Journal of Molecular Evolution

, Volume 70, Issue 5, pp 427–440 | Cite as

Rapid Evolution of Plethodontid Modulating Factor, a Hypervariable Salamander Courtship Pheromone, is Driven by Positive Selection

  • Catherine A. Palmer
  • Richard A. WattsEmail author
  • Amy P. Hastings
  • Lynne D. Houck
  • Stevan J. Arnold


Sexual communication in plethodontid salamanders is mediated by a proteinaceous pheromone that a male delivers to a female during courtship, boosting her receptivity. The pheromone consists of three proteins from three unrelated protein families. These proteins are among a small group of pheromones known to affect female receptivity in vertebrates. Previously, we showed that the genes of two of these proteins (PRF and SPF) are prone to incessant evolution driven by positive selection, presumably as a consequence of coevolution with female receptors. In this report, we focus on the evolution of the third pheromone protein gene family, plethodontid modulating factor (PMF), to determine whether it shows the same pattern of diversification. We used RT-PCR in mental gland cDNA to survey PMF sequences from three genera of plethodontid salamanders (27 spp.) to measure rates of evolution, level of gene diversification, modes of selection, and types of amino acid substitution. Like PRF and SPF, PMF is produced by a multigene family characterized by gene duplication and high levels of polymorphism. PMF evolution is rapid, incessant, and driven by positive selection. PMF is more extreme in these dimensions than both PRF and SPF. Nestled within this extraordinary variation, however, is a signature of purifying selection, acting to preserve important structural and biochemical features of the PMF protein (i.e., secretion signal, cysteine residues, and pI). Although a pattern of persistent diversification exists at the molecular level, the morphological and behavioral aspects of the pheromone delivery system show evolutionary stasis over millions of years.


Pheromone gene Rapid evolution Positive selection Reproductive protein Courtship signal Sexual communication 



We are grateful to R. Feldhoff, P. Wheeler-Feldhoff, and other members of our research team ( for discussion and help with all phases of this project. We would like to extend our appreciation to R. Highton and M. Westphal for their assistance in the field and with tissue samples. This research was supported by NSF predoctoral Fellowship to CAP and by NSF grants IBN-0110666 and IOS-0818554 to LDH and SJA and IOS-0818649 R. Feldhoff. The sequences reported in this manuscript have been deposited in GenBank under the accession numbers DQ882251–DQ882291 and DQ882379–DQ882569.

Supplementary material

239_2010_9342_MOESM1_ESM.doc (32 kb)
Supplementary material 1 (DOC 32 kb)


  1. Andersson M (1994) Sexual selection. Princeton University Press, Princeton, New JerseyGoogle Scholar
  2. Arnold SJ (1977) The evolution of courtship behavior in new world salamanders with some comments on old world salamandrids. In: Taylor DH, Guttman SI (eds) The reproductive biology of amphibians. Plenum Press, New York, pp 141–183Google Scholar
  3. Brown GE, Brown JA (1992) Do rainbow trout and Atlantic salmon discriminate kin? Can J Zool 70:1636–1640CrossRefGoogle Scholar
  4. Chippindale PT, Bonett RM, Baldwin AS, Wiens JJ (2004) Phylogenetic evidence for a major reversal of life-history evolution in plethodontid salamanders. Evolution 58(12):2809–2822PubMedGoogle Scholar
  5. Chivers DP, Kiesecker JM, Wildy EL, Anderson MT, Blaustein AR (1997) Chemical alarm signaling in terrestrial salamanders: intra- and interspecific responses. Ethology 103:599–613CrossRefGoogle Scholar
  6. Christensen TA, Mustaparta H, Hildebrand J (1991) Chemical communication in heliothine moths II: central processing of intra- and interspecific olfactory messages in male corn earworm moth. J Comp Physiol A 169:259–274CrossRefGoogle Scholar
  7. Clark NL, Swanson WJ (2005) Pervasive adaptive evolution in primate seminal proteins. PLoS Genet 1(3):e35CrossRefPubMedGoogle Scholar
  8. Dobzhansky T (1937) Genetics and the origin of species. Columbia University Press, New YorkGoogle Scholar
  9. Dulka JG, Stacey NE, Sorensen PW, Van Der Kraak GJ (1987) A sex steroid pheromone synchronizes male-female spawning readiness in goldfish. Nature 325:251–253CrossRefGoogle Scholar
  10. Dyal LA (2006) Novel courtship behaviors in three small eastern Plethodon species. J Herpetol 40(1):55–65CrossRefGoogle Scholar
  11. Fisher RA (1958) The genetical theory of natural selection. Dover Publication Inc., New YorkGoogle Scholar
  12. Gavriletts S, Hayashi TI (2006) The dynamics of two- and three-way sexual conflicts over mating. Philos Trans R Soc Lond B 361:345–354CrossRefGoogle Scholar
  13. Gershman SN, Verrel PA (2002) To persuade or be persuaded: which sex controls mating in a plethodontid salamander? Behaviour 139:447–462CrossRefGoogle Scholar
  14. Hall TA (1999) Bioedit: a user friendly biological sequence alignment editor and analysis program for Windows 95/97/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  15. Highton R (1962) Revision of North American salamanders of the genus Plethodon. Bull Fla State Mus 6(3):235–367Google Scholar
  16. Highton R (1989) Biochemical evolution in the slimy salamanders of the Plethodon glutinosus complex in the eastern United States. Part I. Geographic protein variation. Ill Biol Monogr 57:1–78Google Scholar
  17. Highton R (1995) Speciation in eastern North American salamanders of the genus Plethodon. Annu Rev Ecol Syst 26:579–600CrossRefGoogle Scholar
  18. Highton R, Larson A (1979) The genetic relationships of the salamanders of the genus Plethodon. Syst Zool 28:579–599CrossRefGoogle Scholar
  19. Highton R, Peabody RB (2000) Geographic protein variation and speciation in salamanders of the Plethodon jordani and Plethodon glutinosus complexes in the southern Appalachian Mountains with the descriptions of four new species. In: Bruce RC, Jaeger RG, Houck LD (eds) The biology of plethodontid salamanders. Plenum, New York, pp 31–94Google Scholar
  20. Houck LD, Arnold SJ (2003) Courtship and mating behavior. In: Sever DM (ed) Phylogeny and reproductive biology of Urodela (Amphibia). Science Publishers, Enfield, New Hampshire, pp 383–424Google Scholar
  21. Houck LD, Reagan NL (1990) Male courtship pheromones increase female receptivity in a plethodontid salamander. Anim Behav 39:729–734CrossRefGoogle Scholar
  22. Houck LD, Arnold SJ, Thisted RA (1985) A statistical study of mate choice: sexual selection in a plethodontid salamander (Desmognathus ochrophaeus). Evolution 39(2):370–386CrossRefGoogle Scholar
  23. Houck LD, Palmer CA, Watts RA, Arnold SJ, Feldhoff PW, Feldhoff RC (2007a) A new vertebrate courtship pheromone that affects female receptivity in a terrestrial salamander. Anim Behav 73:315–320CrossRefGoogle Scholar
  24. Houck LD, Watts RA, Mead LM, Palmer CA, Arnold SJ, Feldhoff PW, Feldhoff RC (2007b) A candidate vertebrate pheromone, SPF, increases receptivity in female salamanders. In: Hurst J, Beynon R, Muller-Schwarze D (eds) Chemical signals in vertebrates XI. Springer, New YorkGoogle Scholar
  25. Houck LD, Watts RA, Arnold SJ, Bowen KE, Kiemnec KM, Godwin HA, Feldhoff PW, Feldhoff RC (2008) A recombinant courtship pheromone affects sexual receptivity in a plethodontid salamander. Chem Senses 33(7):623–631CrossRefPubMedGoogle Scholar
  26. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17(8):754–755CrossRefPubMedGoogle Scholar
  27. Iwasa Y, Pomiankowski A (1994) The evolution of mate preferences for multiple sexual ornaments. Evolution 48:853–867CrossRefGoogle Scholar
  28. Johansson BG, Jones TM (2007) The role of chemical communication in mate choice. Biol Rev 82:265–289CrossRefPubMedGoogle Scholar
  29. Karlson P, Luscher M (1959) Pheromones: a new term for a class of biologically active substances. Nature 183:55–56CrossRefPubMedGoogle Scholar
  30. Kiemnec-Tyburczy K, Watts R, Gregg R, Von Borstel D, Arnold SJ (2009) Evolutionary shifts in courtship pheromone composition revealed by EST analysis of plethodontid salamander mental glands. Gene 432:75–81CrossRefPubMedGoogle Scholar
  31. Kikuyama S, Toyoda F, Iwata T, Takahashi N, Yamamoto K, Hayashi H, Miura S, Tanaka S (1999) Female-attracting peptide pheromone in newt cloacal glands. In: Johnston RE, Muller-Schwarze D, Sorensen PW (eds) Advances in chemical signals in vertebrates. Plenum Press, New York, pp 127–136Google Scholar
  32. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163CrossRefPubMedGoogle Scholar
  33. Lande R (1981) Models of speciation by sexual selection on polygenic traits. Proc Natl Acad Sci USA 78:3721–3725CrossRefPubMedGoogle Scholar
  34. Larson A, Highton R (1978) Geographical protein variation and divergence in the salamanders of the Plethodon welleri group (Amphibia, Plethodontidae). Syst Zool 27:431–448CrossRefGoogle Scholar
  35. Löfstedt C, Herrebout WM, Menken SBJ (1991) Sex pheromones and their potential role in the evolution of reproductive isolation in small ermine moths (Yponomeutidae). Chemoecology 2:20–28CrossRefGoogle Scholar
  36. Macey JR (2005) Plethodontid salamander mitochondrial genomics: a parsimony evaluation of character conflict and implications for historical biogeography. Cladistics 21(2):194–202CrossRefGoogle Scholar
  37. Marvin GA, Hutchison VH (1996) Courtship behavior of the Cumberland Plateau woodland salamander, Plethodon kentucki (Amphibia: Plethodontidae), with a review of courtship in the genus Plethodon. Ethology 102:285–303CrossRefGoogle Scholar
  38. Mason RT, Fales HM, Jones TH, Pannell LK, Chinn JW, Crews D (1989) Sex pheromones in snakes. Science 245:290–293CrossRefPubMedGoogle Scholar
  39. Mathis A, Jaeger RG, Keen WH, Ducey PK, Walls SC, Buchanan BW (1995) Aggression and territoriality by salamanders and a comparison with the territorial behavior of frogs. In: Heatwole H, Sullivan BK (eds) Amphibian biology, vol 2. Social Behaviour Surry Beatty & Sons, Chipping Norton, New South Wales, Australia, pp 634–636Google Scholar
  40. Maxson LR, Highton R, Wake DB (1979) Albumin evolution and its phylogenetic implications in the plethodontid salamander genera Plethodon and Ensatina. Copeia 1979(3):502–508CrossRefGoogle Scholar
  41. Maxwell MR, Barry KL, Johns PM (2010) Examinations of female pheromone use in two praying mantids Stagmomantis limbata and Tenodera aridifolia sinensis (Mantodea: Mantidae). Ann Entomol Soc Am 103(1):120–127CrossRefGoogle Scholar
  42. Mead LS, Arnold SJ (2004) Quantitative genetic models of sexual selection. Trends Ecol Evol 19(5):264–271CrossRefPubMedGoogle Scholar
  43. Min MS, Yang SY, Bonett RM, Vieites DR, Brandon RA, Wake DB (2005) Discovery of the first Asian plethodontid salamander. Nature 435:87–90CrossRefPubMedGoogle Scholar
  44. Nei M, Rooney AP (2005) Concerted and birth-and-death evolution of multigene families. Ann Rev Genet 39:121–152CrossRefPubMedGoogle Scholar
  45. Nei M, Gu X, Sitnikova T (1997) Evolution by the birth-and-death process in multigene families of the vertebrate immune system. Proc Natl Acad Sci USA 94:7799–7806CrossRefPubMedGoogle Scholar
  46. Nielsen R, Yang Z (1998) Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics 148:929–936PubMedGoogle Scholar
  47. Noble GK (1929) The relation of courtship to the secondary sexual characters of the two-lined salamander, Eurycea bislineata (Green). American Museum Novitates, Number 362, American Museum of Natural History, New YorkGoogle Scholar
  48. Organ JA (1960) Studies on the life history of the salamander Plethodon welleri. Copeia 1960(4):287–297CrossRefGoogle Scholar
  49. Palmer CA, Watts RA, Gregg RG, McCall MA, Houck LD, Highton R, Arnold SJ (2005) Lineage-specific differences in evolutionary mode in a salamander courtship pheromone. Mol Biol Evol 22:2243–2256CrossRefPubMedGoogle Scholar
  50. Palmer CA, Watts RA, Houck LD, Picard AL, Arnold SJ (2007a) Evolutionary replacement of components in a salamander pheromone signaling complex: more evidence for phenotypic-molecular decoupling. Evolution 61(1):202–215CrossRefPubMedGoogle Scholar
  51. Palmer CA, Hollis DM, Watts RA, Houck LD, McCall MA, Gregg RG, Feldhoff PW, Feldhoff RC, Arnold SJ (2007b) Plethodontid modulating factor (PMF), a hypervariable salamander courtship pheromone in the three-finger protein superfamily. FEBS J 274(9):2300–2310CrossRefPubMedGoogle Scholar
  52. Park D, Propper CR (2001) Repellent function of male pheromones in the red-spotted newt. J Exp Zool 289:404–408CrossRefPubMedGoogle Scholar
  53. Paterson HEH (1985) The recognition species concept of species. In: Vrba ES (ed) Species and speciation (Transvaal Museum Monograph No 4) pretoria: Transvaal Museum, pp 21–29Google Scholar
  54. Phelan PL (1997) Evolution of mate-signaling in moths: phylogenetic considerations and predictions from the asymmetric tracking hypothesis. In: Choe JC, Crespi BJ (eds) The evolution of mating systems in insects and arachnids. Cambridge University Press, Cambridge, UK, pp 240–256CrossRefGoogle Scholar
  55. Picard AL (2005) Courtship in the zig–zag salamander (Plethodon dorsalis): insights into a transition in pheromone delivery behavior. Ethology 111:799–809CrossRefGoogle Scholar
  56. Pomiankowski A, Iwasa Y (1993) Evolution of multiple sexual preferences by Fisher’s runaway process of sexual selection. Proc R Soc Lond B 253:173–181CrossRefGoogle Scholar
  57. Rice WR (1998) Intergenomic conflict, interlocus antagonistic coevolution, and the evolution of reproductive isolation. In: Howard DJ, Berlocher SH (eds) Endless forms: species and speciation. Oxford University Press, New York, pp 261–270Google Scholar
  58. Roelofs WL, Rooney AP (2003) Molecular genetics and evolution of pheromone biosynthesis in Lepidoptera. Proc Natl Acad Sci USA 100:9179–9184CrossRefPubMedGoogle Scholar
  59. Rollmann SM, Houck LD, Feldhoff RC (1999) Proteinaceous pheromone affecting female receptivity in a terrestrial salamander. Science 285:1907–1909CrossRefPubMedGoogle Scholar
  60. Shirangi TR, Dufour HD, Williams TM, Carroll SB (2009) Rapid evolution of sex pheromone-producing enzyme expression in Drosophila. PLoS Biol 7(8):e1000168. doi: 10.1371/journal.pbio.1000168 CrossRefPubMedGoogle Scholar
  61. Smith RJF (1992) Alarm signals in fish. Rev Fish Biol Fish 2:33–64CrossRefGoogle Scholar
  62. Sorensen PW, Stacey NE, Chamberlain KJ (1989) Differing behavioral and endocrinological effects of two female sex pheromones on male goldfish. Horm Behav 23:317–332CrossRefPubMedGoogle Scholar
  63. Stebbins RC (1949) Courtship of the plethodontid salamander Ensatina eschscholtzii. Copeia 1949:247–281Google Scholar
  64. Stryer L (1995) Biochemistry, 3rd edn. edn. W. H. Freeman and Company, New York, p 23Google Scholar
  65. Swofford DL (2003) PAUP*: phylogenetic analysis using parsimony (* and other methods), Version 4.0b 10 Sinauer Associates, Sunderland, MAGoogle Scholar
  66. Tsetlin V (1999) Snake venom alpha-neurotoxins and other ‘three-finger’ proteins. Eur J Biochem 264:281–286CrossRefPubMedGoogle Scholar
  67. Vitazka ME, Cardenas H, Cruz Y, Fadem BH, Norfold JR, Harder JD (2009) Progesterone receptor in the forebrain of female gray short-tailed opossums: effects of exposure to male stimuli. Horm Behav 55:190–196CrossRefPubMedGoogle Scholar
  68. Wabnitz PA, Bowie JH, Tyler MJ, Wallace JC, Smith BP (1999) Animal behaviour: aquatic sex pheromone of a male tree frog. Nature 401:444–445CrossRefPubMedGoogle Scholar
  69. Watts RA, Palmer CA, Feldhoff RC, Feldhoff PW, Houck LD, Jones AG, Pfrender ME, Rollmann SM, Arnold SJ (2004) Stabilizing selection on behavior and morphology masks positive selection on the signal in a salamander pheromone signaling complex. Mol Biol Evol 21(5):1032–1041CrossRefPubMedGoogle Scholar
  70. Weisrock DW, Kozak KH, Larson A (2005) Phylogeographic analysis of mitochondrial gene flow and introgression in the salamander, Plethodon shermani. Mol Ecol 14:1457–1472CrossRefPubMedGoogle Scholar
  71. 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
  72. Wirsig-Wiechmann CR, Houck LD, Feldhoff PW, Feldhoff RC (2002) Pheromonal activation of vomeronasal neurons in plethodontid salamanders. Brain Res 952:335–344CrossRefPubMedGoogle Scholar
  73. Wirsig-Wiechmann CR, Houck LD, Wood JM, Feldhoff PW, Feldhoff RC (2006) Male pheromone protein components activate female vomeronasal neurons in the salamander Plethodon shermani. BMC Neurosci 7:26CrossRefPubMedGoogle Scholar
  74. Wong WS, Yang Z, Goldman N, Nielsen R (2004) Accuracy and power of statistical methods for detecting adaptive evolution in protein coding sequences and for identifying positively selected sites. Genetics 168:1041–1051CrossRefPubMedGoogle Scholar
  75. Yang Z (1997) PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci 13:555–556PubMedGoogle Scholar
  76. Yang Z (1998) Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution. Mol Biol Evol 15(5):568–573PubMedGoogle Scholar
  77. Yang Z (2007) PAML 4: a program package for phylogenetic analysis by maximum likelihood. Mol Biol Evol 24:1586–1591CrossRefPubMedGoogle Scholar
  78. Yang Z, Bielawski JP (2000) Statistical methods for detecting molecular adaptation. Trends Ecol Evol 15:496–503CrossRefPubMedGoogle Scholar
  79. Yang Z, Wong WS, Nielsen R (2005) Bayes empirical Bayes inference of amino acid sites under positive selection. Mol Biol Evol 22:1107–1118CrossRefPubMedGoogle Scholar
  80. Zhang P, Wake DB (2009) Higher level salamander relationships and divergence dates inferred from complete mitochondrial genomes. Mol Biol Evol 53:492–508Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Catherine A. Palmer
    • 1
  • Richard A. Watts
    • 2
    Email author
  • Amy P. Hastings
    • 3
  • Lynne D. Houck
    • 4
  • Stevan J. Arnold
    • 4
  1. 1.Department of BiologyPortland State UniversityPortlandUSA
  2. 2.CSIRO Plant IndustryCanberraAustralia
  3. 3.Department of Ecology and Evolutionary BiologyCornell UniversityIthacaUSA
  4. 4.Department of ZoologyOregon State UniversityCorvallisUSA

Personalised recommendations