OJGen  Vol.5 No.4 , December 2015
Disjunct Populations of a Locally Common North American Orchid Exhibit High Genetic Variation and Restricted Gene Flow
Abstract: Whether the persistence of natural plant populations is limited by genetic diversity, gene flow, or other ecological and evolutionary factors is an important question in plant population genetics. An assessment of the distribution of genetic variation within and among populations is thus useful for understanding broad-scale gene flow patterns in plants with diverse pollination syndromes. We studied Pogonia ophioglossoides (L.) Ker Gawl., which is self-compatible but a primarily outcrossing species in the Tribe Pogonieae in the family Orchidaceae. Using three self-developed, highly polymorphic nuclear microsatellite (simple sequence repeat, or SSR) markers and two chloroplast microsatellites, we assessed genetic variation in eight populations representing its natural distribution. Relatively high within-population genetic variation (mean An = 9.08, Ho = 0.44, and He = 0.71) was detected in P. ophioglossoides. Eleven different alleles and 13 unique haplotypes were detected for two cpDNA microsatellites. Genetic differentiation based on the hierarchical AMOVA showed that 21% (ФPT = 0.21, P = 0.000) and 63% (ФPT = 0.63, P = 0.000) of the nuclear and cpDNA microsatellite allelic diversity, respectively, was distributed among populations. Pairwise FST values ranged from 0.041 to 0.224 and each was statistically significant at P ≤ 0.05. The isolation by distance estimate did not show an association between genetic differentiation and geographic distance indicating that populations were diverging independently. We documented fine-scale spatial genetic structure (FSGS) up to 40 m distance in Texas. Overall, gene flow across the sampled populations of P. ophioglossoides appears restricted, and the short-distance SGS suggests localized seed dispersal in this locally common North American terrestrial orchid.
Cite this paper: Pandey, M. and Sharma, J. (2015) Disjunct Populations of a Locally Common North American Orchid Exhibit High Genetic Variation and Restricted Gene Flow. Open Journal of Genetics, 5, 159-175. doi: 10.4236/ojgen.2015.54012.

[1]   Dressler, R.L. (1993) Phylogeny and Classification of the Orchid Family. Dioscorides Press, Portland.

[2]   Cozzolino, S., Cafasso, D., Pellegrino, G., Musacchio, A. and Widmer, A. (2003) Molecular Evolution of a Plastid Tandem Repeat Locus in an Orchid Lineage. Journal of Molecular Evolution, 57, S41-S49.

[3]   Tremblay, R.L., Ackerman, J.D., Zimmerman, J.K. and Calvo, R.N. (2005) Variation in Sexual Reproduction in Orchids and Its Evolutionary Consequences: A Spasmodic Journey to Diversification. Biological Journal of the Linnean Society, 84, 1-54.

[4]   Heschel, M.S. and Paige, K.N. (1995) Inbreeding Depression, Environmental Stress, and Population Size Variation in Scarlet Gilia (Ipomopsisaggregata). Conservation Biology, 9, 126-133.

[5]   Frankham, R., Ballou, J.D. and Briscoe, D.A. (2002) Introduction to Conservation Genetics. Cambridge University Press, Cambridge.

[6]   Aguilar, R., Quesada, M., Ashworth, L., Herrerias-Diego, Y. and Lobo, J. (2008) Genetic Consequences of Habitat Fragmentation in Plant Populations: Susceptible Signals in Plant Traits and Methodological Approaches. Molecular Ecology, 17, 5177-5188.

[7]   Cole, C.T. (2003) Genetic Variation in Rare and Common Plants. Annual Review of Ecological Systematics, 34, 213-237.

[8]   Gitzendanner, M.A. and Soltis, P.S. (2000) Patterns of Genetic Variation in Rare and Widespread Plant Congeners. American Journal of Botany, 87, 783-792.

[9]   Honnay, O. and Jacquemyn, H. (2007) Susceptibility of Common and Rare Plant Species to the Genetic Consequences of Habitat Fragmentation. Biological Conservation, 21, 823-831.

[10]   Sokal, R.R. and Oden, N.L. (1978) Spatial Autocorrelation in Biology 1. Methodology. Biological Journal of Linnean Society, 10, 199-228.

[11]   Slatkin, M. and Arter, H.E. (1991) Spatial Autocorrelation Methods in Population Genetics. The American Naturalist, 138, 499-517.

[12]   Young, A., Boyle, T. and Brown, T. (1996) The Population Genetic Consequences of Habitat Fragmentation for Plants. Trends in Ecology and Evolution, 11, 413-418.

[13]   Vekemans, X. and Hardy, O.J. (2004) New Insights from Fine-Scale Spatial Genetic Structure Analyses in Plant Populations. Molecular Ecology, 13, 921-934.

[14]   Chung, M.Y., Chung, G.M., Chung, M.G. and Epperson, B. (1998) Spatial Genetic Structure in Populations of Cymbidium goeringii (Orchidaceae). Genes Genetics Systematics, 73, 281-285.

[15]   Chung, M.Y., Nason, J.D. and Chung, M.G. (2004) Spatial Genetic Structure in Populations of the Terrestrial Orchid Cephalantheralongibracteata (Orchidaceae). American Journal of Botany, 91, 52-57.

[16]   Chung, M.Y., Nason, J.D. and Chung, M.G. (2005) Spatial Genetic Structure in Populations of the Terrestrial Orchid Orchis cyclochila (Orchidaceae). Plant Systematics and Evolution, 254, 209-219.

[17]   Jacquemyn, H., Brys, R., Vandepitte, K., Honnay, O. and Roldan-Ruiz, I. (2006) Fine-Scale Genetic Structure of Life History Stages in the Food-Deceptive Orchid Orchis purpurea. Molecular Ecology, 15, 2801-2808.

[18]   Matsui, K., Ushimaru, A. and Fujita, N. (2001) Pollinator Limitation in a Deceptive Orchid, Pogonia japonica, on a Floating Peat Mat. Plant Species Biology, 16, 231-235.

[19]   Chung, M.Y. and Chung, M.G. (2008) Conservation Genetics of the Endangered Terrestrial Orchid Pogonia minor in South Korea. Annales Botanici Fennici, 45, 455-464.

[20]   Case, M.A., Mlodozeniec, H.T., Wallace, L.E. and Weldy, T.W. (1998) Conservation Genetics and Taxonomic Status of the Rare Kentucky Lady’s Slipper: Cypripedium kentuckiense (Orchidaceae). American Journal of Botany, 85, 1779-1786.

[21]   Forrest, A.D., Hollingsworth, M.L., Hollingsworth, P.M., Sydes, C. and Bateman, R.M. (2004) Population Genetic Structure in European Populations of Spiranthes romanzoffiana Set in the Context of Other Genetic Studies on Orchids. Heredity, 92, 218-227.

[22]   Chung, J.M., Park, K.W., Park, C.-S., Lee, S.-H., Chung, M.G. and Chung, M.Y. (2009) Contrasting Levels of Genetic Diversity between the Historically Rare Orchid Cypripedium japonicum and the Historically Common Cypripedium macranthos in South Korea. Botanical Journal of Linnean Society, 160, 119-129.

[23]   George, S., Sharma, J. and Yadon, V.L. (2009) Genetic Diversity of the Endangered and Narrow Endemic Piperia yadonii (Orchidaceae) Assessed with ISSR Polymorphisms. American Journal of Botany, 96, 2022-2030.

[24]   Swarts, N.D., Sinclair, E.A., Krauss, S.L. and Dixon, K.W. (2009) Genetic Diversity in Fragmented Populations of the Critically Endangered Spider Orchid Caladenia huegelii: Implications for Conservation. Conservation Genetics, 10, 1199-1208.

[25]   Light, M.L. and Sharma, J. (2010) Comparison of in Situ Germination of Pogonia ophioglossoides in the Northern and Southern Parts of Its Range. Native Orchid Conference Journal, 7, 12-24.

[26]   Thien, L.B. and Marcks, B.G. (1972) The Floral Biology of Arethusa bulbosa, Calopogon tuberosus, and Pogonia ophioglossoides (Orchidaceae). Canadian Journal of Botany, 50, 2319-2325.

[27]   Argue, C.L. (2012) Tribe Arethuseae (Calopogon R. Brown and Arethusa L.) and Subfamily Vanilloideae (Part One) (Pogonia Jussieu). Chapter 8. In: Argue, C.L., Ed., The Pollination Biology of North American Orchids: Volume 2, Springer, New York, 147-164.

[28]   NatureServe (2015) NatureServe Explorer: An Online Encyclopaedia of Life (Web Application). Version 7.1, NatureServe, Arlington, Virginia.

[29]   Pandey, M. and Sharma, J. (2012) Efficiency of Microsatellite Isolation from Orchids via Next Generation Sequencing. Open Journal of Genetics, 2, 167-172.

[30]   Pandey, M. and Sharma, J. (2013) Characterization of Microsatellite Loci for Cypripedium kentuckiense (Orchidaceae). Conservation Genetics, 5, 1031-1033.

[31]   Fay, M.F., Bone, R., Cook, P., et al. (2009) Genetic Diversity in Cypripedium calceolus (Orchidaceae) with a Focus on North-Western Europe, as Revealed by Plastid DNA Length Polymorphisms. Annals of Botany, 104, 517-525.

[32]   Micheneau, C., Duffy, K.J., Smith, R.J., Stevens, L.J., Stout, J.C., Civeyrel, L., Cowan, R.S. and Fay, M.F. (2010) Plastid Microsatellites for the Study of Genetic Variability in the Widespread Cephalanthera longifolia, C. damasonium and C. rubra (Neottieae, Orchidaceae), and Cross Amplification in Other Cephalanthera Species. Botanical Journal of Linnean Society, 163, 181-193.

[33]   Taberlet, P., Gielly, L., Pautou, G. and Bouvet, J. (1991) Universal Primers for Amplification of Three Non-Coding Regions of Chloroplast DNA. Plant Molecular Biology, 17, 1105-1109.

[34]   Small, R.L., Ryburn, J.A., Cronn, R.C., Seelanan, T. and Wendel, J.F. (1998) The Tortoise and the Hare: Choosing between Noncoding Plastome and Nuclear Adh Sequences for Phylogeny Reconstruction in a Recently Diverged Plant Group. American Journal of Botany, 85, 1301-1315.

[35]   Oxelman, B., Liden, M. and Berglund, D. (1997) Chloroplast rps16 Intron Phylogeny of the Tribe Sileneae (Caryophyllaceae). Plant Systematics and Evolution, 206, 393-410.

[36]   Micheneau, C. (2002) Relations phylogénétiques des Neottieae (Orchidaceae) etétude de la variabilitégénétique de Cephalanthera longifolia à l’aide de marqueursmoléculaires (séquençage et microsatellites chloroplastiques). DEA mémoire, Muséum National d’Histoire Naturelle, Paris.

[37]   Shaw, J., Lickey, E.B., Schilling, E.E. and Small, R.L. (2007) Comparisons of Whole Chloroplast Genome Sequences to Choose Noncoding Regions for Phylogenetic Studies in Angiosperms: The Tortoise and the Hare III. American Journal of Botany, 94, 275-288.

[38]   Peakall, R. and Beattie, A.J. (1996) Ecological and Genetic Consequences of Pollination by Sexual Deception in the Orchid Caladenia tentaculata. Evolution, 50, 2207-2220.

[39]   Cornuet, J. and Luikart, G. (1996) Description and Power Analysis of Two Tests for Detecting Recent Population Bottlenecks from Allele Frequency Data. Genetics, 144, 2001-2005.

[40]   Beerli, P. and Felsenstein, J. (1999) Maximum Likelihood Estimation of Migration Rates and Population Numbers of Two Populations Using a Coalescent Approach. Genetics, 152, 763-773.

[41]   Nei, M. (1972) Genetic Distance between Populations. The American Naturalist, 106, 283-292.

[42]   Wright, S. (1965) The Interpretation of Population Structure by F-Statistics with Special Regard to Systems of Mating. Evolution, 19, 395-420.

[43]   Excoffier, L., Smouse, P.E. and Quattro, J.M. (1992) Analysis of Molecular Variance Inferred from Metric Distances among DNA Haplotypes: Application to Human Mitochondrial DNA Restriction Sites. Genetics, 131, 479-491.

[44]   Peakall, R. and Smouse, P.E. (2006) GENALEX 6: Genetic Analysis in Excel. Population Genetic Software for Teaching and Research. Molecular Ecology Notes, 6, 288-295.

[45]   Felsenstein, J. (1995) Phylip (Phylogeny Inference Package). Department of Genetics, University of Washington, Seattle.

[46]   Page, R.D.M. (1996) TREEVIEW: An Application to Display Phylogenetic Trees on Personal Computers. Computer Application in Bioscience, 12, 357-358.

[47]   Goudet, J. (1999) PCAGEN. Principal Component Analysis of Gene Frequency Data (Version 1.2).

[48]   Pritchard, J.K., Stephens, M. and Donnelly, P. (2000) Inference of Population Structure Using Multilocus Genotype Data. Genetics, 155, 945-959.

[49]   Evanno, G., Regnaut, S. and Goudet, J. (2005) Detecting the Number of Clusters of Individuals Using the Software Structure: A Simulation Study. Molecular Ecology, 14, 2611-2620.

[50]   Earl, D.A. and von Holdt, B.M. (2012) STRUCTURE HARVESTER: A Website and Program for Visualizing STRUCTURE Output and Implementing the Evanno Method. Conservation Genetics Resources, 4, 359-361.

[51]   Smouse, P.E. and Peakall, R. (1999) Spatial Autocorrelation Analysis of Individual Multiallele and Multilocus Genetic Structure. Heredity, 82, 561-573.

[52]   Gustafson, S. and Thorén, P.A. (2001) Microsatellite Loci in Gymnadenia conopsea, the Fragrant Orchid. Molecular Ecology Notes, 1, 81-82.

[53]   Boonsrangsom, T., Pongtongkam, P., Masuthon, S. and Peyachoknagul, S. (2008) Development of Microsatellite Markers for Dendrobium Orchids. Thai Journal of Genetics, 1, 47-56.

[54]   Pinheiro, F., Santos, M.O., Barros, F., et al. (2008) Isolation and Characterization of Microsatellite Loci in the Brazilian Orchid Epidendrum fulgens. Conservation Genetics, 9, 1661-1663.

[55]   Phuekvilai, P., Pradit, P. and Surin, P. (2009) Development of Microsatellite Markers for Vanda Orchid. Kasetsart Journal of Natural Science, 43, 497-506.

[56]   Hopkins, S.E. and Taylor, D.L. (2011) Microsatellite Loci Development in Mycoheterotrophic Corallorhiza maculata (Orchidaceae) with Amplification in C. mertensiana. American Journal of Botany, 98, e253-e255.

[57]   Cozzolino, S., Cafasso, D., Pellegrino, G., Musacchio, A. and Widmer, A. (2003) Fine-Scale Phylogeographical Analysis of Mediterranean Anacamptis palustris (Orchidaceae) Populations Based on Chloroplast Minisatellite and Microsatellite Variation. Molecular Ecology, 12, 2783-2792.

[58]   Soliva, M. and Widmer, A. (2003) Gene Flow across Species Boundaries in Sympatric, Sexually Deceptive Ophrys (Orchidaceae) Species. Evolution, 57, 2252-2261.

[59]   Campbell, V.V., Rowe, G., Beebee, T.J.C. and Hutchings, M.J. (2007) Genetic Differentiation amongst Fragrant Orchids (Gymnadenia conopseas L.) in the British Isles. Botanical Journal of Linnaeus Society, 155, 349-360.

[60]   Ross, A.A., Aldrich-Wolfe, A., Lance, S., Glenn, T. and Travers, S.E. (2013) Microsatellite Markers in the Western Prairie Fringed Orchid, Platanthera praeclara (Orchidaceae). Application in Plant Sciences, 1, Article ID: 1200413.

[61]   Ebert, D., Hayes, C. and Peakall, R. (2009) Chloroplast Simple Sequence Repeat Markers for Evolutionary Studies in the Sexually Deceptive Orchid Genus Chiloglottis. Molecular Ecology Resources, 9, 673-690.

[62]   Stone, J.L., Crystal, P.A., Devlin, E.E., Le, B., Downer, R.H. and Cameron, D.S. (2012) Highest Genetic Diversity at the Northern Range Limit of the Rare Orchid Isotria medeoloides. Heredity, 109, 215-221.

[63]   Phillips, R., Dixon, K. and Peakall, R. (2012) Low Population Genetic Differentiation in the Orchidaceae: Implications for the Diversification of the Family. Molecular Ecology, 21, 5208-5220.

[64]   Duffy, K.J., Scopece, G., Cozzolino, S., Fay, M.F., Smith, R.J. and Stout, J.C. (2009) Ecology and Genetic Diversity of the Dense-Flowered Orchid, Neotinea maculata, at the Centre and Edge of Its Range. Annals of Botany, 104, 507-516.

[65]   Vitt, P. and Campbell, C.S. (1997) Reproductive Biology of Isotria medeoloides (Orchidaceae). Rhodora, 99, 56-63.

[66]   Murren, C.J. and Ellison, A.M. (1998) Seed Dispersal Characteristics of Brassavola nodosa (Orchidaceae). American Journal of Botany, 85, 675-680.

[67]   Jacquemyn, H., Brys, R., Vandepitte, K., Honnay, O., Roldán-Ruiz, I. and Wiegand, T. (2007) A Spatially-Explicit Analysis of Seedling Recruitment in the Terrestrial Orchid Orchis purpurea. New Phytologist, 176, 448-459.

[68]   Hamrick, J.L. and Nason, J.D. (1996) Consequences of Dispersal in Plants. In: Rhodes Jr., O.E., Chesser, R.K. and Smith, M.H., Eds., Population Dynamics in Ecological Space and Time, University of Chicago Press, Chicago, 203-236.

[69]   Kalisz, S., Nason, J.D., Hanzawa, F.A. and Tonsor, S.J. (2001) Spatial Genetic Structure in Trillium grandiflorum: The Roles of Dispersal, Mating, History and Selection. Evolution, 55, 1560-1568.

[70]   Lande, R. (1988) Genetics and Demography in Biological Conservation. Science, 241, 1455-1460.