Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

Effect of seasonality on the leaf phenology and physiology

of Byrsonima species (Malpighiaceae)

Bárbara Godinho Pereira1; https://orcid.org/0000-0001-5485-8211

Ana Paula de Faria1; https://orcid.org/0000-0001-7043-2480

Vinícius Coelho Kuster2; https://orcid.org/0000-0002-1236-486X

Ana Silvia Franco Pinheiro Moreira1*; https://orcid.org/0000-0001-5090-5527

1. Laboratory of Plant Physiology, Institute of Biology, Universidade Federal de Uberlândia, CEP 38400-902,

Uberlândia, Minas Gerais, Brazil; barbara.godinhop@gmail.com, anapdefaria@gmail.com,

anasilviamoreira@gmail.com (Correspondence*)

2. Laboratory of Plant Anatomy, Universidade Federal de Jataí, CEP 75801-615, Jataí, Goiás, Brazil;

viniciuskuster@ufj.edu.br

Received 07-III-2022. Corrected 20-X-2022. Accepted 24-I-2023.

ABSTRACT

Introduction: Defined seasonality in savanna species can stimulate physiological responses that maximize

photosynthetic metabolism and productivity. However, those physiological responses are also linked to the phe-

nological status of the whole plant, including leaf phenophases.

Objective: To study how physiological traits influence phenophase timing among congeneric and co-occurring

savanna species.

Methods: We evaluated the leaf phenology and physiological traits of populations of Byrsonima intermedia, B.

coccolobifolia, and B. verbascifolia. Physiological measurements were performed at the onset of the dry and

rainy seasons and again late in the season.

Results: B. intermedia and B. coccolobifolia were classified as brevideciduous and B. verbascifolia as evergreen.

The maximum quantum yield for B. intermedia and B. coccolobifolia were lowest during the dry season. At the

onset of the dry period, the highest chloroplastidic pigment levels were observed, which decreased as the season

advanced, total chlorophyll/carotenoid ratios were lowest, and carotenoid contents were highest. We detected

low starch content values at the start of the rainy season, coinciding with the resumption of plant growth. Two

months into this season, the leaves were at their peak structural and functional maturity, with high water-soluble

polysaccharide values and photosynthetic rates, and were storing large amounts of starch.

Conclusions: Physiological and leaf phenological strategies of the Byrsonima species were related to drought

resistance and acclimatization to the seasonality of savanna water resources. The oscillations of the parameters

quantified during the year indicated a strong relationship with water seasonality and with the phenological status

of the leaves.

Key words: carbohydrate balance; Cerrado; drought; tree ecophysiology; leaf phenology; photosynthesis;

woody species.

https://doi.org/10.15517/rev.biol.trop..v71i1.50344

BOTANY AND MYCOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

INTRODUCTION

Low water availability is one of the envi-

ronmental factors that can most alter plant

structures and behavior (Reddy et al., 2004).

Among the strategies developed by plants

to survive dry periods are improvements of

antioxidant systems, changes in leaf osmotic

potentials, as well as in the periods of growth

and developmental of vegetative organs, adjust-

ments of photosynthetic metabolism, and mor-

phological such as increased leaf sclerophylly

(Chaves et al., 2002; Reddy et al., 2004; Reich

et al., 2003; Souza et al., 2015). Water deficits

can induce the development of leaves with

thick cuticles, low investments in mesophyll

formation, and large investments in lignin and

sclerenchyma that confer greater tissue den-

sity (Edwards et al., 2000; Lüttge et al., 1997;

Salleo & Nardini, 2000; Sobrado & Medina,

1980; Souza et al., 2015). Different morpho-

logical characteristics reflect different adaptive

processes to protect against high light intensi-

ties and long dry periods, while still ensuring

efficient photosynthetic levels (Ehleringer &

Mooney, 1978; Hoffmann et al., 2005; Nicotra

et al., 2011; Reich et al., 2003).

The temporal patterns of plant growth

observed in savanna environments are linked

to climatic seasonality especially the rainy and

dry seasons (Williams et al., 1997). The season-

ality of water resources stimulates leaf fall in

several species (Borchert et al., 2005; Morella-

to et al., 2000; Williams et al., 1997), although

the real trigger of leaf phenology remains

uncertain. Rapid decreases in soil moisture,

the activities of the endogenous process such

as stem rehydration (Reich & Borchert, 1984)

and photoperiodism (Rivera et al., 2002) have

been proposed to explain leaf fall. Despite that,

there is a consensus that resource seasonality is

the selective force behind distinct phenologi-

cal strategies (Camargo et al., 2018; Eamus &

Prior, 2001; Goldstein et al., 2008). Decidu-

ousness can have an adaptive advantage when

combined with other functional and structural

characteristics (Rossatto, 2013), as deciduous

species generally have leaves with shorter life

RESUMEN

Efecto de la estacionalidad en la fenología y fisiología de especies de Byrsonima (Malpighiaceae)

Introducción: La marcada estacionalidad en las especies de sabana puede estimular respuestas fisiológicas

que maximicen el metabolismo fotosintético y la productividad. Sin embargo, esas respuestas fisiológicas están

vinculadas al estado fenológico de toda la planta, incluidas las fenofases de las hojas.

Objetivo: Estudiar cómo los rasgos fisiológicos influyen en el tiempo de la fenofase entre especies de sabana

congenéricas y concurrentes.

Métodos: Evaluamos la fenología y características fisiológicas de poblaciones de Byrsonima intermedia, B. coc-

colobifolia y B. verbascifolia. Las mediciones fisiológicas se realizaron al inicio de la estación seca y lluviosa,

y de nuevo al final de la estación.

Resultados: B. intermedia y B. coccolobifolia se clasificaron como brevicaducifolias y B. verbascifolia como

perennifolias. El rendimiento cuántico máximo para B. intermedia y B. coccolobifolia fueron más bajos durante

la época seca. Al inicio del período seco, se observaron niveles de pigmentos cloroplastídicos más altos, aunque

los niveles de clorofila disminuyeron a medida que avanzaba la estación seca, las proporciones clorofila/carot-

enoides totales fueron más bajas y los contenidos de carotenoides más altos. Detectamos valores bajos de con-

tenido de almidón al inicio de la época lluviosa, que coincide con la reanudación del crecimiento de la planta. A

dos meses de esta época, las hojas estaban en su máxima madurez estructural y funcional, con altos valores de

polisacáridos solubles en agua y tasas fotosintéticas, y almacenaban grandes cantidades de almidón.

Conclusiones: Las estrategias fisiológicas y fenológicas de las hojas de las especies de Byrsonima estaban rela-

cionadas con la resistencia a la sequía y la aclimatación a la estacionalidad de los recursos hídricos de la sabana.

Las oscilaciones de los parámetros cuantificados durante el año indicaron una fuerte relación con la estacionali-

dad hídrica y con los estados fenológicos de las hojas.

Palabras clave: balance de carbohidratos; Cerrado; sequía; ecofisiología de árboles; fenología de hojas; foto-

síntesis; especies leñosas.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

spans (and therefore greater investment in pho-

tosynthetic tissues), lower specific leaf masses,

and higher stomatal conductance and carbon

assimilation rates (Eamus et al., 1999; Ishida et

al., 2010). The leaves of evergreen species, on

the other hand, generally are expected to have

greater longevities, lower quantum yields, and

evidence of greater water use efficiencies (Ros-

satto et al., 2013; Somavilla et al., 2014; Wei et

al., 2016; Zhu et al., 2013).

Seasonal leaves may optimize carbon allo-

cation during different seasons (Kitajima et al.,

1997; Newell et al., 2002). Carbohydrates may

be constituents of cell walls, can be stored as

starch, or can be oxidized during respiration (as

small soluble sugars) (Pallardy, 2008). Those

soluble sugars can also decrease the osmotic

potentials of the plants and increase the water-

holding capacities of their tissues (Pallardy,

2008). Roots, stems, and leaves serve as stor-

age sites for carbohydrates, but the storage and

use of those resources appear to be related to

the phenology of carbon gain (Pallardy, 2008).

Maximum non-structural carbohydrate concen-

trations have been found when plant canopies

are at their fullest among seasonal species

(Newell et al., 2002) and, during any given

season (dry or rainy), the leaves may evidence

different patterns of sugar dynamics depending

on their phenological status, which led us to

analyze leaf sugar concentrations at different

times during the same season.

Herein, three congeneric Byrsonima spe-

cies from the Neotropical savanna were used as

a biological study model to analyze variations

in their physiological traits depending on the

timing of their leaf phenophases. Byrsonima

intermedia, B. coccolobifolia, and B. verbas-

cifolia have been described as evergreen (Boas

et al., 2013), deciduous (Silvério & Lenza,

2010), and brevideciduous (Kuster et al., 2017;

Lenza & Klink, 2006) respectively. Phenologi-

cal behaviors may change in different environ-

ments or during different periods (Borchert et

al., 2005; Cuzzuol & Clippel, 2009; Rivera

et al., 2002; Rossatto, 2013), which led us to

examine the phenological patterns of those

selected species in the field and address the

following questions: (i) are there variations in

plant functional traits during the same season?

(ii) Are there relationships between the func-

tional traits and the phenological statuses of the

studied species? We used specific leaf mass to

estimate investments in photosynthetic compo-

nents (Reich & Schoettle, 1988), and their rela-

tive water contents to indicate the water status

of the plant, chloroplastidic pigments, and fluo-

rescence parameters to evaluate the photosyn-

thetic apparatus and photochemical yields. We

also examined the carbohydrate balances of the

plants in each season, as well as the phenologi-

cal stages of the leaves of each species.

MATERIALS AND METHODS

Study area and plant species: The present

study was carried out in savanna areas (Cerrado

sensu lato in Brazil) at the Panga Ecological

Station (19º11’40” S & 48º19’06” W), a Private

Natural Heritage Reserve (Reserva Ecológica

do Panga) belonging to the Universidade Fed-

eral de Uberlândia (UFU) and located 30 km

from Uberlândia in southeastern Brazil. The

area has many Cerrado savanna plant forma-

tions, which include open formations (wetlands

and savannas) and forest formations (dense

Cerrado, evergreen forests, and gallery forests)

(Gonçalves et al., 2021). The local climate is

humid subtropical, with hot austral summers

and dry winters (Cwa) (Alvares et al., 2014),

with an average annual rainfall of apprximately

1 500 mm and a mean annual temperature of

23 ºC. Precipitation and temperature data were

provided by the Meteorological Station of the

Universidade Federal de Uberlândia and are

presented in Fig. 1.

This study was undertaken in a fragment of

Cerrado sensu stricto vegetation, the dominant

phytophysiognomic formation in the reserve

(Gonçalves et al., 2021). Twenty-five adult

individuals of Byrsonima intermedia A. Juss

and B. cocolobifolia H. B. & K., as well as 20

individuals of B. verbascifolia (L.) DC were

randomly marked. Ten individuals of each spe-

cies (n = 10) were used for the physiological

analyses, considering one leaf per individual;

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

the remaining individuals were accompanied

during the phenological analyses (n = 15),

except for B. verbascifolia (with n = 10), due

to the low number of individuals of that species

in the Cerrado fragment.

Fully developed leaves, without signs of

herbivory, were collected both during the dry

season and the rainy season for physiological

analyses. Samples were collected at the onset

of the dry period (June/2016) and again later in

the dry season (August/2016), as well as at the

onset of the rainy season (December/2016) and

again later in the rainy season (February/2017)

(Fig. 1). Only young leaves were collected

from the three species in December/2016, as

leaf sprouting was the principal phenophase

of both B. intermedia and B. coccolobifolia

(Fig. 2, Fig. 3).

Phenological analyses: The vegeta-

tive phenology of the three species studied

was monitored every month. The presence or

absence of foliar events (leaf sprouting, mature

leaves, senescence, and abscission) were

recorded for each plant. This method estimates

the percentage of individuals in the population

evidencing a given phenological event and

indicates the synchronicity of that specific

phenological event during the year (Morellato

et al., 2010). The intensities of the foliar events

(the expression of each phenophase by popula-

tions) were estimated for each plant according

to Fournier (1974), using a semi-quantitative

interval scale of five categories (0 to 4). The

levels of leaf deciduousness were determined

considering: (1) evergreen species, as those that

retain their leaf areas approximately constant,

or immediately recover them throughout the

year (Borges & Prado, 2014; Camargo et al.,

2018); (2) brevideciduous species, as those that

retain approximately half of their leaves and

quickly replace them during the same season

(Borges & Prado, 2014; Lenza & Klink, 2006);

and (3) deciduous species, as those that lose

their leaves for a period of at least 30 days dur-

ing the year (Borges & Prado, 2014; Damascos

et al., 2005). This classification has been used

for many tree species growing in the Brazilian

savanna (Borges & Prado, 2014).

Specific leaf mass and relative water

content: The specific masses and relative water

Fig. 1. The seasonal climate in the Brazilian savanna with its rainy and dry seasons. Mean temperature (line) and monthly

rainfall rates (bars) from April/2016 to August/2017 are indicated. The periods of the physiological analyses are shown for

the dry season (onset of the dry season–June/2016, and near the end of the dry season - August/2016) as well as the rainy

season (onset of the rainy season – December/2016, and during the time of more prolonged rainfall–February/2017). The data

were acquired from the Meteorological Station at the Universidade Federal de Uberlândia (Uberlândia, southeastern Brazil).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

contents of the leaves were obtained consider-

ing mature leaves (completely expanded leaves

on the 2nd or 3rd node), except in December,

when leaf sprouting was the predominant phe-

nophase (and the most expanded leaf of the

same season was used for the determinations).

Specific leaf masses (SLM) and relative water

contents (RWC) were determined using 0.5

cm2 leaf fragments (n = 10 per species). Those

samples were weighed in order to obtain their

fresh masses (FM), and then oven-dried at 60

ºC for 72 h to obtain their dry masses (DM).

The SLM was obtained as the ratio between

the dry mass (DM) and sample area (A) fol-

lowing Witkowski & Lamont (1991), and the

sclerophylly index following Groom & Lamont

(1999). RWC was obtained based on the FM,

using the equation, according to Turner (1986).

Carbohydrate (starch, total soluble sug-

ars, and water-soluble polysaccharides) con-

tents and osmotic potential: Leaf samples

from each species (n = 10) were oven-dried,

ground, and homogenized, and 0.2 mg of

each sample was centrifuged in 15 ml of

methanol:chloroform:water (12:5:3) solution

Fig. 2. Intensities (circular histograms) and activity indices (table) of the leaf phenophases evaluated for Byrsonima

intermedia. The leaf sprouting was presented in green coloration, mature leaves in blue, senescence red, and abscission in

brown coloration. The results of the Rayleigh (z) test, the mean angle concentration index (R), and the mean angle (indicating

the month with the greatest vector) in each phenophase are listed in the table.

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for total soluble sugar (TSS) extraction. The

residues were resuspended in 5 ml of 10

% ethanol to determine their water-soluble

polysaccharide (WSP) contents. Starch con-

tent was determined after further resuspen-

sion of the residue in 5 ml of 30 % perchloric

acid. Carbohydrate content was determined by

the phenol-sulfuric acid colorimetric method

(Chow & Landhäusser, 2004; Dubois et al.,

1956), using glucose as the standard. The

absorbance of the characteristic yellow-orange

color of the extracts was measured at 490

nm for hexoses (Dubois et al., 1956), using a

spectrophotometer (BioSpectro, SP-220, São

Paulo, Brazil).

The osmotic potential (Ψs) was estimated

using van’t Hoff’s equation as proposed by

Niinemets & Kull (1998). In this equation,

CAST is the total soluble sugar concentration

(kmol kg-1), Dw is the dry mass (DM)/fresh

mass (FM) (kg kg-1) ratio, σ is the water density

(kg m-3), R is the universal gas constant, and T

is the temperature in K°.

Chloroplastidic pigment content: To

obtain the chloroplastidic pigment contents,

Fig. 3. Intensities (circular histograms) and activity indices (table) of the leaf phenophases evaluated in Byrsonima

coccolobifolia. The leaf sprouting was presented in green coloration, mature leaves in blue, senescence red, and abscission in

brown coloration. The results of the Rayleigh (z) test, the mean angle concentration index (R), and the mean angle (indicating

the month with the greatest vector) in each phenophase are listed in the table.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

one leaf was collected from each plant (n = 10

per species), stored in a cool environment, and

protected from light until extraction. Leaf disks

(0.5 cm2 area) were immersed (under green

light) in 5 ml of 80 % acetone for 48 hours. The

samples were then grounded, and another 5 ml

of 80 % acetone was added. The extract was

subsequently centrifuged, and the supernatant

subjected to spectrophotometry at 470, 643

and 663 nm (BioSpectro spectrophotometer,

SP-220, São Paulo, Brazil). The Chlorophyll a,

b and carotenoid (xanthophylls and carotenes)

contents were calculated according to Lichten-

thaler & Wellburn (1983).

Photosynthetic efficiency (chlorophyll a

fluorescence): Photosynthetic efficiency was

assessed by the chlorophyll a fluorescence

method, using a modulated fluorometer (MINI-

PAM, Walz, Germany). Measurements were

performed on eight individuals of each species

(n = 8) between 8:00 and 11:30 am. The maxi-

mum quantum yield (Fv/Fm) values of pho-

tosystem II (PSII) were determined after leaf

adaptation to the dark for 30 min, with Fv cor-

responding to the difference between the maxi-

mum (Fm) and minimum (F0) fluorescence of

the dark-adapted PSII (Genty et al., 1989).

Data analysis: The seasonality of activity

and the intensities of the phenology data were

tested, and the mean angles and the concentra-

tion coefficients (r) of the phenophases (con-

sidering their mean periods) were calculated

using the Rayleigh (z) test for circular distribu-

tions (Zar, 1998). The concentration coefficient

refers to the length of the vector, and the mean

angle of that vector corresponds to the mean

date for the period around which a phenophase

activity is most concentrated. When the mean

angle is statistically significant, it means that

the phenophase demonstrates seasonality. The

concentration of the mean angle, indicated by

(r), can be considered a measure of the degree

of seasonality (Morellato et al., 2000). The cir-

cular tests were performed using the R software

package (R Core Team, 2014).

The specific leaf mass, relative water con-

tent, carbohydrate content, osmotic potential,

and chloroplastidic pigment content data were

evaluated by determining the existence of inter-

actions between predictor variables (months

and species). Differences between months and

between species were determined using gen-

eralized linear models (GLM) with Gaussian

distributions, with R software. Fisher’s F test

(R Core Team, 2014) and resources of the

Rcmdr package (Fox, 2005) were used. For the

variables whose F tests were significant, the

means among species and among months were

compared by the RT4Bio package test (Reis-

Junior et al., 2015).

For fluorescence data analysis, the exis-

tence of interactions between predictor variables

(months and species) and differences among

months and species were determined by gener-

alized linear mixed models (GLMM) using R

software (R Core Team, 2014). Data normality

was tested, followed by data log transformation,

and the Tukey test was applied to determine

variations among the different months.

RESULTS

Leaf phenology: Byrsonima intermedia

and B. coccolobifolia were classified as brevi-

deciduous because they conserve up to 50

% of their leaves throughout the year (Fig.

2, Fig. 3); B. verbascifolia was classified as

evergreen because it keeps most of its leaves

throughout the year (Fig. 4). The individuals of

each species showed synchronization of most

of their phenophases, except leaf abscission

in B. coccolobifolia. The concentration index

of the mean angle (r) was low for all three

species during sprouting, mature leaves, and

abscission, thus indicating poor synchrony

of the populations during those phenophases.

Byrsonima intermedia and B. coccolobifolia

produced new leaves throughout the year, but

their production was more concentrated in Sep-

tember-November (Fig. 2, Fig. 3). Byrsonima

verbascifolia, on the other hand, showed clear

seasonal sprouting, senescence, and abscission,

with no leaves retained between August and

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September, and new leaf production starting at

the end of September (P ≤ 0.05, R = 0.541, and

mean angle = 124.0) (Fig. 4).

Variations of functional traits: Inter-

actions between months and species were

observed for many of the functional traits ana-

lyzed (water-soluble polysaccharides - F(6,95)

= 2.95, P ≤ 0.05, total soluble sugar - F(6,95) =

8.89, P ≤ 0.001, and osmotic potential based on

it - F(6,95) = 5.19, P ≤ 0.001, and total chloro-

phyll content - F(6.97) = 2.28, P ≤ 0.5), showing

the connection between the timing of each leaf

phenophase and the leaf phenological pattern

of each species.

No differences in SLM values were

observed when comparing months or species

separately (Fig. 5A), although the relative water

content values of B. intermedia showed lower

values at the onset of the rainy season (Decem-

ber, F(3,34) = 7.60, P ≤ 0.001), as compared to

the other months (Fig. 5B).

Considering the analysis of carbohydrates,

the three species showed differences in their

Fig. 4. Intensities (circular histograms) and activity indices (table) of the leaf phenophases evaluated in Byrsonima

verbascifolia. The leaf sprouting was presented in green coloration, mature leaves in blue, senescence red, and abscission in

brown coloration. The results of the Rayleigh (z) test, the mean angle concentration index (R), and the mean angle (indicating

the month with the greatest vector) in each phenophase are listed in the table.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

starch contents in terms of the timing of the

seasons (Fig. 6A). Byrsonima intermedia and

B. verbascifolia had lower starch contents at

the onset of the rainy season (December) than

during the other periods evaluated (F(3,28) =

3.73, P ≤ 0.05; F(3,35) = 5.93, P ≤ 0.01 respec-

tively). Byrsonima occolobifolia also evidenced

its lowest starch contents during the prolonged

dry season (August) as well as at the onset of

the rainy season (December) (F(3,32) = 3.26,

P ≤ 0.05), showing that this carbon resource

may be more related to the phenological status

of the leaves than to the season. Water-soluble

polysaccharide (WSP) concentrations were also

different only in terms of the timings of the sea-

sons (Fig. 6B). Byrsonima intermedia had the

highest WSP levels during the prolonged dry

season (August) and after the prolonged rainy

season (February) (F(3,28) = 38.46, P ≤ 0.001),

while B. coccolobifolia and B. verbascifolia had

their highest WSP levels during the prolonged

rainy season (February, F(3,32) = 33.82, P ≤

0.001; F(3,35) = 11.33, P ≤ 0.001, respectively).

The lowest values were observed at the onset of

the dry season (June) for all the species.

Regarding the total soluble sugar (TSS)

results (Fig. 6C), differences were observed

among species and among the timings of the

Fig. 5. A. Specific leaf mass–SLM, and B. B. and relative water content–RWC of Byrsonima intermedia-white bars,

Byrsonima coccolobifolia-gray bars, and Byrsonima verbascifolia-black bars. Samples were collected at the onset of the

dry season (June), after the prolonged dry season (August), as well as at the onset of the rainy season (December) and

after the prolonged rainy season (February). Data are reported as the mean ± standard error of the mean. Lower case letters

represent differences between months (seasons). The absence of any letter signifies the absence of any difference between

those factors.

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

seasons. Byrsonima verbascifolia had higher

values than the other species during the pro-

longed dry season (August) (F(2,17) = 7.22, P ≤

0.01) and after the more prolonged rainy season

(February) (F(2,25) = 18.7, P ≤ 0.001), with its

lowest values at the onset of the rainy season

(December) (F(2,27) = 9, 07, P ≤ 0.001). Com-

paring the timing of the seasons, B. intermedia

and B. coccolobifolia showed oscillations dur-

ing the year, with the highest TSS values at the

onset of both the dry season (June) and the rainy

season (December) (F(3,28) = 10.54, P ≤ 0.001;

F(3,32) = 14.76, P ≤ 0.001 respectively). Byrson-

ima verbascifolia had the lowest TSS levels at

the onset of the rainy season (December) (F(3.,5)

= 3.80, P ≤ 0.05). Osmotic adjustments based

on total soluble sugars (Ψs) (Fig. 6D) showed

differences only among months. Byrsonima

intermedia had the lowest Ψs values at the onset

of the rainy season (December) (F(3,28) = 9.61,

P ≤ 0.001), while B. coccolobifolia evidenced

oscillations, with the lowest values at the onset

of the dry season (June) and at the onset of the

rainy season (December) (F(3,32) = 12.50, P ≤

0.001). Byrsonima verbascifolia evidenced no

differences among the different months.

Comparing the three species, differences

could be observed in their total chlorophyll

contents–TCC (Fig. 7A) and total chlorophyll/

carotenoid ratios (Fig. 7B), reinforcing the

different patterns among the different species

and oscillations within the same season. The

different patterns among the different species

could be detected by observing that Byrsonima

Fig. 6. Carbohydrate content in three Byrsonima species. Samples were collected at the onset of the dry season (June),

after the prolonged dry season (August), at the onset of the rainy season (December), and after the prolonged rainy season

(February). A. Starch content. B. Water-soluble polysaccharides (WSP). C. Total soluble sugars (TSS). D. Osmotic potentials

(Ψs). Data are reported here as the mean ± standard error. Lower case letters represent differences between months (seasons),

while upper-case letters represent differences between species. Byrsonima intermedia-white bars; Byrsonima coccolobifolia-

gray bars; and Byrsonima verbascifolia-black bars.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

intermedia had higher TCC values than the

other species during the prolonged dry season

(August) and after the prolonged rainy season

(February) (F(2.22) = 6.26, P ≤ 0.5, F(2.27) =

3.43, P ≤ 0.5, respectively) (Fig. 7A). The high-

est values of the total chlorophyll/carotenoid

ratios were observed in B. verbascifolia at the

onset of the rainy season (December) (F(2.25)

= 5.44, P ≤ 0.5) and in B. intermedia after

the prolonged rainy season (February, F(2.27)

= 7.11, P ≤ 0.5) (Fig. 7B). There were no dif-

ferences in carotenoid contents or chlorophyll

a/b ratios among the different species (Fig. 7C,

Fig. 7D). Comparing the months, it was pos-

sible to detect changes in the total chlorophyll

content (Fig. 7A), total chlorophyll/carotenoid

ratio (Fig. 7B), and carotenoid content (Fig.

7C). Byrsonima intermedia evidenced its low-

est total chlorophyll content value during the

prolonged dry season (August-F(3,34) = 7.82, P

≤ 0.5), while B. coccolobifolia had its highest

chlorophyll levels at the onsets of the dry (June)

and rainy seasons (December) (F(3,30) = 11.30,

P ≤ 0.5) (Fig. 7A); Byrsonima verbascifolia

evidenced oscillating values throughout the

year, with its highest total chlorophyll content

being observed at the onset of the dry season

(June) (F(3,105) = 27,54, P ≤ 0,5) (Fig. 7A). Con-

sidering the chlorophyll/carotenoid ratio, Byr-

sonima intermedia and B. coccolobifolia had

their lowest values during the prolonged dry

season (August - F(3,34) = 6.67, p ≤ 0.5; F(3,30) =

Fig. 7. Chloroplastidic pigment contents of Byrsonima intermedia-white bars, Byrsonima coccolobifolia-gray bars, and

Byrsonima verbascifolia-black bars at the onset of the dry season (June) and after the prolonged dry season (August), and

at the onset of the rainy season (December) and after the prolonged rainy season (February). A. Total chlorophyll content.

B. Chlorophyll/carotenoid content. C. Carotenoid content. D. Chlorophyll a/b ratio. Data are reported here as the mean ±

standard error of the mean. Capital letters represent differences among the different species, and lower-case letters represent

differences among the different months. The absence of any letter signifies the absence of any difference between the factors.

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

5.64, P ≤ 0.5 respectively), while B. verbascifo-

lia evidenced oscillating values during the year,

with its highest values at the onsets of the dry

(June) and rainy (December) seasons (F(3,105) =

4.73, P ≤ 0.5) (Fig. 7B). Carotenoid contents

were higher in B. intermedia and B. verbas-

cifolia at the onset of the dry season (June)

(F(3,34) = 3.18, P ≤ 0.5; F(3,105) = 6.27, P ≤ 0.5

respectively), but B. coccolobifolia evidenced

oscillating values during the different months,

being higher at the onsets of the dry (June) and

rainy (December) seasons (F(3,30) = 7.80, P ≤

0.5) (Fig. 7C). No differences in chlorophyll

a/b ratios were observed among the different

months (Fig. 7D). Despite the observed chang-

es in photosynthetic pigment contents, few

modifications of chlorophyll fluorescence were

detected, highlighting the lowest maximum

quantum yield (Fv/Fm) values for B. intermedia

and B. coccolobifolia during the dry season (χ

= 17.57, df = 3, P < 0.05) (Table 1).

DISCUSSION

The leaf phenology described for the three

Byrsonima species studied here are different

from their previously reported patterns (Boas

et al., 2013; Kuster et al., 2017; Lenza &

Klink, 2006; Silvério & Lenza, 2010), with

B. intermedia and B. coccolobifolia classified

here as brevideciduous and B. verbascifolia

as evergreen. Our data reinforce the view that

environmental seasonality may act differently

on plant growth and development, changing

phenological expression mainly when related

to water resource availability during the year

(Borchert et al., 2005; Cuzzuol & Clippel,

2009; Morellato et al., 2000; Williams et al.,

1997) and to photoperiod differences during

the transition from the dry to the rainy season

(Rivera et al., 2002; Rossatto, 2013). Byr-

sonima intermedia, B. coccolobifolia, and B.

verbascifolia initiated new leaf sprouting in the

transition between the dry to rainy period. The

peak of leaf sprouting occurs at the onset of the

rainy season and, independent of an induction

factor, new leaf production occurred at a time

that guaranteed tissues with high productivity

during the period of greatest water availability.

Additionally, the timing of each leaf pheno-

phase seems to be determinant for whole plant

metabolism in Byrsonima species, adjusting

them to seasonal resource availability.

The discrepancies found between our

phenological data and those reported in the

literature indicate the phenological plasticity

of Byrsonima species, which may alter their

leaf phenology in response to environmental

changes within the same ecosystem. Varia-

tions in the phenological behaviors of plant

species indicate different strategies in response

to similar environmental conditions (Camargo

et al., 2018). The marked seasonality of water

resource availability in the savanna leads to the

predominance of brevideciduous species within

the plant community (Camargo et al., 2018;

Kikusawa, 1991), which, like B. intermedia and

TABLE 1

Maximum quantum yields of photosystem II (Fv / Fm) of the three Byrsonima species. Measurements were made at the onset

of the dry season (June) and after the prolonged dry season (August), as well as at the onset of the rainy season (December)

and after the prolonged rainy season (February), in a savanna environment

Dry season Rainy season

The onset of the

dry season

Prolonged

dry season

The onset of the

rainy season

Prolonged

rainy season

B. intermedia 0.733 ± 0.07b 0.739 ± 0.01b 0.771 ± 0.07ab 0.805 ± 0.01a

B. coccolobifolia 0.710 ± 0.08b 0.721 ± 0.06b 0.796 ± 0.04ab 0.804 ± 0.03a

B. verbascifolia 0.795 ± 0.04ab 0.778 ± 0.01ab 0.812 ± 0.02a 0.808 ± 0.03a

* Lower case letters indicate differences among the different months according to ANOVA, at a 5 % level of probability.

Values are reported as the mean ± standard deviation.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

B. coccolobifolia in our study, lose their leaves

during the dry season as a mechanism of water

stress escape by reducing transpiration (Loh-

beck et al., 2015; Singh & Kushwaha, 2016).

Evergreen species (such as B. verbascifolia in

our study) maintain their leaves during the dry

season, but generally have other strategies to

overcome water stress (such as xylopodia or

well-developed deep root systems with greater

water absorption efficiency) (Franco et al.,

2005; Jackson et al., 1999) that allow them

to maintain their leaves throughout the year

(Franco et al., 2005).

Evergreen species commonly have higher

SLM values than deciduous species, but that

was not observed in our study. SLM represents

a balance between leaf density and leaf thick-

ness (Witkowski & Lamont, 1991), and may

be used to quantify how much matter the plant

invests per unit of photosynthetic area (Villar

et al., 2013); it can also determine differences

in water storage capacities. The similar SLM

values among the different Byrsonima species

may have influenced RWC. Furthermore, the

similar environmental conditions that the three

Byrsonima species studied here were subjected

to affect their SLM values, as their leaves are

more sensitive to the prevailing conditions

during leaf expansion (Witkowski & Lamont,

1991), including light, temperature, water scar-

city, and nutrients (Meziane & Shipley, 1999;

Niinemets, 2001; Poorter et al., 2009; Tholen

et al., 2012). Ishida et al. (2010), in their stud-

ies of evergreen and deciduous species, showed

that differences related to hydraulic conduc-

tance and osmotic adjustments in plants are

more dependent on the species themselves than

on phenological patterns, an observation that

seems to fit for Byrsonima species.

The phenophases of evergreen species are

less dependent on rainfall (Singh et al., 2006),

but can serve as good models for studies of the

effects of water seasonality on the dynamics

of stomatal opening, and its implications for

CO2 assimilation. In general, evergreen species

evidence better control of stomatal conduc-

tance and have leaf characteristics that reduce

transpirational water losses (stomatal hydraulic

resistance may represent 30 to 80 % of the

total hydraulic resistance of the plant body)

(Ishida et al., 2010). The reduction of stomatal

conductance, however, directly affects the entry

of CO2, and therefore reduces assimilation

efficiency (Chaves et al., 2002). The similar

maximum quantum yield (Fv/Fm) values seen

in B. verbascifolia throughout the year suggest

greater water use by that evergreen species

(Givnish, 2002; Ishida et al., 2010) as well as

a sustained photosynthetic rate throughout the

year, allowing the old leaves to persist until

there is complete nutrient translocation to

younger leaves (Jackson, 1978).

The deciduous species studied here showed

higher photosynthetic yields during the season

of abundant water supply (Table 1), mainly

after complete hydric recovery during the pro-

longed rainy season. Low precipitation is usu-

ally responsible for the low photosynthetic

yields observed in savanna plants under water

deficit conditions (Ronquim et al., 2009; Zhang

et al., 2007). A low water potential during

the dry season in Copaifera langsdorffii was

enough to impose significant restrictions on

total photosynthesis (Ronquim et al., 2009). In

the present study, however, the three Byrsonima

species evidenced strategies that guaranteed

the maintenance of the water statuses of their

leaves during the dry season, and despite the

low Fv/Fm values observed during the extended

dry season in the deciduous species, their pho-

tosynthetic yields were not severely affected

by water deficits. Medina & Francisco (1994)

reported that Godmania macrocarpa (and other

plant species) were also able to maintain high

photosynthetic rates even with low relative

water contents during the dry season.

The concentrations of chloroplastidic pig-

ments in savanna plants may vary between

the dry and rainy seasons, as was seen with

B. verbascifolia and Solanum lycocarpum

respectively (Kuster et al., 2017). The high-

est concentrations of chloroplastidic pigments

were observed in our study at the onset of the

dry season (June), although those chlorophyll

levels decreased with the advance of the dry

season. Our data reinforce that chloroplastidic

14 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

pigment concentrations are not related solely

to light variations, but also vary with other

environmental conditions, such as water avail-

ability and phenological conditions. Similar

results were reported for Polylepis tarapa-

cana and other plant species (González et al.,

2007; Inoue, 2010). According to Inoue (2010),

increased pigment contents may be related

to lower solar incidence during the winter

months, although that might not be the case

with vegetation formations in tropical regions

having open canopies. The high concentra-

tions of chloroplastidic pigments seen in the

Byrsonima species studied here appeared to be

a strategy to maintain high photosynthetic rates

in the remaining leaves. Moreover, the lowest

total chlorophyll/carotenoid ratios and the high

carotenoid content values encountered at the

onset of the dry season indicate that, under

water deficit conditions, Byrsonima plants still

invest in mechanisms of photoprotection and

excess energy dissipation in their photosyn-

thetic systems.

The resumption of plant growth at the

end of the dry season/beginning of the rainy

season in the three Byrsonima species studied

here, help explain the low leaf starch contents

observed at the onset of the rainy season. In

contrast, at two months into the rainy season

the leaves are at their peaks of structural and

functional maturity, with high water-soluble

polysaccharide contents (including structural

carbohydrates such as pectins) and high pho-

tosynthetic rates–with the storage of large

amounts of starch. Similar observations were

made of the leaves of Sinningia aghensis (Cuz-

zuol & Clippel, 2009) during the rainy season,

with higher starch contents after prolonged

rains as a consequence of higher photosyn-

thetic yields due to greater water availability

and greater synthesis of photoassimilates and

carbohydrate storage. The high Fv/Fm values

observed here in the prolonged rainy season

demonstrate the maturity of the photosynthetic

tissues in the month following leaf sprouting in

abundantly available water. The high Fv/Fm val-

ues also point to the absence of severe photo-

inhibition and suggest the presence of effective

mechanisms for excess energy dissipation even

during the dry season.

Many studies have reported that savanna

tree species are isohydric (maintaining their

water contents relatively constant throughout

the year and not performing osmotic adjust-

ments), as they have deep root systems and

can extract water from the deepest soil layers

(Bucci et al., 2005; Bucci et al., 2008; Franco,

1998; Meinzer et al., 1999; Prado et al., 2004).

Osmotic adjustment is, however, an acclimati-

zation response and an adaptation that keeps

photosynthetic tissues hydrated under water

deficit conditions, (and may reflect high con-

centrations of soluble carbohydrates in meso-

phyll cells (Chaves et al., 2009; Irigoyen et

al., 1992; Vieira et al., 2017), as was observed

in the present study. Medina and Francisco

(1994) detected osmotic adjustments in Cura-

tella americana and Godmania macrocarpa,

two Venezuelan savanna species with contrast-

ing foliar phenologies. The maintenance of

leaf conductance at low water potential levels

in both species was ensured by reductions of

their osmotic potentials related to increases in

their soluble sugar concentrations (Medina &

Francisco, 1994). Here, only B. coccolobifolia

showed low osmotic potential throughout the

dry season, evidencing osmotic adjustment

as an alternative to the maintenance of their

relative leaf water contents. Byrsonima inter-

media and B. coccolobifolia, on the other hand,

evidenced lower osmotic potential values at

the onset of the rainy season, with high total

soluble sugar values possibly indicating the

mobilization of carbohydrate reserves at the

beginning of the flowering period.

Our data showed divergences in the leaf

phenology of three Byrsonima species in rela-

tion to previous studies, demonstrating the

importance of confirming the phenology of

plants before undertaking ecophysiological

studies. Savanna seasonality did not drastically

affect the photosynthetic efficiencies of the

Byrsonima species studied here, even during

periods of low water availability. The seasonal-

ity of their carbohydrate contents evidenced

osmotic adjustments in B. verbascifolia that

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50344, enero-diciembre 2023 (Publicado 02 de febrero, 2023)

helped conserve its relative water content dur-

ing the dry season and represented a mobili-

zation of soluble sugars at the beginning of

the flowering period. The metabolic differ-

ences between the onsets and peaks of the same

seasons indicate the high importance of the

phenological status of the leaves (or even the

whole plant). Thus, differences in the leaf phe-

nology and physiological strategies among the

Byrsonima species studied here converged on

drought resistance and the seasonality of water

resources in the Brazilian savanna.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

The authors thank CAPES for the scholar-

ships, CNPq and FAPEMIG for their financial

support of the PELD program, Alan N. da

Costa for identifying the species in the field,

Gudryan J. Barônio for assistance with the

statistical analyses, and Roy Funch for English

language revision of the final text.

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