Katherine R. Amato1,2*, Steven R. Leigh2, Angela Kent3, Roderick I. Mackie4,5, Carl J

The gut microbiota compensates for seasonal diet variation in the wild black howler monkey (Alouattapigra)

Katherine R. Amato1,2*, Steven R. Leigh2, Angela Kent3, Roderick I. Mackie4,5, Carl J. Yeoman6, Rebecca M. Stumpf5,7, Brenda A. Wilson5,8, Karen E. Nelson9, Bryan A. White4,5,Paul A. Garber7

1Program in Ecology, Evolution, and Conservation Biology, University of Illinois, Urbana, IL, 61801; 2Department of Anthropology, University of Colorado, Boulder, CO 80309; 3Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, IL, 61801; 4Department of Animal Sciences, University of Illinois, Urbana, IL, 61801; 5Institute for Genomic Biology, University of Illinois, Urbana, IL, 61801;f6Department of Animal and Range Sciences, Montana State University, Bozeman, MT, 59717; 7Department of Anthropology, University of Illinois, Urbana, IL, 80301; 8Department of Microbiology, University of Illinois, Urbana, IL, 61801; 9The J. Craig Venter Institute, Rockville, MD, 20850

*Author for correspondence: , (773)952-0098, fax: (303)492-8425

Microbial Ecology

Supporting Information

Methods

Diet data analysis:For behavioral data collection, the focal individual was chosen pseudo-randomly (no individual was sampled twice consecutively and priority was given to individuals that had been undersampled on previous days). During each twenty minutebehavioral sampling period, the focal individual’s activity was recorded every two minutes. Five activities were recorded: feeding (ingestion of food items), foraging (movement within a feeding tree), resting (inactivity), traveling (movement between trees), and social activity (howling, play, sexual interaction, aggression, etc.). During feeding bouts, the type of food resource (e.g. ripe fruit, unripe fruit, mature leaves, young leaves, flowers, and stems) was recorded as well as the plant species. The number of food items consumed per minute was quantified when possible to provide an estimate of intake rate.

We measured the average wet and dry mass of the top ten food items consumed during each sampling period (based on thepercentageof feeding time)for each howler group and used these data together with intake rates to estimate macronutrient intake based on published values for specific plant species as well as average values for Neotropical fruits, leaves, and flowers(Amato and Garber 2014). We also preserved samples of the top ten food items in 70% methanol for metabolite profiling.Metabolite data were generated using gas chromatography separation and mass spectrometry analysis, as previously described(Poroyko et al 2011). 1.25mM of saturated C31 fatty acid (hentriacontanoic acid) was used as an internal standard in each sample. We reported metabolites in terms of relative concentration (compared to this internal standard) per gram of wet weight and categorized them into amino acids, sugars, and lipids when possible. Due to the method by which the metabolite concentrations were standardized, these data do not provide accurate estimates of the actual amount of each metabolite in a food item, nor can their amounts be accurately compared across categories (i.e. amino acid, sugar, lipid) within a sample (Poroyko et al 2011). However, relative concentrations of the same metabolite across food items and howler diets can be compared to determine their impact on howler nutrition and the gut microbiota. We standardized all diet data by metabolic body weight to control for age and sex differences in the composition of the howler groups.

Fecal sample analysis:We stored fecal samples in 96% ethanol for microbial communitycomposition analyses, 1M NaOH for VFA analyses, and 1M HCl for ammonia analyses. After collection, we shipped the samples to the University of Illinois where they were kept at -80C until processing. Permits to collect and export fecal and plant samples were obtained through the Secretaria del MedioAmbiente y RecursosNaturales (SEMARNAT), the ComisionNacional de Areas NaturalesProtegidas (CONANP), and the Secretaria de Agricultura, Ganaderia, Desarollo Rural, Pesca y Alimentacion (SAGARPA) in Mexico. Permits to import samples to the United States were obtained through the Center for Disease Control (CDC) and the Animal and Plant Health Inspection Service (APHIS). Permission to conduct this research was approved through the IACUC, University of Illinois (#12083).

We amplified the intergenic spacer region of the 16S rRNA gene in all samples using polymerase chain reaction and used automated ribosomal intergenic spacer analysis (ARISA) to create a microbial “fingerprint” for each sample(Kent et al 2007).ARISA PCR products were visualized by denaturing capillary electrophoresis using an ABI 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA) at the UIUC Keck Center for Comparative and Functional Genomics, as described previously (Kent and Bayne 2010). Size-calling and ARISA profile alignment were carried out using GeneMarker version 1.95 (SoftGenetics, State College, PA). We used a signal detection threshold of 500 fluorescence units to exclude background fluorescence and normalized the signal strength (i.e., peak area) of each peak to account for run-to-run variations in signal detection by dividing the area of individual peaks by the total fluorescence (area) detected in each profile(Yannarell and Triplett 2005).

We used 454 FLX-Titanium technology at the J. Craig Venter Institute (Rockville, MD) forpyrosequencing of the V1-V3 region of the 16S ribosomal RNA gene to generate taxonomic data for the microbiotain a subset of samples (N=8 individuals at 15 time points)(Amato et al 2013). We successfully sequenced 119 samples. After we removed sequences shorter than 250nt, with homopolymers longer than 6 nucleotides, containing ambiguous base calls or incorrect primer sequences, there were 3,242.8 ± 1,610.5 reads per sample on average. We aligned sequences against the silva database (Pruesse et al 2007)and pre-clustered using mothur(Schloss et al 2009). We detected and removed potentially chimeric sequences using mothur’s implementation ofuchime(Schloss et al 2009) and assembled sequences using mothur’s average-neighbor algorithm. We defined OTUs as sharing 97 % sequence identity. We produced rarefaction data, Shannon-Weaver and Chao1 indices using mothur(Schloss et al 2009) after rarefying data to 1,600 reads. We generated taxonomic profiles using the RDP Classifier (Wang et al 2007).

Statistical analysis: For all analyses, data were pooled by individual for each sampling block, and only individuals from which data were collected during every sampling block were included in analyses (N=13). Because we detected differences across groups in most measuredvariables, we stratified PERMANOVA models by group, allowing us to test for differences across sampling blocks while controlling for differences among groups. We used Type III sums of squares to determine the significance of each factor in the model and ran all models for 5000 permutations.To ensure that the method by which we were dividing the data into three time periods was not creating artificial patterns, we also ran analyses on data for which samples from each individual were randomly pooled into three groups.

Depending on the distribution of data, Kruskal-Wallis tests or analysis of variance (ANOVA) were used to test for seasonal patterns in total grams of food consumed, total fecal VFA content, and total fecal ammonia content. We also employed a series of Kruskal-Wallis tests to test for temporal patterns in intake of individual diet components, concentration of individual VFAs, and relative abundance of bacterial taxa.For correlations between plant metabolites and microbial OTUs, we tested for correlations using only those metabolites and OTUs that exhibited significant differences in relative consumption or relative abundance across sampling periods. We adjusted p-values for repeated tests using a sequential Bonferroni correction with the initial p = 0.05 (Holm 1979, Rice 1989). However, to detect bacterial taxa that differed in abundance across sampling blocks, we used a p-value of 0.05 since sequencing restrictions reduced the number of individuals for which data were generated, and therefore reduced statistical power. We also used indicator species analysis (R Software, labdsv package) to detect microbial genera (pyrosequencing) characterizing each sampling block based on both abundance and frequency of occurrence (De Caceres and Legendre 2009). Taxa with a significant (p < 0.05) indicator value higher than 0.5 were considered characteristic of each sampling block

RESULTS

Tables

Table S1. Average nutrient and energy intake for each howler group across sampling periods based on literature estimates of food resource nutritional content, and average metabolite intake. Energy and nutrient intake values are expressed in grams per metabolic body weight, energy per metabolic body weight, and percent dry weight ingested. Metabolite values are expressed in relative concentration (compared to a standard) per metabolic body weight. WFD = Wet-Fruit Dominated Period, DLD = Dry-Leaf Dominated Period, DFD = Dry-Fruit Dominated Period

WFD / DLD / DFD
Motiepa / Balam / Motiepa / Balam / Motiepa / Balam
Food consumed / Average / 416.5 / 340.4 / 228.8 / 349.8 / 115.8 / 126.3
SD / 101.2 / 68.6 / 85.8 / 72.1 / 36.0 / 54.0
Available Protein (g/MBW) / Average / 9.4 / 11.9 / 7.7 / 10.0 / 4.7 / 5.3
SD / 3.3 / 3.2 / 2.6 / 3.0 / 1.0 / 1.8
% Available Protein / Average / 10.2% / 11.3% / 12.8% / 11.5% / 9.4% / 9.4%
SD / 1.1% / 0.9% / 1.6% / 0.6% / 0.8% / 0.8%
Total Non-Structural Carbohydrates / Average / 29.0 / 29.7 / 12.4 / 16.2 / 16.3 / 17.0
(g/MBW) / SD / 12.5 / 12.0 / 3.5 / 3.7 / 3.4 / 5.0
% Total Non-Structural Carbohydrates / Average / 30.0% / 27.2% / 21.2% / 18.9% / 32.9% / 32.6%
SD / 4.3% / 6.6% / 3.1% / 3.2% / 3.6% / 10.4%
Lipids / Average / 4.2 / 3.2 / 1.2 / 3.2 / 1.8 / 2.1
(g/MBW) / SD / 1.6 / 0.9 / 0.3 / 1.5 / 0.5 / 0.6
% Lipids / Average / 4.5% / 3.1% / 2.1% / 3.5% / 3.5% / 3.7%
SD / 0.5% / 0.3% / 0.3% / 0.5% / 0.4% / 0.1%
Neutral Detergent Fiber / Average / 42.3 / 50.3 / 27.2 / 39.9 / 21.9 / 24.7
(g/MBW) / SD / 15.9 / 14.5 / 8.8 / 14.0 / 4.9 / 8.0
Total Energy (Kcal/MBW) / Average / 182.9 / 177.4 / 105.5 / 172.4 / 106.6 / 114.5
SD / 64.9 / 56.1 / 30.0 / 56.9 / 26.7 / 30.6
Amino Acid Metabolites / Average / 3,874,740 / 7,655,871 / 4,356,907 / 4,971,969 / 944,692 / 1,952,207
Range / 150,054-13,677,598 / 192,079-30,206,249 / 138,722-15,353,681 / 505,801-14,321,945 / 46,968-5,857,965 / 18,477-10,598,416
Sugar Metabolites / Average / 179,809,901 / 106,698,939 / 46,591,468 / 44,597,474 / 50,043,025 / 96,710,217
Range / 17,355,828-646,243,338 / 5,413,802-782,947,762 / 50,833,383-160,876,872 / 30,250,675-50,793,104 / 6,687,146-29,075,974 / 3,539,931-631,756,346
Lipid Metabolites / Average / 6,521,496 / 4,173,832 / 3,079,078 / 2,637,125 / 2,367,494 / 1,534,664
Range / 959,436-13,868,615 / 800,979-28,930,967 / 507,987-7,645,966 / 416,353-4,813,883 / 251,175-5,618,803 / 102,872-317,739

Table S2. Top ten plant species and parts consumed by each howler group during each sampling block reported in terms of percent of total grams of food ingested. Shannon diversity of plant species and parts consumed by each howler group during each sampling block is also included.WFD = Wet-Fruit Dominated Period, DLD = Dry-Leaf Dominated Period, DFD = Dry-Fruit Dominated Period

MotiepaWFD / Balam WFD
Plant Species / Plant Part / Avg % / SD / Plant Species / Plant Part / Avg % / SD
Dendropanaxarboreus / Ripe Fruit / 12.3 / 11.2 / Brosimumalicastrum / Ripe Fruit / 24.2 / 15.9
Guatteriaanomala / Ripe Fruit / 9.6 / 10.1 / Schizolobiumparahyba / Stem / 14.7 / 10.6
Ficusamericana / Ripe/Unripe Fruit / 9.1 / 4.1 / Dendropanaxarboreus / Ripe Fruit / 8.7 / 8.6
Ficusamericana / Ripe Fruit / 8.3 / 5.4 / Ficusyoponensis / Ripe Fruit / 8.0 / 4.0
Poulseniaarmata / Young Leaf / 7.9 / 5.6 / Guatteriaanomala / Ripe Fruit / 6.0 / 7.5
Schizolobiumparahyba / Stem / 4.4 / 6.0 / Cojobaarborea / Young Leaf / 5.6 / 1.5
Ficusyoponensis / Unripe Fruit / 4.2 / 5.5 / Brosimumalicastrum / Unripe Fruit / 3.9 / 2.9
Ficusinsipida / Ripe Fruit / 3.5 / 3.9 / Cojobaarborea / Stem / 3.7 / 3.5
Ficusaurea / Ripe Fruit / 2.5 / 3.0 / Ficusyoponensis / Young Leaf / 2.7 / 1.1
Ficuspertusa / Ripe Fruit / 2.3 / 2.4 / Ficusyoponensis / Unripe Fruit / 2.1 / 2.0
Shannon Diversity / 2.5 / 0.1 / 2.3 / 0.2
MotiepaDLD / BalamDLD
Plant Species / Plant Part / Avg % / SD / Plant Species / Plant Part / Avg % / SD
Poulseniaarmata / Young Leaf / 19.5 / 10.6 / Ficusyoponensis / Ripe Fruit / 36.4 / 11.1
Schizolobiumparahyba / Stem / 11.2 / 8.8 / Schizolobiumparahyba / Stem / 9.1 / 8.2
Brosimumalicastrum / Unripe Fruit / 10.9 / 5.7 / Brosimumalicastrum / Unripe Fruit / 6.2 / 3.7
Ficusinsipida / Ripe Fruit / 7.5 / 6.0 / Poulseniaarmata / Young Leaf / 6.1 / 3.7
Platymisciumdimorphandrum / Young Leaf / 4.2 / 4.6 / Cojobaarborea / Young Leaf / 5.9 / 5.2
Brosimumalicastrum / Ripe Fruit / 3.4 / 9.0 / Dialiumguianense / Flower / 5.2 / 5.0
Poulseniaarmata / Unripe Fruit / 3.3 / 4.2 / Fabaceaesp. / Young Leaf / 4.2 / 4.6
Brosimumalicastrum / Young Leaf / 2.6 / 3.2 / Platymisciumdimorphandrum / Young Leaf / 4.0 / 2.6
Fabaceae sp. / Young Leaf / 2.5 / 5.1 / Ficusyoponensis / Unripe Fruit / 3.5 / 2.3
Ficusyoponensis / Young Leaf / 1.7 / 1.8 / Brosimumalicastrum / Young Leaf / 2.2 / 2.5
Shannon Diversity / 2.0 / 0.2 / 2.2 / 0.3
MotiepaDFD / BalamDFD
Plant Species / Plant Part / Avg % / SD / Plant Species / Plant Part / Avg % / SD
Poulseniaarmata / Ripe Fruit / 24.5 / 8.5 / Poulseniaarmata / Ripe Fruit / 23.2 / 17.3
Ficusamericana / Ripe Fruit / 15.1 / 5.2 / Ficusyoponensis / Ripe Fruit / 14.1 / 8.5
Ficusaurea / Ripe Fruit / 10.4 / 4.1 / Ficusaurea / Ripe Fruit / 12.3 / 3.2
Ampelocerahottlei / Ripe Fruit / 8.2 / 6.6 / Compsoneurasp. / Ripe Fruit / 9.8 / 7.4
Ficusyoponensis / Ripe Fruit / 7.2 / 6.6 / Cojobaarborea / Young Leaf / 4.9 / 3.5
Poulseniaarmata / Young Leaf / 6.7 / 3.3 / Brosimumalicastrum / Ripe Fruit / 3.8 / 7.0
Brosimumalicastrum / Unripe Fruit / 1.6 / 2.1 / Cojobaarborea / Stem / 2.9 / 2.8
Brosimumalicastrum / Young Leaf / 1.1 / 1.3 / Poulseniaarmata / Young Leaf / 2.7 / 1.9
Cojobaarborea / Young Leaf / 1.1 / 1.3 / Ficus maxima / Ripe Fruit / 1.3 / 2.1
Ficusyoponensis / Stem / 0.9 / 1.6 / Ficusaurea / Unripe Fruit / 1.2 / 1.6
Shannon Diversity / 2.2 / 0.2 / 2.3 / 0.4

Table S3. Relative abundances of bacterial families for each howler group across sampling periods. Values are expressed in percent of total sequences. Taxa highlighted in bold showed significant changes across sampling periods (p < 0.05). Analyses were performed on combined data for both howler groups. WFD = Wet-Fruit Dominated Period, DLD = Dry-Leaf Dominated Period, DFD = Dry-Fruit Dominated Period

WFD / DLD / DFD
Balam / Motiepa / Balam / Motiepa / Balam / Motiepa
Family / AVG / SD / AVG / SD / AVG / SD / AVG / SD / AVG / SD / AVG / SD
Lachnospiraceae / 32.577% / 4.108% / 39.526% / 5.567% / 30.807% / 2.424% / 33.991% / 5.748% / 22.295% / 8.150% / 27.831% / 2.368%
Ruminococcaceae / 15.152% / 0.719% / 14.223% / 1.450% / 20.910% / 5.691% / 16.987% / 6.127% / 29.755% / 9.678% / 22.338% / 4.414%
Prevotellaceae / 8.287% / 3.450% / 8.278% / 4.841% / 10.453% / 7.860% / 12.539% / 5.686% / 7.513% / 3.277% / 7.102% / 3.397%
Veillonellaceae / 2.464% / 3.061% / 0.647% / 0.423% / 0.979% / 0.649% / 1.189% / 0.488% / 0.883% / 0.434% / 1.492% / 0.748%
Coriobacteriaceae / 1.893% / 0.753% / 0.633% / 0.261% / 0.955% / 0.384% / 0.729% / 0.350% / 0.752% / 0.204% / 0.646% / 0.190%
Erysipelotrichaceae / 1.743% / 0.739% / 1.747% / 0.325% / 0.435% / 0.222% / 0.362% / 0.107% / 0.774% / 0.485% / 1.642% / 1.154%
Verrucomicrobiaceae / 1.434% / 1.655% / 0.192% / 0.232% / 0.146% / 0.278% / 0.064% / 0.040% / 0.215% / 0.251% / 0.255% / 0.203%
IncertaeSedis XIII / 0.603% / 0.443% / 0.160% / 0.098% / 0.157% / 0.048% / 0.318% / 0.245% / 0.098% / 0.044% / 0.104% / 0.155%
Synergistaceae / 0.477% / 0.406% / 0.331% / 0.403% / 0.109% / 0.091% / 0.119% / 0.160% / 0.099% / 0.052% / 0.165% / 0.103%
IncertaeSedis XIV / 0.343% / 0.155% / 0.426% / 0.165% / 0.378% / 0.197% / 0.361% / 0.045% / 0.355% / 0.197% / 0.584% / 0.195%
Desulfovibrionaceae / 0.196% / 0.218% / 0.161% / 0.176% / 0.122% / 0.029% / 0.190% / 0.178% / 0.095% / 0.110% / 0.208% / 0.171%
Porphyromonadaceae / 0.174% / 0.143% / 0.147% / 0.156% / 0.210% / 0.126% / 0.362% / 0.453% / 0.211% / 0.217% / 0.669% / 1.034%
Chloroplast / 0.158% / 0.199% / 0.055% / 0.049% / 0.070% / 0.031% / 0.127% / 0.113% / 0.036% / 0.052% / 0.027% / 0.013%
Streptococcaceae / 0.067% / 0.029% / 0.156% / 0.112% / 0.022% / 0.018% / 0.043% / 0.034% / 0.011% / 0.009% / 0.015% / 0.010%
Spirochaetaceae / 0.060% / 0.120% / 0.000% / 0.000% / 0.020% / 0.039% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Sphingomonadaceae / 0.046% / 0.088% / 0.004% / 0.005% / 0.003% / 0.004% / 0.010% / 0.009% / 0.002% / 0.004% / 0.004% / 0.006%
Brachyspiraceae / 0.035% / 0.012% / 0.022% / 0.008% / 0.020% / 0.006% / 0.019% / 0.009% / 0.023% / 0.003% / 0.029% / 0.010%
Oxalobacteraceae / 0.022% / 0.020% / 0.000% / 0.000% / 0.019% / 0.021% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Anaeroplasmataceae / 0.022% / 0.022% / 0.091% / 0.058% / 0.009% / 0.014% / 0.011% / 0.013% / 0.028% / 0.015% / 0.037% / 0.031%
Enterococcaceae / 0.012% / 0.017% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.009% / 0.010% / 0.000% / 0.000%
Hyphomicrobiaceae / 0.012% / 0.012% / 0.021% / 0.032% / 0.004% / 0.005% / 0.006% / 0.009% / 0.000% / 0.000% / 0.004% / 0.005%
Planctomycetaceae / 0.009% / 0.018% / 0.007% / 0.009% / 0.006% / 0.002% / 0.002% / 0.004% / 0.004% / 0.004% / 0.001% / 0.003%
Clostridiaceae / 0.008% / 0.016% / 0.059% / 0.068% / 0.054% / 0.061% / 0.037% / 0.053% / 0.031% / 0.053% / 0.027% / 0.013%
Opitutaceae / 0.008% / 0.006% / 0.054% / 0.045% / 0.015% / 0.026% / 0.050% / 0.054% / 0.016% / 0.011% / 0.203% / 0.169%
Neisseriaceae / 0.008% / 0.015% / 0.039% / 0.061% / 0.000% / 0.000% / 0.023% / 0.023% / 0.003% / 0.007% / 0.016% / 0.015%
Enterobacteriaceae / 0.006% / 0.012% / 0.060% / 0.025% / 0.035% / 0.049% / 0.110% / 0.070% / 0.001% / 0.002% / 0.059% / 0.025%
Nocardioidaceae / 0.006% / 0.011% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.003% / 0.000% / 0.000% / 0.003% / 0.003%
Bradyrhizobiaceae / 0.005% / 0.006% / 0.003% / 0.005% / 0.006% / 0.008% / 0.003% / 0.004% / 0.000% / 0.000% / 0.006% / 0.011%
Phyllobacteriaceae / 0.004% / 0.005% / 0.011% / 0.013% / 0.004% / 0.008% / 0.000% / 0.000% / 0.000% / 0.000% / 0.006% / 0.012%
Flavobacteriaceae / 0.004% / 0.005% / 0.000% / 0.000% / 0.004% / 0.009% / 0.004% / 0.005% / 0.001% / 0.002% / 0.000% / 0.000%
Mycobacteriaceae / 0.004% / 0.007% / 0.004% / 0.008% / 0.002% / 0.004% / 0.004% / 0.005% / 0.000% / 0.000% / 0.001% / 0.002%
Alcaligenaceae / 0.003% / 0.006% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Pseudomonadaceae / 0.002% / 0.005% / 0.006% / 0.007% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000%
Comamonadaceae / 0.002% / 0.004% / 0.002% / 0.003% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.001% / 0.002%
Helicobacteraceae / 0.002% / 0.004% / 0.004% / 0.008% / 0.002% / 0.004% / 0.005% / 0.006% / 0.005% / 0.007% / 0.004% / 0.003%
Pseudonocardiaceae / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Sphingobacteriaceae / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000%
Thiotrichaceae / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.003% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000%
Bacteroidaceae / 0.002% / 0.003% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.001% / 0.002% / 0.006% / 0.012%
Leuconostocaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.006% / 0.011% / 0.000% / 0.000%
Bacillaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000%
Actinomycetaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.003% / 0.003% / 0.005%
Intrasporangiaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000%
Micromonosporaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004% / 0.004% / 0.004% / 0.001% / 0.002% / 0.000% / 0.000%
Nocardiaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000%
Acidimicrobidae / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.002% / 0.004%
Anaerolineaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.003% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000%
Caulobacteraceae / 0.000% / 0.000% / 0.002% / 0.004% / 0.002% / 0.004% / 0.004% / 0.004% / 0.000% / 0.000% / 0.007% / 0.014%
Chitinophagaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.001% / 0.003%
Conexibacteraceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000%
Corynebacteriaceae / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Cystobacteraceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004%
Erythrobacteraceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002%
Gemmatimonadaceae / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
IncertaeSedis XI / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.020% / 0.039% / 0.000% / 0.000% / 0.001% / 0.003%
Kineosporiaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000%
Kofleriaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Microbacteriaceae / 0.000% / 0.000% / 0.004% / 0.005% / 0.000% / 0.000% / 0.004% / 0.007% / 0.000% / 0.000% / 0.002% / 0.002%
Nitrospiraceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004%
Propionibacteriaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004%
Puniceicoccaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.001% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Rhizobiaceae / 0.000% / 0.000% / 0.002% / 0.005% / 0.006% / 0.007% / 0.001% / 0.002% / 0.000% / 0.000% / 0.008% / 0.009%
Rhodobacteraceae / 0.000% / 0.000% / 0.005% / 0.006% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Rubrobacteraceae / 0.000% / 0.000% / 0.003% / 0.005% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Sinobacteraceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.005% / 0.010%
Streptomycetaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Streptosporangiaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.016% / 0.026%
Syntrophomonadaceae / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.003%
Victivallaceae / 0.000% / 0.000% / 0.016% / 0.021% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Xanthomonadaceae / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%

Table S4. Relative abundances of bacterial genera for each howler group across sampling periods. Values are expressed in percent of total sequences. Taxa highlighted in bold showed significant changes across sampling periods (p < 0.05). Analyses were performed on combined data for both howler groups. **again it might be helpful to the reader to define WFD, DLD, and DFD in the table**

WFD / DLD / DFD
Balam / Motiepa / Balam / Motiepa / Balam / Motiepa
Genus / AVG / SD / AVG / SD / AVG / SD / AVG / SD / AVG / SD / AVG / SD
Oscillibacter / 2.847% / 2.154% / 1.427% / 0.272% / 2.282% / 1.829% / 0.838% / 0.226% / 0.634% / 0.356% / 0.827% / 0.283%
Succiniclasticum / 1.703% / 3.047% / 0.242% / 0.459% / 0.183% / 0.148% / 0.334% / 0.482% / 0.042% / 0.029% / 0.100% / 0.180%
Prevotella / 1.608% / 1.458% / 1.680% / 1.184% / 4.605% / 5.733% / 2.682% / 3.331% / 2.098% / 2.672% / 1.928% / 1.480%
Akkermansia / 1.434% / 1.655% / 0.192% / 0.232% / 0.146% / 0.278% / 0.064% / 0.040% / 0.229% / 0.264% / 0.252% / 0.201%
Hallella / 1.356% / 1.189% / 1.741% / 0.641% / 0.743% / 0.513% / 6.294% / 5.168% / 1.352% / 1.257% / 2.589% / 1.640%
Coprobacillus / 1.058% / 0.720% / 1.085% / 0.324% / 0.192% / 0.106% / 0.124% / 0.081% / 0.421% / 0.247% / 1.168% / 0.996%
Subdoligranulum / 0.874% / 0.372% / 0.529% / 0.202% / 0.439% / 0.198% / 0.552% / 0.133% / 0.734% / 0.349% / 0.813% / 0.394%
Faecalibacterium / 0.808% / 0.739% / 1.247% / 0.430% / 0.318% / 0.155% / 1.543% / 1.728% / 1.119% / 1.231% / 1.837% / 1.222%
Xylanibacter / 0.734% / 0.732% / 0.272% / 0.256% / 0.327% / 0.294% / 0.189% / 0.243% / 0.206% / 0.204% / 0.134% / 0.184%
Marvinbryantia / 0.582% / 0.271% / 0.725% / 0.232% / 0.668% / 0.079% / 0.903% / 0.447% / 0.457% / 0.182% / 0.711% / 0.257%
TM7 / 0.392% / 0.500% / 0.015% / 0.019% / 0.181% / 0.215% / 0.070% / 0.058% / 0.440% / 0.414% / 0.238% / 0.150%
Roseburia / 0.371% / 0.141% / 0.998% / 0.821% / 0.751% / 0.361% / 0.584% / 0.500% / 0.386% / 0.355% / 0.415% / 0.182%
Mogibacterium / 0.364% / 0.251% / 0.110% / 0.109% / 0.105% / 0.048% / 0.140% / 0.194% / 0.053% / 0.024% / 0.076% / 0.125%
Blautia / 0.320% / 0.165% / 0.416% / 0.174% / 0.347% / 0.194% / 0.456% / 0.290% / 0.330% / 0.196% / 0.573% / 0.193%
Butyricicoccus / 0.295% / 0.122% / 0.254% / 0.103% / 0.447% / 0.172% / 0.395% / 0.049% / 0.252% / 0.075% / 0.207% / 0.085%
Ruminococcus / 0.263% / 0.099% / 0.117% / 0.074% / 0.090% / 0.033% / 0.085% / 0.042% / 0.357% / 0.468% / 0.091% / 0.033%
Coprococcus / 0.222% / 0.089% / 0.718% / 0.436% / 0.522% / 0.127% / 0.332% / 0.259% / 0.452% / 0.388% / 0.942% / 0.657%
Papillibacter / 0.173% / 0.079% / 0.259% / 0.169% / 0.446% / 0.112% / 0.549% / 0.584% / 0.859% / 0.106% / 0.608% / 0.094%
Atopobium / 0.162% / 0.042% / 0.091% / 0.017% / 0.078% / 0.070% / 0.095% / 0.055% / 0.138% / 0.067% / 0.139% / 0.077%
Streptophyta / 0.158% / 0.199% / 0.055% / 0.049% / 0.070% / 0.031% / 0.125% / 0.114% / 0.036% / 0.052% / 0.027% / 0.013%
Gordonibacter / 0.151% / 0.133% / 0.028% / 0.008% / 0.067% / 0.045% / 0.027% / 0.018% / 0.023% / 0.024% / 0.032% / 0.018%
Desulfovibrio / 0.128% / 0.189% / 0.074% / 0.130% / 0.029% / 0.020% / 0.121% / 0.175% / 0.066% / 0.120% / 0.166% / 0.161%
Anaerovorax / 0.117% / 0.084% / 0.034% / 0.031% / 0.029% / 0.012% / 0.059% / 0.032% / 0.022% / 0.021% / 0.016% / 0.008%
Treponema / 0.105% / 0.210% / 0.000% / 0.000% / 0.011% / 0.022% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Solobacterium / 0.066% / 0.053% / 0.125% / 0.090% / 0.030% / 0.013% / 0.073% / 0.042% / 0.184% / 0.135% / 0.105% / 0.025%
Streptococcus / 0.065% / 0.032% / 0.156% / 0.112% / 0.017% / 0.016% / 0.043% / 0.034% / 0.007% / 0.006% / 0.015% / 0.010%
Collinsella / 0.055% / 0.026% / 0.027% / 0.014% / 0.031% / 0.023% / 0.048% / 0.043% / 0.039% / 0.014% / 0.023% / 0.016%
Moryella / 0.053% / 0.017% / 0.147% / 0.051% / 0.107% / 0.064% / 0.042% / 0.016% / 0.022% / 0.018% / 0.062% / 0.020%
Helicobacter / 0.044% / 0.088% / 0.004% / 0.008% / 0.001% / 0.002% / 0.005% / 0.006% / 0.005% / 0.007% / 0.124% / 0.237%
Anaerotruncus / 0.044% / 0.047% / 0.008% / 0.009% / 0.085% / 0.134% / 0.050% / 0.024% / 0.039% / 0.022% / 0.016% / 0.008%
Brachyspira / 0.033% / 0.012% / 0.022% / 0.008% / 0.020% / 0.006% / 0.019% / 0.009% / 0.023% / 0.003% / 0.029% / 0.010%
Butyrivibrio / 0.031% / 0.022% / 0.028% / 0.010% / 0.022% / 0.018% / 0.024% / 0.014% / 0.018% / 0.004% / 0.018% / 0.013%
Barnesiella / 0.023% / 0.020% / 0.020% / 0.039% / 0.017% / 0.022% / 0.059% / 0.118% / 0.012% / 0.012% / 0.035% / 0.061%
Howardella / 0.023% / 0.017% / 0.009% / 0.013% / 0.031% / 0.009% / 0.025% / 0.019% / 0.025% / 0.005% / 0.011% / 0.005%
Oxalobacter / 0.022% / 0.020% / 0.000% / 0.000% / 0.019% / 0.021% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Anaerostipes / 0.021% / 0.013% / 0.025% / 0.015% / 0.049% / 0.066% / 0.022% / 0.034% / 0.026% / 0.012% / 0.013% / 0.013%
Syntrophococcus / 0.019% / 0.011% / 0.011% / 0.009% / 0.017% / 0.017% / 0.007% / 0.012% / 0.007% / 0.003% / 0.007% / 0.006%
Oribacterium / 0.019% / 0.013% / 0.007% / 0.005% / 0.004% / 0.005% / 0.004% / 0.009% / 0.002% / 0.004% / 0.001% / 0.003%
Dorea / 0.018% / 0.023% / 0.033% / 0.016% / 0.009% / 0.014% / 0.018% / 0.013% / 0.007% / 0.005% / 0.011% / 0.015%
Anaeroplasma / 0.018% / 0.019% / 0.058% / 0.020% / 0.022% / 0.029% / 0.008% / 0.012% / 0.025% / 0.014% / 0.033% / 0.028%
Acetivibrio / 0.016% / 0.015% / 0.010% / 0.010% / 0.011% / 0.005% / 0.002% / 0.003% / 0.001% / 0.003% / 0.000% / 0.000%
Parabacteroides / 0.013% / 0.010% / 0.000% / 0.000% / 0.029% / 0.008% / 0.004% / 0.004% / 0.016% / 0.014% / 0.011% / 0.009%
Enterococcus / 0.012% / 0.017% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.009% / 0.010% / 0.000% / 0.000%
Cryptobacterium / 0.012% / 0.013% / 0.005% / 0.006% / 0.024% / 0.005% / 0.014% / 0.009% / 0.018% / 0.013% / 0.011% / 0.013%
Eggerthella / 0.012% / 0.008% / 0.007% / 0.005% / 0.012% / 0.006% / 0.009% / 0.011% / 0.012% / 0.008% / 0.004% / 0.005%
Dialister / 0.008% / 0.010% / 0.004% / 0.005% / 0.001% / 0.003% / 0.011% / 0.004% / 0.007% / 0.003% / 0.313% / 0.593%
Sarcina / 0.008% / 0.016% / 0.059% / 0.068% / 0.053% / 0.040% / 0.036% / 0.051% / 0.029% / 0.049% / 0.025% / 0.009%
Robinsoniella / 0.005% / 0.006% / 0.007% / 0.014% / 0.002% / 0.003% / 0.004% / 0.003% / 0.003% / 0.004% / 0.003% / 0.003%
Gp6 / 0.005% / 0.006% / 0.002% / 0.004% / 0.007% / 0.003% / 0.004% / 0.004% / 0.002% / 0.004% / 0.004% / 0.008%
Brevundimonas / 0.005% / 0.010% / 0.002% / 0.004% / 0.002% / 0.004% / 0.002% / 0.004% / 0.000% / 0.000% / 0.005% / 0.010%
Devosia / 0.004% / 0.005% / 0.002% / 0.004% / 0.000% / 0.000% / 0.005% / 0.009% / 0.000% / 0.000% / 0.000% / 0.000%
Cloacibacterium / 0.004% / 0.005% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Asteroleplasma / 0.004% / 0.008% / 0.032% / 0.044% / 0.002% / 0.004% / 0.003% / 0.004% / 0.004% / 0.004% / 0.004% / 0.003%
Mycobacterium / 0.004% / 0.007% / 0.004% / 0.008% / 0.002% / 0.004% / 0.004% / 0.005% / 0.000% / 0.000% / 0.001% / 0.002%
Spartobacteria / 0.004% / 0.007% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Schlesneria / 0.003% / 0.006% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Veillonella / 0.003% / 0.006% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Marmoricola / 0.003% / 0.006% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Escherichia/Shigella / 0.003% / 0.006% / 0.055% / 0.022% / 0.022% / 0.040% / 0.108% / 0.070% / 0.000% / 0.000% / 0.057% / 0.026%
Aeromicrobium / 0.002% / 0.005% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Bradyrhizobium / 0.002% / 0.005% / 0.000% / 0.000% / 0.006% / 0.008% / 0.001% / 0.002% / 0.000% / 0.000% / 0.003% / 0.005%
Acetitomaculum / 0.002% / 0.005% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Pseudomonas / 0.002% / 0.005% / 0.004% / 0.004% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000%
Paraprevotella / 0.002% / 0.005% / 0.003% / 0.006% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Olsenella / 0.002% / 0.005% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.002% / 0.004% / 0.000% / 0.000%
Hespellia / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Opitutus / 0.002% / 0.004% / 0.002% / 0.004% / 0.030% / 0.060% / 0.007% / 0.011% / 0.002% / 0.002% / 0.020% / 0.011%
Pirellula / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000%
Aminobacter / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Beggiatoa / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.003% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000%
Blastochloris / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Hyphomicrobium / 0.002% / 0.004% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Pedomicrobium / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Pseudonocardia / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Sphingobacterium / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Bacteroides / 0.002% / 0.003% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.001% / 0.002% / 0.006% / 0.012%
Acetanaerobacterium / 0.002% / 0.003% / 0.000% / 0.000% / 0.005% / 0.006% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Catonella / 0.002% / 0.003% / 0.000% / 0.000% / 0.002% / 0.003% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Gp3 / 0.002% / 0.003% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Lactococcus / 0.002% / 0.003% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Sandaracinobacter / 0.002% / 0.003% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Weissella / 0.000% / 0.000% / 0.003% / 0.006% / 0.000% / 0.000% / 0.000% / 0.000% / 0.006% / 0.011% / 0.000% / 0.000%
Corynebacterium / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.004% / 0.009% / 0.000% / 0.000%
Synergistes / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.004% / 0.007% / 0.000% / 0.000%
Slackia / 0.000% / 0.000% / 0.003% / 0.006% / 0.002% / 0.004% / 0.002% / 0.004% / 0.002% / 0.004% / 0.001% / 0.002%
Bacillus / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000%
Pseudobutyrivibrio / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000%
Mobiluncus / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.003% / 0.000% / 0.000%
Flavobacterium / 0.000% / 0.000% / 0.000% / 0.000% / 0.004% / 0.009% / 0.004% / 0.004% / 0.001% / 0.002% / 0.000% / 0.000%
Gordonia / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.009% / 0.014% / 0.001% / 0.002% / 0.000% / 0.000%
Anaerosporobacter / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.002% / 0.004%
Acidovorax / 0.000% / 0.000% / 0.002% / 0.003% / 0.000% / 0.000% / 0.007% / 0.013% / 0.000% / 0.000% / 0.000% / 0.000%
Actinomyces / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.003% / 0.005%
Allobaculum / 0.000% / 0.000% / 0.005% / 0.010% / 0.000% / 0.000% / 0.016% / 0.032% / 0.000% / 0.000% / 0.031% / 0.063%
Arthrobacter / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Asaccharobacter / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000%
Asticcacaulis / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004%
Balneimonas / 0.000% / 0.000% / 0.003% / 0.005% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000%
Chryseobacterium / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000%
Conexibacter / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000%
Enterobacter / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Erwinia / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.001% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Filomicrobium / 0.000% / 0.000% / 0.003% / 0.005% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Gemmatimonas / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Gp1 / 0.000% / 0.000% / 0.004% / 0.009% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Gp4 / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Ilumatobacter / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.002% / 0.004%
Kofleria / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Leucobacter / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002%
Luteolibacter / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.003% / 0.004%
Mesorhizobium / 0.000% / 0.000% / 0.003% / 0.005% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004%
Microbacterium / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.004% / 0.007% / 0.000% / 0.000% / 0.000% / 0.000%
Nitrospira / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004%
Nocardioides / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.003% / 0.003%
Novosphingobium / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.003% / 0.004%
Parasporobacterium / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002%
Pelospora / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.003%
Phyllobacterium / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Porphyrobacter / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002%
Prosthecomicrobium / 0.000% / 0.000% / 0.003% / 0.005% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Rhizobium / 0.000% / 0.000% / 0.002% / 0.005% / 0.028% / 0.049% / 0.000% / 0.000% / 0.000% / 0.000% / 0.008% / 0.009%
Rhodoplanes / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002%
Rubrobacter / 0.000% / 0.000% / 0.003% / 0.005% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Serratia / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004%
Shuttleworthia / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.002% / 0.004%
Sphingomonas / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002%
Steroidobacter / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.005% / 0.010%
Streptomyces / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.002% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Subdivision 3 / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.001% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Tannerella / 0.000% / 0.000% / 0.000% / 0.000% / 0.003% / 0.005% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Terrimonas / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.001% / 0.003%
Victivallis / 0.000% / 0.000% / 0.013% / 0.016% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%
Xanthomonas / 0.000% / 0.000% / 0.002% / 0.004% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000% / 0.000%

Table S5. Spearman’s ρ values for significant correlations (p < 0.05) between microbial taxa and amounts of different diet components consumed. NDF = Neutral Detergent Fiber, TNC = Total Non-structural Carbohydrates

Taxon / Kcal / Protein / Lipid / NDF / TNC / Young Leaf / Mature Leaf / Unripe Fruit / Stem
Acetivibrio / 0.70 / 0.75 / 0.69 / 0.75 / 0.61 / 0.58
Butyricicoccus / 0.54 / 0.57
Coprobacillus / 0.53
Dialister / -0.53
Hallella / 0.57 / -0.60 / -0.55
Mogibacterium / 0.55
IncertaeSedis XIII / 0.64
Oscillibacter / -0.60 / 0.72 / 0.56
Streptococcus
Streptophyta / 0.59 / 0.68

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Table S6. R values for significant correlations (p < 0.05) between microbial taxa and relative concentration of diet metabolites.

Metabolite / Ruminococcaeeae / Acetivibrio / Akkermanisia / Butyricicoccus / Coprobacillus / Dialister / Gordonibacter / Hallella / Oribacterium / Oscillibacter / Papillibacter / Prevotella / Solobacterium / Streptophyta / TM7 / Xylanibacter
1-hexadecanoylglycerol / 0.95 / 0.68 / 0.66 / 0.89 / 0.88
1-Methyl-alpha-D-glucopyranoside / 0.99 / 0.99
1-Methyl-beta-D-galactopyranoside / 0.68 / 0.76 / 0.72 / 0.66
1-Octedecenoylglycerol / 0.68 / 0.67
1,6-Anhydroglucose / 0.78
2-hydroxyglutaric acid / 0.66
2,3-Dihydroxybutanedioic acid / 0.71 / 0.78 / 0.8 / 0.67
2,3-dihydroxysuccinic acid / 0.67
24-Methylenecycloartanol / 0.74 / 0.75
2-Methylcitric acid / 0.75 / 0.74 / 0.7 / 0.73 / 0.71 / 0.74 / -0.69
2-Methylglutamic acid / 0.69 / 0.72 / 0.7 / 0.68 / 0.73
2-Methylmalic acid / 0.76 / 0.72 / 0.73 / 0.75 / 0.71 / 0.74
2-methylsuccinic acid / 0.73 / 0.65 / 0.66 / 0.68 / 0.79 / 0.69 / -0.68
2-Piperidinecarboxylic acid / 0.68 / 0.67
3,4-Dihydroxybutanoic acid / 0.71 / 0.84 / 0.8 / 0.69 / 0.67
3,4-Dimethoxycinnamic acid / 0.71 / 0.7 / 0.7
3,4,5-Trihydroxypentanoic acid / 0.68
3-Deoxy-arabino-hexaric acid / 0.72 / 0.82 / 0.79 / 0.69 / 0.69 / -0.68
3-methyl-2-hydroxypentanoic acid / 0.69 / 0.68 / 0.68 / 0.67
4,5-dimethyl-2,6-dihydroxypyrimidine / 0.66
4-Caffeoylquinic acid / 0.68 / 0.69 / 0.67 / 0.68 / 0.7 / 0.67
4-Hydroxy-3-methoxyphenethyleneglycol / 0.7 / 0.69 / 0.68
4-Hydroxyproline / 0.66
Adenine / 0.7
Aspartic acid / 0.66 / 0.68 / 0.66 / 0.74
C16:1 / -0.77 / -0.77 / -0.75
Diethyleneglycol / 0.72 / 0.74 / 0.75 / 0.76
Eicosanol / 0.66 / 0.67
Ethanol, (2-(3,4-dihydroxyphenyl)- / 0.66
Erythritol / 0.65 / 0.79 / 0.74
Ferulic acid / 0.69 / 0.67 / 0.78 / 0.67
Gallic acid / 0.68
Glucaric acid / 0.73 / 0.68 / 0.66 / 0.74 / 0.76 / 0.74
Glucuronic acid / 0.7 / 0.65 / 0.65 / 0.69 / 0.66 / 0.7 / -0.72
Glycolic acid / 0.67 / 0.68 / 0.66 / 0.7
Guanine / 0.73 / 0.77 / 0.74 / 0.72 / 0.72 / -0.67
Hentriacontanol / 0.81 / 0.67 / 0.65 / 0.75 / 0.7 / 0.74
Heptacosanol / 0.75 / 0.71 / 0.67 / 0.71 / 0.68 / 0.7
Hexacosanoic acid / 0.73
Isoleucine / 0.75 / 0.76 / 0.77 / 0.74 / 0.74 / -0.69
Kaempferol / 0.72 / 0.73 / 0.72 / 0.71
Lactic Acid / -0.67
Leucine / 0.68
Monomethylphosphate / 0.73 / 0.75 / 0.75 / 0.73 / 0.72
N-Acetyl glucosamine / 0.7 / 0.75 / 0.68 / 0.69 / 0.7 / 0.69 / -0.67
N-Acetylglucosylamine / 0.7
Nicotinic acid / 0.78
Octacosanol / 0.67 / 0.66 / 0.75 / 0.66 / -0.67
p-hydroxyCoumaric acid / -0.7
Protocatechuic acid / 0.7 / 0.65 / 0.67 / 0.73 / 0.71 / 0.72
Quinic acid / 0.67 / 0.71 / 0.71
Ribose / 0.67 / 0.73
Shikimic acid / 0.67
Sorbitol / 0.75
Tocopherol-a / 0.67 / 0.7 / 0.67 / 0.67 / 0.71 / 0.7 / 0.68
Uridine
Xylitol / 0.74 / 0.81 / 0.8 / 0.7 / 0.69 / -0.67

Table S7. Fecal concentrations (mM) of volatile fatty acids and ammoniafor each howler group across sampling periods. *Indicates significant changes across time (p < 0.05). VFA = Volatile Fatty Acid

Sampling Block / Group / Acetic* / Propanoic / Butanoic* / Pentanoic / Isobutanoic / Isopentanoic* / Total VFAs* / Ammonia*
Average / SD / Average / SD / Average / SD / Average / SD / Average / SD / Average / SD / Average / SD / Average / SD
WFD / Balam / 43.0 / 8.2 / 3.4 / 1.1 / 1.9 / 1.0 / 0.3 / 0.1 / 0.2 / 0.1 / 0.2 / 0.1 / 49.0 / 10.3 / 9.6 / 4.0
Motiepa / 36.9 / 4.8 / 3.0 / 0.5 / 1.8 / 0.5 / 0.3 / 0.1 / 0.2 / 0.1 / 0.2 / 0.1 / 42.4 / 4.9 / 11.0 / 7.8
DLD / Balam / 33.0 / 2.1 / 3.6 / 0.4 / 2.4 / 0.4 / 0.4 / 0.2 / 0.1 / 0.0 / 0.1 / 0.0 / 39.7 / 2.4 / 3.9 / 0.8
Motiepa / 36.1 / 2.8 / 3.7 / 0.3 / 2.8 / 0.6 / 0.4 / 0.1 / 0.1 / 0.0 / 0.1 / 0.1 / 43.2 / 3.2 / 4.2 / 1.0
DFD / Balam / 46.5 / 3.0 / 3.7 / 0.5 / 1.7 / 0.2 / 0.3 / 0.1 / 0.1 / 0.0 / 0.2 / 0.1 / 52.5 / 3.1 / 4.2 / 0.9
Motiepa / 48.4 / 4.5 / 4.0 / 0.7 / 2.1 / 0.3 / 0.4 / 0.1 / 0.1 / 0.0 / 0.2 / 0.0 / 55.2 / 5.4 / 4.0 / 0.9

1

Table S8. Spearman’s ρ values for significant correlations (p < 0.05) between individual diet components and fecal VFA and ammonia concentration. NDF = Neutral Detergent Fiber, TNC = Total Non-structural Carbohydrates

Microbial Product / Unripe Fruit / Young Leaves / Mature Leaves / Protein / TNC / NDF
Acetic Acid / -0.58 / -0.65 / 0.50 / -0.51
Butanoic Acid / 0.43
Isobutanoic Acid / 0.46
Isopentanoic Acid / -0.42
Ammonia / 0.43 / 0.51 / 0.50

Table S9. Spearman’s ρ values for significant correlations (p < 0.05) between the relative concentration of ingested metabolites and fecal volatile fatty acid and ammonia concentration.

Metabolite / Acetic / Butanoic / Ammonia
1,2,3-trihydroxybenzene / 0.79
1,2,3-trihydroxybutane / -0.21
1,6-Anhydroglucose / 0.7
1-Methyl-beta-D-galactopyranoside / -0.71
1-Octedecenoylglycerol / 0.65
2,3-Dihydroxybutanedioic acid / -0.67
2,3-dihydroxysuccinic acid / 0.66
2,4,5-Trihydroxypentanoic acid / 0.55
2,5-dihydroxybenzoic acid / 0.56
2,4-Methylenecycloartanol / 0.57
2-Methylcitric acid / 0.56
2-Methylmalic acid / 0.76
2-Oxoisocaproic acid / 0.61
2-Piperidinecarboxylic acid / -0.71
3,4,5-Trihydroxypentanoic acid / 0.73
3,4-Dimethoxycinnamic acid / 0.63
3-Deoxy-arabino-hexaric acid / 0.63
3-hydroxybenzoic acid / 0.57
3-methyl-2-hydroxypentanoic acid / 0.63
3-methyl-2-oxobutanoic acid / 0.65
4-Caffeoylquinic acid / -0.7
4-Hydroxy-3-methoxyphenethyleneglycol / 0.65
4-Hydroxyproline / -0.55
Allantoin / -0.62 / 0.55
beta-Amyrin / 0.62
C16:1 / 0.62
C18:1 / 0.69
chlorogenic acid / -0.71
Citric acid / 0.61
Diethyleneglycol / 0.53
Erythronic acid / 0.63
Ethanol, (2-(3,4-dihydroxyphenyl)- / 0.6
galacturonic acid / -0.63
gallic acid / 0.59
Glucaric acid / 0.64
glucuronic acid / 0.62
Hentriacontanol / 0.71
Inositol, myo- / 0.56
Itaconic acid / 0.64
Kaempferol / 0.62
Mannitol / 0.55
N-Acetylglucosylamine / -0.36
N-Acetyl glucosamine / 0.55
neochlorogenic acid / -0.73
Nonacosanol / 0.58
Octacosanol / 0.71
p-hydroxybenzaldehyde / 0.59
Protocatechuic acid / 0.63
Shikimic acid / 0.59
Sorbitol / -0.63
Thymine / 0.58
Vanillic acid / 0.67
Xylopyranoside / 0.56
Xylose / 0.57

Table S10. Spearman’s ρ values for significant correlations (p < 0.05) between microbial taxa and fecal volatile fatty acid and ammonia concentration.

Taxon / Acetic Acid / Isobutanoic Acid / Isopentanoic Acid / Ammonia
Anaeroplasmataceae / 0.55
Erysipeltotrichaceae / 0.53
Streptococcaceae / 0.61
Acetivibrio / 0.56
Butyricicoccus / -0.61
Coprobacillus / 0.54 / 0.54
Oscillibacter / 0.53
Streptococcus / 0.63
Streptophyta / -0.61
Xylanibacter / 0.53

Table S11.Spearman’s ρ values for significant correlations (p < 0.05) between howler activity and diet components and between howler activity and average daily maximum temperature over the entire study period. NDF = Neutral Detergent Fiber, TNC = Total Non-structural Carbohydrates

Energy (kcal/MBW) / Protein (g/MBW) / NDF (g/MBW) / TNC (g/MBW) / Lipids (g/MBW) / Max Temp(°C)
Resting / -0.5 / -0.67 / -0.68 / 0.87
Feeding / 0.76 / 0.71 / 0.87 / 0.54 / 0.64 / -0.89

Table S12. Volatile fatty acid concentrations and profiles in nonhuman primates and mammals. Table modified from Lambert (2012).

WILD / Total VFA Concentration (mM) / % Acetate / % Butyrate / %Propionate / Source
Procolobusverus / 230 / Ohwaki et al. 1974
Colobusguereza / 107–434 / Ohwaki et al. 1974
Cercopithecusaethiops / 190–229 / Brourton et al. 1991
Cercopithecusmitis / 122–199 / Brourton et al. 1991
138–180 / Clemens and Phillips 1980
Papio cynocephalus / 95–170 / Clemens and Phillips 1980
Pan troglodytes / 44 / 31 / 10 / 3 / Ushida et al. 2006
Alouattapalliata / 94 / 6 / 0.4 / Milton and McBee 1983
Alouattapigra / 40-55 / 83-89 / 3-7 / 7-9 / this study
Homo sapiens / 100 / 57 / 22 / 21 / Cummings 1987
CAPTIVE
Trachypithecuscristatus / 95–133 / 47–56 / 24–26 / 10–18 / Bauchop and Martucci 1968
Semnopithecus entellus / 89–233 / 46–50 / 22–23 / 14–23 / Bauchop and Martucci 1968
Colobusguereza / 53–65 / Kay et al. 1976
Colobusguereza / 79 / 61 / 23 / 10 / Lambert and Fellner 2012
Cercopithecusneglectus / 65 / 47 / 21 / 26 / Lambert and Fellner 2012
Papiohamadryas / 87 / 48 / 36 / 11 / Lambert and Fellner 2012
Pan troglodytes / 81 / 55 / 25 / 12 / Lambert and Fellner 2012
Gorilla gorilla / 89 / 58 / 25 / 11 / Lambert and Fellner 2012
Pongoabelii / 62-67 / 22-27 / 10-14 / Schmidt et al. 2005
Bostaurus / 96–210 / 48–74 / 14–28 / 7–18 / van Soest 1994
Ovisaries / 70–140 / 40–66 / 19–40 / 9–15 / Blaxter et al. 1956

Figures