Multiple factors modulate microbial community assembly in the vertebrate gut, though studies disagree as to their relative contribution. One cause may be a reliance on captive animals, which can have very different gut microbiomes compared to their wild counterparts. To resolve this disagreement, we analyze a new, large, and highly diverse animal distal gut 16 S rRNA microbiome dataset, which comprises 80% wild animals and includes members of Mammalia, Aves, Reptilia, Amphibia, and Actinopterygii. We decouple the effects of host evolutionary history and diet on gut microbiome diversity and show that each factor modulates different aspects of diversity. Moreover, we resolve particular microbial taxa associated with host phylogeny or diet and show that Mammalia have a stronger signal of cophylogeny. Finally, we find that environmental filtering and microbe-microbe interactions differ among host clades. These findings provide a robust assessment of the processes driving microbial community assembly in the vertebrate intestine.

SummaryThe bacterioplankton diversity in large rivers has thus far been under-sampled despite the importance of streams and rivers as components of continental landscapes. Here, we present a comprehensive dataset detailing the bacterioplankton diversity along the midstream of the Danube River and its tributaries. Using 16S rRNA-gene amplicon sequencing, our analysis revealed that bacterial richness and evenness gradually declined downriver in both the free-living and particle-associated bacterial communities. These shifts were also supported by beta diversity analysis, where the effects of tributaries were negligible in regards to the overall variation. In addition, the river was largely dominated by bacteria that are commonly observed in freshwaters. Dominated by the acI lineage, the freshwater SAR11 (LD12) and the Polynucleobacter group, typical freshwater taxa increased in proportion downriver and were accompanied by a decrease in soil and groundwater-affiliated bacteria. Based on views of the metacommunity and River Continuum Concept, we interpret the observed taxonomic patterns and accompanying changes in alpha and beta diversity with the intention of laying the foundation for a unified concept for river bacterioplankton diversity.

A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum Bacteroidetes. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 ؋ 10 9 marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples from a karstic catchment area at levels matching a gradient from negligible to considerable ruminant fecal influence (from not detectable to 10 5

25Multiple factors modulate microbial community assembly in the gut, but the magnitude of 26 each can vary substantially across studies. This may be in part due to a heavy reliance on 27 captive animals, which can have very different gut microbiomes versus their wild counterparts. 28In order to better resolve the influence of evolution and diet on gut microbiome diversity, we 29 generated a large and highly diverse animal distal gut 16S rRNA microbiome dataset, which 30 comprises 80 % wild animals and includes members of Mammalia, Aves, Reptilia, Amphibia, 31 and Actinopterygii. We decoupled the effects of host evolutionary history and diet on gut 32 microbiome diversity and show that each factor explains different aspects of diversity. Moreover, 33we resolved particular microbial taxa associated with host phylogeny or diet, and we show that 34Mammalia have a stronger signal of cophylogeny versus non-mammalian hosts. Additionally, 35 our results from ecophylogenetics and co-occurrence analyses suggest that environmental 36 filtering and microbe-microbe interactions differ among host clades. These findings provide a 37 robust assessment of the processes driving microbial community assembly in the vertebrate 38 intestine. 39 40

Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminant-associated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminant-associated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminant-associated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods.

Aims:  The aim of the study was the development of a sensitive human‐specific quantitative real‐time PCR assay for microbial faecal source tracking (MST) in alpine spring water. The assay detects human‐specific faecal DNA markers (BacH) from 16S rRNA gene sequences from the phylum Bacteroidetes using TaqMan® minor groove binder probes. Methods and Results:  The qualitative and quantitative detection limits of the PCR assay were 6 and 30 marker copies, respectively. Specificity was proved by testing 41 human faeces and waste water samples and excluding cross‐amplification from 302 animal faecal samples from Eastern Austria. Marker concentrations in human faecal material were in the range from 6·6 × 109 to 9·1 × 1010 marker equivalents per gram. The method was sensitive enough to detect a few 100 pg of faeces in faecal suspensions. The assay was applied on water samples from an alpine karstic spring catchment area and the results reflected the expected levels of human faecal influence. Conclusions:  The method exhibited sufficient sensitivity to allow quantitative source tracking of human faecal impact in the investigated karstic spring water. Significance and Impact of the Study:  The developed method constitutes the first quantitative human‐specific MST tool sensitive enough for investigations in ground and spring water.

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The impairment of water quality by faecal pollution is a global public health concern. Microbial source tracking methods help to identify faecal sources but the few recent quantitative microbial source tracking applications disregarded catchment hydrology and pollution dynamics. This quantitative microbial source tracking study, conducted in a large karstic spring catchment potentially influenced by humans and ruminant animals, was based on a tiered sampling approach: a 31-month water quality monitoring (Monitoring) covering seasonal hydrological dynamics and an investigation of flood events (Events) as periods of the strongest pollution. The detection of a ruminant-specific and a human-specific faecal Bacteroidetes marker by quantitative real-time PCR was complemented by standard microbiological and on-line hydrological parameters. Both quantitative microbial source tracking markers were detected in spring water during Monitoring and Events, with preponderance of the ruminant-specific marker. Applying multiparametric analysis of all data allowed linking the ruminant-specific marker to general faecal pollution indicators, especially during Events. Up to 80% of the variation of faecal indicator levels during Events could be explained by ruminant-specific marker levels proving the dominance of ruminant faecal sources in the catchment. Furthermore, soil was ruled out as a source of quantitative microbial source tracking markers. This study demonstrates the applicability of quantitative microbial source tracking methods and highlights the prerequisite of considering hydrological catchment dynamics in source tracking study design.