Effects of sample collection and storage methods on fecal bacterial diversity in California ground squirrels (Otospermophilus beecheyi)

Abstract

Measures of fecal bacterial abundance and taxonomic composition are commonly used as proxies for gut microbial diversity in studies of free-living mammals. Because methods of sample collection and storage may affect measures of bacterial diversity, we evaluated the effects of several procedures on fecal bacterial diversity in a free-living population of California ground squirrels (Otospermophilus beecheyi). Replicate fecal samples from 12 adult female squirrels were collected either from the soil beneath traps in which individuals had been captured or from tubs placed under squirrels during handling. Samples were then either frozen immediately in liquid nitrogen or stored on ice for several hours before being transferred to a −80°C freezer. Sequencing of the bacterial 16s RNA gene revealed no differences among methods with regard to sequence read depth (number of sequences recovered per animal) or alpha (within-individual) diversity of bacterial taxa. While our collection-storage procedures had a significant effect on one of the metrics of beta (among-individual) bacterial diversity examined, this effect was small compared to that of individual identity. Date of sample collection affected alpha and beta diversity; samples collected only 1 week apart differed significantly in bacterial diversity. Overall, these findings suggest that the collection and storage methods employed yield generally comparable information and are capable of capturing potentially important patterns of fecal bacterial composition and diversity in free-living mammals.