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Article

Distinct Patterns of Co-Evolution Among Protist Symbionts of Neoisoptera Termites

by
Serena G. Aguilar
1,
Jordyn Shevat
1,
Daniel E. Jasso-Selles
1,
Kali L. Swichtenberg
1,
Carlos D. Vecco-Giove
2,
Jan Šobotník
3,4,
David Sillam-Dussès
5,
Francesca De Martini
1,6 and
Gillian H. Gile
1,*
1
School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA
2
Facultad de Ciencias Agrarias, Universidad Nacional de San Martín, Tarapoto 22201, Peru
3
Faculty of Tropical AgriSciences, Czech University of Life Sciences, 165 00 Prague, Czech Republic
4
Biology Centre, Czech Academy of Sciences, Institute of Entomology, 370 05 České Budějovice, Czech Republic
5
Laboratory of Experimental and Comparative Ethology (LEEC), University Sorbonne Paris Nord, 93430 Villetaneuse, France
6
Life Science Department, Mesa Community College, Mesa, AZ 85202, USA
*
Author to whom correspondence should be addressed.
Diversity 2025, 17(8), 537; https://doi.org/10.3390/d17080537 (registering DOI)
Submission received: 3 July 2025 / Revised: 25 July 2025 / Accepted: 25 July 2025 / Published: 31 July 2025
(This article belongs to the Special Issue Diversity and Ecology of Termites)

Abstract

Obligate symbionts often exhibit some degree of co-speciation with their hosts. One prominent example is the symbiosis between termites and their wood-feeding hindgut protists. This symbiosis is mutually obligate, vertically inherited by anal feeding, and it predates the emergence of termites from their cockroach ancestors. Termites and their symbiotic protists might therefore be expected to have congruent phylogenies, but symbiont loss, transfer, and independent diversification can impact the coevolutionary history to varying degrees. Here, we have characterized the symbiotic protist communities of eight Neoisoptera species from three families in order to gauge the phylogenetic congruence between each lineage of protists and their hosts. Using microscopy and 18S rRNA gene sequencing of individually isolated protist cells, we identified protists belonging to the Parabasalia genera Pseudotrichonympha, Holomastigotoides, Cononympha, and Cthulhu. Pseudotrichonympha were present in all of the investigated termites, with a strong pattern of codiversification with hosts, consistent with previous studies. The phylogeny of Holomastigotoides indicates several instances of diversification that occurred independently of the hosts’ diversification, along with lineage-specific symbiont loss. Cononympha occurs only in Heterotermitidae and Psammotermes. Surprisingly, the small flagellate Cthulhu is widespread and exhibits cophylogeny with its hosts. This study demonstrates that different symbiont lineages can show different coevolutionary patterns, even within the same host.

1. Introduction

Protist-dependent termites currently comprise 12 families (all but the crown family Termitidae) [1]. They rely on their gut-dwelling microbial eukaryotic symbionts to efficiently digest wood [2]. Without their protists, these termites will eventually starve despite continued feeding [3]. The anaerobic protists cannot live outside their hosts, making this a mutually obligate symbiosis [4]. The establishment of this symbiosis is ancient, predating the divergence between termites and their sister lineage, Cryptocercus, a genus of wood-feeding cockroaches [5], and therefore predating the origin of termites [6,7]. Since then, the protists and their hosts have been evolving in parallel [8]. The termite–protist symbiosis is therefore of particular interest for understanding the evolutionary outcomes of prolonged co-diversification [9].
The protists that participate in this obligate symbiosis are flagellates from the clade Metamonada, specifically various lineages within the phylum Parabasalia and the order Oxymonadida of the phylum Preaxostyla [8]. Parabasalia were traditionally divided into two broad categories: large complex cells with tens to thousands of flagella, called the hypermastigotes, and smaller, simpler cells with up to six flagella, called trichomonads [4]. With the exception of the cockroach symbiont Lophomonas, all hypermastigotes are symbionts of termites or Cryptocercus [8,10]. Trichomonads inhabit the guts of termites, Cryptocercus, and many other vertebrate and invertebrate host animals [4]. Neither category is monophyletic, however, and current taxonomy recognizes 11 classes in the phylum Parabasalia [11]. Three of these classes, Trichonymphea, Spirotrichonymphea, and Cristamonadea, consist entirely of termite or Cryptocercus symbionts [8]. Oxymonadida likewise can be found in termites, Cryptocercus, and other vertebrate and invertebrate hosts, though the vast majority of species are symbionts of termites or Cryptocercus [12].
The protists are faithfully transmitted by proctodeal trophallaxis (anal feeding) in this symbiosis [13]. This behavior not only serves to inoculate newly hatched termite larvae, it also re-inoculates worker hindguts after each molt [14]. Furthermore, the exuded hindgut fluid is packed with protein-rich microbial biomass, making it an essential supplement for the termites’ extremely nitrogen-poor diet of wood [15]. Vertical inheritance is therefore primarily responsible for the general pattern of co-diversification observed between host and symbiont lineages [16,17]. It is expected, and to some extent observed, that closely related hosts harbor closely related symbionts, and that a speciation event in the host will be reflected in their symbiont lineages [18,19,20]. However, this is not always the case, as many factors can contribute to incongruencies between host and symbiont phylogenies [8,21]. Symbiont loss can occur on a host colony or species level, with lasting effects on the congruence between host and symbiont phylogenies [22]. Horizontal transfer of protists between unrelated termites also disrupts the congruence of host and symbiont phylogenies [8,23].
Neoisoptera are a major lineage of termites that are characterized by the presence of a frontal gland [7]. Within Neoisoptera there are six protist-dependent termite families: Stylotermitidae (one genus), Serritermitidae (two genera), Rhinotermitidae (five genera), Termitogetonidae (one genus), Psammotermitidae (two genera), and Heterotermitidae (three genera) [1]. In molecular phylogenies, Stylotermitidae form the deepest branch, and Heterotermitidae branch sister to the crown group Termitidae, which are the protist-independent termites [1,7,8]. Protist-dependent termites within Neoisoptera harbor symbionts exclusively from the phylum Parabasalia, except for Reticulitermes, which acquired Oxymonadida symbionts (family Pyrsonymphidae) via symbiont transfer from an ancestor of the extant genus Hodotermopsis (Teletisoptera) [24,25,26,27]. This is not the only documented occurrence of horizontal transfer within Neoisoptera; the family Serritermitidae also harbors a different protist community than that expected of Neoisoptera. The Serritermitidae fauna was likely received from an extinct species of Stolotermitidae (Teletisoptera), because Stolotermes harbor the closest relatives of the Serritermitidae protists but do not currently inhabit the same continent, South America [23,28].
Aside from these atypical symbiont communities, protist-dependent Neoisoptera have been reported to harbor the hypermastigotes Pseudotrichonympha, Holomastigotoides, Cononympha, and occasionally Cthulhu [8]. Pseudotrichonympha belongs to the class Trichonymphea, Holomastigotoides and Cononympha belong to the class Spirotrichonymphea, and Cthulhu belongs to Trichomonadea [8,11]. Pseudotrichonympha is consistently present in these termites [20,28,29,30]. Holomastigotoides, Cononympha, and Cthulhu have received less study, but seem to have a patchier distribution [8,28,31,32,33]. The vast majority of Neoisoptera species have not had their symbiont communities characterized at all, and still fewer have been investigated using molecular methods [8].
Further study of the symbiont communities in protist-dependent Neoisoptera is therefore required. This study aims to characterize the symbiont communities of additional Neoisoptera host species, including morphology and molecular phylogenetics, to clarify the distribution and cophylogenetic patterns of symbionts in this important termite clade.

2. Materials and Methods

2.1. Termite Collections

Termites were collected in Peru, Cameroon, French Guiana, Namibia, Papua New Guinea, Panama, and Cuba, between 2017 and 2022 (Table 1, Supplementary Table S1), under the following permits: Peru, AUT-IFS-2024-016; Cameroon, research permit No. 0376PRBS/MINFOF/SETAT/SG/DFAP/SDVEF/SC/BJ, export permit No. 0079/P/MINFOF/SG/DFAP/SDVEF/SC/BJ; Namibia, research permit No. RPIV00582019; Papua New Guinea, research and export permit No. 0-8191 (File 9-5-5) issued by Conservator of Fauna, Papua New Guinea, Department of Environment and Wildlife; Panama, research permit No. SC/A-24-17. Prorhinotermes canalifrons, Prorhinotermes inopinatus, and Prorhinotermes simplex from Cuba were from long-term lab breeds, collected in 2001, 2002, and 1964, respectively. One termite colony was sampled from each of the collection locations. To confirm host identities, the mitochondrial 16S rRNA gene was amplified and sequenced using primers LRN and LRJ [34,35] as previously described [33], and subjected to phylogenetic analysis (see below). Sequences were submitted to GenBank under accessions PV863057–PV863065.

2.2. Protist Observation and Sanger Seqeuencing

Several (~7–15) worker individuals were sampled from each termite colony. Hindguts were removed by pulling the anus with forceps. Extracted guts were placed in Ringer’s solution (8.5 g NaCl, 0.20 g KCl, 0.20 g CaCl2, 0.10 g NaHCO3 per liter, HiMedia Laboratories, Maharashtra, India) and gently torn using forceps to release gut protists. Live protists cells were visualized on a Zeiss AxioVert inverted compound microscope and photographed using Axiocam 105 color camera (Zeiss, Jena, Germany). Cells were manually isolated using flame-pulled Pasteur pipettes attached to a syringe by polyethylene tubing. Each isolated cell was rinsed twice with fresh Ringer’s solution, transferred to a 1.5 mL tube, and stored at −20 °C.
Genomic DNA was extracted from individual cells using the MasterPure DNA Purification Kit (Epicentre, Madison, WI, USA) following the manufacturer’s protocol except that DNA was resuspended in 5 µL of purified water (ELGA LabWater, Woodridge, IL, USA). The 18S rRNA gene (18S) was amplified using a nested PCR approach with outer primers SpiroF1/R1 and inner primers GGF/R as previously described for Parabasalia [25,33]. Purified PCR products were ligated into the pCR4-TOPO vector using the TOPO TA Cloning Kit and cloned with One Shot TOP10 chemically competent E. coli (Invitrogen, Carlsbad, CA, USA). Inserts from positive transformant colonies were amplified using the standard sequencing primers M13F/R and submitted for purification and sequencing at the Arizona State University Genomics Core Facility. Each PCR product was sequenced on both strands using standard vector primers M13F/R and internal sequencing primer ParaInF 5′-GCA GCA GGC GCG AAA CTT-3′ [25]. For cell codes, length measurements (from cell apex to posterior pole), host information, and accession numbers, see Supplementary Table S2.

2.3. Hindgut Community 18S Amplicon Sequencing

In order to verify the symbiont community composition, we attempted to amplify the 18S rRNA, 18S rRNA, and ITS or 18S rRNA, ITS, and 28S rRNA regions from whole hindgut DNA templates, as previously described [25]. We were successful for four templates, P. allocerus and P. simplex from Panama (18S rRNA only), R. marginalis (18S rRNA and ITS), and S. putorius (18S rRNA, ITS, and 28S). All PCRs used forward primer SpiroF1. The reverse primers were Spiro R1 for 18S rRNA, NC2 for 18S rRNA + ITS, and Para22R for 18S rRNA + ITS + 28S rRNA [25]. Amplicons were sequenced on the PacBio Sequel II System and demultiplexed by the Arizona Genomics Institute. Primer removal, orientation, dereplicating, denoising, and chimera detection were performed using DADA2 1.26.0 [36]. Sequences were clustered for each sample using VSEARCH 2.27.0 [37] at a 97% threshold. No additional protist phylotypes were detected beyond those already detected from single-cell PCR, with the exception of Cthulhu sequences from R. marginalis and S. putorius, which were included in phylogenetic analyses and submitted to GenBank under accessions PV863137 and PV863138.

2.4. Phylogenetic Analyses

For the host phylogeny, we included sequences for every Neoisoptera species from which protist 18S sequences have been obtained (except for Reticulitermes, for which we only included two commonly studied species). We aligned our mt16S barcode sequences with published mitochondrial genomes, including those with the closest match to our 16S sequences, ensuring that each termite genus was represented by at least one full mitogenome. When possible, we also included mitochondrial genes derived from the same study as the protist sequences (e.g., [20,33]).
For protist phylogenies, single-cell 18S clone sequences were trimmed of vector and assembled using Geneious R9 and Geneious Prime 2023.0 (www.geneious.com). Identities of new sequences were initially checked by BLASTn search against the NCBI NR database and then aligned with previously published sequences from their respective lineage using MAFFT v7.205 using the L-INS-i option [38]. Alignments ends were trimmed flush to exclude the PCR primer binding regions in AliView v1.28 [39].
For all data sets, maximum likelihood phylogenies were estimated using IQ-TREE 1.6.12 [40,41], with support for nodes assessed from 1000 ultrafast bootstrap replicates [42]. Bayesian analyses were carried out using MrBayes v. 3.2.6 [43] using the GTR model, with four evolutionary rate categories approximated by a gamma distribution as previously described [44]. Two chains were run in parallel, sampling every 100 trees until they converged. The first 25% of trees were then discarded as burn-in, and majority rule consensus trees were computed from the remaining trees from both runs for each alignment.

3. Results

3.1. Termite Identification and Phylogeny

We investigated the hindgut protist communities of eight Neoisoptera species belonging to three families (Table 1). Each termite species was identified morphologically and by BLASTn of the mitochondrial 16S rRNA gene against the NCBI NR database, followed by inclusion of the mt16S sequences in a mitochondrial genome phylogenetic analysis (Figure 1, indicated by bold text). All termite species we investigated branched where expected, with full support, confirming their genus-level and in some cases species-level classification. For Psammotermes allocerus, the mt16S did not closely match the previously published sequence, but our termites were collected near the type locality for this species, supporting our species-level identification [45].
In our phylogenetic tree, all termite families and genera were recovered as monophyletic with full support (Figure 1). We also recover a sister relationship between Psammotermitidae and Heterotermitidae. This is consistent with other studies because our analysis did not include the protist-independent Termitidae; otherwise, Termitidae and Heterotermitidae would be sisters [1,6,17,46]. Aside from this, the interfamilial relationships are not well resolved in our analysis, also consistent with previous studies in which the relationships among Termitogetonidae, Serritermitidae, and Rhinotermitidae are somewhat unstable [1,6,17,46]. The intergeneric relationships within Rhinotermitidae in our phylogeny are also consistent with previous work [47].
The symbiont community composition was relatively consistent within host genera, and to some degree, families. In this study, we investigated one species from Heterotermitidae, Heterotermes cardini from Panama, and observed Pseudotrichonympha, Holomastigotoides, and Cononympha, which are typical of Heterotermes and Coptotermes symbiont communities [20,30,32,33,48,49,50,51]. We did not observe Cthulhu, which has only been reported from Heterotermes tenuis in this family so far [31,50]. In Psammotermitidae, we investigated four species, three Prorhinotermes and one Psammotermes. All three Prorhinotermes species harbored Pseudotrichonympha, Holomastigotoides, and Cthulhu, but lacked Cononympha. Psammotermes likewise harbored Pseudotrichonympha and Holomastigotoides, but conversely harbored Cononympha and lacked Cthulhu. Termites from all three Rhinotermitidae genera harbored Pseudotrichonympha and Cthulhu but lacked Holomastigotoides and Cononympha. We never observed Oxymonadida or any other Parabasalia genera in these termites.

3.2. Protist Identification and Phylogeny

3.2.1. Pseudotrichonympha

Pseudotrichonympha were present in all eight of the termite species we investigated. Pseudotrichonympha cells have an anterior rostrum which contains an axial tube-like structure that originates near the cell apex and rapidly widens posteriorly, proceeding outward toward the cell surface [52] (Figure 2). Except for the smooth apical cap, the rostrum is covered with flagella, with a fringe of longer flagella at the posterior boundary of the rostrum. Flagella also cover the cell body posterior to the rostrum (Figure 2). Cells are typically large and spindle-shaped, but this can vary considerably. In the species we investigated, cell length ranged from 88 µm, in a cell from Dolichorhinotermes longilabius, to 316 µm in a cell from Prorhinotermes canalifrons. Most cells measured between 120 and 220 µm in length (Table S2). The proportion of the cell body that forms the rostrum is also variable, from just the anterior 1/10 or so (Figure 2K) to more than 1/5 the cell length (Figure 2A,I). We frequently observed cells in which the rostrum was inverted, forming a cup at the cell apex (Figure 2B–D), though there were always at least a few normally extended cells in the same host (Figure 2B,G). The nucleus was occasionally seen within the rim of the cup formed by this inversion (Figure 2C).
We successfully amplified and sequenced 18S rRNA gene sequences from 29 individually isolated Pseudotrichonympha cells (Table S2). The Pseudotrichonympha 18S sequences obtained from each host species shared high sequence similarity and formed a single, fully supported clade. Although there is no universally applicable sequence identity threshold for protists, the Pseudotrichonympha 18S sequences from each host shared >98% sequence identity, suggesting that only a single species of Pseudotrichonympha inhabits each of the hosts investigated here. Our community 18S amplicon data from Rhinotermes marginalis, Schedorhinotermes putorius, Psammotermes allocerus, and Prorhinotermes simplex (Panama) likewise indicated only a single Pseudotrichonympha species in each host. This pattern of one Pseudotrichonympha species per host is consistent across all other hosts characterized to date [20,29,33] except for Heterotermes tenuis, which harbors two species of Pseudotrichonympha [50,52]. The H. tenuis Pseudotrichonympha species are distinguished on the basis of 18S sequence comparisons (>98% within-species sequence identity, 95–96% between-species identity), and together they form a clade [50,52] (Figure 3). Although it is very likely that each host species we investigated harbors a single Pseudotrichonympha species, it remains possible that we missed an additional Pseudotrichonympha species in one or more of the host species, especially Prorhinotermes inopinatus, for which we only sequenced the 18S from one cell and did not obtain amplicon data.
Our 18S phylogeny of Pseudotrichonympha is broadly congruent with the host phylogeny. For example, the Stylotermes symbiont forms the deepest branch and the Rhinotermitidae symbionts form a clade, with Rhinotermes and Dolichorhinotermes symbionts exhibiting the same sister relationship as their hosts. The Pseudotrichonympha sequences derived from each host genus all form monophyletic groups, with full to moderate support. The Prorhinotermes symbionts show some phylogenetic differentiation according to host species, but they are all so closely related that they could reasonably be considered a single species of Pseudotrichonympha (Figure 3). However, most of the deeper relationships in the Pseudotrichonympha phylogeny are not resolved. Prorhinotermes and Psammotermes symbionts fail to form a clade, as do Coptotermes and Heterotermes symbionts. In sum, the Pseudotrichonympha phylogeny includes parts that align with the host phylogeny and parts that do not, but the parts that do not align have low statistical support.

3.2.2. Holomastigotoides

Holomastigotoides were absent from Rhinotermitidae, but present in all other hosts examined here. Holomastigotoides cells have helical cytoskeletal bands from which the many flagella emerge. These bands surround the cell in a right-handed helix from the cell apex toward the posterior, leaving a naked posterior pole (Figure 4). Flagella emerging from the posterior coils of the helix can be considerably longer than those emerging anteriorly. The nucleus is typically quite large and located near the cell apex (Figure 4). We observed that symbionts from Prorhinotermes had a narrowly pointed cell apex (Figure 4D–G), while symbionts from Psammotermes had a blunt cell apex (Figure 4B,C).
We successfully amplified and sequenced the 18S gene from 35 individually isolated Holomastigotoides cells (Table S2). All symbionts isolated from the same host genus branched together with full support, and the Coptotermes and Heterotermes symbiont clades showed a fully supported sister relationship, consistent with the host phylogeny (Figure 5). Unlike Pseudotrichonympha, we detected multiple Holomastigotoides species per host species. In Prorhinotermes simplex and Prorhinotermes canalifrons, there were two distinct Holomastigotoides size morphs. The sp. 1 clade cells were consistently larger, measuring 97–115 µm in length in P. simplex and 83–151 µm in P. canalifrons, while the sp. 2 clade cells were only 33–71 µm and 41–77 µm, respectively. This is consistent with previous observations of two morphotypes in P. simplex Holomastigotoides, though at the time they were interpreted as a single species [32]. Intriguingly, Prorhinotermes inopinatus appears to show the opposite pattern, in which the sp. 1 cell was only 63 µm long and the sp. 2 cell was 186 µm. In Psammotermes allocerus and Heterotermes cardini, the cell sizes of the two clades are similar (Table S2).
The Prorhinotermes Holomastigotoides form two main clades, though with weak support. Within each of these clades, we find symbionts from all three Prorhinotermes species, with a branching order that mirrors the host phylogeny, i.e., the symbionts of each host species form distinct clades, with P. canalifrons and P. inopinatus symbiont clades as sisters. In one of the clades, there is an additional P. simplex symbiont from Cuba that formed the deepest branch (Figure 5, PSIM6-5), indicating a third species of Holomastigotoides in this population of P. simplex. We also noted some genetic differentiation between Holomastigotoides symbionts of P. simplex from Panama and Cuba in both clades, with strong support in the case of Holomastigotoides sp. 2. This is consistent with the slight divergence we see between the Panama and Cuba populations in the mt16S (Figure 1). Note that the previously published Holomastigotoides sequences Pro1 and Pro2 came from P. simplex collected in Florida, USA. H. cardini and P. allocerus likewise harbored two species each of Holomastigotoides. In Heterotermes, each Holomastigotoides species branched sister to symbionts from Heterotermes aureus, again suggestive of host/symbiont co-diversification in both lineages, but the Psammotermes symbionts were sister to each other. Our community 18S amplicon data from P. allocerus and P. simplex (Panama) had the same two Holomastigotoides phylotypes that we characterized from single cells, while the R. marginalis and S. putorius data had no Holomastigotoides at all.

3.2.3. Cononympha

We only observed Cononympha in Heterotermes and Psammotermes in this study, and we successfully sequenced the 18S from four individually isolated cells, two from each host. This is the first study to demonstrate that Cononympha can inhabit hosts other than Coptotermes and Heterotermes. Cononympha, like Holomastigotoides, have helical flagellar bands that originate at the apex and continue in a right-handed helix toward the posterior, leaving a naked posterior pole (Figure 6B–F). They are distinguished from Holomastigotoides by the presence of an apical columella, which is an axial tube-like structure formed by the tight winding of the flagellar bands at the cell apex. This can be very difficult to see in small, unstained cells, and also difficult to distinguish from the sharply pointed anterior of certain Holomastigotoides cells, notably those from Prorhinotermes hosts. We detected one species of Cononympha each in H. cardini and P. allocerus, though there might be additional species that we failed to sample. Our 18S amplicon data for P. allocerus included the same single phylotype of Cononympha that we obtained from isolated cells but no other Cononympha from any host (we did not obtain amplicon data from H. cardini). The Cononympha cells we observed were considerably smaller than Holomastigotoides, as is typical for Cononympha, though note that Cononympha skunkapei from Coptotermes gestroi is larger than many Holomastigotoides cells [49]. The Psammotermes symbionts were larger than the Heterotermes symbionts (Figure 6, Table S2).
In our 18S phylogeny of Cononympha (Figure 6A), symbionts from each host genus form distinct clades, though without strong support. Cononympha 18S sequences are quite divergent and form long branches in phylogenetic analyses (note the scale bar for substitutions per site is roughly twice the length in the Cononympha tree relative to Holomastigotoides). Because Cononympha is present in so few host genera, there is little in the symbiont tree topology to compare to the host topology. This is compounded by the lack of resolution in the deeper nodes of the tree. Still, Coptotermes and Heterotermes symbionts tend to branch together to the exclusion of Psammotermes, albeit without support.

3.2.4. Cthulhu

We observed Cthulhu cells in all host termites except H. cardini and P. allocerus, the exact opposite distribution of Cononympha in our samples (Table 1). This may or may not be significant; note that H. tenuis harbors both Cononympha and Cthulhu [50]. We obtained sequences from isolated Cthulhu cells from Prorhinotermes species, and we obtained long-read amplicon sequences from R. marginalis and S. putorius (Table 1, Figure 7, Video S1) as well as P. simplex from Panama. All Cthulhu sequences characterized to date come from Neoisoptera, and they form a fully supported clade. Surprisingly, symbionts from all families except Termitogetonidae are represented in the Cthulhu phylogeny, including Serritermitidae, which have lost their “typical” neoisopteran fauna and gained Retractinympha and Heliconympha from an unknown donor instead [23,28]. The phylogeny of Cthulhu is congruent with the host phylogeny, with Stylotermes symbionts forming the deepest branch, followed by Serritermitidae, then Rhinotermitidae, then Psammotermitidae, with losses of Cthulhu inferred from Termitogeton, Psammotermes, and Heterotermitidae. The sole exception is the Heterotermes tenuis symbiont, which branches sister to the Stylotermes symbionts, and might therefore have been acquired by horizontal transfer, again consistent with the inference that Cthulhu was lost in Heterotermitidae.

4. Discussion

4.1. Symbiont Community Composition Across Neoisoptera

Our results, together with previous reports, reveal broad trends in symbiont community composition across protist-dependent Neoisoptera lineages. The deepest branches, Stylotermes, Termitogeton, and Rhinotermitidae, harbor Pseudotrichonympha, and most also harbor Cthulhu. Psammotermitidae and Heterotermitidae harbor Pseudotrichonympha, Holomastigotoides, and Cononympha, though the Prorhinotermes we investigated here lacked Cononympha. Reticulitermes and Serritermitidae are well documented to harbor distinct faunae that were most likely obtained by horizontal symbiont transfer [23,24,25,28,30]; they were not investigated further here (Figure 1). Our results are broadly consistent with previous studies, while providing new insight into the host–symbiont coevolutionary history of Neoisoptera.
The protist symbionts of non-Reticulitermes Heterotermitidae (Coptotermes and Heterotermes) consistently belong to the genera Pseudotrichonympha, Holomastigotoides, and Cononympha. Morphology-linked molecular data have demonstrated their presence in H. aureus, H. tenuis, C. formosanus, and C. gestroi [29,33,49,50,53]. By light microscopy, these same three protist genera have been reported from C. amanii, C. heimi, C. lacteus, C. sjostedti, H. indicola, H. longiceps, H. malabaricus, and H. tenuior [8,30,54,55,56,57,58]. Note that the Cononympha symbionts of these host species are referred to as Spirotrichonympha in the literature. This is because the genus Cononympha was synonymized with Spirotrichonympha in 1921, soon after its description in 1917, and not reinstated until 2017 [33,48,59]. Without direct molecular characterization of these “Spirotrichonympha” species, it remains possible that they do not belong to the Cononympha lineage, but this seems unlikely given the consistency of community composition in other Coptotermes and Heterotermes. Small flagellates like Cthulhu are not typically present. Cthulhu has only been reported from H. tenuis, where it was detected in a minority of the colonies sampled [50]. Similarly, a small trichomonad, probably not Cthulhu, was present in a minority of C. formosanus colonies in Japan [60].
The symbionts of Psammotermitidae are less well studied, but they are reported to include Pseudotrichonympha, Holomastigotoides, and, less consistently, Cononympha and/or Cthulhu. From Psammotermes, one previous study obtained molecular data from a single species of Pseudotrichonympha, in P. allocerus, with no mention of other symbionts [20]. Psammotermes hybostoma was reported to harbor Pseudotrichonympha, Holomastigotoides, and Spirotrichonymphella [61,62]. The morphology of Spirotrichonymphella psammotermitidis is consistent with that of a large Cononympha, but molecular data will be needed to demonstrate its affinities. Trichonympha scortecci was also described from this host, but it was observed in ethanol-preserved termites in which most morphological details were not discernible [63]. It is therefore very likely a misidentification of Pseudotrichonympha. This study is therefore the first to confirm the presence of Holomastigotoides and Cononympha in Psammotermes using molecular data and the first to identify Cononympha outside Heterotermitidae. In Prorhinotermes, Pseudotrichonympha, Holomastigotoides, and Cthulhu have been reported previously from Prorhinotermes simplex using molecular methods; in fact, Cthulhu was first described from P. simplex [29,31,32]. In this study, we confirmed these symbionts in additional P. simplex colonies and observed very similar and closely related symbiont communities in P. canalifrons and P. inopinatus. However, Prorhinotermes flavus and Prorhinotermes japonicus have both been reported to harbor Spirotrichonympha in addition to Pseudotrichonympha and Holomastigotoides, leaving open the possibility that Cononympha might be present in these species [30,64]. No descriptive details are provided to support these identifications, however, and molecular study will be needed to definitively identify these symbionts.
In Rhinotermitidae, Pseudotrichonympha is consistently present, Holomastigotoides and Cononympha are consistently absent, and Cthulhu is typically present. This study is the first to investigate symbionts of Dolichorhinotermes. In Rhinotermes, Pseudotrichonympha has been reported by both morphological and molecular methods [20,65], but this is the first study to report the absence of the spirotrichonymphids and the presence of Cthulhu. We did not investigate any Parrhinotermes symbionts in this study, but they are known to harbor Pseudotrichonympha, and two species were specifically reported to lack any other symbionts [20,30]. In the case of Schedorhinotermes, our observations differ from previous reports. While we only observed Pseudotrichonympha and Cthulhu in Schedorhinotermes putorius from Cameroon, other Schedorhinotermes species are reported to harbor Spirotrichonympha, Spironympha, and Microjoenia [30,55,66], which are otherwise only known from Reticulitermes and Hodotermopsis [8]. Their hosts, Schedorhinotermes intermedius, Schedorhinotermes malaccensis (=S. sawarawkensis), and two unidentified Schedorhinotermes species, are all from Australia or Southeast Asia. These form a clade in phylogenetic analyses to the exclusion of the African species [47]. Perhaps these anomalous spirotrichonymphid symbionts were acquired by horizontal transfer after the divergence of the African Schedorhinotermes clade (Osamu Kitade, pers. comm.)

4.2. Distinct Evolutionary Trajectories Among Neoisoptera Symbionts

The termite phylogeny is the major driver of symbiotic protists’ phylogenies [67], but these phylogenies are not necessarily congruent, due to frequent, lineage-specific symbiont losses, symbiont transfer among distinct host lineages, and independent diversification of symbionts relative to their hosts [8]. Understanding the relative importance of these factors is a central goal of termite symbiont research and host–symbiont coevolution more broadly.
Here, we have seen that the four symbiont phylogenies differ in their congruence with the Neoisoptera phylogeny. The phylogeny of Pseudotrichonympha was perfectly congruent with the host phylogeny in a previous study [20]. In this study, with an expanded taxon selection, some key relationships are highly congruent, such as Stylotermes and their Pseudotrichonympha symbionts forming their respective deepest branches, and Rhinotermitidae and their symbionts having mirror topologies (Figure 1 and Figure 3). Other aspects of host and symbiont phylogenies differ, but these are in weakly supported or unresolved areas of the tree; hence the topologies are actually congruent (Figure 1 and Figure 3). The consistent presence of Pseudotrichonympha suggests that they might provide an essential role in wood digestion that is not easily replaced by other protists. This is consistent with previous work demonstrating that Pseudotrichonympha grassii can degrade higher molecular weight cellulose than the other symbionts in Coptotermes formosanus [68]. Pseudotrichonympha are also known to harbor nitrogen-fixing endosymbionts, with which they have codiversified [20,69]. However, it is unknown whether these factors are sufficient to explain the constant presence and cophylogenetic pattern of Pseudotrichonympha with respect to their hosts.
The phylogeny of Holomastigotoides, by contrast, is more complex, with 2–3 species per host, and its congruence with the host tree is more difficult to evaluate. There is evidence of codiversification, but also plenty of independent diversification and lineage-specific loss. For example, Prorhinotermes symbionts all share a recent common ancestor despite the fact that each host species has two or three Holomastigotoides species. Our phylogeny therefore indicates that the Holomastigotoides lineage split within the stem lineage of Prorhinotermes, after which both Holomastigotoides lineages speciated in parallel with their hosts, with an additional independent split leading to Holomastigotoides sp. 3 in P. simplex that was presumably lost from the other hosts (Figure 5). Within Coptotermes and within Psammotermes, all symbionts likewise form a clade despite each host harboring two species, indicating yet more independent diversifications of Holomastigotoides relative to their hosts. The Heterotermes symbionts fail to form a single clade, indicating a complex coevolutionary history with an unknown number of independent diversifications and lineage-specific losses or perhaps even intrageneric symbiont transfers.
In the Cononympha phylogeny, the symbionts from each host genus form distinct clades, indicating some level of codiversification, but within the Heterotermes and Coptotermes clades, host and symbiont phylogenies are not congruent. There is some evidence of independent diversification, e.g., the two C. formosanus symbionts are sisters, balanced by lineage-specific loss (Figure 6A). Our data also indicate a complete loss of Cononympha from the Prorhinotermes species we sampled.
The Cthulhu phylogeny was surprisingly congruent with that of Neoisoptera (Figure 1 and Figure 7A). Because small flagellates like Cthulhu are closely related to, and resemble, non-termite symbionts, they might have joined the symbiosis more recently than the ancestrally termite-associated lineages. Furthermore, some small flagellates are only sporadically present among colonies within a termite species, suggesting that they are not obligate for the host and might be environmentally acquired [22,50,60]. In contrast with this view, our data suggest that Cthulhu is ancestrally present in Neoisoptera (though lost in Heterotermitidae, Termitogeton and Parrhinotermes, and regained by symbiont transfer in H. tenuis). The Cthulhu phylogeny is quite congruent with the host phylogeny (except for the H. tenuis symbiont), with Stylotermes symbionts forming the deepest branch, and symbionts of Serritermitidae, Rhinotermitidae, and Psammotermitidae each forming a clade. However, Cthulhu from Serritermitidae and Rhinotermitidae do not form a clade, as would be expected from host phylogenomic analyses [1,17] and, weakly, by our host tree (Figure 1), but their separation is only weakly supported (Figure 7). The presence of Cthulhu in Serritermitidae is particularly surprising, as they are the only symbiont genus that is shared by other Neoisoptera. Given the broad congruence of the Cthulhu and Neoisoptera trees, it seems likely that Cthulhu was retained through the symbiont losses and acquisitions that led to the distinct fauna of Serritermitidae. Cthulhu can therefore be seen as a true, obligate, and ancient termite symbiont, having become established in the ancestor of Neoisoptera, ~85 million years ago [6,17]. Consistent with this, Cthulhu is a hypermastigote, having multiplied its flagella in a way that is exclusive to symbionts of termites and cockroaches [4,31].
In sum, it would seem that Pseudotrichonympha and Cthulhu were ancestrally present in Neoisoptera, and, aside from a few losses, have largely co-diversified with their hosts. Holomastigotoides and Cononympha, however, were either lost independently from the deepest branches of Neoisoptera or gained in the ancestor of Psammotermitidae and Geoisoptera (Termitidae + Heterotermitidae). It is not clear where they would have come from, given that Holomastigotoides and Cononympha have not been detected outside of Neoisoptera and they do not have close relatives in the Spirotrichonymphea phylogeny [51]. Further study of termite symbionts is needed to distinguish between these scenarios.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d17080537/s1: Table S1: Termite collections; Table S2: Isolated cells metadata. Video S1: Cthulhu sp. from Schedorhinotermes putorius, DIC.

Author Contributions

Conceptualization, G.H.G., S.G.A., and K.L.S.; methodology, S.G.A., J.S., D.E.J.-S., and K.L.S.; formal analysis, S.G.A. and J.S.; resources, C.D.V.-G., J.Š., and D.S.-D.; writing—original draft preparation, S.G.A., J.S., and G.H.G.; writing—review and editing, S.G.A., J.S., D.E.J.-S., K.L.S., C.D.V.-G., J.Š., D.S.-D., F.D.M., and G.H.G.; supervision, K.L.S., F.D.M., and G.H.G.; funding acquisition, G.H.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the US National Science Foundation, grant number DEB-2045329, and also by Project Peru/Ecos Nord (n° P20A02). JŠ was supported by the project IGA No. 20253134 from the Faculty of Tropical AgriSciences of the Czech University of Life Sciences, Prague.

Data Availability Statement

The original sequence data presented in the study are openly available in GenBank under accession numbers PV863057-PV863138.

Acknowledgments

The authors would like to thank Bradley Bobbett, Katalina Freeman, Mikaela Garcia, Samantha Montoya, Keana Nguyen, LeAnn Nguyen, Tina Piarowski, Xyonane Segovia, and Stephen Taerum for technical support. We thank Thomas Bourguignon and Rudi Scheffrahn for sharing the mitochondrial genome sequences of Heterotermes aureus, Heterotermes longiceps, and Rhinotermes marginalis in advance of publication.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Distribution of protist symbionts across Neoisoptera. Left, mitochondrial genome phylogeny of select Neoisoptera including 16S rRNA gene sequences of termites investigated in this study (bold text). Support values at nodes are % ultrafast bootstrap replicates/Bayesian posterior probabilities, with full support (100/1.0) indicated by filled circles, strong support (>95/>0.99) indicated by open circles, and values below 70/0.9 are not shown. Branches with dashed lines have been reduced in length by half. Gray gradient boxes indicate the two lineages whose symbionts were acquired by horizontal transfer, Reticulitermes and Serritermitidae. Right, presence/absence of protist genera, P = Pseudotrichonympha, H = Holomastigotoides, Con = Cononympha, Cth = Cthulhu. Colored boxes indicate presence of the protist genus in the corresponding host species; numbers indicate the number of species of that genus determined with molecular methods, if greater than 1. Empty spaces indicate known absence of the symbiont in the corresponding host species, and grey boxes indicate lack of information.
Figure 1. Distribution of protist symbionts across Neoisoptera. Left, mitochondrial genome phylogeny of select Neoisoptera including 16S rRNA gene sequences of termites investigated in this study (bold text). Support values at nodes are % ultrafast bootstrap replicates/Bayesian posterior probabilities, with full support (100/1.0) indicated by filled circles, strong support (>95/>0.99) indicated by open circles, and values below 70/0.9 are not shown. Branches with dashed lines have been reduced in length by half. Gray gradient boxes indicate the two lineages whose symbionts were acquired by horizontal transfer, Reticulitermes and Serritermitidae. Right, presence/absence of protist genera, P = Pseudotrichonympha, H = Holomastigotoides, Con = Cononympha, Cth = Cthulhu. Colored boxes indicate presence of the protist genus in the corresponding host species; numbers indicate the number of species of that genus determined with molecular methods, if greater than 1. Empty spaces indicate known absence of the symbiont in the corresponding host species, and grey boxes indicate lack of information.
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Figure 2. Morphological diversity of Pseudotrichonympha symbionts of Neoisoptera, differential interference contrast light micrographs. (A) Symbiont of Psammotermes allocerus. The flagella-free apical cap is relatively wide and blunt and is subtended by the axial tube-like structure. Flagella cover the cell surface, with short flagella on the anterior rostrum and longer flagella on the posterior rim of the rostrum and covering the cell body. (B) Symbiont of Schedorhinotermes putorius with deeply inverted rostrum forming a cup-like structure at the cell apex. The large nucleus is evident in the cytoplasm, along with wood fragments and sub-spherical inclusions. (C,D) Symbiont of Rhinotermes marginalis, two optical sections of the same cell with invaginated rostrum, showing the nucleus position within the wall of the cup (C) and a top view of the apical cap within the cup (D). (E) Symbiont of Prorhinotermes inopinatus with a rounded posterior cell pole, abundant ingested wood particles, and an anterior granular cytoplasm, likely indicative of bacterial endosymbionts. (F) Symbiont of R. marginalis in which the rostrum is not quite inverted; the nucleus is positioned anterolaterally. (G) Symbiont of Schedorhinotermes putorius with normally everted rostrum. The apical cap is relatively narrow and pointed. (HJ) Cells isolated for molecular characterization in this study, DG7 from Dolichorhinotermes longilabius (H), Ram11 from Heterotermes cardini (I), and Prom3 Pseudotrichonympha sp. from Prorhinotermes simplex from Panama (J). (K) Symbiont of Prorhinotermes canalifrons with a relatively short rostrum. All scale bars = 50 µm.
Figure 2. Morphological diversity of Pseudotrichonympha symbionts of Neoisoptera, differential interference contrast light micrographs. (A) Symbiont of Psammotermes allocerus. The flagella-free apical cap is relatively wide and blunt and is subtended by the axial tube-like structure. Flagella cover the cell surface, with short flagella on the anterior rostrum and longer flagella on the posterior rim of the rostrum and covering the cell body. (B) Symbiont of Schedorhinotermes putorius with deeply inverted rostrum forming a cup-like structure at the cell apex. The large nucleus is evident in the cytoplasm, along with wood fragments and sub-spherical inclusions. (C,D) Symbiont of Rhinotermes marginalis, two optical sections of the same cell with invaginated rostrum, showing the nucleus position within the wall of the cup (C) and a top view of the apical cap within the cup (D). (E) Symbiont of Prorhinotermes inopinatus with a rounded posterior cell pole, abundant ingested wood particles, and an anterior granular cytoplasm, likely indicative of bacterial endosymbionts. (F) Symbiont of R. marginalis in which the rostrum is not quite inverted; the nucleus is positioned anterolaterally. (G) Symbiont of Schedorhinotermes putorius with normally everted rostrum. The apical cap is relatively narrow and pointed. (HJ) Cells isolated for molecular characterization in this study, DG7 from Dolichorhinotermes longilabius (H), Ram11 from Heterotermes cardini (I), and Prom3 Pseudotrichonympha sp. from Prorhinotermes simplex from Panama (J). (K) Symbiont of Prorhinotermes canalifrons with a relatively short rostrum. All scale bars = 50 µm.
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Figure 3. Maximum likelihood phylogeny of 18S rRNA gene sequences from Pseudotrichonympha, rooted with outgroup Teranympha and Eucomonympha. New sequences obtained in this study are indicated by shaded boxes with host names at right. Support values at nodes are % ultrafast bootstrap replicates/Bayesian posterior probabilities, with full support (100/1.0) indicated by filled circles, strong support (>95/>0.99) indicated by open circles, and values below 70/0.9 are not shown.
Figure 3. Maximum likelihood phylogeny of 18S rRNA gene sequences from Pseudotrichonympha, rooted with outgroup Teranympha and Eucomonympha. New sequences obtained in this study are indicated by shaded boxes with host names at right. Support values at nodes are % ultrafast bootstrap replicates/Bayesian posterior probabilities, with full support (100/1.0) indicated by filled circles, strong support (>95/>0.99) indicated by open circles, and values below 70/0.9 are not shown.
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Figure 4. Morphological diversity of Holomastigotoides symbionts of Neoisoptera, differential interference contrast light micrographs. (A) A Holomastigotoides cell nestles among the flagella of a Pseudotrichonympha cell in Prorhinotermes inopinatus. The angular refractive contents of the Holomastigotoides cell are ingested wood particles, and the cell apex is sharply pointed, a common characteristic of Holomastigotoides from Prorhinotermes. (B,C) Holomastigotoides cells from Psammotermes allocerus showing the characteristic blunt apex, large anterior nucleus, and long posterior flagella. (D,E) Large and small Holomastigotoides cell types from P. inopinatus. Both morphs exhibit a pointed apex. (F) Small morph Holomastigotoides from Prorhinotermes canalifrons. (G) Small morph Holomastigotoides from Prorhinotermes simplex collected in Panama exhibiting an off-center nucleus. All scale bars = 50 µm.
Figure 4. Morphological diversity of Holomastigotoides symbionts of Neoisoptera, differential interference contrast light micrographs. (A) A Holomastigotoides cell nestles among the flagella of a Pseudotrichonympha cell in Prorhinotermes inopinatus. The angular refractive contents of the Holomastigotoides cell are ingested wood particles, and the cell apex is sharply pointed, a common characteristic of Holomastigotoides from Prorhinotermes. (B,C) Holomastigotoides cells from Psammotermes allocerus showing the characteristic blunt apex, large anterior nucleus, and long posterior flagella. (D,E) Large and small Holomastigotoides cell types from P. inopinatus. Both morphs exhibit a pointed apex. (F) Small morph Holomastigotoides from Prorhinotermes canalifrons. (G) Small morph Holomastigotoides from Prorhinotermes simplex collected in Panama exhibiting an off-center nucleus. All scale bars = 50 µm.
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Figure 5. Maximum likelihood phylogeny of 18S rRNA gene sequences from Holomastigotoides, rooted with outgroup Cononympha. New sequences obtained in this study are indicated by shaded boxes with host names at right. Support values at nodes are % ultrafast bootstrap replicates/Bayesian posterior probabilities, with full support (100/1.0) indicated by filled circles, strong support (>95/>0.99) indicated by open circles, and values below 70/0.9 are not shown. Branches with dashed lines have been reduced in length by half.
Figure 5. Maximum likelihood phylogeny of 18S rRNA gene sequences from Holomastigotoides, rooted with outgroup Cononympha. New sequences obtained in this study are indicated by shaded boxes with host names at right. Support values at nodes are % ultrafast bootstrap replicates/Bayesian posterior probabilities, with full support (100/1.0) indicated by filled circles, strong support (>95/>0.99) indicated by open circles, and values below 70/0.9 are not shown. Branches with dashed lines have been reduced in length by half.
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Figure 6. Phylogeny and morphology of Cononympha symbionts of Neoisoptera. (A) Molecular phylogeny of Cononympha from nearly full-length 18S rRNA gene sequences, rooted with outgroup Holomastigotoides. New sequences obtained in this study are indicated by colored boxes with host names at right. Support values at nodes are % ultrafast bootstrap replicates/Bayesian posterior probabilities, with full support (100/1.0) indicated by filled circles, strong support (>95/>0.99) indicated by open circles, and values below 70/0.9 not shown. (BF) Differential interference contrast light micrographs of Cononympha cells. (BC) Cononympha from Psammotermes allocerus, scale bars 20 µm. (DF) Cononympha from Heterotermes cardini, scale bars 10 µm. (DE) Two cells collected together as Ram29. (F) Cell Ram26.
Figure 6. Phylogeny and morphology of Cononympha symbionts of Neoisoptera. (A) Molecular phylogeny of Cononympha from nearly full-length 18S rRNA gene sequences, rooted with outgroup Holomastigotoides. New sequences obtained in this study are indicated by colored boxes with host names at right. Support values at nodes are % ultrafast bootstrap replicates/Bayesian posterior probabilities, with full support (100/1.0) indicated by filled circles, strong support (>95/>0.99) indicated by open circles, and values below 70/0.9 not shown. (BF) Differential interference contrast light micrographs of Cononympha cells. (BC) Cononympha from Psammotermes allocerus, scale bars 20 µm. (DF) Cononympha from Heterotermes cardini, scale bars 10 µm. (DE) Two cells collected together as Ram29. (F) Cell Ram26.
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Figure 7. Morphology and phylogeny of Cthulhu, symbionts of Neoisoptera. (A) Molecular phylogeny of Cthulhu from nearly full-length 18S rRNA gene sequences, rooted with outgroup Cthylla and relatives. New sequences obtained in this study are indicated by bold text. Cthulhu sequences from Prorhinotermes spp. came from individually isolated cells, while Cthulhu sequences from Rhinotermitidae (Rhinotermes and Schedorhinotermes) were amplified from whole-gut DNA. Support values at nodes are % ultrafast bootstrap replicates/Bayesian posterior probabilities, with full support (100/1.0) indicated by filled circles, strong support (>95/>0.99) indicated by open circles, and values below 70/0.9 are not shown. (BE) Differential interference contrast light micrographs of Cthulhu cells, scale bars 10 µm. (B) Cthulhu from Prorhinotermes simplex collected in Panama. (C) Cthulhu from Prorhinotermes inopinatus. (D) Cthulhu from Prorhinotermes canalifrons. (E) Cthulhu-like cell from Dolichorhinotermes longilabius; no Cthulhu sequences were obtained from this host.
Figure 7. Morphology and phylogeny of Cthulhu, symbionts of Neoisoptera. (A) Molecular phylogeny of Cthulhu from nearly full-length 18S rRNA gene sequences, rooted with outgroup Cthylla and relatives. New sequences obtained in this study are indicated by bold text. Cthulhu sequences from Prorhinotermes spp. came from individually isolated cells, while Cthulhu sequences from Rhinotermitidae (Rhinotermes and Schedorhinotermes) were amplified from whole-gut DNA. Support values at nodes are % ultrafast bootstrap replicates/Bayesian posterior probabilities, with full support (100/1.0) indicated by filled circles, strong support (>95/>0.99) indicated by open circles, and values below 70/0.9 are not shown. (BE) Differential interference contrast light micrographs of Cthulhu cells, scale bars 10 µm. (B) Cthulhu from Prorhinotermes simplex collected in Panama. (C) Cthulhu from Prorhinotermes inopinatus. (D) Cthulhu from Prorhinotermes canalifrons. (E) Cthulhu-like cell from Dolichorhinotermes longilabius; no Cthulhu sequences were obtained from this host.
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Table 1. Termites used in this study and their protist symbiont genera, P = Pseudotrichonympha, H = Holomastigotoides, Con = Cononympha, Cth = Cthulhu. X = present.
Table 1. Termites used in this study and their protist symbiont genera, P = Pseudotrichonympha, H = Holomastigotoides, Con = Cononympha, Cth = Cthulhu. X = present.
Termite FamilyTermite SpeciesCollection LocationPHConCth
RhinotermitidaeRhinotermes marginalisPuerto Maldonado, PeruX X *
Schedorhinotermes putoriusEbogo, Mbalmayo, CameroonX X *
Dolichorhinotermes longilabiusPetit Saut, French GuianaX X
PsammotermitidaePsammotermes allocerusRundu, NamibiaXXX
Prorhinotermes canalifronsRéunion IslandXX X
Prorhinotermes inopinatusBaitabag, Papua New GuineaXX X
Prorhinotermes simplexOmar Torrijos Nt. Park, PanamaXX X
Prorhinotermes simplexPiñar del Rio, Soroa, CubaXX X
HeterotermitidaeHeterotermes cardiniPanama City, PanamaXXX
* Sequence amplified from whole-gut DNA, not isolated cells. Observed but not confirmed by molecular data.
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Aguilar, S.G.; Shevat, J.; Jasso-Selles, D.E.; Swichtenberg, K.L.; Vecco-Giove, C.D.; Šobotník, J.; Sillam-Dussès, D.; De Martini, F.; Gile, G.H. Distinct Patterns of Co-Evolution Among Protist Symbionts of Neoisoptera Termites. Diversity 2025, 17, 537. https://doi.org/10.3390/d17080537

AMA Style

Aguilar SG, Shevat J, Jasso-Selles DE, Swichtenberg KL, Vecco-Giove CD, Šobotník J, Sillam-Dussès D, De Martini F, Gile GH. Distinct Patterns of Co-Evolution Among Protist Symbionts of Neoisoptera Termites. Diversity. 2025; 17(8):537. https://doi.org/10.3390/d17080537

Chicago/Turabian Style

Aguilar, Serena G., Jordyn Shevat, Daniel E. Jasso-Selles, Kali L. Swichtenberg, Carlos D. Vecco-Giove, Jan Šobotník, David Sillam-Dussès, Francesca De Martini, and Gillian H. Gile. 2025. "Distinct Patterns of Co-Evolution Among Protist Symbionts of Neoisoptera Termites" Diversity 17, no. 8: 537. https://doi.org/10.3390/d17080537

APA Style

Aguilar, S. G., Shevat, J., Jasso-Selles, D. E., Swichtenberg, K. L., Vecco-Giove, C. D., Šobotník, J., Sillam-Dussès, D., De Martini, F., & Gile, G. H. (2025). Distinct Patterns of Co-Evolution Among Protist Symbionts of Neoisoptera Termites. Diversity, 17(8), 537. https://doi.org/10.3390/d17080537

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