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

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

Metagenomic analyses highlight the symbiotic association between the glacier stonefly Andiperla willinki and its bacterial gut community.

The glacier stonefly Andiperla willinki is the largest metazoan inhabiting the Patagonian glaciers. In this study, we analysed the gut microbiome of the aquatic nymphs by 16S rRNA gene amplicon and metagenomic sequencing. The bacterial gut community was consistently dominated by taxa typical of animal digestive tracts, such as Dysgonomonadaceae and Lachnospiraceae, as well as those generally indigenous to glacier environments, such as Polaromonas. Interestingly, the dominant Polaromonas phylotypes detected in the stonefly gut were almost never detected in the glacier surface habitat. Fluorescence in situ hybridization analysis revealed that the bacterial lineages typical of animal guts colonized the gut wall in a co-aggregated form, while Polaromonas cells were not included in the aggregates. Draft genomes of several dominant bacterial lineages were reconstructed from metagenomic datasets and indicated that the predominant Dysgonomonadaceae bacterium is capable of degrading various polysaccharides derived from host-ingested food, such as algae, and that other dominant bacterial lineages ferment saccharides liberated by the polysaccharide degradation. Our results suggest that the gut bacteria-host association in the glacier stonefly contributes to host nutrition as well as material cycles in the glacier environment.

Acetogenesis from H2 plus CO2 and nitrogen fixation by an endosymbiotic spirochete of a termite-gut cellulolytic protist.

Symbiotic associations of cellulolytic eukaryotic protists and diverse bacteria are common in the gut microbial communities of termites. Besides cellulose degradation by the gut protists, reductive acetogenesis from H2 plus CO2 and nitrogen fixation by gut bacteria play crucial roles in the host termites' nutrition by contributing to the energy demand of termites and supplying nitrogen poor in their diet, respectively. Fractionation of these activities and the identification of key genes from the gut community of the wood-feeding termite Hodotermopsis sjoestedti revealed that substantial activities in the gut--nearly 60% of reductive acetogenesis and almost exclusively for nitrogen fixation--were uniquely attributed to the endosymbiotic bacteria of the cellulolytic protist in the genus Eucomonympha. The rod-shaped endosymbionts were surprisingly identified as a spirochete species in the genus Treponema, which usually exhibits a characteristic spiral morphology. The endosymbionts likely use H2 produced by the protist for these dual functions. Although H2 is known to inhibit nitrogen fixation in some bacteria, it seemed to rather stimulate this important mutualistic process. In addition, the single-cell genome analyses revealed the endosymbiont's potentials of the utilization of sugars for its energy requirement, and of the biosynthesis of valuable nutrients such as amino acids from the fixed nitrogen. These metabolic interactions are suitable for the dual functions of the endosymbiont and reconcile its substantial contributions in the gut.

Restriction-Modification Systems as Mobile Genetic Elements in the Evolution of an Intracellular Symbiont.

Long-term vertical transmission of intracellular bacteria causes massive genomic erosion and results in extremely small genomes, particularly in ancient symbionts. Genome reduction is typically preceded by the accumulation of pseudogenes and proliferation of mobile genetic elements, which are responsible for chromosome rearrangements during the initial stage of endosymbiosis. We compared the genomes of an endosymbiont of termite gut flagellates, "Candidatus Endomicrobium trichonymphae," and its free-living relative Endomicrobium proavitum and discovered many remnants of restriction-modification (R-M) systems that are consistently associated with genome rearrangements in the endosymbiont genome. The rearrangements include apparent insertions, transpositions, and the duplication of a genomic region; there was no evidence of transposon structures or other mobile elements. Our study reveals a so far unrecognized mechanism for genome rearrangements in intracellular symbionts and sheds new light on the general role of R-M systems in genome evolution.

Dominant ectosymbiotic bacteria of cellulolytic protists in the termite gut also have the potential to digest lignocellulose.

Wood-feeding lower termites harbour symbiotic gut protists that support the termite nutritionally by degrading recalcitrant lignocellulose. These protists themselves host specific endo- and ectosymbiotic bacteria, functions of which remain largely unknown. Here, we present draft genomes of a dominant, uncultured ectosymbiont belonging to the order Bacteroidales, 'Candidatus Symbiothrix dinenymphae', which colonizes the cell surface of the cellulolytic gut protists Dinenympha spp. We analysed four single-cell genomes of Ca. S. dinenymphae, the highest genome completeness was estimated to be 81.6-82.3% with a predicted genome size of 4.28-4.31 Mb. The genome retains genes encoding large parts of the amino acid, cofactor and nucleotide biosynthetic pathways. In addition, the genome contains genes encoding various glycoside hydrolases such as endoglucanases and hemicellulases. The genome indicates that Ca. S. dinenymphae ferments lignocellulose-derived monosaccharides to acetate, a major carbon and energy source of the host termite. We suggest that the ectosymbiont digests lignocellulose and provides nutrients to the host termites, and hypothesize that the hydrolytic activity might also function as a pretreatment for the host protist to effectively decompose the crystalline cellulose components.

Dysgonomonas termitidis sp. nov., isolated from the gut of the subterranean termite Reticulitermes speratus.

A facultatively anaerobic, Gram-stain-negative, non-motile and rod-shaped bacterium, strain N-10(T), was isolated from the gut of the termite Reticulitermes speratus. Strain N-10(T) was closely related to Dysgonomonas gadei JCM 16698(T) according to 16S rRNA gene sequence similarity analysis (98 %) and DNA-DNA relatedness value (≤61.3 %). The optimum growth temperature of strain N-10(T) was 30 °C, which was distinct from that (37 °C) of known species of the genus Dysgonomonas. Growth of strain N-10(T) was inhibited on medium containing 5 or 20 % bile, unlike other species of the genus Dysgonomonas. In addition, acid production in the API 20A system and enzymic reactions in the Rapid ID 32A system of strain N-10(T) differed from those of other species of the genus Dysgonomonas. Based on these characteristics, strain N-10(T) represents a novel species of the genus Dysgonomonas, for which the name Dysgonomonas termitidis sp. nov. is proposed. The type strain is N-10(T) ( = JCM 30204(T) = CCUG 66188(T)).

The nitrogen cycle in cryoconites: naturally occurring nitrification-denitrification granules on a glacier.

Cryoconites are microbial aggregates commonly found on glacier surfaces where they tend to take spherical, granular forms. While it has been postulated that the microbes in cryoconite granules play an important role in glacier ecosystems, information on their community structure is still limited, and their functions remain unclear. Here, we present evidence for the occurrence of nitrogen cycling in cryoconite granules on a glacier in Central Asia. We detected marker genes for nitrogen fixation, nitrification and denitrification in cryoconite granules by digital polymerase chain reaction (PCR), while digital reverse transcription PCR analysis revealed that only marker genes for nitrification and denitrification were abundantly transcribed. Analysis of isotope ratios also indicated the occurrence of nitrification; nitrate in the meltwater on the glacier surface was of biological origin, while nitrate in the snow was of atmospheric origin. The predominant nitrifiers on this glacier belonged to the order Nitrosomonadales, as suggested by amoA sequences and 16S ribosomal RNA pyrosequencing analysis. Our results suggest that the intense carbon and nitrogen cycles by nitrifiers, denitrifiers and cyanobacteria support abundant and active microbes on the Asian glacier.

Molecular diversity of endosymbiotic Nephroselmis (Nephroselmidophyceae) in Hatena arenicola (Katablepharidophycota).

Hatena arenicola (Katablepharidophycota) is a single-celled eukaryote that temporarily possesses a chlorophyte alga of the genus Nephroselmis as an intracellular symbiont. In the present study, we investigated the molecular diversity of the endosymbiont Nephroselmis in a natural population of the host H. arenicola. We sequenced the host's 18S rRNA gene and the endosymbiont's plastid-encoded 16S rRNA gene. The results indicated that almost identical strains of the host harbored at least three distinct strains of the algal endosymbiont affiliated to the clade Nephroselmis rotunda. This finding supports our previous hypothesis that H. arenicola and its symbiotic alga are in an early stage of secondary endosymbiosis.

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

The fidelity of vertical transmission is a critical factor in maintaining mutualistic associations with microorganisms. The obligate mutualism between termites and intestinal protist communities has been maintained for over 130 million years, suggesting the faithful transmission of diverse protist species across host generations. Although a severe bottleneck can occur when alates disperse with gut protists, how protist communities are maintained during this process remains largely unknown. In this study, we examined the dynamics of intestinal protist communities during adult eclosion and alate dispersal in the termite Reticulitermes speratus. We found that the protist community structure in last-instar nymphs differed significantly from that in workers and persisted intact during adult eclosion, whereas all protists disappeared from the gut during moults between worker stages. The number of protists in nymphs and alates was substantially lower than in workers, whereas the proportion of protist species exhibiting low abundance in workers was higher in nymphs and alates. Using a simulation-based approach, we demonstrate that such changes in the protist community composition of nymphs and alates improve the transmission efficiency of whole protist species communities. This study thus provides novel insights into how termites have maintained mutualistic relationships with diverse gut microbiota for generations.

Emergence of putative energy parasites within Clostridia revealed by genome analysis of a novel endosymbiotic clade.

The Clostridia is a dominant bacterial class in the guts of various animals and are considered to nutritionally contribute to the animal host. Here, we discovered clostridial endosymbionts of cellulolytic protists in termite guts, which have never been reported with evidence. We obtained (near-)complete genome sequences of three endosymbiotic Clostridia, each associated with a different parabasalid protist species with various infection rates: Trichonympha agilis, Pseudotrichonympha grassii, and Devescovina sp. All these protists are previously known to harbor permanently-associated, mutualistic Endomicrobia or Bacteroidales that supplement nitrogenous compounds. The genomes of the endosymbiotic Clostridia were small in size (1.0-1.3 Mbp) and exhibited signatures of an obligately-intracellular parasite, such as an extremely limited capability to synthesize amino acids, cofactors, and nucleotides and a disrupted glycolytic pathway with no known net ATP-generating system. Instead, the genomes encoded ATP/ADP translocase and, interestingly, regulatory proteins that are unique to eukaryotes in general and are possibly used to interfere with host cellular processes. These three genomes formed a clade with metagenome-assembled genomes (MAGs) derived from the guts of other animals, including human and ruminants, and the MAGs shared the characteristics of parasites. Gene flux analysis suggested that the acquisition of the ATP/ADP translocase gene in a common ancestor was probably key to the emergence of this parasitic clade. Taken together, we provide novel insights into the multilayered symbiotic system in the termite gut by adding the presence of parasitism and present an example of the emergence of putative energy parasites from a dominant gut bacterial clade.

Insights into the physiological and genomic characterization of three bacterial isolates from a highly alkaline, terrestrial serpentinizing system.

The terrestrial serpentinite-hosted ecosystem known as "The Cedars" is home to a diverse microbial community persisting under highly alkaline (pH ~ 12) and reducing (Eh < -550 mV) conditions. This extreme environment presents particular difficulties for microbial life, and efforts to isolate microorganisms from The Cedars over the past decade have remained challenging. Herein, we report the initial physiological assessment and/or full genomic characterization of three isolates: Paenibacillus sp. Cedars ('Paeni-Cedars'), Alishewanella sp. BS5-314 ('Ali-BS5-314'), and Anaerobacillus sp. CMMVII ('Anaero-CMMVII'). Paeni-Cedars is a Gram-positive, rod-shaped, mesophilic facultative anaerobe that grows between pH 7-10 (minimum pH tested was 7), temperatures 20-40°C, and 0-3% NaCl concentration. The addition of 10-20 mM CaCl2 enhanced growth, and iron reduction was observed in the following order, 2-line ferrihydrite > magnetite > serpentinite ~ chromite ~ hematite. Genome analysis identified genes for flavin-mediated iron reduction and synthesis of a bacillibactin-like, catechol-type siderophore. Ali-BS5-314 is a Gram-negative, rod-shaped, mesophilic, facultative anaerobic alkaliphile that grows between pH 10-12 and temperatures 10-40°C, with limited growth observed 1-5% NaCl. Nitrate is used as a terminal electron acceptor under anaerobic conditions, which was corroborated by genome analysis. The Ali-BS5-314 genome also includes genes for benzoate-like compound metabolism. Anaero-CMMVII remained difficult to cultivate for physiological studies; however, growth was observed between pH 9-12, with the addition of 0.01-1% yeast extract. Anaero-CMMVII is a probable oxygen-tolerant anaerobic alkaliphile with hydrogenotrophic respiration coupled with nitrate reduction, as determined by genome analysis. Based on single-copy genes, ANI, AAI and dDDH analyses, Paeni-Cedars and Ali-BS5-314 are related to other species (P. glucanolyticus and A. aestuarii, respectively), and Anaero-CMMVII represents a new species. The characterization of these three isolates demonstrate the range of ecophysiological adaptations and metabolisms present in serpentinite-hosted ecosystems, including mineral reduction, alkaliphily, and siderophore production.

Toward the functional analysis of uncultivable, symbiotic microorganisms in the termite gut.

Termites thrive on dead plant matters with the aid of microorganisms resident in their gut. The gut microbiota comprises protists (single-celled eukaryotes), bacteria, and archaea, most of which are unique to the termite gut ecosystem. Although this symbiosis has long been intriguing researchers of both basic and applied sciences, its detailed mechanism remains unclear due to the enormous complexity and the unculturability of the microbiota. In the effort to overcome the difficulty, recent advances in omics, such as metagenomics, metatranscriptomics, and metaproteomics have gradually unveiled the black box of this symbiotic system. Genomics targeting a single species of the unculturable microbial members has also provided a great progress in the understanding of the symbiotic interrelationships among the gut microorganisms. In this review, the symbiotic system organized by wood-feeding termites and their gut microorganisms is outlined, focusing on the recent achievement in omics studies of this multilayered symbiotic system.

Improving the nutritional value and bioactivity of soybean meal in solid-state fermentation using Bacillus strains newly isolated from the gut of the termite Termes propinquus.

The present study aimed to isolate and characterize proteolytic Bacillus spp. from termite guts to test the possibility of application for improving the nutritional value and bioactivity of fermented soybean meal (FSBM). Aerobic endospore-forming bacteria were isolated from the gut of the termite Termes propinquus. Ten isolates with high levels of soy milk degradation were selected and tested for extracellular enzyme production. Among them, two isolates, Tp-5 and Tp-7, exhibited all tested hydrolytic enzyme activities (cellulase, xylanase, pectinase, amylase, protease, lipase and phytase), weak alpha hemolytic and also antagonistic activities against fish pathogenic species of Aeromonas and Streptococcus. Both phylogenetic and biochemical analyses indicated that they were closely related to Bacillus amyloliquefaciens. During solid-state fermentation of SBM, Tp-5 and Tp-7 exhibited the highest protease activity (1127.2 and 1552.4 U g-1, respectively) at 36 h, and the resulting FSBMs showed a significant increase in crude protein content and free radical-scavenging ability (P < 0.05), as well as an improvement in the composition of amino acids, metabolites and other nutrients, while indigestible materials such as fiber, lignin and hemicellulose were decreased. The potential strains, especially Tp-7, improved the nutritional value of FSBM by their strong hydrolytic and antioxidant activities, together with reducing antinutritional components.

Agarose gel microcapsules enable easy-to-prepare, picolitre-scale, single-cell genomics, yielding high-coverage genome sequences.

A novel type of agarose gel microcapsule (AGM), consisting of an alginate picolitre sol core and an agarose gel shell, was developed to obtain high-quality, single-cell, amplified genomic DNA of bacteria. The AGM is easy to prepare in a stable emulsion with oil of water-equivalent density, which prevents AGM aggregation, with only standard laboratory equipment. Single cells from a pure culture of Escherichia coli, a mock community comprising 15 strains of human gut bacteria, and a termite gut bacterial community were encapsulated within AGMs, and their genomic DNA samples were prepared with massively parallel amplifications in a tube. The genome sequencing did not need second-round amplification and showed an average genome completeness that was much higher than that obtained using a conventional amplification method on the microlitre scale, regardless of the genomic guanine-cytosine content. Our novel method using AGM will allow many researchers to perform single-cell genomics easily and effectively, and can accelerate genomic analysis of yet-uncultured microorganisms.

Coevolution of Metabolic Pathways in Blattodea and Their Blattabacterium Endosymbionts, and Comparisons with Other Insect-Bacteria Symbioses.

Many insects harbor bacterial endosymbionts that supply essential nutrients and enable their hosts to thrive on a nutritionally unbalanced diet. Comparisons of the genomes of endosymbionts and their insect hosts have revealed multiple cases of mutually-dependent metabolic pathways that require enzymes encoded in 2 genomes. Complementation of metabolic reactions at the pathway level has been described for hosts feeding on unbalanced diets, such as plant sap. However, the level of collaboration between symbionts and hosts that feed on more variable diets is largely unknown. In this study, we investigated amino acid and vitamin/cofactor biosynthetic pathways in Blattodea, which comprises cockroaches and termites, and their obligate endosymbiont Blattabacterium cuenoti (hereafter Blattabacterium). In contrast to other obligate symbiotic systems, we found no clear evidence of "collaborative pathways" for amino acid biosynthesis in the genomes of these taxa, with the exception of collaborative arginine biosynthesis in 2 taxa, Cryptocercus punctulatus and Mastotermes darwiniensis. Nevertheless, we found that several gaps specific to Blattabacterium in the folate biosynthetic pathway are likely to be complemented by their host. Comparisons with other insects revealed that, with the exception of the arginine biosynthetic pathway, collaborative pathways for essential amino acids are only observed in phloem-sap feeders. These results suggest that the host diet is an important driving factor of metabolic pathway evolution in obligate symbiotic systems. IMPORTANCE The long-term coevolution between insects and their obligate endosymbionts is accompanied by increasing levels of genome integration, sometimes to the point that metabolic pathways require enzymes encoded in two genomes, which we refer to as "collaborative pathways". To date, collaborative pathways have only been reported from sap-feeding insects. Here, we examined metabolic interactions between cockroaches, a group of detritivorous insects, and their obligate endosymbiont, Blattabacterium, and only found evidence of collaborative pathways for arginine biosynthesis. The rarity of collaborative pathways in cockroaches and Blattabacterium contrasts with their prevalence in insect hosts feeding on phloem-sap. Our results suggest that host diet is a factor affecting metabolic integration in obligate symbiotic systems.

Complete genome of the uncultured Termite Group 1 bacteria in a single host protist cell.

Termites harbor a symbiotic gut microbial community that is responsible for their ability to thrive on recalcitrant plant matter. The community comprises diverse microorganisms, most of which are as yet uncultivable; the detailed symbiotic mechanism remains unclear. Here, we present the first complete genome sequence of a termite gut symbiont-an uncultured bacterium named Rs-D17 belonging to the candidate phylum Termite Group 1 (TG1). TG1 is a dominant group in termite guts, found as intracellular symbionts of various cellulolytic protists, without any physiological information. To acquire the complete genome sequence, we collected Rs-D17 cells from only a single host protist cell to minimize their genomic variation and performed isothermal whole-genome amplification. This strategy enabled us to reconstruct a circular chromosome (1,125,857 bp) encoding 761 putative protein-coding genes. The genome additionally contains 121 pseudogenes assigned to categories, such as cell wall biosynthesis, regulators, transporters, and defense mechanisms. Despite its apparent reductive evolution, the ability to synthesize 15 amino acids and various cofactors is retained, some of these genes having been duplicated. Considering that diverse termite-gut protists harbor TG1 bacteria, we suggest that this bacterial group plays a key role in the gut symbiotic system by stably supplying essential nitrogenous compounds deficient in lignocelluloses to their host protists and the termites. Our results provide a breakthrough to clarify the functions of and the interactions among the individual members of this multilayered symbiotic complex.

Diversity and genomes of uncultured microbial symbionts in the termite gut.

Termites play a key role in the global carbon cycle as decomposers. Their ability to thrive solely on dead plant matter is chiefly attributable to the activities of gut microbes, which comprise protists, bacteria, and archaea. Although the majority of the gut microbes are as yet unculturable, molecular analyses have gradually been revealing their diversity and symbiotic mechanisms. Culture-independent studies indicate that a single termite species harbors several hundred species of gut microbes unique to termites, and that the microbiota is consistent within a host termite species. To elucidate the functions of these unculturable symbionts, environmental genomics has recently been applied. Particularly, single-species-targeting metagenomics has provided a breakthrough in the understanding of symbiotic roles, such as the nitrogen fixation, of uncultured, individual microbial species. A combination of single-species-targeting metagenomics, conventional metagenomics, and metatranscriptomics should be a powerful tool to dissect this complex, multi-layered symbiotic system.

Single-Cell Genomics of Novel Actinobacteria With the Wood-Ljungdahl Pathway Discovered in a Serpentinizing System.

Serpentinite-hosted systems represent modern-day analogs of early Earth environments. In these systems, water-rock interactions generate highly alkaline and reducing fluids that can contain hydrogen, methane, and low-molecular-weight hydrocarbons-potent reductants capable of fueling microbial metabolism. In this study, we investigated the microbiota of Hakuba Happo hot springs (∼50°C; pH∼10.5-11), located in Nagano (Japan), which are impacted by the serpentinization process. Analysis of the 16S rRNA gene amplicon sequences revealed that the bacterial community comprises Nitrospirae (47%), "Parcubacteria" (19%), Deinococcus-Thermus (16%), and Actinobacteria (9%), among others. Notably, only 57 amplicon sequence variants (ASV) were detected, and fifteen of these accounted for 90% of the amplicons. Among the abundant ASVs, an early-branching, uncultivated actinobacterial clade identified as RBG-16-55-12 in the SILVA database was detected. Ten single-cell genomes (average pairwise nucleotide identity: 0.98-1.00; estimated completeness: 33-93%; estimated genome size: ∼2.3 Mb) that affiliated with this clade were obtained. Taxonomic classification using single copy genes indicates that the genomes belong to the actinobacterial class-level clade UBA1414 in the Genome Taxonomy Database. Based on metabolic pathway predictions, these actinobacteria are anaerobes, capable of glycolysis, dissimilatory nitrate reduction and CO2 fixation via the Wood-Ljungdahl (WL) pathway. Several other genomes within UBA1414 and two related class-level clades also encode the WL pathway, which has not yet been reported for the Actinobacteria phylum. For the Hakuba actinobacterium, the energy metabolism related to the WL pathway is likely supported by a combination of the Rnf complex, group 3b and 3d [NiFe]-hydrogenases, [FeFe]-hydrogenases, and V-type (H+/Na+ pump) ATPase. The genomes also harbor a form IV ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, also known as a RubisCO-like protein, and contain signatures of interactions with viruses, including clustered regularly interspaced short palindromic repeat (CRISPR) regions and several phage integrases. This is the first report and detailed genome analysis of a bacterium within the Actinobacteria phylum capable of utilizing the WL pathway. The Hakuba actinobacterium is a member of the clade UBA1414/RBG-16-55-12, formerly within the group "OPB41." We propose to name this bacterium 'Candidatus Hakubanella thermoalkaliphilus.'

Parallel reductive genome evolution in Desulfovibrio ectosymbionts independently acquired by Trichonympha protists in the termite gut.

Several Trichonympha protist species in the termite gut have independently acquired Desulfovibrio ectosymbionts in apparently different stages of symbiosis. Here, we obtained the near-complete genome sequence of Desulfovibrio phylotype ZnDsv-02, which attaches to the surface of Trichonympha collaris cells, and compared it with a previously obtained genome sequence of 'Candidatus Desulfovibrio trichonymphae' phylotype Rs-N31, which is almost completely embedded in the cytoplasm of Trichonympha agilis. Single-nucleotide polymorphism analysis indicated that although Rs-N31 is almost clonal, the ZnDsv-02 population on a single host cell is heterogeneous. Despite these differences, the genome of ZnDsv-02 has been reduced to 1.6 Mb, which is comparable to that of Rs-N31 (1.4 Mb), but unlike other known ectosymbionts of protists with a genome similar in size to their free-living relatives. Except for the presence of a lactate utilization pathway, cell-adhesion components and anti-phage defense systems in ZnDsv-02, the overall gene-loss pattern between the two genomes is very similar, including the loss of genes responsive to environmental changes. Our study suggests that genome reduction can occur in ectosymbionts, even when they can be transmitted horizontally and obtain genes via lateral transfer, and that the symbiont genome size depends heavily on their role in the symbiotic system.

Increased Mutation Rate Is Linked to Genome Reduction in Prokaryotes.

The evolutionary processes that drive variation in genome size across the tree of life remain unresolved. Effective population size (Ne) is thought to play an important role in shaping genome size [1-3]-a key example being the reduced genomes of insect endosymbionts, which undergo population bottlenecks during transmission [4]. However, the existence of reduced genomes in marine and terrestrial prokaryote species with large Ne indicate that genome reduction is influenced by multiple processes [3]. One candidate process is enhanced mutation rate, which can increase adaptive capacity but can also promote gene loss. To investigate evolutionary forces associated with prokaryotic genome reduction, we performed molecular evolutionary and phylogenomic analyses of nine lineages from five bacterial and archaeal phyla. We found that gene-loss rate strongly correlated with synonymous substitution rate (a proxy for mutation rate) in seven of the nine lineages. However, gene-loss rate showed weak or no correlation with the ratio of nonsynonymous/synonymous substitution rate (dN/dS). These results indicate that genome reduction is largely associated with increased mutation rate, while the association between gene loss and changes in Ne is less well defined. Lineages with relatively high dS and dN, as well as smaller genomes, lacked multiple DNA repair genes, providing a proximate cause for increased mutation rates. Our findings suggest that similar mechanisms drive genome reduction in both intracellular and free-living prokaryotes, with implications for developing a comprehensive theory of prokaryote genome size evolution.