Phylogenetic Position and In Situ Identification of Ectosymbiotic Spirochetes on Protists in the Termite Gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists

Some species of protists inhabiting the hindgut of lower-termites have a large number of ectosymbiotic spirochetes on the cell surface. The phylogenetic positions of the ectosymbiotic spirochetes of three oxymonad protists, Dinenympha porteri in the gut of Reticulitermes speratus, and Pyrsonympha sp. and Dinenympha sp. in Hodotermopsis sjoestedti, were investigated without cultivation of these organisms. Protist fractions carefully collected with a micromanipulator were used as templates for the amplification of small subunit ribosomal RNA genes (SSU rDNA). The phylogenetic tree inferred from the nucleotide sequences of the SSU rDNA showed that they were affiliated with the Treponema cluster of spirochetes and they were divided into two clusters. One was grouped together with the spirochetal sequences reported previously from the gut of termites and the other was related to the Treponema bryantii subgroup of treponemes (denoted as termite Treponema clusters I and II, respectively). Whole-cell in situ hybridization using a fluorescent-labeled oligonucleotide probe specific for the group of sequences in cluster II identified most of the ectosymbiotic spirochetes of the oxymonad protists in the gut of R. speratus and H. sjoestedti. However, not all of the ectosymbiotic spirochetes could be detected by means of this cluster II group-specific probe and the population of ectosymbiotic spirochetes of cluster II was different among the oxymonad species. In the case of D. porteri, an oligonucleotide probe specific for one member of cluster II recognized a portion of the ectosymbiotic spirochetes of cluster II, and their population was also different depending on the cell-type of D. porteri in terms of the attachment of ectosymbiotic spirochetes. The results indicate that the spirochetes of cluster II and probably those of a part of cluster I can be assigned to ectosymbiotic species of oxymonad protists and that the population of ectosymbiotic spirochetes associated with a single protist consists of at least three species of phylogenetically distinct spirochetes.     

Candidatus Symbiothrix dinenymphae: bristle-like Bacteroidales ectosymbionts of termite gut protists

Many reports have stated that flagellated protists in termite guts harbour ectosymbiotic spirochetes on their cell surface. In this study, we describe another bristle-like ectosymbiont affiliated with the order Bacteroidales. The 16S rRNA phylotype Rs-N74 predominates among Bacteroidales clones obtained from the gut of the termite Reticulitermes speratus. An Rs-N74 phylotype-specific probe was designed in this study and used for detection of the corresponding bacteria in the gut by fluorescence in situ hybridization (FISH) analysis. Surprisingly, the signals were detected specifically from the bristle-like 'appendages' of various flagellate species belonging to the genus Dinenympha; these 'appendages' had been believed to be spirochetal ectosymbionts or structures of the protists. The Rs-N74 bacteria attached to the cell surface of the protists by a tip and coexisted with the spirochetal ectosymbionts. An electron micrograph revealed their morphology to be similar to a typical Bacteroidales bacterium. This bacterium is proposed to represent a novel genus and species, 'Candidatus Symbiothrix dinenymphae', phylogenetically affiliated with a cluster consisting exclusively of uncultured strains from termite guts. A Bacteroidales-specific probe for FISH further revealed that this type of symbiosis exists also in various other protists, including parabasalids and oxymonads, and is widespread in termite guts.     

Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach

Bacterial attachments to nearly the entire surface of flagellated protists in the guts of termites and the wood-feeding cockroach Cryptocercus are often observed. Based on the polymerase chain reaction-amplified 16S rRNA gene sequences, we investigated the phylogenetic relationships of the rod-shaped, attached bacteria (ectosymbionts) of several protist species from five host taxa and confirmed their identity by fluorescence in situ hybridizations. These ectosymbionts are affiliated with the order Bacteroidales but formed three distinct lineages, each of which may represent novel bacterial genera. One lineage consisted of the closely related ectosymbionts of two species of the protist genus Devescovina (Cristamonadida). The second lineage comprised three phylotypes identified from the protist Streblomastix sp. (Oxymonadida). The third lineage included ectosymbionts of the three protist genera Hoplonympha, Barbulanympha and Urinympha in the family Hoplonymphidae (Trichonymphida). The ultrastructural observations indicated that these rod-shaped ectosymbionts share morphological similarities of their cell walls and their point of attachment with the protist but differ in shape. Elongated forms of the ectosymbionts appeared in all the three lineages. The protist cells Streblomastix sp. and Hoplonympha sp. display deep furrows and vane-like structures, but these impressive structures are probably evolutionarily convergent because both the host protists and their ectosymbionts are distantly related.     

16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite Mastotermes darwiniensis Froggatt

Abstract We have analyzed the 16S rDNA sequence and the phylogenetic position of an uncultivated spirochete from the hindgut contents of the Australian termite Mastotermes darwiniensis Froggatt. The 16S rRNA genes of bacteria from the hindgut contents of Mastotermes darwiniensis were amplified by polymerase chain reaction. The amplification products were cloned and sequenced. The sequences were compared to known homologous primary structures. Two of the clones (MDS1 and MDS3) had an insert of 1498 nucleotides showing typical signatures of spirochete 16S rRNA sequences. The sequences of the two clones were most similar to the 16S rRNA sequence of Spirochaeta stenostrepta (89.8%) and Treponema sp. strain H1 (90.7%). Phylogenetical analysis positioned the hindgut spirochete sequence with that of the free-living anaerobic Spirochaeta stenostrepta and Treponema sp. strain H1 as its nearest relatives within the cluster of the spirochetes. We conclude that the analyzed SSU rDNA sequences originate from a spirochete related to the genus Treponema . It is possibly one of the uncultivated unique spirochetes symbiotic in termite hindguts.     

Phylogeny of not-yet-cultured spirochetes from termite guts.

Comparisons of 16S rDNA sequences were used to determine the phylogeny of not-yet-cultured spirochetes from hindguts of the African higher termite, Nasutitermes lujae (Wasmann). The 16S rRNA genes were amplified directly from spirochete-rich hindguts by using universal primers, and the amplified products were cloned into Escherichia coli. Clones were screened with a spirochete-specific DNA probe. Analysis of 1,410 base positions of the 16S rDNA insert from one spirochete clone, designated NL1, supported its assignment to the genus Treponema, with average interspecies similarities of ca. 85%. The sequence of NL1 was most closely related (ca. 87 to 88% similarity) to sequences of Spirochaeta stenostrepta and Spirochaeta caldaria and to a previously published sequence (ca. 87% similarity) of spirochetal clone MDS1 from the Australian lower termite, Mastotermes darwiniensis (Froggatt). On the basis of 16S rRNA sequence comparisons and individual base signatures, clones NL1 and MDS1 clearly represent two novel species of Treponema, although specific epithets have not yet been proposed. The gross morphology of NL1 was determined from in situ hybridization experiments with an NL1-specific, fluorescently labeled oligonucleotide probe. Cells were approximately 0.3 to 0.4 by 30 microns in size, with a wavelength and amplitude of about 10 microns and 0.8 to 1.6 micron, respectively. Moreover, electron microscopy of various undulate cells present in gut contents confirmed that they possessed ultrastructural features typical of spirochetes, i.e., a wavy protoplasmic cylinder, periplasmic flagella, and an outer sheath. The sequence data suggest that termite gut spirochetes may represent a separate line of descent from other treponemes and that they constitute a significant reservoir of previously unrecognized spirochetal biodiversity.     

Coevolution of symbiotic spirochete diversity in lower termites.

The phylogenetic relationships of symbiotic spirochetes from five dry-wood feeding lower termites (Cryptotermes cavifrons, Heterotermes tenuis, Kalotermes flavicollis, Neotermes mona, and Reticulitermes grassei) was compared to those described in previous reports. The 16S rDNA bacterial genes were PCR-amplified from DNA isolated from intestinal samples using a spirochete-selective primer, and the 16S amplicons were cloned into Escherichia coli. Sequences of the cloned inserts were then used to determine closest relatives by comparison with published sequences. Clones sharing more than 97% sequence identity were grouped into the same phylotype. Forty-three new phylotypes were identified. These termite whole-gut-spirochetes fell into two previous defined clusters, designated as Treponema Clusters I and II, and one new Cluster III. Thirty-seven phylotypes were grouped in Cluster I. Cluster II comprised three phylotypes, two from Reticulitermes grassei (LJ029 and LJ012) and one from Heterotermes tenuis (LQ016). Three phylotypes, LK057, LK050 and LK028, were affiliated to Cluster III. Members of Cluster I showed the following characteristics: (i) spirochete phylotypes from a particular species of termite were more closely related to each other than to phylotypes of other termite species; (ii) spirochetes obtained from different genera of the same family, such as Cryptotermes sp., Kalotermes sp., and Neotermes sp., all from the family Kalotermitidae, were also related to each other. It was therefore concluded that spirochetes are specific symbionts that have coevolved with their respective species of termites, are stably harbored, and are closely related to members of the same termite family.     

The motility symbiont of the termite gut flagellate Caduceia versatilis is a member of the "Synergistes" group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the "Synergistes" phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the "Synergistes" phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The "Synergistes" phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, "Candidatus Tammella caduceiae," for this unique motility ectosymbiont of Caducaia versatilis.     

Phylogenetic relationships of symbiotic spirochetes in the gut of diverse termites

Phylogenetic relationships of symbiotic spirochetes in the gut of diverse termites were analyzed without cultivation of these microorganisms. A portion of the 16S rDNA (ca. 850 bp) was amplified directly from DNA of the mixed population in the gut by PCR and cloned. A total of 30 spirochetal phylotypes affiliated with the treponemes were identified from four termite species and they were compared with those already reported from other termites. They represented separate lines of descent from any known species of Treponema, and they were divided into two discrete clusters; one was related to Spirochaeta stenostrepta and S. caldaria, and the other was grouped together with members of the Treponema bryantii subgroup. Although some sequences from evolutionarily related termites showed close similarity, most of the sequences of spirochetes were dissimilar among different termite species, and spirochetal sequences from a single termite species occurred in several distinct phylogenetic positions. These findings suggest that termites constitute a rich reservoir of novel spirochetal diversity and that evolution of the symbiosis is not simple.     

Endosymbiotic Bacteroidales Bacteria of the Flagellated Protist Pseudotrichonympha grassii in the Gut of the Termite Coptotermes formosanus

A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.     

Origin of symbiotic gut spirochetes as key players in the nutrition of termites

Termites harbour symbiotic spirochetes in their hindguts, which have long been considered treponemes, although they represent separate lines of descent from known species of Treponema. ‘Termite gut treponemes’ have a mutualistic relationship with the host termites with their physiological properties including CO₂-reductive acetogenesis, from which the resulting acetate fulfils most of the respiratory requirement of the host. Song and co-workers showed that a spirochetal isolate (strain RmG30) from a Madeira cockroach represents the earliest branching lineage of extremely diverse termite (Treponema) cluster I and was a simple homolactic fermenter, suggesting that CO₂-reductive acetogenesis exhibited by some members of termite cluster I originated via horizontal gene transfer. Phylogenomic and 16S rRNA sequence-based phylogenetic analyses indicated a deeply-branched sister clade containing termite cluster I was distinguishable as a family-level lineage. In this context, a new family, ‘Termitinemataceae’ has been proposed for this clade. Strain RmG30 has been designated as the type strain of Breznakiella homolactica gen. nov. sp. nov. named after John A. Breznak, an American microbiologist distinguished in termite gut microbiology. The study has posed important questions for the future, including the actual roles of the termite spirochetes in each termite lineage and the evolutionary process of their physiological properties.     

The Motility Symbiont of the Termite Gut Flagellate Caduceia versatilis Is a Member of the “Synergistes” Group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the “Synergistes” phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The “Synergistes” phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, “Candidatus Tammella caduceiae,” for this unique motility ectosymbiont of Caducaia versatilis.     

Canaleparolina darwiniensis, gen. nov., sp. nov., and other pillotinaceous spirochetes from insects.

We describe two new pillotinaceous spirochetes (Canaleparolina darwiniensis, Diplocalyx cryptotermitidis) and identify for the first time Hollandina pterotermitidis from both the subterranean termite Cryptotermes cavifrons and the wood-eating cockroach Cryptocercus punctulatus based on morphometric analysis of transmission electron micrographic thin sections. C. darwiniensis, gen. nov., sp. nov., limited to near Darwin, Australia, invariably is present on the surface of the treponeme-studded trichomonad Mixotricha paradoxa, a consistent inhabitant of the hindgut of healthy termite Mastotermes darwiniensis. The spirochete both attached to the surface of protists and free-swimming in the paunch (hindgut) lumen of the insect has 16 periplasmic flagella (16:32:16) and imbricated wall structures that resemble flattened crenulations of Pillotina. The flagella surround half the protoplasmic cylinder. C. darwiniensis is the largest (0.5 microm diameter x 25 microm length) of the three epibiotic bacteria (two spirochetes, one rod) that comprise the complex cortex of its host Mixotricha paradoxa. Several criteria distinguish Diplocalyx cryptotermitidis sp. nov. isolated from Cryptotermes cavifrons intestine: smaller diameter, fewer flagella, absence of inner and outer coats of the outer membrane, wider angle subtended by its flagella and, most notably, cytoplasmic tubule-associated centers, which are periodic electron dense spheres within the protoplasmic cylinder from which emanate cytoplasmic tubules up to 24 nm in diameter. This is also the first report of abundant populations of Hollandina in Cryptotermes cavifrons (those populations belong to the species H. pterotermitidis). Morphometric analysis of the first thin sections of any spirochetes (published nearly 40 years ago by A.V. Grimstone) permits us to identify the large (0.9 microm diameter) free-swimming intestinal symbiont of Cryptocercus punctulatus also as Hollandina pterotermitidis.     

Endosymbiotic Bacteroidales bacteria of the flagellated protist Pseudotrichonympha grassii in the gut of the termite Coptotermes formosanus

A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.     

Complex coevolutionary history of symbiotic Bacteroidales bacteria of various protists in the gut of termites

Background  

The microbial community in the gut of termites is responsible for the efficient decomposition of recalcitrant lignocellulose. Prominent features of this community are its complexity and the associations of prokaryotes with the cells of cellulolytic flagellated protists. Bacteria in the order Bacteroidales are involved in associations with a wide variety of gut protist species as either intracellular endosymbionts or surface-attached ectosymbionts. In particular, ectosymbionts exhibit distinct morphological patterns of the associations. Therefore, these Bacteroidales symbionts provide an opportunity to investigate not only the coevolutionary relationships with the host protists and their morphological evolution but also how symbiotic associations between prokaryotes and eukaryotes occur and evolve within a complex symbiotic community.     

Early evolution of microtubules and undulipodia

A critique of both autogeneous and symbiotic hypotheses for the origin of microtubules and cilia and eukaryotic flagella (undulipodia) is presented. It is proposed that spirochetes provided the ancient eukaryotic cell with microtubules twice; cytoplasmic microtubules originated from phagocytosed spirochetes whereas axopodial tubules of undulipodia were transformed from ectosymbiotic spirochetes. A role in transport for microtubules in spirochetes together with a detailed scenario by which free-living spirochetes attached as ectosymbionts and subsequently differentiated into undulipodia is outlined. A mechanism for the continuity of motility in the form of "training" of the novel microtubular axoneme by the ancient spirochete motility apparatus is proposed. Transitional states (missing links) are unlikely to have survived. Constraints regarding the nature of the host cell are discussed. A corresponding flowchart of the early evolution of eukaryotes is presented in which plastids and mitochondria are polyphyletic in their origins.     

GAPDH gene diversity in spirochetes: a paradigm for genetic promiscuity.

In this study we have determined gap sequences from nine different spirochetes. Phylogenetic analyses of these sequences in the context of all other available eubacterial and a selection of eukaryotic Gap sequences demonstrated that the eubacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene diversity encompasses at least five highly distinct gene families. Within these gene families, spirochetes show an extreme degree of sequence divergence that is probably the result of several lateral gene transfer events between spirochetes and other eubacterial phyla, and early gene duplications in the eubacterial ancestor. A Gap1 sequence from the syphilis spirochete Treponema pallidum has recently been shown to be closely related to GapC sequences from Euglenozoa. Here we demonstrate that several other spirochetal species are part of this cluster, supporting the conclusion that an interkingdom gene transfer from spirochetes to Euglenozoa must have occurred. Furthermore, we provide evidence that the GAPDH genes present in the protists Parabasalia may also be of spirochetal descent.     

Thioautotrophic ectosymbiosis in Pseudovorticella sp., a peritrich ciliate species colonizing wood falls in marine mangrove

Ciliates represent a diversified group of protists known to establish symbioses with prokaryotic micro-organisms. They are mainly phagotrophs and symbiotic relationships with bacteria can give them an important advantage in chemosynthetic environments. The aim of this study is to describe the thiotrophic association that occurs between the peritrich ciliate Pseudovorticella sp. and potential sulfur-oxidizing bacteria. Investigations at microscopic scale (LM, SEM, TEM) showed ectosymbiotic bacteria covering the surface of the body of Pseudovorticella sp. According to 16S rDNA phylogenetic analysis, these ectosymbiotic bacteria belong to γ-proteobacteria and are phylogenetically close to the symbiont of the recently described Zoothamnium ignavum, which inhabits shallow-water wood falls. FISH experiments, using symbiont specific probes, clearly indicate that these ectosymbiotic bacteria are also ingested into food vacuoles. Electron lucent granules observed in TEM in the cytoplasm of the ectosymbiotic bacteria have been identified as sulfur granules by Raman microspectrometry analyses. Raman microspectrometry analyses confirmed the thiotrophic nature of this relationship already suggested by the results obtained by TEM and phylogeny. A complete sulfur map was then performed to investigate the sulfur distribution in the zooid. Results show that the relationship between this protist and its bacterial partner is a thiotrophic ectosymbiosis.     

The candidate phylum 'Termite Group 1' of bacteria: phylogenetic diversity, distribution, and endosymbiont members of various gut flagellated protists

The candidate phylum 'Termite Group 1' (TG1) of bacteria, which is abundant in termite guts but has no culturable representative, was investigated with respect to the in situ localization, distribution, and diversity. Based on the 16S rRNA gene sequence analyses and FISH in termite guts, a number of lineages of TG1 members were identified as endosymbionts of a variety of gut flagellated protists from the orders Trichonymphida, Cristamonadida, and Oxymonadida that are mostly unique to termites. However, the survey in various environments using specific PCR primers revealed that TG1 members were also present in termites, a cockroach, and the bovine rumen that typically lack these protist orders. Most of the TG1 members from gut flagellates, termites, cockroaches, and the rumen formed a monophyletic subcluster that showed a shallow branching pattern in the phylogenetic tree, suggesting their recent diversification. Although endosymbionts of the same protist genera tended to be closely related, the endosymbiont lineages were often independent of the higher level classifications of their host protist and were dispersed in the phylogenetic tree. It appears that their cospeciation is not the sole rule for the diversification of TG1 members of endosymbionts.