The desert woodrat (Neotoma lepida) induces a diversity of biotransformation genes in response to creosote bush resin.

Liver biotransformation enzymes have long been thought to enable animals to feed on diets rich in xenobiotic compounds. However, despite decades of pharmacological research in humans and rodents, little is known about hepatic gene expression in specialized mammalian herbivores feeding on toxic diets. Leveraging a recently identified population of the desert woodrat (Neotoma lepida) found to be highly tolerant to toxic creosote bush (Larrea tridentata), we explored the expression changes of suites of biotransformation genes in response to diets enriched with varying amounts of creosote resin. Analysis of hepatic RNA-seq data indicated a dose-dependent response to these compounds, including the upregulation of several genes encoding transcription factors and numerous phase I, II, and III biotransformation families. Notably, elevated expression of five biotransformation families - carboxylesterases, cytochromes P450, aldo-keto reductases, epoxide hydrolases, and UDP-glucuronosyltransferases - corresponded to species-specific duplication events in the genome, suggesting that these genes play a prominent role in N. lepida's adaptation to creosote bush. Building on pharmaceutical studies in model rodents, we propose a hypothesis for how the differentially expressed genes are involved in the biotransformation of creosote xenobiotics. Our results provide some of the first details about how these processes likely operate in the liver of a specialized mammalian herbivore.

Seasonal restructuring facilitates compositional convergence of gut microbiota in free-ranging rodents.

Gut microbes provide essential services to their host and shifts in their composition can impact host fitness. However, despite advances in our understanding of how microbes are assembled in the gut, we understand little about the stability of these communities within individuals, nor what factors influence its composition over the life of an animal. For this reason, we conducted a longitudinal survey of the gut microbial communities of individual free-ranging woodrats (Neotoma spp.) across a hybrid zone in the Mojave Desert, USA, using amplicon sequencing approaches to characterize gut microbial profiles and diet. We found that gut microbial communities were individualized and experienced compositional restructuring as a result of seasonal transitions and changes in diet. Turnover of gut microbiota was highest amongst bacterial subspecies and was much lower at the rank of Family, suggesting there may be selection for conservation of core microbial functions in the woodrat gut. Lastly, we identified an abundant core gut bacterial community that may aid woodrats in metabolizing a diet of plants and their specialized metabolites. These results demonstrate that the gut microbial communities of woodrats are highly dynamic and experience seasonal restructuring which may facilitate adaptive plasticity in response to changes in diet.

Hybridization in the absence of an ecotone favors hybrid success in woodrats (Neotoma spp.).

Hybridization is a common process that has broadly impacted the evolution of multicellular eukaryotes; however, how ecological factors influence this process remains poorly understood. Here, we report the findings of a 3-year recapture study of the Bryant's woodrat (Neotoma bryanti) and desert woodrat (Neotoma lepida), two species that hybridize within a creosote bush (Larrea tridentata) shrubland in Whitewater, CA, USA. We used a genotype-by-sequencing approach to characterize the ancestry distribution of individuals across this hybrid zone coupled with Cormack-Jolly-Seber modeling to describe demography. We identified a high frequency of hybridization at this site with ~40% of individuals possessing admixed ancestry, which is the result of multigenerational backcrossing and advanced hybrid-hybrid crossing. F1, F2, and advanced generation hybrids had apparent survival rates similar to parental N. bryanti, while parental and backcross N. lepida had lower apparent survival rates and were far less abundant. Compared to bimodal hybrid zones where hybrids are often rare and selected against, we find that hybrids at Whitewater are common and have comparable survival to the dominant parental species, N. bryanti. The frequency of hybridization at Whitewater is therefore likely limited by the abundance of the less common parental species, N. lepida, rather than selection against hybrids.

Addressing nontarget amplification in DNA metabarcoding studies of arthropod-feeding rodents.

High-throughput sequencing approaches have revolutionized how we study animal diets by enabling the detection of dietary components from the metabarcoding of DNA in excrement. Mitochondrial cytochrome oxidase C subunit I (mtCOI) DNA metabarcoding is commonly used to study the diets of arthropod-feeding animals; however, this approach is susceptible to nontarget amplification of the consumer species mtCOI locus. Nontarget amplification is often an unforeseen complication that can drastically reduce the quality and utility of the results generated by high-throughput amplicon sequencing. By interrogating the diets of new world rodents in the genus Neotoma (woodrats) in both natural and captive settings, we demonstrate that nontarget amplification can drastically reduce the total read abundance of detected arthropod taxa in fecal samples and inhibit downstream analyses of dietary diversity and composition metrics. Using the results from these investigations, we offer a guide on how to identify concerns for nontarget amplification when selecting degenerate primers for DNA metabarcoding studies and recommend several approaches that can reduce or eliminate nontarget amplification. Lastly, for the community interested in investigating the diets of arthropod-feeding rodents, we generated a database containing the degree of mismatch between publicly available Rodentia mtCOI sequences and four common universal mtCOI primer sets to be used as a resource for inferring the relative risk of nontarget amplification when designing arthropod metabarcoding studies in rodent systems. This guide will be especially useful for researchers working with consumer species that have not previously been studied.

Toxin tolerance across landscapes: Ecological exposure not a prerequisite.

Little is known about the tolerances of mammalian herbivores to plant specialized metabolites across landscapes.We investigated the tolerances of two species of herbivorous woodrats, Neotoma lepida (desert woodrat) and Neotoma bryanti (Bryant's woodrat) to creosote bush (Larrea tridentata), a widely distributed shrub with a highly toxic resin. Woodrats were sampled from 13 locations both with and without creosote bush across a 900 km transect in the US southwest. We tested whether these woodrat populations consume creosote bush using plant metabarcoding of feces and quantified their tolerance to creosote bush through feeding trials using chow amended with creosote resin.Toxin tolerance was analyzed in the context of population structure across collection sites with microsatellite analyses. Genetic differentiation among woodrats collected from different locations was minimal within either species. Tolerance differed substantially between the two species, with N. lepida persisting 20% longer than N. bryanti in feeding trials with creosote resin. Furthermore, in both species, tolerance to creosote resin was similar among woodrats near or within creosote bush habitat. In both species, woodrats collected greater than 25 km from creosote had markedly lower tolerances to creosote resin compared to animals from within the range of creosote bush.The results imply that mammalian herbivores are adapted to the specialized metabolites of plants in their diet, and that this tolerance can extend several kilometers outside of the range of dietary items. That is, direct ecological exposure to the specialized chemistry of particular plant species is not a prerequisite for tolerance to these compounds. These findings lay the groundwork for additional studies to investigate the genetic mechanisms underlying toxin tolerance and to identify how these mechanisms are maintained across landscape-level scales in mammalian herbivores.

Microbiome stability and structure is governed by host phylogeny over diet and geography in woodrats (Neotoma spp.).

The microbiome is critical for host survival and fitness, but gaps remain in our understanding of how this symbiotic community is structured. Despite evidence that related hosts often harbor similar bacterial communities, it is unclear whether this pattern is due to genetic similarities between hosts or to common ecological selection pressures. Here, using herbivorous rodents in the genus Neotoma, we quantify how geography, diet, and host genetics, alongside neutral processes, influence microbiome structure and stability under natural and captive conditions. Using bacterial and plant metabarcoding, we first characterized dietary and microbiome compositions for animals from 25 populations, representing seven species from 19 sites across the southwestern United States. We then brought wild animals into captivity, reducing the influence of environmental variation. In nature, geography, diet, and phylogeny collectively explained ∼50% of observed microbiome variation. Diet and microbiome diversity were correlated, with different toxin-enriched diets selecting for distinct microbial symbionts. Although diet and geography influenced natural microbiome structure, the effects of host phylogeny were stronger for both wild and captive animals. In captivity, gut microbiomes were altered; however, responses were species specific, indicating again that host genetic background is the most significant predictor of microbiome composition and stability. In captivity, diet effects declined and the effects of host genetic similarity increased. By bridging a critical divide between studies in wild and captive animals, this work underscores the extent to which genetics shape microbiome structure and stability in closely related hosts.