3.3 Seed symbiotic and asymbiotic cultures
More germinated seeds with low developmental stages (stage 2 and stage
3) were found after self-pollination (Figure 3A) and nectar addition
(Figure 3B) compared with that after cross-pollination (Figure 3C) and
natural conditions (Figure 3D) under the conditions of 30-day seed
symbiotic culture. There were more seedlings with high developmental
stages (stage 5 and stage 6) after cross-pollination (Figure 4C) and
natural conditions (Figure 4D) compared with that after self-pollination
(Figure 4A) and nectar addition (Figure 4B) under the conditions of
70-day seed asymbiotic culture.
There was no significant difference in seed germination among the four
treatments under the conditions of 60-day symbiotic and 70-day
asymbiotic cultures (all p > 0.05) (Figure 5A). Average fresh weight of
a single germinated seed after self-pollination and nectar addition was
significantly lower than that after cross-pollination and natural
conditions (all p < 0.01). Moreover, there were significant differences
in average fresh weight of a single germinated seed between
self-pollination and nectar addition (p <0.05) but not between
cross-pollination and natural conditions (p>0.05) (Figure 5B). The
results of average developmental stages were similar to that of average
fresh weight of a single germinated seed (Figure 5C).
Under the condition of symbiotic culture, percentage of germinated seeds
with high developmental stages (stage 5 and stage 6) after
cross-pollination and natural conditions was significantly higher than
that after self-pollination and nectar addition, while that with low
developmental stages (stage 2 and stage 3) was significantly lower (all
p < 0.01) (Figure 5D; Table 2). Differences in percentage of germinated
seeds with developmental stage 2-6 did not reach significant level
between cross-pollination and natural conditions (all p > 0.05) (Figure
5D; Table 2). There were significant differences in percentage of
germinated seeds with developmental stage 3 and stage 6 between
self-pollination and nectar addition (all p < 0.01) (Figure 5D; Table
2).
Under the condition of asymbiotic culture, percentage of germinated
seeds with high developmental stages (stage 6) after cross-pollination
and natural conditions was significantly higher than that after
self-pollination and nectar addition, while that with low developmental
stages (stage 3 and stage 4) was significantly lower (all p < 0.01)
(Figure 5E; Table 2). In most cases (except stage 4), percentage of
germinated seeds at different developmental stages showed no significant
difference between cross-pollination and natural conditions (all p >
0.05) (Figure 5D; Table 2). There were significant differences in
percentage of germinated seeds with developmental stage 2, stage 4 and
stage 6 between self-pollination and nectar addition (all p < 0.01)
(Figure 5D; Table 2).
Discussion
Like the other orchids (Johnson & Edwards, 2000; Johnson et al., 2004),
pollinarium bending obviously exists in Papilionanthe teres , and
was reached in 62.43 ± 22.97 s (N = 30). Total time per visit under
natural conditions and after nectar addition were significantly lower
and higher than that of pollinarium bending, respectively (Figure 1A),
suggested that the two conditions could delay and accelerate touching
between pollinia and stigma, respectively. Nectar addition significantly
increased the number of flowers visited in a single inflorescence and
pollinator staying time in a single flower (Figure 1B). Besides, the
proportions of full seeds and seed viability after nectar addition were
significantly lower than that under natural conditions (Table 1; Figure
5). These results indicated that P. teres is similar to most
deceptive orchids that avoid geitonogamy by the two mechanisms of
deception and
pollinarium
bending (Darwin, 1877; Dafni & Ivri, 1979; Johnson & Edwards, 2000;
Johnson et al., 2004), and deception contributes to pollinarium bending
functioning in the aspect of anti-selfing.
Both self-compatibility and inbreeding depression can be checked by
pollination experiments. There was no significant difference in fruit
set between self and cross-pollination in the earlier stage of fruit
development, while self-pollination led to serious fruit abscission in
the late stages (data not shown). Compared with cross-pollination, there
were more shriveled seeds, smaller weight of a single fruit and fewer
seeds per fruit after self-pollination (Figure 2A, C; Table 1). These
results indicated that self and cross-pollination result in different
female reproductive success. Moreover, self-pollination seriously
affects the development of fruits and seeds of P. teres .
Most orchids are self-compatible species, and the seed set and seed
quality are usually greatly reduced after self-pollination (Tremblay et
al., 2005). Compared with cross-pollination and natural conditions,
self-pollination and nectar addition
had more seeds with low developmental stages under the conditions of
30-day (Figure 3A, B), 60-day (Figure 5C, 5D; Table 2) symbiotic culture
and 70-day asymbiotic culture (Figure 4A, B; Figure 5C, E; Table 2),
suggesting that seed viability was greatly reduced after
self-pollination and nectar addition. There were significant differences
in average fresh weight of germinated seeds (Figure 5B), average
developmental stage (Figure 5C), proportions of seeds with high
developmental stage (stage 6) (Figure 5D, E) and shriveled seeds (Table
1) between self-pollination and nectar addition under the conditions of
symbiotic and asymbiotic cultures, suggesting that nectar addition
partially results in geitonogamy. It is important for orchids to reach
higher developmental stage faster under natural conditions to enhance
its survival and resistance to disadvantageous environmental and climate
conditions (Smith et al., 2007; Stewart, 2008). Based on this
conclusion, and combined with our study results, it is confirmed that
germinated seeds after cross-pollination and natural conditions have
higher ability to adapt to the environment than that after
self-pollination and nectar addition.
Seed morphology (Figure 2C, D), and parameter values of fruits and seeds
(Table 1) showed no significant differences between natural conditions
and cross-pollination. Besides, there was no significant difference in
average fresh weight of germinated seeds (Fig. 5B), average
developmental stage (Figure 5C), proportions of seeds with different
developmental stages (Figure 5D, E) between natural conditions and
cross-pollination under the conditions of symbiotic and asymbiotic
cultures. Taken together, these results suggested that P. tereshas high outcrossing rate under natural conditions. Some case studies
confirmed a negative correlation between
inbreeding depression and selfing
rate (Husband & Schemske, 1996), indicating that the lower the selfing
rate, the more obvious the inbreeding depression. Thus, if deceptive
orchid species regularly experience high levels of outcrossing, a higher
cost of selfing is expected in deceptive species than in rewarding ones
(Jersáková et al., 2006). Compared with cross-pollination and natural
conditions, there were more shriveled seeds (Figure 2), lighter weight
of a single fruit and seeds per fruit (Table 1), and lower seed
viability (Figure 5; Table 2) after self-pollination and nectar addition
under the conditions of symbiotic
and asymbiotic cultures, indicating that both self-fertilization and
geitonogamy result in inbreeding
depression. These results further confirmed that P. teres has
high outcrossing rate under the natural conditions.
Nectar addition experiments along with field observation were used in
this study to comparatively analyze the differences of pollinator visit
behavior, and the results confirmed thatP.
teres decreased geitonogamy by the two mechanisms of deception and
pollinarium bending. Deception contributes to pollinarium bending
playing the function of anti-selfing in P. teres . Besides, the
fruits and seeds from four sources (self-pollination, cross-pollination,
nectar addition and natural conditions) were used to compare the
differences in the characteristic parameters of seeds and fruits, and
check the seed viability by symbiotic and asymbiotic culture techniques,
which indirectly proved that P. teres had high level of genetic
load. Both self-fertilization and geitonogamy resulted in inbreeding
depression, such as high proportion of shriveled seeds and lower
developmental speed of germinated seeds. These conclusions support the
outcrossing hypothesis that ecological significance of P. teresdeception is to promote outcrossing and improve the ability of the
offspring to adapt to the environment.