Dioecy is found in 175 flowering plant families and in 7% of flowering plant genera (Renner, 2014). Classic sex ratio theory suggests that when reproductive costs to produce a female vs. a male offspring are equal, natural selection will act to balance the sex ratio of the population (Fisher, 1930). However, numerous investigations have revealed that the population sex ratios of many dioecious plants are biased (Barrett et al., 2010; Sinclair et al., 2012; Munné-Bosch, 2015). Barrett et al. (2010) reported that most plant species exhibit equal or male-biased sex ratios, whereas female-biased sex ratios occur less frequently. Many researchers speculate that this phenomenon is most likely to be associated with the fundamental biological processes of gender dimorphism (Tognetti, 2012). However, we cannot ignore the fact that females have a higher resource requirement for reproduction than males. Obeso (2002) suggested that females invest more resources in reproduction, which would leave them with a smaller amount of resources available for defense. Fujita et al. (2014) suggested that if plants in phosphorus-limited communities invest little in sexual reproduction, plants would be in danger. In addition, sex-specific physiological responses, such as nitrogen uptake and use, would have a great effect on plant stress tolerance. For example, Juniperus thurifera females may use a long-term strategy by increasing N storage to compensate for massive reproductive masting events, whereas males prefer to use current nutrients for promoting gas exchange capacity (Montesinos et al., 2012). If plants grow in nutrient-poor conditions, especially under N shortage, the contrasting responses of males and females may have a direct impact on plant survival.

In Populus, a genus with male-biased sex ratios, females usually experience greater negative effects than do males under environmental stresses. To interpret this important case, a series of studies were carried out. The results suggested that different-sex individuals display distinct morphological and physiological adaptations to environmental stresses (Juvany and Munné-Bosch, 2015). For example, in a water-limited scenario, drought stress could limit poplars' photosynthetic capacity more in females than in males (Xu et al., 2008a). In addition, when the two sexes grow in a combined stress condition, such as elevated temperature (Xu et al., 2008b) or salinity (Chen et al., 2010), some sex-specific reactions will differ from single-stress responses. For instance, the combination of drought and salinity induced greater sex-specific differences in the levels of total chlorophyll, carotenoid, H₂O₂ and Cl⁻ in the leaves than did single stresses (Zhang et al., 2011). Furthermore, when poplars were subjected to low-temperature conditions, males exhibited improved chloroplast structure and more intact plasma membranes than did females, suggesting a better self-protective capacity (Zhang et al., 2011). Populus and Salix, belonging to Salicaceae, display different biased sex ratios that female-biased sex ratios are of approximately 2:1 in many willow species (Alliende and Harper, 1989; Dudley, 2006; Myers-Smith and Hik, 2012). Do female willows have a greater tolerance than males and thus survive multiply stressful conditions? So far, few studies have been performed to confirm the sex-specific mechanisms that produced female-biased sex ratios of willows in nature from an ecophysiological perspective (Jones et al., 1999; Dudley, 2006; Randriamanana et al., 2015). Moreover, previous studies on sexual differences in plant growth were merely based on a single time point, which may miss important information and could not reflect the temporal variation because sexes differ in their timing of development (Glynn et al., 2007; Hultine et al., 2013).

In this study, we employed Salix paraplesia individuals, which were grown with high and low soil fertility for about 140 days over one growing season. Briefly, our study monitored plants' growth parameters at three important time points, which respectively represent the initial-growth stage, rapid-growth stage and late-growth stage according to our observations for many years. The experiment was designed not only for research on how S. paraplesia males and females respond to nutrient limitation but also to provide support for research on willows' tolerance to low nutrients in nature. The latter is important because sporadic desertification has been observed in many of their natural habitats. Temporal variation in the relative growth rate (RGR), net assimilation rate (NAR), stable isotope compositions, non-structural carbohydrates (NSCs), and condensed tannins (CTs) were measured. It is hypothesized that there is a gender-specific trade-off between growth and defense, whereby females have a stronger capacity to overdraw on available resources to satisfy growth requirements (for example, greater biomass accumulation and higher RGR) or defense (for example, higher CT concentrations) than males under nutrient-poor conditions.

Male and female plants play different roles in productive biology and thus have sexually different resource demands imposed upon them. Any decision about resource utilization made by males or females may have different consequences, even contributing to spatial segregation of the sexes (Fujita et al., 2014). Many studies have implied that the population sex ratio of certain species would become female-biased if dioecious plants were placed under high-quality environments. The same reports maintain that the sex ratio would become male biased under stressful or resource-poor habitats, because reproduction is very costly for females (Sanchez-Vilas et al., 2012). However, there are exceptions. Salix often exhibits female-biased population sex ratios in naturally stressful environments (Dudley, 2006; Myers-Smith and Hik, 2012). Thus, we have attempted to find possible explanations that gender-specific trade-offs between growth and defense may be relevant to willow's female-biased sex ratios. In this study, S. paraplesia male and female cuttings were employed. A critical subject is posed: once plants suffer from nutrient limitation, how do males and females make their own choices: growing fast enough to compete or maintaining physiological adaptations necessary for survival?

Estimating growth parameters can help to elucidate whether plants suit the current environment or not. In the present study, female cuttings are likely to accumulate greater biomass than males under either nutrient-rich or nutrient-poor conditions. Statistical analyses have indicated that both sex and nutrients are important factors. Hermans et al. (2006) indicated that plants often allocate biomass to the root system when mineral elements are scarce. However, in our study, nutrient limitation significantly decreased biomass allocation below ground (see RWR) in males, while few effects were observed in females compared with those under nutrient-rich conditions during the initial-growth stage. This suggests that nutrient-limitation did not cause severe effects on the root growth of female cuttings but rather was fatal to males. This may ultimately reduce male cuttings' survival rate and might explain the segregation of the sexes. The largest value of RWR was observed in males under nutrient-poor conditions during the late-growth stage, suggesting that males indeed sensed and experienced greater environmental stress than did females. In other words, nutrient limitation may cause more severe impacts on plant growth in males than in females. In addition to RWR, nutrient limitation increased resource output to the stem in both sexes, and females constantly had higher SWR than males. S. paraplesia females gave priority to vegetative investment at the cost of stem growth during the initial- and late-growth stages, which may have enhanced stem functions including water transport and mechanical stability (Albrectsen et al., 2004; Taneda and Tateno, 2004). Relatively higher levels of nutrient storage in the female cuttings used for propagation may partially explain why female cuttings exhibited stronger capacity than male cuttings during the initial-growth stage.

In addition to plant growth, two compounds' NSC and CTs were investigated. NSC stores are assumed to be an important trait for plant survival under stress (O'Brien et al., 2014), particularly in maintaining basic metabolic functions to optimize growth and defense (Dietze et al., 2014). In general, most carbohydrates are produced in foliage leaves, and some are synthesized in flowers and fruits (Kozlowski, 1992). However, trees in the juvenile stage of development do not flower and, therefore, only have vegetative tissues as carbohydrate sinks. In our study, we analyzed the total NSC contents of entire cuttings (Figure 1). As expected, low soil fertility increased plants' NSC content, conducive to enhancing plant tolerance capacity (Chapin et al., 1990). Interestingly, females had higher NSC levels than males, and statistical analyses indicated a role for sex. This suggests that female cuttings with greater NSC storage were more likely to survive nutrient-poor conditions (Canham et al., 1999). In addition, there was a positive relationship between total plant mass and NSC, suggesting a good balance between photosynthesis and respiration, which ultimately influences carbon availability for growth (Chapin et al., 1990; Dietze et al., 2014).

Recent advances in plant metabolism studies indicate that plants are dependent on the deployment of secondary metabolites for their response to abiotic and biotic stresses (Mumm and Hilker, 2006; Neilson et al., 2013), and a variety of secondary compounds are produced from carbohydrates, amino acids and lipids. For example, Populus tremuloides seedlings increased CT concentrations under conditions of low fertility with competition (Donaldson et al., 2006). In the present study, comparative analysis showed that females produced more CTs than males under nutrient-poor conditions, while producing fewer CTs than males when plants are subjected to nutrient-rich conditions during the initial-growth stage. This suggests that females are likely more sensitive than males to nutrient availability, with regard to chemical defense. Our studies indicate that CTs may play a different role, sexually, in the initial- and late-growth stages rather than the rapid-growth stage (Table 6), which could provide females with greater defense capacity than enjoyed by males. In contrast, CTs are traditionally thought to play a key role in plant defense against herbivorous insects (Agrawal, 2007; Gols, 2014; Agrawal and Weber, 2015). In our study, temporal variation and sex-based differences in the regulatory strategies of CTs were observed in males and females. This may suggest sex-based differences between willows and herbivorous insects and sex-based differences in competition- and resource-mediated trade-offs between growth and defense (Donaldson et al., 2006).

According to the growth-differentiation balance hypothesis (Herms and Mattson, 1992; Glynn et al., 2007), if levels of defense remain stable while resources decrease, fewer resources will be available for growth or reproduction. Stamp (2003) suggested that resources allocated to the synthesis, storage, and regulation of plant secondary metabolites would come at the expense of those allocated for growth and reproduction. In accordance with our hypotheses, the results presented here suggest sex-specific trade-offs between the growth and defense of willows facing nutrient limitation. In the initial-growth stage, male and female strategies when confronting challenges related to nutrient availability differ substantially. Under nutrient-rich conditions, males invested more resources in defense than females (as reflected in the synthesis of more condensed tannins than females, see Table 6), which could be defined as a conservative adaptive strategy. This could impact male cuttings' growth (Table 4). Although high resource availability may diminish allocation costs and allow for growth and defense (Siemens et al., 2002), once plants face limited resources, females' positive regulatory mechanisms seem to be more efficient than those of males. It was found that females synthesized more CTs to enhance defense; levels were greater than those found in males (Table 6). As predicted, nutrient limitation significantly decreased plants' RGR and total plant mass accumulation, but females still had higher RGR and greater total plant mass than males (Table 4). Most explanations of plant stress tolerance indicate an evolutionary trade-off such that plant species with high chemical defense would lower their RGR to maintain relative equilibrium (Herms and Mattson, 1992; Stamp, 2003; Fine et al., 2006; Glynn et al., 2007). However, in our study, females were likely to adopt radical strategies, overdrawing on available resources to satisfy both growth and defense, suggesting sex-based strategy differences. The results also indicated that females may have an extra mechanism to compensate for the investment in growth under nutrient-poor conditions. For example, female Salix may have higher photosynthetic rate than males, or increase allocation to photosynthetic organs, which would enable them to compensate for their higher resource demands (Dudley and Galen, 2007; Ueno et al., 2007). Similar relationships between RGR and CTs were observed when males and females were in the late-growth stage. These results suggest that such aggressive mechanisms could help female individuals win from the start when willows face intersexual competition for limited resources. In the rapid-growth stage, both males and females maintained a high level of growth, and CT levels were significantly decreased when compared with those measured in the initial-growth stage, suggesting an obvious trade-off between growth and defense (Stamp, 2003). The total biomass of females was found to be significantly higher than that of males under nutrient-rich conditions, but nutrient shortages could alter this relationship (the differences between sexes were not substantial). Previous studies have suggested that biomass allocation plays a key role in the plant response to nutrient shortage (Hermans et al., 2006; Agrawal, 2007), and we speculate that different strategies for biomass allocation may result in sex-based differences in defense capacity and trade-offs between growth and defense.

Recent studies have suggested various inter-sexual competition patterns in a male-biased dioecious plant Populus with a sex ratio, in terms of resource utilization within given environments (Chen et al., 2014, 2015). Our study further provided new insights into S. paraplesia, a female-biased sex ratio dioecious plant, in response to nutrient availability. In particular, we demonstrated sex-specific trade-offs between growth and defense. Females are likely to adopt radical strategies, overdrawing on available resources to satisfy both growth and defense, which seems to be more like a gamble compared to the strategies pursued by males. Females may have an extra mechanism to compensate for the investment in growth under nutrient-poor conditions.

HJ designed the experiment and measured the dynamic changes of plant biomass; he was also responsible for the statistical analysis and manuscript writing. SZ contributed largely to language improvement, and provided much useful suggestions and comments for the revision. YL and GX carried out much of the field work and chemical analyses. DZ had the initial research idea and was also doing some of the writing of the paper during initial submission and revisions.