EXAMINING CYPRIPEDIUM () HYBRIDIZATION IN A

PRAIRIE/WOODLAND ECOTONE

Ryan P Walsh

A thesis submitted to Bowling Green State University in

partial fulfillment of the requirements for the Degree of

Masters’ of Science

August 2008

Committee:

Helen J. Michaels, Ph.D. Advisor

John V. Freudenstein, Ph.D.

Moira J. van Staaden, Ph.D.

ii

ABSTRACT

Hybridization has been shown to be an important process in speciation.

Ascertaining the lineage of suspected hybrid individuals can be complicated when using morphological characters alone. This study examined morphological characters as well as nuclear-ribosomal internal transcribed spacers of , C. parviflorum var. pubescens and C. x favillianum in order to test the hypothesis that suspected hybrid individuals growing along a prairie/woodland ecotone in Resthaven

Wildlife Area, Castalia, Ohio indeed had hybrid ancestry. Furthermore, we examined whether C. parviflorum var. pubescens was extirpated from the site. Fifteen morphological characters were employed in a discriminant function analysis which revealed clear separation between the hybrids and the parental taxa. Additionally, analysis of the morphological data showed ten of the fifteen variables measured in the hybrids were intermediate between the parental taxa. Three ITS types were detected in the hybrid populations, two of which were identical to the parental taxa and a third which was identical to C. parviflorum var. pubescens with the exception of a single nucleotide substitution. The results support the hypothesis that the suspected hybrid individuals were indeed hybrids, and C. parviflorum var. pubescens is extirpated from the Resthaven site.

iii

ACKNOWLEDGEMENTS

I would like to thank my committee members, Dr. John Freudenstein and Dr.

Moira van Staaden for their help with this thesis as well as my advisor, Dr. Helen

Michaels for the invaluable lessons she has taught me about molecular biology and science in general. Additional thanks go to the many Graduate students at Bowling

Green State University who have either provided direct help with the thesis or just helped

me maintain sanity throughout my Masters career. Special thanks go to my lab mate,

Mike Plenzler who provided not only scientific but moral support throughout the process.

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TABLE OF CONTENTS

Page

Introduction………………………………………………………………………... 7

Materials and Methods...... 12

Results…………………………………………………………………………….... 15

Discussion………………………………………………………………………….. 18

Tables and Figures………………………………………………………………… 22

Literature Cited……………………………………………………………………. 28

Appendices…………………………………………………………………………. 34

1. Taxa, Accession Numbers and Citations...... 34

2. ITS Sequences………...…………………………………………………. 35

v

LIST OF FIGURES

Figure Page

1 Score Plot for PCA of Parental Taxa………………………………………………...22

2 Canonical Plot for Parental Taxa and Hybrids………………………………………23

3 Strict consensus tree from Maximum Parsimony analysis of ITS sequences……….24

vi

LIST OF TABLES

Table Page

1 Morphological Characters…………………………………………………..25

2 Loading Scores for Principal Components Analysis of Parental Taxa……..25

3 Canonical Discriminant Function Coefficients……………………………..26

4 Means of Morphological Variables………………………………………...27

7

INTRODUCTION

As anthropogenic activities continue to impact natural areas around the world, it is important to know what species we are losing in order to preserve or restore the few of them that remain. Identification of a rare species is the first step in creating an effective management strategy for preservation. Without an appropriate identification no further steps can be taken. Identification of plant species can be complicated when closely related species hybridize along zones of contact. The resulting hybrids may closely resemble the parental taxa and therefore complicate any attempt to establish distinct delineations between species in a given area. When hybrids are present, commonly used morphological-based approaches to plant identification may not be sufficient to adequately separate parental species from hybrids. In such cases, genetic analysis of the population paired with a morphological study can prove invaluable for correctly categorizing individuals.

Hybridization is particularly rampant among plant species (Anderson 1949;

Stebbins 1959; Arnold 1992; Rieseberg and Wendel 1993; Ellstrand et al. 1996; Barkman and Simpson 2002). Arnold (1997) estimated that roughly 50% of all angiosperms are thought to be derived from hybrid ancestry, further underscoring the importance of hybridization in the history of plant speciation. All hybridization events however, do not lead directly to the creation of a new species. In order for the newly created non-sterile hybrids to evolve into distinct species there must be some of reproductive barrier that separates the hybrids from the parental taxa from which they were derived. This barrier may be physical or genetic in nature. Chromosome rearrangements may play an important role in establishing a model in which hybrids remain fertile among themselves, 8

yet are at least partially sterile when crossed back to their parental species (Rieseberg

1997). In addition to genetic isolation, suitable habitat for the hybrids must exist.

Anderson (1948) hypothesized that post-hybridization habitat disturbance may play a

critical role in the creation of habitat suitable for the growth and reproductive isolation of

hybrids. Rieseberg (1997) later commented that many true hybrid species are found in

habitat that is extreme compared to the parental taxa from which they were derived.

The process of hybridization, although extremely important to plant evolution,

may also have a more nefarious side. In addition to the creation of new species,

hybridization events may lead to demographic swamping and/or genetic assimilation

(Wolf et al. 2001), resulting in the potential extirpation of particular hybrid populations

as well as one or both of the constituent parental taxa from a given locale. This process

could occur if no distinct reproductive barriers were in place to prevent introgression and

the hybrid populations occupied a habitat niche close to, or overlapping, that of its parental taxa. In this model, newly created hybrids would grow alongside or even intermingled with the parental taxa, competing both for finite resources and .

In Ohio, orchids of the Cypripedium have patchy distributions wherever

suitable habitat remains. Cypripedium are deciduous, terrestrial orchids with growth

emerging from a subterranean (Stoutamire 1967). Cypripedium candidum Muhl

ex Willd., the Small White Lady’s Slipper, has yellow-green lateral and with

a white labellum spotted with purple (Stoutamire 1967), and occur in calcareous prairies

as well as fens and limestone barrens (Cusick 1980). Due to the rich soil many prairies

offer, most suitable habitat for C. candidum in the state has been lost to agricultural

activities. C. candidum is highly dependent on full sun in open areas and, as with many 9 prairie species, populations begin to decline with the invasion of woody (Curtis

1946). Closely related to C. candidum and with the same general floral morphology, is the Yellow Lady’s Slipper, Salsib. C. parviflorum has greenish-yellow sepals spotted with maroon (Case 1993) and the characteristic yellow labellum. It occurs in a variety of habitats from swamp to upland habitats and calcareous to non-calcareous soils (Case 1993).

C. parviflorum has been the center of much taxonomic debate due to its striking resemblance to the European Yellow Lady’s Slipper, . For the purposes of this paper I will refer to the North American Yellow Lady’s Slipper as C. parviflorum as supported by Sheviak (1992). The North American C. parviflorum is then further separated into three subspecies, var. parviflorum, var. pubescens, and var. planipetalum. These varieties are all highly variable which makes identification of the subspecies often difficult. The parviflorum sub-species is often referred to as the “small”

Yellow Lady’s Slipper, with the pubescens sub-species referred to as the “large” Yellow

Lady’s Slipper. However this size distinction could be hard to recognize in less than ideal habitats. For example a C. parviflorum var. pubescens plant growing in nutrient- poor soil could very well have a small leading one to believe it was a var. parviflorum instead. The lines between the subspecies are further blurred by rampant hybridization among sub-species and the lack of genetic similarity among varieties themselves. Case (1993) found that Nei’s genetic identity within a variety of

Cypripedium was “only slightly more genetically similar than populations belonging to different varieties.” The high level of variation among individuals in a subspecies has been shown to give the species, as a whole, a much higher level of genetic diversity when 10

compared to other Cypripedium species. It was found that 75% of C. parviflorum loci

were polymorphic at the species level with C. candidum having somewhat fewer with

66.7% polymorphic loci (Case 1994).

Identification of Cypripedium species can be made even more difficult by hybridization events in the areas where two species intermingle. C. candidum and C. parviflorum have been reported to hybridize in zones of contact throughout their ranges

(Curtis 1932; Atwood 1975; Luer 1975; Bender 1985; Klier, Leoschke et al. 1991).

These zones of contact usually involve areas which contain prairie/woodland ecotones which provide suitable habitat for both parental species. Curtis (1932) formally

identified the resulting hybrid of these two species as Cypripedium x favillianum.

The primary field site is located at Resthaven Wildlife Area in Sandusky, Ohio.

Resthaven has a large, actively managed prairie area with wooded areas intertwined.

Large, thriving populations of C. candidum (total N ~ 6000) exist in the calcareous soil

and actively burned regions of the prairie. Records from the 1932 and 1982 Ohio

Biological Survey, as well as samples from the Bowling Green State University

Herbarium, indicate C. parviflorum var. pubescens once existed in the Resthaven area

(Schaffner 1932; Cooperrider 1982); however, all plants resembling C. parviflorum

which exist at Resthaven today appear to be of hybrid origin (pers. com. W. Stoutamire,

J. Windus). In order to prevent future confusion over the hybrid/non-hybrid question, it

is necessary to positively identify the plants at Resthaven using both morphological and

genetic approaches.

The internal transcribed spacer (ITS) of the 18S-5.8S-26S nuclear ribosomal

cistron has previously been shown to be extremely useful in the identification of plant 11

species and hybrids (Andreasen and Baldwin 2003; Baldwin et al. 1995). Cox et al.

(1997) and Jo et al. (2005) found ITS to be helpful in differentiating species in the genus

Cypripedium. ITS sequences have become increasingly popular in plant phylogenetic

analyses due to their biparental inheritance, a feature absent in cpDNA markers, and their

ease of use with the availability of universal ITS primers (Álvarez and Wendel 2003).

ITS evolves through concerted evolution which homogenizes sequences through crossing

over and gene conversion, theoretically leaving only species or clade-specific character state changes (Álvarez and Wendel 2003). Álvarez and Wendel (2003) have noted multiple potential problems in the use of ITS markers in constructing plant phylogenies due to the lack of complete sequence homogenization causing both orthologs and paralogs to be present in a single accession, and the differing directions of divergent ITS types following reticulation, introgression and polyploidization events. Both of these situations lead to the loss of significant data, which weakens any evolutionary inferences.

In the case of hybrid ITS sequences, Koch et al. (2003) postulated that there are three different outcomes when ITS sequences of hybrids are examined: one of the parental copies is lost, a new ITS type containing a mixture of parental ITS sequences is brought about, or both parental ITS copies are present.

This study combined genetic and morphological analysis to answer the following

questions: 1) Are the apparent hybrids at Resthaven indeed remnant hybrids from the

previously identified pure C. parviflorum? 2) Are there any pure C. parviflorum

individuals left at Resthaven? and 3) What are the best morphological characters which

may be used to separate Cypripedium x favillianum from its parents in the field?

12

MATERIALS AND METHODS

Thirty plants each from three populations of C. candidum at Resthaven Wildlife

area were surveyed along with two populations (n=17 and 20) of potential Cypripedium x

favillianum hybrids at Resthaven. All C. parviflorum var. pubescens plants at three small

populations (n = 18, 10, and 16) in the Oak Openings Metropark in Swanton, Ohio were

also sampled as the nearest source of data to represent pure C. parviflorum var.

pubescens. Although one population of C. parviflorum was damaged after a storm and

therefore morphological measurements could not be taken, samples for genetic

analysis were still collected. In the C. candidum populations 20-meter line transects

spaced 1 meter apart were used to sample evenly through the population. Photographic

voucher specimens of each species and a hybrid were deposited in the Bowling Green

State University Herbarium (BGSU).

Each population was first surveyed to estimate number of individuals and number

of flowering stems per individual. For C. candidum, stems that were clumped together

were counted as one individual to avoid sampling the same individual twice. In the case

of a clump with multiple stems, the largest (most mature) stem was measured. Each

was measured for fifteen characters (Table 1) that were found by Klier et al.

(1991) to show the most difference between pure parental species and hybrids in Iowa.

Leaf samples were collected from each individual measured. These samples

consisted of no more than 10 cm of the length of a leaf and were separated into several

smaller portions to provide back-up samples in the event of a sample being destroyed.

Sampled tissue was taken from the tip of the third leaf on each plant, transported to the lab in a cooler with dry ice, and stored at -80oC until DNA extraction. DNA was 13

extracted using Qiagen DNeasy® Plant Mini Kit for DNA isolation from plant tissue

(Qiagen Inc., Valencia, California, U.S.A.). DNA fragments of the ITS region were

obtained by PCR amplification using primers ITSL (5’-

TCGTAACAAGGTTTCCGTAGGTG-3’) for forward strand and ITS4 (5’-

TCCTCCGCTTATTGATATGC-3’) for reverse strand following the protocol by Jo et al.

(2005). Prior to sequencing PCR products were run on a 1.5% agarose gel to ensure only one region was amplified and purified using a QIAquick® PCR purification kit (Qiagen

Inc., Valencia, California, U.S.A.) to remove excess primer and nucleotides.

Sequencing was performed by Geneway Research (Hayward, California, U.S.A.).

All sequences were checked and aligned against their complimentary strand using MEGA

version 4.0.2 (Tamura et al. 2007) to check for within individual polymorphisms.

Sequences were deposited in GenBank (Appendix 1). In order to build a comprehensive

phylogeny, we used additional ITS sequences from GenBank (Appendix 1). ITS

sequences from spp. were used for outgroup comparisons. Sequences of

each individual were then aligned and the ragged ends of the sequences were discarded.

The evolutionary history was inferred using the Maximum Parsimony method

(Eck and Dayhoff 1966). Maximum parsimony analysis was performed using PAUP

4.0b10 (Swofford 2001) using a heuristic searches with 100 random addition replicates,

TBR branch swapping and the MulTrees option. Bootstrap analysis was conducted on

the resulting trees with 1000 replicates to examine levels of support for groupings. Due

to the possible confounding nature of hybrid samples in a phylogeny, all hybrid samples

in this study were initially omitted from the analysis. The species only tree was then

compared with the trees obtained from including the hybrid samples. 14

Morphological data was analyzed using JMP 7.0.2 (SAS Institute Inc. Cary, North

Carolina, U.S.A.). Principal components analysis was performed on the parental taxa first in order to confirm that the parental taxa formed two distinct groups based on the morphological characters chosen. A discriminant function analysis (also called canonical variates analysis) was then performed with all samples in order to find a linear combination of variables that maximized the difference between the groups. The discriminant function analysis requires an a priori designation of groups prior to analysis and results in a probability of correct group placement. In order to determine which morphological variables separated the groups, an analysis of variance (ANOVA) was performed with a Tukey-Kramer test for group comparison in order to interpret the data.

15

RESULTS

The principal components analysis of the parental species yielded three

components with an eigenvalue greater than 1. Components 1 and 2 accounted for 71.3%

and 7.3% of the variance respectively. When the eigenvalues were plotted from

components 1 and 2 the two parental species, C. candidum and C. parviflorum var.

pubescens, were well separated from each other (Figure 1). The loading scores for the

first three components are presented in Table 2. The first component separated the

groups based on overall size while the second component described floral traits.

Eigenvalues of the first component were spread relatively evenly across variables which described both vegetative and floral parts. The second component had a negative value

for twists and large positive values for the length and width of the flag leaf.

Component 3, although not represented graphically, also contained large eigenvalues for

flag length, petal twists and leaf length.

When discriminant function analysis was used to determine whether the putative

hybrids could be differentiated from the parental species based on the morphological

traits, the first two components explained 100% of the variation with the first component

explaining 93.43% and the second explaining 6.57%. The discriminant coefficients for

the first two components are presented in Table 3. Unlike the principal component

analysis, when the hybrids were included in the discriminant function analysis, the floral

characters weigh heavily in the separation of all groups. The a priori designation was

correct 95.5% of the time with five C. candidum’s being reassigned as hybrids and two

hybrids being reassigned as C. candidum. Reassigned samples were weakly supported in

the analysis (~55% likelihood) and therefore were omitted from all future morphological 16

and genetic analyses. Figure 2 shows the first two components graphed against each other. As with the principal components analysis, the two parental species show strong separation. Although the hybrid samples plotted closer to the C. candidum grouping, there is a distinction, with some overlap between the hybrids and C. candidum.

Results of the ANOVA of the species and hybrids are presented in Table 4.

Individuals which were re-classified according to the discriminant function analysis were

omitted from the ANOVA in order to prevent possible misclassifications from skewing

the results. Plant height, staminoide length and petal twists did not significantly

differentiate the hybrid samples from C. candidum whereas leaf length and flag length

did not separate them from C. parviflorum var. pubescens. All other variables separated

the three groups at a statistically significant p<0.01. For variables in which all groups

were significantly separated, the hybrid individuals were always intermediate to the two

parental taxa.

C. parviflorum var. pubescens and C. candidum had only one ITS type each in the study, including sequence data acquired from GenBank. The hybrid C. x favillianum had three ITS types, two types identical to the respective parental taxa (identified as candidum and parviflorum type) and a third which was identical to C. parviflorum var.

pubescens with the exception of a single A→G mutation (identified as unique). Of 674

total characters 321 were parsimony-informative. The maximum parsimony analysis

yielded three equally parsimonious trees with a length of 1015 (CI=0.6335, RI=0.8085).

The strict consensus tree is presented in Figure 3. A bootstrap value of 99% supports a

branch grouping C. candidum and C. parviflorum var. pubescens. Two of the C. x

favillianum sequences (parviflorum and unique ITS types) formed a polytomy with C. 17

reginae and C. parviflorum var. pubescens, supported by a 95% bootstrap value. There were a total of 7 nucleotide differences that separated C. candidum from C. parviflorum.

The candidum hybrid ITS type grouped with C. candidum. The general topology of the

tree agreed with a recent phylogeny of Cypripedium spp. reported in Shefferson et al.

(2007).

18

DISCUSSION

Since the advent of ITS as a molecular marker for phylogenetic inference,

numerous studies have examined ITS evolution in hybrid taxa (Álvarez and Wendel

2003). Recombination of two divergent ITS types within an individual hybrid results in

three possible outcomes: 1) Concerted evolution leads to a fixation of one parental ITS

type and the loss of the other, 2) Repeated recombinations and gene conversions during

concerted evolution leads to a new intermediate and complex ITS type that is a mixture

of the two parental types, and 3) Both ITS copies are present in the same individual

(Álvarez and Wendel 2003; Koch et al. 2003). Wendel and Cronn (2003) also demonstrated that concerted evolution may be bi-directional, resulting in hybrid populations containing both parental ITS types but each hybrid individual having only one of those types. The homogenization of ITS types by means of concerted evolution in hybrid individuals may be retarded by longer generation times (Gaut 1998; Álvarez and

Wendel 2003). However it must be cautioned that homogenization may occur in as little as two hybrid generations. In artificially created Armeria hybrids, the expected hybrid intermediacy was detected in the first filial generation, however evidence for sequence homogenization was detected in the second generation (Fuertes Aguilar et al. 1999a;

Álvarez and Wendel 2003).

Results from the morphological analysis provide strong support for the hypothesis

that the unknown individuals at Resthaven are indeed hybrids. There were no suspected hybrid individuals that were reclassified as C. parviflorum var. pubescens in our morphological analysis, lending support to the hypothesis that C. parviflorum var. pubescens is extirpated from the site. In 10 out of the 15 characters measured, the 19

suspected hybrid individuals showed an expected intermediate size between the parental

taxa. These 10 characters should prove useful in further studies when trying to

differentiate suspected hybrids from their parental taxa. When studying a similar hybrid

zone, Klier et al. (1991) found that plants, which were identified as hybrids based on

morphology, tended to be the product of introgression instead of first filial generation

hybrids. Although hybrid intermediacy would be the expected outcome, recent studies

have demonstrated that natural hybrid populations often resemble one of the parents or

display transgressive traits (Rieseberg and Ellstrand 1993; Rieseberg et al. 1999). The

discriminant function analysis showed some evidence for introgression between hybrids

and C. candidum, however not to the extent seen in the Iowa hybrids studied by Klier et al. (1999).

The ITS analysis showed evidence for bi-directional concerted evolution in the

Cypripedium hybrids at Resthaven. Of the hybrid individuals examined, 70% (7

individuals) shared an identical sequence to C. candidum. Only 20% (2 individuals)

showed identical sequences to C. parviflorum var. pubescens, with the remaining 10% (1

individual) showing a unique mutation in an otherwise C. parviflorum var. pubescens ITS

type. This situation, although less common than other scenarios of ITS evolution, has

been documented extensively in the literature (Álvarez and Wendel 2003; Brochmann et

al. 1996, Ferguson et al. 1999; Franzke and Mummenhoff 1999; Fuertes Aguilar et al.

1999a,b; Roelofs et al. 1997). In addition to concerted evolution resulting in the

homogenization of parental ITS types in putative hybrids, Fuertes Aguilar et al. (1999a)

postulated this scenario was the result of introgression in Armeria. Given this evidence,

the morphological and genetic analyses suggest that in the Resthaven Cypripedium 20

hybrids concerted evolution has homogenized the ITS sequences with the C. candidum type being dominant due to introgression events.

Further analysis is necessary in order to come to a clear conclusion on this matter.

The use of a plastid marker would allow us to ascertain the maternal heritage of the

hybrids and along with the ITS data, gain a better understanding of the direction of

hybridization. Inter-Simple Sequence Repeats (ISSR) markers were originally proposed

for this study. ISSR’s are dominant markers of mostly nuclear origin that can be

particularly useful in species delineations. Recent papers have shown their usefulness in

differentiating hybrid individuals from their parental taxa (Conte et al. 2007; Lau et al.

2005; Lee et al. 2006). Hybrid individuals are expected to show additivity of the parental

species specific bands. However when Morris (2002) used ISSR’s on a single C.

candidum/C. parviflorum var. parviflorum hybrid she did not find the additivity that

would be expected in a hybrid individual. Instead it was hypothesized that the particular

individual was the result of hybridization and subsequent backcrossing to the C.

candidum parent. It is possible that further study of the Ohio populations may show both

additivity and introgression due to the long life span of the species and the larger

numbers involved in our study. It would seem very unlikely that there was a single

hybridization event and no subsequent interbreeding, either among hybrids or between

hybrids and C. candidum.

It is interesting to note that although there are no longer any populations of pure

C. parviflorum at the site, there are two populations of hybrids with a total n=56 existing

along the ecotone between the two parental species’ habitat. Although it is beyond the

goal of this paper to examine the underlying biological reasons for the probable 21

extirpation of C. parviflorum from Resthaven, it is possible that hybridization events have

played a role in this situation.

The Resthaven location provides an interesting and unique opportunity to study

plant hybridization. Although there are still many questions that must be answered

before we can get a clear picture of the history of Cypripedium hybridization at the site,

the evidence from this study indicates that C. parviflorum var. pubescens has been extirpated from the site. Further study of the habitat preferences, pollination ecology, and population genetics of the hybrids and both parents may provide interesting insights into

how hybridization affects species at a community level. With our analysis of

morphological traits of 90 C. candidum individuals, there is no evidence to suggest that

the presence of hybrids has had any adverse effect on the vast C. candidum populations at

Resthaven. However, either genetic swamping or resource/habitat competition with the

arrival of hybrids may have adversely affected a declining population of C. parviflorum

var. pubescens. Furthermore, there do not appear to be any clear reproductive barriers

between the hybrids and their parental taxa. Due to the fact that C. parviflorum var.

pubescens is most likely extirpated from the site, any future hybrid individuals created

will either have to be a successive filial generation or back-crosses to the C. candidum parent. A lack of genetic or physical barriers would theoretically lead to eventual assimilation of the hybrids by means of repeated back-crosses.

22

TABLES AND FIGURES

Figure 1 Score Plot for PCA of Parental Taxa ■=candidum X=parviflorum

10

5

0 Prin2

-5

-10 -10 -5 0 5 10 Pr in1

23

Figure 2 Canonical Plot for Parental Taxa and Hybrids

24

Figure 3. Strict consensus tree of three shortest trees (length = 1015, CI = 0.6335, RI = 0.8085) found from Maximum Parsimony analysis of ITS sequences. Taxa marked with “*” indicate samples collected in this study. Fifteen samples of each C. candidum and C. parviflorum var. pubescens along with 10 samples of C. x favillianum were analyzed. Hybrid samples in the phylogeny have their respective ITS types listed in parenthesis. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown under the branches (Felsenstein 1982). Bootstrap values are not provided for basal nodes due to a lack of support in a 50% majority rule consensus tree. Branch lengths are shown above the branches

C. parviflorum var. pubescens* 3 C. reginae 1 95 C. x favillianum (unique)* C. x favillianum (parviflorum)* 7 69 C. montanum

C. candidum* 99 C. x favillianum (candidum)* 44 2 C. macranthos 14 100 4 59 C. himalaicum 2 C. calceolus 58 51 C. segawai

41 C. flavum

100 C. passerinum

33 25 C. guttatum 23 100 C. yatabeanum 17

C. californicum

11 34 C. japonicum

100 C. formosanum

34 11 C. acaule 43 C. lichiangense 18 100

13 C. margaritaceum 128 C. debile

C. plectrochilum 50 52 C. fasciculatum C. irapeanum 6 P. besseae 2 99 P. pearcei 6 80 P. caricinum 12 82 P. longifolium 89 P. lindleyanum P. kovachii

25

Table 1. Morphological Characters. Trait Plant Height(cm) Staminoide Length (cm) Petal Twists Leaf Length (cm) Leaf Width (cm) Dorsal Length (cm) Slipper Teeth Lateral Length (cm) Orifice Width (cm) Dorsal Sepal Width (cm) Flag Length (cm) Flag Width (cm) Lateral Petal Width (cm) Slipper Length(cm) Slipper Width (cm)

Table 2. Loading Scores for Principal Components Analysis of Parental Taxa. Trait Component 1 Component 2 Component 3 Plant Height(cm) 0.29473 -0.08836 0.06944 Staminoide Length (cm) 0.25893 0.07715 -0.20594 Petal Twists 0.14825 -0.60769 0.43149 Leaf Length (cm) 0.20484 -0.12550 0.46083 Leaf Width (cm) 0.26171 -0.19625 0.11839 Dorsal Sepal Length (cm) 0.29350 -0.05361 -0.02320 Slipper Teeth 0.22307 0.16384 -0.21370 Lateral Length (cm) 0.29541 -0.11289 0.00878 Orifice Width (cm) 0.28566 0.01743 -0.19193 Dorsal Sepal Width (cm) 0.28997 0.00791 -0.16184 Flag Length (cm) 0.15236 0.57106 0.57323 Flag Width (cm) 0.24565 0.42859 0.16930 Lateral Petal Width (cm) 0.27618 -0.04536 -0.11979 Slipper Length(cm) 0.28719 -0.06544 -0.16064 Slipper Width (cm) 0.28721 0.06099 -0.18299 Percent Variance 71.323 7.324 6.873

26

Table 3. Canonical discriminant function coefficients. Canon1 Canon2 Plant Height(cm) 0.162781 -0.09365 Staminoide Length (cm) 1.13841 -3.15905 Petal Twists -0.01386 -0.19596 Leaf Length (cm) -0.38359 0.247685 Leaf Width (cm) 0.17528 1.137346 Dorsal Sepal Length (cm) 0.611344 -1.59014 Slipper Teeth -0.03224 0.383647 Lateral Length (cm) 0.431106 0.382614 Orifice Width (cm) 2.881196 1.624689 Dorsal Sepal Width (cm) -0.13459 -0.91318 Flag Length (cm) 0.198606 0.059455 Flag Width (cm) -1.09609 0.248195 Lateral Petal Width (cm) 0.403921 1.340899 Slipper Length(cm) 0.262196 -0.91639 Slipper Width (cm) 1.731118 1.032421

27

Table 4 Means of Morphological Variables Trait C. C. x C. F df parviflorum favillianum candidum Plant 48.29a 28.84b 22.03b 371.74* 2,154 Height(cm) Staminoide 0.825a 0.575b 0.552b 112.98* 2,154 Length(cm) Petal Twists 3.29a 2.14b 2.12b 12.90* 2,154 Leaf Length 14.16a 13.19a 11.48b 22.90* 2,154 (cm) Leaf Width 5.36a 3.92b 2.56c 138.46* 2,154 (cm) Dorsal Sepal 5.11a 3.04b 2.81c 314.87* 2,154 Length(cm) Slipper Teeth 9.03a 7.54b 4.79c 62.21* 2,154 Lateral Length 6.34a 3.83b 3.28c 357.68* 2,154 (cm) Orifice Width 1.26a 0.64b 0.52c 419.96* 2,154 (cm) Dorsal Sepal 2.14a 1.26b 1.07c 312.71* 2,154 Width (cm) Flag Length 6.48a 6.25a 5.51b 9.20* 2,154 (cm) Flag Width (cm) 2.53a 1.99b 1.55c 55.28* 2,154 Lateral Petal 0.78a 0.49b 0.40c 176.35* 2,154 Width (cm) Slipper 3.85a 2.44b 2.22c 371.06* 2,154 Length(cm) Slipper Width 2.45a 1.42b 1.17c 355.72* 2,154 (cm) Means not followed by the same letter are significantly different F-values followed by “*” are significant at P < 0.01

28

LITERATURE CITED

Álvarez, I. and J.F.Wendel. 2003. Ribosomal ITS sequences and plant phylogenetic

inference. Molecular Phylogenetics and Evolution 29: 417-434.

Anderson, E. 1948. Hybridization of the habitat. Evolution 2: 1-9.

Anderson, E. 1949. Introgressive hybridization. New York: Wiley.

Andreasen, K. and B. G. Baldwin. 2003. Nuclear ribosomal DNA sequence

polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae).

Molecular Phylogenetics and Evolution 29: 563-581.

Arnold, M.L. 1992. Natural hybridization as an evolutionary process. Annual Review of

Ecology and Systematics 23: 237-261.

Arnold, M.L. 1997. Natural Hybridization and Evolution. Oxford University Press,

Oxford.

Atwood, J. 1975. The Cypripedium calceolus complex in North America. Proceedings of

the 11th World Orchid Conference: 106-110.

Baldwin, B.G., M.J. Sanderson, J.M. Porter, M.F. Wojciechowski, C.S. Campbell, and

M.J. Donoghue. 1995. The ITS region of nuclear ribosomal DNA: a valuable

source of evidence on angiosperm phylogeny. Ann. Missouri Bot. Gard. 82: 247-

277.

Barkman, T.J. and B. Simpson. 2002. Hybrid origin and parentage of Dendrochilum

acuiferum (Orchidaceae) inferred in a phylogenetic context using nuclear and

plastid DNA sequence data. Systematic Botany 27(2): 209-220.

Bender, J. D. 1985. Organismal Interdependence and Species Survival in Cypripedium

candidum Muhl. ex Willd. MS Thesis. University of Akron. 29

Brochmann, C., T. Nilsson, and T.M. Gabrielsen. 1996. A classic example of postglacial

allopolyploid speciation re-examined using RAPD markers and nucleotide

sequences: Saxifraga osloensis (Saxifragaceae). Symb. Bot. Ups. 31, 75-89.

Case, M. A. 1993. High Levels of Allozyme Variation within Cypripedium calceolus

(Orchidaceae) and Low Levels of Divergence among its Varieties. Systematic

Botany 18(4): 663-677.

Case, M. A. 1994. Extensive Variation in the Levels of Genetic Diversity and Degree of

Relatedess Among Five Species of Cypripedium (Orchidaceae). American

Journal of Botany 81(2): 175-184.

Conte, L., C. Cotti, and G. Cristofolini. 2007. Molecular evidence for hybrid origin of

Quercus crenata Lam. (Fagaceae) from Q. cerris L. and Q. suber L. Plant

Biosystems 141 (2):181-193

Cooperrider, T. S. 1982. Endangered and Threatened Plants of Ohio. Ohio Biological

Survey 16: 40.

Cox, A.V., A.M. Pridgeon, ,V.A. Albert, and M.A.Chase. 1997. Phylogenetics of the

slipper orchids (, Orchidaceae); nuclear rDNA ITS sequences.

Pl. Syst. Evol. 208: 197-223.

Curtis, J. T. 1946. Use of mowing in management of white ladyslippers. Journal of

Wildlife Management 10: 303-306.

Cusick, A. W. 1980. Cypripedium candidum Muhl ex Willd. Ohio Heritage Database.

Eck RV and M.O. Dayhoff. 1966. Atlas of Protein Sequence and Structure. National

Biomedical Research Foundation, Silver Springs, Maryland. 30

Ellstrand, N.C., R. Whitkus, and L.H. Rieseberg. 1996. Distribution of spontaneous plant

hybrids. Proceedings of the National Academy of Sciences USA 93: 5090-5093.

Fay, M. F. and R. S. Cowan. 2001. Plastid Microsatellites in Cyprepedium calceolus

(Orchidaceae): Genetic Fingerprints from Herbarium Specimens. Lindleyana

16(3): 151-156.

Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap.

Evolution 39:783-791.

Ferguson, C.J., F. Krämer, and R.K. Jansen. 1999. Relationships of Eastern North

American Phlox (Polemoniaceae) based on ITS sequence data. Syst. Bot. 24, 616-

631.

Franzke, A., and K. Mummenhoff. 1999. Recent hybrid speciation in Cardamine

(Brassicaceae)-conversion of nuclear ribosomal ITS sequences in statu nascendi.

Theor. Appl. Genet. 98, 831-834.

Fuertes Aguilar, J., J.A. Rosselló, and G. Nieto Feliner. 1999a. Nuclear ribosomal DNA

(nrDNA) concerted evolution in natural and artificial hybrids of Armeria

(Plumbaginaceae). Mol. Ecol. 8, 1341-1346.

Fuertes Aguilar, J., J.A. Rosselló, and G. Nieto Feliner. 1999b. Molecular evidence for

the compilospecies model of reticulate evolution in Armeria (Plumbaginaceae).

Syst. Biol. 44, 735-754.

Gaut, B.S., 1998. Molecular clocks and nucleotide substitution rates in higher plants. In:

Hecht, M.K. (Ed.), Evolutionary Biology. Plenum Press, New York, pp. 93-120. 31

Jo, S., M. Ochiai, K. Furuta, and K. Yagi. 2005. Genetic analyses of genus Cypripedium

found in Northern Japanese islands and related species endemic to Northeast

China. J. Japan Soc. Hort. Sci. 74(3):234-241.

Klier, K., M. J. Leoschke, and J.F. Wendel. 1991. Hybridization and Introgression in

White and Yellow Ladyslipper Orchids (Cypripedium candidum and C.

pubescens). Journal of Heredity 82(4): 305-320.

Koch, M.A., C. Dobeš, and T. Mitchell-Olds. 2003. Multiple hybrid formation in natural

populations: Concerted evolution of the Internal Transcribed Spacers of Nuclear

Ribosomal DNA (ITS) in North American Arabis divaricarpa (Brassicaceae).

Mol. Biol. Evol. 20(3):338-350.

Lau, C.P.Y., L. Ramsden, and R.M.K. Saunders. 2005. Hybrid origin of Bauhinia

blakeana” (Leguminosae: Caesalpinioideae) inferred using morphological,

reproductive, and molecular data. American Journal of Botany 92(3):525-533.

Lee, N.S., SH Yeau, JO Park, and MS Roh. 2006. Confirmation of the hybrid status of

Ilex x wandoensis using molecular markers. Journal of Plant Physiology 49: 491-

497.

Luer, C. 1975. "The Native Orchids of the and Canada, Excluding Florida."

The New York Botanical Garden.

Morris, J.A. 2002. A systematic study of the North American Yellow Lady’s Slipper

Orchids. Master’s Thesis, Kent State University, Kent, OH.

Nei M and S. Kumar. 2000. Molecular Evolution and Phylogenetics. Oxford University

Press, New York. 32

Rieseberg, L.H. 1997. Hybrid origins of plant species. Annu. Rev. Ecol. Syst. 28: 359-

389.

Rieseberg, L.H. and J.F. Wendel. 1993. Introgression and its consequences. Pp. 70-109

in Hybrid Zones and the evolutionary process, ed. R. Harrison. New York:

Oxford University Press.

Rieseberg L.H. and Ellstrand N.C. 1993. What can molecular and morphological markers

tells us about plant hybridization? Crit. Rev. Pl. Sci. 12: 213–241.

Rieseberg L.H., Archer M.A. and Wayne R.K. 1999. Transgressive segregation,

adaptation and speciation. Heredity 83: 363–372.

Roelofs, D., J. van Velzen, P. Kuperus, and K. Bachmann. 1997. Molecular evidence for

an extinct parent of the tetraploid species Microseris acuminata and M.

campestris (Asteraceae, Lactuceae). Mol. Ecol. 6, 641-649.

Schaffner, J. H. 1932. Ohio Biological Survey: Revised Catalog of Ohio Vascular Plants.

The Ohio State University Bulletin: Bulletin 25 5(2).

Saitou N and Nei M. 1987. The neighbor-joining method: A new method for

reconstructing phylogenetic trees. Molecular Biology and Evolution 4:406-425.

Shefferson, R.P., D.L. Taylor, M. Weib, S. Garnica, M.K. McCormick, S. Adams, H.M.

Gray, J.W. McFarland, T. Kull, K. Tali, T. Yukawa, T. Kawahara, K. Miyoshi,

and Y. Lee. 2007. The evolutionary history of mychorrhizal specificity among

Lady’s Slipper Orchids. Evolution 61(6):1380-1390.

Stebbins, G.L. 1959. The role of hybridization in evolution. Proceedings of the American

Philosophical Society 103: 231-251. 33

Stoutamire, W. P. (1967). Flower Biology of the Lady's-Slippers (Ochidaceae:

Cypripedium). The Michigan Botanist. 6(4): 159-175.

Swofford, D.L. 2001. PAUP*: Phylogenetic analysis using parsimony (*and other

methods), version 4. Sinauer, Sunderland, Massachusetts.

Tamura K., Nei M. and S. Kumar 2004 Prospects for inferring very large phylogenies by

using the neighbor-joining method. Proceedings of the National Academy of

Sciences (USA) 101:11030-11035.

Tamura K., J. Dudley, M. Nei and S. Kumar. 2007 MEGA4: Molecular Evolutionary

Genetics Analysis (MEGA) software version 4.0. Molecular Biology and

Evolution 24:1596-1599.

Wendel, J.F., Cronn, R.C., 2003. Polyploidy and the evolutionary history of cotton. Adv.

Agronomy 78, 139-186.

Whitten,W.M., L.M. Damian, and N.H. Williams. 2005. Phragmipedium kovachii:

Molecular Systematics of a New World Orchid. Orchids 74(2): 132-137.

Wolf, D. E., A. Takebayashi, and L.H. Rieseberg. 2001. Predicting the risk of extinction

through hybridization." Conservation Biology 15(2): 1039-1053.

34

Appendix 1 Taxa, Accession Numbers and Citations

Taxa Accession Number Reference Cypripedium x favillianum -- Walsh C. candidum EU814921 Walsh C. parviflorum var. pubescens EU814920 Walsh C. reginae EF370090 Shefferson et al. 2007 C. montanum EF370091 Shefferson et al. 2008 C. segawai Z78520 Cox et al. 1997 C. calceolus AB176594 Jo et al. 2005 C. macranthon AB176593 Jo et al. 2006 C. himalaicum EF370096 Shefferson et al. 2008 C. flavum Z78517 Cox et al. 1997 C. passerinum Z78516 Cox et al. 1998 C. lichiangense Z78529 Cox et al. 1999 C. margaritaceum Z78530 Cox et al. 2000 C. plectrochilum Z78528 Cox et al. 2001 C. acaule EF370099 Shefferson et al. 2008 C. japonicum EF370093 Shefferson et al. 2009 C. formosanum Z78524 Cox et al. 2001 C. californicum EF370095 Shefferson et al. 2009 C. guttatum EF370096 Shefferson et al. 2009 C. yatabeanum Z78527 Cox et al. 2001 C. fasciculatum EF370098 Shefferson et al. 2009 C. irapeanum Z78533 Cox et al. 2001 C. debile EF370097 Shefferson et al. 2009 Phragmipedium kovachii AY918821 Whitten et al. 2005 P. lindleyanum EF156164 Tsai et al. 2006 P. longifolium Z78508 Cox et al. 2001 P. caricinum AY918822 Whitten et al. 2005 P. besseae EF156162 Tsai et al. 2006 P. pearcei EF156163 Tsai et al. 2006

35

Appendix 2 ITS Sequences

C. parviflorum var. pubescens (15 samples):

GGTGGACTTGTGGTTACTCAGCTCGCCATAGGCTTTGCTTTTGCGGTGACCCT

AATTTGTCATTGGGCCTCCTCCAAAGCTTTCCTTGTGGGTTTGAACCTCTAGC

ACGGTGCAGTATGCACCAAGTCATATGAAGCATTGCCGATGAATGACATTAT

TGTCAAAAAGTTGGAGTGGAAGCGTGCTACTGCATGCATGCAAATGAATTTT

TTTATGACTCTCGACAACGGATATCTTGGCTCTTGCATCGATGAAGAACGCAG

CGAAATGCGATAAGTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTT

TGAACGCAAGTTGCGCCCGATGCCATCAGGCTAAGGGCACGCCTGCCTGGGC

GTCGTGTGCTGCGTCTCTCCTGTCAATGCTTTCCCATCATATAGATAGGTTTGC

ATTGTGTGGATGTGAAAGATTGGCCCCTTGTGCCTAGGTGCGGTGGGTCTAA

GAACTTAATGTTTTGATGGTTCGAAACCTGGCAGGAGGTGGAGGATGTTGGC

AGCTATATAAGGCTATCATTTGAATCCCCCAATATTGTCGCGTTTGTTGGACC

TAGAGAAGAACATGTTTGAATCCCAATGGAGGCAAACAACCCTCGGGCGGTT

GATTGCCATTCATAT

36

Appendix 2 (Continued)

C. candidum (15 samples):

GGTGGACTTGTGGTTACTCAGCTCGCCATAGGCTTTGCTTTTGCGGTGACCCT

AATTTGTCATTGGGCCTCCTCCAAAGCTTTCCTTGTGGGTTTGAACCTCTAGC

ACGGTGCAGTATGCACCAAGTCATATGAAGCATCGCCGATGAATGACATTAT

TGTCAAAAAGTTGGAGTGGAAGCGTGCTACTGCATGCATGCAAATGAATTTT

TTTATGACTCTCGACAACGGATATCTTGGCTCTTGCATCGATGAAGAACGCAG

CGAAATGCGATAAGTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTT

TGAACGCAAGTTGCGCCCGATGCCATCAGGCTAAGGGCACGCCTGCCTGGGC

GTCGTGTGCTGCGTCTCTCCTGTCAATGCTTTCCCATCATATAGATAGGTTTGC

ATTGTGTGGATGTGAAAGATTGGCCCCTTGTGCCTAGGTGCGGTGGGTCTAA

GAACTTAGTGTTTTGATGGTTCGAAACCTGGCAGGAGGTGGAGGATGTTGGC

AGCTATAAGGCTATCATTTGAATCCCCCAATATTGTCGTGTTTGTTGGACCTA

GAGAAGAACCTGTTTGAATCCCAATGGGAGGCAAACAACCCTCGGGCGGTTG

ATTGCCATTCATAT