Cones and pollen grains

Adaptation of male reproductive structures to wind pollination in gymnosperms: Cones and pollen grains Y. Lu1,2,4, B. Jin1,2,4, L. Wang1,3, Y. Wang1, D. Wang1, X.-X. Jiang1, and P. Chen1,5 1

College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, People’s Republic of China; College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, People’s Republic of China; and 3Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, People’s Republic of China. Received 26 January 2011, accepted 4 May 2011.

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Can. J. Plant Sci. Downloaded from pubs.aic.ca by 52.2.249.46 on 09/13/15 For personal use only.

Lu, Y., Jin, B., Wang, L., Wang, Y., Wang, D., Jiang, X.-X. and Chen, P. 2011. Adaptation of male reproductive structures to wind pollination in gymnosperms: Cones and pollen grains. Can. J. Plant Sci. 91: 897906. Wind pollination (anemophily) in gymnosperms is thought to be an ancestral state. Previous studies considered wind pollination to be a largely random phenomenon, but recent evidence suggests that wind-pollinated species have evolved different complex reproductive adaptations for controlling and maximizing the success of wind pollination. However, compared with angiosperms, wind pollination in gymnosperms is poorly understood. We investigated the male reproductive structures of 13 representative gymnosperm species using a scanning electron microscope and digital camera, and analyzed how the morphological characteristics of male cones and pollen facilitate pollination. These characteristics showed a surprising variation between different gymnosperm species in improving pollination success. For example, the relationship between the position of the male cone and the surrounding vegetative structures is adjusted to optimize pollen release. The pollen grains have sacs and papilla and exhibit particular shapes after release from microsporangia, including boat-like, saccate, papilla-like and spheroid shapes, which facilitate pollen dispersal in the air. Taken together, our results suggest that the extensive diversity of male reproductive structures within gymnosperms represents an evolutionary response to long-term selection and results in solutions to the physical restraints of anemophily. Key words: Wind pollination, gymnosperms, male cones, pollen grains, reproductive structure Lu, Y., Jin, B., Wang, L., Wang, Y., Wang, D., Jiang, X.-X. et Chen, P. 2011. Adaptation des organes de reproduction maˆles a` la pollinisation par le vent chez les gymnospermes : coˆnes et grains de pollen. Can. J. Plant Sci. 91: 897906. On pense que la pollinisation par le vent (fećondation ane´mophile) chez les gymnospermes est un caracte`re ancestral. Des e´tudes ante´rieures voyaient dans cette forme de pollinisation un pheńome`ne largement aleátoire, cependant des preuves rećentes laissent croire que les espe`ces ainsi fećondeés ont acquis par e´volution des structures de reproduction aussi diffe´rentes que complexes leur permettant de re´guler la pollinisation et de maximiser les chances de fećondation. Quoi qu’il en soit, on comprend mal la pollinisation par le vent des gymnospermes, comparativement a` celle des angiospermes. Les auteurs ont examine´ les structures de reproduction maˆles de 13 espe`ces repre´sentatives des gymnospermes a` l’aide d’un microscope e´lectronique a` balayage et d’un appareil photo nume´rique, puis ils ont analyse´ la façon dont la morphologie des coˆnes maˆles et du pollen favorise la fećondation. Les caracte´ristiques morphologiques varient de manie`re e´tonnante chez les diffe´rentes espe`ces de gymnospermes, en rehaussant les chances de pollinisation. Ainsi, la relation entre l’emplacement du coˆne maˆle et des structures ve´ge´tatives voisines se modifie pour optimiser la libe´ration du pollen. Les grains portent des sacs et des papilles, et pre´sentent une forme particulie`re apre`s leur libe´ration des microsporanges, par exemple celle d’un bateau, une forme dilateé, une forme de papille ou de sphe`re qui facilitent leur dispersion dans l’air. Regroupe´s, les re´sultats donnent a` penser que la grande diversite´ des organes de reproduction maˆles constitue une re´ponse de l’e´volution a` la se´lection a` long terme et engendre des solutions aux contraintes physiques associeés a` la fećondation ane´mophile. Mots cle´s: Pollinisation par le vent, gymnospermes, coˆnes maˆles, grains de pollen, structure de reproduction

Pollination in seed plants occurs through the transfer of pollen by abiotic (wind and water) or biotic (animal) vectors (Faegri and van der Pijl 1979; Proctor et al. 1996). Biotic pollination has been extensively studied, and a rich literature is devoted to it (Harder et al. 1996; Ollerton and Coulthard 2009). Abiotic pollination has received less attention, despite being an important 4 5

These authors contributed equally to this work. Corresponding author (e-mail: [email protected]).

Can. J. Plant Sci. (2011) 91: 897906 doi:10.4141/CJPS2011-020

process in numerous plant families from trees to grasses (Owens et al. 1998; Friedman and Harder 2004). Although abiotic pollination typically involves pollen transfer by wind or water, wind pollination is most common (Ackerman 2000). Wind pollination is closely involved in the ecological adaptation and evolutionary mechanisms of the plants that employ it, and is an

Abbreviations: BL, body length; BW, body width; E, equatorial diameter; P, polar axis length 897


898 CANADIAN JOURNAL OF PLANT SCIENCE

important research topic (Niklas 1985; Culley et al. 2002; Friedman and Barrett 2008). Early studies of wind pollination suggest that it is random and metabolically wasteful, being a relatively passive process in which pollen is released, transported, and deposited largely by chance (Whitehead 1983). However, recent work has shown that wind pollination is more efficient than previously thought (Friedman and Harder 2004; Wang et al. 2010). In wind-pollinated species, morphological adaptations are to be expected in the form of both the pollen-emitting and pollen-receiving part of a plant (Harder and Barrett 2006). Wind-pollinated species have evolved a number of reproductive structural traits to adapt them to anemophily. For example, the anthers of some anemophilous species are typically large, and extend on long, flexible filaments to improve effective pollen dispersal (Niklas 1985; Friedman and Barrett 2009). Their stigmas are often finely divided or feathery to help in pollen collection (Dafni 1992; Friedman and Barrett 2009). In addition, wind-pollinated species must produce large quantities of pollen, usually smooth and uniform in size, to increase pollination success (Cruden 2000). In general, the reproductive structures of windpollinated species show precise morphological and structural adaptations to anemophily, arising from long-term natural selection and ecological evolution for enhanced pollination success. To date, studies on the floral traits that facilitate wind pollination have focused mainly on angiosperms. Gymnosperms represent the majority of windpollinated species, and about 98% of gymnosperm species are wind-pollinated (Faegri and van der Pijl 1979). Unlike angiosperms, in which wind pollination evolved from insect pollination (Culley et al. 2002), wind pollination is the ancestral state in gymnosperms (Owens et al. 1998). Gymnosperms originated during the Late Devonian period and are a very ancient group (Florin 1951; Rothwell and Serbet 1992). In early gymnosprems, male cones and pollen were simple structures that then evolved into a wide diversity of reproductive structures after repeated climate changes and selection for effective wind pollination (Givnish 1980; Pacini et al. 1999; Barrett 2002; Fernando et al. 2010). For example: (1) the ovule may show an erect, variable or inverted orientation, which can help to direct pollen to the micropyle (Owens et al. 1998; Mo¨ller et al. 2000); (2) the specific integument tip of the ovule, such as the wax layer in Thuja and micropylar arms in Pinus, probably contributes to pollen collection (Colangeli 1990; Owens et al. 2001); (3) pollination drops secreted by the ovule may assist in scavenging pollen and transferring it into the micropyle (Gelbart and von Aderkas 2002; Nepi et al. 2009); (4) the geometry of Pinus ovulate cones can generate a complex system of air eddies and thus enhance the probability of pollen entrapment (Niklas and Paw 1982); and (5) the pollen sac can act as an aerodynamic structure, and many

wind-pollinated grains typically have structures to reduce their settling speeds and so increase their dispersal distance (Schwendemann et al. 2007). However, most studies of wind pollination in gymnosperms have focused on aspects of female fitness. Increasingly, researchers have suggested that male fitness and female fitness are equally important in wind pollination (Khanduri and Sharma 2002). Male fitness has been more commonly evaluated, and has more explanatory power (Di-Giovanni and Kevan 1991; Jackson and Lyford 1999). Other studies have focused on saccate pollen (Salter et al. 2002; Doyle 2010). The adaptive role of male cone position and its interactions with the surrounding vegetative structures and the modifications of pollen shape during pollination are unclear. We therefore investigated the characteristics of male cones and pollen, in gymnosperm species from major typical families, and analyzed the adaptive and evolutionary relationships between male reproductive structures and wind pollination in order to better understand wind pollination in gymnosperms. These results will help further our knowledge of pollination diversity and also increase our awareness of pollination biology and reproductive biology in gymnosperms. MATERIALS AND METHODS Study Species and Population We analyzed 13 sympatric gymnosperm species from Yangzhou University and the Shugang National Scenic Area (lat. 32820?N, long. 119830?E), eastern China. All are wind-pollinated, and belong to seven different plant families (Table 1). Male cones were collected during the gymnosperm pollen dispersal period from 2009 Feb. 23 to 2009 Oct. 15, when pollen grains were initially released from microsporangia (Table 1). Morphological data for male cones were recorded. Pollen grains were collected from other, nearly mature male cones after ripening and pollen shedding. These cones were hung upside down in open sulfuric acid paper bags, labeled separately, and left in a hood at room temperature for one day. Fresh pollen released inside the bags, and freshly shed pollen grains were collected and passed through a 150-mm sieve. Unused shed pollen was stored in 10-mL centrifuge tubes sealed with parafilm at 208C. Morphological Characteristics of Male Cones Six male cones (male or hermaphrodite) were selected at random from at least five field specimens of individual trees of each species studied (30 cones at least per species). Thirty male cones were selected from each species and their lengths measured with digital calipers, except for Cycas revoluta, of which only four male cones from four plants could be obtained at the correct stage. Average values and standard deviations were calculated using Microsoft Office Excel 2003.

LU ET AL. * WIND POLLINATION IN GYMNOSPERMS

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Table 1. List of specimens Species


Cycas revoluta Thunb. Ginkgo biloba L. Abies firma Sieb. et Zucc. Cedrus deodara (Roxb.) Loud. Pinus parviflora Sieb. et Zucc. Pinus thunbergii Parl. Cunninghamia lanceolata (Lamb.) Hook. Cryptomeria japonica (L. f.) D. Don Metasequoia glyptostroboides Hu et Cheng Chamaecyparis obtusa (Sieb. et Zucc.) Endl. Sabina chinensis (L.) Ant. (Juniperus chinensis L.) Podocarpus macrophyllus (Thunb.) D. Don Cephalotaxus sinensis (Rehd. et Wils.) Li

Family Cycadaceae Ginkgoaceae Pinaceae Pinaceae Pinaceae Pinaceae Taxodiaceae Taxodiaceae Taxodiaceae Cupressaceae Cupressaceae Podocarpaceae Cephalotaxaceae

Branch tops of 1520 cm bearing mature male cones were clipped from each species except Cycas revoluta, for which photos were taken on the tree. All the other male cones were photographed using a DSC-H7 Sony digital camera using a black background. Male cone description and terminology followed Owens et al. (1998). Morphological Characteristics of Pollen Grains Pollen characteristics from different species were characterized. Dry pollen grains were directly dispersed onto aluminum specimen stubs with adhesive tabs. Samples were examined and imaged with a Hitachi S-4800 scanning electron microscope at 5.0 kV, after coating with gold-palladium using a sputter coater (SCD500) (Lu et al. 2009). Three types of micrographs were digitally recorded: equatorial view of pollen, polar view of pollen and exine ornamentation. Size measurements were based on 30 pollen grains selected randomly from photos of each species. The value of body length (BL) and body width (BW) were measured in five saccate pollen species using AutoCAD 2008 software (Autodesk, Inc.). The body length refers to the maximum distance between the sacci and the body width refers to the width of the sacci. The pollen sizes of polar axis length (P) and equatorial diameter (E) of the other species were measured using the same software. The P:E or BL:BW ratio was calculated with Microsoft Office Excel 2003. In this paper, the generic names are written in full in order to distinguish the different genera more clearly. Pollen description and terminology follows Fernando et al. (2010). RESULTS Male Cone Characteristics We observed a number of characteristics related to wind pollination in the gymnosperm male cones: unisexual cones, catkin-like or spike-like shapes, inconspicuous coloring, generally small size, and spirally arranged microsporophylls (Table 2). These features indicated that male cones were specialized for wind pollination.

Acquisition time 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009

Jul. 20 Apr. 10 Mar. 27 Oct. 15 May 08 Apr. 12 Apr. 01 Feb. 28 Feb. 23 Apr. 08 Apr. 12 May 01 Apr. 03

Locality Shugang National Scenic Area University of Yangzhou campus Shugang National Scenic Area University of Yangzhou campus University of Yangzhou campus University of Yangzhou campus Shugang National Scenic Area University of Yangzhou campus University of Yangzhou campus Shugang National Scenic Area Shugang National Scenic Area University of Yangzhou campus Shugang National Scenic Area

In addition, the gymnosperm species had distinctive features associated with efficient wind pollination during the pollen dispersal phase. The most notable feature was that male cones were borne mainly at the tops of branches or near the top (Fig. 1), except in Cephalotaxus sinensis. Interestingly, Cephalotaxus sinensis cones were situated between two parallel exposed pinnate leaves (Fig. 1m). Furthermore, male gymnosperm cones were mainly aggregated in clusters that resembled a condensated ‘‘inflorescence’’, for example in Ginkgo biloba (Fig. 1b), Abies firma (Fig. 1c), Pinus parviflora (Fig. 1e), Pinus thunbergii (Fig. 1f), Cunninghamia lanceolata (Fig. 1g), Cryptomeria japonica (Fig. 1h), Metasequoia glyptostroboides (Fig. 1i) and Podocarpus macrophyllus (Fig. 1l). In addition, no impediments were observed around the male cones in Pinus thunbergii (Fig. 1f), Cunninghamia lanceolata (Fig. 1g) and Metasequoia glyptostroboides (Fig. 1i). Although some leaves were seen around the male cones in some species, the relationship between the position of male cone and the ambient leaves is very diverse during the pollen dispersal phase. For example, in Cycas revoluta (Fig. 1a), the leaves surrounding male cones were curved to orient the cones outward. For Ginkgo biloba (Fig. 1b) and Podocarpus macrophyllus (Fig. 1l), the leaves were explanate and perpendicular to drooping (Fig. 1b) or erect (Fig. 1l) male cones during pollen release. For Pinus parviflora (Fig. 1e) and Cryptomeria japonica (Fig. 1h), the leaves were at the tip and were not open during the pollen dispersal phase. Finally, the microsporophylls of male cones were arranged in a spiral around the cone axis. Interestingly, microsporophylls of Cedrus deodara cones showed a tight spiral architecture before pollen release (Fig. 1d-1), and then turned outward and showed a curve structure similar to a turbine during pollen release (Fig. 1d-2, arrow). The distribution of male cones is different within the 13 species. The male cones of Cycas revoluta (Fig. 1a) were at the top of the stem, surrounded by a tuft of large feather-like leaves. Some species, such as Ginkgo biloba (Fig. 1b), Cedrus deodara (Fig. 1d) and Cephalotaxus sinensis (Fig. 1m), had male cones distributed throughout

900 CANADIAN JOURNAL OF PLANT SCIENCE Table 2. Male cone characteristics of 13 species Species Cycas revoluta Ginkgo biloba Abies firma Cedrus deodara Pinus parviflora


Pinus thunbergii Cunninghamia lanceolata Cryptomeria japonica Metasequoia glyptostroboides Chamaecyparis obtusa Sabina chinensis Podocarpus macrophyllus Cephalotaxus sinensis

Sexual system

Male cone morphology

Location of male cones

Dioecious Dioecious

A solitary cone at the top of the stem Clustered cones at the top of the short-shoot Monoecious Clustered cones in the upper side of the branch Dioecious, sporadically A solitary cone on the top of Monoecious short-shoot Monoecious Clustered cones in the upper side of the branch Monoecious Clustered cones at the top of the branch Monoecious Clustered cones at the end of the branch Monoecious Clustered cones at the end of the branch Monoecious Clustered cones at the end of the branch Monoecious Solitary cones at the tip of leaves Dioecious, sporadically Monoecious Dioecious, sporadically Monoecious Dioecious

Color

Male cone length (cm)z

Figure

Cylinder-like Catkin-like

Yellow Yellow-green

60.53913.26 2.3790.29

Fig. 1a Fig. 1b

Catkin-like

Yellow

2.0990.23

Fig. 1c

Cylinder-like

Yellow-green

3.5691.11

Fig. 1d

Spike-like

Brown

1.2190.18

Fig. 1e

Cylinder-like

Yellow-brown

1.2790.14

Fig. 1f

Spike-like

Yellow-brown

1.2990.10

Fig. 1g

Spike-like

Brown

1.0090.12

Fig. 1h

Spike-like

Yellow

0.9090.09

Fig. 1i

Spike-like

Brown

0.3490.04

Fig. 1j

Yellow-brown

0.4590.07

Fig. 1k

Yellow

2.5590.24

Fig. 1l

Yellow-brown

1.0790.37

Fig. 1m

Solitary cones in the upper side of the Spike-like branches Clustered cones in the upper side of Spike-like the branch Clustered cones in the middle of Capitulum-like leaves

z

The values are means9SD from five plants (six male cones were randomly selected from each species, except for Cycas revoluta, only four male cones from four plants were obtained).

the tree crown from the interior to the exterior. However, in many species, male cones were oriented away from the crown, as seen for Abies firma (Fig. 1c), Pinus parviflora (Fig. 1e), Pinus thunbergii (Fig. 1f), Taxodiaceae (Fig. 1g, h and i), Chamaecyparis obtusa (Fig. 1j) and Sabina chinensis (Fig. 1k). Pollen Morphological Characteristics The pollen micro-morphology of the investigated species is shown in Fig. 2 and Fig. 3. Pollen shapes, sizes, aperture types and exine ornamentation are compared in Table 3. In all species, pollen grains dehydrated rapidly after release from microsporangia and exhibited particular shapes, including boat-like, saccate, papilla-like, and spheroid forms. The boat-like pollen grains were seen in Cycas revoluta (Fig. 2b) and Ginkgo biloba (Fig. 2e), and both ends of pollen grains were an acute apex after natural dehydration. For saccate pollen, seen in Abies firma (Fig. 2h), Cedrus deodara (Fig. 2k), Pinus parviflora (Fig. 2n) and Pinus thunbergii (Fig. 2q), two sacci were found per pollen grain. When the sacci curled to the ventral side to envelop syncopate during natural dehydration, the shape of the pollen become ellipsoid. Furthermore, the two sacci of the Podocarpus macrophyllus pollen (Fig. 3q) were dented on the surface instead of curled, with a shape similar to the two wings of an airplane. Papilla-like pollen grains were found in Cunninghamia lanceolata (Fig. 3b), Cryptomeria japonica

(Fig. 3d, e), and Metasequoia glyptostroboides (Fig. 3g, h). The pollen grains were obviously dented around the papilla-like protuberance, and transformed into a parachute-like shape during natural dehydration. Spheroid pollen grains were seen in Chamaecyparis obtusa (Fig. 3k), Sabina chinensis (Fig. 3n) and Cephalotaxus sinensis (Fig. 3t). These pollen grains were indented due to natural dehydration and changed into oblate or irregular spheroid. The sizes of the pollen differed significantly within the 13 species, ranging from small to big [P or BLE or BW: (9.0790.96) (69.6396.04) mm (24.1991.67) (86.9995.30) mm] (Table 3). The largest pollen grains were from Abies firma (Fig. 2g, h), and the smallest were from Cycas revolute (Fig. 2a, b). Pinaceae and Podocarpaceae pollen grains were mostly saccate pollen, which were bigger [BL BW: (34.7592.23) (69.639 6.04) mm(39.3093.60) (86.9995.30) mm] (Fig. 2g, h, j, k, m, n, p, q and 3p, q) than non-sacci pollen. The boat-like, papilla-like and spheroid pollen grains were roughly equal in size (2030 mm). All pollen grains had smooth exines with microreticulate or microgranulate patterns, and monocolpate or single apertures or no aperture (Table 3). Pollen from Cunninghamia lanceolata (Fig. 3c), Cryptomeria japonica (Fig. 3f), Metasequoia glyptostroboides (Fig. 3i), Chamaecyparis obtuse (Fig. 3l), Sabina chinensis (Fig. 3o) and Cephalotaxus sinensis (Fig. 3u) had many small orbicules on the surface. Microapertures were



901

Fig. 1. Male cones of 13 wind-pollinated, gymnosperm species. (a) Cycas revolute, with a large male cone at the top of the stem, (b) Ginkgo biloba, with many pendulous male cones clustered at the top of a short shoot, along the branch, (c) Abies firma, with several catkin-like male cones on the upper side of the branch, (d) Cedrus deodara: d-1, male cone before pollen release; d-2, male cone during pollen dispersal with an erect cylindrical male cone on the top of a short shoot, along the branch. Arrow: the spiral direction of microsporophylls is similar to a curve. (e) Pinus parviflora, with spike-like male cones clustered on the upper side of the branch, (f) Pinus thunbergii, with many cylindrical male cones on the top of the branch, (g) Cunninghamia lanceolata, with spike-like male cones in all directions on the top of the branch, (h) Cryptomeria japonica, with spike-like male cones at the end of the branch, (i) Metasequoia glyptostroboides, with many spike-like male cones at the end of the branch, (j) Chamaecyparis obtusa with many small male cones at the tip of the leaves, (k) Sabina chinensis, with many small male cones on the upper side of the branches, (l) Podocarpus macrophyllus, showing many upright male cones on the upper side of the branch, (m) Cephalotaxus sinensis, with many male cones in the middle of two rows of leaves. Scale bars 10 cm (a); 2 cm (bm).

observed on the surface in lots of species (Fig. 2c, f, i, l, o, r, and 3c, r, u), with the microapertures of saccate pollen grains larger than the others. We were unable to observe microapertures on the surface of

Cryptomeria japonica (Fig. 3f), Metasequoia glyptostroboides (Fig. 3i), Chamaecyparis obtuse (Fig. 3l) and Sabina chinensis (Fig. 3o) samples because of many orbicules.



Fig. 2. Scanning electron micrographs of pollen grains from six wind-pollinated gymnosperm species. (a)(c) Cycas revolute, (a) equatorial view exhibiting boat-like shape, (b) polar view with one colpate on the distal face, (c) microfoveolate ornamentation on the surface; (d)(f) Ginkgo biloba, (d) equatorial view exhibiting boat-like shape, (e) polar view with a sunken colpate on the distal face, (f) exine with small striates; (g)(i) Abies firma, (g) equatorial view showing body and two sacci, (h) polar view exhibiting two sacci, (i) smooth surface showing many apertures; (j)(l) Cedrus deodara, (j) equatorial view showing body and two sacci, (k) polar view with two sacci, (l) the ornamentation of microgranulates with a rough surface; (m)(o) Pinus parviflora, (m) equatorial view exhibiting body and two sacci, (n) polar view showing two sacci, (o) smooth surface showing many small apertures; (p)(r) Pinus thunbergii, (p) equatorial view showing body and two sacci, (q) polar view with two sacci, (r) smooth surface with many small apertures. Scale bars 10 mm (a, b, d, e, g, h, j, k, m, n, p q); 5 mm (c, f, i, l, o, r).

DISCUSSION Male Cones are Adapted to Wind Pollination Each part of a ‘‘flower’s’’ design may play a special role in one or more events during pollination (Dafni 1992; Jin et al. 2010). In the case of gymnosperms, a number of morphological features have evolved in different groups that appear to increase the wind pollination success. Among these features are the appearance of pendulous catkin-like or spike-like male cones, light pollen grains, and direct female ovulate organs (Niklas 1985; Farquhar et al. 2000; Owens et al. 2001). In this paper, we not only confirmed the findings of previous studies, but also found that the positional relationship

between male cones and the surrounding vegetative structures may affect pollen dispersal. For example: (1) upward pointing, fan-shaped leaves and downward pointing male cones in Ginkgo biloba (Fig. 1b); (2) antrorse male cones with slightly horizontal pinnate leaves in Podocarpus macrophyllus (Fig. 1l); and (3) upward and downward male cones with parallel pinnate leaves in Cephalotaxus sinensis (Fig. 1m), all serve to reduce the filtration of pollen by leaves. Moreover, while young leaves are found above the male cones in Pinus parviflora (Fig. 1e) and Cryptomeria japonica (Fig. 1h), pollen release occur before these leaves spread. In Metasequoia glyptostroboides (Fig. 1i), pollen release



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Fig. 3. Scanning electron micrographs of pollen grains from seven wind-pollinated gymnosperm species. (a)(c) Cunninghamia lanceolata, (a) equatorial view exhibiting oblate spheroidal shape due to natural dehydration after pollen dispersal in the air, (b) polar view with a sunken papilla-like protuberance in the middle, (c) exine with orbicules on the sculptured surface; (d)(f) Cryptomeria japonica. (d) equatorial view exhibiting ellipsoid shape, (e) polar view with a slightly sunken papilla-like protuberance in the middle, (f) many orbicules on the sculptured surface; (g)(i) Metasequoia glyptostroboides, (g) equatorial view exhibiting oblate spheroidal shape, (h) polar view with a slightly sunken papilla-like protuberance in the middle, (i) many orbicules on the sculptured surface; (j)(l) Chamaecyparis obtusa, (j) pollen grains with indentation due to natural drying after pollen release in the air, (k) single pollen grain showing oblate spheroidal shape with indentation, (l) many orbicules on the sculptured surface; (m)(o) Sabina chinensis, (m) many pollen grains with indentation due to natural drying after dispersal in the air, (n) single pollen grain showing oblate spheroidal shape with indentation, (o) many orbicules on the sculptured surface; (p)(r) Podocarpus macrophyllus, (p) equatorial view showing body and two sacci, (q) polar view with two sacci, (r) many small apertures on the surface; (s)(u) Cephalotaxus sinensis, (s) many pollen grains with indentation due to natural drying after release in the air, (t) single pollen grain showing oblate spheroidal shape with indentation, (u) many orbicules on the sculptured surface. Scale bars50 mm (j, m, s), 10 mm (a, b, d, e, g, h, k, n, p, q, t), 5 mm (c, f, i, l, o, r, u).

occurs after the leaves are shed, which reduces the pollen deposition and wastage caused by leaves and twigs. Those architectures could increase the exposure of male

cones to the wind and prevent vegetative structures obstructing the airflow. Consequently, we suggest that variation within male cones, similar to that in female

3jl 3mo 3pr 3su Fig. Fig. Fig. Fig. Microgranulate with orbicules Microgranulate with orbicules Smooth surface with microapertures Microgranulate with orbicules Single aperture Monocolpate The values are means9SD from 30 pollen grains which were randomly selected from each species.

Metasequoia glyptostroboides Chamaecyparis obtusa Sabina chinensis Podocarpus macrophyllus Cephalotaxus sinensis

Cryptomeria japonica

z

26.2392.55 24.1991.67 39.3093.60 26.3893.14 25.5092.64 24.3592.51 34.7592.23 25.6991.94

0.9890.14 1.0190.15 0.8990.08 0.9890.10

Fig. 3gi Single aperture Microgranulate with orbicules 24.4191.74 11.9991.66

0.4990.07

Fig. 3df Single aperture Microgranulate with orbicules 24.8993.51 16.5092.16

0.6890.17

2ac 2df 2gi 2jl 2mo 2pr 3ac Fig. Fig. Fig. Fig. Fig. Fig. Fig. Microreticulate with microapertures Microreticulate with microapertures Smooth surface with microapertures Microgranulate with microapertures Smooth surface with microapertures Smooth surface with microapertures Microgranulate with orbicules Monocolpate Monocolpate Monocolpate Monocolpate Monocolpate Monocolpate Single aperture 0.3590.05 0.3690.03 0.8090.08 0.9090.06 0.9790.11 1.0490.16 0.5090.09 26.0191.60 33.6891.66 86.9995.30 65.6093.94 47.8994.00 40.0993.99 32.7292.87 9.0790.96 12.0991.28 69.6396.04 59.2795.37 46.2592.93 41.3994.34 16.2093.08

Boat-like Boat-like Ellipsoid with two sacci Ellipsoid with two sacci Ellipsoid with two sacci Spheroid with two sacci Oblate spheroid with a papilla-like protuberance Oblate spheroid with a papilla-like protuberance Oblate spheroid with a papilla-like protuberance Spheroid Spheroid Ellipsoid with two sacci Spheroid Cycas revoluta Ginkgo biloba Abies firma Cedrus deodara Pinus parviflora Pinus thunbergii Cunninghamia lanceolata

Type of aperture E or BW (mm) P (BL)/E (BW) P or BL (mm) Shape Species

Table 3. List of major pollen morphological characteristics


Exine ornamentation

Figure


cones and seed traits (Garcia et al. 2009), can be explained by natural selection and adaptive evolution for facilitated wind pollination. Moreover, we observed an interesting phenomenon whereby sac-like pollen grains are usually present in plants that have male cones pointing upwards, such as Abies firma (Fig. 1c), Cedrus deodara (Fig. 1d), Pinus parviflora (Fig. 1e), Pinus thunbergii (Fig. 1f) and Podocarpus macrophyllus (Fig. 1l). Although the sacs of this type of pollen can help grains to float and drift in the wind (Schwendemann et al. 2007), the grains are relatively large and heavy (Owens et al. 1998). Therefore, they fall more easily in still air, with typically greater sedimentation velocities from downward than from upward cones during pollen dispersal. Thus, upward cones may reduce unnecessary pollen losses from the cone. This may be an important factor contributing to the dispersal of sac-pollen. In addition, downward male cones are usually situated at the ends of long, flexible branches, for example in Cryptomeria japonica (Fig. 1h), Metasequoia glyptostroboides (Fig. 1i), Chamaecyparis obtusa (Fig. 1j) and Sabina chinensis (Fig. 1k). This could be because long flexible branches cannot support upward terminal cones for physicomechanical reasons, such as the effect of gravity. Moreover, slender branches tend to be shaken by the wind and experience greater mechanical turbulence, which may be analogous to the long filaments of anthers in angiosperm species that shed pollen by a stochastic aeroelastic mechanism (Urzay et al. 2009). In angiosperms, inflorescence architecture can interact with wind in a complex mechanistic manner to facilitate pollen dispersal (Farquhar et al. 2000; Friedman and Harder 2004). Niklas and Paw (1982) reported that there were aerodynamically predetermined airflow patterns around ovulate cones with a spiral architecture. In this study, we found that the microsporangia of gymnosperms are arranged in a spiral around the cone axis, which may create aerodynamic patterns that reflect the inflorescence architecture and the spiral ovulate cone to contribute to pollen dispersal. In many cases, the presence of many small flowers in wind-pollinated species may be an adaptive compromise to selection for wind pollination (Friedman and Barrett 2009). In our study, we found that the same pattern exists in gymnosperms. Cycas revoluta is an exception to this condition, and produces only one or two huge male cones per tree. This trait may be related to biotic pollination rather than wind pollination (Ackerman 2000), insect pollination having been recently recorded in some cycad species (Kono and Tobe 2007; Terry et al. 2007). The extant major lineages of insect-pollinated cycads could extend back to the Middle Triassic period or possibly earlier (Terry 2001; Klavins et al. 2003, 2005). In extant cycads, such as Australian Macrozamia cycads (Terry et al. 2005, 2007) and Cycas revoluta (Kono and Tobe 2007), pollination is still mediated by insects.



Pollen Adapted for Wind Pollination Larger pollen grains are generally heavier and have greater inertia. Larger grains are more likely to break away from a deflected airflow and then collide with ovules. Smaller pollen grains are lighter, have lower inertia, are easier to remove from the microsporangium, and are more likely to float in the airstream and travel farther (Niklas 1985; Roussy and Kevan 2000). Thus, the pollen size of wind-pollinated plants reflects an equilibrium selection that balances two contradictory demands. Our results indicated that wind-pollinated gymnosperms exhibit a larger range of pollen sizes (2090 mm) than wind-pollinated angiosperms (1758 mm) (Friedman and Barrett 2009). In wind-pollinated gymnosperms, large pollen grains are more likely to collide with ovules, but are more difficult to disperse over large distances. We suggest that gymnosperm pollen has evolved several traits for floating in the air. First, the pollen dehydrates rapidly through microapertures in the surface after release from the microsporangium (Tekleva et al. 2007), reducing its weight: during the dispersal phase, gymnosperm pollen has a water content of less than 10% (Owens et al. 1998). This enables pollen to travel up to several kilometers to reach its target (Bittencourt and Sebbenn 2007; Bohrerova et al. 2009). Second, pollen grains dehydrated rapidly after release from microsporangia (Owens et al. 1998; Tekleva et al. 2007) and exhibited particular shapes after dehydration, such as boat-like, saccate, parachute-like or oblate, or irregular spheroid forms. The evolution of boat, saccate, or parachute shapes might facilitate their carriage on the wind. Additional aerodynamic models are needed to determine if these pollen shapes are particularily adapted to wind pollination. To the our best of our knowledge, only the saccate shape has been explained using aerodynamic modeling (Schwendemann et al. 2007), and further studies of other pollen shapes are required. Third, special pollen structures include sacci and parachute-like protuberances to balance the pollen in the air. The aerodynamic effects of sacci help the pollen rise and drift farther, facilitating upward movement of drifting pollen to enhance pollination success (Salter et al. 2002; Schwendemann et al. 2007). Papilla-like protuberances were found mainly in Taxodiaceae, producing a parachute shape, which might provide stability and reduce pollen settling speeds, helping dispersal. ACKNOWLEDGMENTS We thank Prof. Z.-X. Zhang of Beijing Forestry University and PostDoc. Y.-L. Wan of Institute of Botany, the Chinese Academy of Sciences for reviewing our manuscript, C. David of International Science Editing for English corrections. This work was supported by the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Scientific Research Foundation for high level talents, Yangzhou University, China (2008).

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