An Integrative Taxonomic Study of Ramps (Allium tricoccum Aiton) Complex

A thesis presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Bina Swasta Sitepu

August 2018

© 2018 Bina Swasta Sitepu. All Rights Reserved. 2

This thesis titled

An Integrative Taxonomic Study of Ramps (Allium tricoccum Aiton) Complex

by

BINA SWASTA SITEPU

has been approved for

the Department of Environmental and Plant Biology

and the College of Arts and Sciences by

Harvey E. Ballard, Jr.

Professor of Environmental and Plant Biology

Joseph C. Shields

Interim Dean, College of Arts and Sciences 3

ABSTRACT

SITEPU, BINA SWASTA, M.S., August 2018, Environmental and Plant Biology

An Integrative Taxonomic Study of Ramps (Allium tricoccum) Complex

Director of Thesis: Harvey E. Ballard, Jr.

The Allium tricoccum complex is native to eastern North America and encompasses broad and confusing morphological variation. Previous studies have led to contrary classifications to account for the diversity of morphologies in the Allium tricoccum complex. Living plants of the complex, leaf tissue samples and soil samples were collected from 28 natural populations in seven states. Plants were cultivated in the

Ohio University common garden for biweekly observations of morphological traits; growth patterns and phenology of leaves, flowers and fruits; and weekly photography of plant structures. Morphology, phenology, ecology, and genetic diversity were analyzed to delineate and compare distinct taxa found in the Allium tricoccum complex. Two new characteristics of the species were described for the first time: scape growth direction

(orientation) and depth of bulb in the ground. Two major groups in the Allium tricoccum complex, Red Ramps (A. tricoccum sensu stricto) and Green Ramps, were broadly distinguished based on many differences in leaf shape and size; pigmentation of leaf, scape and bulb; number of buds, flowers and fruits; scape growth direction, bulb size and depth in the ground. Three distinctive taxa within the broader Green Ramps group were separated based on differences in morphology, ecology, phenology and geography. The

Green Ramps group consisted of A. burdickii (Hanes) G. N. Jones in the Great Lakes and

Great Plains regions; a South Green Ramps taxon in the Interior Highlands of Kentucky 4 and Tennessee, similar to A. burdickii but distinct in its tendencies toward narrower leaves, more flowers, shorter perianth and shorter stamens, consistent retention of leaves during scape elongation, and preference for more silty or clayey soils; and a Highland

Green Ramps taxon in the Appalachian Mountain region, producing broader distinctly petiolate leaves similar to A. tricoccum, with somewhat intermediate flower and fruit traits between A. burdickii and A. tricoccum, absence of reddish-purple pigmentation, and deep bulb and erect scape characteristic of the other Green Ramps taxa. Microsatellite markers failed to provide genetic differentiation among the four taxa, but did provide separation of populations within each, suggesting that loci adapted from cultivated garlic cultivars may be too variable at the species level for the wild species in the A. tricoccum complex. Corroborative evidence from macromorphology, phenology and ecology, and application of the Unified Species Concept, rejected usage of one or two taxa previously proposed, and instead supported the recognition of four distinct evolutionary species to accommodate diversity in the A. tricoccum complex.

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DEDICATION

Magna Vis Fidelitatis,

I dedicate this work for my Lord, owner of the knowledge and my life,

I would like to give special thanks for my wife, Agustina Dwi Setyowati, and my family

for endless support in my effort to achieve my dream. 6

ACKNOWLEDGMENTS

I believe this thesis could be finished as a result of hard work by teamwork, and I am really proud to be part of this team. I express my appreciation to my advisor,

Prof. Dr. Harvey E. Ballard, Jr., for his support, advice, and scientific expertise. I would also like to thank my committee members, Dr. Arthur Trese and Dr. Rebecca

Snell, for their assistance and valuable input for my research and thesis. Special thanks to Dr. Rebecca Snell for her help in statistics and Species Distribution

Modelling. Also, I give my thanks to Dr. Jared DeForest for help in the soil analysis and access to the Soil Analysis room, Prof. Brian C. McCarthy, Ph.D., for granting me access to use his lab and equipment for soil analysis, and Prof. Dr. Morgan L. Vis, for the kindness to provide some lab supplies. Thank you to Harlan Svoboda, Anne

Sternberger, and Jennifer Hastings, for friendship and endless support during my research. I thank Ohio University and the Graduate Student Senate (for an Original

Work Grant) at Ohio University for providing funding for my research. I would like to thank those who have contributed their various time to my research: Danny Wolf,

Colin Kruse, Proma Basu, Yemi Ajayi, Dasmeet Kaur, Tasleem Javaid, and Marion

Holmes. Thank you to the USAID-Prestasi Program for the scholarship and research funding. Additional thanks to botanists in the Tennessee and Kentucky Heritage

Programs for providing Ramps population information, and Bernheim Arboretum and

Research Forest in Kentucky and Eagle Crest Nature Preserve in Indiana for permission and assistance in conducting field work on their lands. Dr. Alan S.

Weakley and Tom Govus provided me invaluable discussion and information about 7

Ramps in the southern Appalachian region. I am indebted to everybody who has taken part in this effort to contribute this new information for science and yield a small colored point in the ocean of knowledge.

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TABLE OF CONTENTS

Page

Abstract ...... 3

Dedication ...... 5

Acknowledgments...... 6

List of Tables ...... 10

List of Figures ...... 11

Chapter 1: Introduction ...... 13

Taxonomic History ...... 17 Types of Data Utilized ...... 21 Objectives ...... 24 Broader Impacts ...... 25 Chapter 2: Morphology ...... 26

Methods...... 26 Results ...... 34 Discussion ...... 44 Chapter 3: Phenology ...... 50

Methods...... 50 Results ...... 52 Chapter 4: Environmental Factors ...... 62

Methods...... 62 Results ...... 63 Discussion ...... 68 Chapter 5: Molecular Analysis ...... 72

Methods...... 72 Results ...... 73 Discussion ...... 75 9

Chapter 6: Species Distribution Models ...... 77

Methods...... 77 Results ...... 78 Discussion ...... 84 Chapter 7: Systematic Review ...... 89

Key to Species in the Allium tricoccum Complex ...... 95 Description of the Taxa ...... 96 Literature Cited ...... 104

Appendix ...... 112

Appendix 1: Morphology examined from 28 populations for four taxa of the Allium tricoccum complex...... 112 Appendix 2: Morphological measurement data from 28 populations for four taxa of the Allium tricoccum complex...... 115 Appendix 3: Phenological data from 20 populations of four taxa of the Allium tricoccum complex...... 126 Appendix 4: Environmental data from 28 populations of four taxa of the Allium tricoccum complex...... 130 Appendix 5: Presence/absence (0/1) data matrix of alleles amplified for twelve microsatellite loci in four taxa of the Allium tricoccum complex...... 133 Appendix 6: Species Distribution Model maps of three Green Ramps taxa of the Allium tricoccum complex utilizing the Maximum Entropy model...... 198 Appendix 7: Geographic coordinates of four taxa of the Allium tricoccum complex used in the Species Distribution Model...... 200

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LIST OF TABLES

Table 1. Field sites for the Allium tricoccum complex ...... 27 Table 2. List of morphological characteristics recorded on OTUs for each of 28 populations and four taxa of the Allium tricoccum complex...... 29 Table 3. Pair-wise Post Hoc Test of morphological traits with 999 permutations among four taxa of the Allium tricoccum complex...... 39 Table 4. Pair-wise Post Hoc Test of phenological traits of scape, flowers and fruits with 999 permutations among four taxa of the Allium tricoccum complex...... 53 Table 5. Pair-wise Post Hoc Test of length periods of leaf emergence to leaf senescence, and from leaf emergence to scape emergence data from phenology observation in 2018 with 999 permutations among four taxa of the Allium tricoccum complex...... 55 Table 6. T-test of comparisons in earliest opening date of flower among co-occuring taxa in two...... 56 Table 7. Pair-wise Post Hoc Test of soil variables and elevation with 999 permutations among four taxa of the A. tricoccum complex...... 65 Table 8. Analysis of molecular variance (AMOVA) based on 12 microsatellite loci in four taxa of the Allium tricoccum complex...... 74 Table 9 Training and test data of AUC values for distribution models of three taxa in the Allium tricoccum complex...... 78 Table 10. Morphological, phenological and ecological traits to distinguish four evolutionary species in the Allium tricoccum complex...... 92

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LIST OF FIGURES

Figure 1. Leaf, stem and bulb morphology of (A) Allium tricoccum, (B) Highland Green Ramps, (C) Allium burdickii, and (D) South Green Ramps...... 38 Figure 2. Non-metric Multidimensional Scaling ordination with Gower’s coefficient from 23 morphological of four taxa in the Allium tricoccum complex...... 40 Figure 3. Canonical Variates Analysis ordination from 23 morphological of four taxa in the Allium tricoccum complex...... 41 Figure 4. Canonical Variates Analysis ordination on 19 morphological of three Green Ramps taxa in the Allium tricoccum complex...... 42 Figure 5 UPGMA Dendogram from Cluster Analysis using Gower's coeffeicient on 23 morphological variables of four taxa in the Allium tricoccum complex and 999 bootstrap replicates...... 43 Figure 6. Two color variants of the Allium tricoccum scape in early development...... 46 Figure 7. Stack bar of phenology period of each taxon in the Allium tricoccum complex for 2017 and 2018 observations...... 52 Figure 8. Non-metric Multidimensional Scaling ordination from length periods of scape emerge to flowering, flowering to fruiting, and from fruiting to Fruiting dehiscence with Gower’s coefficient of four taxa of the Allium tricoccum complex...... 54 Figure 9. Non metric Multidimensional Scaling ordination with Gower’s coefficient on seven environmental variables in four taxa of the Allium tricoccum complex...... 68 Figure 10. Non-metric Multidimensional Scaling ordination with Gower’s coefficient on six environmental variables (altitude excluded) in three Green Ramps taxa of the Allium tricoccum complex...... 68 Figure 11. Extensive population of Allium tricoccum growing in Eagle Crest Nature Preserve, IN, April 28, 2017...... 69 Figure 12. Principal Coordinates Analysis ordination with Dice coefficient based on 12 microsatellite loci in four taxa of the Allium tricoccum complex...... 75 Figure 13. Prediction of A. tricoccum habitat distribution based on presence data with Maximum Entropy method...... 79 Figure 14. The Jackknife test for evaluating the relative importance of environmental variables for Allium tricoccum...... 80 Figure 15. Prediction of Allium burdickii habitat distribution based on presence data with Maximum Entropy method...... 81 Figure 16. Prediction of South Green Ramps habitat distribution based on presence data with Maximum Entropy method...... 82 Figure 17. The Jackknife test for evaluating the relative importance of environmental variables for Allium burdickii...... 83 12

Figure 18. The Jackknife test for evaluating the relative importance of environmental variables for South Green Ramps...... 84 Figure 19. Response curves from variable Bio1 (annual mean temperature) in the model prediction of Allium tricoccum habitat distribution based on presence data with Maximum Entropy model...... 85 Figure 20. Response curve from variable Bio11 (Mean Temperature of Coldest Quarter) in the model prediction of Allium burdickii habitat based on presence data with Maximum Entropy model...... 86 Figure 21. Response curve from variable Bio11 (Mean Temperature of Coldest Quarter) in the model prediction of South Green Ramps habitat distribution based on presence data with Maximum Entropy model...... 87

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CHAPTER 1: INTRODUCTION

The genus Allium L., including cultivated Onion, Garlic, Shallots and other economically important plants, contains from 550 to more than 800 species (Li et al., 2010;

McNeal and Jacobsen, 2002). Traditionally the genus has been submerged in a huge family, the Liliaceae, but molecular phylogenetic studies have broken up the Liliaceae into many smaller families. Recent treatments have placed Allium into a smaller family, Alliaceae, as the largest of 13 genera (e.g., Thorne, 1992), while other treatments such as the

Angiosperm Phylogeny Group have included it in an expanded Amaryllidaceae with 73 genera and over 1600 species (Chase et al., 2009; Stevens, 2001). In North America (north of Mexico), Allium reportedly consists of 96 species (McNeal and Jacobsen, 2002). In traditional systematic studies of Allium, Hanelt (1992) placed most native North American members in New World subgenus Amerallium, with a base chromosome number of x=7, but removed circumboreal A. schoenoprasum L., eastern North American A. tricoccum

Aiton, and Asian and western Alaskan A. victorialis L. to the primarily Old World subgenus Rhizirideum (G. Don ex W. D. J. Koch) Wendelbo, with a base of x=8. Using

ITS sequence data as well as morphological traits, Friesen et al. (2006) reexamined the genus and erected subgenus Anguinum (G. Don ex W. D. J. Koch) N. Friesen for the second lineage including A. tricoccum. More recent molecular phylogenetic studies by Choi et al.

(2012) and others have supported this subdivision of North American taxa into two distantly related evolutionary clades, highlighting intriguing biogeographic disjunctions among the New and Old World taxa. Studies of Herden et al. (2016) and Nguyen et al.

(2008) have specifically placed Allium tricoccum with A. victorialis L. from Europe and A. 14 microdictyon Prokh., A. nerinifolium Baker, A. ovalifolium Hand.-Mazz., A. prattii

C.H.Wright ex Hemsley (among others) from Asia (and Attu Island, Alaska). Separate phylogenetic trees based on the nuclear ribosomal Internal Transcribed Spacer and three chloroplast spacers revealed discordant relationships suggesting the possibility of a hybrid origin for A. tricoccum (Herden et al. 2016) involving two subclades of species. Several accessions of A. tricoccum were included and no cladistic structure was observed among accessions, but it is unclear whether more than one taxon in the Allium tricoccum complex besides A. tricoccum sensu stricto was used. Both A. tricoccum and A. victorialis are unlike other North American Allium species in producing flat broadened leaf blades, resembling several other European and Asian Allium taxa.

Allium tricoccum Aiton, known as Ramps, or Wild leek, is an economically useful wild species that is widely known as a medicinal herb and vegetable. This species was described first by Daniel Carl Solander in 1789, in Aiton’s “Hortus Kewensis” catalog for

Kew Botanical Garden. Allium tricoccum is distinguished from other exclusively American

Allium species by the ephemeral flat broad leaves that shrivel and disappear before or during scape emergence and elongation but generally are completely withered prior to the opening of the flowers. All other North American species of Allium retain their leaves throughout reproduction (McNeal and Jacobsen, 2002). The colloquial name “Ramps” is likely derived from the earlier British English name “Ramson”, applied to the morphologically similar European Bear Leek, A. ursinum L. (Core, 1973). Allium tricoccum as a complex is native to North America, and is widely distributed from Quebec, 15

Canada south to Georgia and west to eastern North Dakota and Missouri, USA (Jones,

1979).

Ramps are described as forest-inhabiting, long-lived perennials, with or without reddish-purple pigmentation on foliage or scape. Plants produce 2-6 bulbs borne along a short rhizome, bulbs with an outer brownish to grayish reticulate finely fibrous coat, and each bulb with an inner white coat. The 2-3 narrowly linear to broadly elliptical leaves have a short indistinct or elongate distinct petiole and a well defined midrib, as well as slightly to somewhat thick and fleshy blades. Leaves emerge in early spring prior to canopy leafout, and some populations (taxa) lose their leaves to senescence nearly or completely before the scape emerges, while others retain their leaves for a period of time before the scape becomes fully erect. The emerging scape revealing the several to numerous flowers arranged in a narrowly conical to nearly spherical umbel. Each flower bears six oblong to ovate erect tepals in two whorls, six slightly to pronouncedly exserted epipetalous stamens, and a three-lobed, three-locular pistil with one style. Ramp populations in Quebec were found to be self-compatible, and seed production was not limited by pollinator activity

(Nault and Gagnon, 1987). Pollination and fertilization of flowers occurs by mid to late spring, although there is some evidence that an appreciable amount of self-pollination contributes to seed set. One large spherical to obovoid seed develops in each of the three pistil locules, and the mature loculicidal capsules dehisce in early summer. In A. tricoccum sensu stricto, colonies are often very dense with numerous plants, while other taxa develop diffuse colonies with individuals and small numbers of plants commonly scattered sporadically over the forest floor. 16

The famous Sulphur-containing compounds of other members of the genus are also found in A. tricoccum (namely, thiosulfinates and cepaenes, Calvey et al. 1998), and the plants are utilized for this reason as a medicinal herb and a food plant over much of the range of the species. Those who eat or cook with Ramps note that all parts of the plant have a flavor combining garlic and onion (Zeldes, 2010). Scattered reports by ramps harvesters and those who regularly eat ramps have mentioned that the different taxa (mainly White and Red ramps) have noticeably different flavors. If this is true, it suggests that the different taxa also diverge in their biochemical composition (Rusalepp, et. al., 2017; Vlase, 2012).

A number of Native American tribes have long used the plants for medicine, as a spring tonic, a cure for colds and croup, to heal earaches, and as an emetic, as well as a food plant

(Witthoft, 1977; Hamel et al. 1975; Perry, 1975; Smith, 1923, 1933; Densmore, 1928;

Waugh, 1916). Several Appalachian towns promote Ramps festivals, in which tons of fresh

Ramps are brought to local markets, as are food products made from Ramps, for tourists and locals. Such festival towns include Cosby and Flag Pond, TN; White Top, VA; and

Elkins, Huntington and Richwood, WV. Traditionally the bulbs are used, although increased reliance on leaves is being encouraged by some as a means to foster more sustainable harvesting practices that leave bulbs in the ground.

Overharvesting has become a serious problem in some states and provinces, including Maine, Rhode Island and Tennessee, where A. tricoccum is considered a species of “Special Concern” (USDA Plants Database, 2018); it is “Threatened” in Quebec. While overharvesting in some areas appears to be a potentially increasing problem, the initial mortality of newly established populations by intentional seed or bulb plantings have been 17 shown to be low and transplantation shock was minimal (Vasseur and Gagnon, 1994).

Seedling emergence and seed dormancy in such experiments were influenced by soil moisture and summer drought, and high soil nutrients fostered proper growth of young and mature plants. A five-year study of demography in Quebec populations indicated that sterile ramets showed high survival rates and vegetative propagation was maintained, but that seedlings largely failed to establish. Most flower scapes died before setting seed in these populations and flowering was rare, but flowering ramets showed high levels of asexual reproduction. Simulations of harvesting levels against measured reproductive success for Quebec populations showed that even low levels (5-15%) were sufficient to compromise population maintenance (P.P. Dion et al., 2016). Whether these results mirror processes in populations elsewhere in the range of A. tricoccum, or if other taxa of the

Allium tricoccum complex demonstrate the same trends, is unclear.

Taxonomic History

In 1952, Moldenke described forma pictum with additional details for the presence of petiole pigmentation (deep-red color of the midrib), but this has since been ignored.

Hanes (1953) proposed var. burdickii for the different “race” that he observed in

Kalamazoo County, Michigan and from herbarium specimens collected and described in a letter by Burdick from Wilton, Wisconsin. In 1979, Almut G. Jones was the first to make extensive range-wide taxonomic studies of the Allium tricoccum complex. She examined approximately 2000 herbarium specimens over the range of the complex and made morphological measurements on numerous specimens. She also conducted one season of weekly or biweekly field observations on morphology and phenology of vegetative and 18 reproductive structures on populations in central Illinois around Indianapolis area.

Documenting many differences in macromorphological traits and phenology of leaves, scapes, flowers and fruits, she raised var. burdickii to species rank, as A. burdickii (Hanes)

A. G. Jones. She also tentatively identified a number of anomalous specimens as potential hybrids, because the encompassed combinations of traits otherwise unique to A. burdickii or A. tricoccum. Koeffman (2001) and McNeal and Jacobsen (2002) decided to retain A. burdickii as a variety of A. tricoccum, without providing any additional study but noting that some specimens outside the main Great Lakes and Great Plains range of A. burdickii appeared to be intermediate between that and A. tricoccum or expressed confounding variation patterns that did not fit either A. burdickii or A. tricoccum as previously delimited

(e.g. unpigmented plant with broad leaves). Bell (2006) conducted research similar to that of Jones, examining and measuring numerous herbarium specimens for some of the same morphological traits used by Jones (and others modified from Jones). She made laboratory observations of morphology and conducted crossing experiments among individuals, populations and taxa to interpret inter-fertility, on living plants brought back from populations in eastern West Virginia, Pennsylvania and New York. She reported apparent overlap of morphological traits and geographic distributions, and found her identified taxa to be fully interfertile. However, it is important to note that Bell assigned all specimens to one of the two previously proposed taxa on the sole basis of presence or absence of reddish- purple pigmentation of the foliage and scape, classifying plants as A. burdickii (non- pigmented) or A. tricoccum (pigmented). Bell furthermore completely ignored Jones’s phenology observations, or failed to document differences. Additionally, the particular 19 methods used by Bell for certain traits, such as blade versus petiole length, diminished the discriminating power of those leaf traits. Bell concluded that there was no evidence to recognize two taxa, and she lumped everything into one variable species, A. tricoccum.

While the taxonomic studies by Jones and Bell relied heavily on macromorphological data,

Jones noted clear phenology shifts in vegetative and reproductive structures of taxa in central Illinois, and Bell examined crossing relationships, no studies to date have included genetic evidence. In addition, neither investigator examined a full range of morphological traits or phenological shifts in every vegetative and reproductive structure from living plants over a substantial portion of the range of the complex. Although both Jones’s and

Bell’s studies utilized numerous herbarium collections, their characterization of total phenotypic diversity in the complex and how they partitioned that diversity were substantially divergent, and neither comprehensively characterized features of leaves, scapes, umbels, flowers, fruits and seeds. Their field studies, conducted in different areas of the range of the complex, very probably detected, characterized and compared quite different sets of taxa.

Presently, many botanists in the central and southern Appalachians remain baffled in applying the taxonomic concepts represented by one or two species to adequately represent phenotypic and phenological diversity of the complex (Dr. Alan Weakley,

University of North Carolina, pers. comm.). This is particularly true of Green Ramps populations that lack pigmentation in the foliage and reproductive structures. While

Midwestern and Great Lakes populations of Green Ramps, attributable to A. burdickii, are immediately recognizable and very distinct from Red Ramps (A. tricoccum sensu stricto), 20

Green Ramps populations outside of that region differ in various ways from the two narrowly delimited and formally recognized species. The contrary taxonomic treatments of previous studies and the continued confusion of apparently unrecognized phenotypic diversity suggest that intensive studies, including field investigations across the range of the complex, common garden observations, and use of genetic evidence, are needed to clarify the taxa and their evolutionary status.

In the last two decades, increasing numbers of taxonomic studies, mostly of animal and fungal groups, have applied a diverse range of evidence, including morphological, environmental, reproductive, geographic, and molecular or genetic data, to delineate sets of populations as distinct taxa. This approach, generally known as Integrative Taxonomy

(Dayrat, 2005), has been successfully employed in challenging groups such as the Coral- root orchids (Corallorhiza striata complex, Barrett and Freudenstein, 2011). Another study of related species of Allium (Choi and Oh, 2011) that used multiple sources of information, but did not explicitly adopt an integrative taxonomic approach by name, distinguished

Allium ochotense Prokh. from Allium victorialis L., citing differences geographic distribution due to environmental variables (East Asia to Alaska vs Eurasia to the

Himalayas), morphology (broad leaves and larger whitish perianth), and phenology

(flowering May to June vs. July to August).

In some instances Integrative Taxonomy has been coupled with explicit application of the General Lineage or Unified Species Concept (USC) by de Queiroz (2007), where the sole criterion of species is a “separately evolving metapopulation”, but detection and delineation of such species is ideally based on multiple lines of evidence (secondary criteria 21 including morphology, ecology, genetics, etc.). A quantitative morphological study of the genus Hedera successfully employed the USC and identified 12 species in two groups. The value of employing the USC as a filter to interpret results from diverse data sets is to have a more objective interpretation of the evolutionary status of potentially diverging sets of populations. Reliance on the USC to detect, delimit and recognize diverging or fully diverged taxa as evolutionary species, the evolutionary status and corresponding taxonomic treatment of sets of populations (as infraspecific taxa, species, or something else) becomes much more subjective and arbitrary. Additionally, the USC expects integration or consideration of all available evidence to make a decision regarding the evolutionary status (and taxonomic treatment) of sets of populations, and encourages recognition of species from any evidence that suggests the presence of separately evolving metapopulations. Although Integrative Taxonomy and the Unified Species Concept address two quite different aspects of biodiversity investigation, they are mutually complementary in fostering comprehensive inquiry into biological diversity of an organismal group, and have the potential to provide a more objective and scientifically valid means of delineating and recognizing the products of evolutionary diversification.

Types of Data Utilized

Comprehensive, methodical characterization of all morphological structures and their phenology through the growing season will include the bulb, leaves, scape, umbel and bract, flowers, fruits and seeds, within and among living populations and across taxa.

Morphological traits are the basic and “traditional” data used to distinguish species

(Dayrat, 2005), resulting in sets of populations known as morphospecies (Cain, 2014). 22

Species delimitation in this approach typically uses several to many morphological variables (both quantitative and qualitative) across the plant body, most or all of which would overlap heavily or completely among populations within a taxon and some of which would not overlap (or overlap extensively) among taxa. Morphological differentiation commonly correlates with geographic range, ecology or other properties of the taxa (Wiens and Servedio, 2000).

Ecological niche data are important to understanding species geographic distributions and habitat preferences. Abiotic environmental variables that contribute to species’ geographic distributions include soil, climate, topography, elevation, and sunlight, and for some plant species biotic interactions such as pollinator or herbivore/predator interactions as well (Gaston 2003). While these broader environmental variables can influence or determine where a species can grow, local factors including substrate variables (e.g., pH, moisture, texture), light availability or slope commonly have tremendous impact on the site-level distribution of populations or taxa. Niche modeling of geographic ranges and analysis of local factors correlated with differential microhabitat occupation provide mutually complementary types of evidence regarding ecological differentiation between taxa. Hanes (1953) and Jones (1979) observed different habitat preferences between A. tricoccum and A. burdickii, with the first inhabiting moister soils and the second, drier soils. However, they also noted several sites where the two species grew side by side or slightly intermingled. Examination of substrate variables will hopefully reveal local ecological niche differences among taxa if the taxa occupy divergent microhabitats. 23

Microsatellite markers are areas (loci) in the DNA of tandemly repeated base pair sequences (1 to 6 bp, generally at least 5 and usually 10-25 repeats long) (Agarwal et al.,

2008). Microsatellites in all eukaryotic organisms occur as hundreds of different locations throughout the nuclear genome, have codominant inheritance, show high allelic diversity within populations and express high differentiation above the population level in non- clonal species, and can readily be assessed for size variation in number of repeats using

PCR. Most of the microsatellite applications in the genus Allium have focused on cultivars of domesticated species, such as A. cepa L. (Onion), A. fistulosum L. (Welsh

Onion) and A. sativum L. (Garlic). None of these are phylogenetically close to the Allium tricoccum complex, although Allium sativum is less phylogenetically distant than the others (Nguyen et al., 2008). The successful application of microsatellite markers to distinguish cultivars of A. sativum led to experimentation with the same loci for investigations of genetic relationships among other Allium species (Lee et al. 2011).

Although microsatellite loci have been isolated and tested in a preliminary way with A. cepa, the application of the primer pairs on other species in the genus Allium is not developed (Chinnappareddy et al. 2013). For this study, 12 apparently variable microsatellite primers developed by Lee et al. (2011) for A. sativum cultivars were chosen to investigate the genetic diversity and variance of taxa in the Allium tricoccum complex.

Species Distribution Models (SDMs), known also as (Ecological) Niche models, utilize associations between environmental factors and known georeferenced occurrences of taxa or populations to define abiotic conditions within which populations can be 24 maintained (Guisan and Thuiller 2005). Application of SDMs in some research indicate the advantage of this method to discover broader ecological parameters that determine the distribution of populations or taxa and then investigate potential gaps where new populations may be found, or to test for differences in ecological parameters that may define distributions of multiple taxa (Raxworthy et al. 2007; Austin et al. 2009). The SDM for each phenotype of the Allium tricoccum complex will enrich our understanding of the biology and evolution of the taxa based on bioclimatic variables. Such investigations will also allow for further predictions of new populations for taxa (if successful), and for modeling future distributions under various scenarios of climate or land use change, as a part of conservation management for the species recognized.

Objectives

Although the Allium tricoccum complex has been studied by a number of investigators, conclusions as to the number of distinct species and their variation have differed substantially. The divergent conclusions have been based on the different methods used, the highly limited geographic areas from which living plants have been observed or studied, and failure of the studies to examine phenotypic and phenological diversity comprehensively. The taxonomic conclusions have thus failed to accommodate the full extent of diversity in the Allium tricoccum complex. Botanists recently have come to realize that many populations over the south-central Midwest and the Appalachian

Mountain regions fail to fit either A. burdickii or A. tricoccum as described, suggesting the presence of more species rather than one or two polymorphic species. This thesis uses an Integrative Taxonomic approach with morphology, phenology, ecology and genetic 25 diversity, applying the Unified Species Concept as an objective filter to detect consistently different taxa that represent evolutionary species as separately evolving metapopulations following the USC.

Broader Impacts

Detection and characterization of additional species, and clarification of those already known, will aid conservationists with valuable information toward developing sustainable harvesting practices of populations, as well as state-level protection and in situ conservation programs for populations and taxa at risk. A revised taxonomic scheme for diversity in the Allium tricoccum complex, in considering the suggestions by Ramps harvesters that different taxa have different flavors, would be a valuable resource for food biochemists to examine chemical properties of the evolutionary species recognized.

Finally, because plant-focused literature employing an integrative taxonomic approach or applying the Unified Species Concept is still quite rare, it is hoped that this study will serve as a model for attempting these pursuits with other plant groups for which previous examinations have remained unsatisfyingly equivocal.

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CHAPTER 2: MORPHOLOGY

Methods

Taxa. A select number of preliminary morphological trait measurements, both qualitative and quantitative, was chosen from bulb, leaves, scape and umbel and applied to populations in the field prior to further study. Red Ramps (A. tricoccum sensu stricto) was immediately distinct from Green Ramps taxa. Green Ramps populations in Michigan and northern Indiana were tentatively assigned to A. burdickii, because plants precisely matched the type specimens and details of descriptions given by Hanes and Jones from living populations. Populations visited and sampled for this study from the Interior

Highlands region diverged in multiple features from A. burdickii populations and were tentatively assigned to South Green Ramps, awaiting further study. Populations sampled from the Appalachian Mountain region diverged even more from A. burdickii and A. tricoccum and were tentatively assigned to Highland Green Ramps. Individual plants, or

Operational Taxonomic Units (OTUs), within each population of one of the four taxa were found in every instance to express highly uniform suites of morphological traits, even in a number of field sites where two taxa were found to commingle. No examples of hybridization were encountered, as results of different flowering period between A. tricoccum and A. burdickii (Jones (1979), Bell (2007).

Field Studies. Initial information for field studies was obtained from specimen data from Ohio University Herbarium (BHO), Ohio State University Herbarium (OS),

University of Michigan Herbarium (MICH), the Consortium of Midwest Herbaria

(http://midwestherbaria.org/portal/), and the Southeast Regional Network of Expertise and 27

Collections (SERNEC, http://www.sernecportal.org/portal/); as well as from professional botanists in Indiana, Kentucky, Michigan, North Carolina, Tennessee, and West Virginia.

Twenty sites in seven states were identified and visited, and found more than one taxon in five sites. Allium tricoccum has 7 sampled population, Allium burdickii has 9 sampled population, South green has 6 sampled population, and Highland green has 6 sampled population, in total there 28 sampled population. At each site (Table 1), all recognizable taxa were tentatively identified, then five living plants of each taxon at each site were dug up, photographed and repotted in a common garden at Ohio University.

For each taxon, fragments of five additional leaves were preserved in silica gel for genetic analysis, bringing the total of DNA samples to 10 for microsatellite marker investigation. Each individual plant represents a separate Operational Taxonomic Unit

(OTU), or taxon, prior to analysis of morphological traits, phenology, ecology, and genetic diversity.

Table 1. Field sites for the Allium tricoccum complex; tentative Taxon: AB = A. burdickii, SGR = South Green Ramps, HGR = Highland Green Ramps, RR = Red Ramps

(A. tricoccum).

Population State Site Taxon Date Number

1 Kentucky Bernheim Arboretum and SGR, RR 4/14/2017 Research Forest 2 " Tara Farm SGR 4/14/2017

28

Table 1 Continued

3-4 " Raven Run Nature SGR 4/15/2017 Sanctuary 5-6 " YMCA Camp Ernst SGR 4/15/2017

7 Michigan Washtenaw County AB 4/26/2017

8 " Calhoun County AB 4/26/2017

9-10 " Kalamazoo County AB 4/26/2017

11 " Warren Dune State Park AB 4/27/2017

12-13 " Warren Wood State Park AB, RR 4/27/2017

14-15 Indiana Eagle Crest Nature AB, RR 4/28/2017 Preserve 16 West Virginia Bear Rocks Lakes Wildlife RR 5/2/2017 Management Area 17 " Pocahontas county RR 5/2/2017

18 North Carolina Craggy Gardens HGR 5/3/2017

19 Tennessee Roan Mountain HGR 5/4/2017

20 " Sullivan county HGR 5/4/2017

21-22 " Standing Stone State Park HGR 5/25-

26/2017

23-24 " Edgar Evins State Park HGR 5/27/2017

25-26 Ohio Howard Collier State AB, RR 6/10/2017 Nature Preserve 27 " A.W. Marion State Park AB 6/10/2017

28 " Strouds Run State Park RR 7/23/2017

29

Morphological characters. Morphological analyses utilized nearly all features described by Jones (1979) and Bell (2006) that potentially provide discrimination among taxa (Table 2), with some modifications based on observations in the field. A few additional traits were observed to provide further differentiation of taxa in the field and common garden. Every garden plant was photographed with a canon 550D digital camera against a white board with a centimeter ruler weekly, until at least two fruits had dehisced and released seeds. Morphological variables were recorded from images using ImageJ software ver. 2.0.0 (Rueden, 2017) after calibration with the ruler to the nearest 0.1 mm, except for RGB measurements. The latter were extracted using the RGB histogram analysis tool from ImageJ. Plants with maturing fruits were bagged in order to capture dehisced seeds for subsequent measurements. Seeds were photographed with a USB 2.0 Digital

Microscope, and measurements were made from images with ImageJ. Seeds were weighed using an analytical balance (Ohaus Pioneer PA3102) to 0.01 g accuracy.

Table 2. List of morphological characteristics recorded on OTUs for each of 28 populations and four taxa of the Allium tricoccum complex.

Units or No Character Description precision 1. Leaf number (NL) Number (count)

Petiole length of 2. Length of petiole 0.1 cm largest leaf (PL)

30

Table 2 Continued

Leaf length of largest 3. Length of the leaf blade 0.1 cm leaf (LL)

Leaf width of largest Width of the leaf blade at the 4. 0.1 cm leaf (LW) widest point

Apical angle of largest Greatest angle at apex of leaf 5. ° leaf (LA) blade

Greatest angle from midvein at Basal angle of largest 6. base of leaf blade above ° leaf (LB) decurrence

7. Bulb diameter (BW) Diameter of bulb 0.1 cm

8. Bulb length (BL) Length of bulb 0.1 cm

Bulb depth in the Depth of bulb embeded in the 9. 0.1 cm ground (DoB) ground

Scape growth Angle of scape growth 10. o orientation orientation

Scape height above 11. Height of scape 0.1 cm ground (SH)

Bract, fully elongated Length of bract just prior to 12. 0.1 cm (BrL) bursting

Number of flowers Number of flowers bud per 13. Number (count) bud (NB) umbel 31

Table 2 Continued

Central flower Height of perianth on tallest 14. 0.1 mm perianth height (PH) central flower in the umbel Central flower stamen Height of stamen on tallest 15. 0.1 mm height (PH) central flower in the umbel Length of pedicel on tallest Central fruiting 16. central flower in the umbel, 0.1 cm pedicel length (PdL) from base of umbel Angle of lowest pedicel from Lowest pedicel 17. vertical (central flower = 0 ° reflexion angle (PA) degrees) Diameter of capsule on central pedicel, measured from above 18. Capsule width (CD) 0.1 mm using circle to delineate capsule

19. Seeds diameter (SD) Diameter of seed 0.1 mm

20. Seeds weight (SW) Weight per seed 0.01 gram

Anthocyanin Relative intensity of RGB values 21. pigmentation in each organ pigmentation in bulb (BR, BG, BB) Anthocyanin Relative intensity of RGB values 22. pigmentation in pigmentation in each organ (MR, MG. MB) midvein Anthocyanin Relative intensity of RGB values 23. pigmentation in pigmentation in each organ (SR. SG, SB) scape/bract

32

From 140 possible OTUs, only 100 were used for morphological analysis, given the lack of leaf and scape morphology information for a few late-collected populations which were already in the stage of scape emergence or the inflorescence bract had split.

From 100 OTUs, 34 OTUs lacked complete morphology data from reproductive traits

(variables no. 10-20) because they failed to develop a scape, or flowers or fruits. To resolve this problem, imputation data were performed with the Mice package in R (van Buuren and

Karin Groothuis-Oudshoorn, 2011). The imputation equation merged Joint Modelling and

Fully Condition Specification approaches, and as a result produced chained equations to handle the missing data in the multivariate data set with incomplete values in more than one variable. MICE use multiple imputation from mean of data set with missing value and combine with value from regression formula developed from the mean value of missing data set and others variables. The cycle of chain equation is repeated several time, which can be set up by the user, until the regression reach stable value and develop complete data set.

Statistical Analyses. While individual plants were treated as OTUs, the fundamental unit of analysis, populations of individual taxa, were the main focus of statistical analyses.

Permutational Multivariate Analysis of Variance (PERMANOVA) is non-parametric statistic test allows for unbalanced sample size of each group and mixed data sets. The test is used to find if there is no difference in the centroids and dispersion among groups

(Anderson, 2017; 2001). PERMANOVA accommodates non-normality of multivariate data with the possibility to use various dissimilarity indices ( e.g. Gower or Jaccard), as well as more than one type of data in single set observations. Pairwise tests were conducted 33 to compare taxa as sets of OTUs, to determine whether each taxon as initially defined differed qualitatively or quantitatively from others in a statistical sense.

Non-metric multidimensional scaling (NMDS) with Gower’s distance was performed, as an ordination to compare OTUs and confirm taxon membership and potential distinctions. NMDS accommodates mixed types of data, allows for flexibility of the distance measure, and use non-parametric approach in the computation (Legendre and

Legendre, 2012). In this ordination approach, the rank order differences among OTUs is used to place the OTUs in multidimensional space, testing different numbers of axes and evaluating a "stress" criterion for fit of the OTUs in each space. No a priori groups are considered in the analysis. The correct number of axes or dimensions has the lowest stress coefficient. The ordination technique tends to reduce divergent variation among OTUs.

Canonical Variates Analysis was performed as a very different ordination approach to the NMDS analysis. Generally, only quantitative multivariate normal variables should be used, although the analysis is very robust to violation of such assumptions. The ordination depends on a priori assignment of OTUs to groups; the four tentatively identified taxa were treated as groups. The algorithm works quite differently from NMDS, using the raw variables to determine the centroids of the groups in multivariate space, then makes pairwise comparisons of OTU membership and the statistical separation of groups based on the variables. The results include F-statistics on individual variables regarding their power to separate groups, variable-variate correlations showing the contribution of individual variables to separation of groups along each axis, and a post hoc classification function of membership of OTUs to assigned versus likely groups. 34

Cluster Analysis was performed on the morphological data set using the

Unweighted Pair Group Method Arithmetic Mean (UPGMA) clustering method and

Gower's distance. This algorithm groups OTUs based on their overall similarity from the coefficient selected, forming a dendrogram or "tree" of similarities. Results of the Cluster

Analysis provided a different comparison to results derived from NMDS and CVA ordinations.

Results

Observations in the field and common garden demonstrated that Allium tricoccum sensu stricto can be easily separated from other taxa by several consistent traits, including the easily observed and common presence of reddish to reddish-purple pigmentation in the bulb, leaf midvein, petiole, and margin, scape, bract, perianth and receptacle. The bulbs mostly have reddish pigment in the upper part and tend to be whitish at the bottom.

Some of the A. tricoccum leaves show a mix of green and reddish pigmentation in the middle part and top of midvein but are strongly reddish or reddish-purple at the base of the midvein. The scape is also usually fully pigmented during emergence until the breaking of the inflorescence bract. Occasional populations throughout the range of A. tricoccum have exceptional plants expressing variable intensity of pigmentation from year to year, for example, a population briefly described below from Strouds Run State

Park, OH as described later, or even sporadic plants totally lacking pigment (A. A.

Reznicek, University of Michigan, pers. comm.). Scarcely pigmented or unpigmented individuals of A. tricoccum sensu stricto will nevertheless be typical in all other distinguishing features of large bulb size and shallow depth, broader petiolate leaves 35 retained through early scape development, curved early scape, broadly hemispherical to nearly spherical umbels with numerous flowers, and larger flowers, capsules and seeds.

Leaf shape of A. tricoccum is broadly elliptical to broadly oblong-lanceolate, the leaf apex tends toward acuminate, and the base is cuneate or narrowly rounded into the abruptly distinct and elongate petiole. The leaf petiole is 2-3 cm long. The bulb is ovoid,

2.5-5 cm long and 1.4-3 cm width. Bulb depth in the ground is very shallow to partially exposed (0-1.5 cm), and plants are typically quite densely packed, commonly forming dense and large colonies, as opposed to the sporadic and diffuse, often quite small colonies of the Green Ramps taxa. The number of buds is much higher compared to other three

Green Ramps taxa, with a range of 11-77 buds per umbel vs 5-27 buds per umbel, arranged in a broadly hemispherical to nearly spherical inflorescence, with the lowest pedicels strongly deflexed below horizontal. The scape always emerges from the ground curved in the beginning, becoming erect or nearly so shortly before inflorescence bract bursting.

Stamens are longer, from 7.1 - 10.6 mm compared to the other three Green Ramps taxa

(4.2-7.5 mm).

A. burdickii and South Green Ramps are similar in having linear to narrowly lanceolate or oblong leaf blades, and are also totally without reddish-purple pigmentation in all structures. Populations of A. burdickii in the field appeared to have more silvery- or gray-green foliage coloration, while foliage in populations of South Green Ramps tended toward medium-green coloration, but these qualitative observations were not measured and analyzed. These two taxa were not fully separated in the NMDS and CVA, although the latter ordination did segregate most OTUs of the two; however, some features, e.g. stamen 36 and perianth size, tended to average smaller in South Green Ramps (mean= 4.21 mm, sd=

0.61 and mean= 4.47 mm, sd= 0.40) compared to A. burdickii (mean= 5.42 mm, sd= 0.40, and mean= 4.91mm, sd = 0.33). A similar pattern was found in leaf traits, A. burdickii tending to have wider leaves with broader apical angle and basal angle (mean= 3.12 mm, sd= 0.78; mean= 31.8o, sd= 6.77; and mean= 31.3o, sd= 7.02, respectively) compared to

South Green Ramps (mean= 2.78 mm, sd= 0.78; mean= 24.4o, sd= 3.87; and mean = 26.5o, sd= 6.49, respectively). The bulbs in both were completely submerged (sometimes deeply so), and were less dense and more scattered over the landscape, rarely forming large colonies. The scapes emerged strictly erect from the ground and remained so throughout fruiting, bearing 5-16 buds in A. burdickii and 7-18 buds in South Green Ramps. Although both taxa can sometimes be found growing in the vicinity of A. tricoccum, they only very rarely completely commingled with the latter; generally, individual colonies at a site were coherent and set apart from the denser colonies of A. tricoccum.

The fourth taxon, Highland Green Ramps, was divergent from A. tricoccum and the other Green Ramps taxa in a broader suite of distinctive characteristics. It completely lacked reddish-purple pigmentation in all structures, resembling the other Green Ramps taxa, but it produced broadly lanceolate to narrowly elliptical leaf blades with a distinct elongate petiole similar to leaves of A. tricoccum (Figure 1). The bulbs were shallowly to deeply submerged (0.5-7 cm) and less densely organized compare to A. tricoccum, forming scattered smaller colonies like other Green Ramps taxa. The scape was strictly erect from emergence through fruiting, like other Green Ramps taxa. The number of buds was 7-27. Overall, certain traits converged on A. tricoccum while others appeared 37 somewhat intermediate between A. burdickii and A. tricoccum, although there was no clear evidence indicating a hybrid origin (especially given that A. burdickii and South

Green Ramps grow outside the geographic range of Highland Green Ramps, at much lower elevations). Unlike the other two Green Ramps taxa, Highland Green Ramps were found quite extensively intermingled with A. tricoccum at some sites. Colonies of this taxon were less coherent and sometimes less separated from A. tricoccum, probably leading to mistaken reports of "polymorphism" in A. tricoccum.

It was noted that all three Green Ramps taxa were completely devoid of the reddish-purple pigmentation commonly (but not always) found in A. tricoccum, suggesting that all pigmented Ramps are A. tricoccum, but not all unpigmented Ramps are taxa of Green Ramps. Fresh plants collected in the field and lacking pigmentation should be examined for traits of the bulb, leaves, scape, flowers or fruits before determining their identity. It is likely that some very faded herbarium specimens of A. tricoccum in which pigmentation is not obvious or is obsolete, have been misidentified as

Green Ramps taxa, when other traits were ignored. 38

A B

C D

Figure 1. Leaf, stem and bulb morphology of (A) Allium tricoccum, (B) Highland Green

Ramps, (C) Allium burdickii, and (D) South Green Ramps. The arrow indicates the position of the soil surface relative to the bulb, as marked with permanent pen in the field prior to plant removal.

39

PERMANOVA test results show significant pairwise differences among taxa based on the morphological variables ( pseudo-F = 36. 13, df = 3, p<0.01), and confirmed by the pair-wise post hoc test (Table 3).

Table 3. Pair-wise Post Hoc Test of morphological traits with 999 permutations among four taxa of the Allium tricoccum complex. Two asterisks indicate 99% confidence level for significant difference.

A. burdickii Highland Green South Green

Highland Green 0.006**

South Green 0.006** 0.006**

A. tricoccum 0.006** 0.006** 0.006**

The NMDS plot (Figure 2) showed strong separation between A. tricoccum and the Green Ramps taxa. The Green Ramps taxa tended to lie closer to or overlap with each other, although the Highland Green Ramps, with its divergent combination of features, was mostly separate from the other Green Ramps taxa. The NMDS1 axis reflected high correlation of Number of buds (NB), followed by the Leaf base angle (LB), Leaf apex angle (LA), and Leaf width (LW), separating A. tricoccum from the Green Ramps taxa, and largely distinguishing Highland Green Ramps from A. burdickii and South Green

Ramps. Depth of bulb in the ground (DOB) and all RGB values from Scape, Midvein, and Bulb (Sc.R, Sc.G, Sc.B, MR, MG, MB, BR, BG, BB) had high variances on the

NMDS2 axis, but little further separation of the taxa occurred along this axis in the ordination. 40

A. tricoccum

A. burdickii Highland Green

South Green

Figure 2. Non-metric Multidimensional Scaling ordination with Gower’s coefficient from

23 morphological of four taxa in the Allium tricoccum complex. Variables acronyms follows table 2

Canonical Variates Analysis provided even greater separation of the four taxa than NMDS (Figure 3); variables contributing heavily to the separation of the taxa were similar to those showing correlatid with NMDS ordination axis 1 and 2. When CVA was conducted without A. tricoccum and excluding the RGB values and scape orientation variables specific to that taxon, the Highland Green Ramps separated fully from the other two Green Ramps taxa, and most individuals of A. burdickii and South Green Ramps separated as well (Figure 4). 41

Figure 3. Canonical Variates Analysis ordination from 23 morphological of four taxa in the Allium tricoccum complex. Variables acronyms follows table 2 42

Figure 4. Canonical Variates Analysis ordination on 19 morphological of three Green

Ramps taxa in the Allium tricoccum complex. Variables acronyms follows table 2.

Both of the NMDS and CVA results were corroborated by the UPGMA dendrogram (Figure 5) that separated OTUs into a coherent and uniform A. tricoccum cluster and a broader Green Ramps cluster. The phenogram also separated the Highland

Green Ramps into a separate cluster. Some smaller subclusters of OTUs of A. burdickii and South Green Ramps were recovered, although significant overlap in the CVA ordination between the latter two taxa was reflected in the phenogram, suggesting several shared or overlapping morphological characteristics between A. burdickii and South

Green Ramps. It is possible that additional morphological features could be found, such as detailed RGB measurements of foliage coloration, that would lead to further 43

A. tricoccum

A. burdickii Highland Green

South Green

Figure 5 UPGMA Dendogram from Cluster Analysis using Gower's coeffeicient on 23 morphological variables of four taxa in the Allium tricoccum complex and 999 bootstrap replicates.

quantitative discrimination of the two taxa. All analyses fully separated A. tricoccum from the Green Ramps taxa, and most analyses nearly or fully separated the Highland 44

Green Ramps from A. burdickii and South Green Ramps OTUs. Certain analyses indicated some segregation, but not full separation, of the latter two taxa, but the

PERMANOVA results indicated that A. burdickii and South Green Ramps were not fully coordinate in their morphological features—they were at least partially differentiated in some morphological traits.

Discussion

Observations and measurements of traits on living plants and populations have given us more information than previously available from herbarium specimens and other field studies. Reddish-purple pigmentation common to structures on A. tricoccum (but not always present) is never found in the Green Ramps taxa, diagnosed using other morphological traits. Occasional unpigmented or weakly pigmented individuals of otherwise typical A. tricoccum can easily be identified using other features, including bigger bulb size and shallow depth, bigger leaf shape, curved shape of scape in the early development, and bigger flower. The somewhat intermediate nature of certain structures in the Highland Green Ramps in comparison to other Green Ramps taxa and A. tricoccum would obviously be grounds for mistaking such plants as representative variants of a polymorphic A. tricoccum, or confusing Highland Green Ramps with unpigmented or weakly pigmented A. tricoccum, which is expected throughout the range of the latter species. However, several uniform morphological distinctions across the plant body fully separate both pigmented and unpigmented A. tricoccum from all Green Ramps taxa and

Highland Green Ramps from other Green Ramps taxa. 45

Observations in the field and common garden have also revealed limited seasonal and year-to-year loss of reddish-purple pigmentation in certain populations of otherwise typical A. tricoccum. In Strouds Run State Park in Athens County, Ohio, the scapes of some otherwise typical A. tricoccum plants were initially noticeably pigmented but became light red or scarcely pigmented when the fruit began to ripen. Observations in the common garden also documented that, after capsule dehiscence, A. tricoccum bulbs in some plants became discolored toward whitish. In one observed population in Stroud run park, OH, all plants possessed strong pigmentation of leaves and bulbs in 2017 but some lacked pigmentation in 2018, whereas plants in another population were initially unpigmented but produced noticeable pigmentation in the inflorescence bract or other structures later in the spring (Figure 6). Bell (2007) apparently observed similar behavior in the A. tricoccum seedlings she studied, where 33% of them were unpigmented compared to 100% in her inferred populations of A. burdickii (some plants of which are inferred here to represent the Highland Green Ramps). All observations and results presented here, from field and garden studies, highlight the importance of taking note of any reddish-purple pigmentation anywhere on the plant, and including such notes on specimen labels. Bulb depth, shown to be diagnostic for separating A. tricoccum from the

Green Ramps taxa, would also be valuable to note on specimen labels. In general, identification of fresh plants or herbarium specimens of the A. tricoccum complex should refer to multiple traits on vegetative and reproductive structures of the population where available, never on one or two features alone. 46

Figure 6. Two color variants of the Allium tricoccum scape in early development. (A)

Unpigmented scape and bract, but showing characteristic strongly curved scape. (B)

Gently curved scape with purple flush at tip of bract.

Dr. A. A. Reznicek (pers. comm.) at the University of Michigan has observed that non-pigmented but otherwise typical A. tricoccum individuals are not uncommonly encountered in the Great Lakes region. We believe that such misidentifications of unpigmented or sparsely pigmented A. tricoccum, as well as unrecognized Highland

Green Ramps populations, have led to erroneous conclusions in the Bell study and sporadic reports of polymorphism in A. tricoccum, and possibly also are the basis of

Jones’s brief allusion to hybridization. The presence/absence of pigmentation as the sole distinguishing factor between Green Ramps taxa and A. tricoccum has driven much of the 47 taxonomic confusion in this complex and should warn taxonomists away from overly simplistic species delimitations (based on pigmentation alone) and reliance on only one or two traits to achieve confident identification. The gain or loss of red-purple pigmentation as a consequence of environmental fluctuations, as suggested by Walker

(1961), may have some credence and deserves study.

Our initial, tentative assignment of individual OTUs and populations to four taxa is generally supported by field and garden observations, and statistical analyses. We have strong morphological evidence to recognize four distinct evolutionary species, with A. burdickii and South Green Ramps clearly not belonging to exactly the same morphospecies, although there is some overlap. Based on morphological traits alone, this four-species scenario fits well both with Jones’s (1979) conclusions for aberrant “A. burdickii” plants reported outside the Great Lakes and Great Plains regions, and also explains the taxonomic difficulties burdening confident identification of Ramps populations in the Appalachian Mountain region (referred to here as Highland Green

Ramps). The much greater biological diversity revealed by the morphological studies is not satisfactorily accommodated by two species or infraspecific taxa as previously delimited.

Two traits not expressly highlighted in previous studies were demonstrated to be of value in this research, namely, emergent scape orientation and depth of bulb. Interestingly, while Jones (1978) illustrated the scapes of A. burdickii and A. tricoccum sensu stricto, she failed to note the relative curvature of the scapes in the two as a diagnostic difference. The difference in scape orientation is only conspicuous early in scape emergence; the older 48 scape in A. tricoccum sensu stricto becomes erect like those of the Green Ramps taxa as the inflorescence bract nears splitting to present the flower buds. The curved emergent scape is an utterly consistent feature of A. tricoccum, compared to the strictly erect emergent scape of the Green Ramps taxa, based on weekly photography over two seasons.

The curved-erect scape distinction is similar to the differences among A. textile, A. geyeri var. tenerum and A. stellatum, but is different in A. cernuum, which exhibits a permanently curved scape apex (Choi and Cota-Sanchez 2010).

Depth of bulb has never been recognized as a diagnostic trait. A few researchers have investigated the shape, size, and texture and color of the bulb tunica (Choi et al., 2012;

Choi and Cota-Sánchez, 2010; Phillips, 2010), however the depth of bulb in the ground has received no notice. A. tricoccum was found to have consistently shallower depth of bulb, with the bulb in many plants at least partially exposed ( -0.73-2.71 cm under ground), as compared to other three Green Ramps taxa in which the bulb is completely covered and often submerged to several centimeters (0.6-7.39 cm under ground). In addition, field studies of many populations of the A. tricoccum complex in several states have confirmed that plant (or bulb) density and overall areal extent (or plant number) of A. tricoccum colonies is much greater than in colonies of the Green Ramps taxa. In fact, bulbs in colonies of A. tricoccum are usually quite densely aggregated in small groups, as though several seeds in a given umbel germinated simultaneously, in contrast to the moderately dispersed plants found in smaller, diffuse colonies of the Green Ramps taxa. Investigations of seed dispersal and germination success in the four taxa might prove illuminating. 49

The morphology information from four taxa of Allium tricoccum complex result several information that can be used for systematic review, which is later will be described.

Generally, A. tricoccum and all Green ramp group clearly have different morphology characteristic almost in all traits. Highland green have intermediate morphology characteristics especially with more broad leaves and longer leaf petiole as in A. tricoccum, however lack of pigmentation, lower number of buds, smaller bulb size and more deeply submerged in the ground as found in A. burdickii and South green. Allium burdickii and

South green are mostly similar in morphological characteristics, and only differ in the leaf apex and basal angle, and stamen and perianth size.

50

CHAPTER 3: PHENOLOGY

Methods

Phenological behavior was inferred from digital images of field-collected plants

(mostly taken during early to mid-leaf emergence but before scape emergence), and continuing after transplantation to a common garden in early to mid-spring 2017.

Photographic observations continued to final fruiting in 2017, and resumed in early to mid- spring during 2018. Only 66 plants from all taxa were available for the reproductive phenology analysis in 2017, given that the rest of plants did not develop reproductive organs or were already in the open bract or flowering stage when collected in the field.

Observations in 2018 yielded information from 130 plants for leaf emergence date, and 35 plants for scape emergence date. Phenological variables recorded for multiple time points on each living plant included time of leaf emergence, full leaf expansion, leaf dieback, scape emergence, inflorescence bract splitting, opening of flowers, fruit maturation, and fruit dehiscence. These chronological categorical variables were plotted on a Julian calendar for populations and taxa, with modification using the following formula: a. MJD= JD-17,000 (data from 2017), b. MJD= JD-18,000 (data from 2018)

MJD: Modification Julian date, JD: Julian Date

A modification was needed to give real date values in a year (365 days) and allow for hypothesis testing of significant differences between dates. A prior test to find significant differences between taxa with original JD always yielded a non-significant result even when the different means between taxa reached 10 days apart. As a result, we 51 followed the formula with modification as suggested from the Astronomical Applications

Department of the U.S. Naval Observatory.

A PERMANOVA test was conducted on the length of periods of scape emergence to flowering, flowering to fruiting, and from fruiting to fruiting dehiscence data from observartions in 2017 to find if there are statistically significant differences of these phenological periods among the taxa. A NMDS ordination plot was performed to show the separation of taxa. Similar tests of the length of periods of leaf emergence to leaf senescence, and leaf emergence to scape emergence from observations in 2018 was conducted, however the NMDS analysis was failed due to insufficient data. The length of periods different for each phenological trait among phenotypes was calculated and presented in a bar stack graphic (Figure 7). A T-test of earliest flowering time in plants was conducted to determine if there was a significant difference among taxa co-occuring at the same site from observationa in 2017. Two sites had two taxa growing locally sympatrically:

Raven Run Nature Sanctuary, KY and Eagle Crest Nature Preserve, IN. Test of normality and equality of variances between the two group in each case indicated that the data were normal, however they expreseed unequal variances, consequently, Welch's t-test was applied. A table with chronological period for each phenological trait was created to give descriptive phenological ordering of structures in each taxon of the A. tricoccum complex

(Appendix 3). 52

Figure 7. Stack bar of phenology period of each taxon in the Allium tricoccum complex for 2017 and 2018 observations. Value in the axis indicates number of days in the Julian calendar.

Results

All Green Ramps taxa showed an earlier flower anthesis period in 2017 than A. tricoccum, with aproximately 15-30 days of separation between the two groups, indicating complete reproductive isolations between A.tricoccum and all Green Ramps taxa in flowering phenology. Highland Green Ramps produced open flowers at a later date in 2017 than A. burdickii and South Green Ramps, and the continuing scape emergence of this taxon in 2018 after overwintering of plants in the common garden indicates that the later flower anthesis may be genetically fixed in the higher-elevation populations providing evidence of additional reproductive isolation in flowering time between A. burdickii and South Green Ramps on one hand and Highland Green Ramps on other hand. Allium tricoccum was similar to the Highland Green Ramps and South 53

Green Ramps taxa in leaf retention during early scape emergence (based on garden observations).

A PERMANOVA test showed that the taxa were significantly different based on phenologies of reproductive structures (Pseudo-F3, 62= 14.46, p<0.0001), and confirmed with pair wise post hoc test that all taxa were significantly different (Table 4). The phenology shifts among the four taxa confirmed and extended Jones's observations and demonstrated the utility of these data to separate taxa taxonomically. Phenological differences among the taxa in flowering time also suggested moderate to absolute reproductive isolation in commingling populations of A. tricoccum and Green Ramps taxa.

Table 4. Pair-wise Post Hoc Test of phenological traits of scape, flowers and fruits with

999 permutations among four taxa of the Allium tricoccum complex. Two asterisks indicate 99% confidence level for significant difference. One asterisk indicates 95% confidence level for significant difference.

A. burdickii Highland Green South Green

Highland Green 0.0012**

South Green 0.0012** 0.0012**

A. tricoccum 0.0012** 0.0100* 0.0012**

The NMDS ordination (Figure 8) showed that A. tricoccum and Highland Green

Ramps overlapped but together were nearly fully separate from A. burdickii and South 54

Green Ramps. There was slight overlap between A. burdickii and South Green Ramps, however most of the samples of the two taxa were separated.

Figure 8. Non-metric Multidimensional Scaling ordination from length periods of scape emerge to flowering, flowering to fruiting, and from fruiting to Fruiting dehiscence with

Gower’s coefficient of four taxa of the Allium tricoccum complex.

A PERMANOVA test showed that the taxa were significantly different based on length periods from leaf emergence to leaf senescence, and from leaf emergence to scape emergence data from phenology observation in 2018 (Pseudo-F3, 31= 3.589, p<0.028). 55

Pair wise post hoc test indicated that A. burdickii significantly different with Highland green and South green only (Table 5). The phenology shifts among the four taxa confirmed and extended Jones's observations and demonstrated the utility of these data to separate taxa taxonomically. Phenological differences among the taxa in flowering time also suggested moderate to absolute reproductive isolation in commingling populations of

A. tricoccum and Green Ramps taxa.

Table 5. Pair-wise Post Hoc Test of length periods of leaf emergence to leaf senescence, and from leaf emergence to scape emergence data from phenology observation in 2018 with 999 permutations among four taxa of the Allium tricoccum complex. One asterisk indicates 95% confidence level for significant difference.

A. burdickii Highland Green South Green

Highland Green 0.018*

South Green 0.018* 0.960

A. tricoccum 0.090 0.982 0.960

The T-test indicated there are statistical differences in flowering time (F3,62=29.33, p<0.001) between the two taxa growing together at each of the two sites (Table 6). These phenological differences in flowering time among co-occuring taxa (in both case involving A. tricoccum provide clear support for absolute reproductive isolation between of A. tricoccum and locally simpatric Green Ramps taxa, altough further studies at many more sites would be desirable.

56

Table 6. T-test of comparisons in earliest opening date of flower among co-occuring taxa in two. Two asterisks indicate 99% confident level for significant difference.

Flowering date Site Taxon df t P-value (mean, sd)

Raven Run Nature South Green 153, 0 3 7 0.00042

Sanctuary, KY A. tricoccum 190, 10.5 3

Eagle Crest Nature A. burdickii 163, 1.73 3 -40.09 1.563e-09

Preserve, IN A. tricoccum 193, 0 4

Allium tricoccum shoots emerged at the end of February 2018 and leaves reached full expansion around early March 2018. Allium tricoccum had the longest flowering and fruiting season. In the study region, most of the scapes emerged around the end of April

2017, with inflorescence bracts bursting and flowers opening around the end of June or early July. Fruiting also had a long period, mature fruits developing in the middle of July and finishing mostly in the September. The last seeds were collected from dehisced capsules on October 15, 2017. Leaves did not senescence during scape emergence but were retained throughout flowering and fruiting. The scape was consistently initially curved (sometimes almost doubled on itself) but ultimately became erect as it elongated, mostly just prior to when the inflorescence bract split and buds are exposed.

Allium burdickii showed late shoot and leaf emergence compared to A. tricoccum.

Shoots emerged mostly at the end of March, and leaves were fully expanded around the end of March through early April. Leaves started to senesce prior to or just as the scape emerged (or sometimes were already withered away by then), and scape growth was erect 57 from the beginning. Scape emergence began in the middle of May, and flowering began in the second week of June. Fruiting occurred from the middle of June to July, and seeds matured and were dispersed in early August.

South Green Ramps leaves mostly started to emerge around the end of February, and leaves were fully expanded by early March. Leaves showed no sign of senescence during scape emergence or during later elongation of the scape (different from A. burdickii), and scape growth was strictly erect from the beginning, similar to A. burdickii.

Scape emergence took place around mid to later April, and flowering began around the end of May. The fruiting period started early June and continued through mid to later

July.

Highland Green Ramps leaves started to emerge around the end of March, and leaves were fully expanded by early to late April. Leaves did not senesce during scape emergence but continued to remain green through the scape elongation (different from A. burdickii). Scapes emerged around the end of April, and flowers reached maturity and opened around the second week of June. Fruiting started shortly after the flowering began to drop off in the middle of June, with fruit ripening continuing into early August. Scape growth was strictly erect from the beginning (different from A. tricoccum).

Discussion

Jones (1979) gave some details of phenology in the leaves, scapes and flowering times of A. tricoccum and A. burdickii based on field work in central Illinois and herbarium records over eastern North America. Compared to Jones's phenological descriptions, the plants in the common garden in 2018 showed earlier leaf emergence and leaf expansion, 58 on March 11, compared to April 8. Interestingly, in general, Allium tricoccum and South

Green Ramps showed leaf emergence earlier compared to A. burdickii and Highland Green

Ramps, where the latter taxon produced fully expanded leaves on April 2, 2018 (Appendix

3).

Results here confirm Jones's observations of leaf senescence in Allium burdickii prior to scape emergence. In the common garden, certain plants from several populations of Allium burdickii (including the type locality) retained withering leaves during and up to one week after scape emergence, although in all plants the leaves fully withered 4-7 days after that. This differs slightly from Jones's observation that leaves are essentially completely withered and missing at the time of scape emergence. Dion (2017) proposed the relationship between canopy closure in the natural habitat with leaf senesces of A. tricoccum in Quebec, Canada. However, this theory may not be applicable for the southern region, where plants are under almost full canopy closure until May 6th in the mountains of

Tennessee and North Carolina.

On the other hand, A. tricoccum, South Green Ramps and Highland Green Ramps retained their leaves for a substantial period of time after the scape emerged, and A. tricoccum kept its leaves throughout the growing season. This finding indicates that Allium burdickii of the Great Lakes and Great Plains regions has evolved genetically fixed ecophysiological responses in leaf, scape, flower and fruit phenologies that are presumably tied to regional photoperiod or other environmental conditions, and that these responses are somewhat or dramatically different from the responses of the other three taxa. Losing the leaves prior to full development of the scape might reduce total plant evaporation as 59 the warm weather arrives after April, and reallocates more nutrients to reproductive structures. However, the South Green Ramps, with similar leaf size and shape, retained their leaves at least through the middle of scape elongation, perhaps indicating that the modified phenological response of that taxon is an adjustment to different climatic conditions in the Interior Highlands region, with overall warmer temperatures in the early spring. Further studies of leaf anatomy, e.g. stomatal size and density or presence of epicuticular layers to prevent too much evaporation, and photosynthetic responses, as they relate to local environmental conditions in the four taxa (especially the three Green Ramps taxa) would potentially yield some interesting hypotheses regarding ecophysiological evolution..

Allium tricoccum had the longest reproductive period from emergence of the scape to splitting of the inflorescence bract and flower anthesis. We recorded that this taxon needed at least two months from scape emergence to flowering, compared to less than one month in the Green Ramps taxa. Jones (1979) stated that A. burdickii preferred to use seeds as the main vehicle for reproduction as opposed to vegetative reproduction by the bulb in

A. tricoccum. This might explain why taxa do not produce flowers and fruits immediately, because the nutrients that are allocated to develop bigger bulbs rather than ensure successful sexual reproduction in late summer and early fall. Nault (1988) also recognized the pattern that plants that do not develop early reproductive structures generally have bigger bulbs compared to plants that reproduce earlier and have smaller bulbs. The Green

Ramps taxa, especially A. burdickii and South Green Ramps, fit this generalized prediction, mostly exhibiting earlier flowering and fruiting phenologies and bearing smaller bulbs 60 compared to A. tricoccum; this may be explained as an impact of differential nutrient allocation for each plant structure.

The phenological patterns in leaves, scapes, inflorescence bract splitting, flowers and fruits were consistent across 2017 and 2018. The patterns were found both in the field and in the common garden, and diverged in various ways in all four taxa, but were not obviously correlated with climatic or environmental variation (given the patterns held up in the common garden) as suggested by Bell (2002) and Walker (1961), who argued for one polymorphic species, A. tricoccum. Divergent phenological patterns among the four taxa support the morphological results that more than two evolutionary species are present in the complex, and that ecophysiological differences as expressed in vegetative and reproductive phenological patterns are genetically fixed.

Field observations in April and May 2018 revealed a large number of A. tricoccum plants in a Stroud Runs population that developed normal bulbs and leaves in the expected phenological pattern, but much delayed scape development compared to the previous year.

This peculiar plant response appeared to correlate with a climate anomaly in February to

April of 2018, in which unusually prolonged lower temperatures (<10oC) were encountered. This climate anomaly potentially postponed scape development or in some plants eliminated it altogether. Further study of the relationship between local climatic conditions and phenological responses should be conducted, perhaps both in the field and in growth chambers, to obtain a better understanding about the genetic basis versus environmental plasticity of vegetative and reproductive phenologies as related to climate, given the possible impacts of climate change. 61

Allium burdickii differs in their phenological response of leaf expansion and senescence, and scape emergence, compared to South Green Ramps, and this is the first report of genetically fixed ecophysiological differences in populations in the two regions.

This differential response, and results from ecological studies below demonstrating different microhabitats occupied by the two taxa, suggest that they evolved under considerably different local and regional climatic conditions and substrate differences.

Walker (1961), Kaufman (2001) and Bell (2002) did not recognize the phenological variation in the taxa, perhaps because they failed to separate the taxa properly from morphological and ecological evidence. However, examination of phenological patterns of vegetative and reproductive structures in living plants in the field and common garden provide compelling support for the recognition of four taxa that have evolved different ecophysiological and developmental responses. On the basis of morphological traits and phenological patterns alone, the taxa could be legitimately considered four separate evolutionary species under the Unified Species Concept.

62

CHAPTER 4: ENVIRONMENTAL FACTORS

Methods

Each field-sampled population was recorded for geographic (latitude-longitude) coordinates and altitude. Three soil cores were removed from the base of plants for each studied population of each taxon at each site (Table 1), sealed in plastic ziplock bags, and taken to Ohio University. Each of the three soil cores from each population were analyzed separately for soil features of moisture content, pH, and texture. Wet weight was recorded within one day of collection, and dry weight was recorded three weeks later from air-dried soils. Calculation of soil moisture (SM), or volumetric water content, used this formula based on wet weight (WW) and dry weight (DW):

SM= 100*((WW-DW)/DW)

Soil pH and soil texture were examined from fully dry soil samples at the

Department of Environmental and Plant Biology Soils Laboratory, using protocols of Dr.

Jared DeForest (2013). Soil pH was measured with the glass electrode method, where for each soil sample, 10 g dried sieved soil was mixed with 20 ml Deionized (DI) water in a

125 ml sample cup. The solution was mixed with an orbital shaker (150 rpm) for 30 min, then allowed to stand for 10 min to equilibrate, then swirled gently. After calibrating the pH meter with pH 4.0, 5.0, and 7.0 buffer solutions, the pH electrode was introduced into the soil slurry to read the pH value.

Soil texture was measured by the Hydrometer method, where 40 g dried, sieved soil from each sample was mixed with 100 ml of Dispersing agent in a 250 ml Erlenmeyer flask, then placed on an orbital shaker (150 rpm) overnight. The resulting soil slurry was 63 introduced into a graduated cylinder, and the cylinder was filled to the 1 liter mark with tap water. The soil suspension was allowed to equilibrate to room temperature. After vortexing, the plunger was removed and the time noted. The hydrometer was placed into the cylinder after 30 secs and a reading was taken after 45 sec (R45s). Twenty four hours after stirring, the hydrometer was inserted into the soil sample again and a second reading

(R24h) was taken. Calculations of percent sand, silt and clay were as follows.

Sand % = 100 – [(R45s – Rblank) * (100/oven dry soil weight in gram)

Clay % = (R24h – Rblank) * (100/oven dry soil weight in gram)

Silt % = 100 – (sand % + clay %)

Site traits and soil texture traits, together with soil pH and moisture, were used to investigate potential differences among taxa in niche differentiation or distinct microhabitat. A PERMANOVA test was used to find statistically significant differences among the taxa based on measured soil traits, and a pairwise test was used to determine which groups were statistically different. NMDS was performed to observe group separation in the ordination plot based on the variables used, and to clarify which environment variables contributed to separation of the taxa.

Results

Generally, all populations occupied various woodland habitats from the study regions, around the southern Great Lakes southward into the Interior Highlands and eastward into the central and southern Appalachians mountains. A PERMANOVA test indicated there were significant differences among taxa for the environmental variables

(Table 5), except between A. tricoccum and Highland Green Ramps (which were 64 commonly found commingling at field sites). NMDS ordination showed considerable environmental factors differentiation between A. burdickii and South Green Ramps, with soil texture as the main variables of separation (Figure 9); however, Allium tricoccum overlapped heavily in ecological features with all three Green Ramps taxa. This was a reasonable interpretation, since A. tricoccum was found in the area or in somewhat close proximity to many populations of Green Ramps taxa, and the geographic distribution of

A. tricoccum is almost completely coordinate with the collective ranges of the Green

Ramps taxa. However, the Green Ramps taxa occupy partially to completely different microhabitats based on the environmental variables measured.

The naturals habitats occupied by Allium tricoccum are diverse, encompassing

Loam Soils to Sandy Soils, with pH of 4.48-7.68, soil moisture of 43.76%- 91.10%, and elevation 191-1,587 m A.S.L. We found the taxon frequently at the same sites in adjacent microhabitats or even abutting colonies of A. burdickii and with the South Green Ramps.

It was nearly always intermingling with plants or small colonies of Highland Green

Ramps. These findings are slightly different from previous reports that A. tricoccum grows mostly separately from A. burdickii (or taxa treated the same by earlier researchers), even in the same location. The frequent local sympatry of A. tricoccum with other taxa, and its extensive geographic range overlap with the Green Ramps taxa, also explain the heavy overlap in ecological variables measured in A. tricoccum and the others. Obviously, ecological differentiation in soil factors and elevation between A. tricoccum on the one hand and the Green Ramps taxa on the other may potentially not be that enlightening, but microhabitat differentiation among the Green Ramps taxa based on 65 those same factors has potential importance in indicating interspecific isolation and the establishment of different ecological responses. A pair-wise post hoc test confirmed statistically significant different in soil traits of microhabitats occupaied by all taxa except for A. tricoccum and Highland Green Ramps (Table 7), probably due to the frequenly intimate association of the latter two at all sites studied for Highland Green

Ramps. Further Studies of soils in locally allopatric populations of the two taxa in the

Applachian Mountains are needed.

Table 7. Pair-wise Post Hoc Test of soil variables and elevation with 999 permutations among four taxa of the A. tricoccum complex. Two asterisk marks indicate 99% confidence level for significant difference.

A. burdickii A. tricoccum Highland Green

A. tricoccum 0.0024 ** - -

Highland Green 0.0020** 0.1900 -

South Green 0.0020** 0.0024** 0.0020**

The natural habitat of Allium burdickii was mostly sandy and silty soils with a pH ranged 4.5-7.6 and soil moisture range 64.2.-91.1%. Populations were found in sand dune forests under pine trees, bog forests under maple and oak, and in mixed oak woodlands.

In southeastern Ohio (Athens County) on the Western Allegheny Plateau, the foothills of the Appalachian Mountains, this taxon grew in mixed maple forest on silt loam soil.

Populations around the Great Lakes inhabited mostly flat topography with little to no 66 slope, however those in Indiana and Ohio occurred on hilly ground. Populations had an altitude range of 200 -300 m AMSL.

The natural habitat of South Green Ramps was mostly on silty loam soil with a pH range 5.66-7.68 and soil moisture range 59.85-86.44%. Populations were mostly found on the slopes leading to, or immediate floodplains along, streams or other water bodies, and were generally found in flat terrain with soil moisture less than 50%. South

Green Ramps had an altitude range of 247 to 452 MASL. The forests were commonly made of mixed maple, oak and ash species characteristic of floodplains.

The natural habitat of Highland Green Ramps was on sandy loam soil with pH of

4.89-6.24, soil moisture of 43.76-61.96%, and elevation of 896-1,587 MASL. The habitat was generally mountain hardwood forests with pine trees and Rhododendron spp..

Highland Green Ramps grew intermingled with A. tricoccum at all field sites, sometimes so extensive that our initial interpretation was of sporadic unpigmented A. tricoccum individuals (until plants were dug up and the deeply submerged bulb was found). An interview with local Ramps harvesters in southern West Virginia revealed that colonies matching the description of Highland Green Ramps often grow separately at even higher elevations than A. tricoccum, for instance, near the top of the ridges. Soil moisture was very high because the sites were always damp from adjacent streams or marshy ground, for instance in the Craggy Mountain site and in the Roan Mountain area, where populations (and A. tricoccum) grew immediately next to small streams.

In the NMDS ordination of all four taxa, Highland Green Ramps was separated mostly from the other Green Ramps taxa by altitude as well as by soil features, but A. 67 tricoccum was broadly overlapping among all three Green Ramps taxa, reflecting its broad ecological amplitude (Figure 9). In a second NMDS ordination restricted to the three Green Ramps taxa, in which altitude was excluded the three taxa overlapped somewhat but were sufficiently segregated that a PERMANOVA test showed they were statistically significant different from each other (Figure 10). This finding enriche our understanding of soil characteristic as they represent modally different microhabitats inhabited by the three Green Ramps taxa, suggesting a substantial degree of ecological differentiation as well as altitudinal separation (in the case of Highland Green Ramps vs

A. burdickii and South Green Ramps). It is also apperant that A. burdickii and South

Green Ramps exhibit microhabitat differentiation in soil traits.

68

Figure 9. Non metric Multidimensional Scaling ordination with Gower’s coefficient on seven environmental variables in four taxa of the Allium tricoccum complex.

Figure 10. Non-metric Multidimensional Scaling ordination with Gower’s coefficient on six environmental variables (altitude excluded) in three Green Ramps taxa of the Allium tricoccum complex.

Discussion

Jones (1979) and Hanes (1958) generally described the habitat differences of Allium tricoccum and A. burdickii, where var. tricoccum prefers moister soils and wetter forested microhabitats and A. burdicikii prefers drier substrates in more upland forests. This study documented that A. tricoccum has a broader environmental range than previously understood, inhabiting upland forests in the Appalachian mountains and wetter, lowland 69 forests in the Great Lakes, expressing not only great diversity in soil moisture but also in pH, texture and altitude. Ordinations and PERMANOVA tests confirmed that the environment range of this taxon overlaps and exceeds that of the Green Ramps taxa with which it is often found locally or (commonly with Highland Green Ramps) even fully commingling. Field visits at Eagle Crest Nature Preserve in central Illinois, and Strouds

Run State Park in southeastern OH, revealed A. tricoccum growing from foothills immediately around streams or creeks to upland situations on hilltops and the steeper slopes between, where the soil was drier. A. tricoccum always grew in large clusters as dense colonies, and in some populations occupied almost the entire face of a slope (Figure 11).

Figure 11. Extensive population of Allium tricoccum growing in Eagle Crest Nature

Preserve, IN, April 28, 2017.

70

Allium burdickii and South Green Ramps were separated by soil texture, which is also characteristic of their regions, with predominately sandy soils around the Great Lakes in the northern region, and more heavily silty and clayey soils in the Interior Highlands, in rich floodplain forests immediately along streams. Allium burdickii was never found close to water, except in Washtenaw County, Michigan, where the plants grew somewhat close to a pond in flat land. South Green Ramps in some Kentucky sites flourished in flat streamside floodplains, but in other Kentucky and Tennessee sites it grew on or near the top of moderate to steep slopes in hilly land (e.g. Standing Stone State Park and Edgar

Evins State Park). This is the first description of the habitats of South Green Ramps, which differed substantially from the habitats of A. burdickii.

The natural habitat of Highland Green Ramps is high-altitude montane forests, and we have determined its geographic distribution from our own field studies, reliable herbarium specimens and botanist reports to be restricted to the Appalachian Mountain region. It grows side by side with or completely intermingled with A. tricoccum as single plants or small colonies in some sites, and prefers moister soil close to a water source such as a stream or drainage. Anecdotal reports by harvesters knowledgeable about the taxa suggest it can also grow near the tops of some mountains, usually above nearby populations of A. tricoccum. Interestingly, at a field site near the Blue Ridge Parkway, the habitat was not dominated by big trees but rather had limited canopy and reduced shade, compared to other populations of Highland Green Ramps mostly found in heavily wooded sites. This is the first description of the ecology of Highland Green Ramps, based on limited field work; more observations over its entire predicted range are needed. 71

Studies of environmental variables in the four taxa of the A. tricoccum complex confirm substantial substrate and elevational differentiation among the three Green Ramps taxa, corroborating morphological and phenological evidence for all three as distinct evolutionary species from each other and from A. tricoccum. However, A. tricoccum shows very broad environment amplitude that largely overlaps with the niches of the Green

Ramps taxa. Our interpretation of morphological, phenological and environmental evidence is that A. tricoccum has long been evolutionarily separated from the Green Ramps taxa, to the extent that ecological isolation is no longer required to maintain the two sets of taxa in nature. Flowering time differences noted in the phenological studies are presumably sufficient to support them as different sublineages.

72

CHAPTER 5: MOLECULAR ANALYSIS

Methods

Genomic DNA was extracted from silica gel-preserved leaf tissue samples using the modified CTAB method and quantified with a UV–Vis spectrophotometer (ND-1000;

NanoDrop, Wilmington, DE, USA). Leaf tissue was ground in a 1.5ml microfuge tube with a minipestle in liquid nitrogen and suspended in 600L extraction buffer [20 mM of EDTA,

0.1 M of Tris-HCl (pH 8.0), 1.4 M of NaCl, 2% CTAB and 40 mM of beta- mercaptoethanol] and vortexed for 3 seconds. Following extraction process were used

Promega Genomic DNA Purification Kit. Extractions results were quantified and adjusted to the same concentration of 10ng/ul prior to PCR amplification.

Twelve primer pairs (Table 2) were selected from Allium sativum studies (Lee et. al., 2011) These were tested on a small subset of samples for each phenotype and grouped to build three four-plex primer sets. PCR reactions were conducted in a total 10L volume, containing including 1L genomic DNA (10 ng/L), 0.5 L multiplex primer (2mmol/L reverse primer, 0.5 mmol/L forward primer with tail, and 2 mmol/L M13 universal primer), 5L 2x Multiplex PCR buffer (Qiagen Corp.) and 3.5L distilled water. DNA amplification were performed using an Applied Biosystems 2720 thermal cycler (Carlsbad,

California, USA). The thermal cycler program consisted of 5 min of denaturing at 95°C, and 35 cycles of 30 sec of denaturing at 94°C, 50 sec of annealing at 57°C and 40 sec of elongation at 72°C. In the following 15 cycles the annealing temperature was decreased to

54°C, with a final elongation step of 10 min at 72°C. Amplified samples were 73 electrophoresed in 1.3% agarose gels, with a 250bp size standard to infer size of PCR fragments.

Successful PCR products were diluted to a ratio of 1L DNA sample : 9 L distilled water and analyzed on a Applied Biosystem 3130xl genetic analyzer at the Ohio University

Genomics Facility. Fragments were scored as present (1, clear and sharp peak) or absent

(0, very weak or not present).

The presence/absence matrix of microsatellite fragments, treating every set of comigrating fragments as a single locus, was analyzed with Principal Coordinates Analysis

(PCoA) using the Jaccard coefficient in PAST 3.x software (Hammer et al. 2001). This analysis first creates a similarity matrix of pairwise comparisons for all samples using the selected coefficient, then applies Principal Components Analysis to the similarity matrix, creating a reduced set of axes into which the sampled points are placed. The algorithm is different from NMDS in that it maximizes the variation in the data set, placing the greatest spread of points in multidimensional space onto the first axis, the next greatest spread on the second axis, and so forth. In addition, an Analysis of Molecular Variance (AMoVA) was conducted using GENALEX software (Peakall and Smouse, 2012), to examine the relative distribution of allelic diversity within and among taxa and populations.

Results

Surprisingly, no significant differences in diversity of 12 microsatellite loci were found among the four taxa of the Allium tricoccum complex. The AMOVA test indicated that only 3% variance was found among the four taxa, and 97% of the variance was within the 74 taxa (Table 8). The AMOVA result were confirmed with the PCoA ordination, where

OTUs of all four taxa fully overlapped (Figure 12).

Table 8. Analysis of molecular variance (AMOVA) based on 12 microsatellite loci in four taxa of the Allium tricoccum complex.

Source df SS MS Est. Var. %

Among Pops 3 502.362 167.454 1.645 3%

Within Pops 276 16492.142 59.754 59.754 97%

Total 279 16994.504 61.399 100%

75

Figure 12. Principal Coordinates Analysis ordination with Dice coefficient based on 12 microsatellite loci in four taxa of the Allium tricoccum complex.

Discussion

The absence of separation of the four taxa based on 12 microsatellite loci was surprising. These results cannot be reconciled with the morphological, phenological or ecological evidence, especially the results based on several to many genetically fixed differences demonstrated in pairwise analyses of the taxa from common garden observations. Preliminary tests showed successful amplification of the 12 primer pairs with 76 a small subset of the four taxa. However, fragment analysis of multiplexed samples for the

280 leaf DNA extracts sample provided little differentiation among taxa.

One reasonable explanation for the failure of the loci to discriminate among the taxa relates to the presence of more than two fragments in many loci found in most samples, when only one or two fragments would be expected in a diploid plant complex. The loci were isolated specifically for use with garlic cultivars, and from a species which is quite distant phylogenetically to the A. tricoccum complex. It is very possible that the primer sites for the loci, surely positioned in noncoding regions, have evolved to the extent that the primer pairs amplified multiple positions in the nuclear genome rather than a single locus when applied to the A. tricoccum complex. This suggests that the applicability of the garlic primer sets limited, and we exceeded the bounds of primer specificity in utilizing the designed primers in this study. Isolation and testing of microsatellite loci for the complex under study would be a worthwhile pursuit.

A previous molecular study conducted by Vasseur (1990) used isoenzymes as a marker system but found low genetic variation between A. tricoccum and A. burdickii.

However, he noted that his study encompassed samples taken only from the northern edge of the range of the complex (and in fact may not have included more than A. tricoccum).

Despite the failure of microsatellite diversity to differentiate the taxa, other lines of evidence mutually support recognition of four taxa as distinct evolutionary species.

77

CHAPTER 6: SPECIES DISTRIBUTION MODELS

Methods

Latitude-longitude coordinates for the taxa were collected during field work, and from confirmed herbarium specimens, as well as additional reports from iNaturalist.org with pictures that gave high confidence for identification of particular taxa. Numbers of population coordinates varied for each taxon, and only A. tricoccum (38 population coordinates) and A. burdickii (31 population coordinates) had population coordinates numbering more than 30 as recommended by Hernandez (2006). The other taxa, South

Green Ramps, had 26 population coordinates, and Highland Green Ramps, only had 8 population coordinates. As a result, distribution modelling was conducted for A. tricoccum,

A. burdickii and South Green Ramps only, due to the insufficient sample size for Highland

Green Ramps.

Species modelling was conducted using Maximum entropy density estimation formulation with the MaxEnt program Ver. 3.4.1(Phillips et al., 2018). The program is widely used in many fields since publication, is known to have higher accuracy in predictive modelling of data compared to other methods or programs, is reportedly user- friendly, and can use presence only data for species under study (Baldwin, 2009; Philips,

2006). Bioclimatic layers at 5 minutes spatial resolution (around 9x9 km square at the equator) were downloaded from WorldClim data sets (www.worldclim.com) and were used in the data modeling as variables to determine hypothetical species distributions.

Modelling computation used 25% of presence data as a random test to compare with 10,000 maximum background points that were interpreted as pseudo-absence of the 78 species. Model robustness is commonly evaluated by AUC values that range from 0 to 1;

AUC values between 0.5–0.7 are considered low, 0.7–0.9 moderate and >0.9 high.

Modelling output classified the habitat distribution prediction into three probability classes: low, medium and high suitability that was represented in the map figure as different colors.

Results

Modeling results with the MaxEnt program gave high AUC values for both the test and prediction training data for three taxa (Table 9). The habitat prediction was similar to the distribution map published by Jones (1979) and Biota of North America program (2014) for A. tricoccum, as shown by the SDM map of A. tricoccum localities provided (Figure 13). For the two other taxa, the SDM map also corresponded somewhat similarly to the actual geographic distribution, but overlap in the climate suitability between South Green Ramps and Highland Green Ramps in the southern U.S.

Table 9 Training and test data of AUC values for distribution models of three taxa in the

Allium tricoccum complex.

Taxon AUC Training data AUC test data A. tricoccum 0.993 0.991 A. burdickii 0.997 0.997 South Green 0.996 0.986

79

Figure 13. Prediction of A. tricoccum habitat distribution based on presence data with

Maximum Entropy method. Color legend indicates habitat suitability from 0 (not suitable) to 1 (very suitable). White squares indicate presence location for training, and purple squares for test location.

Bio1 (annual mean temperature) showed the highest contribution for MaxEnt prediction (71.9%) for the A. tricoccum habitat distribution, followed by Bio12 (annual precipitation) with 12.5% contribution to the prediction. The MaxEnt model’s Jackknife test of variable importance showed that Bio1 and Bio12 also had high gain when used in isolation and when they were omitted (Figure 14). 80

Figure 14. The Jackknife test for evaluating the relative importance of environmental variables for Allium tricoccum.

Allium burdickii and South Green Ramps distribution models were separated in the North (Figure 15) and South region, respectively (Figure 16). Both taxa had similar variables that contributed strongly to development of distribution model, Mean

Temperature of Coldest Quarter (bio11), which contributed 64.3% for A. burdickii and

46,5% for South Green Ramps. However, the Jackknife test for each taxon give different result to evaluate relative importance of environmental variables for distribution model development. 81

Figure 15. Prediction of Allium burdickii habitat distribution based on presence data with

Maximum Entropy method. Color legend indicates habitat suitability from 0 (not suitable) to 1 (very suitable). White squares indicate presence location for training, and purple squares for test location.

The Allium burdickii distribution model jackknife test indicated that Mean

Temperature of Driest Quarter (Bio09) was the most important variable when used independently and when omitted from model formulation (Figure 17). South Green

Ramps’ distribution model Jackknife test indicated that Precipitation Seasonality

(Coefficient of Variation) (bio15) was the most important variable when used independently, and Isothermality (bio3) was the variable with important information when omitted from the model formulation (Figure 18). 82

Figure 16. Prediction of South Green Ramps habitat distribution based on presence data with Maximum Entropy method. Color legend indicates habitat suitability from 0 (not suitable) to 1 (very suitable). White squares indicate presence location for training, and purple squares for test location. 83

Figure 17. The Jackknife test for evaluating the relative importance of environmental variables for Allium burdickii. 84

Figure 18. The Jackknife test for evaluating the relative importance of environmental variables for South Green Ramps.

Discussion

The species distribution models of the three taxa in the A. tricoccum complex generated with Maximum entropy modelling are the first ever presented. The models showed high accuracy as represented by high value of Area Under ROC (receiver operating characteristic curve), or AUC, both for prediction and training tests

(AUC>0.9), with note on the sample size for each taxon that low in number. The modelling, as expected, closely mirrored the distribution of A. tricoccum documented by 85

Jones (1979) from herbarium specimens, and for her reports of A burdickii in the Great

Lakes and Great Plains regions. The modelling also narrowly delineated the anticipated distribution of South Green Ramps in the Interior Highlands region.

Bio1 (Annual Mean Temperature) is the most important variable in the distribution models of Allium tricoccum as explained by the jackknife results diagram for each variable used in the model prediction. Based on the important variable, A. tricoccum has a habitat distribution in the region with Annual Mean Temperature around 10oC

(Figure 19)

Figure 19. Response curves from variable Bio1 (annual mean temperature) in the model prediction of Allium tricoccum habitat distribution based on presence data with

Maximum Entropy model. 86

Allium burdickii and South Green Ramps distribution models indicated that the taxa had habitats in the region with Mean Temperature of Coldest Quarter (Bio11) around 0oC as a variable with high contribution to the models (Figure 20 and 21, respectively). The same bioclimatic variables as a major contributor to distribution can be explained by considering the emerging growth of Allium burdickii and South Green

Ramps growth in the early spring until June when the temperature is still low. The difference, however, is shown y the braoder curve for South Green Ramps compare to that of A. burdickii, indicating a broader range of Mean Temperature of Coldest Quarter for that more southern taxon.

Figure 20. Response curve from variable Bio11 (Mean Temperature of Coldest Quarter) in the model prediction of Allium burdickii habitat based on presence data with

Maximum Entropy model. 87

Figure 21. Response curve from variable Bio11 (Mean Temperature of Coldest Quarter) in the model prediction of South Green Ramps habitat distribution based on presence data with Maximum Entropy model.

Maximum entropy models currently are employed widely as an easy use of the

MaxEnt program and only need presence data (Philips, et el. 2006). The program also allows the user to predict the distribution with future climate data scenario provided by

WorldClim ( http://www.worldclim.org. ). Further model predictions with additional variables used in environmental analysis, e.g. soil and elevation variables, are important to perform to achieve specific model results for each taxon. Future predictions with additional data are also important as a tool for evaluation of species conservation management, and for the potential to locate additional populations of geographically limited taxa. Although quite broad in assessment of bioclimatic variables that closely 88 relate to (and may in fact govern) species distributions, the MaxEnt results for the three taxa generally mirrored inferences from field studies that Allium tricoccum has evolved a broad ecological amplitude over much of eastern North America. Modelling of the A. burdickii and South Green Ramps taxa revealed more narrow or limited niches defined by bioclimatic (or local environmental) variables. Overall, the niche modelling results provided further corroboration, with the site-based ecological studies of substrate and elevation, in support of four taxa as evolutionary species.

89

CHAPTER 7: SYSTEMATIC REVIEW

Systematically, the Allium tricoccum complex is phylogenetically placed in the subgenus Anguinum with Eurasian A. victorialis L. as a sister species. McNeal and

Jacobsen (2002) and, later, Choi and Oh (2011), demonstrated that A. ochotense Prokh. of east Asia and Attu island, Alaska is a different species from A. victorialis L., indicating that the Eurasian and western Alaskan A. victorialis complex and the North American A. tricoccum complex are nearest relatives. Herden et. al. (2016) used a combination of ITS sequence and cpDNA marker sequences to examine subgenus Anguinum more closely and inferred that the A. tricoccum complex (based apparently only on A. tricoccum sensu stricto) was possibly of hybrid origin, because of phylogenetic conflict between the nuclear and chloroplast DNA results. However, absence of fossil evidence of new world

Allium cannot confirm the dispersal time of the species. On the other hand, previous disagreements concerning the distinctness and delineation of A. tricoccum sensu stricto and Green Ramps taxa (Neil and Jacobsen, 2002) also make hard clear explanation about the phylogeny relationship of each initial taxa proposed in this study. Further research is necessary.

Our studies of morphology, ecology (including site-based environmental variables and niche modelling) and phenology comprehensively distinguish four taxa (Table 9). At each field site and in the common garden over two seasons, taxa (even where sympatric) were highly uniform, obviously reproduced consistently, and formed local populations.

The four taxa here are interpreted as evolutionary species under the Unified Species

Concept: A. tricoccum Aiton, A. burdickii (Hanes) A.G. Jones, South Green Ramps and 90

Highland Green Ramps, the latter requiring formal description in a future manuscript.

While A. burdickii and South Green Ramps show fewer sharply distinct morphological or ecological differences, it is clear from the cumulative evidence that they cannot be construed as conspecific sets of populations. Since the Unified Species Concept states that species are “separately evolving metapopulations”—not “separately evolved”—the retention of fully green leaves with early scape emergence, shorter stamens and perianth, preference for more clayey or silty soils, and predominately Interior Highlands geographic distribution argue for distinguishing South Green Ramps from A. burdickii sensu stricto. Treatment of these as infraspecific taxa or, worse, synonymizing one or more of the taxa, amounts to willful ignorance of the processes of biological diversification and acceptance of the products of those processes. Arguably, the scientific discipline of systematics is the detection and characterization of biodiversity, and the research presented here has provided compelling evidence that the most rational scientific approach to recognizing biological diversity in North American Ramps is to formally adopt four evolutionary species.

Consideration of all reliable data is important in consideration of integrative taxonomic studies (Dayrat, 2005). Nevertheless, the genetic results were not considered in the application of the USC to delineate evolutionary species. Unsuccessful separation of the four taxa by microsatellite markers could be considered from two perspectives:

“delayed confirmation” for species delimitation, in that the taxa differentiated by morphology, ecology and phenology have not yet accumulated sufficient microsatellite separation above the population level; or, the loci isolated specifically for distinguishing 91 cultivars of domesticated garlic are so variable above the species level that they are too polymorphic (or non-specific) to provide reliable differentiation. Nevertheless, the field studies, common garden observations on morphology and phenology over two seasons, and laboratory investigations of macromorphology and soil factors, are fully congruent in proposing four consistently distinct taxa as separately evolving metapopulations in the A. tricoccum complex.

Table 10. Morphological, phenological and ecological traits to distinguish four evolutionary species in the Allium tricoccum complex.

Numerical values with minimum-(mean)-maximum presented.

Character Allium tricoccum Allium burdickii South Green Highland Green

Leaf Shape Broadly lanceolate to Narrowly to broadly Narrowly linear to linear- Narrowly ovate-lanceolate elliptical blades with linear or linear-oblong oblong blades gradually or narrowly elliptical abruptly differentiated blades gradually tapering tapering into short blade with abruptly longer petioles, 12.8- into short indistinct indistinct petioles, 15.0- differentiated shorter (18.6)-22.9 cm long, 3.5- petioles, 16.2-(20.8)-27.9 (21.3)-25.6 cm, 1.7-(2.8)- petioles, 15.0-(17.5)-21.7 (6)-8.8 cm wide cm long, 1.6-(3.1)-4.3 4.0 cm wide cm long, 3.6-(4.4)-5.2 cm cm wide wide Pigmentation Bulbs, foliage, scapes Bulbs whitish, foliage, Bulbs whitish, foliage, Bulbs whitish, foliage, and bracts commonly scapes and bracts scapes and bracts medium scapes and bracts green reddish-purple silvery- or gray-green green but lacking reddish- but lacking reddish-purple pigmented but lacking reddish- purple pigment pigment purple pigment

93

Table 10 Continued

Bulb Shallow or partially Deeply set bulbs 0.6- Deeply set bulb 0.8-(2.6)- Deeply set bulb 0.8-(1.7)- exposed bulbs 0.1-(0.5)- (2.7)-7.3 cm deep in the 7.4 cm in the ground, 2.0- 2.6 cm deep in the ground, 2.7 cm deep in the ground, 2.1-(4.0)-5.1 cm (3.9)-6.0 cm long, 1.0-(1.5)- 3.0-(3.7)-4.4 cm long, 1.4-

ground, 2.7-(4.3)-5.5 cm long, 0.9-(1.3)-2.0 cm wide 2.3 cm wide (1.5)-1.7 cm wide long, 1.4-(2.0)-3.1 cm wide Scape Initially weakly to Erect Erect Erect strongly curved but erect at flowering Umbel shape Broadly obconic to Narrowly obconic Narrowly obconic Narrowly to broadly hemispherical obconic Buds/flowers/fruits 11-(35)-77 1-(9)-16 7-(13)-18 7-(13)-27 per umbel Pedicels Lowest ascending to Lowest ascending to Lowest ascending to Lowest ascending to strongly reflexed, angled strongly ascending, strongly ascending, strongly ascending, 75o-(103o)-132o upward angled 121o-(135o)-156o angled 118o-(136o)-156o angled 111o-(129o)-155o from scape upward from scape upward from scape upward from scape Perianth length 4.8-(5.9)-7.1 mm long 4.3-(4.9)-5.8 mm long 3.3-(4.5)-5.1 cm long 4.9-(5.9)-6.6 mm long 94

Table 10 Continued Stamens Strongly exserted, 7.1- Barely exserted, 4.7- Slightly exserted, 4.2- Slightly exserted, 6.0- (8.8)-10.6 mm long (5.4)-6.3 mm long (5.2)-6.7 mm long (7.0)-7.5 mm long Capsule diam 9.0-(9.6)-10.6 mm 7.7-(8.8)-10.1 mm 7.9-(8.6)-9.5 mm 7.9-(8.8)-9.9 mm Seed diam 2.9-(3.1)-3.5 mm 2.6-(2.9)-3.3 mm 2.5-(2.9)-3.1 mm 2.7-(3.0)-3.4 mm Colony form Usually large, plants Small and scattered, Small and scattered, Small and scattered, very dense plants solitary or loosely plants solitary or loosely plants solitary or loosely aggregated aggregated aggregated (often intermingled with A. tricoccum) Phenology Intact leaves retained Leaves typically Intact leaves retained Intact leaves retained through scape withered away prior during early scape during early scape emergence, later scape scape emergence, earlier emergence, earlier bud emergence, earlier bud emergence, bud opening bud opening and fruit opening and fruit opening and fruit and fruit maturation maturation maturation maturation Environment Occurs in broad range of Mostly in sandy loam Mostly in silt loam soils, Mostly in clay loam soils, woodland habitats soils, alt. 191-295 MASL alt. 247-452 MASL alt. 896-1,587 MASL Descriptions of each taxon delineated and recognized here as a distinct species use all information documented from the studies presented in this thesis or confirmed to be reliable from earlier studies. It is important to note that the geographic distributions of the three Green Ramps taxa presented below are preliminary, due to difficulties in confidently identifying some herbarium specimens. New taxa noted or described in this thesis are not intended for effective online publication here. One or more separate manuscripts will be submitted to scientific journals to publish the new names.

Key to Species in the Allium tricoccum Complex

1. Leaves, bulb, and scape commonly reddish-purple pigmented; leaf blades narrowly to

broadly elliptical or ovate-lanceolate, sharply differentiated from the long petiole;

scape curved in early development; bulb partially exposed or shallow in ground;

buds, flowers and fruits 15-(35)-77, in a broadly obconic to hemispherical

umbel…………………………………………………………...…...Allium tricoccum

1. Leaves, bulb, and scape green, never with reddish-purple pigmentation; leaf blades

narrowly linear to lanceolate or oblong tapering into short indistinct petiole, or

narrowly elliptical to narrowly ovate-lanceolate and somewhat differentiated from the

short petiole; scape strictly erect; bulb totally submerged up to 7 cm in the ground;

buds, flowers and fruits 1-(12)-35, in a narrowly obconic

umbel……………………...……………..……...... …2

2. Leaf blades broadly elliptical to broadly lanceolate, contracted abruptly into a more or

less distinct elongate petiole; perianth height 5-6.5 mm; stamen height 6-7.5 mm; 96

green leaves always retained during scape emergence; Appalachian Mountain

region...... Highland Green Ramps

2. Leaf blades linear to narrowly lanceolate or oblong, gradually tapering into a short

indistinct petiole; perianth height 3-5.8 mm; stamen height 4-6.6 mm; green leaves

retained or senescent to obsolete during scape emergence; lower elevations west of

the Appalachian Mountain

region...... ……………...... …3

3. Intact leaves retained during scape emergence, medium green life; flowers 7-18 (avg.

13); perianth height 3.3-5.1 mm; stamens slightly exserted, 4.2-6.7 mm long; growing

in heavily silty and clayey soils in the Interior Highlands region of KY and

TN……...... …………………………South Green Ramps

4. Leaves usually fully withered and obsolete at scape emergence (rarely retained but

partially withered), silvery- or gray-green in life; flowers 1-16 (avg. 9); perianth

height 4.3-5.8 mm; stamens barely exserted, 4.7-6.3 mm; growing in sandy loam soils

in the Great Plains and Great Lakes region, possibly contacting the northernmost

range of South Green Ramps in southern IL and IN…………………Allium burdickii

Description of the Taxa

Allium burdickii (Hanes) A. G. Jones, Syst. Bot. 4: 32. 1979. Allium tricoccum Aiton var.

burdickii Hanes, Rhodora 55: 243. 1953.—TYPE: USA. Michigan, McCreary

woods in Sec. 16 Prairie Ronde Twp., 30 Apr 1951, C. R. & F. N. Hanes 2051

(lectotype designated here: WMU1000001; isolectotypes: GH00029750, 97

MICH1259018; all three specimens confirmed as JSTOR Global Plants Database

images!).

The whole plant always devoid of reddish-purple pigmentation, scattered as individuals or loosely aggregated small colonies. Bulb 2.1-(avg. 4.0)-5.1 cm long, 0.9-

(avg. 1.3)-2.0 cm wide, 0.6-(2.7)-7.3 cm deep in the ground. Leaves 2-3, at least the blades commonly silvery- or gray-green in life, blades rather indistinctly grading into short petioles; petiole 1.3-(avg. 1.6)-2.1 cm long; blade narrowly to broadly linear or linear-oblong, 16.2-(avg. 20.8)-27.9 cm long, 1.6-(avg. 3.1)-4.3 cm wide, apex narrowly acute, 21°-(avg. 32°)-48°, base narrowly acute, 17°-(avg. 31°)-51°. Scape emerging strictly erect from the ground, in nature always following complete withering of the leaves, 9.9-(avg. 16)-26.5 cm tall at anthesis; inflorescence bract 1.4-(avg. 1.9)-2.4 cm long; inflorescence a narrowly obconic umbel with lowest pedicels angled 121°-(avg.

135°)-156° upward from scape. Flowers 1-(avg. 9)-16; pedicels 1.3-(avg. 1.6)-2.1 cm long; perianth whitish, 4.3-(avg. 4.9)-5.8 mm long; stamens barely exserted, 4.7-(avg.

5.4)-6.3 mm long. Mature capsules green, 7.7-(avg. 8.8)-10.1 mm in diam; mature seeds black, 2.6-(avg. 2.9)-3.3 mm in diam, weight 1.7-(avg. 1.9)-2.9 g.

Jones (1979) mentioned that no type collection was cited by Hanes (1953) in the description of var. burdickii, yet she designated Hanes and Hanes 2051 at WMU as the holotype, in part because the collection was labeled by the Hanes’s as “Type specimens”.

The current International Code of Nomenclature, and clarifications offered by McNeill

(2014), reject holotype designations; however, the collection is accepted as original 98 material, and the WMU sheet is designated here as the lectotype, while sheets at GH and

MICH are accepted as isolectotypes.

This and other Green Ramps taxa are wholly divergent from A. tricoccum in numerous morphological and phenological traits, are reproductively isolated due to apparently fully separated flower anthesis, occupy a narrower range of microhabitats, have smaller regional distributions, and show a different growth pattern in colony formation (being more sporadically distributed over the forest floor and much less dense).

Lumping of taxa into one (or two) species grossly obscures biological diversity in the complex and ignores the evolutionary significance of these patterns of divergence.

Several morphological distinctions, limited phenological separation of leaves and flowers, different ranges, and divergent substrate factors demonstrate the uniqueness of all three Green Ramps taxa. This species exhibits sharp separation in phenology of intact leaves and emergence of the scape in nature, although plants in the common garden occasionally retain withering leaves in the earliest stage of scape emergence. Other Green

Ramps taxa retain intact leaves through early scape emergence. The southern range limit of A. burdickii and the northern range limit of South Green Ramps are still uncertain; living populations early in scape emergence should be examined in the intervening region of the Lower Midwest to clarify geographic ranges.

South Green Ramps

The whole plant always devoid of reddish-purple pigmentation, scattered as individuals or loosely aggregated small colonies. Bulb 2.1-(avg. 3.9)-6.1 cm long, 1.0- 99

(avg. 1.5)-2.3 cm wide, 0.8-(avg. 2.6)-7.4 cm deep in the ground. Leaves 2-3(-4), at least the blades green in life, blades rather indistinctly grading into short petioles; petiole 1.4-

(avg. 1.8)-2.3 cm long; blade narrowly linear to linear-oblong, 15.0-(avg. 21.3)-25.6 cm long, 1.7-(avg. 2.8)-4.0 cm wide, apex narrowly acute, 17°-(avg. 24°)-34°, base narrowly acute, 12°-(avg. 27°)-37°. Scape emerging strictly erect from the ground while intact leaves are initially retained (leaves later wither prior to inflorescence bract breakage),

11.9-(avg. 15.6)-21.0 cm tall at anthesis; inflorescence bract 1.2-(avg. 2.1)-2.7 cm long; inflorescence a narrowly obconic umbel with lowest pedicels angled 118°-(avg. 136°)-

156° upward from scape. Flowers 7-(avg. 13)-18; pedicels 0.6-(avg. 1.6)-2.2 cm long; perianth whitish, 3.3-(avg. 4.5)-5.1 cm long; stamens slightly exserted, 4.2-(avg. 5.2)-6.7 mm long. Mature capsules green, 7.9-(avg. 8.6)-9.5 mm in diam; mature seeds black, 2.5-

(avg. 2.9)-3.1 mm in diam, weight 1.7-(avg. 2.0)-3.0 g.

South Green Ramps resembles A. burdickii in many features but is distinct in producing generally narrower green leaf blades that remain fully intact during early scape emergence, and umbels with typically more flowers. This species also often inhabits low flat terraces of floodplains in close proximity to streams, with more strongly silty soils than the sandy substrates preferred by A. burdickii.

Highland Green Ramps

The whole plant always devoid of reddish-purple pigmentation, scattered as individuals or loosely aggregated small colonies; very often intermingled with A. tricoccum, sometimes in separate colonies at slightly higher elevations than that species. 100

Bulb 3.0-(avg. 3.7)-4.4 cm long, 1.4-(avg. 1.5)-1.7 cm wide, 0.8-(avg. 1.7)-2.6 cm deep in the ground. Leaves 2-3, at least the blades green in life, petioles rather sharply distinct from blades, petiole 1.2-(avg. 1.5)-2.2 cm long; blade narrowly ovate-lanceolate or narrowly elliptical, 15.0-(avg. 17.5)-21.8 cm long, 3.6-(avg. 4.4)-5.2 cm wide, apex narrowly acute to acute, 28°-(avg. 40°)-50°, base narrowly acute to acute, 34°-(avg. 46°)-

60°. Scape emerging strictly erect from the ground while intact leaves are initially retained (leaves later wither prior to inflorescence bract breakage), 19.0-(avg. 21.7)-24.8 cm tall at anthesis; inflorescence bract 1.4-(avg. 1.6)-1.9 cm long); inflorescence a narrowly obconic umbel with lowest pedicels angled 111°-(avg. 129°)-155° upward from scape. Flowers 7-(avg. 13)-27; pedicels 1.0-(avg. 1.2)-1.3 cm long; perianth whitish, 4.9-

(avg. 5.9)-6.6 mm long; stamens slightly exserted, 6.0-(avg. 7.0)-7.5 mm long. Mature capsules green, 7.9-(avg. 8.8)-9.9 mm in diam; mature seeds black, 2.7-(avg. 3.0)-3.4 mm in diam, weight 1.7-(avg. 1.9)-2.0 g.

Highland Green Ramps differs in many morphological features from the other two Green Ramps taxa. It also prefers more clayey soils, grows in somewhat different forest communities, and inhabits significantly higher elevations than A. burdickii and

South Green Ramps. It likely represents much of the basis for reports of hybridization or intergradation between A. burdickii and A. tricoccum in the Appalachian Mountain region. The northeastern reports of A. burdickii within the Appalachian Mountain ecoregion and associated highlands are attributed to this taxon, based on examinations of herbarium specimens. It often grows fully intermingled with A. tricoccum at many sites, and botanists have suggested that the anomalous plants were merely a phenotypic 101 extreme of A. tricoccum on this basis. Nevertheless, all Green Ramps taxa are reproductively isolated by sharp differences in flower anthesis phenology from A. tricoccum, and the many morphological distinctions between the two groups expressed in nature wherever Green Ramps taxa grow near A. tricoccum are maintained in the common garden. This evidence, demonstrating numerous genetically fixed morphological and phenological differences, clearly indicates separate evolutionary status between the two major groups, as well as sharp distinctions between Highland Green Ramps and the other Green Ramps taxa.

Allium tricoccum Aiton, Hort. Kew., vol. 1, ed. 1: 428. 1789. “Nat. of North America.

Mr. William Young. Introd. 1770. Fl. July”.—TYPE: USA. Hort. Kew. (ex

america sept.) (lectotype designated here: BM001066816; specimen confirmed as

JSTOR Global Plants Database image!).

Plant commonly with faint to deep reddish-purple pigmentation on one or more parts in life (bulb, leaf petiole or midrib, scape, inflorescence bract or perianth), forming dense colonies; often growing in the same area or vicinity as one of the Green Ramps taxa, overall occupying a broad range of habitats over its range. Bulb 2.7-(avg. 4.3)-5.5 cm long, 1.4-(avg. 2.1)-3.1 cm wide, 0.1-(avg. 0.5)-2.7 cm deep in the ground. Leaves 2-

3, petioles sharply distinct from blades, petioles 1.5-(avg. 2.0)-2.9 cm long; blade narrowly to broadly ovate-lanceolate or elliptical, 12.8-(avg. 18.6)-22.9 cm long, 3.5-

(avg. 6.0)-8.8 cm wide, apex narrowly to broadly acute, 40°-(avg. 57°)-77°, base narrowly to broadly acute or obtuse, 38°-(avg. 66°)-92°. Scape emerging shallowly to 102 strongly (re)curved, becoming strictly erect only a short time prior to breakage of the inflorescence bract, leaves fully intact and retained into fruit maturation, 10.5-(avg. 21.1)-

30.5 cm tall at anthesis; inflorescence bract 1.1-(avg. 1.9)-2.2 cm long; inflorescence a broadly obconic to hemispherical umbel with lowest pedicels spreading or reflexed, angled 75°-(avg. 103°)-131° upward from scape. Flowers 11-(avg. 35)-77; pedicels 1.3-

(avg. 2.0)-2.9 cm long; perianth whitish or flushed with reddish-purple pigmentation, 4.8-

(avg. 5.9)-7.1 mm long; stamens strongly exserted, 7.1-(avg. 8.8)-10.6 mm long. Mature capsules green, 9.0-(avg. 9.6)-10.6 mm in diam; mature seeds black, 2.9-(avg. 3.1)-3.5 mm in diam, weight 1.9-(2.0)-2.1 g.

Jones (1979) argued that no collections were extant to typify A. tricoccum, and she elected to designate a neotype at GH. However, a specimen at BM annotated for

Hortus Kewensis and taken from North America is available that represents A. tricoccum and is presumed to be original material. In accordance with the International Code of

Nomenclature, the lectotype is designated here and unequivocally identifies the broad- leaved, pigmented taxon as detailed in this thesis as A. tricoccum. Since other specimens provided by William Young cited Pennsylvania and Virginia for provenance, it is assumed that the present lectotype probably came from one of those states as well.

This species occupies a wide range of habitats, spanning the ecological amplitude of the Green Ramps taxa collectively. It characteristically forms very dense, often quite large colonies, and may well have a different vegetative mode of propagation and/or a different seed dispersal strategy to explain the obvious and consistent difference in colony formation. Allium tricoccum often grows in the same area as, or in the immediate 103 vicinity of, one of the Green Ramps taxa, occasionally loosely intermingling where individual colony boundaries overlap. In the case of the Highland Green Ramps it is found intimately intermixed with individuals of that species at some montane (usually lower-elevation) sites, but reportedly forms pure colonies at high elevations near ridgetops. Allium tricoccum is distinct from Green Ramps taxa in most morphological features measured, expresses a non-overlapping floral anthesis shift in nature and in the common garden (indicating reproductive isolation), and no intermediates or hybrids have been found in field studies. The intact leaves are retained for a longer time period than in

South Green Ramps or Highland Green Ramps, the other species in which leaf phenology and reproductive phenology overlap. This species is on a completely different evolutionary trajectory and deserves species status separate from the Green Ramps taxa.

104

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APPENDIX

Appendix 1: Morphology examined from 28 populations for four taxa of the Allium tricoccum complex.

Highland Traits A. tricoccum A. burdickii South Green Green Leaf number 2-3 2-3 2-3 2-3 Broad, elliptical Linear or narrow Linear or narrow Broad, elliptical to lanceolate elliptical elliptical to lanceolate Leaf blade Length: 12.8- Length: 16-(21)- Length: 15- Length: 15- (18.6)-22.9 cm 28 cm (21.3)-25.6 cm (17.5)-21.7 cm Width: 3.5-(6)- Width: 1.6-(3)-4.3 Width: 1.7-(2.8)- Width: 3.6- 8.8 cm cm 4 cm 94.3)-5.2 cm Exposed, or Always exposed Exposed, or almost Exposed above above ground, almost submerged Leaf Petiole submerged in the ground, 1.2-2.1 1.5-3 cm in in the ground, 0.1 ground, 0.1 – 1.8 in length length – 2 cm in length cm in length Ovoid, Slenderly ovoid Slenderly ovoid Slenderly ovoid Length:2.7-(4.3)- Length: 2.1-(4)- Length: 3-(3.7)- Length: 2-(3.9)-6 Bulbs 5.5 cm 5.1 cm 4.4 cm Width: 1-(1.5)- Width: 1.4-(2)- Width: 0.9-(1.3)-2 Width: 1.4- 2.3 cm 3.1 cm cm (1.5)-1.7 cm Curved during Straight development of Straight Straight Scape height: bract, turn Scape height: 9.9- Scape height: 12- 19-(21.7)-24.8 Scape and straight after (16.3)-26.5 cm (15.6)-21 cm cm bract bract broken Scape orientation: Scape Scape Scape height:10.5 180 orientation: 180 orientation: 180 –(21)-30.5 cm 113

Scape orientation: 76o- (134o)-161o Stamen relatively Stamen mostly Stamen shorter, Stamen shorter, longer, and long, exposed, only slightly only slightly exposed to almost double in exposed, usually exposed, usually perianth, shorter size with at same height at same height than A. perianth. with perianth with perianth tricoccum. Flowers Bud numbers: Bud numbers: Bud numbers: Bud numbers: Stamen height: 7- Stamen height: Stamen height: 7-(13)-27 (8.8)-10.6 mm 4.7-(5.4)-6.3 mm 4.2-(5)-6.6 mm Stamen height: Perianth height: Perianth height: Perianth height: 6-(7)-7.5 mm 4.8-(6)-7 mm 4.3-(4.9)-5.8 mm 3.3-(4.8)-5.1 mm Perianth height: 4.9-(5.9)-6.6 mm One to three One to three One to three One to three locus, locus, one seed locus, one seed locus, one seed one seed each each each each Fruit diameter: Fruit/capsule Fruit diameter: 9- Fruit diameter: Fruit diameter: 7.7-(8.8)-10 cm 10 mm 7.8-(8.6)-9.5 mm 7.9-(9.9)-9.9 cm Pedicle length: Pedicle length: Pedicle length: Pedicle length: 1.3-(1.7)-2.1 cm 1.3-(1.90)-2.8 cm 0.5-(1.5)-2.2 cm 1-(1.2)-1.3 cm Rounded, black Rounded, black Rounded, black Rounded, black Seed Weight: Seeds Seed weight: 1.8- Seed Weight: 1.7- Seed Weight: 1.72-(1.85)-2 2.1 gram (1.9)-2.9 gram 1.7-(2)-3 gram gram Present, turning Absent Absent Anthocyanin Absent light or R: 155-(210)-254 R: 137-(189)-244 pigmentation R: 165-(175)- unpigmented in G: 151-(207)-253 G: 125-(179)-232 in bulb 193 the below ground B: 16-(179)-228 B: 62-(132)-182 114

R: 90-(188)-229 G: 152-(162)- G: 68-(164)-215 182 B: 70-(155)-200 B: 111-(120)- 144 Present, in the mature leaf Absent Absent Absent Anthocyanin occasionally R: 73-(124)-182 R: 72-(123)-242 R: 50-(98)-147 pigmentation mixed with green G:105-(153)-201 G: 111-(150)-235 G: 79-(125)-166 in midvein R: 11-(35)-77 B: 27-(76)-141 B: 29-(54)-146 B: 20-(35)-54 G: 60-(111)-156 B: 23-(63)-96 Present, fully Absent bract or at the tip Absent Absent R: 113-(156)- Anthocyanin only R: 141-(195)-239 R: 146-(189)-224 205 pigmentation R: 54-(111)-164 G: 90-(196)-236 G: 138-(185)-216 G: 133-(159)- in bract G: 21-(54)-95 B: 88-(154)-216 B:72-(121)-195 201 B: 31-(57)-108 B: 47-(87)-170

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Appendix 2: Morphological measurement data from 28 populations for four taxa of the

Allium tricoccum complex. Pop.: Population number; Taxon; group of taxon; NL:

Number of Leaf; BL: Bulb length (cm);BW: Bulb width (cm); DoB: Depth of bulb

Underground (cm); LL: Leaf length (cm); LW: Leaf wide (cm); PL: Petiole length (cm);

ScG: Scape growth orientation (degree); SH: Scape height (cm); SL: Stamen Length; PH:

Perianth height (cm); PdL: Pedicle length (cm); CD: Capsule Diameter (mm); SW: Seed

Weight (gram); LA: Leaf apex (degree); LB: Leaf base (degree); NB: Number of Bud;

MR: Midvein RGB (R value); MG: Midvein RGB (G Value); MB: Midvein RGB (B value); SR: Scape RGB (R value); SG: Scape RGB (G value); SB: Scape RGB (B value); BR: Bulb RGB (R value); BG: Bulb RGB (G value); BB: Bulb RGB (B value).

Pop. Taxon NL BL BW DoB LL LW PL ScG SH SL 4 A_tricoccum 3 4.36 2.58 0.08 19.21 4.22 1.81 104 27.8 8.87 4 A_tricoccum 3 5.06 2.45 0.1 17.28 4.68 2.08 134 10.47 8.5 4 A_tricoccum 2 4.98 2.06 0.1 20.83 4.75 1.72 141 18.91 9.48 4 A_tricoccum 3 4.18 2.71 0.1 16.53 5.77 1.58 125 27.27 8.93 4 A_tricoccum 3 4.86 2.34 0.1 19.44 4.44 2.08 139 10.47 8.54 12 A_tricoccum 2 5.12 2.1 0.92 22.88 8.82 2.45 150 18.31 10.61 12 A_tricoccum 2 4.75 2.51 1.96 22.38 7.72 1.49 152 25.22 10.39 12 A_tricoccum 2 3.74 2.22 0.21 19.56 6.77 1.81 76 27.27 9.24 12 A_tricoccum 2 4.23 1.68 0.29 19.82 6.82 1.76 152 23.91 9.51 12 A_tricoccum 3 3.5 1.62 0.61 21.32 8.25 1.98 144 22.52 8.68 15 A_tricoccum 3 5.02 2.6 0.25 21.11 7.09 2.46 139 25.36 9.08 15 A_tricoccum 3 4.18 2.35 0.51 16.56 7.35 2.08 76 25.92 10.59 15 A_tricoccum 2 4.1 1.9 0.7 16.83 6.42 1.76 98 23.91 9.75 15 A_tricoccum 2 4.4 2.35 2.71 17.79 5.51 2.31 123 11.93 9.55 15 A_tricoccum 3 4.37 3.07 1.32 20.98 7.76 2.9 122 22.46 9.65 16 A_tricoccum 3 3.69 1.77 0.1 15.45 5.01 1.74 154 19.96 8.17 16 A_tricoccum 2 3.07 1.49 0.1 13.7 4.73 1.82 152 10.47 7.85 16 A_tricoccum 2 3.77 1.71 0.1 17.56 5.49 1.78 135 21.6 7.38 16 A_tricoccum 3 4.87 1.62 0.1 21.44 7.19 2.1 161 17.86 7.67 16 A_tricoccum 3 2.69 1.62 0.1 12.76 5.38 1.55 123 18.24 7.74 116

Pop. Taxon NL BL BW DoB LL LW PL ScG SH SL

17 A_tricoccum 3 5.48 1.74 0.48 19.33 4.91 2.45 154 18.31 8.95 17 A_tricoccum 3 4.13 1.95 0.1 17.22 5.2 1.92 153 28.05 7.59 17 A_tricoccum 3 4.88 1.86 0.1 17.74 5.72 1.82 135 30.54 7.56 17 A_tricoccum 2 4.27 1.41 0.68 16.74 3.46 2.08 153 18.31 7.96 17 A_tricoccum 3 4.79 1.63 0.1 20.49 6.18 1.52 159 22.24 7.11

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Pop. Taxon PH PcL CD SW LA LB NB MR MG MB SR 4 A_tricoccum 6.4 1.7 9.7 1.97 40 69 70 134 96 73 90 4 A_tricoccum 5.08 2 9.54 2.01 53 65 41 68 42 23 74 4 A_tricoccum 4.79 1.61 9.82 1.89 46 91 51 93 54 40 94 4 A_tricoccum 5.53 1.48 10.64 2.05 50 92 77 105 98 53 79 4 A_tricoccum 5.02 1.53 8.97 2.05 61 81 52 84 45 39 54 12 A_tricoccum 6.37 2.33 9.78 1.99 59 69 31 129 120 80 98 12 A_tricoccum 6.78 1.32 9.95 2.09 70 70 32 132 111 76 157 12 A_tricoccum 7.05 2.09 9.37 2.05 66 59 77 111 108 73 96 12 A_tricoccum 6.18 1.48 9.19 2 68 67 16 128 128 72 140 12 A_tricoccum 6.14 2.69 9.6 1.86 58 73 36 146 127 88 93 15 A_tricoccum 6.42 2.69 9.83 2 49 75 51 145 160 92 152 15 A_tricoccum 6.92 2.25 10 2.04 77 87 54 100 105 86 108 15 A_tricoccum 6.01 1.83 9.84 1.91 61 55 30 127 148 63 125 15 A_tricoccum 5.62 2.46 10.1 2.02 55 53 17 122 127 70 161 15 A_tricoccum 5.69 2.86 9.5 2.1 55 88 48 114 128 82 137 16 A_tricoccum 5.78 1.79 9.49 1.92 49 63 25 110 125 65 122 16 A_tricoccum 5.03 1.75 9.46 1.91 55 74 31 105 128 54 108 16 A_tricoccum 5.36 2.15 9.53 1.96 59 58 22 123 147 73 164 16 A_tricoccum 5.55 1.53 10.39 2.01 55 59 20 156 166 96 135 16 A_tricoccum 6.73 1.53 9.67 1.9 62 72 22 143 148 80 134 17 A_tricoccum 6 1.82 9.56 2.05 53 41 11 76 78 40 80 17 A_tricoccum 5.82 1.77 9.28 1.99 59 46 16 91 89 51 91 17 A_tricoccum 6.37 1.48 9.19 1.88 53 38 18 90 88 35 82 17 A_tricoccum 6.6 1.53 9.23 2.09 60 56 18 60 68 32 87 17 A_tricoccum 5.19 1.53 8.98 1.98 57 54 11 85 71 29 109

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Pop. Taxon SG SB BR BG BB 4 A_tricoccum 33 43 215 182 162 4 A_tricoccum 33 38 184 163 120 4 A_tricoccum 45 49 184 163 120 4 A_tricoccum 31 35 90 68 70 4 A_tricoccum 21 34 156 128 125 12 A_tricoccum 39 41 180 115 115 12 A_tricoccum 76 85 181 147 144 12 A_tricoccum 46 39 173 134 110 12 A_tricoccum 60 55 198 174 168 12 A_tricoccum 33 33 124 115 197 15 A_tricoccum 90 89 220 193 192 15 A_tricoccum 93 108 176 160 167 15 A_tricoccum 67 82 217 197 189 15 A_tricoccum 94 105 201 184 157 15 A_tricoccum 95 89 205 166 161 16 A_tricoccum 51 53 188 173 157 16 A_tricoccum 46 46 212 194 186 16 A_tricoccum 61 64 229 215 200 16 A_tricoccum 66 64 186 168 160 16 A_tricoccum 68 67 214 195 184 17 A_tricoccum 36 38 175 147 124 17 A_tricoccum 39 37 229 215 200 17 A_tricoccum 30 31 186 168 160 17 A_tricoccum 41 43 214 195 184 17 A_tricoccum 49 50 175 147 124

119

Pop. Taxon NL BL BW DoB LL LW PL ScG SH 7 A_burdickii 2 4.51 1.09 4.83 21.03 2.65 1.48 180 18.31 7 A_burdickii 2 3.4 0.98 1.4 16.3 1.98 1.42 180 9.92 7 A_burdickii 2 3.94 1.26 3.31 17.56 1.96 1.76 180 12.73 7 A_burdickii 3 3.11 0.99 1.79 16.15 1.69 1.56 180 15.97 7 A_burdickii 2 3.45 1.19 5.13 18.67 1.63 1.46 180 11.4 8 A_burdickii 3 4.6 1.2 3.99 20.08 2.46 1.26 180 18.71 8 A_burdickii 2 2.78 1 2.38 16.7 4.27 1.46 180 18.29 8 A_burdickii 3 4.12 1.39 3.22 18.06 4.08 1.81 180 17.41 8 A_burdickii 2 4.69 0.86 3.49 18.18 2.58 1.46 180 14.99 8 A_burdickii 2 4.37 0.934 2.93 17.59 2.32 1.46 180 14.15 9 A_burdickii 3 3.58 0.97 1.93 22.49 2.7 1.28 180 12 9 A_burdickii 3 3.97 1.22 2.41 23.32 3.91 1.64 180 14.91 9 A_burdickii 3 3.96 1.43 1.67 21.65 3.75 1.66 180 14.99 9 A_burdickii 2 3.82 0.98 3.6 21.91 3.63 1.42 180 14.15 9 A_burdickii 3 2.99 1.18 1.59 19.65 3.44 2.05 180 13.58 10 A_burdickii 3 3.56 1.44 0.6 21.37 3.81 1.76 180 12.73 10 A_burdickii 3 4.04 1.59 0.89 20.78 4.12 1.26 180 26.52 10 A_burdickii 3 2.14 1.27 0.89 16.44 3.22 1.71 180 13.38 10 A_burdickii 3 2.62 1.55 0.73 20.28 3.67 2.09 180 15.83 10 A_burdickii 3 3.68 1.48 1.43 19.12 4.05 1.93 180 14.79 11 A_burdickii 2 3.95 1.59 1.81 22.91 3.35 1.88 180 18.48 11 A_burdickii 2 5.05 1.36 1.41 23.14 2.59 1.48 180 18.29 11 A_burdickii 2 4.78 1.27 0.72 20.59 3.08 1.82 180 18.71 11 A_burdickii 3 4.17 1.22 2.87 21.8 3.15 1.65 180 18.71 11 A_burdickii 2 4.4 1.17 2.83 18.26 1.66 1.7 180 26.52 13 A_burdickii 2 4.35 1.66 2.85 24.85 4 1.61 180 18.19 13 A_burdickii 2 3.48 1.34 1.21 18.49 2.92 1.53 180 12.73 13 A_burdickii 2 5.02 1.34 1.85 23.31 2.79 2.01 180 21.52 13 A_burdickii 2 4.02 1.46 1.46 22.96 2.98 1.32 180 16.23 13 A_burdickii 2 3.19 1.34 0.73 17.71 3.07 1.7 180 16.22 14 A_burdickii 3 4.32 1.63 3.49 22.44 4.17 1.7 180 13.96 14 A_burdickii 3 5.07 1.93 5.63 27.91 3.77 1.6 180 18.29 14 A_burdickii 2 4.69 1.67 7.28 26.24 3.25 1.44 180 13.16 14 A_burdickii 2 3.84 1.64 6.75 25.01 2.67 1.44 180 18.29 14 A_burdickii 2 4.97 2.01 4.46 23.45 3.73 1.55 180 16.91

120

Pop. Taxon SL PH PcL CD SW LA LB NB MR 7 A_burdickii 5.27 4.53 1.45 9.37 1.96 26.5 36.67 5 109 7 A_burdickii 4.66 4.72 1.36 9.2 1.89 27.91 25.96 5 160 7 A_burdickii 4.71 4.46 1.57 8.98 1.82 34.09 25.24 6 95 7 A_burdickii 5.15 4.49 1.6 8.95 1.81 25.8 19.38 5 128 7 A_burdickii 5.19 4.3 1.65 8.46 1.71 21.83 17.07 8 153 8 A_burdickii 6.1 4.98 1.75 9.68 1.89 28.37 39.9 1 117 8 A_burdickii 5.13 4.92 1.5 9.6 1.96 47.54 50.53 15 115 8 A_burdickii 6.07 4.98 1.36 10.13 1.99 42.9 34.05 8 182 8 A_burdickii 5.27 4.93 1.37 8.46 1.89 28.29 21 6 152 8 A_burdickii 5.06 4.97 1.87 8.79 1.96 24.86 28.32 5 126 9 A_burdickii 5.42 4.87 1.32 9.05 1.88 26.2 29.59 7 156 9 A_burdickii 5.9 5.42 1.65 8.94 1.82 30.2 32.82 13 109 9 A_burdickii 5.43 5.26 1.59 8.73 1.81 31.5 43.49 9 108 9 A_burdickii 5.28 5.15 1.37 8.46 1.82 30.93 33.81 6 139 9 A_burdickii 5.37 4.89 1.99 8.33 1.92 30 33.95 11 137 10 A_burdickii 5.46 5.41 1.73 9.23 1.92 37.77 32.69 7 137 10 A_burdickii 5.81 5.29 1.37 9.69 2.91 36.92 36.25 9 136 10 A_burdickii 4.88 5.48 1.77 9.69 1.91 37.46 36.54 8 105 10 A_burdickii 4.94 5.1 2.03 9.16 1.87 35.54 35.81 13 115 10 A_burdickii 4.93 5.8 1.86 9.63 1.85 40.67 37.27 11 98 11 A_burdickii 5.92 4.46 1.8 8.17 1.84 27.42 36.74 12 129 11 A_burdickii 5.63 4.99 1.78 8.36 1.81 22.5 25.13 6 154 11 A_burdickii 6.28 4.55 1.87 8.16 1.83 26.66 31.61 8 116 11 A_burdickii 5.57 5.14 1.75 8.04 1.88 35.28 31.22 13 179 11 A_burdickii 5.39 4.75 2.03 8.02 1.99 28.77 21.69 13 81 13 A_burdickii 5.8 4.79 1.69 8.93 1.99 41.64 33.35 9 123 13 A_burdickii 5.6 5.02 1.71 8.42 1.96 35.89 29.04 1 126 13 A_burdickii 5.65 4.97 2.14 8.75 1.95 38.76 27.5 13 129 13 A_burdickii 4.91 4.74 1.5 8.58 1.89 41.14 25.55 6 116 13 A_burdickii 5.8 5.06 1.75 7.67 1.81 38.93 33.4 8 120 14 A_burdickii 5.63 4.91 1.87 9 1.95 32.32 40.31 9 93 14 A_burdickii 5.27 4.87 1.65 8.9 1.82 29.03 24.5 16 117 14 A_burdickii 5.55 4.46 1.51 8.93 1.89 24.68 26.86 7 84 14 A_burdickii 5.41 4.69 1.77 8.18 1.92 23.34 26.19 13 127 14 A_burdickii 5.2 4.63 1.65 7.92 1.82 20.7 31.39 15 73

121

Pop. Taxon MG MB SR SG SB BR BG BB 7 A_burdickii 138 58 193 181 150 172 162 123 7 A_burdickii 181 104 175 176 114 172 162 123 7 A_burdickii 117 31 173 162 117 172 162 123 7 A_burdickii 151 76 207 195 156 175 160 135 7 A_burdickii 174 95 198 187 165 175 160 135 8 A_burdickii 144 99 216 218 206 172 205 181 8 A_burdickii 155 95 160 195 161 245 228 217 8 A_burdickii 201 95 226 220 192 200 198 182 8 A_burdickii 174 99 188 187 173 155 151 133 8 A_burdickii 145 141 168 166 117 155 151 133 9 A_burdickii 189 112 175 187 126 227 228 216 9 A_burdickii 139 71 160 170 122 228 231 228 9 A_burdickii 137 40 189 208 149 190 189 166 9 A_burdickii 176 99 205 217 171 207 207 191 9 A_burdickii 164 60 224 227 161 252 237 210 10 A_burdickii 162 73 185 189 100 254 253 206 10 A_burdickii 165 85 184 181 136 254 253 206 10 A_burdickii 130 55 156 168 116 187 188 16 10 A_burdickii 147 69 141 162 88 209 205 181 10 A_burdickii 130 52 189 206 139 209 205 181 11 A_burdickii 159 79 229 228 206 231 227 211 11 A_burdickii 167 109 181 178 149 185 222 155 11 A_burdickii 144 67 213 210 193 217 211 190 11 A_burdickii 198 132 225 225 207 226 224 210 11 A_burdickii 111 38 239 236 216 234 230 213 13 A_burdickii 157 84 222 221 197 213 219 198 13 A_burdickii 165 90 204 215 161 213 219 198 13 A_burdickii 166 91 211 218 169 215 219 199 13 A_burdickii 151 78 216 218 175 221 215 190 13 A_burdickii 155 80 219 222 183 226 222 204 14 A_burdickii 130 45 163 154 119 188 175 153 14 A_burdickii 142 43 212 206 163 241 227 203 14 A_burdickii 122 40 226 212 153 245 238 217 14 A_burdickii 155 66 220 215 162 245 238 217 14 A_burdickii 105 27 152 90 89 245 238 217

122

Pop. Taxon NL BL BW DoB LL LW PL ScG SH 1 SouthGreen 4 3.84 1.82 1.87 23.22 2.02 1.55 180 15.88 1 SouthGreen 3 3.57 2.26 1.68 20.89 4.04 2.27 180 14.34 1 SouthGreen 3 3.21 1.84 3.39 21.6 1.82 2.03 180 11.93 1 SouthGreen 3 2.14 1.03 7.39 15.89 1.96 2.03 180 15.77 1 SouthGreen 3 3.89 1.58 4.33 21.93 3.22 2.27 180 12.9 2 SouthGreen 3 3.39 1.99 1.13 21.51 2.45 1.56 180 14.83 2 SouthGreen 2 3.44 1.07 2 19.3 2.22 2.14 180 11.93 2 SouthGreen 3 3.52 1.7 1.13 19.6 2.12 1.7 180 14.34 2 SouthGreen 3 3.47 1.49 2.1 15.04 2.07 2.03 180 14.66 2 SouthGreen 3 3.94 1.34 4.6 22.1 2.07 1.39 180 12.9 3 SouthGreen 3 4.29 1.32 1.01 20.2 1.87 2.27 180 14.66 3 SouthGreen 3 4.3 1.84 1.74 22.87 3.85 1.99 180 14.66 3 SouthGreen 2 3.91 1.5 2.13 23.53 3.98 1.74 180 15.82 3 SouthGreen 2 3.29 1.57 1.21 22.13 3.95 1.74 180 15.27 3 SouthGreen 2 4.75 1.59 3.28 25.64 3.92 1.79 180 19.32 5 SouthGreen 2 3.53 1.26 3.36 21.75 2.87 2.27 180 19.32 5 SouthGreen 3 5.15 1.91 1.46 21.6 3.83 1.42 180 15.06 5 SouthGreen 3 3.63 1.32 4 22.4 2.52 1.45 180 15.03 5 SouthGreen 3 5 1.4 2.04 22.48 3.46 2.03 180 13.67 5 SouthGreen 3 4.65 1.68 3.28 21.32 3.37 1.59 180 11.93 6 SouthGreen 2 3.5 1.44 0.8 17.2 2.26 1.99 180 12.9 6 SouthGreen 2 3.86 1.68 3.19 23.84 2.43 1.45 180 14.83 6 SouthGreen 2 4.97 1.2 2.15 24.5 2.43 1.46 180 21.04 6 SouthGreen 2 6.14 1.33 3.73 23.07 2.14 1.46 180 15.88 6 SouthGreen 3 4.1 0.95 1.89 17.35 1.71 2.14 180 14.83 20 SouthGreen 2 4.11 1.65 3.19 24.13 4.03 2.14 180 20.55 20 SouthGreen 2 3.74 1.35 2.15 25.5 2.95 1.99 180 21.04 20 SouthGreen 2 4.08 1.12 3.49 19.97 2.67 1.46 180 16.58 20 SouthGreen 2 2.76 1.32 1.88 16.71 2.52 1.71 180 15.77 20 SouthGreen 2 3.34 1.15 2.69 22.36 2.66 1.46 180 19.32

123

Pop. Taxon SL PH PcL CD SW LA LB NB MR 1 SouthGreen 5.36 4.53 1.01 8.4 1.74 19.47 15.55 15 115 1 SouthGreen 4.98 4.74 1.85 8.68 1.71 30.28 36.08 15 151 1 SouthGreen 5.24 4.08 1.32 7.93 2.97 18.79 17.89 14 242 1 SouthGreen 6.22 4.85 1.32 8.29 2.65 31.27 19.75 15 163 1 SouthGreen 5.27 4.45 1.93 8.45 1.87 24.34 29.49 14 102 2 SouthGreen 6.06 4.78 1.4 8.69 1.68 20.68 18.63 14 171 2 SouthGreen 5.66 4.68 1.6 9.16 1.71 21.52 20.16 15 151 2 SouthGreen 5.9 4.2 1.4 8.11 2.71 23.61 27.73 16 123 2 SouthGreen 4.28 4.15 1.01 8.27 1.7 19.45 24.91 7 123 2 SouthGreen 5.23 3.82 0.624 8.34 1.7 16.5 12.34 9 139 3 SouthGreen 5.31 4.57 2.23 9.51 1.73 24.59 31.4 15 88 3 SouthGreen 5.52 4.82 1.73 9.11 1.73 27.02 37.27 12 93 3 SouthGreen 5.16 5.1 1.63 8.87 2.97 25.18 30.72 13 118 3 SouthGreen 5.05 4.6 1.79 7.86 1.87 28.12 33.4 14 107 3 SouthGreen 4.29 4.66 1.4 7.93 1.78 25.58 32.63 18 145 5 SouthGreen 4.74 4.73 1.34 8.83 2.65 22.07 23.76 18 143 5 SouthGreen 4.4 4.12 1.43 8.14 1.92 29.28 34.91 9 124 5 SouthGreen 4.2 4.22 1.908 8.39 1.71 25.81 21.25 10 114 5 SouthGreen 4.37 4.52 0.624 8.94 1.8 23.46 33.53 10 114 5 SouthGreen 4.26 5.13 1.34 8.63 1.84 25.9 28.76 8 133 6 SouthGreen 4.98 5.13 1.34 9.51 2.97 25.51 29.64 8 72 6 SouthGreen 4.93 4.82 0.624 8.94 1.7 33.74 33.77 14 114 6 SouthGreen 4.8 5.13 1.34 9.21 1.68 22.5 20.5 15 136 6 SouthGreen 4.89 4.85 1.01 9.21 1.68 24.08 23.06 8 108 6 SouthGreen 5.35 4.08 1.01 7.93 1.92 23.43 30.5 10 100 20 SouthGreen 6.65 4.43 1.93 9.21 1.81 25.08 27.36 14 91 20 SouthGreen 5.72 3.32 1.85 8.78 2.65 20.81 28.77 13 115 20 SouthGreen 5.75 4.19 1.32 8.9 1.73 23.21 20.31 8 99 20 SouthGreen 5.42 4.76 1.52 8.69 1.75 28.75 25.8 9 93 20 SouthGreen 5.11 4.3 1.67 8.74 2.71 22.62 25.73 18 103

124

Pop. Taxon MG MB SR SG SB BR BG BB 1 SouthGreen 145 38 192 189 139 208 192 148 1 SouthGreen 164 56 202 197 112 228 213 172 1 SouthGreen 235 146 209 184 148 208 196 146 1 SouthGreen 173 84 222 215 195 228 213 172 1 SouthGreen 136 46 201 198 136 208 196 146 2 SouthGreen 188 78 209 205 134 189 172 129 2 SouthGreen 179 67 224 216 148 216 197 151 2 SouthGreen 148 55 222 208 156 156 142 116 2 SouthGreen 153 52 187 172 89 203 204 164 2 SouthGreen 161 53 189 183 116 137 125 100 3 SouthGreen 118 45 169 177 94 178 162 127 3 SouthGreen 124 44 169 178 77 244 232 182 3 SouthGreen 148 56 179 184 92 220 212 165 3 SouthGreen 129 56 178 178 72 213 199 151 3 SouthGreen 166 69 171 164 106 217 208 173 5 SouthGreen 168 57 215 214 164 156 128 125 5 SouthGreen 154 72 183 180 123 172 162 123 5 SouthGreen 146 36 197 190 133 172 162 123 5 SouthGreen 144 49 198 192 148 172 162 123 5 SouthGreen 162 67 200 197 136 172 162 123 6 SouthGreen 111 31 160 167 75 172 162 123 6 SouthGreen 148 50 205 210 110 172 162 123 6 SouthGreen 164 54 212 209 164 172 162 123 6 SouthGreen 139 37 146 138 95 172 162 123 6 SouthGreen 132 41 164 157 105 172 162 123 20 SouthGreen 122 33 153 151 76 198 187 149 20 SouthGreen 146 43 169 166 98 160 172 62 20 SouthGreen 125 41 179 168 131 187 187 97 20 SouthGreen 125 29 178 169 98 176 179 86 20 SouthGreen 135 37 189 184 150 187 187 97

125

Pop . Taxon NL BL BW DoB LL LW PL ScG SH 18 Highland Green 2 4.42 1.69 1.13 16.28 4.9 1.56 180 22.2 18 Highland Green 2 3.49 1.38 2.58 18.61 3.56 1.21 180 19.53 18 Highland Green 3 3.75 1.61 1.28 15.75 3.9 1.6 180 23.59 18 Highland Green 2 3.6 1.63 2.13 16.7 4.34 1.56 180 19.53 18 Highland Green 2 4.21 1.47 2.08 16.89 4.24 1.26 180 23.38 19 Highland Green 2 2.96 1.39 1.41 15.04 4.78 1.26 180 22.2 19 Highland Green 2 3.69 1.56 1.68 15.4 5.24 1.26 180 23.38 19 Highland Green 2 3.27 1.36 2.18 17.15 4.5 1.3 180 24.8 19 Highland Green 2 3.96 1.43 0.84 21.24 3.71 1.56 180 19.01 19 Highland Green 3 3.67 1.72 1.64 21.75 4.47 2.18 180 19.01

Pop . Taxon SL PH PcL CD SW LA LB NB MR 18 Highland Green 7.48 6.25 1.27 9.58 1.87 42 51 10 50 18 Highland Green 7.51 6.59 1.17 9.71 1.76 28 46 15 147 18 Highland Green 7.31 6.32 1.29 8.12 1.8 48 42 19 81 18 Highland Green 7.47 5.45 1.13 7.89 1.72 32 44 8 124 18 Highland Green 6.77 5.91 1.34 7.97 1.76 38 40 16 71 19 Highland Green 6.76 6.22 1.12 9.93 2 50 50 8 139 19 Highland Green 6.83 6.38 1.12 9.27 1.72 47 60 8 60 19 Highland Green 7.3 5.27 1.02 9.17 1.91 50 54 7 121 19 Highland Green 6.9 5.73 1.11 8.27 1.91 33 39 16 92 19 Highland Green 5.98 4.94 0.99 8.27 2 32 34 27 98

Pop. Taxon MG MB SR SG SB BR BG BB 18 Highland Green 79 22 128 136 59 186 171 124 18 Highland Green 166 52 195 192 126 186 171 124 18 Highland Green 107 20 134 133 59 170 155 111 18 Highland Green 152 41 171 175 82 170 155 111 18 Highland Green 98 31 113 133 47 186 171 124 19 Highland Green 157 54 205 201 170 193 182 144 19 Highland Green 96 23 141 135 63 165 154 117 19 Highland Green 144 44 203 198 157 165 154 117 19 Highland Green 122 32 143 153 55 166 152 119 19 Highland Green 133 29 126 136 48 168 152 114

126

Appendix 3: Phenological data from 20 populations of four taxa of the Allium tricoccum complex.

Date Alliun tricoccum Allium burdickii South Green Highland Green

24-Feb Shoot Emerge Shoot emerge Shoot emerge, Leaves Shoot not present already emerge from yet the shoot bract

28-Feb Leaves emerge from Shoot emerge Leaves fully shoot bract expanded

2-Mar Leaves fully Leaves fully expanded expanded

5-Mar Shoot emerge, Leaves not emerge from shoot bract until March 15

9-Mar Leaves emerge from shoot bract

11-Mar Leaves fully expanded

15-Mar Leaves Emerge from shoot bract, leaves not fully expanded until April 2

2-Apr Leaves fully expanded 127

28-Apr Scape emerge, Scape not emerge Scape emerge, Scape Emerge, Leaves not turning yet until May 5-8 Leaves not turning Leaves not yellow yellow turning yellow

5-May Scape Emerge, Leaves not turning yellow

8-May Leaves turning yellow

15-May Leaves turning Leaves fully Leaves turning Leaves turning yellow, scape start to senesces, but not yellow yellow have curved pattern disintegrated from plants

20-May Leaves fully Leaves fully degraded senesces, but not disintegrated from plants

25-May Leaves fully Bract broken and Leaves fully senesces, but not flower bud emerge senesces, but not disintegrated from disintegrated plants. Scape have from plants full curved pattern, some plants have scape like a hook.

4-Jun Bract broken and Bract broken and flower bud emerge Flower anthesis flower bud emerge

11-Jun Flower anthesis Fruit developed, Flower anthesis some fruits come in 128

the same time flower fully anthesis

14-Jun Scape start to reduce Fruit developed curved pattern and back to erect pattern

18-Jun Fruit developed

25-Jun Bract broken and flower bud emerge, the scape back in the erect mode.

8-Jul Flower Anthesis

16-Jul Fruit developed

31-Jul Fruit capsule dehisced, seed exposed

4-Aug Fruit capsule dehisced, seed exposed

10-Aug Fruit capsule dehisced, seed exposed

25-Aug All fruit capsule All fruit capsule fully All fruit capsule fully dehisced, dehisced, seed fully dehisced, seed collected collected seed collected 129

5-Sep Fruit capsule dehisced, seed exposed

25-Sep All fruit capsule fully dehisced, seed collected

130

Appendix 4: Environmental data from 28 populations of four taxa of the Allium tricoccum complex.

Pop. Taxon Sand Clay Silt Ph Moisture Altitude 1 South Green 22.69 13.15 64.17 5.99 74.68 300 1 South Green 35.19 13.15 51.67 7.68 67.91 300 1 South Green 30.19 13.15 56.67 5.74 77.48 300 2 South Green 20.81 13.15 66.04 5.66 79.32 300 2 South Green 25.19 5.65 69.17 6.58 74.37 300 2 South Green 37.06 8.15 54.79 6.59 60.24 300 3 South Green 30.19 16.9 52.92 7.27 73 300 3 South Green 22.69 15.65 61.67 7.33 75.25 265 3 South Green 39.56 16.9 43.54 7.31 71.34 265 4 A. tricoccum 40.19 10.65 49.17 7.07 64.84 265 4 A. tricoccum 30.19 8.15 61.67 6.11 71.75 265 4 A. tricoccum 19.56 14.4 66.04 6.67 66.99 265 5 South Green 51.44 14.4 34.17 6.93 59.85 265 5 South Green 20.19 21.9 57.92 6.88 70.41 248 5 South Green 27.69 15.65 56.67 6 70.81 248 6 South Green 35.19 8.15 56.67 7.05 68.7 248 6 South Green 26.44 15.65 57.92 7.01 73.72 248 6 South Green 27.06 16.9 56.04 6.85 74.98 248 7 A. burdickii 70.19 5.65 24.17 6.18 64.2 291 7 A. burdickii 67.69 5.65 26.67 5.65 74.84 291 7 A. burdickii 89.56 3.15 7.29 6.45 80.1 291 8 A. burdickii 89.56 3.15 7.29 6.03 75 282 8 A. burdickii 76.44 0.65 22.92 5.83 77.78 282 8 A. burdickii 84.56 0.65 14.79 7.32 74.95 282 9 A. burdickii 58.31 6.9 34.79 6.47 79.76 271 9 A. burdickii 62.69 5.65 31.67 7.27 75.65 271 9 A. burdickii 66.44 8.15 25.42 7.04 66.62 271 10 A. burdickii 55.19 5.65 39.17 6.38 74.02 271 10 A. burdickii 37.69 6.9 55.42 7.06 71.81 271 10 A. burdickii 30.81 8.15 61.04 6.33 71.49 271 11 A. burdickii 87.69 5.65 6.67 6.77 73.07 220 11 A. burdickii 90.19 4.4 5.42 7.53 66.39 220 131

11 A. burdickii 90.19 5.65 4.17 7.25 74.36 220 12 A. tricoccum 85.19 4.4 10.42 5.6 77.82 191 12 A. tricoccum 82.06 3.15 14.79 5.7 77.63 191 12 A. tricoccum 66.44 3.15 30.42 6.49 61.71 191 13 A. burdickii 83.94 4.4 11.67 5.62 83.4 191 13 A. burdickii 71.44 1.9 26.67 5.31 81.5 191 13 A. burdickii 83.94 3.15 12.92 5.43 81.53 191 14 A. burdickii 31.44 5.65 62.92 6.75 68.84 295 14 A. burdickii 26.44 8.15 65.42 6.29 69.1 295 14 A. burdickii 27.69 9.4 62.92 6.19 73.4 295 15 A. tricoccum 28.31 6.9 64.79 6.29 68.29 295 15 A. tricoccum 34.56 9.4 56.04 6.6 70.48 295 15 A. tricoccum 31.44 4.4 64.17 6.07 73.68 295 16 A. tricoccum 65.19 3.15 31.67 6.57 71.66 303 16 A. tricoccum 43.94 13.15 42.92 7.54 62.9 303 16 A. tricoccum 68.94 6.9 24.17 7.58 73.7 303 17 A. tricoccum 72.69 5.65 21.67 4.48 64.21 1112 17 A. tricoccum 63.94 14.4 21.67 4.65 61.49 1112 17 A. tricoccum 70.81 4.4 24.79 4.92 57.6 1112 18 Highland Green 53.31 6.9 39.79 5.31 55.18 1587 18 Highland Green 65.81 10.65 23.54 4.89 52.41 1587 18 Highland Green 69.56 8.15 22.29 5.33 43.76 1587 19 Highland Green 42.69 15.65 41.67 5.61 61.96 896 19 Highland Green 75.81 5.65 18.54 6.24 58.47 896 19 Highland Green 53.94 8.15 37.92 5.95 57.73 896 20 South Green 36.44 8.15 55.42 5.81 72.1 452 20 South Green 38.31 8.15 53.54 6.45 74.23 452 20 South Green 36.44 10.65 52.92 6.47 70.78 452 21 South Green 42.69 0.65 56.67 6.33 86.44 316 21 South Green 40.81 3.15 56.04 6.24 69.67 316 21 South Green 49.56 4.4 46.04 6.44 72.6 316 22 South Green 27.69 8.15 64.17 6.71 72.99 316 22 South Green 32.06 8.15 59.79 6.9 82.22 316 22 South Green 38.94 5.65 55.42 6.59 77.47 316 23 South Green 34.56 9.4 56.04 6.66 82.28 247 23 South Green 45.19 4.4 50.42 6.68 77.8 247 23 South Green 33.31 10.65 56.04 6.78 82.4 247 132

24 South Green 47.06 5.65 47.29 6.77 82.48 247 24 South Green 53.94 6.9 39.17 6.2 74.65 247 24 South Green 47.06 10.65 42.29 7.02 71.99 247 25 A. tricoccum 32.69 8.15 59.17 6.63 80.36 256 25 A. tricoccum 35.81 8.15 56.04 6.24 75.57 256 25 A. tricoccum 35.19 10.65 54.17 6.65 78.98 256 26 A. burdickii 40.19 5.65 54.17 5.93 79.77 256 26 A. burdickii 42.06 5.65 52.29 5.55 87.08 256 26 A. burdickii 33.94 5.65 60.42 6.53 80.01 256 27 A. burdickii 53.31 3.15 43.54 6.7 87.42 275 27 A. burdickii 51.44 5.65 42.92 5.53 91.1 275 27 A. burdickii 47.69 5.65 46.67 7.08 82.47 275 28 A. tricoccum 38.31 10.65 51.04 5.36 87.42 226 28 A. tricoccum 65.19 5.65 29.17 5.88 91.1 226 28 A. tricoccum 45.19 8.15 46.67 5.58 82.47 226

133

Appendix 5: Presence/absence (0/1) data matrix of alleles amplified for twelve microsatellite loci in four taxa of the Allium tricoccum complex.

Locus: G011 (Allele size below) 10 10 10 11 11 11 11 12 12 12 13 14 14 # Sample 98 1 4 7 0 3 6 9 2 5 8 1 0 3 1 1A 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2 1B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 1C 0 1 0 0 0 0 0 0 0 0 0 0 0 0 4 1D 0 1 0 0 0 0 1 0 0 0 0 0 0 0 5 1E 1 0 0 0 0 0 1 0 0 0 0 0 0 0 6 1F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 1G 1 0 0 0 1 0 0 0 0 0 0 0 0 0 8 1H 0 1 1 0 0 1 0 0 0 0 0 0 0 0 9 1I 0 0 0 1 0 0 0 0 0 0 0 0 0 0 10 1J 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11 2A 1 0 1 0 0 0 0 0 0 0 0 0 0 0 12 2B 1 1 0 0 0 0 0 1 0 0 0 0 0 0 13 2C 0 0 1 0 0 0 1 0 1 0 0 0 0 0 14 2D 1 1 0 0 0 0 1 0 0 0 0 0 0 0 15 2E 0 0 1 0 0 0 0 1 1 0 0 0 0 0 16 2F 1 0 0 0 0 0 0 1 0 0 0 0 0 0 17 2G 0 0 1 0 0 0 0 0 1 0 0 0 0 1 18 2H 1 0 1 0 0 0 0 0 1 0 0 0 0 0 19 2I 0 1 0 0 0 0 1 0 0 0 0 0 0 0 20 2J 0 1 0 0 0 0 1 0 0 0 0 0 0 0 21 3A 0 0 0 0 1 0 0 0 1 0 1 0 0 0 22 3B 0 1 1 0 0 0 0 0 0 0 0 0 0 0 23 3C 1 0 0 0 1 0 0 0 1 0 0 1 0 0 24 3D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 25 3E 0 0 0 1 0 0 0 0 1 1 0 0 0 0 26 3F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 27 3G 0 0 0 1 0 0 0 0 0 0 0 0 1 1 28 3H 0 0 0 1 0 0 0 0 0 0 0 0 1 1 29 3I ? ? ? ? ? ? ? ? ? ? ? ? ? ? 30 3J ? ? ? ? ? ? ? ? ? ? ? ? ? ? 31 4A 0 0 1 0 0 0 0 1 0 0 0 0 0 0 32 4B 0 1 0 0 0 0 0 0 1 0 1 0 1 0 33 4C 0 1 1 0 0 0 0 0 1 0 0 0 1 0 34 4D 0 1 1 0 0 0 0 0 1 0 0 0 1 0 35 4E 0 1 0 0 0 0 0 0 1 0 0 0 0 0 36 4F 0 0 1 0 0 0 0 0 1 0 0 0 0 0 37 4G 0 0 1 1 0 0 0 0 1 0 0 0 1 0 134

14 15 16 17 17 17 17 19 19 20 20 20 21 27 28 29 29 # Sample 6 5 7 0 3 6 9 4 7 0 3 6 2 8 4 0 3 1 1A 0 0 0 1 1 0 0 0 0 0 0 1 0 0 1 0 0 2 1B 0 0 0 1 0 0 0 0 0 0 1 0 1 1 0 0 0 3 1C 0 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 4 1D 0 0 1 1 0 1 1 0 0 1 0 0 0 0 1 0 0 5 1E 0 0 0 1 0 0 0 0 0 1 0 0 0 0 1 0 0 6 1F 0 1 0 0 1 1 0 0 0 0 0 1 0 1 0 0 1 7 1G 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 8 1H 0 1 0 1 0 1 1 0 0 0 0 1 0 0 0 1 0 9 1I 0 0 0 0 1 0 0 0 0 0 0 1 0 0 1 0 0 10 1J 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 11 2A 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 12 2B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 13 2C 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 2D 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 15 2E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 16 2F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 17 2G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 18 2H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 19 2I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20 2J 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 21 3A 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 22 3B 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 1 23 3C 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 24 3D 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 25 3E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 26 3F 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 27 3G 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 28 3H 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 29 3I ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 30 3J ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 31 4A 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 32 4B 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 33 4C 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 34 4D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 35 4E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 36 4F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 37 4G 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0

135

G041 32 32 33 33 33 10 10 10 11 11 11 11 12 13 13 14 # Sample 98 6 9 2 5 8 1 4 7 0 3 6 9 5 1 4 0

1 1A 0 0 0 1 0 0 0 0 0 1 0 0 1 0 0 1 0 2 1B 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 1 0 3 1C 1 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 4 1D 1 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 5 1E 1 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 6 1F 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 7 1G 0 0 0 0 0 1 0 1 0 0 1 1 0 1 1 0 1 8 1H 0 1 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 9 1I 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 10 1J 0 0 0 1 0 ? ? ? ? ? ? ? ? ? ? ? ? 11 2A 0 0 0 0 0 1 1 0 1 0 0 0 0 1 0 0 0 12 2B 0 0 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 13 2C 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 14 2D 0 0 0 0 0 1 1 0 1 0 0 0 1 0 1 0 0 15 2E 0 0 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 16 2F 0 0 0 0 0 0 1 0 1 0 0 0 0 0 1 0 0 17 2G 0 0 0 0 0 0 1 0 1 0 1 0 0 1 0 1 0 18 2H 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 19 2I 0 0 0 0 0 0 1 0 1 0 0 0 1 1 0 0 0 20 2J 0 0 0 0 0 1 0 0 0 0 0 0 1 1 0 0 0 21 3A 0 0 0 0 0 0 1 0 1 0 1 0 0 1 1 0 0 22 3B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23 3C 0 0 0 0 0 0 1 0 1 0 1 0 1 0 1 0 0 24 3D 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 25 3E 0 0 0 0 0 1 0 1 0 1 0 1 0 0 0 0 0 26 3F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 27 3G 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 28 3H 0 0 1 0 0 1 0 1 0 0 0 0 0 1 0 0 0 29 3I ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 30 3J ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 31 4A 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 32 4B 0 0 0 0 0 0 1 1 0 0 0 0 0 1 0 0 0 33 4C 0 0 0 0 0 0 1 1 0 1 0 0 0 1 0 0 0 34 4D 0 0 0 0 0 0 1 1 0 0 0 0 0 1 1 0 0 35 4E 0 0 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 36 4F 0 0 0 0 0 0 1 1 0 1 0 0 0 1 0 0 0 37 4G 0 0 0 0 0 0 1 1 1 0 0 0 0 1 1 0 0

136

G028 14 15 16 16 17 17 21 23 23 27 39 10 10 10 10 11 11 # Sample 6 5 4 7 0 6 2 0 3 5 8 0 4 6 8 0 2

1 1A 0 0 0 0 1 0 0 1 0 0 1 0 0 0 0 0 0 2 1B 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 3 1C 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 4 1D 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 1E 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 1F 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 7 1G 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 1H 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 9 1I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 1J ? ? ? ? ? ? ? ? ? ? ? 0 0 0 0 0 0 11 2A 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 12 2B 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 13 2C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 2D 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 15 2E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 16 2F 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 17 2G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 18 2H 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 19 2I 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 20 2J 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 21 3A 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 22 3B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23 3C 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 24 3D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 25 3E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 26 3F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 27 3G 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 28 3H 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 29 3I ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 30 3J ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 31 4A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 32 4B 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 33 4C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 34 4D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 35 4E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 36 4F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 37 4G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

137

11 12 12 13 13 13 13 13 14 14 17 19 20 20 21 28 28 # Sample 6 6 8 0 2 4 6 8 0 4 6 8 6 8 0 2 6

1 1A 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 2 1B 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 3 1C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 4 1D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 5 1E 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 6 1F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 7 1G 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 8 1H 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 9 1I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 1J 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 11 2A 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 12 2B 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 13 2C 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 14 2D 0 0 0 0 0 0 0 1 0 1 0 0 0 1 0 0 0 15 2E 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 16 2F 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 17 2G 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 18 2H 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 19 2I 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 20 2J 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 21 3A 1 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 22 3B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23 3C 1 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 24 3D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 25 3E 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 26 3F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 27 3G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28 3H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 29 3I ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 30 3J ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 31 4A 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 32 4B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 33 4C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 34 4D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 35 4E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 36 4F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 37 4G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

138

G027 29 51 51 51 52 52 53 54 10 10 10 11 16 17 17 17 24 # Sample 0 2 4 6 2 4 0 0 1 4 7 3 7 0 3 6 2

1 1A 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 2 1B 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 3 1C 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 1D 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 5 1E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 1F 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 7 1G 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 8 1H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9 1I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 10 1J 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11 2A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 12 2B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 13 2C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 2D 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 15 2E 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 16 2F 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 17 2G 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 18 2H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 19 2I 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 20 2J 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 21 3A 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 22 3B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23 3C 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 24 3D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 25 3E 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 26 3F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 27 3G 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 28 3H 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 29 3I ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 30 3J ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 31 4A 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 32 4B 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 33 4C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 34 4D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 35 4E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 36 4F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 37 4G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

139

G036 26 26 27 28 32 33 33 33 10 10 10 10 11 11 11 11 # Sample 3 9 2 7 9 2 5 8 0 2 4 8 0 2 6 8

1 1A 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 2 1B 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 3 1C 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 4 1D 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 5 1E 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 6 1F 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 7 1G 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 8 1H 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 9 1I 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 10 1J 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 11 2A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 12 2B 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 13 2C 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 14 2D 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 15 2E 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 16 2F 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 17 2G 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 18 2H 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 19 2I 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 20 2J 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 21 3A 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 22 3B 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 23 3C 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 24 3D 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 25 3E 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 26 3F 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 27 3G 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 28 3H 0 0 0 1 0 0 0 0 ? ? ? ? ? ? ? ? 29 3I ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 30 3J ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 31 4A 0 0 0 0 0 0 0 0 1 0 1 1 1 1 0 0 32 4B 0 0 0 0 0 0 0 0 1 0 1 1 1 0 0 0 33 4C 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 34 4D 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 35 4E 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 36 4F 0 0 0 0 0 0 0 1 0 0 0 0 1 0 1 0 37 4G 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0

140

12 12 12 12 13 13 15 15 16 16 17 17 17 24 25 25 25 # Sample 0 2 4 6 2 4 0 4 2 8 0 6 8 6 0 6 8

1 1A 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 2 1B 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 1C 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 4 1D 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 1E 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 6 1F 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 1G 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 8 1H 0 0 1 1 0 0 0 0 0 0 1 1 0 0 0 0 0 9 1I 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 10 1J 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11 2A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 12 2B 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 13 2C 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 14 2D 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 15 2E 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 16 2F 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 17 2G 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 18 2H 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 19 2I 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 20 2J 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 21 3A 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 22 3B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23 3C 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 24 3D 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 25 3E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 26 3F 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 27 3G 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 28 3H ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 29 3I ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 30 3J ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 31 4A 1 0 1 0 0 0 1 1 1 1 0 0 1 0 0 0 0 32 4B 0 0 1 0 0 1 1 0 1 1 0 0 1 0 0 0 0 33 4C 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 34 4D 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 35 4E 0 1 0 0 1 0 0 1 0 1 0 0 0 0 0 0 1 36 4F 0 0 0 0 0 1 0 1 1 0 1 0 0 0 0 0 0 37 4G 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0

141

G089 26 26 28 28 32 32 33 33 34 36 37 40 10 10 10 11 # Sample 98 0 2 0 2 2 8 6 8 0 0 8 6 1 4 7 0

1 1A 0 0 0 0 0 0 1 0 1 0 0 0 1 1 0 1 0 2 1B 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 3 1C 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 1 0 4 1D 1 0 0 0 0 0 1 0 1 0 0 0 0 1 1 0 0 5 1E 0 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 0 6 1F 1 0 0 0 0 0 0 0 1 0 0 0 0 1 1 1 0 7 1G 0 0 0 0 0 0 1 0 0 0 0 0 1 1 0 0 0 8 1H 1 0 0 0 0 0 0 1 1 0 0 0 0 0 1 1 0 9 1I 0 0 0 0 0 0 1 0 1 0 0 0 1 1 0 1 0 10 1J 1 0 0 0 0 0 0 0 1 0 0 0 0 1 1 0 1 11 2A 0 0 0 0 0 0 1 0 1 0 0 0 0 1 0 0 0 12 2B 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 13 2C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 2D 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 15 2E 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 16 2F 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 17 2G 0 0 0 0 0 0 0 1 1 0 0 0 0 1 1 0 0 18 2H 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 19 2I 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 20 2J 0 0 0 0 0 0 1 0 0 0 0 0 1 1 0 1 0 21 3A 0 0 0 0 0 0 1 0 0 0 0 0 1 1 0 1 0 22 3B 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 23 3C 0 0 0 0 0 0 1 0 1 0 0 0 0 1 0 1 0 24 3D 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 0 0 25 3E 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 26 3F 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 0 1 27 3G 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 1 28 3H ? ? ? ? ? ? ? ? ? ? ? ? 0 1 1 0 1 29 3I ? ? ? ? ? ? ? ? ? ? ? ? 0 1 1 0 0 30 3J ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 31 4A 0 0 0 1 0 0 0 0 0 0 1 0 0 1 0 0 1 32 4B 0 0 1 0 0 0 0 0 0 0 1 0 1 0 0 1 0 33 4C 0 0 0 0 0 0 0 0 0 1 0 0 1 0 1 1 1 34 4D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 35 4E 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 36 4F 0 1 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 37 4G 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0

142

G088 11 11 11 12 12 13 13 14 14 15 15 16 16 17 17 10 10 # Sample 3 6 9 2 5 1 7 3 9 2 5 1 4 0 9 0 2

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143

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Locus: G011 (Allele size below) 10 10 10 11 11 11 11 12 12 12 13 13 13 14 14 # Sample 98 1 4 7 0 3 6 9 2 5 8 1 4 7 0 3 76 8F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 77 8G 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 78 8H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 79 8I 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 80 8J 0 0 1 1 0 0 0 0 1 0 0 0 0 0 0 0 81 9A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 82 9B 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 83 9C 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 84 9D 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 85 9E 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 86 9F 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 87 9G 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 88 9H 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 89 9I 0 0 1 0 1 0 0 0 0 0 1 0 0 0 0 0 90 9J 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 91 10A 0 0 1 0 0 0 0 1 0 0 0 0 0 1 0 0 92 10B 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 93 10C 0 1 0 0 0 0 1 0 1 0 0 0 1 0 0 0 94 10D 0 1 1 0 0 0 1 0 0 0 0 1 0 0 0 0 95 10E 1 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 96 10F 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 97 10G 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 98 10H 1 1 0 0 0 0 1 0 0 0 0 0 1 0 1 0 99 10I 0 0 0 0 0 1 0 0 1 1 0 1 0 0 0 1 100 10J 1 0 0 0 0 0 1 1 0 0 0 0 1 0 0 0 101 11A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 102 11B 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 103 11C 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 104 11D 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 105 11E 0 0 1 0 0 0 0 0 1 0 1 0 0 1 1 0 106 11F 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 107 11G 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 108 11H 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 109 11I 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 110 11J 0 1 0 0 0 0 1 0 0 0 0 0 0 1 0 0 111 12A 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 112 12B 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 113 12C 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 114 12D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

180

14 15 16 17 17 17 17 19 19 20 20 20 21 27 27 28 29 # Sample 6 5 7 0 3 6 9 4 7 0 3 6 2 5 8 4 0

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181

G0 41 32 32 33 33 33 10 10 10 11 11 11 11 12 12 13 13 # Sample 98 6 9 2 5 8 1 4 7 0 3 6 9 2 5 1 4 76 8F 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 77 8G 0 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? 78 8H 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? 79 8I 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? 80 8J 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? 81 9A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 82 9B 0 0 0 0 0 0 1 0 1 0 0 0 1 0 1 0 0 83 9C 0 0 0 0 0 0 1 0 0 0 1 0 0 0 1 0 0 84 9D 0 0 0 0 0 0 1 0 0 0 1 0 0 0 1 0 0 85 9E 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 86 9F 0 0 0 0 0 0 0 0 1 1 0 1 1 0 1 0 0 87 9G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 88 9H 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 89 9I 0 0 0 0 0 0 1 0 1 0 0 0 0 0 1 0 0 90 9J 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 91 10A 0 0 0 0 0 0 0 1 1 0 0 1 1 0 1 0 0 92 10B 0 0 0 0 0 0 0 1 1 0 0 0 0 0 1 0 0 93 10C 0 0 0 0 0 0 0 1 1 0 0 0 1 0 1 0 0 94 10D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 95 10E 0 0 0 0 0 0 1 1 0 0 0 1 0 0 1 0 0 96 10F 0 0 0 0 0 0 0 1 0 0 0 1 0 0 1 0 0 97 10G 0 0 0 0 0 0 1 1 1 0 1 0 1 1 1 0 0 98 10H 0 0 0 0 0 0 0 0 1 1 0 1 0 1 1 0 0 99 10I 0 0 0 0 0 0 0 0 0 1 0 0 1 0 1 0 0 100 10J 0 0 0 0 0 0 1 1 0 0 0 0 0 0 1 0 0 101 11A 0 0 0 0 0 0 0 0 1 0 0 0 0 1 1 0 0 102 11B 0 0 0 0 0 0 0 1 0 0 0 0 1 0 1 0 0 103 11C 0 0 0 0 0 0 0 0 1 1 0 0 0 1 1 0 0 104 11D 0 0 0 0 0 0 1 0 0 1 0 0 1 0 1 0 0 105 11E 0 0 0 0 0 0 1 0 1 1 0 0 1 1 1 0 1 106 11F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 107 11G 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 108 11H 0 0 0 0 0 0 1 0 0 1 0 0 0 0 1 0 0 109 11I 0 0 0 0 0 0 0 0 1 0 0 0 0 1 1 0 0 110 11J 0 0 0 0 0 0 1 1 1 0 0 0 1 0 1 0 0 111 12A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 112 12B 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 113 12C 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 114 12D 0 0 0 0 0 0 0 1 0 0 0 0 1 0 1 0 0 182

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185

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G106 24 25 25 25 27 28 28 32 37 40 11 11 12 12 13 22 26 # Sample 6 0 2 4 6 4 6 8 6 8 6 8 0 6 0 0 0 76 8F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 77 8G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 78 8H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 79 8I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 80 8J 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 81 9A 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 82 9B 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 83 9C 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 84 9D 1 0 1 0 0 0 0 0 1 0 1 0 0 0 0 0 0 85 9E 1 0 0 0 1 0 0 0 1 0 1 0 0 0 0 1 0 86 9F 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 87 9G 1 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 88 9H 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 89 9I 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 90 9J 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 91 10A 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 92 10B 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 93 10C 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 94 10D 1 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 95 10E 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 96 10F 1 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 97 10G 1 0 0 0 1 0 0 0 1 0 1 0 0 0 0 0 0 98 10H 1 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 99 10I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 10J 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 101 11A 0 0 1 0 0 0 0 0 1 0 1 0 0 0 0 0 0 102 11B 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 103 11C 1 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 104 11D 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 105 11E 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 106 11F 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 107 11G 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 108 11H 1 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 109 11I 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 110 11J 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 111 12A 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 112 12B 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 1 113 12C 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 114 12D 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 196

GBAS109 10 10 10 11 13 13 14 14 16 16 16 20 20 22 25 34 34 # Sample 2 5 8 4 2 8 4 7 2 5 8 1 7 8 0 5 8 76 8F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 77 8G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 78 8H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 79 8I 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 80 8J 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 81 9A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 82 9B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 83 9C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 84 9D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 85 9E 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 86 9F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 87 9G 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 88 9H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 89 9I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 90 9J 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 91 10A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 92 10B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 93 10C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 94 10D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 95 10E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 96 10F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 97 10G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 98 10H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 99 10I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 10J 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 101 11A 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 102 11B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 103 11C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 104 11D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 105 11E 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 106 11F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 107 11G 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 108 11H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 109 11I 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 110 11J 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 111 12A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 112 12B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 113 12C 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 114 12D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 197

39 45 48 48 # Sample 6 6 3 6 76 8F 0 0 0 0 77 8G 0 0 0 0 78 8H 0 0 0 0 79 8I 0 0 0 0 80 8J 0 0 0 0 81 9A 0 0 0 0 82 9B 0 0 0 0 83 9C 0 0 0 0 84 9D 0 0 0 0 85 9E 0 0 0 0 86 9F 0 0 0 0 87 9G 0 0 0 0 88 9H 0 0 0 0 89 9I 0 0 0 0 90 9J 0 0 0 0 91 10A 0 0 0 0 92 10B 0 0 0 0 93 10C 0 0 0 0 94 10D 0 0 0 0 95 10E 0 0 0 0 96 10F 0 0 0 0 97 10G 0 0 0 0 98 10H 0 0 0 0 99 10I 0 0 0 0 100 10J 0 0 0 0 101 11A 0 0 0 0 102 11B 0 0 1 1 103 11C 0 0 0 0 104 11D 0 0 0 0 105 11E 0 0 0 0 106 11F 0 0 0 0 107 11G 0 0 0 0 108 11H 0 0 0 0 109 11I 0 0 0 0 110 11J 0 0 0 0 111 12A 0 0 0 0 112 12B 0 0 0 0 113 12C 0 0 0 0 114 12D 0 0 0 0 198

Appendix 6: Species Distribution Model maps of three Green Ramps taxa of the Allium tricoccum complex utilizing the Maximum Entropy model. (A) South Green Ramps; (B)

Allium burdickii; (C) Highland Green Ramps.

A

B

199

C

200

Appendix 7: Geographic coordinates of four taxa of the Allium tricoccum complex used in the Species Distribution Model.

# Species Longitude Latitude 1. Allium tricoccum -83.181524 35.575989 2. Allium tricoccum -83.002309 35.51135 3. Allium tricoccum -83.082819 35.426029 4. Allium tricoccum -83.589355 35.000119 5. Allium tricoccum -73.768345 42.507595 6. Allium tricoccum -74.122059 42.751595 7. Allium tricoccum -76.569031 42.327832 8. Allium tricoccum -72.33675 43.824803 9. Allium tricoccum -78.904652 43.009347 10. Allium tricoccum -94.522254 47.541965 11. Allium tricoccum -95.200233 46.449772 12. Allium tricoccum -92.841261 46.164429 13. Allium tricoccum -95.773551 44.994629 14. Allium tricoccum -92.173889 46.744804 15. Allium tricoccum -94.34584 45.47524 16. Allium tricoccum -93.341163 44.709155 17. Allium tricoccum -92.45469 45.37733 18. Allium tricoccum -86.823063 46.518918 19. Allium tricoccum -86.823063 45.411553 20. Allium tricoccum -87.107633 45.254297 21. Allium tricoccum -87.345154 45.056202 22. Allium tricoccum -87.345154 44.726976 23. Allium tricoccum -86.211036 44.526114 24. Allium tricoccum -85.898526 44.424776 25. Allium tricoccum -93.050156 44.322001 201

26. Allium tricoccum -85.243058 43.201447 27. Allium tricoccum -85.696122 43.246667 28. Allium tricoccum -85.847208 42.915342 29. Allium tricoccum -84.396944 37.8980556 30. Allium tricoccum -86.622326 41.8350817 31. Allium tricoccum -86.312854 39.8834031 32. Allium tricoccum -80.666915 40.1085065 33. Allium tricoccum -79.642757 38.5771374 34. Allium tricoccum -82.391854 35.6998371 35. Allium tricoccum -82.086104 36.1464981 36. Allium tricoccum -83.20186 41.0037421 37. Allium tricoccum -82.061503 39.3503398 38. Allium burdickii -88.86468 40.54798 39. Allium burdickii -88.37793 40.20423 40. Allium burdickii -82.26210 39.74064 41. Allium burdickii -75.46877 40.54899 42. Allium burdickii -82.96394 40.11950 43. Allium burdickii -84.75066 39.49736 44. Allium burdickii -85.83227 38.54038 45. Allium burdickii -85.01453 41.40025 46. Allium burdickii -85.15550 39.45631 47. Allium burdickii -85.03747 41.58589 48. Allium burdickii -84.97358 39.78580 49. Allium burdickii -85.19502 40.77390 50. Allium burdickii -85.52404 41.11314 51. Allium burdickii -85.49577 40.85476 52. Allium burdickii -85.02402 41.08368 53. Allium burdickii -87.90282 42.92953 54. Allium burdickii -87.94603 42.44834 202

55. Allium burdickii -92.57745 45.43323 56. Allium burdickii -92.66325 45.38506 57. Allium burdickii -84.11004 42.18635 58. Allium burdickii -84.80723 42.30839 59. Allium burdickii -85.64593 42.11653 60. Allium burdickii -86.59017 41.91771 61. Allium burdickii -86.62233 41.83508 62. Allium burdickii -86.31285 39.88340 63. Allium burdickii -83.20186 41.00374 64. Allium burdickii -82.87412 39.63365 65. South Green -85.619722 39.901389 66. South Green -85.700249 42.126114 67. South Green -84.396944 37.898056 68. South Green -84.721389 38.998889 69. South Green -83.498207 35.8475 70. South Green -85.419214 36.471518 71. South Green -85.807072 35.974287 72. South Green -89.118013 36.254149 73. South Green -80.400824 38.474394 74. South Green -82.251332 38.448937 75. South Green 84.881513 37.840742 76. South Green -85.812231 34.790428 77. South Green -85.708028 35.408494 78. South Green -84.248657 39.121005 79. South Green 85.8133931 36.0876321 80. South Green 85.4192144 36.4715177 81. South Green -85.8358481 36.0705947 82. South Green -85.388061 35.5741738 83. South Green -85.817618 36.0870034 203

84. South Green -85.388061 35.7541738 85. South Green -79.645506 36.4448009 86. South Green -80.159369 39.3816501 87. South Green -79.396673 39.2543502 88. South Green -79.491481 38.8065757 89. South Green -83.631719 37.8332107 90. South Green -85.459796 36.8504324 91. South Green -82.201946 36.1538715

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