Pollen tube development in two species of Trithuria - Springer Link

Sex Plant Reprod (2012) 25:83–96 DOI 10.1007/s00497-012-0183-6

ORIGINAL ARTICLE

Pollen tube development in two species of Trithuria (Hydatellaceae) with contrasting breeding systems Mackenzie L. Taylor • Joseph H. Williams

Received: 2 November 2011 / Accepted: 8 February 2012 / Published online: 25 February 2012 Ó Springer-Verlag 2012

Abstract Trithuria (Hydatellaceae; Nymphaeales) is unique among early-divergent angiosperms in that its species are extremely small and most have exceptionally short, annual life histories. Given the evolution of these extremes of size and development, we sought to understand whether post-pollination processes still varied predictably with breeding system in Trithuria. To address this question, we studied two Western Australian species, Trithuria austinensis (dioecious, obligately outcrossing) and Trithuria submersa (bisexual, highly selfing). To document developmental timing, carpels were hand-pollinated, collected at sequential time points, and examined with light and fluorescence microscopy. In both species, pollen tubes first entered ovules \1 h after pollination, but the pollen tube pathway of outcrossing T. austinensis was almost four times longer and its pollen tube growth rates were up to six times faster (B2,166 vs. 321 lm/h) than those of T. submersa. T. austinensis also exhibited greater male investment, slower pollen germination, and greater pollen tube attrition. These differences in male gametophyte development are predicted for outcrossers versus selfers in phylogenetically derived angiosperms. These new data for Hydatellaceae reinforce the idea that an acceleration of pollen tube development occurred in the Nymphaeales stem lineage, before the origin of Hydatellaceae. We infer

Communicated by Scott Russell. M. L. Taylor (&) Department of Biology, Creighton University, 2500 California Plaza, Omaha, NE 68178, USA e-mail: [email protected] J. H. Williams Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 37996, USA

that a recent evolutionary transition to selfing in T. submersa has been accompanied by predictable modifications to reproductive development, which, because of the ancient relationship between Hydatellaceae and all other angiosperms, suggests that traits underlying the lability of flowering plant post-pollination biology were present early in their history. Keywords Breeding system Dioecy Hydatellaceae Pollen tube growth rate Progamic phase Self-fertilization

Introduction Trithuria, the only genus within Hydatellaceae, is a deeply divergent lineage within the ancient angiosperm order Nymphaeales, where it is sister to all other water lilies (Cabombaceae ? Nymphaeaceae; Saarela et al. 2007; Sokoloff et al. 2008). Because of its distant relationship to other flowering plants, Trithuria is of particular interest for reconstructing early stages of angiosperm evolution. Several potentially pre-angiospermous traits have been characterized in the genus, such as pre-fertilization seed provisioning (Friedman 2008) and the enigmatic organization of its potentially pre-floral reproductive structures, or ‘‘reproductive units’’ (Rudall et al. 2007, 2009a; Rudall and Bateman 2010). On the other hand, Trithuria is highly reduced in size and exhibits a much shorter overall life history than any other ANA-grade angiosperm (Amborella, Nymphaeales, Austrobaileyales), with ten of the twelve species reported to possess an exclusively annual life history (Sokoloff et al. 2008; Taylor et al. 2010). Trithuria also exhibits great diversity in sexual systems: four of the twelve species typically have bisexual reproductive units, four are dioecious, and four can be described

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as being cosexual or monoecious (Yadav and Janarthanam 1995; Sokoloff et al. 2008). Breeding systems are equally variable, ranging from primarily selfing in Trithuria submersa (Taylor et al. 2010) to obligately outcrossing in the dioecious species. Breeding system is intimately linked with the developmental events that occur during the life history stage between pollination and fertilization (the progamic phase). In contrast to non-flowering plants, substantial mate sorting occurs during this phase in angiosperms, either via selfincompatibility systems (de Nettancourt 1977; Allen and Hiscock 2008) or via intraspecific or interspecific pollen competition (Jones 1920; Hogenboom 1975, 1984; Mulcahy and Mulcahy 1987; Walsh and Charlesworth 1992; Skogsmyr and Lankinen 2002). Pollen competition is thought to have been a pervasive force in angiosperms relative to other seed plants (Willson and Burley 1983; Mulcahy 1979). If so, one might expect to see its effects in both ancient and modern angiosperm lineages. Pollen competition is expected to be stronger in outcrossers than in long-term selfers, because outcrossers should have more genetically diverse stigmatic pollen loads (Mazer et al. 2010). Outcrossers, in turn, should have faster pollen tube growth rates (Smith-Huerta 1996; Kerwin and SmithHuerta 2000), but greater pollen tube attrition (Plitmann 1993, 1994; Smith-Huerta 1997), and might also have evolved larger flowers with longer styles (Mazer et al. 2010). Few studies have explicitly investigated the consequences of breeding system divergence on the evolution of pollen tube development. Many of the post-pollination traits that are associated with outcrossing are also hypothesized to have played a role in early flowering plant diversification (e.g., Whitehouse 1950; Stebbins 1957; Willson and Burley 1983; Mulcahy 1979; Williams 2008). With the origin of bisexual reproductive structures in the angiosperm lineage, male and female organs were brought into close proximity and early flowers might have experienced regular self-pollination and self-fertilization (Lloyd and Webb 1986; Bernhardt and Thien 1987; Lloyd and Wells 1992; Dilcher 2000). The disadvantages of long-term self-fertilization are thought to have subsequently caused the evolution of a host of mechanical and perhaps also biochemical barriers to self-fertilization among early angiosperms (Stebbins 1974; Dilcher 2000). Thus, understanding the relationship between breeding system and progamic phase biology in ancient lineages can provide insight into both the nature of early angiosperm reproduction and its capacity for change. In this study, we describe and compare progamic phase development in two Trithuria species: Trithuria austinensis D.D. Sokoloff, Remizowa, T.D. Macfarl. & Rudall and T. submersa Hook.f. These are not sister species, but are within sister clades of annual species (Iles et al. 2012). We

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Sex Plant Reprod (2012) 25:83–96

characterize reproductive morphology and timing of developmental events in both species and then consider species differences in light of their contrasting breeding systems. Our results bear on two questions: (1) how have progamic phase ontogenies evolved within Hydatellaceae relative to other Nymphaeales and (2) have small size and rapid development constrained the evolution of post-pollination traits in Trithuria?

Materials and methods Field sites Trithuria austinensis and T. submersa occur in ephemeral wetlands that are characterized by standing water in the wet season, followed by a brief period of water drawdown lasting a few days to a few weeks, and complete desiccation during the dry season (Hill et al. 1996; Sokoloff et al. 2008). Seeds of these two species germinate and plants grow vegetatively while completely submerged. As the water level drops, plants become exposed and must complete flowering and fruit-set before the habitat dries out completely (Taylor et al. 2010). Experimental pollinations and collections were undertaken in southwest Western Australia (shire of Manjimup) during November–December of 2008 and 2009. Laboratory work was conducted at the Department of Environment and Conservation Science Division facility in Manjimup, WA, and at the University of Tennessee, Knoxville (UTK). Voucher specimens have been deposited in the UTK Herbarium (TENN). Experimental pollinations and collections for T. austinensis were conducted at Branchinella Lake (34°210 S; 116°430 E), and for T. submersa at Frying Pan Swamp (34°160 S, 116°420 E), and Kulunilup Swamp, Kulunilup Nature Reserve (34°190 S, 116°460 E). Reproductive development Plants of both species were collected en masse and fixed whole in FAA (2:1:10:7 of 40% formaldehyde, glacial acetic acid, 95% ethanol, distilled water) for 24 h and stored in 70% ethanol for analysis of reproductive morphology and natural pollen loads. Living plants were observed (*75 h for each species) to determine timing of anther dehiscence and degree of insect visitation. Experimental pollinations were conducted to document the timing of developmental events. Submerged female plants of T. austinensis were covered with pollen exclusion cages constructed from small plastic cups staked into the ground with wire to prevent external pollination. Reproductive units of covered plants developed normally. Mature


anthers were collected between 0930 and 1400 h, stored (\1 h) on filter paper until fully open, and then gently brushed across mature stigmatic hairs to achieve pollination. Each cross was from a single pollen donor. Plants were re-caged until reproductive units were collected. Over the course of the study, 243 female reproductive units were pollinated and collected at either 5, 10, 15, 20, 30, or 45 min or 1, 1.5, 2, 2.5, or 3 h after pollination (hap). Only one reproductive unit was collected per plant per time point. Trithuria submersa anthers were removed from submerged, immature reproductive units, and plants were covered with pollen exclusion cages until emergent and mature. Mature anthers were collected from nearby plants between 0830 and 1130 h, stored on filter paper until fully open (\1 h), and brushed gently over mature stigmatic hairs. Plants were re-caged until reproductive units were collected. Over the course of the study, 115 reproductive units were pollinated and collected at 5, 15, or 30 min or at 1, 1.5, 2, or 3 hap. All reproductive units collected were fixed in FAA or 3:1 (95% ethanol: glacial acetic acid) for 24 h and stored in 70% ethanol. Carpels were dissected out of reproductive units, stained 4–8 h with 0.1% aniline blue, rinsed in distilled water, and then stained with 40 ,6-diamidino-2-phenylindole (DAPI) for 4 h. These were viewed under UV light with a Zeiss Axioplan II compound microscope (Carl Zeiss, Oberkochen, Germany) for simultaneous visualization of callose in pollen tubes and DNA in nuclei. For histological analysis, carpels were dehydrated with 95% EtOH, then infiltrated, and embedded in JB-4 polymer (Polysciences, Inc., Warrington, PA, USA) following standard protocols. Serial sections (5 lm) were cut with a Sorvall Dupont JB-4 microtome (Newtown, CT, USA), using glass knives, mounted on glass slides, and stained with 0.1% toluidine blue O (TBO) for general histology, 0.1% aniline blue for visualization of callose, or 0.01% Auramine O for visualization of the cuticle (Heslop-Harrison 1977). Because ungerminated pollen grains often washed off stigmatic hairs during fixation, the percentage of germinated grains per stigma could not be calculated. Instead, the timing of pollen germination was assessed by calculating the percentage of reproductive units at each time point that exhibited germinated pollen grains (which do not wash off stigmas). Individual pollen tube growth rates were determined by measuring the length of the longest pollen tube on each carpel and dividing it by the time since pollination, less time to germination. In T. austinensis, 15 min was subtracted to account for minimum time to pollen germination, whereas in T. submersa, no time was subtracted because pollen germinated in less than 5 min (see ‘‘Results’’). Pollen tube growth rates were averaged across carpels on

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each maternal plant, and then across maternal plants, to obtain the average for each time point. Unless otherwise noted, rates and sizes were compared between species with a one-way ANOVA conducted in SPSS 16.0 (SPSS Inc., Chicago, IL, USA). All measures of variation in the text are standard deviations unless otherwise noted.

Results Reproductive morphology and ecology of T. austinensis Plants grew at very high densities in monoculture carpeting the bottom of Branchinella Lake at depths of up to approximately 40 cm of water. The habitat was very open, with little surrounding vegetation and no canopy. Large numbers of adjacent T. austinensis plants became emergent at the same time as the lake margin gradually receded. However, not all reproductive units on a plant emerged simultaneously because reproductive units developed sequentially and peduncles were at different heights (Fig. 1a). Plants of T. austinensis are unisexual (Fig. 1a–b), with multiple reproductive units, each supported by a peduncle (9.0 ± 2.9 mm; n = 15). On average, female plants produced more reproductive units than male plants (Table 1). Female reproductive units measured 3.3 ± 0.3 mm from base to tip of bracts and contained an average of 14.8 uniovulate carpels that exhibited an average of 5.5 uniseriate stigmatic hairs each (Table 1; Figs. 1d, 2b). Anthers or carpels were completely enclosed in bracts of immature reproductive units. As female reproductive units matured, stigmatic hairs elongated and emerged between the bracts of female reproductive units. Mature stigmatic hairs were 2.06 mm long on average (maximum length = 3.89 mm; Table 2) and extended far beyond the tips of the bracts (Fig. 1d). As male reproductive units matured, filaments elongated and pushed anthers out of the bracts, which never became strongly reflexed (Fig. 1b, e). Male reproductive units contained an average of 7.9 anthers that matured consecutively and produced over 3,500 pollen grains each (Table 1). Anthers opened via two longitudinal slits that extended the entire length of the anther (Fig. 1b), and pollen was observed being released into the air. Insects were never observed landing on emergent reproductive units nor did any anthers show signs of herbivory. Anthers abscised after they dehisced, leaving behind a persistent filament (Fig. 1e). When plants were completely emergent, peduncles typically no longer supported reproductive units and plants lay prostrate in the mud. Insects, primarily flies, did contact reproductive units at this time as they crawled along the shoreline to drink, but by that time, anthers were typically empty or abscised.

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Fig. 1 Reproductive morphology of Trithuria species. a T. austinensis female plant mostly submerged, but with one fully emergent (arrow) and one newly emergent reproductive unit (asterisk) borne on peduncles (P). Stigmatic hairs are emerging from bracts (arrow). A few linear leaves are visible (L). Scale bar = 2 mm. b T. austinensis male plant with a few leaves (L) and an emergent reproductive unit. Multiple anthers (arrowhead) are dehiscing. Scale bar = 2 mm. c T. submersa plants with multiple peduncles (P) shortly after water drawdown. Stigmatic hairs (arrow) are emerging from bisexual reproductive units. Scale bar = 2 mm. d T. austinensis female reproductive unit with many uniovulate carpels (C). Each carpel

exhibits several elongated stigmatic hairs (arrow) that extend beyond bracts (B). Scale bar = 0.5 mm. e T. austinensis male reproductive unit with several stamens of varying developmental stages. Several filaments (F) have elongated and one anther (A) has abscised (arrowhead), while at least one more is dehiscent. Immature stamens are enclosed in bracts (B). Scale bar = 0.5 mm. f T. submersa reproductive unit with bracts (B) surrounding two central stamens [anthers have abscised, leaving persistent filaments (F)] and several uniovulate carpels (C) that are peripheral to the stamens. Each carpel has three stigmatic hairs (arrow). Scale bar = 0.5 mm

Table 1 Comparison of male (M) and female (F) reproductive investment in two Trithuria species Species

RUs per plant

T. austinensis

M: 5.4 ± 2.4a

T. submersa

F: 8.6 ± 4.6 4.3 ± 4.0h

Carpels per RU

Anthers per RU

No. of stigmatic hairs

Pollen grains per anther

Pollen grains per RU

Pollen-to-ovule ratio

F: 14.8 ± 4.2c

M: 7.9 ± 1.7d

F: 5.5 ± 0.9e

M: 3,525 ± 1,609f

M: 26,818 ± 9,193f

R: 1,569.1g

b i

19.3 ± 6.4

1.1 ± 0.3

i

3.0 ± 0.0

j

k

426 ± 149

k

468 ± 164

P:1,137.8ab 23.9k

The number of carpels per reproductive unit (RU) equals the number of ovules per RU Values are mean ± SD; n = a 20;

b

20

c

60;

d

10;

e

103; f 17;

g

11;

The pollen-to-ovule ratio was 1,569 at the reproductive unit level (pollen produced by male units relative to ovules produced by female units) and 1,138 at the whole plant level (pollen produced by male plants relative to ovules

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h

85; i 280; j 30;

k

27. R reproductive unit; P plant

produced by female plants; Table 1). The average natural pollen load in T. austinensis was 29.1 ± 14.1 grains per reproductive unit or 2.1 ± 1.5 germinated pollen grains per ovule.


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from the carpel mouth and were 0.56 mm long at maturity (maximum length = 1.05 mm; Fig. 2a; Table 2). Anthers each produced 426 pollen grains, and the pollen-to-ovule ratio was 23.9 (Table 1; Taylor et al. 2010). Pollen and pollen germination

Fig. 2 Carpel morphology in Trithuria. a Longitudinal section through T. submersa carpel (C) with two stigmatic hairs in section (arrow). A single ovule (O) is enclosed within the carpel, with the micropyle (arrowhead) of the anatropous ovule oriented adjacent to the stylar canal, near the carpel mouth (M). b Longitudinal section through T. austinensis carpel (C) with a single ovule (O) and four to five stigmatic hairs (arrow) at least partially in the plane of section. As in T. submersa, the micropyle (arrowhead) is oriented near the carpel mouth (M). a and b are at the same scale. Scale bar = 100 lm

Reproductive morphology of T. submersa At both T. submersa sites, plants grew at a lower density than T. austinensis. Plants grew intermixed with other plant species, and the surrounding vegetation created a closed environment (Taylor et al. 2010). Adjacent plants became emergent at different times due to the uneven ground surface (Taylor et al. 2010). Trithuria submersa plants produced bisexual reproductive units that were each borne on long peduncles (26.7 ± 6.8 mm; n = 15; Fig. 1c; Table 1). Bracts measured 1.8 ± 0.3 mm in length and enclosed an average of 19.3 carpels and 1.1 anthers (Fig. 1f; Table 1). Carpels exhibited exactly three uniseriate stigmatic hairs that radiated

Pollen grains of both species were small, mostly spherical and lacked ornamentation (Fig. 3f, h). Hydrated T. austinensis pollen on stigmas measured 16.3 ± 0.7 lm perpendicular to the aperture and 17.6 ± 1.5 lm, parallel to the aperture, whereas hydrated T. submersa pollen on stigmas measured 15.8 ± 1.8 9 18.5 ± 1.7 lm, respectively (n = 20). Pollen grains germinated along the entire stigmatic hair, but were most often located within 150 lm of the tip. The single aperture of germinated pollen was often not in direct contact with the stigmatic surface (Fig. 3h). At germination, fluorescence of the pollen tube wall following aniline blue staining was continuous with a fluorescent layer in the inner wall of the pollen grain, particularly near the aperture (Fig. 3g), indicating the presence of a continuous callose layer (1,3-b-glucan; Stone and Clarke 1992). In hand pollinations, no individuals of T. austinensis had germinated pollen at 5 and 10 min after pollination (Fig. 4). Germinated pollen was first observed at 15 min and was present in nearly one-third of individuals (Fig. 4). In T. submersa, 12.5% of individuals exhibited germinating pollen at 5 min after pollination (Fig. 4). Stigmatic receptivity was assessed after the fact, and stigmatic hairs in many hand-pollinated individuals in T. submersa consisted of short, compact cells that had not yet fully expanded and therefore were likely not receptive at the time of experimental pollination. This may explain why the percentage of reproductive units that exhibited pollen germination was consistently low in T. submersa (Fig. 4). Pollen tube growth and the pollen tube pathway The pollen tube pathway of T. austinensis was about 2.2 mm long, which is almost four times as long as that of T. submersa (0.6 mm; Fig. 2; Table 2). The majority of the pathway comprises the stigmatic hair. In T. austinensis,

Table 2 Pollen tube pathway lengths of two Trithuria species Species

Distance from pollen grain to carpel mouth (lm)

Mean stigmatic hair length (lm)

Mean distance from carpel mouth to FMG (lm)a

Total pathway length (lm)

T. austinensis

1,917.9 ± 575.9b

2,062.8 ± 593.4b

160.9 ± 19.5

2,223.7 (2,078.8)

c

556.2 ± 223.4d

175.6 ± 11.5

731.77 (636.2)

T. submersa

460.6 ± 219.9

Stigmatic hair length and internal carpel measurements were taken on a separate set of carpels. Actual distance pollen tubes traveled is in parentheses in the total pathway length column Values are mean ± SD; n = a 10,

b

66, c 38,

d

64. FMG female gametophyte

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pollen germinated 0.33–3.24 mm away from the carpel mouth, but the average pollen tube had to grow about 93% of the length of the stigmatic hair to reach the carpel (Table 2). Pollen of T. submersa germinated between 0.10 and 1.00 mm from the carpel mouth and on average grew about 83% of the length of the stigmatic hair.

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Pollen tubes in both species emerged from the aperture and if necessary grew around the grain to reach the stigmatic hair surface (Fig. 3f–i). Pollen tubes commonly branched at the point where the tube first contacted the stigmatic hair (Fig. 3i). Branching was not observed at any other location, and one branch was always much shorter

Sex Plant Reprod (2012) 25:83–96 b Fig. 3 Pollen germination and pollen tube growth on the stigmatic

hairs of two Trithuria species. a Naturally pollinated T. austinensis carpel with multiple pollen tubes (arrow) with callose plugs (arrowhead) growing along stigmatic hairs (S). Scale bar = 200 lm. b Self-pollinated T. submersa carpel with multiple pollen tubes (arrow) with callose plugs (arrowhead) growing along stigmatic hairs and entering carpel mouth (M). Scale bar = 100 lm. c T. austinensis reproductive unit with several carpels enclosed in bracts (B) and many intertwined stigmatic hairs (S). Many stigmatic hairs support 1–2 pollen tubes (arrows), but no pollen tubes cross between hairs. Scale bar = 200 lm. d Close-up of T. austinensis stigmatic hair (S) with a single tightly associated pollen tube (arrow). Scale bar = 20 lm. e Stigmatic hair (S) from D following aniline blue staining. Pollen tube walls (arrow) are callosic, and a callose plug is developing (arrowhead) at 1 h after pollination (hap). Note that fluorescence in the cell wall of the stigmatic hair indicates that callose is present after the pollen tube has passed. Scale bar = 20 lm. f Germinated T. austinensis pollen grains at 1 hap. The relatively thick exine (E) is apparent, and pollen tubes have emerged from the pollen aperture (A). The pollen tube tip (arrow) is growing along the stigmatic hair. Scale bar = 10 lm. g Pollen grains from F following aniline blue staining. Callose is present in the pollen tube wall and also in the inner pollen grain wall (arrowhead), but not at the growing tip (arrow). As it nears the tip, callose staining is only visible on the top of the pollen tube. Scale bar = 10 lm. h Germinated T. austinensis pollen grain at 45 min after pollination. The pollen tube (arrows) has emerged from an outward-facing aperture and grown around the grain to contact the stigmatic hair. The tip has penetrated the cuticle of the stigmatic hair cell (arrowheads indicate cuticle layer). Scale bar = 10 lm. i T. austinensis pollen tube at 1 hap. Walls are callosic (arrow), and branching occurred at point where the pollen tube first contacted the stigmatic hair. Scale bar = 10 lm. j Section through a naturally pollinated stigmatic hair of T. austinensis stained with auramine O. The outer layer of the stigmatic hair cell wall fluoresces around the entire hair cell, indicating the presence of a lipid layer (arrowheads), which also extends around the pollen tube as a thinner layer. Scale bar = 5 lm. k Close-up of a T. austinensis stigmatic hair cell and pollen tube cross-section stained with auramine O. Two layers are apparent, an inner layer, corresponding to the stigmatic hair primary wall, continuing beneath the pollen tube (arrow) and an outer lipid layer surrounding the pollen tube (arrowhead). The lipid layer that encloses the pollen tube (PT) is clearly continuous with the cuticle of the stigmatic hair cell Scale bar = 5 lm. l Cross-section of a T. austinensis stigmatic hair supporting two pollen tubes (PT). The inner layer of the stigmatic hair cell wall is continuous around the entire cell, including under the pollen tubes (arrow), while the translucent outer layer that corresponds to the cuticle in H and K is also visible (arrowhead). Scale bar = 10 lm. m Cross-section in L stained with aniline blue, indicating the presence of callose in the pollen tube (PT) wall only. The bilayered stigmatic cell wall is faintly visible (arrow, arrowhead). Scale bar = 10 lm. Stains: aniline blue (a–c, e, g, i, m), auramine O (j, k)

than the other. Pollen tube diameters on the stigma were significantly larger in T. austinensis (mean = 4.46 ± 0.90 lm; n = 162) than in T. submersa (mean = 3.89 ± 0.71 lm; n = 38; P = 0.0001; Fig. 3d–m). The stigmatic cell wall was distinctly bilayered. The outer layer was translucent under light and fluoresced brightly following auramine O staining, indicating the presence of a lipid cuticle (Heslop-Harrison 1977; Fig. 3j–k). Pollen tubes penetrated the cuticle very near the

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pollen grain attachment site (Fig. 3h), and growing pollen tubes tunneled under the outer layer of the stigmatic cell wall but did not disrupt the inner opaque layer (Fig. 3k–m). The outer layer was thickened at the pollen tube edge and thinner over the pollen tube wall (Fig. 3j–k). Callose was present in the pollen tube wall (Fig. 3e, i, m) and occasionally in the stigmatic hair cell walls (Fig. 3e). Callose in the stigmatic hair cell walls was not consistently present after pollen tube passage and was sometimes present in hairs without pollen tubes. The round shape of most pollen tubes in cross-section (Fig. 3h, j–m) indicates that they were young and turgid. Pollen tubes never pulled away from the stigmatic hairs (Fig. 3d–e). Although the ends of stigmatic hairs in T. austinensis became entangled and had to be physically pulled apart during dissections, pollen tubes never crossed from one stigmatic hair to another (Fig. 3c), and loose pollen tubes were never observed in dissected material. Pollen tubes did not grow laterally around stigmatic hairs (Fig. 3a–c) but instead always grew parallel to the hair axis, in either direction. When growing away from the carpel mouth, pollen tubes grew over the top of the hair before growing down to the ovule. In both species, more than one stigmatic hair per carpel often supported pollen tubes, and a single hair could support several pollen grains. Pollen tube walls stained strongly with aniline blue at the exit point from the pollen grain aperture (Fig. 3g, i) and along the length of the extended pollen tube (Fig. 3e). Relative to older parts of the pollen tube, the intensity of the callose fluorescence became reduced near the tip and was absent in the apex (Fig. 3f–g; note that lower side of tube is out of view). At the base of the stigmatic hair, pollen tubes turned sharply to enter the carpel mouth (Fig. 5a, c). In T. submersa, more than one pollen tube was often observed entering the carpel mouth (Fig. 5a). This occurred less frequently in T. austinensis. Once in the carpel, pollen tubes grew through a short, extremely narrow stylar canal lined with small, slightly elongated, and densely cytoplasmic cells (Fig. 5h–m). We never observed more than one pollen tube growing through the stylar canal in either species even when multiple tubes were present at the mouth. Upon reaching the ovary, pollen tubes entered the long neck of the micropyle and grew to a 2–3-cell-layer-thick nucellus (Fig. 5j). The micropyle was formed by integuments both in T. submersa and in T. austinensis (as also in Rudall et al. 2008 for T. submersa and Friedman 2008 for T. inconspicua). Pollen tubes were not observed to grow freely in the ovarian cavity. At the time of pollination, the female gametophyte was four-celled and four-nucleate, with a single central cell nucleus located at the chalazal pole [as also reported by Rudall et al. (2008, 2009b) for T. submersa and Friedman (2008) for T. inconspicua]. Within 30 min of pollination,

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volume of the tube between internal callose plugs was 1,295 ± 519 lm3 (n = 10). Pollen cell mitosis In T. austinensis, pollen was bicellular at pollination. Sperm nuclei were never observed in pollen tubes \1 hap, whereas undivided generative nuclei or sperm nuclei (Fig. 5d) were observed in pollen tubes at 1 and 1.5 hap. At 2 hap and later, sperm nuclei were observed in all tubes. Pollen of T. submersa was also bicellular at pollination, but two sperm nuclei were observed in pollen tubes as early as 15 min after pollination, and most tubes had formed sperm within 60 min. Fig. 4 Timing of pollen germination in T. austinensis and T. submersa. Sample sizes for each time point in ascending order are: T. austinensis, n = 5, 18, 27, 25, 26, 27, 25, 23, 18, 11, 13 individual plants, respectively, and T. submersa, n = 16, 12, 22, 20, 8, 13, respectively

the central cell nucleus was observed directly adjacent to the egg cell (Fig. 5j). Callose plugs In both species, callose plugs were present in pollen tubes on the stigmatic hair (see also Prychid et al. 2011 for T. submersa). However, callose plugs generally did not form until pollen tubes had traversed most or all of the stigmatic hair. In T. austinensis, callose plugs were first observed at 45 min after pollination, and at 1 hap, callose plugs were present in 39% of tubes. There were few plugs per tube at these times. Plugs were more abundant within single tubes at later times, as there was an increase in both the number of plugs within each tube and the percentage of tubes exhibiting plugs (43% at 1.5 hap, 88% at 2 hap). Callose plugs were present in all pollen tubes that had reached the carpel mouth. The first callose plug developed 205.9 ± 85.2 lm (n = 30) from the pollen grain, and subsequent plugs were spaced at intervals of 153.8 ± 55.1 lm (n = 42). In T. submersa, callose plugs appeared as early as 30 min after pollination and were present in every tube by 2 h, as well as in every tube that had reached the carpel mouth. They first formed at 84.9 ± 56.0 lm (n = 18) from the pollen grain and then occurred at intervals of 85.7 ± 34.3 lm (n = 27). Plugs formed through thickening of the inner pollen tube wall (Fig. 5a–b, e–g). Pollen grains of T. austinensis had a volume of 2,897 ± 750 lm3 (n = 5), whereas the volume of the tube between internal callose plugs was 3,059 ± 1,096 lm3 (n = 10). The volume of germinating pollen grains in T. submersa was 2,486 ± 733 lm3 (n = 5), whereas the

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Carpel and ovule entry Trithuria austinensis pollen tubes first entered the carpel mouth at 30 min after pollination and reached an ovule at 45 min (only one pollen tube had entered a micropyle at that time). At 1 hap, 6.5% of longest pollen tubes had entered a micropyle, and at 1.5 hap, 20.6% of pollen tubes had reached a micropyle. In T. austinensis, the majority of pollen tubes did not successfully reach an ovule during the first 3 h after pollination, and the percentage of pollen tubes that successfully reached the carpel mouth leveled off between 20 and 30% after 2 h (Fig. 6b). T. submersa pollen tubes had first entered the carpel mouth at 15 min after pollination, and by 30 min, 42.9% of leading pollen tubes had reached the carpel mouth and entered the micropyle. At 2 hap, over 90% of pollen tubes had entered the carpel (Fig. 6b) and reached a micropyle. Pollen tube growth rate The average pollen tube growth rate (PTGR) of all leading pollen tubes of T. austinensis was 499.4 ± 435.1 lm/h (Fig. 6a). However, at all time points after 45 min, it was apparent that there were two groups of pollen tubes: those that had reached an ovule and those that seemed to have arrested growth on the stigmatic hair. Pollen tubes that were successful in reaching a micropyle from 0.75 to 3 hap had significantly faster growth rates than those that were not (paired t test, P = 0.028; N = 6 time points; Fig. 6a). Over 70% of T. austinensis pollen tubes did not reach the carpel mouth within 3 h (Fig. 6b). To account for this potential confounding effect in estimating mean PTGR of successful pollen tubes, mean PTGR was recalculated after removing the tubes that had not reached ovules. PTGR of successful pollen tubes averaged over all time points was 1,046.7 ± 604.5 lm/h (n = 6). However, this calculation underestimates PTGR because pollen tubes at later time points were increasingly likely to have reached carpels


Fig. 5 Late pollen tube growth of two Trithuria species. a T. submersa carpel at 30 min after pollination (map). Three pollen tubes (arrows) have reached the based of the stigmatic hair (S) and entered the carpel mouth (M). Callose plugs are present (arrowheads). Scale bar = 20 lm. b Longitudinal section through a T. submersa stigmatic hair (S) and carpel mouth (M). One pollen tube with a callose plug (arrow) is in section. Scale bar = 20 lm. c Longitudinal section through a T. submersa carpel showing stigmatic hairs (S) and the transmitting tract (TT). A pollen tube (arrow) has turned 90° to enter the carpel mouth (M) (note developing callose plug at bend in pollen tube). Scale bar = 20 lm. d T. austinensis pollen tube at 1.5 h after pollination (hap). The second mitotic division has occurred, and two sperm nuclei (SN) are present in the tube. Scale bar = 10 lm. e T. austinensis pollen tube with two callose plugs (arrows). Scale bar = 20 lm. f T. austinensis pollen tube at 2.5 hap with a callosic thickening of the pollen tube wall (arrow), indicting early callose plug formation. Scale bar = 5 lm. g Fully developed callose plug in T. austinensis at 45 map. Scale bar = 5 lm. h Cross-section through a T. submersa carpel just at the base of stigmatic hairs (S). Large vacuolate cells at the base of stigmatic hairs surround the mouth of the carpel and the smaller, densely cytoplasmic cells that line the open stylar canal (arrow). Scale bar = 10 lm. i Cross-section as in H, but in T. austinensis and

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below the mouth. Note larger cells of the carpel wall (C) and the extracellular space of stylar canal (arrow). Scale bar = 10 lm. j Longitudinal section of a T. austinensis carpel (C) at 30 map. The carpel mouth (M) and stylar canal through the transmitting tract (TT) lead directly to the micropyle of the single, anatropous ovule, very near the top of the ovarian cavity. Several layers of nucellar tissue (N) are present above the micropylar pole of the female gametophyte, and a thickened tegument layer is present just above the nucellus. Scale bar = 50 lm. k Longitudinal section of a T. submersa carpel 30 map. The carpel mouth (M) opens into a very short stylar canal through a thin layer of transmitting tissue (TT) with the bistomal micropyle (arrowhead) of the ovule positioned at the top of the ovary. Several layers of nucellar tissue (N) are present, as is a thickened tegument layer. Scale bar = 20 lm. l Longitudinal section of a naturally collected T. austinensis carpel with two pollen tubes on a stigmatic hair (S) and one shown having entered the carpel mouth (M) and the stylar canal (arrows) surrounded by the transmitting tract (TT). Scale bar = 25 lm. m Close-up of the open stylar canal of T. austinensis at 30 map. A pollen tube (arrow) has entered the carpel mouth (M) and is growing through the extracellular space between the densely cytoplasmic cells that line the canal (TT). Scale bar = 25 lm. Stains: aniline blue (a–c, e–g, l), DAPI (d), toluidine blue O (h–k, m)

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Pollen tube length (mm)

2.5

a

T. austinensis

2.0 1.5 1.0 0.5 0.0 0.0

T. submersa 0.5

1.0

1.5

2.0

2.5

3.0

% pollen tubes at carpel mouth

92 100 90 80 70 60 50 40 30 20 10 0

b

0.0

T. submersa

T. austinensis 0.5

1.0

1.5

2.0

2.5

3.0

Hours after pollination Fig. 6 Comparison of pollen tube growth in T. austinensis and T. submersa. a Mean (±SE) length of pollen tubes averaged over individual plants at different times after pollination. Pollen tubes of T. austinensis were considered two ways: all pollen tubes (closed circles; mean n = 8.1 plants/time point) and just those that had reached an ovule (open circles; mean n = 4.5 plants/time point). Pollen tubes of T. submersa are indicated by triangles (mean n = 6.4 plants/time point). Slopes of black lines indicate mean growth rates within each interval, and those of gray lines are mean sustained growth rates calculated for the species, as discussed in text. Gray

dotted lines represent the average distance from pollen to female gametophyte (realized pollen tube pathway length). b The percentage of leading pollen tubes that had entered the carpel mouth in T. austinensis (circles) and T. submersa (triangles) at each collection point. Note that less than 35% of T. austinensis pollen tubes reach the mouth in the first 3 h, compared to over 90% in T. submersa. In nearly all cases, pollen tubes that were observed to have entered the carpel mouth had also entered a micropyle. The trend line indicates the moving average

much earlier, and therefore, an overly long time was used to calculate their growth rates. As evidence, PTGR decreased progressively from a maximum of 2,165.9 ± 568.6 lm/h at 1 hap to 664.5 ± 81.6 lm/h at 3 hap (Fig. 6a). The earliest time points most accurately reflect the average PTGR of successful tubes in T. austinensis, since these pollen tubes in these time points had just arrived at ovules and therefore their growth periods were only slightly overestimated. Thus, the best estimate of mean growth rate of successful pollen tubes in T. austinensis is between the maximum of 2,166 lm/h and the mean of 1,047 lm/h. Pollen tubes did not fall into two classes in T. submersa, because there was little attrition and [90% of tubes reached the carpel mouth within 1.5 hap (Fig. 6b). Thus, the corresponding measure of PTGR for T. submersa was 321.1 ± 281.0 lm/h over the first 2 h, the period in which most pollen tubes reached the ovule. This was significantly slower than even the minimum estimate of average pollen tube growth rate in T. austinensis of 499 lm/h (P = 0.01).

Breeding system

Discussion Trithuria austinensis and T. submersa are extremely small ephemerals that have evolved strongly outcrossing and strongly selfing breeding systems. Below, we first briefly establish the nature of breeding systems in these two species. We then discuss differences in male reproductive effort, reproductive morphology, and pollen tube development between Trithuria species with respect to their divergent breeding systems and consider their bearing on progamic phase evolution in Nymphaeales.

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Trithuria submersa is a primarily selfing species, since natural pollen loads were mostly of self-pollen and both natural and artificially selfed seed production was high (Taylor et al. 2010). Outcross pollination is by wind, but is ineffective, and a system of autonomous self-pollination and self-fertilization has evolved, as reflected by a very low pollen-to-ovule ratio of 24 (Taylor et al. 2010). Trithuria austinensis is dioecious and therefore obligately outcrossing. Outcrossing predominantly occurs via wind pollination in T. austinensis, as the species has many of the traits characteristic of wind pollination (see Whitehead 1969; Friedman and Barrett 2009). In the populations we observed, plants were densely spaced across an open habitat with large numbers of plants emerging and flowering in synchrony along the receding edge of the water. Anthers opened via two large slits through which pollen was observed dispersing into the wind. Pollen was not sticky and lacked ornamentation. As is typical in outcrossing, wind-pollinated plants, the stigmatic surface area was relatively large and comprised 5 or more stigmatic hairs, each over 2 mm long. Reproductive units lacked morphological features associated with insect pollination, such as a colorful or showy perianth, a landing platform, or nectaries. Finally, many hours of direct observation recorded no insect visitation. Therefore, we consider it unlikely that insects play a significant role in pollination of T. austinensis. Outcrossing by wind was effective in the T. austinensis population studied, with over 86% of carpels per reproductive unit receiving some pollen and an average pollen


load of two grains per ovule. Such success was linked to high pollen production. Compared to T. submersa, plants of T. austinensis produced more pollen grains per anther and more anthers per plant, resulting in a 50-fold higher pollen production level per plant (or 25-fold, after accounting for dioecy in T. austinensis). Higher pollen production, not lower ovule production, was the primary cause of the much higher pollen-to-ovule ratio of 1,138 in T. austinensis (ovule production was 127 per female plant versus 85 per plant in T. submersa; Table 1). Selfing populations are expected to exhibit lower male investment than related outcrossing populations because, as higher levels of selfing evolve and fewer pollen grains are exported to outcross ovules, the fitness gain from maintaining high pollen production diminishes (Charnov 1982; Brunet 1992; Barrett et al. 1996; Sato and Yahara 1999). Self-pollinators might also gain from investing less in individual pollen grains. There is some evidence that this was the case in T. submersa as its pollen grains were 14% smaller in volume and its pollen tubes were 13% narrower in diameter than those of T. austinensis. Reproductive unit morphology The reproductive units of the two Trithuria species exhibited strikingly different morphologies (c.f. Fig. 1d, f). The bracts that enclose the reproductive organs of T. austinensis are much longer than those of T. submersa (3.3 vs. 1.8 mm), and the entire reproductive unit has a distinctly vertical orientation, versus a more bowl-shaped structure in T. submersa. In T. austinensis, carpels within reproductive units overtop each other, whereas the carpels of T. submersa are positioned primarily in the same plane. The contrasting carpel packaging strategies reflect the different breeding systems and pollen reception strategies of the two species. The bowl shape of T. submersa reproductive units allows all carpels equal access to self-pollen from the overarching anthers. In contrast, the vertical orientation of both male and female reproductive units in T. austinensis might reflect an advantageous organ arrangement for both pollen export and pollen reception via wind. However, the bracts in T. austinensis interfere with both pollen dispersal and reception by wind. As such, the vertical carpel arrangement constrains stigmatic hairs and anther filaments to be long in order to emerge from deep reproductive units. This is strikingly similar to what must have occurred during the evolution of wind-pollinated, cultivated maize (Zea mays subspecies mays) from its presumed wild ancestor. Styles in cultivated maize are 10–30 cm or more in length, whereas those of Balsas teosinte (Zea mays subspecies parviglumis), thought to be similar to a wild ancestor of maize, average only 3.9 cm in length (Wu et al. 2011). Longer style length had to evolve in cultivated maize because adding more and

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larger carpels to an ensheathed inflorescence (the cob) caused ovules to be placed far from the point of emergence of stigmatic surfaces (the ends of the silks). This comparison indicates that style length is highly labile in angiosperms, accommodating changes in floral shape and structure between closely related species or populations from the smallest to the largest flowers. Rates of pollen tube germination Pollen germination in both T. austinensis and T. submersa occurred very shortly after pollen reception. Such rapid pollen germination appears to be a trait shared by most water lilies. Substantial pollen germination occurs within 15 min of pollination in Brasenia schreberi and Cabomba caroliniana (Cabombaceae; Taylor and Williams 2009), as well as in Nymphaea odorata (Nymphaeaceae; Williams et al. 2010). These taxa also exhibit rapid pollen tube growth, suggesting that the origin of the aquatic habit in Nymphaeales was accompanied by an overall shift to faster development during this stage. Trithuria austinensis exhibited slightly slower pollen germination than T. submersa, with no germination occurring until 15 min after pollination in the former and abundant germination within 5 min in the latter. This finding is consistent with the hypothesis that pollen germination should be slower in outcrossing than in selfing populations (Mulcahy 1979; Snow and Spira 1991; Acar and Kakani 2010). Stigmas must interact with a variety of pollen genotypes in outcrossing populations, and thus, favorable epistatic pollen–stigma interactions that facilitate rapid pollen germination are not as likely to evolve as they are in selfing populations where stigmas receive pollen from the same single donor across many generations (Plitmann and Levin 1990; Kerwin and Smith-Huerta 2000; Mazer et al. 2010). Pollen tube growth rates Trithuria austinensis exhibited a significantly faster average pollen tube growth rate than T. submersa by any of several measures. The best measure of mean PTGR was taken from tubes that had reached ovules within 1 hap. In these, PTGR was about 2 mm/h, or up to six times as fast as that in T. submersa (Fig. 6a). However, ovule penetration continued after this time, as well, indicating that the average PTGR of successful tubes was between 1 and 2 mm/h. It is important to note that in comparing PTGRs, single-donor crosses were used in both species. Pollen tube growth rates of T. austinensis are comparable to those of other water lilies, which range from 589 lm/h in Nuphar polysepala to about 1 mm/h in others (Williams 2008; Taylor and

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Williams 2009; Williams et al. 2010), whereas average PTGR in T. submersa was much slower (321 lm/h). The markedly slower PTGR of T. submersa relative to T. austinensis might have been caused by (1) an acceleration of PTGR in T. austinensis relative to an ancestrally slow PTGR in either Trithuria, Nymphaeales or all angiosperms (slow PTGR may be plesiomorphic in angiosperms; Williams 2008, 2009) or (2) a deceleration of PTGR in T. submersa due to the origin of its derived, selfing mating system. We favor the second scenario for the following reasons. First, both Trithuria species and all other taxa studied in Nymphaeales exhibit much faster PTGRs than those exhibited by other early-divergent angiosperms, all of which are woody (see Williams 2009). The faster rates in Nymphaeales correspond to a conspicuously shorter progamic phase, consistent with the finding that many aquatic lineages are marked by shifts to a shorter fertilization process (Williams 2009). Thus, the fast PTGR of T. austinensis may be a plesiomorphic feature of Nymphaeales. Trithuria submersa also has a short progamic phase, despite having a much slower PTGR than the rest of Nymphaealean taxa. In T. submersa, an exceptionally short pollen tube pathway allows rapid fertilization to be maintained. The stigmatic hair of T. submersa is 0.56 mm long (among the shortest in the genus), whereas seven of ten Trithuria species, including all that are closely related to T. submersa (Iles et al. 2012), have stigmatic hair lengths of 2 mm or greater (Yadav and Janarthanam 1995; Sokoloff et al. 2008). The only other species with short hairs, T. cookeana and T. inconspicua (Sokoloff et al. 2008), are distantly related (Iles et al. 2012). Thus, the short pollen tube pathway in T. submersa is likely a derived character within Trithuria. The evolution of shorter stigmatic hairs in T. submersa would be consistent with a loss of outcrossing by wind pollination (origin of selfing), reduced intermale pollen competition, and selection for maintaining a short time to fertilization as a PTGR slowdown evolved. Thus, the predominantly selfing breeding system of T. submersa (Taylor et al. 2010) likely evolved from an aquatic Trithuria ancestor that had a plesiomorphic fast PTGR. By any measure of PTGR, our results are consistent with two main predictions about variability between selfers and outcrossers in derived angiosperms (Mazer et al. 2010). T. austinensis had faster and more variable PTGRs than did T. submersa. Variability in T. austinensis was primarily caused by a high incidence of either pollen tube pausing or attrition. This also resulted in an apparently lower fertility. The evolution of PTGR is expected to be biased toward ever faster rates due to the effects of pollen competition, unless opposed by some other force (Walsh and Charlesworth 1992). Self-fertilization is one such force, since

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selection is relaxed in the face of increasing relatedness of competing pollen tubes (the penalty to mutants that incur slow growth is diminished). Pollen tube growth We observed pollen tube branching in both species, as did Prychid et al. (2011) in Trithuria cowieana, Trithuria konkanensis, Trithuria lanterna, and T. submersa. Interestingly, branching only occurred just after the pollen tube exited the pollen grain. Pollen tube branching is uncommon, but widespread in angiosperms, and perhaps most often occurs within the ovule (Johri et al. 1992). Branched pollen tubes are also present in some gymnosperms. Ginkgo and some conifers have highly branched pollen tubes, whereas other conifers, Gnetales, and cycads exhibit little or no branching (Friedman 1993). The Carboniferous seed fern Callistophyton also produced a branched pollen tube (Rothwell 1972). Pollen tube branching is thought to be the ancestral condition in seed plants, related to a haustorial or anchoring function such as in many extant non-flowering seed plants (Friedman 1993; Rudall and Bateman 2007). Whereas pollen tube branching in Trithuria may or may not be homologous to branching in gymnosperms, the pattern of early branching, just after exit from the pollen grain, is reminiscent of the early branching of some gymnosperm pollen tubes. However, the haustorial function of branches is clearly precluded in Trithuria and other angiosperms because the pollen tube wall is largely sealed with callose and because callose plugs prevent transfer of nutrients from the non-fertilizing tube branch (Abercrombie et al. 2011). Pollen tube branching in Trithuria may serve to better anchor the pollen tube to the stigmatic hair as it penetrates the cuticle. However, early branching in T. submersa may also be a consequence of the unique nature of the pollen tube pathway in this species: after pollen tubes penetrate the cuticle, they grow within the outer cell walls of the stigmatic hair to the stylar canal (Prychid et al. 2011). Our data extend this finding to T. austinensis, in which pollen tube tips entered the cuticle just after branching, displacing the outer but not the inner layer of the lateral stigmatic cell walls (Fig. 4j–m). Prychid et al. (2011) suggested that pollen tube growth within the stigmatic hair cell walls in Trithuria may be constrained to grow only parallel to the hair axis by microfibril orientation. We also observed that pollen tubes only grew directly either up or down the hair, never laterally, until they reached the base of the stigmatic hair. Early branching may allow initial growth in the only two directions possible, thereby increasing the chance that the pollen tube is positioned to take the shortest route to the ovary. However, we also observed that successful pollen tubes, containing the male germ unit, often had not taken the shortest route.


After entering the carpel mouth, Trithuria pollen tubes grow through an extremely narrow and short stylar canal in which the secretory inner carpel surfaces are nearly appressed. However, these surfaces do not become interlocked to form a solid tissue as they do in Nymphaea or Nuphar (Igersheim and Endress 1998; Endress 2001). Pollen tubes grew only in the narrow space of the stylar canal and were not seen within the carpel ground tissue adjacent to the canal.

Conclusion Trithuria is known for its great variability in sexual systems, and by a complete range of breeding systems—from autonomous selfing in T. submersa to obligate outcrossing in T. austinensis. The differences in post-pollination biology between these two closely related species correspond to their divergent breeding systems and are consistent in many ways with what one would predict from the derived angiosperm systems that have been studied. First, morphological traits that promote outcrossing by wind in many angiosperms are present in T. austinensis, which displays greater pollen production, deeper reproductive units, and longer stigmatic hairs than T. submersa. In contrast, selfing T. submersa exhibits all the hallmarks of reduced investment in male organs while maintaining a high number of carpels in an arrangement that maximizes autonomous self-fertilization and contributes to high self-seed set. Secondly, a number of predictions for the effects of breeding system on the evolution of pollen tube growth rates among populations were met. Outcrossing T. austinensis had slightly slower pollen germination times, faster PTGRs, and a greater variance in PTGR, due to higher attrition and/or pollen tube slowdowns (Willson and Burley 1983; Mazer et al. 2010). In both species, rapid pollen tube ontogenies resulted in a progamic phase whose duration is among the shortest known in angiosperms. Yet, despite the extremeness of their reproductive cycles, we found considerable developmental and morphological variation that tracked their divergent breeding and pollination systems. Such variability in reproductive processes of Trithuria, nested within a flowering plant lineage separated from the main line of angiosperms for over 120 million years, suggests there are features of male gametophyte development and growth underlying that lability that were present early in angiosperm history. Acknowledgments The authors thank the Western Australia Department of Environment and Conservation in Manjimup, particularly Robyn Bowles and Rich Robinson for providing logistical support and use of facilities, as well as Roger Hearn for insightful conversations about the ecology of the area wetlands. We especially thank Terry Macfarlane for his assistance in locating populations and

95 identifying plants, as well as for valuable discussion. We also thank Anna Becker and M. Steven Furches for assistance with field collections, Andrew Moffat for laboratory assistance, and Nicholas Buckley and Matthew Lettre for comments on early drafts of this manuscript. This work was supported by a National Science Foundation (NSF) Doctoral Dissertation Improvement Grant to M. L. T. (DEB 0910171) and by NSF award IOS 1052291 to J. H. W.

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