. In this study, protist communities were observed under light microscopy; cells were counted under hemocytometer and characterized at the molecular level using Illumina amplicon sequencing. When comparing sequence read abundances to cell abundances, some general trends were found in both analysis methods. Cononmypha repeatedly formed the majority of the community, while Holomastigotoides and Pseudotrichonympha were responsible for a smaller yet similar portion of the population. Cell counts and sequence reads were also compared using an assumed linear model, with R2 values generated to quantify the relationship between both. The results suggest that Illumina sequencing can be used to obtain rough estimates of community diversity, but the high variability within the data suggest that the read abundances should be treated with caution.