We show that a citizen science, self-selected cohort shipping samples through the mail at room temperature recaptures many known microbiome results from clinically collected cohorts and reveals new ones. Of particular interest is integrating n = 1 study data with the population data, showing that the extent of microbiome change after events such as surgery can exceed differences between distinct environmental biomes, and the effect of diverse plants in the diet, which we confirm with untargeted metabolomics on hundreds of samples.

Over the past decade several studies have reported that the gut microbiomes of mammals with similar dietary niches exhibit similar compositional and functional traits. However, these studies rely heavily on samples from captive individuals and often confound host phylogeny, gut morphology, and diet. To more explicitly test the influence of host dietary niche on the mammalian gut microbiome we use 16S rRNA gene amplicon sequencing and shotgun metagenomics to compare the gut microbiota of 18 species of wild non-human primates classified as either folivores or closely related non-folivores, evenly distributed throughout the primate order and representing a range of gut morphological specializations. While folivory results in some convergent microbial traits, collectively we show that the influence of host phylogeny on both gut microbial composition and function is much stronger than that of host dietary niche. This pattern does not result from differences in host geographic location or actual dietary intake at the time of sampling, but instead appears to result from of differences in host physiology. These findings indicate that mammalian gut microbiome plasticity in response to dietary shifts over both the lifespan of an individual host and the evolutionary history of a given host species is constrained by host physiological evolution. Therefore, the gut microbiome cannot be considered separately from host physiology when describing host nutritional strategies and the emergence of host dietary niches.

word count: 203 68 3 Main text word count: 3280 69 70 Abstract: Although much work has linked the human microbiome to specific phenotypes and 71 lifestyle variables, data from different projects have been challenging to integrate and the extent 72 of microbial and molecular diversity in human stool remains unknown. Using standardized 73 protocols from the Earth Microbiome Project and sample contributions from over 10,000 citizen-74 scientists, together with an open research network, we compare human microbiome specimens 75 primarily from the USA, UK, and Australia to one another and to environmental samples. Our 76 results show an unexpected range of beta-diversity in human stool microbiomes as compared to 77 environmental samples, demonstrate the utility of procedures for removing the effects of 78 overgrowth during room-temperature shipping for revealing phenotype correlations, uncover 79 new molecules and kinds of molecular communities in the human stool metabolome, and 80 examine emergent associations among the microbiome, metabolome, and the diversity of plants 81 that are consumed (rather than relying on reductive categorical variables such as veganism, 82 which have little or no explanatory power). We also demonstrate the utility of the living data 83 resource and cross-cohort comparison to confirm existing associations between the microbiome 84 and psychiatric illness, and to reveal the extent of microbiome change within one individual 85 during surgery, providing a paradigm for open microbiome research and education. 86 87Importance: We show that a citizen-science, self-selected cohort shipping samples through the 88 mail at room temperature recaptures many known microbiome results from clinically collected 89 cohorts and reveals new ones. Of particular interest is integrating n=1 study data with the 90 population data, showing that the extent of microbiome change after events such as surgery can 91 4 exceed differences between distinct environmental biomes, and the effect of diverse plants in the 92 diet which we confirm with untargeted metabolomics on hundreds of samples. 93 94 Introduction 95The human microbiome plays a fundamental role in human health and disease. While 96 many studies link microbiome composition to phenotypes, we lack understanding of the 97 boundaries of bacterial diversity within the human population, and the relative importance of 98 lifestyle, health conditions, and diet, to underpin precision medicine or to educate the broader 99 community about this key aspect of human health. 100 We launched the American Gut Project (AGP; http://americangut.org) in November of 101 2012 as a collaboration between the Earth Microbiome Project (EMP) (1) and the Human Food 102 Project (HFP; http://humanfoodproject.com/) to discover the kinds of microbes and microbiomes 103 "in the wild" via a self-selected citizen-scientist cohort. The EMP is tasked with characterizing 104 the global microbial taxonomic and functional diversity, and the HFP is focused on 105 understanding microbial diversity a...

A major bottleneck for metagenomic sequencing is rapid and efficient DNA extraction. Here, we compare the extraction efficiencies of three magnetic bead-based platforms (KingFisher, epMotion, and Tecan) to a standardized column-based extraction platform across a variety of sample types, including feces, oral, skin, soil, and water. Replicate sample plates were extracted and prepared for 16S rRNA gene amplicon sequencing in parallel to assess extraction bias and DNA quality. The data demonstrate that any effect of extraction method on sequencing results was small compared with the variability across samples; however, the KingFisher platform produced the largest number of high-quality reads in the shortest amount of time. Based on these results, we have identified an extraction pipeline that dramatically reduces sample processing time without sacrificing bacterial taxonomic or abundance information.

Recent studies suggest that variation in diet across time and space results in changes in the mammalian gut microbiota. This variation may ultimately impact host ecology by altering nutritional status and health. Wild animal populations provide an excellent opportunity for understanding these interactions. However, compared to clinical studies, microbial research targeting wild animals is currently limited, and many published studies focus only on a single population of a single host species. In this study we utilize fecal samples from two species of howler monkey (Alouatta pigra and A. palliata) collected at four sites to investigate factors influencing the gut microbiota at three scales: taxonomic (host species), ecosystemic (forest type), and local (habitat disturbance/season). The results demonstrate that the effect of host species on the gut microbiota is stronger than the effect of host forest type, which is stronger than the effect of habitat disturbance or seasonality. Nevertheless, within host species, gut microbiota composition differs in response to forest type, habitat disturbance, and season. Variations in the effect size of these factors are associated both with host species and environment. This information may be beneficial for understanding ecological and evolutionary questions associated with Mesoamerican howler monkeys, as well as determining conservation challenges facing each species. These mechanisms may also provide insight into the ecology of other species of howler monkeys, non-human primates, and mammals.