Sex‐specific floral morphology, biomass, and phytohormones associated with altitude in dioecious Populus cathayana populations

Abstract Relationships between sex‐specific floral traits and endogenous phytohormones associated with altitude are unknown particularly in dioecious trees. We thus examined the relationships between floral morphology or biomass and phytohormones in male and female flowers of dioecious Populus cathayana populations along an altitudinal gradient (1,500, 1,600, and 1,700 m above sea level) in the Xiaowutai Nature Reserve in northern China. The female and male flowers had the most stigma and pollen at 1,700 m, the largest ovaries and least pollen at 1,500 m, and the smallest ovaries and greater numbers of anthers at 1,600 m altitude. The single‐flower biomass was significantly greater in males than in females at 1,600 or 1,700 m, but the opposite was true at 1,500 m altitude. The biomass percentages were significantly higher in anthers than in stigmas at each altitude, while significantly greater gibberellin A3 (GA 3), zeatin riboside (ZR), indoleacetic acid (IAA), and abscisic acid (ABA) concentrations were found in female than in male flowers. Moreover, most flower morphological traits positively correlated with IAA in females but not in males. The biomass of a single flower was significantly positively correlated with ABA or IAA in males but negatively with ZR in females and was not correlated with GA 3 in both females and males. Our results demonstrate a distinct sexual adaptation between male and female flowers and that phytohormones are closely related to the size, shape, and biomass allocation in the pollination or fertilization organs of dioecious plants, although with variations in altitude.

Altitude is an important abiotic factor associated with temperature, precipitation, light, and soil physicochemical properties (Körner, 2007), and studies have shown that floral characteristics are significantly affected by altitude (Bodson & Outlaw, 1985;Nagano et al., 2014). For instance, Duan, He, and Liu (2005) demonstrated that the floral display in Gentiana straminea increased with increasing altitude, and Baonza and Malo (1997) found that the floral size of Cytisus scoparius showed a clinal variation with larger flowers at higher altitudes ranging from 700 m to 1,500 m. Kudo and Molau (1999) observed that the floral size (as well as anthesis) in Astragalus alpinus was significantly greater in higher population. In contrast, Nagano et al. (2014) found that the floral size of Campanula punctata var. hondoensis decreased with increasing altitude. These inconsistent variations in the floral characteristics of different species at different altitudes may be related to changes in biomass allocation (Li, Xu, Zang, Korpelainen, & Berninger, 2007;Pickering, 2000;Zhao, Du, Zhou, Wang, & Ren, 2006) as plants may allocate more carbon to reproductive organs (i.e., the flower; Fabbro & Körner, 2004;Hautier, Randin, Stöcklin, & Guisan, 2009) or to flower physiological traits at higher altitudes (Chandler, 2011;van Doorn & van Meeteren, 2003).
Populus cathayana Rehd., a dioecious woody tree, is widely distributed in northern, central, and southwestern China, including mountainous areas at altitudes from 1,000 to 3,000 m above sea level (a.s.l.). Our previous studies addressed the different growth and floral performance responses to elevated temperatures and UV-B radiation (Xu et al., 2008(Xu et al., , 2010 and relationships among twig components between male and female P. cathayana saplings (Yang, He, Xu, & Yang, 2015). To further address whether sexual differences in the floral traits of P. cathayana could vary with altitude, this study aimed to determine (1) how sex-related differences in the morphology, biomass, and phytohormones of flowers could respond to altitude changes and

| Study site
The study site is located in the Xijin River Valley of Xiaowutai Mountain Nature Reserve in Hebei, China (39°50′-40°07′N, 114°47′-115°30′E; 1,142-2,882 m a.s.l.). This site area is characterized by a warm-temperate continental monsoon climate with mean annual precipitation of 528 mm and a mean annual temperature of 3.5°C. The major soil types are Alfisols, Aridisols, and Inceptisols (USDA soil taxonomy). There are five distinct forest zones along the western slope of Xiaowutai Mountain: the deciduous shrub zone, the deciduous broad-leaved forest zone, the mixed coniferous and broadleaved forest zone, the conifer forest zone, and the subalpine meadow zone (Liu, Zheng, & Fang, 2004). The forest vegetation is dominated by species in the Acer, Birch, Cerasus, Corylus, Quercus, Populus, Tilia, or Ulmus genus. The natural secondary P. cathayana population is generally distributed throughout the deciduous broad-leaved forest zone (1,400-1,800 m a.s.l.), but it has been gradually replaced by Betula platyphylla above 1,700 m a.s.l. (Yu, Liu, & Cui, 2002).

| Measurements of floral morphological traits
The inflorescence length was measured with a micrometer, and the number of flowers per inflorescence was then counted before the flowers were removed from the inflorescence. Five randomly selected male or female flowers (one middle, two terminal, and two basolateral flowers) per inflorescence were then dissected with the aid of a stereoscope (Leica, M205C; Leica Microsystems, Wetzlar, Germany), and the pedicel, sepal, floral disk, anther, and filament (or pedicel sepal, floral disk, stigma, and ovary) of each flower were then dissected under a stereoscopic microscope equipped with a charge-coupled device (CCD) camera (MoticamPro285A; Motic, Xiamen, China). The number of anthers per flower, pollen grains per anther, and ovules per ovary was recorded, and the sizes (length, width, or diameter) of the individual parts (pedicel, sepal, ovary, and stigma) were measured to the nearest 0.01 mm using an ocular reticle.
To calculate the number of pollen grains, 50 randomly selected undehisced anthers (one anther per flower) were soaked in 1.0 mol HCl solution for 1 hr at 60°C to dispose of the anther wall, and 10.0 ml 0.9% NaCl solution was added after grinding (method modified from Guo, Wang, and Weber (2013)). A 2.0μl suspension was plated on a hemocytometer (with a blood-cell counting chamber with 400 small, square grids in a central 1.0-mm square), and the pollen grains per anther was calculated. After dissecting the ovary on a slide, ovules were counted under the above-mentioned stereoscopic microscope equipped with a CCD camera.

| Measurement of flower biomass traits
The biomass production of the male and female flowers measured for their morphological traits was recorded. The samples were oven-dried at 70°C for 48 hr to a constant weight. The biomass of the individual anther, stigma, or flower was then determined, and the weight of the anthers or stigma per single flower was accordingly calculated as a percentage.

| Phytohormone measurements
The five male or female inflorescences measured for their morphological traits were also used to measure the concentrations of ABA,

| Statistical analysis
Data (means ± SE, n = 5) analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA was used to determine differences in the flower morphological traits among altitudes, and Duncan's multiple range tests were employed to detect significant differences among means at p ≤ .05. Two-way ANOVAs were used to separate the effects of sex, altitude, and their combination. Pearson's correlation coefficients were calculated to determine the relationships between the biomass and phytohormone concentrations of male or female flowers, and a simple linear regression was used to examine these relationships.

| Variations in the morphological traits of female and male flowers
Almost all tested flower morphological traits were significantly affected by altitude (Table 1, Figure 1). Among the female flowers, the number of flowers per inflorescence and stigma width increased with altitude elevation, while flowers at 1,600 m had the lowest values for inflorescence length, pedicel length, sepal size, ovary diameter, and number of ovules per ovary compared to their counterparts at other two altitudes (Table 1)

Male
Inflorescence length (  α -.005** -Different letters following the data (means ± SE, n = 5) in the same row denote significant differences between altitudes among females (a, b, c) and males (α, β, γ) or between sexes at the same altitude ( values for these traits (Table 1). However, no significant effects of altitude were observed on the number of male flowers per inflorescence (p = .17). In addition, compared with other altitudes, male plants at 1,600 m had the shortest inflorescence length, the longest pedicel, and the greatest number of anthers per flower (Table 1). Moreover, compared with the females, male flowers had a significantly larger sepal size at the same altitude, a longer pedicel length at 1,600 or 1,700 m, and a higher number of flowers per inflorescence at 1,500 m (p < .05; Table 1).

| Variations in single-flower biomass and its allocation in the two sexes
Significantly greater single-flower biomass was observed among altitudes in the order of 1,500 m > 1,700 m ≈ 1,600 m for the female flowers, whereas the order was 1,700 m ≈ 1,600 m > 1,500 m for the male flowers ( Figure 2a). Moreover, males had greater singleflower biomass than females at altitude of 1,600 or 1,700 m but less biomass at 1,500 (p < .05). In addition, a significantly greater stigma biomass percentage was observed among altitudes in the order of 1,600 m > 1,700 m > 1,500 m, whereas the biomass percentage of the anthers was similar among altitudes ( Figure 2b).
The anther biomass percentage was always significantly higher than the stigma biomass percentage at all altitudes (p < .001; Figure 2b).

| Relationships between morphological traits and phytohormone concentrations in the two sexes
In female flowers, significantly positive correlations were observed between ABA concentrations and the sepal size or the number of ovules per ovary as well as between IAA concentrations and the pedicel length, sepal size, ovary length, ovary diameter, or the number of ovules per ovary (bold values in Table 2). In contrast, significantly negative correlations were exhibited between ZR concentrations and the pedicel length or ovary length, and no correlations were observed between GA 3 and any of the female flower trait examined (Table 2). In male flowers, significantly positive correlations were found between ABA concentrations and sepal size, between GA 3 concentrations and pedicel length, and between ZR concentrations and the pedicel length or number of anthers per flower (bold values in Table 3). However, no correlations were observed between IAA and any of the tested female flower traits (Table 3). In addition, GA 3 significantly positively correlated with ZR in both female and male flowers (Tables 2 and 3). In female flowers, ABA significantly positively correlated with IAA (Table 2) but significantly negatively correlated with GA 3 or ZR in male flowers (Table 3).

| Relationships between flower biomass and phytohormone concentrations in the two sexes
The biomass of a single flower was significantly positively related to concentrations of ABA and IAA in male but not in female flowers (p < .01; Figure 4a or c), while was negatively related to ZR concentrations in female but not in male flowers (p < .05; Figure 4d). In addition, no relationships were observed between the biomass of a single

| DISCUSSION
In the present study, the variations in floral morphology between male and female P. cathayana plants were quite different along an altitudinal gradient (Tables 1 and 2, Figure 1). For example, the number of flowers per P. cathayana inflorescence significantly increased with increasing altitude in female but not in male plants. The longest inflorescences were observed on female plants at an altitude of 1,700 m but in male plants at 1,500 m, while the pedicel and sepal sizes were largest in female but smallest in male plants at 1,500 m. These results indicated that there were no consistent responses to an increase in altitude in floral morphology between sexes (Table 1, Figure 1). As a general rule, the sexual dimorphism in floral display is a consequence of selection for sex-specific optimal strategies and resource allocation (Delph, 1990;Obeso, 2002;Pickering & Hill, 2002). We assume that a longer inflorescence and more flowers would be beneficial to high- biomass percentage at all three altitudes (Figure 2). These results suggested that male P. cathayana plants at a higher altitude invest more mass in flower and allocate more biomass to the anthers than the females, and these may be beneficial to enhance pollen dispersal.
Also compared with females as a wind-pollinated plant, most and/ or more male P. cathayana plants are distributed at higher-altitude sites (Wang, Xu, Li, Yang, & Yuan, 2011), which would benefit their exposure to higher wind speeds and longer pollen dispersal distances (Hesse & Pannell, 2011;Van Drunen & Dorken, 2014). Consequently, maintaining an adequate quantity of pollen could improve the rate of successful fertilization. Similar results from Dombeya ciliata showed that the flower size was larger in males than in females at a higher altitude (Humeau et al., 2000). On the other hand, the female plants  (Knight, 2003;Oleques & de Avila, 2014), and plants in high-density populations have fewer ovules per flower and smaller inflorescences (Weber & Kolb, 2011  These results suggested that intrinsic relationships between floral morphology and endogenous phytohormones could lead to sex-specific morphological changes in response to altitude. As low-molecular mass-signaling substances in plants, phytohormones (e.g., ABA, GA 3 , IAA, and ZR) are known to function in intercellular regulation in multicelled organisms (Sonnewald, 2013). Studies have reported that the levels of these endogenous hormones in flower tissues vary during flowering (Chen, Du, Zhao, & Zhou, 1996;Villacorta et al., 2008) and are related to the initiation and development of floral organs (Arrom & Munné-Bosch, 2012;Meilan, 1997). Consistent with these findings, morphological traits were closely related to phytohormone contents in both male and female flowers (e.g., between sepal size and ABA or between pedicel length and ZR, see Tables 2 and 3