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\begin{document}

\title{Do larger pollinators have higher pollination efficiency for a
generalist plant Hibiscus mutabilis?}



\author[1]{Xiaoqing Shi}%
\author[2]{Bin Zheng}%
\author[1]{Xiaoli Liu}%
\author[3]{Qiumei Quan}%
\author[2]{Yunxiang Li}%
\affil[1]{Chengdu Botanical Garden (Chengdu Institute of Park City Plant Research)}%
\affil[2]{China West Normal University}%
\affil[3]{China West Normal Univ}%


\vspace{-1em}



  
  \date{June 11, 2024}


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\selectlanguage{english}
\begin{abstract}
Many insect species provide pollination services for flowering plants.
However, the pollination efficiency of various pollinator species varies
greatly due to differences in body size, foraging behavior, and
visitation rate. Here, we investigated a generalist plant Hibiscus
mutabilis, and we compared the body size, visitation rate, and
pollination efficiency of five pollinator species. Our observations
revealed that the pollination efficiency of the moth species was
relatively lower than all bee species. The body length of five
pollinator species increased the amount of transferred pollen, and
larger bees tended to remove more pollen from anthers and deposit more
pollen on stigmas, resulting in higher pollination efficiency. However,
there was no correlation between the intertegular distance and
pollination efficiency of visitors in H. mutabilis. Body length may be a
better index of body size than intertegular distance.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Ninety percent of flowering plants are pollinated by animals
(Ollerton\emph{et al} . 2011; Tong \emph{et al} . 2023). Without
pollinators, many plants could not set seed and reproduce; and without
plants to provide pollen, nectar, and other floral rewards, many animal
populations would decline (Kearns \emph{et al} . 1998). Pollinators tend
to move both within and among neighboring plants, and the pollen
transfer efficiency of pollinators varies markedly, so that most plants
end up with a mixed mating strategy at the point of being pollinated
(Harder and Barrett 1996; Goodwillie \emph{et al} . 2005). Vogler and
Kalisz (2001) reviewed available data to show that most plants achieve a
mixture of selfing and outcrossing. Plants can control their mating
opportunities by the way they manipulate their visitors. Attracting a
larger number of pollinators is mainly determined by the advertising
traits and floral rewards of plants. Therefore, understanding the
pollination efficiency of various pollinators can help predict how
different floral visitors could influence plant reproduction.

In animal-pollinated plants, the contribution of different pollinator
species to plant's reproductive fitness differs significantly because
their pollination efficiency varies greatly due to differences in body
size, foraging behavior, and visitation rate (Chang \emph{et al} .
2023). Fewer pollinator visits or less pollen delivered per visit may
reduce the reproductive success of plants (Olsen 1996; Ashman \emph{et
al} . 2004; Sahl and Conner 2007). Body size is an important functional
characteristic that underlies pollination-related ecological processes
(Kendall \emph{et al} . 2019). The body size of pollinators such as body
length and intertegular distance can influence the amount of deposited
pollen (Goulson \emph{et al} . 2002; F\selectlanguage{ngerman}öldesi\emph{et al} . 2020). The
intertegular distance is a useful body size index established by Cane
(Cane, 1987). Goulson \emph{et al} . (2002) found that the amount of
pollen transferred by bees was influenced by the intertegular distance.
Larger bees tended to deposit more pollen on stigmas at each visit than
smaller bees, and the interspecific body length and intertegular
distance of bees had a positive effect on pollen deposition (Földesi
\emph{et al} . 2020; Chang \emph{et al} . 2023). There was no
correlation between intertegular distance and pollen deposition among
individuals of the same species (Chang \emph{et al} . 2023). However,
whether pollen removal and pollination efficiency (pollination
efficiency = pollen deposition/pollen removal, see Li \emph{et al} .
2022) are affected by body size of pollinators is still unknown.

In this study, we used a generalist plant\emph{Hibiscus mutabilis} as an
example to investigate the impact of pollinators' body size on
pollination efficiency. We measured the floral traits of plants and the
body length and intertegular distance of various pollinator species. We
identified the breeding system of \emph{H. mutabilis} by imposing four
pollination treatments. We compared the visitation rates of various
pollinator species. To assess pollination efficiency, we examined pollen
deposition and pollen removal of each pollinator species. Based on field
investigations, we aimed to address the following three questions: (1)
Are there differences in visitation rate and pollination efficiency
among various pollinator species in \emph{H. mutabilis} ? (2) Does the
species \emph{H. mutabilis} have a mixed mating system? (3) Is the body
size of pollinators related to pollination efficiency?

\textbf{Materials and Methods}

\textbf{Study species and site}

\emph{Hibiscus mutabilis} (Malvaceae) is a hermaphroditic perennial
shrub or small tree cultivated in many areas of both north and south
China. This species is almost certainly native to China but was
domesticated a long time ago. Flowering individuals can grow up to 2\selectlanguage{english}~5
m high. Flowers are solitary and axillary on the upper branches. Each
flower is actinomorphic, with five petals, one staminal column, five
styles, and one campanulate calyx. The corolla is white or reddish. The
nectary is located at the base of the calyx. Flowering generally occurs
from July to November. The flower longevity of one single flower lasts
only 1\selectlanguage{english}~2 days. This study was conducted during the flowering season of
2023 in a field located at 104deg45\selectlanguage{english}'29\selectlanguage{english}"E, 30deg40\selectlanguage{english}'45\selectlanguage{english}"N, 433.1 m above sea
level, about 39 km southeast of Jintang County, Sichuan Province, China.

\textbf{Floral traits}

To understand the floral traits of \emph{H. mutabilis} , 30 bagged
flowers from 15 plants were examined on 10 October 2023. The Flowers are
single- or double-flowered on a 5\selectlanguage{english}~8 cm pedicel on the upper branches
(Fig. 1A). The \emph{H. mutabilis} flower displays a color change from
white to red (Fig. 1B). First, corolla diameter, corolla height, sepal
length, sepal width, petal length, petal width, stamen length, pistil
length, ovary height, and ovary diameter of each flower were measured
with a digital calipre to 0.01 mm. The base circumference was measured
by a thread, then the longth of the thread was determined by a digital
caliper to 0.01 mm. The anther and ovule number of each flower were
counted. To examine the production of pollen grains per flower, the
anthers of each flower were dissected and washed in 400 mL of distilled
water to dislodge the pollen grains from the anthers. The suspensions
were stirred for 2 min, and three 1-mL samples of suspension were drawn
respectively on the plankton counting chambers, after which the number
of pollen grains in the samples was counted under a light microscope at
x10 magnification. the pollen count of the three sub-samples (1 mL each)
was averaged and multiplied by the dilution factor (400) to obtain the
total number of pollen grains per flower. To evaluate the production of
nectar volume per flower (open one day), nectar volume was measured
using 10-\selectlanguage{greek}μ\selectlanguage{english}L glass microcapillary tubes during the period of 17:00~18:00
p.m. on 10 October.

\textbf{Breeding system}

To identify the breeding system of \emph{H. mutabilis} , 160 flowers
from 30 individual plants experienced four pollination treatments in
September 2023. Flowers were randomly chosen from different plants to
reduce the possible effect of resource reallocation on the fruit set,
and were enclosed with fine-mesh polyester bags to exclude any visitors
before the artificial treatments. To test for potential autogamy, 40
flowers were caged to exclude any insects. In addition, 40 flowers were
hand-pollinated with self-pollen grains from flowers of the same
individual, and another 40 flowers were hand-pollinated with outcross
pollen grains from multiple flowers of other individuals to test for any
differences in seed production between selfing and outcrossing. The
remaining 40 flowers without any treatment were exposed to open
pollination as a natural control. Four weeks later, the fruits produced
by these flowers were harvested, and the seeds and undeveloped ovules in
each fruit were counted.

\textbf{Foraging behavior observations}

To estimate the types of floral visitors and their foraging behaviors
(for pollen or/and nectar) in\emph{H. mutabilis} , we (four observers)
observed the foraging behaviors of various visitors in the periods from
08:30 to 17:30 on sunny days in October 2023. Four plots (2m \selectlanguage{ngerman}× 2m) were
randomly established, each including 10 flowers. These plots were
observed daily for 30 min. A total of 128 observation sessions were
conducted. Using a camera (Nikon D7000), we recorded the foraging
behaviors of various visitors. Moreover, we recorded the number of
visits and the number of flowers visited by various visitors per 30 min,
after which we calculated the visitation rates of each visitor (visits
per flower per 30 min) by dividing the total number of observed flowers
by the number of flowers visited per 30 min. The main pollinators were
determined by the number of visits and the foraging behaviors.

\textbf{Measurements of body size of pollinators}

To measure the body size of various pollinators, we captured 10
individual insects of each species visiting the flowers of \emph{H.
mutabilis} during peak flowering. The insect specimens were pinned and
dried in a specimen box. Specimens were photographed using a
stereoscopic microscope JSZ6S (Nanjing Jiangnan Yongxin Optical Co.
Ltd., Nanjing, China) with a digital camera. After imaging, the body
length and intertegular distance were measured using ImageJ 1.8.0
software (NIH Image, Research Services Branch, USA) (Fig. 2).

\textbf{Pollination efficiency}

Our field surveys showed that there were five pollinator species
of\emph{H. mutabilis} during our experiments. To compare the pollen
removal, pollen deposition, and pollination efficiency of various
pollinator species, more than 200 flowers of \emph{H. mutabilis} were
selected randomly from different individuals and were bagged with
fine-mesh polyester bags before blooming in October 2023. When the
flowers bloomed, all flowers were exposed to pollinators. When the
flowers had been visited a single time in the field, we harvested the
flowers immediately. To estimate pollen grains deposited on the stigmas
of the flowers, each stigma was dissected and washed in 5 mL of 75\%
alcohol to dislodge the pollen grains from the stigma. The suspension
was stirred for 2 min, and the sample of suspension was drawn on the
plankton counting chamber, after which the number of pollen grains in
the sample was counted under a light microscope at ×10 magnification.
Moreover, we counted pollen grains remaining in the anthers of the
flowers following the methods described above. Pollen removal per flower
was calculated as the mean number of pollen grains per flower minus the
remaining grains per flower. Finally, pollination efficiency was
calculated by dividing pollen deposition by pollen removal.

\textbf{Statistical analyses}

For the pollination treatments, a generalized linear model (GLM) with
binomial distribution and logistic-link function was used to detect the
effects of the treatments on the seed set (with seed number as event
variable, total ovule number as trial variable and different treatments
as factors) and fruit set (with fruit number as event variable, total
treated flower number as trial variable, and different treatments as
factors). We also performed a GLM with normal distribution and
identity-link function to test for differences in the visitation rates
(with visitation rate as the dependent variable, and pollinator species
as factors) and pollination efficiency (with pollination efficiency as
the dependent variable, and pollinator species as factors) of various
pollinator species. To compare pollen removal and pollen deposition
among pollinator species, data were analyzed with a GLM with Poisson
distribution and loglinear-link function (with pollen number as a
dependent variable and different treatments as factors). We carried out
a Pearson correlation analysis to determine the relationship between the
body size and the pollination efficiency of various pollinator species
in \emph{H. mutabilis} . All statistical analyses were performed in SPSS
V. 19.0 (SPSS Inc., USA).

\textbf{Results}

\textbf{Floral traits}

On average, each\emph{H. mutabilis} flower produced 72813 ± 1221 pollen
grains and 24.61 ± 1.08 \selectlanguage{greek}μ\selectlanguage{english}L nectar volume (Table 1), suggesting that
flowers offered plenty of floral rewards for visitors. Each flower
produced 241 \selectlanguage{ngerman}± 3 ovules, and the pollen/ovule ratio was 303 ± 6. The
Corolla diameter is 128.79 ± 0.54 mm and the corolla height is 30.97 ±
1.15 mm, suggesting that this species has a large corolla.

\textbf{Breeding system}

The GLM analysis showed that pollination treatments have significant
influences on fruit set (Wald \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 9.348, df = 3, P =
0.009) and seed set (Wald \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 22.018, df = 3, P
\textless{} 0.001) (Fig. 3). Under natural conditions, the fruit set and
seed set were 65 \selectlanguage{ngerman}± 7.6 \% (N = 40) and 80.4 ± 2.6 \% (N = 26)
respectively, which were significantly lower than hand self-pollination
(87.5 ± 5.3 \%, N = 40, \emph{P} = 0.004, and 90 ± 0.9 \%, N = 35,
\emph{P} \textless{} 0.001, respectively) and cross-pollination (85 ±
5.7 \%, N = 40,\emph{P} = 0.014, and 87.3 ± 1 \%, N = 34, \emph{P} =
0.002, respectively). It suggested that there was a pollen limitation
under natural conditions. There was no significant difference in the
fruit set between self-pollination and cross-pollination, indicating
that \emph{H. mutabilis} is highly self-compatible (Fig. 3). The fruit
set of bagged flowers was zero (N = 40), which indicated that neither
spontaneous autogamy nor apomixis occurs in this species.

\textbf{Foraging behavior of pollinators}

At our study site,\emph{H. mutabilis} was visited by five insect species
(Fig. 4), including four bee species (\emph{Xylocopa appendiculata} ,
\emph{Xylocopa dissimilis} ,\emph{Bombus breviceps} , and \emph{Apis
mellifera} ) (Fig. 4A\selectlanguage{english}~F) and one moth species (\emph{Macroglossum
pyrrhosticta} ) (Fig. 4G and H). The body length (12.58 +- 0.18 mm, N =
10) and intertegular distance (3.29 +- 0.07 mm, N = 10) of \emph{A.
mellifera} were the smallest among five visitor species (Wald
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 4738.98, df = 4, \emph{P}\textless{} 0.001, and
Wald \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 1877.37, df = 4,\emph{P} \textless{} 0.001)
(Fig. 5B and C), but the visitation rate (0.92 \selectlanguage{ngerman}± 0.08 visits per flower
per 30 min, N = 128) of\emph{A.mellifera} was the highest among five
visitor species (Wald \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 253.16, df = 4, \emph{P}
\textless{} 0.001) (Fig. 5D). The body length (31.72 \selectlanguage{ngerman}± 0.18 mm, N = 10)
and intertegular distance (6.52 ± 0.08 mm, N = 10) of \emph{M.
pyrrhosticta} were relatively larger than that of \emph{X. dissimilis}
(21.37 ± 0.22 mm and 5.59 ± 0.09 mm, N = 10, \emph{P} \textless{} 0.001)
(Fig. 5A and B), \emph{B. breviceps} (17.96 ± 0.22 mm and 5.52 ± 0.03
mm, N = 10, \emph{P}\textless{} 0.001) (Fig. 5B and C), and \emph{A.
mellifera} (\emph{P}\textless{} 0.001) (Fig. 5B and C), but the
visitation rate (0.12 ± 0.02 visits per flower per 30 min, N = 128) of
\emph{M. pyrrhosticta} was relatively lower than that of \emph{B.
breviceps} (0.38 ± 0.05 visits per flower per 30 min, N = 128, \emph{P}
\textless{} 0.001) (Fig. 5D), and \emph{A. mellifera} (\emph{P}
\textless{} 0.001) (Fig. 5D). All visitor species did not actively
collect the pollen grains of \emph{H. mutabilis} . When they entered the
corolla of flowers to forage for nectar, they would remove pollen grains
from mature anthers and deposit pollen grains on the stigma. Therefore,
all visitor species served as effective pollinators of \emph{H.
mutabilis} .

\textbf{Pollination efficiency}

The GLM analysis showed that the pollen removal (22750 ± 456, N = 20)
and pollen deposition (748 ± 33, N = 20) of \emph{X. appendiculata} were
the most among five visitor species (Wald \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 428.94,
df = 4, \emph{P}\textless{} 0.001, and Wald \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} =
369.91, df = 4,\emph{P} \textless{} 0.001) (Fig. 6A and B). The
pollination efficiency of \emph{X. appendiculata} (0.033 \selectlanguage{ngerman}± 0.002) and
\emph{X. dissimilis}(0.032 ± 0.002) were relatively higher than that of
the other three pollinator species (Wald \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 187.35,
df = 4, \emph{P} \textless{} 0.001) (Fig. 6C). In terms of four bee
species, the pollen removal (11556 \selectlanguage{ngerman}± 548, N = 20) and pollen deposition
(306 ± 19, N = 20) of \emph{A. mellifera} were the fewest (Wald
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 181.87, df = 4, \emph{P}\textless{} 0.001, and
Wald \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 164.78, df = 3,\emph{P} \textless{} 0.001)
(Fig. 6A and B), and the pollination efficiency of \emph{A. mellifera}
were the lowest (Wald \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 10.17, df = 4, \emph{P} =
0.02) (Fig. 6C). The pollen removal (5896 \selectlanguage{ngerman}± 459, N = 20) and pollen
deposition (19 ± 4, N = 20) of \emph{M. pyrrhosticta} were relatively
fewer than that of four bee species (\emph{P} \textless{} 0.001) (Fig.
6A and B), and the pollination efficiency (0.004 ± 0.001, N = 20) of
\emph{M. pyrrhosticta}was also relatively lower than that of four bee
species (\emph{P}\textless{} 0.001) (Fig. 6C).

\textbf{Relationships between pollen transfer and body size of
pollinators}

Pearson correlation analysis showed that there were significant positive
correlations between the body length of five pollinator species and
pollen removal, pollen deposition, and pollination efficiency
(\emph{P}\textless{} 0.05) (Table 2), suggesting that the body length
increased the amount of transferred pollen. When the moth species was
excluded, the intertegular distance of four bee species was
significantly and positively correlated to pollen removal and pollen
deposition (\emph{P} \textless{} 0.05) (Table 2), suggesting that the
larger bees tended to remove more pollen from anthers and deposit more
pollen on stigmas. However, there was no correlation between the
intertegular distance and pollination efficiency (Table 2).

\textbf{Discussion}

Our investigations demonstrated that the species \emph{H.
mutabilis}showed a mixed mating system, and its sexual reproduction
depended on pollinators. The floral reward (nectar) of \emph{H.
mutabilis} was consumed by various visitors, including four bee species
and one moth species. The pollination efficiency of the moth species was
relatively lower than all bee species. The body length of five
pollinator species increased the amount of transferred pollen, and
larger bees tended to remove more pollen from anthers and deposit more
pollen on stigmas, resulting in higher pollination efficiency. However,
there was no correlation between the intertegular distance and
pollination efficiency of visitors in \emph{H. mutabilis} . Body length
may be a better index of body size than intertegular distance.

The nectar of\emph{H. mutabilis} was not displayed at the base of the
petals but at the base of the ovary. So the flower structure of \emph{H.
mutabilis} shielded visitors with shorter proboscis. The best match
between the length of the proboscis and the circumference of the nectar
secretion at the base of the flower is the \emph{M. pyrrhosticta} ,
which suggests the \emph{M. pyrrhosticta} should be the most important
pollinator in its native habitat (Fig. 5A). However, the investigation
results found that bees were still important pollinators of \emph{H.
mutabilis} . Although the proboscis length does not match the
circumference of the basal nectar, they can suck in nectar from the gap
between the front petals. Different proboscis lengths of bees could
absorb different amounts of nectar, which can improve the chances of
pollen removal and pollen deposition.

Although the moth species\emph{M. pyrrhosticta} had the longest body
length among the five visitor species, its pollination efficiency was
relatively lower than all bees. The interpretation of the relationship
between body size and pollination efficiency may depend on the influence
of other morphological traits of \emph{M. pyrrhosticta} . Unlike bees,
the moth without hairiness may carry a few pollen grains. Moreover, the
moth has a long proboscis so that they can get nectar easily without
entering deep into the corolla of flowers. In this case, the moth may
have little contact with floral sex organs. In terms of four bee
species, the body length of \emph{X. appendiculata} was 1.8× longer than
that of honey bee \emph{A. mellifera} (Fig. 5B). Moreover, the pollen
removal and pollen deposition of \emph{X. appendiculata} were 1.9× and
2.4× more than that of \emph{A. mellifera}(Fig. 6A and B), and the
pollination efficiency of \emph{X. appendiculata} was 1.2× more than
that of\emph{A. mellifera} (Fig. 6C). These results demonstrated that
larger bees removed and deposited more pollen than smaller bees in a
single visit, and larger bees were more efficient pollinators in
\emph{H. mutabilis} . The reason was that larger bees were more likely
to come into contact with floral sex organs. This finding is partly
consistent with a previous study, which indicated that larger bee
species deposited more pollen grains onto stigmas than smaller bees in
\emph{Brassica napus} (Chang \emph{et al} . 2023), but they did not
consider the effect of body size on pollen removal and pollination
efficiency. Besides, we found that the intertegular distance was not an
effective index of body size, because there was no correlation between
the intertegular distance and pollination efficiency of visitors in
\emph{H. mutabilis} . Only the moth species was excluded, the
intertegular distance of four bee species was significantly and
positively correlated to pollen removal and pollen deposition. However,
there were always significant positive correlations between the body
length of four or five pollinator species and pollen removal, pollen
deposition, and pollination efficiency. We concluded that body length
may be a better index of body size than intertegular distance.

The visitation rate of bees is another important behavioral parameter.
Honey bee \emph{A. mellifera} was the most frequent visitor to flowers
of \emph{H. mutabilis} . The visitation rate of \emph{A. mellifera} was
5.1× more than that of \emph{X. appendiculata} , even though the
pollination efficiency of \emph{A. mellifera} was relatively lower. The
high visitation rate of bees to flowers may increase the pollinator's
interaction with the stigma or pollen transfer, so \emph{A.
mellifera}was also a very efficient pollinator in \emph{H. mutabilis} .
Although the pollination efficiency of \emph{X. appendiculata} was
higher than honey bees, they visited fewer flowers than honey bees.
Therefore, it is difficult to comprehensively assess which insect
species contributes more pollination service to flowers in \emph{H.
mutabilis} , and further study is needed to investigate the pollination
service of various pollinators to flowers throughout the flowering
period of plants.

The flowers of \emph{H. mutabilis} had a generalist pollination system
and attracted various pollinator species by providing plenty of nectar
rewards. Although \emph{H. mutabilis} was highly self-compatible, there
was pollen limitation under natural conditions. The sexual reproduction
of \emph{H. mutabilis} depended on pollinators. The visitation rate and
pollination efficiency of various pollinator species were greatly
different. The species \emph{H. mutabilis} with a mixed mating system
may be an adaptation to variable pollinator visiting.

\textbf{Acknowledgements}

The authors sincerely thank Shuang-Quan Huang and Deng-Fei Li for their
comments on this article.

\textbf{Funding}

This research was supported by the Project of Chengdu Botanical Garden
(NO. 401627).

\textbf{Conflicts of interests}

The authors declare that they have no confict of interest.

\textbf{Authors contribution}

Authors of the article include: Xiao-Qing Shi, Bin Zheng, Xiao-Li Liu,
Qiu-Mei Quan, Yun-Xiang Li. The experimental design of this article is
guided by Qiu-Mei Quan; Experimental site survey, receipt collection and
analysis were completed by Xiao-Qing Shi and Bin Zheng; The article was
illustrated by Xiao-Li Liu; Yun-Xiang Li provided guidance and objective
proofreading for the article, and all authors contributed to further
revisions. All authors read and approved the final manuscript.

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