Seed collection
Mature seeds of G. officinalis were collected from the natural-growing plant community at the Hezuo alpine meadow and wetland ecosystem research station of Lanzhou University on the southeast Qinghai-Tibet Plateau (lat. 34°53′ N, long. 101°53′ E, alt. 2900 m) in 2014 and grown in a nursery. Robust seedlings were selected and transplanted to the Haibei Alpine Meadow Ecosystem Research Station of the Chinese Academy of Sciences on the northeast Qinghai-Tibet Plateau (lat. 37°37′N, long. 101°19′ E, alt. 3200 m) and Datong ecological agriculture experimental station of the Northwest Institute of Plateau Biology (lat. 34°53′ N, long. 101°53′ E, alt. 2900 m). Transplantation was also performed in a natural environment (the Hezuo alpine meadow and a wetland ecosystem research station of Lanzhou University).
Study plots and transplanting
The naturally studied population is located at the Hezuo alpine meadow and wetland ecosystem research station of Lanzhou University on the southeast Qinghai-Tibet Plateau (henceforth referred to as the natural environment (NE)), China (lat. 34°53′ N, long. 101°53′ E, alt. 2900 m). The third transplantation site was created in a natural environment and was termed “natural transplant” (NT). The average annual air temperature is 2 °C, with extremes of 11.5 °C (maximum) and –8.9 °C (minimum). The annual precipitation is approximately 550 mm, 80% of which falls in the short summer growing season between May and September. Hezuo station is dominated by Kobresia humilis, Pedicularis kansuensis, Heteropappus altaicus, Stellera chamaejasme, Aconitum gymnandrum and Nepeta pratti, which bloom at the same time as G. officinalis.
The higher-elevation transplanted plot was located at the Haibei Alpine Meadow Ecosystem Research Station of the Chinese Academy of Sciences on the northeast Qinghai-Tibet Plateau (henceforth referred to as the high-elevation environment (HE) (lat. 37°37′N, long. 101°19′ E, alt. 3200 m). The average annual air temperature was –1.7 °C, with extremes of 27.6 °C (maximum) and –37.1 °C (minimum). The annual precipitation ranged between 426 and 860 mm, mainly in July and August.
The lower-elevation transplanted plot was located at the Datong ecological agriculture experimental station of the Northwest Institute of Plateau Biology on the transition zone between Qinghai-Tibet Plateau and loess plateau (henceforth referred to as the low-elevation environment (LE)) (lat. 34°53′ N, long. 101°53′ E, alt. 2900 m). The average annual air temperature was 7.6 °C, with extremes of 34.6 °C (maximum) and –18.9 °C (minimum). The annual precipitation was approximately 380 mm, mainly in July and August. The study area was dominated by cultivated crops.
Robust seedlings with floral buds were selected for transplantation. The density of G. officinalis under the NE was approximately 1.5 plants/m2; therefore, we planted individuals at the same plant density in all transplanted plots. Moreover, more than 300 robust seedlings of G. officinalis were transplanted to each transplanted plot. The total planting area was greater than 200 m2 at each plot. The transplanted seedlings flowered in the summer, and we conducted our experiments during the following 2 years (2016–2017).
Flowing phenology and flower duration
To observe flowering phenology, three 1 × 10-m areas were created within each experimental plot in 2016. In each plot, flower opening and duration were monitored and recorded every morning until all flowers withered.
At the full anthesis phase of G. officinalis in 2016, 10 plants from each plot were randomly selected. On each plant, two buds at the middle position of the inflorescence were selected, and the floral duration of all the selected buds was monitored and recorded. The pollen (male phase) and stigma (female phase) presentations were monitored and recorded.
Floral display and reproductive allocation
At the full-bloom stage, 50 single plants were selected from each plot to test the inflorescence traits. Stem length (the distance from the stem base to apex) was measured by a straightedge. The number of sprays on each plant and the average flower numbers (including buds and fruits) on each spray were counted.
We selected 100–150 fully open flowers on different plants in each population to test the flower sizes at each plot. To avoid the position effect as much as possible, we did not choose terminal flowers. The length and width (diameter) of the flowers in each plot were measured by Vernier calipers. To test the sexual allocation changes in G. officinalis among the three plots, 30 buds on different plants in each plot were selected randomly. Then, the pollen numbers (PNs) and ovule numbers (ONs) were counted. The pollen/ovule ratios (P/O) were calculated as P/O = pollen numbers in all five anthers/ovule numbers21.
Sampling dates corresponded to the height of the flowering season at each site (mid-August in the LE and early September in the NE and HE) before fruiting had occurred. While fresh, the aboveground parts of 30 fully flowering plants per site were dissected into inflorescences, peduncles, leaves, and stems. Plant material was oven-dried at 70 °C for 3 days, and the dry weights were obtained to the nearest 0.1 mg on an analytical balance (Ohaus). The inflorescence and peduncle fractions of each plant were summed to provide a measure of reproductive biomass (R), and the leaf and stem fractions of each plant were summed to provide a measure of vegetative biomass (V). The reproductive allocation (RA) was calculated as RA = R/(R + V).
Observation of pollinators
The floral visitors to G. officinalis were recorded in the three plots. Ten neighbouring inflorescences on different individual plants were selected at random and labelled. Before observation, we counted all the open flowers on one inflorescence and then recorded the number of flowers visited by pollinators. We observed these flowers between 9:00 a.m. and 6:00 p.m. in each plot during 2016 and 2017. In total, observations were carried out for 65 h in each plot over the 2 years. While carrying out these observations, we stayed 2 m away from the focal flowers to observe all of the floral visitors without disturbing their foraging behaviours. The visitor species, behaviour in the flower, and visiting times of each species were recorded, and the visit frequencies of each visitor species were calculated. The visit frequency was calculated as visit frequency = visit times/visit flower numbers/hour.
To identify whether flower visitors were legitimate pollinators of G. officinalis, collected visitors were observed and photographed with a stereomicroscope to identify whether G. officinalis pollen was attached to their bodies. Additionally, each visitor was observed to determine whether the reproductive structures of flowers had been touched. Visitors that were positive for all these factors were considered legitimate pollinators.
Seed production
To test the self-compatibility of G. officinalis, flowers subjected to self-pollination treatment (unopened flowers were isolated with paper bags) in 2017 on the three plots were subjected. To further analyse self-compatibility, we conducted outcrossing pollination. In addition, 30 individual inflorescences on different plants were bagged, and two buds at the same position on each inflorescence were selected. Both buds on each inflorescence were emasculated before the flowers opened. When the stigma opened, one flower was pollinated with fresh pollen from the same inflorescence or different inflorescences on the same plant (selfing), and the other was pollinated with fresh pollen from a plant 5 m away (outcrossing). To test whether facilitated selfing occurred, 30 individual plants in each plot were tagged. On each tagged plant, two individual buds were selected: one was assigned to natural pollination, and the other was assigned to emasculation (removal of all anthers before stigma lobe opening). To test whether agamospermy occurred, the flowers were subjected to emasculation treatment and isolated in three plots. Thirty buds on different plants were randomly selected, and all the anthers were removed before the flowers opened, and then all the buds were isolated with paper bags. At maturity, all fruits were collected, and all of the seeds (including mature and abortive seeds) were counted. Seed-set ratios were used to assess the reproductive success of each treatment, which were calculated by the number of mature seeds divided by the total ovules in each ovary. The facilitated selfing data were calculated as the natural seed-set ratio minus the emasculated seed-set ratio.
Similarly, 30 inflorescences were tagged on different plants in each plot, and two buds were then tagged at the same position on each inflorescence; one bud was assigned to natural pollination, and the other was assigned to supplemental hand pollination when stigmas opened. For supplemental hand pollination, pollen was collected randomly from unmarked individuals at a minimum distance of 5 m from the recipient individual. Supplemental hand pollination events were conducted every day until the flower was permanently closed. When mature, all seeds were counted, and seed-set ratios were calculated. For each plot, we calculated an index of pollen limitation (IPL): IPL = 1 − (Po/Ps), where Po is the natural seed-set ratio and Ps is the supplemental hand-pollination seed-set ratio. As the seed-set ratios showed no significant difference between natural and supplemental hand pollination in the natural environment, we considered the IPL at this plot to be 0. The IPL data at the other two plots were compared using an independent-samples t test.
Statistical analysis
The normality of the data was tested using one-sample Kolmogorov–Smirnov (1-K-S) tests, and then one-way ANOVAs (with Tukey’s multiple contrasts) were used to test differences in all traits among the three environments.
Source: Ecology - nature.com
