Pollen performance traits reveal prezygotic nonrandom mating and interference competition in Arabidopsis thaliana.

AJB Advance Article published on February 29, 2016, as 10.3732/ajb.1500172. The latest version is at http://www.amjbot.org/cgi/doi/10.3732/ajb.1500172 RESEARCH ARTICLE A M E R I C A N J O U R N A L O F B O TA N Y

I N V I T E D PA P E R For the Special Issue: Ecology and Evolution of Pollen Performance

Pollen performance traits reveal prezygotic nonrandom mating and interference competition in Arabidopsis thaliana1 Robert J. Swanson2,6, Adam T. Hammond3, Ann L. Carlson2, Hui Gong4, and Thad K. Donovan5

PREMISE: The lack of ability to measure pollen performance traits in mixed pollinations has been a major hurdle in understanding the mechanisms of differential success of compatible pollen donors. In previous work, we demonstrated that nonrandom mating between two accessions of Arabidopsis thaliana, Columbia (Col) and Landsberg (Ler), is mediated by the male genotype. Despite these genetic insights, it was unclear at what stage of reproduction these genes were acting. Here, we used an experimental strategy that allowed us to differentiate different pollen populations in mixed pollinations to ask: (1) What pollen performance traits differed between Col and Ler accessions that direct nonrandom mating? (2) Is there evidence of interference competition? METHODS: We used genetically marked pollen that can be visualized colorimetrically to quantify pollen performance of single populations of pollen in mixed pollinations. We used this and other assays to measure pollen viability, germination, tube growth, patterns of fertilization, and seed abortion. Finally, we assessed interference competition. RESULTS: In mixed pollinations on Col pistils, Col pollen sired significantly more seeds than Ler pollen. Col pollen displayed higher pollen viability, faster and greater pollen germination, and faster pollen tube growth. We saw no evidence of nonrandom seed abortion. Finally, we found interference competition occurs in mixed pollinations. CONCLUSION: The lack of differences in postzygotic processes coupled with direct observation of pollen performance traits indicates that nonrandom mating in Arabidopsis thaliana is prezygotic, due mostly to differential pollen germination and pollen tube growth rates. Finally, this study unambiguously demonstrates the existence of interference competition. KEY WORDS Arabidopsis; Brassicaceae; interference competition; mate choice; nonrandom mating; pollen; pollen competition; pollination

When pollinations include pollen from more than one donor, pollen often sire seeds disproportionate to the amounts initially present on the stigma. This process is called postpollination nonrandom mating. Nonrandom mating can be the result of self incompatibility if some pollen fails because it is self pollen in an obligately outcrossing plant. The genetics and physiology of self incompatibility

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Manuscript received 15 April 2015; revision accepted 1 October 2015. Department of Biology, Valparaiso University, Valparaiso, Indiana 46383 USA; 3 Biophysical Sciences, The University of Chicago, Chicago, Illinois 60637 USA; 4 Department of Mathematics and Computer Science, Valparaiso University, Valparaiso, Indiana 46383 USA; and 5 Smith Donovan Marketing & Communications, Chesterton, Indiana 46304 USA 6 Author for correspondence (e-mail: [email protected]) doi:10.3732/ajb.1500172 2

systems are heavily studied (for recent reviews, see Chapman and Goring, 2010; Nasrallah, 2011; Wang and Kao, 2012; Nasrallah and Nasrallah, 2014; Wilkins et al., 2014). On the other end of the genetic spectrum, pollen may fail because of incongruity—mismatched physiology between pollen and pistil because they are from genetically diverged populations (Hogenboom, 1973, 1975; de Nettancourt, 2001). A more difficult phenomenon to explain lies between these two extremes when there is differential siring ability of compatible pollen, often revealed when pollen are in competition. This process is of great interest because it has the potential to select for advantageous traits that function in both gametophytes and resulting sporophytes. Pollen competition has been linked to the evolutionary success of angiosperms, and its action is consistent with the evolution of rapid reproduction early in angiosperm history (Mulcahy, 1979; Williams, 2008, 2009, 2012).

A M E R I C A N J O U R N A L O F B OTA N Y 103(3): 1–16, 2016; http://www.amjbot.org/ © 2016 Botanical Society of America • 1

Copyright 2016 by the Botanical Society of America

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Empirical studies make it clear that the ability to mate nonrandomly among compatible mates is widespread in plants. More than 90 years ago, D. F. Jones was demonstrating nonrandom mating using mixtures of corn pollen (Jones, 1920, 1922, 1924). Jones used endosperm-color morphs in strains of maize to ask whether equal amounts of pollen sired equal amounts of seed. They did not. Since then, similar but varied experimental strategies have been used to demonstrate nonrandom mating in a vast array of plants (reviewed by Bernasconi, 2003). Nonrandom mating is present in agricultural, as well as wild species (e.g., Allium cepa and Viola tricolor, respectively) (Currah, 1981; Skogsmyr and Lankinen, 1999; Lankinen and Skogsmyr, 2002). It occurs in plants that display different life history traits, such as animal and wind pollination (e.g., Raphanus sativus and Betula pendula, respectively) (Marshall and Ellstrand, 1986; Pasonen et al., 1999). It occurs in plants that predominately outcross, predominately self, and plants that display mixed mating systems (e.g., Lesquerella fendleri, Arabidopsis thaliana, and Erythronium grandiflorum, respectively) (Rigney et al., 1993; Mitchell and Marshall, 1998; Carlson et al., 2009, 2013). It occurs in every major clade of eudicots, including basal tricolpates, core tricolpates, asterids, rosids (e.g., Persoonia mollis, Silene latifolia, Campsis radicans, and Cucurbita pepo, respectively) (Bertin and Sullivan, 1988;

Bertin, 1990; Quesada et al., 1991; Taylor et al., 1999; Krauss, 2000) and monocots (e.g., Allium cepa and Eichhornia paniculata) (Currah, 1981; Cruzan and Barrett, 1996). It has even been shown in gymnosperms (e.g., Pseudotsuga menziesii) (Apsit et al., 1989). Many of these studies explore how pre- and postzygotic nonrandom mating can lead to higher quality offspring in response to evolutionary pressures such as inbreeding depression and sexual selection (Charnov, 1979; Mulcahy, 1979; Willson, 1979; Queller, 1983; Stephenson and Bertin, 1983; Willson and Burley, 1983; Marshall and Ellstrand, 1986; Charlesworth and Charlesworth, 1987; Mulcahy and Mulcahy, 1987; Cruzan, 1990b; Quesada et al., 1993; Snow, 1994; Paschke et al., 2002; Skogsmyr and Lankinen, 2002; Stephenson et al., 2003; Armbruster and Rogers, 2004; Bernasconi et al., 2004; Lankinen and Armbruster, 2007; Lankinen and Green, 2015). Despite these studies, we have yet to develop an integrated mechanistic and genetic model of nonrandom mating that, in turn, may help us understand the possibilities and limits of sexual selection in plants. In this introduction, we provide an overview of what is known about pollen performance traits and how they may direct nonrandom mating. This overview will focus on Brassicaceae pollination, especially Arabidopsis thaliana, where a combination of facile genetics, well-characterized cell physiology, and long history of study has led to considerable understanding of pollen behavior in the pistil (Preuss et al., 1993; Thorsness et al., 1993; Kandasamy et al., 1994; Swanson et al., 2004; Ma et al., 2012). At the same time, little work has been done in Arabidopsis thaliana in regard to the mechanisms of nonrandom mating. The bulk of studies on nonrandom mating have been performed using a variety of different systems, including Hibiscus moscheutos, Raphanus sativus, Cucurbita pepo, Collinsia heterophylla, and Viola tricolor, among others (Schlichting et al., 1990; Snow and Spira, 1991b, a, 1993, 1996; Lankinen, 2000, 2001; Marshall and Diggle, 2001; Lankinen et al., 2009).

POLLEN PERFORMANCE TRAITS THAT INFLUENCE NONRANDOM MATING Reproduction in plants is a sequential process that involves pollen adhesion, pollen tube germination, pollen tube growth through the style and into the ovary, fertilization of ovules and seed development (Figs. 1–3). Each stage of fertilization has the potential to influence nonrandom mating.

FIGURE 1 Standard mixed pollination experiment in Arabidopsis thaliana. Pollen from two different accessions (red and blue) are placed in equal amounts on the pistil. Nonrandom mating occurs when seed counts statistically differ from a 50:50 ratio.

Pollen viability—At maturity, pollen grains of Arabidopsis thaliana are triploid structures, with two haploid sperm cells enclosed in a haploid vegetative cell surrounded by a highly specialized double cell wall. The outer layer, the sculptured exine, houses the pollen coat, a mixture of lipids, carbohydrates, and protein.

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FIGURE 2 The events that lead to pollen germination. (A) A desiccated pollen grain binds to a stigma papilla. (B) The pollen coat migrates to the interface between the pollen and the stigma papilla, forming the foot (red). (C) The pollen hydrates with liquid released from the stigma papilla. (D) The pollen tube breaks through the outer wall of the pollen grain either through the aperture or directly out of the exine and invades the cell wall (yellow) of the stigma papilla. The tube then grows down to the subdermal cells of the stigma.

In several tested species, some amount of pollen is aborted on dehiscence, and it has long been recognized that differential pollen viability can affect the results of mixed pollinations (Marshall and Ellstrand, 1986; Stone et al., 1995). Simply put, when equal amounts of pollen from different accessions are placed on a pistil and a

substantial number of pollen from one accession are nonviable, nonrandom mating would result, even if all subsequent events in fertilization were random. Pollen development of Arabidopsis thaliana is fairly robust; we have previously demonstrated greater than 95% viability in pollen from multiple accessions (Carlson et al., 2013). Pollen tube germination—Mature pollen grains of Arabidopsis

FIGURE 3 Fertilization in Arabidopsis thaliana. The pollen tube exits the transmitting tract and grows on the septum to reach the ovule. One synergid cell degenerates, as does the pollen tube tip, to release the two sperm cells that fertilize the egg cell (to form the embryo) and the central cell (to form the endosperm).

thaliana are released from the anther in a desiccated and metabolically inert form. It is not until pollen bind the distil end of the pistil, the stigma, that the series of events occurs that results in the germination of a pollen tube (Fig. 2). Arabidopsis thaliana sports a dry stigma (Elleman et al., 1992), topped with a dense array of papillar cells that serve to capture pollen in an initial adhesion event (Fig. 2A). Once pollen grains are bound to a papillar cell, the pollen coat migrates to the interface between the pollen and the stigma papilla, forming the “foot” (Fig. 2B) (Dickinson and Lewis, 1973; Elleman and Dickinson, 1990; Elleman et al., 1992). During the formation of the foot, there are changes to the nature of the pollen coat called coat conversion that likely act to facilitate hydration of the pollen grain from the stigma papilla (Fig. 2C) (Elleman and Dickinson, 1986; Mayfield and Preuss, 2000; Murphy, 2006; Updegraff et al., 2009). In Arabidopsis thaliana, this process typically occurs within 5 min of pollination (Kandasamy et al., 1994; Mayfield and Preuss, 2000). Once the grain is hydrated, its internal structures reorganize to support the polarized growth of the germinating pollen tube. The pollen tube exits through specialized holes in the pollen wall (apertures) or directly through the exine (Fig. 2D) (Edlund et al., 2004; A. F. Edlund, Lafayette College, personal communication). In Arabidopsis thaliana, the pollen tube grows through the foot to penetrate the stigma papilla cuticle within 20 min, and then enters the outer layer of the papillae cell wall where it continues to grow (Fig. 2D). Within 50 min, pollen tubes have grown to the subdermal cells of the stigma (Kandasamy et al., 1994).

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Much like pollen viability, pollen germination has long been recognized as an important component of nonrandom mating in mixed pollinations, and for much the same reason (Snow and Spira, 1991b; Sari-Gorla et al., 1992; Smith-Huerta, 1996; Jolivet and Bernasconi, 2007). Unequal numbers of pollen germinating and/or the speed of pollen germination influence nonrandom mating, as both dictate which pollen tubes may transit into the ovary to gain first access to unfertilized ovules (Bertin, 1988). A host of studies have demonstrated that pollen germination can be influenced by paternal identity, maternal identity, and environmental factors, such as temperature, humidity, and pH (e.g., Cook and Walden, 1967; Barnes and Cleveland, 1972; Campbell, 1982; Nemeth and Smith-Huerta, 2002; Hedhly et al., 2003; Boavida and McCormick, 2007; Jolivet and Bernasconi, 2007; Smith-Huerta et al., 2007; Austerlitz et al., 2012). Pollen germination can also be influenced by other pollen. For example, as early as 1924, Brink was demonstrating positive density-dependent pollen germination in vitro, a finding later verified in vivo (Brink, 1924; Brewbaker and Majumder, 1961; Cruzan, 1986). However, high pollen loads can also interfere with germination (e.g., Erythronium grandiflorum; Thomson, 1989). In several species, the genotype of interacting pollen influences germination frequency, with single pollinations showing greater germination in Raphanus sativus and Clarkia unguiculata compared with mixed pollinations (Marshall et al., 1996; Nemeth and Smith-Huerta, 2002), while Betula pendula displays the opposite effect (Pasonen and Kapyla, 1998). Pollen tube attrition—From the subdermal stigma, pollen tubes converge in the style. Arabidopsis thaliana sports a short solid style with a core of transmitting tract that is continuous with the septum that divides the two chambers of the ovary (Fig. 1). The number of growing pollen tubes decreases dramatically during growth through the style, a phenomenon termed pollen tube attrition (Herrero and Dickinson, 1981; Cruzan, 1986, 1989, 1990a; Snow, 1986; Scribailo and Barrett, 1991; Montalvo, 1992; Cruzan and Barrett, 1993, 1996; Plitmann, 1993; Winsor and Stephenson, 1995; Hormaza and Herrero, 1996; Smith-Huerta, 1997; Erbar, 2003; Stephenson et al., 2003; Mazer et al., 2016, in this issue). Nonrandom mating will occur if pollen tube attrition differentially affects one pollen population in a mixed pollination. As with pollen germination, pollen tube attrition can depend on the maternal and paternal identities of the plants involved (Cruzan, 1990a, 1993; Hormaza and Herrero, 1996). There are also indications that some level of pollen tube attrition may be independent of the identity of the pollen (Cruzan, 1986; Herrero, 1992). For example, in this special issue, Mazer et al. (2016) demonstrate a clear association between the number of pollen deposited on stigmas and pollen tube attrition in natural populations of Clarkia. This association could be due to antagonistic interactions or stylar constriction, a physical limitation on the width of the transmitting tissue of the style. Alternatively, there could be limitations on the nutrient supplies available to pollen during heterotrophic growth, resulting in later pollen failing (Herrero and Dickinson, 1981; Mulcahy and Ottaviano, 1983; Cruzan, 1986; Marshall and Folsom, 1991; Nemeth and Smith-Huerta, 2002; Stephenson et al., 2003). Consistent with this idea, in Erythronium grandiflorum, there seems to be an upper limit on the number of pollen tubes that can pass through the style (Cruzan, 1989). Pollen tube growth—Once pollen tubes traverse the stigma and

style, they enter the ovary which, in Arabidopsis thaliana, houses

50–70 ovules. Differential pollen tube growth rates were identified very early as a potential cause of nonrandom mating and are the most studied aspect of postpollination nonrandom mating (Jones, 1922). Theoretically, faster-growing pollen tubes are more likely to gain access to unfertilized ovules and thus be sires, an idea supported by quite a few studies (Bateman, 1956; Pfahler, 1967; Ottaviano et al., 1988; Snow and Spira, 1991a, b, 1996; Cruzan and Barrett, 1993, 1996; Marshall, 1998; Pasonen et al., 1999; Skogsmyr and Lankinen, 1999, 2000; Lankinen and Skogsmyr, 2001; Aronen et al., 2002; Lankinen, Maad, and Armbruster, 2009). As seen in other variables, pollen tube growth rates are influenced by female and male identity, and the environment (Sarigorla et al., 1975; Fenster and Sork, 1988; Snow and Spira, 1991b, a; Mazer and Gorchov, 1996; Smith-Huerta, 1996; Delph et al., 1997; Jóhannsson and Stephenson, 1997; Pasonen et al., 1999; Kerwin and Smith-Huerta, 2000). There are also studies that indicate pollen–pollen interactions influence pollen tube growth rates (Cruzan, 1986, 1990a; Lankinen and Skogsmyr, 2002). For example, by growing different donor pollen in different channels of the same style in Erythronium grandiflorum, Cruzan (1990a) demonstrated that pollen tube growth rate was influenced by other pollen. As with pollen tube attrition, it is possible that pollen–pollen interactions may be the result of pollen directly influencing growth dynamics of other pollen, or it may be a passive effect of crowding or shared resources. For example, when many pollen tubes are present, competition among pollen tubes for limited stylar resources could result in a slower overall rate of pollen tube growth for some or all tubes (Cruzan, 1986). Ovule fertilization patterns—The female tissue provides chemical

gradients and nutrients for pollen tube growth and guidance. Chemoattractant gradients in the pistil play an important role in guiding pollen tubes to ovules, but the molecular details of these signals are beyond the scope of this introduction (Higashiyama et al., 2001; Kasahara et al., 2005; Crawford and Yanofsky, 2008; Higashiyama and Hamamura, 2008; Okuda et al., 2009; Chapman and Goring, 2010; Higashiyama and Takeuchi, 2015). Once the pollen tube emerges from the transmitting tract, it grows over the surface of the septum to the funiculus (the attachment between the septum and the ovule) (Fig. 3). The pollen tube grows along the funicular surface to the micropylar opening of the ovule, where it degenerates and releases the two sperm. One 1n sperm nucleus fuses with the 1n egg nucleus of the embryo sac to form the 2n nucleus of the zygote. The second 1n sperm nucleus fuses with two 1n nuclei of the central cell of the embryo sac to form the 3n primary endosperm nucleus. In Arabidopsis thaliana, fertilization occurs within 8–10 h (Kandasamy et al., 1994). It is likely that pollen tubes exit the transmitting tract when they receive signals from unfertilized ovules. In theory, this process should lead to the orderly fertilization of ovules, with the fastestgrowing pollen tubes fertilizing the ovules closest to the stigma. Later pollen tubes would progress further down the transmitting tract and fertilize ovules deeper in the ovary, until the ovules at the base of the ovary are reached. Yet several studies have demonstrated nonrandom distribution of seeds in fruits, possibly related to the order of fertilization of the ovules within an ovary, which in turn may experience differential levels of female provisioning or predation (Cooper and Brink, 1940; Jaranowski, 1962; Hill and Lord, 1986; Marshall and Ellstrand, 1986; Marshall, 1991; Quesada et al., 1991; Ibarra-Perez et al., 1996), although see (Mazer et al.,

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1986; Marshall and Diggle, 2001). Indeed, in work published in this issue, Marshall and Evans (2016, in this issue) show that seed position in mixed pollinations is heritable and responsive to selection. What occurs in Arabidopsis thaliana is unclear; different studies provide contradictory results, possibly related to the fact that different accessions were used (Hülskamp et al., 1995; Crawford et al., 2007). Different genotypes of pollen donors may have different patterns of ovule fertilization in ovaries that influence the results of mixed pollinations; faster pollen tubes may bypass unfertilized ovules for ovules farther toward the base of the ovary, allowing slower tubes opportunities. Thus, different pollen populations would sire seeds, even if one population has slowly growing tubes. Seed development and abortion—After fertilization, the zygote divides to form the embryo, the endosperm divides to form the endosperm tissue, while the outer layer of the ovule hardens to form a seed coat. Once the embryo reaches maturity, growth and development ceases, and the seed dessicates. Selective abortion of seeds is well documented in plants and can also be involved in nonrandom mating (Willson and Burley, 1983; Marshall and Ellstrand, 1988; Bertin, 1990; Montalvo, 1992; Rigney et al., 1993; Rigney, 1995; Cruzan and Barrett, 1996; Mahy and Jacquemart, 1999; Travers and Holtsford, 2000; Obeso, 2004), perhaps as a result of incompatible genetic combinations. For example, in outcrossing plants that have the capacity to self, self pollinations often (but not always) yield less seed than outcross pollinations, likely from early-acting inbreeding depression that results in self fertilized embryo lethality (Weller and Ornduff, 1977; Levin, 1984; Cheliak et al., 1985; Charlesworth, 1988; Cruzan, 1989; Quesada et al., 1991; Snow and Spira, 1993; Ibarra-Perez et al., 1996; Bell et al., 2010). This is not the case for Arabidopsis thaliana, where self pollinations or pollination with genetically similar pollen do not result in fewer progeny (Carlson et al., 2013). Generally, Arabidopsis thaliana plants grown under ideal conditions display extremely low levels of seed abortion (less than 1%) (Meinke, 1994; Meinke et al., 2008).

OUR SYSTEM Studying nonrandom mating in a largely selfing plant such as Arabidopsis thaliana provides both theoretical and practical advantages. First, outcrossing plants carry higher levels of heterozygosity and thus produce more variable pollen phenotypes because of segregating alleles. Such variation complicates phenotypic analysis. Also, outcrossing plants often display higher inbreeding depression such that pollinations with self pollen or pollen from genetically similar plants lead to poor reproductive success, changing nonrandom mating patterns depending on the genetic relatedness of the parents (Bateman, 1956; Weller and Ornduff, 1977; Bowman, 1987; Eckert and Barrett, 1994; Jones, 1994; Hauser and Siegismund, 2000; Teixeira et al., 2009; but see also Sork and Schemske, 1992; Johnston, 1993). Thus, in outcrossing plants, population-specific gene variants that influence reproductive success are confounded with parental relatedness and segregating heterozygosity. The latter two factors are essentially eliminated by studying plant populations that have historically selfed. As outcrossing populations become increasingly self-fertilizing, they both lose heterozygosity, and their genetic load is purged (Lande and Schemske, 1985; Schemske and

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Lande, 1985; Charlesworth and Charlesworth, 1987; Lande et al., 1994; Byers and Waller, 1999; Crnokrak and Barrett, 2002). This is the case for Arabidopsis thaliana, whose tested populations show relatively low levels of heterozygosity and no evidence for earlyacting inbreeding (or outbreeding) depression (Bakker et al., 2006; Bomblies et al., 2010; Platt et al., 2010; Carlson et al., 2013). Finally, the increased levels of purifying and positive selection seen in pollen and pollen tube-specific genes in Arabidopsis thaliana are consistent with the influences of prezygotic sexual selection such as by pollen competition (Gossmann et al., 2013). Thus, Arabidopsis thaliana provides a practical system to identify and explore nonrandom mating that develops or persists in plant populations unrelated to genetic effects such as parental relatedness or inbreeding depression (although the variations found may not represent variations seen in plants with differing reproductive strategies). In our previous work, we demonstrated consistent and strong nonrandom mating in genetically and geographically distinct accessions of Arabidopsis thaliana (Carlson et al., 2009, 2013). We also developed an experimental strategy for separating male and female influence in nonrandom mating. For example, in mixed pollinations with pollen from Columbia (Col) and Landsberg erecta (Ler) accessions on Col pistils, Col pollen sires 89% of the progeny, while Ler pollen sires only 11%. While in other systems, nonrandom mating is influenced by female identity, this difference in siring ability is primarily male-mediated, as it is independent of the identity of the pistil. Genetic dissection of this process has revealed two male loci and no detectable female loci (Carlson et al., 2009, 2011). Importantly, despite demonstrating that the difference in siring ability between Col and Ler pollen is directed by the male genotype, we don’t know what aspects of pollen or seed performance mediate it. It is unclear why Col pollen performs better than Ler pollen. Studying pollen performance is not trivial because of the technical difficulty in distinguishing pollen identity during competition in mixed pollinations. In this paper, we describe an experimental design that allowed us to accomplish this in Arabidopsis thaliana. We used this method and others to investigate nonrandom mating between Col and Ler pollen and asked two questions. First, what pollen performance traits, such as pollen viability, pollen germination, pollen tube growth, patterns of ovule fertilization and seed abortion differ between Col and Ler pollen? Second, do we see evidence for interference competition?

MATERIALS AND METHODS Study species and plant growth—Arabidopsis thaliana (L.) Heynh.

(Brassicaceae) is an annual weed found widely distributed throughout the northern hemisphere (Al-Shehbaz and O’Kane, 2002; Hoffmann, 2002). Arabidopsis thaliana displays the characteristic floral morphology of a self-fertilizing species: small flowers, lack of strong scent, and close juxtaposition of anthers to stigma. Selfing rates in natural populations have been estimated to be greater than 97% (Abbott and Gomes, 1989; Bergelson et al., 1998; Bakker et al., 2006; Picó et al., 2008; Platt et al., 2010). All plants were grown in identical, controlled environments. Seeds were imbibed and cold-treated at 4°C for 7 d in the dark to break dormancy and promote uniform germination. Plants were grown in 4.5-inch pots with generally 5 plants per pot in Percival growth chambers (Percival Scientific, Perry, Indiana, USA) in Shultz

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premium potting soil with watering every second day and fertilized (18-18-21 at 200 ppm) twice per week. Plants were subjected to 16 h of 130 μE fluorescent lighting at 22°C. For scoring BASTA resistance, seeds were grown as normal and at 4 wk were sprayed with glufosinate ammonium. Experimental pollinations—The methods for single-donor and

mixed pollinations (Fig. 1) have been previously described (Carlson et al., 2009, 2011, 2013; Gerald et al., 2014). Briefly, all pollinations were performed on primary inflorescences of newly bolting Col plants. We emasculated buds during stages 11 and 12 of development (Smyth et al., 1990). We allowed pistils to mature to stage 14 before performing pollinations. We harvested anthers from stage 14 flowers and visually inspected them for levels of dehiscence. We chose two anthers from each potential father and readied them on forceps. We used a stereomicroscope (Leica ZOOM2000) to better visualize the stigma when we applied pollen on half the available surface area of the stigma. We then applied pollen from the competing accession on the remaining surface area. It has been shown that different methods of pollen application in mixed pollinations can lead to different results (Mitchell and Marshall, 1995; Nemeth and Smith-Huerta, 2002). We applied pollen on different stigma hemispheres because it yields highly reproducible results. In singledonor pollinations, we performed pollinations as described above, but all anthers were taken from a single accession. “Full pollinations” involved 1040 ± 236 pollen (for counting method, see below). For “limiting pollinations”, we used only one anther and touched it once to the stigma. Visually inspecting the stigmas revealed that limiting pollinations involved 22.3 ± 11.3 pollen. We completed each pollination within 1 min. For visualizing pollen tubes, we substituted the wild-type Col accession (Col-4) for a Col accession (Col-3 background, CS16336) transformed with the pCSA110 T-DNA containing an integrated pollen specific LAT52 promoter fused to the colorimetric marker β-glucuronidase (GUS) gene, as well as resistance to the herbicide BASTA (Twell et al., 1989, 1990). Originally, this strain contained the qrt mutation, which we removed by backcrossing to Col-0 and selfing the progeny to isolate a strain lacking the qrt mutation but homozygous for BASTA resistance (Johnson et al., 2004). We refer to this strain as Col-GUS. We introgressed the T-DNA into the Ler background by mating CS16336 to CS20 (Ler) and backcrossing the progeny 9 times to Ler. We then selfed the resulting line to isolate a strain homozygous for BASTA resistance. We refer to this strain as Ler-GUS. The presence of T-DNA marker did not change the siring success of the pollen. We showed this by performing mixed pollinations of Col-GUS and Col pollen on virgin Col pistils. These pollen are genetically identical except for the addition of the T-DNA and did not differ from the expected 1:1 ratio of progeny (48.5% ± 15.4% BASTA resistant seeds) and showed no statistical difference in siring success (Z = 16.5, P > 0.05). We demonstrated equal delivery of pollen in mixed pollinations by manually counting the number of pollen of each type. We performed mixed pollinations on virgin Col pistils using Col-GUS and Ler pollen as described already. We immediately washed pollen from pistils using 0.5% Triton X-100 for 5 min (Zinkl et al., 1999). We visually verified that all pollen grains were removed. We centrifuged (13,300 × g) the pollen for 1 min and replaced the 0.5% Triton with X-Gluc substrate of GUS (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 50 mM NaPO4 pH 7, 0.5 mg/mL

5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid), incubating pollen overnight at 37° (Johnson et al., 2004). Col-GUS pollen turned blue. We counted blue pollen, unstained pollen, and total pollen using a Nikon Eclipse E200 microscope. We saw a 1:1 ratio of ColGUS and Ler pollen (46 ± 8% Col pollen, N = 9), and there was no statistical difference between the number of pollen deposited from both populations (Z = −0.700, p > 0.05). As noted, pollinations involved 1040 ± 236 total pollen. Scoring pollen viability—Several methods have been devised to

test pollen viability that include stains for pollen enzymatic activity and germination. There is a long history of focusing on aborted pollen using stains that demonstrate proper pollen shape and intact plasma membranes (reviewed by Stone et al., 1995). Since we tested germination separately in this study, we focused our viability counts on numbers of aborted pollen grains using Alexander’s stain (Alexander, 1969). To assay pollen viability, we collected pollen from stage 14 flowers. We mounted pollen from anthers directly in Alexander’s stain, flaming the slide to hasten the staining. Developmentally normal pollen grains stain dark blue or purple. Pollen grains that are inviable stain pale blue. A minimum of 500 pollen grains were counted from each plant, with 9 plants counted for Col and Ler. Measuring germination speed and amount—Pollen germination has been tested in vitro on pollen growth media, providing the opportunity to measure pollen traits in the absence of female confounds (e.g., Jolivet and Bernasconi, 2007). In vitro pollen germination frequencies are not always good predictors of in vivo pollen germination (Hedhly et al., 2005). In vitro germination tests can be unreliable in A. thaliana because the composition of the pollen growth media can elicit accession-specific behaviors from pollen (Johnson-Brousseau and McCormick, 2004; Qin et al., 2011). As an alternative, pollen germination is often quantified in vivo via microscopy, as judged by the presence of a pollen tube, or the absence of the cytoplasm in the pollen grain (Cruzan and Barrett, 1993). Yet we find visualizing pollen tubes or empty pollen via microscopy is difficult when stigma heads contain over 1000 pollen grains. We have developed another way of quantifying this process based on the adhesion properties of the pollen grain. In plants like Arabidopsis thaliana, with dry stigma, pollen adhesion has been characterized in stages (Fig. 2) (Heslop-Harrison and Shivanna, 1977; Swanson et al., 2004). First, the exine binds the stigma papilla. Later, once the pollen tube has germinated and migrated down the stigma, the pollen is tethered to the pistil via the growing pollen tube. The initial adhesion between the pollen exine and stigma papilla can be disrupted with detergent, but as pollen germination occurs, the pollen become resistant to detergent washes (Zinkl et al., 1999). We have used these properties to quantify the time it takes for pollen to germinate and the total amount of pollen germination. We quantified germination by washing pistils with detergent after fixed time periods. We then counted the pollen remaining on the stigma or counted the pollen in washes via microscopy. This method is similar to the method used by Cruzan (1986) to quantify pollen germination in Nicotiana glauca using NaOH washes. Col pistils were emasculated and pollinated as described with either Col-GUS, Ler-GUS or mixed pollinations of Col-GUS/Ler. After designated times, pistils were harvested and incubated for 5 min in either 400 μL of 0.5% Triton X-100 or 0.1% sodium dodecyl

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sulfate (SDS). We found that SDS washes were more stringent but destroyed GUS activity, while Triton X-100 washes did not completely remove ungerminated pollen but left GUS activity intact. Washed pistils were either fixed in 80% acetone, stained with X-GLUC and mounted in 90% glycerol, or simply mounted in 90% glycerol. Pollen grains remaining on the stigma were counted using an Olympus BH-2 microscope. Sample sizes were N = 9 for each accession at each time point. Measuring pollen tube growth rate—There are several methods of

measuring pollen tube growth, each with advantages and disadvantages. Several studies have correlated pollen tube growth in vitro with success in mixed pollinations. In some instances, in vitro pollen tube growth rates co vary with success in mixed pollinations, in some instances they do not (e.g., Pasonen et al., 1999; Skogsmyr and Lankinen, 1999; Aronen et al., 2002; Haileselassie et al., 2005). Alternatively, pollen tube growth rates in single-donor pollinations, visualized with aniline blue, have been used to predict performance in mixed pollinations, again with mixed success (e.g., (Marshall and Diggle, 2001; Pasonen et al., 2002). Finally, pollen tube growth rates have been measured in some protandrous plants that have separate styles that lead to the same ovary, such as Dianthus chinensis, Hibiscus moscheutos, or species that have different channels in the style, such as Erythronium grandiflorum (Aizen et al., 1990; Cruzan, 1990a; Snow and Spira, 1991b, a). Generally, these methods are unable to account for pollen–pollen interactions, such as crowding or direct interference. We have developed a method of measuring the speed of pollen tube growth of a single population of pollen in mixed pollinations. We pollinated virgin Col pistils with two types of donor pollen as described (e.g., with Col/Ler-GUS pollen, or Col-GUS/Ler pollen). We then visualized GUS pollen in mixed competitions, while the competing pollen remained invisible. At designated times after pollination, pistils were harvested and fixed for 1 h in 80% acetone before staining in X-GLUC. Pistils were then mounted in 90% glycerol and digitally photographed using a Zeiss Axiovert Zoom Hal100 inverted widefield microscope. The longest pollen tube was measured by drawing a line across the base of the style and measuring the distance between that line and the leading pollen tube tip. Measurements were made using the program ImageJ (Schneider et al., 2012). Sample sizes were as follows: mixed pollinations, ColGUS measured after 3, 6, 9, and 24 h, N = 11, 10, 17, and 9, respectively; mixed pollinations, Ler-GUS measured, N = 9, 9, 9, and 9, respectively; single pollinations, Col-GUS measured, N = 9, 8, 9, and 9, respectively; single pollinations, Ler-GUS measured, N = 9, 8, 8, and 9 respectively. Measuring patterns of ovule fertilization and seed abortion—We

performed single-donor pollinations on Col pistils with limiting pollen as described. After 7–9 d, we opened siliques and mapped fertilized ovules onto a silique grid (Meinke, 1994; Meinke et al., 2008). To visualize pollen tubes, we performed single-donor pollinations with limiting pollen. After 24 h, we harvested pistils and stained for GUS activity. The lengths of the longest pollen tubes were measured. Sample sizes were as follows: Col-GUS, N = 9, Ler-GUS, N = 9. To measure seed abortion, we performed mixed pollinations on Col pistils. After 9–10 d, we opened siliques and counted aborted seeds, identified by their small size and seed coat that has turned

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prematurely brown (Meinke, 1994; Meinke et al., 2008). A second way of assessing whether abortion contributes to nonrandom mating is to compare seed yields in single-donor pollinations and mixed pollinations. If nonrandom mating is due to differential abortion in mixed pollinations, we expected mixed pollinations to have lower seed yield than single-donor pollinations. Col pistils were pollinated as described already with single-donor pollinations of LerGUS or Col-GUS, or mixed pollinations of Col-GUS/Ler. We collected mature siliques and counted seeds. Sample sizes were as follows: Col-GUS, N = 9, Ler-GUS, N = 9. Data analysis—The plants for this study were not selected at random, so all independent variables in our statistical analyses were treated as fixed. Given the nonnormality of the data, we used a series of nonparametric statistics to compare our measured parameters. We performed a Wilcoxon signed rank test for number of progeny sired in mixed pollinations and ratio of pollen in mixed pollinations. We performed the Mann–Whitney–Wilcoxon test for pollen viability, pollen germination, and pollen tube length in mixed and single-donor pollinations and pollen tube length in limiting pollinations. To test for differences in seed yield, we performed a Kruskal–Wallis test. All statistics were done in Microsoft Excel (Microsoft, Redmond, Washington, USA).

RESULTS Col pollen sires significantly more seeds than Ler pollen in mixed pollinations—In previous work, we demonstrated that when Col

pollen and Ler pollen competed on a Col female, Col sired 89% of the progeny, while Ler sired only 11% (Carlson et al., 2009; 2011). The Col and Ler accessions we used for this study contained a different transgenic marker than we previously used, so we verified this nonrandom mating pattern by performing mixed pollinations and paternity testing the resulting seeds. When Col pollen and Ler-GUS pollen were placed on virgin Col pistils, they showed a pattern similar to that seen in previous studies; Col sired 93.1 ± 5.4% progeny, while Ler-GUS sired 6.8 ± 5.4% progeny (Z = 3.78, P < 0.05). Col pollen displays higher viability than Ler pollen—In Alexander’s

stain to identify dead or aborted pollen, Col-GUS pollen displayed 99.7% ± 0.3% viability while LER-GUS displayed 97.9% ± 2.7% viability. Despite this small difference, pollen viability was significantly different between Col and Ler (U = −1.99, P < 0.05). Col pollen germinates faster and displays greater total germination than Ler pollen—We quantified the speed of pollen germina-

tion in vivo by pollinating Col pistils with Col-GUS or Ler-GUS pollen and counting germinated pollen (Fig. 4). At 10 min, there was no significant difference between the number of Col and Ler germinated pollen, while at 20, 30, and 40 min, Col pollen had significantly more germinated pollen (Fig. 4A). Similarly, we found that Col pollen germinated faster in mixed pollinations and showed similar germination kinetics as single-donor pollinations (Fig. 4B–D). Quantifying the total number of pollen germinated is challenging; fully pollinated pistils can carry more than 1000 pollen. Instead, we quantified the number of pollen that failed to germinate in Col and Ler by washing ungerminated pollen from stigmas after

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FIGURE 4 Pollen of the Col accession of Arabidopsis thaliana has higher germination rates than pollen of the Ler accession. (A) Col pistils were pollinated with either Col-GUS pollen (circles) or Ler-GUS pollen (squares). At designated times, ungerminated pollen were washed from the stigmas with detergent. The remaining pollen grains were counted. Error bars = standard deviations; an asterisk (*) indicates differences between Col and Ler were statistically different at P < 0.05 for time point. (B) Col pistils were pollinated with Col-GUS and Ler. At 10 min, the reaction was halted and stained for GUS. Col-GUS pollen stain blue (black arrow), Ler pollen do not stain (white arrow). (C) Col pistils pollinated with Col-GUS and Ler, stained after 20 min. (D) Col pistils pollinated with Col-GUS and Ler, stained after 40 min. Scale bars = 100 μM.

24 h. When we performed single-donor pollinations, Ler-GUS pollen displayed significantly higher numbers of ungerminated pollen (313 ± 149.1) than Col-GUS pollen (107 ± 56.9) (U = −3.0, P < 0.05). When we took these numbers and divided by the average number of pollen in pollinations (see Materials and Methods), 69.9% of Ler pollen germinated, while 89.7% of Col pollen germinated. Col pollen tubes grow faster than Ler pollen tubes in mixed pollinations—We measured length of Col-GUS pollen tubes in mixed

pollinations with Ler pollen, and Ler-GUS pollen tubes in mixed pollinations with Col pollen (Fig. 5). Col-GUS pollen tubes grew faster and had significantly longer mean pollen tube lengths at all time points (Fig. 6). Col and Ler pollen have similar patterns of ovule fertilization—If

Col and Ler pollen show different patterns of ovule fertilization, we would expect, in limiting, single-donor pollinations, for Col and Ler pollen tubes to populate different parts of the pistil and to fertilize seeds in different parts of the pistil. We saw neither. To measure ovule fertilization preferences, we performed limiting, single-donor pollinations with Col-GUS and Ler-GUS on Col pistils. After 9–10 d, we split open the siliques and counted fertilized and

unfertilized ovules. The majority of seeds for both accessions were in the top half of the pistil. Col-GUS averaged 16.1 ± 9.6 seeds per pollination. The lower half of the pistil had only 0.9 ± 1.2 seeds per pollination. Ler-GUS averaged 21.7 ± 10.5 seeds per pollination, with only 3.5 ± 3.2 seeds per pollination in the lower half of the pistil. We also measured Col-GUS and Ler-GUS pollen tube lengths after limiting pollinations to test whether they grow to different areas in the pistil. Col-GUS and Ler-GUS pollen tubes grew to the same length and were confined to the top half of the pistil. Their final tube lengths did not differ (U = 0.31, P > 0.05). There is no evidence of nonrandom abortion—If nonrandom mat-

ing between these two accessions were due to nonrandom abortion, we would expect mixed pollinations to have fewer seeds than singledonor pollinations and a substantial number of aborted seeds. We saw neither. A standard way of measuring nonrandom abortion is to compare seed yield from mixed pollinations and single-donor pollinations. We performed single-donor pollinations with Col-GUS and Ler-GUS pollen and mixed pollinations with Col-GUS/Ler-GUS pollen, collected the mature siliques and counted seeds. Though single-donor pollinations had a higher mean seed yield (Col = 48.9 ± 15.5, Ler = 47.8 ± 13.2, mixed pollinations = 41.1 ± 13.5), the means

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FIGURE 5 Visualizing individual populations of pollen in mixed pollinations. Col pistils were pollinated with either Col-GUS/Ler pollen (labeled ColGUS) or Col/Ler-GUS pollen (labeled Ler-GUS). Pollinations were halted after (A) 3, (B) 6, or (C) 9 h, and stained for GUS activity. Pollen tube lengths were then measured from the tip of the leading pollen tube to the end of the style.

were not statistically significant, supporting the idea that nonrandom abortion does not contribute to nonrandom mating (H = 2.91, df = 2, P > 0.05). If nonrandom abortion contributed to nonrandom mating, we also expected to see substantial numbers of aborted seeds in mixed pollinations. We did not. Mixed pollinations yielded, on average, only 0.7 ± 1.1 aborted seeds.

Col and Ler pollen display interference competition in mixed pollinations—We quantified pollen length in single-donor pollinations

of Col-GUS and Ler-GUS (Fig. 7). Similar to mixed pollinations, Col-GUS pollen tubes grew faster and had significantly longer mean pollen tube lengths at all time points. If interference competition occurs, we expected pollen tube lengths in mixed pollinations to be shorter than in single-donor

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Col pollen tubes grow faster than Ler pollen tubes in mixed pollinations. Mixed pollinations were performed on Col pistils with either Col-GUS/Ler pollen (circles) or Col/Ler-GUS pollen (squares). At designated times, pistils were fixed and stained for GUS activity. The length of the longest GUS pollen tubes were measured. Error bars = standard deviations; an asterisk (*) indicates differences between Col and Ler were statistically different at P < 0.05 for time point. FIGURE 6

pollinations. This was the case for Ler-GUS pollen. Ler-GUS pollen tubes in mixed pollinations (Ler-GUS/Col) were significantly shorter than in single-donor pollinations at all time points (Fig. 8). The pattern of interference in Col pollen was more complex (Fig. 9). At 3, 6, and 24 h, there was no significant difference in Col-GUS

FIGURE 7 Col pollen tubes grow faster than Ler pollen tubes in singledonor pollinations. Single-donor pollinations were performed on Col pistils with either Col-GUS pollen (circles) or Ler-GUS pollen (squares). At designated times, pistils were fixed and stained for GUS activity. The length of the longest GUS pollen tubes were measured. Error bars = standard deviations; an asterisk (*) indicates differences between Col and Ler were statistically different at P < 0.05 for time point.

FIGURE 8 Comparison of Ler-GUS pollen tube growth in single-donor and mixed pollinations. Single-donor pollinations were performed with Ler-GUS pollen (triangles). Mixed pollinations were performed with LerGUS/Col pollen (squares). Error bars = standard deviations; an asterisk (*) indicates differences were statistically significant at P < 0.05 for time point.

pollen tube lengths in single-donor and mixed pollinations. At 9 h, however, Col-GUS pollen tubes in single-donor pollinations were significantly longer. DISCUSSION There have been very few studies that systematically measured all internal stages of the reproductive process (see for example, Palser et al.,

FIGURE 9 Comparison of Col-GUS pollen tube growth in single-donor and mixed pollinations. Single-donor pollinations were performed with Col-GUS pollen (triangles). Mixed pollinations were performed with ColGUS/Ler pollen (circles). Error bars = standard deviations; an asterisk (*) indicates differences were statistically significant at P < 0.05 for time point.

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1989; Rigney, 1995; Marshall and Diggle, 2001). As far as we are aware, there have been no studies that have measured pollen behavior of two competing conspecific pollen donors in mixed pollinations in the same style because of the difficulty in visualizing different populations of gametes (although Havens and Delph [1996] quantified performance at the final stage of fertilization, while Williams et al. [1999] visualized pollen populations in con- and heterospecific mixed pollinations). This issue has hampered research into both pollen and sperm competition. For example, one of the most popular animal models for understanding postcopulatory sexual selection is Drosophila melanogaster. It was only recently that movement dynamics of different populations of sperm were first visualized in flash frozen and dissected seminal receptacles in Drosophila melanogaster using red and green fluorescently labeled sperm heads (Manier et al., 2010; Lupold et al., 2011; Manier et al., 2013; Ala-Honkola et al., 2014). Unlike visualizing moving sperm, visualizing the elongating pollen tubes offers us the opportunity of not just seeing where gametophytes are, but also tracing the history of growth of the pollen tube since germination. For this study, we developed a method of quantifying pollen tube growth of individual populations in mixed pollinations that offers us unprecedented insight into the physical mechanisms that influence competition. Differential pollen germination and tube growth—The goal of this study was to determine what pollen performance traits differed between Col and Ler pollen that may contribute to nonrandom mating. While the pattern of ovule fertilization and abortion did not differ between the two accessions, many other parameters did to differing degrees. Pollen germination accounted for one of the larger differences, with Col pollen displaying 19.8% greater germination than Ler pollen (89.7% pollen germination in Col vs. 69.9% in Ler). This number likely includes the 1.8% difference in pollen viability (99.7% viable pollen in Col vs. 97.9% in Ler), as inviable pollen likely do not germinate. If this performance trait were the only one acting in pollen competitions between Col and Ler, we would have predicted that Col pollen should sire 77.9% of seeds in mixed pollinations, while Ler pollen should sire 22.1% of seeds (8.97:6.99 ratio of fertilization). These are not, however, the ratios we saw. In mixed pollinations, Col pollen sired 93.1% of the progeny; other pollen performance traits must account for the remaining 15.2% seed siring advantage Col pollen showed over Ler. Differential pollen tube growth rate is the second major performance trait that differs between Col and Ler pollen. When comparing speed of pollen tube growth in mixed pollinations, Ler pollen displayed, at 9 h, a 1.8-h delay compared with Col pollen (Fig. 6). This number includes the 12.8-min delay in pollen germination in Ler compared with Col pollen (Fig. 4). This finding reinforces the correlation between faster-growing pollen tubes and siring success in mixed pollinations seen in Viola tricolor, Hibiscus moscheutos, and many others (Snow and Spira, 1991a; Skogsmyr and Lankinen, 1999). And while undoubtedly differential pollen tube growth is a major factor in accounting for the remaining 15.2% siring success Col pollen have over Ler pollen, it is unclear whether it accounts for all of it, as we were unable to quantify one aspect of pollen performance for this study: pollen tube attrition. A number of methods have been applied to quantifying attrition in a number of different species, but all rely on the ability to reliably discriminate and count individual pollen tubes or callose plugs in pollen tubes in vivo (Cruzan, 1986, 1989, 1990a, 1993;

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Winsor and Stephenson, 1995; Smith-Huerta, 1997; Nemeth and Smith-Huerta, 2002; Mazer et al., 2016, in this issue). We have found this to be difficult in Arabidopsis thaliana due to the large number of pollen involved and the inability to clearly delineate pollen tubes in the solid style. What is clear from other studies (see introduction) is that differential attrition can have a profound impact on nonrandom mating and will need to be addressed in any system quantifying the major parameters of nonrandom mating. It is hypothesized, however, that predominately selfing plants such as A. thaliana should experience less attrition compared to outcrossing plants, possibly due to shorter styles or lower genetic load. This hypothesis is supported by data from Clarkia tembloriensis and others (Plitmann, 1993, 1994; Smith-Huerta, 1997; Mazer et al., 2010). Pollen interference—Many studies have described the impact pollen populations have on one another in mixed pollinations. For example, pollen germination is influenced by interactions between different donors, in some cases leading to increased germination, while other cases, leading to lower germination (Marshall and Folsom, 1992; Mitchell and Marshall, 1995; Marshall et al., 1996; Nemeth and Smith-Huerta, 2002). Interference can also occur at the level of pollen tube growth. For example, in Dianthus chinensis differences between self and outcross pollen tube growth rates are increased when both pollen types are found together in the same pistil (Aizen et al., 1990). In our case, pollen tube growth rates show robust interference, with significant differences between single-donor and mixed pollinations. Ler pollen tubes grew slower in mixed pollinations than in single-donor pollinations at all time points, demonstrating that the presence of Col pollen interfered with Ler pollen tube growth. Col pollen tube growth in mixed pollinations shows a more complex pattern, with interference only evident at 9 h. This interference is not likely related to initial ovule fertilization, as at 9 h, Col pollen tubes have not only reached the first ovules, they have traversed more than half the pistil. Ler pollen tubes are not far behind, having traversed almost half the pistil. While mechanisms of interference could include (1) structural or physiological limits in the pistil, (2) pollen directly affecting other pollen, or (3) pistil tissue hindering or helping different pollen populations, how we might distinguish among these mechanisms may require a better baseline understanding of pollen tube growth in the absence of competition between different genotype pollen. For example, in this special issue, Harder et al. (2016) describes methods that distinguish and inform about pollen facilitation, competition, and attrition. Finally, it is possible that multiple mechanisms of interference occur in Brassicaceae. For example, interference in Raphanus sativus is detectable at germination, but could not be assayed during tube growth (Marshall et al., 1996). In A. thaliana, we see interference during pollen tube growth, without clear indication of interference in germination. Indeed, if the interference is chemical, both germination interference and pollen tube growth interference may rely on the same mechanism. The genetics to pollen performance—One challenge in under-

standing the genetics of nonrandom mating lies in its complexity, potentially involving multiple distinct genetic pathways specific to either female or male tissues. In previous work, we developed a procedure to separately map female and male genes that influence nonrandom mating (Carlson et al., 2011; Gerald et al., 2014). Using

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this procedure, we identified two loci, qMNRM1 (Male-mediated NonRandom Mating 1) and qMNRM2 that influence pollen success in competitions between Col and Ler pollen (Carlson et al., 2011). When qMNRM1 is present as a Col allele, it provides, on average, a 14% increase in siring ability in competitions compared to when the Ler allele is present. qMNRM2 has a much smaller effect (4.6%), and it interacts epistatically with qMNRM1. It is likely that either the differential pollen germination and tube growth behaviors we see between Col and Ler pollen will be mediated by the gene(s) present at qMNRM1. While knowing the phenotype of qMNRM1 may provide a useful tool in narrowing candidate genes in this region, this study also makes clear that the genetic diversity at the loci we identified translates phenotypically into differential rates of prezygotic pollen performance traits. This represents an important step in developing an integrated mechanistic and genetic model of nonrandom mating.

CONCLUSION Previously, we demonstrated the widespread ability of A. thaliana accessions to mate nonrandomly. We have genetically defined loci that direct nonrandom mating and demonstrated for Col and Ler accessions the process is male-mediated. Now, we have implicated differential pollen germination and pollen tube growth as major performance traits influencing nonrandom mating patterns and demonstrated the existence of interference competition. Developing connections between the mechanisms and genetics of nonrandom mating will help us understand the possibilities and limits of selection on this process in plants. These studies will add new dimensions to our understanding of this widespread and important phenomenon. ACKNOWLEDGEMENTS We thank our anonymous reviewers for critical comments that greatly improved this manuscript. We especially thank Joe Williams for his time, comments, and encouragement. This work was supported by a grant from the National Science Foundation to R.J.S. (1020325). LITERATURE CITED Abbott, R. J., and M. F. Gomes. 1989. Population genetic structure and outcrossing rate of Arabidopsis thaliana (L.) Heynh. Heredity 62: 411–418. Aizen, M. A., K. B. Searcy, and D. L. Mulcahy. 1990. Among-flower and within-flower comparisons of pollen tube growth following self-pollinations and cross-pollinations in Dianthus chinensis (Caryophyllaceae). American Journal of Botany 77: 671–676. Al-Shehbaz, I. A., and S. L. O’Kane. 2002. Taxonomy and phylogeny of Arabidopsis (Brassicaceae). The Arabidopsis Book 1: e0001. doi:10.1199/ tab.0001 Ala-Honkola, O., M. K. Manier, S. Lupold, E. M. Droge-Young, W. F. Collins, J. M. Belote, and S. Pitnick. 2014. No inbreeding depression in sperm storage ability or offspring viability in Drosophila melanogaster females. Journal of Insect Physiology 60: 1–6. Alexander, M. P. 1969. Differential staining of aborted and non-aborted pollen. Stain Technology 44: 117–122. Apsit, V. J., R. R. Nakamura, and N. C. Wheeler. 1989. Differential male reproductive success in Douglas fir. Theoretical and Applied Genetics 77: 681–684. Armbruster, W. S., and D. G. Rogers. 2004. Does pollen competition reduce the cost of inbreeding? American Journal of Botany 91: 1939–1943.

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New Arabidopsis advanced intercross recombinant inbred lines reveal female control of nonrandom mating.

Phytosulfokine peptide signaling controls pollen tube growth and funicular pollen tube guidance in Arabidopsis thaliana.

Pollen-Specific Aquaporins NIP4;1 and NIP4;2 Are Required for Pollen Development and Pollination in Arabidopsis thaliana.

Inbreeding effects in a mixed-mating vine: effects of mating history, pollen competition and stress on the cost of inbreeding.

Effective size of nonrandom mating populations.

Structure and evolution analysis of pollen receptor-like kinase in Zea mays and Arabidopsis thaliana.

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana.

Genomic Conflicts that Cause Pollen Mortality and Raise Reproductive Barriers in Arabidopsis thaliana.

Exocyst subunit SEC3A marks the germination site and is essential for pollen germination in Arabidopsis thaliana.

PCP-B class pollen coat proteins are key regulators of the hydration checkpoint in Arabidopsis thaliana pollen-stigma interactions.

Analyses of Catharanthus roseus and Arabidopsis thaliana WRKY transcription factors reveal involvement in jasmonate signaling.

Host-parasite interactions and the evolution of nonrandom mating.

"Out of pollen" hypothesis for origin of new genes in flowering plants: study from Arabidopsis thaliana.

Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement.

Selection for fresh weight in Arabidopsis thaliana under two mating systems.

Testing models for the leaf economics spectrum with leaf and whole-plant traits in Arabidopsis thaliana.

High humidity partially rescues the Arabidopsis thaliana exo70A1 stigmatic defect for accepting compatible pollen.

HISTONE DEACETYLASE 9 represses seedling traits in Arabidopsis thaliana dry seeds.

High-Throughput Non-destructive Phenotyping of Traits that Contribute to Salinity Tolerance in Arabidopsis thaliana.

Molecular evolution of candidate genes involved in post-mating-prezygotic reproductive isolation.

Bacterial Traits Involved in Colonization of Arabidopsis thaliana Roots by Bacillus amyloliquefaciens FZB42.

Sulfenome mining in Arabidopsis thaliana.

Gene targeting in Arabidopsis thaliana.

Involvement of GPI-anchored lipid transfer proteins in the development of seed coats and pollen in Arabidopsis thaliana.