Conservation Genetics

, Volume 20, Issue 5, pp 947–960 | Cite as

A morphologically cryptic salamander reveals additional hidden diversity: evidence for ancient genetic divergence in Webster’s salamander, Plethodon websteri

  • Sheena FeistEmail author
  • Thomas Mann
  • Sean Graham
  • Jessica Wooten
  • Cory Toyota
  • Debora Mann
  • Madeline Balius
  • Jose Polanco
  • Peyton Wolwehender
  • Je’Coiya Moore
Research Article


We investigated the genetic diversity and phylogenetic structure of Webster’s salamander (Plethodon websteri), an understudied species of conservation concern and one of the first morphologically cryptic salamander species described solely on the basis of molecular techniques. Using a combination of mitochondrial and microsatellite data, we discovered significant genetic differentiation across the known distribution (ɸST > 0.868 and FST = 0.261−0.652, p < 0.001), and identified at least two highly divergent clades. These clades, referenced herein as the Semlitsch and Type Locality clades, are of late Miocene origin (approximately 5.3 million years old) and are composed of several geographically and genetically distinct populations. Together, our observations suggest that previously unrecognized species-level diversity may exist in P. websteri, with populations potentially representing distinct but undescribed taxa. We hypothesize that divergence within P. websteri likely resulted from a culmination of radical changes in climate, hydrology, and geology over deep time. Moreover, our results add to mounting evidence suggesting that P. websteri does not belong within the P. welleri group, but instead forms its own discrete species complex separate and genetically distant from P. welleri. This study provides a starting point for future work and reiterates that relationships among, and species-level diversity within, eastern species of Plethodon require additional investigation and potential reevaluation.


Phylogeography Population genetics Cryptic Plethodon Webster’s salamander Rock 



Salamanders were handled and tail clips collected under the authority granted by the following scientific collection permits: United States National Park Service #NATR-2011-SCI-0005 and #NATR-2013-SCI-0001; United States Fish and Wildlife Service (USFWS) #43665-15-001 and #43665-16-001; State of Alabama #2014012054668680, #2015050109068680, #2016062270668680. Transport of tissues and DNA extracts were covered under an injurious salamander permit from USFWS (MA19723C-0). This project was made possible by State Wildlife Grant funds provided by USFWS, and was additionally partially supported by the Mississippi INBRE, funded by an Institutional Development AWARD (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant #P20GM103476. We thank the following people for either contributing to fieldwork or for supplying tissue samples utilizing their own permits/authority: Elizabeth Timpe, John Jensen, Megan Gibbons, Ken Marion, Adrienne Duvall, Jeff Boundy, Jennifer Lamb, Jennifer Frey, Mike Stegall, K. Bakkegard, and Will Dillman.


Funding was provided by U.S. Fish and Wildlife Service (Grant Nos. MS-T-F17AF01210, MS-T-F16AF01209, MS-T-F15AF00032, MS-T-F14AF01350, MS-T-F13AF01252) and Foundation for the National Institutes of Health (Grant No. P20GM103476)


  1. Bergsten J (2005) A review of long-branch attraction. Cladistics 21:163–193CrossRefGoogle Scholar
  2. Bouckaert R, Heled J, Kühnert D et al (2014) BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Comput Biol 10:e1003537CrossRefPubMedPubMedCentralGoogle Scholar
  3. Cameron AC, Anderson JJ, Page RB (2017) Assessment of intra and interregional genetic variation in the eastern red-backed salamander, Plethodon cinereus, via analysis of novel microsatellite markers. PLoS ONE 12:e0186866CrossRefPubMedPubMedCentralGoogle Scholar
  4. Camp C (1986) Distribution and habitat of the southern red-back salamander, Plethodon serratus Grobman (Amphibia: Plethodontidae), in Georgia. Ga J Sci 44:136–146Google Scholar
  5. Camp CD, Bozeman LL (1981) Foods of two species of Plethodon (Caudata: Plethodontidae) from Georgia and Alabama. Brimleyana 6:163–166Google Scholar
  6. Chippindale PT, Bonett RM, Baldwin AS, Wiens JJ (2004) Phylogenetic evidence for a major reversal of life-history evolution in plethodontid salamanders. Evol Int J Org Evol 58:2809–2822CrossRefGoogle Scholar
  7. Combellas-Bigott RI, Galloway WE (2006) Depositional and structural evolution of the middle Miocene depositional episode, east-central Gulf of Mexico. AAPG Bull 90:335–362CrossRefGoogle Scholar
  8. Connors LM, Cabe PR (2003) Isolation of dinucleotide microsatellite loci from red-backed salamander (Plethodon cinereus). Mol Ecol Notes 3:131–133CrossRefGoogle Scholar
  9. Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772CrossRefPubMedPubMedCentralGoogle Scholar
  10. Deitloff J, Floyd C, Graham SP (2016) Examining head-shape differences and ecology in morphologically similar salamanders at their zone of contact. Copeia 104:233–242CrossRefGoogle Scholar
  11. Edgington HA, Ingram CM, Taylor DR (2016) Cyto-nuclear discordance suggests complex evolutionary history in the cave-dwelling salamander, Eurycea lucifuga. Ecol Evol 6:6121–6138CrossRefPubMedPubMedCentralGoogle Scholar
  12. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software structure: a simulation study. Mol Ecol 14:2611–2620CrossRefPubMedPubMedCentralGoogle Scholar
  13. Excoffier L, Lischer HEL (2010) Arlequin suite v. 3.5: a new series of programs to perform population genetics analyses under Linux and windows. Mol Ecol Resour 10:564–567CrossRefPubMedPubMedCentralGoogle Scholar
  14. Feist SM, Mann TM, Mann DL (2017) Genetic characterization of Webster’s salamander (Plethodon websteri) within a fragmented landscape, using novel microsatellites. Herpetol Conserv Biol 12:85–95Google Scholar
  15. Folt B, Garrison N, Guyer C et al (2016) Phylogeography and evolution of the red salamander (Pseudotriton ruber). Mol Phylogenet Evol 98:97–110CrossRefPubMedGoogle Scholar
  16. Galloway WE, Whiteaker TL, Ganey-Curry P (2011) History of Cenozoic North American drainage basin evolution, sediment yield, and accumulation in the Gulf of Mexico basin. Geosphere 7:938–973CrossRefGoogle Scholar
  17. Graham SP, Timpe EK, Durso AM et al (2012) The second known contact zone between Plethodon websteri and P. ventralis, and additional records for Bibb County, Alabama. USA Herpetol Rev 43:311–312Google Scholar
  18. Grant WAS, Bowen BW (1998) Shallow population histories in deep evolutionary lineages of marine fishes: insights from sardines and anchovies and lessons for conservation. J Hered 89:415–426CrossRefGoogle Scholar
  19. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704CrossRefGoogle Scholar
  20. Highton R (1979) A new cryptic species of salamander of the genus Plethodon from the southeastern United States (Amphibia: Plethodontidae). Brimleyana 1:31–36Google Scholar
  21. Highton R (1985) The width of the contact zone between Plethodon dorsalis and P. websteri in Jefferson County. Alabama. J Herpetol 19:544–546CrossRefGoogle Scholar
  22. Highton R (1995) Speciation in eastern North American salamanders of the genus Plethodon. Annu Rev Ecol Syst 26:579–600CrossRefGoogle Scholar
  23. Highton R, Larson A (1979) The genetic relationships of the salamanders of the genus Plethodon. Syst Biol 28:579–599CrossRefGoogle Scholar
  24. Highton R, Hastings AP, Palmer C et al (2012) Concurrent speciation in the eastern woodland salamanders (Genus Plethodon): DNA sequences of the complete albumin nuclear and partial mitochondrial 12 s genes. Mol Phylogenet Evol 63:278–290CrossRefPubMedGoogle Scholar
  25. Jockusch EL, Wake DB (2002) Falling apart and merging: diversification of slender salamanders (Plethodontidae: Batrachoseps) in the American West. Biol J Linn Soc 76:361–391CrossRefGoogle Scholar
  26. Kalinowski ST (2005) Hp-rare 1.0: a computer program for performing rarefaction on measures of allelic richness. Mol Ecol Notes 5:187–189CrossRefGoogle Scholar
  27. Kearse M, Moir R, Wilson A et al (2012) Geneious basic: an integrated and extendable desktop platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kolaczkowski B, Thornton JW (2009) Long-branch attraction bias and inconsistency in Bayesian phylogenetics. PLoS ONE 4:e7891CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kopelman NM, Mayzel J, Jakobsson M et al (2015) Clumpak: a program for identifying clustering modes and packaging population structure inferences across K. Mol Ecol Resour 15:1179–1191CrossRefPubMedPubMedCentralGoogle Scholar
  30. Kozak KH, Weisrock DW, Larson A (2006) Rapid lineage accumulation in a non-adaptive radiation: phylogenetic analysis of diversification rates in eastern North American woodland salamanders (Plethodontidae: Plethodon). Proc R Soc Lond B Biol Sci 273:539–546CrossRefGoogle Scholar
  31. Kuchta SR, Brown AD, Converse PE, Highton R (2016) Multilocus phylogeography and species delimitation in the Cumberland Plateau salamander, Plethodon kentucki: incongruence among data sets and methods. PLoS ONE 11:e0150022CrossRefPubMedPubMedCentralGoogle Scholar
  32. Larson A, Highton R (1978) Geographic protein variation and divergence in the salamanders of the Plethodon Welleri group (Amphibia, Plethodontidae). Syst Biol 27:431–448CrossRefGoogle Scholar
  33. Larson A, Wake DB, Yanev KP (1984) Measuring gene flow among populations having high levels of genetic fragmentation. Genetics 106:293–308PubMedPubMedCentralGoogle Scholar
  34. Li Y-L, Liu J-X (2018) StructureSelector: a web-based software to select and visualize the optimal number of clusters using multiple methods. Mol Ecol Resour 18:176–177CrossRefPubMedGoogle Scholar
  35. Mann TM, Mann DL (2017) Seasonal migration by a terrestrial salamander, Plethodon websteri (Webster’s Salamander). Herpetol Conserv Biol 12:96–108Google Scholar
  36. Mann TM, Mann DL, Toyota CG et al (2017) Clarification of the nature of the contact zone between Plethodon websteri and P. ventralis at the Cahaba River National Wildlife Refuge in Bibb County, Alabama. USA Herpetol Rev 48:522–525Google Scholar
  37. Moritz C, Schneider C, Wake DB (1992) Evolutionary relationships within the Ensatina eschscholtzii complex confirm the ring species interpretation. Syst Biol 41(3):273–291CrossRefGoogle Scholar
  38. NatureServe (2016) Comprehensive Report Species—Plethodon websteri. Accessed 26 Jun 2018
  39. Newman CE, Austin CC (2015) Thriving in the cold: glacial expansion and post-glacial contraction of a temperate terrestrial salamander (Plethodon serratus). PLoS ONE 10:e0130131CrossRefPubMedPubMedCentralGoogle Scholar
  40. Omernik JM, Griffith GE (2014) Ecoregions of the conterminous United States: evolution of a hierarchical spatial framework. Environ Manage 54:1249–1266CrossRefPubMedGoogle Scholar
  41. Palmer CA, Watts RA, Gregg RG et al (2005) Lineage-specific differences in evolutionary mode in a salamander courtship pheromone. Mol Biol Evol 22:2243–2256CrossRefPubMedGoogle Scholar
  42. Parra-Olea G, Wake DB (2001) Extreme morphological and ecological homoplasy in tropical salamanders. Proc Natl Acad Sci 98:7888–7891CrossRefPubMedGoogle Scholar
  43. Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research—an update. Bioinformatics 28:2537–2539CrossRefPubMedPubMedCentralGoogle Scholar
  44. Petranka JW (1998) Salamanders of the United States and Canada. Smithsonian Books, Washington, DCGoogle Scholar
  45. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959PubMedPubMedCentralGoogle Scholar
  46. Radomski TP (2016) Biogeography and climatic niche evolution in the eastern red-backed salamander (Plethodon cinereus). Thesis, Ohio University, AthensGoogle Scholar
  47. Rice WR (1989) Analyzing tables of statistical tests. Evolution 43:223–225CrossRefPubMedGoogle Scholar
  48. Rissler LJ, Smith WH (2010) Mapping amphibian contact zones and phylogeographical break hotspots across the United States. Mol Ecol 19:5404–5416CrossRefPubMedGoogle Scholar
  49. Rousset F (2008) Genepop’007: a complete re-implementation of the genepop software for Windows and Linux. Mol Ecol Resour 8:103–106CrossRefPubMedPubMedCentralGoogle Scholar
  50. Rovito SM, Parra-Olea G, Hanken J et al (2013) Adaptive radiation in miniature: the minute salamanders of the Mexican highlands (Amphibia: Plethodontidae: Thorius): Thorius Phylogeny. Biol J Linn Soc 109:622–643CrossRefGoogle Scholar
  51. Semlitsch RD, West CA (1983) Aspects of the life history and ecology of Webster’s salamander, Plethodon websteri. Copeia 1983:339–346CrossRefGoogle Scholar
  52. Shepard DB, Burbrink FT (2011) Local-scale environmental variation generates highly divergent lineages associated with stream drainages in a terrestrial salamander, Plethodon caddoensis. Mol Phylogenet Evol 59:399–411CrossRefPubMedGoogle Scholar
  53. Snedden JW, Galloway WE, Milliken KT et al (2018) Validation of empirical source-to-sink scaling relationships in a continental-scale system: the Gulf of Mexico basin Cenozoic record. Geosphere 14:768–784CrossRefGoogle Scholar
  54. Spotila JR (1972) Role of temperature and water in the ecology of lungless salamanders. Ecol Monogr 42:95–125CrossRefGoogle Scholar
  55. Suzuki Y, Glazko GV, Nei M (2002) Overcredibility of molecular phylogenies obtained by Bayesian phylogenetics. Proc Natl Acad Sci USA 99:16138–16143CrossRefPubMedGoogle Scholar
  56. Swofford DL (2003) PAUP*. Phylogenetic analysis using parsimony (*and other methods). Sinauer Associates, SunderlandGoogle Scholar
  57. Taylor DJ, Piel WH (2004) An assessment of accuracy, error, and conflict with support values from genome-scale phylogenetic data. Mol Biol Evol 21:1534–1537CrossRefPubMedGoogle Scholar
  58. Untergasser A, Cutcutache I, Koressaar T et al (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40:e115–e115CrossRefPubMedPubMedCentralGoogle Scholar
  59. Villesen P (2007) FaBox: an online toolbox for fasta sequences. Mol Ecol Notes 7:965–968CrossRefGoogle Scholar
  60. Wake DB (1987) Adaptive radiation of salamanders in middle American cloud forests. Ann Mo Bot Gard 74:242–264CrossRefGoogle Scholar
  61. Wake DB (2006) Problems with species: patterns and processes of species formation in salamanders. Ann Mo Bot Gard 93:8–23CrossRefGoogle Scholar
  62. Wiens JJ (2004) Speciation and ecology revisited: phylogenetic niche conservatism and the origin of species. Evolution 58:193–197CrossRefPubMedGoogle Scholar
  63. Wiens JJ, Engstrom TN, Chippindale PT (2006) Rapid diversification, incomplete isolation, and the “speciation clock” in North American salamanders (genus Plethodon): testing the hybrid swarm hypothesis of rapid radiation. Evolution 60:2585–2603PubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Mississippi Department of Wildlife, Fisheries, and Parks, Museum of Natural ScienceJacksonUSA
  2. 2.Department of Biology, Geology, and Physical SciencesSul Ross State UniversityAlpineUSA
  3. 3.Department of BiologyPiedmont CollegeDemorestUSA
  4. 4.Department of Chemistry and BiochemistryMillsaps CollegeJacksonUSA
  5. 5.Department of BiologyMillsaps CollegeJacksonUSA
  6. 6.Department of BiologyCentre CollegeDanvilleUSA

Personalised recommendations