BIOLOGICAL INVESTIGATIONS IN THE GENUS PLATANTHERA (ORCHIDACEAE): CONSERVATION ISSUES IN PLATANTHERA LEUCOPHAEA AND EVOLUTIONARY DIVERSIFICATION IN SECTION LIMNORCHIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Lisa Ellen Wallace, MA. * * * * * The Ohio State University 2002 Dissertation Committee: Approved by Dr. Daniel Crawford, Co-adviser _______________________ Co-adviser Dr. Andrea Wolfe, Co-adviser Dr. Kent Holsinger _______________________ Co-adviser Department of Evolution, Ecology, and Organismal Biology i ABSTRACT Orchidaceae boasts incredible floral and habit diversity, and many species are quite rare due to natural causes or human activities. In this dissertation, the diversity and rarity of orchids are investigated in studies on species in the genus Platanthera, a primarily temperate group with centers of diversity in North America and eastern Asia. Population genetic structure and the potential for inbreeding depression are examined in Platanthera leucophaea, a threatened species found in wet prairie fragments in the Midwestern US. Populations harbor little allozyme variability (AP = 1.18; PP = 12%; HO = 0.008), but higher levels of diversity were found at RAPD loci (PP = 45%; HNei = 0.159). Both data sets suggested populations are quite differentiated (allozyme FST = 0.75; RAPD ΦST = 0.21), which may indicate little interpopulational gene flow and the potential for inbreeding within populations. In a subsequent study, the potential for inbreeding depression was determined in populations of differing size and genetic structure. The results of these studies indicated that inbreeding depression could be strong as a result of geitonogamous pollination. However, because this species has mechanisms to promote outcrossing, it is expected that biparental inbreeding would more likely lead to inbreeding depression, especially in small populations. In the latter set of studies, taxonomic relationships within Platanthera section Limnorchis are examined, with special emphasis on the evolutionary origin and placement of the polyploid species Platanthera huronensis within the section. ii Taxonomic classification in Limnorchis is difficult due to intraspecific morphological variability and the purported appearance of interspecific hybrids. Molecular markers correlate with morphological markers and suggest the presence of at least two primary lineages corresponding to green or white flower color. Subtle changes in floral morphology are hypothesized to be important mechanisms of speciation as is divergence of isolated populations allopatrically separated from other conspecific populations. In this analysis, it was also found that P. huronensis is most closely related to both P. dilatata and P. aquilonis, the progenitors suggested by morphological characteristics. Parental genotypes are not strictly additive in the polyploid, but there is evidence of bidirectional evolution of nuclear rDNA towards each of the parental species, with most individuals showing greater similarity to P. dilatata. Molecular data from ISSR and RAPD data also support an allopolyploid origin of P. huronensis from P. aquilonis and P. dilatata, but again, the patterns observed in the polyploid were not strictly additive of parental genotypes. Extant polyploid lineages are thought to have diverged substantially since the initial formation of polyploid lineages. A survey of chloroplast RFLP patterns indicated that P. aquilonis was the maternal parent for eastern samples of P. huronensis, whereas, P. dilatata was the most likely maternal parent of western samples of P. huronensis. The strong geographic differences found in RFLP patterns were mirrored by banding patterns at ISSR and RAPD loci as well as morphological differences. Eastern populations of P. huronensis may represent an evolutionarily unique entity worthy of iii taxonomic recognition, but further studies are needed to better understand the relationship between eastern and western populations of P. huronensis because similar levels of divergence were observed in P. aquilonis and P. dilatata. Although polyploid species often show increased or novel diversity relative to their diploid progenitors, this result was not found in P. huronensis. Instead, populations of P. huronensis are similar to populations of P. dilatata in the level of diversity, and populations of both of these species are on average significantly more diverse than populations of P. aquilonis. However, at the species level, P. aquilonis is more diverse than either of the other two species. It is hypothesized that differences in breeding system are largely responsible for these patterns. Platanthera aquilonis is capable of self-fertilization while P. dilatata and P. huronensis are thought to be primarily outcrossing. Thus, gene flow within and between populations of P. aquilonis is expected to be limited, which would result in the patterns observed in this study, highly differentiated populations and relatively low levels of intrapopulation variation. iv Dedicated to my family v ACKNOWLEDGMENTS I would like to thank my co-advisers, Dan Crawford and Andrea Wolfe, and committee member, Kent Holsinger for giving me the freedom to develop my own research program and offering guidance and assistance when I needed it. I thank Mark Wallace for many entertaining road trips, navigating, help in locating and collecting specimens, and artistic contributions to this dissertation. I thank Shannon Datwyler and TJ Jones for providing help in conducting field studies, and John Freudenstein for collecting plant material from Alaska. Additionally, Jennifer Windus, Phyllis Higman, Michael Penskar, Kyle Stockwell, and personnel at the Ohio Division of Wildlife aided in locating and collecting samples of Platanthera leucophaea. I am grateful to Charles Sheviak for sharing his knowledge of orchids and location information with me in the early stages of this project. I also wish to thank past and present members of the plant systematics and ecology labs at Ohio State, especially Theresa Culley, Shannon Datwyler, Mingjuan Huang, Andrew Lutz, Chris Randle, and Sarena Selbo for thoughtful discussions. Additionally, the expertise of Shannon Datwyler, Kay Havens, and Nancy Cowden provided a great aid to the development of laboratory protocols. I thank Amber, Cleo, Shannon Datwyler, Sarena Selbo, Chris Randle, Jeff Morawetz, Shawn Krosnick, Mark Mort, Sibyl Bucheli, and TJ Jones for friendship, humor, and encouragement during my time at Ohio State. vi This research was supported by grants from the American Orchid Society, The Ohio Chapter of The Nature Conservancy, Ohio Department of Natural Resources, and The Beatley Herbarium Award. vii VITA May 16, 1972 ………………………….Born- Nassawadox, Virginia 1994 …………………………………...B.S. Biology, College of William and Mary 1997 ……...............................................M.A. Biology, College of William and Mary 1997-2001 ………………………….... Graduate Teaching Associate, The Ohio State University 2002-present ………………………..... Presidential Fellow, The Ohio State University PUBLICATIONS 1. Wallace, L. E. 2002. Examining the effects of fragmentation on genetic variation in Platanthera leucophaea (Orchidaceae): inferences from allozyme and random amplified polymorphic DNA markers. Plant Species Biology 17: 37-49. 2. Culley, T. M., L. E. Wallace, K. M. Gengler-Nowak, and D. J. Crawford. 2002. A comparison of two methods of calculating GST, a genetic measure of population differentiation. American Journal of Botany 89: 460-465. 3. Wallace, L.E. and M.A. Case. 2000. Contrasting allozyme diversity between northern and southern populations of Cypripedium parviflorum (Orchidaceae): implications for Pleistocene refugia and taxonomic boundaries. Systematic Botany 25: 281-296. 4. Case, M.A., H.T. Mlodozeniec, L.E. Wallace, and T.W. Weldy. 1998. Conservation genetics and taxonomic status of the rare Kentucky Lady’s Slipper: Cypripedium kentuckiense (Orchidaceae). American Journal of Botany 85:1779-1786. 5. Weldy, T.W., H.T. Mlodozeniec, L.E. Wallace, and M.A. Case. 1996. The current status of Cypripedium kentuckiense (Orchidaceae), including a morphological analysis of a newly discovered population in Eastern Virginia. Sida 17: 423-435. viii FIELDS OF STUDY Major Field: Evolution, Ecology, and Organismal Biology ix TABLE OF CONTENTS Page Dedication ……………………………………………………………….……………... v Acknowledgments ……………………………………………………..……………….. vi Vita …………………………………………………………………….……………... viii List of Tables ………………………………………………………….………………. xiii List of Figures ……………………………………………………….……………….. xviii Chapters: 1. Introduction ……………………………………………………………………… 1 2. Examining the effects of fragmentation on genetic variation in Platanthera leucophaea (Orchidaceae): inferences from allozyme and random amplified polymorphic DNA markers ……………………………………………………… 9 2.1 Introduction ……………………………………………………………… 9 2.2 Materials and Methods …………………………………………………. 12 2.2.1 Allozyme analysis ……………………………………………… 12 2.2.2 Allozyme data analysis ....……………………………………… 13 2.2.3 RAPD analysis …………………………………………………. 14 2.2.4 RAPD data analysis ……………………………………………. 15 2.3 Results ………………………………………………………………….. 17 2.3.1 Variation at allozyme loci ……………………………………… 17 2.3.2 Variation at RAPD loci ………………………………………… 18 2.4 Discussion …………………………………………………………....… 20 2.4.1 Allozyme variation within and among populations ………….… 20 2.4.2 RAPD variation within and among populations ……………….. 23 2.4.3 Conclusions