Hybridization Between Castilleja Levisecta and C. Hispida: Implications for Pacific Northwest Prairie Management

AN ABSTRACT OF THE THESIS OF

Isaac Jerome Sandlin III for the degree of Master of Science in Botany and Plant Pathology presented on March 15, 2018.

Title: Hybridization between Castilleja levisecta and C. hispida: Implications for Pacific Northwest Prairie Management

Abstract approved: ______Thomas N. Kaye

Conservation conflicts may develop on restoration sites with multiple species recovery objectives. For example, on Pacific Northwest prairies, the co-planting of the diploid cytotype of the common native wildflower Castilleja hispida with the endangered wildflower C. levisecta has resulted in putative Castilleja hybrids on restoration sites, prompting fears that genetic swamping could threaten C. levisecta. Because C. hispida is a larval host for the endangered

Taylor’s checkerspot butterfly (Euphydryas editha taylori), this situation puts the recovery of both of these species at risk. However, hybrid fertility in this system is unknown.

To assess hybrid fertility and introgression in Castilleja hybrids, we conducted a series of controlled reciprocal crosses between C. levisecta and C. hispida, and backcrosses between F1 hybrids and their progenitors. We measured the resulting fruit set, seed set, and seed germination to look at post pollination barriers to reproduction. Because populations of C. hispida can be diploid (2n = 2x = 24), tetraploid (2n = 4x = 48), or hexaploid (2n = 6x = 72), and C. levisecta is only diploid (2n = 2x = 24), this project explores mixed ploidy crosses between the two species.

Reproductive isolation from C. levisecta was between 32 – 61% in crosses between C. levisecta and diploid C. hispida, 89 – 100% in C. levisecta and tetraploid C. hispida crosses, and

98 – 99% in C. levisecta and hexaploid C. hispida crosses. Reproductive isolation from C. levisecta was between -22 – 0% in backcrosses between C. levisecta and diploid F1 hybrids, suggesting higher diploid hybrid fitness. Reproductive isolation from C. levisecta was between

99 – 100% in backcrosses between C. levisecta and triploid F1 hybrids, suggesting interploidy crosses act as a barrier to gene flow.

In addition, in order to prevent introgression, the eradication of putative hybrids is critically important to the conservation of the C. levisecta genome, but hybrids can appear extremely morphologically close to either parental species, or highly distinct. Because ploidy differences between two interspecific mating partners can be a potent isolation mechanism in plants, land managers might choose to co-plant a polyploid C. hispida cytotype with C. levisecta at recovery sites in order to mitigate hybridization between these two species, but little morphological data exists on C. hispida cytotypes. To better identify hybrids and C. hispida polyploids, we looked at whether measurable differences are detectable in 15 bract, calyx, and floral characteristics of C. levisecta, three cytotypes of C. hispida, and their diploid F1 hybrids, and if so, whether those differences are distinct enough for field technicians to distinguish them.

Using multivariate analyses and univariate ANOVA, we found groups of traits that distinguished between hybrids and their progenitors, and C. hispida cytotypes, that could prove useful for field biologists.

Hybridization between Castilleja levisecta and C. hispida: Implications for Pacific Northwest Prairie Management

by Isaac Jerome Sandlin III

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Presented March 15, 2018 Commencement June 2018

Master of Science thesis of Isaac Jerome Sandlin III presented on March 15, 2018

APPROVED:

Major Professor, representing Botany and Plant Pathology

Head of the Department of Botany and Plant Pathology

Dean of the Graduate School

I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request.

Isaac Jerome Sandlin III, Author

ACKNOWLEDGEMENTS

I would first like to thank our many collaborators in this project: Peter Dunwiddie, Sierra Smith and the staff of the Center for Natural Lands Management in Olympia, WA for general guidance, help, and sourcing and collecting seeds for the C. hispida parent generation; Jeremy Fant, Kay Havens, Pattie Vitt, Andrea Kramer, and the Plant Science and Conservation Staff at the Chicago Botanic Gardens for guidance and help; Amy Bartow and the staff of the USDA NRCS Plant Materials Center, Corvallis, OR for teaching me how to grow Castilleja; Sean Logan, Gloria O’Brian, James Ervin, and the staff at the Oregon State University greenhouses for keeping my plants warm, happy, and pest free; Ryan Contreras and the good folks at the Contreras Lab for access to and guidance with flow cytometry; and Dale Brown and the staff of the OSU Seed Lab for cold stratification and germination resources, and all other seed-related knowledge and know-how.

So many people have provided personal and professional help, assistance, and inspiration to me over the course of this project. Foremost, I’d like to thank my father, Jerry Sandlin, for providing many, many years of support and patience while I pursued a “sorta funny” profession in botany (he’s an engineer...); Caitlin Lawrence and Andrew Esterson for watering my plants, and being great friends; Sarai Carter for being my closest friend and confidant during the hardest times, and helping me count so many seedlings; Danielle Agular, Christina Partipilo, and Camille Eckel for helping with plant care and counting seeds; and all of my fellow graduate students at the Department of Botany and Plant Pathology at OSU, I couldn’t have done this without their support.

The opportunity for me to study botany at OSU simply would not have been possible without serious help from colleagues and mentors: Dr. Johnny Randall and the staff at the North Carolina Botanical Garden, who gave me my first job out of university and taught me all about plant conservation; Dr. Ed Guerrant, director of the Rea Selling Berry Seed Bank & Plant Conservation Program, who convinced me that I was indeed cut out for graduate school (and introduced me to my future major professor); and, of course, Dr. Tom Kaye, executive director of the Institute for Applied Ecology, my major professor, and my mentor of many years, who has provided me with endless opportunities. Finally, this thesis is dedicated to TLH, my heart…

TABLE OF CONTENTS

Page

Chapter 1: Introduction ...... 1 Pacific Northwest Prairies ...... 2 Taylor’s Checkerspot Butterfly ...... 3 The Castilleja System ...... 4 Research Goals and Objectives ...... 5 Literature Cited ...... 6

Chapter 2: Hybridization between Castilleja levisecta and C. hispida: Implications for Rare Plant and Butterfly Management ...... 9 Abstract ...... 10 Introduction ...... 11 Materials and Methods ...... 16 PARENT PLANT MATERIAL ...... 16 PLOIDY and GENOME SIZING ...... 17 SEED GERMINATION ...... 18 GREENHOUSE CULTURE ...... 19 CREATION OF F1 HYBRIDS ...... 19 ASSESSMENT OF F1 HYBIRD FERTILITY ...... 21 FITNESS MEASURES ...... 22 Results ...... 27 PLOIDY DIFFERENCES ...... 27 FRUIT SET, SEED SET, and SEED GERMINATION ...... 28 REPRODUCTIVE ISOLATION BETWEEN C. LEVISECTA and F2 HYBRIDS ...... 34 Discussion ...... 36 HOMOPLOID CROSSES ...... 38 HETEROPLOID CROSSES ...... 38 MANAGEMENT IMPLICATIONS ...... 41 Literature Cited ...... 42

Chapter 3:Morphological Analysis of the endangered wildflower Castilleja levisecta, a native congener C. hispida, and their F1 hybrids ...... 51 Abstract ...... 52 Introduction ...... 53

Page

Materials and Methods ...... 57 PARENT PLANT MATERIAL ...... 57 SEED GERMINATION ...... 57 GREENHOUSE CULTURE ...... 58 CYTOLOGY ...... 59 CREATION OF F1 HYBRIDS ...... 61 MORPHOMETRICS ...... 61 STATISTICAL ANALYSIS ...... 63 Results ...... 64 CYTOLOGY ...... 64 THE HYBRID GROUP ...... 65 THE CAHI IPV GROUP ...... 72 Discussion ...... 79 THE HYBRID GROUP ...... 79 THE CAHI IPV GROUP ...... 80 Future Directions ...... 82 Conservation Implications ...... 83 Literature Cited ...... 84

Chapter 4: Conclusions ...... 90 Crosses between C. levisecta and diploid cytotypes of C. hispida ...... 91 Crosses between C. levisecta and polyploid cytotypes of C. hispida ...... 91 Morphological analysis of C. levisecta, diploid C. hispida, and their F1 Hybrids ...... 92 Morphological analysis of three C. hispida cytotypes ...... 93 Literature Cited ...... 94

Bibliography ...... 96 APPENDIX ...... 109

LIST OF FIGURES

Figure Page

2.1 Fruit set, seed set, and germination percentage F1 hybrid crosses relative to conspecific C. levisecta crosses...... 22

2.2 Fruit set, seed set, and germination percentage F2 hybrid crosses relative to conspecific C. levisecta crosses...... 31

3.1 Illustration of Castilleja bract, calyx, and corolla...... 58

3.2 Principal component analysis of 10 morphological traits from diploid C. hispida, C. levisecta, and their F1 diploid hybrids...... 63

3.3 Linear discriminant analysis of 10 morphological traits, from diploid C. hispida, C. levisecta, and their F1 diploid hybrids...... 65

3.4 Morphological differences between C. levisecta, diploid C. hispida, and their hybrids...... 67

3.5 Principal component analysis of seven morphological traits sampled from diploid C. hispida, triploid C. hispida, and hexaploid C. hispida...... 69

3.6 Linear discriminant analysis of seven morphological traits sampled from diploid C. hispida, triploid C. hispida, and hexaploid C. hispida...... 71

3.7 Differences between three cytotypes of C. hispida for calyx pubescence length, corolla length, and galea length...... 73

3.8 Differences between three cytotypes of C. hispida for floral angle and stigma diameter...... 73

LIST OF TABLES

Table Page

2.1 Taxa, origin populations, number of maternal lines, and number of plants from each maternal line used in the study...... 17

2.2 Number of crosses performed between C. levisecta and C. hispida in order to create F1 hybrids...... 20

2.3 Number of F2 crosses between C. levisecta, diploid hybrids, and triploid hybrids...... 22

2.4 Number of F1 crosses between C. levisecta and diploid C. hispida, tetraploid C. hispida, and hexaploid C. hispida...... 22

2.5 2C DNA content and monoploid genome size of Castilleja populations and hybrids....27

2.6 Fruit set, seed set, and germination percentage for F1 crosses...... 28

2.7 Fruit set, seed set, and germination percentage for F2 crosses...... 30

2.8 Results of ANOVAs to determine whether crossing treatment influenced the number of seeds per fruit or the proportion of seeds that germinated...... 31

2.9 Measures of reproductive isolation (RI) for F1 crosses...... 32

2.10 Measures of reproductive isolation (RI) for F2 crosses ...... 33

3.1 Taxa, origin populations, number of maternal lines, and number of plants from each maternal line used in the study ...... 54

3.2 2C DNA content and monoploid genome size of Castilleja populations and hybrids....60

3.3 Results of principal components analysis, and linear discriminant analysis of morphological variation between C. levisecta, C. hispida and their hybrids...... 62

3.4 Mean values ± S.E. for morphological characters in C. levisecta, diploid F1 hybrids, and diploid C. hispida...... 66

3.5 Results of principal components analysis, and linear discriminant analysis of morphological variation between three ploidy races of C. hispida...... 68

3.6 Mean values ± S.E. for morphological characters in diploid, triploid, and hexaploid C. hispida...... 72

LIST OF APPENDIX TABLES

Table Page

2.11 Fruit set, seed set, and germination percentage F1 hybrid crosses relative to conspecific C. levisecta crosses...... 106

2.12 Fruit set, seed set, and germination percentage F1 hybrid crosses relative to conspecific C. levisecta crosses...... 107

Chapter 1:

Introduction

Pacific Northwest Prairies

The prairies and oak savannahs of the Willamette Valley-Puget Trough-Georgia Basin

(WPG) ecoregion are among the most endangered ecosystems in the region (Floberg et al., 2004;

Noss et al., 1995). For ca. 10,000 years, these landscapes were maintained through frequent, low- intensity burning for food, hunting, and other material resources by Native peoples (Whitlock and Knox, 2002; Walsh et al., 2010; Hamman et al., 2011). Today, fire suppression, species invasion, conifer encroachment, habitat fragmentation, and development have contributed to a dramatic decline in prairie oak obligate species (Dennehy et al., 2011), and increased concerns for what Kearns et al. (1998) termed “endangered mutualisms” with regard to lost host/pollinator interactions.

The field of restoration ecology attempts to reverse habitat degradation, increase biodiversity and supplement ecosystem services. In the WPG ecoregion, this often means restoring small, highly-fragmented parcels of remnant prairie – a situation that can be especially consequential for the conservation and recovery of rare species. Because so little is known about the biology, ecology, and reason behind species rarity (Dunwiddie et al., 2016), this deficit in knowledge can expose heretofore unknown or understudied antagonistic interactions on restoration sites where multiple species recovery efforts take place. This is the scenario currently playing out in the south Puget Sound region of Washington State, where the federally threatened wildflower Castilleja levisecta (golden paintbrush) is sometimes co-planted with a native congener, C. hispida (harsh paintbrush). Starting in 2007, C. hispida was planted in close proximity and in high densities for the benefit of Taylor’s checkerspot butterfly (Euphydryas editha taylori), another iconic endangered species of the Pacific Northwest. 3

Though C. levisecta and C. hispida share a historic range, the only documented co- occurrence was at Rocky Prairie in Thurston County in the 1980s (Kaye and Blakeley-Smith,

2008). More recently, the restoration process has brought these two species into close proximity on remnant prairies because the listing status of C. levisecta requires a minimum of twenty populations of at least 1,000 flowering individuals on protected land (USFWS, 2000, 2010;

Caplow, 2004; Clark, 2015). These same prairies are important for the recovery of Taylor’s checkerspot, and C. hispida is often included in prairie restoration seedings where the endangered butterfly is slated for recovery.

Taylor’s Checkerspot Butterfly

Taylor’s checkerspot is a subspecies of Edith’s checkerspot (E. editha) belonging to the family Nymphalidae, or Brush-footed butterflies (Stinson, 2005). As recently as 50 years ago, the species could be frequently encountered in the glacial outwash prairies, low elevation balds and coastal grasslands from south British Columbia to central Oregon (Severns and Grosboll,

2011) . However, contemporary populations have declined dramatically and the butterfly can be found in only 13 extant sites ranging in size from a few to 1000+ individuals (Black and

Vaughan, 2005).

Larval food preference for checkerspots varies across the closely related Orobanchaceae and Plantaginaceae plant families. Historically, Taylor’s checkerspot may have relied on several native species that occur in lowland prairies, including C. hispida, C. levisecta, Plectritis congesta, Collinsia parviflora, Collinsia grandiflora, and species of Veronica as larval host plants, but at some point, the butterflies were able to switch hosts in many sites to a common invasive plant, English plantain (Plantago lanceolata) (Vaughan and Black, 2002; Aubrey, 4

2013). Some land managers today are reluctant to plant an invasive weed as part of prairie restorations to support the butterfly, and instead have widely planted C. hispida.

The Castilleja System

Castilleja levisecta and C. hispida are both short-lived perennial forbs in the family

Orobanchaceae, with overlapping geographic species distributions. As with other Castilleja species, they are facultative hemi-parasites but are capable of reproduction without a host plant

(Wentworth, 2001). Most Castilleja species are mostly or entirely self-incompatible, producing minimal seed without cross-pollination via animal pollinators (Kaye and Lawrence, 2003).

Castilleja levisecta is known to be a self-incompatible species primarily pollinated and out- crossed by Bombus spp. (bumble-bees) and halictids (sweat bees), and is known to reproduce only by seed (Wentworth, 2001). Chromosomal analyses have only found golden paintbrush in a diploid form (2n = 2x =24), whereas C. hispida exists in diploid (2n = 2x =24), tetraploid (2n =

4x = 48), and hexaploid (2n = 6x = 72) forms (Kaye and Blakeley-Smith, 2008).

The current C. levisecta geographical distribution is quite narrow, and its habitat is limited to the glacial outwash prairies and coastal balds of the Puget Trough of Washington and southwestern British Columbia, and in the remnant prairies of the Willamette Valley in Oregon.

The historical geographic distribution and habitat preference of the species in unclear due to agricultural conversion of the WPG ecoregion following European settlement. It was last collected in the Willamette Valley in 1938 and is considered extirpated from the state of Oregon

(Gamon, 1995), though reintroduction is currently underway. Until reintroduction began in 2006, only 11 extant C. levisecta populations remained in Washington and British Columbia. Castilleja hispida is much more common and widespread in its range, and inhabits open meadows, oak woodlands, forest openings, and grassy slopes from California, east to Idaho and Montana, and 5 north to Alberta and Vancouver Island in Canada.

Castilleja species hybridize readily and can experience subsequent genome duplication, which causes speciation to commonly take place within the genus (Heckard and Chuang, 1977).

The base haploid chromosome number for the genus is n = 12, but ploidal variation is broad both within and among species (Heckard, 1968; Heckard and Chuang, 1977). In sympatric Castilleja species, ploidal differences might be the only barrier to reproduction besides pollinator selection in hybrid zones (Heckard and Chuang, 1977). The Castilleja genus is considered to be recently diverged, and most species share morphological characteristics that often make field identification challenging (Tank and Olmstead, 2008). Therefore, hybridization is often inferred from field observations but verified via cytological studies or molecular markers (Heckard and

Chuang, 1977; Tank et al., 2009).

At restoration sites where C. levisecta and C. hispida have been planted, biologists have documented the presence of abundant spontaneous putative hybrids. This hybridization creates a threat to C. levisecta and its recovery through introgression (Levin et al., 1996), possibly altering the genetic makeup of the species.

Research Goals and Objectives

To assess the hybridization threat to C. levisecta, we performed a series of reciprocal greenhouse crosses between C. levisecta, three cytotypes of C. hispida, and their F1 hybrids. We combined these studies with post pollination fitness measures and a morphometric analysis to assess hybrid fertility, relative fitness, and morphological differentiation within the C. levisecta x

C. hispida system. Specifically, we asked the following questions related to hybrid fertility and fitness:

1. Are hybrids between C. levisecta and C. hispida fertile? 6

2. Does hybrid fertility depend on the difference in ploidy between the parental individuals?

3. Does reproductive isolation between C. levisecta and C. hispida increase as C. hispida

ploidy increases?

Related to morphological analyses we asked:

1. Are the floral characteristics of C. levisecta, diploid C. hispida, and their hybrids distinct

enough for measurable differences to distinguish them?

2. Is there morphological evidence of differences in ploidy between the three cytotypes of

C. hispida?

Literature Cited

AUBREY, D. 2013. Oviposition Preference in Taylor’s Checkerspot Butterflies (Euphydryas editha taylori): Collaborative Research and Conservation with Incarcerated Women. Thesis. The Evergreen State College.

BLACK, S., and D. VAUGHAN. 2005. Species Profile: Euphydryas editha taylori . Xerces Society for Invertebrate Conservation.

CAPLOW, F. 2004. Reintroduction Plan for Golden Paintbrush (Castilleja levisecta). Washington Natural Heritage Program, Olympia, WA.

CAROL A. KEARNS, DAVID W. INOUYE, and N.M. WASER. 1998. Endangered Mutualisms: The Conservation of Plant-Pollinator Interactions. Annual Review of Ecology and Systematics 29: 83–112.

CLARK, L.A. 2015. Bee-crossed lovers and a forbidden Castilleja romance: Cross-breeding between C. hispida and endangered C. levisecta in prairie restoration sites. Thesis. The University of Washington.

DENNEHY, C., E.R. ALVERSON, H.E. ANDERSON, D.R. CLEMENTS, R. GILBERT, and T.N. KAYE. 2011. Management Strategies for Invasive Plants in Pacific Northwest Prairies, Savannas, and Oak Woodlands. Northwest Science 85: 329–351.

DUNWIDDIE, P.W., N.L. HAAN, M. LINDERS, J.D. BAKKER, C. FIMBEL, and T.B. THOMAS. 2016. Intertwined Fates: Opportunities and Challenges in the Linked Recovery of Two Rare Species. Natural Areas Journal 36: 207–215. 7

FLOBERG, J., T. HORSMAN, D. ROLPH, P. SKIDMORE, G. WILHERE, C. CHAPPELL, P. LACHETTI, ET AL. 2004. Willamette Valley - Puget Trough - Georgia Basin Ecoregional Assessment.

GAMON, J. 1995. Report on the Status of Castilleja levisecta. Washington Natural Heritage Program, Department of Natural Resources, Olympia, WA.

HAMMAN, S.T., P.W. DUNWIDDIE, J.L. NUCKOLS, and M. MCKINLEY. 2011. Fire as a Restoration Tool in Pacific Northwest Prairies and Oak Woodlands: Challenges, Successes, and Future Directions. Northwest Science 85: 317–328.

HECKARD, L.R. 1968. Chromosome Numbers and Polyploidy in Castilleja (Scrophulariaceae). Brittonia 20: 212–226.

HECKARD, L.R., and T.-I. CHUANG. 1977. Chromosome Numbers, Polyploidy, and Hybridization in Castilleja (Scrophulariaceae) of the Great Basin and Rocky Mountains. Brittonia 29: 159–172.

KAYE, T.N., and M. BLAKELEY-SMITH. 2008. An Evaluation of the Potential for Hybridization Between Castilleja levisecta and C. hispida. Research Gate. Available at: https://www.researchgate.net/publication/265478592_An_Evaluation_of_the_Potential_f or_Hybridization_Between_Castilleja_levisecta_and_C_hispida [Accessed November 30, 2016].

KAYE, T.N., and B.A. LAWRENCE. 2003. Fitness Effects of Inbreeding and Outbreeding on Golden Paintbrush (Castilleja levisecta): Implications for recovery and reintroduction. Washington Department of Natural Resources and Institute for Applied Ecology.

LEVIN, D.A., J. FRANCISCO-ORTEGA, and R.K. JANSEN. 1996. Hybridization and the Extinction of Rare Plant Species. Conservation Biology 10: 10–16.

NOSS, R.F., E.T. LAROE, and J.M. SCOTT. 1995. Endangered Ecosystems of the United States: A Preliminary Assessment of Loss And Degradation. US Dept. of Interior, Washington, D.C.

SEVERNS, P., and D. GROSBOLL. 2011. Patterns of reproduction in four Washington State Populations of Taylor’s checkerspot (Euphydryas editha taylori) During the Spring of 2010. The Nature Conservancy, Olympia Washington.

STINSON, D.W. 2005. Washington State Status Report for the Mazama Pocket Gopher, Streaked Horned Lark, and Taylor’s Checkerspot. Washington Department of Fish and Wildlife, Olympia, WA. Available at: https://wdfw.wa.gov/publications/00390/wdfw00390.pdf [Accessed February 26, 2018].

TANK, D.C., J.M. EGGER, and R.G. OLMSTEAD. 2009. Phylogenetic Classification of Subtribe Castillejinae (Orobanchaceae). Systematic Botany 34: 182–197.

TANK, D.C., and R.G. OLMSTEAD. 2008. From Annuals to Perennials: Phylogeny of Subtribe Castillejinae (Orobanchaceae). American Journal of Botany 95: 608–625. 8

USFWS. 2000. Recovery Plan for the Golden Paintbrush (Castilleja levisecta). U.S. Fish and Wildlife Service, Portland, OR.

USFWS. 2010. Recovery Plan for the Prairie Species of Western Oregon and Southwestern Washington. U.S. Fish and Wildlife Service, Portland, OR.

VAUGHAN, M., and S. BLACK,. 2002. Petition to Emergency List Taylor’s (whulge) Checkerspot Butterfly (Euphydryas editha taylori) as an Endangered Species Under the U.S. Endangered Species Act. The Xerces Society. Available at: http://www.xerces.org/wp- content/uploads/2008/09/taylors_checkerspot_petition.pdf [Accessed January 21, 2018].

WALSH, M.K., C. WHITLOCK, and P.J. BARTLEIN. 2010. 1200 Years of Fire and Vegetation History in the Willamette Valley, Oregon and Washington, Reconstructed Using High- Resolution Macroscopic Charcoal and Pollen Analysis. Palaeogeography, Palaeoclimatology, Palaeoecology 297: 273–289.

WENTWORTH, J.B. 2001. The demography and population dynamics of Castilleja levisecta, a federally threatened perennial of Puget Sound Grasslands. In R. S. Reichard, P. Dunwiddie, J. Gamon, A. Kruckeberg, and D. Salstrom [eds.], Conservation of Washington’s Native Plants and Ecosystems., 49–51. Washington Native Plant Society, Seattle, WA.

WHITLOCK, C., and M. KNOX. 2002. Prehistoric burning in the pacific northwest: human versus climatic influences. In T. Vale [ed.], Fire, Native Peoples, and the Natural Landscape, Island Press, Washington, UNITED STATES. Available at: http://ebookcentral.proquest.com/lib/osu/detail.action?docID=3317362 [Accessed February 22, 2018]

Chapter 2:

Hybridization between Castilleja levisecta and C. hispida: Implications for Rare Plant and Butterfly Management

Abstract

Conservation conflicts may develop on restoration sites with multiple species recovery objectives. For example, the recovery of Taylor’s checkerspot butterfly (Euphydryas editha taylori) and Castilleja levisecta (golden paintbrush), two federally listed species, have come into conflict on several remnant prairies in southwest Washington. This conflict arises from the planting of C. hispida (harsh paintbrush), which is a favored pre-diapause larval host plant for

Taylor’s checkerspot, as well as C. levisecta, which has been introduced in recovery efforts in some of the same sites. Recently, biologists have confirmed putative Castilleja hybrids on recovery sites, prompting fears that genetic swamping could threaten C. levisecta.

To assess hybrid fertility and introgression, we conducted a series of controlled reciprocal crosses between C. levisecta and C. hispida, and backcrosses between F1 hybrids and their progenitors. We measured the resulting fruit set, seed set, and seed germination to look at post pollination barriers to reproduction. Because populations of C. hispida can be diploid (2n = 2x =

24), tetraploid (2n = 4x = 48), or hexaploid (2n = 6x = 72), and C. levisecta is known only to be diploid (2n = 2x = 24), this project explores mixed ploidy crosses between the two species.

Reproductive isolation from C. levisecta was between 32 – 61% in crosses between C. levisecta and diploid C. hispida, 89 – 100% in C. levisecta and tetraploid C. hispida crosses, and

99 – 100% in backcrosses between C. levisecta and triploid F1 hybrids, suggesting interploidy crosses act as a barrier to gene flow. 11

Hybrid crosses between the diploid types pose a potential threat to C. levisecta through introgression and genetic swamping and should not be co-planted. Crosses with polyploid C. hispida have lower risk of successful hybridization, but more research is needed before co- planting these taxa.

Introduction

Habitat destruction is a significant threat to biodiversity (Leemans and Groot, 2003;

Giam et al., 2010). As a result, the number of endangered species has increased (Pimm et al.,

2014), while habitat to support their recovery has decreased. In reaction, the field of restoration ecology is experiencing rapid growth as humanity grapples with the unforeseen environmental consequences of its success, and strives to make amends (Zedler, 1999; D’Antonio and

Meyerson, 2002; Suding, 2011; Suding et al., 2015). Reintroduction of rare species has emerged as one possible solution to biodiversity loss (Maunder, 1992; Hodder and Bullock, 1997; Rout et al., 2009; Godefroid et al., 2011), but we often do not understand the reasons behind a species’ rarity, let alone its biology or ecology (Dunwiddie et al., 2016). These knowledge gaps can create confusion and tension among agencies and managers working to prioritize recovery efforts, and can even lead to deleterious interactions between cohabitating endemic species (Lee et al., 2007; Oro et al., 2009; Gumm et al., 2011; Chadès et al., 2012; Raimondi et al., 2015).

Habitat scarcity coupled with the drive to protect endemic species from extinction promotes the crowding of multiple species recovery efforts onto scarce habitat fragments. This process can expose hitherto unknown or understudied antagonistic species interactions and lead to conservation conflicts among protected species. These antagonistic interactions between protected species usually focus on competition for resources (Lee et al., 2007; Oro et al., 2009) or predator-prey dynamics (Gumm et al., 2011; Chadès et al., 2012; Raimondi et al., 2015). 12

However, there are very few examples in the literature of hybridization driving a conservation conflict between protected species (Riley et al., 2003; Fredrickson and Hedrick, 2006;

Fitzpatrick and Shaffer, 2007; Fitzpatrick et al., 2010, 2015).

In angiosperms, hybridization is commonly known as an agent of diversification, and plays a central role in the expansion of phylogenetic lineages, development of novel traits, and/or introgression of traits across species boundaries (Anderson, 1953; Grant, 1981; Abbott, 1992;

Arnold, 1997; Rieseberg, 1997; Ellstrand and Schierenbeck, 2000; Mallet, 2007; Soltis and

Soltis, 2009; Abbott et al., 2013; Frankham, 2015). Plant hybrids can also have ecological and evolutionary impacts on their associated communities and ecosystems (Whitham et al., 1999,

2006). Therefore, hybridization can play an important role in the evolution and ecology of plant taxa, but its importance varies by taxon and location (Ellstrand et al., 1996).

Despite its role as catalyst for diversification, hybridization can also decrease diversity when reproductive barriers are eased or eliminated, resulting in the combination of previously separate evolutionary lines. This process can ultimately result in the extinction of populations or species (Vuillaume et al., 2015; Rieseberg et al., 1989; Ellstrand, 1992; Levin et al., 1996;

Rhymer and Simberloff, 1996; Allendorf et al., 2001; Buerkle et al., 2003; Todesco et al., 2016).

Human activities are exacerbating these genetic threats to biodiversity by bringing previously isolated populations and species into contact, thus promoting gene flow that had previously been precluded. Such novel genetic interchange can contribute to species decline and extinction via genetic swamping or demographic swamping (Wolf et al., 2001; Kramer and Havens, 2009;

Todesco et al., 2016). Genetic swamping is a process through which one or more distinct evolutionary lineages are replaced by hybrids, and occurs in systems without severe outbreeding depression, that is, in systems where hybrid fitness is increased relative to that of the parental 13 individuals (Todesco et al., 2016). In occurrences of genetic swamping, the hybrid population growth rate exceeds replacement rate, thus replacing one or both parental species with hybrids.

Demographic swamping takes place in systems where hybridization is common, but hybrid fitness is strongly reduced via outbreeding depression. In this case, if population growth rates of the parental species decline below replacement rates, extinction of one or both conspecifics may occur due to wasted reproductive effort (Wolf et al., 2001). Species decline might be attributed to both genetic and demographic swamping, and it may be difficult to distinguish between them

(Wolf et al., 2001). These mechanisms are particularly important to conservation biologists because human-mediated artificial gene flow can provide an opportunity for common congeners to alter rare and endangered species’ genomes, thus resulting in lower fitness, extirpation, or extinction (Levin et al., 1996; Rhymer and Simberloff, 1996; Wolf et al., 2001; Maunder, 2004).

Polyploidy, when coupled with interspecific hybridization, can be both a driver of angiosperm diversity (Wood et al., 2009), and a potent isolating mechanism (Stebbins, 1950; de

Wet, 1971; Grant, 1981; Otto and Whitton, 2000; Ramsey and Schemske, 2002). Defined as the possession of three or more sets of chromosomes, polyploidy is estimated to occur in 30 to 80% of plant species (Otto and Whitton, 2000; Soltis et al., 2014; Barker et al., 2016), and can arise either from crosses within or between populations of a single species (autopolyploidy), or from hybrids between species (allopolyploidy). In addition, hybridization can occur between species with or without a change in chromosome number. The latter is referred to as homoploid hybridization (Welch and Rieseberg, 2002; Rieseberg et al., 2003). Ploidy difference between two interspecific mating partners can be a potent isolation mechanism in plants because offspring often have an odd number of homologous chromosomes (Ramsey and Schemske, 1998; Levin,

2002; Comai, 2005; Köhler et al., 2010), rendering them sterile, inviable, or inferior to their 14 progenitors (Stebbins, 1950; de Wet, 1971; Grant, 1981; Otto and Whitton, 2000; Ramsey and

Schemske, 2002; Köhler et al., 2010; Pekkala et al., 2012). Postzygotic hybrid inviability in interploidy crosses arise from a process known as “triploid block,” which is manifested in the endosperm, a region particularly sensitive to ploidy misbalances (Köhler et al., 2010). In wild populations, if individuals with an odd number of chromosomes survive to reproductive maturity, they are often subject to frequency dependent mating disadvantages in a process known as minority cytotype exclusion (Levin, 1975; Husband, 2000). That said, despite being recognized as a major evolutionary and ecological force in plants, we know very little about how ploidy variation in interspecies crosses affects realized gene-flow (Soltis et al., 2010).

The Castilleja genus is an ideal system for studying how ploidy affects gene flow and hybridization because homoploid hybridization (Clay et al., 2012), autopolyploidy, and allopolyploidy (Heckard and Chuang, 1977; Heckard et al., 1980; Mathews and Lavin, 1998;

Hersch-Green and Cronn, 2009; Clay et al., 2012; Hersch-Green, 2012) have been shown to contribute to the diversification of the genus. Furthermore, species within this genus are known to be highly promiscuous, and have been shown to hybridize readily where two or more species overlap in range (Egger, 1994; Hersch and Roy, 2007; Hersch-Green and Cronn, 2009; Clay et al., 2012). In addition, Castilleja species can be composed of multiple ploidy races (Heckard and

Chuang, 1977; Heckard et al., 1980), and ploidy has been proposed as contributing to the evolution of discrete Castilleja species (Heckard and Chuang, 1977). Importantly, differences in chromosome number have been shown to shape, but not necessarily prevent, Castilleja hybridization (Hersch-Green, 2012).

Here, we examine how differences in chromosome number can affect interspecies gene flow in Castilleja using a unique study system that has recently developed in the Pacific 15

Northwest (PNW). Declining native PNW prairie habitat has led to a conservation conflict between the two federally-listed native species Castilleja levisecta (golden paintbrush;

Orobanchaceae) and Taylor’s checkerspot butterfly (Euphydryas editha taylori; Nymphalidae)

(Dunwiddie et al., 2016). The conflict originates in the dense co-planting of C. hispida, a native congener, on southwest Washington prairie restoration sites where the parallel but independent recovery efforts of C. levisecta and Taylor’s checkerspot take place. Castilleja hispida is readily incorporated into regional seed mixes because it is known to be a native larval host for Taylor’s checkerspot (Vaughan and Black, 2002; Aubrey, 2013). In 2007, the observation of putative

Castilleja hybrids prompted concern that hybridization could alter the rare C. levisecta genome via genetic swamping, thus jeopardizing its recovery. Like many paintbrush species, C. levisecta is diploid with a chromosome number of (2n = 2x = 24) (Godt et al., 2005; Kaye and Blakeley-

Smith, 2008). Most populations of C. hispida are also diploid, but the species also has polyploid races with populations fixed for ploidy level. These two species have been shown to hybridize in greenhouse experiments (Kaye and Blakeley-Smith, 2008), but hybrid fertility is unknown.

To explore Castilleja hybrid fertility and fitness in this system, we performed F1 and F2 reciprocal hand pollinations between C. levisecta, and diploid, tetraploid, and hexaploid races of

C. hispida, and resultant F1 hybrids in a greenhouse, then measured fruit set, seed set, seed germination, and the genome size of parental plants and their offspring. We performed these actions to investigate three hypotheses:

1. Crosses between C. levisecta (2n = 2x = 24) and diploid cytotypes of C. hispida (2n = 2x

= 24) will produce fertile hybrids.

2. Reproductive isolation from C. levisecta will be lower in crosses between C. levisecta

and the diploid C. hispida cytotype, and the diploid F1 hybrids, than crosses between C. 16

levisecta and polyploid cytotypes of C. hispida. Interploid crosses will have reduced

fitness and higher reproductive isolation due to meiotic problems associated with odd

numbers of chromosomes.

3. Fitness in interploidy crosses will be further reduced and reproductive isolation will

increase with a growing difference in ploidy between the interspecific crosses. This

hypothesis is based on the assumption that: 1) occasional spontaneous non-reduced

gametes in diploid plants are diploid, and reduced gametes in tetraploid plants are

diploid, therefore, some gene-flow could occur in these systems, and 2) reduced and non-

reduced gametes from diploid plants will fail to produce viable offspring when united

with triploid reduced gametes from hexaploid plants, therefore halting seed creation and

therefore gene-flow.

Materials and Methods

PARENT PLANT MATERIAL

Germplasm for the parental generation in this study was collected in August and

September of 2015. Castilleja levisecta (CALE) germplasm was obtained from NRCS Plant

Materials Center (PMC) in Corvallis, OR. The C. levisecta population at PMC Corvallis is part of a larger reintroduction taking place throughout the Willamette Valley, and is composed of a mixture of three Washington populations. Castilleja hispida (CAHI) seeds used in this study were collected from wild populations in southwestern Washington (Table 2.1), and all plant materials were collected by separate maternal lines.

Table 2.1 Taxa, origin populations, number of maternal lines, and number of plants from each maternal line used in the study.

Taxa Population Origin Population Maternal Plants Location Lines Included Acquired Castilleja levisecta NRCS Plant Materials Corvallis, OR 28 28 Center Castilleja hispida Training Area 14 Joint Base Lewis- 13 13 (JBLM) McChord, Pierce County, WA Castilleja hispida Scatter Creek Wildlife Thurston County, WA 4 10 Area Castilleja hispida Johnson Prairie Joint Base Lewis- 12 12 (JBLM) McChord, Pierce County, WA Castilleja hispida Wolf Haven Thurston County, WA 1 9 International Castilleja hispida Bald Hill Natural Area Thurston County, WA 5 5 Preserve Castilleja hispida Yellow Island San Juan County, WA 20 20

PLOIDY and GENOME SIZING

Of the six C. hispida population accessions received, two populations were thought to be of diploid origin and four populations were thought of be of tetraploid origin based on previous chromosome counts (Kaye and Blakeley-Smith, 2008). In order to estimate the ploidy level of each population of C. hispida included in the study, we used flow cytometry to estimate ploidy of a randomly selected sample of plants from each population of C. hispida.

We assessed the 2C genome size of individual Castilleja plants using flow cytometry compared to an internal standard. Terminology regarding ploidy and genome size follows that proposed by Greilhuber et al. (2005). To represent a random sample of nuclei, we prepared 1-2 cm2 tissue samples from three young, fully expanded leaves from each Castilleja plant chosen for ploidy analysis. We then co-chopped Castilleja samples with a 1-2cm2 sample of the internal standard Pisum sativum ‘Ctirad’, which has a known 2C genome size of 8.76 pg (Bai et al., 18

2012). We chopped samples with a razor in a polystyrene petri dish containing 400 µL of nuclei extraction buffer solution (Cystain Ultraviolet Precise P Nuclei Extraction Buffer; Sysmex,

Görlitz Germany). We then passed the buffer and chopped leaf tissue mixture through a 30-µm gauze filter (Partec Celltrics, Münster, Germany) into a 3.5-mL plastic tube (Sarstedt Ag & Co.,

Nümbrecht, Germany) and combined the mixture with 1.6 mL of fluorochrome stain (4’,6- diamidino-2-phenylindole) (Cystain Ultraviolet Precise P Staining Buffer; Partec). We used a flow cytometer to analyze a minimum of 3000 nuclei per sample (CyFlow Ploidy Analyzer;

Partec). We accepted samples only if average CV for each florescence histogram was under 10.

We calculated relative 2C genome size as:

mean florescence value of sample 2� = DNA Content of standard × mean florescence value of standard

Monoploid (1Cx) genome size was calculated as:

2C 1Cx = ploidy

SEED GERMINATION

To break seed dormancy, we cold stratified Castilleja seeds on moist germination paper

(Anchor Paper, St. Pail, MN, USA) kept in clear plastic boxes with tight fitting lids at 5°C for 6 weeks at the Oregon State University Seed Lab following the methods of Lawrence and Kaye

(2005). We kept seeds damp during cold stratification using deionized water. Once cold stratification was complete, we placed seeds in a germination chamber at alternating 15°C /25°C with alternating 12-hr dark, 12-hr light for seven days.

GREENHOUSE CULTURE

In January 2016, we randomly selected 100 C. levisecta seedlings and up to 40 C. hispida seedlings from each maternal line from the six population accessions to be placed into seedling flats using Metro-Mix 840 professional soil mix (Sungro Horticulture, Agwam, MA, USA).

Because Castilleja species are hemi-parasitic, seedlings were grown with Eriophyllum lanatum, which is a known host plant (Lawrence and Kaye, 2008). Seedling flats were placed into a climate controlled greenhouse at Oregon State University in Corvallis, OR. After six weeks, a random subset of seedlings with hosts were transplanted into 4L pots. We watered plants every other day, and fertilized every two weeks using Miracle Grow (24-8-16) at a rate of 2 grams per four liters of water. Parental plants reached reproductive maturity approximately 4 months after germination, in April 2016.

Plants were randomly distributed throughout the greenhouse and rotated to a different location in the greenhouse every two weeks in order to minimize differences in growth associated with greenhouse microclimates.

CREATION OF F1 HYBRIDS

To examine hybrid fertility between C. levisecta and the three cytotypes of C. hispida, we created F1 hybrids in May 2016 by establishing six interspecific cross types (Table 2.2).

Between six and twenty-five replicates for each cross type were performed.

Table 2.2 Number of 2016 crosses performed between C. levisecta (CALE) and C. hispida (CAHI) in order to create F1 hybrids. The first species in the cross type name indicates the maternal species.

2016 Heterospecific Cross Types n CALE(2x) x CAHI(2x) 23 CAHI(2x) x CALE(2x) 25 CALE(2x) x CAHI(4x) 14 CAHI(4x) x CALE(2x) 14 CALE(2x) x CAHI(6x) 25 CAHI(6x) x CALE(2x) 6

Half of the interspecific cross type replicates were repeated with the parent taxon as either the pollen recipient or the pollen donor in order to evaluate possible imbalances in gene flow directionality (Table 2.2). For each cross type replicate, plants were chosen at random without replacement. However, due to high parental plant mortality, some parental plants were re-used as either pollen donors or pollen recipients.

For each cross type replicate, pollen was harvested from a single flower on pollen donors and placed on the stigma of one single flower on pollen recipients. Because flowering in these two species occurs on multiple indeterminate racemes, we chose flowers to be involved in the crosses at random from the first six fully mature flowers closest to the apical meristem. Because these species are likely protogynous (Lawrence and Kaye, 2008; Clark, 2015), we judged flowers to have reached maturity once anthers were dehiscent. Past research has confirmed that stigmas are still receptive once the anthers are dehiscent (Kaye and Blakeley-Smith, 2008). Prior to performing the cross, the stigma and anthers were also visually inspected. Anthers that were indehiscent and stigmas that were curling or that otherwise appeared senescent were excluded from crosses. When flowers were rejected for these reasons, another flower was chosen at random for crossing. 21

Pollen was harvested by extracting the entire corolla from the chosen flower with forceps.

This method appeared to do minimal damage to the plant while preserving the maximum amount of pollen for transfer. We attempted to remove individual anthers first, but found a large amount dehiscent pollen to be wasted in this process. The cross was performed immediately after pollen harvest. Pollen was thoroughly smeared throughout the entire stigma to ensure maximum surface contact.

We tied colored embroidery floss loosely at the base of each flower used in a given cross to indicate the pollen donor plant. Fertilized ovules were allowed to ripen for four months, and capsules were harvested in August 2016. Fitness measures (fruit set, seed set, and germination percentage) were recorded. The 2016 F1 crosses produced 22 diploid hybrid maternal lines, and

2 triploid hybrid maternal lines (F1 hybrids were propagated using the methods described above). We randomly chose six seedlings from the 22 diploid hybrid maternal lines for the study.

Only nine putative triploid seeds resulted from our 2016 crosses and all nine seedlings were used in the second half of the study.

ASSESSMENT OF F1 HYBIRD FERTILITY

To assess F1 hybrid fertility, we created aforementioned diploid and triploid F1 hybrids in 2016, then we back crossed the F1 hybrids with the parental plants in 2017. To create diploid

F1 hybrids, we made 48 crosses (Table 2.2) between C. levisecta and diploid C. hispida. We used 22 F1 hybrid maternal lines from these crosses, from which we randomly chose 6 siblings for grow-out for a total of 132 diploid hybrids to choose from. We randomly chose two of the six diploid F1 hybrid siblings from each maternal line to participate in the study. To create triploid hybrids, we made 28 total crosses (Table 2.2) between C. levisecta and tetraploid C. hispida in

2016. Two triploid F1 hybrid maternal lines survived to reproductive maturity. One maternal line 22 yielded one seedling, the second maternal line yielded eight seedlings. F2 backcrosses were performed in May 2017 using the methods described above (Table 2.3). In addition, we repeated all F1 crosses in 2017 in order to assess F1 and F2 crosses under identical growing conditions

(Table 2.4). The number of F1 crosses differed from 2016 due to high parent plant mortality. We assessed fitness measures in the F1 and F2 hybrids using post pollination fitness measures of proportion fruit set per cross type, proportion seed set per fruit, and proportion seed germination using a subset of seeds per fruit. Detailed methods for assessing fitness are described below.

Table 2.3 Number of 2017 F2 crosses between C. levisecta (CALE), diploid hybrids [H(2x)], and triploid hybrids [H(3x)]. Reciprocal crosses are indicated by maternal then paternal species.

2017 F2 Hybrid Crosses n CALE(2x) x CALE(2x) 26 CALE(2x) x H(2x) 14 H(2x) x CALE(2x) 14 CALE(2x) x H(3x) 6 H(3x) x CALE(2x) 9 H(2x) x H(2x) 20

Table 2.4. Number of 2017 F1 crosses between C. levisecta (CALE) and diploid C. hispida [CAHI(2x)], tetraploid C. hispida [CAHI(4x)], and hexaploid C. hispida [CAHI(6x)]. Reciprocal crosses are indicated by maternal then paternal species.

2017 F1 Cross Types n CALE(2x) x CAHI(2x) 6 CAHI(2x) x CALE(2x) 6 CALE(2x) x CAHI(4x) 6 CAHI(4x) x CALE(2x) 9 CALE(2x) x CAHI(6x) 8 CAHI(6x) x CALE(2x) 6

FITNESS MEASURES

We examined fruit set, seed set, and seed germinability as measures of fitness in F1 and

F2 crosses. The type of cross served as the independent variable in this study, with reciprocal 23 crosses indicated by maternal then paternal species to indicate direction of gene flow. Parent plant population of origin was not included as a random effect due to imbalances in the number of plants from a given population. For instance, only five maternal lines were acquired from the

Bald Hill population, compared to 20 from the Yellow Island population. For F1 crosses, we compared CALE(2x) x CALE(2x) crosses to the six heterospecific crosstypes (Table 2.4). For the F2 crosses, we compared CALE(2x) x CALE(2x) to the four hybrid backcrosses and the

H(2x) x H(2x) crosstype (Table 2.3). All analyses were done with R version 3.3.2 (R Core Team,

2017).

Fruit Set

Fruit set was measured as presence/absence of a developed capsule, and proportion fruit set was measured as the number of capsules in a given cross type divided by the total number of crosses performed in a given cross type. Even unfertilized Castilleja fruit will produce a small, primordial capsule (personal observation), so we counted any capsule over 5mm as a viable fruit for the purposes of this study, following Kaye and Blakeley-Smith (2008). To test for differences in fruit set between conspecific (CALE(2x) x CALE(2x)) crosses and heterospecific crosses, we used logistic regression utilizing the glm function in base R (R Core Team, 2017) with a binomial model that included a logit link function to account for explanatory variables. A likelihood ratio test using the anova() function with the additional option test=”Chisq” in R was used to determine whether at least one of the cross types has a statistically different fruit set than any of the other groups. The estimates for the effect of a given cross type generated in the glm function were subtracted from the estimate for the effect of the CALE(2x) x

CALE(2x) crosstype. These parameters were exponentiated and reported as odds ratios.

Seed Set

Because Castilleja seeds are very small (<2mm), all seed set assessments were performed using a Leica S6 D stereo microscope (Leica Microsystems, Wetzlar, Germany). Capsules were opened with a dissecting spatula, and the contents were carefully extracted onto graph paper, which was then placed on the stage of the stereo microscope. Due to the reticulate seed coat surrounding Castilleja seeds, the contents of each capsule were lit from below in order to differentiate between live seeds, aborted seeds, and unfilled ovules. We recorded proportion seed set as proportion of live seeds to the sum of live seeds, aborted seeds and unfilled ovules in each of the fruits produced by the crosses. This resulted in counted proportion data, which served as the response variable. The type of cross served as the explanatory variable. To test for differences in seed set between conspecific (CALE(2x) x CALE(2x)) crosses and heterospecific crosses, we used logistic regression utilizing the glm function in base R (R Core Team, 2017) with a quasibinomial model that included a logit link function to account for explanatory variables. A quasibinomial distribution was utilized because of overdispersion in the data set. A drop-in-deviance F-test using the anova() function in R was used to determine whether at least one of the crosstypes has a statistically different proportion seed set than any of the other groups.

The estimates for the effect of a given cross type generated in the glm function were subtracted from the estimate for the effect of the CALE x CALE crosstype. These parameters were exponentiated and reported as odds ratios.

Germination percentage

We used a random sample of fifty seeds from each capsule, or fewer if less were available, to estimate germination percentage from each cross type. We broke seed dormancy 25 using the same methods described above. Once seeds were removed from the germination chamber, we calculated germination percentage. We considered seedlings as germinated if the emergent radical was >5mm. We calculated germination percentage as the number of germinated seeds divided by the total number of seeds attempted. This resulted in counted proportion data, which served as the response variable in statistical tests. To test for differences in seed set between conspecific (CALE(2x) x CALE(2x)) crosses and heterospecific crosses, we used logistic regression with a quasibinomial model that included a logit link function to account for explanatory variables. A quasibinomial distribution was utilized because of overdispersion in the data set. A drop-in-deviance F-test using the anova function in R was used to determine whether at least one of the crosstypes has a statistically different germination percentage than any of the other groups. Estimates of the effects of the parameters generated in the glm function were exponentiated and reported as odds ratios. The estimates for the effect of a given cross type generated in the glm function were subtracted from the estimate for the effect of the CALE(2x) x CALE(2x) crosstype. These parameters were exponentiated and reported as odds ratios.

REPRODUCTIVE ISOLATION

The relative strength of three reproductively isolating barriers – fruit set, seed set, and germination percentage – was assessed in order to estimate how much gene flow is reduced by a given barrier. We used the following equation to describe the relationship between isolation and mating preference:

�� ℎ�� = 1 − ��

Now widely adopted (Sobel and Chen, 2014), this equation was first presented in Coyne and Orr

(1989), and can be abbreviated as:

� �� = 1 − �

The resulting metric produces an isolation index equal to zero when there is no difference in relative fitness between the heterospecific cross and the conspecific cross. The metric equals 1 when there is total isolation between the two species. A negative value indicates that the heterospecific cross is more fit than the conspecific cross.

For the F1 crosses, we calculated reproductive isolation between C. levisecta, the three cytotypes of C. hispida, and their hybrids by estimating the amount of reproductive isolation attributed to each nth barrier to reproductive isolation, RIn. For example, fruit set for the F1 crosses was calculated as:

�� ℎ�� = 1 − ( ) ��

We calculated the RI metric separately for the barriers fruit set (RI fruit), seed set (RI seed) and germination percentage (RI germination). We calculated these metrics for each maternal by paternal species combination in F1 and F2 crosses to estimate the percentage reproductive isolation per barrier.

The post pollination barriers measured in this study occur in sequence, so we used the multiplicative approach to estimate the strength of reproductive isolation between species

(Coyne and Orr, 1989; Ramsey et al., 2003; Husband and Sabara, 2004; Hersch-Green, 2012). 27

The multiplicative approach starts with calculating the absolute contribution of the nth component of RI, ACn,

AC = RI 1 − AC thus reflecting the isolation due to previous measured barriers in the sequence. At each step, the previous absolute contribution values (ACi) were summed and subtracted from one. For the final step, all AC values were summed, yielding a measure of cumulative reproductive isolation (RI cum) between species pairs ranging from no reproductive isolation (RIcum = 0) to complete isolation (RIcum = 1). Negative values indicate higher fitness in heterospecific crosses.

Results

PLOIDY DIFFERENCES

In this study, 2C genome size ranged from 1.39±0.65 pg in C. hispida plants from the

TA-14 and Scatter Creek populations, to 4.39±0.21 pg in C. hispida plants from the Yellow

Island population (Table 2.5). The average mass of nuclear DNA differed significantly across parental taxa (F5 = 3670.1, p < 0.0001), although differences in taxa depended on population origin (F3 = 4.1, p < 0.007). The TA-14 and Scatter Creek C. hispida populations are estimated to be diploid, with a 2C genome size 1.39±0.65 pg. The Johnson Prairie, Wolf Haven, and Bald

Hills C. hispida populations were estimated to be tetraploid with a 2C genome size of 2.91±0.16 pg. The Yellow Island population of C. hispida was estimated to be hexaploid, with a 2C genome size of 4.39±0.21 pg. Castilleja levisecta was estimated to be diploid, with a 2C genome size of 1.44±0.084 pg.

Hybrid genome sizes were estimated using flow cytometry. Crosses between C. levisecta and diploid C. hispida plants were estimated to be diploid, with a 2C genome size of 1.43±0.49 28 pg (Table 2.5). Crosses between C. levisecta and tetraploid C. hispida plants were estimated to have produced triploid hybrids with a 2C genome size of 2.13±0.05 pg. Significant differences were found among taxa 1Cx genome size (F5 = 3.7, P = 0.002). Values ranged from 0.69±0.03 pg in diploid C. hispida plants, to 0.73±0.03 pg in hexaploid C. hispida plants.

Table 2.5 The 2C DNA content (pg) and monoploid genome size (pg) of 9 Castilleja populations, and two hybrids from hand pollinations involving different maternal and paternal species. The predicted ploidy of F1 hybrids based upon their 2C DNA contents.

Taxon Accession 2C genome size 1Cx genome size Estimated [mean ± SE (pg)] [mean ± SE (pg)] Ploidy C. levisecta PMC Corvallis 1.45±0.01 0.72±0.007 2x C. hispida Scatter Creek S. 1.39±0.02 0.69±0.01 2x C. hispida JBLM TA-14 1.41±0.01 0.71±0.007 2x C. hispida Bald Hill 3.01±0.04 0.75±0.009 4x C. hispida JP 2.87±0.03 0.72±0.007 4x C. hispida Wolf Haven Int’l 3.04±0.05 0.76±0.013 4x C. hispida Yellow Is. 4.40±0.03 0.73±0.005 6x Hybrid CALE(2x) x CAHI(2x) 1.44±0.01 0.72±0.003 2x Hybrid CALE(2x) x CAHI(4x) 2.14±0.002 0.71±0.001 3x

FRUIT SET, SEED SET, and SEED GERMINATION

Differences in fruit set, seed set, and germination percentage for F1 and F2 crosses are presented as odds ratios (Appendix Table 2.11; 2.12). Only statistically significant results are presented below.

F1 Crosses

Cross type significantly affected fruit set (p < 0.024 for X2 with df=9). CAHI (2x) x

CALE(2x) crosses had 6.5 times smaller (95% CI 0.97 to 43.2) odds of setting fruit than

CALE(2x) x CALE(2x) crosses (P=0.003) (Appendix Table 2.11). One-hundred per cent of the

CALE(2x) x CAHI(4x), and CALE(2x) x CAHI(6x) crosses set fruit. Because there is no variance around a mean of 1.00, a p-value cannot be obtained to infer significance between these crosses and the CALE(2x) x CALE(2x) cross. 29

Cross type also significantly affected seed set (F5 = 9.09, p <0.001) (Table 2.8).

CALE(2x) x CAHI(4x) crosses had 18.9 times lower odds (95% CI 5.12 to 69.7) of setting seed than CALE(2x) x CALE(2x) crosses (p < 0.0001) (Appendix Table 2.11). CAHI(4x) x CALE(2x) crosses had 4.04 times lower odds (95% CI 1.83 to 8.89) of setting seed than CALE(2x) x

CALE(2x) crosses (p = 0.001). CAHI(6x) x CALE(2x) crosses had 214.58 times lower odds

(95% CI 12.7 to 3606.79) of setting seed than CALE(2x) x CALE(2x) crosses (p < 0.0001)

(Appendix Table 2.11). Seed set was lower in polyploid crosses. In CALE(2x) x CAHI(4x) crosses, seed set was 9%, and in CAHI(4x) x CALE(2x) crosses seed set was 36% (Table 2.6).

Seed set in CALE(2x) x CAHI(6x) crosses was 0.8%, and seed set in CAHI(6x) x CALE(2x) crosses was 0.2% (Table 2.6). For reference, seed set was 76% in CALE(2x) x CALE(2x) crosses

(Table 2.6).

Cross type significantly affected germination percentage (F4 = 5.31, p <0.001) (Table

2.8). CALE(2x) x CAHI(4x) crosses had 5.45 times lower odds (95% CI 1.39 to 21.40) of germinating than CALE(2x) x CALE(2x) crosses (p = 0.019). One-hundred percent of the seeds produced by the CALE(2x) x CAHI (6x) and CAHI (6x) x CALE(2x) germinated (Appendix

Table 2.11). Because there is no variance around a mean of 1.00, a p-value cannot be obtained to infer significance between these crosses and the CALE(2x) x CALE(2x) cross.

Table 2.6 Mean proportion fruit set ±1 S. E., seed set ± 1 S.E. and germination percentage ± 1 S.E. for the different F1 crosses. Crosses are identified by the maternal (pollen recipient) and paternal (pollen donor). Castilleja species include C. levisecta (CALE), diploid C. hispida, and triploid C. hispida. Sample size (n) for each cross is listed.

Mean Mean Mean Germination 2017 F1 Cross Fruit Set(n) S.E. Seed Set(n) S.E. Percentage(n) S.E. CALE(2x) x CALE(2x) 0.76(26) 0.07 0.6326) 0.047 0.91(12) 0.02 CALE(2x) x CAHI(2x) 0.83(6) 0.17 0.61(5) 0.07 0.9(2) 0.04 CAHI(2x) x CALE(2x) 0.33(6) 0.21 0.56(2) 0.12 0.56(5) 0.09 CALE(2x) x CAHI(4x) 1(6) 0.00 0.095(6) 0.02 0.7(5) 0.08 CAHI(4x) x CALE(2x) 0.67(9) 0.17 0.36(6) 0.10 0.0(6) 0.00 CALE(2x) x CAHI(6x) 1(8) 0.00 0.008(8) 0.001 1.0(3) 0.00 CAHI(6x) x CALE(2x) 0.83(6) 0.17 0.002(5) 0.004 1.0(2) 0.00

F1 Fruit Set 1 0.8 * 0.6 0.4 0.2 0 C*C C*Ch(2x) Ch(2x)*C C*Ch(4x) Ch(4x)*C C*Ch(6x) Ch(6x)*C

F1 Seed Set 1 0.8

0.6 ** 0.4 0.2 *** *** *** 0 C*C C*Ch(2x) Ch(2x)*C C*Ch(4x) Ch(4x)*C C*Ch(6x) Ch(6x)*C

F1 Germination Percentage 1 * 0.8 0.6 0.4 0.2 0 C*C C*Ch(2x) Ch(2x)*C C*Ch(4x) Ch(4x)*C C*Ch(6x) Ch(6x)*C

Figure 2.1 Proportion fruit set, mean proportion seed set ± 1 per fruit, and mean germination percentage ±1 per fruit for hand pollination in F1 hybrid crosses (C = C. levisecta, Ch(2x) = diploid C. hispida, Ch(4x) = tetraploid C. hispida, Ch(6x) = hexaploid C. hispida), relative to CALE x CALE (C*C) crosses. Stars indicate significant difference from CALE x CALE crosses, with a=0.05 (* = 0.1, ** = 0.001, *** = 0.0001).

F2 Crosses

All diploid hybrid seedlings survived to reproductive maturity. There were only nine total triploid seedlings that germinated, four of which were sterile, and two of which produced flowers 32 very late in the growing season. This left three plants from two maternal lines from which to make crosses. Because of the low sample sizes in the triploid F2 backcrosses, these crosses were excluded from statistical inference.

Cross type significantly affected fruit set (p < 0.0003 for X2 with df=5) (Table 2.8). All of the H(2x) x CALE(2x) and H(2x) x H(2x) crosses set fruit. Because there was no variance around a mean of 1.0, a p-value could not be obtained to infer significance in fruit set between these crosses and the CALE(2x) x CALE(2x) cross. For comparison, fruit set in CALE(2x) x

CALE(2x) crosses was 0.76.

Cross type significantly affected seed set (F4 = 5.18; p = 0.0016)(Table 2.8). The odds of seed set for CALE(2x) x H(3x) crosses were 72.98 times smaller than for CALE(2x) x

CALE(2x) crosses (Appendix Table 2.12). H(3x) x CALE(2x) crosses did not set seed (Table

2.7).

Cross type significantly affected germination percentage (F4= 5.31, p = 0.0014) (Table

2.8). The odds of seed germination for H(2x) x CALE(2x) crosses were 4.17 times lower (95%

CI 1.7 to 10.15) than the odds of seeds germinating from a cross between CALE(2x) x CALE(2x)

(p < 0.001) (Appendix Table 2.12). The odds of seeds germinating for crosses between H(2x) and H(2x) was 3.9 times lower (95% CI 1.8 to 8.77) than the odds of a cross between CALE(2x) x CALE(2x) (p < 0.001) (Appendix Table 2.12). No other factors significantly affected proportion seed set per fruit.

Table 2.7 Mean proportion fruit set (±1 S.E.), seed set (±1 S.E.) and germination percentage (±1 S.E.) for the different F2 crosses. Crosses are identified by the maternal (pollen recipient) and paternal (pollen donor). Castilleja species and include C. levisecta (CALE), diploid hybrids [H(2x)], and triploid hybrids [H(3x)]. Sample size (n) for each cross is listed.

F2 Cross Fruit Set (n) S.E. Seed Set (n) S.E. Germination (n) S.E. CALE(2x) x CALE(2x) 0.76(26) 0.07 0.63(26) 0.05 0.91(12) 0.02 CALE(2x) x Hybrid(2x) 0.786(14) 0.17 0.72(11) 0.07 0.9(7) 0.03 Hybrid(2x) x CALE(2x) 1.00(14) 0.00 0.57(13) 0.07 0.74(9) 0.05 CALE(2x) x Hybrid(3x) 0.5(6) 0.22 0.22(3) 0.01 0.5(2) 0.50 Hybrid(3x) x CALE(2x) 1.00(9) 0.00 0.0(9) 0.00 -- -- Hybrid(2x) x Hybrid(2x) 1.00(20) 0.00 0.61(20) 0.04 0.75(20) 0.04

Table 2.8 Results of ANOVAs to determine whether crossing treatment, as specified by F1 and F2 crosses, influenced (A) the number of seeds per fruit or (B) the proportion of seeds that germinated.

Seed Set Germination percentage Source of Variation df F P df F P F1 Cross Type 5 9.09 <0.0001 4 5.31 0.0014 ** F2 Cross Type 4 1.11 0.35 4 5.18 0.0016 **

F2 Fruit Set 1 0.8 0.6 0.4 0.2 0 C*C C*H(2x) H(2x)*C C*H(3x) H(3x)*C H(2x)*H(2x)

F2 Seed Set 1 0.8 0.6 0.4 *** 0.2 0 C*C C*H(2x) H(2x)*C C*H(3x) H(3x)*C H(2x)*H(2x)

F2 Germination Percentage 1 ** ** 0.8 0.6 0.4 0.2 -- 0 C*C C*H(2x) H(2x)*C C*H(3x) H(3x)*C H(2x)*H(2x)

Figure 2.2 Proportion fruit set, mean proportion seed set ± 1 per fruit, and mean germination percentage ±1 per fruit for hand pollination in F2 hybrid crosses (C = C. levisecta, H(2x) = diploid hybrids, H(3x) = triploid hybrids) relative to CALE x CALE (C*C) crosses. Stars indicate significant difference from CALE x CALE crosses, with a=0.05 (* = 0.1, ** = 0.001, *** = 0.0001).

REPRODUCTIVE ISOLATION BETWEEN C. LEVISECTA and F2 HYBRIDS

In F1 crosses, the three measured barriers to reproductive isolation – fruit set, seed set, and germination percentage – all differed in their contributions to reproductive isolation (Table 35

2.9). Average cumulative reproductive isolation was 41.31% between C. levisecta and the C. levisecta x diploid C. hispida cross, 94.74% between C. levisecta and the C. levisecta x tetraploid

C. hispida cross, and 98.68% between C. levisecta and C. levisecta x hexaploid C. hispida cross.

Table 2.9 Measures of reproductive isolation (RI) attributed to three barriers (fruit set, seed set, seed germination) for crosses involving Castilleja levisecta, diploid C. hispida, tetraploid C. hispida, and hexaploid C. hispida. Negative values indicate comparisons in which hybrids are more fit than parents. RI values have been multiplied by 100 to give percentage reproductive isolation.

C. levisecta x diploid C. C. levisecta x tetraploid C. levisecta x hexaploid hispida C. hispida C. hispida Barrier RI C*Ch(2x) RI Ch(2x)*C RI C* Ch(4x) RI Ch(4x)*C RI C*Ch(6x) RI Ch(6x)*C Fruit Set -5.62% 57.75% 15.50% -26.74% -26.74% -5.62% Seed Set -1.16% 8.61% 84.31% 40.29% 98.61% 99.60% Germ. Perc. 36.34% -1.93% 20.52% 100.00% -20.48% -20.48% Cumulative 31.98% 60.65% 89.47% 100.00% 97.88% 99.49% Average 41.31% 94.74% 98.68%

In F2 crosses, the three measured barriers to reproductive isolation – fruit set, seed set, and germination percentage – all differed in their contributions to reproductive isolation (Table

2.10). Broadly, seed set had more influence as a barrier to reproductive isolation, followed closely by germination percentage. Average cumulative reproductive isolation was -10.81% between C. levisecta and the F1 diploid hybrid backcross, -9.10% between C. levisecta and diploid hybrids crossed among themselves (H(2x)x H(2x)), and 99.34% between C. levisecta and triploid hybrid backcrosses. There were asymmetries in crosses depending on maternal taxa and paternal taxa. For instance, reproductive isolation between H(2x) x CALE(2x) crosses and

CALE(2x) x CALE(2x) crosses was 0.01%, yet reproductive isolation between CALE(2x) x

H(2x) crosses and CALE(2x) x CALE(2x) crosses was -21.62%.

Table 2.10 Measures of reproductive isolation (RI) attributed to three barriers (fruit set, seed set, seed germination) for crosses involving Castilleja levisecta, diploid hybrids, and tetraploid hybrids. Reciprocal crosses are indicated by maternal and then paternal species where C. levisecta = CALE, diploid hybrids = H(2x), and tetraploid hybrids = H(3x). Negative values indicate comparisons in which hybrids are more fit than parents. RI values are multiplied by 100 to give percentage reproductive isolation.

C. levisecta X Diploid F1 C. levisecta x Triploid Diploid F1 Hybrid x Hybrid F1 Hybrid Diploid F1 Hybrid RI CALE x RI H(2x) x RI CALE x RI H(3x) x Barrier H(2x) CALE H(3x) CALE RI H(2x) x H(2x) Fruit Set 0.42% -26.74% 36.63% -26.74% -26.74% Seed Set -18.70% 6.08% 96.33% 100.00% -0.94% Germ. Perc. -2.90% 16.01% 43.37% 14.72% Cumulative -21.62% 0.01% 98.68% 100.00% -9.10% Average -10.81% 99.34%

Discussion

In this experiment, we found that diploid hybrids and triploid hybrids were both fertile, but that the diploid hybrids (homoploid hybrids) had much higher post pollination fitness than the triploids – in fact, these hybrids had higher fitness that C. levisecta itself.

Past work has found evidence for both homoploid hybridization (Clay et al., 2012) and interploidy hybridization (Hersch-Green, 2012) in Castilleja. In addition, post-pollination barriers to hybridization are weaker when Castilleja species have the same number of chromosomes (Hersch-Green, 2012). For these reasons, we hypothesized that crosses between C. levisecta (2n = 2x = 24) and plants from the diploid race of C. hispida (2n = 2x = 24) would produce fertile hybrids and that both diploid C. hispida and the diploid F1 hybrids would have lower reproductive isolation from C. levisecta. Indeed, we found that diploid hybrids were fertile, backcrossed readily with C. levisecta to form F2 hybrids, and had higher fitness measures than their progenitors. 37

Ploidy differences have been found to shape, but not necessarily prevent gene flow between Castilleja species (Hersch-Green, 2012), and there is a growing body of evidence indicating that differences in ploidy are not a complete barrier to reproduction in plants in general (Brochmann, 1993; Norrmann et al., 1997; Hagen et al., 2002; Park et al., 2002; Tel-Zur et al., 2004; Bleeker and Matthies, 2005; Kapralov et al., 2006; Lihová et al., 2007). Given this evidence, we hypothesized that interploidy crosses between C. levisecta and C. hispida would have reduced fitness and higher reproductive isolation due to meiotic problems associated with odd numbers of chromosomes. We found that crosses between C. levisecta and polyploid races of C. hispida had lower seed set than homoploid crosses, which ultimately contributed to much greater reproductive isolation between C. levisecta and polyploids.

We also hypothesized that fitness in these crosses would be further reduced and that reproductive isolation would increase with a growing difference in ploidy in the interspecific crosses. To that end, we found seed set in CALE(2x) x CAHI(4x) crosses was 9%, and that seed set in CAHI(4x) x CALE(2x) was 36%, compared to 63% in CALE(2x) x CALE(2x) crosses.

Furthermore, we found that seed set in CALE(2x) x CAHI(6x) crosses was 0.8%, and that seed set in CAHI(6x) x CALE(2x) crosses was 0.2%, suggesting that average seed set decreases as differences in ploidy increase. However, the germination percentage in F1 CALE(2x) x CAHI

(6x) and CAHI(6x) x CALE(2x) crosses was 100%, which ultimately contributed to very similar reproductive isolation measures in the tetraploid and hexaploid crosses (94.7% vs. 98.6%, respectively). Given these similarities, we did not find evidence that reproductive isolation increases ploidy distance increases in interploidy crosses.

HOMOPLOID CROSSES

Postzygotic reproductive isolation in homoploid hybrid crosses between C. levisecta and

C. hispida was substantially less than one, ranging from 32% when C. levisecta was the maternal plant, to 61% when C. hispida was the maternal plant for cumulative isolation attributed to fruit set, seed set and seed germination. A value of 100% indicates total reproductive isolation. This suggests that, in absence of prezygotic barriers reproduction such as pollinator fidelity or ecogeographic isolation, hybridization is likely between these two species. This also reinforces the idea that hybridization is more likely to occur when two closely-related parent species share the same number of chromosomes.

Backcrosses between C. levisecta and diploid F1 hybrids confirm that the diploid hybrids are in fact fertile and have the same or better relative fitness than the C. levisecta parents.

Reproductive isolation in crosses between C. levisecta and diploid hybrids was -21.62% when C. levisecta served as pollen recipient, suggesting that these crosses had higher fitness than conspecific C. levisecta crosses (Table 2.10). When diploid hybrids served as pollen recipient in these crosses, reproductive isolation was 0, suggesting that there are no post pollination barriers to reproduction.

Taken collectively, these data show that, in absence of prezygotic reproductive barriers,

C. levisecta, the diploid race of C. hispida, and their hybrids have a high likelihood of gene flow and introgression, confirming that genetic swamping could threaten the C. levisecta genome when these two species exist at the same site.

HETEROPLOID CROSSES

Differences in ploidy between C. levisecta and polyploid races of C. hispida severely curtailed gene flow. In crosses between C. levisecta and tetraploid C. hispida plants, 39 reproductive isolation was 89.5% where C. levisecta served as maternal plant and reproductive isolation was complete (100%) in cases where tetraploid C. hispida plants served as pollen recipient. Average reproductive isolation in these crosses was 94.7%. Reproductive isolation was comparable in crosses between C. levisecta and hexaploid C. hispida. Reproductive isolation was

97.8% when C. levisecta served as pollen recipient, and 99.5% when it was pollen donor, for an average reproductive isolation of 98.6%. This suggests that post pollination reproductive isolation between diploids and polyploids is strong, but that it does not necessarily increase as ploidy differences in interploidy crosses increase in this system.

When creating F1 hybrid stock in 2016, crosses between C. levisecta and tetraploid C. hispida produced nine seeds, and we were thus able to propagate only nine triploid hybrids. One maternal line yielded three reproductively mature triploids, and the other yielded only one reproductively mature triploid. Therefore, the measures of reproductive isolation in this study are limited by small sample sizes resulting from low F1 interfertility between these taxa.

Reproductive isolation in backcrosses between C. levisecta and triploid hybrids was

98.68% when C. levisecta served as pollen recipient, and 100% when the triploid hybrids served as pollen recipient. Of the 16 crosses performed between C. levisecta and the triploid hybrids, we were able to get seven seeds from one fruit to germinate, suggesting that fertile crosses between these types is a rare event compared to diploid backcrosses.

The 2016 crosses between C. levisecta and hexaploid C. hispida plants produced no seeds, and thus no putative F2 hybrids with which to work. However, of the 22 crosses performed between these two species in 2017, five set seed to produce 23 total seeds – of which all 23 germinated. The high germination percentage coupled with very low seed set suggests issues with seed formation possibly due to issues related to endosperm formation. The 40 endosperm in most flowering plants is triploid, resulting from a haploid sperm cell fusing with a diploid central cell within a maternal embryo sac. The endosperm is particularly sensitive to ploidy misbalances (Kohler et al., 2010) because deviations from the two maternal (2m) to one paternal (1p) genomes in endosperm cause endosperm failure (Brink and Cooper, 1947; Johnston et al., 1980; Lin, 1984). Deviations from this ratio could explain low seed set in diploid/polyploid crosses.

In summary, the genetic threat to C. levisecta on prairies where C. hispida is co-planted dramatically increases when C. hispida seed stock originates from a diploid population. Diploid cytotypes of C. hispida should not be co-planted with C. levisecta. Gene flow is greatly reduced between these two species when C. hispida seed stock originates from polyploid populations, but more research is needed before co-planting polyploid cytotypes of C. hispida with C. levisecta.

There are three primary reasons for this. First, the successful creation of fertile triploid F1 hybrids could facilitate the creation of allotetraploid hybrids, defined as tetraploids derived from hybrids between species, and thus gene flow between C. levisecta and tetraploid C. hispida cytotypes (Ramsey and Schemske 1998). In this process, called “triploid bridge,” tetraploid hybrids can result through backcrosses with diploid plants (which in this case would be C. levisecta), depending on the ploidy of the triploid’s functional gametes (e.g. n=x, 2x, or 3x), fertility of the triploids, and frequency of unreduced gametes in the diploid plant’s population

(Ramsey and Schemske, 1998; Husband, 2004). This process could also explain the formation of

F2 hybrids in crosses between C. levisecta and the hexaploid cytotype of C. hispida. In this case, haploid reduced gametes from C. levisecta could meet with triploid reduced gametes from C. hispida, forming tetraploid individuals, making this caveat applicable to both tetraploids and hexaploids. 41

The second reason more research is needed before co-planting polyploids is because more sampling is needed to determine how hybrid ploidy is manifested in interploidy crosses.

For instance, ploidy analysis shows that homoploid F1 crosses (C. levisecta x diploid C. hispida) are diploid (2n = 2x = 24), and that heteroploid F1 crosses (C. levisecta x tetraploid C. hispida) are triploid (2n = 3x = 36). However, heteroploid crosses yielded only two maternal lines from which to sample. In her cytological analysis of interploidy matings between C. miniata

(octoploid), C. rhexiifolia (tetraploid), and C. sulphurea (diploid), Hersch-Green (2012) found hybrids to have additive or intermediate ploidy levels, and that some interploidy Castilleja hybrids have ploidy levels identical to one parent species. This suggests that more investigation is warranted in order to discern whether crosses between C. levisecta and tetraploid C. hispida plants are reliably triploid, or if ploidy level varies in these crosses.

A third reason more research is needed is because the frequency of unreduced gametes is unknown in C. levisecta and C. hispida wild populations. Due to the presence of multiple ploidy races in C. hispida, and the ease with which these two species hybridize, a thorough analysis of non-reduced gametes in these populations will help determine the origin and maintenance of autopolyploid populations, and the expected frequency of allopolyploidy in populations where C. levisecta and polyploid races of C. hispida occur in sympatry.

MANAGEMENT IMPLICATIONS

This research shows that C. levisecta and diploid C. hispida can form fertile diploid and triploid hybrids under laboratory conditions. Because these two species have little to no reproductive isolation as diploids, these taxa should not be co-planted. More research is needed before polyploid C. hispida cytotypes are co-planted with C. levisecta. Despite the need for further research, the logic behind co-planting with polyploids is sound because diploids and their 42 polyploid derivatives can be isolated due to strong, post pollination genetic barriers (Marks,

1966; Schluter, 2001; Futuyma and Kirkpatrick, 2017). Furthermore, this experiment confirms gene flow is greatly reduced between C. levisecta and polyploid races of C. hispida.

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Chapter 3:

Morphological Analysis of the endangered wildflower Castilleja levisecta, a native congener C. hispida, and their F1 hybrids

Abstract

The co-planting of the diploid cytotype of the common native wildflower Castilleja hispida with the endangered wildflower C. levisecta has resulted in putative Castilleja hybrids on

Pacific Northwest prairie restoration sites, prompting fears that genetic swamping could threaten

C. levisecta. The eradication of these putative hybrids is critically important to the conservation of the C. levisecta genome, but hybrids can appear extremely morphologically close to either parental species, or highly distinct. Because ploidy differences between two interspecific mating partners can be a potent isolation mechanism in plants, land managers might choose to co-plant a polyploid C. hispida cytotype with C. levisecta at recovery sites in order to mitigate hybridization between these two species, but little morphological data exists on C. hispida cytotypes. In this article, we investigate whether there are measurable differences in 15 bract, calyx, and floral characteristics of C. levisecta, three cytotypes of C. hispida, and their diploid F1 hybrids, and if so, whether those differences are distinct enough for field technicians to distinguish them.

Using multivariate analyses and univariate ANOVA, we found groups of traits that distinguished between hybrids and their progenitors and C. hispida cytotypes. We found C. levisecta flowers tend to grow at tighter angles to the stem, and have large stigmas that rest against the galea. Analysis of C. hispida traits showed diploids’ corollas and galeas are shorter, and stigma diameters are smaller than their tetraploid and hexaploid counterparts. The flowers of hexaploid C. hispida plants project at a wider angle from the stem on the raceme, and calyx pubescence is about one millimeter longer, on average, than tetraploids. We found that these morphological characters might prove useful for field biologists, especially when attempting to differentiate C. levisecta from hybrids. However, we found that C. hispida cytotypes could not 53 be unambiguously distinguished from each other with certainty bases solely on morphological features. A thorough cytological analysis of populations considered as seed sources is necessary to ensure that ploidy levels are accurately determined.

Introduction

Hybridization and polyploidy have jointly and independently shaped the evolution and diversity of flowering plant species. Hybridization, defined here as the production of viable offspring from the mating of two or more species (Levin et al., 1996; Vilà et al., 2000), occurs frequently among flowering plants and is estimated to occur in 25% of angiosperm families

(Mallet, 2005; Whitney et al., 2010). The failure of hybrids to become completely reproductively isolated from their parent species can result in introgression, which is the incorporation of genetic material from one species into another via repeated backcrossing (Baack and Rieseberg,

2007). Introgression can be especially harmful when one parent species is rare or endangered

(Levin et al., 1996; Fant et al., 2010; Ma et al., 2010). Harm to rare species via introgression can manifest in several ways, including the disappearance of genetic distinctiveness (Todesco et al.,

2016), the reduction of mating opportunities leading to a decline in population growth (Wolf et al., 2001), or the swamping of rare plant traits leading to lowered fitness via outbreeding depression (Ellstrand and Elam, 1993).

Likewise, polyploidy has played an important role in early flowering plant diversification and radiation, and may be responsible for up to 15% of contemporary angiosperm species (De

Bodt et al., 2005; Soltis et al., 2007; Wood et al., 2009). Systematists generally recognize two forms of polyploidy, allopolyploidy and autopolyploidy. Following Ramsey and Schemske

(1998, 2002), we define allopolyploids as polyploids derived from interspecific hybridization. 54

Autopolyploids are considered to be derived from within or between populations of a single species. Autopolyploids have traditionally been understudied because they were thought to occur with much lower frequency than allopolyploids (Soltis et al., 2007, 2014). However, the advent of flow cytometry coupled with accelerating advances in genetics and genomics, has led to a body of research suggesting that 12%-16% of angiosperm species contain intraspecific ploidy variation (IPV) (Soltis et al., 2007; Wood et al., 2009; Husband et al., 2013; Rice et al., 2015).

IPV is often coupled with complex geographic partitioning (Lafuma et al., 2003; Suda et al., 2007; Halverson et al., 2008; Cires et al., 2010; Laport et al., 2012; McAllister et al., 2015;

Tompkins et al., 2015; Etterson et al., 2016; Wallace et al., 2017), and mixed-ploidy populations are generally rare (Ramsey and Schemske, 1998; Burton and Husband, 2000; Stuessy et al.,

2004; Baack, 2005; Suda et al., 2007; Halverson et al., 2008; Španiel et al., 2008; Kolář et al.,

2009). The rarity of mixed-ploidy populations is due to the usual, near complete reproductive isolation between non-matching ploidy states. Plants with mismatched ploidy states only occasionally interbreed successfully (Ramsey and Schemske, 1998; Petit et al., 1999) due to pre- pollination reproductive barriers (Husband and Sabara, 2004) and if pollination does occur, seed set is reduced, seeds have low germination percentages, and the odd-ploidy level progeny are often partially infertile (Ramsey and Schemske, 1998). This results in reduced fitness among cytotypes leading to population instability, with one cytotype becoming fixed and dominating a population, even if both cytotypes are initially equally common and fit (Levin, 1975; Husband,

2000).

In the field of restoration ecology, there is a growing recognition that genetic diversity of native plant materials must be actively expanded in response to global climate change. These practices bring previously genetically isolated populations together, which can increase the 55 likelihood of outbreeding depression, especially when restoring species with IPV (Kramer et al.,

2018). Ploidy variation of rare species has been shown to be an important aspect in creating management and recovery plans (Brown and Young, 2000; Severns and Liston, 2008; Frankham et al., 2011; Severns et al., 2013) but it is rarely considered in restoration projects involving common species. Kramer et al. (2018) found IPV present in one-third of the 115 most common species used in ecological restoration in the United States.

The Castilleja genus provides ample opportunity for studying the effects of both hybridization and interspecific ploidy variation. Allopolyploidy, autopolyploidy and homoploid hybridization have all played important roles in the diversification of Castilleja (Heckard and

Chuang, 1977; Heckard et al., 1980; Mathews and Lavin, 1998; Clay et al., 2012; Hersch-Green,

2012). In addition, Castilleja species are known to be promiscuous, and readily hybridize between species (Egger, 1994; Hersch and Roy, 2007; Hersch-Green and Cronn, 2009). In addition, multiple ploidy races are commonly found within Castilleja species (Heckard and

Chuang, 1977; Heckard et al., 1980). Castilleja species’ morphology can overlap in nearly every character, attributing to fascinating and complex trait profiles observed within the genus

(Holmgren, 1984; Chuang and Heckard, 1991; Tank and Olmstead, 2008).

On Pacific Northwest (PNW) prairies, a new opportunity to study the morphological complexity of Castilleja in a restoration context has recently emerged. Castilleja levisecta

Greenm. (golden paintbrush), one of the characteristic endangered endemic plants of the PNW, is widely planted on highly-fragmented, remnant prairies throughout southwest Washington and

Oregon. When introduced with a common, native congener, C. hispida Benth. (harsh paintbrush), the risk of genetic pollution and subsequent fitness loss becomes a major concern.

The observation of putative hybrids on prairies where these two species have been co-planted 56 inspired experiments to confirm the creation of potentially fertile hybrids (Kaye and Blakeley-

Smith, 2008). Biologists have also observed morphologically variable populations of C. hispida, raising the suspicion that the species has more than one cytological variant.

Here we explore whether morphological signals of hybridization and polyploidy can be detected in overall phenotypes in greenhouse-grown plants from wild populations. Putative hybrids identified on restoration sites where C. levisecta (CALE) and C. hispida (CAHI) have been planted in tandem have highly variable morphology, with a suite of traits that can look extremely close to either parental species, or highly distinct. Furthermore, morphological variation between the ploidy races of C. hispida has not been explored, providing an opportunity to explore how IPV shapes morphological diversity. Land managers attempting to preserve reproductive isolation between C. levisecta and C. hispida on prairie restoration sites would benefit from the ability to eliminate putative hybrids without risking the accidental eradication of rare C. levisecta plants. Therefore, the ability to identify a suite of morphological traits of hybrids would be valuable. In addition, because ploidy differences between two interspecific mating partners can be a potent isolation mechanism in plants (Ramsey and Schemske, 1998;

Levin, 2002; Comai, 2005; Kohler et al., 2010), land managers might choose to co-plant a polyploid C. hispida cytotype with C. levisecta at recovery sites. A set of morphological traits used in polyploid identification would serve as a useful tool for the initial identification of polyploid populations from which to source seeds to this end.

In this study, we combine cytogenetic and morphological techniques to assess how polyploidy and hybridization affect morphology in Castilleja. We measured 15 morphological traits from multiple individuals of C. levisecta, three ploidy races of C. hispida, and hybrids between the two species. We used these morphological and cytological data to explore two 57 questions regarding the identification of putative hybrid individuals and cytotype variants. First, can hybrids between C. levisecta and diploid C. hispida be reliably distinguished from their parents based on their morphology? Second, can the ploidy races of C. hispida be distinguished base on their morphology?

These questions are addressed in greenhouse-grown plants grown from seeds sourced from wild populations, as well as F1 hybrids created from reciprocal crossing of C. levisecta and the diploid cytotype of C. hispida. The morphological comparison reported here will provide a foundation for further studies regarding the field identification of C. levisecta x C. hispida hybrids, and C. hispida ploidy races. Furthermore, this comparison will also supplement genetic analyses while providing further evidence for differences among the taxa.

Materials and Methods

PARENT PLANT MATERIAL

All germplasm for this study was collected in August and September 2015. These seeds originated from six populations from western Washington (Table 1). We obtained C. levisecta

(CALE) germplasm from NRCS Plant Materials Center in Corvallis, OR. The C. levisecta population at PMC Corvallis provides seed for the C. levisecta reintroduction taking place throughout the Willamette Valley, and is composed of a mixture of four Washington populations. In some cases, our accessions arrived cleaned. In other cases, accessions arrived in capsules, in which case the capsules were opened and the seeds were manually extracted.

SEED GERMINATION

In October 2015, we cold stratified C. levisecta and C. hispida seeds representing three ploidy races at the Oregon State University Seed Lab for six weeks to break dormancy. Seeds were placed on moist germination paper (Anchor Paper, St. Paul, MN, USA) kept in clear plastic 58 boxes with tight fitting lids at 5°C. We kept seeds damp during cold stratification using deionized water. We germinated the seeds once cold stratification was complete in a light chamber set to 15°C/25°C on an alternating 12-hr dark, 12-hr light regimen for seven days.

Table 3.1 Taxa, origin populations, number of maternal lines, and number of plants from each maternal line used in the study.

Taxa Population Origin Population Maternal Plants Location Lines Included Castilleja levisecta NRCS Plant Corvallis, OR 28 28 Materials Center Castilleja hispida Training Area 14 Joint Base Lewis- 13 13 (JBLM) McChord, Pierce County, WA Castilleja hispida Scatter Creek Thurston County, 4 10 Wildlife Area WA Castilleja hispida Johnson Prairie Joint Base Lewis- 12 12 (JBLM) McChord, Pierce County, WA Castilleja hispida Wolf Haven Thurston County, 1 9 International WA Castilleja hispida Bald Hill Natural Thurston County, 5 5 Area Preserve WA Castilleja hispida Yellow Island San Juan County, WA 20 20

GREENHOUSE CULTURE

In January 2016, we randomly selected a total of 100 C. levisecta seedlings from 28 different maternal lines, and up to 40 C. hispida seedlings from 55 different maternal lines representing six population accessions to be placed into seedling flats using Metro-Mix 840 professional soil mix (Sungro Horticulture, Agwam, MA, USA). Castilleja species are hemiparasitic and Eriophyllum lanatum has been identified as an excellent host plant in C. levisecta propagation (Kaye and Lawrence, 2003). As a result, all seedlings from both species were co- planted with Eriophyllum lanatum. We placed seedling flats into a climate controlled greenhouse 59 with day time temperatures set to 13°C and night time temperature set to 10°C. All plants were grown under high pressure sulfur lamps.

Plant mortality was very high in the seedling stage due to damping-off disease. After six weeks, seedlings with hosts were transplanted into 1-gallon (US) pots and greenhouse temperatures were raised to mimic seasonal temperature averages while avoiding extremes

(approximately 25°C days and 12°C nights). We watered plants every other day, and fertilized every two weeks using Miracle Grow (24-8-16) at a rate of 2 grams per four liters of water.

Parental plants reached reproductive maturity approximately 4 months after germination, in April

2016.

CYTOLOGY

Based on morphological estimations, two of the six C. hispida populations we received for the parental generation were thought to be diploid and four populations were thought of be tetraploid. We used flow cytometry (FCM) to estimate genome size for each C. hispida population by randomly choosing a subset of parental plants from each population. Using FCM,

WEconcluded that one of the putative tetraploid populations is hexaploid.

All terminology regarding ploidy and genome size follows that proposed by Greilhuber et al. (2005). We used flow cytometry to assess the 2C genome size of individual Castilleja plants compared to an internal standard. We prepared 1-2 cm2 samples from three different young, fully expanded leaves chosen from each Castilleja plant to represent a random sample of nuclei for ploidy analysis. We co-chopped Castilleja samples with a 1-2cm2 sample of the internal standard 60

Pisum sativum ‘Ctirad’, which has a known 2C genome size of 8.76 pg (Bai et al., 2012). We chopped the samples in a polystyrene petri dish containing 400 µL of nuclei extraction buffer solution (Cystain Ultraviolet Precise P Nuclei Extraction Buffer; Sysmex, Görlitz Germany) with a razor. We then used a 30-µm gauze filter (Partec Celltrics, Münster, Germany) to remove excess biomass from the buffer and chopped leaf tissue mixture, passing it into a 3.5-mL plastic tube (Sarstedt Ag & Co., Nümbrecht, Germany). We then combined the mixture with 1.6 mL of fluorochrome stain (4’,6-diamidino-2-phenylindole) (Cystain Ultraviolet Precise P Staining

Buffer; Partec). We used a flow cytometer to analyze a minimum of 3000 nuclei per sample

(CyFlow Ploidy Analyzer; Partec). We accepted samples only if average CV for each fluorescence histogram was under 10.

We calculated relative 2C genome size as:

mean florescence value of sample 2� = DNA Content of standard × mean florescence value of standard

Monoploid (1Cx) genome size was calculated as:

2C 1Cx = ploidy

Following estimation of 2C genome size via FCM, all plants from diploid, tetraploid, and hexaploid C. hispida populations were pooled into C. hispida (2x), C. hispida (4x), and C. hispida (6x) groups, respectively. In addition, the C. levisecta group consisted of all C. levisecta plants, and the Hybrid (2x) group consisted of all diploid F1 hybrids created from the C. levisecta x C. hispida (2x) crosses.

CREATION OF F1 HYBRIDS

We created diploid F1 hybrids for this study in May 2016 by crossing C. levisecta (2n =

2x = 24) with the diploid cytotype of C. hispida (2n = 2x = 24), with 60 total replicates. Half of the interspecific cross-type replicates were repeated with the parent taxa as either the dam (pollen recipient) or the sire (pollen donor) in case of imbalances in gene flow directionality. For each replicate, plants were chosen at random without replacement. However, due to parental plant mortality, some surviving parental plants were re-used as either pollen donors or pollen recipients. The 2016 F1 crosses produced 22 diploid hybrid maternal lines. We randomly chose six seedlings from each of the 22 diploid hybrid maternal lines for grow-out and subsequent participation in morphometric analysis.

MORPHOMETRICS

We consulted Clark (2015), Weidner and Fant (2017), Crosswhite and Crosswhite

(1970), and Hitchcock et al. (1969) to determine four bract traits, four calyx traits, and seven floral traits to distinguish C. levisecta from C. hispida, for a total of 15 optimal diagnostic quantitative traits. Fifteen morphological measurements were collected for each plant, and one measurement for each of the 15 traits was taken per plant. We used these traits to determine whether there are clear morphological differences between Castilleja taxa.

All traits were measured to the nearest millimeter. Bract traits included bract width, total bract length, total bract lobe depth, and total bract lobe depth as a percentage of bract length

(called “bract-lobe ratio”) (Figure 3.1). Clark (2015) found no obvious differences between C. hispida and C. levisecta calyces, based on observation. However, with the addition of two ploidy races to this study, we chose to measure calyx length, calyx pubescence length, and deepest and shallowest calyx clefts (Figure 3.1). Floral traits assessed in this study included total corolla 62

Figure 3.1 Illustration of Castilleja bract, the flower (with corolla and calyx combined), and corolla on its own with location of floral measurements indicated. Image adapted from Widener and Fant (2017).

length, total galea length, and galea length as a percentage of the total corolla (called “corolla- galea ratio”) (Figure 3.1). In addition, we also measured the length of the lower lip region, the diameter of the stigma, and the length of the portion of the style protruding from the galea.

Finally, to capture differences in the overall “look” of the species, we measured the angle of the flower as it protruded from the stem, because C. levisecta flowers tend to press against the stem, and C. hispida flowers tend to protrude away from the stem. All traits from each species were 63 measured on the same day and sample sizes differed based on the number of plants that were flowering at the time of measurement and parent plant mortality.

We took measurements from a single flower on each plant in the study. The flower was taken from among the first six flowers below the top-most whirl. To take the measurements, we carefully removed the bract, calyx and corolla from a single flower, arranged them on a white background, then photographed the structures next to a ruler. Once photographed, we then measured the structures, and pressed one stem from each plant for herbarium specimens to be preserved the Oregon State University Herbarium. We assessed all floral traits on live plants except for floral angle, which was measured once the specimens had been pressed and dried.

STATISTICAL ANALYSIS

All morphological analyses were performed using R Statistical Software version 3.3.2 (R

Core Team, 2017). We created two experimental groups to test my hypotheses. The first group

(the Hybrid group) consisted of CALE(2x), CAHI(2x) and their F1 hybrids. The second group

(the CAHI IPV group) consisted of the diploid, tetraploid, and hexaploid variants of C. hispida.

Both groups received identical statistical evaluation.

We applied a multivariate analysis of variance (MANOVA) to test the effective morphological differentiation among taxa. To determine similarity between species, performed univariate ANOVA on each morphological trait, then used the R package Agricolae (de

Mendiburu, 2017) to perform a Tukey’s honest significant difference test. To explain the maximal amount of morphological variation produced by multiple continuous explanatory trait variables, we used principle components analysis to reduce the variables into fewer dimensions.

PCA was performed using the prcomp() function in base R (R Core Team, 2017). To evaluate 64 which morphological traits best separated the taxa and are therefore useful in identifying hybrids, we used the R package MASS (2002) to perform linear discriminant analysis (LDA).

Results

CYTOLOGY

2C genome size in this study ranged from 1.39±0.65 pg in C. hispida plants from the TA-

14 and Scatter creek populations, to 4.39±0.21 pg in C. hispida plants from the Yellow Island population (Table 3.2). The average mass of nuclear DNA differed significantly across parental taxa (F5 = 3670.1, p < 0.0001), although differences in taxa depended on population origin (F3 =

4.1, p < 0.007). The TA-14 and Scatter Creek C. hispida populations are estimated to be diploid, with a 2C genome size 1.39±0.65 pg (Table 3.2).. The Johnson Prairie, Wolf Haven, and Bald

Hills C. hispida populations are estimated to be tetraploid with a 2C genome size of 2.91±0.16 pg (Table 3.2). The Yellow Island population of C. hispida is estimated to be hexaploid, with a

2C genome size of 4.39±0.21 pg (Table 3.2). Castilleja levisecta is estimated to be diploid based on our study, with a 2C genome size of 1.44±0.084 pg (Table 3.2).

Hybrid genome sizes were evaluated based on genome size of the parental taxa. Crosses between C. levisecta and diploid C. hispida plants are estimated to have produced diploid hybrids with a 2C genome size of 1.43±0.49 pg (Table 3.2). Significant differences were found among taxa 1Cx genome size (F5 = 3.7, P = 0.002). Values ranged from 0.69±0.03 pg in diploid

C. hispida plants, to 0.73±0.03 pg in hexaploid C. hispida plants (Table 3.2).

Table 3.2 The 2C DNA content (pg) and monoploid genome size (pg) of 9 Castilleja populations, and two hybrids from hand pollinations involving different maternal and paternal species. The predicted ploidy of F1 hybrids based upon their 2C DNA contents.

2C genome size Estimated 1Cx genome size Taxon Accession [mean ± SE (pg)] Ploidy [mean ± SE (pg)] C. levisecta PMC Corvallis 1.45±0.01 2x 0.72±0.007 C. hispida Scatter Cr. South 1.39±0.02 2x 0.69±0.01 C. hispida JBLM TA-14 1.41±0.01 2x 0.71±0.007 C. hispida Bald Hill 3.01±0.04 4x 0.75±0.009 C. hispida Johnson Prairie 2.87±0.03 4x 0.72±0.007 C. hispida Wolf Haven Int’l. 3.04±0.05 4x 0.76±0.013 C. hispida Yellow Is. 4.40±0.03 6x 0.73±0.005 Hybrid CALE(2x) x CAHI(2x) 1.44±0.01 2x 0.72±0.003

THE HYBRID GROUP

The MANOVA analysis showed significant morphological differences among taxa

(Pillai’s trace = 1.5692, F2,30 = 6.997, P < 0.001). The F1 hybrid plants showed many intermediate morphological traits between C. levisecta and C. hispida. Univariate ANOVA of individual traits showed strong evidence that trait differences depend on taxon or hybrid history for 9 of the 15 traits measured (Table 3.4). Because of low samples sizes in our study, we only included traits with a p-value lower than 0.001 for multivariate analyses.

Hybrid Principle Components Analysis

A plot of the data in the space of the first two principal components (Figure 3.2), which did not take taxon groupings into account, shows individuals of like taxa grouped together with very little overlap. This indicates an association between taxon and the primary axes of multivariate phenotypic variation.

The first PC axis explained 39% of morphological variation between individuals, with C. levisecta having lower eigenvalues than the diploid hybrids, and C. hispida individuals broadly overlapping with both. Five of the 10 traits had factor loadings with an absolute value between

0.34 and 0.45: bract length, calyx length, corolla length, deepest calyx cleft, and galea length 66

(Table 3.3). The second PC axis explained 24.5% of morphological variation between individuals, with C. hispida having higher eigenvalues that did not overlap with C. levisecta and the hybrids, which broadly shared the same low eigenvalues on PC2. Five of the 10 traits had loadings with absolute values between 0.33 and 0.54 (Table 3.3): floral angle, stigma diameter, bract length, corolla-galea ratio, and style length.

Table 3.3 Results of principal components analysis (PC1 and PC2), and linear discriminant analysis (LDA; LD1 and LD2) of morphological variation between C. levisecta, C. hispida (2x) and their hybrids. Bolded values indicate traits that had the highest absolute values for loadings.

Hybrid Traits PC1 PC2 LD1 LD2

bract.length 0.347 -0.365 0.295 -0.116

calyx.length 0.455 0.049 -0.153 0.254

corolla.galea.ratio 0.089 0.42 -20.939 69.714

corolla.length 0.409 -0.22 -0.254 1.307

deepest.calyx.cleft 0.41 0.011 -0.603 -0.252

floral.angle -0.184 -0.426 0.167 0.028

galea.length 0.4 0.127 0.335 -2.921

lobe.ratio -0.26 0.189 3.162 -6.346

stigma.diam 0.024 -0.545 1.331 1.424

style.length 0.26 0.33 -0.051 0.122

Figure 3.2 Principal component analysis of 10 morphological traits sampled from greenhouse- grown diploid C. hispida (CAHI 2x) (red triangles), C. levisecta (CALE) (green circles), and their F1 diploid hybrids (blue squares) (n = 58) derived from natural populations. Percentage overall morphological variation explained by each axis is shown in parentheses.

Hybrid linear discriminant analysis

The LDA of C. levisecta, C. hispida, and hybrid morphological traits formed well- separated clusters (Figure 3). The first linear discriminant axis (LD1) accounted for 80.4% of the variance among groups, and 19.5% of the variance was spread along the second linear discriminant axis (LD2). The model had a very low rate of misclassification (3.5%) and correctly assigned 100% of the C. levisecta and diploid C. hispida individuals. Only two hybrid individuals were classified as C. levisecta (9.09% misclassified). Using leave-one-out cross- validation, the LDA correctly classified 93% of all individuals (54 out of 58). Three of the four misclassified individuals were diploid hybrids incorrectly classified as C. levisecta. In brief, leave-one-out cross-validation is an estimate of the generalization performance of a model trained on n−1 samples of data, which is a more conservative estimate of the performance of a model trained on n samples. On LD1, hybrids were located at an intermediate position between their parent taxa (Figure 3.3). Whereas both parental taxa showed very similar positions on the

LD2 axis, hybrids did not locate at an intermediate place, but apart (upward), indicating morphological novelty.

Univariate analysis showed average style length of C. levisecta was 0.63±0.29mm (S.E.), with the stigma often appearing to rest upon the tissue of the galea (Table 3.4). Hybrids had the next longest average style length at 2.64mm±0.45mm, while diploid C. hispida had the longest average style length of the three taxa at 3.92±0.63mm. The average floral angle of C. levisecta plants was 76.2°±0.63°, giving the species a “closed” look. The hybrids looked slightly more

“open,” having a floral angle of 72.17°±1.05°. Diploid C. hispida looked much more open, with an average floral angle of 63.52°±0.92°. On average, hybrid plants had significantly longer corollas than their progenitors at 28.93±0.62mm, compared to 23.56±0.61mm and 69

24.36±1.61mm in C. levisecta and diploid C. hispida, respectively. Finally, hybrid plants also had significantly longer bracts than their progenitors at 27.33±0.88mm, compared to

24.00±0.65mm for C. levisecta and 20.84±1.35mm for diploid C. hispida.

Figure 3.3 Linear discriminant analysis of 10 morphological traits, showing differences between diploid C. hispida (CAHI 2x) (red triangles), C. levisecta (CALE) (green circles) and their diploid F1 hybrids (Hybrid 2x) (blue squares) individuals (n = 58). Confidence ellipses (95%) are shown for each taxa.

Table 3.4 Mean values ± S.E. for morphological characters in C. levisecta, diploid F1 hybrids, and diploid C. hispida. C. levisecta Hybrid(2x) C. hispida(2x) p-value Bract Length (mm) 24±0.65 27.33±0.88 20.84±1.35 <0.0001 Bract Lobe Length (mm) 8.24±0.29 7.01±0.42 6.99±0.56 0.03 Bract Length-Lobe Ratio 0.34±0.01 0.26±0.02 0.35±0.04 0.0008 Bract Width (mm) 9.27±0.44 9.79±0.55 8.58±0.80 0.4 Calyx Length (mm) 13.7±0.55 19.32±0.52 18.27±0.96 <0.0001 Calyx Pubescence Length (mm) 1.84±0.13 2.05±0.23 1.28±0.19 0.05 Deepest Calyx Cleft (mm) 6.34±0.20 8.12±0.30 8.05±0.38 <0.0001 Shallowest Calyx Cleft (mm) 3.03±0.24 3.9±0.26 2.62±0.35 0.009 Corolla Length (mm) 23.56±0.61 28.93±0.62 24.36±1.16 <0.0001 Galea Length (mm) 8.62±0.24 10.81±0.34 10.98±0.64 <0.0001 Corolla-Galea Ratio 0.37±0.008 0.37±0.012 0.45±0.012 <0.0001 Lower Lip Length (mm) 2.32±0.15 2.82±0.18 1.92±0.30 0.0127 Stigma Diameter (mm) 1.62±0.09 1.7±0.07 0.66±0.09 <0.0001 Style Length (mm) 0.63±0.29 2.63±0.45 3.92±0.63 <0.0001 Floral Angle (degrees) 76.2±0.63 72.17±1.05 63.53±0.92 <0.0001

Bract Length (mm) Corola-Galia Ratio 30.00 a 0.5 a b 0.45 b b 25.00 b 0.4 20.00 0.35 0.3 15.00 0.25 0.2 10.00 0.15 5.00 0.1 0.05 0.00 0 C. hispida(2x) C. levisecta Hybrid(2x) C. hispida(2x) C. levisecta Hybrid(2x)

Calyx Length (mm) Corolla Length (mm) 25 35.00 a a a 30.00 b 20 b b 25.00 15 20.00

10 15.00 10.00 5 5.00

0 0.00 C. hispida(2x) C. levisecta Hybrid(2x) C. hispida(2x) C. levisecta Hybrid(2x)

Floral Angle (degrees) Stigma Diameter (mm)

90.00 2 a a a 80.00 c 1.8 1.6 70.00 b 1.4 60.00 1.2 50.00 1 b 40.00 0.8 30.00 0.6 20.00 0.4 10.00 0.2 0.00 0 C. hispida(2x) C. levisecta Hybrid(2x) C. hispida(2x) C. levisecta Hybrid(2x)

Figure 3.4 Morphological differences between C. levisecta, diploid C. hispida, and their hybrids for six of the ten characters that were significantly different with p<0.0001. Error bars indicate ± one standard error.

THE CAHI IPV GROUP

The MANOVA analysis showed significant morphological differences among cytotypes

(Pillai’s trace = 1.4538, F2,27 = 3.771, P < 0.001). Subsequent univariate ANOVA analysis of the individual morphological characters indicated that ploidy state significantly affected seven of the

15 traits tested (Table 3.6). These seven traits were included in the subsequent PCA and LDA analyses.

CAHI IPV Principle Components Analysis

A plot of the data in the space of the first two principal components (Figure 3.5) shows individuals of like genome size grouped together with some notable overlap. The first PC axis explained 46.8% of morphological variation between individuals, with diploid C. hispida having negative axis scores, tetraploid C. hispida having intermediate scores, and hexaploid C. hispida individuals having positive scores. Three of the seven traits had factor loadings with an absolute value between 0.42 and 0.50: calyx pubescence length, corolla length, and galea length (Table

3.5). The second PC axis explained 15.6% of morphological variation between individuals, with the three ploidy races broadly overlapping. Three of the seven traits had loadings with absolute values between 0.27 and 0.80 (Table 3.5): bract width, lower lip length, and style length.

Table 3.5 Results of principal components analysis (PC1 and PC2), and linear discriminant analysis (LDA; LD1 and LD2) of morphological variation between three ploidy races of C. hispida. Bolded values indicate the factor loadings of the top three traits.

CAHI Traits PC1 PC2 LD1 LD2 bract.width 0.153 0.809 -0.105 0.415 calyx.pub.length 0.422 0.040 0.053 0.598 corolla.length 0.507 0.030 0.413 -0.167 galea.length 0.458 0.028 -0.118 0.045 lower.lip.length 0.377 -0.491 -0.419 0.608 stigma.diam 0.342 -0.168 2.548 -0.636 style.length 0.271 0.271 0.039 -0.025

Figure 3.5 Principal component analysis of seven morphological traits sampled from greenhouse-grown diploid C. hispida (CAHI 2x; red), triploid C. hispida (CAHI 4x; green), and hexaploid C. hispida plants (CAHI 6x; blue) (n = 58) derived from natural populations. Percentage overall morphological variation explained by each axis is shown in parentheses.

CAHI IPV linear discriminant analysis

All three C. hispida cytotypes formed overlapping clusters based on the LDA of floral, bract, and calyx traits (Figure 3.6). The first linear discriminant axis (LD1) accounted for 88.2% of the variance among groups, whereas 11.7% of the variance was spread along the second linear discriminant axis (LD2). On LD1, diploids were located apart from the polyploids, whereas both 75 polyploid cytotypes showed very similar positions on the LD1 axis (Figure 3.6). Diploids occupied an intermediate position between tetraploids and hexaploids on the LD2 axis. Diploid plants generally showed smaller trait values, with tetraploid and hexaploid plants showing traits with similar values. The combined morphological variables supported a highly correct taxonomic classification: the model had a very low rate of misclassification (3.3%) and correctly assigned

100% of the diploid C. hispida and hexaploid C. hispida individuals. One observed tetraploid individual was predicted to be a hexaploid individual (11.11% misclassified). Using leave-one- out cross-validation, the LDA correctly classified 63% of all individuals (19 out of 30). Three out of 11 diploids were misclassified, five out of nine tetraploids were misclassified, and three out of 10 hexaploids were misclassified.

Diploids had a significantly smaller average corolla length at 24.36±1.16mm, compared to 31.22±0.79mm for tetraploids and 33.90±0.75mm for hexaploids (Table 3.6; Figure 3.7).

Likewise, diploids had smaller average galea lengths at 10.98±0.64mm, while tetraploids and hexaploids had galea lengths of 14.50±1.17mm and 15.80±0.70mm, respectively (Table 6;

Figure 7). Calyx pubescence length was 1.28±0.19mm for diploids, 1.86±0.35mm for tetraploids, and 3.00±0.33mm for hexaploids (Table 6; Figure 8). On average, diploid plants had significantly smaller stigmas. Diploid stigmas were 0.66±0.09mm in diameter, tetraploid stigmas were 0.93±0.07mm, and hexaploid stigmas were 1.04±0.04mm (Table 3.6; Figure 3.8). Finally, the flowers of hexaploid plants extended from the flowering stem at a wider angle, 57.98°±1.44°, whereas tetraploid flower angle was 64.82°±1.49°, and diploid flower angle was 63.52°±0.92°.

Figure 3.6 Linear discriminant analysis of seven morphological traits, showing differences between diploid C. hispida (CAHI 2x; red triangles), triploid C. hispida (CAHI 4x; green circles), and hexaploid C. hispida plants (CAHI 6x; blue squares) (n = 58) derived from natural populations. Confidence ellipses (95%) are shown for each taxa.

Table 3.6. Mean values ± S.E. for morphological characters in diploid, tetraploid, and hexaploid C. hispida. C. hispida(2x) C. hispida(4x) C. hispida(6x) p-value Bract Len. (mm) 20.84±1.35 20.83±1.16 21.00±1.06 0.994 Bract Lobe Len. (mm) 6.99±0.56 8.7±0.92 8.18±0.69 0.238 Lobe Ratio 0.35±0.04 0.41±0.03 0.39±0.02 0.301 Bract Width (mm) 8.59±0.80 7.39±0.51 10.1±0.66 0.038 Calyx Len. (mm) 18.27±0.96 20.44±1.50 21±1.13 0.228 Calyx Pubescence Len. (mm) 1.28±0.19 1.85±0.35 3.00±0.33 <0.0001 Deepest Calyx Cleft (mm) 8.05±0.38 9.5±0.88 8.7±0.63 0.288 Shallowest Calyx Cleft (mm) 2.62±0.35 2.89±0.51 3.1±0.31 0.671 Corolla Len. (mm) 24.36±1.16 31.22±0.79 33.9±0.75 <0.0001 Galea Len. (mm) 10.98±0.64 14.5±1.17 15.8±0.70 <0.0001 Corola-Galea Ratio 0.45±0.01 0.46±0.03 0.46±0.01 0.852 Lower Lip Len. (mm) 1.93±0.30 2.39±0.25 2.8±0.13 0.047 Stigma Diameter (mm) 0.66±0.09 0.93±0.07 1.04±0.04 0.002 Style Len. (mm) 3.92±0.63 6.5±1.25 6.9±1.18 0.088 Floral Angle (degrees) 63.52±0.92 64.82±1.49 57.98±1.44 0.0019

Calyx Pubescence Length (mm) Corolla Length (mm) a 3.50 40.00 a a 3.00 35.00 30.00 2.50 b b 25.00 2.00 b 20.00 1.50 15.00 1.00 10.00

0.50 5.00

0.00 0.00 C. hispida(2x) C. hispida(4x) C. hispida(6x) C. hispida(2x) C. hispida(4x) C. hispida(6x)

Galea Length (mm)

18.00 a a 16.00 14.00 b 12.00 10.00 8.00 6.00 4.00 2.00 0.00 C. hispida(2x) C. hispida(4x) C. hispida(6x)

Figure 3.7 Differences between three cytotypes of C. hispida for the morphological characters that were significantly different with p<0.0001. Traits include calyx pubescence length (mm), corolla length (mm), and galea length (mm). Error bars indicate ± one standard error.

Stigma Diameter (mm) Floral Angle (degrees) 1.20 a 68.00 a a 66.00 a 1.00 64.00 b 0.80 62.00 b 60.00 0.60 58.00 0.40 56.00 54.00 0.20 52.00 0.00 50.00 C. hispida(2x) C. hispida(4x) C. hispida(6x) C. hispida(2x) C. hispida(4x) C. hispida(6x)

Figure 3.8. Differences between three cytotypes of C. hispida for the morphological characters that were significantly different with p<0.001. Traits include floral angle (degrees) and stigm diameter (mm). Error bars indicate ± one standard error.

Discussion

THE HYBRID GROUP

This study shows that C. levisecta, C. hispida, and their hybrids are morphologically distinct, and provides insight into traits that can distinguish these taxa and their hybrids. We set out to investigate whether there are measurable differences in bract, calyx, and floral characteristics of C. levisecta, C. hispida, and their hybrids, and if so, whether those differences are distinct enough for field technicians to distinguish them. Most hybrid traits measured were intermediate between C. levisecta and C. hispida, and there was no one trait that easily distinguished the hybrids from their parent taxa. However, there were combinations of traits that could serve as a guide to land managers who need to distinguish C. levisecta from the diploid hybrids.

A unique challenge in the co-recovery of C. levisecta and Taylor’s checkerspot on Pacific

Northwest prairies is the fact that C. levisecta hybridizes with C. hispida, a species that is widely planted by managers as a host plant for Taylor’s checkerspot. This has caused concern for the recovery of both endangered species (Dunwiddie et al., 2016). To compromise, managers agreed that new plantings of one Castilleja species occur 200m from populations of the other, but this spatial separation is not easily maintained, as putative hybrids continue to be found in the buffer zones (Dunwiddie et al., 2016) and individuals of mixed morphology continue to appear on restoration sites. The expeditious eradication of these putative hybrids is critically important to prevent genetic swamping because hybrids facilitate gene flow and thus introgression from C. hispida to C. levisecta, and vice versa, which greatly increases the risk of genetic pollution in the endangered C. levisecta genome. Because these Castilleja traits are highly variable, and can look 80 extremely close to either parental species, eradication is only possible with the ability to identify hybrids from C. levisecta with a high degree of certainty.

Though bract color variation was not addressed in this study, it is important to note that

Clark (2015), using Royal Botanic Society color charts to identify the range of color variation, found flower color to overlap between C. levisecta, diploid C. hispida, and their hybrids. In that study, putative F1 hybrid bract coloration in wild populations was highly variable ranging from salmon, orange, yellow-orange and yellow. Likewise, C. hispida had highly varied bract coloration within populations, ranging from red, dark orange, orange, gold, salmon yellow- orange, and yellow. However, there was no color variation recorded in C. levisecta, with all populations appearing yellow.

In this study, C. levisecta appears more “closed,” with flowers tending to grow at tighter angles to the stem. The closed look, when combined with the characteristic yellow color, and a large (>1.50mm) stigma that rests against (or very close to) the galea, could serve to help distinguish C. levisecta from hybrids. Our multivariate analysis confirmed that, given small sample sizes, F1 CALE(2x) x CAHI(2x) hybrids are morphologically distinct from their progenitors, but with distinct morphological overlap. Some Castilleja hybrids can be identified in the field (Eggar 1994), but with repeated backcrossing it will become more difficult or impossible to distinguish some hybrid genotypes from parental genotypes.

THE CAHI IPV GROUP

The second phase of this study investigated morphological differences between three cytotypes in the C. hispida species complex. Polyploidy has been shown to increase morphological diversity in wild plant populations (Levin et al., 1996; Otto and Whitton, 2000;

Chansler et al., 2016), and in this study we found considerable morphological variation between 81 ploidy races, despite small sample sizes and identical growing conditions. Visualizing the data with PCA and LDA, tetraploids are situated between and overlap with the diploids and the hexaploids on the primary axes of variation, but cytotypes form discrete clusters in both analyses. This suggests that the complex contains significant morphological variation, that trait values operate on a spectrum that spans cytotypes, and that the ploidy races form distinct morphological units when all traits are considered.

Individuals of two differing ploidy states can be discerned with a high success rate

(Hardy et al., 2000; Španiel et al., 2008), but the typical success rate is between 50% and 80%

(Hodálová et al., 2007; Mandáková and Münzbergová, 2008). But in systems with three or more ploidy levels, discerning cytotypes based on morphology has rarely been attempted and is considered quite challenging (Chansler et al., 2016). Our linear discriminant analysis model correctly classified 63% of all individuals using leave-one-out cross validation, suggesting moderate success in predicting the ploidy of an individual C. hispida plant given the suite of characters incorporated. Without using leave-one-out cross validation, our model correctly classified 96.7% of all individuals suggesting that high rates of multivariate morphological discrimination among three ploidy levels is possible.

Identification of specific traits that can be used to discern between C. hispida cytotypes will help managers identify polyploids in preparation for full cytological analysis of a given population. Castilleja hispida seeds sourced from polyploid populations could be useful in restoration projects where co-planting with C. levisecta is necessary, because ploidy differences between two interspecific mating partners can be a potent isolation mechanism in plants

(Ramsey and Schemske, 1998; Levin, 2002; Comai, 2005; Kohler et al., 2010), but more research is needed before co-planting polyploid cytotypes of C. hispida with C. levisecta. A set 82 of morphological traits used in polyploid identification would serve as a useful tool for the initial identification of polyploid populations from which to source seeds to this end, but is no substitute for a thorough cytological evaluation of a source population.

The three variables that were most useful in distinguishing the cytotypes were calyx pubescence length, corolla length, and galea length. Post-hoc analysis suggests that diploid corollas are, on average, about five millimeters shorter, diploid galeas are three millimeters shorter, and diploid stigma diameters are 0.27 millimeters smaller than their tetraploid and hexaploid counterparts. In hexaploids, the floral angle in pressed specimens is 6.84° wider and calyx pubescence is about one millimeter longer, on average, than tetraploids. It is important to note that floral angle in wild plants will differ from pressed specimens used in this study. With the addition of environmental variation, ecogeographic differences, and phenotypic plasticity, these rather minute differences might not prove helpful in cytotype delimitation.

Future Directions

This study addressed whether morphology varies solely with either taxa or ploidy in a highly controlled environment, and incorporated no additional variables to help explain observed trait variation. For future work, a good starting point to address this would be the addition of origin population of parental taxa as a fixed effect. In this study, we couldn't address this question due to variation in sample sizes. For instance, we either received or were only able to propagate 4 maternal lines from the Scatter Creek population, 5 maternal lines from the Bald Hill population, and one maternal line from the Wolf Haven population (Table 1). Compare this to abundant plant material available for both C. levisecta and hexaploid C. hispida populations.

However, the incorporation of additional parent population germplasm into an ex situ, 83 controlled-environment study would only go so far in accounting for variation between populations.

In order to get a fine-scaled accounting of morphological variation between C. hispida cytotypes, or to explore morphological differences between C. levisecta, C. hispida, and their hybrids, traits must be sampled directly from wild populations. A data set of this magnitude would go well beyond simple taxa identification. It would add to a growing body of work attempting to explain exactly how hybridization and especially ploidy are affected by environmental variation, and how morphology is partitioned along ecogeographic clines.

Furthermore, our study relied on the assumption that populations of C. hispida are static for ploidy level. However, wider sampling from within C. hispida populations could very-well reveal what many plant population ecologists know to be true: that interspecific ploidy variation can exist at both low, intermediate, and high frequencies within populations, and can depend on multiple environmental factors, pollinator constancy, selection processes, and the frequency of unreduced gametes. Here, another important research opportunity exists for examining the environmental and ecological mechanisms contributing to the development of and selection for autopolyploid populations arising from originally diploid populations.

Conservation Implications

In this article, we presented a set of traits that can assist managers in identifying diploid hybrids from C. levisecta, and in distinguishing between three cytotypes of C. hispida. In this study, C. levisecta racemes and flowers appear more “closed” than hybrids, with flowers tending to grow at tighter angles to the stem. The closed look, when combined with the characteristic yellow color, and a large (>1.50mm) stigma that rests against (or very close to) the galea, could serve to help distinguish C. levisecta from hybrids. When identifying C. hispida cytotypes, 84 diploid corollas are about five millimeters shorter, galeas are three millimeters shorter, and stigma diameters are 0.27 millimeters smaller, on average, than their tetraploid and hexaploid counterparts.

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Chapter 4:

Conclusions

Crosses between C. levisecta and diploid cytotypes of C. hispida

We hypothesized that crosses between C. levisecta (2n = 2x = 24) and plants from the diploid race of C. hispida (2n = 2x = 24) would produce fertile hybrids and that both diploid C. hispida and the diploid F1 hybrids would have lower reproductive isolation from C. levisecta.

Castilleja species are known to be highly promiscuous, and have been shown to hybridize readily where two or more species overlap in range (Egger, 1994; Hersch and Roy, 2007;

Hersch-Green and Cronn, 2009; Clay et al., 2012). Indeed, we found that diploid hybrids were fertile, backcrossed readily with C. levisecta to form F2 hybrids, and had higher fitness measures than their progenitors.

Cumulative reproductive isolation in crosses between C. levisecta and diploid hybrids was -21.68% when C. levisecta served as pollen recipient, suggesting that these crosses had higher fitness than conspecific C. levisecta crosses. If mixed populations between these two species are left unmanaged, these hybrids could facilitate introgression between C. levisecta and

C. hispida, which could ultimately lead to genetic swamping of the C. levisecta genome. When diploid hybrids served as pollen recipient in these crosses, reproductive isolation was 0, suggesting that there are no post pollination barriers to reproduction. Taken collectively, these data show that C. levisecta and the diploid race of C. hispida hybridize, and that the hybrids are fertile, confirming that genetic swamping could threaten the C. levisecta genome on sites where these two species are co-planted.

Crosses between C. levisecta and polyploid cytotypes of C. hispida

Diploid-polyploid matings between other closely related plant species resulting in viable progeny have been documented in wild populations (Petit et al., 1997, 1999; Ramsey and Schemske,

1998; Husband, 2004; Kohler et al., 2010; Laport et al., 2016). In addition, differences in 92 chromosome number have been shown to shape, but not necessarily prevent, Castilleja hybridization (Hersch-Green, 2012). We hypothesized that interploidy crosses between C. levisecta and C. hispida would have reduced fitness and higher reproductive isolation due to meiotic problems associated with odd numbers of chromosomes. We found that heteroploid crosses had lower seed set than homoploid crosses, which ultimately contributed to much greater reproductive isolation between C. levisecta and polyploids.

We also hypothesized that fitness in interploidy crosses would be further reduced and that reproductive isolation would increase with a growing difference in ploidy in the interspecific crosses. To that end, we found seed set in CALE(2x) x CAHI(4x) crosses was 9%, and that seed set in CAHI(4x) x CALE(2x) was 36%, compared to 63% in CALE(2x) x CALE(2x) crosses. We found that seed set in CALE(2x) x CAHI(6x) crosses was 0.08%, and that seed set in CAHI(6x) x CALE(2x) crosses was 0.02%, suggesting that average seed set decreases as differences in ploidy increase. However, though seed set decreased as interploidy distance increased, reproductive isolation did not. Germination percentage in F1 CALE(2x) x CAHI (6x) and

CAHI(6x) x CALE(2x) crosses was 100%, which ultimately contributed to very similar reproductive isolation measures in the tetraploid and hexaploid crosses (94.7% vs. 98.6%, respectively). Given these similarities, we did not find evidence that reproductive isolation increases ploidy distance increases in interploidy crosses.

Morphological analysis of C. levisecta, diploid C. hispida, and their F1 Hybrids

Hybridization between C. levisecta and C. hispida presents a unique challenge to the co- recovery of Taylor’s checkerspot and C. levisecta on PNW prairies (Dunwiddie et al., 2016). The eradication of putative hybrids on restoration sites is critically important to the endangered C. levisecta genome. Because these Castilleja traits are highly variable, and can look extremely 93 close to either parental species, eradication is only possible with the ability to identify hybrids from C. levisecta with a high degree of certainty. Multivariate morphological analysis shows that

C. levisecta, C. hispida, and their hybrids are morphologically distinct, and provides insight into traits that distinguish these taxa and their hybrids. We set out to investigate whether there are measurable differences in bract, calyx, and floral characteristics of these taxa, and if so, whether those differences are distinct enough for field technicians to distinguish them. Most hybrid traits measured were intermediate between C. levisecta and C. hispida, there was no one trait that easily distinguished the hybrids from their parent taxa. However, there were combinations of traits that could serve as a guide to land managers who need to distinguish C. levisecta from the diploid hybrids.

Morphological analysis of three C. hispida cytotypes

Multivariate morphological analysis of three cytotypes in the C. hispida species complex revealed morphological variation between ploidy races. Polyploidy has been shown to increase morphological diversity in wild plant populations (Levin et al., 1996; Otto and Whitton, 2000;

Chansler et al., 2016), and in this study we found considerable morphological variation between ploidy races. In trait space, tetraploids are situated between the diploids and the hexaploids on the primary axes of variation in both LDA and PCA, and cytotypes form overlapping clusters in 94 both analyses. This suggests that the complex contains significant morphological variation, that trait values operate on a spectrum.

The three variables that most clearly distinguished the cytotypes were calyx pubescence length, corolla length, and galea length. Post-hoc analysis suggests that diploid corollas are, on average, about five millimeters shorter, diploid galeas are three millimeters shorter, and diploid stigma diameters are 0.27 millimeters smaller than their tetraploid and hexaploid counterparts. In hexaploids, the floral angle in pressed specimens is 6.84° wider and calyx pubescence is about one millimeter longer, on average, than tetraploids. However, with the addition of environmental variation, ecogeographic differences, and phenotypic plasticity, these rather minute differences might not prove helpful in cytotype delimitation.

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