Fine‐scale genetic diversity and putative ecotypes of oxymonad protists coinhabiting the hindgut of Reticulitermes speratus

Abstract

The hindgut of lower termites is generally coinhabited by multiple morphologically identifiable protist species. However, it is unclear how many protist species truly coexist in this miniaturized environment, and moreover, it is difficult to define the fundamental unit of protist diversity. Species delineation of termite gut protists has therefore been guided without a theory‐based concept of species. Here, we focused on the hindgut of the termite Reticulitermes speratus, where 10 or 11 morphologically distinct oxymonad cell types, that is, morphospecies, coexist. We elucidated the phylogenetic structure of all co‐occurring oxymonads and addressed whether their diversity can be explained by the “ecotype” hypothesis. Oxymonad‐specific 18S rRNA gene amplicon sequencing analyses of whole‐gut samples, combined with single‐cell 18S rRNA sequencing of the oxymonad morphospecies, identified 210 one‐nucleotide‐level variants. The phylogenetic analysis of these variants revealed the presence of microdiverse clusters typically within 1% sequence divergence. Each known oxymonad morphospecies comprised one to several monophyletic or paraphyletic microdiverse clusters. Using these sequence data sets, we conducted computational simulation to predict the rates of ecotype formation and periodic selection, and to demarcate putative ecotypes. Our simulations suggested that the oxymonad genetic divergence is constrained primarily by strong selection, in spite of limited population size and possible bottlenecks during intergenerational transmission. A total of 33 oxymonad ecotypes were predicted, and most of the putative ecotypes were consistently detected among different colonies and host individuals. These findings provide a possible theoretical basis for species diversity and underlying mechanisms of coexistence of termite gut protists.

Transmission dynamics of symbiotic protist communities in the termite gut: association with host adult eclosion and dispersal

“…Before the lysis step, a sterile stainless-steel bead (5 mm, Qiagen, Hilden, Germany) was placed in each tube, and the sample tubes were attached to a Vortex Adapter (24 tubes, Qiagen) and homogenized using a vortex mixer (Vortex-GENIE 2, Scientific Industries, Bohemia, NY, USA) at maximum speed for 10 min. The V3–V4 regions of 18S rRNA genes were PCR-amplified using the oxymonad-specific primers [40], with some modifications (RsOx_Sc432: 5′-GCGCAAATTACCCACTGGCA-3′; RsOx_Sc789_2Y: 5′-TTCAGCYGCGARACGCCYTG-3′), and the parabasalid-specific primers [23] (Par_18S-F: 5′-GCAGCAGGCGYGAAAC-3′; Par_18S-R: 5′-CCTACTCTCGCYCTTGATCG-3′). The PCR mixture contained 1 μL of whole-gut DNA, 1× HF buffer, 0.2 mM dNTPs, 0.5 μM primer set, and 0.4 U of Phusion high-fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA).…”

“…The rarefied reads were combined (i.e., 470,000 and 450,000 read pairs in total for Oxymonadida and Parabasalia, respectively), followed by denoising, merging of paired-end reads, identification of chimeras, and sorting into single-nucleotide-level amplicon sequence variants (ASVs) using dada2. ASVs with a relative abundance of <0.1% of the total reads in a given sample and those detected in only one sample were excluded based on previous control experiments [40]. ASVs were subsequently grouped into operational taxonomic units (OTUs) based on 97% nucleotide similarity using the DECIPHER package in R [43].…”

“…For all castes/groups, 20 μL of the resulting solution was loaded into a C-chip haemocytometer (NanoEntek, Seoul, Korea), and the protist cells were counted for each oxymonad and parabasalid species under a differential interference contrast microscope (IX81; Olympus, Tokyo, Japan). Reticulitermes speratus workers harbour up to 16 morphologically distinguishable species of protists in the hindgut [38][39][40], and the protists were categorized into the following categories for convenience, as some protist species can be difficult to discriminate using live specimens: Pyrsonympha spp. (P. grandis and P. modesta), Dinenympha exilis, D. porteri type III and IV, Dinenympha spp.1 (D. rugosa and D. porteri type I and II), Dinenympha spp.2 (D. leidyi and D. parva), Tr.…”

Protist symbionts of termites: diversity, distribution, and coevolution

“…These approaches have revealed cryptic protist species in many termites, even in such well‐studied termites as Coptotermes formosanus (Nishimura et al ., 2020; Jasso‐Selles et al ., 2020). In a ground‐breaking study that combined single‐cell 18S sequencing with whole‐gut metabarcoding data, 33 species‐level lineages of oxymonads were counted in Reticulitermes speratus , far higher than the 11 morphospecies described from the same host (Koidzumi, 1921; Igai et al ., 2022). Molecular studies occasionally reveal lower diversity than was reported by morphology (Strassert et al ., 2009), but on the whole, termite protist diversity is higher when informed by sequence data than when informed by morphology alone.…”

“…Only three oxymonad phylotypes have been characterised from H. sjostedti so far, despite the recognition of six morphotypes. It seems likely that many additional phylotypes will be uncovered by a more focused analysis, given that molecular data have recently demonstrated a very high diversity of Pyrsonympha and Dinenympha in Reticulitermes speratus (Igai et al ., 2022).…”