Bacterial gut symbionts are tightly linked with the evolution of herbivory in ants
Abstract
Ants are a dominant feature of terrestrial ecosystems, yet we know little about the forces that drive their evolution. Recent findings illustrate that their diets range from herbivorous to predaceous, with “herbivores” feeding primarily on exudates from plants and sap-feeding insects. Persistence on these nitrogen-poor food sources raises the question of how ants obtain sufficient nutrition. To investigate the potential role of symbiotic microbes, we have surveyed 283 species from 18 of the 21 ant subfamilies using molecular techniques. Our findings uncovered a wealth of bacteria from across the ants. Notable among the surveyed hosts were herbivorous “turtle ants” from the related genera Cephalotes and Procryptocerus (tribe Cephalotini). These commonly harbored bacteria from ant-specific clades within the Burkholderiales, Pseudomonadales, Rhizobiales, Verrucomicrobiales, and Xanthomonadales, and studies of lab-reared Cephalotes varians characterized these microbes as symbiotic residents of ant guts. Although most of these symbionts were confined to turtle ants, bacteria from an ant-specific clade of Rhizobiales were more broadly distributed. Statistical analyses revealed a strong relationship between herbivory and the prevalence of Rhizobiales gut symbionts within ant genera. Furthermore, a consideration of the ant phylogeny identified at least five independent origins of symbioses between herbivorous ants and related Rhizobiales. Combined with previous findings and the potential for symbiotic nitrogen fixation, our results strongly support the hypothesis that bacteria have facilitated convergent evolution of herbivory across the ants, further implicating symbiosis as a major force in ant evolution.
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Identifying the mechanisms underlying adaptation and diversification is a central goal of evolutionary biology. Great strides have been made toward this end through the development of fast and affordable molecular tools, and the joint application of molecular phylogenetics and comparative methods (1–3). One unanticipated theme that has emerged from this work is that bacterial symbionts have played key roles in the evolution and diversification of eukaryotes, starting with endosymbiotic origins of mitochondria and chloroplasts (4). Bacterial symbionts are also prevalent among insects that feed on inaccessible or nutritionally marginal diets such as blood, wood, xylem, and phloem (5). Given the demonstrated nutritional roles of these bacteria, their near-ubiquity in insects that specialize on nutrient-poor diets, their long histories of coevolution, and the diversity of the many groups that harbor nutritional symbionts, it is clear that bacteria have had a strong impact on the dietary evolution and diversification of their insect hosts (6).
Symbiosis has played an integral role in the evolution of the ants (Hymenoptera: Formicidae). Throughout the course of their 115–168 million year history (7, 8), these diverse and ecologically dominant insects have repeatedly evolved symbiotic relationships with sap-feeding insects (9), plants (10), and microbes (11, 12, 13), including nitrogen-recycling and upgrading Blochmannia species harbored by ants from the Camponotini (carpenter ants) (14, 15). With only a few exceptions outside of this group (16–20) and the tribe Attini (13), we know little about the identities and significance of bacteria across >12,000 described ant species. However, researchers have recently hypothesized nutritional roles for microbes in a number of ants (21), suggesting that bacteria have shaped the evolution of ant diets. Moreover, the discovery of bacteria in the guts of several exudate-feeding species (17, 22–24) suggests that ants represent an under-explored habitat for potentially unique microbial lineages.
Results and Discussion
Diverse and Bacterial Communities of Ants.
Through a combination of PCR amplification, sequencing, and DNA sequence analyses, we have examined the diversity and distributions of bacteria in ants collected from 375 colonies spanning over 283 species from 141 genera, 46 tribes, and 18 of the 21 subfamilies within the Formicidae (SI). Our approach focused on bacterial 16S rRNA genes: We first used universal primers (Table S1) to identify unique and potentially significant bacteria from a smaller subset of species; we then designed diagnostic primers (Table S1) that enabled us to explore the distributions of these bacteria across the full range of our collections.
Our initial sample comprised 52 ant species targeted with universal primers: 45 were chosen randomly, and seven were chosen after diagnostic screening identified them as potential hosts of gut symbionts (see SI for ants and selection criteria, see Table S2 for information on all sequences). The RDP II Classifier tool (25) grouped 258 bacterial 16S rRNA fragments from these ants into 16 distinct orders from nine classes and seven phyla (Fig. S1a). Overall, 155 sequences belonged to the Proteobacteria, making this the most abundantly represented phylum. The phylum Verrucomicrobia was also well represented, with 54 sequences, albeit from only two ant species targeted as hosts of gut symbionts. Out of 90 sequences from randomly selected ants, 31 (from 21 ant species) were classified to the genus Wolbachia (Proteobacteria: Alphaproteobacteria: Rickettsiales), reflecting previous findings of high Wolbachia prevalence across the ants (18–20).
Among 119 nonredundant (SI) 16S rRNA sequences obtained through the universal approach, the average divergence from their closest GenBank relatives was 5.2% (median = 4.5%). Furthermore, 75 (63%) of these groups differed from their nearest GenBank relatives by >3%, revealing that most ant-associated bacteria belong to divergent lineages and, potentially, to unique species (26).
Individual ants typically harbored a diverse assemblage of bacteria, consisting of microbes from several orders (Fig. S1b). To further assess within- and between-host bacterial diversity, we calculated uncorrected pairwise distance values for 103 cloned 16S rRNA sequences from four related ants from the tribe Cephalotini. The analyzed sequences (n = 12–38 per ant) grouped into 30 phylotypes, which we defined as exclusive groups with members sharing ≥97% sequence similarity (SI and Table S3). Between six and ten phylotypes were found in each individual ant host. Although the taxonomic distributions of the identified microbes revealed some similarities between ant species (Fig. S1b), only 2/30 (6.7%) phylotypes were found in more than one Cephalotini species. Furthermore, 20/30 (67%) phylotypes had just one representative among the 103 analyzed clones. Considering these two observations and our initially modest sampling within individuals and across species, it is clear that we have only begun to uncover the wealth of diverse microbial communities within the Formicidae (SI).
Identifying Unique, Ant-Specific Bacterial Lineages.
Among the microbes discovered through amplification and sequencing with universal bacterial primers, several grouped into ant-specific lineages in preliminary phylogenetic analyses. These included bacteria from the orders Rhizobiales, Burkholderiales, Xanthomonadales, and Verrucomicrobiales, along with a clade of microbes from an unclassified lineage of Gammaproteobacteria that consistently grouped with the Pseudomonadales.
To further investigate their prevalence and distributions, we designed diagnostic PCR primers (Table S1), targeting the identified ant-associates and their close relatives. Rhizobiales were the most prevalent and broadly distributed of the targeted lineages, associating with 5.2% (19/362) of the surveyed ants. Their hosts belonged to ≈13–19 species from nine genera, seven tribes, and three subfamilies within the family Formicidae (Table S4). Additional screening within the ant genera Dolichoderus (2 colonies: 10 workers each) and Tetraponera (1 colony: 11 workers) detected Rhizobiales in all adult workers (Table S4). Screening across 55 colonies of Cephalotes varians found similar evidence for these bacteria in 100% of adult workers (Table S5), suggesting that Rhizobiales are ubiquitous associates of the worker caste within some ant species.
Parsimony, likelihood, and Bayesian phylogenetic analyses each revealed that Rhizobiales bacteria identified in our study formed an ant-specific clade with previously identified bacteria from Tetraponera and Dolichoderus ants (Fig. 1) (16). This lineage was, itself, most closely related to microbes in the genus Bartonella and, more distantly, to mutualistic nitrogen-fixing rhizobia symbionts from leguminous plants. Within this exclusively ant-associated lineage, Rhizobiales from ants of the genus Tetraponera and the tribe Cephalotini (turtle ants) each formed well-supported monophyletic groups, suggesting diffusely coevolved and specialized relationships. But the lack of monophyly for Rhizobiales from other ant genera (Cardiocondyla, Dolichoderus, and Procryptocerus) indicated that these bacteria have undergone occasional, and possibly recent, host shifts. Perhaps most illustrative were Rhizobiales from two geographically isolated Cardiocondyla species (USA and Thailand), which belonged to two separate clades on the 16S rRNA phylogeny. Interestingly, the Thai strain was 0%–0.25% divergent in 16S rRNA sequence from Rhizobiales strains found in Tetramorium and Dolichoderus ants sampled from the same geographic location. So despite some evidence for specialized relationships, these latter findings suggest that Rhizobiales are occasionally acquired through: (i) horizontal transfer between species, or (ii) acquisition from free-living microbial populations found in unidentified environmental sources.
Fig. 1.
Compared with Rhizobiales, the other targeted bacteria were found at lower frequencies (Table S1 and Table S4). Verrucomicrobiales and Pseudomonadales lineages were the least abundant, associating with only 2.2% (4/183) and 2.7% (5/182) of the surveyed ants, whereas Xanthomonadales and Burkholderiales were respectively found in 3.2% (7/217) and 4.5% (10/224) of the surveyed ant collections. Most of these bacteria were found in ants from the tribe Cephalotini, typically coinhabiting individual hosts alongside Rhizobiales.
Like the Rhizobiales, phylogenetic analyses grouped nearly all of these microbes into ant-specific clades, including three within the Burkholderiales and one each within the three additional orders (Fig. S2). Analyses of uncorrected pairwise distance values mirrored our phylogenetic results, further illustrating that these ant-associated microbes were more closely related to each other than to bacteria from other environments (SI and Table S2). Combined with our findings on Rhizobiales, these results suggest loosely specialized relationships between ants and bacteria that bear resemblance to those between termites and their gut microbes (27).
Ant-Specific Bacteria Are Symbiotic Residents of Ant Guts.
To assess the localization, prevalence, and persistence of ant-specific bacteria, we performed diagnostic PCR screening on DNA extracted from multiple host tissues and from whole ants reared in the lab on artificial diets. Tissue-specific screening revealed that the targeted Burkholderiales, Pseudomonadales, Rhizobiales, Verrucomicrobiales, and Xanthomonadales microbes were confined to the guts of their hosts—they were not detectable in DNA extracted from head or other nongut tissues from multiple species (Table S4). These findings raise at least two possibilities: (i) these bacteria are transients acquired from their hosts' diets, or (ii) they are persistent, symbiotic bacteria that colonize the alimentary canal. To distinguish between these alternatives, we performed extensive PCR screening across lab-reared colonies of Cephalotes varians (SI and Table S5).
Our initial screens identified these bacteria in all, or nearly all, mature workers from 55 C. varians colonies reared on sugar water for only three weeks after field-collection (55/55 for Burkholderiales, Rhizobiales, and Xanthomonadales; 54/54 for Pseudomonadales; and 53/55 for Verrucomicrobiales). To measure symbiont persistence, we screened a total of 37 mature workers from 33 colonies after 11 months of lab rearing on sugar water diets (SI). With the exception of Pseudomonadales (34/37 workers), all microbes were detected in all surveyed individuals. Considering the duration of this persistence and the fact that these microbes are highly related to those from other, wild-caught Cephalotini, we surmise that these bacteria are symbiotic residents of ant guts.
Potential Routes of Bacterial Transmission in Cephalotes Varians.
PCR screening across different C. varians life stages provided clues about potential routes of bacterial transmission (Table S5). Transovarial transmission in this species can be ruled out, because we did not detect any of the five groups of microbes in surveys of small, early instar larvae. In contrast, each of the five bacterial groups was found in later instar larvae, suggesting that they are acquired at some point during juvenile development. Previous observations of workers from related C. rowheri showed that ant guts were microbe-free upon eclosion from the pupal stage (24); similarly, we failed to detect any of the five microbial lineages in C. varians pupae. Moreover, only half of recently eclosed, unmelanized adult workers (callows) tested positive for each of the five surveyed groups; the remaining young adults were symbiont-free.
These association patterns suggest that bacterial gut symbionts of Cephalotes varians are acquired during larval development, lost during pupation (likely caused by the shedding of the gut lining during the final larval molt), and then reacquired in adulthood. As previously suggested by Wheeler (28), the most likely route of within-colony transmission is oral-anal (abdominal) trophallaxis, a behavior that has previously been documented in C. varians (29). Because our findings revealed that queens are infected with all five bacterial groups (Table S5), we further hypothesize that between-colony spread is achieved primarily through phoresis with founding queens, followed by behavioral transmission to offspring produced early in the colony's development. Nevertheless, incongruent host and symbiont phylogenies (Fig. 1 and Fig. S2) indicate that these gut symbionts occasionally move through alternative routes.
Rhizobiales Distributions Are Correlated with Ant Trophic Level.
As noted, Rhizobiales were the most prevalent and broadly distributed of the identified bacterial groups (Fig. S2, Table S4, and Table S5), being generally found in groups of related ants. In addition to this phylogenetic trend, their distributions also showed a striking ecological pattern that was largely independent of their hosts' phylogeny. Specifically, 75% (14/19) of the ants harboring Rhizobiales [including Cataulacus (one species), Cephalotes (four species), Dolichoderus spp. (seven colonies), Procryptocerus batesi, and Tetraponera attenuata] belonged to clades of known exudate-feeding arboreal ants that have previously been shown to harbor gut bacteria (16, 17, 22–24). Because stable isotope measures had previously classified relatives of these hosts as herbivores (30, 31), our findings suggested a relationship between trophic level and the distribution of these symbionts.
To calibrate this relationship, we used stable isotope data from two previous publications that assessed the trophic position of ants by comparing the relative amounts of heavy and light nitrogen in ant tissues (15N/14N, calculated as δ15N) with those found in primary producers (low δ15N), herbivores (intermediate δ15N), and predators (high δ15N) from the same regions (30, 31). To compare between locations sampled in these studies (Peru, Brunei, and Australia), we separately calculated the average δ15N for plants (δ15Nplant) studied at each site. We then estimated the relative trophic level of ants compared with sympatric plants by computing δ15Nant − δ15Nplant for each ant species in each location. Using these values, we calculated the average δ15Nant − δ15Nplant value for each ant genus included in our Rhizobiales screen. These standardized averages were subsequently compared with within-genus Rhizobiales frequencies to determine the relationship between ant trophic level and symbiont prevalence (SI).
We found that congeneric ant relatives of Rhizobiales hosts are consistently found at the herbivorous end of the trophic scale (Fig. 2 and SI). The relationship between Rhizobiales frequencies and trophic level was highly significant according to logistic regression (regression equation: Y = 0.9625 - 0.8323 X; where X = δ15Nant − δ15Nplant averaged for each genus, and Y = ln[p/(1-p)], where P = probability of harboring Rhizobiales; R2 = 0.2392; pwhole-model <0.0001; pslope < 0.0001) and weighted regression statistics (regression line equation: Y = 0. 4952 − 0.0894 X, where Y = Rhizobiales frequency within genera and X = δ15Nant − δ15Nplant averaged for each genus; R2 = 0.3706; pwhole-model <0.0001; pslope < 0.0001).
Fig. 2.
A consideration of Rhizobiales bacteria distributions across the ant phylogeny revealed that this pattern is phylogenetically independent, as the five herbivorous host genera were not closely related (Fig. 3). Furthermore, a concentrated changes test (32) provided strong support for the correlated evolution between the presence of Rhizobiales symbionts and herbivorous diets across the ant phylogeny (P = 0.001), indicating that this association is not the result of a single, shared phylogenetic history. We, thus, conclude that herbivorous ants have independently evolved associations with related gut symbionts from the order Rhizobiales on at least five occasions throughout the history of the Formicidae.
Fig. 3.
Functional Roles for Symbiotic Gut Bacteria of Herbivorous Ants.
Although most ants are not herbivorous in a strict sense, those at the low end of the trophic scale feed largely on plant exudates and hemipteran excrement, or honeydew (31), and studies of their stable isotope signatures reveal that they obtain little nitrogen from predation or scavenging (30, 31). Because honeydew and plant exudates are rich in sugar but poor in essential amino acids (33–35), these ants may require additional nitrogen from alternative sources (30, 31, 36). Based on the incidence of gut microbes in exudate-feeding ants, Cook and Davidson hypothesized that they rely on the metabolic activities of bacteria to make up for their dietary shortcomings (21).
Nitrogen fixation has previously been proposed as a mechanism for dietary supplementation by bacterial symbionts (16). To examine this possibility, we screened for bacterial nifH genes in herbivorous ants. The nifH gene encodes the dinitrogenase reductase subunit of the nitrogen-fixing nitrogenase enzyme, which converts atmospheric N2 into NH3, a form of nitrogen that can be modified for use by eukaryotes. Stoll and colleagues first detected nifH sequences in ants from the genera Echinopla, Dolichoderus, and Tetraponera (16). Focusing on ants that hosted Rhizobiales, we amplified and sequenced nifH genes from a broader range of herbivorous ants, including Cephalotes, Dolichoderus, Procryptocerus, and Tetramorium species (SI and Table S2). We similarly identified a nifH gene from dissected C. varians gut tissues, suggesting that gut bacteria are capable of nitrogen fixation.
These results provide evidence of a means by which bacteria could compensate for the lack of sufficient nitrogen in their hosts' diets, suggesting that “herbivorous” ants (i.e., those at the low end of the trophic scale) engage in mutualistic symbioses resembling those between termites and their gut bacteria (37, 38). However, acetylene reduction assays have, thus far, failed to detect nitrogen-fixation activity in adult workers of Cephalotes varians and Cephalotes atratus (SI). So although a role of diet enrichment seems likely, future investigations on the nutritional significance of gut bacteria should consider the possibilities of nitrogen recycling and upgrading as alternative means of supplementation (15, 17, 21). These studies will benefit from the development of methods to manipulate the microbes and measure their metabolic activities. Unfortunately, attempts to eliminate bacteria from C. varians by rearing them on diets with antibiotics have, to date, been unsuccessful (SI and Table S5). However, our initial attempts at bacterial cultivation have met with some success, revealing the potential to study these gut microbes in vitro (SI).
Implications for the Evolution of the Ants and Their Diets.
Discoveries of ant trophic levels and feeding habits have raised the question of how some taxa derive sufficient nutrition from their nitrogen poor diets (30, 31, 39). Our work has identified an impressive pattern in the prevalence and diversity of gut bacteria in herbivorous ants, suggesting that symbionts have helped these insects to overcome their dietary deficiencies. When we consider our findings of multispecies bacterial communities in light of the limited sampling across the ants (283 species surveyed out of >12,000), it is evident that many influential bacteria await discovery.
Although we have only begun to identify the importance of bacterial symbionts as sources of evolutionary novelty, previous research on ants from the tribes Attini (fungus-growing ants) and Camponotini (carpenter ants) has underscored the services provided by their microbial residents (see ref. 11 for review). Ants from this latter group harbor heritable Blochmannia symbionts (14) that supplement host nutrition through a combination of nitrogen recycling and upgrading of nonessential amino acids (15). Interestingly, the two camponotine ant genera considered in our analyses exhibited low trophic levels (δ15Nant − δ15Nplant), ranking second (genus Polyrhachis) and third (genus Camponotus), out of 47 analyzed ant genera (Fig. 2 and Fig. S3a). Combined with our findings on the distributions of Rhizobiales across unrelated ant herbivores, these observations further support the hypothesis that symbiotic bacteria supplement the nitrogen-poor diets of herbivorous ants, facilitating the convergent evolution and maintenance of herbivory across this diverse group of insects. Not only does this implicate bacteria as driving agents of dietary evolution across the Formicidae, it also suggests that microbes enabled the radiation of ants into tropical rainforest canopies (31), revealing a significant instance of innovation through symbiosis.
Materials and Methods
PCR Screening and Analyses of 16S rRNA Sequences.
In this study we used both general (universal) and targeted (diagnostic) screening of 16S rRNA genes to survey bacterial diversity and distributions. Our surveys included 375 ant samples spanning 283 species, 141 genera, 46 tribes, and 18 of the 21 subfamilies within the ant family Formicidae (SI). Using universal eubacterial primers (9Fa and 1513R; Table S1), we amplified and sequenced 258 16S rRNA fragments from 52 ant species. These sequences were classified using the RDP II Classifier tool (25) to provide insight into the taxonomic distributions of bacteria found across species (Fig. S1a) and within individual ant hosts (Fig. S1b).
To further investigate bacterial diversity, sequences were aligned using the RDP II Sequence Aligner (40) and manual adjustments were made in MacClade (41). Pairwise distances were computed for sequences with no missing data between nucleotides 28–411 of E. coli U00096 using the dnadist program (42). We used these distances to identify related groups, or phylotypes, from clone libraries of cephalotine ants, defining phylotypes as sharing ≥97%, ≥98%, or ≥99% sequence identity. A consideration of the number and distribution of phylotypes within and across species provided insight into the diversity and similarities of bacterial communities in ants from the tribe Cephalotini (Table S3).
Pairwise distances were also computed for the larger dataset of 16S rRNA sequences to facilitate the selection of representatives from species with multiple sequenced clones. Single representative sequences from groups with ≥99% sequence identity were submitted to GenBank and used in phylogenetic analyses (one representative, per species group, per ant).
Preliminary analyses of sequences obtained with universal primers identified several ant-specific clades. Alignments of these sequences with their closest GenBank relatives enabled us to design diagnostic PCR primers to specifically screen for these ant-associates across a broader range of ant taxa. Positive PCR results were confirmed through DNA sequencing, and the resulting sequences were used in phylogenetic and distance analyses. Diagnostic screening was also used: (i) to establish the symbiotic status of bacteria by measuring their persistence in lab-reared ant colonies; (ii) to localize bacteria through surveys of dissected ant tissues (Table S4); (iii) to describe the frequency of bacteria within ant colonies and species (Table S4 and Table S5); and (iv) to determine the distributions of these microbes across multiple castes and developmental stages as a means to study their transmission (Table S5).
A total of 169 nonredundant 16S rRNA sequences were submitted to GenBank under the accession numbers FJ477550-FJ477670 and GQ275098-GQ275146, including representatives from sequence groups identified with universal PCR and sequencing and sequences obtained with diagnostic PCR primers (Table S2). Ten sequences from cultured bacteria were submitted to GenBank under the accession numbers FJ477671-FJ477680. Each sequence was compared with the nr/nt GenBank database through BLASTn, and top hits were downloaded and aligned with our sequences. From this alignment, we computed pairwise distances between ant-associates and their closest GenBank relatives, contrasting these with distances between pairs of related ant-associates to highlight the overall pattern of relatedness among bacteria from ants (Table S2).
Finally, to infer evolutionary relationships, we performed phylogenetic analyses using parsimony (43), maximum likelihood (44), and Bayesian methods (SI; ref. 45). For these analyses, we included nonredundant ant sequences (≥1000 bp) from the Rhizobiales (Fig. 1), Gammaproteobacteria (Fig. S2a), Verrucomicrobiales (Fig. S2b), and Burkholderiales (Fig. S2c), along with their top BLASTn matches and other representative relatives.
Ant Trophic Levels.
To assess trophic position of the ants, we calculated the average trophic level for each ant genus studied in the Blüthgen and Davidson papers (SI; refs. 30 and 31), finding that variation was considerably lower within vs. between genera (Fig. S3a). In our calculations, we corrected for between-site differences in nitrogen isotope ratios by subtracting the average δ15N of plants from each individual value of δ15N recorded for sympatric ant species (δ15Nant − δ15Nplant). We then plotted the within-genus frequency of Rhizobiales (y axis) against the average δ15Nant − δ15Nplant for 47 analyzed genera (x axis) (Fig. 2). We used logistic and weighted linear regression to examine the relationship between trophic position and the prevalence of Rhizobiales bacteria across genera within the Formicidae (SI).
To test for a correlation between presence of Rhizobiales and tropic level, we implemented the concentrated changes test (32) in MacClade (41). We first generated a pruned version of the ant phylogeny (8), with nodes corresponding to ant genera with data from stable isotope analyses and Rhizobiales screening (Fig. 3). We then tested for an association between presence of Rhizobiales bacteria and trophic position (herbivore vs. predator; SI), to determine whether the presence of Rhizobiales is concentrated on branches leading to herbivorous ant taxa. This analysis enabled us to determine whether the relationship between these two variables was independent of the ant phylogeny.
Acetylene Reduction Assays, nifH PCR, nifH Sequencing.
To explore the potential for nutritional contributions by gut-associated bacteria from herbivorous ants, we performed acetylene reduction assays on adult workers from colonies of Cephalotes varians and C. atratus. We also amplified nifH genes using a nested PCR approach. Sequences (Accession numbers FJ462366-FJ462374) were compared to the GenBank database through BLASTn searches to identify related genes from other bacteria (Table S2).
Data Availability
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. FJ462366–FJ462374, FJ477550–FJ477680, and GQ275098–GQ275146).
Acknowledgments.
We thank John Tjepkema and Christa Schwintzer for running acetylene reduction assays on Cephalotes ants and providing useful insights into our research; Michael Giampapa and Colin Funaro for technical support and help with data analyses; Nancy Moran, Mariana Mateos, and Sascha Stoll for giving generous and valuable advice that helped to shape the directions of this project; Brian Farrell for kindly donating space in his insect rearing chambers; Fred Ausubel, Adam Bahrami, Andrew Berry, Stefan Cover, Cameron Currie, Ada Kaliszewska, Sue Kilham, Daniel Kronauer, Michael O'Connor, Richard Ree, Susannah Russell, Jon Sanders, Noah Whiteman, Eugenia Zandona, and three anonymous reviewers for providing us with important insights and criticisms that helped to improve our study; the National Research Council of Thailand and the Department of National Parks, Wildlife, and Plant Conservation for granting us permits to work at Khao Ban Thad Wildlife Sanctuary; Sarayudh Bunyavejchewin, Chaweewan Hutacherern, Watana Sakchoowong, and Janya Jarearnrattanawong from the Department of National Parks, Wildlife and Plant Conservation for their help in facilitating our field work at Khao Chong Wildlife Conservation Promotion Station; Lloyd Davis, Jr. and Mark Deyrup for assisting in collecting live ant colonies; Ysabel Milton for assisting with ant rearing; Diana Wheeler for providing advice on dietary supplements; Kathy Horton for generously assisting with file formatting; and Edward O. Wilson for his inspiration, advice, and support. This work was funded by grants from Baker Fund, Tides Foundation, Harvard University Center for the Environment, Green Memorial Fund of Harvard University, the Putnam Expeditionary Fund of the Museum of Comparative Zoology, and National Science Foundation Social and Economic Sciences 0750480. J.A.R. was supported by the Green Memorial Fund and a National Science Foundation Postdoctoral Fellowship in Microbiology; C.S.M. was supported by a graduate fellowship from the Department of Organismic and Evolutionary Biology at Harvard; the Miller Institute for Basic Research in Science, University of California Berkeley; and the Department of Zoology, Field Museum of Natural History; and D.J.L. was supported by Grant R-154–000-270–112 from the Singapore Ministry of Education.
Supporting Information
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References
1
J Felsenstein, Phylogenies and the comparative method. Am Nat 125, 1–15 (1985).
2
T Garland, PH Harvey, AR Ives, Procedures for the analysis of comparative data using phylogenetically independent contrasts. Syst Biol 41, 18–32 (1992).
3
M Pagel, Inferring the historical patterns of biological evolution. Nature 401, 877–884 (1999).
4
L Margulis, R Fester Symbiosis as a Source of Evolutionary Innovation (The MIT Press, Cambridge, MA, 1991).
5
P Buchner Endosymbiosis of Animals with Plant Microorganisms (Interscience, New York, NY, 1965).
6
N Moran, P Baumann, Phylogenetics of cytoplasmically inherited microorganisms of arthropods. Trends Ecol Evol 9, 15–20 (1994).
7
SG Brady, TR Schultz, BL Fisher, PS Ward, Evaluating alternative hypotheses for the early evolution and diversification of ants. Proc Natl Acad Sci USA 103, 18172–18177 (2006).
8
CS Moreau, CD Bell, R Vila, SB Archibald, NE Pierce, Phylogeny of the ants: Diversification in the age of angiosperms. Science 312, 101–104 (2006).
9
B Stadler, AGF Dixon, Ecology and evolution of aphid-ant interactions. Ann Rev Ecol Evol Syst 36, 345–372 (2005).
10
M Heil, D McKey, Protective ant-plant interactions as model systems in ecological and evolutionary research. Ann Rev Ecol Evol Syst 34, 425–453 (2003).
11
E Zientz, H Feldhaar, S Stoll, R Gross, Insights into the microbial world associated with ants. Arch Microbiol 184, 199–206 (2005).
12
UG Mueller, RA Rehner, TR Schultz, The evolution of agriculture in ants. Science 281, 2034–2038 (1998).
13
CR Currie, JA Scott, RC Summerbell, D Malloch, Fungus-growing ants use antibiotic-producing bacteria to control garden parasites. Nature 398, 701–704 (1999).
14
C Sauer, E Stackebrandt, J Gadau, B Hölldobler, R Gross, Systematic relationships and cospeciation of bacterial endosymbionts and their carpenter ant host species: Proposal of the new taxon Candidatus Blochmannia gen. nov. Int J Syst Evol Microbiol 50, 1877–1886 (2000).
15
H Feldhaar, et al., Nutritional upgrading for omnivorous carpenter ants by the endosymbiont Blochmannia. BMC Biology 5, 48 (2007).
16
S Stoll, J Gadau, R Gross, H Feldhaar, Bacterial microflora associated with ants of the genus Tetraponera. Biol J Linn Soc 90, 399–412 (2007).
17
S van Borm, A Buschinger, JJ Boomsma, J Billen, Tetraponera ants have gut-symbionts related to nitrogen-fixing symbionts. Proc R Soc Lond B 269, 2023–2027 (2002).
18
S van Borm, T Wenseleers, J Billen, JJ Boomsma, Wolbachia in leafcutter ants: a widespread symbiont that may induce male killing or incompatible matings. J Evol Biol 14, 805–814 (2001).
19
T Wenseleers, et al., Widespread occurrence of the micro-organism Wolbachia in ants. Proc R Soc Lond B 265, 1447–1452 (1998).
20
JA Russell, et al., Specialization and geographic isolation among Wolbachia symbionts from ants and lycaenid butterflies. Evolution 63, 624–640 (2009).
21
SC Cook, DW Davidson, Nutritional and functional biology of exudate-feeding ants (Hymenoptera: Formicidae). Ent Exp et Appl 118, 1–10 (2006).
22
FH Caetano, C da Cruz-Landim, Presence of microorganisms in the alimentary canal of ants of the tribe Cephalotini (Myrmicinae: Location and relationship with intestinal structures). Naturalia 10, 37–47 (1985).
23
K Jaffe, et al., Sensitivity of ant (Cephalotes) colonies and individuals to antibiotics implies feeding symbiosis with gut microorganisms. Can J Zool 79, 1120–1124 (2001).
24
RK Roche, DE Wheeler, Morphological specializations of the digestive tract of Zacryptocerus rohweri. J Morphol 234, 253–262 (1997).
25
Q Wang, GM Garrity, JM Tiedje, JR Cole, A naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73, 5261–5267 (2007).
26
E Stackebrandt, BM Goebel, A place for DNA-DNA reassociation and 16S ribosomal-RNA sequence-analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849 (1994).
27
Y Hongoh, et al., Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host. Appl Environ Microbiol 71, 6590–6599 (2005).
28
D Wheeler, Behavior of the ant Procryptocerus scabriusculus Hymenoptera Formicidae with comparisons to other cephalotines. Psyche 91, 171–192 (1984).
29
EO Wilson, Social ethogram of neotropical arboreal ant Zacryptocerus varians (FR Smith). Anim Behav 24, 354 (1976).
30
N Blüthgen, G Gebauer, K Fiedler, Disentangling a rainforest food web using stable isotopes: Dietary diversity in a species-rich ant community. Oecologia 137, 426–435 (2003).
31
DW Davidson, SC Cook, RR Snelling, TH Chua, Explaining the abundance of ants in lowland tropical rainforest canopies. Science 300, 969–972 (2003).
32
WP Maddison, A method for testing the correlated evolution of two binary characters: Are gains or losses concentrated on certain branches of a phylogenetic tree? Evolution 44, 539–557 (1990).
33
JL Auclair, Aphid feeding and nutrition. Ann Rev Entomol 8, 439–490 (1963).
34
HG Baker, PA Opler, I Baker, A comparison of the amino acid complements of floral and extrafloral nectars. Bot Gaz 139, 322–332 (1978).
35
MK Fischer, W Völkl, R Schopf, KH Hoffmann, Age-specific patterns in honeydew production and honeydew composition in the aphid Metopeurum fuscoviride: Implications for ant-attendance. J Ins Physiol 48, 319–326 (2002).
36
DW Davidson, Ecological stoichiometry of ants in a New World rain forest. Oecologia 142, 221–231 (2005).
37
JR Benemann, Nitrogen fixation in termites. Science 181, 164–165 (1973).
38
JA Breznak, WJ Brill, JW Mertins, HC Coppel, Nitrogen fixation in termites. Nature 244, 577–580 (1973).
39
J Tobin Nourishment and Evolution in Insect Societies, eds JH Hunt, CA Nalepa (Westview Press, Boulder, CO), pp. 279–308 (1994).
40
JR Cole, et al., The Ribosomal Database Project (RDP-II): Previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucl Acids Res 31, 442–443 (2003).
41
WP Maddison, DR Maddison, MacClade: Analysis of phylogeny and character evolution. (Sinauer Associates, Sunderland, MA, Version 4.06. (2003).
42
J Felsenstein, PHYLIP—Phylogeny Inference Package (Version 3.2). Cladistics 5, 164–166 (1989).
43
DL Swofford, PAUP*. (Sinauer, Sunderland, MA, Version 4.03b10. (2001).
44
DJ Zwickl, GARLI, Genetic Algorithm for Rapid Likelihood Inference, Version 0.94., Available at http://www.bio.utexas.edu/faculty/antisense/garli/Garli.html. (2006).
45
JP Huelsenbeck, F Ronquist, MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17, 754–755 (2001).
Information & Authors
Information
Published in
Classifications
Copyright
© 2009. Freely available online through the PNAS open access option.
Data Availability
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. FJ462366–FJ462374, FJ477550–FJ477680, and GQ275098–GQ275146).
Submission history
Received: March 12, 2009
Published online: December 15, 2009
Published in issue: December 15, 2009
Keywords
Acknowledgments
We thank John Tjepkema and Christa Schwintzer for running acetylene reduction assays on Cephalotes ants and providing useful insights into our research; Michael Giampapa and Colin Funaro for technical support and help with data analyses; Nancy Moran, Mariana Mateos, and Sascha Stoll for giving generous and valuable advice that helped to shape the directions of this project; Brian Farrell for kindly donating space in his insect rearing chambers; Fred Ausubel, Adam Bahrami, Andrew Berry, Stefan Cover, Cameron Currie, Ada Kaliszewska, Sue Kilham, Daniel Kronauer, Michael O'Connor, Richard Ree, Susannah Russell, Jon Sanders, Noah Whiteman, Eugenia Zandona, and three anonymous reviewers for providing us with important insights and criticisms that helped to improve our study; the National Research Council of Thailand and the Department of National Parks, Wildlife, and Plant Conservation for granting us permits to work at Khao Ban Thad Wildlife Sanctuary; Sarayudh Bunyavejchewin, Chaweewan Hutacherern, Watana Sakchoowong, and Janya Jarearnrattanawong from the Department of National Parks, Wildlife and Plant Conservation for their help in facilitating our field work at Khao Chong Wildlife Conservation Promotion Station; Lloyd Davis, Jr. and Mark Deyrup for assisting in collecting live ant colonies; Ysabel Milton for assisting with ant rearing; Diana Wheeler for providing advice on dietary supplements; Kathy Horton for generously assisting with file formatting; and Edward O. Wilson for his inspiration, advice, and support. This work was funded by grants from Baker Fund, Tides Foundation, Harvard University Center for the Environment, Green Memorial Fund of Harvard University, the Putnam Expeditionary Fund of the Museum of Comparative Zoology, and National Science Foundation Social and Economic Sciences 0750480. J.A.R. was supported by the Green Memorial Fund and a National Science Foundation Postdoctoral Fellowship in Microbiology; C.S.M. was supported by a graduate fellowship from the Department of Organismic and Evolutionary Biology at Harvard; the Miller Institute for Basic Research in Science, University of California Berkeley; and the Department of Zoology, Field Museum of Natural History; and D.J.L. was supported by Grant R-154–000-270–112 from the Singapore Ministry of Education.
Notes
This article contains supporting information online at www.pnas.org/cgi/content/full/0907926106/DCSupplemental.
Authors
Competing Interests
The authors declare no conflict of interest.
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