an ecophylogenetic approach to determine the evolutionary history of the mammalian gut microbiome...
TRANSCRIPT
An Ecophylogenetic Approach to Determine the Evolutionary History of the Mammalian Gut MicrobiomeChristopher A. Gaulke(1), Holly Arnold(1), Steven W. Kembel(2), James P. O’Dwyer(3), Thomas J. Sharpton (1,4)
(1) Department of Microbiology, Oregon State University, Corvallis, OR, USA (2) Département des Sciences Biologiques, Université du Québec à Montréal, Montreal, QC, Canada(3) Department of Plant Biology, University of Illinois, Urbana, IL, USA (4) Department of Statistics, Oregon State University, Corvallis, OR, USA
Abstract
ClaaTU: An Ecophylogenetic Microbial Community Analysis Framework
Identifying those gut microbes that co-diversify with mammals is important to our un-derstanding of the mechanisms and health implications of host-microbiome interac-tions. For example, microbiota that are conserved across mammalian species may ex-press a trait that has been subject to selection throughout the evolution of these mam-mals, possibly because it is critical to health. While advances in environmental DNA se-quencing have transformed our understanding of how enteric microbes are distributed across mammalian species, these data are frequently analyzed using phylogenetically agnostic approaches. Such approaches can obscure the detection of diverged groups of bacteria that have been conserved across mammalian species. To provide enhanced res-olution into evolutionary associations between gut microbiota and mammals, we inno-vated a high-throughput ecophylogenetic method, known as ClaatTU (Cladal Taxonom-ic Units). ClaaTU analyzes phylogenies assembled from environmental DNA sequences collected from a set of microbial communities and pro�les the presence and abundance of each monophyletic clade in each community. As a result, it enables the identi�cation of speci�c microbial clades that are distributed across host communities in a manner in-dicative of being associated with mammalian evolution. To demonstrate this, we ap-plied ClaaTU to a mammalian microbiome dataset and (1) identi�ed clades of gut bacte-ria that are unique to groups of mammals based on their taxonomy or dietary regime, (2) found that there exists ecophylogenetic structure in the mammalian gut microbiome, indicating that gut bacterial phylogenetic diversity associates with host phylogeny, and (3) discovered speci�c clades that are present in a larger number of mammals than ex-pected by chance, some of which may co-diversify with their hosts. Our �ndings indi-cate that some mammalian gut microbiota may have been anciently acquired and sub-sequently retained in extant lineages, indicating that they may play an important role in mediating host-microbiome interactions and maintaining host health.
Case Study: Conserved Responses to Dietary Variation Among Monophyletic
Clades of Gut Bacteria
Summary
Acknowledgements
Ecophylogenetic Structure in the MammalianGut Microbiome
Monophyletic Clades of Gut BacteriaAre Conserved across Mammalian
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Figure 1: Analysis of the ecological distribu-tion of monophyletic clades (ecophylogenet-ics) can reveal groups of organisms that ex-hibit evolutionarily conserved functions that impact their ecology. 16S sequences derived from three di�erent various communities (”Sam-ples”) can be related via a phylogeny (solid lines) or clustered into OTUs (red circles). In this exam-ple, no OTU is common to all communities (red arrows), but a monophyletic clade is (shaded area), indicating that the common ancestor may have evolved and subsequently maintained a function critical to occupying these communi-ties. Note that interesting clades (e.g., core clades) may also be discovered at the sub-OTU
Figure 2: The ClaaTU work�ow. The Cladal Taxonomic Units (ClaaTU) work�ow quanti�es the abundance of speci�c clades in a user provided phylogenetic tree. ClaaTU �rst assigns identi�ers to all internal clades on a phylogenetic tree and then conducts a root-to-tip traversal of the tree to quantify the abundance of each clade by summing the counts of the de-scendants. The output of ClaaTU is a clade abundance matrix, which logs the abundance of each clade in each sample. Much like an OTU abundance matrix, these data can be used to identify clades that associate with samples, indicating their interaction. Notably, ClaaTU can use data �les produced by third party microbiome analysis software (e.g., QIIME and Mothur) and analyze phylogenies assembled from OTU clustered sequences.
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ArtiodactylaCarnivoraDiprotodontiaHyracoideaLagomorphaPerissodactylaPrimatesProboscidaeRodentiaXenarthra
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ArtiodactylaCarnivoraDiprotodontiaHyracoideaLagomorphaPerissodactylaPrimatesProboscidaeRodentiaXenarthra
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ArtiodactylaCarnivoraDiprotodontiaHyracoideaLagomorphaPerissodactylaPrimatesProboscidaeRodentiaXenarthra
BushDogBlack BearPolar BearSpectacled BearHyenaLionHorseZebraBlack RhinoOkapiGiraffeUrialBig HornGazelleSpringbackPigCapybaraSquirrelRabbitOrangutanGorillaChimpanzeColobusBaboonSakiCallimicosRed Tailed LemurBlack LemurArmadilloHyraxAfrican ElephantKangaroo
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Figure 5: Gut microbiomes from 32 mammals were pro-cessed with ClaaTU. 16S rRNA sequences that were originally published in [1] were processed with ClaaTU as in Fig. 3. (Left) The phylogeny of the host taxa as provided by [2]. (Right) The distribution of clades unique to members of each host Order (only Artiodactyla, Carnivora, Primates shown due to limited sampling in other Orders) or diet (Herbivore, Carnivore, Omni-vore) were quanti�ed. Unique clades were normalized by number of hosts to correct for sampling disparities. This analysis reveals that di�erent groups of hosts have acquired specialized clades of bacteria.
Figure 6: Highly abundant gut microbiome clades sepa-rate hosts by dietary preference while lowly abundant clades separate by host taxonomy. The clade abundance matrix produced by ClaaTU from the analysis of the 32 mam-mals was used to calculate the weighted and unweighted Bray-Curtis dissimilarity (BCD). PCoA was used to visualize re-sults. Ellipses represent 95% con�dence intervals and are sup-pored by PERMANOVA analysis (p < 0.05). Separation among groups is highest for unweighted BCD when clustering by Order, and for weighted BCD when clustering by diet.
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Clade 1888 - A Subclade within GammaproteobacteriaDe�ned Diet
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Figure 3: A Zebra�sh Dietary Exposure Study Design. Forty-�ve 5D line zebra�sh were fed one of three diets (3 tanks/diet): a standard lab diet (Gemma Micro 300, a 130 mg/kg zinc), a de�ned diet with su�cient levels of zinc (33 mg/kg), and a de�ned diet with de�cient levels of zinc (12.46 mg/kg). Stool was collected through temporary isola-tion of �sh. Gut microbiomes were interro-gated using V4 16S rRNA MiSeq sequencing, QIIME (97% identity OTUs), and ClaaTU with FastTree.
Figure 4: An example of a monophyletic clade that strati-�es high and low zinc diets. A speci�c clade (1888, ingroup above blue line) signi�cantly di�ers in abundance between the high (orange and blue) and low (green) zinc diets (krus-kal-wallis p < 0.05). Notably, no single OTU reveals a signi�-cant di�erence in association with zinc status.
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Figure 6: Several clades of mammalian gut bacteria are more conserved across host samples than expected by chance. A phyloge-netic permutational analysis was used to quanti-fy whether the frequency with which a clade was observed across Mammalian hosts (i.e., clade conservation rate) was greater than ex-pected by chance. (Top) The clade conservation rate is moderately inversely proportional to the distance of the clade from the root of the 16S phylogeny. Each point represents a clade and is colored by its q-value corrected permutational test p-value. The line of best �t is shown in black. Many signi�cantly conserved clades are ob-served near the middle of the 16S phylogeny in-dicating that they represent broad groups of bacteria. Others appear closer to the tips, indi-cating that they contain more specialized groups of bacteria. (Bottom) The taxonomy of each signi�cantly conserved clade was charac-terized based on the consensus of the taxonom-ic annotations of all descendents. The Green-Genes taxonomy was used as a reference in this analysis. Bars correspond to taxonomic groups that contain signi�cantly conserved clades and are colored by their taxonomic level. Notably, the species Prevotella copri is signi�cantly con-served among the Primates.
Figure 7: Possible patterns of co-diversi�cation be-tween conserved gut microbiome clades and mam-mals may exist. (Top) A co-phylogeny of gut microbi-ome OTUs (left) and host mammals (right). Edges indi-cate an OTU was found in a host. Only edges from OTUs that are member of signi�cantly conserved clades are visualized (q < 0.2). (Bottom) An example of one clade,
which contains Ruminococcus avefaciens, which is enriched among the herbivores and known to be im-portant to degrading plant cell walls. [3]. Future work will quantify correla-tions between host and clade phylog-
1. ClaaTU reveals how monophyletic clades of bacteria are distributed across samples (https://github.com/chrisgaulke/Claatu).
2. This can clarify how bacteria respond to environmental variation
3. Applying this approach to the mammalian microbiome reveals highly conserved clades of bacteria and potential patterns of co-diversi�cation and �nds
We are grateful to E. Ho, R. Tanguay, C. Wong, L. Beaver, C. Barton, N. Kircho� and C. Armour for their assistance with varisous aspects of this project. This work is generously supported by the National Science Foundataion (DEB 1557192).References[1] Muegge BD, et al. Diet drives convergence in gut microbiome functions across mam-malian phylogeny and within humans. Science. 332(6032). 2011.[2] Hedges SB, et al. Tree of Life Reveals Clock-Like Speciation and Diversi�cation. Mol Biol Evol. 32. 2015.[3] Rincon MT, et al. A novel cell surface-anchored cellulose-binding protein encoded by the sca gene cluster of ruminococcus avefaciens. Journal of bacteriology. 189 (13) 2007.