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Reproductive Biology |
2Department of Botany, University of Toronto, Toronto, Ontario, Canada M5S 3B2 3Department of Biology, University of California, Riverside, California 92521 USA; and Rocky Mountain Biological Laboratory, Crested Butte, Colorado 81224 USA
Received for publication July 28, 2005. Accepted for publication November 1, 2005.
ABSTRACT
Based on previous studies, extreme (>99%) self-sterility in scarlet gilia (Ipomopsis aggregata) appears to be involved in late-acting ovarian self-incompatibility (OSI). Here, we confirm this suggestion by comparing structural events that follow from cross- vs. self-pollinations of I. aggregata. Growth of cross- and self-pollen tubes in the style at 11 h and growth in the ovary at 24 h was equivalent. Nonetheless, by 24 h, cross-pollen effected a significantly higher percentage of both ovule penetration and fertilization. Ovules in self-pollinated flowers showed pronounced changes, including an absence of embryo sac expansion and reduced starch in the integument, by 11 h post-pollination, well before pollen tube entry into the ovary. In addition, the integumentary tapetum and adjacent 1–3 cell layers exhibited abnormal cell division, pronounced deposition of thick, pectin-rich cell walls, and cellular collapse. Ovules and embryo sacs from cross-pollinated flowers rarely showed such features. Developmental changes in ovules from self-pollinated flowers eventually resulted in integument and embryo sac collapse, a process not observed in ovules of unpollinated flowers. We suggest that OSI involves long-distance signaling between self-pollen or self-pollen tubes and carpel tissue that reduces availability of receptive ovules for fertilization before pollen tubes arrive in the ovary.
Key Words: histology • long-distance signaling • ovarian self-incompatibility (OSI) • ovular collapse • Polemoniaceae • Rocky Mountain Biological Laboratory • self-incompatibility
Self-incompatibility (SI) is a genetically controlled process in angiosperms that results in the recognition and rejection of self- or self-related pollen and pollen tubes (de Nettancourt, 1977
, 1997
, 2001
). In some cases, rejection occurs at the stigma through instantaneous post-pollination reactions that inhibit or retard pollen hydration and germination or growth of pollen tubes soon after pollen germination (Hecht, 1964
; Knox, 1973
; Owens, 1981
; Heslop-Harrison, 1982
; Franklin-Tong et al., 1994
; Pontieri and Sage, 1999
; Sage et al., 2001
; Bernhardt et al., 2003
). Depending on the species, such stigmatic SI may be sporophytic (SSI; incompatibility type determined by diploid S-genotype of the sporophytic pollen parent) or gametophytic (GSI; incompatibility determined by the haploid pollen genotype; de Nettancourt, 1977
, 1997
, 2001
). In other cases, rejection of self-pollen occurs in the style through reactions that inhibit growth of self-pollen tubes (Crane and Lawrence, 1929
; Crane and Brown, 1937
; Herrero and Dickinson, 1980
). To date, all stylar SI is documented to be gametophytic (de Nettancourt, 1977
, 1997
, 2001
).
Self-rejection in some species may not affect self-pollen germination and tube growth even though selfing is followed by sterility (Seavey and Bawa, 1986
; Sage et al., 1994
). In one version of such late-acting ovarian SI (OSI), self-pollen tubes enter ovules but do not penetrate embryo sacs (Kenrick et al., 1986
). In other cases, self-pollen tubes deposit sperm into the embryo sac but double fertilization is not complete (Cope, 1962
). In still other instances, double fertilization occurs but zygotes arising following self-pollination never divide (Sears, 1937
; Sparrow and Pearson, 1948
; Williams et al., 1984
; Sage and Williams, 1991
; Gibbs and Bianchi, 1993
; 1999
; Gibbs et al., 1999
; Lewis and Gibbs, 1999
; Sage and Sampson, 2003
; Bittencourt and Semir, 2005
). Because inbreeding depression is expected to cause embryo failure at a variety of developmental stages, the uniform failure of zygotes arising from self-pollination at an initial stage prior to cell division differentiates this last form of OSI (postzygotic OSI) from inbreeding depression (Charlesworth, 1985
; Seavey and Bawa, 1986
). Furthermore, the high degree of self-sterility of some OSI species also precludes inbreeding depression as an explanation of developmental failure (see Appendix I of Waser and Price, 1991
). Once considered to be an anomalous phenomonon (de Nettancourt, 1977
), OSI is now known to be widely distributed amongst flowering plants (Seavey and Bawa, 1986
; Sage et al., 1994
; Gibbs and Bianchi, 1999
; Gibbs et al., 1999
; Bittencourt et al., 2003
; Pound et al., 2003
; Sage and Sampson, 2003
; Bittencourt and Semir, 2005
) and hence, to play an important role, as do other forms of SI, in reducing inbreeding and its harmful effects (Charlesworth and Charlesworth, 1987
; Barrett, 1988
; de Nettancourt, 1997
; Waser and Williams, 2001
). A limited number of studies indicate that OSI is under genetic control (Cope, 1962
; Lipow and Wyatt, 2000
; LaDoux, 2004
).
Self-sterility in the montane perennial Ipomopsis aggregata (Pursh) V. Grant (Polemoniaceae) is extremely high, with greater than 99% of ovules failing to develop following self-pollination (Waser and Price, 1991
). Self-sterility in this species is not due to SI reactions that prevent self-pollen from germinating on the stigma, growing in the style, or penetrating ovules (Waser and Price, 1991
). Rather, self-sterility appears to be due to late-acting OSI because ovules that are penetrated by self-pollen tubes appear to degenerate as indicated by reduced size and appearance of a milky substance interpreted to be callose (Waser and Price, 1991
). Since self-pollen tubes of I. aggregata enter ovules, self-sterility may be postzygotic, although this has not been demonstrated.
We compared ovule and seed development following cross-, self-, and no pollination in I. aggregata to assess (1) whether SI is pre- or postzygotic; (2) temporal aspects of ovule and seed degeneration following self-pollination; and (3) the anatomical and histochemical features of ovule and seed degeneration. Our findings confirm OSI in I. aggregata and indicate that the phenomenon is prezygotic, apparently involving a modification of long-distance signaling between pollen or pollen tubes and stigmatic or stylar tissue on the one hand and ovarian tissue on the other.
MATERIALS AND METHODS
Study organism
Scarlet gilia, Ipomopsis aggregata, is a monocarpic herbaceous perennial that is widespread in montane areas of western North America (Grant and Wilken, 1986
). Plants may grow for 2–7 yr as a vegetative rosette prior to producing up to several hundred nectar-producing red, tubular flowers. Populations of I. aggregata used for this experiment were located at or near the Rocky Mountain Biological Laboratory (RMBL) in western Colorado. The predominant pollinators of I. aggregata growing at RMBL are hummingbirds, although flowers are also visited by insects from several orders, some of them effective pollinators, including bumble bees, solitary bees, syrphid and muscoid flies, and hawkmoths and butterflies (Price et al., 2005
).
Pollination treatments
Two separate sets of controlled pollinations were conducted to determine whether there were differences in the time course and nature of ovule and seed development following cross- and self-pollination. In late June 1998, we potted five adult plants from each of two sites separated by ca. 10 m within a population at the RMBL. These plants were potted before flowering and were maintained in a greenhouse at the RMBL. Similarly, in late June 1999, we potted six adult plants from each of two sites separated by ca. 10 m within a population 4 km south of the RMBL. These plants were taken to the University of Toronto where they were maintained in a glasshouse at 25°C until flowering was complete. In both experiments, flowers were emasculated in the bud stage so that stigmas were free of any pollen when they became receptive several days later, as indicated by reflexed stigma lobes. On each plant, controlled hand pollinations were conducted by applying either pure cross- or self-pollen on all three stigmatic lobes of separate flowers, alternating the treatments from one flower to the next. All cross-pollinations were between plants from the two sites in the source population separated by ca. 10 m. Pollinated carpels were harvested at 11 h, 24 h, 48 h, and 120 h post-pollination. Unpollinated carpels were also harvested at the same times. Time zero for emasculated, unpollinated carpels was determined as the time when stigmas became receptive. Harvested pollinated and unpollinated carpels were fixed in either formalin–acetic acid–alcohol (FAA; 1998 experiment) or in glutaraldehyde (1999 experiment) as described by Sage and Williams (1995)
, dehydrated in ethanol, embedded in LR White resin (Electron Microscopy Supplies, Fort Washington, Pennsylvania, USA), and serially sectioned at 1.5 µm with a diamond histoknife (Delaware Diamond Knives, Wilmington, Delaware, USA). Carpel tissue fixed in glutaraldehyde only was used for quantitative observations because of the absence of cell distortion.
Quantitative analysis of pollen tube growth
Stigmas, styles, and ovaries from serially sectioned carpels were examined to assess pollen tube growth following cross- vs. self-pollination to insure that OSI was indeed functioning as previously described (Waser and Price, 1991
). To determine whether or not there were differences between cross- vs. self-pollen tube growth within the carpel at 11 and 24 h post-pollination, the number of germinated pollen grains on the stigma, pollen tubes at the base of the style and mid-ovary, and micropyles penetrated by pollen tubes were quantified for a subset of sectioned carpels (N = 1 carpel per plant per pollination treatment per sample time; five plants). These times were selected because Waser and Price (1991)
noted that ovule degeneration had been initiated by 24 h. Sectioned tissue was used to quantify pollen tube growth because I. aggregata does not deposit callose in the pollen tube walls within the style (Waser and Price, 1991
; T. Sage, personal observations), thereby preventing the use of squashes of softened pollinated carpels to detect callose in pollen tube walls with fluorescence microscopy (Martin, 1959
). For each carpel, the number of cross- or self- pollen tubes at the base of the style or mid-ovary divided by the number of germinated pollen grains per stigma was multiplied by 100. The number of penetrated micropyles was divided by the number of ovules within the carpel and multiplied by 100. The values for cross- vs. self-pollen tubes at the base of the style were contrasted with a Student's t test as were those for cross- vs. self- pollen tubes at mid-ovary and micropyles penetrated by cross- vs. self-pollen tubes. All statistical analyses were performed using Sigma Stat (version 2.03; SPSS, Inc., Chicago, Illinois, USA).
Ovule, seed, and embryo sac development following cross-, self-, and no pollination
Anatomical and histochemical features of ovule, seed, and embryo sac development following each pollination treatment were assessed after staining the majority of sections with periodic acid-Schiff's reagent (PAS; O'Brien and McCully, 1981
) and counterstaining with toluidine blue (O'Brien and McCully, 1981
). To characterize the nature of the milky ovular occlusion reported to be present in the ovule micropyle following self- vs. cross-pollination (Waser and Price, 1991
), a subset of sections (N = 3 carpels per plant per pollination treatment; five plants) was stained with either aniline blue to detect callose (Martin, 1959
), phloroglucinol to detect lignin (O'Brien and McCully, 1981
), or ruthenium red to detect pectins (O'Brien and McCully, 1981
).
Microscopic examination of stained serial ovary sections to assess general structural and histochemical features of ovule and seed development following cross- vs. self-pollination (N = 14 carpels per plant per pollination treatment per harvest time; 11 plants) revealed a qualitative difference in the amount of starch in the integument of ovules from cross- vs. self-pollinated flowers at 11 h. Furthermore, although small plastids were present in the integument of unpollinated flowers, starch grains were absent. Therefore, the amount of integument area occupied by starch at 11 h was quantified with imaging software (Image J; National Institutes of Health, Bethesda, Maryland, USA) at the median longitudinal section of a subset of sectioned carpels fixed in glutaraldehyde (N = 3 ovules per ovary; one ovary per plant per pollination treatment; four plants). Initially, a one-way ANOVA was used to determine if there were any significant differences in integumentary starch between ovaries developing on different plants within a pollination treatment. No differences were observed. Therefore, all data within treatments were pooled. Statistical analyses were then performed to determine whether there were differences in the amount of starch in the integument at 11 h between pollination treatments using an ANOVA on ranks with multiple comparison procedures (Dunn's or Tukey's).
Serially sectioned carpels were also used to quantitatively determine the timing of ovule and seed collapse following cross- vs. self-pollination that was reported by Waser and Price (1991)
. The median longitudinal section of ovules and seeds and the enclosed embryo sacs from cross-, self-, and unpollinated flowers of glutaraldehyde-fixed tissue at 11 h, 24 h, and 120 h (N = 6 ovules–seeds–embryo sacs per ovary; one ovary per plant per pollination treatment per sample time; five plants) were used to quantify the area of ovules, seeds, and embryo sacs with the 10x objective of a Zeiss Axioplan microscope (Carl Zeiss, Göttingen, Germany) equipped with an image analysis system (Northern Exposure; Empix Imaging, Mississauga, Ontario, Canada). Initially, a one-way ANOVA was used to determine whether there were any significant differences in ovule, seed, and embryo sac area between ovules, seeds, and embryo sacs from different ovaries developing on different plants within a pollination treatment at each harvest time. No differences were observed. Therefore, all data within treatments for each harvest time were pooled. Statistical analyses were then performed to determine whether there were differences between ovule, seed, and embryo sac area between pollination treatments at each harvest time using one-way ANOVA on ranks with multiple comparison procedures (Dunn's or Tukey's). Serial sections from unpollinated carpels used for quantification of ovule, seed, and embryo sac area as well as starch occupied on an area basis were examined to insure that pollination had not occurred.
Quantification of double fertilization
Serial sections of ovaries were used to assess the percentage of ovules with double fertilization (N = 3–4 ovaries per plant; three plants) at 24 and 48 h after cross- vs. self-pollination. The mean percentage of double fertilization following cross- vs. self-pollination was then compared using a Mann-Whitney rank sum test. Double fertilization was indicated by the presence of: (1) pollen tube penetration of the embryo sac; (2) a resting zygote or embryo; and (3) one or more endosperm nuclei.
RESULTS
Pollen tube growth following cross- vs. self-pollination
Pollen tubes initially grew intercellularly amongst ground cells adjacent to stigmatic epidermal cells (Fig. 1) and then to the base of the style by 11 h following cross- and self-pollination. Pollen tubes contained continuous cytoplasm along their entire length from the stigma to the ovary. No significant differences were observed between the mean percentage of pollen tubes from germinated cross- vs. self-pollen at the base of the style at 11 h (cross = 72.0 ± 7.9%; self = 64.6 ± 10.9% [mean ± 95% confidence interval]; df = 8, t = 1.518, P = 0.167) or at the mid-ovary at 24 h (cross = 25.8 ± 9.8%; self = 30.6 ± 10.9%; df = 8; t = –0.892; P = 0.399). While both cross- and self-pollen tubes entered micropyles by 24 h (Fig. 2), significant differences were observed in the percentage of micropyles penetrated (cross = 29.2 ± 8.5%; self = 7.0 ± 6.2%; df = 8; t = –5.833; P 
0.001).
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0.001). The innermost layer of the integument adjacent the embryo sac, hereafter referred to as the integumentary tapetum (Kapil and Tiwari, 1978
), was characterized by hypertrophied cells with localized regions of cell divisions that were periclinal to the surface of the embryo sac at 11 h following self-pollination (Fig. 6). These localized cell divisions were in some instances organized into proembryos (Fig. 7; adventitious embryony; Koltunow, 1993
). Cellular collapse of integumentary tapetum cells was common at 11 h following self-pollination (Figs. 6–9). In addition, the integumentary tapetum and 1–3 adjacent layers of integumentary cells developed thick cell walls (Figs. 6– 9) that were PAS- and ruthenium red-positive but phloroglucinol- and aniline blue-negative. As observed for the integumentary tapetum, adjacent integumentary cells also exhibited cellular collapse (Figs. 7, 8). The cellular features apparent in ovules at 11 h following self-pollination became more pronounced at 24 and 48 h. By 120 h, the embryo sac and cells comprising the integument collapsed (Figs. 3, 10). Histochemical studies indicated an absence of micropyle exudate in all ovules examined from self-pollinated carpels.
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0.001). A four-celled embryo (Fig. 14) with greater than 32 endosperm nuclei was apparent in all fertilized seeds by 120 h following cross-pollination only. Ovule, seed, and embryo sac area at the median longitudinal section increased from 11 to 120 h following cross-pollination (Fig. 3). DISCUSSION
We have demonstrated that the recognition of self-pollen in I. aggregata flowers is prezygotic and is associated with abnormal development of the integument and embryo sac of ovules. This abnormal development begins before self-pollen tubes penetrate the ovary and results ultimately in the collapse of ovules, even though self-pollen tubes may subsequently enter the micropyle, and in rare cases, effect double fertilization. We begin by discussing the implications of these findings for the mechanisms of self-recognition and rejection in I. aggregata. Because OSI in this species results in prezygotic initiation of events that lead to abortion of ovules, we next address the functional consequences for reproduction. We conclude by comparing our results with what is known about SI in other genera within the Polemoniaceae.
Mechanism of OSI in I. aggregate
The observations reported here indicate that the process of self-recognition and rejection in I. aggregata entails long-distance signaling between self-pollen or self-pollen tubes and stigmatic or stylar tissue on the one hand and ovarian tissues on the other, that results in the modification of one or more post-pollination stimulatory functions of pollen or pollen tubes on ovule tissue development. This signaling, and the reactions to it, occur at least by 11 h following pollination and are distinct from those that occur in the absence of pollination and after cross-pollination because the histological and anatomical results are so clearly different. Whereas cross-pollen or cross-pollen tubes elicit significant starch storage in the integument, induce an orderly development of the integumentary tapetum and adjacent cell layers before pollen tubes enter the ovary, the presence of self-pollen or self-pollen tubes instead elicits little starch storage, disorderly development of the integumentary tapetum and adjacent cells, and a reduction in embryo sac area in comparison to both cross-pollinated and unpollinated flowers. In the absence of pollination, no starch storage occurred even though integument development was not significantly different from that following cross-pollination.
There is good evidence from other flowering plants for long-distance signaling in which the presence of compatible pollen or pollen tubes influences ovule development, embryo sac viability, starch metabolism, and transmitting tissue secretion in ovarian tissue prior to entry by pollen tubes (Pimienta and Polito, 1982
, 1983
; Herrero and Gascon, 1987
; Herrero and Arbeloa, 1989
; O'Neill, 1997
; Pontieri and Sage, 1999
; Koehl, 2002
). And, although the distances of signaling are less than stigma-to-ovule, a form of pollen-to-carpel signaling has been documented in I. aggregata whereby pollen application results in closure of stigma lobes (Waser and Fugate, 1986
).
Ipomopsis aggregata represents the first eudicot OSI species and second flowering plant in which SI reactions result not in altered pollen tube development, but instead in altered ovule development prior to entry of ovaries by pollen tubes. Self-pollination in the monocotyledonous OSI species Narcissus triandrus also resulted in failed ovule development that was initiated well before pollen tubes entered the ovary (Sage et al., 1999
). These results are important because they broaden the taxonomic range of species reported to exhibit OSI that is prezygotic in its action. In contrast to the negative prezygotic affect of self-pollen on ovule development reported here for I. aggregata and elsewhere for N. triandrus (Sage et al., 1999
), a study on SI in the basal angiosperm lizard's-tail (Saururus cernuus) indicated that even in the presence of SI mechanisms that operate to prevent self-pollen hydration and germination at the stigma, long-distance signaling from self-incompatible pollen still stimulates ovule development, a phenomenon that also occurs following cross-compatible pollinations but not in the absence of pollination (Pontieri and Sage, 1999
). Our results for I. aggregata reinforce the view proposed by Sage et al. (1999)
that such OSI phenomena be included within a broadened functional view of SI that recognizes prezygotic maternal recognition of self as the fundamental feature of all incompatibility systems, regardless of the underlying molecular and physiological mechanisms involved.
Functional consequences of self-pollination on reproduction in I. aggregate
Our data show that self-sterility following application of only self-pollen in I. aggregata was due to an absence of fertilization. This absence in fertilization occurred as a result of a reduction in the number of ovules penetrated by self-pollen tubes even though the number of pollen tubes present in the ovary was not significantly different between the two pollination treatments. We posit that developmental changes in ovules that occurred following self-pollination had a negative impact on the ability of ovules to attract pollen tubes. A similar reduction in self-fertilization due to the negative influence of self-pollen on ovule development has also been reported for Narcissus triandrus (Sage et al., 1999
). Significantly, self-pollination in N. triandrus resulted in a low level of self-seed set (Sage et al., 1999
). Self-seed set occurred because ovule development in N. triandrus was asynchronous, thereby placing some ovules outside of a developmental window where they could not be negatively influenced by self-pollen or self-pollen tubes (Sage et al., 1999
). We posit that the very low rate of self-fertilization observed in the present study and low levels of seed set following self-pollination in I. aggregata (Waser and Price, 1991
) may also be due to physiological asynchrony in ovule development, which cannot be observed at the anatomical level. Staggered ovule development may provide reproductive assurance (Sage et al., 1999
).
Stigmas in natural populations may experience mixed cross- and self-pollen loads and the role of mixed cross- and self-pollen loads on fecundity has been a long-standing topic of interest (Galen et al., 1989
). A common observation in OSI species is that the application of self-pollen either before or mixed with cross-pollen results in reduced seed set (Cope, 1962
; Dulberger, 1964
; Waser and Price, 1991
; Lloyd and Wells, 1992
; Broyles and Wyatt, 1993
; Barrett et al., 1996b). This reduction has been referred to as "ovule usurpation" by Waser and Price (1991)
and "ovule-discounting" by Barrett et al. (1996b)
. In the case of I. aggregata, Waser and Price (1991)
reported a substantial decrease in seed set when self-pollen was applied to the stigma (separate lobes) either at the same time as cross-pollen or 9 h before. Notably, only a small percentage of the reduction in seed set could be accounted for by a decline in cross-pollen deposition or adherence of cross-pollen to stigmas, suggesting that additional mechanisms result in lower seed set with mixtures of self- and cross-pollen. The developmental consequences following application of only self-pollen to stigmas of I. aggregata, ovule collapse during the first 120 h following pollination, may serve as a valuable indicator to confirm whether or not prezygotic reduction in ovule availability also occurs in the presence of pollen loads containing both cross- and self-pollen on the stigma in natural populations.
Given the common presence of resource limitations on reproduction in flowering plants, the wastage of ovules in OSI species following self-pollination or pollination with mixed pollen loads appears to be maladaptive because of the loss in reproductive potential that it may entail (Seavey and Bawa, 1986
). However, it has been posited that by minimizing investment of maternal resources in selfed-seed during a reproductive episode, plants with OSI may ultimately save resources, compared with those that invest in selfed-seeds that make little contribution to fitness because of inbreeding depression (Sage et al., 1999
). It is therefore significant that the first histological and developmental signs of prezygotic reduction in ovule availability following self-pollination in I. aggregata include a reduction in integumentary starch in addition to collapse of integumentary cells adjacent the embryo sac and deposition of thick pectin-rich integumentary cell walls that results in a boundary between the integument (sporophyte) and embryo sac (gametophyte). Notably, these microscopic changes occur prior to a metabolically costly two- to three-fold increase in embryo sac area that was observed between 11 h and 24 h following cross- and no pollination. Integumentary carbohydrates are essential for successful ovule and seed growth (Pimienta and Polito, 1983
; Sage and Webster, 1990
; Zinselmeier et al., 1999
). In addition, the integumentary tapetum and adjacent cells in angiosperms function in the regulation of assimilates from the sporophyte to the gametophyte, two generations that do not share a common symplast (Kapil and Tiwari, 1978
; Cheng et al., 1996
; Wobus and Weber, 1999
; Weschke et al., 2003
). Along these lines, we report the presence of transfer cell walls in the embryo sac adjacent the integumentary tapetum of I. aggregata ovules at 24 h following cross-fertilization only. Transfer cell walls are common at the sporophyte-to-gametophyte junction in all land plants, where they are posited to be the site of metabolite transfer between the two symplastically isolated generations, and they can be induced to develop in the presence of sugars (Offler et al., 2002
). Cellular hypertrophy, hyperplasia, and collapse of the integument are also correlated with seed abortion in other species, whereby the deviant integument development is posited to form a physical barrier preventing the flow of assimilates to the embryo sac (Cooper and Brink, 1945
; Satina et al., 1950
; Miller and Chourey, 1992
). Embryo abortion in almond is accompanied by callose deposition around the embryo sac and a decrease in metabolite supply to the embryo sac (Pimienta and Polito, 1982
).
Self-incompatibility in the Polemoniaceae
Self-incompatibility is widespread in the Polemoniaceae (Grant and Grant, 1965
). A unique feature of the taxon is the presence of multiple types of SI with apparent differences in spatial and genetic control. Self-incompatibility in Polemonium viscosum, Phlox drummondii, and Linanthus parviflorus results in failure of germination of bicellular pollen at a dry stigmatic surface (Levin, 1975
, 1993
; Galen et al., 1989
; Goodwillie, 1997
). Self-incompatibility in P. drummondii is likely under gametophytic control (Levin, 1975
,1993
) whereas SI in L. parviflorus is reportedly under sporophytic control (Goodwillie, 1997
). In addition, a multi-locus SI system is present in Ipomopsis tenuifolia and has been classified as late-acting due to the failure of self-pollen tube growth in the ovary (LaDoux, 2004
). On the one hand, different types of SI may have evolved on multiple independent occasions in the taxon (Barrett et al., 1996a
). Alternatively, Goodwillie (1997)
has proposed that SI in the Polemoniaceae may be a complex genetic system whereby mechanisms of recognition and rejection under sporophytic control are acting in tandem with an ancestral gametophytic system.
Results from the present study on OSI in I. aggregata add another layer of complexity to the SI phenomenon in the Polemoniaceae because self-recognition and rejection does not result in failure of pollen germination at the stigma as observed in Polemonium viscosum, Phlox drummondii, and L. parviflorus. Rather, self-recognition results in failure of ovule development in the absence of differential cross- and self-pollen tube growth. Barrett (1988)
has proposed that within the angiosperms, ancestral SI systems may be some form of OSI. Does OSI in I. aggregata represent yet another type of SI to have evolved within the Polemoniaceae? Or, does it represent an ancestral condition within the taxon? If OSI is ancestral in the Polemoniaceae, would OSI still be acting in tandem with stigmatic SI? Interestingly, application of self-pollen to stigmas 24 h prior to cross-pollen application resulted in a reduction of cross-seed set in Polemonium viscosum (Galen et al., 1989
). Only a portion of the decline in seed set in P. viscosum could be attributed to interference from self-pollen on cross-pollen performance at the stigma (Galen et al., 1989
). Similar studies conducted on I. aggregata yielded the same results, and Waser and Price (1991)
attributed the remaining reduction in cross-seed set to an additional post-pollination effect. Future studies examining the influence of self-pollen on ovule development in stigmatic SI species will be required to address the question as to whether OSI is acting in tandem with stigmatic SI in the Polemoniaceae.
An outstanding difference between I. aggregata and other SI species within the Polemoniaceae is the observation that both cross- and self-pollen tubes growing within the style of I. aggregata do not deposit callose plugs or callose in their cell wall (Waser and Price, 1991
; T. Sage, personal observations). In contrast, callose plugs and a callose cell wall is present in compatible pollen tubes in other species in the Polemoniaceae (Levin, 1975
, 1993
; Galen et al., 1989
; Goodwillie et al., 2004). Amongst seed plants, callose deposition in pollen tubes is unique to angiosperms (Bell, 1995
). The absence of callose in developing angiosperm pollen tubes is a rare trait and indicates that the pollen tubes of I. aggregata are physiologically different from those of other species within the taxa. Is the unique feature of pollen tube growth in I. aggregata important for understanding OSI as it compares to SI in other species within the Polemoniaceae? The complex and seemingly contradictory nature of SI in the Polemoniaceae demands that biology of SI in this family be examined in greater detail.
FOOTNOTES
The authors thank Peter Drobac for technical assistance. Research was funded by a Natural Sciences and Engineering Research Council of Canada grant to T.L.S. 
4Author for correspondence (tsage{at}botany.utoronto.ca
)
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