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Abstract The gut microbial communities of mammals provide numerous benefits to their hosts. However, given the recent development of the microbiome field, we still lack a thorough understanding of the variety of ecological and evolutionary factors that structure these communities across species. Metabarcoding is a powerful technique that allows for multiple microbial ecology questions to be investigated simultaneously. Here, we employed DNA metabarcoding techniques, predictive metagenomics, and culture-dependent techniques to inventory the gut microbial communities of several species of rodent collected from the same environment that employ different natural feeding strategies [granivorous pocket mice (Chaetodipus penicillatus); granivorous kangaroo rats (Dipodomys merriami); herbivorous woodrats (Neotoma albigula); omnivorous cactus mice (Peromyscus eremicus); and insectivorous grasshopper mice (Onychomys torridus)]. Of particular interest were shifts in gut microbial communities in rodent species with herbivorous and insectivorous diets, given the high amounts of indigestible fibers and chitinous exoskeleton in these diets, respectively. We found that herbivorous woodrats harbored the greatest microbial diversity. Granivorous pocket mice and kangaroo rats had the highest abundances of the genus Ruminococcus and highest predicted abundances of genes related to the digestion of fiber, representing potential adaptations in these species to the fiber content of seeds and the limitations to digestion given their small body size. Insectivorous grasshopper mice exhibited the greatest inter-individual variation in the membership of their microbiomes, and also exhibited the highest predicted abundances of chitin-degrading genes. Culture-based approaches identified 178 microbial isolates (primarily Bacillus and Enterococcus), with some capable of degrading cellulose and chitin. We observed several instances of strain-level diversity in these metabolic capabilities across isolates, somewhat highlighting the limitations and hidden diversity underlying DNA metabarcoding techniques. However, these methods offer power in allowing the investigation of several questions concurrently, thus enhancing our understanding of gut microbial ecology. 

Abstract Measurements of fecal pellet size can provide important information about wild mammals, such as body size and demographic information. Previous studies have not rigorously tested whether diet can confound these measurements. Furthermore, it is unknown whether diet might alter fecal dimensions directly or through changes in animal physiology. Here, we studied three closely related rodent species that differ in natural feeding strategies. Individuals were fed diets that varied in protein and fiber content for 5 weeks. We then measured body size, fecal widths and lengths, and the radius of the large intestine. Diet composition significantly changed fecal widths in all species. High-fiber content significantly increased fecal widths and would cause overestimations of body size if applied to wild feces. Using path analysis, we found that fiber can increase fecal widths both directly and indirectly through increasing the large intestine radius. Protein affected each species differently, suggesting that protein effects vary by species feeding strategy and existing physiology. Overall, diet and large intestine morphology can alter fecal pellet measurements. Studies using fecal measurements therefore must consider these effects in their conclusions. 

The field of microbial ecology is increasingly recognizing the need for methods to isolate and culture gut microbes to better understand how these microorganisms impact animal physiology, especially in mammalian hosts. Currently, there is a lack of clear methods to store microbial samples for cultivability, especially when samples are collected from the field, transported to the laboratory, and preserved under long-term storage for weeks to months compared to mere days in the biomedical field. Here, the cecal contents of groundhogs (Marmota monax) were processed and stored with or without various preservation solutions at −80 °C for at least 2 months. All microbial samples were then grown in distinct nutrient media in liquid and plate conditions and were incubated under anaerobic and aerobic environments. Treatment comparisons revealed that the samples stored in preservation solutions containing 1 or more cryoprotectants provided the greatest and most consistent bacterial densities. To test the long-term storage efficacy of the preservation solutions, we inventoried taxonomic identities and abundances of these cultures using 16S rRNA amplicon sequencing. Our findings highlight that: (1) preserved samples containing cryoprotectants exhibited the highest microbial richness and diversity and resembled the original cecal samples the most when grown under anaerobic conditions; and (2) the effect of individual animal identity was detectable in the membership of cultured communities, irrespective of preservation solutions. Our study is the first to demonstrate the importance of preservation solutions containing multiple cryoprotectants for long-term storage and further microbial culturing and novel isolation. Understanding and improving storage methods that preserve microbial physiology and conserve their compositional diversity is essential for field-collected samples useful in mammalian microbiome and culturomics studies, promoting a better comprehension of the identity and function of wild host-associated microbiomes. 

Convergent evolution is a widespread phenomenon. While there are many examples of convergent evolution at the phenotypic scale, convergence at the molecular level has been more difficult to identify. A classic example of convergent evolution across scales is that of the digestive lysozyme found in ruminants and Colobine monkeys. These herbivorous species rely on foregut fermentation, which has evolved to function more optimally under acidic conditions. Here, we explored if rodents with similar dietary strategies and digestive morphologies have convergently evolved a lysozyme with digestive functions. At the phenotypic level, we find that rodents with bilocular stomach morphologies exhibited a lysozyme that maintained higher relative activities at low pH values, similar to the lysozymes of ruminants and Colobine monkeys. Additionally, the lysozyme of Peromyscus leucopus shared a similar predicted protonation state as that observed in previously identified digestive lysozymes. However, we found limited evidence of positive selection acting on the lysozyme gene in foregut-fermenting species and did not identify patterns of convergent molecular evolution in this gene. This study emphasizes that phenotypic convergence need not be the result of convergent genetic modifications, and we encourage further exploration into the mechanisms regulating convergence across biological scales. 

Diet selection is a fundamental aspect of animal behavior with numerous ecological and evolutionary implications. While the underlying mechanisms are complex, the availability of essential dietary nutrients can strongly influence diet selection behavior. The gut microbiome has been shown to metabolize many of these same nutrients, leading to the untested hypothesis that intestinal microbiota may influence diet selection. Here, we show that germ-free mice colonized by gut microbiota from three rodent species with distinct foraging strategies differentially selected diets that varied in macronutrient composition. Specifically, we found that herbivore-conventionalized mice voluntarily selected a higher protein:carbohydrate (P:C) ratio diet, while omnivore- and carnivore-conventionalized mice selected a lower P:C ratio diet. In support of the long-standing hypothesis that tryptophan—the essential amino acid precursor of serotonin—serves as a peripheral signal regulating diet selection, bacterial genes involved in tryptophan metabolism and plasma tryptophan availability prior to the selection trial were significantly correlated with subsequent voluntary carbohydrate intake. Finally, herbivore-conventionalized mice exhibited larger intestinal compartments associated with microbial fermentation, broadly reflecting the intestinal morphology of their donor species. Together, these results demonstrate that gut microbiome can influence host diet selection behavior, perhaps by mediating the availability of essential amino acids, thereby revealing a mechanism by which the gut microbiota can influence host foraging behavior.