Abstract
Proteinaceous pheromones that diversify through gene duplication can result in shifts in courtship cocktails that may serve as a mechanism for reproductive isolation. The molecular evolution of pheromones has been extensively studied in salamanders, but how these genes and associated novel courtship glands have codiversified has not been evaluated. In this study we used transcriptional analyses to examine the relationship between pheromone diversification and gland type in three divergent lineages of plethodontid salamanders. Our results revealed that plethodontid salamanders express up to eight divergent Sodefrin Precursor-like Factor genes (spf, representing both alpha and beta subfamilies) along with Plethodontid Modulating Factor (pmf) and Plethodontid Receptivity Factor (prf). Expression of pheromone genes is tissue specific with pmf, prf, and some spf genes restricted to the mental gland. In contrast, the caudal gland shows strong expression of the other spf genes. We found evidence for punctuated changes in pheromone cocktail composition related to the loss of metamorphosis, and subsequent extreme reduction of the mental gland, in a paedomorphic lineage. Our study provides insight into how pheromone diversification can be partitioned into unique glands, which may lead to cocktail specificity in behavioral modules during courtship.
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Data Availability
Gene transcripts are available on GenBank. Histological slides, specimens, and tissue samples are available from RMB.
References
Abramoff MG, Magalhaes PJ, Ram S (2004) Image processing with ImageJ. Biophotonics Intern 11:36–42
Allison JD, Cardé RT (2016) Pheromones: reproductive isolation and evolution in moths. In: Allison JD, Cardé RT (eds) Pheromone communication in moths. University of California Press, Berkeley, pp 11–23
Anderson JS (2012) Fossils, molecules, divergence times, and the origin of Salamandroidea. PNAS 109:5557–5558. https://doi.org/10.1073/pnas.1202491109
Aran RP, Steffen MA, Martin SD, Lopez OI, Bonett RM (2014) Reduced effects of thyroid hormone on gene expression and metamorphosis in a paedomorphic plethodontid salamander. J Exp Zool Part B 322:294–303. https://doi.org/10.1002/jez.b.22580
Arnold SJ (1977) The evolution of courtship behavior in new world salamanders with some comments on old world salamanders. In: Taylor DH, Guttman SI (eds) The reproductive biology of amphibians. Plenum Press, New York, pp 141–183
Arnold SJ, Kiemnec-Tyburczy KM, Houck LD (2017) The evolution of courtship behavior in plethodontid salamanders, contrasting patterns of stasis and diversification. Herpetologica 73:190–205. https://doi.org/10.1655/Herpetologica-D-16-00068.1
Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de Castro E, Duvaud S, Flegel V, Fortier A, Gasteiger E, Grosdidier A, Hernandez C, Ioannidis V, Kuznetsov D, Liechti R, Moretti S, Mostaguir K, Redaschi N, Rossier G, Xenarios I, Stockinger H (2012) ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 40:W597–W603. https://doi.org/10.1093/nar/gks400
Bonett RM, Blair AL (2017) Evidence for complex life cycle constraints on salamander body form diversification. PNAS 114:9936–9941. https://doi.org/10.1073/pnas.1703877114
Bossuyt F, Maex M, Treer D, Schulte LM, Van Bocxlaer I, Janssenswillen S (2019) Chemistry between salamanders: evolution of the SPF courtship pheromone system in Salamandridae. In: Buesching CD (ed) Chemical signals in vertebrates 14. Springer, Cham, pp 205–220. https://doi.org/10.1007/978-3-030-17616-7_15
Bouckaert R, Heled J, Kühnert D, Vaughan T, Wu CH, Xie D, Suchard MA, Rambaut A, Drummond AJ (2014) BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Comput Biol 10:e1003537. https://doi.org/10.1371/journal.pcbi.1003537
Bridgham JT, Carroll SM, Thornton JW (2006) Evolution of hormone-receptor complexity by molecular exploitation. Science 312:97–101. https://doi.org/10.1126/science.1123348
Clay TA, Steffen MA, Treglia ML, Torres DT, Trujano-Alvarez AL, Bonett RM (2019) Multiple stressors produce differential transcriptomic patterns in a stream-dwelling salamander. BMC Genom 20:482. https://doi.org/10.1186/s12864-019-5814-y
Denoël M, Poncin P, Ruwet JC (2001) Sexual compatibility between two heterochronic morphs in the Alpine newt, Triturus alpestris. Anim Behav 62:559–566. https://doi.org/10.1006/anbe.2001.1793
Doty KA, Wilburn DB, Bowen KE, Feldhoff PW, Feldhoff RC (2016) Co-option and evolution of non-olfactory proteinaceous pheromones in a terrestrial lungless salamander. J Proteom 135:101–111. https://doi.org/10.1016/j.jprot.2015.09.019
Eick GN, Thornton KW (2011) Evolution of steroid receptors from an estrogen-sensitive ancestral receptor. Mol Cell Endocrinol 334:31–38. https://doi.org/10.1016/j.mce.2010.09.003
Emel SL, Bonett RM (2011) Considering alternative life history modes and genetic divergence in conservation: a case study of the Oklahoma salamander. Conserv Genet 12:1243–1259
Fischer AH, Jacobson KA, Rose J, Zeller R (2008) Hematoxylin and eosin staining of tissue and cell sections. CSH Protoc 2008:pdb.prot4986
Freilich S, Massingham T, Blanc E, Goldovsky L, Thornton JM (2006) Relating tissue specialization to the differentiation of expression of singleton and duplicate mouse proteins. Genom Biol 7:R89. https://doi.org/10.1186/gb-2006-7-10-r89
Gao KQ, Shubin NH (2012) Late Jurassic salamandroid from western Liaoning, China. PNAS 109:5767–57772. https://doi.org/10.1073/pnas.1009828109
Gu Z, Rifkin S, White K, Li WH (2004) Duplicate genes increase gene expression diversity within and between species. Nat Genet 36:577–579. https://doi.org/10.1038/ng1355
Henikoff S, Henikoff JG (1992) Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci 89:10915–10919. https://doi.org/10.1073/pnas.89.22.10915
Houck LD (2009) Pheromone communication in amphibians and reptiles. Annu Rev Physiol 71:161–176
Houck LD, Arnold SJ (2003) Courtship and mating. In: Sever DM (ed) Phylogeny and reproductive biology of Urodela (Amphibia). Science Publishers, Enfield, pp 383–424
Houck LD, Palmer CA, Watts RA, Arnold SJ, Feldhoff PW, Feldhoff RC (2007) A new vertebrate courtship pheromone, PMF, that affects female receptivity in a terrestrial salamander. Anim Behav 73:315–320. https://doi.org/10.1016/j.anbehav.2006.07.008
Houck LD, Watts RA, Mead LM, Palmer CA, Arnold SJ, Feldhoff PW, Feldhoff RC (2008) A candidate vertebrate pheromone, SPF, increases female receptivity in a salamander. In: Hurst JL, Beynon RJ, Roberts SC, Wyatt TD (eds) Chemical signals in vertebrates 11. Springer, New York, pp 213–221
Hughes AL (1994) The evolution of functionally novel proteins after gene duplication. Proc R Soc B 256:119–124. https://doi.org/10.1098/rspb.1994.0058
Huminiecki L, Wolfe KH (2004) Divergence of spatial gene expression profiles following species-specific gene duplications in human and mouse. Genom Res 14:1870–1879. https://doi.org/10.1101/gr.2705204
Janssenswillen S, Vandebergh W, Treer D, Willaert B, Maex M, Van Bocxlaer I, Bossuyt F (2015a) Origin and diversification of a salamander sex pheromone system. Mol Biol Evol 32:472–480. https://doi.org/10.1093/molbev/msu316
Janssenswillen S, Willaert B, Treer D, Vandebergh W, Bossuyt F, Van Bocxlaer I (2015b) High pheromone diversity in the male cheek gland of the red-spotted newt Notophthalmus viridescens (Salamandridae). BMC Evol Biol. https://doi.org/10.1186/s12862-015-0333-1
Kiemnec-Tyburczy KM, Watts RA, Gregg RG, Borstal V, Arnold SJ (2009) Evolutionary shifts in courtship pheromone composition revealed by EST analysis of plethodontid salamander mental glands. Gene 432:75–81. https://doi.org/10.1016/j.gene.2008.11.007
Kikuyama S, Toyoda F, Ohmiya Y, Matsuda K, Tanaka S, Hayashi H (1995) Sodefrin: a female attracting peptide pheromone in newt cloacal glands. Science 267:1643–1645
Krenz JD, Verrell PA (2002) Integrity in the midst of sympatry: does sexual incompatibility facilitate the coexistence of metamorphic and paedomorphic mole salamanders (Ambystoma talpoideum)? J Zool 258:435–440. https://doi.org/10.1017/S0952836902001589
Li WH, Yang J, Gu X (2005) Expression divergence between duplicate genes. Trends Genet 21:602–607. https://doi.org/10.1016/j.tig.2005.08.006
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550. https://doi.org/10.1186/s13059-014-0550-8
Maddison WP, Maddison DR (2018) Mesquite: a modular system for evolutionary analysis. Available at http://mesquiteproject.org
Maex M, Van Bocxlaer I, Mortier A, Proost P, Bossuyt F (2016) Courtship pheromone use in a model urodele, the Mexican Axolotl (Ambystoma mexicanum). Sci Rep 6:20184. https://doi.org/10.1038/srep20184
Makova KD, Li WH (2003) Divergence in the spatial pattern of gene expression between human duplicate genes. Genom Res 13:1638–1645. https://doi.org/10.1101/gr.1133803
Nei M, Maruyama T, Wu CI (1983) Models of evolution of reproductive isolation. Genetics 103:557–579
Oromi N, Michaux J, Denoël M (2016) High gene flow between alternative morphs and the evolutionary persistence of facultative paedomorphosis. Sci Rep 6:32046. https://doi.org/10.1038/srep32046
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 22:2243–2256. https://doi.org/10.1093/molbev/msi219
Palmer CA, Hollis DM, Watts RA, Houck LD, McCall MA, Gregg RG, Feldhoff PW, Feldhoff RC, Arnold SJ (2007a) Plethodontid modulating factor, a hypervariable salamander courtship pheromone in the three-finger protein superfamily. FEBS J 274:2300–2310. https://doi.org/10.1111/j.1742-4658.2007.05766.x
Palmer CA, Watts RA, Houck LD, Picard AL, Arnold SJ (2007b) Evolutionary replacement of components in a salamander pheromone signaling complex: more evidence for phenotypic-molecular decoupling. Evolution 61:202–215. https://doi.org/10.1111/j.1558-5646.2007.00017.x
Palmer CA, Picard AL, Watts RA, Houck LD, Arnold SJ (2010) Rapid evolution of plethodontid modulating factor (PMF), a hypervariable salamander courtship pheromone, is driven by positive selection. J Mol Evol 70:427–440
Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Meth 8:785–786
Phillips JG, Fenolio DB, Emel SL, Bonett RM (2017) Hydrologic and geologic history of the Ozark Plateau drive phylogenomic patterns in a cave-obligate salamander. J Biogeogr 44:2463–2474.. https://doi.org/10.1111/jbi.13047
Prince V, Pickett F (2002) Splitting pairs: the diverging fates of duplicated genes. Nat Rev Genet 3:827–837. https://doi.org/10.1038/nrg928
Proulx SR (2012) Multiple routes to subfunctionalization and gene duplicate specialization. Genetics 190:737–751. https://doi.org/10.1534/genetics.111.135590
Rambaut A, Drummond AJ (2007) Tracer v1.5. Available at http://beast.bio.ed.ac.uk/Tracer
Rollmann SM, Houck LD, Feldhoff RC (1999) Proteinaceous pheromone affecting female receptivity in a terrestrial salamander. Science 285:1907–1909
Rupp AE, Sever DM (2018) Histology of mental and caudal courtship glands in three genera of plethodontid salamanders (Amphibia: Plethodontidae). Acta Zool 99:20–31. https://doi.org/10.1111/azo.12188
Sever D (2003) Courtship and mating glands. Reproductive biology and phylogeny of Urodela (Amphibia). Science Publishers Inc., Enfield, pp 323–381. https://doi.org/10.1111/azo.12188
Sever DM, Siegel DS, Taylor MS, Beachy CK (2016) Phylogeny of mental glands, revisited. Copeia 104:83–93. https://doi.org/10.1643/CH-14-210
Shen XX, Liang D, Chen MY, Mao RL, Wake DB, Zhang P (2016) Enlarged multilocus data set provides surprisingly younger time of origin for the Plethodontidae, the largest family of salamanders. Syst Biol 65:66–81. https://doi.org/10.1093/sysbio/syv061
Siegel DS, Long CL, Waltz JT, Wren SA, Pereira KE, McClelland SJ, Murray CM, Sever DM (2020) Sexually dimorphic heads of Eurycea bislineata. Copeia 108:578–592. https://doi.org/10.1643/CH2020014
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. https://doi.org/10.1038/msb.2011.75
Swofford DL (2003) PAUP*. Phylogenetic analysis using parsimony (*and other Methods). Sinauer Associates, Sunderland
Symonds MRE, Elgar MA (2008) The evolution of pheromone diversity. Trends Ecol Evol 23:220–228. https://doi.org/10.1016/j.tree.2007.11.009
Thornton JW (2001) Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions. Proc Natl Acad Sci 98:5671–5676. https://doi.org/10.1073/pnas.091553298
Treer D, Maex M, Van Bocxlaer I, Proost P, Bossuyt F (2018) Divergence of species-specific protein sex pheromone blends in two related, nonhybridizing newts (Salamandridae). Mol Ecol 27:508–519. https://doi.org/10.1111/mec.14398
Van Bocxlaer I, Treer D, Maex M, Vandebergh W, Janssenswillen S, Stegen G, Kok P, Willaert B, Matthijs S, Martens E, Mortier A, de Greve H, Proost P, Bossuyt F (2015) Side-by-side secretion of Late Palaeozoic diverged courtship pheromones in an aquatic salamander. Proc R Soc B 282:20142960. https://doi.org/10.1098/rspb.2014.2960
Watts RA, Palmer CA, Feldhoff RC, Feldhoff PW, Houck LD, Jones AG, Pfrender ME, Arnold SJ (2004) Stabilizing selection on behavior and morphology masks positive selection on the signal in a salamander pheromone signaling complex. Mol Biol Evol 21:1032–1041. https://doi.org/10.1093/molbev/msh093
Whiteman HH, Krenz JD, Semlitsch RD (2006) Intermorph breeding and the potential for reproductive isolation in polymorphic mole salamanders (Ambystoma talpoideum). Behav Ecol Sociobiol 60:52–61
Wicker-Thomas C (2011) Evolution of insect pheromones and their role in reproductive isolation and speciation. Ann Soc Entomol Fr 47:55–62. https://doi.org/10.1080/00379271.2011.10697696
Wilburn DB, Feldhoff RC (2019) An annual cycle of gene regulation in the red-legged salamander mental gland: from hypertrophy to expression of rapidly evolving pheromones. BMC Dev Biol. https://doi.org/10.1186/s12861-019-0190-z
Wilburn DB, Swanson WJ (2016) From molecules to mating: rapid evolution and biochemical studies of reproductive proteins. J Proteom 135:12–15. https://doi.org/10.1016/j.jprot.2015.06.007
Wilburn DB, Bowen KE, Gregg RG, Cai J, Feldhoff PW, Houck LD, Feldhoff RC (2012) Proteomic and UTR analyses of a rapidly evolving hypervariable family of vertebrate pheromones. Evolution 66:2227–2239. https://doi.org/10.1111/j.1558-5646.2011.01572.x
Wilburn DB, Bowen KE, Doty KA, Arumugam S, Lane AN, Feldhoff PW, Feldhoff RC (2014) Structural insights into the evolution of a sexy protein: novel topology and restricted backbone flexibility in a hypervariable pheromone from the red-legged salamander, Plethodon Shermani. PLoS ONE 9:e96975. https://doi.org/10.1371/journal.pone.0096975
Wilburn DB, Arnold SJ, Houck LD, Feldhoff PW, Feldhoff RC (2017) Gene duplication, co-option, structural evolution, and phenotypic tango in the courtship pheromones of plethodontid salamanders. Herpetologica 73:206–219. https://doi.org/10.1655/Herpetologica-D-16-00082.1
Woodley SK (2010) Pheromonal communication in amphibians. J Comp Physiol A 196:713–727. https://doi.org/10.1007/s00359-010-0540-6
Woodley SK, Staub NL (2021) Pheromonal communication in Urodelan amphibians. Cell Tissue Res 383:327–345. https://doi.org/10.1007/s00441-020-03408-1
Zhang J (2003) Evolution by gene duplication: an update. Trends Ecol Evol 18:292–298. https://doi.org/10.1016/S0169-5347(03)00033-8
Acknowledgements
We thank N. Ledbetter and B. Molone for assisting with field work and salamander collection. We would also like to thank A. Trujano, who helped with RNA extractions and cDNA synthesis and C. Burleson who annotated gene transcripts. Finally, we thank M. Howery of the Oklahoma Department of Wildlife Conservation and K. Irwin of the Arkansas Game and Fish Commission for permitting logistics.
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This study was in part funded by the National Science Foundation (DEB1050322 and DEB1840987 to RMB; DEB1210859 to RMB and MAS) as well as by the University of Tulsa through the Student Research Grant program.
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MAH, MAS, and RMB designed the study and performed analyses. All authors participated in collecting data and preparing the manuscript.
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Specimens were handled in accordance with Institutional Animal Care and Use Committee (IACUC) protocols at the University of Tulsa (TU-0028 and TU-0029).
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Herrboldt, M.A., Steffen, M.A., McGouran, C.N. et al. Pheromone Gene Diversification and the Evolution of Courtship Glands in Plethodontid Salamanders. J Mol Evol 89, 576–587 (2021). https://doi.org/10.1007/s00239-021-10026-0
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DOI: https://doi.org/10.1007/s00239-021-10026-0