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Centre for Forest Biology, University of Victoria, P.O. Box 3020, Victoria, British Columbia, Canada, V8W 3N5
Received for publication January 22, 1998. Accepted for publication July 13, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Picea • Pinaceae • pollen • pollination • sacci
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). These functions may be exaptations as defined by Gould and Vrba (1982)
, however, most evidence supports the conclusion that sacci function primarily as floatation devices and should more appropriately be called "bladders." The case has been made that by retaining air, sacci make pollen buoyant to increase the efficiency of pollination (Doyle and O'Leary, 1935
; Doyle, 1945
; Sporne, 1965
; Tomlinson, 1994
; Runions and Owens, 1996
). Floating saccate pollen occurs in all species of spruce but one. Oriental spruce [P. orientalis (L.) Link] has an exceptional pollination mechanism in which pollen is saccate but sinks.
In general, a suite of pollination mechanism characteristics evolve in concert. Conifers with floating, saccate pollen have anatropous ovules in ovulate cones that are erect on the branch during the pollination period (Singh, 1978
). A pollination drop secreted by the ovule exudes from the micropyle. Wind-blown pollen adheres to surfaces near the micropyle and, when contacted by the pollination drop, float upwards into the ovule (Runions and Owens, 1996
). In oriental spruce, ovule position at pollination and pollen buoyancy remain correlated characters, but the character states are opposite those in other spruce species. Because the ovulate cone is pendant at the time of pollination, anatropous ovules open upwards during pollination drop secretion. Pollen with sacci would be expected to remain floating in this pollination drop. In fact, pollen floats briefly and then sinks into the upright ovule (Doyle, 1945
). Pollination in this exceptional species has called into question the established connection between pollen floatation and ovule position and made necessary an evaluation of saccus function in pollen floatation.
Because the physical attributes that differentiate saccate floating from saccate sinking were unknown, we used a variety of anatomical techniques to compare the sinking pollen of oriental spruce with the floating pollen of white spruce [P. glauca (Moench) Voss]. Our hypothesis was that the wall layer of the saccus, the exine, must be anatomically different or must function differently during pollen hydration between the species. The conventional method used to prepare pollen samples for transmission electron microscopy (TEM) is dehydrating and so is not practical for examination of the saccus exine in a hydrated state. A technique that enabled examination of hydrated pollen wall layers was adapted from Kurmann (1990)
. By this technique, ultrathin sections of pollen exine in hydrated condition were obtained. Hydration of pollen was studied by confocal microscopy (CM) and found to be a dynamic process. In oriental spruce, pollen hydration results in a reduction in buoyancy. Loss of buoyancy is correlated in our observations with morphology of the hydrated ektexine.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Pollen hydration
Observation of pollen hydration was done by dissecting microscope. Pollen was generally dusted onto a drop of water on a microscope slide.
Scanning electron microscopy (SEM)
Pollen specimens (which are naturally dehydrated so that a drying procedure is not required) were prepared for SEM observation by dusting them onto sticky-tape coated aluminum stubs and gold coating. Specimens were viewed with a JEOL JSM 35U SEM (JEOL, Ltd., Tokyo, Japan) operated at 15 kV.
Light microscopy (LM) and transmission electron microscopy (TEM)
To prepare hydrated pollen samples for LM and TEM, freeze-fixation/freeze substitution was used. Pollen was first hydrated in a 0.3% agar solution at 35°C for 10 min. Silver wire loops were coated with 0.6% formvar film and dipped twice into the hydrated-pollen and agar mixture. Coated loops were then plunged rapidly into liquid propane at -190°C in a Reichert KF80 immersion cryofixation system (Leica Inc., Toronto, Canada). Loops with frozen pollen were transferred to a Reichert CSauto cryosubstitution apparatus and freeze substituted at -90°C in a mixture of dry acetone with 1.5 % osmium tetroxide for 70 h. Freeze-substituted samples were warmed at a rate of 5°C/h to 15°C, infiltrated with Spurr's resin for 48 h, and polymerized at 60°C for 18 h.
For LM, sections between 0.5 and 1.0 µm thick were cut with a Reichert Ultracut E microtome and stained with toluidine blue (Color Index #52040) at pH 11.1 (O'Brien and McCully, 1981). Stained sections were mounted in distilled H2O, coverslipped, and observed with a Leitz Labrolux S microscope and camera system (Leica Inc., Toronto, Canada). Hydration of pollen was studied by fluorescence microscopy. To do this, pollen was hydrated in 0.01% aqueous calcofluor white M2R (C.I. #40622) and observed with a Leitz Orthoplan microscope equipped with a BP 350–460 excitation filter block G.
For TEM, sections of the Spurr's resin embedded pollen were cut at 65 nm and collected on formvar-coated, 75-mesh copper grids. Sections were stained with uranyl acetate and lead citrate. Observation of stained sections was with an Hitachi H-7000 TEM (Hitachi Instruments Inc., Montreal, Canada) operated at 75 kV.
Confocal microscopy (CM): image acquisition
Pollen hydrated in various fluorochrome solutions (see next subsection) was examined using a Zeiss LSM 410 confocal microscope (Carl Zeiss Inc., Thornwood, New York) equipped with krypton and argon laser excitation at wavelengths of 488, 568, and 647 nm. Two types of image were recorded, surface projections and extended depth of focus sections. In each case, a stained pollen grain was first scanned in incremental steps along the Z-axis to produce a stack of optical sections representing different depths within it. Each optical section was created as an 8-s scan with 4x line averaging (32 s). For surface projections, which resemble SEM images, 30 x 0.5 µm Z sections were projected with maximum overlay, i.e., each Z section blocks the part of the adjacent image that it overlays to produce a representation of surface features only. For extended depth of focus sections, 4 x 0.5 µm median Z sections were composited to produce an image in which fluorescent emission from above and below the section plane was eliminated.
Confocal microscopy: staining
For surface projections, pollen was hydrated in a 0.01% aqueous solution of phosphine 3R (stain specificity for neutral lipids, C.I. 46045) for >5 min. To prevent pollen from moving during microscopy, the staining solution was mixed dropwise with Farrant's medium (BDH Ltd., Toronto, Canada), a viscous, water-soluble mounting medium containing glycerin and gum arabic. For extended depth of focus sections, pollen was hydrated in a solution containing equal parts of (1) 1.0 x 10-4% rhodamine B (C.I. 45170) in 0.05 mol/L phosphate buffer at pH 5.8 and (2) 1.0 x 10-1% fluorescein diacetate stock in 0.05 mol/L phosphate buffer at pH 5.8. Rhodamine B stains the exine and is incapable of penetrating the plasmalemma while fluorescein diacetate is used as a vital stain that fluoresces only if esterases of the living cell cleave the acetate. A stock solution of fluorescein diacetate was made by dissolving 2.0 mg/mL in acetone and this solution was then mixed with phosphate buffer to the required concentration. Pollen hydrated fully in 
1 min in this solution. To extend the hydration time for CM and to prevent pollen from moving during microscopy, the staining solution was mixed dropwise with Farrant's medium as described above. The resulting solution has higher osmotic potential than the stain solution and caused plasmolysis of the stained pollen grains, which rehydrated again completely over the next 15 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Wind-blown pollen adhered to the micropylar arms prior to cone closure and pollination drop exudation. As the cones began to close, pollination drops were secreted from the ovules (Fig. 2) and pollen moved into the ovules. Pollen applied to an exposed pollination drop in an experimental set-up floated for 1 min within the pollination drop and then sank into the micropyle. Figure 3 contrasts the pollination mechanisms of oriental spruce and white spruce.
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When dry pollen of each species was compared by SEM, they appeared similar (Figs. 4–5) although pollen of oriental spruce were slightly smaller (Ho and Szikalai, 1972
). Even at high magnification (Figs. 6–7), no differences that could be interpreted as having functional significance were observed, although the sculptured exine patterns were different.
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), in its proximal region and sacci. An approximately I-shaped region between the sacci remained relatively unstained. Hydrated pollen grains appeared similar between species in anatomical detail when examined by LM (Figs. 10–11). The large tube cell was bounded by the intine wall layer and was filled with small vacuoles. Body and stalk cells were bounded by primary cell walls, which appeared continuous with the intine at the proximal pole. Remnants of prothallial cells were embedded in the intine adjacent to the site of stalk cell attachment. In each species, the exine stained similarly with toluidine blue and was continuous around the pollen, although very thin at the distal pole. Sacci appeared similar between species. In relative terms, the sacci of oriental spruce might be smaller than those of white spruce, but the difference is slight.
Hydrated saccus exine appeared different when the two types of pollen were compared in TEM micrographs (Figs. 12–13). The sacci of white spruce pollen consist only of a homogenous appearing ektexine layer 0.25 µm thick. Inward projections of the ektexine form a reticulate network when viewed in cross section (Fig. 12). Enclosed (as judged from serial sections) and partly enclosed spaces, which vary from 0.25 to 5.0 µm across, were formed by this reticulate network. In contrast, the ektexine of oriental spruce pollen sacci was thinner (0.15–0.20 µm) and porous (Fig. 13). Elaborations of the ektexine formed inward projections, but enclosed spaces, capable of trapping air, were very uncommon.
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Pollen hydration resulted in rapid swelling of the tube cell and surrounding intine. Exine layers of the pollen stained with rhodamine B. Fluorescein diacetate was used as a vital stain within the cytoplasm at the same time. Rehydrating pollen grains were scanned with the CM to produce median optical sections periodically during a 15-min period following immersion in staining solution. In aqueous solutions with low osmotic potential, full hydration of pollen required only 1 min. Addition of Farrant's medium to slow pollen hydration and to prevent pollen movement for the purposes of CM seemed to prevent quenching of the fluorochrome. Farrant's medium was not toxic to pollen even after 3 h of immersion as indicated by fluorescein diacetate staining. Figure 17 shows median sections of the same pollen grain of oriental spruce at 1, 8, and 15 min during hydration. Exine layers including the ektexine of the sacci fluoresced red, and the cytoplasm of the three cells within the pollen body fluoresced green. Esterase activity was concentrated around the nuclei of the stalk, body, and tube cells. Cytoplasm within the pollen grain was predominately that of the tube cell (largest nucleus), which surrounds the smaller stalk and body cells. As pollen hydrated, the tube cell swelled, resulting in a reduction of the saccate air space (arrowheads in Fig. 17).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), and thus indicates that although pollen is buoyant (at least briefly, in the case of oriental spruce), the pollen surface is hydrophilic and wettable. Our hypothesis, that anatomical differences must be a factor governing pollen behavior in water, was correct, but the differences were only visible when hydrated pollen was compared by TEM. Freeze-fixation/freeze-substitution proved invaluable as the hydrated pollen was not subsequently subject to the dehydration that routinely results in tissue shrinkage and artifacts when standard embedment protocols are used.
Sacci of oriental spruce pollen can be considered porous when compared to the relatively nonporous, air-trapping sacci of white spruce. The exine layer that forms the sacci is the ektexine as defined by Kurmann (1990)
. Deposition of the ektexine in her study of Tsuga canadensis (L.) Carr. pollen was mediated by orientation of cellulose microfibrils in the microspore surface coating. If orientation of microfibrils can be considered the precursor to ektexine pattern formation, there is a possible genetic basis for species differences that might not be well defined if the ektexine was randomly deposited.
Existence of holes in the sacci of oriental spruce pollen suggested the reason why this saccate pollen should sink, but the reason why air is displaced when pollen is added to water remained unclear. These holes are of very small diameter and, at least initially, air is retained within the sacci. The confocal microscope allowed visualization of details of pollen hydration. Pollen tube cell and intine expansion into the saccate space occurs rapidly once pollen is added to water. This rapid reduction in saccus volume necessarily results in displacement of air from the sacci, otherwise the pressure and temperature increase associated with gas compression would seem detrimental. White spruce pollen retains enough air within the reticulate network of ektexine extensions and enclosed spaces in the sacci to remain buoyant, while air is not trapped in the porous ektexine of oriental spruce pollen. Ektexine of the sacci of other conifer pollen has been described as porous (e.g., Pocknall, 1981
) and, indeed, pores occasionaly occur in the sacci of white spruce pollen. In no case, however, do photomicrographs of other species give evidence of the concentration of pores observed in oriental spruce.
Water enters through the distal pole of the pollen in an I-shaped area between the sacci. In dehydrated pollen, the sacci close together to hide this flexible region, the leptolemma (Kurmann, 1990
), which, upon hydration, becomes the site of pollen tube emergence. During the course of hydration in calcofluor white M2R, the fluorochrome stained the intine most intensely in this I-shaped region. As water entered the pollen, stain molecules were excluded and accumulated at the plasmalemma within the intine at the site of water entry. Canny (1990)
described this sort of stain accumulation, in sumps, as water enters the symplast of a cell. In germinated pollen, the staining was clearly localized to the intine and pollen tube. In this case, the bright fluorescence in the region between sacci highlights the movement of water into the tube cell, subsequent inflation of the tube cell, and expansion of the intine reduce the saccate volume, and the porosity of the ektexine determines the sinking or floating nature of the pollen.
Saccus function and evolution of pollination mechanisms in Pinaceae
Characters integrated in pinaceous pollination mechanisms include (1) orientation of the ovulate cone and, therefore, ovule position at the time of pollination, (2) pollen with or without sacci, and (3) a pollination drop involved in pollination or not. Evolutionary change in one of these characters requires compensatory change in the others if the pollination mechanism is to function efficiently. Since all members of Pinaceae that secrete a pollination drop, except oriental spruce, have downwardly positioned ovules at pollination and floating pollen, we consider this character set to be the ancestral condition (Doyle, 1945
; Mapes, 1987
; Osborn and Taylor, 1994
). In oriental spruce, the relationship between ovule position and pollen floatation has changed. Change in ovule position has been effected by a change in ovulate cone orientation at the time of pollination. Ovules open upwardly when ovulate cones are pendant. The importance of ovule orientation in the pollination mechanism is highlighted by the fact that ovulate cone stalks bend to orient cones vertically before they become receptive to pollen (unpublished observation). Significantly, pollen buoyancy would be maladaptive in oriental spruce and saccus morphology has evolved accordingly.
Brief floatation of oriental spruce pollen may be adaptive and retained, or might represent an intermediate step towards complete loss of saccus function. Pollen scavenging, in which buoyant pollen that lands on distal ovulate cone structures enters the micropyle upon contact with a large pollination drop, has been described for other conifer species (Tomlinson, Braggins, and Rattenbury, 1991
; Runions and Owens, 1996
). Retention of sacci in oriental spruce may confer a selective advantage by allowing a brief period of floatation from the point of pollen capture on the ovulate cone to the micropyle, thus extending the possibility of pollination temporally and spatially. At the same time, saccate pollen of sympatric conifer species, because it floats, would be excluded from the micropyle during the time that sinking pollen of oriental spruce occupied the site of germination on the nucellus within the ovule. Pollen selection mechanisms that discriminate pollen types based on floatation have been termed "exclusion mechanisms" by Tomlinson (1991)
. Conifer species from families other than Pinaceae (e.g., Cupressaceae) that have upright ovules that secrete a pollination drop have nonsaccate pollen. This pollen lands in the pollination drop and sinks into the ovule (Tison, 1911
; Owens and Molder, 1980
). In these species, because pollen lands directly in the pollination drop, there is no requirement for pollen floatation.
Diversifying selection has resulted in two pollination mechanisms within Picea. Intermediate ovule orientations and within-species variation in pollen buoyancy are unknown, although evolution of this phenotypic gap has probably proceeded through a series of intermediate steps (Maynard Smith et al., 1985
). How the shift in ovulate cone position at pollination and loss of saccus function occurred can only be speculated upon. In one scenario, the erect position of ovulate cones was lost in an ancestral oriental spruce, but the loss was not completely maladaptive. Buoyant, saccate pollen floating within pollination drops would have been taken into the more or less upright ovules as the pollination drops receded. Genetic control of the upright ovulate cone position, once lost, would be unlikely to have been regained and selection on sinking pollen and inverted ovulate cone position to increase pollination efficiency would have driven the concerted evolution of these traits.
Loss of the erect character of ovulate cones at pollination would be maladaptive in large populations or where sympatric species created competition. Stabilizing selection (Charlesworth, Lande, and Slatkin, 1982
) would tend to maintain the basal condition in ancestral populations under adaptive constraint due to competition. The proposed scenario requires a reduction in selective pressure as might occur if a founder species was relatively isolated and in a small population (Jernigan, Culver, and Fong, 1994
). Modern oriental spruce is native to the Caucasus mountains of northeastern Turkey and Georgia (Davis, 1965
) where it is isolated from other spruce species. Isolation has allowed not only evolutionary change in the pollination mechanism but in vegetative characters as well. Oriental spruce is distinct from other spruces in leaf form. Despite its desirable appearance (Dallimore and Jackson, 1974
), hybrids with other Picea are unknown. Many Picea species hybridize readily and the lack of oriental spruce hybrids might reflect not genetic incompatibility but an unrecognized incompatibility in pollination mechanisms. Hybridization of oriental spruce with other spruce species was attempted without success by Wright (1955)
and apparently with success by Mergen, Burley, and Furnival (1965)
, but these authors claim that the progeny were unverified and could have been the result of self-fertilization.
Several genera have arisen within Pinaceae since establishment of the pines (Pinus) and spruces (Chase et al., 1993
; Hart, 1987
; Price, Olsen-Stojkovich, and Lowenstein, 1987
). In each case, the key innovation (Hunter, 1998
) necessary for origin or subsequent success of the new taxonomic group seems to be a change in the pollination mechanism. These more modern genera, e.g., Pseudotsuga, have diverged in pollination mechanism but in a manner different from oriental spruce. In these cases, evolutionary loss of the exuded pollination drop means that pollen is not required to float and sacci have been lost or are vestigial and do not function (Owens, Simpson, and Molder, 1981
). In Abies, no pollination drop is exuded by the ovule, but pollen is saccate and floats. This seemingly contradictory situation might be explained by field observations of Abies, which suggest that atmospheric moisture in the form of condensation or rain can fill the micropyle and cause pollen to float into the ovule (Luke Chandler, University of Victoria, personal communication). The possibility that Abies, and to a lesser extent even species with pollination drops, use rainwater in pollination is under investigation. Whatever the factors governing speciation in each case, retention of sacci correlates with the requirement for pollen that float, even if only briefly as observed in oriental spruce, as a component of the pollination mechanism.
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FOOTNOTES |
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2 Author for correspondence, Current address: Section of Ecology and Systematics, Corson Hall, Cornell University, Ithaca, NY 14853.
3 Current address: Iriomote Station, Tropical Biosphere Research Center, University of the Ryukyus, 870 Uehara, Taketomi-cho, Okinawa 907-15, Japan.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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———, S. J. Simpson, and M. Molder. 1981 The pollination mechanism and the optimal time of pollination in Douglas-fir (Pseudotsuga menziesii). Canadian Journal of Forest Research 11: 36–50.
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———, and J. N. Owens. 1996 Pollen scavenging and rain involvement in the pollination mechanism of interior spruce. Canadian Journal of Botany 74: 115–124.
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