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Expression of Secondary Sexual Dimorphism in the Diurnal Course of Leaf Gas

Exchanges Is Modified by the Rhythmic Growth of Ilex paraguariensis Under
Monoculture and Agroforestry

Article in Forests · January 2025

DOI: 10.3390/f16010161

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Article

Expression of Secondary Sexual Dimorphism in the Diurnal

Course of Leaf Gas Exchanges Is Modified by the Rhythmic
Growth of Ilex paraguariensis Under Monoculture
and Agroforestry
Miroslava Rakočević 1,2,3, * , Eunice Reis Batista 2 , Rafael Leonardo de Almeida 1 , Ivar Wendling 3
and Rafael Vasconcelos Ribeiro 1

1 Laboratory of Crop Physiology, Department of Plant Biology, Institute of Biology, State University of
Campinas (UNICAMP), Campinas 13083-862, SP, Brazil; rafael.leonardo.iac@gmail.com (R.L.d.A.);
rvr@unicamp.br (R.V.R.)
2 Laboratory of Ecophysiology, Embrapa Meio Ambiente, Jaguariúna 13918-110, SP, Brazil;
eunice.reis@embrapa.br
3 Department of Research and Development, Embrapa Florestas, Colombo 83411-000, PR, Brazil;
ivar.wendling@embrapa.br
* Correspondence: mima.rakocevic61@gmail.com; Tel.: +55-19-97161-8918

Abstract: Dioecious species show a division of labor expressed through the differentiated
manifestation of resource acquisition. We hypothesized that the expression of secondary
sexual dimorphism (SSD) in the leaf gas exchange of yerba mate would be more intensive
in females than in males to permit females the carbon investments necessary to finish the
reproductive cycle. This species can present two growth units annually (GU1-fall and
GU2-spring) intercalated with two rest periods (R1-summer and R2-winter). The leaf area
index (LAI) and the diurnal courses of leaf photosynthesis (Anet ), stomatal conductance (gs ),
leaf transpiration (E), intercellular CO2 concentration (Ci ), water use efficiency (WUE), and
instantaneous carboxylation efficiency (Anet /Ci ) were estimated in female and male plants
of yerba mate during four periods of annual rhythmic growth in monoculture (MO) and
agroforestry (AFS). Leaf gas exchanges varied over the annual rhythmic growth and were
more intensive under MO than under AFS. Anet , Anet /Ci ratios, and WUE were higher in
Received: 7 December 2024
females than in males during the summer (R1) and spring (GU2). Also, gs and E were more
Revised: 13 January 2025
Accepted: 13 January 2025
intensive in females than males during the summer. Oppositely, higher WUE in males than
Published: 16 January 2025 in females was observed during the fall (GU1) and winter (R2), with males also showing a
Citation: Rakočević, M.; Batista, E.R.;
higher Anet /Ci ratio during the winter and higher E during the spring (GU2). Despite the
de Almeida, R.L.; Wendling, I.; strong effect of the cultivation system on LAI and leaf gas exchange traits over the diurnal
Ribeiro, R.V. Expression of Secondary course, SSD expression was rarely modified by the cultivation system, being expressed
Sexual Dimorphism in the Diurnal only in MO for E during the spring (GU2) and WUE during the winter (R2). High WUE
Course of Leaf Gas Exchanges Is in males during the winter would benefit plants during cold and dry periods, improving
Modified by the Rhythmic Growth of
the balance between carbon acquisition and water loss through transpiration. On the other
Ilex paraguariensis Under Monoculture
and Agroforestry. Forests 2025, 16, 161.
hand, high Anet during the summer and spring could be considered as a general fitness
https://doi.org/10.3390/f16010161 strategy of female plants to improve photoassimilate supply and support their additional
reproduction costs.
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
Keywords: Anet ; LAI; stomatal conductance; transpiration; WUE; yerba mate
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/
licenses/by/4.0/).

Forests 2025, 16, 161 https://doi.org/10.3390/f16010161

Forests 2025, 16, 161 2 of 21

1. Introduction
Yerba mate (Ilex paraguariensis A. St.-Hil., family Aquifoliaceae) is an evergreen
broadleaf tree species [1] native to South American subtropical humid forests [2], today
growing in Brazil, Argentina, Paraguay, and Uruguay [3]. It is widely used for South
American tea preparations [4] and, recently, for cosmetics and pharmaceutic industries [2]
due to its recognized phytochemical properties [5–8]. This dioecious tree has rhythmic
growth [1,9]. Yerba mate is usually pruned every 18 or 24 months, in ‘plate form’ as a
shrub of various geometrical forms [10], and its raw material is used intensively for tea,
medicines, and pharmaceuticals. Some rare specimens are grown freely up to a potential
height of 15 m, known as female mother trees, with the aim of producing seeds for further
propagation [11]. In field conditions, forest clearing of various tree and understory species
improves yerba mate production, and complete forest clearing is usual, for cultivating
yerba mate in monoculture (MO), in consortium with an annual crop species, or for growing
other crop species [12,13].
Decreases in forest biodiversity, as in MO systems, are accompanied by a reduction
of soil and water conservation capacity [14] and thermal regulation capacity, increasing
the risk of frost, drought, and other environmental stresses [15]. Then, yerba mate plants
are more vulnerable to environmental stresses when grown in MO, a scenario that can be
more limited under climate change [16]. In fact, higher radiation use efficiency in yerba
mate sprouts is obtained in the intercropping system, as compared to MO [17]. Despite
the ecological benefits of forest and intercropping systems, MO is the most intensive and
productive system for yerba mate. Biomass harvested in MO is about ten times higher
compared to anthropized forest understory or agroforestry (AFS) [1,9]. Adult yerba mate
plants cultivated in AFS differ from those in MO in leaf-area density up to six times, in
plant leaf area (LA) up to seven times, and in leaf area index (LAI) up to twelve times [1,10].
In AFS, yerba mate forms longer internodes and bigger, wider individual leaves than plants
cultivated in MO.
More than 70% of tree species have a rhythmic growth [18], meaning that organo-
genesis and elongation are characterized by periods of active growth and rest [19]. The
appearance of growth pauses defines rhythmic growth, which is widely observed in
trees [18]. It is characterized by the succession of periods of uninterrupted organ emission
and extension and periods of rest. In yerba mate, ideally, two growth flushes are observed
when portions of leafy branches, called growth units (GUs), are formed, occurring in
spring and fall, interrupted with periods of rest (R) occurring in summer and winter [1,9].
When several successive GUs are formed in the same annual vegetative cycle, they most
often present distinctive characteristics, as observed in yerba mate. In the first year after
harvest (pruning), the most vigorous individuals do not stop growing during the summer,
especially in MO, where such uninterrupted metamer emission is likely due to high light
conditions [20]. The useful yerba mate biomass, composed of leaves and fine branches, is
harvested according to growth pauses, generally during the winter growth pause (rest) or
during the summer rest, and sometimes, the harvest is conducted all year long, depend-
ing on industry needs [1]. The end-of-spring flush corresponds to flowering (males and
females), while the summer growth pause corresponds to fruit ripening in females [1].
In dioecious species, the differences between the genders in characters not directly
related to gamete production are known as secondary sexual dimorphism (SSD) [21]. SSD
is generally based on the difference in the use of resources between males and females,
which is caused by varying reproductive costs between the genders [22]. While females
require larger amounts of resources due to seed and fruit production, males just produce
flowers and pollen, which are usually less costly [23]. Sexual reproduction in Sagittaria
latifolia imposes asymmetry between the genders, with greater biomass costs for females
Forests 2025, 16, 161 3 of 21

and greater nitrogen costs for males [24]. Curiously, this difference in nitrogen costs
between the genders was not associated with differences in photosynthetic rates [24]. On
the other hand, males of Silene latifolia produce small flowers in abundance and have higher
stomatal conductance (gs ) and leaf transpiration (E) than females, leading to a higher cost
of reproduction for males [25]. In some dioecious species and environments, biomass
allocation to reproduction is three times higher in female plants than in male ones [22].
In other species, as in Fraxinus mandshurica, there is no biomass difference between the
genders during early ontogeny [26]. SSD can be expressed in morphological, physiological,
and life-history traits [22,27–29] and can promote differential leaf and trunk biomass
production [30,31], or variation in leaf chemical composition [7,32] between genders. The
complex sexual phenotypic differentiation in angiosperms can be understood through
genetic regulation and the spatial and temporal role of plant growth regulators [33]. The
single-gene sex-determining genetic systems could be common in dioecious species that
evolved via monoecy [34].
In yerba mate, SSD expression is higher under stressful conditions [9,28,30] and
depends on phenology, genetic material, and study conditions [1,10,35,36]. In the literature,
SSD expressions in yerba mate are still contradictory, and we believe this is partially
caused by phenology, as is the case for photosynthetic capacity [35]. In other words, SSD
expressions likely change along the annual cycle in field-grown plants. Increasing net
photosynthetic rate (Anet ) in dioecious Populus spp. during the early summer is related to
an increase in the chlorophyll pool during leaf development [37]. However, differences
between the genders in terms of photosynthetic gas exchanges are not always observed [37].
The adult female yerba mate trees cultivated in MO presented higher Anet , gs , and E than
male trees during the summer rest pause and spring GU emission [36]. However, such
sexual segregation is always perceived in sun leaves, not in self-shaded ones, and gs is
the most responsive trait to SSD. When evaluating yerba mate progeny, the Anet of fully
expanded leaves in female trees grown in AFS was higher than in males, but only during
the winter growth pause, while gs did not segregate between genders. In an experiment
with clonal yerba mate plants, Anet was higher in females than in males under AFS (but not
in MO) over a large part of the diurnal period at the beginning of summer growth pause
and beginning of fruit ripening, while SSD was not expressed in terms of carbon gain at
the plant scale, irrespective of cultivation system [35]. Then, SSD in yerba mate seems to
depend on the environment, phenophase, and cultivation system [10,35].
As the pattern of SSD expression in yerba mate trees varies largely in the literature, we
followed the diurnal dynamics of leaf gas exchange during one annual rhythmic growth in
males and females. Additionally, we evaluated the SSD expression in terms of leaf water
use efficiency [38] and the instantaneous carboxylation efficiency [39], two traits rarely
studied in dioecious species [40] and never studied in yerba mate trees in the scope of SSD.
Due to SSD, we hypothesized that leaf gas exchange would be more intensive in females
than in males to support female carbon investments during the reproductive period.

2. Materials and Methods

2.1. Experimental Area and Cultivation Systems
The experiment was established in an area of the Federal University of Santa Catarina
(UFSC), near Curitibanos (27◦ 19′ 09′′ S, 50◦ 42′ 39′′ W, 836 m a.s.l.), Southern Brazil. The
climate is defined as subtropical humid (Cfa), according to the Köppen-Geiger classification,
with regularly distributed rains and an average air temperature of 20.8 ± 0.3 ◦ C in the
warmest month [41].
Two contrasting cultivation systems of yerba mate were established in neighboring
plots, with about 150 m distance between plots. One system was the anthropized rainforest
Forests 2025, 16, 161 4 of 21

in recovery enriched with yerba mate in its understory (agroforestry system, AFS), and
the second was the full-sun cultivation (monoculture, MO). Each cultivation system with
yerba mate was established at plots of about 75 × 20 m. The rainforest was composed
of native tree species (Araucaria angustifolia, Cordia trichotoma, Cordia ecalyculata, Eugenia
involucrata, Luehea divaricata, Ocotea spp., Peltophorum dubium, and Syagrus romanzoffiana),
non-native tree species (Pinus elliottii and Eucalyptus spp.), and understory vegetation. The
MO was established at the initial stage of forest regeneration in AFS. The soil is classified
as Dystric Cambisol [35], and lime was applied at a depth of 50 cm only in the planting
lines. The fertilization in both cultivation systems was carried out according to soil analyses
and species recommendations [42], with mechanical weed control and no pest/disease
protection.
In both cultivation systems, young clonal plants issued from vegetative reproduction
were planted in June 2018. They were ~10 cm in height, and each clone originated from
one plant of the same sex. The planting arrangement was about 3 m distance between
rows and 1.5 m between plants in rows. When plants were three years old, six male
(♂) and six female (♀) plants were chosen for morpho-physiological evaluations, totaling
24 plants. Plant selection was based on the presence of 5–6 visible growth units (GU),
using the chronological age as a reference. In December 2021, at the beginning of the
summer rest period, the formation pruning was performed at 40 cm of stem height, leaving
about 10%–20% of photosynthesizing leaf area for plant recovery [35], with the regrowth
being where the branches of the 1st order tend to grow perpendicular to the supporting
structure [10].

2.2. Microclimate, Leaf Area Index, and Physiological Measurements

Microclimate, leaf area, and leaf gas exchange were measured in four seasons, fol-
lowing the plant growth rhythmicity after pruning in December 2021: growth rest 1 (R1,
summer—February 2022); growth unit 1 (GU1, fall—May 2022); growth rest 2 (R2, winter—
August 2022); and growth unit 2 (GU2, spring—November 2022).
Photosynthetic photon flux density (PPFD, µmol photons m−2 s−1 ) was measured
using silicon pyranometer smart sensors (S-LIx-M003, HOBO, Bourne MA, USA). They
captured the sun radiation in a 400–700 nm waveband. Two light sensors were used from
summer 2022 to spring 2022 and maintained at two heights from the soil, 0.4 and 2 m (above
and below the canopy of representative yerba mate plants), in each cultivation system.
PPFD values were recorded with a datalogger (H21-USB Data Logger USB, HOBO, Bourne
MA, USA) every 10 s. Here, we used hourly mean values. As we had only one datalogger,
the sensors were moved every two to three weeks from one to the other system, AFS and
MO, during the experimental period. Simultaneously with PPFD measurements, the air
temperature was registered in both systems during the four seasons with the U23-003
HOBO 2x external temperature data logger (HOBO, Bourne MA, USA, Figure S1). The
temperature sensor was positioned at 2 m height from the soil, near the upper PPFD sensor.
Rainfall was obtained from the local meteorological station.
Leaf area index (LAI) was measured with an LAI–2000 plant canopy analyzer (LICOR,
Lincoln, NE, USA). The procedure comprised a set of six readings: the first one was taken
above the yerba mate plants; and the next four below the plants at 0.2 m from the trunk
and oriented to the four cardinal points; the sixth reading was again taken above the yerba
mate plants. In AFS, the 1st and 6th readings were taken inside the forest, capturing the
canopy above the yerba mate plants. The LAI estimated in AFS was expected to be higher
than in reality because the reference readings were not taken under a clear sky, but rather
under the forest upper canopy layer.
Forests 2025, 16, 161 5 of 21

The responses of leaf gas exchange to light in yerba mate were measured over four
seasons. Twenty-three PPFD levels were used: 1500, 1200, 800, 500, 200, 100, 90, 80, 70,
and every 5 µmol m−2 s−1 between 70 and 0 µmol m−2 s−1 . The 6-cm2 leaf cuvette was
fitted with a red–blue light source (6400-02B, LICOR, Lincoln NE, USA, Figure S1). For
respiration in the light (RL ), we used the Kok method [43], computing the y-axis intercept
of a first-order linear regression fitted to Anet /PPFD when PPFD varied between 25 and
65 µmol m−2 s−1 [44]. All gas exchange data were corrected for the increase in intercellular
CO2 concentrations (Ci ) when decreasing PPFD [44].
By using the response curves of leaf CO2 assimilation to PPFD and hourly PPFD values
(Figure S1), we estimated diurnal variation of leaf CO2 assimilation (Anet, µmol m−2 s−1 ) in
MO and AFS. As variables from light response curves [45], we had the apparent quantum
efficiency of CO2 assimilation (Φ, µmol CO2 µmol photons−1 , calculated from the initial
linear slope of the modelled light response curves for PPFD < 30 µmol m−2 s−1 ), the
maximum net photosynthesis (Amax , µmol m−2 s−1 , a proxy of photosynthetic capacity),
RL , and the convexity factor of the curve (θ), as shown in Equation (1) [46]:
q
(Φ ∗ PPFD + Amax )2 − 4 ∗ Φ ∗ θ ∗ PPFD ∗ Amax
Anet = Φ ∗ PPFD + Amax − − RL (1)
2∗θ

The stomatal conductance (gs , mol m−2 s−1 ), leaf transpiration rate (E, mmol m−2 s−1 ),
and intercellular CO2 concentration (Ci , µL L−1 ) dependences on PPFD were also esti-
mated by using a second-order polynomial equation, which tended to a linear regres-
sion in the case of a null quadratic coefficient. The instantaneous leaf water use effi-
ciency (WUE, µmol mmol−1 ) was calculated as Anet /E. Additionally, the Anet /Ci ratio
(µmol m−2 s−1 Pa−1 ), as a proxy of the instantaneous carboxylation efficiency, was also
calculated. The last two parameters were calculated for diurnal hours when Anet > 0.

2.3. Experimental Design and Statistical Analysis

A complete randomized design was used, which considered two genders (female
and male) grown in two cultivation systems (AFS and MO) with six replications (plants)
over four seasons. Two-way ANOVA was applied to analyze effects of factors, such as
genders and cultivation systems (separately for each season) on LAI. Three-way ANOVA
was applied to analyze effects of factors, such as genders and cultivation systems observed
over the diurnal period (separately for each season) on leaf gas exchange. ANOVAs
considered a mixed linear model (lme function from the ‘nlme’ package) and maximum
likelihood to test the significance of studied variables. The Bartlett homogeneity test and
the Shapiro normality test were performed for each variable in each season. Genders,
cultivation systems and evaluation time were considered as fixed factor effects, while
plant number (repetitions) was considered a random effect observed separately for each
season/growth period. If interaction was non-significant, the model reduction was applied,
starting from the interaction of two or three factors. After that, the model was fitted again.
For comparison of variables among the four seasons, one-way ANOVA was performed,
in which the seasons were considered as fixed factor effect, while total plant number as
a random effect. All ANOVAs were performed at 95% confidence, followed by a Tukey
Honestly Significant Difference test, where p < 0.05 for mean comparisons was considered
significant. P-values, estimated means, and standard errors (SE) are shown in figures. All
statistical analyses were performed using R. 4.1.0 software [45], with ‘nlme’, ‘emmeans’,
and ‘multcomp’ packages for ANOVA analyses.
Forests 2025, 16, 161 6 of 21

3. Results
3.1. Climate Conditions
At midday, the mean PPFD values coming to the top of the yerba mate canopy in
MO (2 m from the soil) were 1120, 850, 790, and 950 µmol m−2 s−1 during the summer,
fall, winter, and spring, respectively (Figure 1A–D). The maximum hourly PPFD values
ranged between 1510 and 2000 µmol m−2 s−1 in MO at midday (from 10:45 h to 14:45 h)
during the experimental period. In MO and at 40 cm from the soil surface (below plant
canopies), the mean PPFD values at midday were about 160, 70, 85, and 45 µmol m−2 s−1 ,
and represented 14.3%, 8.2%, 10.8%, and 4.7% of the upcoming PPFD in the summer, fall,
winter, and spring, respectively. The decreased light transmission in MO was primarily
related to high absorbance and reflectance of light by increased leaf area of yerba mate
plants (Figure 2) and less to seasonal variation of PPFD. In AFS, the mean PPFD coming at
2 m of height from the soil (above plant canopy) was about 46, 39, 53, and 75 µmol m−2 s−1 ,
being reduced by 96%, 95%, 93%, and 92% when compared to PPFD coming to 2 m of
height from the soil in MO (Figure 1A–D). The values of PPFD coming at 2 m of height
from the soil in AFS were additionally reduced by 43%, 57%, 57%, and 63% at a height of
40 cm (below the plant canopy). Compared to MO, lower PPFD absorbance in AFS was
certainly related to low LAI (Figure 2).
During the summer, the mean daily temperatures were 18.9 ◦ C in MO and 17.3 ◦ C
in AFS, while the lowest/highest mean hourly temperatures were 14.4/26.4 ◦ C and
15.0/20.8 ◦ C in MO and AFS, respectively (Figure 1E). The absolute minimum/maximum
hourly temperatures during the experimental period were 6.8/35.7 ◦ C and 4.2/24.2 ◦ C
in MO or AFS, respectively. During the fall, the mean daily temperatures were 13.2 ◦ C
in MO and 11.0 ◦ C in AFS, while the lowest/highest mean hourly temperatures were
8.8/21.3 ◦ C and 8.4/15.6 ◦ C in MO and AFS, respectively (Figure 1E). The absolute mini-
mum/maximum hourly temperatures during the fall were −3.6/35.4 ◦ C and −0.4/26.4 ◦ C
in MO and AFS, respectively. During the winter, the mean daily temperatures were 13.6 ◦ C
in MO and 12.4 ◦ C in AFS, while the lowest/highest mean hourly temperatures were
8.6/22.5 ◦ C and 8.5/17.8 ◦ C, occurring early in the morning and at midday in MO and
AFS, respectively (Figure 1F). The absolute minimum/maximum hourly temperatures
of −4.3/33.4 ◦ C and −2.1/27.1 ◦ C were registered during the winter in MO and AFS,
respectively. During the spring, the mean daily temperatures were 18.3 ◦ C in MO and
15.5 ◦ C in AFS, while the lowest/highest mean hourly temperatures of 12.4/26.6 ◦ C and
13.6/18.6 ◦ C occurred early in the morning and at midday in MO or AFS, respectively
(Figure 1F). The absolute minimum/maximum hourly temperatures during the spring
were −0.9/38.5 ◦ C and 9.7/26.3 ◦ C in MO and AFS, respectively.
The precipitation summed 1957 mm in 2022, with 364 mm in summer, 491 mm in fall,
494 mm in winter, and 608 mm in spring. Precipitations were relatively well-distributed
over the year, except for May 3 and 4 and October 10, when rainfall was excessive, i.e., 82,
102, and 115 mm, respectively.
ForestsForests 16, 161
2025, 2025, 16, x FOR PEER REVIEW 7 of7 21
of 21

Figure
Figure 1. Microclimate
1. Microclimate inin
thethe monoculture(MO)
monoculture (MO)and and agroforestry
agroforestry (AFS)
(AFS) of ofyerba
yerbamate.
mate.Mean
Mean± SE± SE
(n =(n18–72)
= 18–72)forfor
photosynthetic
photosyntheticphoton photonfluxfluxdensity
density (PPFD) measuredover
(PPFD) measured overthethediurnal
diurnal cycle
cycle at at
twotwo
heights
heightsfromfromthethesoil surface
soil surface(2(2and
and0.40.4m)m)during
during thethe summer
summer (A), (A),fall
fall(B),
(B),winter
winter(C),
(C), and
and spring
spring
(D),(D),
andand air air
temperature
temperature measured
measuredover overthethedaily
daily cycle at at 22 m
m height
heightfromfromthethesoil
soilsurface
surface during
during
the the
summer
summer andandfallfall
(E)(E)and
andduring
duringthethewinter
winter and
and spring (F).(F). In
In(G),
(G),daily
dailyprecipitation
precipitation during
during thethe
experimental
experimental period
period (year
(year2022)
2022)isisshown.
shown.Red Red arrows
arrows indicate whenphysiological
indicate when physiologicalmeasurements
measurements
were taken
were during
taken seasons
during andand
seasons growth
growthperiods [(rests
periods 1 and
[(rests 2 (R12 and
1 and R2) and
(R1 and R2) growth unit formations
and growth unit for-
(GU1 and GU2)].
mations (GU1 and GU2)].
Forests 2025,
Forests 2025,16,
16,x 161
FOR PEER REVIEW 8 8of of
21 21

Figure2.
Figure Yerba mate
2. Yerba mate leaf
leaf area
area index
index (LAI)
(LAI)estimated
estimatedinintwo
twocultivation
cultivationsystems
systems (monoculture—MO
(monoculture—MO
and agroforestry system—AFS) for two genders (female— ♀
and agroforestry system—AFS) for two genders (female—♀ and male—♂) inseasons
and male—♂) in four [summer—
four seasons [sum-
rest 1 (R1); fall—growth unit 1 (GU1); winter—rest 2 (R2); spring—growth unit 2 (GU2)]. Estimated
mer—rest 1 (R1); fall—growth unit 1 (GU1); winter—rest 2 (R2); spring—growth unit 2 (GU2)]. Es-
mean ± SE and P-values (bold when significant) are shown (n = 6). Lowercase black letters compare
timated mean ± SE and P-values (bold when significant) are shown (n = 6). Lowercase black letters
genders (Gen) in each cultivation system (Sys) and each season, whereas uppercase black letters
compare
compare genders (Gen)
cultivation in each
systems for cultivation
each gendersystem
in each(Sys) andThe
season. each season, green
uppercase whereas uppercase
bold black
letters at the
letters
bottom compare
positioncultivation systems
compare seasons (n for each
= 24, P <gender
0.0001).in each season. The uppercase green bold letters
at the bottom position compare seasons (n = 24, P < 0.0001).
3.2. Expression of SSD in LAI
3.2. Expression of SSD
LAI increased in LAIthe 1st year after pruning, from season to season, and it was
during
much higher
LAI in MOduring
increased than inthe
AFS
1st(Figure 2). LAI
year after difference
pruning, frombetween
season tocultivation systems
season, and it was
could be even higher than shown here (about 70%–100%, depending on
much higher in MO than in AFS (Figure 2). LAI difference between cultivation the season), because,
systems
in AFS,
could bethe sensors
even alsothan
higher captured
showna fraction of the70%–100%,
here (about forest canopy, includingon
depending notthe
only the LAIbe-
season),
of yerba mate plants.
cause, in AFS, the sensors also captured a fraction of the forest canopy, including not only
The gender effects on LAI had been significant only in the summer, after formation
the LAI of yerba mate plants.
pruning, with males showing higher LAI than females in MO. The opposite was detected
The gender effects on LAI had been significant only in the summer, after formation
in AFS, with LAI being higher in females than in males in the summer (Figure 2). Such a
pruning, with males showing higher LAI than females in MO. The opposite was detected
situation was highly dependent on the remaining leaf area after pruning, as a new leaf area
in AFS, with LAI being higher in females than in males in the summer (Figure 2). Such a
was not formed between the R1 (summer) and GU1 (fall). From the fall, SSD in LAI was
situation was highly dependent on the remaining leaf area after pruning, as a new leaf
not expressed anymore.
area was not formed between the R1 (summer) and GU1 (fall). From the fall, SSD in LAI
was
3.3. not expressed
Expression anymore.
of SSD in Diurnal Leaf Gas Exchanges
The estimated leaf CO2 assimilation rate (Anet , leaf photosynthesis) was the highest
3.3. Expression
during the fallofand
SSDwinter
in Diurnal Leaflowest
and the Gas Exchanges
during the spring, with intermediate values
Thethe
during estimated leaf CO3).
summer (Figure 2 assimilation rate (A
Anet was impacted by , leaf
netthe photosynthesis)
cultivation wasallthe
system over highest
seasons,
during
showing the fall and
higher values winter
in MOand the
than lowest
AFS, exceptduring themorning
for early spring, and
withlateintermediate values
afternoon hours
in all seasons
during (interactions
the summer 3). Anet×was
(FigureSystem Hour). The difference
impacted between the
by the cultivation two systems
system over allforsea-
A
sons, increased over the diurnal hours, being the most expressive at
net showing higher values in MO than AFS, except for early morning and late afternoon the midday period,
from 10:45
hours in all hseasons
to 14:45(interactions
h. The SSD in Anet was
System expressed
× Hour). during the between
The difference summer theR1 and
twospring
systems
GU2 periods, expressed in higher values of females than in males (Figure
for Anet increased over the diurnal hours, being the most expressive at the midday period, 3A,D).
from The
10:45estimated
h to 14:45stomatal
h. The SSDconductance (gsexpressed
in Anet was ) was the highest
duringin thesummer
the winter, R1
followed by
and spring
generally similar
GU2 periods, expressed g s in fall and spring (two periods of growth unit formations,
in higher values of females than in males (Figure 3A,D). GU1 and
GU2),The estimated stomatalwere
and the lowest values found in (g
conductance the summer R1 period (Figure S2). The gs wasby
s) was the highest in the winter, followed
higher in MO than in AFS, except during
generally similar gs in fall and spring (two periods the fall of GU1, when itunit
of growth was formations,
similar between
GU1the and
two cultivation systems (Figure S2B). The gs showed low variations during the diurnal
GU2), and the lowest values were found in the summer R1 period (Figure S2). The gs was
course in AFS, while in MO, such diurnal variation was significant. At midday, gs was
higher in MO than in AFS, except during the fall of GU1, when it was similar between the
two cultivation systems (Figure S2B). The gs showed low variations during the diurnal
course in AFS, while in MO, such diurnal variation was significant. At midday, gs was up
Forests 2025, 16, 161 9 of 21

up to 4–5 times higher than one registered at the beginning or end of the day during R1
and R2 (Figure S2A,C). The SSD in gs was expressed only during summer R1, being higher
in females than in males (Figure S2A) and allowing higher Anet in females than in males
(Figure 3A).
The estimated leaf transpiration rate (E) was the highest in spring GU2 as compared to
all three previous growth periods (Figure 4). It was impacted by the cultivation system over
all four annual growth periods, being higher in MO than in AFS. Stronger variations over
the diurnal course were observed in MO when compared to AFS. Hourly E variations in AFS
were non-significant during GU1 and GU2 periods—interaction Sys × Hour (Figure 4B,D).
The SSD was expressed for E and females presented higher values than males during the
summer R1 period (Figure 4A). The opposite situation in SSD expression was observed
during the spring GU2, when males had higher E than females in MO, without any gender
segregation in AFS (Figure 4D). In addition, no gender differentiation in E was observed
during the fall (GU1) and winter (R2) seasons (Figure 4B,C).
During fall R1, the intercellular CO2 concentration (Ci ) was the lowest among the four
growth periods (Figure S3). Ci was modified by the cultivation system over all seasons,
generally being higher in AFS than in MO, with the exception of early morning and late
afternoon hours (Figure S3). Significant variations in Ci were observed over the diurnal
cycle, especially in MO, and very low variations were registered in AFS. Ci showed gender
segregation during the four rhythmic growth periods. Ci was higher in males than in
females in the summer R1 and spring GU2 periods (Figure S3A,D), while it was higher in
females than in males during the fall GU1 and winter R2 periods (Figure S3B,C).
The estimated Anet /Ci ratio was the highest during the fall GU1 and diminished
gradually during the winter and spring GU2 (Figure 5). Anet /Ci ratio was always higher in
MO than in AFS, and it varied significantly over the four rhythmic growth periods in MO,
but only slightly in AFS. In the summer R1 and spring GU2 periods, the Anet /Ci ratio was
statistically higher in females than in males in both cultivation systems (Figure 5A,D), as
happened with SSD in Ci (Figure S3A,D). During fall GU1, no significant SSD expression
in the Anet /Ci ratio was observed (Figure 5B), while Anet /Ci was higher in males than in
females during the winter R2 period (Figure 5C).
During the spring, the estimated instantaneous water use efficiency (WUE) was the
lowest among the four seasons (Figure 6). WUE was higher in MO than in AFS during the
summer R1 and the fall GU1 periods (Figure 6A,B). During winter R2, WUE was higher in
MO than in AFS for males but not for females (Figure 6C). However, no differences among
the two cultivation systems were observed during the spring GU2 period (Figure 6D). The
SSD was expressed in WUE in all growth periods, with females showing higher values
than males in both systems during the summer R1 and spring GU2 periods (Figure 6A,D).
Males showed higher WUE than females during the fall GU1 period in both cultivation
systems (Figure 6B), while such gender segregation was observed only in the MO during
the winter R2 period (Figure 6C).
Forests 2025,
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2025, 16,161
x FOR PEER REVIEW 10 of 2110 of 21

Yerba
Figure3.3.Yerba
Figure mate
mate leafleaf net2 CO
net CO 2 assimilation
assimilation rate (Arate (Anet ) estimated
net) estimated in two cultivation
in two cultivation systems (mon-systems
(monoculture—MO
oculture—MO and agroforestry system—AFS) for two genders (female—♀ and male—♂) duringduring
and agroforestry system—AFS) for two genders (female— ♀ and male—♂)
four seasons:
four seasons:(A)
(A)summer—rest
summer—rest 1 (R1),
1 (R1), (B)(B) fall—growth
fall—growth unitunit 1 (GU1),
1 (GU1), (C) winter—rest
(C) winter—rest 2 (R2),2 and
(R2), and
(D) spring—growth
(D) spring—growth unit
unit 2 (GU2)]
2 (GU2)] alongalong the diurnal
the diurnal period.period. Estimated
Estimated mean ± SEmean ± SE and
and P-values P-values
(bold
(bold
when significant) are shown (n = 6). Lowercase, colored letters compare genders (Gen) under eachunder
when significant) are shown (n = 6). Lowercase, colored letters compare genders (Gen)
each cultivation system (Sys) for each hour and each season; uppercase, colored letters compare
cultivation system (Sys) for each hour and each season; uppercase, colored letters compare cultiva-
cultivation systems for each gender for each hour and for each season. The uppercase bold green
tion systems for each gender for each hour and for each season. The uppercase bold green letters in
letters in parenthesis on the right side compare general Anet seasonal responses (P < 0.0001).
parenthesis on the right side compare general Anet seasonal responses (P < 0.0001).
Forests 2025,
Forests 16,16,
2025, 161x FOR PEER REVIEW 11 of 21
11 of 21

Figure 4.
Figure 4. Yerba
Yerbamate
mateleaf
leaftranspiration
transpiration rate (E)(E)
rate estimated
estimatedin two cultivation
in two systems
cultivation (monoculture—
systems (monoculture—
MO
MO and agroforestrysystem—AFS)
and agroforestry system—AFS) forfor
twotwo genders
genders (female—
(female—♀ and♀and male—♂)
male—♂) duringduring four seasons:
four seasons:
(A)
(A) summer—rest
summer—rest 11(R1),(R1),(B)(B)fall—growth
fall—growth unit
unit 1 (GU1),
1 (GU1), (C) (C) winter—rest
winter—rest 2 (R2),
2 (R2), andspring—
and (D) (D) spring—
growth unit 22 (GU2)]
growth unit (GU2)]for
foreach
eachhour.
hour.Estimated
Estimated mean
mean ± and
± SE SE and P-values
P-values (bold(bold
whenwhen significant)
significant) are are
shown (n = 6). Lowercase colored letters compare gender (Gen) responses
shown (n = 6). Lowercase colored letters compare gender (Gen) responses under each cultivation under each cultivation
system (Sys) for
system (Sys) foreach
eachhour
hourandandeach
each season;
season; uppercase
uppercase colored
colored letters
letters compare
compare cultivation
cultivation systems
systems
for
for each gender,hour,
each gender, hour,and
andseason.
season.The
The uppercase
uppercase bold
bold green
green letters
letters in parenthesis
in parenthesis onright
on the the right
side side
compare generalEEseasonal
compare general seasonalresponses
responses (P(P < 0.0001).
< 0.0001).
 Forests 2025, 16, x FOR PEER REVIEW 12 of 21
Forests 2025, 16, 161 12 of 21

Figure 5. Yerba mate Anet /Ci ratio estimated in two cultivation systems (monoculture—MO and agro-
Figure 5. Yerba mate Anet/Ci ratio estimated in two cultivation systems (monoculture—MO and ag-
forestry system—AFS) for two genders (female—♀and male—♂) during four seasons: (A) summer—
roforestry system—AFS) for two genders (female—♀ and male—♂) during four seasons: (A) sum-
rest 1 (R1), (B) fall—growth unit 1 (GU1), (C) winter—rest 2 (R2), and (D) spring—growth unit 2
mer—rest 1 (R1), (B) fall—growth unit 1 (GU1), (C) winter—rest 2 (R2), and (D) spring—growth
(GU2)] for each hour. Estimated mean ± SE and P-values (bold when significant) are shown (n = 6).
unit 2 (GU2)] for each hour. Estimated mean ± SE and P-values (bold when significant) are shown
Lowercase colored letters compare gender (Gen) responses under each cultivation system (Sys) for
(n = 6). Lowercase colored letters compare gender (Gen) responses under each cultivation system
each hour and season; uppercase, colored letters compare cultivation systems for each gender, hour,
(Sys) for each hour
and season. and season; uppercase,
The uppercase, bold, greencolored
lettersletters compare cultivation
in parenthesis systems
on the right sidefor each gen-
compare general
der, hour, and season. The uppercase, bold, green letters in parenthesis on the right
Anet /Ci seasonal responses (P < 0.0001). Different y-axis scales for seasons are used toside compare
observe the
gender segregations.
 Forests 2025, 16, x FOR PEER REVIEW 13 of 21

Forests 2025, 16, 161 13 of 21

general Anet/Ci seasonal responses (P < 0.0001). Different y-axis scales for seasons are used to observe
the gender segregations.

Figure 6. Yerba
Figure Yerbamate
mateinstantaneous
instantaneous water
water useuse
efficiency (WUE)
efficiency estimated
(WUE) estimatedin twoin cultivation systems
two cultivation systems
(monoculture—MO
(monoculture—MO and andagroforestry
agroforestrysystem—AFS)
system—AFS)for fortwo
two genders
genders (female—and
(female—♀ ♀and male—♂)
male—♂) during
dur-
four seasons:
ing four (A)(A)
seasons: summer—rest
summer—rest 1 (R1), (B)
1 (R1), (B)fall—growth
fall—growthunitunit 11 (GU1),
(GU1), (C) winter—rest
winter—rest 22(R2),
(R2), and
(D)
and spring—growth
(D) spring—growth unit 2 (GU2)]
unit 2 (GU2)]for
foreach
each hour. Estimatedmean
hour. Estimated mean ± SE
± SE andand P-values
P-values (bold(bold
whenwhen
significant)
significant) are shown
shown (n == 6).
6). Lowercase, colored letters
letters compare
compare gender
gender (Gen)
(Gen) responses
responses under
under each
cultivation system (Sys) for each hour and season; uppercase, colored letters compare
each cultivation system (Sys) for each hour and season; uppercase, colored letters compare cultiva- cultivation
systems for each
tion systems gender,
for each hour,
gender, andand
hour, season.
season.TheTheuppercase,
uppercase,bold,
bold, green lettersin
green letters inparenthesis
parenthesisonon the
right side compare general WUE seasonal responses (P < 0.0001). Different y-axis scales for seasons
are used to observe the gender segregations.
 Forests 2025, 16, x FOR PEER REVIEW 14 of 21

Forests 2025, 16, 161 14 of 21

the right side compare general WUE seasonal responses (P < 0.0001). Different y-axis scales for sea-
sons are used to observe the gender segregations.

4. Discussion
4. Discussion
The leaf gas exchanges of yerba mate were more intense in MO than in AFS for all
The leaf gas exchanges of yerba mate were more intense in MO than in AFS for all
traits, with the exception of Ci . The SSD in yerba mate was expressed over all four annual
traits, with the exception of Ci. The SSD in yerba mate was expressed over all four annual
periods of rhythmic growth for physiological traits such as Ci and WUE, while it was
periods of rhythmic growth for physiological traits such as Ci and WUE, while it was less
less frequent in other traits. SSD was modified by the cultivation system only in two
frequent in other traits. SSD was modified by the cultivation system only in two cases,
cases, being expressed in E only in MO during the spring (Figure 4D) and in WUE in MO
being expressed in E only in MO during the spring (Figure 4D) and in WUE in MO during
during
the the (Figure
winter winter (Figure 6C). Indioecious
6C). In higher higher dioecious
species, species,
females females have
have larger larger reproductive
reproductive ef-
efforts compared with males, especially during the summer R1
forts compared with males, especially during the summer R1 and spring GU2 growth and spring GU2pe- growth
periods.
riods. OverOver
thethe phenology
phenology andand seasonal
seasonal variations
variations in environmental
in environmental conditions,
conditions, malesmales
and females would express different strategies to finish the phenophases
and females would express different strategies to finish the phenophases and cope with and cope with any
limiting
any condition.
limiting Females
condition. Femaleswere more
were moreintensive
intensive Anet
in in Anet(R1
(R1and
andGU2) andgsgs(R1),
GU2)and (R1),while
the superiority
while of male
the superiority or female
of male individuals
or female was was
individuals expressed
expressedin different growth
in different growthperiods
in otherintraits
periods otherstudied here (Figure
traits studied 7). Such
here (Figure greatgreat
7). Such variation in SSD
variation altering
in SSD thethe
altering gender
gender
dominancedominance overand
over time timeseasons
and seasons was observed
was observed previously
previously for photosynthetic
for photosynthetic ca-
capacity of
pacity mate
yerba of yerba
andmate and for physiological
for physiological responses responses
in various in other
various other dioecious
dioecious trees
trees [29,40,47,48]
[29,40,47,48]
and grasses and[49].grasses [49].

Summaryofofthe
Figure 7.7.Summary
Figure thegender
gender dominance
dominance in yerba
in yerba mate
mate leaf leaf gas exchange
gas exchange (net2 assimilation
(net CO CO2 assimilation
rate—Anetnet
rate—A , stomatal
, stomatal conductance—g
conductance—g s , transpiration
s, transpiration rate—E,
rate—E, intercellular
intercellular CO2 concentration—C
CO2 concentration—Ci, in- i,
instantaneous
stantaneous carboxylation
carboxylation efficiency—A
efficiency—A net/Cnet /Ci ratio,
i ratio, and instantaneous
and instantaneous leaf water
leaf water use efficiency—
use efficiency—
WUE) during
WUE) during thethe annual
annual rhythmic
rhythmic growthgrowth [summer—rest
[summer—rest 1 (R1), fall—growth
1 (R1), fall—growth unit 1 (GU1),
unit 1 (GU1), winter—
winter—rest
rest 2 (R2), and2 spring—growth
(R2), and spring—growth
unit 2 (GU2)]unit 2 (GU2)]
under under two
two contrasting contrasting
cultivation cultivation
systems (monocul-systems
(monoculture—MO and agroforestry—AFS).
ture—MO and agroforestry—AFS). The black
The black upper upper
arrows arrows
indicate theindicate the cultivation
cultivation system withsystem
with
higherhigher
valuesvalues
for eachforofeach of leaf
leaf gas gas exchange
exchange parameters.
parameters. The dominance
The dominance of females
of females (red) (red)
or males or males
(blue)
(blue)
is marked with upper arrows, while ‘n.s.’ means no significant gender dominance. Two cases when cases
is marked with upper arrows, while ‘n.s.’ means no significant gender dominance. Two
when SSD expression
SSD expression was modified
was modified by the cultivation
by the cultivation system,
system, being being expressed
expressed only in MOonly
within MOdom-
male with male
dominance
inance in EWUE,
in E and and WUE, are tagged
are tagged withovals.
with yellow yellow ovals.

4.1. Expression of SSD in Yerba Mate Leaf Gas Exchanges During the Annual Rhythmic Growth
4.1. Expression of SSD in Yerba Mate Leaf Gas Exchanges During the Annual Rhythmic
Under Contrasting Cultivation Systems
Growth Under Contrasting Cultivation Systems
Adult yerba mate growth strongly responds to open areas and high light conditions,
Adult yerba mate growth strongly responds to open areas and high light conditions,
showing intensive flushing in MO [9]. In AFS, they form longer internodes and bigger,
showing intensive flushing in MO [9]. In AFS, they form longer internodes and bigger,
wider individual leaves than plants cultivated in MO. Both the cultivation system and
wider individual leaves than plants cultivated in MO. Both the cultivation system and the
the developmental
developmental stage stage
act onact
the on
sexthe sex expression
expression ofarchitectural
of various various architectural
parametersparameters
in yerba in
yerba mate, as in LA distribution over the plant profile, light interception, leaf, and canopy
photosynthesis [10,35]. When cultivated in AFS, yerba mate plants reduce leaf area density
up to six times, total leaf area up to seven times, and, in LAI, up to twelve times compared
to those in MO [10]. Such a difference was less pronounced herein (Figure 2), which is likely
Forests 2025, 16, 161 15 of 21

due to the methods and equipment used for evaluating plants, the genetic background of
populations/clones, and environmental conditions.
Yerba mate can be classified as a “less shade-tolerant species” [50] because it clearly
responds to decreased light intensity, as seen here under the low light availability of AFS
(Figures 1–6), a kind of environmental pressure. On the other hand, high light conditions
in MO, especially in the early stages after planting and after pruning, can be equally
considered as a stressful factor due to species origin in the forest shade [2]. A higher
degree of environmental degradation is indicated by a higher leaf C/N ratio in MO than in
AFS, with females showing leaves with a lower C/N ratio and higher photosynthesis than
males in both systems [35]. Herein, the two cases of gender segregation for E and WUE
occurred only in MO conditions (Figures 4D and 6C), suggesting the MO as the system of
higher environmental pressure for yerba mate and then causing higher gender segregation
than AFS.
Gas exchange of two genders of Silene latifolia do not respond differently to low
resource availability (light, water, nitrogen, phosphorus, and potassium), and higher
female reproductive effort relative to males does not differentially affect their ability to
assimilate carbon, despite male Anet and gs being slightly, but consistently, higher than those
of females [51]. Varying gender acclimation to low light can be observed in some dioecious
species, as in Amaranthus palmeri females [52]. They respond to shading by stem elongation,
whereas the male plants respond by increasing specific leaf area. A. palmeri genders showed
a differential response to stressful conditions because of differences in their ontogeny and
physiology, and possibly due to the cost of reproduction. In yerba mate, males are more
sensitive to environmental changes than females, especially in MO. Females optimize
foliage structure and phenology in MO, which is determined intrinsically by high fruit
loading and modified by night length [11]. Fruit load varies tremendously among yerba
mate genotypes [1]. On average, the fruit yield is 1.6 kg tree−1 , varying from 0.57 kg tree−1
in a Brazilian progeny to 14.6 kg in the M35 genotype from breeding experiments in
Argentina. As a matter of fact, the demand for photosynthetic products will be different in
males as compared with females because of sink investments. Even with optimized foliage
structure and physiology in females compensating for greater reproductive costs in early
developmental stages, females and males are similar in terms of canopy photosynthesis
after 2 years of regrowth [10]. Sexual specialization in leaf and plant photosynthesis is
related to early vegetative and reproductive stages in yerba mate, which is when females
have shown higher carbon assimilation than males [10,35]. Interestingly, leaves in MO have
higher stomatal density in females than in male plants, and this trait shows higher values
when leaves are formed during the vegetative growth period compared to the flowering
period [53]. Herein, we noticed higher Anet in females than in males at early vegetative
(R1) and early reproductive (GU2) stages (Figures 2 and 7). Despite that genetic variation
could have a prominent role in sex expression in dioecious species [21,35], the findings
from three independent experiments with varying genetic material indicate a generalized
SSD expression in yerba mate, and this could be associated with a fitness strategy of female
plants in their additional reproduction efforts.
Values of Anet , gs , and E were relatively low in yerba mate in some experiments that did
not study seasonal variation or SSD [54,55]. The Anet estimated in females cultivated in MO
is higher than in males, differing only at the fruit ripening stage during the first year after
pruning [10]. The opposite situation in SSD is found under AFS, with the Anet of females
being lower than that of males at spring flush and fruit ripening [10]. Similarly, the greater
photosynthetic capacity of female plants compared with male ones was noticed under
high light conditions (compared here to MO) in Amaranthus palmeri, and such differential
photosynthetic performance diminished in both genders with progressed phenology [56].
Forests 2025, 16, 161 16 of 21

Even the quality of light can contribute to gender differentiation in a number of plants
(gender ratio), as observed in Drynaria roosii ferns [57]. The red light promoted more males
than blue light did in D. roosii, with the last contributing to a female-biased trend. Carbon
gain of entire female plants in MO is higher than that of male ones during fruit ripening,
while this sexual differentiation occurred during spring flushing in AFS [10]. In our study,
the cultivation systems modified the expression of SSD, and MO was the system where
males had more intensive E and WUE during spring GU2 and winter R2, respectively
(Figure 7). Such rare cases, when compared to other case studies, could be related to leaf
ontogeny and evaluated traits.

4.2. Expression of SSD in Yerba Mate Leaf Gas Exchanges Under Environmental Pressures
During Ontogeny
Sexual specialization in yerba mate biomass production is more expressed under
environmental pressures [31]. The ontogeny, together with environmental stressors (e.g.,
drought and light stresses), play a role in the SSD expression of Pistacia lentiscus [40].
When environmental conditions are optimal under controlled environments (e.g., growth
chambers), just a few consistent SSD expressions in ecophysiological responses are found
at the leaf level. In older communities, the ecological advantage of male plants is due to
higher competition for water uptake, while in the youngest open areas, it is the higher WUE
of female plants that confers an ecological advantage. In our experiment, WUE was higher
in females than in males during the early vegetative (summer R1) and early reproductive
(spring GU2) periods (Figure 6), conferring an ecological advantage of female yerba mate
plants. On the other hand, higher WUE was observed in males in fall (GU1) and winter
(R2), conferring the ecological advantage of male plants due to higher competition for water
uptake during colder and drier periods (Figure 7). Longer observations, i.e., at least during
10–20 years, could reveal whether the ontogeny changes the male or female dominance in
leaf gas exchange expressions in yerba mate, while hormonal balance and genetic markers
could help to elucidate the pathways of gender dominance.
Females of Taxus baccata have similar photosynthetic capacity to males, and the higher
reproductive efforts of females do not happen at the expense of their photosynthetic ca-
pacity [58]. In such species, SSD fluctuates seasonally and is remarkable under nutrient
deficiency, being related to gender’s ability to protect the photosynthetic apparatus against
photoinhibition through antioxidants. Under stresses caused by heavy metal (lead) and low
water availability, Populus cathayana males exhibit greater plasticity in the photosynthetic
capacity than females [59], with females allocating more carbon and nitrogen to leaf defense
chemical components than males after long-term severe defoliation under nitrogen defi-
ciency [60]. Even the recently described phenomenon of leaf water uptake by P. euphratica
is significantly greater in female than in male plants, and it increases from the initial to the
final stages of one vegetative period [61]. The Anet and gs in P. cathayana females responded
more gradually in water-related traits than males under drought and heat stress, with
growth and photosynthesis being mainly driven by soil moisture in females, while male
performance is mainly related to temperature [62]. Female roots of P. cathayana release
diverse phenolic allelochemicals into the soil environment, resulting in growth inhibition
of same-sex neighbors when grown in sex monoculture, but their growth with males is
consistently enhanced, especially root growth [63]. However, the dioecious species of the
genus Populus do not always present SSD and the gender superiority is not the same [64].
Such discrepancies in literature could be associated with the experimental design, varying
species and genotypes, stress conditions, and sampling.
In field-grown Pistacia lentiscus, SSD observed in Anet and gs can be related to low
water availability during the summer [40]. A similar trend was observed in Anet and gs of
yerba mate (Figures 3A and S1A). In Acer negundo, canopy gs is higher in females during the
Forests 2025, 16, 161 17 of 21

spring but higher in males during the summer [29], with seasonal fluctuations comparable
to those of E for yerba mate (Figure 4). Ci of Pistacia lentiscus is lower in females than
males during the summer [40], as observed in yerba mate leaves during the summer and
spring (Figures S3A,D and 7). Summer water stress significantly reduces the growth of Ilex
aquifolium female trees in the following growing season, revealing greater vulnerability of
female trees to increasing drought [65]. Salix glauca females have lower drought tolerance
than males under years of extreme aridity, while males grew at a consistent rate regardless
of habitat aridity, which was observed during a ten-year period [66]. Such responses
indicate that one annual cycle probably would not be enough for a full generalization
about yerba mate. The spatial gender segregation could shift under global warming, and
female plants of various dioecious species would lose their dominance in high-resource
habitats, and males would increase their dominance in relatively low-resource habitats [66].
Accordingly, males of P. cathayana exhibit higher Anet , biomass accumulation, and carbon
balance mechanisms under elevated CO2 than females [67]. No experiment about the
physiology of yerba mate was performed under elevated CO2 , but some simulations
projected that current favorable areas for yerba mate cultivation would reduce significantly
in Brazil [3,16]. How gender dominance in yerba mate carbon acquisition and WUE would
change under elevated CO2 is a topic for further research.

5. Conclusions
As a novelty, we found a modification of SSD expression in leaf gas exchange traits
over the rhythmic growth period, which was unknown in yerba mate. Then, our initial
hypothesis that females would have higher photosynthetic performance than males was
strongly modified by growth rhythmicity. In fact, Anet , Anet /Ci , and WUE were higher in
females than males during the summer rest (R1) and spring growth unit (GU2) periods.
Also, gs and E were higher in females than males during summer R1. Oppositely, higher
WUE in males than in females was observed during the fall growth unit (GU1) and winter
rest (R2), together with the Anet /Ci ratio during winter R2 and E during the spring GU2
periods. This means that the gender dominance changed over the growth periods and
phenology of yerba mate. All those leaf gas exchange traits were modified by the light
environment, with higher values found generally under MO as compared with AFS. Despite
the strong effect of the cultivation system on LAI and leaf gas exchange traits over the
diurnal course, SSD expression was rarely modified by the cultivation system, being
expressed for E and WUE only in MO during spring GU2 and winter R2, respectively.
Higher WUE of females during early vegetative (R1) and early reproductive (GU2) growth
periods would confer an ecological advantage during warmer periods. On the other
hand, higher WUE in males during fall GU1 and winter R2 would benefit plants during
colder and drier annual periods. The superior Anet in females during early vegetative (R1)
and early reproductive (GU2) stages can be underlined as a generalized SSD expression
in yerba mate and could be considered as a fitness strategy of female plants in their
additional reproduction efforts. While long-term experiments would reveal if the ontogeny
changes the gender dominance in leaf gas exchanges of yerba mate, our study highlights
the importance of the period of rhythmic growth when the ecophysiological traits were
evaluated.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/f16010161/s1, Figure S1: Flowchart showing measurements
of light (photosynthetically active photon flux density, PPFD) and Anet /PPFD curves for further
modeling the instantaneous leaf gas exchanges along the diurnal period in monoculture (MO) and
agroforestry (AFS); Figure S2: Yerba mate stomatal conductance (gs ) estimated in two cultivation
systems (monoculture—MO and agroforestry system—AFS), for two genders (female—♀and male—
Forests 2025, 16, 161 18 of 21

♂), during four seasons: (A) summer—rest 1 (R1), (B) fall—growth unit 1 (GU1), (C) winter—rest 2
(R2), and (D) spring—growth unit 2 (GU2)] for each hour. Estimated mean ± SE, and P-values (bold
when significant) are shown (n = 6). Lowercase colored letters compare gender (Gen) responses under
each cultivation system (Sys), for each hour, and each season; uppercase colored letters compare
cultivation systems for each gender, for each hour, and for each season. The uppercase bold green
letters in parenthesis on the right side compare general gs seasonal responses (P < 0.0001). Different
y-axis scales for seasons are used to observe the gender segregations; Figure S3: Intercellular CO2
concentration (Ci ) estimated in two cultivation systems (monoculture—MO and agroforestry system—
AFS), for two genders (female—♀and male—♂), during four seasons: (A) summer—rest 1 (R1), (B)
fall—growth unit 1 (GU1), (C) winter—rest 2 (R2), and (D) spring—growth unit 2 (GU2)] for each
hour. Estimated mean ± SE, and P-values (bold when significant) are shown (n = 6). Lowercase
colored letters compare gender (Gen) responses under each cultivation system (Sys), for each hour,
and each season; uppercase colored letters compare cultivation systems for each gender, for each
hour, and for each season. The uppercase bold green letters in parenthesis on the right side compare
general Ci seasonal responses (P < 0.0001).

Author Contributions: Conceptualization, M.R.; data curation, M.R. and E.R.B.; formal analysis, M.R.;
funding acquisition, M.R. and I.W.; investigation, M.R., E.R.B., R.L.d.A. and R.V.R.; methodology, M.R.
and E.R.B.; project administration, M.R. and I.W.; resources, M.R.; E.R.B., R.V.R. and I.W.; validation,
all authors; writing—original draft, M.R.; writing—review and editing: all authors. All authors have
read and agreed to the published version of the manuscript.

Funding: This research was funded by the National Council for Scientific and Technological Devel-
opment (CNPq, Brazil) with the fellowship for Invited Researcher awarded to Miroslava Rakočević
(350509/2020-4). R.V.R. is a CNPq fellow (3042950/2022-1).

Data Availability Statement: The authors can provide the experimental data for all interested
researchers.

Acknowledgments: To Mário Dobner, Kelen Haygert Lencina, and Ana Clara Dondoerfer Teixeira
from UFSC, Brazil, for assisting us in the data collection and maintenance of the experimental field.

Conflicts of Interest: Authors Miroslava Rakočević, Eunice Reis Batista and Ivar Wendling were
employed by Embrapa. The remaining authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed as a potential conflict of interest.

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