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\begin{document}

\title{Molecular sexing reveals ontogenetic shifts in sex ratios and underlying
processes in a dioecious tree species}



\author[1]{Wei Lin}%
\author[2]{Yonghua Zhang}%
\author[3]{Simon  Queenborough}%
\author[4]{Ming Ni}%
\author[5]{Qing He}%
\author[5]{Bu-Hang Li}%
\author[5]{Chengjin Chu}%
\affil[1]{Sun Yat-Sen University}%
\affil[2]{Wenzhou University}%
\affil[3]{Yale University}%
\affil[4]{Université de Sherbrooke}%
\affil[5]{Sun Yat-sen University}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Most dioecious plants are trees. However, because of the difficulty in
determining sex from vegetative morphology, previous investigations of
the sex ratios of dioecious trees were limited to flowering individuals,
leading to inadequate and potentially unreliable data on patterns of sex
ratios and the underlying mechanisms driving their variation. Here, we
applied sex-specific molecular markers to investigate the sex ratio of a
fully mapped population of the dioecious tree \emph{Diospyros
morrisiana} (Ebenaceae) in a subtropical forest. We also investigated
the sexual dimorphism of life-history traits and spatial association
between male and female trees to determine potential processes shaping
the sex ratio at different life stages. Molecular sexing revealed a
female-biased population sex ratio for this \emph{D. morrisiana}
population, contrasting with the male-biased operational (i.e.,
flowering) sex ratio. The sex ratio of \emph{D. morrisiana} shifted from
female-biased to male-biased over older life stages. We found that
reproduction had a larger impact on the growth of female trees, which
may account for the ontogenetic shift in sex ratio. There was no
evidence of spatial segregation of the sexes beyond a scale of 2 m.
Through molecular sexing of all individuals across all life stages, our
work revealed for the first time a shift from a female- to a male-biased
sex ratio in a huge population of a dioecious tree species. To better
understand variation in sex ratios and the underlying mechanisms in
dioecious trees, the sex of non-flowering and juvenile individuals
should be included in future studies.%
\end{abstract}%



\sloppy


\textbf{Molecular sexing reveals ontogenetic shifts in sex ratios and
underlying processes in a dioecious tree species}

Wei Lin\textsuperscript{1\#}, Yonghua Zhang\textsuperscript{2\#}, Simon
A. Queenborough\textsuperscript{4}, Ming Ni\textsuperscript{5}, Qing
He\textsuperscript{1}, Buhang Li\textsuperscript{1}, Chengjin
Chu\textsuperscript{1,3*}

1 State Key Laboratory of Biocontrol and School of Life Sciences, Sun
Yat-sen University, Guangzhou, 510275, China

2 College of Life and Environmental Sciences, Wenzhou University,
Wenzhou, 325035, China

3 State Key Laboratory of Biocontrol and School of Ecology, Sun Yat-sen
University, Guangzhou, 510275, China

4 Yale School of the Environment, Yale University, New Haven, CT 06511,
USA

5 D\selectlanguage{ngerman}épartement de Biologie, Université de Sherbrooke, Sherbrooke, J1K
2R1, Canada

\# Authors contributed equally to this paper.

* Corresponding author: Chengjin Chu; Mailing address: Xingang Xi Road
135, Sun Yat-sen University, Guangzhou, 510275, China; Tel:
(+86)2084111541; E-mail:\emph{chuchjin@mail.sysu.edu.cn}.

\textbf{Keywords}

Dioecy, operational sex ratio (OSR), sex-specific molecular marker,
reproduction cost, sex allocation theory, spatial point pattern analysis

\section*{Abstract}

{\label{abstract}}

Most dioecious plants are trees. However, because of the difficulty in
determining sex from vegetative morphology, previous investigations of
the sex ratios of dioecious trees were limited to flowering individuals,
leading to inadequate and potentially unreliable data on patterns of sex
ratios and the underlying mechanisms driving their variation. Here, we
applied sex-specific molecular markers to investigate the sex ratio of a
fully mapped population of the dioecious tree \emph{Diospyros
morrisiana} (Ebenaceae) in a subtropical forest. We also investigated
the sexual dimorphism of life-history traits and spatial association
between male and female trees to determine potential processes shaping
the sex ratio at different life stages. Molecular sexing revealed a
female-biased population sex ratio for this \emph{D.
morrisiana}population, contrasting with the male-biased operational
(i.e., flowering) sex ratio. The sex ratio of \emph{D. morrisiana}
shifted from female-biased to male-biased over older life stages. We
found that reproduction had a larger impact on the growth of female
trees, which may account for the ontogenetic shift in sex ratio. There
was no evidence of spatial segregation of the sexes beyond a scale of 2
m. Through molecular sexing of all individuals across all life stages,
our work revealed for the first time a shift from a female- to a
male-biased sex ratio in a huge population of a dioecious tree species.
To better understand variation in sex ratios and the underlying
mechanisms in dioecious trees, the sex of non-flowering and juvenile
individuals should be included in future studies.

\section*{1 Introduction}

{\label{introduction}}

Dioecious plants are phylogenetically widespread among angiosperms
(Renner 2014) and often play crucial roles in various ecosystems,
especially forests (Chazdon et al. 2003, Lin et al. 2020, Wang et al.
2020). Dioecy prevents selfing and potentially improves reproductive
efficiency, but also raises a greater risk of population decline driven
by an imbalance in the sex ratio (Charlesworth and Charlesworth 1981,
Henry et al. 2018), which may be exacerbated by climate change (Eckert
et al. 2010, Petry et al. 2016, Hultine et al. 2016). Thus, it is
imperative to undertake more thorough investigations into variation in
the sex ratios of dioecious plants as well as the underlying mechanisms
that drive variation in maturity and other life-history traits that
differ between male and female individuals.

The majority of dioecious species are trees with a long life-span
(Renner and Ricklefs 1995, Wang et al. 2021b), but studies of the sex
ratios of these taxa suffer from several difficulties. One primary
challenge is that the sex of immature or non-flowering individuals is
difficult to identify through vegetative traits (Lloyd and Webb 1977,
Barrett and Hough 2013). Most previous studies have therefore been able
to use only flowering individuals (i.e., the operational sex ratio, OSR)
in order to estimate the population sex ratio (Barrett and Hough 2013,
Field et al. 2013). However, using the OSR could generate unreliable and
biased results because the sex ratio of all non-flowering individuals is
not necessarily the same as the sex ratio of flowering individuals. This
problem is especially acute for long-lived tree species, since their
populations often include a high proportion of these immature and
non-flowering plants (Nanami et al. 1999, Ueno et al. 2007, Queenborough
et al. 2007). To address this issue, sex-specific molecular markers can
be used to provide more accurate and reliable information on population
sex ratios, because they are not restricted to flowering individuals
(Eppley 2001, Stehlik and Barrett 2005, Shelton 2010). These markers
have mostly been developed for applied research in agriculture,
forestry, horticulture, and medicine, and few studies have applied
molecular approaches to provide accurate and comprehensive assessments
of the sex ratios of dioecious trees in natural field environments (Ling
et al. 2003, Zhang et al. 2016, Liao et al. 2017, Zhou et al. 2018).

Interestingly, sex ratios can change across plant life stages from
juveniles to adults, the study of which is critical for addressing when
and how sex ratio biases are established (Eppley 2001, Stehlik and
Barrett 2005, Stehlik et al. 2007). Because trees are often most
vulnerable to competition, herbivory, and pathogen attack at juvenile
stages (Moles and Westoby 2004, Comita et al. 2014), the population sex
ratio could change dramatically at these stages if one sex is more
likely to survive than the other. However, the OSR cannot be used across
a number of these significant stages, because the sex of juvenile trees
(seeds, seedlings, and saplings) and other non-flowering individuals are
not included in the OSR. Several studies have applied the molecular
sexing approach to reveal shifts in the ontogenetic sex ratio (Taylor
1994, Eppley 2001, Stehlik et al. 2007, Shelton 2010). For example,
Stehlik et.al (2007) found that the perennial herb \emph{Rumex nivalis}
had a slightly female-biased sex ratio using flow cytometry, which
shifted to be even more female-biased in later life stages (Stehlik et
al. 2007). However, these studies have been limited to herbaceous plants
with a relatively short life-span, and variation in the sex ratios
across the life stages of trees with a long life-span has not been
investigated. Sex allocation theory predicts that the juvenile sex ratio
is negatively correlated with the adult sex ratio in species with
overlapping generations like trees (West 2009). In species with
overlapping generations, individuals not only compete for mates within
their own generation but also with individuals from the previous
generation. For example, if females are less available among adult
individuals, female offspring will face less competition for mates
compared to males, resulting in an increased reproductive value for
females. Consequently, when the sex ratio of adult individuals is biased
towards males, a primary sex ratio biased towards females would be
preferred (Werren and Charnov 1978, López and Domínguez 2003, Booksmythe
et al. 2018). But, to our knowledge, this hypothesis has not obtained
any empirical support from tree species.

Understanding the mechanisms that drive variation in sex ratio is
critical, and can be broadly grouped into two categories: genetic and
ecological (Stehlik and Barrett 2005, Che-Castaldo et al. 2015). A
variety of genetic mechanisms could drive divergence from a 1:1 sex
ratio at the seed stage, including segregation distortion, Y chromosome
degeneration, and sex-selective abortion (Taylor 1994, Stehlik and
Barrett 2005, Stehlik et al. 2007). The early life stages of dioecious
trees, such as seedlings, can better reflect such gene-determined sex
ratios. Variation in sex ratio at later life stages is mainly influenced
by ecological mechanisms involved in sex-related differences in resource
allocation trade-offs towards growth, reproduction, and defense
(Queenborough et al. 2007, Timerman and Barrett 2019). For example,
females generally allocate more energy to reproduction, resulting in
later maturity, lower flowering frequency, and higher mortality of
female individuals and thus a male-biased operational sex ratio (Lloyd
and Webb 1977, Delph 1999, Barrett and Hough 2013, Field et al. 2013).
Furthermore, if one sex has a different environmental preference
compared to another due to different reproductive inputs, a biased sex
ratio could occur in heterogeneous environments (Freeman et al. 1976),
and may even lead to spatial segregation of the sexes (Nanami et al.
1999). It should be noted that spatial segregation between two sexes
would also occur when inter-sexual competition is stronger than
intra-sexual competition (Bierzychudek and Eckhart 1988). Multiple
studies have compared the growth pattern and spatial distributions
between sexes to explore mechanisms influencing the operational sex
ratios in dioecious trees based on flowering individuals (Nanami et al.
1999, Queenborough et al. 2007, Jácome-Flores et al. 2016, Timerman and
Barrett 2019). However, as stated above, these results might be biased
because of the understandable exclusion of non-flowering and immature
individuals.

To address this gap in current studies of the sex ratios of dioecious
trees, we applied a molecular sexing approach to investigate variation
in the sex ratio across life stages and to determine the underlying
mechanisms in the dioecious tree \emph{Diospyros morrisiana} (Ebenaceae)
in the Heishiding 50-ha forest dynamics plots located in southern China.
Specifically, we identified the sex of all 2,255 tagged individuals in
the plot and 349 germinated seedlings through sex-specific molecular
markers. Combined with analyses of growth dynamics and the spatial
patterns of tagged individuals, we address the following questions:

(1) Does the field-observed OSR accurately reflect the overall
population sex ratio of \emph{D. morrisiana} , and how does the sex
ratio change across life stages? We hypothesize that OSR differs
significantly from the overall population sex ratio. Additionally, we
expect considerable changes in the sex ratio of \emph{D. morrisiana}
across life stages. Specifically, the adult trees are more likely
male-biased, but juvenile trees are more likely female-biased.

(2) What processes are responsible for the shift in the sex ratio
of\emph{D. morrisiana} in later life stages? We hypothesize that the
sex-specific difference in the growth--reproduction trade-off will lead
to a slower growth rate and higher mortality in female trees, resulting
in a sex ratio that is increasingly male-biased in later life stages; We
also tested whether male and female trees are spatially separated and
discussed the potential cause of spatial pattern.

\section*{2 Materials and Methods}

{\label{materials-and-methods}}

\subsection*{\texorpdfstring{\emph{2.1 Study site and study
species}}{2.1 Study site and study species}}

{\label{study-site-and-study-species}}

The study was conducted in a 50-ha (1000 × 500 m) forest dynamics plot
within the Heishiding (HSD) Nature Reserve, Guangdong Province, southern
China. The region has a subtropical moist monsoon climate with an
average annual temperature of 19.6°C. The mean annual precipitation is
1743.8 mm, the majority of which falls from April to September. The HSD
50-ha plot was initiated in 2011 and all woody stems with diameter at
breast height (DBH, 1.3m) \selectlanguage{english}[?] 1 cm were tagged, identified, measured, and
georeferenced following the field protocols of the ForestGEO network
(Condit 1998). A second census was conducted from January 2016 to March
2017, and all living stems and new recruits were surveyed as above.

\emph{Diospyros morrisiana} Hance ex Walp. (Ebenaceae) is a dioecious
deciduous shrub or small tree inhabiting slopes, ravines, or streamsides
in East Asian monsoon subtropical forests between 100 and 1000 m a.s.l
(Wu and Raven 1997). It is a dominant species in early and
mid-successional subtropical forests in southern China and can grow up
to 20 m in height (Abbas et al. 2021). The flowers are small (about 7mm
long), white, and urn-shaped. Male plants have axillary cymose
inflorescence with 2--3 flowers, whereas female flowers are solitary.
The fruits are fleshy berries, yellow when ripe, with a sweet flavor.
The flowering season is from May to June, and fruits are ripe in
November. The flowers are bee-pollinated, whereas seeds are dispersed by
civets (\emph{Paguma larvata} and \emph{Viverricula indica} ,
Viverridae) (Corlett 1996, 2001).

\subsection*{\texorpdfstring{\emph{2.2 Phenotypic sexing and molecular
sexing}}{2.2 Phenotypic sexing and molecular sexing}}

{\label{phenotypic-sexing-and-molecular-sexing}}

In May--June and September 2017, all tagged individuals of \emph{D.
morrisiana} in the plot were censused. Flowering individuals were
recorded and leaves of all individuals were simultaneously collected for
molecular sexing. Phenotypic sexing process in the field was undertaken
by visual identification, based on flower structure and pedicels or
fruit stalks remaining on the branches. Flowering individuals were
revisited in May--June 2018 and their reproductive condition was
recorded.

To obtain the sex ratio at the seedling stage, we collected seeds from
the canopies of 16 female \emph{D. morrisiana} in the HSD 50-ha plot in
December 2017. After soaking in distilled water for 1 h and then
sterilizing with 25\% perhydrol for 1 h, a total of 2,202 seeds from 16
female trees were planted in plastic seedling trays (28 cells, 7 x 7 x
14 cm each) filled with peat soil in March 2018. The plants were
routinely irrigated with Hoagland solution. After 3 months, we recorded
the number of germinated seedlings for each parent tree, collected the
leaves of all seedlings for molecular sexing.

We used a molecular marker to determine the sex of each individual tree
and seedling as follows. Total genomic DNA of \emph{D}
.\emph{morrisiana} was extracted from the silica-gel dried leaves using
DNA Plantzol (Lifefeng, Hangzhou, China) according to the manufacturer's
protocol with slight modifications. Silica-dried leaves (0.3 g) were
ground by a high-through tissue grinder (Scientz-48, Ningbo, China) with
magnetic beads and 1\% PVP for 400 s, and mixed with 850 \selectlanguage{greek}μ\selectlanguage{english}L Plantzol.
After incubating at 65\selectlanguage{ngerman}°C for 30 min at 600 rpm in MTH-100 Shaker
Incubator (Thermo Scientific, Waltham, USA), an equal volume of
phenol:chloroform (24:1) was added, and the tubes were turned upside
down for 80 times and centrifuged (12,000 rpm, 20 min, 4°C). DNA was
precipitated by mixing the aqueous phase with 520 \selectlanguage{greek}μ\selectlanguage{english}L isopropanol, turned
upside down gently for 100 times and centrifuged (10,000 rpm, 20 min,
4\selectlanguage{ngerman}°C). The DNA pellet was washed with 800 \selectlanguage{greek}μ\selectlanguage{english}L 75\% ethanol, dried and
dissolved in an 80 \selectlanguage{greek}μ\selectlanguage{english}L TE buffer {[}10 mM Tris-HCl (pH 8.0), 1mM EDTA{]}.
After isolation, DNA quality was checked by gel electrophoresis and
qualified samples were stored in a -20\selectlanguage{ngerman}°C refrigerator.

For molecular sexing, we used the primers
\emph{OGI-candF1}(5'-CACAGTAGTCATATATTTTTAGC-3')/
\emph{OGI-spR}(5'-CTGGCACACAAAATATTTTCAACCCT-3') developed by Akagi
(Akagi et al. 2014). The PCR reaction mixture contained a total volume
of 20 \selectlanguage{greek}μ\selectlanguage{english}L including 1 \selectlanguage{greek}μ\selectlanguage{english}L template DNA, 1 \selectlanguage{greek}μ\selectlanguage{english}L forward and reverse primers
(TsingK, Beijing, China), and 10 \selectlanguage{greek}μ\selectlanguage{english}L 2 \selectlanguage{ngerman}× EasyTaq PCR SuperMix (+dye)
(Transgene, Guangzhou, China). Amplifications were performed using the
following program: initial denaturation step at 94°C for 3 min; 30
cycles consisting of denaturation step at 94°C for 30 s, primer
annealing step at 58°C for 30 s and primer extending step at 72°C for 90
s; and final extending step at 72°C for 7 min. Additionally, we designed
and used the primers \emph{OGI\_500-F} (5'-ACATACAACCAAGCGGAACTG--3')
and\emph{OGI\_500\_R} (5'-AACAGTGCCACCTTCCTTGA -3'), specific to the
homologous regions of the \emph{OGI} and \emph{MeGI} genes. Notably,
\emph{MeGI} is an autosomal homolog of \emph{OGI} and present in both
male and female individuals. The reaction mixture and program were the
same as described above except for the temperature of the primer
annealing step (52°C) and extension time (60 s). The PCR amplification
products were examined on a 1.0\% agarose gel contaning ethidium
bromide. The gels were imaged using a Tangunon 4100 Gel Image Analysis
System (Tanon Science49 \& Technology Co., Ltd.). The PCR products of
\emph{OGI-candF1/OGI-spR} showed an approximately 1000bp band only for
males, while the PCR products of\emph{OGI\_500\_F/OGI\_500\_R} showed
bands for both sexes. Using two primer sets simultaneously limits false
negative results and thus improves the credibility of the sex
identification. We examined the results of molecular sexing for
reproductive individuals, and found it was in full agreement with field
observation, which proved the feasibility of our molecular approach.

\subsection*{\texorpdfstring{\emph{2.3 Sex ratios and sexual dimorphism
in life-history
traits}}{2.3 Sex ratios and sexual dimorphism in life-history traits}}

{\label{sex-ratios-and-sexual-dimorphism-in-life-history-traits}}

To track the ontogenetic shift in the sex ratio of this population
of\emph{D. morrisiana} , we chose 5 and 10 cm as cut-off points to
divide the tagged individuals of \emph{D. morrisiana} alive in the
second census into three DBH classes, and trees \selectlanguage{english}[?] 2 cm DBH were further
divided out because most flowering individuals are \textgreater{} 2 cm
DBH (98.7\%), resulting in the following five stages: seedlings,
saplings (1 \textless{} DBH \selectlanguage{english}[?] 2 cm), small trees (2 \textless{} DBH \selectlanguage{english}[?] 5
cm), medium trees (5 \textless{} DBH \selectlanguage{english}[?] 10 cm), and large trees (DBH
\textgreater{} 10 cm).

Following the results of determining the sex of individuals using
phenotypic and molecular methods, we calculated the OSR and population
sex ratio for the whole population and for each size class. Sex ratios
were expressed as the proportion of male plants to all individuals. To
infer the sex ratios in population of \emph{D. morrisiana} , we used
Bayes' theorem to estimate the posterior distribution of sex ratios
based on binomial likelihood and a uniform prior. We calculated the mean
value, standard deviation, and 95\% credible interval to summarize the
posterior distribution and determine whether each sex ratio is biased.
Calculations were performed with the statistical software R (R Core Team
2020) in the package \emph{rethinking} (McElreath 2020).

To assess for sex-related differences in flowering, growth, and
survival, we compared the flowering proportion, DBH, relative growth
rate (RGR, log difference of DBH in two censuses divided by 5 years),
and mortality between the two sexes. The flowering proportion was
expressed as the number of flowering individuals as a proportion of all
individuals of a given sex. For flowering individuals, we compared
flowering proportion in 2017 between the sexes. In addition, we used a
generalized linear model to evaluate the effects of sex and DBH on
whether individuals flowered or not. Further, we compared DBH and RGR
for males and females among flowering individuals and molecular-sexed
individuals, as well as RGR between flowering and non-flowering
individuals using a Kolmogorov--Smirnov test. We also compared RGR of
males and females in each size classes and used a generalized linear
model to evaluate the effects of sex and DBH on RGR.

\subsection*{\texorpdfstring{\emph{2.4} \emph{Spatial association between
the
sexes}}{2.4 Spatial association between the sexes}}

{\label{spatial-association-between-the-sexes}}

To infer processes responsible for the sex ratio pattern of \emph{D.
morrisiana} population from its spatial distribution, we performed
spatial point pattern analyses for all tagged individuals. We first
tested if there was spatial segregation between male and female trees
through a mark connection function. We calculated the mark connection
function \emph{p(r)} , which gives the probability that two trees
separated by a distance \emph{r} are of the same sex or opposite (Illian
et al. 2008). It follows that the sum of all mark connection functions
equals one. If the sexes are randomly distributed over the trees, the
mark connection functions are constants derived from the proportions of
male, \emph{p\textsubscript{M}} , and female, \emph{p\textsubscript{F}}
, and \emph{p\textsubscript{MM}(r) = p\textsubscript{M}}
,\emph{p\textsubscript{FF}(r)} = \emph{p\textsubscript{F}}
,\emph{p\textsubscript{MF}(r)} = \emph{p\textsubscript{FM}(r)}
=\emph{p\textsubscript{M}} * \emph{p\textsubscript{F}} . To test if the
observed pattern deviated from the expected value, we compared the mark
connection functions for the observed pattern to a random labeling null
model which fixed the locations of all trees and shuffled the sexes. It
indicates the existence of spatial segregation of the sexes
if\emph{p\textsubscript{MF}(r)} is smaller than the expected value of
null model. To detect small-scale spatial structure more precisely, we
additionally used a local random labeling null model that switched only
sexes of trees located within 30 m, a scale somewhat larger than the
typical range of plant--plant interactions that is widely affirmed in
tropical forest and our study sites (Harms et al. 2001, Uriarte et al.
2004, Wiegand et al. 2007, Wang et al. 2021a).

To determine whether the observed spatial patterns deviated from the
null models, we calculated point-wise simulation envelopes with a
significance level \selectlanguage{greek}\emph{α} \selectlanguage{english}= 0.05 from the 25th highest and 25th lowest
values of 999 Monte Carlo simulations of each null model. To avoid the
Type I error inflation caused by simultaneous inference, we also
constructed a global envelope to jointly evaluate whether observed
pattern deviated from the null models significantly (Myllym\selectlanguage{ngerman}äki et al.
2017). All spatial point pattern analyses were performed in the
software\emph{Progamita} (Wiegand and Moloney 2013).

\section*{3 Results}

{\label{results}}

\subsection*{\texorpdfstring{\emph{3.1 Sex ratio shifted from
female-biased to male-biased during
ontogeny}}{3.1 Sex ratio shifted from female-biased to male-biased during ontogeny}}

{\label{sex-ratio-shifted-from-female-biased-to-male-biased-during-ontogeny}}

In 2017, a total of 297 \emph{D. morrisiana} trees on the plot flowered,
181 males and 116 females, giving a male-biased operational sex ratio
(OSR) of 0.61 (95\% CI: 0.56--0.65). Among flowering trees, 45.3\% of
males and 34.5\% of females were observed flowering again the following
year. In contrast, among the 2,255 tagged individuals \textgreater{}1 cm
DBH, the molecular sexing technique identified 1,068 male and 1,187
female trees (Figure 1), giving a significantly female-biased population
sex ratio of 0.47 (95\% CI: 0.46--0.49) (Figure 2a).

The flowering OSR of \emph{D. morrisiana} was male-biased in each size
class from small to large trees (Figure 2a). Although more male saplings
(1--2cm) flowered, the number of total flowering individuals (n = 4) at
this stage was too small to give a significant result (Table S1). In
contrast, molecular sexing revealed that the population sex ratio
shifted from female-biased in saplings to male-biased in large trees
(Figure 2b).

The sex ratio of seedlings followed this same general pattern. Of the
2,203 seeds we collected from 16 mother trees, only 359 seeds from 12
mother trees germinated and survived to produce leaves. We were able to
determine the sex of 349 seedlings. The overall sex ratio of seedlings
from all the mother trees was strongly female-biased (0.38, 0.34--0.43)
(Table S2). Because of the low numbers of seedlings from each mother
tree, most sex ratios were statistically insignificant, except for
seedlings of one mother tree (C) which were significantly female-biased.

\subsection*{\texorpdfstring{\emph{3.2 Sexual dimorphism in life-history
traits}}{3.2 Sexual dimorphism in life-history traits}}

{\label{sexual-dimorphism-in-life-history-traits}}

We found significant differences in flowering proportion, tree size
(DBH), and growth (RGR) between the two sexes (Figure 3). Male trees
exhibited a higher flowering proportion than females (Figure 3a). Model
results also showed that male trees had higher probability of flowering
than female trees (Figure S1). When comparing all individuals together,
male trees had a larger DBH than females, although this difference was
not statistically significant (\emph{D} = 0.05, \emph{p}= 0.07) (Figure
3b, S2b). In contrast, the DBH of flowering female trees was slightly,
but not significantly, larger than the DBH of flowering males (\emph{D}
= 0.07, \emph{p} = 0.92) (Figure 3b, S2a).

Across all individuals, there was no significant difference between the
RGR of males and females (\emph{D} = 0.03, \emph{p} = 0.70) (Figure 3c,
S2d). However, in flowering individuals only, RGR was significantly
higher in males than in females (\emph{D} = 0.19, \emph{p} \textless{}
0.01) (Figure S2a). The RGR of flowering females was significantly lower
than that of non-flowering females (\emph{D} = 0.20, \emph{p}
\textless{} 0.01), whereas the RGR of flowering and non-flowering males
did not differ significantly (\emph{D}= 0.07, \emph{p} = 0.46). In the
comparison of RGR at each life stage for flowering individuals,
flowering male trees had higher RGR than flowering females at the
medium-sized stage (\emph{D} = 0.27, \emph{p}\textless{} 0.01), while
there was no significant difference between RGR of males and females in
small flowering trees (\emph{D} = 0.18, \emph{p}= 0.78) (Figure S3). The
RGR of male trees was also higher than that of females in flowering
large-sized trees, but it was not significant (\emph{D} = 0.20, \emph{p}
= 0.45) (Figure S3). Model results also showed that the RGR of male
trees was higher than that of females only in flowering individuals but
not in all individuals pooled (Figure S4).

During the period between the two censuses, 465 trees died, but only 43
of these trees were sexed, 21 of which were females and 22 of which were
males, yielding a mortality rate of 1.77\% for females and 2.06\% for
males.

\subsection*{\texorpdfstring{\emph{3.3 Spatial segregation of
sexes}}{3.3 Spatial segregation of sexes}}

{\label{spatial-segregation-of-sexes}}

For all tagged individuals of \emph{D. morrisiana} in the HSD plot, we
found that males and females were spatially segregated only at small
scales (Figure 4, S5). The mark connection
function\emph{p\textsubscript{MF}} (\emph{r} ) was below the simulation
envelopes within 2 m, which indicated that the probability of both
female and male trees appearing together at this scale was lower than
the expectation of the random labeling null model (Figure 4b). On the
other hand,\emph{p\textsubscript{MM}} (\emph{r} )
and\emph{p\textsubscript{FF}} (\emph{r} ) lay well inside the global
simulation envelopes for all distances \emph{r} , exhibiting no
repulsion or attraction within the sexes.

\section*{4 Discussion}

{\label{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 \emph{Diospyros morrisiana} in a 50-ha forest plot, which
was significantly different from the male-biased flowering sex ratio (or
OSR). More importantly, \emph{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 \emph{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 \textgreater{} 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 \emph{D.
morrisiana}population 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 \emph{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 \emph{D.
morrisiana}shifted 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
genus\emph{Diospyros} 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,
B\selectlanguage{polish}ł\selectlanguage{english}ocka-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
\emph{Diospyros}produce 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 \textless{}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 \emph{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 \emph{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).

\section*{Conclusion}

{\label{conclusion}}

In summary, through the novel application of molecular sexing of all
individuals in a large forest plot, our study revealed an ontogenetic
shift in the sex ratio of a dioecious tree population for the first
time, and shed light on the factors driving this change. A higher cost
of reproduction for females likely leads to an increasingly male-biased
sex ratio in later life stages, and Y-chromosome degeneration may
account for the pronounced female-biased sex ratio at early stages. The
opposite sex ratios of seedlings and large trees were in accordance to
the expectations of sex allocation theory. The difference between OSR
and true population sex ratio supported previous results based on the
sexing of flowering individuals that males invest less in reproduction,
and so can flower at smaller sizes, more frequently, and may grow faster
than females. Further studies of sex ratio in a wider variety of
dioecious tree species using sex-specific markers are needed to obtain
more comprehensive knowledge for sex ratio patterns and process of
dioecious trees, and to improve our understanding of the ecology and
evolution of breeding systems in plants.

\section*{Authors' contributions}

{\label{authors-contributions}}

C.J. Chu, Y.H. Zhang and W. Lin conceived and designed the study. Y.H.
Zhang and W. Lin performed the experiments and data analysis. B.H. Li
conducted the forest census. Y.H. Zhang and W. Lin contributed materials
collection. W. Lin wrote the paper. C.J. Chu, S.A. Queenborough, M. Ni,
Q. He, and Y.H. Zhang revised the paper.

\section*{Acknowledgement}

{\label{acknowledgement}}

This study was funded by the National Natural Science Foundation of
China (31800309 to YHZ, 31925027 to CC) and the Scientific Research
Project of Baishanzu National Park (Grant No. 2021KFLY06).

\section*{Data Availability
Statement:}

{\label{data-availability-statement}}

Data are provided as private-for-peer review via the following link
https://datadryad.org/stash/share/S153-14snQidELyJ-Uy3wr2\_SwNIfenljDIbyIelj0A
and will be permanently archived in Dryad (https://doi.org/
10.5061/dryad.vt4b8gtzj, not active yet) once the manuscript is
accepted.

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\textbf{Figure 1} Spatial point pattern for male (black points) and
female (red points) individuals of \emph{Diospyros morrisiana} at the
HSD 50-ha plot. Opaque points represent flowering individuals, while the
transparent points represent non-flowered individuals that have been
molecular-sexed. The grey scale represents the elevation.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 2} Posterior probability distribution of the operational
sex ratio (OSR) and sex ratio for all tagged individuals (a) and sex
ratio at each life stage (b) of \emph{Diospyros morrisiana} at the HSD
50-ha plot. The dashed line and shaded area in (a) refer to the mean
value and 95\% credible interval of distribution. Values plotted in (b)
are the sex ratio at different life stages (with 95\% credible
interval). Intervals on the x-axis in (b) are the range of diameter at
breast height (DBH) for each life stage in cm. The long dark dashed line
indicates an unbiased ratio.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 3.} The sex-based difference in life-history traits
including flowering proportion (a), diameter at breast height (DBH) (b),
and relative growth rate (RGR) (c) of \emph{Diospyros morrisiana} at the
HSD 50-ha plot. The size of points on (a) represents the number of
molecular-sexed individuals in each size class. Notably, flowering
individuals were also molecular-sexed. The boxplots show difference in
DBH (b) and RGR (c) between males and females in flowering and
molecular-sexed individuals, with each individual represented as a dot.
The thick horizontal line indicates the median, the box represents
interquartile range (IQR), and the whiskers reach the maximum and
minimum value within 1.5 * IQR. \emph{p} -value significance level: not
significant (ns) \textgreater{} 0.05, * \textless{} 0.05, ** \textless{}
0.01.\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

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\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

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\textbf{Figure 4} Spatial association within and between sexes
in\emph{Diospyros morrisiana} based on mark connection function\emph{p}
(\emph{r} ). \emph{pMM} (r) and \emph{pFF} (\emph{r} ) detect the
strength of association within males (a) and females (c),
while\emph{pMF} (\emph{r} ) detects spatial segregation between sexes
(b). Local random labelling was used as the null model, which switches
the sexes of points closer than 30m. Observed and expected mark
connection function \emph{p} (\emph{r} ) under the null model are
represented by solid (black) and dashed (red) lines respectively. Shaded
areas represent 95\% pointwise simulation envelopes (dark grey boundary)
and global envelopes (light grey boundary) constructed using the 25th
highest and lowest values of 999 Monte Carlo simulations of the null
model. Values outside the envelopes indicate significant departure from
the randomness.

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