- Email: [email protected]
Flower development of different genders in the morphologically andromonoecious but functionally monoecious plant Acer elegantulum Fang et P. L. Chiu
Flora 233 (2017) 179–185
Contents lists available at ScienceDirect
Flora journal homepage: www.elsevier.com/locate/flora
Flower development of different genders in the morphologically andromonoecious but functionally monoecious plant Acer elegantulum Fang et P. L. Chiu Yi-Bo Luo a , Jin-Liang Yu c , Zai-Kang Tong a , Hong-Bo Zhao a,b,∗ a
State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, 311300 Lin’an, Zhejiang, China Department of Ornamental Horticulture, School of Landscape Architecture, Zhejiang Agriculture and Forestry University, 311300 Lin’an, Zhejiang, China c Hangzhou Botanical Garden, 310013 Hangzhou, Zhejiang, China b
a r t i c l e
i n f o
Article history: Received 17 June 2016 Received in revised form 9 June 2017 Accepted 9 June 2017 Edited by Alessio Papini Available online 13 June 2017 Keywords: Acer elegantulum Flower development Staminate flower Hermaphrodite flower Anther dehiscence failure Monoecy
a b s t r a c t Acer elegantulum is morphologically andromonoecious, and hermaphrodite flowers are protogynous. 25.73% of flowers studied were hermaphrodite, and these did not differ in petal length, petal width or flower diameter from staminate flowers, though the lengths of anthers, filaments, styles and ovaries differed significantly. Staminate flowers are functionally male; their staminate flowers developed, dehisced and shed normally, but their ovules and ovaries degenerated gradually at the megasporogenesis stage, and the mature ovaries contained only aborted ovules. Surprisingly, however, despite developing normally until tapetum formation, the anthers of hermaphrodite flowers failed to dehisce when mature, despite developing normally through tapetum formation and dissolution; these flowers’ pistil primordia developed normally, finally forming eight-nucleic embryo sacs. The morphologically hermaphrodite flowers of A. elegantulum are therefore functionally female, and the species is functionally monoecious. We discuss the evolution of staminate and hermaphrodite flowers and suggest that both evolved from ancestrally hermaphrodite flowers, by complete abortion of the pistil or incomplete and late-stage stamen abortion. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction To mate successfully, plants have evolved complex and diverse breeding systems (Barrett, 1998, 2002, 2003). Hermaphroditic species include those with hermaphrodite (perfect) flowers and monoecious individuals with unisexual flowers. Approximately 72% of angiosperms are hermaphroditic, and 6% are dioecious and 7% are monoecious (Renner, 2014). Fossil records suggest that the most primitive breeding system is hermaphroditism (Sun et al., 1998), but separation of the sexes, of various kinds, has evolved in many angiosperm taxa (Renner and Ricklefs, 1995). Dioecy evolved from hermaphroditism multiple, independent evolutionary times, through different pathways (Spigler and Ashman, 2012; Dufay et al., 2014). Dioecy can arise from hermaphroditism: (1) via gynodioecy, (2) via androdioecy, (3) via monoecy and (4) via heterostyly (Webb, 1999; Barrett, 2003; Rubén and Marcos, 2011). These pathways involve either the invasion of a male or female-sterile mutant
∗ Corresponding author at: State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, 311300 Lin’an, Zhejiang, China. E-mail addresses: [email protected], [email protected] (H.-B. Zhao). http://dx.doi.org/10.1016/j.flora.2017.06.006 0367-2530/© 2017 Elsevier GmbH. All rights reserved.
(gynodioecy and androdioecy, respectively) or disruptive selection acting on existing variation (distyly, heterodichogamy and monoecy) (Ashman, 2006). The Aceraceae includes two genera (Acer and Dipteronia) and approximately 202 species worldwide (Xu, 1996, 1998). These plants primarily grow in the temperate regions of Asia, Europe and North America with China having the highest diversity (Xu, 1996), with more than 160 species, approximately 75% of the worldwide total. Acer species exhibit sexual diversity both among and within species. Morphologically, the flowers of Acer species can be divided into unisexual and hermaphrodite flowers (Jong, 1976); while most species are andromonoecious (Renner et al., 2007), a few are androdioecious (A.japonicum belonging to Sect. Palmata; Sato, 2002), trioecious (A.triflorum belonging to Sect. Trifoliata; Gabriela and Miguel, 2005), sub-dioecious (A.wardii belonging to sect. Macrantha; Gabriela and Miguel, 2005) or dioecious (A.argutum belonging to Sect. Glabra). Renner et al. (2007) described 124 species of Acer in the northern hemisphere, most of them monoecious, but including 13 dioecious species that either evolved in three clades or as one of two phylogenetically isolated species; none of the dioecious species were phylogenetically close to the heterodichogamous species, such as A. japonicum and A. monoin Sect.
180
Y.-B. Luo et al. / Flora 233 (2017) 179–185
Fig. 1. The process of inflorescence development in A. elegantulum. A, floral bracts of the inflorescence loosen and the flower buds become visible (1st day of inflorescence development); B, flowers spread out gradually and middle hermaphrodite flowers beginning to bloom (8th day); C, middle flowers in full bloom (13th day); D, end of blooming of the middle flowers (18th day); E, young samaras emerging (21th day). Hf, hermaphrodite flower; Sf, staminate flower; Sa, samara. Scale bars = 1 cm.
Table 1 Comparison of floral traits between staminate flowers and hermaphrodite flowers in Acer elegantulum. Morph
Staminate flower Hermaphrodite flower Fstatistic * **
Sample size
15 15
Petal (mm)
Diameter of disc (mm)
Length of petal
Width of petal
2.88 ± 0.17 2.39 ± 0.11 4.774
2.01 ± 0.12 2.41 ± 0.12 5.451
2.27 ± 0.09 2.34 ± 0.04 0.171
Stamen (mm)
Pistil (mm)
Length of anther
Length of filament
Length of ovary
Length of style
0.99 ± 0.03 1.10 ± 0.03 7.884*
3.27 ± 0.23 1.44 ± 0.05 61.013**
1.10 ± 0.07 2.89 ± 0.13 164.573**
0.81 ± 0.05 3.97 ± 0.13 550.298**
p < 0.05. p < 0.01.
Platanoidea (Sato, 2002); this state commonly includes protogyny, protandry and duodichogamy (monoecious individuals with duodichogamous flowering, male-female-male sequence) (Shang et al., 2012), in which gender expression in protandry and duodichogamy is unstable (Sato, 2002). Gabriela and Miguel (2005) established three comprehensive molecular phylogenetic trees within Acer, and found that the ancestral state was heterodichogamous androdioecy and hypothesized that dioecy evolved from heterodichogamous androdioecy, from heterodichogamous trioecy and from dichogamous subdioecy. Based on a molecular phylogeny, Renner et al. (2007) suggested that the breeding system in Acer evolved from duodichogamous monoecy to dioecy or heterodichogamy (but not from heterodichogamy to dioecy). We here studied A. elegantulum (Sect. Palmata, per Xu et al., 2008), whose breeding system has not previously been reported. This species is native to northwestern Zhejiang and the southern areas of the Anhui and Jiangxi province. In Sect. Palmata, A. campbellii, A. erianthum and A. kweilinense are andromonoecious (Renner et al., 2007). We studied morphological characteristics, sex ratios, flowering phenology and the differentiation and development of flowers with different genders. 2. Materials and methods 2.1. Experimental location and materials The 20 A. elegantulum plants selected for this study were collected from wild habitats and were cultivated for more than five years in the garden of Zhejiang A & F University; they grew well and bloomed and fruited normally. The site is located in a subtropical region with a subtropical monsoon climate, with an average annual temperature of 16.4 ◦ C and average annual rainfall of 1628.6 mm.
Staminate and hermaphrodite flowers from random inflorescences were sampled, and the anthers, filaments and styles were separated, observed, measured and photographed under a stereoscopic microscope (Stemi 2000-C, Zeiss, Germany). 2.3. Flowering phenology Three plants with well growing inflorescences at the same developmental stage were further selected and labeled to investigate flowering phenology. The developmental status of flowers of different genders and inflorescences was noted and photographed every day. The peripheral tissues were removed under the stereo microscope to reveal structural features, which were photographed. 2.4. Development of anthers and ovaries Further, staminate and hermaphrodite flowers of different developmental stages were collected for sectioning (15–20 flowers per stage, fixed using FAA solution including 5 mL formalin, 5 mL acetic and 90 mL 50% ethanol). The fixed flowers were sectioned according to the method of Xu et al. (2014). Specifically, the samples underwent gradient alcohol dehydration, xylene treatment and paraffin penetration and embedding before being sectioned using a microtome (Leica RM2235). Longitudinal sections of 8–10 m in thickness were generated and stained with safranin and fast green before being sealed with neutral resin. The sections were observed and photographed under an Axio Imager A2 (Zeiss, German) upright fluorescence microscope. 3. Results 3.1. Floral traits and sex ratio
2.2. Floral traitsandsex ratio Every two days during floral bud emergence, development and blooming (March–May 2015), 15 intact inflorescences were collected from each of 15–20 plants, dissected, observed and measured under a microscope, and the gender of each flower was recorded.
Acer elegantulum bloomed simultaneously with robust leaf growth. The inflorescences are paniculate, and blooming started from the top. Individual inflorescences had an average of 10–30 flowers, with one or three flowers on each branch of the inflorescences. Staminate and hermaphrodite flowers were observed on
Y.-B. Luo et al. / Flora 233 (2017) 179–185
181
Fig. 2. The process of staminate flower development in A. elegantulum. A1–A5, young flower buds (the 9th day of inflorescence development); B1–B5, flowers first blooming (13rd day); C1–C5; flowers blooming and anthers beginning to disperse pollen (16th day); D1–D5, flowers full blooming (18th day). 1, front view of the flower; 2, overhead view of the flower; 3, front view of the stamens and ovary; 4, overhead view of the stamens and ovary; 5, the ovary. Ca: calyx; Pe:petal; An: anther; Ov: ovary; St: style. Scale bars = 1000 m.
the same inflorescence with no obvious distribution pattern (Fig. 1). On average, 25.73 ± 1.99% of the total flowers were hermaphroditic. Morphologically, there were no differences in petal length, petal width or diameter of flower disc between staminate and hermaphrodite flowers. Staminate flowers contained only anthers and completely aborted pistils (Fig. 2). Hermaphrodite flowers had intact male and female organs (Fig. 3). The morphology of their anthers was similar to anthers of staminate flowers; however, the filaments were significantly shorter (1.44 ± 0.05 mm versus 3.27 ± 0.23 mm) (Table 1).The anthers were larger (1.10 ± 0.03 mm) than those of staminate flowers (0.99 ± 0.03 mm) but they never dehisced, indicating that the morphologically hermaphrodite flowers are functionally female. Therefore, although the species is morphologically andromonoecious, it is functionally monoecious.
3.2. Flowering phenology The blooming period of individual trees lasted approximately 24 ± 2.64 d, defining the 1st day as the point when the inflorescence loosens and the flowers became visible. The anthesis of each inflorescence lasted approximately 20 ± 1.42 d. Hermaphrodite flowers matured 8 days earlier than staminate flowers, exhibiting protogyny. On the 8th day, the floral buds of hermaphrodite flowers loosened, and the stigmas extended to their maximum length (Fig. 1B); the petals of staminate flowers were either tightly wrapped or had recently loosened. On the 13th day, the hermaphrodite flowers were in full bloom (Fig. 1C). On the 18th day, staminate flowers were in full bloom and shed pollen (Fig. 1D). On the 21st day, the majority of hermaphrodite flowers were spent; stamens had fallen off, and ovaries began to swell. On the 27th day, all staminate flowers fell off.
In the early developmental stage of floral buds (before the 2nd day of anthesis), the morphology of staminate and hermaphrodite flowers showed no significant differences. At this stage, petals of these two genders were tightly packed, and each flower contained eight yellow anthers; small stigmas had not yet stretched and could be observed in the middle of flowers (Figs. 2A1–A5 and 3A1–A5). After this stage (from 3rd day), there were obvious differences between staminate and hermaphrodite flowers. In staminate flowers, the corolla gradually opened up, and the filaments elongated and bent inwardly (Fig. 2B1 and C1). After pollen was shed (18th day), the filaments stopped bending and were arranged radially, forming angles of 60◦ to the flower disk, and the pistil remained only as small dichotomous stigmas and degenerated ovaries (Fig. 2D1–D5). In hermaphrodite flowers, the styles elongated gradually and remained erect or bent at maturity (13th day); stigmas slightly dehisced and turned brown after pollination, and there was no marked elongation of the anther filaments (Fig. 3B1–D5).
3.3. Anther and pollen development When the floral bud emerged and bud scales cracked, male and female gametophytes began to develop (as the 1st day of anther and pollen development, 7 days before the flowers began to bloom). In both staminate and hermaphrodite flowers, microspore development was normal and yielded mature pollen. The anther development of these two genders differed only in the late developmental stage (10th day) during anther wall formation. Microspore mother cells underwent meiosis to generate tetrads (6th day) that were arranged as tetrahedrons within the anthers (Fig. 4B). Then, the callose walls of the tetrads dissolved gradually, and the microspores were released from the tetrads and formed pollen
182
Y.-B. Luo et al. / Flora 233 (2017) 179–185
Fig. 3. The process of hermaphrodite flower development in A. elegantulum. A1–A5, young flower buds (1st day of inflorescence development); B1–B5, big flower buds, with styles beginning to elongate (3rd day); C1–C5; blooming but without pollination of the stigmas (8th day); D1–D5, stigma after pollination (13th day). 1, front view of the flower; 2, overhead view of the flower; 3, front view of the stamens and ovary; 4, overhead view of the stamens and ovary; 5, the ovary. Ca: calyx; Pe: petal; An: anther; Ov: ovary; St: style. Scale bars = 1000 m.
grains, each with a single central nucleus (Fig. 4C). At this time (8th day), the tapetum started to decompose drastically. The nutrients released by the dissolved tapetum were taken up by the microspores, which were continuously enlarged, and large vacuoles were generated in the center of each cell, while the nucleus was pushed to one side. Microspores then entered the late uninucleate stage (10th day) (Fig. 4D). Prior to this stage, it was impossible to distinguish the stamen morphology between staminate and hermaphrodite flowers. Microspores continued to develop; each microspore underwent mitosis and formed two nuclei, one each for reproductive cells and vegetative cells (Fig. 4E and G). When flowers entered the binucleate pollen stage (13th day), staminate and hermaphrodite flowers could be clearly distinguished by their external morphologies. After the maturation of staminate flower anthers (15th day), the fibrous layer dehydrated, secondary thickening occurred in the inner walls of the anthers, and the outer tangential wall of the fibrous layer did not thicken. The outer wall wrinkled, generating the mechanical force that dehisced the anther. Then the stomium was generated (Fig. 4H) and the pollen grains released (Fig. 4I). However, in the hermaphrodite flowers, the fibrous layer of the mature anthers showed obvious thickening at the anther connection and did not form the stomium (Fig. 4F), resulting in the failure of anther dehiscence.
3.4. Ovary and ovule development Before the tetrad stage (8th day), pistil (or pistillode) development was similar in staminate and hermaphrodite flowers. In hermaphrodite flowers, the ovary contained two carpels, two locules and anatropous ovules. During early development, a protrusion formed at the axile placentation and later developed into the ovule primordium (Fig. 5A). The ovule primordium further devel-
oped into the megasporocyte (7th day) (Fig. 5B), which formed four longitudinally-aligned megaspores via meiosis (Fig. 5C). Three of the four ultimately degenerated, leaving one functional megaspore (Fig. 5D). Then, the megaspore sequentially developed into the di-nucleate embryo sac (12th day; Fig. 5E and F), tetra-nucleate embryo sac (13th day; Fig. 5G) and finally the mature eightnucleate embryo sac (14th day; Fig. 5H). The mature embryo sac was Polygonum-type, consisting of one egg cell and two helper cells at the micropylar end, two polar nuclei of the central cells and three antipodal cells at the chalazal end. In staminate flowers, ovule development stopped upon the formation of the functional megaspore (Fig. 5I). The boundaries of the ovule gradually blurred; ultimately, only the aborted ovule remained (Fig. 5J and K), although the ovary wall continued to swell (Fig. 5L).
4. Discussion Sex differentiation in plants can occur at any developmental stage. Two categories of unisexual flowers have been recognized according to the timing difference of sex differentiation (Mitchell and Diggle, 2005). Organ abortion can occur in any of four stages: stage 0, before the differentiation of stamen and carpel primordia; stage 1, early development of stamens and pistils; stage 2, prophase of meiosis; and stage 3, after meiosis (Diggle et al., 2011). Unisexual flowers are classified into type I, which are bisexual at initiation and become unisexual by termination of male or female sexual organ at some developmental stage, and type II, in which flowers become unisexual very early, before initiation of stamens and carpels. Diggle et al. (2011) surveyed 292 taxa with unisexual flowers and found sex organ abortion with equal frequency at each of the four stages in both male and female flowers, and in monoecious and dioecious taxa. Although all combinations of
Y.-B. Luo et al. / Flora 233 (2017) 179–185
183
Fig. 4. The development of anthers and pollen in A. elegantulum. A–C, the common developmental process in both staminate and hermaphrodite flowers; D–F, the developmental process of anthers and pollen in hermaphrodite flowers; G–I, the developmental process of anthers and pollen in staminate flowers. A, tetrahedral tetrads forming; B, microspores are released from the tetrads; C, microspores absorb the nutrients from the tapetum; D, late uninucleate stage; E, development of two-celled pollen; F, mature anthers of hermaphrodite flowers; G, development of two-celled pollen; H, mature anthers of staminate flowers; I, anthers dehiscing. Mmc, Microspore mother cell; Ms, microspore; Ta, Tapetum; Gc, Generative cell; Vc, Vegetative cell; St, Stomium; Fl, Fibrous layer; Pg, pollen grains. Scale bars = 20 m.
184
Y.-B. Luo et al. / Flora 233 (2017) 179–185
Fig. 5. The development of ovaries and ovules in A. elegantulum. A, longitudinal section of the normal ovary during early development stage in both staminate and hermaphrodite flowers; B–H, development of ovaries and ovules in hermaphrodite flower. B, integuments arose from the ovule primordium, and the megaspore mother cells formed; C, linear mega-spore tetrads formed as result of meiosis of the megaspore mother cells; D, functional megaspore; E, F, bi-nucleate embryo sac; G, tetra-nucleate embryo sac; H, eight-nucleate embryo sac; I–L, the process of ovule abortion in staminate flower. I, functional megaspore; J, boundaries of the ovule gradually blurred; K, abortive ovule; L, abortive ovary. Op, ovule primordium; Mmc, megaspore mother cell. Mt, megaspore tetrad; Fm, functional megaspore; Bes: bi-nucleate embryo sac; Tes: tetra-nucleate embryo sac; Sy, synergid; Pn, polar nucleus; Ec, egg cell; Art, antipodal cell. Scale bars = 10 m for A–J and 20 m for K and L.
stages of androecium and gynoecium abortion are seen, taxa in which the androecium and gynoecium abort at the same stage are over-represented (Diggle et al., 2011; Fig. S2). More recent examples (Table S1) yield similar results (Fig. S1), and in A. elegantulum, both androecium and gynoecium abort at stage 3. The pistil primordium of staminate flowers was selectively aborted at the phase of functional megaspore formation. The anthers of hermaphrodite flowers did not dehisce, resulting in functional female flowers. Such late and concurrent of androecium and gynoecium abortion in A. elegantulum suggests that the species may have evolved monoecy recently, from andromonoecy. If A. elegantulum is animal-pollinated, the retention of well-developed, yet
indehiscent, anthers could attract pollinators, ensuring reproductive success (Podolsky, 1992). Monoecy may reduce interference between male and female functions (dispersing pollen vs. accepting pollen) and avoid self-pollination (Barrett et al., 2000). After the anthers in staminate flowers of A. elegantulum mature, the fibrous layer cells dehydrate, and secondary thickening occurs in the inner wall, but not the outer tangential wall of the fibrous layer, which causes the anther wall to wrinkle. The mechanical forces this induces lead to anther dehiscence of staminate flowers. Anther dehiscence in other plants is induced by programmed cell death that leads to degeneration of the inner wall and connective tissues of the anther, the dehydration of cells, and the outward
Y.-B. Luo et al. / Flora 233 (2017) 179–185
bending of the anthers (Sanders et al., 1999; Varnier et al., 2005; Senatore et al., 2009; Parish and Li, 2010). When programmed cell death does not occur normally, or secondary thickening fails to develop in the inner wall of the anthers, dehiscence fails (Sanders et al., 1999; Parish and Li, 2010; Mizuno et al., 2007; Thvenin et al., 2011). In Acer, incompletely dissolved tapetum or a lack of lip cell structure (Xu et al., 2012; Zhang et al., 2011) and a lack of significant lignification can also cause anther dehiscence failure (Hu et al., 2009). In A. elegantulum, the tapetum was completely dissolved in both staminate and hermaphrodite flowers, and significant lignification was not observed, so these factors did not result in anther dehiscence failure in the hermaphrodite flowers. In the anthers of A. elegantulum hermaphrodite flowers, anther development is mostly similar to that in staminate flowers, but normal programmed cell death does not occur in the inner walls of the anthers and tapetum, and the anthers fail to dehisce. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.flora.2017.06. 006. References Ashman, T.L., 2006. The evolution of separate sexes: a focus on the ecological context. In: Ecology & Evolution of Flowers. Oxford University Press, Oxford, pp. 204–222. Barrett, S.C.H., Jesson, L.K., Baker, A.M., 2000. The evolution and function of stylar polymorphisms in flowering plants. Ann. Bot. 85 (3), 253–265. Barrett, S.C.H., 1998. The evolution of mating strategies in flowering plants. Trends Plant Sci. 3 (9), 335–341. Barrett, S.C.H., 2002. The evolution of plant sexual diversity. Nat. Rev. Genet. 3 (4), 274–284. Barrett, S.C.H., 2003. Mating strategies in flowering plants: the outcrossing–selfing paradigm and beyond. Philos. Trans. R. Soc. B Biol. Sci. 358 (1434), 991–1004. Diggle, P.K., Stilio, V.S.D., Gschwend, A.R., Golenberg, E.M., Moore, R.C., Russell, J.R., Sinclair, J.P., 2011. Multiple developmental processes underlie sex differentiation in angiosperms. Trends Genet. 27 (9), 368–376. Dufay, M., Champelovier, P., Käfer, J., Henry, J.P., Mousset, S., Marais, G.A.B., 2014. An angiosperm-wide analysis of the gynodioecy-dioecy pathway. Ann. Bot. 114 (3), 539–548. Gabriela, G., Miguel, V., 2005. Repeated evolution of dioecy from androdioecy in Acer. New Phytol. 165 (2), 633–640. Hu, Q., Li, F.L., Du, Y.L., Guo, H.H., 2009. Morphological and cytological study on floral sex differentiation of Acer truncatum. Bull. Bot. Res. 29 (02), 136–140. Jong, P.C.D., 1976. Flowering and Sex Expression in Acer L. A Biosystematic Study., pp. 76. Mitchell, C.H., Diggle, P.K., 2005. Evolution of unisexual flowers: morphological and functional convergence results from diverse developmental transitions. Am. J. Bot. 92 (7), 1068–1076. Mizuno, S., Osakabe, Y., Maruyama, K., Ito, T., Osakabe, K., Sato, T., Shinozaki, K., Yamaguchi-Shinozaki, Kazuko, 2007. Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana. Plant J. 50 (5), 751–766.
185
Parish, R.W., Li, S.F., 2010. Death of a tapetum: a programme of developmental altruism. Plant Sci. 178 (2), 73–89. Podolsky, R.D., 1992. Strange floral attractors: pollinator attraction and the evolution of plant sexual systems. Science 258 (5083), 791–793. Renner, S.S., Ricklefs, R.E., 1995. Dioecy and its correlates in the flowering plants. Am. J. Bot. 82 (5), 596–606. Renner, S.S., Beenken, L., Grimm, G.W., Kocyan, A., Ricklefs, R.E., 2007. The evolution of dioecy, heterodichogamy, and labile sex expression in Acer. Evolution 61 (11), 2701–2719. Renner, S.S., 2014. The relative and absolute frequencies of angiosperm sexual systems: dioecy, monoecy, gynodioecy, and an updated online database. Am. J. Bot. 101 (10), 1588–1596. Rubén, T., Marcos, M., 2011. Where do monomorphic sexual systems fit in the evolution of dioecy? Insights from the largest family of angiosperms. New Phytol. 190 (1), 234–248. Sanders, P.M., Bui, A.Q., Weterings, K., McIntire, K.N., Hsu, Y.C., Lee, P.Y., Truong, M.T., Beals, T.P., Goldberg, R.B., 1999. Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex. Plant Reprod. 11 (6), 297–322. Sato, T., 2002. Phenology of sex expression and gender variation in a heterodichogamous maple, Acer japonicum. Ecology 83 (5), 1226–1238. Senatore, A., Trobacher, C.P., Greenwood, J.S., 2009. Ricinosomes predict programmed cell death leading to anther dehiscence in tomato. Plant Physiol. 149 (2), 775–790. Shang, H., Luo, Y.B., Bai, W.N., 2012. Influence of asymmetrical mating patterns and male reproductive success on the maintenance of sexual polymorphism in Acer pictum subsp. mono (Aceraceae). Mol. Ecol. 21 (15), 3869–3878. Spigler, R.B., Ashman, T.L., 2012. Gynodioecy to dioecy: are we there yet? Ann. Bot. 109 (3), 531–543. Sun, G., Dilcher, D.L., Zheng, S., Zhou, Z., 1998. In search of the first flower: a urassic angiosperm, archaefructus, from northeast china. Science 282 (5394), 1692–1695. Thvenin, J., Pollet, B., Letarnec, B., Saulnier, L., Gissot, L., Alessandra, Maia-Grondard, Lapierre, C., Jouanin, L., 2011. The simultaneous repression of CCR and CAD, two enzymes of the lignin biosynthetic pathway, results in sterility and dwarfism in Arabidopsis thaliana. Molecular Plant 4, 70–82. Varnier, A.L., Mazeyrat-Gourbeyr, e., Florenc, e., Sangwan, R.S., Clément, C., 2005. Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation. J. Struct. Biol. 152, 118–128. Webb, C.J., 1999. Empirical Studies: Evolution and Maintenance of Dimorphic Breeding Systems. Springer, Berlin, Heidelberg. Xu, T.Z., Chen, Y.S., de Jong, P.C., Oterdoom, H.J., Chang, C.S., 2008. Flora of China, vol. 11. Science Press and Missouri Botanical Garden Press, Beijing and St. Louis, pp. 515–533. Xu, X.L., Jin, H.X., Chen, C.B., Tian, Q., Zhu, M.L., Chen, X.Y., 2012. Studies on the formation of microspores and development of male gametophyte in Acer yanjuechi (Aceraceae). Plant Divers. Resour. 34 (04), 339–346. Xu, Y.C., Zhou, L.H., Hu, S.Q., Hao, R.M., Huang, C.J., Zhao, H.B., 2014. The differentiation and development of pistils of hermaphrodites and pistillodes of males in androdioecious Osmanthus fragrans L. and implications for the evolution to androdioecy. Plant Syst. Evol. 300 (5), 843–849. Xu, T.Z., 1996. Phytogeography of the family Aceraceae. Acta Bot. Yunnanica 18 (01), 43–50. Xu, T.Z., 1998. The systematic evolution and distribution of the genus Acer. Acta Bot. Yunnanica 20 (04), 383–393. Zhang, L.Z., Shang, H., Luo, Y.B., Cheng, X., Bai, W.N., 2011. Morphology and cytology of three flower phenotypes in a duodichogamous tree species, Acer mono. Biodivers. Sc. 19 (05), 551–557.
Contents lists available at ScienceDirect
Flora journal homepage: www.elsevier.com/locate/flora
Flower development of different genders in the morphologically andromonoecious but functionally monoecious plant Acer elegantulum Fang et P. L. Chiu Yi-Bo Luo a , Jin-Liang Yu c , Zai-Kang Tong a , Hong-Bo Zhao a,b,∗ a
State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, 311300 Lin’an, Zhejiang, China Department of Ornamental Horticulture, School of Landscape Architecture, Zhejiang Agriculture and Forestry University, 311300 Lin’an, Zhejiang, China c Hangzhou Botanical Garden, 310013 Hangzhou, Zhejiang, China b
a r t i c l e
i n f o
Article history: Received 17 June 2016 Received in revised form 9 June 2017 Accepted 9 June 2017 Edited by Alessio Papini Available online 13 June 2017 Keywords: Acer elegantulum Flower development Staminate flower Hermaphrodite flower Anther dehiscence failure Monoecy
a b s t r a c t Acer elegantulum is morphologically andromonoecious, and hermaphrodite flowers are protogynous. 25.73% of flowers studied were hermaphrodite, and these did not differ in petal length, petal width or flower diameter from staminate flowers, though the lengths of anthers, filaments, styles and ovaries differed significantly. Staminate flowers are functionally male; their staminate flowers developed, dehisced and shed normally, but their ovules and ovaries degenerated gradually at the megasporogenesis stage, and the mature ovaries contained only aborted ovules. Surprisingly, however, despite developing normally until tapetum formation, the anthers of hermaphrodite flowers failed to dehisce when mature, despite developing normally through tapetum formation and dissolution; these flowers’ pistil primordia developed normally, finally forming eight-nucleic embryo sacs. The morphologically hermaphrodite flowers of A. elegantulum are therefore functionally female, and the species is functionally monoecious. We discuss the evolution of staminate and hermaphrodite flowers and suggest that both evolved from ancestrally hermaphrodite flowers, by complete abortion of the pistil or incomplete and late-stage stamen abortion. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction To mate successfully, plants have evolved complex and diverse breeding systems (Barrett, 1998, 2002, 2003). Hermaphroditic species include those with hermaphrodite (perfect) flowers and monoecious individuals with unisexual flowers. Approximately 72% of angiosperms are hermaphroditic, and 6% are dioecious and 7% are monoecious (Renner, 2014). Fossil records suggest that the most primitive breeding system is hermaphroditism (Sun et al., 1998), but separation of the sexes, of various kinds, has evolved in many angiosperm taxa (Renner and Ricklefs, 1995). Dioecy evolved from hermaphroditism multiple, independent evolutionary times, through different pathways (Spigler and Ashman, 2012; Dufay et al., 2014). Dioecy can arise from hermaphroditism: (1) via gynodioecy, (2) via androdioecy, (3) via monoecy and (4) via heterostyly (Webb, 1999; Barrett, 2003; Rubén and Marcos, 2011). These pathways involve either the invasion of a male or female-sterile mutant
∗ Corresponding author at: State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, 311300 Lin’an, Zhejiang, China. E-mail addresses: [email protected], [email protected] (H.-B. Zhao). http://dx.doi.org/10.1016/j.flora.2017.06.006 0367-2530/© 2017 Elsevier GmbH. All rights reserved.
(gynodioecy and androdioecy, respectively) or disruptive selection acting on existing variation (distyly, heterodichogamy and monoecy) (Ashman, 2006). The Aceraceae includes two genera (Acer and Dipteronia) and approximately 202 species worldwide (Xu, 1996, 1998). These plants primarily grow in the temperate regions of Asia, Europe and North America with China having the highest diversity (Xu, 1996), with more than 160 species, approximately 75% of the worldwide total. Acer species exhibit sexual diversity both among and within species. Morphologically, the flowers of Acer species can be divided into unisexual and hermaphrodite flowers (Jong, 1976); while most species are andromonoecious (Renner et al., 2007), a few are androdioecious (A.japonicum belonging to Sect. Palmata; Sato, 2002), trioecious (A.triflorum belonging to Sect. Trifoliata; Gabriela and Miguel, 2005), sub-dioecious (A.wardii belonging to sect. Macrantha; Gabriela and Miguel, 2005) or dioecious (A.argutum belonging to Sect. Glabra). Renner et al. (2007) described 124 species of Acer in the northern hemisphere, most of them monoecious, but including 13 dioecious species that either evolved in three clades or as one of two phylogenetically isolated species; none of the dioecious species were phylogenetically close to the heterodichogamous species, such as A. japonicum and A. monoin Sect.
180
Y.-B. Luo et al. / Flora 233 (2017) 179–185
Fig. 1. The process of inflorescence development in A. elegantulum. A, floral bracts of the inflorescence loosen and the flower buds become visible (1st day of inflorescence development); B, flowers spread out gradually and middle hermaphrodite flowers beginning to bloom (8th day); C, middle flowers in full bloom (13th day); D, end of blooming of the middle flowers (18th day); E, young samaras emerging (21th day). Hf, hermaphrodite flower; Sf, staminate flower; Sa, samara. Scale bars = 1 cm.
Table 1 Comparison of floral traits between staminate flowers and hermaphrodite flowers in Acer elegantulum. Morph
Staminate flower Hermaphrodite flower Fstatistic * **
Sample size
15 15
Petal (mm)
Diameter of disc (mm)
Length of petal
Width of petal
2.88 ± 0.17 2.39 ± 0.11 4.774
2.01 ± 0.12 2.41 ± 0.12 5.451
2.27 ± 0.09 2.34 ± 0.04 0.171
Stamen (mm)
Pistil (mm)
Length of anther
Length of filament
Length of ovary
Length of style
0.99 ± 0.03 1.10 ± 0.03 7.884*
3.27 ± 0.23 1.44 ± 0.05 61.013**
1.10 ± 0.07 2.89 ± 0.13 164.573**
0.81 ± 0.05 3.97 ± 0.13 550.298**
p < 0.05. p < 0.01.
Platanoidea (Sato, 2002); this state commonly includes protogyny, protandry and duodichogamy (monoecious individuals with duodichogamous flowering, male-female-male sequence) (Shang et al., 2012), in which gender expression in protandry and duodichogamy is unstable (Sato, 2002). Gabriela and Miguel (2005) established three comprehensive molecular phylogenetic trees within Acer, and found that the ancestral state was heterodichogamous androdioecy and hypothesized that dioecy evolved from heterodichogamous androdioecy, from heterodichogamous trioecy and from dichogamous subdioecy. Based on a molecular phylogeny, Renner et al. (2007) suggested that the breeding system in Acer evolved from duodichogamous monoecy to dioecy or heterodichogamy (but not from heterodichogamy to dioecy). We here studied A. elegantulum (Sect. Palmata, per Xu et al., 2008), whose breeding system has not previously been reported. This species is native to northwestern Zhejiang and the southern areas of the Anhui and Jiangxi province. In Sect. Palmata, A. campbellii, A. erianthum and A. kweilinense are andromonoecious (Renner et al., 2007). We studied morphological characteristics, sex ratios, flowering phenology and the differentiation and development of flowers with different genders. 2. Materials and methods 2.1. Experimental location and materials The 20 A. elegantulum plants selected for this study were collected from wild habitats and were cultivated for more than five years in the garden of Zhejiang A & F University; they grew well and bloomed and fruited normally. The site is located in a subtropical region with a subtropical monsoon climate, with an average annual temperature of 16.4 ◦ C and average annual rainfall of 1628.6 mm.
Staminate and hermaphrodite flowers from random inflorescences were sampled, and the anthers, filaments and styles were separated, observed, measured and photographed under a stereoscopic microscope (Stemi 2000-C, Zeiss, Germany). 2.3. Flowering phenology Three plants with well growing inflorescences at the same developmental stage were further selected and labeled to investigate flowering phenology. The developmental status of flowers of different genders and inflorescences was noted and photographed every day. The peripheral tissues were removed under the stereo microscope to reveal structural features, which were photographed. 2.4. Development of anthers and ovaries Further, staminate and hermaphrodite flowers of different developmental stages were collected for sectioning (15–20 flowers per stage, fixed using FAA solution including 5 mL formalin, 5 mL acetic and 90 mL 50% ethanol). The fixed flowers were sectioned according to the method of Xu et al. (2014). Specifically, the samples underwent gradient alcohol dehydration, xylene treatment and paraffin penetration and embedding before being sectioned using a microtome (Leica RM2235). Longitudinal sections of 8–10 m in thickness were generated and stained with safranin and fast green before being sealed with neutral resin. The sections were observed and photographed under an Axio Imager A2 (Zeiss, German) upright fluorescence microscope. 3. Results 3.1. Floral traits and sex ratio
2.2. Floral traitsandsex ratio Every two days during floral bud emergence, development and blooming (March–May 2015), 15 intact inflorescences were collected from each of 15–20 plants, dissected, observed and measured under a microscope, and the gender of each flower was recorded.
Acer elegantulum bloomed simultaneously with robust leaf growth. The inflorescences are paniculate, and blooming started from the top. Individual inflorescences had an average of 10–30 flowers, with one or three flowers on each branch of the inflorescences. Staminate and hermaphrodite flowers were observed on
Y.-B. Luo et al. / Flora 233 (2017) 179–185
181
Fig. 2. The process of staminate flower development in A. elegantulum. A1–A5, young flower buds (the 9th day of inflorescence development); B1–B5, flowers first blooming (13rd day); C1–C5; flowers blooming and anthers beginning to disperse pollen (16th day); D1–D5, flowers full blooming (18th day). 1, front view of the flower; 2, overhead view of the flower; 3, front view of the stamens and ovary; 4, overhead view of the stamens and ovary; 5, the ovary. Ca: calyx; Pe:petal; An: anther; Ov: ovary; St: style. Scale bars = 1000 m.
the same inflorescence with no obvious distribution pattern (Fig. 1). On average, 25.73 ± 1.99% of the total flowers were hermaphroditic. Morphologically, there were no differences in petal length, petal width or diameter of flower disc between staminate and hermaphrodite flowers. Staminate flowers contained only anthers and completely aborted pistils (Fig. 2). Hermaphrodite flowers had intact male and female organs (Fig. 3). The morphology of their anthers was similar to anthers of staminate flowers; however, the filaments were significantly shorter (1.44 ± 0.05 mm versus 3.27 ± 0.23 mm) (Table 1).The anthers were larger (1.10 ± 0.03 mm) than those of staminate flowers (0.99 ± 0.03 mm) but they never dehisced, indicating that the morphologically hermaphrodite flowers are functionally female. Therefore, although the species is morphologically andromonoecious, it is functionally monoecious.
3.2. Flowering phenology The blooming period of individual trees lasted approximately 24 ± 2.64 d, defining the 1st day as the point when the inflorescence loosens and the flowers became visible. The anthesis of each inflorescence lasted approximately 20 ± 1.42 d. Hermaphrodite flowers matured 8 days earlier than staminate flowers, exhibiting protogyny. On the 8th day, the floral buds of hermaphrodite flowers loosened, and the stigmas extended to their maximum length (Fig. 1B); the petals of staminate flowers were either tightly wrapped or had recently loosened. On the 13th day, the hermaphrodite flowers were in full bloom (Fig. 1C). On the 18th day, staminate flowers were in full bloom and shed pollen (Fig. 1D). On the 21st day, the majority of hermaphrodite flowers were spent; stamens had fallen off, and ovaries began to swell. On the 27th day, all staminate flowers fell off.
In the early developmental stage of floral buds (before the 2nd day of anthesis), the morphology of staminate and hermaphrodite flowers showed no significant differences. At this stage, petals of these two genders were tightly packed, and each flower contained eight yellow anthers; small stigmas had not yet stretched and could be observed in the middle of flowers (Figs. 2A1–A5 and 3A1–A5). After this stage (from 3rd day), there were obvious differences between staminate and hermaphrodite flowers. In staminate flowers, the corolla gradually opened up, and the filaments elongated and bent inwardly (Fig. 2B1 and C1). After pollen was shed (18th day), the filaments stopped bending and were arranged radially, forming angles of 60◦ to the flower disk, and the pistil remained only as small dichotomous stigmas and degenerated ovaries (Fig. 2D1–D5). In hermaphrodite flowers, the styles elongated gradually and remained erect or bent at maturity (13th day); stigmas slightly dehisced and turned brown after pollination, and there was no marked elongation of the anther filaments (Fig. 3B1–D5).
3.3. Anther and pollen development When the floral bud emerged and bud scales cracked, male and female gametophytes began to develop (as the 1st day of anther and pollen development, 7 days before the flowers began to bloom). In both staminate and hermaphrodite flowers, microspore development was normal and yielded mature pollen. The anther development of these two genders differed only in the late developmental stage (10th day) during anther wall formation. Microspore mother cells underwent meiosis to generate tetrads (6th day) that were arranged as tetrahedrons within the anthers (Fig. 4B). Then, the callose walls of the tetrads dissolved gradually, and the microspores were released from the tetrads and formed pollen
182
Y.-B. Luo et al. / Flora 233 (2017) 179–185
Fig. 3. The process of hermaphrodite flower development in A. elegantulum. A1–A5, young flower buds (1st day of inflorescence development); B1–B5, big flower buds, with styles beginning to elongate (3rd day); C1–C5; blooming but without pollination of the stigmas (8th day); D1–D5, stigma after pollination (13th day). 1, front view of the flower; 2, overhead view of the flower; 3, front view of the stamens and ovary; 4, overhead view of the stamens and ovary; 5, the ovary. Ca: calyx; Pe: petal; An: anther; Ov: ovary; St: style. Scale bars = 1000 m.
grains, each with a single central nucleus (Fig. 4C). At this time (8th day), the tapetum started to decompose drastically. The nutrients released by the dissolved tapetum were taken up by the microspores, which were continuously enlarged, and large vacuoles were generated in the center of each cell, while the nucleus was pushed to one side. Microspores then entered the late uninucleate stage (10th day) (Fig. 4D). Prior to this stage, it was impossible to distinguish the stamen morphology between staminate and hermaphrodite flowers. Microspores continued to develop; each microspore underwent mitosis and formed two nuclei, one each for reproductive cells and vegetative cells (Fig. 4E and G). When flowers entered the binucleate pollen stage (13th day), staminate and hermaphrodite flowers could be clearly distinguished by their external morphologies. After the maturation of staminate flower anthers (15th day), the fibrous layer dehydrated, secondary thickening occurred in the inner walls of the anthers, and the outer tangential wall of the fibrous layer did not thicken. The outer wall wrinkled, generating the mechanical force that dehisced the anther. Then the stomium was generated (Fig. 4H) and the pollen grains released (Fig. 4I). However, in the hermaphrodite flowers, the fibrous layer of the mature anthers showed obvious thickening at the anther connection and did not form the stomium (Fig. 4F), resulting in the failure of anther dehiscence.
3.4. Ovary and ovule development Before the tetrad stage (8th day), pistil (or pistillode) development was similar in staminate and hermaphrodite flowers. In hermaphrodite flowers, the ovary contained two carpels, two locules and anatropous ovules. During early development, a protrusion formed at the axile placentation and later developed into the ovule primordium (Fig. 5A). The ovule primordium further devel-
oped into the megasporocyte (7th day) (Fig. 5B), which formed four longitudinally-aligned megaspores via meiosis (Fig. 5C). Three of the four ultimately degenerated, leaving one functional megaspore (Fig. 5D). Then, the megaspore sequentially developed into the di-nucleate embryo sac (12th day; Fig. 5E and F), tetra-nucleate embryo sac (13th day; Fig. 5G) and finally the mature eightnucleate embryo sac (14th day; Fig. 5H). The mature embryo sac was Polygonum-type, consisting of one egg cell and two helper cells at the micropylar end, two polar nuclei of the central cells and three antipodal cells at the chalazal end. In staminate flowers, ovule development stopped upon the formation of the functional megaspore (Fig. 5I). The boundaries of the ovule gradually blurred; ultimately, only the aborted ovule remained (Fig. 5J and K), although the ovary wall continued to swell (Fig. 5L).
4. Discussion Sex differentiation in plants can occur at any developmental stage. Two categories of unisexual flowers have been recognized according to the timing difference of sex differentiation (Mitchell and Diggle, 2005). Organ abortion can occur in any of four stages: stage 0, before the differentiation of stamen and carpel primordia; stage 1, early development of stamens and pistils; stage 2, prophase of meiosis; and stage 3, after meiosis (Diggle et al., 2011). Unisexual flowers are classified into type I, which are bisexual at initiation and become unisexual by termination of male or female sexual organ at some developmental stage, and type II, in which flowers become unisexual very early, before initiation of stamens and carpels. Diggle et al. (2011) surveyed 292 taxa with unisexual flowers and found sex organ abortion with equal frequency at each of the four stages in both male and female flowers, and in monoecious and dioecious taxa. Although all combinations of
Y.-B. Luo et al. / Flora 233 (2017) 179–185
183
Fig. 4. The development of anthers and pollen in A. elegantulum. A–C, the common developmental process in both staminate and hermaphrodite flowers; D–F, the developmental process of anthers and pollen in hermaphrodite flowers; G–I, the developmental process of anthers and pollen in staminate flowers. A, tetrahedral tetrads forming; B, microspores are released from the tetrads; C, microspores absorb the nutrients from the tapetum; D, late uninucleate stage; E, development of two-celled pollen; F, mature anthers of hermaphrodite flowers; G, development of two-celled pollen; H, mature anthers of staminate flowers; I, anthers dehiscing. Mmc, Microspore mother cell; Ms, microspore; Ta, Tapetum; Gc, Generative cell; Vc, Vegetative cell; St, Stomium; Fl, Fibrous layer; Pg, pollen grains. Scale bars = 20 m.
184
Y.-B. Luo et al. / Flora 233 (2017) 179–185
Fig. 5. The development of ovaries and ovules in A. elegantulum. A, longitudinal section of the normal ovary during early development stage in both staminate and hermaphrodite flowers; B–H, development of ovaries and ovules in hermaphrodite flower. B, integuments arose from the ovule primordium, and the megaspore mother cells formed; C, linear mega-spore tetrads formed as result of meiosis of the megaspore mother cells; D, functional megaspore; E, F, bi-nucleate embryo sac; G, tetra-nucleate embryo sac; H, eight-nucleate embryo sac; I–L, the process of ovule abortion in staminate flower. I, functional megaspore; J, boundaries of the ovule gradually blurred; K, abortive ovule; L, abortive ovary. Op, ovule primordium; Mmc, megaspore mother cell. Mt, megaspore tetrad; Fm, functional megaspore; Bes: bi-nucleate embryo sac; Tes: tetra-nucleate embryo sac; Sy, synergid; Pn, polar nucleus; Ec, egg cell; Art, antipodal cell. Scale bars = 10 m for A–J and 20 m for K and L.
stages of androecium and gynoecium abortion are seen, taxa in which the androecium and gynoecium abort at the same stage are over-represented (Diggle et al., 2011; Fig. S2). More recent examples (Table S1) yield similar results (Fig. S1), and in A. elegantulum, both androecium and gynoecium abort at stage 3. The pistil primordium of staminate flowers was selectively aborted at the phase of functional megaspore formation. The anthers of hermaphrodite flowers did not dehisce, resulting in functional female flowers. Such late and concurrent of androecium and gynoecium abortion in A. elegantulum suggests that the species may have evolved monoecy recently, from andromonoecy. If A. elegantulum is animal-pollinated, the retention of well-developed, yet
indehiscent, anthers could attract pollinators, ensuring reproductive success (Podolsky, 1992). Monoecy may reduce interference between male and female functions (dispersing pollen vs. accepting pollen) and avoid self-pollination (Barrett et al., 2000). After the anthers in staminate flowers of A. elegantulum mature, the fibrous layer cells dehydrate, and secondary thickening occurs in the inner wall, but not the outer tangential wall of the fibrous layer, which causes the anther wall to wrinkle. The mechanical forces this induces lead to anther dehiscence of staminate flowers. Anther dehiscence in other plants is induced by programmed cell death that leads to degeneration of the inner wall and connective tissues of the anther, the dehydration of cells, and the outward
Y.-B. Luo et al. / Flora 233 (2017) 179–185
bending of the anthers (Sanders et al., 1999; Varnier et al., 2005; Senatore et al., 2009; Parish and Li, 2010). When programmed cell death does not occur normally, or secondary thickening fails to develop in the inner wall of the anthers, dehiscence fails (Sanders et al., 1999; Parish and Li, 2010; Mizuno et al., 2007; Thvenin et al., 2011). In Acer, incompletely dissolved tapetum or a lack of lip cell structure (Xu et al., 2012; Zhang et al., 2011) and a lack of significant lignification can also cause anther dehiscence failure (Hu et al., 2009). In A. elegantulum, the tapetum was completely dissolved in both staminate and hermaphrodite flowers, and significant lignification was not observed, so these factors did not result in anther dehiscence failure in the hermaphrodite flowers. In the anthers of A. elegantulum hermaphrodite flowers, anther development is mostly similar to that in staminate flowers, but normal programmed cell death does not occur in the inner walls of the anthers and tapetum, and the anthers fail to dehisce. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.flora.2017.06. 006. References Ashman, T.L., 2006. The evolution of separate sexes: a focus on the ecological context. In: Ecology & Evolution of Flowers. Oxford University Press, Oxford, pp. 204–222. Barrett, S.C.H., Jesson, L.K., Baker, A.M., 2000. The evolution and function of stylar polymorphisms in flowering plants. Ann. Bot. 85 (3), 253–265. Barrett, S.C.H., 1998. The evolution of mating strategies in flowering plants. Trends Plant Sci. 3 (9), 335–341. Barrett, S.C.H., 2002. The evolution of plant sexual diversity. Nat. Rev. Genet. 3 (4), 274–284. Barrett, S.C.H., 2003. Mating strategies in flowering plants: the outcrossing–selfing paradigm and beyond. Philos. Trans. R. Soc. B Biol. Sci. 358 (1434), 991–1004. Diggle, P.K., Stilio, V.S.D., Gschwend, A.R., Golenberg, E.M., Moore, R.C., Russell, J.R., Sinclair, J.P., 2011. Multiple developmental processes underlie sex differentiation in angiosperms. Trends Genet. 27 (9), 368–376. Dufay, M., Champelovier, P., Käfer, J., Henry, J.P., Mousset, S., Marais, G.A.B., 2014. An angiosperm-wide analysis of the gynodioecy-dioecy pathway. Ann. Bot. 114 (3), 539–548. Gabriela, G., Miguel, V., 2005. Repeated evolution of dioecy from androdioecy in Acer. New Phytol. 165 (2), 633–640. Hu, Q., Li, F.L., Du, Y.L., Guo, H.H., 2009. Morphological and cytological study on floral sex differentiation of Acer truncatum. Bull. Bot. Res. 29 (02), 136–140. Jong, P.C.D., 1976. Flowering and Sex Expression in Acer L. A Biosystematic Study., pp. 76. Mitchell, C.H., Diggle, P.K., 2005. Evolution of unisexual flowers: morphological and functional convergence results from diverse developmental transitions. Am. J. Bot. 92 (7), 1068–1076. Mizuno, S., Osakabe, Y., Maruyama, K., Ito, T., Osakabe, K., Sato, T., Shinozaki, K., Yamaguchi-Shinozaki, Kazuko, 2007. Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana. Plant J. 50 (5), 751–766.
185
Parish, R.W., Li, S.F., 2010. Death of a tapetum: a programme of developmental altruism. Plant Sci. 178 (2), 73–89. Podolsky, R.D., 1992. Strange floral attractors: pollinator attraction and the evolution of plant sexual systems. Science 258 (5083), 791–793. Renner, S.S., Ricklefs, R.E., 1995. Dioecy and its correlates in the flowering plants. Am. J. Bot. 82 (5), 596–606. Renner, S.S., Beenken, L., Grimm, G.W., Kocyan, A., Ricklefs, R.E., 2007. The evolution of dioecy, heterodichogamy, and labile sex expression in Acer. Evolution 61 (11), 2701–2719. Renner, S.S., 2014. The relative and absolute frequencies of angiosperm sexual systems: dioecy, monoecy, gynodioecy, and an updated online database. Am. J. Bot. 101 (10), 1588–1596. Rubén, T., Marcos, M., 2011. Where do monomorphic sexual systems fit in the evolution of dioecy? Insights from the largest family of angiosperms. New Phytol. 190 (1), 234–248. Sanders, P.M., Bui, A.Q., Weterings, K., McIntire, K.N., Hsu, Y.C., Lee, P.Y., Truong, M.T., Beals, T.P., Goldberg, R.B., 1999. Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex. Plant Reprod. 11 (6), 297–322. Sato, T., 2002. Phenology of sex expression and gender variation in a heterodichogamous maple, Acer japonicum. Ecology 83 (5), 1226–1238. Senatore, A., Trobacher, C.P., Greenwood, J.S., 2009. Ricinosomes predict programmed cell death leading to anther dehiscence in tomato. Plant Physiol. 149 (2), 775–790. Shang, H., Luo, Y.B., Bai, W.N., 2012. Influence of asymmetrical mating patterns and male reproductive success on the maintenance of sexual polymorphism in Acer pictum subsp. mono (Aceraceae). Mol. Ecol. 21 (15), 3869–3878. Spigler, R.B., Ashman, T.L., 2012. Gynodioecy to dioecy: are we there yet? Ann. Bot. 109 (3), 531–543. Sun, G., Dilcher, D.L., Zheng, S., Zhou, Z., 1998. In search of the first flower: a urassic angiosperm, archaefructus, from northeast china. Science 282 (5394), 1692–1695. Thvenin, J., Pollet, B., Letarnec, B., Saulnier, L., Gissot, L., Alessandra, Maia-Grondard, Lapierre, C., Jouanin, L., 2011. The simultaneous repression of CCR and CAD, two enzymes of the lignin biosynthetic pathway, results in sterility and dwarfism in Arabidopsis thaliana. Molecular Plant 4, 70–82. Varnier, A.L., Mazeyrat-Gourbeyr, e., Florenc, e., Sangwan, R.S., Clément, C., 2005. Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation. J. Struct. Biol. 152, 118–128. Webb, C.J., 1999. Empirical Studies: Evolution and Maintenance of Dimorphic Breeding Systems. Springer, Berlin, Heidelberg. Xu, T.Z., Chen, Y.S., de Jong, P.C., Oterdoom, H.J., Chang, C.S., 2008. Flora of China, vol. 11. Science Press and Missouri Botanical Garden Press, Beijing and St. Louis, pp. 515–533. Xu, X.L., Jin, H.X., Chen, C.B., Tian, Q., Zhu, M.L., Chen, X.Y., 2012. Studies on the formation of microspores and development of male gametophyte in Acer yanjuechi (Aceraceae). Plant Divers. Resour. 34 (04), 339–346. Xu, Y.C., Zhou, L.H., Hu, S.Q., Hao, R.M., Huang, C.J., Zhao, H.B., 2014. The differentiation and development of pistils of hermaphrodites and pistillodes of males in androdioecious Osmanthus fragrans L. and implications for the evolution to androdioecy. Plant Syst. Evol. 300 (5), 843–849. Xu, T.Z., 1996. Phytogeography of the family Aceraceae. Acta Bot. Yunnanica 18 (01), 43–50. Xu, T.Z., 1998. The systematic evolution and distribution of the genus Acer. Acta Bot. Yunnanica 20 (04), 383–393. Zhang, L.Z., Shang, H., Luo, Y.B., Cheng, X., Bai, W.N., 2011. Morphology and cytology of three flower phenotypes in a duodichogamous tree species, Acer mono. Biodivers. Sc. 19 (05), 551–557.