Antifungal Bacteria on Woodland Salamander Skin Exhibit High Taxonomic Diversity and Geographic Variability

Carly R. Muletz-Wolz, Graziella V. DiRenzo, Stephanie A. Yarwood, Evan H. Campbell Grant, Robert C. Fleischer, Karen R. Lips

Diverse bacteria inhabit amphibian skin, some of which inhibit growth of the fungal pathogen, Batrachochytrium dendrobatidis (Bd). Yet, there has been no systematic survey of anti-Bd bacteria across localities, species and elevations. This is important given geographic and taxonomic variation in amphibian susceptibility to Bd. Our sites were within the Appalachian Mountains where previous sampling indicated low Bd prevalence. We determined the number and identity of anti-Bd bacteria on 61 Plethodon salamanders (37 P. cinereus, 15 P. glutinosus, 9 P. cylindraceus) using culturing methods and 16S rDNA sequencing. We sampled co-occurring species at three localities, and P. cinereus along an elevational gradient (700 - 1000 masl) at one locality. We identified 50 anti-Bd bacterial OTUs and found that the degree of Bd inhibition was not correlated with relatedness. Five anti-Bd bacteria occurred on multiple species at multiple localities, but none were shared among all species and localities. Prevalence of anti-Bd bacteria was higher at Shenandoah NP, VA, with 96% (25/26) of salamanders hosting at least one anti-Bd bacteria compared to 50% (7/14) at Catoctin MP, MD and 38% (8/21) at Mt. Rogers NRA, VA. At the individual level, salamanders at Shenandoah NP had more anti-Bd 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 Bd. Anti-Bd bacteria are diverse in central Appalachian Plethodon salamanders, and their distribution varied geographically. The antifungal bacteria we identified may play a protective role for these salamanders.IMPORTANCE Amphibians harbor skin bacteria that can kill an amphibian fungal pathogen, Batrachochytrium dendrobatidis (Bd). Some amphibians die from Bd infection, whereas others do not. The bacteria that can kill Bd, called anti-Bd bacteria, are thought to influence Bd infection outcome of the amphibian. Yet, how anti-Bd bacteria vary among amphibian species and populations is unknown. We determined the distribution of anti-Bd bacteria among three salamander species (n = 61) sampled at three localities. We identified 50 unique anti-Bd bacteria, and found that all salamanders were negative for Bd. Five anti-Bd bacteria were commonly detected, suggesting a stable, functional association with these salamanders. The number of anti-Bd 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-Bd bacterial community. These anti-Bd bacteria may serve a protective function for their salamander hosts.