4
Discussion
To our knowledge, the present
study represents a significant contribution to understanding variation
in sex ratios across life stages and the underlying mechanisms in the
dioecious trees. Sex-specific molecular markers were applied for the
first time to the study of sex ratios in a fully mapped population of a
dioecious tree species and revealed a female-biased sex ratio among all
tagged and mapped individuals of Diospyros morrisiana in a 50-ha
forest plot, which was significantly different from the male-biased
flowering sex ratio (or OSR). More importantly, D. morrisiana was
distinctly female-biased in early life stages, but gradually shifted to
be male-biased in later life stages. We also tested several ecological
mechanisms known to shape sex-specific differences in both life-history
traits and spatial pattern. We found that the onset of reproduction had
a larger impact on the growth of female trees than that of males, which
may account for the ontogenetic shift in sex ratio. Moreover, there was
no spatial segregation between male and female trees, except for the
scale smaller than 2 m.
The OSR of flowering individuals of D. morrisiana in the HSD
50-ha plot was significantly male-biased (Figure 2a), consistent with
previous findings in congeners (House 1992, Somanathan and Borges 2000,
Venkatasamy et al. 2007). A systematic review also reported that the
mean sex ratio based on flowering individuals of 88 dioecious tree
species was significantly male-biased (Field et al. 2013). However, in
our study we also identified the sex of all tagged individuals in the
plot through sex-specific molecular markers and found that the
population sex ratio (including all juvenile, immature, and
non-flowering trees > 1 cm DBH) was actually female-biased
(Figure 2a), confirming for the first time that the OSR does not
represent the true overall population sex ratio in dioecious tree
population. The disparity between the sex ratios of the OSR and the full
population stemmed from sex-specific differences in flowering
probability. We found that male trees were more likely to flower than
females (Figure 3a, S1), which resulted in the D. morrisianapopulation with a female-biased population sex ratio displaying a
male-biased OSR. In particular, saplings had a low flowering probability
and were therefore extremely underrepresented in the OSR, but saplings
were the most female-biased of all life stages. The gap between OSR and
population sex ratio in D. morrisiana supports the results and
inferences of previous studies on sex ratios in dioecious trees, showing
male-biased flowering sex ratios and the effects of a greater
reproductive investment by females in long-lived growth forms such as
trees (Lloyd and Webb 1977, Field et al. 2013). Even so, it would be
beneficial to apply sex-specific molecular markers more extensively when
investigating sex ratios in dioecious plants, especially in trees.
Another important finding was that the sex ratio of D. morrisianashifted from female- to male-biased through later life stages (Figure
2b). The sex ratio in the seedling stage and for large trees was biased
in opposite directions, which is consistent with predictions of sex
allocation theory for species with overlapping generations (Werren and
Charnov 1978, West 2009). Our study provides the first observational
evidence supporting this prediction in dioecious trees. The proportion
of female individuals gradually declined across life stages, likely
resulting in more intense mate competition occurring among males than
females.
Thus,
it could be suggested that the female-biased primary sex ratio might
have been favored by selection. Through variation in the sex ratio
across life stages, we could postulate how genetic and ecological
mechanisms have jointly driven the ontogenetic shift of sex ratio. A
distinctly female-biased sex ratio in seedlings may potentially suggest
the involvement of genetic mechanisms at the early stage. As the genusDiospyros has an XY sex-determination system, restricted
recombination between X and Y chromosome would lead to the accumulation
of deleterious genes in Y chromosome, which could potentially affect the
survival rate of malesn (Charlesworth and Charlesworth 1981, Ming et al.
2011, Akagi et al. 2014, Pilkington et al. 2019). Multiple studies have
found that Y-bearing pollen would be less produced in meiosis or be
inferior than X-bearing pollen in certation, while male ovules may also
have a lower probability of development and
germination (Correns 1922,
Błocka-Wandas and Sliwinska 2007, Stehlik et al. 2007).
Different costs of reproduction between male and female trees likely
explains much of why the population sex ratio was increasingly
male-biased in later life stages (Figure 2). Female Diospyrosproduce large fruit, with a greater investment of carbon, nitrogen, and
other nutrients than the male individuals invest in flowers and pollen.
This differential cost prohibits many females from maturing at the same
small size as males (e.g., Figure 3a, Figure S1), from flowering as
frequently as males, and also leads to lower growth rates in larger
mature females compared to similar-sized males (Figure S3).
Specifically, in flowering individuals, male trees at the medium-tree
stage that experienced more reproduction events had a significantly
larger RGR than females at the same stage, while there was no difference
in RGR between male and female saplings or small trees that seldom
flowered (Figure S3). Our results are in line with previous studies of
dioecious animal-dispersed trees in which females exhibit a greater
reproductive investment (Cipollini and Whigham 1994, Obeso 2002,
Queenborough et al. 2007, Barrett and Hough 2013, Field et al. 2013).
Among flowering individuals, male
trees had a higher probability of reflowering, so it was quite likely
that males reproduced more times than females during the five-year
census interval. However, the lower flowering frequency of female trees
did not offset their higher reproduction costs, which suggested that the
difference in the costs of one single reproduction event between males
and females would be greater.
We may have underestimated the effects of this difference in the costs
of reproduction on the population dynamics in this species. Mature
females had lower growth than males, but a higher cost of reproduction
in females could also lead to lower survival.
Unfortunately, due to the timing
of our sampling was too close to the second census, there were
insufficient molecular-sexed individuals died during the intervening
period. Therefore, we were unable to draw definitive conclusions about
potential mortality differences between the sexes. Both lower growth and
survival rate of females caused by higher reproductive costs would
result in a decreasing proportion of females as size increases, and
subsequent plot census will help us decouple the influence of
sex-specific growth and survival on the shift in sex ratio. Besides,
there are signs that sex-specific mortality unrelated to reproductive
investment could be also responsible to the shift of sex ratio (Shelton
2010). We found that the sex ratio of individuals became less
female-biased from saplings to small trees (Figure 2b), a period when
flowering individuals was scarce and RGR between males and females were
not significantly different. This shift suggests that female saplings
(i.e., juveniles) may incur higher mortality than males, which was not
driven by reproduction cost per se. Further monitoring of the saplings
is needed to confirm a sex-specific difference in mortality at this
stage.
Sex-based differences in life-history traits could lead to sex-specific
habitat preferences, and may be manifested as sexual segregation in
spatial pattern (Bierzychudek and Eckhart 1988). However, we did not
detect any evidence of sexual spatial segregation among the tagged
individuals, except for distances <2 m (Figure 4, Figure S1).
Generally, because the scale at
which environmental factors operate is always broader than the scale at
which individuals interact, this small-scale spatial segregation is less
likely to be attributed to sex-specific preferences for microhabitats,
but was instead more likely caused by inter-sexual competition (Harms et
al. 2001, Valencia et al. 2004, Wiegand and Moloney 2013, Timerman and
Barrett 2019). Specifically, for D. morrisiana trees, the crown
and underground root system spread far beyond 2 m, which indicates that
environmental variation occurring only within 2 m would not lead to a
difference in the response of male and female trees. In support of this
inference, He et al. (2021) demonstrated that seedlings of D.
morrisiana were more competitive when grown with opposite-sex neighbors
than with same-sex ones in a greenhouse experiment, and suggested that
differences in root exudates between the sexes may mediate the
inter-sexual competition (He et al. 2021).