Pollen limitation negatively impacts endangered and endemic plants with small fragmented populations, such as Sinocalycanthus chinensis, an endangered plant endemic to China. In this study, we analyzed the pollen limitation of the S. chinensis Damingshan (DMS) population in 2006, 2009, and 2010, and crossed plants with mates separated by different distances, both within and between populations. The DMS population exhibited strong pollen limitation in fruit set, seed set, and seeds per fruit in 2006, 2009, and 2010. The average accumulated pollen limitation (for fruit set times seeds per fruit) was 0.510 ± 0.180. Progeny crossed with pollen from intermediate neighboring plants within the same population (separated by 30 – 50 m from pollen recipients) had the lowest fitness. No optimal outcrossing distance was found within the DMS population. Progeny from crosses with the SXW and DLS populations performed relatively better, while those from crosses with QLF and LXS populations performed worse. Compared with average reproductive success, outbreeding depression was found in progeny from crosses with the LXS and QLF populations. Reproductive success from pure self-pollination indicated S. chinensis is self-compatible. Geitonogamous selfing increased reproductive success. Based on geitonogamous selfing, the proportion of selfed offspring was relatively high. These results provide basic references for the conservation of this species.

Study site

This study was conducted at Damingshan Mountain (DMS, 30º02′N, 118º59′E), Linan City, Zhejiang Province, China (Table 1), the location of the largest known extant population (Liu, Zhou, Huang, Bao & Zhao, 2016). The pollen donor plants were located in the main DMS population and the other five populations were located at Tashajiang Village (TSJ), Shunxiwu Village (SXW), and Qingliangfeng Mountain (QLF) in Linan City, Zhejiang Province, Daleishan Mountain (DLS) in Tiantai County, Zhejiang Province, and Longxushan Mountain (LXS) in Jixi County, Anhui Province (Table 1). Among these populations, the population size of SXW and QLF were relatively large (Table 1).

Experimental design

Pollen limitation

As a woody shrub, the age class of S. chinensis can be estimated from the volume calculated as d from the formula d=a×b×c, where a is the height of the tallest stem, b is the crown width along the longitudinal axis, and c is the width along the perpendicular axis (Yang et al., 2006). For S. chinensis in the DMS population, the age class ranged from 0 to 3 (Li, Jin & Liu, 2012). In May 2006, 19 mature plants in age class III (2 < d £ 3) and with similar phenologies that were located in the center of the DMS population and separate from each other by distances of more than 10 m were chosen as pollen recipients. Pollen grains were also collected from 15 pollen donor plants in age class III and with similar phenologies that were located within the same population and separated from each other by a distance of more than 10 m by rubbing a toothpick against newly dehisced anthers. The pollen grains were then mixed in a small plastic vial and stored at 4°C on ice bags in a plastic box, and then the pollen grains were transferred to recipient stigmas within 2 hours (Irwin, 2001). In total, 30 flowers were used for each pollen supplementation, and 30 flowers among 19 naturally open-pollinated DMS plants were selected as the control (Bossuyt, 2007; Holmes, James & Hoffmann, 2008). In October 2006, fruits from all tested plants were collected. Fruit set was calculated as the number of mature fruits divided by the number of treatment flowers. Seed set was estimated as the average number of mature seeds divided by the number of fruits within a treatment. (Seeds have a dark, shiny, thickened seed coat when mature. If ovules are not fertilized or aborted, the seeds are very thin, transparent, and whitish in color). The mean number of seeds per fruit was also measured.

Similar experiments were conducted in May and October in 2009 and 2010 to obtain two biological replicates. In 2009 and 2010, 100 and 30 flowers for each treatment were used, respectively. We calculated the pollen limitation of the species by averaging PL values over the three years.

Optimal outcrossing distance

In May 2009, before S. chinensis flowers were open, emasculation and bagging was conducted to protect the stigma, then a pollen supplementation experiment was conducted by pollinating flowers with pollen grains collected from different populations separated by nine different distances (Table 1) to determine the optimal outcrossing distance. Nine treatments were conducted as follows. (1) Geitonogamous selfing was conducted by supplementary hand-pollination with geitonogamous pollen from the same plant. (2) Immediate neighbor treatments were conducted by supplementary hand-pollination with xenogamous pollen collected from plants within the same population separated by distances of 1 to 20 m. (3) Intermediate neighbor treatments were conducted by supplementary hand-pollination with xenogamous pollen collected from plants within the same population separated by distances of 30 to 50 m. (4) Remote neighbor treatments were conducted by supplementary hand-pollination with xenogamous pollen collected from plants within the same population separated by distances of 80 to 100 m and thus unlikely to interact via open pollination. The within-population distances from the center of the DMS population were measured with tape measures. (5–9) Between population treatments were conducted by supplementary hand-pollination with xenogamous pollen collected from plants in the TSJ, SXW, QLF, LXS, and DLS populations, respectively. Geographical distances were calculated using Earth Explorer 4.0, and a detailed summary of the nine treatments is provided in Table 1. Thirty flowers from 19 mature plants in age class III (2 < d £ 3), with similar phenologies, and that were located in the center of the DMS population and separated from each other by a distance of more than 10 m were chosen as pollen recipients for each treatment. Pollen grains were collected from 15 pollen donor plants in age class III, with similar plant phenologies, separated from each other by a distance of more than 10 m, and located in the different populations in Table 1 by rubbing a toothpick against newly dehisced anthers. The pollen grains were then mixed in a small plastic vial and stored at 4°C on ice bags in a plastic box, and then the pollen grains were transferred immediately to recipient stigmas. According to a previous study by Zhang and Jin (2007), the pollen viability of S. chinensis lasts for five days after collection (no significant difference was detected among pollen of different days). The within-population pollen grains were used for hand-pollination within 2 hours, while the between-population pollen grains were used for hand-pollinations within 6 hours, without an observed decrease in the viability of the pollen grains. All hand-pollinated stigmas were saturated with pollen grains. After treatment, bagging was conducted on the flowers to avoid the contamination of other pollens.