Towards an integrated understanding of the consequences of fungus domestication on the fungus‐growing termite gut microbiota

Approximately 30 million years ago (MYA), the subfamily of higher termites Macrotermitinae domesticated a fungus, Termitomyces, as the main plant decomposer and food source for the termite host. The origin of fungiculture shifted the composition of the termite gut microbiota, and some of the functional implications of this shift have recently been established. I review reports on the composition of the Macrotermitinae gut microbiota, evidence for a subfamily core gut microbiota, and the first insight into functional complementarity between fungal and gut symbionts. In addition, I argue that we need to explore the capacities of all members of the symbiotic communities, including better solidifying Termitomyces role(s) in order to understand putative complementary gut bacterial contributions. Approaches that integrate natural history and sequencing data to elucidate symbiont functions will be powerful, particularly if executed in comparative analyses across the well‐established congruent termite–fungus phylogenies. This will allow for testing if gut communities have evolved in parallel with their hosts, with implications for our general understanding of the evolution of gut symbiont communities with hosts.     

Comparison of gut-associated and nest-associated microbial communities of a fungus-growing termite (Odontotermes yunnanensis)

Abstract The digestion of cellulose by fungus-growing termites involves a complex of different organisms, such as the termites themselves, fungi and bacteria. To further investigate the symbiotic relationships of fungus-growing termites, the microbial communities of the termite gut and fungus combs of Odontotermes yunnanensis were examined. The major fungus species was identified as Termitomyces sp. To compare the micro-organism diversity between the digestive tract of termites and fungus combs, four polymerase chain reaction clone libraries were created (two fungus-targeted internal transcribed spacer [ITS]– ribosomal DNA [rDNA] libraries and two bacteria-targeted 16S rDNA libraries), and one library of each type was produced for the host termite gut and the symbiotic fungus comb. Results of the fungal clone libraries revealed that only Termitomyces sp. was detected on the fungus comb; no non-Termitomyces fungi were detected. Meanwhile, the same fungus was also found in the termite gut. The bacterial clone libraries showed higher numbers and greater diversity of bacteria in the termite gut than in the fungus comb. Both bacterial clone libraries from the insect gut included Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, Nitrospira, Deferribacteres, and Fibrobacteres, whereas the bacterial clone libraries from the fungal comb only contained Firmicutes, Bacteroidetes, Proteobacteria, and Acidobacteris.     

Diversity of fungus‐growing termites (Macrotermes) and their fungal symbionts (Termitomyces) in the semiarid Tsavo Ecosystem,Kenya

Fungus‐growing termites of the subfamily Macrotermitinae together with their highly specialized fungal symbionts (Termitomyces) are primary decomposers of dead plant matter in many African savanna ecosystems. The termites provide crucial ecosystem services also by modifying soil properties, translocating nutrients, and as important drivers of plant succession. Despite their obvious ecological importance, many basic features in the biology of fungus‐growing termites and especially their fungal symbionts remain poorly known, and no studies have so far focused on possible habitat‐level differences in symbiont diversity across heterogeneous landscapes. We studied the species identities of Macrotermes termites and their Termitomyces symbionts by excavating 143 termite mounds at eight study sites in the semiarid Tsavo Ecosystem of southern Kenya. Reference specimens were identified by sequencing the COI region from termites and the ITS region from symbiotic fungi. The results demonstrate that the regional Macrotermes community in Tsavo includes two sympatric species (M. subhyalinus and M. michaelseni) which cultivate and largely share three species of Termitomyces symbionts. A single species of fungus is always found in each termite mound, but even closely adjacent colonies of the same termite species often house evolutionarily divergent fungi. The species identities of both partners vary markedly between sites, suggesting hitherto unknown differences in their ecological requirements. It is apparent that both habitat heterogeneity and disturbance history can influence the regional distribution patterns of both partners in symbiosis.     

Diet is the primary determinant of bacterial community structure in the guts of higher termites

The gut microbiota of termites plays critical roles in the symbiotic digestion of lignocellulose. While phylogenetically ‘lower termites’ are characterized by a unique association with cellulolytic flagellates, higher termites (family Termitidae) harbour exclusively prokaryotic communities in their dilated hindguts. Unlike the more primitive termite families, which primarily feed on wood, they have adapted to a variety of lignocellulosic food sources in different stages of humification, ranging from sound wood to soil organic matter. In this study, we comparatively analysed representatives of different taxonomic lineages and feeding groups of higher termites to identify the major drivers of bacterial community structure in the termite gut, using amplicon libraries of 16S rRNA genes from 18 species of higher termites. In all analyses, the wood‐feeding species were clearly separated from humus and soil feeders, irrespective of their taxonomic affiliation, offering compelling evidence that diet is the primary determinant of bacterial community structure. Within each diet group, however, gut communities of termites from the same subfamily were more similar than those of distantly related species. A highly resolved classification using a curated reference database revealed only few genus‐level taxa whose distribution patterns indicated specificity for certain host lineages, limiting any possible cospeciation between the gut microbiota and host to short evolutionary timescales. Rather, the observed patterns in the host‐specific distribution of the bacterial lineages in termite guts are best explained by diet‐related differences in the availability of microhabitats and functional niches.     

Distribution pattern of Termitomyces types symbiotic with the fungus‐growing termite Odontotermes formosanus on Okinawa Island

Fungus‐growing termites (subfamily Macrotermitinae) cultivate the symbiotic basidiomycete fungus Termitomyces in their fungus comb to digest cellulosic materials and to supply nitrogen‐rich fungal diet. In Japan, the fungus‐growing termite Odontotermes formosanus is found on the Yaeyama Islands and Okinawa Island, Okinawa Prefecture. Odontotermes formosanus is thought to have been recently and artificially introduced to Okinawa Island as its distribution is discontinuous and restricted to small areas. Previous DNA analyses revealed that two types of Termitomyces, namely Termitomyces sp. Type A and Termitomyces sp. Type B, whose fruiting bodies correspond to Termitomyces microcarpus‐like pseudorhiza‐lacking small mushroom and Termitomyces intermedius, respectively, are cultivated by O. formosanus on the Yaeyama Islands. However, information about the Termitomyces types cultivated by O. formosanus on Okinawa Island is limited. To define the fungal types cultivated by O. formosanus on Okinawa Island, I developed a diagnostic polymerase chain reaction method using primer sets specific to the nuclear ribosomal DNA sequences consisting of the internal transcribed spacers (ITS1 and ITS2) and 5.8S rDNA of Termitomyces not using fungal mycelium, but using the termite gut metagenome including fungal DNA as a template. The results indicated that the same two Termitomyces types from Iriomote Island are cultivated by O. formosanus on Okinawa Island. The distribution pattern of Termitomyces types on Okinawa Island showed that Termitomyces sp. Type A is limited to the mountainous side of Sueyoshi Park, despite Termitomyces sp. Type B being widely distributed in the area in which O. formosanus is found. This finding implies that O. formosanus on Okinawa Island was recently introduced from Iriomote Island to Sueyoshi Park.     

Open relationships in the castles of clay: high diversity and low host specificity of Termitomyces fungi associated with fungus-growing termites in Africa

In the African and Asian tropics, termites of the subfamily Macrotermitinae play a major role in the decomposition of dead plant material. Their ecological success lies in the obligate mutualism of the termites with fungi of the genus Termitomyces. Before the advent of molecular studies, the interaction with these fungi was poorly understood. Here, we combined available ITS sequence data from West, Central, and South Africa with data of 39 new samples from East Africa to achieve the most comprehensive view of the diversity and host specificity of Termitomyces symbionts across Africa to date. A high amount of sequence divergence in the ITS sequences was found; 11 different Termitomyces lineages in East Africa and >30 lineages across Africa were identified, and the expected diversity is estimated to be about 41 lineages. The fungal lineages belong to four major clades, each almost exclusively associated with one termite host genus. Analysis of molecular variance revealed that 40% of the ITS sequence variation occurred between host genera, indicating close co-evolution at this level. However, within host genera, fungal lineages and haplotypes were frequently shared among host species and sampling localities, except for fungal symbionts of Odontotermes. Horizontal transmission of fungal symbionts may facilitate the transfer of haplotypes and species among hosts. However, at present, we have little understanding of the maintenance of specificity at the genus level. Possible explanations range from substrate specificity of fungi to an active selection of fungi by termites.     

Intracolony variation of bacterial gut microbiota among castes and ages in the fungus-growing termite Macrotermes gilvus

The fungus-growing termites Macrotermes cultivate the obligate ectosymbiontic fungi, Termitomyces. While their relationship has been extesively studied, little is known about the gut bacterial symbionts, which also presumably play a crucial role for the nutrition of the termite host. In this study, we investigated the bacterial gut microbiota in two colonies of Macrotermes gilvus, and compared the diversity and community structure of bacteria among nine termite morphotypes, differing in caste and/or age, using terminal restriction fragment length polymorphism (T-RFLP) and clonal analysis of 16S rRNA. The obtained molecular community profiles clustered by termite morphotype rather than by colony, and the clustering pattern was clearly more related to a difference in age than to caste. Thus, we suggest that the bacterial gut microbiota change in relation to the food of the termite, which comprises fallen leaves and the fungus nodules of Termitomyces in young workers, and leaves degraded by the fungi, in old workers. Despite these intracolony variations in bacterial gut microbiota, their T-RFLP profiles formed a distinct cluster against those of the fungus garden, adjacent soil and guts of sympatric wood-feeding termites, implying a consistency and uniqueness of gut microbiota in M. gilvus. Since many bacterial phylotypes from M. gilvus formed monophyletic clusters with those from distantly related termite species, we suggest that gut bacteria have co-evolved with the termite host and form a microbiota specific to a termite taxonomic and/or feeding group, and furthermore, to caste and age within a termite species.     

Diversity and resilience of the wood‐feeding higher termite Mironasutitermes shangchengensis gut microbiota in response to temporal and diet variations

Termites are considered among the most efficient bioreactors, with high capacities for lignocellulose degradation and utilization. Recently, several studies have characterized the gut microbiota of diverse termites. However, the temporal dynamics of the gut microbiota within a given termite with dietary diversity are poorly understood. Here, we employed 16S rDNA barcoded pyrosequencing analysis to investigate temporal changes in bacterial diversity and richness of the gut microbiota of wood‐feeding higher termite Mironasutitermes shangchengensis under three lignocellulose content‐based diets that feature wood, corn stalks, and filter paper. Compositions of the predominant termite gut residents were largely constant among the gut microbiomes under different diets, but each diet caused specific changes in the bacterial composition over time. Notably, microbial communities exhibited an unexpectedly strong resilience during continuous feeding on both corn stalks and filter paper. Members of five bacterial phyla, that is, Spirochaetes, Firmicutes, Actinobacteria, Tenericutes, and Acidobacteria, were strongly associated with the resilience. These findings provide insights into the stability of the gut microbiota in higher termites and have important implications for the future design of robust bioreactors for lignocellulose degradation and utilization.     

Environmental and biological determinants of <Emphasis Type="Italic">Termitomyces</Emphasis> species seasonal fructification in central and southern Côte d’Ivoire

The Macrotermitinae subfamily is characterized by its symbiosis with fungi of the genus Termitomyces. The most common and presumably primitive mode of reproduction for these fungi is to produce basidiocarps on the mounds of the host termite colony. The seasonal fructification pattern of the fungi seems to depend on the habitat type and termite ecology. We examined Termitomyces diversity and distribution in two phytogeographic zones in central and southern Côte d’Ivoire. Data were collected in different habitats in exhaustive searches with standardized methods for termites and basidiocarps as well. The respective findings were complemented with behavioral and life cycle data of the associated termite species. Basidiocarps occur species-specifically either during the long or the short rainy season in wooded habitats. The diversity and abundance of termites and their associated basidiocarps were significantly correlated only with woody plant species richness. Nuptial flight of termites, comb biomass and Termitomyces fructification periods were correlated. Termitomyces appear to fructify during (1) the period of strong precipitation, (2) in habitats with appropriate microclimatic characteristics.     

Evolutionary trend of phylogenetic diversity of nitrogen fixation genes in the gut community of wood-feeding termites

Nitrogen fixation by gut microorganisms is one of the crucial aspects of symbiosis in wood-feeding termites since these termites thrive on a nitrogen-poor diet. In order to understand the evolution of this symbiosis, we analysed the nitrogenase structural gene nifH in the gut microbial communities. In conjunction with the published sequences, we compared approximately 320 putatively functional NifH protein sequences obtained from a total of 19 termite samples that represent all the major branches of their currently proposed phylogeny, and from one species of the cockroach Cryptocercus that shares a common ancestor with termites. Using multivariate techniques for clustering and ordination, a phylogeny of NifH protein sequences was created and plotted variously with host termite families, genera, and species. Close concordance was observed between NifH communities and the host termites at genus level, but family level relationships were not always congruent with accepted termite clade structure. Host groups examined included basal families (Mastotermitidae, Termopsidae, Kalotermitidae, as well as Cryptocercus), the most derived lower termite family Rhinotermitidae, and subfamilies representing the advanced and highly diverse apical family Termitidae (Macrotermitinae, Termitinae, and Nasutitermitinae). This selection encompassed the major nesting and feeding styles recognized in termites, and it was evident that NifH phylogenetic divergence, as well as the occurrence of alternative nitrogenase-type NifH, was to some extent dependent on host lifestyle as well as phylogenetic position.     

Metagenomic Insights into Metabolic Capacities of the Gut Microbiota in a Fungus-Cultivating Termite (Odontotermes yunnanensis)

Macrotermitinae (fungus-cultivating termites) are major decomposers in tropical and subtropical areas of Asia and Africa. They have specifically evolved mutualistic associations with both a Termitomyces fungi on the nest and a gut microbiota, providing a model system for probing host-microbe interactions. Yet the symbiotic roles of gut microbes residing in its major feeding caste remain largely undefined. Here, by pyrosequencing the whole gut metagenome of adult workers of a fungus-cultivating termite (Odontotermes yunnanensis), we showed that it did harbor a broad set of genes or gene modules encoding carbohydrate-active enzymes (CAZymes) relevant to plant fiber degradation, particularly debranching enzymes and oligosaccharide-processing enzymes. Besides, it also contained a considerable number of genes encoding chitinases and glycoprotein oligosaccharide-processing enzymes for fungal cell wall degradation. To investigate the metabolic divergence of higher termites of different feeding guilds, a SEED subsystem-based gene-centric comparative analysis of the data with that of a previously sequenced wood-feeding Nasutitermes hindgut microbiome was also attempted, revealing that SEED classifications of nitrogen metabolism, and motility and chemotaxis were significantly overrepresented in the wood-feeder hindgut metagenome, while Bacteroidales conjugative transposons and subsystems related to central aromatic compounds metabolism were apparently overrepresented here. This work fills up our gaps in understanding the functional capacities of fungus-cultivating termite gut microbiota, especially their roles in the symbiotic digestion of lignocelluloses and utilization of fungal biomass, both of which greatly add to existing understandings of this peculiar symbiosis.     

高效降解木质纤维素的白蚁肠道微生物组

木食性白蚁是自然界木质纤维素的高效降解者,在长期进化过程中白蚁与其肠道微生物组协同作用发展出不同的纤维素降解机制。木食性白蚁具有分别来源于白蚁和共生微生物的两套纤维素酶系统。在低等白蚁中,木质颗粒经过白蚁前、中肠分泌的内源性酶初步消化后,在后肠共生鞭毛虫中被降解为乙酸、二氧化碳和氢。高等木食性白蚁在进化中丢失了鞭毛虫,木质颗粒经白蚁自身分泌的酶初步消化后,在后肠大量共生细菌的帮助下被有效降解。培菌类白蚁利用其菌圃中的蚁巢伞菌和肠道微生物协同作用降解木质纤维素。共生微生物在白蚁的氮素固定与循环、中间产物代谢及纤维素降解等过程中发挥了重要作用。学习和模拟白蚁高效降解木质纤维素的体系,对生物质能源的产业化发展具有积极的意义。     

The Enterobacterium Trabulsiella odontotermitis Presents Novel Adaptations Related to Its Association with Fungus-Growing Termites

Fungus-growing termites rely on symbiotic microorganisms to help break down plant material and to obtain nutrients. Their fungal cultivar, Termitomyces, is the main plant degrader and food source for the termites, while gut bacteria complement Termitomyces in the degradation of foodstuffs, fixation of nitrogen, and metabolism of amino acids and sugars. Due to the community complexity and because these typically anaerobic bacteria can rarely be cultured, little is known about the physiological capabilities of individual bacterial members of the gut communities and their associations with the termite host. The bacterium Trabulsiella odontotermitis is associated with fungus-growing termites, but this genus is generally understudied, with only two described species. Taking diverse approaches, we obtained a solid phylogenetic placement of T. odontotermitis among the Enterobacteriaceae, investigated the physiology and enzymatic profiles of T. odontotermitis isolates, determined the localization of the bacterium in the termite gut, compared draft genomes of two T. odontotermitis isolates to those of their close relatives, and examined the expression of genes relevant to host colonization and putative symbiont functions. Our findings support the hypothesis that T. odontotermitis is a facultative symbiont mainly located in the paunch compartment of the gut, with possible roles in carbohydrate metabolism and aflatoxin degradation, while displaying adaptations to association with the termite host, such as expressing genes for a type VI secretion system which has been demonstrated to assist bacterial competition, colonization, and survival within hosts.     

Nest specificity of the bacterial community in termite guts (Hodotermes mossambicus)

While recent results have provided strong evidence for the presence of a stable gut microbiota among several termite species, little is known about variations at the colony or individual level. Using a cultivation-independent approach, we investigated the structure of the bacterial community in the gut of termites from four different colonies of Hodotermes mossambicus. 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis of the bacterial gut microbiota revealed (1) a high consistency of the gut microbiota among nestmates and (2) subtle but distinct differences in community structure between individuals from different colonies. Since products of bacterial metabolism may contribute to a colony odor that can be used as discriminatory signal, the presence of a colony-specific bacterial community adds support to the hypothesis that the gut microbiota of termites is involved in nestmate recognition. Received 12 July 2005; revised 10 February and 15 March 2006; accepted 7 April 2006.     

The Cockroach Origin of the Termite Gut Microbiota: Patterns in Bacterial Community Structure Reflect Major Evolutionary Events

Termites digest wood and other lignocellulosic substrates with the help of their intestinal microbiota. While the functions of the symbionts in the digestive process are slowly emerging, the origin of the bacteria colonizing the hindgut bioreactor is entirely unknown. Recently, our group discovered numerous representatives of bacterial lineages specific to termite guts in a closely related omnivorous cockroach, but it remains unclear whether they derive from the microbiota of a common ancestor or were independently selected by the gut environment. Here, we studied the bacterial gut microbiota in 34 species of termites and cockroaches using pyrotag analysis of the 16S rRNA genes. Although the community structures differed greatly between the major host groups, with dramatic changes in the relative abundances of particular bacterial taxa, we found that the majority of sequence reads belonged to bacterial lineages that were shared among most host species. When mapped onto the host tree, the changes in community structure coincided with major events in termite evolution, such as acquisition and loss of cellulolytic protists and the ensuing dietary diversification. UniFrac analysis of the core microbiota of termites and cockroaches and construction of phylogenetic tree of individual genus level lineages revealed a general host signal, whereas the branching order often did not match the detailed phylogeny of the host. It remains unclear whether the lineages in question have been associated with the ancestral cockroach since the early Cretaceous (cospeciation) or are diet-specific lineages that were independently acquired from the environment (host selection).     

Variations in Diversity and Richness of Gut Bacterial Communities of Termites (Reticulitermes flavipes) Fed with Grassy and Woody Plant Substrates

Diets shape the animal gut microbiota, although the relationships between diets and the structure of the gut microbial community are not yet well understood. The gut bacterial communities of Reticulitermes flavipes termites fed on four individual plant biomasses with different degrees of recalcitrance to biodegradation were investigated by 16S rRNA pyrosequencing analysis. The termite gut bacterial communities could be differentiated between grassy and woody diets, and among grassy diets (corn stover vs. sorghum). The majority of bacterial taxa were shared across all diets, but each diet significantly enriched some taxa. Interestingly, the diet of corn stover reduced gut bacterial richness and diversity compared to other diets, and this may be related to the lower recalcitrance of this biomass to degradation.     

Resolving relationships in some African fungus-growing termites (Termitidae, Macrotermitinae) using molecular phylogeny, morphology, and field parameters

The Macrotermitinae are a large and successful subfamily of fungus-growing termites, characterised by their symbiotic association with white-rot fungi of the genus Termitomyces. The taxonomy of the subfamily, and in particular of the largest genus Odontotermes, is problematic. We used sequences of the mitochondrial 16S gene from termites occurring in East Africa and Malawi to explore the phyletic relationships within the genus Odontotermes and to place the genus in the broader context of other fungus-growing termites. We also interpret this phylogeny in relation to classical morphological taxonomy in the form of absolute and relative dry weights of the sterile castes, and in relation to innate behaviour as shown by nest architecture and fungus comb structure. This work lays the foundations of a complete taxonomic revision of the Macrotermitinae. The phylogeny supports one clear subdivision, here called the “tanganicus” group, within the genus Odontotermes. It also supports the significance of field observations on the structure of fungus combs, as the whole “tanganicus” group builds fungus combs of the sponge type (or modified forms thereof) which are hardly known elsewhere. Other phyletic relationships are less clear, the residual sequences being referred to as the “latericius” group. We recognise three probable miniature species within the “tanganicus” group and another possible one in the residual “latericius group”. Received 16 January 2007; revised 30 October 2007 and 11 March 2008; accepted 4 April 2008.     

Phylogenetic relationships in Termitomyces (Family Agaricaceae) based on the nucleotide sequence of ITS: a first approach to elucidate the evolutionary history of the symbiosis between fungus-growing termites and their fungi

Termitomyces constitutes a very poorly known genus of fungi whose essential characteristic is that all representatives of the genus are cultivated by termites (Macrotermitinae) in their nest and that all the fungi cultivated by termites belong to this genus. For the first time, the phylogenetic relationships of several African Termitomyces species was studied by the sequencing of their internal transcriber spacer region (ITS1--5.8S--ITS2). It appeared that this group is clearly monophyletic and belongs to the Tricholomataceae family. The total homology of the ITS zone of several Termitomyces symbionts of different termite genera indicated that the specific diversity of this group is in fact less important than previously supposed. Finally, the comparison between the Termitomyces phylogenetic tree and the taxonomic tree of Macrotermitinae showed that if for certain genera the hypothesis of termite/fungus coevolution is acceptable, it should not be applied for all symbiosis.     

Molecular phylogenetic profiling of prokaryotic communities in guts of termites with different feeding habits

Termites are an important group of terrestrial insects that harbor an abundant gut microbiota, many of which contribute to digestion, termite nutrition and gas (CH(4), CO(2) and H(2)) emission. With 2200 described species, termites also provide a good model to study relationships between host diet and gut microbial community structure and function. We examined the relationship between diet and gut prokaryotic community profiles in 24 taxonomically and nutritionally diverse species of termites by using nucleic acid probes targeting 16S-like ribosomal RNAs. The relative abundance of domain-specific 16S-like rRNAs recovered from gut extracts varied considerably (ranges: Archaea (0-3%); Bacteria (15-118%)). Although Bacteria were always detectable and the most abundant, differences in domain-level profiles were correlated with termite diet, as evidenced by higher relative abundances of Archaea in guts of soil-feeding termites, compared to those of wood-feeding species in the same family. The oligonucleotide probes also readily distinguished gut communities of wood-feeding taxa in the family Termitidae (higher termites) from those of other wood-feeding termite families (lower termites). The relative abundances of 16S-like archaeal rRNA in guts were positively correlated with rates of methane emission by live termites, and were consistent with previous work linking high relative rates of methanogenesis with the soil (humus)-feeding habit. Probes for methanogenic Archaea detected members of only two families (Methanobacteriaceae and Methanosarcinaceae) in termite guts, and these typically accounted for 60% of the all archaeal probe signal. In four species of termites, Methanosarcinaceae were dominant, a novel observation for animal gut microbial communities, but no clear relationship was apparent between methanogen family profiles and termite diet or taxonomy.     

Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic "termite clusters" comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.