Cutaneous bacterial community diversity of Plethodon salamanders along an elevational gradient in Shenandoah National Park, VA

Background/Question/Methods: Amphibians support diverse communities of cutaneous bacteria thought to offer protection from microbial pathogens. Studies suggest that there is some degree of host specificity of bacterial community structure across taxa. Species of terrestrial Appalachian salamanders (genus Plethodon) are associated with particular elevational ranges so, we expected to see consistent bacterial community composition across elevational ranges. We compared the composition of cutaneous bacterial communities of two species of terrestrial salamanders P. cinereus and P. cylindraceus along an elevational gradient (702 – 974 m) in Shenandoah National Park, VA. Of the 10 P. cinereus populations sampled, four populations co-occurred with P. cylindraceus. If cutaneous bacteria were taxon-specific we expected to find species-specific communities even where species co-occur. If bacterial community structure is influenced by environmental conditions (e.g., temperature, soil pH) then we expected to find that co-occurring individuals would have more similar communities.  We used traditional culturing methods to isolate bacteria from the skin of 19 P. cinereus and 7 P. cylindraceus. We used new high-throughput sequencing methods (454 pyrosequencing) of 16s rRNA amplicons to identify all bacteria from skin swabs of 53 P. cinereus and 7 P. cylindraceus.

Results/Conclusions: Using culturing methods we isolated 197 bacterial morphospecies from P. cinereus (µ = 8, range 1 –16) and P. cylindraceus (µ = 6.4, range 2 – 9).  When we compared the number of culturable bacterial morphospecies between the two species, we found no significant difference (Mann-Whitney U-test: p > 0.05). From the first 454 run of 28 P. cinereus we generated 58,000 sequences representing 1,172 distinct OTUs from 14 phyla . The most common OTU found on every individual was from the family Moraxellaceae (43% of generated sequences) indicating a close association between P. cinereus and Moraxellaceae species. When we compared the number of bacterial OTUs among P. cinereus populations, we found that there was a significant difference between the lowest and highest elevations (ANOVA: p = 0.003). Furthermore, both methods detected a weak positive correlation between P. cinereus culturable bacterial morphospecies diversity and elevation (Shannon-Wiener: p = 0.03, R² = 0.25) and P. cinereus OTU richness and elevation (chao1: p = 0.001, R² = 0.32). Taken together, our results suggest that salamander microbiome composition is influenced by environmental (elevation) factors.