Seed germination rate and germination energy
Both of seed germination energy (F=7.508, P <0.01; Figure 3c)
and germination rate (F=7.931, P <0.01; Figure 3d) had a
significant correlation with pollination methods. And the above two
indicators showed the same trend among different pollination modes.
The germination energy (27.6%) of xenogamy (X) was significantly higher
than autogamy (SP, 15.2%) and geitonogamy (G, 16.0%), and there was no
significant difference between open pollination (OP, 24.4%). Similarly,
the seed germination rate of xenogamy (X, 60.0%) was significantly
higher than autogamy (SP, 32.8%) and geitonogamy (G, 33.6%), and there
was no significant difference between open pollination (OP, 55.2%).
There was no statistical difference in seed germination energy and
germination rate between autogamy (SP) and geitonogamy(G).
Discussion
Through controlled pollination experiments, it was found that V.
negundo L. var. heterophylla can produce mature fruits under
both outcrossing (the highest fruit-setting rate) and selfing
strategies, indicating that V. negundo L. var.heterophylla presents a mixed mating system with mainly
outcrossing and self-compatibility. It is consistent with the conclusion
of Sun et al. (2020) based on the hybridization index and pollen ovule
ratio.
The maintenance of the mixed mating system is the result of continuous
evolution under the selective pressure of outcrossing and selfing. It
can not only adapt to the plant itself and environmental conditions, but
also ensure the successful reproduction of plants under environmental
mutations. The dire state of insect biodiversity in the world due to the
serious decline in insect diversity due to the widespread use of
agricultural pesticides, as almost half of species are rapidly declining
and one-third are threatened with extinction
(Sánchez-Bayo & Wyckhuys, 2019). When
outcrossing cannot be carried out under the conditions of habitat change
or restricted pollination, choosing selfing can provide a certain amount
of protection for the reproduction of plants
(Lloyd, 1992). Other species of the genusVitex have also been reported to have mixed mating systems in
which selfing and outcrossing coexist, such as V. lucens(Barrell, Richardson, & Gardner, 1997),V. doniana (Sinebou, Quinet,
Ahohuendo, & Jacquemart, 2016), and V. rotundifolia(Murren et al., 2014) and V.
fischeri (Lengkeek, Mwangi, Agufa,
Ahenda, & Dawson, 2006).
In present artificial pollination experiments, we also found that
autonomous selfing cannot happen to V. negundo L. var.heterophylla , and there is no apomixis.
Anemophily (WP) can only produce very
few offspring (fruit setting rate is 0.5%), and the effect on allogamy
is minimal. Therefore, despite the coexistence of entomophily and
anemophily flower morphology, this species eventually reproduces
successfully. It still depends entirely on the service level of
zoophily. In order to adapt to insect pollination, V. negundo L.
var. heterophylla also exhibits a series of special floral
morphology: (1) In most Vitex species, the stamens and styles are
located near the upper lip, conforming to the esophageal type of flower
described by Van der Pijl (1972). V. negundo L. var.heterophylla has a bilabial flower that is symmetrical, and the
lower lip of the bilabial corolla is flat and broad, which can be used
as a comfortable platform for insects to forage (Sun et al. ,
2021). (2) The terminal panicle tightly gathers the flowers together,
reducing the time for insects to fly and search for nectar or pollen,
which is conducive to the maximum use of their own energy for foraging;
(3) The flower secretes nectar and releases fragrance during the
flowering process to attract insects (Van der Pijl, 1972).
A similar situation also occurred in Thalictrum pubescens , an
ambophilous (both zoophily and anemophily) species that probably
represents a transitional state in the evolution of anemophily. And the
ambophily was also studied in Olea ferruginea , over a period of
three years. It was an andromonoecious and out-crossing tree species of
the olive complex. The species was predominately pollinated by wind
because it showed fruit set even when the insect pollinators were
excluded (Khan, Kumari, & Verma, 2022).Mallotus spp. (Euphorbiaceae) japonicus and Mallotus
wrayi exhibited floral characteristics that were adapted to both wind
and insect pollination, ambophily may be actively maintained in the two
species at the study sites and perhaps
elsewhere(Yamasaki & Sakai, 2013). Other
studies had shown that the flowers of Crateva adansonii(Capparaceae) exhibited traits conducive to a mixture of wind and insect
pollination (ambophily). Although a variety of insects visited the
flowers, they were ineffective in pollinating. Nevertheless, active
foraging by the honeybees (Apis dorsata, A. mellifera andA. cerana indica ) facilitated enhanced pollen dispersal in the
air and resulted in indirect pollination by wind
(Mangla & Tandon, 2011). While zoophily
mediate, the advantage of anemophily will be weakened
(Labouche, Richards, & Pannell, 2017).
In addition, the seed setting rate of V. negundo L. var.heterophylla is a constantly 25%, which means that only one of
the four ovules can fully develop and bear fruit, and the other three
will be aborted due to late zygotic competition. The same phenomenon is
found in V. fischeri (Lengkeek et
al., 2006) and V. Doniana (Sinebou
et al., 2016) is also reported. This is not due to pollen limitation,
but may be related to resource allocation. Research by K.C. Zoeller et
al. shows that artificial pollination of two species of geoflorusProtea species (Proteaceae) has not improved their seed setting
rate, and resources are limited (Zoeller,
Midgley, Johnson, & Steenhuisen, 2017).
Inbreeding depression was defined as the reduced fitness of selfing
progeny relative to outcrossed progeny, it was a frequent phenomenon in
plants that can be expressed at various stages in the life-cycle
(Charlesworth D & B Charlesworth 1987; Husband & Schemske, 1996). It
was a common experimental technique to judge the degree of inbreeding
depression by germination rate and germination energy. Ramsey et al,
germinated selfing and outcrossed seeds from the same Blandfordia
grandiflora plants and estimated inbreeding depression in germination
under benign laboratory conditions and adverse field conditions. They
found that 60% of Blandfordia grandiflora expressed inbreeding
depression in the laboratory, whereas in the field all plants expressed
inbreeding depression. A similar example would be Echium
wildpretii (Boraginaceae) on Tenerife, Canary Islands. Researchers
investigated the fitness of progeny from experimental self- and
cross-pollination in eight populations of different size of Echium
wildpretii , and found that seed set of open- and
hand-outcrossed-pollinated flowers was higher in large than in small
populations, possibly due to more frequent biparental inbreeding in the
latter (Sedlacek, Schmid, Matthies, &
Albrecht, 2012). Through the study on combining the analysis of floral
morphology, behavior of flower visitors, and artificial pollination, the
reproductive characteristics of the Ziziphus jujuba var.spinosa were revealed (Wang et al.,
2021). In this study, through artificial pollination experiment, we
determined that the inbred progeny of V. negundo L. var.heterophylla had serious inbreeding decline in early seed setting
and germination stage, and it was difficult to grow into propagators.
We conclude that there was still inbreeding depression during the
process of V. negundo L. var. heterophylla seedlings. The
inbreeding depression occurs in various stages of life history of an
individual to varying degrees (Van Etten,
Soble, & Baucom, 2021; Voillemot &
Pannell, 2017). Species with different mating systems may have
different stages and degrees of inbreeding depression
(Van Etten et al., 2021). Inbreeding
depression of some species is mainly manifested in the early stage of
seed setting and germination, while others are mainly manifested in the
later stage of survival and growth
(Sinebou et al., 2016). When we evaluated
the quality of the progeny of V. negundo L. var.heterophylla under different pollination treatments, we found
that the fruit weight of V. negundo L. var. heterophyllaoutcrossing progeny was significantly higher than that of the selfing
progeny. Similarly, in the process of seed germination, the seed
germination rate and germination energy of outcrossing progeny were
significantly higher than that of selfing progeny, which indicated thatV. negundo L. var. heterophylla had different degrees of
inbreeding depression during seed maturation and germination.
From the above results, it can be inferred: the V. negundo L.
var. heterophylla is mainly autogamy, as well as be allogamy; It
has the phenomenon of selfing but cannot finish the autonomous selfing.
The V. negundo L. var. heterophylla depends on entomophily
for their pollination; The inbreeding progeny of V. negundo L.
var. heterophylla had serious inbreeding depression at early seed
setting and germination stage. It is worth to be discussed that in this
study, only the general observation of the visiting population ofV. negundo L. var. heterophylla was carried out during the
experiment, and there is no data to support a comprehensive
understanding of the species and number of pollinators and their
visiting behavior. Further studies are needed to investigate the types
and numbers of visitors, visiting frequency, staying time, and effective
pollinators. Whether the herkogamy in flower evolution of V.
negundo L. var. heterophylla is mediated by pollinators needs to
be further investigated by various experimental methods.
In this study, we conducted artificial pollination experiments to
further understand the mating system and other reproductive strategies
of V. negundo L. var. heterophylla. The results will
provide scientific basis for the protection of plant resources, the
effective use of slope protection function and the selection of wild
varieties of this species.
Study we explored only a general observation was made on the flower
visiting population of V. negundo L. var. heterophylla ,
without data to support a comprehensive understanding of pollinator
groups and their visiting behaviors, etc. Later, further studies should
be conducted on the species and number of flower visitors, visiting
frequency, stay time and effective pollinators. In addition, it is
necessary to pay more attention to the pollinator mediated floral
evolution of some herkogamy in Vitex sinensis population.