Introduction
Plant breeding system is the sum of various physiological and
morphological mechanisms that control the relative frequency of
outcrossing or self-crossing, which is an important part of reproductive
ecology and a hotspot of evolutionary ecology
(Andrus, 1963). On present trends, more
and more ecological researches focus on the ecology of plant
reproduction (McCallen et al., 2019). As
the core of the breeding system, the mating system represents the mating
pattern of individuals in a population, which fundamentally answers the
question of how individuals mate and the relative frequency of
selfing-outcrossing (Barrett, 1998). The
study of plant mating systems and pollination ecology plays a vital role
in the cultivation and utilization of plants and the understanding of
evolutionary processes. For example, by research on the reproductive
biology of Clematis vitalba L., a process of reproductive
limitations could be used to establish an intrusion barrier, as reducing
the damage caused by the invasive plant on the original
(Redmond & Stout, 2018). Another is that
by the research of the breeding system and inbreeding depression in a
translocated population of the endangered plant Dianthus
morisianus (Caryophyllaceae), the cause of its protection could be
understood and as well as the development of appropriate management
measures (Nebot, Cogoni, Fenu, &
Bacchetta, 2020). Reproductive ecology, with plant reproduction as its
core, organically combines the interaction and mutual adaptation between
plant and environment, biological and abiotic factors
(Baucom, Heath, & Chambers, 2020). Due to
the characteristic of fixed growth of plants, pollen must reach the
stigma successfully through certain media to achieve reproductive
success (Thomson & Page, 2020).
Therefore, pollination system is also significant in the process of
plant reproduction. Combining flower morphology and pollination system
research can objectively understand flower characteristics and their
evolutionary driving forces. Research on the reproductive ecology of
species is helpful to understand the pollination mechanism of plants,
reproductive strategies and adaptive evolution to the environment. It
provides a theoretical basis for formulating scientific and feasible
measures for biodiversity conservation and management to make better use
of germplasm resources. As people pay more attention to the protection
of plant diversity, the reproduction characteristics and flowering
biological characteristics of more and more species have been revealed
step by step (Montagna, Silva, Pikart, &
Reis, 2018; Pool-Chale, Ramirez-Morillo,
Fernandez-Concha, & Hornung-Leoni, 2018;
Roccotiello et al., 2009;
Yamasaki & Sakai, 2013).
Although completely outcrossing or completely selfing mating systems are
generally regarded as a stable reproduction strategy, there are few
plant groups that are absolutely outcrossing or selfing in nature
(Goodwillie, Kalisz, & Eckert, 2005).
Both models have their own advantages and disadvantages. Selfing will
reduce the genetic diversity of individuals and the fitness of offspring
due to the expression of harmful traits with higher gene homozygosity
(Takebayashi & Morrell, 2001).
Outcrossing can accumulate higher levels of genetic variation, but it
must be at the cost of increasing investment in reproductive resources
(reflected in increasing the attraction of zoophily)
(Barrett, 1998;
Prasifka et al., 2018). Therefore, in
order to weigh the pros and cons of selfing and outcrossing, most plants
choose a mixed mating system model
(Goodwillie et al., 2005). Mating between
self and closely related individuals is called inbreeding, and the
degree of inbreeding between individuals varies due to differences in
genetic history (Schoen & Baldwin,
2022). When plants cannot avoid being selfing due to various
mechanisms, inbreeding depression becomes a selective force that
regulates the mating dynamics of the population
(T. Han et al., 2021;
Husband & Schemske, 1996), which
contributes to the evolution and maintenance of the mating system that
plays an important part.
V. negundo var. heterophylla is widely used in slope
restoration projects for water and soil conservation because of its
developed root system, drought tolerance, and barren tolerance
(Xu et al., 2019). It is a common water
and soil conservation shrub in northern mountainous and hilly areas and
an important nectar plant in China especially in summer
(Du, Guo, Zhang, & Wang, 2010). Flowers
of Vitex species are attractive to diverse pollinators, including
butterflies, honeybees, and bumblebees (Ashoke and Sudhendu 2012, Jain
2013, Murren 2014). The flowers of Vitex species are usually
hermaphroditic (Schmidt, 2000). Sinébou et al.
(2016) had studied the reproductive ecological characteristics ofVitex doniana (the endangered African tree species), that this
species had hermaphrodite flowers, as well as were adichogamy. A variety
of insects of Hymenoptera were effective pollinators forVitex doniana , and there was no obstacle for seed germination in
the offspring of flower visitors. Vitex rotundifolia was found to
be capable of both zoogamy and agamogenesis, with strong invasive
potential, and pollinator activity directly affecting fruit set and seed
setting rate, which may be the mechanism leading to the current
successful sexual reproduction (Murren et al. 2014). Compared with other
species in the same genus, V. negundo L. var. heterophyllaexhibited a shorter single flowering period of only 1-2 days, and the
previous flower was usually withered when the next flower opened, which
greatly reduced the occurrence of geitonogamy
(Sun et al., 2020). Sun et al. also
observed in their study that the single flowering period of V.
negundo L. var. heterophylla was indeed prolonged by rainy
weather, and the same phenomenon was observed in Vitex doniana(Sinébou et al. 2016). The above mechanisms were
beneficial for plants to increase the time and probability of
entomophily in the absence of pollinators, and was considered to be for
ensure reproductive success.
However, recent pharmacological studies of V. negundo L. var.heterophylla had shown a variety of its activities, such as the
antioxidative effect (Hu et al., 2017),
the antidiabetic effect (Djimabi et al.,
2022), the anti-inflammatory (Xu et al.,
2019), and the wound-healing activities
(L. Han et al., 2008) and few reproductive
researches have been conducted (Yan et
al., 2023).
Global pollinator decline and land-use change may lead to pollination
limitation (Goulson, Nicholls, Botias, &
Rotheray, 2015). In animal-pollinated plants, the intensity of
interactions with pollinators can be quantified by the degree of pollen
limitation of female fitness, that is, the extent to which seed
production is limited by insufficient pollination (Trunschke et al.,
2017). Manual pollination as a valuable tool can improve pollination
control. In recent years, research on V. negundo var.heterophylla has mostly focused on environmental adaptation,
chemical composition, clinical application and seed germination
(Sun et al., 2020). However, there are
few studies on the mating system of V. negundo var.heterophylla . Reddy and Reddi (1994) found that the flowers ofVitex negundo (Lamiaceae) were homogamous, herkogamy,
self-compatible and exhibited a facultative xenogamous breeding
system. The scented flowers ofVitex negundo (Verbenaceae) were studied to be hermaphrodite and
pollinated by entomophily (Reddy et al., 1994). Kumar et al. (2017)
carried out a detailed observation of the breeding system of Vitex
chinensis in India, and the results were similar to the previous
studies. The flowers of Vitex chinensis were homogamous,
herkogamy, self-compatible and exhibited a facultative xenogamous
breeding system. Flowers ofVitex negundo L. (Lamiaceae) were found to be self-compatible and
promote xenogamy with narrow chance of spontaneous selfing in nature.
(Khan S et al., 2021). The RAPD and cpDNA (chloroplast DNA) molecular
markers were used to study the genetic structure of 10 populations ofVitex negundo (Verbenaceae) in two regions along the Three Gorges
River (Zhang, Zheng, & Ge, 2007). At
present, studies on the genetic diversity of V. negundo var.heterophylla are new but relatively few. Liu et al. (2018)
combined functional traits with two molecular markers, which were MSAP
(methylation sensitive amplification polymorphism) and AFLP, to analyze
phenotypic, genetic and epigenetic variation in natural populations ofV. negundo var. heterophylla in different habitats. And
the results showed that there were significant relationships between
epigenetics and genetic variation, epigenetics and phenotypic variation.
Liu et al. (2019) have developed 14 pairs of expressed sequence tags and
simple repeat sequence (EST-SSR) marker primers for this species, which
provided useful resources for the study of reproduction and genetic
ecology of V. negundo var. heterophylla . Therefore, there
are not many studies on the reproductive ecological characteristics ofV. negundo var. heterophylla at present. Our study plans
to conduct a standardized understanding of reproduction information such
as the mating system of V. negundo var. heterophylla ,
which is implemented through artificial pollination experiments.
In order to clarify the reproduction strategy of V. negundo var.heterophylla, different kinds of artificial pollination were
conducted in the present study. Artificial pollination refers to the
artificial transfer of plant pollen to the stigmata in order to improve
seed yield, or directional change of plant species. Four indicators of
fruit setting rate, fruit weight, germination rate and germination
energy were detected to compare the quality of the progeny of different
artificial pollination to evaluate the success of their reproduction.
This study makes up for the lack of research on the breeding system ofV. negundo var. heterophylla in China and even in the
world, and also provides a theoretical background and technical
reference for the subsequent research on the pollination biology and the
mating systems of relatives of this species.