Successes and limitations of quantitative diet metabarcoding in a small, herbivorous mammal.

DNA metabarcoding is widely used to determine wild animal diets, but whether this technique provides accurate, quantitative measurements is still under debate. To test our ability to accurately estimate the abundance of dietary items using metabarcoding, we fed wild-caught desert woodrats (Neotoma lepida) diets consisting of constant amounts of juniper (Juniperus osteosperma, 15%) and varying amounts of creosote (Larrea tridentata, 1%-60%), cactus (Opuntia sp., 0%-100%) and commercial chow (0%-85%). Using metabarcoding, we compared the representation of items in the original diet samples to that in the faecal samples to test the sensitivity and accuracy of diet metabarcoding, the performance of different bioinformatic pipelines and our ability to correct sequence counts. Metabarcoding, using standard trnL primers, detected creosote, juniper and chow. Different pipelines for assigning taxonomy performed similarly. While creosote was detectable at dietary proportions as low as 1%, we failed to detect cactus in most samples, probably due to a primer mismatch. Creosote read counts increased as its proportion in the diet increased, and we could differentiate when creosote was a minor and major component of the diet. However, we found that estimates of juniper and creosote varied. Using previously suggested methods to correct these errors did not improve accuracy estimates of creosote, but did reduce error for juniper and chow. Our results indicate that metabarcoding can provide quantitative information on dietary composition, but may be limited. We suggest that researchers use caution when quantitatively interpreting diet metabarcoding results unless they first experimentally determine the extent of possible biases.

Plant secondary compound- and antibiotic-induced community disturbances improve the establishment of foreign gut microbiota.

Fecal transplants are a powerful tool for manipulating the gut microbial community, but how these non-native communities establish in the presence of an intact host gut microbiome is poorly understood. We explored the microbiome of desert woodrats (Neotoma lepida) to determine whether disrupting existing microbial communities using plant secondary compounds (PSCs) or antibiotics increases the establishment of foreign microbes. We administered two fecal transplants between natural populations of adult woodrats that harbor distinct gut microbiota and have different natural dietary exposure to PSCs. First, we administered fecal transplants to recipients given creosote resin, a toxin found in the natural diet of our "donor" population, and compared the gut microbial communities to animals given fecal transplants and control diet using 16S rRNA gene sequencing. Second, we disrupted the gut microbial community of the same recipients with an antibiotic prior to fecal transplants. We found that gut microbial communities of woodrats disrupted with PSCs or antibiotics resembled that of donors more closely than control groups. PSC treatment also enriched microbes associated with metabolizing dietary toxins in transplant recipients. These results demonstrate that microbial community disturbances by PSCs or antibiotics are sufficient to facilitate establishment of foreign microbes in animals with intact microbiomes.

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.