Protist-dependent termites participate in an obligate symbiosis with the community of protists in their hindgut. The termite host and protist symbionts are interdependent, with the host relying on their symbionts to digest the cellulose in their wood-based diet. Cospeciation is commonly observed between hosts and their symbiotic protists due to the strict vertical inheritance of symbionts via a process called proctodeal trophallaxis (anus-to-mouth feeding). While codiversification is an expected pattern, not all protist lineages follow this trend and must be investigated on a case-by-case basis. Protist hindgut communities were characterized from termite hosts of the major lineage Neoisoptera (excluding Termitidae, which lack symbiotic protists) in order to observe the differential pattern of cospeciation between protist lineages and host. 18S sequencing was performed on isolated protist cells from the phylum Parabasalia, specifically the genera: Pseudotrichonympha, Holomastigotoides, Cononympha, and Cthulhu and used to infer their phylogenies, evaluate their presence/absence across Neoisoptera hosts, and assess their congruency with the host tree. Pseudotrichonympha is observed to be present in almost all investigated termites and displays a strong pattern of codiversification with hosts, all having a single, unique species present (except one host with two). Holomastigotoides and Cononympha are missing in many host species and subfamilies and are generally found to have 2 species per host (sometimes 3-4 Holomastigotoides species). Cthulhu was present at the lowest frequency which may suggest it is not host specific. This investigation highlights variability of termite and protist coevolution, even when examining symbionts from the same host lineage.

The symbiotic relationship between wood-eating termites and hindgut protists is crucial for termite digestion, with protists aiding in lignocellulose degradation. This relationship, dating back to the late Jurassic, resembles the ancestral association between termites and wood roaches, Cryptocercus, established over 150 million years ago. Paraneotermes simplicicornis and Kalotermes flavicollis, members of the Kalotermitidae family, harbor diverse symbiotic communities pivotal for wood digestion and nitrogen fixation. Parabasalians, such as Cristamonadea, exhibit morphological diversity, with some taxa being joeniids, calonymphids, or devescovinids, residing primarily in termite guts. To explore the coevolutionary history and morphological evolution, this study aims to describe devescovinid communities in P. simplicicornis and K. flavicollis using morphological and molecular approaches. Phylogenetic analysis reveals the relationships among Devescovina, Metadevescovina, Macrotrichomonas, and Calonympha. A misidentification of published sequence AB458854 Joenia annectens provides valuable insights into how species are classified, while the discovery of previously unknown symbionts demonstrates the extent of diversity within these ecosystems. Notably, Clade 2 was named Prototermanova, where novel Cristamonadea species were identified, exhibiting genetic and morphological similarities to Devescovina. Similarly, Clade 4 was labelled Trichoterm, where two novel Devescovina species challenged existing taxonomic classifications. DNA sequencing analyses provided additional validation, highlighting the genetic diversity and potential novelty of symbionts within the termite gut. Morphological examination aligns with previously identified genera, and BLAST analysis supports observations of potential novelty in certain symbionts. Protists from P. simplicicornis and K. flavicollis show close relation to Joenia and Devescovina, respectively. This study sheds light on the complexity of termite symbiotic relationships and underscores the need for continued research to fully comprehend protist diversity within termite guts.