Evolution of Mammals and Their Gut Microbes

Our “metagenome” is a composite of Homo sapiens genes and genes present in the genomes of the trillions of microbes that colonize our adult bodies (1). The vast majority of these microbes live in our distal guts. “Our” microbial genomes (microbiomes) encode metabolic functions that we have not had to evolve wholly on our own, including the ability to extract energy and nutrients from our diet. It is unclear how distinctively human our gut microbiota is, or how modern H. sapiens' ability to construct a wide range of diets has affected our gut microbial ecology. Here, we address two general questions concerning the evolution of mammals: How do diet and host phylogeny shape mammalian microbiota? When a mammalian species acquires a new dietary niche, how does its gut microbiota relate to the microbiota of its close relatives?

The acquisition of a new diet is a fundamental driver for the evolution of new species. Coevolution, the reciprocal adaptations occurring between interacting species (2), produces physiological changes that are often recorded in fossil remains. For instance, although mammals made their first appearance on the world stage in the Jurassic [∼160 million years ago (Ma)], most modern species arose during the Quaternary [1.8 Ma to the present (3)], when C4 grasslands (dominated by plants that use for photosynthesis the Hatch-Slack cycle rather than the Calvin cycle typical of C3 plants) expanded in response to a fall in atmospheric CO₂ levels and/or climate changes (4–6). The switch to a C4 plant–dominated diet led to selection for herbivores with high-crowned teeth (7, 8) and longer gut retention times necessary for the digestion of lower-quality forage (9). However, these adaptations may not suffice for the exploitation of a new dietary niche. The community of microbes in the gut constitutes a potentially critical yet unexplored component of diet-driven speciation.

Because we cannot interrogate extinct gut microbiotas directly, past evolutionary processes can only be inferred from comparative analyses of extant mammalian gut microbial communities. Therefore, we analyzed the fecal microbial communities of 106 individual mammals representing 60 species from 13 taxonomic orders, including 17 nonhuman primates. To isolate the effects of phylogeny and diet, we included multiple samples from many of the mammalian species, as well as species that had unusual diets compared to their close phylogenetic relatives. For example, the majority of the nonhuman primate species studied were omnivores (12 of 17), but the leaf-eating (folivorous) East Angolan colobus, Eastern black-and-white colobus, Douc langur, and François langur were also sampled. In addition, the herbivorous giant panda and red panda were included from the Carnivora. Most animals were housed at the San Diego Zoo and the San Diego Zoo's Wild Animal Park (n = 15) or the St. Louis Zoo (n = 56). Others were examined in the wild (n = 29) or domesticated (n = 6; table S1). To test the reproducibility of host species–associated gut microbiotas and to gauge the effects of animal provenance, we represented mammalian species by multiple individuals from multiple locales where possible, and chose wild animals to match captive animals. We generated a data set of >20,000 16S rRNA gene sequences; for comparison of the human, primate, and nonprimate mammalian gut microbiotas (10), the 106 samples also included published fecal bacterial 16S rRNA sequences (>3000) from wild African gorilla (11), Holstein cattle (12), Wistar rats (13), and healthy humans of both sexes, ranging in age from 27 to 94, living on three continents and including a strict vegetarian (14–18) (table S1).

We used network-based analyses to map gut microbial community composition and structure onto mammalian phylogeny and diet, thereby complementing phylogeny-based microbial community comparisons. These analyses were used to bin 16S rRNA gene sequences into operational taxonomic units (OTUs) and to display microbial genera partitioning across hosts. Genus-level OTUs (sets of sequences with ≥96% identity) and animal hosts were designated as nodes in a bipartite network, in which OTUs are connected to the hosts in which their sequences were found (Fig. 1A). To cluster the OTUs and hosts in this network, we used the stochastic spring-embedded algorithm, as implemented in Cytoscape 2.5.2 (19), where nodes act as physical objects that repel each other, and connections act as a spring with a spring constant and a resting length; the nodes are organized in a way that minimizes forces in the network.

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The ensemble of sequences in this study provides an overarching view of the mammal gut microbiota. We detected members of 17 phyla (divisions) of Bacteria (10). The majority of sequences belong to the Firmicutes [65.7% of 19,548 classified sequences (10)] and to the Bacteroidetes (16.3%); these phyla were previously shown to constitute the majority of sampled human (and mouse) gut-associated phylotypes (10, 20). The other phyla represented were the Proteobacteria (8.8% of all sequences collected; 85% in the Gamma subdivision), Actinobacteria (4.7%), Verrucomicrobia (2.2%), Fusobacteria (0.67%), Spirochaetes (0.46%), DSS1 (0.35%), Fibrobacteres (0.13%), TM7 (0.13%), deep-rooting Cyanobacteria [0.10%; these are not chloroplasts (20)], Planctomycetes (0.08%), Deferribacteres (0.05%), Lentisphaerae (0.04%), and Chloroflexi, SR1, and Deinoccus-Thermus (all 0.005%). We were unable to assign 1985 16S rRNA gene sequences that passed a chimera-checking algorithm (21) to known phyla on the basis of BLAST searches against the Greengenes database (22) and the Ribosomal Database Project taxonomy annotations (23). Of the phyla that were detected, only Firmicutes were found in all samples (fig. S1). However, each mammalian host harbored OTUs (96% sequence identity) not observed in any other sample (at this level of sampling, on average, 56% and 62% of OTUs were unique within a sample and species, respectively; table S1).

The network-based analyses disclosed that overall, the fecal microbial communities of same-species (conspecific) hosts were more similar to each other than to those of different host species: Host nodes were significantly more connected within than between species (G test for independence, G = 11.9, P = 0.0005; Fig. 1B). Shown in fig. S2 is a tree-based analysis where similarity is defined using the UniFrac metric; this metric is based on the degree to which individual communities share branch length on a common (master) phylogenetic tree constructed from all 16S rRNA sequences from all communities being compared (24, 25). The results are consistent with the network-based analysis; that is, they show that UniFrac distances are smaller within conspecific hosts than between nonconspecific hosts (P < 0.005 by one-tailed t test, confirmed by matrix permutation and corrected for multiple comparisons).

The impact of host species on community composition is most evident when considering conspecific hosts living separately, because co-housing may confound any species effect. For example, the two Hamadryas baboons clustered together (fig. S2), although one is from Namibia and the other from the St. Louis Zoo; similarly, the red pandas housed in different zoos clustered together. All 16 human samples also clustered together. Nonetheless, some conspecifics with different origins did not cluster (e.g., the two Western lowland gorillas), which suggests that diet and other environmental exposures [“legacy effects” (26)] play roles in addition to host phylogeny (taxonomic order).

The clustering by diet (herbivore, omnivore, and carnivore) was highly significant in both the tree-based (fig. S2) and network-based analyses (Fig. 1B). In the network-based analysis, host nodes are significantly more connected to other host nodes from the same diet group (G = 115.8; P = 5.1 × 10^–27) (10). Similarly, hosts within the same taxonomic order are more connected in the network to hosts within the same order (Fig. 1C; G = 356; P = 2.1 × 10^–79). Likewise, UniFrac-based principal coordinates analysis (PCoA) showed clustering by diet (Fig. 2, A and B) and by taxonomic order (Fig. 2D). (UniFrac distances are smaller for within versus between diet categories, and for within versus between orders, P < 0.005.) There was no significant clustering according to the provenance of the animals (including humans) in either the network- or UniFrac-based analyses (P >0.05 for both; Fig. 1D and fig. S2, respectively), nor in a randomized network (Fig. 1E).

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Classification of the mammals into herbivore, omnivore, and carnivore groups was based on diet records and natural history. Heavy isotopes of carbon and nitrogen bioaccumulate in the food chain (27). Therefore, to obtain a more objective marker of diet, we measured stable isotope ratios of carbon and nitrogen, δ¹³C and δ¹⁵N, in the feces (where δ = 1000 × [(R_sample – R_standard)/R_standard] and R = ratio of atom percentages ¹³C/¹²C and ¹⁵N/¹⁴N). The results were consistent with the original diet group classification. Heavy isotopes were enriched in the order herbivore < omnivore < carnivore (Fig. 3A). The protein and fat contents of the diets of animals in captivity (obtained from diet records) were positively correlated with δ¹³C and δ¹⁵N fecal values (R² values for fat versus δ¹³C and δ¹⁵N were 0.51 and 0.45, respectively, and for protein, 0.36 and 0.38).

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To test for a direct link between diet and microbial community composition, we mapped stable isotope values onto the coordinates that explained the largest proportion of the variance in the microbial communities, as determined by PCoA of the UniFrac distances between hosts (Fig. 3B). Principal coordinate 1 (PC1) separates carnivores from herbivores and omnivores (mean is significantly lower for carnivores than herbivores, which are equivalent to omnivores; F_80,2 = 9.9, P < 0.001) and is also correlated with δ¹³C and δ¹⁵N values (multiple regression R² = 0.25, F_80,2 = 12.7, P < 0.001). Together, these results support an association between microbial community membership and diet, and provide an independent validation of the dietary clustering observed in the network diagrams that is free of bias in assigning hosts to one of the three diet categories.

Underlying the correlation between bacterial community composition and diet is the partitioning of bacterial phyla among hosts according to diet. Herbivore microbiotas contained the most phyla (14), carnivores contained the fewest (6), and omnivores were intermediate (12) (fig. S1). Phylogenetic trees constructed from 16S rRNA sequences from the feces of herbivores also had the greatest amount of total branch length (phylogenetic diversity; fig. S3A). Consistent with this finding, herbivores had the highest genus-level richness, followed by omnivores and carnivores (fig. S3B).

Ancestral mammals were carnivores (9). We used an analysis based on the Fitch parsimony algorithm (10) to test whether bacterial lineages found in herbivores were derived from lineages found in carnivores. The results did not support this notion; hence, gut bacterial communities required to live largely on a plant-based diet were likely acquired independently from the environment.

Adaptation to a plant-based diet was an evolutionary breakthrough in mammals that resulted in massive radiations: 80% of extant mammals are herbivores, and herbivory is present in most mammalian lineages (9). To access the more complex carbohydrates present in plants, such as celluloses and resistant starches, disparate mammalian lineages lengthened gut retention times to accommodate bacterial fermentation; this occurred via enlargement of the foregut or hindgut (9). We found that herbivores clustered into two groups that corresponded generally to foregut fermenters and hindgut fermenters: the foregut-fermenting sheep, kangaroo, okapi, giraffe, and cattle clustered together to form herbivore group 1 in fig. S2, whereas the hindgut-fermenting elephant, horse, rhinoceros, capybara, mole rat, and gorilla clustered together in herbivore group 2. The strong impact of gut morphology on bacterial community composition is also evident in PCoA of the UniFrac data: Herbivores separate into fore- and hindgut groups, and omnivores separate into hindgut fermenters and those with simple guts (Fig. 2, C and D).

Differences between the fecal communities of foregut and hindgut fermenters are likely due to host digestive physiology: In foregut fermenters, the digesta is moved into the equivalent of the monogastric stomach after fermentation, so that part of the microbiota is also digested; in hindgut fermenters, the fermentative microbes are more likely to be excreted in the feces. Fermentation requires microbial interactions such as cross-feeding and interspecies hydrogen transfer (28). Our results suggest that as mammals underwent convergent evolution in the morphological adaptations of their guts to herbivory, their microbiota arrived at similar compositional configurations in unrelated hosts with similar gut structures.

The diet outliers in our study were folivores. Despite their herbivorous diet, red and giant pandas have simple guts, cluster with other carnivores, and have carnivore-like levels of phylogenetic diversity (figs. S2 and S3). In folivorous primates, the simple gut has evolved pouches for fermentation of recalcitrant plant material (9). The fecal microbiota of the two colobus monkeys and the François langur cluster together by UniFrac with the three pig species (Red River hog, Visayun warty pig, babirusa) and the flying fox, baboon, chimpanzee, gorilla, and orangutan, forming a phylogenetically mixed group whose diets include a large component of plant material. This cluster occupies an intermediate position between other primates and herbivorous foregut fermenters in fig. S2. This observation suggests that the colobus monkeys and the François langur harbor microbial lineages typical of omnivores but have a greater representation of the lineages driving the breakdown of a plant-based diet. Such host-level selection of specific members of a microbiota has been demonstrated under laboratory conditions by reciprocal transplantations of gut microbiota from one host species to germ-free recipients of a different species: Groups of bacteria were expanded or contracted in the recipient host to resemble its “normal” microbiota through a process that may have been influenced by diet (26).

Coevolution has been hypothesized to occur in animal species whose parental care enables vertical transmission of whole gut communities, and where the properties of the community as a whole confer a fitness advantage to the host (29). Although coevolution has been inferred from observations of bacterial host specificity (30), these observations could also be explained by dietary preference. Therefore, we searched for evidence of codiversification, a special case of coevolution (2) that would be manifest in this case by a clustering of fecal microbial communities that mirrors the mammalian phylogeny. A UniFrac analysis was performed recursively (10) on the entire mammalian fecal bacterial tree, using a procedure that had the effect of asking whether the bacterial lineages stemming from each tree node mirrored the mammalian phylogeny (31). The results were compared to those using a randomized version of the mammalian phylogeny. The patterns of community similarity matched the mammal phylogeny more often than would be expected if no codiversification had occurred (fig. S4; P = 1.79 × 10^–11; t = –6.73, df = 88).

Although mammalian gut microbes are highly adapted to life in this body habitat, and many lineages are extremely rare outside of it (29), they appear to be fairly promiscuous between hosts. This could account for the spectacular success of mammals and herbivores in particular: Acquiring a gut microbiota was not a constraint, and morphological and behavioral adaptations were likely far more restrictive. One implication of this work is that the tolerance of the immune system to gut microbes is a basal trait in mammal evolution.

The global success of humans is based in part on our ability to control the variety and amount of food available using agriculture and cookery. These capabilities have not appreciably affected the major bacterial lineages that constitute our gut microbiota: As noted above, fecal samples from unrelated healthy human samples cluster with other omnivores (Fig. 1 and fig. S2), with interpersonal differences (UniFrac distances) being significantly smaller than the distances between humans and all other mammalian species (G = –47.7, P < 0.005, n = 106). Although our interpersonal differences appear to be smaller than interspecies differences among mammals, deeper sampling and analysis will be required to circumscribe the gut microbial diversity inherent to humans. This is one of the early goals of the recently initiated international human microbiome project (1).