Ecological Correlates of Large-Scale Turnover in the Dominant Members of Pseudacris crucifer Skin Bacterial Communities

  • Myra C. HugheyEmail author
  • Eric R. Sokol
  • Jenifer B. Walke
  • Matthew H. Becker
  • Lisa K. Belden
Environmental Microbiology


Animals host a wide diversity of symbiotic microorganisms that contribute important functions to host health, and our knowledge of what drives variation in the composition of these complex communities continues to grow. Microbiome studies at larger spatial scales present opportunities to evaluate the contribution of large-scale factors to variation in the microbiome. We conducted a large-scale field study to assess variation in the bacterial symbiont communities on adult frog skin (Pseudacris crucifer), characterized using 16S rRNA gene amplicon sequencing. We found that skin bacterial communities on frogs were less diverse than, and structurally distinct from, the surrounding habitat. Frog skin was typically dominated by one of two bacterial OTUs: at western sites, a Proteobacteria dominated the community, whereas eastern sites were dominated by an Actinobacteria. Using a metacommunity framework, we then sought to identify factors explaining small- and large-scale variation in community structure—that is, among hosts within a pond, and among ponds spanning the study transect. We focused on the presence of a fungal skin pathogen, Batrachochytrium dendrobatidis (Bd) as one potential driver of variation. We found no direct link between skin bacterial community structure and Bd infection status of individual frog hosts. Differences in pond-level community structure, however, were explained by Bd infection prevalence. Importantly, Bd infection prevalence itself was correlated with numerous other environmental factors; thus, skin bacterial diversity may be influenced by a complex suite of extrinsic factors. Our findings indicate that large-scale factors and processes merit consideration when seeking to understand microbiome diversity.


Microbiota Microbiome Amphibian Chytrid fungus Symbiosis Metacommunity 



We thank P. Shirk, J. Vonesh, J. Wyderko, and S. Zemmer for assistance in the field; P. Sattler at Liberty University, G. Eaton at Claytor Nature Study Center, and J. Vonesh at Virginia Commonwealth University for recommending sites in Lynchburg, Bedford, and Richmond, respectively; J. Touchon for R code consultations; C. Herbold for stats advice; and Z. Herbert at the Dana Farber Cancer Institute Molecular Biology Core Facility for Illumina MiSeq amplicon sequencing. We are also thankful for the comments of anonymous reviewers that improved the manuscript.


This work was supported by the Morris Animal Foundation [D10ZO-028] and the National Science Foundation [DEB-1136640].

Compliance with Ethical Standards

This research was conducted with approval from Virginia Tech’s Institutional Animal Care and Use Committee (protocol 10-029-BIOL) and with permission from the Virginia Department of Game and Inland Fisheries (permit 44303).

Supplementary material


  1. 1.
    Adair KL, Douglas AE (2017) Making a microbiome: the many determinants of host-associated microbial community composition. Curr Opin Microbiol 35:23–29CrossRefGoogle Scholar
  2. 2.
    Griffiths SM, Harrison XA, Weldon C, Wood MD, Pretorius A, Hopkins K, Fox G, Preziosi RF, Antwis RE (2018) Genetic variability and ontogeny predict microbiome structure in a disease-challenged montane amphibian. ISME J 12:2506–2517CrossRefGoogle Scholar
  3. 3.
    SanMiguel A, Grice EA (2015) Interactions between host factors and the skin microbiome. Cell Mol Life Sci 72:1499–1515CrossRefGoogle Scholar
  4. 4.
    Ricklefs RE (1987) Community diversity: relative roles of local and regional processes. Science 235:167–171CrossRefGoogle Scholar
  5. 5.
    Lindström ES, Langenheder S (2012) Local and regional factors influencing bacterial community assembly. Environ Microbiol Rep 4:1–9CrossRefGoogle Scholar
  6. 6.
    Nemergut DR, Schmidt SK, Fukami T, O’Neill SP, Bilinski TM, Stanish LF, Knelman JE, Darcy JL, Lynch RC, Wickey P, Ferrenberg S (2013) Patterns and processes of microbial community assembly. Microbiol Mol Biol Rev 77:342–356CrossRefGoogle Scholar
  7. 7.
    Costello EK, Stagaman K, Dethlefsen L, Bohannan BJM, Relman DA (2012) The application of ecological theory toward an understanding of the human microbiome. Science 336:1255–1262CrossRefGoogle Scholar
  8. 8.
    Fierer N, Ferrenberg S, Flores GE, González A, Kueneman J, Legg T, Lynch RC, McDonald D, Mihaljevic JR, O’Neill SP, Rhodes ME, Song SJ, Walters WA (2012) From animalcules to an ecosystem: application of ecological concepts to the human microbiome. Annu Rev Ecol Evol Syst 43:137–155CrossRefGoogle Scholar
  9. 9.
    Mihaljevic JR (2012) Linking metacommunity theory and symbiont evolutionary ecology. Trends Ecol Evol 27:323–329CrossRefGoogle Scholar
  10. 10.
    Leibold MA, Holyoak M, Mouquet N, Amarasekare P, Chase JM, Hoopes MF, Holt RD, Shurin JB, Law R, Tilman D, Loreau M, Gonzalez A (2004) The metacommunity concept: a framework for multi-scale community ecology. Ecol Lett 7:601–613CrossRefGoogle Scholar
  11. 11.
    Holyoak M, Leibold MA, Holt RD (eds.) (2005) Metacommunities: spatial dynamics and ecological communities. University of Chicago PressGoogle Scholar
  12. 12.
    Venkataraman A, Bassis CM, Beck JM, Young VB, Curtis JL, Huffnagle GB, Schmidt TM (2015) Application of a neutral community model to assess structuring of the human lung microbiome. MBio 6:e02284–e02214CrossRefGoogle Scholar
  13. 13.
    Burns AR, Stephens WZ, Stagaman K, Wong S, Rawls JF, Guillemin K, Bohannan BJ (2016) Contribution of neutral processes to the assembly of gut microbial communities in the zebrafish over host development. ISME J 10:655–664CrossRefGoogle Scholar
  14. 14.
    Bletz MC, Loudon AH, Becker MH, Bell SC, Woodhams DC, Minbiole KPC, Harris RN (2013) Mitigating amphibian chytridiomycosis with bioaugmentation: characteristics of effective probiotics and strategies for their selection and use. Ecol Lett 16:807–820CrossRefGoogle Scholar
  15. 15.
    Rebollar EA, Antwis RE, Becker MH, Belden LK, Bletz MC, Brucker RM, Harrison XA, Hughey MC, Kueneman JG, Loudon AH, McKenzie V (2016) Using “omics” and integrated multi-omics approaches to guide probiotic selection to mitigate chytridiomycosis and other emerging infectious diseases. Front Microbiol 7:68CrossRefGoogle Scholar
  16. 16.
    Woodhams DC, Bletz M, Kueneman J, McKenzie V (2016) Managing amphibian disease with skin microbiota. Trends Microbiol 24:161–164CrossRefGoogle Scholar
  17. 17.
    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 2:140377CrossRefGoogle Scholar
  18. 18.
    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
  19. 19.
    Becker CG, Longo AV, Haddad CF, Zamudio KR (2017) Land cover and forest connectivity alter the interactions among host, pathogen and skin microbiome. Proc R Soc B 284:20170582CrossRefGoogle Scholar
  20. 20.
    Hughey MC, Pena JA, Reyes R, Medina D, Belden LK, Burrowes PA (2017) Skin bacterial microbiome of a generalist Puerto Rican frog varies along elevation and land use gradients. PeerJ 5:e3688CrossRefGoogle Scholar
  21. 21.
    Muletz Wolz CR, Yarwood SA, Campbell Grant EH, Fleischer RC, Lips KR (2018) Effects of host species and environment on the skin microbiome of Plethodontid salamanders. Anim Ecol 87:341–353CrossRefGoogle Scholar
  22. 22.
    Jani AJ, Briggs CJ (2014) The pathogen Batrachochytrium dendrobatidis disturbs the frog skin microbiome during a natural epidemic and experimental infection. 2014. Proc Natl Acad Sci 111:E5049–E5058CrossRefGoogle Scholar
  23. 23.
    Belden LK, Hughey MC, Rebollar EA, Umile TP, Loftus SC, Burzynski EA, Minbiole KPC, House LL, Jensen RV, Becker MH, Walke JB, Medina D, Ibáñez R, Harris RN (2015) Panamanian frog species host unique skin bacterial communities. Front Microbiol 6:1171CrossRefGoogle Scholar
  24. 24.
    Walke JB, Becker MH, Loftus SC, House LL, Teotonio TL, Minbiole KP, Belden LK (2015) Community structure and function of amphibian skin microbes: an experiment with bullfrogs exposed to a chytrid fungus. PLoS One 10:e0139848CrossRefGoogle Scholar
  25. 25.
    Rebollar EA, Hughey MC, Medina D, Harris RN, Ibáñez R, Belden LK (2016) Skin bacterial diversity of Panamanian frogs is associated with host susceptibility and presence of Batrachochytrium dendrobatidis. ISME J 10:1682–1695CrossRefGoogle Scholar
  26. 26.
    Jani AJ, Knapp RA, Briggs CJ (2017) Epidemic and endemic pathogen dynamics correspond to distinct host population microbiomes at a landscape scale. Proc R Soc B 284:20170944CrossRefGoogle Scholar
  27. 27.
    Longo AV, Zamudio KR (2017) Environmental fluctuations and host skin bacteria shift survival advantage between frogs and their fungal pathogen. ISME J 11:349–361CrossRefGoogle Scholar
  28. 28.
    Longo AV, Zamudio KR (2017) Temperature variation, bacterial diversity and fungal infection dynamics in the amphibian skin. Mol Ecol 26:4787–4797CrossRefGoogle Scholar
  29. 29.
    Bates KA, Clare FC, O’Hanlon S, Bosch J, Brookes L, Hopkins K, McLaughlin EJ, Daniel O, Garner TW, Fisher MC, Harrison XA (2018) Amphibian chytridiomycosis outbreak dynamics are linked with host skin bacterial community structure. Nat Commun 9:693CrossRefGoogle Scholar
  30. 30.
    Kilpatrick AM, Briggs CJ, Daszak P (2010) The ecology and impact of chytridiomycosis: an emerging disease of amphibians. Trends Ecol Evol 25:109–118CrossRefGoogle Scholar
  31. 31.
    Lips KR (2016) Overview of chytrid emergence and impacts on amphibians. Philos Trans R Soc B 371:20150465CrossRefGoogle Scholar
  32. 32.
    Voyles J, Rosenblum EB, Berger L (2011) Interactions between Batrachochytrium dendrobatidis and its amphibian hosts: a review of pathogenesis and immunity. Microbes Infect 13:25–32CrossRefGoogle Scholar
  33. 33.
    Harris RN, Brucker RM, Walke JB, Becker MH, Schwantes CR, Flaherty DC, Lam BA, Woodhams DC, Briggs CJ, Vredenburg VT, Minbiole KPC (2009) Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus. ISME J 3:818–824CrossRefGoogle Scholar
  34. 34.
    Hughey MC, Becker MH, Walke JB, Swartwout MC, Belden LK (2014) Batrachochytrium dendrobatidis in Virginia amphibians: within and among site variation in infection. Herpetol Rev 45:428–438Google Scholar
  35. 35.
    Gahl MK, Longcore JE, Houlahan JE (2012) Varying responses of northeastern north American amphibians to the chytrid pathogen Batrachochytrium dendrobatidis. Conserv Biol 26:135–141CrossRefGoogle Scholar
  36. 36.
    Hyatt AD, Boyle DG, Olsen V, Boyle DB, Berger L, Obendorf D, Dalton A, Kriger K, Hero M, Hines H, Phillott R, Campbell R, Marantelli G, Gleason F, Colling A (2007) Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis. Dis Aquat Org 73:175–192CrossRefGoogle Scholar
  37. 37.
    McKenzie VJ, Bowers RM, Fierer N, Knight R, Lauber CL (2012) Co-habiting amphibian species harbor unique skin bacterial communities in wild populations. ISME J 6:588–596CrossRefGoogle Scholar
  38. 38.
    Walke JB, Becker MH, Loftus SC, House LL, Cormier G, Jensen RV, Belden LK (2014) Amphibian skin may select for rare environmental microbes. ISME J 8:2207–2217CrossRefGoogle Scholar
  39. 39.
    Boyle DG, Boyle DB, Olsen V, Morgan JAT, Hyatt AD (2004) Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Dis Aquat Org 60:141–148CrossRefGoogle Scholar
  40. 40.
    Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6:1621–1624CrossRefGoogle Scholar
  41. 41.
    Meisel JS, Hannigan GD, Tyldsley AS, SanMiguel AJ, Hodkinson BP, Zheng Q, Grice EA (2016) Skin microbiome surveys are strongly influenced by experimental design. J Invest Dermatol 136:947–956CrossRefGoogle Scholar
  42. 42.
    Aronesty E. (2011) Ea-utils: “command-line tools for processing biological sequencing data”.
  43. 43.
    Caporaso JG, Kuczynski J, Stombaugh JJ, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336CrossRefGoogle Scholar
  44. 44.
    Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461CrossRefGoogle Scholar
  45. 45.
    DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72:5069–5072CrossRefGoogle Scholar
  46. 46.
    Caporaso JG, Bittinger K, Bushman FD, DeSantis TZ, Andersen GL, Knight R (2010) PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics 26:266–267CrossRefGoogle Scholar
  47. 47.
    Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267CrossRefGoogle Scholar
  48. 48.
    Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, Knight R, Mills DA, Caporaso JG (2013) Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Methods 10:57–59CrossRefGoogle Scholar
  49. 49.
    R Core Team (2014) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna Google Scholar
  50. 50.
    Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin PR, O’Hara RB, Simpson GL, Solymos P, Stevens MHH, Szoecs E, Wagner H (2015) Vegan: community ecology package.
  51. 51.
    Anderson MJ (2001) A new method for non-parametric multivariate analysis of variance. Aust Ecol 26:32–46Google Scholar
  52. 52.
    Whittaker RH (1972) Evolution and measurement of species diversity. Taxon 21:213–251CrossRefGoogle Scholar
  53. 53.
    Fournier DA, Skaug HJ, Ancheta J, Ianelli J, Magnusson A, Maunder M et al (2012) AD model builder: using automatic differentiation for statistical inference of highly parameterized complex nonlinear models. Optim Methods Softw 27:233–249CrossRefGoogle Scholar
  54. 54.
    Skaug H, Fournier D, Bolker B, Magnusson A, Nielsen A (2014) Generalized linear mixed models using AD model builder. R package version 0.8.0Google Scholar
  55. 55.
    Legendre P, Legendre L (2012) Numerical ecology. 3rd English edition. ElsevierGoogle Scholar
  56. 56.
    Tuomisto H, Ruokolainen K (2006) Analyzing or explaining beta diversity? Understanding the targets of different methods of analysis. Ecology 87:2697–2708CrossRefGoogle Scholar
  57. 57.
    Paliy O, Shankar V (2016) Application of multivariate statistical techniques in microbial ecology. Mol Ecol 25:1032–1057CrossRefGoogle Scholar
  58. 58.
    Legendre P, Gallagher ED (2001) Ecologically meaningful transformations for ordination of species data. Oecologia 129:271–280CrossRefGoogle Scholar
  59. 59.
    Campbell BJ, Yu L, Heidelberg JF, Kirchman DL (2011) Activity of abundant and rare bacteria in a coastal ocean. Proc Natl Acad Sci U S A 108:12776–12781CrossRefGoogle Scholar
  60. 60.
    Walke JB, Becker MH, Hughey MC, Swartwout MC, Jensen RV, Belden LK (2017) Dominance-function relationships in the amphibian skin microbiome. Environ Microbiol 19:3387–3397CrossRefGoogle Scholar
  61. 61.
    Austin JD, Lougheed SC, Neidrauer L, Chek AA, Boag PT (2002) Cryptic lineages in a small frog: the post-glacial history of the spring peeper, Pseudacris crucifer (Anura: Hylidae). Mol Phylogenet Evol 25:316–329CrossRefGoogle Scholar
  62. 62.
    Christian N, Whitaker BK, Clay K (2015) Microbiomes: unifying animal and plant systems through the lens of community ecology theory. Front Microbiol 6:869CrossRefGoogle Scholar
  63. 63.
    Burns AR, Miller E, Agarwal M, Rolig AS, Milligan-Myhre K, Seredick S, Guillemin K, Bohannan BJ (2017) Interhost dispersal alters microbiome assembly and can overwhelm host innate immunity in an experimental zebrafish model. Proc Natl Acad Sci 114:11181–11186CrossRefGoogle Scholar
  64. 64.
    Fitzpatrick BM, Allison AL (2014) Similarity and differentiation between bacteria associated with skin of salamanders (Plethodon jordani) and free-living assemblages. FEMS Microbiol Ecol 88:482–494CrossRefGoogle Scholar
  65. 65.
    Banning JL, Weddle AL, Wahl GW, Simon MA, Lauer A, Walters RL, Harris RN (2008) Antifungal skin bacteria, embryonic survival, and communal nesting in four-toed salamanders, Hemidactylium scutatum. Oecologia 156:423–429CrossRefGoogle Scholar
  66. 66.
    Walke JB, Harris RN, Reinert LK, Rollins-Smith LA, Woodhams DC (2011) Social immunity in amphibians: evidence for vertical transmission of innate defenses. Biotropica 43:396–400CrossRefGoogle Scholar
  67. 67.
    Hughey MC, Delia J, Belden LK (2017) Diversity and stability of egg-bacterial assemblages: the role of paternal care in the glassfrog Hyalinobatrachium colymbiphyllum. Biotropica 49:792–802CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Myra C. Hughey
    • 1
    • 2
    Email author
  • Eric R. Sokol
    • 2
    • 3
    • 4
  • Jenifer B. Walke
    • 2
    • 5
  • Matthew H. Becker
    • 2
    • 6
  • Lisa K. Belden
    • 2
  1. 1.Biology DepartmentVassar CollegePoughkeepsieUSA
  2. 2.Department of Biological SciencesVirginia TechBlacksburgUSA
  3. 3.Battelle, National Ecological Observatory Network (NEON)BoulderUSA
  4. 4.Institute of Arctic and Alpine Research (INSTAAR)University of Colorado BoulderBoulderUSA
  5. 5.Department of BiologyEastern Washington UniversityCheneyUSA
  6. 6.Department of Biology and ChemistryLiberty UniversityLynchburgUSA

Personalised recommendations