The impacts of flowering phenology on the reproductive success of the narrow endemic Nouelia insignis Franch. (Asteraceae)

Abstract Nouelia insignis Franch. (Asteraceae) is a short, narrow endemic and endangered tree, growing with a natural population in the dry and hot valley of the Jinsha River in the southwest area of China. In this work, flowering phenology (time and duration), floral biology, visit frequency and behavior of pollinators, and pollination characteristics were studied based on investigation in the field and analysis in the laboratory with the help of a stereomicroscope, and the relationship between seed setting rate and reproductive traits, as well as the relationship between flowering time and rainfall before flowering, was tested using the method of general linear regression model. The results showed that natural population of N. insignis exhibited high flowering synchrony with relatively stable flowering duration, and the flowering time fluctuated greatly depending on the rainfall 5 months before flowering. The pollination of N. insignis required pollinators, and insect activities played a very important role in the pollination process. However, lack of the pollinators was not a limitation for reproductive fitness in N. insignis, although the number of pollinators was small and the frequency of visits was low. In addition, no pollen limitation was found during pollination. The average seed setting rate of N. insignis in the natural condition was only 1.52%–3.73%, and it was generally affected by changes in flowering phenology between years and had a higher seed set in early flowering year. The annual variation of seed set might be related to the annual variations of stamen and pistil functions, such as changes of pollen viability and stigma receptivity, which were closely related to flowering time. The results of this study are of value for further conservation actions on natural population of this threatened endemic plant.


| INTRODUC TI ON
Flowering is an important phase of plant life cycle, which strongly affects plant fitness (Hafdahl & Craig, 2014;Rathcke & Lacey, 1985;Sandring et al., 2007). Flowering phenology at the community level is often affected by several ecological factors, and the period during which flowering produces the maximum seed setting rate may vary from year to year, depending on the availability of resources (Mahoro, 2002;Rathcke & Lacey, 1985). For example, it is possible to observe different flowering phenology patterns for species in dry, harsh, and variable environments because of temporally limited water resources, and reproductive success usually depends on the time of onset of flowering (Cortés-Flores et al., 2017;Tarayre et al., 2007), which can be related to plant strategies to access water sources deep in the soil (Borchert et al., 2004). Flower-visiting insect is another factor affected by flowering time, constituting one of the most important interactions for seed production. This interaction may influence the evolution of flowering time through competition for pollinators, leading to the selection of asynchronous flowering, or promoting pollinators to improve service through synchronous flowering (Cortés-Flores et al., 2017;Rathcke & Lacey, 1985). For example, in many seasonal dry forests, most canopy plants flower simultaneously during droughts (van Schaik et al., 1993), and attracting more pollinators and exposure to pollinators have been proposed to explain this pattern. Furthermore, changes in flowering time and synchronization have been associated with regularity and behavior patterns of insects' visits to flowers, and the activity patterns of insects are usually consistent with the flowering phenology of associated types of plants ( De Jong & Klinkhamer, 1991;Fuchs et al., 2003;Hafdahl & Craig, 2014). Insect-specific flowering plants must rely on the activity of pollinators to complete the ovule fertilization process, and temporal overlap with pollinators is an important factor in the evolution of flowering phenology (Tarasjev, 1997;Totland, 2001). Therefore, changes in the behavior patterns of flower-visiting insects have a particularly serious impact on the reproduction of these plants (Aguirre & Dirzo, 2008). Exploring the relationship between flowering phenology and insect visitors and their behavior, seed production, and some ecological factors, as well as the degree of temporal and spatial variation in these relationships, can provide insight into the selection power that affects the evolution of flowering time.
Several features of the floral biology, such as pollen vitality and stigma receptivity, are particularly important in reproductive success of a population, and they were also affected by flowering time (Clivati et al., 2014;Gao et al., 2004;Hong et al., 2011;Thompson, 2001). Strong evidence has shown that there are significant differences in pollen viability, longevity, and stigma receptivity as well as the encounter periods of pollen and stigma among different flowering periods and habitats (Cui et al., 2008;Liu et al., ;Nebot et al., 2016;Nelizabeth & Sedoniad, 2010;Rymer et al., 2005;Wei & Huang, 2006). However, little information is available on the precise relations between those major components, or particular features as the relationship between pollen viability and stigma receptivity of the plant and flowering time, and the changes in pollen longevity and stigma receptivity through flowering time. Studies of the interaction between these factors are fundamental prerequisites for an understanding of the reproductive constraints which affect a given population.

Nouelia insignis
Franch. is a species of Mutisieae (Asteraceae), and it is characterized by an unusual woody growth form. Nouelia insignis is a small tree with abundant branches, a height of 3-5 m, and a diameter at breast height (DBH) of 10-20 cm ( Figure 1). It grows in dry valleys within 1,000 to 2,800 m a. s. l. in the Jinsha and Nanpan drainage areas in southwestern China (Peng et al., 2003).
Nouelia insignis has become endangered, and most of the populations are seriously threatened. The species suffers from reproductive failure because of low seed productivity and seed germination rates, especially along the Jinsha River drainage. Therefore, very few seedlings could be located in the natural habitats (Peng et al., 2003). According to field observations, all N. insignis populations are fragmented and patchy. These populations are mostly distributed in habitats with steep slopes and poorly developed soil (Gong et al., 2011). Some of populations are even on the brink of extinction. Most N. insignis populations do not exceed 80 individuals, and the total number of individuals in all populations does not exceed 5,000 (Luan et al., 2006). Regeneration failure of endangered N. insignis trees in the dry and hot valleys of southwestern China is an important ecological issue, which is attracting more attention than in the past. Studies have shown that inherent factors (e.g., lower fertility, lower viability, and lower adaptability) of endangered plants are fundamental drivers of their endangered status (Zhang et al., 2002). In recent years, research on the flowering phenology and flowering biology of rare and endangered plants has F I G U R E 1 Nouelia insignis blooming in its natural environment F I G U R E 2 The floral morphological characteristics observed during flowering of Nouelia insignis. a-g: Morphological changes during flowering; h: The anther tube covered with pollen; i: the stigma sliver spread a "Y" type; j-l: Morphological structure of the florets

| Flowering characteristics and phenological records
Nouelia insignis blooms from late February to early April. The capitulum is axillary or terminal on shoots with lengths of 4.1 ± 0.6 (mean ± SD) cm long. The maximum diameter is 1. As the capitulum of the Asteraceae plant is considered to be a single flower, the observation of flowering dynamics is generally carried out for the whole capitulum (Burtt, 1961;Mani & Saravanan, 1999).
According to Burtt (1961) Flowering synchrony was calculated using the method of Primack (1980). The index of synchrony (X) for an individual plant was estimated as: where e j is the number of weeks the flowering periods of individual i and j overlapped; f i is the total flowering period of individual i in weeks, and n is the number of individuals in the sample. X varies from 1 (plant flowering overlaps with that of all other individuals) to 0 (no overlap with any other individuals).

| Floral biology
Floral morphology was observed in the field and also in the laboratory with the help of a stereomicroscope (Leica M80). The pollen viability and stigma receptivity were estimated according to the methods of Dafni (1992) and Nebot et al. (2016). In terms of pollen viability, 12 anthers were randomly selected from different capitula at different developmental stages.
According to the TTC (2, 3, 5-triphenyl tetrazolium chloride) method, the staining of pollen grains was observed under the microscope (×10), and the number of stained pollen grains was counted (×40). The appearance of a red color indicated vitality, and light red, no change, black, or yellow indicated no vitality.
Pollen viability was assessed by red pollen staining rate. Stigma receptivity was checked by the benzidine-hydrogen peroxide method (benzidine: hydrogen peroxide: water = 4:11:22). In the experiment, 12 stigmas were randomly selected from different capitula at different developmental stages and placed on 12 concave glass slides. After dropping a small amount of benzidine-hydrogen peroxide reaction solution, cover the glass slide and observe the receptivity of the stigmas under the optical microscope (×40). The appearance of a blue color and a large number of bubbles indicates receptivity. These insects were then identified by an entomologist. The reproductive success of each pollination treatment was compared in terms of seed setting rate, assessed as the proportion of treated flowers that eventually produced seeds.

| Flowering phenology
The

| Floral biology
All florets were protandrous, and the mature pollen dispersed in the anther canister. The pollen grains had a strong vitality when they were pushed out from the anther canister by stigmata, and the high- In the early stage of the stigmata protruding from the anther canister, the stigmata lobes were unopened, and the stigmata were not receptive at this time. During the second day, the stigma lobes showed a Y-shaped pattern of separation, and the stigma had the greatest receptivity ( Figure 5). This indicated that the best time for pollination was on the second day when the stigmata protruded from the anther canister. From the third day, the receptivity of the stigma declined rapidly until it reached zero. The stigma was keep receptive for 4-5 days, and significant differences were found among different years. Specifically, it lasted for 4 days in 2014, 2015, 2016, and 2020, and 5 days in 2017, 2018, and 2019.

| Seed production
There was a significant difference in seed setting rate under the natural pollination condition across different years (F = 7.534, df = 6, p < .01). The highest seed setting rate was 3.73% in 2018, and the lowest was 1.52% in 2014 (Figure 6), which showed that the seed setting rate might be affected by the environmental changes in different years. The seed setting rates of geitonogamy treatments (GE), xenogamy treatments (XE), and supplementary pollination treatments (SP) were 1.77%, 1.84%, and 1.82%, respectively, which were slightly higher than those of the natural pollination treatments (NP) (1.64%) ( Figure 7). However, no significant differences were found between them (p > .05). Obligated autogamy treatments (OA) and spontaneous autogamy treatments (SA) had less seed formation, with seed setting rates of 0.08% and 0.25%, respectively, which were significantly lower than those of other treatments (p < .05). Significant differences in seed setting rate were observed between obligated autogamy treatments and spontaneous autogamy treatments (p < .05).  F I G U R E 7 Comparison of seed setting rate of Nouelia insignis between different pollination treatments. SA: spontaneous autogamy, in which capitulum buds were bagged with a fine nylon mesh net to exclude insect interactions; OA: obligated autogamy, in which capitulum buds were pollinated with their own pollen and bagged with sulfuric acid paper; GE: geitonogamy, in which capitulum buds were pollinated with pollen of capitulum from the same plant and bagged with sulfuric acid paper; XE: xenogamy, in which capitulum buds were pollinated with pollen of capitulum from the other plant and bagged with sulfuric acid paper; SP: supplementary pollination, in which capitulum buds were pollinated with outcross pollen without bagging; NP: natural pollination (2020), in which capitulum buds were not manipulated.

| Factors influencing seed setting rate and flowering time
The regression analysis ( Figure 10)  The regression analysis showed that there was a significant negative linear relationship between the flowering time and the rainfall 5 months before flowering (p < .05; Figure 11 ). No significant linear relationship was found between the flowering time and the rainfall 1, 2, 3, 4, 6, and 7 months before flowering (p > .05). It was showed that the rainfall of 5 months before flowering might affect the flowering time of N. insignis.

| D ISCUSS I ON
This study demonstrates that there are significant interannual differences in flowering phenology of N. insignis. The natural seed setting "Mass-flowering pattern" or "cornucopia-flowering pattern" is generally considered as an effective adaptation mechanism to resist adverse environments (Herrerías-Diego et al., 2006). Due to the flowering synchrony, each plant can exchange genes with most plants of the population, increasing the genetic diversity of the same population (Augspurger, 1981;Martínez-Sánchez et al., 2011).
We found that this flowering pattern also existed in N. insignis.
Several studies have documented the concentrated flowering patterns of plants helped attract more pollinating insects, which was a kind of reproductive protection to adapt to difficult environment during long-term evolution (Buide et al., 2002;Herrerías-Diego et al., 2006), and people have explained many possible reasons for this phenomenon, from plant characters and environmental factors, such as size, flowering duration, and growing season length (Austen et al., 2017). But no significant pollinator-mediated selection on phenology was detected in some experimental quantified studies (Jiang & Li, 2017). Furthermore, it has been proposed that high seed set was also dependent on high pollinator service, especially for plants that required insect pollination for reproduction (Kudo, 1993;Kudo & Hirao, 2006).
As a matter of fact, there might be significant obstacles to the sexual reproduction process of N. insignis. Under natural conditions, the average seed setting rate of N. insignis in different years was only 1.52%-3.73%. It was obviously different from other Asteraceae plants that usually had a higher seed setting rate (Grombone-Guaratini et al., 2004;Hao et al., 2015;Li & Dang, 2007). In our research, insect activities might play a very important role in the pollination process of N. insignis ( Figure 11). However, we did not detect pollinator limitation in N. insignis, and R. yasumatsui, A. cerana, S. lunata, and C. megacephaia were all effective pollinators. Our study also showed that there was no significant difference in the number and the behavior of flower visitors across years, whether N. insignis bloomed early or late, and no significant relevant relationship was found between visit frequencies of insects and seed setting rate (Figure 9). It suggested that lack of the pollinators was not a limitation for reproductive fitness in N. insignis, although the number of pollinators was small and the frequency of visits was low.
Simultaneously, artificial auxiliary pollination experiments, including experiments of geitonogamy, xenogamy, and supplementary pollination, did not significantly increase the seed setting rate of N. insignis, indicating that pollen limitation was not important factor affecting the reproductive success of N. insignis (Figure 11). This study added the evidence of the relationship between reproductive fitness and insect activity, as well as the relationship between reproductive fitness and pollen limitation.
Floral biology in N. insignis was detected significant differences among different years (Figures 4 and 5), and a significant linear relationship between some parameters of floral biology such as pollen vitality and stigma receptivity and seed setting rate was observed previous studies, in which it was believed that the duration of pollen vitality and stigma receptivity directly affected the seed setting rate (Mani & Saravanan, 1999;Wyatt, 1983). Furthermore, we found that the pollen vitality of N. insignis was always low (41.3% at the highest), and the pollen maintained its vitality for a very short period (only 3-5 days). At the same time, the highest proportion of stigmas with vitality was only 39.7%, and the stigma kept receptivity for 4-5 days. In Asteraceae plants with reported breeding systems, pollen activity reached up to 90%. For example, B. pilosa reached 98.83%, E. breviscapus reached 95%, and C. lanceolata reached 94% for pollen vitality. Even on the day before the flower faded, C. lanceolata pollen vitality remained around 80% (Grombone-Guaratini et al., 2004;Li & Dang, 2007;Zeng et al., 2010). The pollen vitality of A. artemisiifolia and S. canadensis was relatively low, but they also reached 56.8% and 67.0%, respectively. In addition, their pollen vitality was maintained for 8 days and 7 days, respectively. In terms of stigma receptivity, the active stigma of A. artemisiifolia reached up to 55.6%, and the viability was maintained for about 12 days.
The stigma receptivity of S. canadensis was above 50.0%, and the viability was maintained for about 10 days (Hao et al., 2015;Li & Dang, 2007). Compared with other Asteraceae plants, the pollen vitality and stigma receptivity of N. insignis were relatively low, and the pollen vitality was maintained for a very short time, which would limit the success of pollination, resulting in the inability to produce a large number of effective seeds.
Flowering time is explained mainly by environmental variation, and it has been proposed that flowering time is a plastic trait that responds to various environmental cues (Lessard-Therrien et al., 2013;Silva et al., 2011). The plasticity of plant reproductive phenology, especially the flowering time, reflects the adaptability of plant reproduction to environmental changes (Davis et al., 2010;Matthews & Mazer, 2016;Rathcke & Lacey, 1985;Siegmund et al., 2016). For example, in seasonally arid areas, water is a temporally limited resource, and this seasonal variation is one of the most important abiotic factors influencing flowering time (Bullock, 1995;van Schaik et al., 1993). The present study demonstrated that N. insignis had big fluctuations in flowering time across years (Figure 3), and flowering time was significantly related to the seed setting rate (Figure 9). Further researches showed that there was a significant linear relationship between flowering time and rainfall 5 months before flowering ( Figure 10). This was inconsistent with what has been documented in other seasonally dry tropical forests, where the flowering time depended on the first heavy rains following the dry season (Domínguez & dirzo, 1995;Lampe et al., 1992), and suggested that the rainfall from the end of the rainy season to the middle of the dry season (from October to February of the following year) had a great influence on the flowering time of N. insignis. This result might be explained by considering that flowering time was affected by regional environmental variation (Lessard-Therrien et al., 2013;Silva et al., 2011), because there was almost no heavy rainfall, or even very little rainfall, 2-3 months before flowering of N. insignis in the dry-hot valley. This observation provided the first description of the relationship between flowering time of N. insignis and rainfall before flowering.

| CONCLUSION
Our results indicate that the seed setting rate of N. insignis is low in the natural condition and varies greatly from year to year. Neither pollinator limitation nor pollen limitation causes the low seed setting rate of N. insignis. The obstacles in sexual reproduction process of this species may be attributable to its low stamen and pistil functions, such as poor pollen viability and stigma receptivity.
The flowering time of N. insignis depends on the rainfall 5 months before flowering, which has a significant effect on seed setting rate. In addition, our study represents a relatively short period of time, especially in terms of factors affecting seed setting rate and flowering time, and it is uncertain whether our results will differ in a longer time series. Consequently, long-term studies are necessary, including other phases of the reproductive cycle such as seed germination, analyzed from the functional and phylogenetic perspectives.
ceived for this study. We would like to thank LetPub (www.letpub. com) for providing linguistic assistance during the preparation of this manuscript.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data used for the analysis are uploaded in a Dryad repository (https://doi.org/10.5061/dryad.fbg79 cnv2).