Materials and Methods
Study site and
species
E. trifoliatus is
a perennial herb which is long day crop and after-ripening of seed physiology.
Small yellow-green flowers are borne on an umbel, terminal racemes, or compound umbels. The pistil is
uneven in length. The ovary is inferior, with two or three styles. The flowers
bloom in the autumn. The fruit is a depressed-globose berry, developing into
black fruits that mature in September or October. This study was conducted in
the Nanchong West Mountain Scenic Area, China West Normal University (30°48'54'' N, 106°03'47'') from August to October
2014–2017. Three 20×20 m field sites (I, II, and III) were selected for the
study with significantly different relative light intensities (Table 1, P<0.05). In each site, there are
about twenty plant of E. trifoliatus were recorded, and soil samples were taken
back to the laboratory. Within this region, the average annual precipitation
ranges from 820 mm to 1,100 mm, and the average annual temperature is
approximately 17.6°C; the average annual relative humidity is 73 to 83%, an
annual average frost-free period of 312.4 days, and 1,292.9 sunshine h. In
addition to the light relative intensity, the environmental factors of the
three field sites were not different.
Table 1: Comparison
of three environmental factors of E. trifoliatus from three field sites
|
Site
|
Altitude (m)
|
Temperature (°C)
|
Relative light intensity (%)
|
Relative humidity (%)
|
Soil moisture content (%)
|
Soil organic content (%)
|
|
I
|
150
|
22.62±1.31a
|
32.30±1.48c
|
60.54±4.73a
|
17.23±2.14a
|
3.55±0.14a
|
|
II
|
350
|
23.42±1.30a
|
79.28±3.05a
|
50.54±3.81a
|
18.15±2.29a
|
3.73±0.12a
|
|
III
|
456
|
20.84±1.02b
|
54.05±2.40b
|
50.76±4.66a
|
18.58±2.22a
|
3.32±0.35a
|
Significant differences are indicated by
different letters within columns (ANOVA, p <
0.05)
Table 2: Flowering
phenology of E. trifoliatus
at the three study sites
|
Parameter
|
Field site
|
Individual plant
|
|
|
I
|
II
|
III
|
I
|
II
|
III
|
|
First
flowering date (month-day)
|
9–20
|
9–3
|
9–7
|
9–25
|
9–7
|
9–10
|
|
Duration
(day)
|
20
|
37
|
25
|
10
|
12
|
15
|
|
Peak flowering
date (month-day)
|
10–1
|
9–25
|
9–12
|
10–3
|
9–10
|
9–17
|
|
Flowering
amplitude
|
—
|
—
|
—
|
23
|
27
|
20
|
|
Flowering
synchrony index
|
—
|
—
|
—
|
0.719
|
0.288
|
0.404
|
|
Last
flowering date (month-day)
|
10–10
|
10–9
|
10–3
|
10–5
|
9–19
|
9–25
|
Floral
characteristics
During the breeding period, more than ten
flowering plants were randomly selected of each site. The flowering dynamics of
three sites were observed and photographed.
Pollen viability
and stigma receptivity
We selected 60 individuals and counted
flowers per inflorescence and inflorescences per plant, and one unopened bud
was selected on each individual to record flower longevity. We also quantified
pollen grains and the number of ovules by selecting 30 flower buds and fixing
the dissected anthers and ovaries separately informaldehyde-ethanol-glacial
acetic acid (FAA) solution. 3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide
(MTT) was used to test the
presence of dehydrogenase in pollen as an assay for pollen viability (Gobet
et al. 2005). Following the methods described
by Dafni (1992), stigma receptivity was determined during each of the six
flowering stages by placing styles into hydrogen peroxide (H2O2)
with forceps and examining the resulting reaction with a hand lens (10×). A
stigma that released oxygen bubbles was considered receptive.
Flower biology
To observe
the dynamic process of flowering on the inflorescence, we recorded the number
of flowering, littering, or fruiting individuals per day. The flowering amplitudes curves was calculated according to
Herrera's statistical method on per plant individual as
the number of flowers open per day and number of flowers open per unit time
(represented by number of flowers plant-1 day-1).
Flowering intensity was calculated as follows: the relative flowering intensity
of the plant was equal to the number of flowers
produced on the peak flowering day of the plant and the ratio of the maximum
number of flowers per plant produced on the peak flowering day.
Manipulated pollination
experiments
We explored the breeding
system of E. trifoliatus
using five different pollination treatments. Three different field sites of E. trifoliatus were
selected and 240 individuals with buds were selected from each site. Artificial
pollination tests were conducted in the three different sites over 2014–2017. Following
the methods described by Dafni (1992), five treatments were set up: (1) natural
control, without any treatment, observed until the fruit ripened; (2) bagging
control, without treatment, checked to determine whether self-pollination was
possible; (3) after artificial self-pollination, the stamens were removed and
the flowers were bagged; this treatment was used to identify whether the
species is self-compatible; (4) artificial cross-pollination of the same
species; (5) artificial cross pollination. All treatments were conducted in
triplicate.
Pollinator visitation
During the peak flowering
season, we selected three 2×2 m samples to determine the main pollinators and
organisms visiting E. trifoliatus.
Five clear and sunny days were chosen to observe the pollinators, and
observations recorded were conducted from 8:00 to 18:00 each day. A total of 55
h of in-person observations were made at three sites. After each instance of an
organism visiting a flower, insect specimens were collected with an insect net
and brought back to the laboratory. Experts were then invited to identify the
collected insects. In order to detect the pollination efficiency of insects
visiting E. trifoliatus,
we examined the pollen removal rate and the number of pollen grains on the
stigma after an insect visited a flower. To do this, we removed the stigma and
anthers with tweezers, and placed them in a centrifuge tube with 1.5 mL FAA
solution. The samples were then taken back to the laboratory and counted the
number of pollen grains under an optical microscope (×40).
Statistical analyses
One-way
analysis of variance (ANOVA) was used to test for significant differences in
various parameters and sites. Post-hoc
multiple comparisons of means were performed using Tukey’s least significant
difference test (P<0.05). A correlation analysis of field site factors with
flowering period, number of inflorescences, number of flower buds, flower
visiting frequency, and fruit setting rate was conducted. All statistical tests were conducted using the IBM SPSS Statistics V22.0 computer
software; the level of significance used was P<0.05 for all tests.
Floral features and
flowering phenology
The flowers were compound racemes consisting of six to ten inflorescences; they were located in the top branches of the current
year’s growth. The main inflorescence was surrounded by inflorescences with
relatively thin peduncles and fewer flowers. Within the compound raceme, the
main inflorescence flowered and set fruit earlier (1–2 months) than the accompanying
inflorescences (Fig. 1). Within the same inflorescence, flowers positioned near
the bottom flowered earlier than flowers higher up on the inflorescence.
Phenological
investigations of E. trifoliatus showed that the
flowering season lasted from early September to late October; peak flowering occurred
from late September to early October (Table 2). The numbers of flowers per inflorescence and flowering times
were significantly (P<0.05)
different under different light intensities. The duration
of flowering was 20 to 37 days; it was shortest at site I and longest at site II (Table 2). The duration of flowering of individuals was 10
to15 days, and was also shortest at site I, but it was the longest at site III
(Table 4). Comparisons of the flowering dynamics
of E.
trifoliatus at all
three sites showed that E. trifoliatus population at site II flowered and set fruits earlier than
the populations at the other field sites; flowering and fruiting at site II was
approximately 10 to15 days earlier than E. trifoliatus at site III (high light intensity). At
site I (low light intensity), the number of flowers was relatively low and many
plants had no flowers.
%201053-1060_files/image002.png)
Fig. 1: Flowering in E. trifoliatus
%201053-1060_files/image004.jpg)
Fig. 2: The variations of (A) flowering amplitudes curves and (B)
relative flowering intensity of E. trifoliatus at the three study sites. Significant
differences are indicated by different lowercase letters within groups (ANOVA, P<0.05)
Fig. 3: The effective floral pollinators of E. trifoliatus during the
flowering season
Pollen
viability and stigma receptivity
Pollen
viability generally increased within 4 days after the start of flowering and
was above 83% at all sites (Table 3). Pollen viability then decreased rapidly and the
rate of decrease was significantly higher at site II (P<0.05). Similarly, stigma receptivity increased and quickly
became highly receptive 1 day after the start of flowering at all sites.
Interestingly, stigma receptivity decreased 4 days after the start of flowering
at sites I and III, but the stigma remained receptive for 6 days at site II,
suggesting that a longer duration of stigma receptivity was beneficial for the
pollination of E. trifoliatus at site II.
Floral
biology
Daily
flowering ratio indicated that the flowering amplitude was significantly
different among the three sites (P<0.05). The mean flowering
amplitude at sites I and II displayed a bell
curve that peaked at 15 and 26 days of flowering, respectively (Fig. 2A);
however, site III displayed two peaks, at 20 and 24 days of flowering (Fig.
2A). The flowering synchrony index of sites I, II, and III were 0.719, 0.288,
and 0.404 (Table 2), respectively, which indicated that the flowering period of E. trifoliatus was more
concentrated at sites I and III, possibly because of relative light
restrictions during flowering.
The relative
flowering intensity of E. trifoliatus was significantly different among the three
sites (P<0.05, Fig. 2B); it was highest at site II (20–50%) and
lowest at site III (< 20%). At all three sites the flowering amplitude
curves were largely characterized by a single-peak, and the relative flowering
intensities were reasonably high than 50% (Fig. 2), which suggested that the
flowering period of E. trifoliatus was short and centralized.
Table 3: Pollen viability and stigma
receptivity of E. trifoliatus
at the three study sites
%201053-1060_files/image008.jpg)
Fig. 4: Variations of visitation frequency of
effective pollinators at the three sites.
Manipulated
pollination experiments
The results of
the manipulated pollination experiments are displayed in Table 4. Pollen interference in E. trifoliatus resulted in
low rates of natural fruit setting. The control inflorescences set fruits at a rate of 42.7–65.5%, and the fruit set was highest at site II
(P<0.05). In all the bagged
inflorescences in which self-pollination was allowed and artificial
geitonogamy performed, E. trifoliatus did not fruit. However, the fruit setting rate of the natural control and artificial xenogamous pollination treatments were from 42.7% to 88.57%. The
highest fruit setting rate was in the natural control at site II, whereas the lowest was for the same treatment at site III (P<0.05). These results
demonstrated that E. trifoliatus had an outcrossing breeding system.
Floral
visitors
The observation of visitors to E. trifoliatus flowers at the three sites suggested that the main visitors were Diptera and Hymenoptera during the flowering season (Fig. 3).
A total of nine insect taxa were identified on E. trifoliatus as floral visitors. The effective pollinators were determined by electron microscopy, which were Syrphidae (Diptera: Syrphidae;
Fig. 3A, B), tachinid fly (Diptera: Tachinidae; Fig. 3C, D), and bees Apis sp. (Hymenoptera: Apidae;
Fig. 3E, F). The visitors fed on the nectar and
transferred pollen between flowers. Within days of flowering, for the purpose of feeding nectar, those effective pollinators grasped the stamens on the forefoot, the later master grabbed the
flower holder, and its mouthpiece extended out on the flower holder to eat
nectar. During the period, the head and mouthpiece were polluted with pollen.
At the same time, the head and mouthpiece contacted the stigma to complete the
pollination event. A small amount of pollen was found on the mouthparts and
front and back feet of these effective visitors by microscopy. We observed no significant differences in pollinator diversity among sites.
The visiting
frequency of effective pollinators was recorded
only on clear days (Fig. 4). The visiting frequency was highest at site II and
lowest at site I, and the visitation frequency between these sites was significantly different (Fig. 4). We observed that with greater light intensity there were fewer
visits, yet we expected flies and bees to be more active with more light. In
this study, plants at site II showed the greatest number of flower visitors was observed at site II. During the day, visitor frequency
initially increased and then decreased later in the day. Visitor frequency was
highest from 12:00 to 14:00 (Fig. 4).
Table 4: Effects of pollination treatments on the fruit setting
rate of E. trifoliatus
at the three study sites
Table 5: Correlations
of plant reproductive and environmental factors at three sites
Correlation
analysis between reproduction factors and environmental factors
Correlations
were found between the reproductive factors and environmental factors of E. trifoliatus
(Table 5). Specifically, the flowering period
and temperature at the field site were significantly
positively correlated with light intensity, indicated that temperature and light affected the flowering of E.
trifoliatus. The number of flower buds was
significantly positively correlated with temperature and light intensity, which indicates that temperature and light intensity
affect the behavior of floral visitors. The
natural fruit set rate at all field sites were
significantly positively correlated with altitude and light, indicating that
the reproductive characteristics of E. trifoliatus were closely
related to altitude and light intensity in the field site.
Discussion
In addition,
the pollination experiments provided insight into the floral biology of E. trifoliatus.
Specifically, we found that reproduction in E. trifoliatus was
dependent on animal pollinators and that E.
trifoliatus was xenogamous.
The cross-pollination of angiosperms evolved in ancient natural ecosystems,
from entomophily to anemophily, which was likely to reduce the dependence on
organisms that were strongly affected by erratic climatic conditions (Bawa 1995). This was also supported by the rudimentary and
inefficient nectarines in wind-pollinated species (Culley et al. 2002).
Our hand-pollination treatments showed significantly greater fruit set than we
observed in the open-pollinated flowers (Table 4), which suggested possible
pollen limitations under natural conditions. This was further supported by our
field observations that pollinator visitation
frequency was low (Fig. 4). Floral morphology and pollinator foraging behavior
in E. trifoliatus
may reduce self-pollination and pollinations between
flowers with the same stylar deflection. E. trifoliatus has several unique floral traits, such as long-lived flowers and small
inflorescences (Table 2; Fig. 1); therefore, it is reasonable to expect that the
longevity of flowers ensures pollination success (Bingham and Orthner 1998; Rathcke 2003). This
study suggests that the long duration of flowering has nevertheless allowed the
plants to survive in environments with reduced populations of pollinator (Dixon
2009).
According to
the concept of pollination syndromes, floral traits reflect specialization to a
particular pollinator or set of pollinators (Zhang et al. 2011).
Pollination specialization has long been considered an important process
underlying the evolution of floral diversity. Floral traits have been viewed as
adaptations to attract specific pollinators and to enhance the efficiency of
pollen transfer (Stebbins 1970). Although nine taxa were floral visitors of E. trifoliatusin Sichuan
Province, they had varying visitation frequencies. In this study, we collected
floral visitors as they exited the flower, and some were found with a pollen
mass of E. trifoliatus on the
thorax. These results suggest that Syrphidae, tachinid flies, and Apis sp. are effective
pollinators (Fig. 4). Bees were considered the most effective pollinators of plants, but flies have also been shown to provide
substantial pollination services in natural systems (Power and Stout 2011). The
importance of flies for plant reproduction was based on flower visitation
observations (Goulson and Wright 1998), conspecific
pollen load counts (Vance et al. 2004), and experimentally
(Fontaine et al. 2006). To date, most Araliaceae species have been
found to be pollinated by various bees and flies, and flies were considered the
primary pollinators of Araliaceae (Jacobs
et al. 2010). As adults, the flies rely on nectar for carbohydrates,
pollen as a source of carbohydrates and lipids, and proteins for egg formation
(Rotheray and Gilbert 2011).
The structure
of pollinator assemblages was consistent in the three study sites (P>0.05), but the visitation
frequency was significantly different between sites (Fig. 4). High activity
levels (i.e., more species and individuals caught) were recorded around midday
(from 12:00 to 14:00), with
highest activity at site II (Fig. 4), which was in accordance with the
population of E. trifoliatus at each site
(Table 3 and 5). The differences can be explained in terms of the temporal
variation in floral resources and physiological limits of the species, which
determine the time of day the flower-pollinator interactions occur. At midday,
favorable conditions (sunshine and temperature) encouraged the opening of more
flowers, increasing the availability of pollen and nectar; the amount of nectar
secreted per flower tends to decrease towards the evening (Pleasants and
Chaplin 1983). Greater availability of food around midday likely contributed to
higher visitation rates. We found that E. trifoliatus had more
flowers and less fruit, which was estimated to be related to the insufficient
number of pollinators, especially considering the little amount of nectar
produced by E. trifoliatus. The
attractive for flower visiting is insufficient.
The
reproduction activity of E. trifoliatus generally
occurred from August to October. Climate conditions
were more favorable during that time because there was sufficient water and
higher temperatures. In addition, the reproduction
of pollinators was also most
active at this time (Lucas et al. 2017). We found that the pollination
of E. trifoliatus was centralized, which provided effective access for
pollinators and allows plants to achieve reproductive success
(Rollin et al. 2016). We recorded discordant
abundance, species richness, and co-occurrence patterns in the three study
sites, and we determined that site II was the most favorable for the pollination of E. trifoliatus (Table 2–5) because the natural fruit set rate was higher compared with the other field sites. This may be due to the more suitable
light intensity at this field site. The moderate light intensity at site II was
the most beneficial for pollination and reproduction of E. trifoliatus, which suggest that moderate
sunshine can protect the plant from injury due to photo-oxidation (Lin et al.
1998).
The
correlation analysis showed that temperature and light
were important limiting factors (Table 5). In natural environments, insect
pollinators were impacted by several limiting factors, including climate
change, human disturbance, agricultural intensification, and landscape
fragmentation (Földesi et al. 2016), which
lead to less effective pollination and a reduction in agricultural production
(Garibaldi et al. 2013). Different species or functional groups respond differently to environmental change (Kremen
et al. 2004; Brittain et al. 2013).
Considering the variable projected responses of bees and flies to future
climate change, their value as pollinators, and the increasing threats they
currently face, Radenković et al. (2017)
showed that the ranges of all species, and the abundances of many
species, are projected to decrease in the future. In addition, outcrossing
plant species that are reliant on the declining pollinator groups have
themselves declined relative to other plant species. Taken together, our
findings strongly suggest a causal connection between local extinctions of
functionally-linked plant and pollinator species.
Conclusion
Acknowledgements
We
acknowledge the National Natural Science
Foundation of China (31700387), the Research Fund for
Excellence Project of China West Normal University (17YC144), and the Doctoral
Scientific Research Foundation of China West Normal University (14E011).
Author Contributions
Juan Xiao conceived
and designed the experiments; Xiao Xiao and Lanying Chen
performed the experiments; Xiao Xiao and Lanying Chen
analyzed the data and wrote the manuscript.
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