Antifungal Bacteria on Woodland Salamander Skin Exhibit High Taxonomic Diversity and Geographic Variability
Authors: Muletz-Wolz Carly R. , DiRenzo Graziella V. , Yarwood Stephanie A. , Campbell Grant Evan H. , Fleischer Robert C. , Lips Karen R. 
Published: February 2017

Diverse bacteria inhabit amphibian skin; some of those bacteria inhibit growth of the fungal pathogen
Batrachochytrium dendrobatidis
. Yet there has been no systematic survey of anti-
B. dendrobatidis
bacteria across localities, species, and elevations. This is important given geographic and taxonomic variations in amphibian susceptibility to
B. dendrobatidis
. Our collection sites were at locations within the Appalachian Mountains where previous sampling had indicated low
B. dendrobatidis
prevalence. We determined the numbers and identities of anti-
B. dendrobatidis
bacteria on 61
Plethodon
salamanders (37
P. cinereus
, 15
P. glutinosus
, 9
P. cylindraceus
) via culturing methods and 16S rRNA gene sequencing. We sampled co-occurring species at three localities and sampled
P. cinereus
along an elevational gradient (700 to 1,000 meters above sea level [masl]) at one locality. We identified 50 anti-
B. dendrobatidis
bacterial operational taxonomic units (OTUs) and found that the degree of
B. dendrobatidis
inhibition was not correlated with relatedness. Five anti-
B. dendrobatidis
bacterial strains occurred on multiple amphibian species at multiple localities, but none were shared among all species and localities. The prevalence of anti-
B. dendrobatidis
bacteria was higher at Shenandoah National Park (NP), VA, with 96% (25/26) of salamanders hosting at least one anti-
B. dendrobatidis
bacterial species compared to 50% (7/14) at Catoctin Mountain Park (MP), MD, and 38% (8/21) at Mt. Rogers National Recreation Area (NRA), VA. At the individual level, salamanders at Shenandoah NP had more anti-
B. dendrobatidis
bacteria per individual (μ = 3.3) than those at Catoctin MP (μ = 0.8) and at Mt. Rogers NRA (μ = 0.4). All salamanders tested negative for
B. dendrobatidis
. Anti-
B. dendrobatidis
bacterial species are diverse in central Appalachian
Plethodon
salamanders, and their distribution varied geographically. The antifungal bacterial species that we identified may play a protective role for these salamanders.

IMPORTANCE
Amphibians harbor skin bacteria that can kill an amphibian fungal pathogen,
Batrachochytrium dendrobatidis
. Some amphibians die from
B. dendrobatidis
infection, whereas others do not. The bacteria that can kill
B. dendrobatidis
, called anti-
B. dendrobatidis
bacteria, are thought to influence the
B. dendrobatidis
infection outcome for the amphibian. Yet how anti-
B. dendrobatidis
bacterial species vary among amphibian species and populations is unknown. We determined the distribution of anti-
B. dendrobatidis
bacterial species among three salamander species (
n
= 61) sampled at three localities. We identified 50 unique anti-
B. dendrobatidis
bacterial species and found that all of the tested salamanders were negative for
B. dendrobatidis
. Five anti-
B. dendrobatidis
bacterial species were commonly detected, suggesting a stable, functional association with these salamanders. The number of anti-
B. dendrobatidis
bacteria per individual varied among localities but not among co-occurring salamander species, demonstrating that environment is more influential than host factors in structuring the anti-
B. dendrobatidis
bacterial community. These anti-
B. dendrobatidis
bacteria may serve a protective function for their salamander hosts.