Genomic erosion and extensive horizontal gene transfer in gut-associated Acetobacteraceae.

BACKGROUND: Symbiotic relationships between animals and bacteria have profound impacts on the evolutionary trajectories of each partner. Animals and gut bacteria engage in a variety of relationships, occasionally persisting over evolutionary timescales. Ants are a diverse group of animals that engage in many types of associations with taxonomically distinct groups of bacterial associates. Here, we bring into culture and characterize two closely-related strains of gut associated Acetobacteraceae (AAB) of the red carpenter ant, Camponotus chromaiodes. RESULTS: Genome sequencing, assembly, and annotation of both strains delineate stark patterns of genomic erosion and sequence divergence in gut associated AAB. We found widespread horizontal gene transfer (HGT) in these bacterial associates and report elevated gene acquisition associated with energy production and conversion, amino acid and coenzyme transport and metabolism, defense mechanisms, and lysine export. Both strains have acquired the complete NADH-quinone oxidoreductase complex, plausibly from an Enterobacteriaceae origin, likely facilitating energy production under diverse conditions. Conservation of several lysine biosynthetic and salvage pathways and accumulation of lysine export genes via HGT implicate L-lysine supplementation by both strains as a potential functional benefit for the host. These trends are contrasted by genome-wide erosion of several amino acid biosynthetic pathways and pathways in central metabolism. We perform phylogenomic analyses on both strains as well as several free living and host associated AAB. Based on their monophyly and deep divergence from other AAB, these C. chromaiodes gut associates may represent a novel genus. Together, our results demonstrate how extensive horizontal transfer between gut associates along with genome-wide deletions leads to mosaic metabolic pathways. More broadly, these patterns demonstrate that HGT and genomic erosion shape metabolic capabilities of persistent gut associates and influence their genomic evolution. CONCLUSIONS: Using comparative genomics, our study reveals substantial changes in genomic content in persistent associates of the insect gastrointestinal tract and provides evidence for the evolutionary pressures inherent to this environment. We describe patterns of genomic erosion and horizontal acquisition that result in mosaic metabolic pathways. Accordingly, the phylogenetic position of both strains of these associates form a divergent, monophyletic clade sister to gut associates of honey bees and more distantly to Gluconobacter.

In it for the long haul: evolutionary consequences of persistent endosymbiosis.

Phylogenetically independent bacterial lineages have undergone a profound lifestyle shift: from a free-living to obligately host-associated existence. Among these lineages, intracellular bacterial mutualists of insects are among the most intimate, constrained symbioses known. These obligate endosymbionts exhibit severe gene loss and apparent genome deterioration. Evolutionary theory provides a basis to link their unusual genomic features with shifts in fundamental mechanisms - selection, genetic drift, mutation, and recombination. This mini-review highlights recent comparative and experimental research of processes shaping ongoing diversification within these ancient associations. Recent work supports clear contributions of stochastic processes, including genetic drift and exceptionally strong mutational pressure, toward degenerative evolution. Despite possible compensatory mechanisms, genome degradation may constrain how persistent endosymbionts (and their hosts) respond to environmental fluctuations.

Ancient bacterial endosymbionts of insects: Genomes as sources of insight and springboards for inquiry.

Ancient associations between insects and bacteria provide models to study intimate host-microbe interactions. Currently, a wealth of genome sequence data for long-term, obligately intracellular (primary) endosymbionts of insects reveals profound genomic consequences of this specialized bacterial lifestyle. Those consequences include severe genome reduction and extreme base compositions. This minireview highlights the utility of genome sequence data to understand how, and why, endosymbionts have been pushed to such extremes, and to illuminate the functional consequences of such extensive genome change. While the static snapshots provided by individual endosymbiont genomes are valuable, comparative analyses of multiple genomes have shed light on evolutionary mechanisms. Namely, genome comparisons have told us that selection is important in fine-tuning gene content, but at the same time, mutational pressure and genetic drift contribute to genome degradation. Examples from Blochmannia, the primary endosymbiont of the ant tribe Camponotini, illustrate the value and constraints of genome sequence data, and exemplify how genomes can serve as a springboard for further comparative and experimental inquiry.

Deep divergence and rapid evolutionary rates in gut-associated Acetobacteraceae of ants.

BACKGROUND: Symbiotic associations between gut microbiota and their animal hosts shape the evolutionary trajectories of both partners. The genomic consequences of these relationships are significantly influenced by a variety of factors, including niche localization, interaction potential, and symbiont transmission mode. In eusocial insect hosts, socially transmitted gut microbiota may represent an intermediate point between free living or environmentally acquired bacteria and those with strict host association and maternal transmission. RESULTS: We characterized the bacterial communities associated with an abundant ant species, Camponotus chromaiodes. While many bacteria had sporadic distributions, some taxa were abundant and persistent within and across ant colonies. Specially, two Acetobacteraceae operational taxonomic units (OTUs; referred to as AAB1 and AAB2) were abundant and widespread across host samples. Dissection experiments confirmed that AAB1 and AAB2 occur in C. chromaiodes gut tracts. We explored the distribution and evolution of these Acetobacteraceae OTUs in more depth. We found that Camponotus hosts representing different species and geographical regions possess close relatives of the Acetobacteraceae OTUs detected in C. chromaiodes. Phylogenetic analysis revealed that AAB1 and AAB2 join other ant associates in a monophyletic clade. This clade consists of Acetobacteraceae from three ant tribes, including a third, basal lineage associated with Attine ants. This ant-specific AAB clade exhibits a significant acceleration of substitution rates at the 16S rDNA gene and elevated AT content. Substitutions along 16S rRNA in AAB1 and AAB2 result in ~10 % reduction in the predicted rRNA stability. CONCLUSIONS: Combined, these patterns in Camponotus-associated Acetobacteraceae resemble those found in cospeciating gut associates that are both socially and maternally transmitted. These associates may represent an intermediate point along an evolutionary trajectory manifest most extremely in symbionts with strict maternal transmission. Collectively, these results suggest that Acetobacteraceae may be a frequent and persistent gut associate in Camponotus species and perhaps other ant groups, and that its evolution is strongly impacted by this host association.

Genome evolution in an ancient bacteria-ant symbiosis: parallel gene loss among Blochmannia spanning the origin of the ant tribe Camponotini.

Stable associations between bacterial endosymbionts and insect hosts provide opportunities to explore genome evolution in the context of established mutualisms and assess the roles of selection and genetic drift across host lineages and habitats. Blochmannia, obligate endosymbionts of ants of the tribe Camponotini, have coevolved with their ant hosts for ∼40 MY. To investigate early events in Blochmannia genome evolution across this ant host tribe, we sequenced Blochmannia from two divergent host lineages, Colobopsis obliquus and Polyrhachis turneri, and compared them with four published genomes from Blochmannia of Camponotus sensu stricto. Reconstructed gene content of the last common ancestor (LCA) of these six Blochmannia genomes is reduced (690 protein coding genes), consistent with rapid gene loss soon after establishment of the symbiosis. Differential gene loss among Blochmannia lineages has affected cellular functions and metabolic pathways, including DNA replication and repair, vitamin biosynthesis and membrane proteins. Blochmannia of P. turneri (i.e., B. turneri) encodes an intact DnaA chromosomal replication initiation protein, demonstrating that loss of dnaA was not essential for establishment of the symbiosis. Based on gene content, B. obliquus and B. turneri are unable to provision hosts with riboflavin. Of the six sequenced Blochmannia, B. obliquus is the earliest diverging lineage (i.e., the sister group of other Blochmannia sampled) and encodes the fewest protein-coding genes and the most pseudogenes. We identified 55 genes involved in parallel gene loss, including glutamine synthetase, which may participate in nitrogen recycling. Pathways for biosynthesis of coenzyme A, terpenoids and riboflavin were lost in multiple lineages, suggesting relaxed selection on the pathway after inactivation of one component. Analysis of Illumina read datasets did not detect evidence of plasmids encoding missing functions, nor the presence of coresident symbionts other than Wolbachia. Although gene order is strictly conserved in four Blochmannia of Camponotus sensu stricto, comparisons with deeply divergent lineages revealed inversions in eight genomic regions, indicating ongoing recombination despite ancestral loss of recA. In sum, the addition of two Blochmannia genomes of divergent host lineages enables reconstruction of early events in evolution of this symbiosis and suggests that Blochmannia lineages may experience distinct, host-associated selective pressures. Understanding how evolutionary forces shape genome reduction in this system may help to clarify forces driving gene loss in other bacteria, including intracellular pathogens.

Endosymbiont evolution: predictions from theory and surprises from genomes.

Genome data have created new opportunities to untangle evolutionary processes shaping microbial variation. Among bacteria, long-term mutualists of insects represent the smallest and (typically) most AT-rich genomes. Evolutionary theory provides a context to predict how an endosymbiotic lifestyle may alter fundamental evolutionary processes--mutation, selection, genetic drift, and recombination--and thus contribute to extreme genomic outcomes. These predictions can then be explored by comparing evolutionary rates, genome size and stability, and base compositional biases across endosymbiotic and free-living bacteria. Recent surprises from such comparisons include genome reduction among uncultured, free-living species. Some studies suggest that selection generally drives this streamlining, while drift drives genome reduction in endosymbionts; however, this remains an hypothesis requiring additional data. Unexpected evidence of selection acting on endosymbiont GC content hints that even weak selection may be effective in some long-term mutualists. Moving forward, intraspecific analysis offers a promising approach to distinguish underlying mechanisms, by testing the null hypothesis of neutrality and by quantifying mutational spectra. Such analyses may clarify whether endosymbionts and free-living bacteria occupy distinct evolutionary trajectories or, alternatively, represent varied outcomes of similar underlying forces.

Thermally adaptive tradeoffs in closely related marine bacterial strains.

Time series studies have shown that some bacterial taxa occur only at specific times of the year while others are ubiquitous in spite of seasonal shifts in environmental variables. Here, we ask if these ubiquitous clades are generalists that grow over a wide range of environmental conditions, or clusters of strain-level environmental specialists. To answer this question, vibrio strains isolated at a coastal time series were phylogenetically and physiologically characterized revealing three dominant strategies within the vibrio: mesophiles, psychrophiles and apparently generalist broad thermal range clades. Thermal performance curves from laboratory growth rate experiments help explain field observations of relative abundances: the mesophilic clade grows optimally at temperatures 16°C higher than the psychrophilic clade. Strains in the broad thermal range clade all have similar optimal growth temperatures but also exhibit temperature-related tradeoffs with faster growth rates for warm temperature strains and broader growth ranges for strains from cool temperatures. Moreover, the mechanisms of thermal adaptation apparently differ based on evolutionary time scales: shifts in the temperature of maximal growth occur between deeply branching clades but thermal performance curve shape changes on shorter time scales. Thus, apparently ubiquitous clades are likely not generalists, but contain subclusters with distinct environmental preferences.

First impressions in a glowing host-microbe partnership.

Despite the clear significance of beneficial animal-microbe associations, mechanisms underlying their initiation and establishment are rarely understood. In this issue of Cell Host & Microbe, Kremer et al. (2013) reveal that first contact within the squid-vibrio symbiosis triggers profound molecular and chemical changes that are crucial for bacterial colonization.

Can't take the heat: high temperature depletes bacterial endosymbionts of ants.

Members of the ant tribe Camponotini have coevolved with Blochmannia, an obligate intracellular bacterial mutualist. This endosymbiont lives within host bacteriocyte cells that line the ant midgut, undergoes maternal transmission from host queens to offspring, and contributes to host nutrition via nitrogen recycling and nutrient biosynthesis. While elevated temperature has been shown to disrupt obligate bacterial mutualists of some insects, its impact on the ant-Blochmannia partnership is less clear. Here, we test the effect of heat on the density of Blochmannia in two related Camponotus species in the lab. Transcriptionally active Blochmannia were quantified using RT-qPCR as the ratio of Blochmannia 16S rRNA to ant host elongation factor 1-α transcripts. Our results showed that 4 weeks of heat treatment depleted active Blochmannia by >99 % in minor workers and unmated queens. However, complete elimination of Blochmannia transcripts rarely occurred, even after 16 weeks of heat treatment. Possible mechanisms of observed thermal sensitivity may include extreme AT-richness and related features of Blochmannia genomes, as well as host stress responses. Broadly, the observed depletion of an essential microbial mutualist in heat-treated ants is analogous to the loss of zooanthellae during coral bleaching. While the ecological relevance of Blochmannia's thermal sensitivity is uncertain, our results argue that symbiont dynamics should be part of models predicting how ants and other animals will respond and adapt to a warming climate.

Sequence context of indel mutations and their effect on protein evolution in a bacterial endosymbiont.

Indel mutations play key roles in genome and protein evolution, yet we lack a comprehensive understanding of how indels impact evolutionary processes. Genome-wide analyses enabled by next-generation sequencing can clarify the context and effect of indels, thereby integrating a more detailed consideration of indels with our knowledge of nucleotide substitutions. To this end, we sequenced Blochmannia chromaiodes, an obligate bacterial endosymbiont of carpenter ants, and compared it with the close relative, B. pennsylvanicus. The genetic distance between these species is small enough for accurate whole genome alignment but large enough to provide a meaningful spectrum of indel mutations. We found that indels are subjected to purifying selection in coding regions and even intergenic regions, which show a reduced rate of indel base pairs per kilobase compared with nonfunctional pseudogenes. Indels occur almost exclusively in repeat regions composed of homopolymers and multimeric simple sequence repeats, demonstrating the importance of sequence context for indel mutations. Despite purifying selection, some indels occur in protein-coding genes. Most are multiples of three, indicating selective pressure to maintain the reading frame. The deleterious effect of frameshift-inducing indels is minimized by either compensation from a nearby indel to restore reading frame or the indel's location near the 3'-end of the gene. We observed amino acid divergence exceeding nucleotide divergence in regions affected by frameshift-inducing indels, suggesting that these indels may either drive adaptive protein evolution or initiate gene degradation. Our results shed light on how indel mutations impact processes of molecular evolution underlying endosymbiont genome evolution.

Animals in a bacterial world, a new imperative for the life sciences.

In the last two decades, the widespread application of genetic and genomic approaches has revealed a bacterial world astonishing in its ubiquity and diversity. This review examines how a growing knowledge of the vast range of animal-bacterial interactions, whether in shared ecosystems or intimate symbioses, is fundamentally altering our understanding of animal biology. Specifically, we highlight recent technological and intellectual advances that have changed our thinking about five questions: how have bacteria facilitated the origin and evolution of animals; how do animals and bacteria affect each other's genomes; how does normal animal development depend on bacterial partners; how is homeostasis maintained between animals and their symbionts; and how can ecological approaches deepen our understanding of the multiple levels of animal-bacterial interaction. As answers to these fundamental questions emerge, all biologists will be challenged to broaden their appreciation of these interactions and to include investigations of the relationships between and among bacteria and their animal partners as we seek a better understanding of the natural world.

Proteomic analysis of an unculturable bacterial endosymbiont (Blochmannia) reveals high abundance of chaperonins and biosynthetic enzymes.

Many insect groups have coevolved with bacterial endosymbionts that live within specialized host cells. As a salient example, ants in the tribe Camponotini rely on Blochmannia, an intracellular bacterial mutualist that synthesizes amino acids and recycles nitrogen for the host. We performed a shotgun, label-free, LC/MS/MS quantitative proteomic analysis to investigate the proteome of Blochmannia associated with Camponotus chromaiodes. We identified more than 330 Blochmannia proteins, or 54% coverage of the predicted proteome, as well as 244 Camponotus proteins. Using the average intensity of the top 3 "best flier" peptides along with spiking of a surrogate standard at a known concentration, we estimated the concentration (fmol/µg) of those proteins with confident identification. The estimated dynamic range of Blochmannia protein abundance spanned 3 orders of magnitude and covered diverse functional categories, with particularly high representation of metabolism, information transfer, and chaperones. GroEL, the most abundant protein, totaled 6% of Blochmannia protein abundance. Biosynthesis of essential amino acids, fatty acids, and nucleotides, and sulfate assimilation had disproportionately high coverage in the proteome, further supporting a nutritional role of the symbiosis. This first quantitative proteomic analysis of an ant endosymbiont illustrates a promising approach to study the functional basis of intimate symbioses.

Strategies of genomic integration within insect-bacterial mutualisms.

Insects, the most diverse group of macroorganisms with 900,000 known species, have been a rich playground for the evolution of symbiotic associations. Symbionts of this enormous animal group include a range of microbial partners. Insects are prone to establishing relationships with intracellular bacteria, which include the most intimate, highly integrated mutualisms known in the biological world. In recent years, an explosion of genomic studies has offered new insights into the molecular, functional, and evolutionary consequences of these insect-bacterial partnerships. In this review, I highlight some insights from genome sequences of bacterial endosymbionts and select insect hosts. Notably, comparisons between facultative and obligate bacterial mutualists have revealed distinct genome features representing different stages along a shared trajectory of genome reduction. Bacteria associated with the cedar aphid offer a snapshot of a transition from facultative to obligate mutualism, illustrating the genomic basis of this key step along the symbiotic spectrum. In addition, genomes of stable, dual bacterial symbionts reflect independent instances of astonishing metabolic integration. In these systems, synthesis of key nutrients, and perhaps basic cellular processes, require collaboration among co-residing bacteria and their insect host. These findings provide a launching point for a new era of genomic explorations of bacterial-animal symbioses. Future studies promise to reveal symbiotic strategies across a broad ecological and phylogenetic range, to clarify key transitions along a spectrum of interaction types, and to fuel new experimental approaches to dissect the mechanistic basis of intimate host-symbiont associations.

Endosymbiosis.

The phenomenon of endosymbiosis, or one organism living within another, has deeply impacted the evolution of life and continues to shape the ecology of countless species. Traditionally, biologists have viewed evolution as a largely bifurcating pattern, reflecting mutations and other changes in existing genetic information and the occasional speciation and divergence of lineages. While lineage bifurcation has clearly been important in evolution, the merging of two lineages through endosymbiosis has also made profound contributions to evolutionary novelty. Mitochondria and chloroplasts are relicts of ancient bacterial endosymbionts that ultimately extended the range of acceptable habitats for life by allowing hosts to thrive in the presence of oxygen and to convert light into energy. Today, the sheer abundance of endosymbiotic relationships across diverse host lineages and habitats testifies to their continued significance.

Mutualism meltdown in insects: bacteria constrain thermal adaptation.

Predicting whether and how organisms will successfully cope with climate change presents critical questions for biologists and environmental scientists. Models require knowing how organisms interact with their abiotic environment, as well understanding biotic interactions that include a network of symbioses in which all species are embedded. Bacterial symbionts of insects offer valuable models to examine how microbes can facilitate and constrain adaptation to a changing environment. While some symbionts confer plasticity that accelerates adaptation, long-term bacterial mutualists of insects are characterized by tight lifestyle constraints, genome deterioration, and vulnerability to thermal stress. These essential bacterial partners are eliminated at high temperatures, analogous to the loss of zooanthellae during coral bleaching. Recent field-based studies suggest that thermal sensitivity of bacterial mutualists constrains insect responses. In this sense, highly dependent mutualisms may be the Achilles' heel of thermal responses in insects.

Purifying selection, sequence composition, and context-specific indel mutations shape intraspecific variation in a bacterial endosymbiont.

Comparative genomics of closely related bacterial strains can clarify mutational processes and selective forces that impact genetic variation. Among primary bacterial endosymbionts of insects, such analyses have revealed ongoing genome reduction, raising questions about the ultimate evolutionary fate of these partnerships. Here, we explored genomic variation within Blochmannia vafer, an obligate mutualist of the ant Camponotus vafer. Polymorphism analysis of the Illumina data set used previously for de novo assembly revealed a second Bl. vafer genotype. To determine why a single ant colony contained two symbiont genotypes, we examined polymorphisms in 12 C. vafer mitochondrial sequences assembled from the Illumina data; the spectrum of variants suggests that the colony contained two maternal lineages, each harboring a distinct Bl. vafer genotype. Comparing the two Bl. vafer genotypes revealed that purifying selection purged most indels and nonsynonymous differences from protein-coding genes. We also discovered that indels occur frequently in multimeric simple sequence repeats, which are relatively abundant in Bl. vafer and may play a more substantial role in generating variation in this ant mutualist than in the aphid endosymbiont Buchnera. Finally, we explored how an apparent relocation of the origin of replication in Bl. vafer and the resulting shift in strand-associated mutational pressures may have caused accelerated gene loss and an elevated rate of indel polymorphisms in the region spanning the origin relocation. Combined, these results point to significant impacts of purifying selection on genomic polymorphisms as well as distinct patterns of indels associated with unusual genomic features of Blochmannia.

Reduced selective constraint in endosymbionts: elevation in radical amino acid replacements occurs genome-wide.

As predicted by the nearly neutral model of evolution, numerous studies have shown that reduced N(e) accelerates the accumulation of slightly deleterious changes under genetic drift. While such studies have mostly focused on eukaryotes, bacteria also offer excellent models to explore the effects of N(e). Most notably, the genomes of host-dependent bacteria with small N(e) show signatures of genetic drift, including elevated K(a)/K(s). Here, I explore the utility of an alternative measure of selective constraint: the per-site rate of radical and conservative amino acid substitutions (D(r)/D(c)). I test the hypothesis that purifying selection against radical amino acid changes is less effective in two insect endosymbiont groups (Blochmannia of ants and Buchnera of aphids), compared to related gamma-Proteobacteria. Genome comparisons demonstrate a significant elevation in D(r)/D(c) in endosymbionts that affects the majority (66-79%) of shared orthologs examined. The elevation of D(r)/D(c) in endosymbionts affects all functional categories examined. Simulations indicate that D(r)/D(c) estimates are sensitive to codon frequencies and mutational parameters; however, estimation biases occur in the opposite direction as the patterns observed in genome comparisons, thereby making the inference of elevated D(r)/D(c) more conservative. Increased D(r)/D(c) and other signatures of genome degradation in endosymbionts are consistent with strong effects of genetic drift in their small populations, as well as linkage to selected sites in these asexual bacteria. While relaxed selection against radical substitutions may contribute, genome-wide processes such as genetic drift and linkage best explain the pervasive elevation in D(r)/D(c) across diverse functional categories that include basic cellular processes. Although the current study focuses on a few bacterial lineages, it suggests D(r)/D(c) is a useful gauge of selective constraint and may provide a valuable alternative to K(a)/K(s) when high sequence divergences preclude estimates of K(s). Broader application of D(r)/D(c) will benefit from approaches less prone to estimation biases.

Unprecedented loss of ammonia assimilation capability in a urease-encoding bacterial mutualist.

BACKGROUND: Blochmannia are obligately intracellular bacterial mutualists of ants of the tribe Camponotini. Blochmannia perform key nutritional functions for the host, including synthesis of several essential amino acids. We used Illumina technology to sequence the genome of Blochmannia associated with Camponotus vafer. RESULTS: Although Blochmannia vafer retains many nutritional functions, it is missing glutamine synthetase (glnA), a component of the nitrogen recycling pathway encoded by the previously sequenced B. floridanus and B. pennsylvanicus. With the exception of Ureaplasma, B. vafer is the only sequenced bacterium to date that encodes urease but lacks the ability to assimilate ammonia into glutamine or glutamate. Loss of glnA occurred in a deletion hotspot near the putative replication origin. Overall, compared to the likely gene set of their common ancestor, 31 genes are missing or eroded in B. vafer, compared to 28 in B. floridanus and four in B. pennsylvanicus. Three genes (queA, visC and yggS) show convergent loss or erosion, suggesting relaxed selection for their functions. Eight B. vafer genes contain frameshifts in homopolymeric tracts that may be corrected by transcriptional slippage. Two of these encode DNA replication proteins: dnaX, which we infer is also frameshifted in B. floridanus, and dnaG. CONCLUSIONS: Comparing the B. vafer genome with B. pennsylvanicus and B. floridanus refines the core genes shared within the mutualist group, thereby clarifying functions required across ant host species. This third genome also allows us to track gene loss and erosion in a phylogenetic context to more fully understand processes of genome reduction.

Slip into something more functional: selection maintains ancient frameshifts in homopolymeric sequences.

Mutational hotspots offer significant sources of genetic variability upon which selection can act. However, with a few notable exceptions, we know little about the dynamics and fitness consequences of mutations in these regions. Here, we explore evolutionary forces shaping homopolymeric tracts that are especially vulnerable to slippage errors during replication and transcription. Such tracts are typically eliminated by selection from most bacterial sequences, yet persist in genomes of endosymbionts with small effective population sizes (N(e)) and biased base compositions. Focusing on Blochmannia, a bacterial endosymbiont of ants, we track the divergence of genes that contain frameshift mutations within long (9-11 bp) polyA or polyT tracts. Earlier experimental work documented that transcriptional slippage restores the reading frame in a fraction of messenger RNA molecules and thereby rescues the function of frameshifted genes. In this study, we demonstrate a surprising persistence of these frameshifts and associated tracts for millions of years. Across the genome of this ant mutualist, rates of indel mutation within homopolymeric tracts far exceed the synonymous mutation rate, indicating that long-term conservation of frameshifts within these tracts is inconsistent with neutrality. In addition, the homopolymeric tracts themselves are more conserved than expected by chance, given extensive neutral substitutions that occur elsewhere in the genes sampled. These data suggest an unexpected role for slippage-prone DNA tracts and highlight a new mechanism for their persistence. That is, when such tracts contain a frameshift, transcriptional slippage plays a critical role in rescuing gene function. In such cases, selection will purge nucleotide changes interrupting the slippery tract so that otherwise volatile sequences become frozen in evolutionary time. Although the advantage of the frameshift itself is less clear, it may offer a mechanism to lower effective gene expression by reducing but not eliminating transcripts that encode full-length proteins.

One nutritional symbiosis begat another: phylogenetic evidence that the ant tribe Camponotini acquired Blochmannia by tending sap-feeding insects.

BACKGROUND: Bacterial endosymbiosis has a recurring significance in the evolution of insects. An estimated 10-20% of insect species depend on bacterial associates for their nutrition and reproductive viability. Members of the ant tribe Camponotini, the focus of this study, possess a stable, intracellular bacterial mutualist. The bacterium, Blochmannia, was first discovered in Camponotus and has since been documented in a distinct subgenus of Camponotus, Colobopsis, and in the related genus Polyrhachis. However, the distribution of Blochmannia throughout the Camponotini remains in question. Documenting the true host range of this bacterial mutualist is an important first step toward understanding the various ecological contexts in which it has evolved, and toward identifying its closest bacterial relatives. In this study, we performed a molecular screen, based on PCR amplification of 16S rDNA, to identify bacterial associates of diverse Camponotini species. RESULTS: Phylogenetic analyses of 16S rDNA gave four important insights: (i) Blochmannia occurs in a broad range of Camponotini genera including Calomyrmex, Echinopla, and Opisthopsis, and did not occur in outgroups related to this tribe (e.g., Notostigma). This suggests that the mutualism originated in the ancestor of the tribe Camponotini. (ii) The known bacteriocyte-associated symbionts of ants, in Formica, Plagiolepis, and the Camponotini, arose independently. (iii) Blochmannia is nestled within a diverse clade of endosymbionts of sap-feeding hemipteran insects, such as mealybugs, aphids, and psyllids. In our analyses, a group of secondary symbionts of mealybugs are the closest relatives of Blochmannia. (iv) Blochmannia has cospeciated with its known hosts, although deep divergences at the genus level remain uncertain. CONCLUSIONS: The Blochmannia mutualism occurs in Calomyrmex, Echinopla, and Opisthopsis, in addition to Camponotus, and probably originated in the ancestral lineage leading to the Camponotini. This significant expansion of its known host range implies that the mutualism is more ancient and ecologically diverse than previously documented. Blochmannia is most closely related to endosymbionts of sap-feeding hemipterans, which ants tend for their carbohydrate-rich honeydew. Based on phylogenetic results, we propose Camponotini might have originally acquired this bacterial mutualist through a nutritional symbiosis with other insects.