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

Large-scale metagenome assemblies of human microbiomes have produced a vast catalogue of previously unseen microbial genomes; however, comparatively few microbial genomes derive from other vertebrates. Here, we generated 4374 metagenome assembled genomes (MAGs) from gut samples of 180 predominantly wild animal species representing 5 classes. Combined with existing datasets, we produced 5596 non-redundant, quality MAGs and 1522 species-level genome bins (SGBs). Most SGBs were novel at the species, genus, or family levels, and the majority were enriched in host versus environment metagenomes. Many traits distinguished SGBs enriched in host or environmental biomes, including the number of antimicrobial resistance genes. We identified 1986 diverse and largely novel biosynthetic gene clusters. Gene-based assembly revealed tremendous gene diversity, much of it host or environment specific. Our MAG and gene datasets greatly expand the microbial genome repertoire and provide a broad view of microbial adaptations to life within a living host.

51Outbreaks of emerging coronaviruses in the past two decades and the current pandemic 52 of a novel coronavirus (SARS-CoV-2) that emerged in China highlight the importance of this 53 viral family as a zoonotic public health threat. To gain a better understanding of coronavirus 54 presence and diversity in wildlife at wildlife-human interfaces in three southern provinces in Viet 55Nam 2013-2014, we used consensus Polymerase Chain Reactions to detect coronavirus 56 sequences. In comparison to previous studies, we observed high proportions of positive samples 57 among field rats (34.0%, 239/702) destined for human consumption and insectivorous bats in 58 guano farms (74.8%, 234/313) adjacent to human dwellings. Most notably among field rats, the 59 odds of coronavirus RNA detection significantly increased along the supply chain from field rats 60 sold by traders (reference group; 20.7% positivity, 39/188) by a factor of 2.2 for field rats sold in 61 large markets (32.0%, 116/363) and 10.0 for field rats sold and served in restaurants (55.6%, 62 84/151). Coronaviruses were detected in the majority of wildlife farms (60.7%, 17/28) and in the 63 Malayan porcupines (6.0%, 20/331) and bamboo rats (6.3%, 6/96) that are farmed. We identified 64 six known coronaviruses in bats and rodents, clustered in three Coronaviridae genera, including 65 the Alpha-, Beta-, and Gammacoronaviruses. Our analysis also suggested either mixing of 66 animal excreta in the environment or interspecies transmission of coronaviruses, as both bat and 67 avian coronaviruses were detected in rodent feces in the trade. The mixing of multiple 68 coronaviruses, and their apparent amplification along the wildlife supply chain into restaurants, 69 suggests maximal risk for end consumers and likely underpins the mechanisms of zoonotic 70 spillover to people. 71 72

Commonly used 16S rRNA gene primers do not detect the full range of archaeal diversity present in the vertebrate gut. As a result, several questions regarding the archaeal component of the gut microbiota remain, including which Archaea are host-associated, the specificities of such associations and the major factors influencing archaeal diversity. Using 16S rRNA gene amplicon sequencing with primers that specifically target Archaea, we obtained sufficient sequence data from 185 gastrointestinal samples collected from 110 vertebrate species that span five taxonomic classes (Mammalia, Aves, Reptilia, Amphibia and Actinopterygii), of which the majority were wild. We provide evidence for previously undescribed Archaea–host associations, including Bathyarchaeia and Methanothermobacter, the latter of which was prevalent among Aves and relatively abundant in species with higher body temperatures, although this association could not be decoupled from host phylogeny. Host phylogeny explained archaeal diversity more strongly than diet, while specific taxa were associated with both factors, and cophylogeny was significant and strongest for mammalian herbivores. Methanobacteria was the only class predicted to be present in the last common ancestors of mammals and all host species. Further analysis indicated that Archaea–Bacteria interactions have a limited effect on archaeal diversity. These findings expand our current understanding of Archaea–vertebrate associations.