Nies EXCESS AND LIMITATION OF SOIL

EXCESS AND LIMITATION OF SOIL RESOURCES IN ILLINOIS COMMUNITIES OF CYPRIPEDIUM CANDIDUM (ORCHIDACEAE), THE UNEXPECTED RELATIONSHIP BETWEEN SOIL CHARACTER, MYCORRHIZAL COLONIZATION AND PHOTOSYNTHESIS

A THESIS SUBMITTED TO THE FACULTY OF THE PROGRAM IN PLANT BIOLOGY AND CONSERVATION

BY ANNE NIES

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PLANT BIOLOGY AND CONSERVATION FROM NORTHWESTERN UNIVERSITY AND THE CHICAGO BOTANIC GARDEN

MAY 2, 2014

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SIGNATURE PAGE

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ABSTRACT The environmental and plant physiological correlates of plant growth and reproductive effort in the locally threatened orchid, Cypripedium candidum Muhl. ex Willd were examined in the context of a conceptual model of demographic and reproductive trade-offs, focusing on three Illinois populations. This study addresses the current status and long-term trends of the populations, investigated the soil characteristics and fungal colonization of root tissues, and measured the physiological characteristics of each population through carbon uptake and allocation. The tests of the conceptual model revealed the following: I.

Increased soil fertility did not increase reproductive effort nor did it decrease mycorrhizal root colonization.

II. Leaf tissue analyses for δ15N were inconclusive, with no significant difference in δ15N found across sites. Mycorrhizal colonization was negatively correlated with ramets per clump and reproductive effort. III. Water use efficiency corresponded negatively with mycorrhizal colonization suggesting that fungi are facilitating up-regulated photosynthesis, via increased water availability. IV. For the plants at Sites A and B carbon gained through up-regulated photosynthesis appears to be transferred to the colonizing fungi. This is supported by the evidence of little or no root fungal colonization at Site C, which had lower photosynthetic rate and larger clump size. Differences in fungi and in their functionality was likely driven by the very high N content of the soils. V. Primary carbon gain for all plants was through photosynthesis, evidenced by δ13C values, which did not vary across site. Increased photosynthetic rate did not result in increased ramet production, nor in increased reproductive effort. At all sites as the number of flowers in a clump increased so did the carbon assimilation rate of that clump. The major driving factor in the differences between populations is likely the fungal commuNies

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ABSTRACT nity and its response the variation in soil N. C. candidum does not carry over a reproductive debt from year to year, yet declines in size were found at two of the three sites. Thus, it is concluded that mycorrhizal colonizations observed in the roots of these plants do not have the expected beneficial effect on fitness. This study has further confirmed the conclusions of Whigham and Willems (2003), habitat conservation alone is not sufficient for the conservation of our native orchid species.

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ACKNOWLEDGEMENTS Without the assistance and support of many individuals, this project would not have been possible. Although I have not had the pleasure of meeting everyone whose work and time has contributed to this project, I would like to include them with those whose direct support was received. First, I must express my most sincere gratefulness for the guidance and encouragement of my thesis advisor, Dr. Pati Vitt. Without her patience and belief in me, I would not have achieved as much as I did. She lent me books, use of the Licor 6400 XT and most valuable, her time and feedback. I have truly been fortunate to have an advisor that has pushed me so far. I could not have survived the soils portion of this project without the help of my committee member, Dr. Louise Egerton-Warburton. Not only did she allow me and my interns to use her lab, but she helped to guide me through the complex and fascinating world of soil science. My final committee member, Dr. Hormoz BassiriRad, helped me with use of the Licor 6400 XT and gave me great advice and insight into using and translating the data from the Licor. In setting up this project, the input of Dr. Richard Shefferson and Susanne Masi was extremely helpful. Dr. Shefferson’s feedback on doing MF work helped me to focus my approach to understanding Cypripedium candidum’s interactions with MF in a way that made completing this project on time possible. After talking to Dr. Gregg Muller, I had a conversation with Susanne Masi that set the ground work for this study. Her passion for the work done by the Plants of Concern volunteers inspired me to use their data. With the help of Rachel Goad and her team I was able to analyze the Plants of Concern data early in this project. Shortly after I let her know I would be working on C. candidum they digitized all of the data, for all of the years, that had been collected. Additionally, the years of monitoring that Plants of Concern volunteers have done has been invaluable to this project. Over the summer of 2013, the diligence of my interns ensured that the project stayed on schedule. Not only did Geralle Powell, my REU intern, work without complaint, but she was Nies

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ACKNOWLEDGEMENTS thorough, doing her work so well that I was able to focus on other parts of the project. She put in long hours at the lab. Often she was there before I arrived and after I left. She did an excellent job of supervising our College First Intern, Isobel Araujo. Before Geralle started, Rayna Benzeev interned on this project, helping me complete field work. It was a real pleasure to go out with her and she did most of the rather tedious work of collecting leaf area measurements. The Site managers and land stewards were also very helpful in this endeavour. I was allowed to make extra visits to collect my base level photosynthesis readings, at Site C. Jeff Wepprecht was endlessly patient with me and scheduling site visits. I never would have found the plants at Site A without the help of Joyce Proper, the steward. Joyce also drove me from the parking area to the populations. She even rescued me once in inclement weather. Also, Ken Klick, the land manager was kind enough to take me on a tour of the site early in the year and discuss the populations and site with me. During my second set of site visits, I was helped by my husband and classmates, Lynnaun Johnson and Jessamine Finch. Having company in the field always makes the time pass faster, both literally and metaphorically. Special thanks to my mother, who at the very last moment helped with grammar checking this entire document. She’s always been there to correct my English for me, especially when I need it the most. Finally I must also thank my husband for his encouragement and motivation I could not have finished this project without. Not only did he suffer the domestic consequences of my constant focus on this project, but he did so while making sure that I had everything I needed to stay focused.

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TABLE OF CONTENTS Title Page.........................................................................................................................................1 Signature Page.................................................................................................................................2 Abstract..........................................................................................................................................3-4 Acknowledgements........................................................................................................................5-6 Table of Contents..........................................................................................................................7-8 List of Equations...............................................................................................................................9 List of Figures............................................................................................................................10-11 List of Tables...................................................................................................................................12 Chapter 1: Introduction..............................................................................................................13-35 References.................................................................................................................29-35 Chapter 2: The current status of Cypripedium candidum in Illinois: Analysis of 13 years of volunteer collected data.........................................................................................................36-66 Introduction...............................................................................................................37-42 Materials and Methods..............................................................................................44-47 Results.......................................................................................................................50-51 Discussion.................................................................................................................58-60 References.................................................................................................................61-66 Chapter 3: Soil characteristics of Cypripedium candidum populations, with differing reproductive effort, in three Illinois prairies...........................................................................................67-99 Introduction...............................................................................................................68-73 Materials and Methods..............................................................................................75-78 Results.......................................................................................................................80-82 Discussion.................................................................................................................91-93 References..................................................................................................................95-99 Chapter 4: Characteristics of leaf photosynthesis in three populations of Cypripedium candidum Nies

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TABLE OF CONTENTS and their implications for fecundity..............................................................................100-127 Introduction............................................................................................................101-105 Materials and Methods..........................................................................................106-109 Results....................................................................................................................112-114 Discussion..............................................................................................................121-124 References..............................................................................................................125-127 Chapter 5: Conclusions..........................................................................................................128-136 References..............................................................................................................135-136 Appendix A: List of Variables.......................................................................................................137 Appendix B: Summary of Site Details..................................................................................138-139 Appendix C: Leaf Nitrogen Data..........................................................................................140-141 Appendix D: Suggestions for Plants of Concern...................................................................142-144

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LIST OF EQUATIONS Equation 2-1: Density of Ramets in a Population..........................................................................45 Equation 2-2: Average Number of Ramets per Clump...................................................................46 Equation 2-3: Average Number of Flowers per Clump..................................................................46 Equation 3-1: Soil Moisture...........................................................................................................75 Equation 3-2: C:N Ratio.................................................................................................................76 Equation 3-3: N:P Ratio..................................................................................................................76 Equation 3-4: δ13C........................................................................................................................77 Equation 3-5: δ15N........................................................................................................................77 Equation 3-6: Carbon Isotope Discrimination (Δ).........................................................................78 Equation 4-1: Leaf Area of a Clump.............................................................................................106 Equation 4-2: Area of a Leaf........................................................................................................106 Equation 4-3: Number of Leaves in a Clump from Flowering Ramets.......................................108 Equation 4-4: Number of Leaves in a Clump from non-Flowering Ramets................................108 Equation 4-5: Mean Photosynthetic Rate for Flowering Ramets.................................................108 Equation 4-6: Mean Photosynthetic Rate for non-Flowering Ramets..........................................108 Equation 4-7: Photosynthetic Carbon Assimilation Rate.............................................................108

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LIST OF FIGURES Figure 1.1: Cypripedium candidum Flower....................................................................................25 Figure 1.2: Cypripedium candidum Botanical Illustration.............................................................26 Figure 1.3: Maps of Confirmed Populations (c. 1975, c. 1981).....................................................27 Figure 1.4: Conceptual Model........................................................................................................28 Figure 2.1: Sites with Level 2 POC Monitoring of Cypripedium candidum.................................43 Figure 2.2: Tagged Clump of Cypripedium candidum...................................................................48 Figure 2.3: Map of Cypripedium candidum clumps at Site B.......................................................49 Figure 2.4: Number of Ramets per m2 by Year..............................................................................52 Figure 2.5: Sites with Level 2 POC Monitoring of Cypripedium candidum.................................53 Figure 2.6: Average Number of Flowers per Clump by Year.........................................................54 Figure 2.7: Average Number of Ramets per Clump by Site for 2013............................................55 Figure 2.8: Average Number of Flowers per Clump by Site for 2013...........................................56 Figure 2.9: Percent of Clumps Flowering After a Burn.................................................................57 Figure 3.1: Hypothetical Cypripedium candidum Niche................................................................74 Figure 3.2: Mycorrhizal Fungal Colonization of a Cypripedium candidum Root Cell.................77 Figure 3.3: Maps of Near and Far Sample Points for Each Site....................................................79 Figure 3.4: pH by Site....................................................................................................................83 Figure 3.5: Potassium Concentration by Site and Distance...........................................................84 Figure 3.6: Percent Carbon by Site and Distance...........................................................................85 Figure 3.7: Percent Nitrogen by Site and Distance.........................................................................86 Figure 3.8: C:N Ratio by Site........................................................................................................87 Figure 3.9: Nitrogen Concentration by Site...................................................................................88 Figure 3.10: N:P Ratio by Site.......................................................................................................89 Figure 3.11: Mycorrhizal Colonization by Site and Season...........................................................90 Figure 3.12: Revised Hypothetical Cypripedium candidum Niche................................................94 Nies

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LIST OF FIGURES Figure 4.1: Estimated versus Actual Leaf Area............................................................................110 Figure 4.2: Photosynthetic Light Response Curve.......................................................................111 Figure 4.3: Estimated Area of a Leaf by Site...............................................................................115 Figure 4.4: Average Number of Leaves per Clump by Site.........................................................116 Figure 4.5: Photosynthetic Rate of Flowering and non-flowering Ramets by Site......................117 Figure 4.6: Water Use Efficiency by Ramet Status......................................................................118 Figure 4.7: Estimated Mean Photosynthetic Carbon Assimilation Rate per Clump by Site........119 Figure 4.8: Photosynthetic Carbon Assimilation Rate by Number of Flowers in a Clump.........120 Figure 5.1: Conceptual Model with Results.................................................................................134

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LIST OF TABLES Table 2.1: Site Information for the Study Sites..............................................................................42 Table 2.2: Result of Spatial Analysis of Populations at Each Site.................................................50 Table 2.3: Summary of Population Data by Site............................................................................51 Table 3.1: Results of 2-way MANOVAs for Soil Characteristics by Site and Distance................81 Table 4.1: Leaf Characteristics of Cypripedium candidum at Three Sites...................................112 Table 4.2: Results of 2-way ANOVAs for Gas Exchange and Leaf Characteristics....................113 Table 4.3: Gas Exchange Characteristics and Carbon Isotope Discrimination............................113

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CHAPTER 1

CHAPTER 1

INTRODUCTION

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CHAPTER 1 The Orchidaceae is one of the largest and most diverse families of plants and thus is an important contributor to global biodiversity (Dressler 1981). Orchids are exceptionally diverse because most are niche specialists, with narrowly specific relationships between their habitat, pollinators, and fungal partners (Arditti 1992; Waterman and Bidartondo 2008). These traits, together with a long life span (sometimes measured in decades), makes orchids excellent indicator species for monitoring ecosystem health (Carignan and Villard 2002). The utility of orchids as ecosystem indicators has been demonstrated previously in a number of studies. For example, in North American bogs, Laroche et al. (2012) demonstrated that the white-fringed orchid (Platanthera blephariglottis) was an effective indicator of sphagnum bog integrity. In order for an indicator species to be truly effective, it is necessary to identify the baseline conditions in which it exists and its responses to changes in those conditions so that it can be used to monitor ecosystems and to direct management and restoration activities. A vast literature exists on the ecology and propagation of terrestrial orchids, but there are only a small number of studies in which the environmental and physiological correlates of orchids have been used to define species’ requirements. In addition, most of these are for species found in southwestern Australia or Europe. To date, there are few studies of the environmental or habitat requirements of North American orchids. Thus, a primary challenge in North American orchid conservation is knowing which environmental factors should be considered. In North America, terrestrial orchids often occur within more temperate climates in which rainfall ranges from 50 to 90 cm per annum, rarely occurring in the semi-arid zones of the southwest. By entering seasonal dormancy, terrestrial orchids survive cold winters, while their active growing season takes place between spring and fall, varying by species. Like many other plants, the life cycle of the terrestrial orchid is closely linked to seasonal changes in soil moisture and temperature. Two other factors that also significantly influence orchid growth and persistence are soil fertility and mycorrhizal abundance. Nies

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CHAPTER 1 Based on previous studies, it can be anticipated that soil nutrient availability will be directly linked to orchid plant fitness and photosynthetic activity (Domingues et al. 2010; Willby et al. 2001). For example, soil nitrogen and phosphorus are often correlated with above ground biomass and flower production. Another important factor influencing the productivity of orchid plants is their mycorrhizal fungal associates. An extensive literature shows that mycorrhizal fungi are vital to the germination of orchid seeds and plant growth. These associations start during seed germination when the germinant is reliant on the fungus for carbon, this dependency sometimes persists into adulthood (Dearnaley 2007). The primary mycobionts of orchids are from the phylum Basidiomycota belonging to the genus Rhizoctonia. Mycorrhizal associates can be found in three clades of the polyphyletic Rhizoctonia group, which includes Sebacinaceae, Tulasnellaceae, and Ceratobasidiales. The specificity of this relationship varies between orchid species (Dearnaley 2007). North American terrestrial orchids may associate with a wide phylogenetic array of Rhizoctonia species, and hence, some show little specificity. Other green orchids, and all studied achlorophyllous orchids, associate with narrow phylogenetic groups of fungi, and show significant specificity. For example, Cephalanthera austinae and Corallorhiza trifida form mycorrhiza exclusively with fungi belonging to Thelephoraceae (Taylor & Bruns 1997; McKendrick et al. 2000a), Corallorhiza maculata and Corallorhiza mertensiana associate uniquely with fungi in the Russulaceae (Taylor & Bruns 1999), and Goodyera pubescens and Liparis lilifolia associate with fungi belonging to the single genus Tulasnella (McCormick et al.). By contrast, species such as Neottia nidus-avis in Europe (Selosse et al. 2002a) and Hexalectris spicata in North America (Taylor et al. 2003) specialize in fungi from the Sebacinaceae, which are known to be ectomycorrhizal in tree species (Selosse et al. 2002b). In several species, this tight specificity has been shown to apply from seed germination through adulthood under natural conditions (Arditti 1992; Martos et al. 2012; Shefferson et al. 2005). In other species, there are shifts in the mycorrhizal associates with plant ontogenetic Nies

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CHAPTER 1 change. Molecular phylogenetic studies show that many orchid taxa also associate with ectosmycorrhizal (EM) Basidiomycete fungi, such as the Russulaceae, and a number of orchids contain vesicles and/or arbuscles, indicating associations with arbuscular mycorrhizal (AM) fungi. (Bidartondo et al. 2004). The fact that most orchid plants display substantial variations in specificity from highly specialized to more generalist interactions (Molina et al. 1992) makes it important to determine the functional role(s) of their mycorrhizal associates. Orchid plants can show mycoheterotrophy and mixotrophy. In mycoheterotrophy, the orchid is completely dependent on the fungus (heterotrophic) for C and nutrients, some of which may be transferred from neighboring trees to the orchid (Dearnaley 2007; Rasmussen 2002). Achlorophyllous orchids and some photosynthetic forest orchids of the Neottieae tribe appear to acquire carbon from their mycorrhizal fungi as suggested by their stable isotopic composition (Gebauer & Meyer 2003; Bidartondo et al. 2004). The term ‘mixotrophy’ was proposed by Selosse et al. (2004) to indicate a dual strategy (photosynthetic and mycoheterotrophic), which is demonstrated in the ectomycorrhizal associations of the orchid genera Cephalanthera and Epipactis (Bidartondo et al. 2004; Selosse et al. 2004). This variation in interactions means that assumptions into the relationships between mycorrhizae and orchids should be made cautiously. Orchid mycorrhizal associates are most commonly associated with the acquisition of nitrogen and water for their host orchid. Acquisition of N can be measured through the accumulation of δ15N and total %N in leaf tissue. This is because the stable nitrogen isotope δ15N is retained within the fungal mycelia so that orchids obtaining N from fungal sources show significantly lower δ15N concentrations than orchids obtaining N directly from the soil alone (Dearnaley et al. 2012). Furthermore, it has been demonstrated through the measure of water use efficiency across the stomatal boundary that orchids with fungal colonization of their root tissue demonstrate significantly lower water use efficiency than orchids that do not have fungal colonization of their Nies

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CHAPTER 1 root tissue. The measurement of gas exchange across the stomatal boundary reveals the photosynthetic behavior of plants. In general, increased photosynthetic rates are positively correlated with increased above ground biomass production and probability of survival (Wendel and Antlfinger 1996). Also, in many cases, flowering structures (stems, branches, and flowers) demonstrate significantly higher photosynthetic rates than non-flowering structures. This is one way in which plants pay for the cost of reproduction. In this study, the environmental and plant physiological correlates of plant growth and reproductive effort in the locally threatened orchid, Cypripedium candidum Muhl. ex Willd are examined and the resulting data was used to test a conceptual model of these correlates. To do this, the study examined three major aspects of orchid ecology, focusing on three Illinois populations. First, this study addresses the current status and long-term trends of these populations. Second, the study investigates the soil characteristics and fungal colonization of root tissue within the populations. Third, the study looks at the physiological characteristics of each population through its photosynthetic behavior. About Cypripedium candidum Habitat Cypripedium candidum, commonly known as the white lady’s slipper orchid, typically grows in calcareous, mesic prairies and fens across the northeast and north-central United States and Ontario, Canada (Case 1989; Sheviak 1974; Swink and Wilhelm 1994). The habitat features that appear to be most important for C. candidum are: regular flooding leading to periods of high soil moisture; full sunlight, especially early in the year; and high limestone concentration in the soil (Case 1989; Sheviak 1974). The pH range is considered quite broad for a plant species. Across its native distribution, populations are found in soils where the pH varies from 7.0 to 8.2 (Curtis 1943; Bowles 1983). Because the lowest observed pH is 7.0, it is unlikely that any of the Nies

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CHAPTER 1 populations recorded as being in “bogs” can actually be considered to be in true bogs. Instead, it is more likely this definition represents a misidentification of the habitat, as bogs are very acidic with pH < 5 (Swink and Wilhelm 1994; Jeffords et al. 1995). Soil types recorded vary from fine sand to granular silt and clay loams, and soil moisture varies from mesic to wet-mesic which likely reflects the capacity of C. candidum to persist in seasonally or periodically inundated environments (Sheviak 1974). Morphology Like all orchids, C. candidum is a monocot (Arditti 1992). It grows in clonal clumps (genets) of stems (ramets), which are all descended from, and may still be attached to, the same rhizome. Typically there are many ramets, 7 to 30 cm tall, per genet, but few ramets flower. The inflorescences present a single (rarely two) flower that is characterized by a white slipper-shaped labellum with green, sometimes spirally twisted, sepals and petals (Figure 1.1). There is only one inflorescence per ramet and ramets are unbranched with leaves clasping the stem (Figure 1.2). There is a floral bract above each flower. In most cases, this bract is perpendicular to the basal leaves. Wide fluctuations in flowering between years are common (Curtis 1954). The leaves are hypostomatous with adaxial sticky hairs (personal observation). Roots are un-branching and grow laterally in a sinusoidal pattern up to 1.5 m from the plant and 1-5 cm deep (Stoutamire 1989; Case 1989). Life History Cypripedium candidum is a slow growing and long-lived species, with time from seed germination to first flowering ranging from 10 to 16 years (Curtis 1954; Curtis 1943). As is typical for many orchid species, mycorrhizal fungi are required for seed germination and protocorm development (Arditti 1992; detailed below). Each spring, new growth appears and each fall, the above ground growth dies back entirely, leaving a rhizome below ground to generate the next year’s growth. All ramets and leaves are produced early in the spring before flowering, so that Nies

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CHAPTER 1 after flowering there is no additional above ground growth (personal observation). Once plants are mature, reproduction can take place both vegetatively and sexually. Vegetative reproduction and clonal spread takes place through the rhizome where branching is common and where up to 10 sympoidal buds may form each year (Stoutamire 1989), each resulting in a potential new ramet. Sexual reproduction is through pollination by bees, and both Andrenid and Halictine bees have been observed visiting C. candidum flowers (Catling and Knerer 1980; Curtis 1954; Bowles 1983; Stoutamire 1989). Pollination success is generally very low. This is because C. candidum employs a deception strategy, using fragrance as a lure for the promise of nectar when there is none (Catling and Knerer 1980; Stoutamire 1967; Waterman and Bidartondo 2008). Structurally, self-pollination cannot occur (Brownell 1981; Catling and Knerer 1980). Flowers are present during the spring and are open for approximately two weeks. Pollinated flowers develop over the spring and summer and in late summer the fruits open and seeds disperse. Cypripedium candidum plants can be dormant for up to 6 years and subsequently appear above ground. The probability of transition to dormancy from flowering or from vegetative is about 30%, and decreases as the number of ramets produced in the previous year increases (Shefferson 2006; Shefferson and Simms 2007). It is not clear if C. candidum requires mycorrhizal associations to survive dormancy, although it has been hypothesized that it does; since dormancy can last years (Shefferson 2006; Stoutamire 1989). Mycorrhizal Fungal Relationships Cypripedium candidum has been included in a number of mycorrhizal studies and colonization is frequently found in adult plants. When present, fungal colonization in adult plants is found in mature (but not decaying) roots (Shefferson et al. 2005). Like other members of the genus Cypripedium (Dearnaley 2007), known fungal associates of adult C. candidum plants include members of the Tulasnellaceae, Thelephoraceae, and species of Phialophora (Shefferson et al. Nies

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CHAPTER 1 2005). Members of the Thelephoraceae are classed as the medium-distance smooth hyphal exploration types, because their external hyphae explore and exploit the soil matrix and may form hyphal networks (Agerer 2001). Tulasnellaceae belongs to the contact exploration type, while members of Phialophora may belong to either the contact exploration or short-distance hyphal exploration types. In both exploration types the external hyphae emanate only a small distance in the soil (Tedersoo and Smith 2013). These differences are important because the fungal hyphae of orchid mycorrhizas effectively operate as a de facto root system (Egerton-Warburton 2014). This differentiation in hyphal exploration also influences their functional capacity. A number of studies have shown that terrestrial orchids have the capacity to take up organic N directly from soils in the form of free amino acids (Nurfadilah et al. 2013), as well as to switch the N source uptake from NO3 to NH4 (Wu et al. 2013). Furthermore, it was recently found that the extraradical mycelium of fungi from the medium-distance exploration type might facilitate the uptake of inorganic and organic N from deeper soil (Wu et al. 2013). Genetic Identity It has been hypothesized that Cypripedium candidum is derived from C. calceolus because: they share 0.794 of their genetic identity, C. candidum readily hybridizes with C. calceolus, and C. candidum has lower genetic variation within populations than C. calceolus (Case 1994; Luer 1975). Threats to Cypripedium candidum populations Historically, Cypripedium candidum was much more prevalent than it is today. Loss of habitat due to wetland drainage, especially for agriculture, mining and development, has caused dramatic declines in the number and size of populations across North America (Bowles 1983; Curtis 1946, 1954; Sheviak 1974). Using historic county records, Bowles (1983) demonstrated that populations of C. candidum had declined 52%, across its range (Figure 1.3). This trend has been consistent over the past 70 years and has resulted in most of its habitat being destroyed. Nies

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CHAPTER 1 Today, C. candidum holds state-level protected status in nine of the seventeen states in which it occurs. Although it is protected at the state level, it is not protected at the federal level due to the broadness of its current range (USDA 2013). In addition to loss of habitat, Cypripedium candidum has faced reproductive issues due to destruction of ripening fruits by a small black weevil (Stoutamire 1989). Although there may be some pollinator limitation, C. candidum is also able to reproduce and spread vegetatively (Catling and Knerer 1980). Surprisingly, there is no record of poaching contributing to the decline of this species. This may be because the populations are generally difficult to access, and within its habitat C. candidum is difficult to identify unless in flower (personal observation). Furthermore, during the early 1900’s C. candidum was so plentiful that it may have been uninteresting to poachers, although there are records of it being introduced in the United Kingdom in 1826 (Correll 1950). Damage from deer has also been reported, but it appears that C. candidum is not a first food of choice for deer. Instead, deer may nest in a site without actually eating any of the plants (personal observation). Herbivory by other mammals and by insects is rare. The most prominent threat to populations is woody brush encroachment. Woody brush encroachment is the primary threat facing grasslands across the world, especially in the North American Great Plains (Briggs et al. 2005). This threat has two affects on the survival of C. candidum. The first is that shrubs create shade, under which C. candidum cannot survive (Case 1989; Sheviak 1974). The second is a change in community structure, most visible through decreased richness of the herbaceous plant community (Briggs et al. 2005). Cultivation Cypripedium candidum has proven difficult to propagate from seed leaving only very invasive and potentially destructive forms of propagation available (Hadley 1989). Growth of seeds in vitro has had very little success, with no published records of germinated seeds surviving long Nies

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CHAPTER 1 enough to become reproductive adults. Progress has been made with in vitro seed germination and growing protocorms, by using a modified Norstog (1973) medium (De Pauw et al. 1995). Yet, less than 50% of seeds germinate in culture, and 25% of protocorms die due to ineffective culture conditions (De Pauw and Remphrey 1993; De Pauw et al. 1995). Cultivation of C. candidum is most frequently met with failure. There has been some success with transplanting whole plants although there is often a high plant mortality rate after removal from their habitat (From 2007). Removal of plants from their native habitat has been successful when enough of the root system is undamaged. However, this means creating a considerable disturbance to the site from which they are removed (Stoutamire 1989). Conceptual Model and Hypotheses This study addresses knowledge gaps in our current understanding of the functional links between plant productivity, environmental factors, and plant physiology in the conservation of rare and endangered orchids. These factors are visualized in a conceptual model (Figure 1.1). Cypripedium candidum was used as a model species to test the interactions among plant productivity, soil fertility, mycorrhizal abundance in root tissue, and plant physiological capacity in three populations (see Conceptual Model). These data were then used to test the following five hypotheses: I.

Soil fertility exerts a strong positive effect on plant productivity and negative effects on mycorrhizal root colonization;

II. Mycorrhizal colonization of root tissue results in increased uptake of N, which will decrease the concentration of δ15N in leaf tissue, by plants and thus increase ramet production; III. Mycorrhizal colonization of root tissue will increase water uptake and photosynthetic rate, thereby decreasing water use efficiency; IV. Some, but not all, of the additional carbon gained by upregulated photosynthetic rate, Nies

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CHAPTER 1 due to mycorrhizal colonization, will be transferred to fungi; V. Primary carbon gain is through photosynthesis so δ13C should be constant across sites and increased carbon gain through increased photosynthetic rate will result in increased ramet and flower production. In order to test these hypotheses, C. candidum was studied at three sites considered high quality natural areas in Northeastern Illinois. Site A is located in Grant Woods Forest Preserve in Lake County, Illinois and is classified as a high quality wet prairie. The Grant Woods Forest Preserve is 1070 acres and, from the 1830’s to 1958, the site was farmed and grazed (Grant Woods 2014). This part of the state is characterized by glacial landforms including natural lakes and areas of poor drainage. Depressions, including a nearby 35-acre bog, are scattered throughout the tract (Heidorn 2009). The area around Site A contains many recreational lakes and residential neighborhoods. The land managers are very active at this site in restoring and maintaining the preserve and there are many forbs growing in the prairie, which is surrounded by a forest on three sides. According to the site steward, Joyce Proper (2012), the population of C. candidum at this site is small and scattered. She expressed concern over the long-term survival of the population. The broader area enclosing the population is comprised of a mosaic of different soil types. The most prevalent among them are: Houghton muck, 0 to 2 percent slopes (~ 25%); Peotone silty clay loam, 0 to 2 percent slopes (~ 25%); and Grays silt loam, 0 to 2 percent slopes (~10%) (NRCS 2014). Site B is located in Meissner Prairie-Corron Forest Preserve, Kane County, Illinois and is classified as a fen (Kane Co. 2014). The site was evaluated in the 1970’s Natural Areas Inventory, but was privately held property. In 2002, the Forest Preserve District of Kane County purchased the 229 acre site, 19 of which were wetland and 13 of which were woodland (Kane Co. 2002). It is directly downhill from a horse farm and is surrounded by agricultural fields. This site has not been burned in many years and the population is surrounded by tall woody brush. The Nies

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CHAPTER 1 plant community is typically dominated by C4 grasses and prairie dock (Silphium terebinthinaceum). The broader area enclosing the population is categorized as Drummer silty clay loam, 0 to 2 percent slopes (NRCS 2014). Site C is located in the Grant Creek Prairie Nature Preserve in Will County, Illinois and is classified as a wet prairie (Illinois D.N.R. 2014). It was established in September 1978 and was once grazed by cattle (Illinois D.N.R. 2014). Site C has multiple populations, but only the largest population was used for this study. Although it is only 78 acres, it borders Midewin National Tallgrass Prairie which is comprised of 19,000 acres. It is down hill from an oil refinery and a large rail yard, east of a major inter-state highway, and it is just north of the Kankakee River. This Site is centered in a large prairie with no nearby shrubs or trees. The broader area enclosing the population is categorized as Joliet silt loam, 0 to 2 percent slopes (NRCS 2014). The following three chapters each present one of the three major aspects of orchid ecology addressed in this study. In these three chapters the background knowledge used to build the hypotheses tested, the materials and methods used, the results of the analysis done, and a discussion interpreting the results are presented. After these three chapters, there is a fifth and final chapter that reviews the results and implications of the major hypotheses presented in this chapter and integrates them with the conceptual model proposed in Figure 1.4.

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CHAPTER 1: FIGURE 1.1

Figure 1.1: Cypripedium candidum flower at Site C.

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Figure 1.2: Cypripedium candidum: 1, Flowering ramet. 2, Sepals and petals, spread out. 3, Staminode from above, spread out. 4, Column with staminode removed, front view. Drawn by Gordon W. Dillon (Correll 1950) Nies

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Figure 1.3: Adapted from Brownell 1981. Top: dots represent documented populations prior to 1975, line shows the area with populations according to Luer (1975). Bottom: confirmed populations by Brownell (1981). Nies

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CHAPTER 1: FIGURE 1.4 Figure 1.4: Conceptual Model: Cypripedium candidum’s interactions between plant productivity,

Figure 1.1: Cypripedium candidum’s interactions betweenrate plant productivity, soil nitrogen, mycorrhizal colonization, soilConceptual nitrogen, Model: mycorrhizal colonization, and photosynthetic and photosynthetic rate

III Water Use Efficiency

I Ramets/Clump Reproductive Effort

Mycorrhizal Colonization of Roots

Soil Nitrogen Concentration II

Photosynthetic Rate IV

δ15Nitrogen

Carbon V δ13Carbon

Figure 1.1: Conceptual Model: Cypripedium candidum’s interactions between plant productivity, soil nitrogen, mycorrhizal colonization, and photosynthetic rate. Blue arrows represent steps in the research process, green arrows indicate a two-way relationship, red arrows represen Figure 1.4: Conceptual Model: Cypripedium candidum’s interactions between plant productivity, transfer of carbon from plant to fungi, black arrows represent processes that effect stable isotope concentrations in C. candidum leaf tissue.

soil nitrogen, mycorrhizal colonization, and photosynthetic rate. Blue arrows represent steps in the research process, green arrows indicate a two-way relationship, red arrows represent transfer of carbon from plant to fungi, black arrows represent processes that effect stable isotope concentrations in C. candidum leaf tissue

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CHAPTER 1: REFERENCES Agerer, R. (2001) Exploration types of ectomycorrhizae - a proposal to classify ectomycorrhizal mycelial systems according to their patterns of differentiation and putative ecological importance. Mycorrhiza, 11: 107-114. Arditti, J. (1992) Fundamentals of Orchid Biology. John Wiley & Sons, New York. Bidartondo, M.I., Burghardt, B., Gebauer, G., Bruns, T.D. and Read, D.J. (2004) Changing partners in the dark: Isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and trees. Proceedings of the Royal Society B - Biological Sciences, 271: 1799-1806. Bowles, M.L. (1983) The tallgrass prairie orchids: Platanthera leucophaea (Nutt.) Lindl. and Cypripedium candidum Muhl. ex Willd.: Some aspects of their status, biology, and ecology, and implications towards management. Natural Areas Journal, 3: 14-37. Briggs, J.M., Knapp, A.K., Blair, J.M., Heisler, J.L., Hoch, G.A., Lett, M.S. and McCarron, J.K. (2005) An ecosystem in transition: Causes and consequences of the conversion of mesic grassland to shrubland. BioScience, 55: 243-254. Brownell, V.R. (1981) Status report on the small white lady’s slipper Cypripedium candidum in Canada. Committee on the Status of Endangered Wildlife in Canada, Ottawa, Ontario. Carignan, V. and Villard, M.A. (2002) Selecting indicator species to monitor ecological integrity: A review. Environmental Monitoring and Assessment, 78: 45-61. Case, F.W.J. (1989) Native orchid habitats: The horticulturist’s viewpoint. In: North American Native Terrestrial Orchid Propagation & Production. Brandywine Conservancy, Mt. Cuba Center, New Engalnd Wildflower Society, pp. 1-14. Case, M.A. (1994) Extensive variation in the levels of genetic diversity and degree of relatedness Nies

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CHAPTER 1: REFERENCES among five species of Cypripedium (Orchidaceae). American Journal of Botany, 81: 175-184. Catling, P.M. and Knerer, G. (1980) Pollination of the small white lady’s-slipper (Cypripedium candidum) in Lambton County, Southern Ontario. Canadian Field-Naturalist, 94: 435-438. Correll, D.S. (1950) Native Orchids of North America North of Mexico. Chronica Botanica Company, Waltham MA. Curtis, J.T. (1943) Germination and seedling development in five species of Cypripedium L. American Journal of Botany, 30: 199-206. Curtis, J.T. (1946) Use of mowing in management of White Ladyslipper. The Journal of Wildlife Management, 10: 303-308. Curtis, J.T. (1954) Annual fluctuation in rate of flower production by native Cypripediums during two decades. Bulletin of the Torrey Botanical Club, 81: 340-352. Dearnaley, J.D.W. (2007) Further advances in orchid mycorrhizal research. Mycorrhiza, 17: 475486. Dearnaley, J.D.W., Martos, F. and Selosse, M.A. (2012) Orchid mycorrhizas; molecular ecology, physiology, evolution and conservation aspects. In: The Mycota IX. Ed., Hock, B. Springer-Verlag, Berlin Heidelberg pp. 207-230. De Pauw, M.A. and Remphrey, W.R. (1993) In vitro germination of three Cypripedium species in relation to time of seed collection, media, and cold treatment. Canadian Journal of Botany, 71: 879-885. De Pauw, M.A., Remphrey, W.R. and Palmer, C.E. (1995) The cytokinin preference for in vitro germination and protocorm growth of Cypripedium candidum. Annals of Botany, 75: 267-275. Nies

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CHAPTER 1: REFERENCES Domingues, T.F., Meir, P., Feldpausch, T.R., Saiz, G., Veenendaal, E.M., Schrodt, F., Bird, M., Djagbletey, G., Hien, F., Compaore, H., Diallo, A., Grace, J. and Lloyd, J. (2010) Co-limitation of photosynthetic capacity by nitrogen and phosphorus in west Africa woodlands. Plant Cell and Environment, 33: 959-980. Dressler, R. (1981) The Orchids: Natural History and Classification. Harvard University Press, Cambridge MA. Egerton-Warburton, L.M., Johnson, N.C. and Allen, E.B. (2007) Mycorrhizal community dynamics following nitrogen fertilization: A cross-site test in five grasslands. Ecological Monographs, 77: 527-544. From, M.M. (2007) Drought, peril, and survival in the Great Plains: Cypripedium candidum. North American Native Orchid Journal, 13: 66-74. Gebauer, G. and Meyer, M. (2003) 15N and 13C natural abundance of autotrophic and mycoheterotrophic orchids provides insight into nitrogen and carbon gain from fungal association. New Phytologist, 160: 209-223. Grant Woods. (2014) Your forest preserves: Grant Woods. Lake County Forest Preserves. May 7 2014. http://www.lcfpd.org/preserves/index.cfm?fuseaction=home.view&object_id=189 Hadley, G. (1989) The role of mycorrhizae in orchid propagation. In: North American Native Terrestrial Orchid Propagation & Production. Brandywine Conservancy, Mt. Cuba Center, New Engalnd Wildflower Society, pp. 15-24. Heidorn, R. (2009) Gavin Bog and Prairie. Illinois DNR. March 16, 2014. http://www.dnr.illinois.gov/INPC/Pages/Area2LakeGavinBogAndPrairie.aspx

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CHAPTER 1: REFERENCES Illinois, D.N.R. (2014) Grant Creek Prairie. http://www.dnr.illinois.gov/inpc/pages/area3willgrantcreekprairie.aspx Jeffords, M.R., Post, S.L. and Robertson, K.R. (1995) Illinois Wilds. Phoenix Publishing, Urbana IL. Kahmen, S., Poschold, P. and Schreiber, K.-F. (2002) Conservation management of calcerous grasslands: Changes in plant species composition and response of functional traits during 25 years. Biological Conservation, 104: 319-328. Kane Co., F.P.D. (2002) Chapter 2: Inventory and analysis of existing facilities. In: Comprehensive Master Plan. Ed. Forest Preserve District of Kane County, Kane County, IL pg. 148. Kane Co., F.P.D. (2014) Meissner Prairie - Corron Forest Preserve. Forest Preserve District of Kane County. March 16, 2014. http://www.kaneforest.com/ForestPreserveView.aspx?ID=46 Kohler, B., Gigon, A., Edwards, P.J., Krusi, B., Langenauer, R., Luscher, A. and Ryser, P. (2005) Changes in the species composition and conservation value of limestone grasslands in northern Switzerland after 22 years of contrasting managements. Perspectives in Plant Ecology, Evolution and Systematics, 7: 51-67. Kohring, M. (1981) Response of white lady’s-slipper to a fall burn. Restoration & Management Notes, 1: 10. Laroche, V., Pellerin, S. and Brouillet, L. (2012) White fringed orchid as indicator of sphagnum bog integrity. Ecological Indicators, 14: 50-55. Luer, C.A. (1975) The native orchids of the United States and Canada excluding Florida. Ed. The New York Botanical Garden. W.S. Cowell Ltd, Ipswich England. Nies

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CHAPTER 1: REFERENCES Martos, F., Munoz, F., Pailler, T., Kottke, I., Gonneau, C. and Selosse, M.A. (2012) The role of epiphytism in architecture and evolutionary constraint within mycorrhizal networks of tropical orchids. Molecular Ecology, 21: 5098-5109. McKendrick, S.L., Leake, J.R., Taylor, D.L. and Read, D.J. (2000) Symbiotic germination and development of myco-heterotrophic plants in nature: Ontogeny of Corallorhiza trifida and characterization of its mycorrhizal fungi. New Phytologist, 145: 523-537. Moog, D., Poschold, P., Kahmen, S. and Schreiber, K.-F. (2002) Comparison of species composition between different grassland management treatments after 25 years. International Association for Vegetation Science, 5: 99-106. Norstog, K. (1973) New synthetic medium for culture of premature barley embryos. In VitroJournal of the Tissue Culture Association, 8: 307-308. NRCS (2014) Web Soil Survey. 25 March 2014. http://websoilsurvey.sc.egov.usda.gov/App/ HomePage.htm?TARGET_APP=Web_Soil_Survey_application_4efjrnjj0cmvhtfbptdiba3b Nurfadilah, S., Swarts, N.D., Dixon, K.W., Lambers, H. and Merritt, D.J. (2013) Variation in nutrient-acquisition patterns by mycorrhizal fungi of rare and common orchids explains diversification in a global biodiversity hotspot. Annals of Botany, 111: 1233-1241. Rasmussen, H.N. (2002) Recent developments in the study of orchid mycorrhiza. Plant and Soil, 244: 149-163. Selosse, M.A., Bauer, R. and Moyersoen, B. (2002a) Basal hymenomycetes belonging to the Sebacinaceae are ectomycorrhizal on temperate deciduous trees. New Phytologist, 155: 183-195. Selosse, M.A., Weiss, M., Jany, J.L. and Tillier, A. (2002b) Communities and populations of Nies

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CHAPTER 1: REFERENCES sebacinoid Basidiomycetes associated with the achlorophyllous orchid Neottia nidus-avis (L.) LCM Rich. and neighbouring tree ectomycorrhizae. Molecular Ecology, 11: 1831-1844. Shefferson, R.P., Weiss, M., Kull, T. and Taylor, D.L. (2005) High specificity generally characterizes mycorrhizal association in rare lady’s slipper orchids, genus Cypripedium. Molecular Ecology, 14: 613-626 Shefferson, R.P. (2006) Survival costs of adult dormancy and the confounding influence of size in lady’s slipper orchids, genus Cypripedium. Oikos, 115: 253-262. Shefferson, R.P. and Simms, E.L. (2007) Costs and benefits of fruiting to future reproduction in two dormancy-prone orchids. Journal of Ecology, 95: 865-875. Sheviak, C.J. (1974) An introduction to the ecology of the Illinois Orchidaceae. Illinois State Museum Scientific Papers, XVI. Illinois State Museum, Springfield IL. Stoutamire, W.P. (1967) Flower biology of the lady’s slippers. Michigan Botanist, 6: 159-175. Stoutamire, W. (1989) Eastern American Cypripedium species and the biology of Cypripedium candidum. North American Native Terrestrial Orchid Propagation & Production. Brandywine Conservancy, Mt. Cuba Center, New England Wildflower Society, pp. 40-48. Swink, F. and Wilhelm, G. (1994) Plants of the Chicago Region. The Indiana Academy of Science, Indianapolis IN. Taylor, D.L. and Bruns, T.D. (1997) Independent, specialized invasions of ectomycorrhizal mutualism by two nonphotosynthetic orchids. Proceedings of the National Academy of Sciences of the United States of America, 94: 4510-4515. Taylor, D.L., Bruns, T.D., Szaro, T.M. and Hodges, S.A. (2003) Divergence in mycorrhizal speNies

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CHAPTER 1: REFERENCES cilazation within Hexalectris spicata (Orchidaceae), a nonphotosynthetic desert orchid. American Journal of Botany, 90: 1168-1179. Tedersoo, L. and Smith, M.E. (2013) Lineages of ectomycorrhizal fungi revisited: Foraging strategies and novel lineages revealed by sequences from belowground. Fungal Biology Reviews, 27: 83-99. USDA (2013) Cypripedium candidum Muhl. ex Willd. USDA. February 11. http://plants.usda. gov/java/profile?symbol=CYCA5 USGS (2013) US Topo Quadrangles — Maps for America. http://nationalmap.gov/ustopo/index. html Waterman, R.J. and Bidartondo, M.I. (2008) Deception above, deception below: Linking pollination and mycorrhizal biology of orchids. Journal of Experimental Botany, 59: 1085-1096. Wendel, L.F. and Antlfinger, A.E. (1996) Characteristics of leaf photosynthesis in Spiranthes cernua: A field study. Lindleyana, 11: 1-11. Whigham, D.F. and Willems, J.H. (2003) Demographic studies and life-history strategies of temperate terrestrial orchids as a basis for conservation. In: Ed., Dixon, K.W., Kell, S.P., Barrett, R.L. and Cribbs, P.J. Natural History Publications (Borneo), Kota Kinabalu, Sabah pp. 137-158 Willby, N.J., Pulford, I.D. and Flowers, T.H. (2001) Tissue nutrient signatures predict herbaceous-wetland community responses to nutrient availability. New Phytologist, 152: 463-481. Wu, J.R., Ma, H.C., Xu, X.L., Qiao, N., Guo, S.T., Liu, F., Zhang, D.H. and Zhou, L.P. (2013) Mycorrhizas alter nitrogen acquisition by the terrestrial orchid Cymbidium goeringii. Annals of Botany, 111: 1181-1187. Nies

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CHAPTER 2

CHAPTER 2

THE CURRENT STATUS OF CYPRIPEDIUM CANDIDUM IN ILLINOIS: ANALYSIS OF 13 YEARS OF VOLUNTEER COLLECTED DATA

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CHAPTER 2: INTRODUCTION The Orchidaceae, one of the largest and most diverse families of plants, is an important contributor to global biodiversity (Dressler 1981). Although much information has been gathered on the ecology and propagation of terrestrial orchids in general, few species have been studied in detail and most management plans focus on habitat conservation alone (Whigham and Willems 2003). Sadly, this strategy has proven ineffective and wild orchid species continue to be lost (Whigham and Willems 2003). Nevertheless, habitat conservation is still an important part of orchid conservation since without their habitat, native orchids cannot survive. In order to assess the effect of management on Cypripedium candidum and its long-term population trends in Illinois data, collected through the Chicago Botanic Garden’s Plants of Concern program (POC) was analyzed for three Illinois populations. Cypripedium candidum Muhl. ex Willd., commonly known as the white lady’s slipper orchid grows in calcareous, mesic prairies and fens across the North East and North Central United States and Ontario, Canada (Case 1989; Sheviak 1974; Swink and Wilhelm 1994). Habitat features that appear to be most important for this species are: regular flooding leading to periods of high soil moisture; full sunlight, especially early in the year; and high limestone concentration in the soil (Case 1989; Sheviak 1974). Cypripedium candidum is a slow growing and long-lived species, and time from seed germination to first flowering is 10 to 16 years (Curtis 1943, 1954). Thus, long-term data, such as that provided by the Chicago Botanic Garden’s Plants of Concern program (POC), is necessary for an accurate assessment of population growth trends. Each spring, new growth appears early in the year and each fall above ground growth dies back entirely, leaving a rhizome below ground to generate the next year’s growth (Chapter 1). All ramets and leaves are produced early in the spring, before flowering, so that after flowering there is no additional above-ground growth (Chapter 1). Once plants are mature, reproduction can take place both vegetatively and sexually. VegNies

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CHAPTER 2: INTRODUCTION etative reproduction and spread takes place through the rhizome where branching is common and where up to 10 sympoidal buds may form each year (Stoutamire 1989), each resulting in a potential new ramet. Sexual reproduction is through pollination by bees, both Andrenid and Halictine bees have been observed visiting C. candidum flowers (Bowles 1983; Catling and Knerer 1980; Curtis 1954; Stoutamire 1989). Pollination success is generally very low, probably because C. candidum uses a deception strategy; promising nectar, through fragrance, when there is none (Catling and Knerer 1980; Stoutamire 1967; Waterman and Bidartondo 2008). Structurally, selfpollination cannot occur (Brownell 1981; Catling and Knerer 1980). Flowers are present during the spring and are open for approximately two weeks. Pollinated flowers develop over the spring and summer and in late summer the fruits open and seeds disperse. Cypripedium candidum plants can be dormant for up to 6 years and subsequently appear above ground. The probability of transition to dormancy from flowering or from vegetative is about 30% and decreases as the number of ramets produced in the previous year increases (Shefferson 2006). Neither flower nor fruit production increase the chance of entering dormancy or effect sprout production (Shefferson 2006; Shefferson and Simms 2007). Population Monitoring Since 2001, the Chicago Botanic Garden’s Plants of Concern program has been conducting “Level 2” (their most intensive) monitoring of Cypripedium candidum at six sites in Illinois. Although a lot of data has been collected, much of it was not fully analyzed. Among the data collected are annual details at the population level, including: number of clumps, percent of population flowering, management completed, and threats to the population; and biannual details at the plant level (for tagged plants), including: number of ramets per clump, number of flowers per clump, diameter of clump, fruits per clump, and damage by herbivory. Threats to Populations Surprisingly, there is no record of poaching contributing to the decline of this species. This Nies

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CHAPTER 2: INTRODUCTION may be because the populations are generally difficult to access, and within its habitat, Cypripedium candidum is difficult to identify unless in flower (personal observation). POC volunteers have often listed unauthorized trail as a threat to local populations, but these trails are not associated with missing plants. Instead, they appear to be the result of site walk-through and do not impact more than 25% of any population in any year. Damage from deer has been reported, but it appears that Cypripedium candidum is not a first choice for deer. Deer may nest in a site without actually eating any of the plants (Chapter 1) and the threats recorded by POC volunteers shows that surrounding vegetation is often more damaged than the Cypripediums are. Herbivory by other mammals and by insects is rare. The most frequently reported threat to local populations by POC volunteers is woody brush encroachment. Woody brush encroachment is the primary threat facing grasslands across the world, especially in the North American Great Plains (Briggs et al. 2005). This threat has two affects on the survival of Cypripedium candidum. The first is that shrubs create shade, under which C. candidum cannot survive (Case 1989; Sheviak 1974). The second is a change in community structure, most visible through decreased richness of herbaceous species (Briggs et al. 2005). Management Active habitat conservation and management has contributed to population retention of Cypripedium candidum. Not only is C. candidum an obligate-wetland species, it also requires full sun conditions for survival (Curtis 1946; Case 1989; USDA 2013). Therefore, woody brush removal is essential to maintaining viable populations. In a fen habitat, the natural method for controlling the encroachment of shrubs is periodic flooding, but this is not a practical method for land managers (Case 1989). Instead a common method of maintaining the full sun condition is prescribed burning. However, only one study, limited to one population for one year, has been conducted where burning increased ramet and flower production (Kohring 1981). Although not a current method, historically, mowing has proven a very effective method of management over Nies

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CHAPTER 2: INTRODUCTION longer periods of time (Curtis 1946). Additionally, in a number of European studies, it has been demonstrated that, for calcareous grasslands, mowing is a much more effective management method for habitat conservation than burning (Kahmen et al. 2002; Kohler et al. 2005; Moog et al. 2002). Hypotheses The purpose of this study was to assess the in-site distribution, long-term population trends, and management responses of Cypripedium candidum populations in the Chicago Region. To do this four hypotheses were tested: I.

Clumps are clustered and flowering is random. Populations do not seem to be widespread over apparently suitable habitat. Yet historical reports discuss wide coverage of populations. Since orchids tend to be habitat specialists, it may be that clumps are clustered within the population.

II. At all three sites, the populations are demonstrating at least one sign of decline; decreased clump ramet production, decreased clump flower production, and/or decreased number of ramets per meter square. As a native North American orchid, Cypripedium candidum has faced significant habitat loss. Often locally rare, populations persist in prairie wetlands facing encroachment by woody brush. It is likely that for this species, like many other native orchids, habitat conservation is proving insufficient for longterm population conservation. The long life span of this species makes it difficult to make short-term assessments of population growth. Thus, the long-term monitoring by POC provides an exceptional opportunity to assess our local Illinois populations. III. Management is correlated with increased population size and increased percent of the population flowering. A short-term assessment of the affect of a prescribed burn on Cypripedium candidum growth and flowering showed a significant increase in population size and flowering (Kohring 1981). Furthermore, Curtis (1946) has demonstrated, Nies

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CHAPTER 2: INTRODUCTION in a six-year study, that both spring and fall mowing significantly increases population size. The POC data provides a unique opportunity to assess the effect of four management types across over 11 years for multiple populations across the Chicago Region. IV. Reproductive effort varies between sites. Conversations with land managers, stewards, and volunteer monitors revealed different population assessments for each site. Furthermore, a preliminary overview of the POC data revealed that the populations were very different in size from one another and have demonstrated different percent of population flowering during the last POC monitor visits. The sites are spread across a band that runs approximately 70 miles north to south and 20 miles east to west. Additionally, the sites are different in size, age, and land use history. Thus, there is no reason to assume that the populations within them will behave in the same manner. In order to test these hypotheses, three sites at which Level 2 POC monitoring was completed were available for this study and were therefore used (Figure 2.1). These three sites are in Northeastern Illinois and considered high quality natural areas, accessible by permit only and requiring an off-trail hike of at least 10 minutes. Site A is located in the 1070 acre Grant Woods Forest Preserve in Lake County, Illinois (Grant Woods 2014). The land managers are very active at this site in restoring and maintaining the preserve and there are many forbs growing in the prairie, which is surrounded by a forest on three sides (Chapter 1). According to the site steward, Joyce Proper (2012), the population of Cypripedium candidum at this site is small and scattered. Mrs. Proper expressed concern over the long-term survival of the population. A preliminary review of the POC data for 2012 revealed that this population was the smallest among the three sites, 63 clumps, and had the lowest percent flowering at 46%. Site B is located in the 229 acre Meissner Prairie-Corron Forest Preserve in Kane County, Illinois (Kane Co. 2014). This site has not been burned in many years, and the population is Nies

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CHAPTER 2: INTRODUCTION surrounded by woody brush over three meters tall (Chapter 1). The community is dominated by grasses and prairie dock (Silphium terebinthinaceum). It was not monitored in 2012, but what it was in 2011 the site had 117 clumps, 85% of which flowered. Site C is located in the 78 acre Grant Creek Prairie Nature Preserve in Will County, Illinois that borders the Midewin National Tallgrass Prairie (Illinois D.N.R. 2014). This Site is centered in a large prairie with no nearby shrubs or trees (Chapter 1). In 2013, POC monitors recorded 291 clumps, 80% of which flowered.

Table 2.1: Site information for each of the study sites used. Site A B Grant Woods Forest Meissner Prairie-CorLocation Preserve ron Forest Preserve County Lake Kane Established 1958 1970’s Land use history Farming & grazing Unknown Acres 1070 229 Number of clumps at 63 117 last monitoring % Flowering at last 46% 85% monitoring Last year of monitor2012 2011 ing (before 2013) Burn Frequency Frequently Rare Site soil type(s) - from muck, silty clay loam, silty clay loam USGS survey silt loam Nies

C Grant Creek Prairie Nature Preserve Will 1978 Grazing 78 291 80% 2012 Intermediate silt loam Page 42

CHAPTER 2: FIGURE 2.1 Figure 2.1: Sites Siteswith withLevel Level2 2POC POCMonitoring Figure 1.4: Monitoring of Cypripedium of Cypripedium candidum candidum Site A

Site B

Site C

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CHAPTER 2: MATERIALS AND METHODS Data on individual clumps for 2013 were collected during spring site visits. This data was combined with long-term data collected by POC volunteers. Which included: management history, number of individuals, percent reproductive, and percent fruiting. All of the data collected was compiled into data sets for each population. Data Collection - Plants of Concern Plants of Concern data collection is done by trained volunteers at two levels. Level 1 Monitoring uses standardized data collection forms to annually record: location of the population, area covered by a population, number of plants within a population, management activity, threats to the population (from invasive plants, herbivory and human activity), and associate species (POC 2012b). Level 1 Monitoring takes place in the spring for Cypripedium candidum. This means that if the visit that monitoring is taking place is vn then management activity noted is management that took place between the current visit (vn) and the previous visit (vn-1). For three target species, one of which is Cypripedium candidum, POC completes Level 2 Monitoring (demographic), which relies on annual relocation of plants marked by permanent tags (Figure 1.5) that are found at each “clump” and whose location is recorded on a coordinate system for the site (POC 2012b; POC 2012c). A clump is a group of ramets that are at least 15 cm from any other ramet (POC 2012c). Individual genets cannot be identified without serious disruption to the plant, so clumps provide a practical way to track the population. Level 2 Monitoring takes place two times a year, once in the spring (typically May) and once in the summer (typically August) (POC 2012c). These visits are timed to coincide with the spring flowering and summer fruit production, respectively. In the spring tags are added to new plants and their location is recorded, clump size and number of flowers at each clump are recorded, and damage from herbivory is noted. In the fall the clumps that were flowering during the spring visit are revisited and number of fruits is recorded along with any signs of herbivory. Data from Plants of Concern monitoring is stored both in paper and electronic format. Nies

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CHAPTER 2: MATERIALS AND METHODS The Level 1 Monitoring information is available through secure login to the POC website (POC 2012a). The Level 2 Monitoring information, collected over the past 13 years, was entered into a database during 2013 and is now available in electronic format through the POC department. Meta-Data Analysis Among the data collected by POC was approximate area covered by the population (N-S meters and E-W meters), total number of clumps, and number of ramets for each clump. The area (m2) of the population was not the same for all years at each site. Furthermore, the long life history of C. candidum makes it unlikely that adult plants would spontaneously appear from one year to the next. On the other hand, occasionally, there were notes that clumps had grown into one another so that they were no longer distinguishable and so they were combined into a single clump in the database. This means that clump count is likely not an actual reflection of number of genets in the population. So, rather than using total number of clumps as a measure of population size, ramet density was calculated as a measure of population size. Ramet density is calculated as the total number of ramets in the population divided by the area covered by the population. Rt ρR = (2-1) DEW DNS If a tag had no ramets for seven or more consecutive years, then that clump was considered dead and removed from the list of tags for that site. There were no instances in the POC reports for which a tag had no ramets for seven years and then ramets appeared in a subsequent year. For each site, the average number of ramets per clump and average number of flowers per clump were calculated for each year. Although collected by POC volunteers, clump diameter was not used as there was no apparent relationship between clump diameter and number of ramets; indicating some inconsistency in the measurement of clump diameter. If a clump had no ramets for a given year but was not considered dead for that year, then it was assumed to be dormant. Dormant clumps were not used to calculate average number of ramets per clump for a given year. Average number of ramets per clump (CR) was calculated as Nies

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CHAPTER 2: MATERIALS AND METHODS the sum of ramets per clump, for each clump with ≥1 ramet, divided by the sum of clumps with ≥1 ramet. CR =

(2-2)

∑R ∑C

Average number of flowers per clump (CF) was calculated as sum of flowers per clump, for each clump with ≥1 ramet, divided by the sum of clumps with ≥1 ramet. CF =

(2-3)

∑F ∑C

Using linear models and MANOVA’s in R, data were analyzed for effect of management upon change in population size and percent of population flowering. Of the four types of management recorded by POC volunteers, “Burning,” “Brush or Invasive Tree Removal,” and “Herbaceous Invasive Removal” were used. “Mowing” was so rare that it was statistically meaningless. For the three management types used, effects of each management type alone and in conjunction with other management types was modeled. Also, whether any type of management was done was analyzed. Spatial Data Analysis POC data included a GPS location for stakes within each population. Then distance from the stake and degrees north from east were recorded for each tagged plant (POC 2012c). This information was used to map the population in ARC GIS (Figure 2.3). Using the coordinates for each tagged plant listed in the POC data, along with information on individual clumps, ARC GIS was used to analyze the sites, and to calculate clump density, flowering distribution, and stem distribution across each population. These were completed using the Euclidean distance Average Nearest Neighbor Summary Report function to analyze the density of clumps and the Euclidean distance Spatial Autocorrelation Report function to analyze the distribution of clumps and the distribution of flowering clumps. Topography maps from the USGS were overlaid onto each site Nies

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CHAPTER 2: MATERIALS AND METHODS map to over-view the relationship between the site and the surrounding area and to determine potential pollution sources (USGS 2013).

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Figure 2.2: Cypripedium candidum clump at Site C. The metal tag with yellow flag is a permanent tag identifying the clump for the Plants of Concern Program. Nies

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Figure 2.3: Map of Cypripedium candidum clumps at Site B.

Figure 2.3: Map of Cypripedium candidum clumps at Site B. This map was generated in ARC GIS using the clump coordinates and site location as recorded by POC volunteers. Each dot represents a tagged clump of C. candidum ramets.

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CHAPTER 2: RESULTS According to the Average Nearest Neighbor Summary report from ARC GIS, at all sites, distribution of clumps is significantly clustered (p < 0.001). According to the Spatial Autocorrelation Report, at all sites, flowering clump distribution is random (Site A: p = 0.13, Site B: p = 0.77, Site C: p = 0.97; Table 2.2). The Spatial Autocorrelation Report also showed that number of ramets per clump is significantly clustered at Sites A (p < 0.01) and B (p < 0.01) while random at Site C (p = 0.79;Table 1.2). At Site A the mean distance between clumps is about 2.3 meters, at Site B the mean distance between clumps is about 1.0 meters, and at Site C the mean distance between clumps is about 0.7 meters. Flowers per clump, ramets per clump, percent of tagged clumps observed, and percent of observed clumps with flowers is different at each site (Table 2.3). At Sites B and C the number of ramets per m2 (ρR) has been steadily declining for the past ten years (Figure 2.4). Site C has been declining in ρR more rapidly than Site B. Whereas at Site A ρR has been constant, mean ρR = 0.0161 ± 0.0004 (1 s.e., p < 0.0001). Sites B and C have demonstrated dramatic changes in ramets per clump (CR) and flowers per clump (CF) between years, while Site A has had relatively smaller annual variations (Figure 2.5, Figure 2.6). Site A appears to be constant in regards to both CR and CF, but over all CR and CF are declining at a rate of 0.04 CR per year and 0.05 CF per year. Site B is showing a more dramatic average annual decline of 0.41 CR and 0.13 CF. While Site C is increasing at a rate of 0.37 CR and 0.28 CF. Furthermore, Sites B and C are decreasing in ρR, while Site A is stable (Figure 2.4). Table 2.2: Results of spatial analysis of populations at each site. Average nearest neighbor compares distance between clumps. Stem spatial autocorrelation compares number of stems in a clump with clump distribution. Flower spatial autocorrelation compares number of flowers in a clump with clump distribution. Stem Flower Site Average Nearest Neighbor Spatial Autocorrelation Spatial Autocorrelation A Clustered, p < 0.001 Clustered, p < 0.01 Random, p = 0.13 B Clustered, p < 0.001 Clustered, p < 0.01 Random, p = 0.77 C Clustered, p < 0.001 Random, p = 0.79 Random, p = 0.97 Nies

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CHAPTER 2: RESULTS For 2013 Site C had significantly higher ramets (p < 0.001) and flowers ( p