Supplementary Methods
Animals and Sample Collection
Northern leopard frog (Lithobates pipiens) embryos were purchased from Nasco
(Ft. Atkinson, WI, USA), transferred to the laboratory and allowed to hatch. Once freeswimming [Gosner Stage (GS) 25] [1], tadpoles were distributed in glass aquaria and fed
ad libitum a diet of ground alfalfa. Tadpoles (n = 7 GS = 36.1 ± 1.8) were euthanized in
1% buffered tricaine methanesulfonate, and digesta was collected and frozen. Another
group of tadpoles was allowed to develop through metamorphosis and, as frogs, were fed
a diet of crickets and mealworms for 16 weeks. At 16 weeks post-metamorphosis, frogs
(n = 8) were euthanized in 1% buffered tricaine methanesulfonate and intestinal contents
were collected and frozen. Due to the small amount of material present, we collected and
inventoried total digesta from the whole intestine (small and large) of tadpoles and frogs.
Samples of tank water, ground alfalfa, and whole crickets were also collected for
microbial inventories. The University of Wisconsin’s Institutional Animal Care and Use
Committee approved all experimental procedures involving tadpoles and frogs.
Sequencing
A previously established technique was used to amplify the V4 region of the 16SrRNA
gene with the primers 515F and 806R [2]. The reverse primer also contained a 12 bp
barcode sequence, allowed for pooling of samples. PCR reactions were conducted in
triplicate and resulting products were pooled within a sample. DNA was quantified using
Invitrogen’s PicoGreen and a plate reader and cleaned using the UltraClean PCR CleanUp Kit (MoBIO). Amplicons were sequenced on an Illumina MiSeq machine using
previously described techniques [3].
Sequence Analysis
Sequences were analyzed using the QIIME software package [4]. Sequences
underwent standard quality control and were split in to libraries using default parameters
in QIIME. Sequences were grouped into operational taxonomic units (OTUs) using
UCLUST [5] with a minimum sequence identity of 97%. The most abundant sequences
within each OTU were designated as a ‘representative sequence’, and then aligned
against the Greengenes core set [6] using PyNAST [7] with default parameters set by
QIIME. A PH Lane mask supplied by QIIME was used to remove hypervariable regions
from aligned sequences. FastTree [8] was used to create a phylogenetic tree of
representative sequences. OTUs were classified using the Ribosomal Database Project
(RDP) classifier with a the standard minimum support threshold of 80% [9]. Sequences
identified as chloroplasts or mitochondria were removed from analysis. We compared the
relative abundances of bacterial phyla in tadpoles and frogs using a Student’s t-test.
Several  diversity measurements were calculated for each sample. We calculated
the Shannon Diversity Index, a biodiversity measure that incorporates both richness and
evenness. We calculated evenness, or how similar in abundance the OTUs in a sample
are, as well as Chao1, which estimates the asymptote on a species accumulation curve to
estimate OTU richness. However, these diversity metrics weight all OTUs equally
regardless of phylogenetic relationships. Therefore, we calculated a measurement of
phylogenetic diversity [10], which measures the cumulative branch lengths from
randomly sampling OTUs from each sample. For each sample, we calculated the mean of
20 iterations for a sub-sampling of 9,500 sequences. All diversity metrics were then
compared using a Student’s t-test.
We compared community memberships (presence or absence of lineages, and not
their relative abundances) of treatment groups. We calculated unweighted UniFrac
scores, which measures diversity shared between treatment groups ( diversity) by
determining the fraction of branch length shared between two samples in the
phylogenetic tree created from all representative sequences. We then conducted Principal
Coordinates Analysis (PCoA) on unweighted UniFrac scores to investigate similarities
between tadpole and frog samples.
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