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.