\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[ngerman,polish,english]{babel}



\usepackage{float}








\begin{document}

\title{Sexual dimorphism and phenology interplay determines arthropod
communities}



\author[1]{Ixchel González-Ramírez}%
\author[2]{Zenón Cano-Santana}%
\author[3]{Araceli Araceli Romero}%
\author[4]{Víctor López-Gómez}%
\author[5]{Johnattan Hernandez Cumplido}%
\affil[1]{University of California Berkeley}%
\affil[2]{Universidad Nacional Autonoma de Mexico}%
\affil[3]{Universidad Nacional Autónoma de México}%
\affil[4]{México Instituto Nacional de Ecología y Cambio Climático}%
\affil[5]{Universidad Nacional Autonoma de Mexico Facultad de Ciencias}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Intraspecific variation in plants is expected to have profound impacts
on the arthropod communities associated with them. Because sexual
dimorphism in plants is expected to provide consistent variation among
individuals of the same species, researchers have often studied the
effect it has on associated arthropods. Nevertheless, most studies have
focused on the effect of sexual dimorphism in a single or a few
herbivores, thus overlooking the potential effects on the whole
arthropod community. Our main objective was to evaluate effects of
Buddleja cordata's plant-sex on its associated arthropod community. We
surveyed 13 pairs of male and female plants every two months during a
year (June 2010-April 2011). Every sampling date, we measured plant
traits (water content and leaf thickness), herbivory, and the arthropod
community. We did not find differences in herbivory between plant sex or
through time. However, we found differences in water content through
time, with leaf water-content matching the environmental seasonality.
For arthropod richness, we found 68 morphospecies associated with female
and 72 with male plants, from which 53 were shared by both sexes. We did
not observe differences in morphospecies richness; however, we found
sex-associated differences in the diversity of all species and
differences on the diversity of the most abundant species with an
interesting temporal component. During peak flowering season male plants
showed higher values on both parameters, but during the peak
fructification season female plants showed the higher values on both
diversity parameters. Our research exemplifies the interaction between
plant-phenology and plant-sex as drivers of arthropod communities'
diversity, even when plant sexual-dimorphism is inconspicuous, and
highlighting the importance of accounting for seasonal variation. We
stress the need of conducting more studies that test this time-dependent
framework in other dioecious systems, as it has the potential to
reconcile previous contrasting observations reported in the literature.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

When studying interspecific interactions, we often ignore the potential
effects of intraspecific variation. But in the context of the
interactions between a plant and its associated arthropod community, the
individual-plant traits can have a profound impact on the arthropod
community because of the multiple ways in which a plant affects its
resident arthropods (Mooney et al. 2012, Abdala-Roberts et al. 2016,
Nell et al. 2018, Moreira et al. 2019). For example, for herbivore
arthropods, a plant not only constitutes a nutrient resource, but it
also provides shelter by modifying microenvironmental conditions like
temperature and humidity (Obeso 2002). Because a single plant commonly
interacts with more than one individual arthropod, it also influences
the arthropod community assemblage; thus, determining the
arthropod-arthropod interactions that individual arthropod residents
experience (Strong et al. 1984, Schoonhoven et al. 2005).

Because sexual dimorphism in plants is expected to provide consistent
variation among groups of individuals of the same species (i.e., males
vs females), researchers have often studied the effect that this type of
intraspecific variation has on arthropods. Differences between male and
female plants have been well documented on vegetative (Nell et al.
2018), reproductive (Delph 1999, Eckhart 1999, Barret and Hough 2013),
and defensive traits (Agren et al. 1999, Cornelissen and Stiling 2005,
Sargent and McKeough 2022). The effects of these differences on
arthropod communities have been explored, primarily through the study of
plant-herbivore interactions, like foraging preferences and densities
(Danell et al. 1985, Agren 1988, Elmqvist and Gardfjell 1988, Boecklen
et al.1990, Hjalt\selectlanguage{ngerman}én et al. 1993), or herbivory bias. For a long time,
studies pointed to a male-biased herbivory preference as the rule (Agren
1988, Agren et al. 1999, Cornelissen and Stiling 2005), but recently
Sargent and McKeough (2022) challenged that view through a meta-analysis
that revealed no consistent differences on chemical defenses and
herbivory between male and female plants. Even if this study suggests
that the effect of sexual dimorphism is not consistent across sexes or
plant-lineages, most studies still find an effect on herbivores due to
sexual dimorphism which may be given by the differences on the resource
allocation to reproduction in each sex (Cornelissen and Stiling 2005).

Why the effects of sexual dimorphism are not consistent across plant
groups might be due to sexual dimorphism being only one of the axes of
plant-phenotypic variation. One of the most drastic and consistent
changes that plants, particularly deciduous ones, experience are
phenological changes (Bawa and Opler 1975), and these have the potential
to interact and conflate the effects of sexual dimorphism on the
arthropod communities. Furthermore, most of the work regarding the
effect of sexual dimorphism on arthropod-plant interaction has focused
on understanding how differences in plant resource allocation affect
pairwise interactions (i.e., plant herbivore interactions). But by
affecting the herbivore populations, plant-sex might indirectly affect
arthropod carnivores through bottom-up forces (Oksanen1988, Chen and
Wise 1999, Gruner 2004; Han et al. 2022), so a community approach might
help to unravel patterns on the effect of plant sexual dimorphism in
arthropods. So far, very few studies have explored the effect of dioecy
on a multitrophic approach (Nell et al. 2018, Tsuji and Fukami 2018).
Nell et al. (2018) found that, in \emph{Baccharis
salicifolia}(Asteraceae), plant sex influenced plant traits like flower
number, relative growth of the plant, predator density, and arthropod
community composition. Tsuji and Fukami (2018) evaluated the effects of
dioecy in the shrubs \emph{Eurya emarginata} and \emph{E. japonica} on
the microbiome community associated with plant nectar. They found that
the microbes and fungi that colonize nectar were more abundant in male
flowers compared to female flowers; also they found that the composition
of microbial species was different between sexes.

The objective of this study was to use a community and year-long
(phenological) approach to evaluate the effects of plant sex in the
shrub \emph{Buddleja cordata} Kunth (Scrophulariacae), 1) on several
plant traits, 2) on the arthropod community associated to this plant
species and 3) on the guild of herbivores and carnivores that inhabit
these plants.

\textbf{METHODS}

\textbf{Study site}

We conducted the fieldwork in the Pedregal de San Angel Ecological
Reserve (PSAER or REPSA for its name in Spanish; 237 ha). This reserve
belongs to the National Autonomous University of Mexico and is located
on its main campus in Mexico City (19°18'-19°19' N, 99°10'-99°11' W). In
this reserve the most common vegetation type is a native xeric shrubland
dominated by \emph{Pittocaulon praecox} Cav. (Rzedowski 1954,
Cano-Santana 1994). The climate is temperate sub-humid with summer
rainfall (Castillo-Argüero et al. 2004). We report the temperature and
rainfall during the study year in Supplemental Materials I.

\textbf{Plant species}

\emph{Buddleja cordata} is a dioecious tree that is widely distributed
across the valley of Mexico in elevations between 2,250 to 3,000 m
(Vargas 2001). Its leaves are pubescent, elliptical, and 5.5 to 24 cm
long. Flowers are small, organized in large panicles and present from
June to February. This plant produces small fruits (2.5 to 6 mm) from
October to April (Norman 2000, Vargas 2001). Its height varies between 1
to 20 m, but in the REPSA, most individuals are shorter than 7 m. During
this study, \emph{B. cordata} flowered from June 2010 to September 2010,
and fructified from September 2010 to January 2011.

\textbf{Tree selection}

We conducted a paired design to compare arthropod communities associated
to male and female trees of \emph{B. cordata} . We selected thirteen
pairs of male and female individuals of \emph{B. cordata} with similar
traits: diameter at breast height (DBH), tree coverage (measured as a
vertical projection of exposed leaf area), and the tree height. Because
the environment in the reserve is highly heterogeneous, we chose pairs
of trees with individuals that were within 10 m of each other, to
control for any neighborhood effect and for any microclimate effect. We
report and statistically compare the characteristics of the trees in
Supplemental Materials II.

\textbf{Plant traits survey}

Every two months, from June 2010 to April 2011, we measured two
morphological traits known to potentially affect arthropod
plant-interactions in \emph{B. cordata} : leaf thickness and water
content (Agren et al. 1999).~ Leaf thickness was measured with a Vernier
on five leaves per tree. Water content was measured in five leaves per
plant, controlling for leaf age and size. Water content proportion was
obtained as:

Water content = (fresh weight-dry weight) / fresh weight

We also recorded herbivory rate as it is known to be related to plant
defense and it is by itself a record of plant-herbivore interactions
(Kersch-Becker et al. 2017). Herbivory rate depends on many traits such
as secondary compound concentration, nutrient content, and trichomes
abundance, and it is thus a proxy for herbivore's plant preference and
performance. Every two months, we marked six young undamaged leaves per
plant. Three weeks later, herbivory was estimated on those leaves as
follows:

HR= CLA/TLA

Where CLA is the consumed leaf area and TLA is the total leaf area. The
unit is leaf-area consumed for 21 days. Additionally, \emph{B.
cordata}sex-ratio was measured because differences in this trait may
indicate differences in the survival rate between sexes (Cornelissen and
Stiling 2005, Barrett and Hough 2013, Sargent and McKeough 2022) and
therefore differences in reproduction investment (Sinclair et al. 2012).

\textbf{Arthropod community sampling}

Every 2 months, from June 2010 to April 2011, we collected four terminal
branches of each tree (30-40 cm) in plastic bags and all the arthropods
were manually removed from the vegetal tissue in the laboratory.
Sampling was performed between 8:00 and 10:00 h, because arthropod
sampling is affected by the sampling time (Schoonhoven et al. 2005,
Schowalter 2006). Arthropods were classified into morphospecies, and
then identified to the finest taxonomic level possible. Finally,
herbivore, carnivore, and detritivore categories were assigned to each
morphospecies according to specialized literature and field observation.
Although we did not perform any formal test on this regard, we consider
that the effect of branch removal on this shrub had a negligeable effect
on the tree fitness given the small relative size of the branches that
were sampled in comparison with the plant.

\textbf{Statistical analysis}

\emph{Plant traits.} For leaf thickness and herbivory rate, we performed
a generalized linear model (GLM) with Poisson distribution and link: log
due to our data not meeting the assumption of a normal distribution even
after transformation. In the case of the leaf water content, we
performed a GLM with normal distribution. In the three cases, the effect
of sex (male and female), time, and their interaction were included in
the model as fixed factors. Finally, for sex-ratio, frequencies
of\emph{B. cordata} were compared to a null model (1:1 male-female
ratio) by using a \selectlanguage{english}?\textsuperscript{2} test.

\emph{Arthropod traits.} Abundance was calculated by tree and by time;
however, our data did not meet the assumptions of normality, then we
performed a GLM with a Poisson distribution and a log-link function. We
treated the effect of sex (male and female), time, and their interaction
as fixed factors.

For the diversity parameters, we first estimated sampling completeness
estimates using the iNext website (Hsieh et al. 2016). We evaluated the
effect of plant sex on the arthropod community. To evaluate the
richness, diversity, and evenness of the community of adult arthropods
between plant sex (male or female), we used the Hill number
(\textsuperscript{0}\emph{D} , \textsuperscript{1}\emph{D} ,
and\textsuperscript{2}\emph{D} ). The diversity of order 0
(\textsuperscript{0}\emph{D} ) correspond to the richness of observed
species, order 1 (\textsuperscript{1}\emph{D} ) corresponds to the
exponential of the Shannon-Wiener entropy index (\emph{H} '), and order
2 (\textsuperscript{2}\emph{D} ) is the diversity of the most dominant
species, which is equivalent to the inverse of the Simpson dominance
index (Jost 2006, Sanjuan-Trejo et al. 2021). Similarity among plants
and sex was estimated by using Jaccard's coefficient indices. We
assigned functional groups by categorizing the species into guilds
(herbivores, carnivores, and detritivores) based on literature and field
observations. We used a \emph{t} -test to compare the richness of
species in the guilds \emph{per} plant sex per collection time, but in
case our data did not meet the assumptions of normal distribution we
performed a Wilcoxon rank test.

\textbf{RESULTS}

\textbf{Plant traits}

There were no significant differences in leaf thickness of male and
female plants (\selectlanguage{english}?\textsuperscript{2}=0.11, P=0.73), across time
(\selectlanguage{english}?\textsuperscript{2}=2.93, P=0.70), or the interaction between both
variables (\selectlanguage{english}?\textsuperscript{2}=0.74, P=0.98). Herbivory rates
in\emph{B. cordata} did not show significant differences between plant
sex (\selectlanguage{english}?\textsuperscript{2}=0.02, P=0.87), across time
(\selectlanguage{english}?\textsuperscript{2}=2.90, P=0.40), or the interaction between both
variables (\selectlanguage{english}?\textsuperscript{2}=0.08, P=0.99). For the water content in
leaves, we found significant differences only across time
(\selectlanguage{english}?\textsuperscript{2}=0.31, P\textless{}0.001), whereas plant sex
(\selectlanguage{english}?\textsuperscript{2}=0.001, P=0.67) and the interaction between sex and
time (\selectlanguage{english}?\textsuperscript{2}=0.002, P=0.825) were not significantly
different. The highest amount of water in the leaves was observed in
August (male=0.64+-0.009; female=0.65+-0.007) and the lowest was during
February for both sexes (male=0.54+-0.009; female=0.54+-0.01)
(\textbf{Fig. 1} ). Finally, we detect significant differences on sex
ratio, showing a female-biased frequency of 3:2
(\selectlanguage{english}?\textsuperscript{2}=8.48, P=0.003, N=283).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} . Water content (mean \selectlanguage{ngerman}± s.e.) of male and
female\emph{B. cordata} plants from June 2010 to April 2011. Different
letters correspond to significant differences.

\textbf{Arthropods association to plant sex}

We collected a total of 1,961 arthropods in female and male trees
of\emph{B. cordata} . From these arthropods, we identified 87
morphospecies in 15 different orders--the list of morphospecies is in
Supplemental Materials III. We only considered adult morphospecies for
the subsequent analyses.~For arthropod abundance, we did not detect
significant differences between plant sex (P=0.62); however, time and
the interaction between sex*time had a significant effect on arthropod
abundance (\selectlanguage{english}?\textsuperscript{2}=1488.9, P\textless{}0.0001 and
\selectlanguage{english}?\textsuperscript{2}=16.08, P=0.006 respectively). The highest abundance
of arthropods was recorded during the first collection date decreasing
with time (\textbf{Fig 2} ). When we compared species richness by plant
sex, 68 morphospecies were found in female plants and 72 in male plants,
from which 53 species were shared between sexes, which corresponds to a
0.61\% Jaccard's similarity index. Then, according to Hill numbers, we
did not detect differences on the effective number of species per plant
sex (\textsuperscript{0}\emph{D} ) across time (\textbf{Fig. 3} ). When
we tested the exponential of Shannon's entropy
(\textsuperscript{1}\emph{D} ) and the inverse of Simpson's index
(\textsuperscript{2}\emph{D} ), they present an interesting reverse
pattern. Diversity was higher in male plants for both metrics early in
the season, then during the middle of the season they show no
differences between sexes whereas by October-November both metrics
display a reverse pattern, having females showing higher diversity and
higher evenness (\textbf{Fig. 3} ).

Sixty four out of 87 morphospecies found in this study were classified
into a particular guild. These morphospecies contained 1,810 specimens
(92\% of all arthropods collected). When compared with a\emph{t}
-student-paired test, neither herbivores nor carnivores showed
significant differences (\selectlanguage{english}?\textsuperscript{2}=0.05, P=0.81;
\selectlanguage{english}?\textsuperscript{2}=0.14, P=0.70; Figure 4). When we evaluated richness
per guild per time, again herbivores did not show significant
differences between males and females at any time; however, carnivores
did show significant differences in June and December. Indeed, more
carnivores were found in male plants in June (\selectlanguage{english}?\textsuperscript{2}=4.98,
d.f.= 1, P=0.02; Figure 4), then after five months we found a reverse
pattern with more carnivore species found in female plants
(\selectlanguage{english}?\textsuperscript{2}=3.94, d.f.= 1, P=0.04; Figure 4). Regarding
detritivores, only seven morphospecies (24 individuals) were found in
this guild, so a statistical analysis was not possible.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2} . Abundance of arthropods (mean \selectlanguage{ngerman}± s.e.) collected on
male and female \emph{Buddleia cordata} plants from June 2010 to April
2011. Different letters correspond to significant differences.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} . Interpolation and extrapolation curves of richness
and arthropod diversity on male and female trees of \emph{Buddleia
cordata} across three times. Continuous line in each curve corresponds
to the interpolated portion, the dotted line represents the extrapolated
portion and the symbol in each graph represents the observed diversity.
Watermark bands represent the 95\% confidence intervals. We show the q
of diversity expressed as the effective number of species. 0D
corresponds to the richness of morphospecies (a, d, g), 1D corresponds
to the diversity of all morphospecies (b, e, h) and finally, 2D
corresponds to the diversity of the most abundant morphospecies. Curves
were extrapolated until 492 individuals. This value corresponds to the
highest abundance registered in our study (first collection).

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/671900/articles/673573-sexual-dimorphism-and-phenology-interplay-determines-arthropod-communities}

\textbf{Figure 4.} Species richness of carnivores (top row) and
herbivores (bottom row) in male and female plants of \emph{Buddleia
cordata} . When looking at the average (right column) there are no
differences between male and females. But when observed across
collection dates (left column), significant differences between male and
females are marked with an asterisk.

\textbf{DISCUSSION}

Even if we did not find direct evidence of sexual dimorphism in \emph{B.
cordata} , our results suggests that plant-sex affects arthropod
communities through both top-down and bottom-up mechanisms; and that
this effect has a strong temporal component linked to the plant
phenology. We suggest that seasonal variation on plant sex- arthropod
interactions might be particularly important for plants that experience
seasonal environments and that accounting for plant seasonality effects
has the potential to reconcile previous debates about plant-sex
preference and performance.

\textbf{Sexual dimorphism in\emph{B}} \emph{uddleia cordata} \textbf{and
its implications on sex-biased herbivory}

Differences between sexes (i.e., sexual dimorphism) are common in
dioecious plants. These differences that are the result of sex-specific
gene expression and physiological trade-offs affect the arthropod
communities associated with plants of different sexes (Mooney and Singer
2001, Nell et al. 2018). But contrary to our initial expectation, we did
not find sex-related differences in the two plant traits that we
evaluated: leaf thickness and water content (Fig. 1). These results are
consistent with the observations by Moreira et al. (2019) who found no
sex-related differences in water content, and in nitrogen and
phosphorous concentrations in \emph{B. cordata} during a study conducted
in September 2017. Nevertheless, we observed a strong temporal variation
in the water content of \emph{B. cordata} 's leaves that is consistent
with the highly seasonal climate of the preserve, having the highest
leaf water content during the rainy season in August, and the lowest
leaf water content during the dry season in February (Fig. 1). It is
important to highlight that while our results do not suggest sexual
dimorphism, there are multiple important axis of variation that we did
not evaluate. For example, other studies have found differences in the
secondary metabolites content of \emph{B. cordata'} s males and females
(Moreira et al. 2019). Furthermore, the female-biased sex-ratio that we
found in \emph{B. cordata} 's population at PSAER is consistent with
differences in life-history traits between individuals of different sex.

The response of arthropods to plant sexual dimorphism has been studied
during the last century mostly in the context of herbivore preference
and performance, showing a great support for the hypothesis of
sex-biased herbivory (Danell et al. 1985, Boecklen et al. 1990, Hjalt\selectlanguage{ngerman}én
1992, Agren et al. 1999, Cornelissen and Stiling 2005, Granados-Sánchez
et al. 2008, Kabir et al. 2014). This hypothesis suggests that male
plants experience higher herbivory than female plants due to male plants
allocating more resources on vegetative growth, while female plants
allocate more resources into reproduction and defensive traits
(Cornelissen and Stiling 2005, Ribeiro-Mendes et al. 2012). For example,
in a review, Agren et al. (1999) showed that in 25 out of 39 studies
male plants were preferred over female plants by herbivores, and
herbivore densities were higher in male plants than in females during
field experiments. Nevertheless, Sargent and McKeough (2022) in a recent
meta-analysis, that included 58 new observations of herbivory and 41 of
secondary chemistry compared to the original database used by
Cornelissen and Stiling (2005), found evidence that a male-sex bias in
herbivory is not the rule. Our results did not show differences in
herbivory rates between plants of different sex, and thus constitute
another example against the herbivory-bias hypothesis. Nevertheless, it
is important to notice that our quantification method for herbivory
might not adequately capture the damaged produced by sap-sucking
insects, such as Hemiptera, and this group was the second most important
group of herbivores in our sample.

Overall, recent studies contrast with the previously proposed pattern of
male-preference in sex-biased herbivory, by either finding no
differences in herbivory or female-preference examples. Even if we
accept the expectation of female plants allocating more resources on
reproduction compared to male plants, there are many compensatory
mechanisms that may reduce the effect of a higher reproductive resource
assignment on vegetative traits (Delph 1999). Furthermore, in some
scenarios, we might expect male-plant reproductive allocation to be
higher than in female plants, for example in wind-pollinated plants with
a large production of nitrogen-rich pollen (Delph et al. 1993, Harris
and Pannell 2008). In fact, there is evidence of cases in which male
plants show higher concentration of secondary metabolites associated
with defense against herbivores (Hjaltén 1992, Bañuelos et al. 2004,
Zhang et al. 2019, Yang et al. 2020); including our study system\emph{B.
cordata} (Moreira et al. 2019). Finally, even if we did not find
sex-bias in leaf water content and thickness, we found a strong seasonal
pattern observed in these metrics that raises the question of whether
other variables that show sex-dependence (like the secondary compounds
recorded by Moreira et al. 2019) would also display a temporal variation
pattern. A seasonal dependent pattern of plant traits could explain some
of the contradictory results regarding which plant sex displays more
defensive traits found in the literature (i.e., Sargent and McKeough
2022). It could also suggest that the effect of plant sex on arthropod
communities varies through time, and it highlights the need for
conducting studies in dioecious plants, especially those experiencing
seasonal environments, while accounting for seasonal variation.

\textbf{The effect of plant sex on arthropod communities through time}

Although in recent years it has become evident that there is not a
universal pattern in terms of plant-sex preference by herbivores (i.e.,
male vs female plants), many studies show that sexual dimorphism can
modify the availability and quality of resources for arthropod
herbivores, ultimately determining herbivore density on plants (Danell
et al. 1985, Hjaltén 1992, Boecklen et al.1990, Agren et al. 1999,
Carneiro et al. 2006). These effects on herbivores have the potential to
affect the rest of the arthropod community (i.e., carnivores) through
bottom-up forces (Chen y Wise 1999, Utsumi et al. 2009, Gruner 2004),
but most studies have only focused on understanding pairwise
interactions between a dioecious plant and one herbivore. In this study,
not only we evaluated the effect of \emph{B. cordata} 's sex on the
whole community of arthropods, but we also accounted for the potential
temporal variation of this interaction by studying the community every
two months during a year. Although we did not detect differences in the
total richness of species (\textsuperscript{0}\emph{D} ) between
plant-sexes at any point in time, our results showed that \emph{B.
cordata} 's sex affects the diversity of all the species
(\textsuperscript{1}\emph{D} ) and the diversity of the most abundant
species (\textsuperscript{2}\emph{D} ) (Fig. 3). We also found that the
sex-associated differences in arthropod communities have a strong
temporal component; depending on the date of observation, male or female
plants hosted more diverse arthropod communities (Fig. 3). During the
highest peak of the flowering season (June-July), the male plants showed
a more diverse arthropod community (\textsuperscript{1}\emph{D
and}\textsuperscript{2}\emph{D} ), and during the peak fructification
season in October-November the female plants had higher diversity of
arthropods (\textsuperscript{1}\emph{D and} \textsuperscript{2}\emph{D}
). These two periods of plant-sex related differential diversity are
separated by a period of no statistical differences between the
arthropod communities (August-September). Our results highlight the
dynamism of arthropod communities associated with \emph{B. cordata} ,
most likely driven by the high seasonality of the whole PSAER ecosystem.

This finding exemplifies the importance of accounting for seasonal
effects when trying to understand the ecological interactions among
species that inhabit seasonal ecosystems. In fact, other studies have
recorded seasonal variation in metabolite contents in nutrients and
metabolite contents in plants inhabiting seasonal regions like the
savannas in South Africa (Scogings et al. 2015), the Atlantic rainforest
of Brazil (Suguiyama et al, 2014), and alpine regions in China (Dai et
al. 2014). Furthermore, Dai et al. (2014) showed that this variation
influences the preference of one herbivore. In fact, Rabska et al.
(2020) observed that differences in carbohydrates levels between male
and female plants of \emph{Juniperus communis} varied through time, thus
constituting an example of the potential seasonal variation of sexual
dimorphism expression. Based on our results, we hypothesize that
dioecious species that experience seasonal environments might offer
different resources and conditions to the resident arthropods at
different times, which might translate into time-dependent diversity
patterns. For example, if chemical defense correlates with reproduction,
we might expect different peaks of chemical defense for males and
females; males having the higher concentration of defenses during
pollination, and females having the highest defense during
fructification season. This hypothesis is difficult to evaluate given
the current state of knowledge, which highlights the need of studies
that address the temporal component of sex-plant effect on arthropods.

\textbf{Beyond herbivory, plant sex effects on arthropod communities}

By studying the effect of \emph{B. cordat} a's plant sex on specific
groups of arthropods, we found that the only significant differences
occurred on the carnivores: male plants hosted significantly more
carnivores than female plants during June, while the opposite pattern
was true during December (Figure 4). Observations of effects present
only on higher trophic levels have been recorded before
in\emph{Baccharis salicifolia} whose female plants had 50\% more
predators than male plants, while there were no differences in
herbivores (Nell et al. 2018). Consistent with these results, previous
studies found examples of female plants hosting more natural enemies
(Mooney et al. 2012, Petry et al. 2013) by increasing the availability
of floral resources that provide nutritional benefits (i.e., nectar) to
associated arthropod communities (Ashman and King 2005, Wäckers et al.
2005, Pacini and Nepi 2007). Such effects can increase indirect defenses
from predators and parasitoids attracted to these floral resources
(Cepeda-Cornejo and Dirzo 2010). Plants commonly use this strategy to
mechanistically employ indirect plant defenses, increasing top--down
effects (Kessler and Heil 2011). This dynamic is leveraged in
agricultural systems, where they intercrop flowering plants as a
biological control method (Letourneau et al. 2011, Bickerton and
Hamilton 2012). Our findings, thus, lend support to the growing
consensus that floral resources for natural enemies and predators are a
key trait driving sexual dimorphism in the structure of plant-associated
multitrophic communities.

Even if we did not find evidence for bottom forces occurring in \emph{B.
cordata} 's associated arthropod community (i.e., no differences in
plant traits and herbivory), there are many axes of variation that we
did not capture in our study. In fact, a previous laboratory experiment
reported that the looper \emph{Acronyctodes mexicanaria} (Lepidoptera:
Geometridae) preferred to feed from leaves of female \emph{B.
cordata}plants than from males and that caterpillars nourished with
leaves of\emph{B} . \emph{cordata} female plants were less susceptible
to parasitoidism and developed faster, in comparison to caterpillars
nourished in male leaves in the laboratory (García-García and
Cano-Santana 2015). Because García-García and Cano-Santana's study was
conducted during October 2015, their results are consistent with a
scenario in which female plants of \emph{B. cordata} are preferred by
herbivores during the fructification season, ultimately affecting the
carnivore guild through bottom-up forces; consistent with the
significant differences between carnivores we found in our study (Fig.
4). It is possible that our study didn't capture the differences in the
herbivore guild due to the expectation that, in terrestrial ecosystems,
changes in the productivity of an ecosystem (i.e., the plant) will
affect carnivores ten times more than herbivores according to the
Lindeman rule (Lindeman 1942).

Not only the resources that dioecious plants offer to arthropods
yearlong (like leaves) can vary in their quality, but the reproductive
structures of plants are a resource in themselves (flower, pollen,
fruits) and their availability is highly tied to time. Furthermore,
pollen and fruits are resources produced exclusively in one of the two
sexes and may favor different specific groups of arthropods such as
pollinivores and fruit-eaters. For example, we noticed a higher
abundance of thrips in male plants during August and October, probably
associated with pollen consumption in male flowers. Pollen as a resource
might be particularly important in plants with wind-dispersal strategies
(Delph 1999), like \emph{B. cordata} (dispersal type infered by floral
morphology and personal observations). Unfortunately, the sample size
for arthropod guilds, like pollinivores and fruit-eaters, in our study
wasn't big enough to conduct statistical analyses, but we hypothesize
that the availability of flowers and fruits is a determining force of
arthropod communities through bottom-up forces.

\textbf{CONCLUSIONS}

Our research exemplifies the interaction between plant-phenology and
plant-sex as drivers of arthropod community diversity. These effects can
be explained both through bottom-up forces --through changes in the
diversity, quantity, and quality of resources-- and through top-down
mechanisms. These results highlight the importance of accounting for
seasonal variation when studying sexual dimorphism in plants and its
effect on resident arthropods. Although, the generality of our findings
is unknown given the paucity of such studies, we expect that variation
on plant-sex and arthropods interaction is important at least for
dioecious plants inhabiting seasonal environments. This time-dependent
framework has the potential to reconcile previous contrasting
observations reported in the literature. If we understand plant sexual
dimorphism as a time-dependent variable, and we consider the highly
dynamic nature of plant arthropod communities, as evidenced by our
results, it is possible that contrasting results about the effect of
plant sexual dimorphism on arthropod communities are the result of
observations made at different phenological stages. Further work is
needed to mechanistically link plant trait genetic variation (i\emph{.}
e\emph{.} , genetic sex-determination), to the structure of associated
arthropod communities to predict how plant responses to abiotic and
biotic conditions (like seasonality) can in turn affect the broader
ecological community.

\textbf{DATA AVAILABILITY STATEMENT}

Data has been uploaded in the section of file upload with the name

RamirezGonzalezet al2023rawdata.xlsx as a Supporting Information for
review and publication.

\textbf{REFERENCES}

Abdala-Roberts, L., Hernández-Cumplido, J., Chel-Guerrero, L.,
Betancur-Ancona, D., Benrey, B. and Moreira, X. 2016. Effects of plant
intra-specific genetic diversity across three trophic levels: Underlying
mechanisms and plant traits. -- Am. J. Bot. 103: 1810--1818.

Agren, J. 1988. Sexual differences in biomass and nutrient allocation in
the dioecious \emph{Rubus chamaemorus} . -- Ecology 69: 962-973.

Agren, J., Danell, K., Elmqvist, T., Ericson, L. and Hjaltén, J. 1999.
Sexual dimorphism and biotic interactions. -- In: Geber, M. A., Dawson,
T. E. and Delph, L. F. (eds), \emph{Gender and Sexual Dimorphism in
Flowering Plants} . Springer, pp. 123-148.

Ashman, T. L. and King, E. 2005. Are flower-visiting ants mutualists or
antagonists? A study in a gynodioecious wild strawberry. -- Am. J. Bot.
92: 891-5. doi: 10.3732/ajb.92.5.891

Bañuelos, M. J., Sierra, M. and Obeso, J. R. 2004. Sex, secondary
compounds and asymmetry. Effects on plant-herbivore interaction in a
dioecious shrub. -- Acta Oecol. 25: 151--157.

Barrett, S. C. H. and Hough, J. 2013. Sexual dimorphism in flowering
plants. -- J. Exp. Bot. 64:67--82. doi:10.1093/jxb/ers308

Bawa, K. S. and Opler, P. A. 1975. Dioecism in tropical forest trees. --
Evolution 29: 167--179.

Boecklen, W. J., Mopper, S. and Price, P. W. 1990. Sex and drugs and
herbivores: Sex-biased herbivory in arroyo willow (\emph{Salix
lasiolepis} ). -- Ecology 71: 581-588. doi:10.2307/1940311

Bickerton, M. W. and Hamilton, G. C. 2012. Effects of intercropping with
flowering plants on predation of \emph{Ostrinia nubilalis} (Lepidoptera:
Crambidae) eggs by generalist predators in bell peppers. -- Environ.
Entomol. 41: 612-620.

Cano-Santana, Z. 1994. Flujo de energía a través de \emph{Sphenarium
purpurascens} (Orthoptera: Acrididae) y productividad primaria neta
aérea en una comunidad xerófita. -- PhD thesis, Univ. Nal. Autón. Méx.

Carneiro, M. A., Fernandes, G. W., de Souza, O. F. and Souza, W. V.
2006. Sex-mediated herbivory by galling insects on \emph{Baccharis
concinna} (Asteraceae). -- Rev. Bras. Entomol. 50: 394--398.
doi:10.1590/S0085-56262006000300009

Castillo-Argüero, S., Montes-Cartas, G., Romero-Romero, M. A.,
Martínez-Orea, Y., Guadarrama-Chávez, P., Sánchez-Gallén, I. and
Nuñez-Castillo, O. 2004. Dinámica y conservación de la flora del
matorral xerófilo de la Reserva Ecológica del Pedregal de San Ángel
(D.F., México). -- Bol. Soc. Bot. Mex. 74: 51-75.

Cepeda-Cornejo, V. and Dirzo, R. 2010. Sex-related differences in
reproductive allocation, growth, defense and herbivory in three
dioecious neotropical palms. -- PLoS ONE 5: e9824.

Chen, B. and Wise, D. 1999. Bottom-up limitation of predaceous
arthropods in a detritus-based terrestrial food web. -- Ecology 80:
761-772. doi:10.1890/0012-9658

Cornelissen, T. and Stiling, P. 2005. Perfect is best: low leaf
fluctuating asymmetry reduces herbivory by leaf miners. -- Oecologia
142: 46-56.

Danell, K., Elmqvist, T., Ericson, L. and Salomonson, A. 1985. Sexuality
in willows and preference by bark-eating voles: defence or not? -- Oikos
44: 82-90.

Dai, X., et al. 2014. Seasonal changes in the concentrations of plant
secondary metabolites and their effects on food selection
by\emph{Microtus oeconomus} . -- Mamm. Biol. 79: 215-220.

Delph, L. F. 1999. Sexual dimorphism in life history. -- In: Geber, M.
A., Dawson, T. E. and Delph, L. F. (eds) \emph{Gender and Sexual
Dimorphism in Flowering Plants} . Springer, pp. 149--174.

Delph, L. F., Ying, L. and Jayne, L. D. 1993. Patterns of resource
allocation in a dioecious Carex (Cyperaceae). -- Am. J. Bot. 80:
607--615. doi:10.2307/2445429

Eckhart, V. M. 1999. Sexual dimorphism in flowers and inflorescences. --
In: Geber, M. A., Dawson, T. E. and Delph, L. F. (eds) \emph{Gender and
Sexual Dimorphism in Flowering Plants} . Springer, pp 123--148.

Elmqvist, T. and Gardfjell, H. 1988. Differences in response to
defoliation between males and females of \emph{Silene dioica} . --
Oecologia 77: 225-230. doi:10.1007/BF00379190

García-García, P. L. and Cano-Santana, Z. 2015. Nutritional ecology,
growth and density of \emph{Acronyctodes mexicanaria} (Lepidoptera:
Geometridae) on a dioecious plant \emph{Buddleja
cordata}(Scrophulariaceae). -- Rev. Mex. Biodivers. 86: 172-177.
doi:10.7550/rmb.44382

Granados-Sánchez, D., Ruíz-Puga, P. and Barrera-Escorcia, H. 2008.
Ecología de la herbivoria. -- Rev. Chapingo Ser. Cienc. For. Ambiente.
14: 51-64.

Gruner, D. 2004. Attenuation of top-down and bottom-up processes in a
complex terrestrial community. -- Ecology 85: 3010-3022.
doi:10.1890/04-0020

Han P., Lavoir A.V., Rodriguez-Saona C., and Desneux N. 2022. Bottom-Up
Forces in Agroecosystems and Their Potential Impact on Arthropod Pest
Management. -- Ann. Rev. Entomol. 7;67: 239-259. doi:
10.1146/annurev-ento-060121-060505.

Harris, M. S. and Pannell, J. R. 2008. Roots, shoots and reproduction:
sexual dimorphism in size and costs of reproductive allocation in an
annual herb. -- P Roy Soc B-Biol Sci 275: 2595--2602.
doi:10.1098/rspb.2008.0585

Hjaltén, J. 1992. Plant sex and hare feeding preferences. -- Oecologia
89: 253-256. doi:10.1007/BF00317225

Hjaltén, J., Ástrom, M., Áberg, E. and Danell, K. 1993. Biased sex
ratios in Spanish populations of
\emph{Pistacialentiscus}(Anacardiaceae): The possible role of herbivory.
-- An. Jard. Bot. Madr. 51: 49-53.

Hsieh, T. C., Ma, K. H. and Chao, A. 2016. iNEXT: An R package for
rarefaction and extrapolation of species diversity (Hill numbers). --
Methods Ecol. Evol. 7: doi:10.1111/2041-210X.12613

Jost, L. 2006. Entropy and diversity. -- Oikos 113: 363--375.
doi:10.1111/j.2006.0030-1299.14714.x

Kabir, M. F., K. M. Moritz, and Stenberg, J. A. 2014. Plant-sex-biased
tritrophic interactions on dioecious willow. -- Ecosphere 5: 153.
doi:10.1890/ES14-00356.1

Kersch-Becker, M. F., Kessler, A. and Thaler, J. S. 2017. Plant defences
limit herbivore population growth by changing predator-prey
interactions. -- Proc. R. Soc. B: Biol. Sci. 284: 20171120.
doi:10.1098/rspb.2017.1120

Kessler, A. and Heil, M. 2011. The multiple faces of indirect defences
and their agents of natural selection. -- Funct. Ecol. 25: 348-357.
doi:10.1111/j.1365-2435.2010.01818.x.

Letourneau, D. K., Armbrecht, I., Salguero-Rivera, B., Montoya-Lerma,
J., Jiménez-Carmona, E., Constanza Daza, M., Escobar, S., Galindo, V.,
Gutiérrez, C., Duque-López, S., López-Mejía, J., Acosta-Rangel, M. A.,
Herrera-Rangel, J., Rivera, L., Saavedra, C. A., Torres, A. M.,
Reyes-Trujillo, A. 2011. Does plant diversity benefit agroecosystems? A
synthetic review. -- Ecol. Appl. 21: 9-21. doi10.2307/29779633.

Lindeman, R. L. 1942. The trophic-dynamic aspect of ecology. -- Ecology
23: 399-417. doi:10.1007/BF02464428

Mooney, K. A., Pratt, R. T. and Singer, M. S. 2012. The tri-trophic
interactions hypothesis: interactive effects of host plant quality, diet
breadth and natural enemies on herbivores. -- PLoS ONE 7: e34403.
doi:10.1371/journal.pone.0034403

Mooney, K. A. and Singer, M. S. 2001. Plant variation in herbivore-enemy
interactions in natural systems. -- Cambridge University Press.

Moreira, X., et al. 2019. Impacts of urbanization on insect herbivory
and plant defences in oak trees. -- Oikos 128: 113--123.

Nell, C. S., Abdala-Roberts, L., Parra-Tabla, V. and Mooney, K. A. 2018.
Tropical tree diversity mediates foraging and predatory effects of
insectivorous birds. -- Proc. R. Soc. B: Biol. Sci. 285: 20181842.
doi:10.1098/rspb.2018.1842

Norman, M. 2000. Buddlejaceae. -- In: Luteyn, J. L., Cary, M. F. and
Gradstein, S. R. (eds), \emph{Flora Neotropica. Monografía 81} . The New
York Botanical Garden, pp. 1-16

Obeso, J. R. 2002. The cost of reproduction in plants. Tansley review
no. 139. -- New Phytol. 155: 321-348.

Oksanen, L. 1988. Ecosystem organization: mutualism and cybernetics of
plain Darwinian struggle for existence. -- Am. Nat. 131: 424--444.

Pacini, E. and Nepi, M. 2007. Nectar production and presentation. -- In:
Nicolson, S. W., Nepi, M. and Pacini, E. (eds), \emph{Nectaries and
nectar} . Springer, pp. 167--214.

Petry, W.K., et al. 2013. Mechanisms underlying plant sexual dimorphism
in multi-trophic arthropod communities. -- Ecology 94: 2055--2065.

Rabska, M., Pers-Kamczyc, E., \selectlanguage{polish}Ż\selectlanguage{english}ytkowiak, R., Adamczyk, D., Iszku\selectlanguage{polish}ł\selectlanguage{english}o, G.
2020. Sexual dimorphism in the chemical composition of male and female
in the dioecious tree, \emph{Juniperus communis} L., growing under
different nutritional conditions. -- Int. J. Mol. Sci. 21: 8094.
doi:10.3390/ijms21218094

Ribeiro-Mendes, H. Nt., Marques, E. S. A., Silva., I. M. and Fernandes,
G. W. 2002. Influence of host-plant sex and habitat on survivorship of
insect galls within the geographical range of the host-plant. -- Trop.
Zool. 15: 5-15. doi:10.1080/03946975.2002.10531163

Rzedowski, J. 1954. Vegetaci\selectlanguage{ngerman}ón del Pedregal de San Ángel (Distrito
Federal, México). -- An. Esc. Nal. Cienc. Biol. Inst. Pol. Nal 8:
59-129.

Sanjuan-Trejo, G., Mejia, D. and Moreno, C. E. 2021 Ensambles de
artrópodos asociados a los frutos de garambullo (\emph{Myrtillocactus
geometrizans} ) en dos localidades del valle del Mezquital, Hidalgo,
México. -- Rev. Mex. Biodivers. 92: e923487.

Sargent, R. D. and McKeough, A.D. 2022. New evidence suggests no sex
bias in herbivory or plant defense. -- Am. Nat. 200: 435--447.

Schoonhoven, L. M., Van Loon, J. J. A. and Dicke, M. 2005. Insect-Plant
Biology. -- Oxford University Press.

Schowalter, T. D. 2006. Insect Ecology. An Ecosystem Approach. --
Elsevier.

Scogings, P. F., Hattas, D., Skarpe, C., Hjaltén, J., Dziba, L., Zobolo,
A. and Rooke, T. 2015. Seasonal variations in nutrients and secondary
metabolites in semi-arid savannas depend on year and species. -- J. of
Arid Environ. 114: doi:10.1016/j.jaridenv.2014.11.003.

Sinclair, J. P., Emlen, J. and, Freeman, D. C. 2012. Biased sex ratios
in plants: theory and trends. -- Bot. Rev. 78: 63--86.
doi:10.1007/s12229-011-9065-0

Strong, D. R., Lawton, J. H. and Southwood, R. 1984. Insects on plants:
community patterns and mechanisms. -- Blackwell Scientific.

Suguiyama, V., Silva, E., Meirelles, S., Centeno, D. and Braga, M. 2014.
Leaf metabolite profile of the Brazilian resurrection
plant\emph{Barbacenia purpurea} Hook. (Velloziaceae) shows two
time-dependent responses during desiccation and recovering. -- Front.
Plant Sci. 5: 96. doi:10.3389/fpls.2014.00096.

Tsuji, K. and Fukami, T. 2018. Community-wide consequences of sexual
dimorphism: evidence from nectar microbes in dioecious plants. --
Ecology 99: 2476-2484. doi:10.1002/ecy.2494

Utsumi, S., Nakamura, M. and Ohgushi, T. 2009. Community consequences of
herbivore-induced bottom-up trophic cascades: The importance of resource
heterogeneity. -- J. Anim. Ecol. 78: 953--963.
doi:10.1111/j.1365-2656.2009.01570.x1

Vargas, N. A. 2001. Longaniaceae. -- In: Rzedowski, J and Rzedowski, G.
(eds), \emph{Flora Fanerogámica del Valle de México} . Instituto de
Ecología A.C. y Comisión Nacional para el Conocimiento y Uso de la
Biodiversidad pp. 546-550.

Wackers, F. L., van Rijn, P. C. J., and Bruin, J. 2005.Plant-provided
food for carnivorous insects: A protective mutualism and its
applications. -- Cambridge University Press.

Yang, W., et al. (2020). Liquid chromatography--mass spectrometry-based
metabolomics analysis of \selectlanguage{english}flavonoids and anthraquinones in \emph{Fagopyrum
tataricum} L. Gaertn. (Tartary buckwheat) seeds to trace morphological
variations. -- Food Chem. 331: 127354.
doi:10.1016/j.foodchem.2020.127354

Zhang, L., Rana, I., Shaffer, R.M., Taioli, E., and Sheppard, L. 2019.
Exposure to glyphosate-based herbicides and risk for non-Hodgkin
lymphoma: a meta-analysis and supporting evidence. -- Mutation
Research/Reviews in Mutation Research 781: 186-206.
doi:10.1016/j.mrrev.2019.02.001

\selectlanguage{english}
\FloatBarrier
\end{document}

