Bouteloua Curtipendula (Poaceae): Reproductive Biology, Phenotypic Plasticity, and the Origins of an Apomictic Species Complex

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Authors Halbrook, Andronike Kandres

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BOUTELOUA CURTIPENDULA (POACEAE): REPRODUCTIVE BIOLOGY, PHENOTYPIC PLASTICITY, AND THE ORIGINS OF AN APOMICTIC SPECIES COMPLEX

Andronike Kandres Halbrook

______

A Dissertation Submitted to the Faculty of the

SCHOOL OF NATURAL RESOURCES AND THE ENVIRONMENT

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

2012

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Andronike Kandres Halbrook entitled:

Bouteloua curtipendula (Poaceae): Reproductive Biology, Phenotypic Plasticity, and the Origins of an Apomictic Species Complex and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy

______Date: 07/20/2012 Steven R. Archer

______Date: 07/20/2012 D. Lawrence Venable

______Date:

Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

______Date: 07/20/2012 Dissertation Director: Steven E. Smith

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED: Andronike Kandres Halbrook

TABLE OF CONTENTS ABSTRACT ...... 5

CHAPTER 1 INTRODUCTION ...... 7

Explanation of the problem and its context ...... 7

Explanation of the dissertation format ...... 14

CHAPTER 2 PRESENT STUDY ...... 16

REFERENCES ...... 20

APPENDIX A: HABITAT SUITABILITY PALEODISTRIBUTION MODELING REVEALS SPECIES RANGE DYNAMICS IN THE BOUTELOUA CURTIPENDULA (POACEAE) APOMICTIC COMPLEX ...... 27

APPENDIX B: PHENOTYPIC PLASTICITY OF KEY TRAITS THAT DEFINE INFRASPECIFIC VARIETIES IN BOUTELOUA CURTIPENDULA (POACEAE) . . 87

APPENDIX C: PSEUDOGAMOUS APOMIXIS, RELATIVE PLOIDY, AND FERTILITY IN BOUTELOUA CURTIPENDULA (POACEAE) ...... 129

ABSTRACT

Bouteloua curtipendula (Michx.) Torr. is one of the most broadly distributed C4 grass species in the USA and Mexico and is a key component of grassland and shrubland communities throughout its range. It is also part of a wider species complex, commonly referred to as the Bouteloua curtipendula complex (BCC), composed of 11 species and five varieties distributed in North and South America with its center of diversity in

Mexico. The BCC is characterized by tremendous phenotypic variation and taxonomic complexity that is most likely due to reticulate evolution, phenotypic plasticity, and the development of asexual reproduction (apomixis).

Present day biodiversity and distributions are a reflection of past ecological and evolutionary events. There is evidence that climate oscillations affected community composition and the genetic structure of populations as species retreated and advanced in response to glacial and interglacial periods. Climate-induced range changes may explain the origin of morphologically diverse species complexes such as the BCC as suites of species came into contact over time, hybridized, and created new species, cytotypes, and reproductive modes. I investigated the origins of the BCC by creating habitat suitability models based on present-day occurrence records for eight BCC taxa and hindcast these models to paleoclimate reconstructions for the Last Glacial Maximum (c. 21,000 YBP) and Last Interglacial (c. 120,000 YBP). By estimating range dynamics over time, coupled with phylogenetic data, I inferred the locations of contact zones among taxa and identified likely progenitor taxa for various cytotypes found in the BCC. 6

Species with large and environmentally heterogeneous distributions may have large ranges due to plastic responses to environmental variation for adaptive traits or they may maintain differently adapted ecotypes to specific habitats within their distribution. I evaluated phenotypic plasticity for stolon and rhizome production in the three taxonomic varieties of B. curtipendula. My results indicate that expression of these traits is correlated with local environmental conditions and not to broadly defined environments in geographic space and that phenotypic plasticity accounts for a greater proportion of trait expression than does total genetic variance. These data suggest that plastic responses to heterogeneous environments may partially explain the ecological breadth of B. curtipendula. They also suggest that presence/absence of stolons and rhizomes are not reliable to distinguish the infraspecific varieties.

Apomixis, asexual reproduction via seed, most likely results from interploidy hybridization. There are many possible pathways that lead to asexual seed formation and understanding these pathways is important to understanding genetic diversity, demography, and evolutionary potential in apomictic populations and in particular, in mixed apomictic-sexual populations. I discovered that B. curtipendula var. caespitosa, the only recognized apomictic taxon in the BCC, is pseudogamous, indicating that although fertilization is unnecessary to produce the embryo, it is necessary to produce the endosperm. These data also indicate that meiosis is highly irregular and results in sperm nuclei with variable chromosome numbers, which in turn affects endosperm production and, ultimately, fertility and demographics in apomictic B. curtipendula.

INTRODUCTION

Explanation of the problem and its context

Bouteloua curtipendula (Michx.) Torr. is one of the most broadly distributed C4 grass species in the USA and Mexico (Gould, 1979) and is a key component of grassland and shrubland communities in a number of ecosystems within its range (Smith et al.,

2004). It is also part of a wider species complex, commonly referred to as the Bouteloua curtipendula complex (BCC), composed of 11 species and five varieties (Columbus et al., 1998; Columbus et al., 2000) distributed in North and South America with its center of diversity in Mexico (Gould and Kapadia, 1964). The BCC is characterized by tremendous phenotypic variation and concomitant taxonomic complexity that is most likely due to reticulate evolution and apomixis (Gould and Kapadia, 1964; Siqueiros,

2001; Siqueiros et al., in press). The goal of this dissertation is to better comprehend the evolutionary dynamics in the BCC by understanding the reproductive biology that underlies apomixis, the biogeographical circumstances affecting the complex, and the genetic basis for adaptively significant traits within its members.

Apomixis is defined as asexual reproduction through seeds (Nogler, 1984). It is a process that mimics many of the cytological steps of sexual reproduction, but the fundamental difference between the two processes is in the origin of the embryo. Sexual individuals produce an embryo derived from fusion of male and female gametes while apomictic individuals produce an embryo derived solely from maternal tissue. Sexual reproduction is further characterized by double fertilization where, in addition to fertilization of the egg cell to form the embryo, a second sperm nucleus fertilizes the 8 central cell to create the endosperm. In apomictic reproduction the endosperm can develop with fertilization (pseudogamy) or without fertilization (autonomous) (Nogler,

1984; Asker, 1992). In both sexual and apomictic plants, endosperm generally requires a

2:1 ratio of maternal to paternal genomes for normal endosperm development (Johnston et al., 1980; Haig and Westoby, 1991; Gehring et al., 2004; Kinoshita et al., 2008) and failure to achieve this ratio often leads to seed abortion (Thompson, 1930; Cooper and

Brink, 1945; Hakansson and Ellerstrom, 1950; Hakansson, 1956; Lin, 1984; Kermicle and Alleman, 1990).

There are many possible pathways that lead to asexual seed formation. These pathways can be grouped into two basic forms: adventitious embryony and gametophytic apomixis (Nogler, 1984; Richards, 1986; Asker, 1992). In the former, an embryo is derived directly from a somatic cell in the ovary. In the latter, an embryo is derived from an egg cell with an unreduced embryo sac. Gametophytic apomixis can be further subdivided into categories based on the origin of the embryo sac. An embryo sac that develops from a megaspore that fails to initiate or complete meiosis is diplosporic while an embryo sac derived from a somatic cell is aposporic. There are a number of variations to be found within these broad categories and it can be said that there are just about as many mechanisms of apomixis as there are apomictic species (Richards, 1986).

The mechanistic pathway leading to an asexual seed would seem to be irrelevant if the seed is simply a clone of the maternal parent. Although the maternal genotype is generally maintained in progenies derived from apomixis (Ozias-Akins and van Dijk,

2007), there is mounting evidence that offspring may not be identical to the parent due to 9 mutation and developmental differences, such as epigenetic effects associated with function of regulatory genes (Loxdale and Lushai, 2003; Lushai et al., 2003). As a result, understanding and describing mechanistic pathways is important to understanding sources and patterns of genetic diversity in apomictic populations and species.

Apomictic species generally develop within complexes comprised of closely related diploid and polyploid species. A wide array of ploidy levels can be found in such complexes resulting from chromosome doubling (autoploidy), hybridization that results in chromosome doubling (alloploidy), recurrent formation of polyploids from more than one pair of progenitor taxa, and introgression between polyploids and their progenitors

(Stace, 2000; Coyne and Orr, 2004; Soltis et al., 2004). The vast majority of apomictic species are polyploid and evidence suggests that they may result from hybridization disturbing biochemical pathways in the sexual reproduction system (Barcaccia et al.,

1998; Schallau et al., 2010; Pupilli and Barcaccia, 2012). Apomicts can be dynamic members of the broader species complex because they often produce functional pollen that can fertilize sexual species, which may introgress apomixis into these populations.

Apomixis is also generally facultative and not obligatory (Koltunow and Grossniklaus,

2003; Sartor et al., 2011; Burson et al., 2012); thus, an apomictically derived egg cell can be fertilized to create an entirely new genotype and perhaps, cytotype, in the population.

The extraordinary genetic and cytological diversity possible in apomictic species complexes is reflected in the nearly continuous array of phenotypes seen in such complexes (McDade, 1995; Richards, 2003). Molecular and morphological phylogenetic analyses of apomictic species complexes often reveal extremely discordant gene and 10 species trees, which leave taxonomists in a quagmire: either they must lump all phenotypic forms into one species or split them into a potentially infinite number of species (Stebbins, 1950; Asker, 1992; Soltis et al., 2007). Designating species and taxonomic varieties help reveal biological diversity and broaden our understanding of evolution and speciation within a species complex (Soltis et al., 2007; Peirson et al.,

2012), but taxa that have overlapping morphology, ploidy, and geographic distributions can obscure our understanding of the patterns and processes that create this diversity.

A complicating factor for species circumscriptions is our generally limited understanding the role phenotypic plasticity plays in taxonomically diagnostic traits.

Phenotypic plasticity is reflected in the ability of a genotype to exhibit a range of phenotypes in response to environmental variation (Bradshaw, 1965; Agrawal, 2001;

Pigliucci, 2001). It is most commonly associated with morphological traits (Schlichting and Pigliucci, 1998), but many aspects of physiology, biochemistry, and life history also have been shown to be environmentally dependent (Sultan, 2000; Nicotra et al., 2010;

Davidson et al., 2011; Levin, 2012). Differences in plastic responses to heterogeneous environments may partially explain the ecological breadth of a species (Sultan, 2001;

Gonzalez and Gianoli, 2004). For example, species with restricted ranges would be expected to have less plastic responses to environmental variation while those with widespread distributions would exhibit either greater tolerance through plasticity

(Futuyma and Moreno, 1988; van Tienderen, 1997) or maintain differently adapted ecotypes to specific habitats within its geographic range (Baskauf and Eichmeier, 1994;

Sultan et al., 1998). If a phenotypically plastic response is adaptive, it can broaden a 11 species’ geographic range and contribute to divergence and speciation via genetic and phenotypic accommodation (West-Eberhard, 2003; Schlichting, 2004; Pigliucci et al.,

2006). However, phenotypic plasticity that affects adaptation can greatly complicate understanding of phenotype-based systematics within a group. This is especially true in cases where selection for local adaptation may act to stabilize the expression of certain traits within individual environments, but retain the ability to express that trait differently in different environments.

Circumscription of many taxa in the BCC is problematic due to the complex’s large and environmentally heterogeneous range, continuous variation, intermediate forms, ploidy variation, and overlapping distributions (Gould and Kapadia, 1964;

Siqueiros, 2001). Eight of the 11 species are sexual diploids (2n = 2x = 20) and seven have relatively restricted distributions in Mexico, the Caribbean, and northwest South

America. These latter taxa include B. distans Swallen, B. disticha (Kunth) Benth., B. media (E. Fourn.) Gould and Kapadia, B. pedicellata Swallen, B. reflexa Swallen, B. triaena (Trin.) Scribn., and B. vaneedenii Pilg. in Urb. (Gould, 1979; Siqueiros, 2001).

The diploid B. uniflora Vasey is found in south-central USA and northeastern Mexico.

There is one sexual tetraploid taxon in Mexico, B. purpurea Gould and Kapadia (2n =

40) (Gould and Kapadia, 1964; Siqueiros, 2001) and two taxa with distributions that span the USA and Mexico and have a range of ploidy levels. Bouteloua warnockii Gould and

Kapadia has aneuploid chromosome numbers ranging from diploid to tetraploid (2n =

20–40) (Gould and Kapadia, 1962; Gould and Kapadia, 1964; Siqueiros, 2001), but its 12 mode of reproduction is unknown. Bouteloua curtipendula (Michx.) Torr. sensu stricto has both sexual and apomictic forms and a vast range of ploidy levels (2n = 20–103).

In a taxonomic revision of the BCC, Gould and Kapadia (1964) ascribed three infraspecific varieties of B. curtipendula based predominately on phenotype (presence or absence of stolons and rhizomes), ploidy, and geographic distribution. Bouteloua curtipendula var. curtipendula Gould and Kapadia, is described as sexual and predominately tetraploid (2n = 40), but chromosome numbers that range from 2n = 20–64 have been reported (Nielsen and Humphrey, 1937; Fults, 1942; Freter and Brown, 1955;

Gould and Kapadia, 1964; Reeder, 1977; Siqueiros, 2001). It produces rhizomes and it found in the short- and tallgrass prairies of USA. Bouteloua curtipendula var. tenuis is generally diploid or tetraploid (2n = 20, 40) (Gould and Kapadia, 1964; Gould and

Soderstrom, 1970), but there are records of aneuploids from 2n = 40–90 (Siqueiros,

2001). Its reproductive mode has not been assessed. It produces both stolons and rhizomes and is found in desert, matorral, and mesquital regions of Mexico, although occurrences have been recorded in Texas (Hatch et al., 2009) and Arizona (see ARIZ

319938). Bouteloua curtipendula var. caespitosa Gould and Kapadia is a facultative pseudogamous apomictic species distributed in the Sonoran and Chihuahuan Deserts of southwestern USA and northern Mexico and produces neither stolons nor rhizomes. A nearly continuous series of aneuploid chromosome numbers have been found (2n = 58–

103) (Fults, 1942; Gould and Kapadia, 1964; Reeder, 1977; Halbrook et al., in review).

Molecular, phenotypic, and cytogenetic evidence indicate that there is auto- and alloploidy, recurrent hybridization, and introgression in the BCC (Gould and Kapadia, 13

1964; Siqueiros, 2001). This is particularly evident in the apomictic aneuploid polyploid

B. curtipendula where there is tremendous morphological and cytotype variation within and among populations (Siqueiros, 2001; Halbrook et al., in review). Based on phenotypic traits and current distribution, it has been hypothesized that B. warnockii and

B. uniflora contributed to the creation of B. curtipendula sensu stricto or through introgression to the apomictic B. curtipendula var. caespitosa (Freter and Brown, 1955;

Gould and Kapadia, 1962). However, the southern limits of B. curtipendula potentially overlap with a number of diploid congeners and specimens referable to B. curtipendula have been found scattered throughout Central and South America. Thus, multiple diploids may have been involved in the formation of B. curtipendula.

Present day biodiversity and distributions are a reflection of past ecological and evolutionary events. In the Quaternary, there is evidence that climate oscillations affected community composition and the genetic structure of populations as species continually retreated and advanced in response to glacial and interglacial periods (Bennett, 1997;

Williams et al., 1998; Hewitt, 2004). We know from B. curtipendula’s present broad distribution that it occupies climatically and topographically heterogeneous environments. Some of these environments were once glaciated and others were impacted by periodic climatic fluctuations during glacial and interglacial periods (Wells, 1966;

Axelrod, 1979; Van Devender and Spaulding, 1979; Van Devender, 1990; Thompson and

Anderson, 2000; Hunter et al., 2001; Holmgren et al., 2007). Paleobotanical analyses indicate that community composition changed in an individualistic and uneven manner as range expansion and contraction occurred in response to changing climate (see regional 14 summaries in Betancourt et al., 1990). As a result, changes in species distributions may have permitted certain related species to come into contact in mutual refugia as ranges contracted or meet at hybrid zones when expanding from separate refugia (Nason et al.,

2002; Fehlberg and Ranker, 2009). The genetic consequences of these climate-induced range changes may be seen today and might explain the origin of morphologically and genetically diverse species complexes such as the BCC.

Explanation of the dissertation format

The goal of this dissertation is to understand (i) the biogeographic processes that may have led to the development of the BCC, (ii) the quantitative genetics and phenotypic plasticity underlying a key diagnostic trait distinguishing three BCC taxa, and

(iii) the reproductive biology of the BCC’s apomictic taxon, B. curtipendula var. caespitosa. The results of this dissertation are presented as three separate, appended manuscripts. Appendix A estimates BCC range dynamics since the Last Interglacial period (c. 120 000 years before present), infers locations of contact zones among taxa and likely diploid progenitors for polyploid taxa, and evaluates B. curtipendula var. caespitosa for geographical parthenogenesis. Appendix B determines if stolons and rhizomes, two key diagnostic traits circumscribing the infraspecific varieties of B. curtipendula, are correlated to local environmental conditions regardless of their location within the geographic range of the species and if stolons exhibit phenotypic plasticity under controlled conditions. Appendix C determines the mode of reproduction in apomictic B. curtipendula var. caespitosa, including whether endosperm develops from 15 fertilization or autonomously, and assesses the relationship between fertility and embryo and endosperm characteristics.

PRESENT STUDY

The specific research questions asked, methodologies used, results obtained, and conclusions drawn from this study are presented in the manuscripts appended to this dissertation. The following is a brief summary of each manuscript. Although the appendices have co-authors, the dissertation as a whole represents my original, independent work. I designed the research, collected data, performed analyses, and wrote the manuscripts. The contribution of each co-author is described below

Appendix A: Habitat suitability paleodistribution modeling reveals species range dynamics in the Bouteloua curtipendula (Poaceae) apomictic complex is a manuscript to be submitted to the American Journal of Botany. In this research we (i) developed habitat suitability models with occurrence records for eight BCC taxa using Maxent, (ii) hindcast these models to paleoclimate reconstructions for the Last Glacial Maximum and Last

Interglacial, and (iii) addressed questions regarding species range dynamics, contact zones among species, and geographical parthenogenesis. Our models revealed contact zones between diploid and polyploid taxa in southeastern New Mexico, southwestern

Texas and northern Coahuila that may represent zones of repeated hybridization. The majority of the evidence does not support geographical parthenogenesis for the apomictic taxon. Even so, describing range dynamics over time can help identify relationships among diploid and polyploid BCC taxa and aid interpretation of phylogenetic analyses.

Co-author Steven Smith, dissertation director, assisted with model development and GIS techniques while Matthew Lee assisted with initial model development for five of the 11 taxa modeled. Both co-authors read and commented on manuscripts drafts. 17

Appendix B: Phenotypic plasticity of key traits that define infraspecific varieties in Bouteloua curtipendula (Poaceae) is a manuscript to be submitted to the journal

Taxon. This study investigated phenotypic plasticity, the ability of a genotype to exhibit a range of phenotypes in response to environmental variation. Differences in plastic responses to heterogeneous environments may partially explain the ecological breadth of widespread species such as B. curtipendula. We asked if the growth forms distinguishing

B. curtipendula’s infraspecific varieties are the result of a phenotypically plastic response to different environments that is present within genotypes or due to relatively non-plastic local adaptation. We evaluated 1381 georeferenceable B. curtipendula herbarium accessions from throughout the species’ range for presentation of stolons and rhizomes and correlated trait expression to elevation and 19 bioclimatic variables. We also used quantitative genetic analyses to directly partition intra-population variation for the stoloniferous growth form into components associated with genetically based variation in plasticity and non-plastic genetic variation. Our results indicate that expression of stolons and rhizomes is correlated to local environmental conditions and not to broadly defined environments in geographic space and that phenotypic plasticity accounts for a greater proportion of stolon trait expression than does total genetic variance. The data suggest that presence/absence of stolons and rhizomes do not reliably distinguish among the infraspecific varieties and that perhaps these varieties are not valid.

Co-author Steven Smith, dissertation director, provided experimental design advice, reviewed data analyses, and commented on manuscripts drafts. Co-authors 18

Kathryn Lacey, Mathew Benson, Leawna Broduer, and Eric Wagner helped maintain greenhouse plants, collect stolon data, and commented on manuscript drafts.

Appendix C: Pseudogamous apomixis, relative ploidy, and fertility in Bouteloua curtipendula (Poaceae) is a manuscript to be submitted to the American Journal of

Botany. This study investigates reproductive biology and its relationship to fertility in B. curtipendula var. caespitosa. We used flow cytometric seed screens to determine relative ploidy among individuals, estimate embryo and sperm nucleus relative DNA content, calculate embryo:endosperm ratios, and then correlated these data with fertility in five populations of Bouteloua curtipendula var. caespitosa (Poaceae). We confirmed that this taxon is aneuploid and reproduces by pseudogamous apomixis. Although embryo:endosperm ratios varied among individuals, there was no relationship between mean endosperm ratio and fertility. The evidence suggests that maternal effects play a more prominent role in fertility than paternal effects and that paternal contribution to the endosperm, while necessary for grain formation, is not the primary factor determining grain production. The results of this study will help us better understand reproductive biology, and thereby population dynamics and evolution in apomictic species.

Co-author Steven Smith, dissertation director, assisted with the development of a new technique to evaluate flow cytometric data and commented on manuscript drafts.

The research required long-term maintenance plants in a greenhouse and dissection of over 87,000 flowers. Kathryn Lacey assisted with flower emasculation for one of the experiments. She, along with, Matthew Binney, Mathew Benson, Leawna Broduer, and 19

Colin Rigden helped dissect flowers, maintain plants, and commented on manuscript drafts.

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HALBROOK ET AL., RANGE DYNAMICS IN BOUTELOUA CURTIPENDULA COMPLEX

APPENDIX A: HABITAT SUITABILITY PALEODISTRIBUTION MODELING

REVEALS SPECIES RANGE DYNAMICS IN THE BOUTELOUA CURTIPENDULA

(POACEAE) APOMICTIC COMPLEX

TO BE SUBMITTED TO: American Journal of Botany

1 1 1 KANDRES HALBROOK , STEVEN E. SMITH , AND MATTHEW LEE

1 Manuscript received ______; revision accepted ______.

2 School of Natural Resources and the Environment, University of Arizona. Tucson,

Arizona 85721, USA

The authors thank Dan Warren, Andrew Honaman, and Mickey Reed for their assistance in this research.

3 Author for correspondence, [email protected]

ABSTRACT

Premise of the study: Apomictic species complexes are often characterized by a series of ploidy levels and a nearly continuous array of phenotypes, usually resulting from polyploidization, recurrent hybridization, and introgression among related taxa.

Phylogenetic analyses of such species complexes may have poor resolution. The

Bouteloua curtipendula apomictic complex (BCC) is composed of 11 species and five varieties widely distributed in USA and Mexico in areas that experienced Quaternary climatic oscillations. Circumscription of taxa in the BCC is problematic due to its large and heterogeneous range, continuous variation, intermediate forms, ploidy variation, and overlapping distributions. Habitat suitability models (HSMs) can suggest species range dynamics over time and support phylogenetic descriptions, especially in genetically complex groups. We used HSM to address the following questions: Do climate-induced changes in suitable habitat among BCC taxa suggest locations for glacial refugia and contact zones among taxa? and Can likely diploid progenitors for polyploid taxa be inferred from HSMs?

Methods: We developed HSMs with occurrence records for eight BCC taxa using Maxent and then hindcast these models to paleoclimate reconstructions for the Last Glacial

Maximum and Last Interglacial.

Key results: HSMs reveal contact zones between diploid and polyploid taxa in southeastern New Mexico, southwestern Texas and northern Coahuila that may represent zones of repeated hybridization. 30

Conclusions: Describing range dynamics over time helps identify relationships among diploid and polyploid BCC taxa and aids interpretation of phylogenetic analyses.

Keywords: climate; ENMTools; Last Glacial Maximum; Last Interglacial; niche;

Maxent; polyploidy

INTRODUCTION

The terms contact, hybrid, and suture zones have all been used to describe geographic regions where the distributions of many biotic assemblages overlap and species and subspecies within the assemblages hybridize (Remington, 1968; Avise, 2000;

Swenson and Howard, 2005; Hewitt, 2011). These zones are often thought to reflect colonization and recolonization events as plants and animals migrated to and from glacial refugia in response to climate oscillations, particularly in the Quaternary (Barton and

Hewitt, 1989; Hewitt, 2001). In a meta-analysis of hybrid zones, contact zones, and phylogeographic breaks in North America, Swenson and Howard (2005) identified a number of Pleistocene glacial refugia and locations where multiple biotic assemblages cluster (hotspots). In southwestern North America, refugia were identified in central Baja

California, and near the borders of Sonora, Chihuahua, and Sinaloa, Mexico and in southeastern Texas, USA. Hotspots were located in the Sierra Madre Occidental and

Oriental Mountains of Mexico, the Peloncillo Mountains of southeastern Arizona and southwestern New Mexico, and the northern portion of the Edwards Plateau and Central

Oklahoma/Texas Plains ecoregions in north-central Texas.

Considerable geologic, paleoclimatic, and paleobotanical evidence suggest that the southwestern USA and northern Mexico was wetter and cooler in the late Pleistocene than it is today (Wells, 1966; Axelrod, 1979; Van Devender and Spaulding, 1979;

Thompson and Anderson, 2000). The cooler, more mesic climate supported woodland communities at much lower elevations than found today while many xeric-adapted species were relegated to restricted distributions in the southern portions of their modern 32 range or had more southerly distributions (Van Devender, 1990; Thompson and

Anderson, 2000; Hunter et al., 2001). Paleobotanical analyses indicate that community composition changed in an individualistic and uneven manner as range expansion and contraction occurred in response to changing climates over the last 40 000 years before present (YBP) (see regional summaries in Betancourt et al., 1990). These range shifts may have permitted related species to come into contact in mutual refugia as ranges contracted or to meet at hybrid zones when expanding from separate refugia (Nason et al., 2002;

Fehlberg and Ranker, 2009). The general trend in region-wide aridification began at the end of the last glacial maximum (LGM; c. 21 000 YBP) and most communities in this region did not develop their modern composition and elevational gradients—pine forests, xeric woodlands, shrub- and grasslands, and desert scrub—until after c. 8 000 YBP (Van

Devender and Spaulding, 1979; Holmgren et al., 2003; Holmgren et al., 2006).

Phylogeographic studies have been influential in identifying the locations of potential refugia, colonization routes, and hybrid zones in western USA and northern

Mexico (Nason et al., 2002; Fehlberg and Ranker, 2009; Sosa et al., 2009; Ruiz et al.,

2010; Gugger et al., 2011; Barton and Wisely, 2012). However, interpreting the results from phylogenetic analyses can be challenging in cases of recent or rapid speciation where incomplete lineage sorting can create discordance between different gene trees

(Felsenstein, 2004; Maddison and Knowles, 2006). This is particularly troublesome in species complexes exhibiting reticulate evolution due to hybridization, introgression, and recurrent polyploidization (Soltis and Soltis, 1999; Linder and Rieseberg, 2004; Soltis,

2005). A wide array of ploidy levels can be found in such complexes resulting from 33 recurrent formation of tetraploids from more than one pair of diploids, introgression between tetraploids and their diploid progenitors to create triploids, and the production of higher ploidy levels via unreduced gametes (Stace, 2000; Coyne and Orr, 2004; Soltis et al., 2004). This situation is often further compounded by apomixis, asexual reproduction via seed (Grant, 1981). The vast majority of apomictic species are also polyploid (Asker,

1992) and they may have been formed by a number of progenitor species. Because of asexual reproduction, apomictic polyploid genotypes are not homogenized and can appear as multiple single lineages in a phylogenetic tree. However, apomixis is generally facultative and occasional outcrossing events have the potential to create even more haplotype diversity and add complexity to phylogenetic analyses. The genic diversity possible in apomictic species complexes can lead to a nearly continuous array of phenotypes (McDade, 1995; Richards, 2003) that make species circumscription and concordance between gene and species trees difficult.

The Bouteloua curtipendula apomictic complex (BCC) is composed of 11 species and five varieties (Columbus et al., 1998; Columbus et al., 2000) widely distributed in

North and South America with its center of diversity in Mexico. Circumscription of many taxa in the BCC is problematic due to its large and environmentally heterogeneous range, continuous variation, intermediate forms, ploidy variation, and overlapping distributions

(Gould and Kapadia, 1964; Siqueiros, 2001). Eight of the 11 species are sexual diploids

(2n = 2x = 20) and seven have relatively restricted distributions in Mexico, the

Caribbean, and northwest South America. These taxa include B. distans Swallen, B. disticha (Kunth) Benth., B. media (E. Fourn.) Gould and Kapadia, B. pedicellata Swallen, 34

B. reflexa Swallen, B. triaena (Trin.) Scribn., and B. vaneedenii Pilg. in Urb. (Gould,

1979; Siqueiros, 2001). The diploid B. uniflora Vasey is found in south-central USA and northeastern Mexico. There is one sexual tetraploid taxon in Mexico, B. purpurea Gould and Kapadia (2n = 40) (Gould and Kapadia, 1964; Siqueiros, 2001) and two taxa with distributions that span USA and Mexico and have a range of ploidy levels. Bouteloua warnockii Gould and Kapadia has aneuploid chromosome numbers ranging from diploid to tetraploid (2n = 20–40) (Gould and Kapadia, 1962b; Gould and Kapadia, 1964;

Siqueiros, 2001), but its mode of reproduction is unknown. Bouteloua curtipendula

(Michx.) Torr. sensu stricto has both sexual and apomictic forms and a vast range of ploidy levels (2n = 20–103).

In a taxonomic revision of the BCC, Gould and Kapadia (1964) ascribed three infraspecific varieties of B. curtipendula based predominately on phenotype (presence or absence of stolons and rhizomes), ploidy, and geographic distribution. Bouteloua curtipendula var. curtipendula Gould and Kapadia, is sexual and predominately tetraploid (2n = 40), but chromosome numbers that range from 2n = 20–64 have been reported (Nielsen and Humphrey, 1937; Fults, 1942; Freter and Brown, 1955; Gould and

Kapadia, 1964; Reeder, 1977; Siqueiros, 2001). It produces rhizomes and it found in the short- and tallgrass prairies of USA. Bouteloua curtipendula var. tenuis is generally diploid or tetraploid (2n = 20, 40) (Gould and Kapadia, 1964; Gould and Soderstrom,

1970), but there are records of aneuploids from 2n = 40–90 (Siqueiros, 2001). Its reproductive mode has not been assessed. It produces both stolons and rhizomes and is predominately found in desert, matorral, and mesquital regions of Mexico, but 35 occurrences have been recorded also in Texas (Hatch et al., 2009) and Arizona (see herbarium accession ARIZ 319938). Bouteloua curtipendula var. caespitosa Gould and

Kapadia is a facultative pseudogamous apomictic species distributed in the Sonoran and

Chihuahuan Deserts of southwestern USA and northern Mexico and produces neither stolons nor rhizomes. A nearly continuous series of aneuploid chromosome numbers have been found (2n = 58–103) (Fults, 1942; Gould and Kapadia, 1964; Reeder, 1977;

Halbrook et al., A in review).

Molecular, phenotypic, and cytogenetic evidence indicate that there is auto- and alloploidy, recurrent hybridization, and introgression in the BCC (Gould and Kapadia,

1964; Siqueiros, 2001). This is particularly evident in the apomictic aneuploid polyploid

B. curtipendula where there is tremendous morphological and cytotype variation within and among populations (Siqueiros, 2001; Halbrook et al., A in review). Based on phenotypic traits and current distribution, it has been hypothesized that B. warnockii and

B. uniflora contributed to the creation of B. curtipendula sensu stricto or through introgression to the apomictic B. curtipendula var. caespitosa (Freter and Brown, 1955;

Gould and Kapadia, 1962b). However, the southern limits of B. curtipendula potentially overlap with a number of diploid congeners and specimens referable to B. curtipendula have been found scattered throughout Central and South America. Thus, multiple diploids may have been involved in the formation of B. curtipendula.

Habitat suitability modeling (HSM) can be used to infer species range dynamics and provide support for phylogenetic patterns, especially in groups with a complex genetic history. These models are correlative and estimate the characteristics of the 36 physical environment that best predict the known distribution of a group based on data for occurrence, and sometimes absence (Pearson and Dawson, 2003). The resulting model, variously termed habitat suitability, species distribution, ecological niche, climatic niche, or climate envelope, can then be projected onto geographic space to identify locations that have similar environmental conditions (Soberón and Peterson, 2005). The model attempts to approach the concept of Hutchinson’s (1957) fundamental ecological niche by estimating the environmental characteristics that impose physiological limits to a species’ distribution. Climatic data are perhaps the most widely used predictor variables for HSM because climate is perceived to have a significant influence on species distributions at coarse spatial scales (Svenning et al., 2011). Models developed and calibrated on present-day distributions can be hindcast to earlier time periods where paleoclimate reconstructions are available, e.g., Last Interglacial (LIG; c. 120 000 YBP) and Last Glacial Maximum (LGM; c. 21 000 YBP). The fundamental assumption of hindcasting models is that a species’ niche requirements are stable through time and that evolutionary or ecological shifts in niches are relatively insignificant. Evidence from a number of theoretical studies suggests that ecological niches remain reasonably constant over time and space (see reviews: Nogues-Bravo, 2009; Svenning et al., 2011). Thus, hindcast HSMs can be used to investigate species range dynamics over time and identify potential contact zones and glacial refugia (Benito Garzon et al., 2007; Waltari et al.,

2007; Benito Garzon et al., 2008; Rodriguez-Sanchez and Arroyo, 2008; Waltari and

Guralnick, 2009). They can also be used to investigate climatic niche stability, expansion, and conservatism among closely related taxa in the past and present (Martínez-Meyer et 37 al., 2004; Martínez-Meyer and Peterson, 2006; Ruegg et al., 2006; Pearman et al., 2008;

Peterson and Nyári, 2008; Levsen et al., 2012).

In this study we reassess existing phylogenetic analyses and then relate these to range dynamics in the BCC using spatially explicit climatic HSMs hindcast to the LGM and LIG time periods to approach species relationships from a climatic-geographic perspective. Specifically, we ask the following questions: (i) Do climate-induced changes in suitable habitat among BCC taxa suggest locations for glacial refugia and contact zones among taxa? (ii) Can likely diploid progenitors for polyploid taxa be inferred from

HSMs?

MATERIALS AND METHODS

Phylogenetic analysis—Evolutionary history in the BCC was estimated using 83 cpDNA trnT-L-F and 94 nrDNA ITS sequences, including outgroup taxa, from GenBank

(for methodology see Siqueiros, 2001; for GenBank accession numbers see Table S1).

These sequences represent the geographic range of the BCC. Although we included sequences from B. distans, B. pedicellata, and B. vaneedenii, we did not include these in other analyses (see below). Only sequences with accession locality information were used. Sequences were aligned with MUSCLE with UPGMB clustering (Edgar, 2004).

Phylogenetic relationships among taxa in trnT-L-F were inferred using the Maximum

Likelihood method based on the Tamura (1992) 3-parameter model and the Kimura 2- parameter model for ITS. Bootstrap consensus trees were inferred from 100 replicates

(Felsenstein, 1985) using MEGA5 (Tamura et al., 2011). Initial trees for the heuristic 38 search were obtained from algorithms in MEGA5. The maximum parsimony method was used if the number of common sites was < 100 or less than 0.25 of the total number of sites. In all other cases, the BIONJ (Gascuel, 1997) method with Maximum Composite

Likelihood distance matrix was used (Tamura and Nei, 2004). For trnT-L-F and ITS, a discrete Gamma distribution was used to model evolutionary rate differences among sites using 5 categories +G (trnT-L-F parameter = 0.11; ITS parameter = 0.66) and included a rate variation model that allowed some sites to be evolutionarily invariable (+I, 52% sites) for trnT-L-F analysis. All positions with > 0.05 alignment gaps, missing data, and ambiguous bases were eliminated.

Study species occurrences—We derived occurrence data for B. curtipendula complex from records with stated latitude/longitude from within the region bounded by

10.5–51.0° N, − 121.5–− 87.5° W within the Global Biodiversity Information Facility

(accessed through GBIF Data Portal, data.gbif.org 2010-12-1). Also included were locations from herbarium records not in GBIF (Halbrook et al., B in review). Our primary interest is in understanding range dynamics in the northern distribution of BCC and therefore we limited our focus primarily to North and Central America (Figure 1).

Accessions east of – 87.5° W in North America were excluded primarily because their occurrence is sporadic and may represent recent introduction (Smith et al., 2004).

Similarly, we did not include B. vaneedenii, B. distans, and B. pedicellata because their distributions are primarily Caribbean and South American and there were < 10 georeferencable locations for each taxon (Gould, 1979; Siqueiros, 2001). The two infraspecific varieties of B. uniflora, B. uniflora var. uniflora and B. uniflora var. 39 coahuilensis were modeled as one taxon because their distributions overlap and there are limited occurrence records (n = 9) for var. coahuilensis. We eliminated duplicate occurrences, leaving a total of 4145 locations. Occurrence data for non-B. curtipendula taxa (B. uniflora, B. warnockii, B. media, B. purpurea, B. disticha, B. reflexa, and B. triaena) were compiled and handled in a similar manner except that georeferencing was done using locality information for occurrences without latitude/longitude data in GBIF.

This was done using BioGeomancer Workbench version 1.2.6 (Guralnick et al., 2006) following georeferencing recommendations of Chapman and Wieczorek (2006).

Occurrences were excluded if georeferencing uncertainty exceeded 4 km.

Environmental variables—Environmental data used in habitat suitability modeling included elevation and the 19 bioclimatic (BIO) variables from the WorldClim

1.4 dataset for the period 1950-2000 (= “Current”) at 2.5-minute resolution (Hijmans et al., 2005). Individual records from this dataset are referred to here as grids (c. 4.35 × 4.63 km at 30° N) and values within this database (other than elevation) are based on predicted monthly means. Final models were also projected to past environmental layers based on simulations from general circulation models (Poelchau and Hamrick, 2011). For the last glacial maximum we used the Model for Interdisciplinary Research on Climate (MIROC

3.2, Hasumi and Emori, 2004) and the Community Climate System Model (CCSM3,

Kiehl and Gent, 2004). We also used a CCSM3 model for the last interglacial (Otto-

Bliesner et al., 2008). These environmental layers were downloaded from the WorldClim site (worldclim.org/past, 2012-1-15) (Hijmans et al., 2005) and used in modeling at 2.5- minute resolution. 40

Habitat suitability modeling and analyses—Optimized individual HSMs (Warren et al., 2010; Warren and Seifert, 2011) were developed using Maxent version 3.3.3a

(Phillips et al., 2006) for the production of suitable habitat maps. Maxent is one of the best-performing HSM platforms that uses “presence-only” data (Elith et al., 2006;

Hernandez et al., 2006; Mateo et al., 2010) and has been extensively utilized for this purpose (Elith et al., 2011). We assumed that the sampled area represented by occurrence records equaled that of an individual environmental grid. Before model development we used the Trim Duplicate Occurrences tool within ENMTools (Warren et al., 2010) so that only a single occurrence record existed for each modeled group within a given environmental grid. This resulted in a total of 2238 B. curtipendula and 308 non- B. curtipendula accessions. To better account for the bias associated with non-random movement of collectors we used target-group background occurrences (pseudo-absences) rather than randomly selected background locations in these models (Phillips et al., 2009;

Elith et al., 2011). The target group involved 10 000 randomly selected occurrence records for Poaceae from GBIF (accessed through GBIF Data Portal, data.gbif.org 2012-

1-2). The Trim Duplicate Occurrences tool (Warren et al., 2010) was also used to insure that only a single occurrence was included in each environmental grid before selection of the target-group background began. For each group modeled, we used Maxent to generate

SWD files including altitude and BIO1–BIO19 that were used in all analyses.

Environmental data in habitat suitability modeling are often highly correlated and this redundancy may reduce accuracy of predictions (van Zonneveld et al., 2009; Comas et al., 2011). For each modeled group we used hierarchical clustering of environmental 41 variables on oblique centroid components to objectively identify redundancies among environmental variables (PROC VARCLUS in SAS under default settings)

Institute. SAS and all other SAS Institute Inc. Product or service names are registered trademarks of SAS Institute, Cary, NC, USA). This procedure is designed to identify non-overlapping clusters of variables and maximize total variation across clusters explained by cluster components. Initial Maxent model development began with a single variable from each variable cluster from the VARCLUS output. In multiple-variable clusters, this primary variable was the one most highly correlated with other members of that cluster and most weakly correlated with other clusters (i.e., smallest ratio of correlation within the cluster to next closest cluster). We also calculated correlation coefficients among all variables for each grid of the study area using the Correlation tool in ENMTools (Warren et al., 2010). No two variables were included within any model if | r ≥ 0.7 between them. Selection of the variable used among such pairs was accomplished using the same procedure used to identify primary variables described above.

Bouteloua curtipendula has a large (> 3500 km north-south, > 2200 km east-west in our sample) and environmentally heterogeneous range; and attempting to develop a single HSM over this range may yield overly complex models with relatively low regional predictive value. Separate models for environmentally similar regions of a species range may be justified (Nakazato et al., 2010). We therefore examined patterns of environmental variability at occurrence locations of all taxa by conducting a hierarchical cluster analysis (PROC CLUSTER, Ward's minimum-variance method in SAS). In this 42 analysis, each occurrence represented an observation and clustering was done using the primary variables from each intrataxon cluster derived from the VARCLUS analysis described above (standardized to mean = 0 and standard deviation = 1). In the cluster analysis, examination of the cubic clustering criterion, and the pseudo F and t 2 statistics

(P ≤ 0.05) was used to determine whether there was evidence of distinguishable intrataxon clusters (Cooper and Milligan, 1988). When significant support for multiple clusters within a taxon was established, separate HSMs were developed for occurrences within each cluster, which were named based on their relative locations. Multiple clusters were supported for B. curtipendula (Fig. 1) and analyses did not support the generation of multiple models for non-B. curtipendula taxa (Fig. 2).

When running Maxent we used default settings for features, convergence threshold, regularization, clamping, extrapolation, and maximum iterations. To reduce bias associated with training-testing fold selection during model selection, omission values from final models represented means of four replicate runs from cross-validation where all occurrences were present within a single test fold (Marmion et al., 2009). As optimized model selection proceeded, decisions regarding variable inclusion were made using a variety of inputs. These included results of VARCLUS analysis of variables, and jackknife tests and response curves for environmental variables generated by Maxent

(Phillips et al., 2006). In rare cases we also used existing knowledge of the apparent environmental requirements of the group involved (e.g., van Zonneveld et al., 2009).

Final models were selected primarily based on mean values for the Akaike information 43

criterion (AICc) (Warren and Seifert, 2011) as calculated in the Model Selection tool within ENMTools over four replicate runs (Warren et al., 2010).

We converted the continuous cumulative probability maps for final optimized models produced by Maxent into binary maps (all maps: GCS WGS 1984) presenting suitable-unsuitable habitat (e.g., Fig. 3). This was done using the relatively conservative equal test sensitivity (true predicted presences) and specificity (true predicted absences) threshold from the four replicate runs in DIVA-GIS version 7.3.0 (diva-gis.org, accessed

2010-8-1). Examination of threshold maps generated for each replicate run were also used to identify the final Maxent model, especially with fewer than 50 occurrences in the group. We report mean receiver operating characteristic curve (AUC) values (Lobo et al.,

2008) (all > 0.915) and training and testing omissions over four cross-validation replicates (Table S2) and permutation importance values for each model (Table S3).

For B. curtipendula, where cluster analysis justified generation of five separate models (Fig. 1), we generated whole-taxon suitable habitat maps (rasters) by overlaying the binary maps so as to display the maximum value per pixel from the individual maps in the overlay map (Fig. 3). These and all species or current-past comparison maps were similarly produced using Grid Overlay options in DIVA-GIS.

Climatic niche comparisons—Using non-optimized Maxent HSMs we calculated niche overlap, identity and similarity among taxa in the current time period and niche overlap for individual taxa among time periods. Niche identity and similarity tests require taxa occurrence data, which is only available for the current time period, therefore these tests could not be conducted for LGM or LIG. Our objective is to use these metrics to 44 infer phylogenetic relationships using spatially explicit niche information. We used routines within ENMTools (Warren et al., 2010) for these calculations. Niche overlap between pairs of taxa is calculated using Schoener’s D, which ranges from 0 (modeled habitat has no overlap) to 1 (complete overlap) and is based on a pixel-by-pixel comparison of model-derived predicted suitable habitat values (Warren et al., 2008).

Niche identity tests whether two HSMs are drawn from the same underlying distribution.

It tests this using randomization tests where pairs of pseudotaxa are created by pooling occurrences of both taxa and then randomly selecting occurrences to generate each pseudotaxon. The number used for each pseudotaxon is equal to that the actual number of occurences found in the original taxon. HSMs are created for each pseudotaxon and niche overlap values calculated for the pair as described above. We generated 100 pseudotaxon pairs and the resulting D values are compared with the actual observed via a one-sample t-test (PROC TTEST in SAS) and P values assigned using a one-tailed test. The hypothesis of niche identity is rejected when D values from pseudoreplicates are significantly larger than the actual observed D value (Warren et al., 2008).

HSMs between pairs of taxa (A and B, below) may appear different because the taxa actually occupy very different environments (backgrounds) or they may appear different because despite sharing environmental background they actually inhabit different locations within the environment (McCormack et al., 2008). To distinguish between these circumstances, niche similarity (background) tests evaluate the effect that different backgrounds have on comparisons of niche overlap between two taxa. We used background randomization procedures within ENMTools for these tests (Warren et al., 45

2010). Background was defined for a taxon as any point on land within 0.5 degrees from an actual occurrence of that taxon (Warren et al., 2010). The test is conducted by calculating an niche overlap value (D) based on a HSM for taxon A and a HSM based on a sample of the background points for taxon B, where the number of randomly selected background points is equal to the number of occurrences for taxon B. This was done for

100 random samples from taxon B’s background. The entire process is then replicated for taxon B and the background of taxon A. The overlap values from each of these two random sets are compared to the actual observed overlap value using a two-sided, one- sample t-test (PROC TTEST in SAS). In cases where this test is significant and values are less than the actual observed overlap, this indicates that both taxa are within niches that are as similar as possible under the available background (=conservatism).

Alternatively, when values are greater than the actual observed overlap then the taxa occupy niches that are more different than expected given the background environments available (=divergence). We identified taxa for background tests based on overlapping distributions predicted by optimized HSMs. Comparisons between climatic niches require generation of HSMs using the same environmental layers for all taxa instead of models with optimized parameters for each taxon. As a result, we used the 19 BIO variables and altitude for niche comparison. This likely generated over-parameterized HSMs relative to the optimized HSMs discussed above (Warren and Seifert, 2011); however, Maxent is considered relatively insensitive to autocorrelation among environmental variables

(Galbreath et al., 2011).

RESULTS

Phylogenetic analysis—The Maximum Likelihood bootstrap consensus trees estimated in this study were consistent with those developed in PAUP* using a strict consensus of over 100 000 most-parsimonious trees (Siqueiros, 2001). Three major clades were identified in both cpDNA trnT-L-F and nrDNA ITS trees (Figs. 4 and 5).

The data indicate that the BCC is monophyletic, but monophyly is only supported for B. reflexa in trnT-L-F, which forms a subclade with 64% bootstrap support. The majority of taxa are para- or polyphyletic. There is some evidence for an association between rare diploid B. curtipendula cytotypes and B. warnockii and B. uniflora in the two basal clades in both trees, but the relationships are not at all resolved. There are multiple polytomies in all clades, but they are especially prominent in clade A that predominately contains polyploid taxa.

Optimized HSMs—Optimized binary HSMs depicting suitable-unsuitable habitat for non-B. curtipendula taxa and the five B. curtipendula clusters are shown in Figs. 3, 6–

8. For the LGM, we modeled taxa to both the MIROC and CCSM3 paleoclimate reconstructions. Collection locations of B. curtipendula macrofossils recovered from packrat middens and dated to c. 11–45 600 YBP (Van Devender, 1995; Holmgren et al.,

2003; Holmgren et al., 2006; Holmgren et al., 2007), which spans the LGM (c. 21 000

YBP), were plotted on the B. curtipendula combined cluster maps for both paleoclimate reconstructions (Fig. 6). The map based on MIROC shows suitable habitat at fossil midden locations, but the CCSM3 map does not. Similarly, MIROC maps for B. warnockii and B. uniflora overlap midden locations in Texas, but CCSM3 maps do not 47

(Supplemental Fig. S1). Therefore, we only consider MIROC reconstructions of the LGM for the subsequent comparisons. Last Interglacial paleoreconstructions are based on a

CCSM3 model. Given the lack of fossils for the LIG period, we cannot estimate its accuracy in modeling BCC taxa.

Climatic niche comparisons—Niche identity tests among all pairs of taxa were significant (P ≤ 0.05), indicating that the actual observed overlap is less than would be expected if pairs of niches were equivalent and that HSMs were drawn from different underlying distributions. Niche similarity (background) tests between 10 pairs of taxa from areas where optimized HSMs suggested there are overlapping distributions indicate that three taxa pairs exhibit niche conservatism (B. media vs. B. disticha, B. media vs. B. triaena, and B. warnockii vs. B. curtipendula Southwest cluster) and two clearly exhibit niche divergence (B. media vs. B. purpurea and B. triaena vs. B. purpurea) (Table 1).

The results are equivocal in the other five pairs of taxa, where in one direction (e.g., taxa

A into random samples of taxon B’s background) the result suggests conservatism and in the other divergence or a non-significant difference.

In the current climate, the vast majority of Schoener’s D overlap values for all pairwise combinations of taxa were < 0.2 (mean D = 0.151; Table 2). However, 13/66 comparisons have values > 0.3 and these are mostly among taxa with central and southern Mexico distributions. For example, the B. curtipendula Tropical cluster had D- values > 0.3 in combination with B. uniflora, B. triaena, B. disticha, B. media, and B. purpurea. Comparisons among these taxa also often produced overlap values > 0.3.

Among taxa with more northerly distributions, D-values > 0.3 were found among B. 48 curtipendula Southwest cluster, B. warnockii, and B. uniflora. The LGM shows a similar pattern to the current climate (12/66 with D > 0.3), in that taxa that had relatively large current overlap values also show large overlap at the LGM (Table 2). The mean D-value among all taxa pairs is slighter higher in the LGM than the current (mean D = 0.176). In contrast, the LIG has the lowest mean overlap (D = 0.126), and only 7/16 pairwise comparisons have D-values > 0.3 (Table 2). However, many taxa with large overlap values in the other time periods are also prominent in the LIG, namely, B. curtipendula

Tropical cluster, B. uniflora, B. media, B. purpurea, B. disticha, and B. triaena.

We compared each taxon’s Schoener’s D overlap value among time periods, i.e., current-LGM, LGM-LIG, and current-LIG (Table 3). This analysis reveals that climatic niches suitable for some taxa were relatively stable through time (e.g., mean ± SE values for tropical taxa: B. reflexa D = 0.682 ± 0.028 and B. media D = 0.811 ± 0.025), while climatic niches for other taxa were much more variable among time periods (e.g., temperate taxa: B. curtipendula Shortgrass cluster D = 0.305 ± 0.241 and Tallgrass D =

0.224 ± 0.135).

DISCUSSION

BCC range dynamics—Optimized HSMs and climatic niche comparisons reveals two general patterns. Bouteloua curtipendula complex taxa with current distributions above c. 21˚ N latitude (e.g., central Mexico) experienced a reduction in suitable habitat and a southward latitudinal shift in estimated distributions from the LIG to the LGM

(Figs. 7 and 8, B. media). Taxa with current distributions below c. 21˚ N latitude 49 experienced a south and eastward expansion in suitable habitat (Fig. 8, B. triaena, B. disticha).

Bouteloua uniflora and B. purpurea are two exceptions to this trend. The range of suitable habitats for B. uniflora expanded northward from the LIG to LGM. Values for permutation importance in B. uniflora models indicate that the most important feature for predicting suitable habitat for this taxon is minimum temperature of the coldest month

(BIO6). Based on species occurrences in the current climate, optimal habitat occurs within a relatively narrow range of c. 3–5°C for this variable (data not shown). Although the LIG was warmer overall compared to the LGM (mean temperature over entire study area, 10.5 Vs. 0.3°C) locations for this variable, in combination with the other model parameters, were optimized in the LGM for B. uniflora. In the LIG there is very little suitable habitat for B. purpurea, which is tetraploid, but by the LGM its potential distribution expands greatly northward through south-central Mexico (Fig. 8). This taxon’s expansion might represent increased availability of suitable habitat in the LGM or it might suggest that it was created from auto- and alloploid speciation from the LIG to

LGM.

In the north, the extent of suitable habitat for B. curtipendula, as a whole, was nearly as large as it is in the current climate (Fig. 7). However, at the LGM, when temperatures were colder than they were in the LIG, approximately 50% of the northern suitable habitat had disappeared as had habitat in the Sierra Madre Occidental that runs along the border of Sonora and Chihuahua. Suitable habitat remained in portions of southern Arizona, the junction of New Mexico, Texas, and Chihuahua, and much of 50

Coahuila and the east coast of Baja California. New suitable habitats opened to the west in a swath from southern Nevada-southeastern California, in what is now the Mojave

Desert, to southwestern Arizona-northwestern Sonora. In the south, suitable habitats were now found in central Mexico in a band transecting the country from approximately

Nayarit in the west to Tamaulipas in the east. Although B. warnockii has a much smaller distribution in the LIG compared to B. curtipendula (Fig. 7), it shows a similar pattern of range dynamics in response to cooling temperatures. Suitable habitat remains in Baja

California, but is eliminated in New Mexico and Texas, the northern portion of its LIG range, while new potential suitable habitats open in isolated locations in south-central

Mexico.

From the LGM to current climate, suitable habitat for B. curtipendula, B. warnockii, and B. uniflora expanded (Fig. 7). For B. curtipendula, the taxon essentially reclaimed past suitable habitat in the northern portion of its range. Bouteloua warnockii also reclaimed past suitable habitat in far western Texas, but also expanded into New

Mexico, Chihuahua, and west-central Texas. Bouteloua uniflora maintained its predicted

LGM range and also expanded into most of Texas in the current climate. It is interesting to note that these three taxa also have patchy distributions in the Baja California peninsula in each of the three time periods. However, of these three taxa only collections referring to B. curtipendula have been found here.

Species range dynamics inferred from the optimized HSM maps are supported by the within-taxon across-time Schoener’s D overlap values, which are calculated from models using all 19 BIO variables and altitude and not HSMs optimized for each taxon 51

(Table 3). The overlap values indicate that suitable habitat for the Short- and Tallgrass clusters of B. curtipendula were greatly reduced in the LGM compared to either of the other two periods. The Southwest cluster was also reduced in size, but the Highland and

Tropical clusters increased in the LGM and further expanded in the current climate.

Similarly, B. uniflora shows more suitable habitat in the LGM and current compared to the LIG.

Bouteloua reflexa occurs at similar latitudes to B. warnockii, B. uniflora, and the southern portions of B. curtipendula’s range. Like the other taxa, suitable habitat for B. reflexa contracted from the LIG to the LGM (Fig. 8). However, the degree of contraction was minor and suitable habitat for this taxon remains stable through time, based on both optimized HSMs and suitable habitat overlap (D) among time periods (Table 3).

Bouteloua reflexa is also the only taxon that has clear phylogenetic support (Fig. 5).

Bouteloua media has suitable habitat that extends above and below 21˚ N latitude.

In the LIG, suitable habitat for B. media extended north into Baja California and coastal

Sonora and Sinaloa and southeast into the Yucatán (Fig. 8). In the LGM, suitable habitat for B. media contracted to the southwest coast of Mexico. From the LGM to current climate, B. media expanded north and east again, but habitat was no longer suitable on the east coast. Suitable habitat overlap among time periods suggests more stable climatic niches across time than the optimized HSM maps would suggest (Table 4). This indicates that the full models (19 BIO variables and altitude) used in niche comparisons picked up low values for suitable habitat that were excluded from binary threshold maps produced from optimized models. 52

In the south, B. triaena and B. disticha both have small areas of suitable habitat on

Mexico’s southwest coast in the LIG (Fig. 8). In the LGM, both taxa expand south and eastward and there is very little change in suitable habitat from the LGM to the current climate. Range dynamics in HSM maps are also seen in the across-time overlap values for each taxon (Table 3).

Contact zones, glacial refugia, diploid progenitors of polyploid taxa—Present day biodiversity and distributions are a reflection of past ecological and evolutionary events. In the Quaternary, there is evidence that climate oscillations affected community composition and the genetic structure of populations as species continually retreated and advanced in response to glacial and interglacial periods (Bennett, 1997; Williams et al.,

1998; Hewitt, 2004). We know from B. curtipendula’s present broad distribution that it occupies climatically and topographically heterogeneous environments. Assuming it had occupied its hypothesized distribution based on availability of suitable habitat in the LIG, then populations would be expected to have adapted to local conditions in different parts of the range. When climate changed and suitable habitat was reduced or shifted in space, some genetic variation would be lost as populations and lineages went extinct either from stochastic events or selection pressure, especially if climate change was rapid (Hewitt,

2004). If climate change progressed gradually then some populations might have adapted to new conditions in situ or migrated to more favorable environments. Animals transporting plant propagules as they moved in search of suitable habitats would have assisted plant migration (Jansen, 1986). Different animal species within communities may have responded to climate change differently and as a result, plant propagules from a 53 community may have ended up in very different environments leading to contact between different species, subspecies, varieties, or forms.

Hybridization followed by polyploidization could occur in these contact zones and create a mosaic of diploid and polyploid cytotypes in sympatric and parapatric populations. Habitat differentiation among cytotypes and further migrations as climate oscillated could allow repeated contact among cytotypes at different points in time and lead to introgression and reformation of polyploids from diploid progenitors. Populations at the periphery of a distribution would be expected to have complex histories, adapt to local conditions in new, refugial environments, and provide individuals for the next range expansion or recolonization. Evidence suggests that polyploids are more likely to have larger geographic ranges and colonize deglaciated regions than their diploid congeners

(Stebbins, 1985; Brochmann et al., 2004).

Patterns in the change in suitable habitat over time suggest possible contact zones among taxa and locations for glacial refugia. The far northern BCC taxa (B. curtipendula,

B. warnockii, and B. uniflora) have overlapping distributions and appear to contract and expand in a similar manner as changing climates alter availability of suitable habitat. For these taxa, contraction and expansion appear to occur in a south-southeasterly route from

USA into east-central Mexico and possibly a southwesterly route from USA into southwestern Arizona and adjacent States. As a result, contact zones for these taxa from the LIG to LGM appear in a swath from southeastern New Mexico through west Texas and into Coahuila in the east and in northern Baja California in the west. In the LGM all three taxa have overlapping distributions in northern Coahuila and states to the south. 54

These locations could represent zones of repeated contact and hybridization. It also appears that southwestern Arizona and adjacent regions of California and northern Baja

California had suitable habitat for these taxa in the LGM. Although we do not have any fossil or present collections of B. warnockii or B. uniflora from Baja California, there are two collections of B. uniflora from northwestern Arizona (Fig. 2). Therefore, it is possible that these taxa may have been present in the peninsula at some point in the past.

Clusters of biotic assemblages have been reported for the Sierra Madre Occidental and Oriental Mountains of Mexico, Peloncillo Mountains of southeastern Arizona and southwestern New Mexico, and the north-central Texas (Swenson and Howard, 2005).

Our evidence supports contact zones for all but the Sierra Madre Occidental. Locations for glacial refugia for each taxon can be inferred from their potential LGM distributions

(see LGM, Figs. 7 and 8). These locations are consistent with refugia proposed for other species (Swenson and Howard, 2005; van Devender and Spaulding 1979; Hunter, 2001;

Holmgren et al., 2007).

Evidence from HSMs supports inferences made from the phylogenetic analysis suggesting that there is a relationship between diploid B. warnockii and B. uniflora and rare occurrences of diploid B. curtipendula. These taxa were most likely involved in recurrent formation and introgression of polyploid B. curtipendula and in particular, apomictic B. curtipendula var. caespitosa. In the current climate niche similarity

(background) tests among B. warnockii, B. uniflora, and B. curtipendula Southwest,

Shortgrass, and Tallgrass clusters were conducted to assess conservatism and divergence between pairs of climatic niches. The results indicate that the Southwest cluster and B. 55 warnockii exhibit conservatism indicating that both occupy habitats as similar as possible given the available background (Table 1). Theory predicts that recently derived sister taxa would exhibit ecological similarity (Losos, 2008) and this result might support a phylogenetic relationship between B. warnockii and the B. curtipendula Southwest cluster. Alternatively, niche divergence suggests that related taxa have adapted to new environments and expanded their niches. With the exception of the Southwest vs. B. warnockii comparison (Table 1), all comparisons showed equivocal results depending on which taxon provided locality points and which provided background environmental points. This suggests that there may be an issue in selecting the appropriate background buffer size for the comparisons and/or the degree of environmental heterogeneity between climatic niches. For example, in these data, there is a trend for the taxon occupying a more heterogeneous environment to exhibit conservatism when compared to the background of a taxon occupying a more homogeneous environment, while the converse leads to a result of divergence (cf McIntyre, 2012).

Phylogenetic evidence suggests there may be a relationship between B. media and

B. curtipendula (Figs. 4 and 5) and some investigators have treated B. media as a synonym for B. curtipendula var. caespitosa (Tovar, per. comm. in Brako and Zarucchi,

1993). Bouteloua media also has a wide array of morphological forms, as does B. curtipendula (Siqueiros, 2001). Niche overlap values indicate fairly high and consistent overlap between these taxa since the LIG (D = 0.391–0.605). This suggests that it might be fruitful to examine contact zones between these taxa and look for fine-scale evidence of hybridization. 56

In the LIG, suitable habitat for B. media extended into Baja California and coastal

Sonora and Sinaloa where it appears to overlap with B. reflexa (Figs. 7 and 8). Overlap values between B. media and B. reflexa remain stable through time (D = 0.119–0.136) despite showing very little overlap in the optimized HSMs in the LGM. There is some evidence to suggest a relationship between these taxa, particularly in clade C of the ITS tree (Fig. 4, see also Siqueiros et al., in press). However, the majority of B. media individuals in clade C are from Central and South America, which would suggest a relationship further back in time than the present study considers.

Bouteloua media, B. triaena, and B. disticha have similar distribution patterns, particularly since the LGM, and have consistently high overlap values across all time periods (Fig. 8, Table 3). Niche similarity tests indicate there is niche conservatism between B. media and each of the other two taxa, but an equivocal finding between B. triaena and B. disticha (Table 1). Although phylogenetic resolution is very poor, there does not appear to be a close relationship among these taxa. However, these taxa are diploids and occur in the tropics and may lend support to the tropical conservatism hypothesis where clades in the tropics are expected to be older and more diverse than relatives in temperate zones (Francis and Currie, 2003; Wiens and Donoghue, 2004). It should be noted again that three BCC taxa with tropical distributions were not modeled because of insufficient occurrence data.

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Table S1. Taxon, collector, locality, and GenBank accessions used in phylogenetic analyses of the Bouteloua curtipendula complex. (Available on-line at http://cals.arizona.edu/research/azalfalf/GenBankAccessions.xlsx)

FIGURE LEGENDS

Fig. 1. Occurrences of Bouteloua curtipendula accessions in five environmental clusters used in this research.

Fig. 2. Occurrences of accessions from seven non-Bouteloua curtipendula taxa used in this research.

Fig. 3. Predicted suitable habitat maps derived from optimized models under current conditions for accessions from five environmental clusters of Bouteloua curtipendula and the combined map for all clusters.

Fig. 4. Maximum Likelihood tree based on the Kimura 2-parameter model inferred from

MEGA5 analysis of Bouteloua curtipendula complex nrDNA ITS sequences. Bouteloua juncea, B. americana, B. hirsuta, and B. Williamsii serve as an outgroup. Values adjacent to nodes represent bootstrap consensus from 100 replicates, branches with < 50% bootstrap support are collapsed. Species name appears at the branch tips followed by collector number and locality (see Siqueiros, 2001). Abbreviations: curt curt = B. curtipendula var. curtipendula, curt caesp = B. curtipendula var. caespitosa, curt tenuis =

B. curtipendula var. tenuis, uniflora unif = B. uniflora var. uniflora, uniflora coah = B. uniflora var. coahuilensis, A = Axelrod, C = Columbus, EP = Evans & Pablo, H =

Herrera, LD = La Doux, R = Rebman, S = Siqueiros, Sou = Sousa. State names in

Mexico and USA follow standard abbreviations.

Fig. 5. Phylogenetic relationships among Bouteloua curtipendula complex taxa inferred from Maximum Likelihood tree based on the Kimura 3-parameter model in MEGA5 analysis of the trnT-L-F region cpDNA sequences. Bouteloua juncea, B. eludens, B. 78 johnsonii, B. rigidiseta, B. karwinskii, B. americana, B. hirsuta, and B. Williamsii serve as an outgroup. Values adjacent to nodes represent bootstrap consensus from 100 replicates, branches with < 50% bootstrap support are collapsed, bootstrap percentages >

50% are shown next to the branches. Species name appears at the branch tips followed by collector name or number (see Siqueiros, 2001). Abbreviations are defined in Figure 1 legend.

Fig. 6. Predicted suitable habitat maps for the combination of five environmental clusters of Bouteloua curtipendula derived from optimized models under conditions for the last glacial maximum based on predictions of CCSM3 model (A) and MIROC model (B).

Green dots represent collection locations of B. curtipendula macrofossils recovered from packrat middens and dated to ~ 11–45 600 YBP (Van Devender, 1995; Holmgren et al.,

2003; Holmgren et al., 2006; Holmgren et al., 2007).

Fig. 7. Predicted suitable habitat maps derived from optimized models under conditions for the last interglacial, last glacial maximum (MIROC), and current conditions for the combination of five environmental clusters of Bouteloua curtipendula, B. warnockii, B. uniflora, and B. reflexa.

Fig. 8. Predicted suitable habitat maps derived from optimized models under conditions for the last interglacial, last glacial maximum (MIROC), and current conditions for

Bouteloua media, B. triaena, B. disticha, and B. purpurea.

Fig. S1. Predicted suitable habitat maps derived from optimized models under conditions for the last glacial maximum (CCSM3) for the combination of five environmental clusters of Bouteloua curtipendula and seven non-B. curtipendula taxa. 79

Fig. 1

Fig. 2

Fig. 3

curt caesp S4468 GUA MX curt caesp C2276 AZ MX 52 curt caesp S4451 PUE MX 66 curt caesp C2326 SLP MX curt caesp S4487 COA MX curt caesp C2449 AZ USA curt caesp C2500 AZ USA curt tenuis C2331 HID MX warnockii S4492 COA MX curt caesp C2191 COA MX disticha S4456 PUE MX 59 disticha C2376 MIC MX curt curt LD IA USA purpurea S4504 QUE MX purpurea S4470 GUA MX curt curt C3234 KS USA curt curt C3207 MO USA curt caesp C2909 CA USA curt caesp H1458 COA MX curt caesp C2124 TX USA curt caesp H1348 DUR MX curt caesp H1334 SON MX curt caesp C3247 AZ USA curt caesp H1343 CHH MX curt caesp S4472 ZAC MX 87 curt tenuis C3566 PERU 68 curt caesp C3189 ARGENTINA curt tenuis C2275 AZ USA 59 curt caesp S4530 PUE MX media S4442 MIC MX 76 media S436 MIC MX media S4441 MIC MX media S457 PUE MX media G12689 CHP MX 81 media S417 JAL MX media S4416 NAY MX media EP476 OAX MX 65 media S4464 DIF MX 99 media S4511 PUE MX curt caesp C2213 NM USA curt caesp S4475 CHH MX curt caesp H1345 DUR MX vaneedenii E1013 CUBA disticha C3471 PERU disticha S4537 ECUADOR 77 disticha S4539 ECUADOR 99 70 disticha S4535 COSTA RICA uniflora unif S4496 COA MX curt caesp H1307 AGU MX uniflora coah S4484 ZAC MX curt caesp S4530 PUE MEXICO 51 uniflora unif C3319 TX USA 94 72 uniflora coah C2190 COA MX uniflora coah S4489 COA MX uniflora coah C2319 ZAC MX 81 pedicellata S4526 PUE MX 61 pedicellata C2634 PUE MX distans S4449 OAX MX distans S4454 PUE MX 99 triaena C2338 DIF MX 85 99 triaena C2357 JAL MX media C2420 OAX MX warnockii C2907 TX USA 81 uniflora unif C2901 TX USA 98 warnockii C3282 NM USA 66 curt caesp S4490 COA-MX 98 reflexa S4395 SON MX 90 reflexa S4394 SON MX reflexa R3561 BCS MX 73 reflexa R3510 BCS MX 85 reflexa C2436 BCS MX 98 reflexa Sou141 CAM MX reflexa S4404 SIN MX reflexa S4401 SIN MX 54 media S4533 COSTA RICA media C3500 PERU 96 media C3513 PERU 96 65 media C3468 PERU juncea A8856 99 juncea A8862 75 williamsii C2353 99 americana W22775 hirsuta C2900

Fig. 4

curt curt 3207 curt curt 3209 disticha 3445 disticha 4534 disticha 3471 52 disticha 4421 disticha 2376 disticha 4456 disticha 3425 disticha Baltra disticha N Seymour 66 64 disticha Santiago purpurea 2337 purpurea 4470 purpurea 4504 curt curt LD warnockii 4492 curt curt 3234 curt curt 3226 curt curt 3357 media 4457 76 media 4511 media 4441 media 4464 78 61 curt tenuis 4455 media 4436 curt caesp 3247 curt caesp 4475 curt caesp 4472 curt caesp 1334 51 curt caesp 1345 curt caesp 2276 curt caesp 2326 curt caesp 2213 uniflora unif 4496 curt caesp 2500 curt caesp 4451 curt caesp 1343 curt caesp 3283 curt tenuis 4500A media 4417 curt caesp 3189 70 curt caesp 4471 curt tenuis 2499 curt tenuis 2316 curt tenuis 2331 curt x unif 2191 62 69 curt caesp 4503 uniflora unif 4486 66 curt caesp 1307 curt caesp 2124 81 curt caesp 3487 curt tenuis 3566 uniflora coah 4489 uniflora unif 3319 uniflora coah 4484 52 pedicellata 2634 74 pedicellata 4526 uniflora coah 2190 90 distans 4449 curt caesp 4530 63 uniflora coah 4531 77 pedicellata 4501 100 66 distans 4454 triaena 2338 99 100 triaena 2357 media 4416 media 2420 curt caesp 4483 51 curt caesp 4500 64 media 4533 curt caesp 3314 curt caesp 4490 86 warnockii 1492 65 warnockii 3282 uniflora unif 2901 52 86 warnockii 2907 media 3500 curt tenuis 3465 67 media 3513 media 3468 reflexa S141 78 reflexa R3510 64 reflexa 4404 reflexa 4394 80 94 reflexa 4401 juncea 8856 eludens 2272 johnstonii 2851 rigidiseta 2231 hirsuta 2900 55 89 karwinskii 2208 americana W22775 100 williamsii 2353 Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. S1

APPENDIX B: PHENOTYPIC PLASTICITY OF KEY TRAITS THAT DEFINE

INFRASPECIFIC VARIETIES IN BOUTELOUA CURTIPENDULA (POACEAE)

TO BE SUBMITTED TO: Taxon

Authors: Halbrook, Kandres1; Smith, Steven E.1; Lacey, Kathryn E.2; Benson, Matthew

J.1; Broduer, Leawna M.1; Wagner, Eric V.1

Affiliations: 1: School of Natural Resources and the Environment, University of

Arizona, 1311 E. 4th Street, Tucson, AZ 85721, U.S.A.; Email: [email protected] 2: University of New Mexico, Albuquerque, NM 87131,

U.S.A.

ABSTRACT Phenotypic plasticity is the ability of a genotype to exhibit a range of phenotypes in response to environmental variation. Differences in plastic responses to heterogeneous environments may partially explain the ecological breadth of a species. Bouteloua curtipendula (Poaceae) is a C4 perennial widespread in USA and Mexico that exhibits three growth forms—rhizomatous, stoloniferous, and caespitose, each designated as an infraspecific variety. We sought to determine if the growth forms distinguishing the infraspecific varieties represent relatively non-plastic local adaptations or if they are the result of a phenotypically plastic response to environmental conditions. We evaluated

1381 georeferenceable B. curtipendula herbarium accessions from throughout the species’ range for presentation of stolons and rhizomes and correlated trait expression to elevation and 19 bioclimatic variables. We also evaluated stolon production in four populations of the caespitose taxon experimentally in a controlled environment. Using quantitative genetic analyses, we partitioned intra-population variation in this trait into plastic and non-plastic genetic variance components. Results from herbarium collections indicate that expression of stolons and rhizomes is correlated to local environmental conditions but not to broadly defined environments in geographic space. Accessions from each of the four greenhouse-grown populations exhibited some form of stoloniferous or rhizomatous growth, with the percentage of ramets with stolons in each population varying across years, but always exceeding 50%. Accordingly, quantitative genetic analysis of stolon production indicates that phenotypic plasticity accounts for a greater proportion of stolon trait expression than does total genetic variance. Our data indicate

90 that presence/absence of stolons and rhizomes are not sufficiently reliable to distinguish the infraspecific varieties and that varietal designations based on these traits are not valid.

Keywords defoliation, caespitose, heritability, local adaptation, rhizome, stolon

INTRODUCTION

Phenotypic plasticity is reflected in the ability of a genotype to exhibit a range of phenotypes in response to environmental variation (Bradshaw, 1965; Agrawal, 2001;

Pigliucci, 2001). It is most commonly associated with morphological traits (Schlichting

& Pigliucci, 1998), but many aspects of physiology, biochemistry, and life history also have been shown to be environmentally dependent (Sultan, 2000; Nicotra & al., 2010;

Davidson & al., 2011; Levin, 2012). Differences in plastic responses to heterogeneous environments may partially explain the ecological breadth of a species (Sultan, 2001;

Gonzalez & Gianoli, 2004). For example, species with restricted ranges would be expected to have less plastic responses to environmental variation while those with widespread distributions would exhibit either greater tolerance through plasticity

(Futuyma & Moreno, 1988; van Tienderen, 1997) or maintain ecotypes differently adapted to specific habitats (Baskauf & Eichmeier, 1994; Sultan & al., 1998). If a phenotypically plastic response is adaptive, it can broaden a species’ geographic range and contribute to divergence and speciation via genetic and phenotypic accommodation

(West-Eberhard, 2003; Schlichting, 2004; Pigliucci & al., 2006). However, phenotypic plasticity that affects adaptation can greatly complicate understanding of phenotype- based systematics within a group. This is especially true in cases where selection for local adaptation may act to stabilize the expression of certain traits within individual environments, but retain the ability to express that trait differently in different environments.

The production of stolons and rhizomes are two related morphological traits

(above and belowground horizontally growing stems, respectively) known to exhibit phenotypic plasticity by modifying the spacing and timing of ramet development in response to environmental heterogeneity (Doust, 1981; de Kroon & Hutchings, 1995;

Hutchings & Wijesinghe, 1997). These traits, or growth forms, are well-known in the grasses and show environmental specificity based on abiotic and biotic factors such as soil moisture (Nelson, 1934), light (Dong & de Kroon, 1994; Dong & Pierdominici,

1995), and defoliation (Augustine & McNaughton, 1998; Briske & Derner, 1998).

Grazing-tolerant stoloniferous and rhizomatous species are found in relatively moist grasslands and both growth forms can be an effective mode of dispersal and persistence in environments with large populations of grazing animals (McNaughton, 1979, 1984), where stolons growing low to the ground avoid herbivory and rhizomes growing belowground escape it. In contrast, caespitose grasses grow in compact clusters of ramets and do not produce stolons or rhizomes. Caespitose grasses are found in many environments, but are particularly common in arid regions (Tomlinson & O'Connor,

2004). Some evidence suggests that this growth form is perpetuated because soil nutrients are disproportionately consolidated beneath ramet clusters rather than between them

(Briske & Derner, 1998). As a result, caespitose ramets can more effectively acquire belowground nutrients.

Bouteloua curtipendula (Michx.) Torr. (Poaceae) is a widespread C4 perennial that exhibits all three growth forms. Its range extends from southern Canada through the

Great Plains of USA, west into southwestern USA and through south-central Mexico

(Gould & Kapadia, 1964). It is a key component of grassland and shrubland communities in a number of ecosystems and an important forage species in some environments (Smith

& al., 2004). Three infraspecific varieties have been identified based on overlapping morphology, cytology, and geography (Table 1; for distributions see Gould & Kapadia,

1964), with the primary morphological character distinguishing the varieties being the presence or absence of stolons and rhizomes (Gould & Kapadia, 1964; Gould, 1979).

According to Gould and Kapadia (1964), Bouteloua curtipendula var. curtipendula is identified by “creeping” rhizomes. It is primarily distributed in the central grasslands of

USA and southern Canada. Bouteloua curtipendula var. tenuis is described as being endemic to Mexico with the plants in eastern Mexico having stolons or stoloniferous culms while those in western Mexico rarely having stolons but often presenting rhizomes.

Bouteloua curtipendula var. caespitosa is distinguished by its caespitose habit with rhizomes and stolons “not developed.” Variety caespitosa is found in southwestern USA through central Mexico.

Bouteloua curtipendula is part of a broader species complex, identified as the

Bouteloua curtipendula complex (BCC) (Gould & Kapadia, 1964) presently composed of

11 species and five varieties (Columbus & al., 1998; Columbus & al., 2000) distributed in

North and South America with its center of diversity in Mexico. Phylogenetic studies indicate that the BCC is monophyletic (Columbus & al., 1998; Columbus & al., 2000;

Siqueiros, 2001), but monophyly is supported for only Bouteloua triaena (Trin. ex

Spreng.) Scribn. (Siqueiros, 2001). As with Bouteloua curtipendula, circumscription in the BCC is problematic due to its large and environmentally heterogeneous range

(Halbrook & al., A in review), continuous variation, intermediate forms, ploidy variation, including aneuploidy and apomixis (Halbrook & al., B in review), and overlapping distributions (Siqueiros, 2001). The remarkable variation and the fact that two or more taxa have been found in the same population (Gould & Kapadia, 1962, 1964) have led some to question the validity of the infraspecific varieties of B. curtipendula (McVaugh,

1983; R. Felger, pers. comm.) and recent character state reconstruction analysis of stolon and rhizome presence suggests homoplasy for these traits (Siqueiros, 2001).

Given the broad ecological breadth of B. curtipendula, we asked if the growth forms distinguishing the infraspecific varieties are the result of a phenotypically plastic response to different environments experienced by genotypes from throughout the taxon’s range or due to relatively non-plastic local adaptation. We explored this question by first evaluating B. curtipendula specimens from throughout its distribution for expression of each of these traits and correlating trait expression to environmental factors at collection locations. We hypothesized that if taxon-differentiating traits are phenotypically plastic, then their expression should be consistently associated with local environmental characteristics regardless of their location within the range. This contention would also be supported by the observation of multiple character states within individuals in close proximity, although this could still simply reflect selection for local adaptation. We then examined stolon expression experimentally in four populations of the caespitose taxon, B. curtipendula var. caespitosa, when grown in greenhouse conditions significantly different from their original collection environment. We used quantitative genetic analysis to partition intra-population variation in this trait into plastic

95 and non-plastic genetic variance components. Demonstrating phenotypic plasticity within a population in this way represents the most definitive demonstration of phenotypic plasticity (Scheiner 1993).

MATERIALS AND METHODS

Herbarium collections. — We examined 1892 B. curtipendula accessions—with or without an infraspecific variety designated—from seven herbaria (ARIZ, KANU, RM,

SDC, TAES, UNM, UTC) (Table S1) and used the BioGeomancer WorkBench

(http://bg.berkeley.edu/latest/) to georeference collection locations or to confirm latitude/longitude values provided with the specimen if necessary. Specimens collected from plants labeled as having been grown under cultivation were excluded. Only accessions with verifiable map coordinates or sufficiently detailed locality information to permit georeferencing within a radius of less than 4 km were used in analysis of morphological characters (n = 1381). We excluded collections from east of –86.5° W and south of 15° N as these were often isolated at great distance from other collections and we believe may reflect introductions of B. curtipendula as a pasture crop (Smith & al.

2004) rather than sites of native occupation.

Morphological characters. — In B. curtipendula, as with other grasses, stolons and rhizomes develop from extravaginal growth where axillary buds located adjacent to the tiller axis emerge laterally through the leaf sheath (Briske, 1991). The resulting horizontal tillers elongate away from the crown where adventitious roots and new tillers can develop from axillary buds at the nodes. Stoloniferous culms are also found in B.

96 curtipendula where densely clustered buds can develop at nodes on erect culms (Miller &

Donart, 1979). Over the course of one or more growing seasons (typically spring, summer and early autumn) stoloniferous culms become decumbent placing the nodes in close proximity to the soil surface where adventitious roots can develop. Stolons were identified in greenhouse-grown and herbarium accessions based on the presence of one or more phytomers (modular component of a tiller) or buds at a node. Each developed node on a stolon or stoloniferous culm was counted as a “stolon” present on that accession.

Stolons were also grouped into classes based on the number of phytomers or buds they contained: 1–3, 4–9, ≥ 10. Rhizomes were identified by their scalelike leaves and horizontal growth or, in some cases, vertical growth into the soil profile. Length of the longest rhizome on each plant was measured.

We classified each herbarium accession as belonging to one of the three infraspecific taxa based only on the presentation of morphological characters on the specimen or simply B. curtipendula when a infraspecific variety designation could not be determined. Following Gould (1979) (Table 1), any accession exhibiting a stolon was identified as B. curtipendula var. tenuis while accessions with rhizomes and no stolons were identified as B. curtipendula var. curtipendula. In the taxonomic key (Gould, 1979)

B. curtipendula var. caespitosa is described as being in large or small clumps, some forms have a “knotty” base, and stolons or creeping rhizomes not developed. The term

“not developed” is never explicitly defined so we interpreted it as “absent.” Given that var. caespitosa is then distinguished by an absence of a trait, we arbitrarily determined that specimens had to have a basal (crown) diameter ≥ 6 cm in addition to a lack of

97 stolons and rhizomes to be classified as B. curtipendula var. caespitosa. Although this is a conservative designation, we wanted to limit the possibility of misidentifying small specimens as var. caespitosa when they may actually just be too small or young at the time of collection to exhibit stolons or rhizomes, in which case, they would belong to a different taxon. Specimens that did not have stolons or rhizomes and were < 6 cm at the base were classified as “B. curtipendula” and not included in further analyses.

Field collections. — For experimental analysis of stolon and rhizome production, grain was harvested in 2006 from approximately 60 randomly selected individual plants

(accessions) identified as B. curtipendula var. caespitosa (Gould, 1979) from each of four unique populations in southern Arizona (Table 2). In 2007 grain from each accession was sown into individual pots (15.2 x 14.6 cm) filled with 1835 mL of Sunshine Professional

Growing Mix #3 (Sun Gro Horticulture, Bellevue, Washington, USA) supplemented with sand in a 3:1 ratio (v/v) of mix to sand. This planting mix was used throughout the experiment (2007--2011). Accessions were grown in a 6.1 x 11.0 m greenhouse in the

Campus Greenhouse Facility at the University of Arizona, Tucson, Arizona, USA with hydronic heating and evaporative cooling to maintain optimal growth conditions year round. High-pressure sodium and metal halide lamps (1000W) provided supplemental light. Mean annual temperature over all years was 29˚C (Table 3). Accessions were irrigated as needed and fertilized about once per month with a 0.25% (w/v) Hoagland’s

Solution (Hoagland & Arnon, 1950) or 0.25% (w/v) Peters Professional Fertilizer 20-20-

20 (Everris, Geldermalsen, The Netherlands).

Evaluation of stolon and rhizome production under controlled conditions. —

For quantitative genetic analysis, production of stolons was assessed, as described above, in all greenhouse-grown accessions that were originally established in 2007 and still alive in 2009. The first replication consisted of years 2009 and 2010 where 43--63 accessions from four populations (Cochise, Graham, Pima-East, and Pima-West) were analyzed. At the end of 2010, all accessions were clonally propagated and grown in a completely randomized design with two replications and one year of data collection. Tillers were clipped to a height of approximately 6 cm at the end of each growing season. In 2012, accessions used for BSH estimation were removed from their pots and also examined for rhizome production using the method described for herbarium specimens.

Data analysis. —Environmental variables for collection locations (elevation and

19 bioclimatic variables, Hijmans & al., 2005) are derived from the WorldClim 1.4 dataset for the period 1950-2000 at 2.5-min resolution (worldclim.org, accessed 2010-12-

1). Linkage of environmental variables with collection locations was done using techniques for the generation of “SWD files” for use by the species distribution modeling software Maxent (Phillips & al., 1996). Distribution maps were generated using DIVA-

GIS vers. 7.3.0 (diva.gis.org, accessed 2010-12-1) and are presented using WGS 1984.

All statistical analysis was done using procedures (“PROCs”) within

Institute. SAS and all other SAS Institute Inc. Product or service names are registered trademarks of SAS Institute, Cary, NC, USA). Statistical significance of differences among multiple taxa or environments were evaluated using analysis of variance (PROC

GLM) and mean separation was with Duncan’s multiple range test. Pairwise comparisons were made using t-tests. Fisher’s Exact Chi-square test within PROC FREQ was used to analyze the frequency distribution of presence or absence of stolons and rhizomes across all populations while Spearman’s rank correlation within PROC CORR was used to assess the association between stolon and rhizome production. Statistical significance was assigned at P ≤ 0.05 for all tests.

Greenhouse environmental conditions were recorded using HOBO® data loggers

(Onset, Pocasset, Massachusetts, USA). We identified B. curtipendula collection locations with annual temperature profiles that were most similar to those of our greenhouse using Mahalanobis distances based on five temperature variables (annual mean temperature, maximum temperature of the warmest month, minimum temperature of the coldest month, and mean temperatures of the warmest and coldest quarters).

Distance calculations are based on the approach described by Gray & Hamann (2011) where Mahalanobis distance for each environmental variable was estimated from each of the 1381 collection locations to the mean of all locations and from greenhouse environmental values to that of all collection locations

(http://support.sas.com/kb/30/662.html, accessed 2012-5-1).

For quantitative genetic analysis, PROC TRANSREG was used to locate the optimum power (Box-Cox) transformation for each population and variable. Broad-sense

2 heritability estimates (H ) were computed as the ratio of VG ⁄ (VG + VE + VG x E + VW), where VG is the within-population genetic variance expected under mass selection on genotype means, VE is the environmental variance over years/environments, VE x G is the

100

genotype x environment variance, and VW is the residual variance (Holland & al., 2003).

Phenotypic plasticity was estimated as the ratio of (VE + VG x E ) ⁄ (VG + VE + VG x E + VW)

(Lavergne & Molofsky, 2007). Variance components were estimated using PROC

MIXED in SAS with all class variables (replications, years and genotypes) considered random effects. These variance components were then used to calculate phenotypic plasticity and broad-sense heritability estimates following Lavergne and Molofsky

(2007). Final estimates and confidence intervals for both values (α = 95%) were generated using delete-one genotype jackknife (non-parametric) procedures following protocols described by (Knapp & al., 1989).

RESULTS

Of the 1892 herbarium accessions examined, 1381 were georeferenceable and 820 were originally classified as belonging to one of the three infraspecific varieties while

561 had no infraspecific variety determined (Table S1). Only 810 accessions could be placed in one of the infraspecific varieties using our reevaluation scheme where specimens expressing a stolon were classified as var. tenuis, those with rhizomes and no stolons as var. curtipendula, and those without stolons or rhizomes and ≥ 6 cm diameter crown as var. caespitosa (Figs. 1, 2). Mean values for eight environmental layers based on collection locations for varieties as originally classified showed significant differences for all variables with five of eight variables—annual mean temperature, minimum temperature of the coldest month, mean temperature of the coldest quarter, annual precipitation, and precipitation of the warmest quarter—showing significant differences

101 among all three varieties (Duncan’s multiple range test, P ≤ 0.05; Table 4). In the original classification, var. tenuis was associated with the warmest and wettest environments

(annual mean temperature 19.9˚C, annual precipitation 721.7 mm), var. curtipendula the coldest (annual mean temperature 12.4˚C), and var. caespitosa the driest environments

(annual precipitation 472.7 mm). In contrast, under our reevaluated classification, there are significant differences among taxa for seven of the eight variables but only between two taxa for each variable. The general patterns for most variables remained the same, but the amplitude among environmental variables was moderated by our reevaluation scheme. Comparisons between original and reevaluated classifications for each taxon show that there are no statistical differences between environmental variables for var. caespitosa classifications, only one difference between var. curtipendula classifications, but six differences between var. tenuis classifications (Table 4).

We analyzed environmental layers for possible association with trait expression within each reevaluated taxon. For var. tenuis, we examined environmental conditions based on presence/absence of stolons, total number of stolons produced per accession as grouped into three classes consisting of 1–2 stolons, > 3 stolons, or no stolons, and the number of buds per stolon: 1–3, 4–6, and > 7. These analyses show that stolons are found in relatively warmer environments (annual mean temperature: 15.6 vs. 12.7˚C, respectively, t-test, P ≤ 0.05) and where temperatures are seasonally moderate, i.e., cooler in the warmest months and warmer in the coolest months as compared to where stolons are not found (Table 5). The number of stolons produced per accession follows this temperature trend, but the number of buds per stolon does not. Here, the only significant

102 difference between many and few buds is annual precipitation where stolons with many buds are found in significantly wetter locations (annual precipitation: 847.6 vs. 541.2 and

524.2 mm, respectively, Duncan’s multiple range test, P ≤ 0.05). Similarly, more stolons per accession are found in environments that have a significantly greater amount of precipitation in the warmest quarter (246.3 vs. 205.5 and 197.5 mm, respectively,

Duncan’s multiple range test, P ≤ 0.05). For var. curtipendula, we looked at environmental conditions based on presence/absence of rhizomes and total length of rhizomes, where rhizome length was grouped into classes consisting of no rhizomes, < 2 mm, 2.5--5 mm, 5.5--8 mm, and > 8.5 mm. Results indicate that rhizomes are generally found in cooler (annual mean temperature: 12.1 vs. 16.3˚C, t-test, P ≤ 0.05) and moister

(annual precipitation: 587.4 vs. 491.9 mm, t-test, P ≤ 0.05) locations. However, the longest rhizomes (> 8.5 cm) were found in significantly warmer and wetter locations than the other rhizome length classes (Table 6).

Greenhouse environmental conditions were significantly warmer for all variables except maximum temperature in the warmest month (35.7 vs. 36.2˚C, respectively, single-sample t-test, P < 0.05) compared to the mean environmental conditions found at the four collection locations in southern Arizona (Tables 2, 3). Based on Mahalanobis distances between temperature conditions recorded in the greenhouse and temperature values obtained for each herbarium collection location, greenhouse conditions most closely resemble environments in southeastern Texas, USA and Tamaulipas, Mexico

(e.g., TAES 236416, 151239, 160967, 99548, 171480).

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Accessions from each of the four greenhouse-grown populations exhibited some form of stoloniferous or rhizomatous growth. The percentage of ramets with stolons in each population varied across years, but was always > 50% (Table 7). In 2011 and 2012, respectively, 351/383 ramets had stolons (92 %) and 148/383 ramets had rhizomes (37

%). Only 21/383 ramets had neither stolons or rhizomes (5 %) while 137/383 exhibited both traits (36 %). Assuming stolon and rhizome presence/absence would be unassociated, Fisher’s Exact Chi-square tests indicate no relationships between the distribution of these traits among or within populations. However, there is a significant negative correlation between number of stolons produced and rhizome length among all ramets (rs = – 0.139, df = 381, P = 0.0064) with the primary difference driving this negative correlation in the Cochise population (rs = – 0.215, df = 88, P = 0.0423). There was no significant correlation between stolon and rhizome production by a given ramet when examining only the 148 ramets that had rhizomes, of which 137/148 (93 %) also had stolons. However, there was a significant negative correlation when examining all

351/383 ramets that had stolons, of which 137/351 also had rhizomes (rs = -0.1825, df =

349, P = 0.0006), but there were no significant effects within populations indicating that a sample size effect changed the significance threshold.

Analysis of variance among populations for number of stolons and rhizome length in 2011 and 2012 showed significant differences for both variables (stolons: F = 10.06, df

= 3, P < 0.0001; rhizomes: F = 36.51, df = 3, P < 0.0001). Duncan’s multiple range test

(P ≤ 0.05) revealed that Pima-East and Pima-West were significantly different from each other and the other two populations for both traits. Pima-West produced the greatest

104 mean number of stolons per accession (12.9) but had rhizomes of only moderate length

(mean: 1.1 cm). In contrast, Pima-West produced the fewest mean number of stolons

(5.8), but had the longest mean rhizome length (2.5 cm). Cochise and Graham had similar responses for both variables (stolons: 9.5 and 9.0, respectively; rhizomes: 0.2 and 0.5 cm, respectively). Cochise and Pima-West showed significant within population differences for mean rhizome length based on ANOVA (Cochise: F = 2.86, df = 24, P < 0.0004;

Pima-West: F = 1.97, df = 18, P < 0.0252). Estimates for phenotypic plasticity for the number of stolons were significantly greater than zero in all four populations. Estimates of broad-sense heritability for the same trait were greater than zero in three of the four populations (Table 7).

DISCUSSION

Our review of nearly 1900 herbarium specimens indicates that many infraspecific variety determinations were made based on geography (Gould and Kapadia, 1964; Gould,

1979) and not on the presentation of morphological characters either on the specimen or described in the record label. For example, reproductive traits overlap in these varieties

(e.g., anther color, number of spikelets per branch; Table 1; Gould & Kapadia, 1964;

Gould, 1979), but dozens of accessions containing only an inflorescence and no tissue from the root-shoot transition region were given an infraspecific variety determination. In other cases, there were specimens classified as var. curtipendula or var. caespitosa that each contained 1-cm rhizomes. The only substantive difference between these specimens was collection location. Our conservative definition of var. caespitosa for the

105 reevaluation exercise reduced the number of accessions with this designation from 255 to

114 (Table 4). Some of these specimens were reclassified as B. curtipendula with no infraspecific designation because they did not have stolons or rhizomes and the basal area was < 6 cm in diameter, but others were moved to vars. tenuis or curtipendula because they contained a stolon or rhizome, respectively. As a result, the number of var. tenuis designations increased from 26 to 105 specimens and var. curtipendula increased from

539 to 591.

Collection locations for the infraspecific varieties as originally classified indicate that var. curtipendula was clustered in the temperate grasslands of North America (Sims

& Risser, 2000), var. caespitosa is in the desert grasslands and shrublands of North

America, and var. tenuis is predominately in the matorral ecoregions of Mexico (Ricketts,

1999) (Fig 1). These ranges are quite different in their environmental characteristics. The originally described range of Bouteloua curtipendula var. curtipendula is significantly colder in the winter than that of the other two taxa and this is largely responsible for its lower annual mean temperature (Table 4). Bouteloua curtipendula var. tenuis occurs in the warmest and wettest of the environmental clusters and although the subtropical deserts where var. caespitosa is found are known for their high summer temperatures, this region is significantly colder in the winter compared to tropical regions further south.

The more xeric environments where var. caespitosa are found receive significantly less precipitation than either of the other two geographic regions. These regions and their associated environmental characteristics relate well to the morphological traits seen in the plants as one would expect rhizomatous growth in temperate grasslands that have evolved

106 with large, grazing animals, stolons in more mesic environments, and caespitose plants in more arid regions.

Collection locations of herbarium specimens evaluated and classified based on presentation of stolons and rhizomes and not geography, show that the original geographical clusters do no break apart per se but individuals representing the three taxa now appear in each of the geographic regions (Fig 2). For example, specimens with stolons (= var. tenuis) were identified in collections made in the desert and temperate grasslands, while specimens with rhizomes and no stolons (= var. curtipendula) were found in both the desert grasslands and matorral. These reclassifications moderate the environmental differences among taxa that were seen in the original classifications (Table

4). Our analysis of trait expression based on environmental layers of collection locations show that on average, stolon-producing plants are preferentially found in warm and wet environments (Table 4, reevaluated classification; Table 5, presence/absence). However, they also occur in drier environments. For example, of the 105 accessions that had stolons, 88 had only 1--2 stolons per accession and these accessions occurred in environments receiving only 519.9 mm annual precipitation compared to 653.2 mm for accessions with ≥ 3 stolons. (t-test, P ≤ 0.05). Many accessions formerly classified as var. caespitosa (arid environments; annual precipitation 465.1 mm) were reevaluated as var. tenuis (wet environments; annual precipitation 556.3 mm), which is most likely a reflection of both timing and amount of precipitation in the months prior to collection and suitable microhabitats for stolon production. For example, stolons, and especially

107 stoloniferous culms, are susceptible to loss by grazing so for these features to persist the plant would need to be in a safe site protected from defoliation.

These data indicate that the presentation of these morphological characters is not fixed in all populations of a particular environment, but rather that they show some degree of genetic variation and/or phenotypic plasticity. A herbarium specimen is a reflection of the environmental conditions experienced during the plant’s growth and development up to the point of collection. Thus, more xeric environments where var. caespitosa is typically found are generally too arid to promote stolon or rhizome production, but moist microhabitats, unusually wet growing seasons, environmental conditions that permit tiller perenniation, grazing exclusion, or precisely the right collection time (e.g., before stolons have been severed from the maternal plant) may reveal specimens displaying stolons or rhizomes.

Results from our greenhouse study support the evidence from collected specimens that stolon and rhizome production are influenced by local environmental conditions. In the field, the four populations of B. curtipendula var. caespitosa did not exhibit any stolons or stoloniferous culms, but accessions originating from grain harvested from these populations exhibited stoloniferous growth, either as typical stolons or more commonly as stoloniferous culms, when grown in a greenhouse. The percent of ramets with stolons in each population was > 50% each year from 2009--2011 and ramets with rhizomes was

> 50% in three of four populations representing growth from 2010--2012. Five environmental variables relating to temperature were all significantly different between the mean values for the field collection locations and greenhouse conditions (Table 3).

108

This difference is especially prominent in the winter months where greenhouse temperatures were warmer because hydronic heaters automatically kept temperatures above 19.0˚C to prevent low-temperature damage. Greenhouse accessions were irrigated daily approximately six months of the year and every other day for the remainder.

Although we did not measure the amount of irrigation applied, it was without a doubt tremendously greater than what field-grown plants would receive from annual precipitation. Similarly, the greenhouse was evaporatively cooled and not air- conditioned, which caused the relative humidity to be higher inside the facility than what field-grown plants would experience. Greenhouse accessions were also provided supplemental light, which had the effect of accelerating growth and development, including, most likely, stolon production. Based on Mahalanobis distances between greenhouse temperature and those from all herbarium specimens, the greenhouse temperature regime most closely resembled conditions at collection locations in southern

Texas and northeastern Mexico rather than southern Arizona.

Although we trimmed tillers to approximately 6 cm above the base of the crown at the end of each year (generally November), our observations from greenhouse-grown plants not used in the study (data not shown) indicate that individual stolons can develop many buds and become quite large if conditions allow them to perenniate (Miller &

Donart, 1979). This suggests that increased moisture as well as protection from defoliation and absence of below-freezing temperatures are important factors in the production and development of stolons. Based on our analyses of environmental variables, individulals with stolons and rhizomes occur in environments with similar

109 precipitation. All things being equal, one would expect rhizomes to predominate in environments exposed to frequent grazing, where the tissue can escape defoliation, while stolons would predominate in areas without grazing. This would be true especially for stoloniferous culms because they would be highly susceptible to defoliation when they are in their erect state. Given this, one might expect an individual plant to preferentially exhibit one trait over the other. Our analysis did find a significant negative correlation between number of stolons produced and rhizome length among all ramets (rs = – 0.139, df = 381, P = 0.0064). However, the associations were slight or non-significant when analyzed by population or by subsets of ramets with and without stolons and rhizomes indicating that the relationship is not strong.

Broad-sense heritability estimates the proportion of the phenotypic variance within a population that is the result of genetic factors, which in this case includes additive, dominant, epistatic, and maternal effects (Holland et al., 2003). This estimates genetic differences among genotypes within a population within individual environments.

In contrast, phenotypic plasticity estimates the proportion of the phenotypic variance that is the result of genotypes performing relatively differently in different environments compared to others within their population (Scheiner & Goodnight, 1984). Our analysis of stolon production in greenhouse conditions indicates that phenotypic plasticity accounts for a greater proportion of the expressed phenotype than total genetic variance

(Table 7). These data indicate that the four desert grassland populations evaluated have retained the ability to express stoloniferous growth when exposed to a suitable environment. Although there is genetic variation for this trait based on broad-sense

110 heritability and differential expression within populations, the plastic response is greater.

These data, combined with the analysis of herbarium specimens, suggest that B. curtipendula’s widespread distribution is more a reflection of phenotypic plasticity than the result of locally adapted genotypes. More extensive studies encompassing reciprocal transplant experiments with more diverse populations would be necessary to fully investigate this question.

Phylogenetic analysis of the BCC indicates that while the complex is monophyletic there is little support for monophyly of the constituent taxa (Siqueiros

2001). Three major clades were identified and B. curtipendula was represented in each clade and in particular, the three varieties of B. curtipendula were poly- or paraphyletic in all the molecular trees analyzed. Many portions of the trees contain unresolved polytomies and four of the six character states reconstructed, including stolons and rhizomes, are homoplasic. Within the BCC, and B. curtipendula in particular, there is evidence for auto- and alloploidy with ploidy levels ranging from diploid to high polyploid (10x) including aneuploids and at least one apomictic taxon (Halbrook & al., B in review). Based on molecular and morphological evidence, there is recurrent hybridization in the group. The term variety is generally used to denote a morphologically and geographically distinct population that can hybridize with other varieties if brought into contact (Singh, 2010). Careful reevaluation of herbarium specimens revealed that while the majority of accessions fit the Gould & Kapadia (1964) circumscription of varieties based on presentation of morphological characters and geographic distribution, many do not (see also Gould & Kapadia, 1962). Our greenhouse

111 evidence indicates that the presentation of stolons and rhizomes is spatatially and temporally dynamic in the four populations we examined and is most likely the result of phenotypic plasticity. Given these data, we believe the presence/absence of stolons and rhizomes are not sufficiently reliable to distinguish the varieties; and that varietal designations based on these traits are not valid. Designated varieties help reveal biological diversity and broaden our understanding of evolution and speciation within a group, but misapplied designation or varieties with overlapping morphology, ploidy, and distributions can obscure our understanding of environmental adaptation, phenotypic plasticity, and ultimately evolution. It is clear that there is a tremendous amount of variability in these taxa and the species complex is still very much in flux. A more concerted effort needs to be applied to understanding diversity, distributions, and phylogeny in this group and then make a well-informed decision regarding taxonomic circumscriptions.

ACKNOWLEDGEMENTS

The authors gratefully thank the R.L. McGregor Herbarium (University of Kansas),

Rocky Mountain Herbarium (University of Wyoming), South Dakota State University

Herbarium, S.M. Tracy Herbarium (Texas A & M University), University of New

Mexico Herbarium, and Intermountain Herbarium (Utah State University) for collection loans and M.M. McMahon, S. Hunkins, B. Brandt, and P. Jenkins of the University of

Arizona Herbarium for collections management assistance. We also thank A. Simons, A.

Anouti, & C. Loveless of the Arizona Crop Improvement Association and A. Baez & T.

112

Schmidt of the College of Agriculture and Life Sciences for use of their greenhouse facilities and S.R. Archer, J.L. Hayes, and B.M.B. Haybrook for helpful manuscript comments.

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Table 3. Comparison between mean environmental variables for original field collections and greenhouse used in analysis of stolon and rhizome expression.

Four collection Environmental variables (˚C) Greenhouse (˚C) P-value2 locations (mean) (˚C)1

Annual mean temperature 18.2 29.0 0.0029 Maximum temp warmest 36.2 35.7 0.0067 month Minimum temp coldest month 1.3 19.7 0.0046

Mean temp warmest quarter 27.2 30.6 0.0037

Mean temp coldest quarter 9.6 26.4 0.0007

1 Environmental variables for collection locations are derived from the WorldClim 1.4 dataset for the period

1950–2000 at 2.5-min resolution (worldclim.org, accessed 2010-12-01).

2 Single-sample t-test (P ≤ 0.05). Null hypothesis: mean value from four locations are equal to greenhouse value.

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Table 6. Rhizome expression in 810 reevaluated herbarium accessions of Bouteloua curtipendula.

Presence/absence2 Rhizome length3

Environmental variables1 Present Absent None < 2 mm 2.5--5 mm 5.5--8 mm ≥ 8.5 mm (n = 616)4 (n = 194) (n = 194) (n = 122) (n = 409) (n = 53) (n = 32)

Annual mean temperature 12.1b 16.3a 16.3a 11.9c 11.7c 12.7c 16.9a

Maximum temp. warmest month 32.1b 32.6a 32.6b 31.8b 32.1b 32.4b 33.7a

Minimum temp. coldest month -7.7b -0.1a -0.1a -7.6b -8.3b -6.8b -0.8a

Mean temp. warmest quarter 23.2b 23.9a 23.9b 22.8b 23.0b 23.5b 25.8a

Mean temp. coldest quarter 0.6b 8.4a 8.4a 0.6b -0.1b 1.5b 7.3a

Annual precipitation 587.7a 491.9b 491.9c 596.0b 570.1b 606.4b 743.8a

Precip. of warmest quarter 212.0a 182.9b 182.9b 219.6a 210.1a 205.3a 217.8a

Precip. of coldest quarter 67.9b 74.7a 72.6b 67.8b 63.6b 73.2b 115.1a

1 Temperatures (temp.) are in ˚C and precipitation (precip.) is in mm.

2 Means followed by the same letter are not significantly different based on a two-sample t-test (P ≤ 0.05).

3 ANOVA-derived Duncan’s multiple range test comparing differences among length classes. Means followed by the same letter are not significantly different (P ≤ 0.05).

4 Analysis includes 591 accessions reevaluated as B. curtipendula var. curtipendula and 25 accessions reevaluated as B. curtipendula var. tenuis that exhibited rhizomes in addition to stolons.

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Table S1. List of herbaria and accessions examined with original and reevaluated determinations (bocu = Bouteloua curtipendula, caes = B. curtipendula var. caespitosa, ten = B. curtipendula var. tenuis, curt = B. curtipendula var. curtipendula). (Available on-line at: http://cals.arizona.edu/research/azalfalf/Evaluated_BOCU_accessions.pdf)

FIGURE LEGENDS

Fig. 1. Collection locations for 820 Bouteloua curtipendula herbarium specimens containing infraspecific variety determinations.

Fig. 2. Collection locations for herbarium specimens of Bouteloua curtipendula based on a strict reevaluation of taxa where accessions with stolons = var. tenuis, rhizomes = var. curtipendula, and neither stolons nor rhizomes and > 6 cm basal diameter = var. caespitosa.

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Fig. 1

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Fig. 2

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HALBROOK ET AL., PSEUDOGAMOUS APOMIXIS IN BOUTELOUA CURTIPENDULA.

APPENDIX C: PSEUDOGAMOUS APOMIXIS, RELATIVE PLOIDY, AND

FERTILITY IN BOUTELOUA CURTIPENDULA (POACEAE)1

TO BE SUBMITTED TO: American Journal of Botany

KANDRES HALBROOK,2, 4 STEVEN E. SMITH,2 KATHRYN E. LACEY,3 MATTHEW S.A.

BINNEY,2 LEAWNA M. BRODUER,2 MATHEW J. BENSON,2 AND COLIN L. RIDGEN2

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1 Manuscript received ______; revision accepted ______.

2 School of Natural Resources and the Environment, University of Arizona. Tucson,

Arizona 85721, USA

3 University of New Mexico

The authors thank the following University of Arizona individuals, institutions and grants: P. Campbell, J.C. Fitch, and B. Carolus of the AZCC/ARL-Division of

Biotechnology Cytometry Core Facility for flow cytometry assistance; R. Ryan, A. Baez, and T.M. Schmidt of the College of Agriculture and Life Sciences for laboratory and greenhouse facilities; and Cancer Center Support Grant (CCSC-CA 023074).

4 Author for correspondence, [email protected].

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ABSTRACT

Premise of the study: Most sexual angiosperms produce an endosperm with a ratio of 2 maternal to 1 paternal genomes (2Cm:1Cp). This ratio has been shown to be generally necessary for normal endosperm development and failure to achieve it often leads to seed abortion. Pseudogamous apomictic plants can produce an embryo without fertilization, but require fertilization to produce the endosperm. Aneuploidy in pseudogamous species may result in irregular meiosis and variable sperm chromosome numbers that may affect endosperm development and seed formation. We asked how one putative pseduogamous apomictic aneuploid species produces endosperm and whether there is a relationship between m:p contribution to the endosperm and grain set (fertility).

Methods: We used flow cytometric seed screens to evaluate reproductive pathway, estimate embryo and sperm nucleus relative DNA content, calculate m:p endosperm ratios and then correlated these data with fertility in five populations of Bouteloua curtipendula var. caespitosa (Poaceae).

Key results: We confirmed that this taxon is aneuploid and reproduces by pseudogamous apomixis. Endosperm ratios varied from 2Cm:1.7Cp to 2Cm:2.4Cp, but there was no relationship between mean endosperm ratio and fertility. However, accessions with higher fertility also had a larger number of grain that aborted early in development.

Conclusions: Evidence suggests that maternal effects play a more prominent role in fertility than paternal effects and that paternal contribution to the endosperm, while necessary for grain formation, is not the primary factor determining grain production. The

132 results of this study will help us better understand reproductive biology, and thereby population dynamics and evolution in apomictic species.

Keywords: aneuploidy; endosperm; flow cytometry; polyploidy; sideoats grama

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INTRODUCTION Double fertilization in diploid sexual angiosperms typically yields a diploid 2C

(where C represents DNA content of the holoploid genome and Cx represents the monoploid genome with chromosome base number x; Greilbuber et al., 2005) embryo formed by the fusion of a haploid egg cell (1C) with haploid sperm nucleus (1C) and a triploid (3C) endosperm that is the product of fusion of the polar nuclei of the central cell

(1C + 1C) with a sperm nucleus (1C). In plants, genomic imprinting—expression of maternal and paternal alleles in a parent-of-origin dependent manner—has been primarily observed in endosperm where a 2:1 ratio of maternal to paternal genomes (2Cm:1Cp) is generally required for normal endosperm development (Johnston et al., 1980; Haig and

Westoby, 1991; Gehring et al., 2004; Kinoshita et al., 2008). Failure to achieve this ratio often leads to seed abortion and the phenomenon has been well documented in interploidy crosses (Thompson, 1930; Cooper and Brink, 1945; Hakansson and

Ellerstrom, 1950; Hakansson, 1956; Lin, 1984; Kermicle and Alleman, 1990) and intraploidy manipulations in maize (Lin, 1982, 1984) and Arabidopsis, where some endosperm genomic ratio imbalance is tolerated (Scott et al., 1998). Empirical and mechanistic models have been developed to predict outcomes for certain interspecific crosses and understand the molecular genetic underpinnings of the phenomenon. Some gene dosage models include polar nuclei activation/endosperm balance number

(Nishiyama and Yabuno, 1978; Johnston et al., 1980; Katsiotis et al., 1995), intragenomic parental conflict theory (Haig and Westoby, 1989, 1991), and a differential dosage hypothesis (Scott et al., 1998; Dilkes and Comai, 2004; Gehring et al., 2004).

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Plants that reproduce by aposporous gametophytic apomixis produce an unreduced megagametophyte (embryo sac) derived from mitotic divisions of a somatic cell (typically from the nucellus) without undergoing meiosis (apospory). The 2C egg cell develops into an embryo (2Cm:0Cp) without fertilization (parthenogenesis). The endosperm can develop with fertilization of the polar nuclei in the central cell of the embryo sac (pseudogamy) or autonomously without fertilization (Nogler, 1984; Asker,

1992). Pseudogamy is by far the most common process observed in apomictic plants

(Mogie, 1992). Microsporogenesis in most pseudogamous plants is regular and produces microspores with the reduced number of chromosomes (1C). However, chromosome number in each of the cells in the embryo sac is unreduced (2C). Fertilization of fused polar nuclei (4Cm) with a reduced sperm nucleus (1Cp) would yield an endosperm ratio that is out of balance with the 2Cm:1Cp seemingly necessary for normal endosperm development (Table 1). Some apomictic grass species, such as in the genera Poa and

Tripsacum, are insensitive to endosperm genome dosage and produce functional endosperm with unbalanced ratios, including 4Cm:1Cp (Grimanelli et al., 1997; Wieners et al., 2006). More commonly, apomictic plants have developed a number of adaptations to restore functional 2Cm:1Cp endosperm (Nogler, 1984; Koltunow and Grossniklaus,

2003). For example, apomictic Panicum (Poaceae) produce a four-celled embryo sac that has only one polar nucleus (2C) and when fertilized produces a 2Cm:1Cp endosperm

(Nogler, 1984). In Ranunculus auricornus (Rosaceae), two sperm nuclei fertilize the fused, polar nucleus resulting in a 4Cm:2Cp endosperm (Hörandl, 2010), while in

Dichanthium annulatum (Poaceae) one sperm nucleus fertilizes each polar nucleus

135 individually, also resulting in a 4Cm:2Cp endosperm (Reddy and d'Cruz, 1969; Nogler,

1984). Quantifying the degree of maternal to paternal genome imbalance that can be tolerated or identifying developmental modifications that restore 2m:1p functionality has practical applications for understanding reproduction and population biology in apomictic species.

Bouteloua curtipendula (Michx.) Torr. (Poaceae) is an apomictic polyploid series of three putative infraspecific varieties circumscribed by geography, continuous and discrete characters, and cytotype (Gould and Kapadia, 1964; Gould, 1979). Bouteloua

curtipendula is one of the most widespread C4 grass species in USA and Mexico (Gould,

1979) and is a key component in grassland and shrubland communities in a number of ecosystems within its broad range (Smith et al., 2004). Ploidy levels range from diploid to high polyploid with aneuploids reported for all three taxa. Bouteloua curtipendula var. tenuis is generally diploid (2n = 2x = 20), but there are records of tetraploids and aneuploids (2n = 42) (Gould and Kapadia, 1964; Gould and Soderstrom, 1970).

Similarly, B. curtipendula var. curtipendula is generally tetraploid (2n = 40), but chromosome numbers have been reported that range from 2n = 20–64 (Nielsen and

Humphrey, 1937; Fults, 1942; Freter and Brown, 1955; Gould and Kapadia, 1962;

Reeder, 1977). Variety caespitosa has chromosome numbers estimated to be from 2n =

58–103 (Fults, 1942; Gould and Kapadia, 1964; Reeder, 1971). However, given the small size and large number of chromosomes in this species, cytological study is difficult

(Harlan, 1949; Bryant, 1952) and the higher counts might best be considered estimates.

Both sexual and apomictic forms have been identified in B. curtipendula and a number of

136 cytological investigations have characterized sporo- and gametogenesis. In apparently sexually reproducing plants, meiotic pairing and divisions appear normal in microsporogenesis (Freter and Brown, 1955; Gould and Kapadia, 1962). In higher chromosome-numbered plants, there is no- to low chromosome pairing and meiotic divisions are unbalanced. During division I only a few chromosomes separate at the metaphase plate and move toward one pole, while the remainder stay at the plate. The result is one cell containing a large percentage of the total chromosomes and one cell with but a few chromosomes (Harlan, 1949; Bryant, 1952; Freter and Brown, 1955;

Gould and Kapadia, 1962). Freter and Brown (1955) also observed instances where a spindle forms in division I, but no chromosomes separate; two cells result, but one is anucleate. Division II can be regular with each cell’s chromatids separating and moving to opposite poles. The resulting tetrads consist of two small cells with only a few chromosomes each and two large cells containing nearly the unreduced chromosome number. However, it is common for the larger of the two cells in division I to produce multiple, irregularly shaped sporads in division II (Harlan, 1949; Freter and Brown,

1955). The ultimate result of meiosis can be anything from diads to octads (Harlan,

1949). Pollen diameter in apomictic plants has been shown to have a bimodal distribution with wide variance (Gould, 1959; Kapadia and Gould, 1964), which suggests that there are two classes of sporads.

Past cytogenetic studies agree that megasporogenesis initiates with the differentiation of an archesporial cell within the nucellus (Harlan, 1949; Bryant, 1952;

Mohamed and Gould, 1966). As with microsporogenesis, division I in megasporogensis

137 is asymmetrical with the majority of chromosomes going to one cell (Harlan, 1949;

Bryant, 1952). Division II, if it occurs, is apparently regular. Mohamed and Gould (1966) observed the initiation and development of a nucellar cell into an eight-nucleate embryo sac; however, they report that the megasporocyte collapsed before the onset of meiosis or before the onset of division II. Bryant (1952) observed instances of the disintegration of the four megaspores both before and, concurrently with, the development of a nucellar cell into an eight-nucleate embryo sac. Mature three-nucleate embryo sacs that appear to lack antipodals and synergids have also been reported (Mohamed and Gould, 1966).

Evidence from these studies indicate that high polyploid-aneuploid B. curtipendula, generally identified as B. curtipendula var. caespitosa, reproduce via gametophytic apomixis whereby the embryo sac develops without meiosis (apomeiosis) and the egg cell develops parthenogenetically (Nogler, 1984; Asker, 1992).

The mechanism for endosperm formation in B. curtipendula has been less well characterized. Bryant (1952) reported that the polar nuclei fused and divided to form a cellular endosperm, but she could not determine if fertilization was necessary for endosperm formation. Mohamed and Gould (1966) did not observe fusion or fertilization of the polar nuclei, but they did observe two sperm nuclei in the central cell. These results are inconclusive and leave open the possibility that endosperm could form autonomously, i.e., without fertilization. However, evidence from emasculation experiments suggests that fertilization is required for endosperm development and therefore, the apomictic plants are pseudogamous (Freter and Brown, 1955).

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A number of species in the genus Bouteloua, including sexual B. curtipendula var. caespitosa, are known for their high variability in fertility—as measured by number of grains produced per floret—and germination percentage (Harlan, 1949; Freter and

Brown, 1955). The apomictic forms have been reported to have higher fertility, but our observations (Smith et al., 2000), and others (H. Dial, USDA-Natural Resource

Conservation Service, personal communication), suggest that only certain individual genotypes of putative apomictic B. curtipendula var. caespitosa have fertility similar to cultivated varieties of B. curtipendula var. curtipendula (Boe and Gellner, 1990).

Polyploidy is recognized as an important mechanism in plant evolution (Soltis and Soltis, 1999), but our knowledge of reproduction in polyploid and apomictic species complexes is more limited (Whitton et al., 2008), especially in apomictic aneuploids.

Bouteloua curtipendula is widely distributed in North America and inhabits diverse environments (Halbrook et al., in prep.), but without a detailed understanding of

Bouteloua reproductive biology we cannot begin to understand its population dynamics and possible evolutionary trajectories. In our research we asked whether there is a relationship between fertility and either the embryo to endosperm DNA ratio or the ratio of maternal to paternal contribution to the endosperm. Also, are fertile apomictic genotypes euploid and do they produce endosperm autonomously or are they pseudogamous? To address these questions we used flow cytometric seed screens (Matzk et al., 2000) to evaluate reproductive pathway and estimate embryo and sperm nucleus relative DNA content and correlated these data with grain set experiments in putative apomictic plants from five B. curtipendula var. caespitosa populations from Arizona.

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MATERIALS AND METHODS

Plant material —Grain was harvested in 2006 from approximately 60 randomly selected individual plants (accessions) in each of five populations identified as B. curtipendula var. caespitosa (Gould, 1979) from southern Arizona (Table 2). In 2007 grain from each accession was sown into individual pots (15.2 x 14.6 cm) filled with

1835 mL of Sunshine Professional Growing Mix #3 (Sun Gro Horticulture, Bellevue,

Washington, USA) supplemented with sand in a 3:1 ratio (v/v) of mix to sand. This planting mix was used throughout the experiment (2007–2011). Accessions were grown in a 6.1 x 11.0 m greenhouse in the Campus Greenhouse Facility at the University of

Arizona, Tucson, Arizona, USA with hydronic heating and evaporative cooling to maintain optimal growth conditions year round. High-pressure sodium and metal halide lamps (1000W) provided supplemental light. Mean yearly temperature over all years was

29˚C; during the March–October primary flowering season the mean temperature was

30˚C (daily mean low and high: 25˚C and 35˚C). Accessions were irrigated as needed and fertilized about once per month with a 0.25% (w/v) Hoagland’s Solution (Hoagland and

Arnon, 1950) or 0.25% (w/v) Peters Professional Fertilizer 20-20-20 (Everris,

Geldermalsen, The Netherlands).

Flow cytometry—The distribution of relative fluorescence intensity of nuclei from grain (flow cytometric seed screen, FCSS) was used to infer reproductive mode (Matzk et al., 2000) and estimate embryo and sperm nucleus relative DNA content. Grain from 51 accessions were initially run as replicates in sets of three separate days, but given the abnormal meiosis and potential for facultative apomixis in this species, the majority of

140 grain replicates were run in a single day to eliminate day-to-day variation that might complicate analysis of DNA content. Additionally, grain from accessions where we had an ample (> 10) grain sample was dissected and embryos and endosperm were analyzed separately. Twelve ovaries (ca. 5–28 d post-anthesis) from one accession were analyzed to estimate reproductive mode and relative DNA content of embryos in physiologically immature grain. Two external grain controls of B. curtipendula cv. Vaughn (2n = 2x = 40 confirmed by chromosome counts of root tip squashes) were included in each day’s analysis. The order that grains or ovaries were analyzed was randomized each day.

Individual grains (mean 680 μg) or, separated, mature embryos and endosperm, were ground (10–15 s) between the flat sides of two diamond-coated cutting discs

(Dremel, Mount Prospect, Illinois, USA; Chicago Electric Power Tools, Camarillo,

California, USA). This technique yielded more nuclei from single grains in a shorter amount of sample preparation time than did chopping with a razor blade (Matzk et al.,

2000). It also did not leave residue, as did grinding using various grades of sandpaper

(Matzk et al., 2005) and emery cloth. Ground tissue was rinsed from the disks with 150

μL of ice-cold Otto I buffer (0.1 M citric acid, 0.5% (v/v) Tween 20), incubated on ice for 90 s, filtered through a 50-μm nylon mesh, and suspended in 600 μL Otto II buffer

(0.4 M Na2HPO4•12H20) supplemented with propidium iodide (100 μg/mL) and RNAse

(50 μg/mL). Ovaries representing different stages of development were harvested between 0930 and 1030 h, chopped 30-75 times—depending on stage of development, e.g., early milk to late dough—with a sharp razor blade in 100 μL Otto I, incubated and filtered, as described above, and added to 400 μL Otto II containing propidium iodide (50

141

μg/mL) and RNAse (50 μg/mL). Optimal concentrations of stain and volumes of buffer were determined empirically. Partec CyStain PI Absolute P nuclei extraction and staining kit (05-5022) was also suitable for nuclei isolation (Partec GmbH, Münster, Germany).

All nuclei suspensions were stored on ice for 2.5 h before acquisition of generally 5500 nuclei (range 2500–10 000) with a FACScan (Becton Dickinson, San Jose, California) equipped with a 488 nm argon ion laser and CELLQuest software. Flow cytometer standard (FCS) file data were gated with FlowJo (version 7.6.5, Ashland, Oregon, USA) and exported as a text file for data analysis.

Self-compatibility and autonomous endosperm production—Self-compatibility and autonomous endosperm production were assessed with a bagging and emasculation experiment performed on 37 randomly selected accessions from the five populations. For each accession, one immature inflorescence (with > 25 spicate branches) was left intact and covered with a maize ear shoot bag (selfed) and one was emasculated by removing the distal 1–2 cm from each spikelet with fine scissors and extracting immature anthers with forceps. Emasculated inflorescences were then bagged to limit moisture loss. All other inflorescences on each accession were left open pollinated. Accessions were placed in close proximity to each other and in an area of the greenhouse where air movement would facilitate pollen dispersal. Bagging and emasculations were performed during a two-week period in late July and early August and data were collected in mid-November

2011. We dissected all spikelets (each spikelet contains one fertile floret) from emasculated and selfed inflorescences and a random sample of spikelets from open- pollinated inflorescences equal to the number of selfed spikelets analyzed (mean per

142 inflorescence = 88.1). For each inflorescence we recorded the number of physiologically mature and immature grains, i.e., grains that had collapsed in the milk or dough stage of development (relating to globular, scutellar, or early coleoptilar stages of embryo development).

Euploidy and fertility—Relative Cx-values for embryo G0/G1 were binned into multiple euploid classes. For octoploids (8x) the bins classes contained grains with embryo G0/G1 values 7.5–8.5Cx, 7.6–8.4Cx, 7.7–8.3Cx and 7.8–8.2Cx. Only one class,

9.5–10.5Cx, was tested for the decaploids due to the small number of individuals in this range.

Broad-sense heritability of fertility—We used broad-sense heritability (BSH) to estimate the degree to which phenotypic variation in fertility within a population is due to

(maternal) genetic factors. Randomly selected individual accessions were clonally propagated and grown in 1) a randomized-complete block design with 20 accessions

(Yuma) and 15 accessions (Pinal) with 12 replications and two years of data collection and 2) a completely randomized design with 12 accessions each (Pima-West, Pima-East, and Graham) with two replications and two years of data collection. Replicates were organized to promote pollen dispersal, as above. Data on mature and immature grain were recorded for over 87 000 harvested spikelets.

Data analysis—Flow cytometer data are typically depicted as histograms showing the frequency distribution of particles interrogated and analyzed by the cytometer for an individual parameter, e.g., relative fluorescence (Robinson and Grégori, 2007). Ideally, there will be a single distinct peak occupying a single fluorescence channel that

143 represents the population of interest. In most situations histograms involve a distribution of relative fluorescence intensities around a peak that can be described by summary statistics. Analysis of flow cytometry results that include all individual events (rather than simply means for selected channels within a distribution) are ideal since variation within the distribution may be useful in interpreting results. However, the large sample sizes

(number of events) associated with this approach limits the utility of typical inferential statistics where even very small absolute differences will be shown to differ significantly using common statistical tests. Recognizing this, we calculated effect sizes using Cohen’s d (Cohen, 1988) to assess the strength of relationship between variables among different distributions. Cohen’s d was calculated using a script written for SAS (SAS/STAT® software, version 9.3 of the SAS System for Windows. Copyright© SAS Institute. SAS and all other SAS Institute Inc. Product or service names are registered trademarks of

SAS Institute, Cary, NC, USA).

Flow cytometric seed screens of sexually produced grain generally reveal a minimum of three peaks that represent embryo G0/G1 (2C), endosperm G0/G1 (3C), and embryo G2 (4C) (Fig. 1). In aneuploid, pseudogamous apomictic plants, it is possible for the endosperm G0/G1 and embryo G2 peaks to overlap (Fig. 2A). To distinguish partially overlapping peaks (Fig. 2B) we devised the following protocol. Exported Flow

Cytometry Standard (FCS) file data summarizing each FCSS (relative fluorescence intensity binned into channels and number of events) were visualized as a histogram using PROC UNIVARIATE in SAS (SAS Institute Inc. 2010). Approximate channel limits for regions of interest (i.e., embryo G0/G1, embryo G2, endosperm G0/G1) were

144 identified from this initial histogram. Each region was delineated by iteratively inspecting histograms that excluded channels outside the approximate boundaries of a normal distribution with mean and median values most similar to those in the observed peak.

After identifying the embryo G0/G1 region, a second histogram was produced that focused on possible endosperm G1/embryo G2 events. These typically included the channel that equaled twice the embryo G0/G1 mean channel (hence, embryo G2). In cases where the mean of this distribution was close to that of twice the embryo G0/G1

(Cohen’s d < 0.35; Cohen, 1988) and only a single peak was apparent, no further analysis was done. In all other cases, this distribution was reconstructed by generating two partially overlapping normal distributions using the RAND function in SAS with number of events, mean, and standard deviation as variables. The total number of events was initially divided equally with half placed in each of the two distributions and their means and standard deviations estimated based on the appearance of the combined histogram.

The goal was to iteratively reconstruct the combined distribution by simultaneously adjusting the three variables (number of events, mean, standard deviation) within each of the overlapping distributions. Because each iteration represented a new random sample, at least four consecutive iterations were required where the mean, standard deviation, peak channel, and median values of the combined overlapping distributions each differed by < 1% from that of the original combined histogram.

To generate the ratios endosperm:embryo G0/G1 and embryo G2:embryo G0/G1 we generated random pairings of individual events in each distribution using the

SURVEYSELECT function (with replacement) within SAS. The number of ratios

145 produced equaled the number of events in the smaller of the two distributions. The deviation between the mean of all these ratios and an expected ratio (i.e., 1.5 for endosperm:embryo G0/G1 and 2.0 for embryo G2:embryo G0/G1) was evaluated based on the effect size measured with Cohen’s d. Relative DNA content, expressed as mean fluorescence intensity in a peak, was estimated by dividing the observed mean channel number by the mean embryo G0/G1 value observed for tetraploid controls over all runs.

Cohen’s (1988) general rules regarding approximate effect size strength and values of d are 0–0.3 small, 0.4–0.6 medium, and > 0.8 large.

Mean fertility from self- and open-pollinated inflorescences among populations was analyzed using the Wilcoxon signed rank sum test (population pairs) or Kruskal-

Wallis test (> two populations) within PROC NPAR1WAY of SAS. Mean separation in

Kruskal-Wallis tests was done using a SAS macro implementation of a multiple comparison post hoc (Dunn’s) test (Elliott and Hynan, 2011). This test was also used to analyze mean fertility among aneuploids and euploid classes and mean embryo G0/G1 relative DNA content between counts of mature and immature grain from all experiments. Spearman’s rank correlation coefficients generated by PROC CORR of

SAS was used to assess the association between self- and open-pollinated fertility, production of mature and immature grains, and the ratio of endosperm:embryo DNA content to fertility. Statistical significance was assigned at P ≤ 0.05 for all tests.

For BSH estimates before any genetic analysis, PROC TRANSREG was used to locate the optimum power (Box-Cox) transformation for each population and variable

(SAS Institute Inc. 2010). BSH estimates are those expected under mass selection on

146 genotype means over years (Holland et al., 2003). Computations of heritability estimates and their standard errors were done using PROC MIXED and IML routines in SAS based on example code presented by Holland et al. (2003) and with all class variables

(replications, years and genotypes) considered random effects. Given that typical standard errors associated with heritability estimates may be unreliable, we also used the initial estimation routines to generate delete-one genotype jackknife (non-parametric) heritability estimates and confidence intervals following protocols described by (Knapp et al., 1989), which are the values reported.

RESULTS

Grains from the tetraploid sexual control (B. curtipendula cv. Vaughn; n = 19) consistently showed three peaks representing the embryo G0/G1 peak (mean = 4.0Cx), endosperm G0/G1 (mean = 6.1Cx), and embryo G2 (mean = 8.2Cx). The 154 apomictic plants (embryo G0/G1 mean = 8.8Cx, range: 7.5–10.3Cx; embryo G2 mean = 17.6Cx, range: 15.0–20.5Cx) show either two or three peaks (Table 3). Analyses from dissected grain, where embryo and endosperm were screened separately, indicate that the endosperm DNA content varies considerably among grains and that the endosperm overlaps the embryo G2 in two-peak histograms (Fig. 3A, B). Ninety-four of 154 grains exhibited three discernable peaks (e.g., Fig. 2B, Table 3) with mean endosperm 16.9Cx

(range: 11.0–22.3Cx). Expressed as a ratio of endosperm to embryo, the mean endosperm value was 1.9 (range: 1.3 to 2.4) (Fig. 4). There were 60 two-peak histograms. The variance associated with the embryo G2 peak in these grains (CV, mean = 5.11%, range:

147

3.02–7.29%) was considerably greater than seen in the embryo G0/G1 peak (CV, mean =

4.02%, range: 2.90–5.24%) (mean CVs, Wilcoxon paired-sample (signed rank sum) test,

S = –2223.5, n = 94, P ≤ 0.0001), which suggests that there are two underlying peaks that have similar, but not identical, DNA content.

Of the 12 ovaries (physiologically immature grain) analyzed, two were collapsed at the time of harvest and flow cytometry data showed only a scattering of cells for these samples. Analysis of the remaining 10 ovaries, two of which were deflating and in the process of collapsing, revealed that a much broader range of embryo G0/G1 relative

DNA content is found in ovaries than in mature grain (immature grain mean 7.9Cx, range: 5.5–9.6Cx; mature grain mean 8.2Cx, range: 8.1–8.4Cx) (Table 4). Three of the lowest values in the immature grain range (5.5Cx, 6.5Cx, 6.7Cx) were outside of the range found in any of the 154 apomictic grains analyzed. However, all comparisons of mean Cx between immature collapsed, immature not collapsed, and mature grain were insignificant at P ≤ 0.05 using the Wilcoxon signed rank sum test. Five of the 10 samples had quantifiable endosperm and the endosperm:embryo ratios for each of these was likely above 2.0 (see Table 4).

There was no difference in mean self-fertility among the five populations (Χ2 =

7.34, df = 5, P = 0.197), but there was a significant difference in mean open-pollinated fertility (Χ2 = 11.76, df = 5, P = 0.03). Dunn’s post hoc test indicated that only Pima-East and Yuma had significantly different means (q = 3.13, P ≤ 0.05). There were no significant differences in fertility between aneuploids and any of the euploid classes. The

Χ2-statistic among the various bin classes ranged from 0.731–4.79 for three-bin

148 comparisons (df = 2, P > 0.05) and the W-statistic from 71–176 in paired bin comparisons

(df = 1, P > 0.05). There was no correlation between fertility and the ratio

endosperm:embryo for grains with three-peak histograms (rs = 0.094, df = 62, P = 0.464) or grains with two-peak histograms where the embryo G2 served as a proxy for the

endosperm value (rs = – 0.177, df = 35, P = 0.295). However, there was a significant positive correlation between self- and open-pollinated fertility (rs = 0.326, df = 35, P =

0.049) and between mature and immature grain production (rs = 0.330, df = 972, P ≤

0.0001). Jackknife estimates of broad-sense heritability for fertility indicate that this trait is heritable in four of the five populations. Estimates range from 0.53 (Pinal, n = 166,

95% confidence limits (CL) = 0.36–0.70) to 0.96 (Graham, n = 18, CL: 0.88–1.0) (Table

5).

DISCUSSION

Flow cytometry data allow us to make inferences about the structure of the embryo sac in apomictic B. curtipendula var. caespitosa. Embryological studies have reported nucellar cells developing into eight-nucleate (Bryant, 1952; Mohamed and

Gould, 1966) and three-nucleate (Mohamed and Gould, 1966) embryo sacs that appeared to lack synergids and antipodals. In both of these conditions, fusion of two polar nuclei in an unreduced embryo sac would create a 4Cm central cell. If, for example, this cell were fertilized by a reduced sperm nucleus (1Cp), the result would be a 5C endosperm (Table

1). Our data suggest that a) only one of the two polar nuclei is fertilized or b) embryo sacs actually have one-nucleate central cells because estimated endosperm relative DNA

149 content ranged from 3.7C to 4.4C (Fig. 4). Microsporogenesis in this taxon generally produces two microspores containing nearly the unreduced number of chromosomes

(Harlan, 1949; Bryant, 1952; Freter and Brown, 1955; Gould and Kapadia, 1962).

Therefore, fertilization of one unreduced polar nucleus (2C) with one nearly unreduced sperm nucleus (e.g., 1.7C) would yield a 3.7C endosperm. Grains where endosperm is above 4C, or a ratio of 2.0 endosperm:embryo, suggest that the pollen donor was an individual with a higher chromosome number than the maternal parent.

Flow cytometry of grain nuclei support previous cytoembryological evidence that

B. curtipendula var. caespitosa reproduces by apomixis and our data indicate that the mode of reproduction is pseudogamous apomixis (Fig. 2B). The grains where there was no discernable endosperm peak might suggest autonomous endosperm production, where fusion of polar nuclei in an unreduced embryo sac followed by parthenogenesis would lead to a 4C endosperm. A 4C embryo G2 and 4C endosperm would overlap and be indistinguishable in a histogram from a whole-grain preparation. However, the relatively wide embryo G2 peaks (cf. Fig. 1, Fig. 2A) and non-normal distributions found in combined two-peak histograms suggest that there are two overlapping peaks in the embryo G2 vicinity. This, coupled with the fact that the majority of grains examined had an evident endosperm peak, strongly suggests pseudogamy.

Sperm contribution to the endosperm varied considerably in apomictic accessions with endosperm:embryo ratios ranging from 1.7–2.4 in grains with three discernable peaks (mean = 1.92) (Fig. 4). There is no evidence for a 2Cm:1Cp endosperm ratio.

Given that the observed aneuploidy is most certainly due to the irregular meiosis in the

150 microsporocyte, the ratio would best be described as 1:1 (2Cm:2Cp) with a tolerance for genomic imbalance away from the typical 2m:1p. Genomic imprinting and absolute requirements for maternal and paternal contributions to endosperm have been found to be quite flexible (reviewed in Raissig et al., 2011) and 1m:1p ratios have been reported in

Tulipa spp. (Liliaceae) (Mizuochi et al., 2009) and Arabidopsis (Scott et al., 1998).

Assuming that all grains analyzed by flow cytometry were germinable had they not been destructively sampled, each analysis shows the outcome of a functional reproductive event. Therefore, we cannot predict what endosperm dosage ratios, if any, would be intolerable. To address this question one would need to analyze grains early in development during the peak stage of endosperm proliferation.

Evidence for facultative apomixis is suggested by the presence of one grain that may have been the product of sexual reproduction (cf. Fig. 2C, D). The embryo DNA content of Yuma 26-3 (8.4Cx) was considerably smaller than Yuma 26-2 (9.0Cx,

Cohen’s d = 1.65) and moderately smaller than Yuma 26-1 (8.7Cx, Cohen’s d = 0.86).

Flow cytometry from root tissue of the maternal plant producing these grains indicates that the somatic Cx-value for this accession is 9.0Cx (8.3–9.8Cx, n = 3; data not shown,

Halbrook and Smith, in prep.) and most closely matches Yuma 26-1 and Yuma 26-2.

Yuma 26-3 also had an endosperm to embryo ratio of only 1.3. The smaller embryo size coupled with a smaller endosperm than that seen in all other grains suggests Yuma 26-3 was the product of a meiotically reduced embryo sac. Previous studies in this taxon report that megasporogenesis, like microsporogenesis, produce two megaspores with nearly the unreduced chromosome complement and two with but a few chromosomes each (Harlan,

151

1949; Bryant, 1952). Therefore, if a nearly unreduced egg cell were fertilized by a nearly unreduced sperm nucleus that originated from a plant with a similar somatic Cx-value

(from self- or, cross-pollen), the result would be an embryo with a Cx-value less than that of the maternal parent. If the egg cell were fertilized by a sperm nucleus from a plant with a larger somatic Cx-value, the result might be an embryo with a larger Cx-value than the maternal plant, depending on the relative Cx-values of the two gametes. Likewise, if the central cell contained two nearly unreduced polar nuclei fertilized by a nearly unreduced sperm nucleus, the endosperm to embryo ratio would more closely approach the 1.5 ratio seen in sexual reproduction from a meiotic embryo sac. Alternatively, Yuma 26-3 might have been the product of an aposporous embryo sac and the central cell fertilized by a sperm nucleus with a very small DNA content (e.g., a microspore containing only a few chromosomes). However, this does not explain the presence of the lower DNA content embryo.

Apomictic populations have been shown to contain substantial genetic variation derived from initial and introgressive hybridization, facultative apomixis, polyploidization, mutation, and immigration (Ellstrand and Roose, 1987; Hamrick and

Godt, 1989; Widén et al., 1994; Hörandl and Paun, 2007; Silvertown, 2008). Molecular

(Siqueiros, 2001), morphological, and cytological evidence (Freter and Brown, 1955;

Gould, 1959; Gould and Kapadia, 1962, 1964; Mohamed and Gould, 1966) suggest B. curtipendula var. caespitosa has had multiple origins and recurrent formation via auto- and allopolyploidy and introgression among related species. Our evidence suggests that a rare sexual event (1/154 grains) can create a unique cytotype. Over time, facultative

152 apomixis can increase cytotype diversity in a population, which may allow the population to adapt to new environments (Asker, 1992; Richards, 2003) while maintaining fit genotypes through apomixis. Each population studied had a relative embryo G0/G1 Cx- value that spanned 1.5–2 Cx: Graham: 7.6–9.5Cx, Pima-East: 7.5–9.0Cx, Pima-West:

7.7–9.7Cx, Pinal: 8.2–10.3Cx, and Yuma: 7.7–9.7Cx.

Our relative Cx-value estimates, when converted to chromosome number

(chromosome number = apomictic Cx-value/tetraploid control Cx-value • 40; 2n = 76–

104), are consistent with chromosome numbers reported for this taxon from microsporocytes (2n = 53–103; Fults, 1942; Gould and Kapadia, 1964a; Reeder, 1971).

However, both of these values are estimates due to potential error in counting large numbers of chromosomes from squashes and the assumption that all chromosomes are a similar size in flow cytometric analysis. In the beginning we asked if fertile apomictic accessions are euploid. Although estimation of euploidy in aneuploid individuals is limited by the variance inherit in flow cytometric data, our binning procedure where we pooled relative Cx-values that straddled the euploid values (e.g., 2n = 8x = 80, 2n = 10x =

100) indicates euploids or near euploids are not significantly more fertile than aneuploids.

Broad-sense heritability estimates are specific to a population and an environment and measure the proportion of the phenotypic variance that is the result of genetic factors

(Holland et al., 2003). An estimate for levels of grain production greater than zero indicates that there is a maternal component to the observed differences in fertility. These estimates assess average performance of the maternal parent given the same pollen population. Under these conditions, certain maternal plants will, on average, better

153 execute a cascade of events leading to grain development and provisioning than other plants.

We also asked if there was relationship between mean fertility and the ratios endosperm:embryo or maternal:paternal contribution to the endosperm. We found no correlation between fertility and these ratios and, given the evidence that apparently only one polar nucleus is fertilized or, the central cell is uninucleate, the two ratios are ultimately the same. We did find a significant positive association between self- and open-pollinated fertility with some accessions showing high relative fertility under both mating types while others were completely infertile. The presence of self-fertile accessions reveals that self-incompatibility barriers have broken down in these individuals (reviewed in Hörandl, 2010). Sexual relatives of apomictic species are usually self-incompatible (Asker, 1992) and evidence from the sexual tetraploid B. curtipendula var. curtipendula, the most agronomically important and studied close relative to B. curtipendula var. caespitosa, indicates that it is cross-pollinated (Harlan, 1950; Smith et al., 2004). The fact that only certain individuals showed self-compatibility suggests that either this trait has not developed in all individuals within these populations or that some other factor limits its expression, such as lack of a functional embryo sac. We did not test whether the endosperm in grain derived from open-pollination was from self- or cross- pollen.

There was a positive association between the production of mature and immature grain in all accessions used in the various experiments. Thus, accessions with higher fertility (mature grain production) also produced a larger number of grain that aborted

154 early in development (immature grain production). It appears that the most fertile accessions have the ability to initiate more embryo sacs—sexual or aposporous—than less fertile accessions, because if the aborted grain were due solely to endosperm imbalance one would expect to see no correlation or a negative correlation between the two factors. However, there is some evidence that aborted grain might be the result of an extreme endosperm imbalance. Preliminary evidence from flow cytometric analysis of ovaries (immature grain) reveals that 4/10 immature grain were most likely the result of a meiotic embryo sac because their mean embryo G0/G1 relative DNA content was much lower than that seen in mature grain from this accession (6.4Cx vs. 8.2Cx, respectively).

Two of these four immature grain had quantifiable endosperm peaks and in both of these cases their endosperm:embryo ratios were above 2.0. If these embryos were the result of a meiotic embryo sac, one would expect a functioning endosperm:embryo ratio close to

1.5. Of the remaining six immature grain, four had embryo G0/G1 relative DNA contents

> 9.0Cx (mean 9.4Cx). One of these immature grains, with an embryo G0/G1 of 9.6Cx, was collapsing at the time of preparation (no identifiable endosperm peak), but so too was an immature grain with an embryo G0/G1 of 8.1Cx and an endosperm:embryo ratio of 2.1. This latter immature grain had embryo and endosperm values consistent with those seen in mature grain from this accession. Thus, neither the relative DNA content of the embryo or the endosperm:embryo ratio can easily explain why some grains survive to physiological maturity and others do not. We know that 4/12 immature grains were collapsed or collapsing at the time of harvest, but we have no way of knowing how many of the remainder would have survived. Given a mean of 0.22 grains/spikelet for this

155 accession over two years, we would expect only 3/12 immature grains examined to survive. Of course, the ovary sample size was small and taken from only one accession, but the results are striking because they suggest that there is tremendous range in reproductive behavior in this accession and perhaps in this species.

The combined results from BSH estimates of fertility, the positive correlation between immature and mature grain production, and the breadth of embryo relative DNA content found in immature grain suggest that maternal effects play a more prominent role in fertility than do paternal effects because without a functional embryo sac, there is nothing to be fertilized. It would appear that paternal contribution to the endosperm, while necessary for grain formation, is not the primary factor determining grain production. These results, combined with our evidence that a range of endosperm:embryo values can produce functional endosperm and that some accessions are self fertile, are important because they provide insight into the little known reproductive behavior of an aneuploid apomictic species. This will help guide studies in population dynamics and evolution in apomictic species as well as help restoration ecologists develop effective strategies to improve grain production in B. curtipendula for use in rehabilatation of grassland and shrubland communities in North America.

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FIGURE LEGENDS

Fig. 1. Flow cytometric grain screen of a sexually produced Bouteloua curtipendula var. curtipendula cv. Vaughn.

Fig. 2. Flow cytometric grain screens of Bouteloua curtipendula var. caespitosa showing,

A. embryo G0/G1 (left arrow) and overlapping endosperm G0/G1 and embryo G2 peaks

(right arrow), B. embryo G0/G1 (left arrow) and partially overlapping endosperm G0/G1 and embryo G2 peaks (right arrows), C. a sexually produced grain (Yuma 26-3) with a smaller embryo G0/G1 (left arrow) and endosperm G0/G1 (right arrow) than that found in apomictically produced sibling grains, D. an apomictically produced sibling grain

(Yuma 26-1) from the same accession as (C) with partially overlapping endosperm

G0/G1 and embryo G2 (right arrow).

Fig. 3. Flow cytometric analysis of an embryo (A) and endosperm (B) dissected from a

Bouteloua curtipendula var. caespitosa grain showing that endosperm G0/G1 overlaps with embryo G2.

Fig. 4. Endosperm:embryo ratios calculated from relative Cx-values derived from flow cytometric grain screens of Bouteloua curtipendula var. caespitosa grain.

174

350 2C

300

250

200

150

Number of nuclei of nuclei Number 100

4C 50 3C 8C

0 0 50 100 150 200 250 300 350 400 450 500 550 600 Fluorescence (channel number)

Fig. 1

175

180 A 120 160 B 140 100 120 80 100 80 60

60 40 40 Number of nuclei of nuclei Number Number of nuclei of nuclei Number 20 20

0 0 0 50 100 150 200 250 300 350 400 450 500 550 600 0 50 100 150 200 250 300 350 400 450 500 550 600 Fluorescence (channel number) Fluorescence (channel number)

300 C 200 D 250 150 200

150 100

100 50 Number of nuclei of nuclei Number 50 of nuclei Number

0 0 0 50 100 150 200 250 300 350 400 450 500 550 600 0 50 100 150 200 250 300 350 400 450 500 550 600 Fluorescence (channel number) Fluorescence (channel number) Fig. 2 176

60 A

20 of nuclei Number

0 0 50 100 150 200 250 300 350 400 450 500 550 Florescence (channel number)

20 B

of nuclei Number

0 0 50 100 150 200 250 300 350 400 450 500 550 Florescence (channel number)

Fig. 3 177

50 Embryo G2 and endosperm inseparable

40 Endosperm peak evident

20 Number of grains Number 10

0 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 Endosperm:Embryo Fig. 4