Microbial Ecology

, Volume 74, Issue 3, pp 745–756 | Cite as

Intraspecific Variation in the Skin-Associated Microbiome of a Terrestrial Salamander

  • Sofia R. Prado-IrwinEmail author
  • Alicia K. Bird
  • Andrew G. Zink
  • Vance T. Vredenburg
Host Microbe Interactions


Resident microbial communities living on amphibian skin can have significant effects on host health, yet the basic ecology of the host-microbiome relationship of many amphibian taxa is poorly understood. We characterized intraspecific variation in the skin microbiome of the salamander Ensatina eschscholtzii xanthoptica, a subspecies composed of four genetically distinct populations distributed throughout the San Francisco Bay Area and the Sierra Nevada mountains in California, USA. We found that salamanders from four geographically and genetically isolated populations harbor similar skin microbial communities, which are dominated by a common core set of bacterial taxa. Additionally, within a population, the skin microbiome does not appear to differ significantly between salamanders of different ages or sexes. In all cases, the salamander skin microbiomes were significantly different from those of the surrounding terrestrial environment. These results suggest that the relationship between E. e. xanthoptica salamanders and their resident skin microbiomes is conserved, possibly indicating a stable mutualism between the host and microbiome.


Amphibian Microbiome Symbiosis Ensatina eschscholtzii 



The authors would like to thank S. Ellison for his great help with lab work and troubleshooting. We would also like to thank F. Cipriano and A. Swei for advice on methodology. J. de la Torre provided valuable advice as a member of SPI’s Master’s thesis committee. SPI would also like to thank her classmates from OEB210 for valuable feedback on the manuscript. We would also like to thank four anonymous reviewers whose comments greatly improved the manuscript. The National Science Foundation provided funding through a research grant (IOS-1258133) awarded to AGZ and VTV as well as a GRFP (DGE-1144152) awarded to SPI. The National Institutes of Health also provided financial support through MBRS-RISE fellowships awarded to SPI and AKB (R25-GM059298).

Compliance with Ethical Standards

Ethical Approval

All procedures performed with live animals in this study were approved by the Institutional Animal Care and Use Committee at San Francisco State University (Protocol no. A12-07). All sampling of wild salamanders was performed with approval from the California Department of Fish and Wildlife (SC-12920) and California State Parks.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

248_2017_986_MOESM1_ESM.xlsx (9 kb)
Supplementary Table 1 Sequences of Illumina primers used for amplicon sequencing (Illumina Inc., San Diego, CA, USA). (XLSX 8 kb)
248_2017_986_MOESM2_ESM.xlsx (18 kb)
Supplementary Table 2 Complete list of core bacterial OTUs and average abundances by sample group. (*) indicates an OTU with previously-documented antifungal properties. (XLSX 18 kb)
248_2017_986_MOESM3_ESM.xlsx (15 kb)
Supplementary Table 3 List of bacterial OTUs unique to one population. All unique OTUs were present at <0.01% abundance. The East Bay population had the most unique OTUs. (XLSX 15 kb)


  1. 1.
    McFall-Ngai M, Hadfield MG, Bosch TCG, et al (2013) Animals in a bacterial world, a new imperative for the life sciences. Proc. Natl. Acad. Sci. U. S. A. 110:3229–3236CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Spor A, Koren O, Ley R (2011) Unravelling the effects of the environment and host genotype on the gut microbiome. Nat Rev Microbiol 9:279–290CrossRefPubMedGoogle Scholar
  3. 3.
    Rosenberg E, Zilber-Rosenberg I (2011) Symbiosis and development: the hologenome concept. Birth Defects Res Part C - Embryo Today Rev 93:56–66CrossRefGoogle Scholar
  4. 4.
    Naik S, Bouladoux N, Wilhelm C, et al (2012) Compartmentalized control of skin immunity by resident commensals. Science 337:1115–1119CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Loudon AH, Woodhams DC, Parfrey LW, et al (2014) Microbial community dynamics and effect of environmental microbial reservoirs on red-backed salamanders (Plethodon cinereus). ISME J 8:830–840CrossRefPubMedGoogle Scholar
  6. 6.
    Küng D, Bigler L, Davis LR, et al (2014) Stability of microbiota facilitated by host immune regulation: informing probiotic strategies to manage amphibian disease. PLoS One 9:e87101CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kueneman JG, Parfrey LW, Woodhams DC, et al (2013) The amphibian skin-associated microbiome across species, space and life history stages. Mol. Ecol. 23:1238–1250CrossRefPubMedGoogle Scholar
  8. 8.
    McKenzie VJ, Bowers RM, Fierer N, et al (2012) Co-habiting amphibian species harbor unique skin bacterial communities in wild populations. ISME J 6:588–596CrossRefPubMedGoogle Scholar
  9. 9.
    Wardziak T, Luquet E, Plenet S, et al (2013) Impact of both desiccation and exposure to an emergent skin pathogen on transepidermal water exchange in the palmate newt Lissotriton helveticus. Dis. Aquat. Org. 104:215–224CrossRefPubMedGoogle Scholar
  10. 10.
    Harris RN, James TY, Lauer A, et al (2006) Amphibian pathogen Batrachochytrium dendrobatidis is inhibited by the cutaneous bacteria of amphibian species. EcoHealth 3:53–56CrossRefGoogle Scholar
  11. 11.
    Becker MH, Harris RN (2010) Cutaneous bacteria of the redback salamander prevent morbidity associated with a lethal disease. PLoS One 5:e10957CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Loudon A, Holland J (2014) Interactions between amphibians’ symbiotic bacteria cause the production of emergent anti-fungal metabolites. Front. Microbiol. 5:1–8CrossRefGoogle Scholar
  13. 13.
    Becker MH, Harris RN, KPC M, et al (2011) Towards a better understanding of the use of probiotics for preventing chytridiomycosis in Panamanian golden frogs. EcoHealth 8:501–506CrossRefPubMedGoogle Scholar
  14. 14.
    Becker MH, Richards-Zawacki CL, Gratwicke B, Belden LK (2014) The effect of captivity on the cutaneous bacterial community of the critically endangered Panamanian golden frog (Atelopus zeteki). Biol. Conserv. 176:199–206CrossRefGoogle Scholar
  15. 15.
    Bletz MC, Loudon AH, Becker MH, et al (2013) Mitigating amphibian chytridiomycosis with bioaugmentation: characteristics of effective probiotics and strategies for their selection and use. Ecol. Lett. 16:807–820CrossRefPubMedGoogle Scholar
  16. 16.
    Walke JB, Becker MH, Loftus SC et al (2014) Amphibian skin may select for rare environmental microbes. ISME J 1–11Google Scholar
  17. 17.
    Fitzpatrick BM, Allison AL (2014) Similarity and differentiation between bacteria associated with skin of salamanders (Plethodon jordani) and free-living assemblages. FEMS Microbiol. Ecol. 88:482–494CrossRefPubMedGoogle Scholar
  18. 18.
    Markle JGM, Frank DN, Mortin-toth S, et al (2013) Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity. Science (80- ) 339:1084–1088CrossRefGoogle Scholar
  19. 19.
    Fierer N, Hamady M, Lauber CL, Knight R (2008) The influence of sex, handedness, and washing on the diversity of hand surface bacteria. Proc. Natl. Acad. Sci. U. S. A. 105:17994–17999CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Gomez A, Luckey D, Taneja V (2014) The gut microbiome in autoimmunity: sex matters. Clin. Immunol. 159:154–162CrossRefGoogle Scholar
  21. 21.
    Moore FL, Boyd SK, Kelley DB (2005) Historical perspective: hormonal regulation of behaviors in amphibians. Horm. Behav. 48:373–383CrossRefPubMedGoogle Scholar
  22. 22.
    Houck LD, Arnold SJ (2003) Courtship and mating behavior. In: Reprod Biol Phylogeny Urodela, pp 383–424Google Scholar
  23. 23.
    Houck LD (2009) Pheromone communication in amphibians and reptiles. Annu. Rev. Physiol. 71:161–176CrossRefPubMedGoogle Scholar
  24. 24.
    Kueneman JG, Woodhams DC, Van Treuren W, et al (2015) Inhibitory bacteria reduce fungi on early life stages of endangered Colorado boreal toads (Anaxyrus boreas). ISME J 10:1–11Google Scholar
  25. 25.
    Carey C, Cohen N, Rollins-Smith L (1999) Amphibian declines: an immunological perspective. Dev. Comp. Immunol. 23:459–472CrossRefPubMedGoogle Scholar
  26. 26.
    Rollins-Smith LA (1998) Metamorphosis and the amphibian immune system. Immunol. Rev. 166:221–230CrossRefPubMedGoogle Scholar
  27. 27.
    Longo AV, Savage AE, Hewson I, Zamudio KR (2015) Seasonal and ontogenetic variation of skin microbial communities and relationships to natural disease dynamics in declining amphibians. R Soc Open Sci 2:140377CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Kuchta SR, Parks DS, Wake DB (2009) Pronounced phylogeographic structure on a small spatial scale: geomorphological evolution and lineage history in the salamander ring species Ensatina eschscholtzii in central coastal California. Mol. Phylogenet. Evol. 50:240–255CrossRefPubMedGoogle Scholar
  29. 29.
    Stebbins RC (1954) Natural history of the salamanders of the plethodontid genus Ensatina. University of California PressGoogle Scholar
  30. 30.
    Caporaso JG, Lauber CL, Walters WA, et al (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. U. S. A. 108:4516–4522CrossRefPubMedGoogle Scholar
  31. 31.
    Bokulich NA, Subramanian S, Faith JJ, et al (2013) Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat. Methods 10:57–59CrossRefPubMedGoogle Scholar
  32. 32.
    Flechas SV, Sarmiento C, Cárdenas ME, et al (2012) Surviving chytridiomycosis: differential anti-Batrachochytrium dendrobatidis activity in bacterial isolates from three lowland species of Atelopus. PLoS One 7:e44832CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Becker MH, Walke JB, Cikanek S, et al (2015) Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus. Proc. R. Soc. B 282:20142881CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Woodhams DC, Alford RA, Antwis RE, et al (2015) Antifungal isolates database of amphibian skin-associated bacteria and function against emerging fungal pathogens. Ecology 96:595–595CrossRefGoogle Scholar
  35. 35.
    Davenport ER, Mizrahi-Man O, Michelini K, et al (2014) Seasonal variation in human gut microbiome composition. PLoS One 9:e90731CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Meyer EA, Cramp RL, Bernal MH, Franklin CE (2012) Changes in cutaneous microbial abundance with sloughing: possible implications for infection and disease in amphibians. Dis. Aquat. Org. 101:235–242CrossRefPubMedGoogle Scholar
  37. 37.
    Hajishengallis G, Liang S, Payne MA, et al (2011) Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe 10:497–506CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Brucker RM, Bordenstein SR (2012) Speciation by symbiosis. Trends Ecol. Evol. 27:443–451CrossRefPubMedGoogle Scholar
  39. 39.
    Shade A, Handelsman J (2012) Beyond the Venn diagram: the hunt for a core microbiome. Environ. Microbiol. 14:4–12CrossRefPubMedGoogle Scholar
  40. 40.
    Jani AJ, Briggs CJ (2014) The pathogen Batrachochytrium dendrobatidis disturbs the frog skin microbiome during a natural epidemic and experimental infection. Proc. Natl. Acad. Sci. 111:E5049–E5058CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Lauer A, Simon MA, Banning JL, et al (2007) Common cutaneous bacteria from the eastern red-backed salamander can inhibit pathogenic fungi. Copeia 2007:630–640CrossRefGoogle Scholar
  42. 42.
    Walke JB, Becker MH, Hughey MC et al (2015) Most of the dominant members of amphibian skin bacterial communities can be readily cultured. Appl Environ Microbiol AEM. 01486–15Google Scholar
  43. 43.
    Krynak KL, Burke DJ, Benard MF (2016) Landscape and water characteristics correlate with immune defense traits across Blanchard’s cricket frog (Acris blanchardi) populations. Biol. Conserv. 193:153–167CrossRefGoogle Scholar
  44. 44.
    Walke JB, Becker MH, Hughey MC, et al (2015) Most of the dominant members of amphibian skin bacterial communities can be readily cultured. Appl. Environ. Microbiol. 81:6589–6600CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Savage K (2015) Comparative analysis of anti-Bd bacteria from six Malagasy frog species of Ranomafana National ParkGoogle Scholar
  46. 46.
    Krynak KL, Burke DJ, Benard MF (2015) Larval environment alters amphibian immune defenses differentially across life stages and populations. PLoS One 10:e0130383CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Belden LK, Hughey MC, Rebollar EA, et al (2015) Panamanian frog species host unique skin bacterial communities. Front. Microbiol. 6:1–21CrossRefGoogle Scholar
  48. 48.
    Sabino-Pinto J, Bletz MC, Islam MM et al (2016) Composition of the cutaneous bacterial community in Japanese amphibians: effects of captivity, host species, and body region. Microb Ecol. doi: 10.1007/s00248–016–0797-6
  49. 49.
    Murray R, Brenner DJ, Bryant MP (1984) Bergey’s manual of systematic bacteriology. Williams and WilkinsGoogle Scholar
  50. 50.
    Johansen JE, Binnerup SJ, Kroer N, Molbak L (2005) Luteibacter rhizovicinus gen. nov., sp. nov., a yellow-pigmented gammaproteobacterium isolated from the rhizosphere of barley (Hordeum vulgare L.) Int. J. Syst. Evol. Microbiol. 55:2285–2291CrossRefPubMedGoogle Scholar
  51. 51.
    Harris RN, Lauer A, Simon MA, et al (2009) Addition of antifungal skin bacteria to salamanders ameliorates the effects of chytridiomycosis. Dis. Aquat. Org. 83:11–16CrossRefPubMedGoogle Scholar
  52. 52.
    Harris RN, Brucker RM, Walke JB, et al (2009) Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus. ISME J 3:818–824CrossRefPubMedGoogle Scholar
  53. 53.
    Wake DB, Vredenburg VT (2008) Colloquium paper: are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proc. Natl. Acad. Sci. U. S. A. 105(Suppl):11466–11473CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Civitello DJ, Cohen J, Fatima H, et al (2015) Biodiversity inhibits parasites: broad evidence for the dilution effect. Proc. Natl. Acad. Sci. 112:8667–8671CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Winter C, Bouvier T, Weinbauer MG, Thingstad TF (2010) Trade-offs between competition and defense specialists among unicellular planktonic organisms: the “killing the winner” hypothesis revisited. Microbiol. Mol. Biol. Rev. 74:42–57CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Koeppel AF, Wu M (2013) Surprisingly extensive mixed phylogenetic and ecological signals among bacterial operational taxonomic units. Nucleic Acids Res. 41:5175–5188CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Loudon AH, Venkataraman A, Van Treuren W, et al (2016) Vertebrate hosts as islands: dynamics of selection, immigration, loss, persistence, and potential function of bacteria on salamander skin. Front. Microbiol. 7:1–11CrossRefGoogle Scholar
  58. 58.
    Longbottom D, Coulter LJ (2003) Animal chlamydioses and zoonotic implications. J. Comp. Pathol. 128:217–244CrossRefPubMedGoogle Scholar
  59. 59.
    Blumer C, Zimmermann DR, Weilenmann R, et al (2007) Chlamydiae in free-ranging and captive frogs in Switzerland. Vet. Pathol. 44:144–150CrossRefPubMedGoogle Scholar
  60. 60.
    Hamdi C, Balloi A, Essanaa J, et al (2011) Gut microbiome dysbiosis and honeybee health. J. Appl. Entomol. 135:524–533CrossRefGoogle Scholar
  61. 61.
    Bresciano JC, Salvador CA, Paz-y-Miño C, et al (2015) Variation in the presence of anti-Batrachochytrium dendrobatidis bacteria of amphibians across life stages and elevations in Ecuador. EcoHealth. doi: 10.1007/s10393-015-1010-y PubMedGoogle Scholar
  62. 62.
    Shine RS (1979) Sexual selection and sexual dimorphism in the Amphibia. Copeia 1979:297–306CrossRefGoogle Scholar
  63. 63.
    Klein SL (2000) Hormones and mating system affect sex and species differences in immune function among vertebrates. Behav. Process. 51:149–166CrossRefGoogle Scholar
  64. 64.
    Walke JB, Harris RN, Reinert LK, et al (2011) Social immunity in amphibians: evidence for vertical transmission of innate defenses. Biotropica 43:396–400CrossRefGoogle Scholar
  65. 65.
    Rebollar EA, Simonetti SJ, Shoemaker WR, Harris RN (2016) Direct and indirect horizontal transmission of the antifungal probiotic bacterium Janthinobacterium lividum on green frog (Lithobates clamitans) tadpoles. Appl Environ Microbiol AEM 04147–15Google Scholar
  66. 66.
    Banning JL, Weddle AL, Wahl GW, et al (2008) Antifungal skin bacteria, embryonic survival, and communal nesting in four-toed salamanders, Hemidactylium scutatum. Oecologia 156:423–429CrossRefPubMedGoogle Scholar
  67. 67.
    Brucker RM, Bordenstein SR (2012) The roles of host evolutionary relationships (genus: Nasonia) and development in structuring microbial communities. Evolution 66:349–362CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Sofia R. Prado-Irwin
    • 1
    Email author
  • Alicia K. Bird
    • 2
  • Andrew G. Zink
    • 3
  • Vance T. Vredenburg
    • 3
  1. 1.Department of Organismic and Evolutionary BiologyHarvard UniversityCambridgeUSA
  2. 2.Department of Evolution and EcologyUniversity of California, DavisDavisUSA
  3. 3.Department of BiologySan Francisco State UniversitySan FranciscoUSA

Personalised recommendations