Seed Polymorphism and Domestication in the Lost Crop Chenopodium berlandieri A thesis presented to the faculty of the Voinovich School of Leadership and Public Affairs of Ohio University In partial fulfillment of the requirements for the degree Master of Science Daniel R. Williams May 2019 © 2019 Daniel R. Williams. All Rights Reserved. 2 This thesis titled Seed Polymorphism and Domestication in the Lost Crop Chenopodium berlandieri by DANIEL R. WILLIAMS has been approved for the Program of Environmental Studies and the Voinovich School of Leadership and Public Affairs by Sabrina Curran Assistant Professor of Sociology and Anthropology Mark Weinberg Dean, Voinovich School of Leadership and Public Affairs 3 Abstract WILLIAMS, DANIEL R., M.S., May 2019, Environmental Studies Seed Polymorphism and Domestication in the Lost Crop Chenopodium berlandieri (97 pp.) Director of Thesis: Sabrina Curran Domesticated Chenopodium taxa are distinguished from their wild relatives on the basis of seed morphology including thin seed coats, truncate seed margins, and light-colored appearance. Wild chenopods produce polymorphic seeds with a range of seed coat thicknesses, colors, and margin shapes. Rather than assuming a homogeneous and smooth morphological transition gradient during domestication, the effects of seed polymorphism must be considered. To compare polymorphism of wild seeds to domesticated seeds, wild chenopod seeds were collected and separated into morphological types by color. From each plant, ratios of seed morphs were counted, morphological measurements were taken, and germination was tested under a range of stratification and germination conditions. The same measurements and tests were conducted on the seeds of domesticated chenopods. Those seeds with lighter colors and thinner seed coats, called red morphs, are morphologically similar to seeds of domesticated chenopods. Red morphs, like domesticated chenopods, also have lower dormancy and lower over-wintering viability than do black morphs. Differences in polymorphic heterogeneity among wild chenopods implies a model of domestication. Under conditions of high disturbance and spring planting, chenopods producing higher proportions of red morph seeds may have a selective advantage. The role of heterogeneity in the domestication of chenopodium can inform modern crop improvement programs. Additionally, the yield of experimental plots of wild chenopods compares favorably with quinoa and modern agricultural crops. Re-domestication of the lost Chenopodium domesticate has the potential to create a new high value agricultural crop for eastern North America. 4 Dedication To my parents and my brother who always listened 5 Acknowledgements Dr. Paul E. Patton provided funding, resources, and guidance which made this work possible. Dr. Sabrina Curran was always available to talk about methods and data analysis. Dr. Sarah E. Wyatt helped me see the connection between seed colors and seed development. Dr. Harvey Ballard helped with taxonomy, adding necessary complexity to my understanding of the genus. Dr. Jonathan Fresnedo Ramirez provided thoughtful timely feedback on the design and requirements of plant breeding programs, helping to shape the direction of my research. Harold Blazier and Dr. Art Trese allowed me to grow weeds in the greenhouse and learning gardens. Undergraduates students and volunteers also helped at many stages; Jacob Ballas, Skylar Adkins, and Brenna Tippie deserve special recognition. Funding was provided by the Sugar Bush Foundation, the Ohio University Voinovich School of Leadership and Public Affairs, and the Ohio University Food Studies Theme. Land, laboratory space, and material resources were provided by the Department of Anthropology and Sociology and the Department of Environmental and Plant Biology. Michael Lloyd provided additional land for experimental plots. Seed accessions were provided by the U.S. National Plant Germplasm System. 6 Table of Contents Page Abstract.........................................3 Dedication........................................4 Acknowledgements...................................5 List of Tables......................................8 List of Figures......................................9 Foreword........................................ 11 Chapter 1: Seed Polymorphism in Chenopodium berlandieri and C. missouriense .. 12 Introduction..................................... 12 The wild precursor of C. berlandieri var. jonesianum ............. 12 Indicators and artifacts of domestication.................... 17 The red morph problem............................. 18 Maternal regulation of seed phenotype..................... 20 Seed polymorphism as environmental adaptatation............... 21 Hypotheses................................... 21 Methods....................................... 22 Counting morphs in seed samples....................... 24 Morphological analysis............................. 27 Results........................................ 30 Seed morphology................................ 30 Variance of morph proportion.......................... 35 Discussion...................................... 37 Potential environmental causes of seed polymorphism............. 38 Potential genetic causes of seed polymorphism................. 39 Hypotheses for seed development and implications for domestication..... 40 Future work................................... 43 Conclusion..................................... 44 Chapter 2: The role of seed polymorphism in the domestication of C. berlandieri .. 46 Introduction..................................... 46 Domestication.................................. 47 Environmental adaptations and effects..................... 49 Day length and harvest time........................ 49 Stratification requirements......................... 50 7 Adaptations to freezing........................... 50 Spring planting and weed competition................... 50 Hypotheses................................... 51 Stratification requirement varies by morph................. 51 Freezing susceptibility varies by morph.................. 51 Germination speed varies by morph.................... 51 Seedling growth speed varies by morph.................. 52 Methods....................................... 52 Preparation and taxonomic identification of samples.............. 52 Stratification................................... 54 Freezing..................................... 55 Germination speed............................... 56 Seedling growth................................. 57 Results........................................ 58 Stratification................................... 58 Freezing..................................... 59 Germination speed............................... 59 Seedling growth................................. 60 Results summary................................ 60 Discussion...................................... 61 Interaction of human behavior and polymorphism genetics.......... 63 Conclusion..................................... 65 Chapter 3: Lost Crop and Future Food......................... 67 Introduction..................................... 67 An open economic and environmental niche.................. 67 Endemic Chenopodium spp. as a future crop.................. 68 Use as a crop in prehistory......................... 69 Prehistoric economics and yield...................... 70 Quinoa as a proxy and proof of market................... 71 Industrial production of quinoa outside the Altiplano is problematic.... 72 C. berlandieri has the potential to address several concerns........ 73 Methods....................................... 73 Field plots.................................... 73 Calculation and estimation of yield....................... 74 Results........................................ 74 Discussion...................................... 75 The modern economic potential of Chenopodium spp.............. 75 Conclusions..................................... 76 Chapter 4: Takeaways.................................. 78 References........................................ 80 8 List of Tables Page Table 1 Chenopodium populations used to study variation in the distribution of red and black seed morphs.......................... 26 Table 2 Chenopodium populations in seed morphology study. Cleaned seed samples were separated into categories by color. Red seeds were separate from black seeds. Red sees were sometimes also separated into light red (LR) and dark red (DR) categories...................... 29 Table 3 Chenopodium populations used in stratification, freezing, germination speed, and growth speed experiments. Seeds of populations except B80 were sorted into three color categorical bins: light red, dark red, and black. B80 was sorted into red and black categorical bins. The total number of seeds from each population included in each experiment is listed. Abbreviations: nstrat. = stratification, nfreeze = freezing susceptibility, ngerm. = germination speed, ngrow. = seedling growth speed................................. 53 9 List of Figures Page Figure 1 Distribution of Chenopodium spp. Adapted from Jellen et al. (2011) and Steven E. Clemants & Mosyakin (2004).................. 15 Figure 2 The morphology of Chenopodium seeds. A line diagram shows the tissue types and internal structure of a generic Chenopodium seed (a). Whole seeds and their cross-section are shown for C. berlandieri subsp. nuttalliae (b), C. quinoa (c), C. berlandieri var. macrocalycium (d), and red (e) and black (f) morphs of C. missouriense.............. 19 Figure