Received: 8 September 2020 Revised: 10 January 2021 Accepted: 26 January 2021

DOI: 10.1002/jemt.23727

RESEARCH ARTICLE

Leaf anatomy of two medicinal Croton species: Contribution

to plant recognition

Aline Camargo Rosa1 | Alexandre Ferraro2 | Rosa Helena da Silva3 |

Vali Joana Pott3 | Cristiane Pimentel Victório4 | Rosani do Carmo de Oliveira Arruda5

1
Programa de Pós-Graduaç~ao em Biologia
Vegetal, Universidade Federal de Mato Grosso Abstract
do Sul, Campo Grande, Mato Grosso do Sul, Croton L. (Euphorbiaceae) is the second largest genus of the Euphorbiaceae s.l. family,
Brazil
which is composed of about 1,200 taxa. Several species are widely used medicinally
2
Programa de Pós-Graduaç~ao em Biologia
Vegetal, Universidade Estadual de Campinas, in various parts of the world to treat cancer, digestive problems, skin diseases,
Campinas, Sao Paulo, Brazil
malaria, ulcers, and obesity. The leaves and stems are sold as dehydrated fragments,
3
Laboratório de Ecologia Vegetal, Instituto de
Biociências, Universidade Federal de Mato or in powder form, and used in the preparation of teas and various types of formula-
Grosso do Sul, Campo Grande, Mato Grosso tions. The aim of this study was to accurately characterize the leaf anatomy of Croton
do Sul, Brazil
4
bonplandianus Baill. and Croton gracilipes Baill. and to perform histochemical analysis
Laboratório de Pesquisa em Biotecnologia
Ambiental, Fundaç~ao Centro Universitário of laticifers to provide subsidies for botanical certification. To accomplish this, leaf
Estadual da Zona Oeste (UEZO), Campo
blade and petiole samples were fixed and prepared following standard techniques for
Grande, Rio de Janeiro, Brazil
5
Laboratório de Anatomia Vegetal, Instituto de
scanning electron and light microscopy. Histochemical tests were carried out on sec-
Biociências, Universidade Federal de Mato tions to detect the compounds present in the secretions. Results showed neutral and
Grosso do Sul, Campo Grande, Mato Grosso
do Sul, Brazil
acidic lipids, as well as phenolic compounds, in the latex. In addition, leaf anatomical
structures of these two species were studied for specific identification of morpholog-
Correspondence
Cristiane Pimentel Victório, Laboratório de
ical characters and for quality assessment of these medicinal plants.
Pesquisa em Biotecnologia Ambiental,
Fundaç~ao Centro Universitário Estadual da KEYWORDS
Zona Oeste (UEZO), Av. Manuel Caldeira de Croton bonplandianus, Croton gracilipes, Crotoneae, Euphorbiaceae, latex, secretory structures,
Alvarenga, 1.203 Campo Grande, RJ, Brazil. trichomes
Email: cris.pvictor@gmail.com

Review Editor: Alberto Diaspro

1 | I N T RO DU CT I O N Pantanal and Chaco ecosystems in clayish, sandy, rocky or flooded soils

Croton L. is one of the richest genera of Angiospermae, the second larg-
est of the Euphorbiaceae family, with about 1,200 species described and
disseminated throughout the world, including trees, shrubs, and herbs
(Caruzo & Cordeiro, 2007; Govaerts, Frodin, & Radcliffe-Smith, 2000).
The taxonomy and systematics of Croton are difficult owing to the large
size of the genus with its wide geographic distribution in the tropics,
subtropics and temperate zones in the Northern Hemisphere, as well as
the lack of distinct morphological synapormorphies (Caruzo, Cordeiro,
Berry, & Riina, 2013; Riina, Berry, Van, & W, 2009). The genus occurs
predominantly in the Americas with its center of diversification in Brazil
where 350 species occur in Cerrado, Caatinga, Atlantic Rain Forest,
and both dry and humid climates. In Brazil, Croton's diversity is vulnera-
ble to ecosystems, such as the Cerrado, where many species are sub-
jected to intense agricultural exploitation.
Croton species, as well as other euphorbiaceous plants, are mar-
ked by the presence of laticifers in the entire body of the plant. These
structures specialize in the secretion and accumulation of latex, a vis-
cous liquid that can present different white, yellow, orange, brown,
and red colors, or can even be transparent. Several taxa are also char-
acterized by chemical constituents with biological activity, mainly
diterpenoids, triterpenoids, alkaloids, and phenolic substances, in addi-
tion to volatile aromatic substances (Salatino, Salatino, & Negri, 2007;
Vitarelli et al., 2015).

Microsc Res Tech. 2021;1–11. wileyonlinelibrary.com/journal/jemt © 2021 Wiley Periodicals LLC 1

2 ROSA ET AL.
In Brazil, Croton cajucara, popularly known as “sacaca,” is used as
a traditional medicinal plant in the Amazon region. Its leaves and barks
are used in the form of pills or teas for treatment of diabetes, high
cholesterol, liver and gastrointestinal disorders, weight loss, and anti-
oxidant activity (Maciel et al., 2009; Salatino et al., 2007).
In many regions of Brazil and other parts of the world, indigenous
populations have no easy access to pharmaceuticals, making medicinal
plants the only botanical resource. In some communities or ethnic groups,
medicinal plants are purchased at local markets or even found in back-
yards for domestic planting (Maciel et al., 2002). Botanical certification
through histological analysis is an essential tool for confirming the iden-
tity of medicinal plants sold in the form of powder, capsules or fragments,
allowing the detection of fraud or forgery, and they can also indicate the
places of synthesis and accumulation of chemical substances of interest.
The Croton genus presents high medicinal, industrial and eco-
nomic relevance. Croton bonplandianus Baill. is a medicinal plant
widely commercialized in several parts of the world, including Ayurve-
dic medicine (Ghosh, Biswas, Roy, & Guin, 2018). It is also used as an
antidiarrheal agent and for stomachache by folk people in the north-
western Argentine Chaco (Scarpa, 2004). Croton gracilipes Baill. is pop-
ularly used in South America to treat skin conditions, such as
dermatitis and mycoses caused by Microsporum gypseum and
Trichophyton mentagrophytes (Portillo, Vila, Freixa, Adzet, & (Soriano & Alves, 2003). The climate is subhumid tropical type Aw
Cañigueral, 2001). Both species occur spontaneously in Brazil in areas
of ferruginous mountains, in the Pantanal and in the Brazilian Chaco.
In this study, we aimed to evaluate the leaf anatomy of two medicinal
plants, Croton—C. bonplandianus and C. gracilipes, in order to establish
the morphological characters useful for specific recognition and for
quality assessment of these medicinal plants that belong to one of the
most complex and broad genera of the Euphorbiaceae family.
2 | MATERIAL AND METHODS
2.1 | Collection area and sampling
Plants investigated were collected in the western region of Brazil,
Mato Grosso do Sul state. In this study, we collected leaves from at
least 10 individuals in each occurrence environment. C. bonplandianus
leaves were collected in the Brazilian Humid Chaco, in Porto
Murtinho municipality (21
410 5600 S, 57
520 5700 W, Figure 1). In this
region, the climate is marked by strong seasonality with hot and rainy
summers and temperatures reaching 49
C. The winters are dry and
cold with occasional frost. During and after the rainy season, flooding
may occur owing to the poor drainage of compact soil (Pennington,
Prado, & Pendry, 2000). The rainy season occurs from November to
February (rainfall ≥100 mm) with soil flooding in subsequent months.
C. gracilipes leaf samples were collected in two sites. The first is an
ironstone area in Ladário municipality (19
080 2700 S, 57
340 0300 W,
Fazenda Banda Alta, Figure 1) where plants are exposed to intense
sunlight and high temperature most of the year (Paggi et al., 2015).
The same species was also collected in Corumbá municipality, Panta-
nal do Miranda-Abobral subregion. From November to April, the aver-
age annual rainfall in this region ranges between 1,000 and 1,400 mm
with dry winters and rainy summers, having an average annual tem-
perature of 25
C, with highs reaching 40
C and lows near 10
C. Rep-
resentative vouchers of specimens are deposited in the CGMS
herbarium collection (C. gracilipes CGMS 57125 and C. bonplandianus
CGMS 57126). For methodologic standardization, fully expanded leaf
blades were collected from the third stem node from the
branch apex.
F I G U R E 1 Map of Mato do
Grosso do Sul (MS) State (Brazil)
where samples of Croton
bonplandianus (triangle) and
Croton gracilipes (circle) were
collected: Porto Murtinho (humid
Chaco) and Ladário (ironstone
area) municipality, respectively
ROSA ET AL. 3

2.2 | Light microscopy analysis
After collection, leaf samples were immediately fixed in buffered neu-
tral formalin for 48 hours (Johansen, 1940), washed with water,
dehydrated in ethanolic series and stored in ethanol 70% (Kraus &
Arduin, 1997). After fixation, the samples were infiltrated in
historresin (Leica Historresin®, prepared according to the manufac-
turer's instructions) and cut 7 μm thick using a rotary microtome
(Leica RM-2145; Leica Microsystem, Germany). The sections were
submitted to the usual treatments in plant anatomy. For staining, we
used Toluidine blue 1%, followed by mounting on permanent slides. To
describe the epidermis, epidermal peels were prepared using Franklin
solution Franklin (1945), stained in 0.25% basic fuchsin dye dissolved
in ethanol 50%, and mounted on slides in 50% glycerin. All slides were
analyzed under light microscopy (Olympus CX-41F). Photomicro-
graphs were obtained with aid of a Leica DMLB (Leica Microsystem,
Germany) coupled to an image capture system and camera (Leica DC
300F, Leica Suite Application v.3.0.8).
for calcium oxalate crystals (Strassburger, 1943), Nile blue for acid
lipids (Cain, 1947) and 1% ruthenium red for mucilage and pectins
(Gregory & Baas, 1989).
2.4 | Scanning electron microscopy analysis (SEM)
For scanning electron microscopic (SEM) analyses, two fragments
about 0.5 cm2, representing adaxial and abaxial leaf surfaces, were
obtained from fixed leaves. The samples were dehydrated in ethanolic
series, critical point dried, fixed on stubs, and coated with a thin layer
of gold (Dentum Vaccum Desk IV Standard Sputter Coater, LLC).
Observation and images were obtained using a JEOL JSM-6880LV
scanning electron microscope (JEOL, Japan).
3 | RE SU LT S
3.1 | Epidermis

2.3 | Histochemical analysis Frontal views of both species analyzed show both sides of the leaf
surface covered by a layer of striated epicuticular wax (Figures 2a–d
Histochemical tests were performed from cross sections on fresh and 3a,b). Striations are comparatively more evident in
leaves using Sudan IV for neutral lipids (Pearse, 1985), ferric chloride C. bonplandianus than C. gracilipes and well developed in the region of
for phenolics (Johansen, 1940), 5% sulfuric acid and hydrochloric acid anticlinal cell walls (Figures 2d and 3b).

F I G U R E 2 Croton bonplandianus (Euphorbiaceae). Micromorphology of the leaf blade in surface electronic microscopy. (a, b) Non-glandular,
pluricellular trichome-like emergence (tle). (c) Detail of the central cell in the trichome-like emergence (arrow). (d) Leaf abaxial side showing
common epidermal cells, stomata (st) and circular epidermal cells (arrows). (e) Abaxial side, detail of the circular epidermal cell (arrow). (f) Abaxial
side showing subsidiary cells of the paracytic stomatal type (asterisks)
4 ROSA ET AL.

F I G U R E 3 Croton gracilipes
(Euphorbiaceae).
Micromorphology of the leaf
blade in surface electronic
microscopy. (a) Adaxial side
showing trichome-like emergence
(tle) and unicellular (ut) trichomes.
(b) Detail of the single-celled
trichome and the anticlinal cell
walls of common epidermal cells
(acw). (c) Detail of trichome-like
emergence showing a long
central cell. (d) Abaxial face
showing trichomes covering the
leaf surface. (e) Detail of the
dendritic trichome, short central
cell, basal cell with elevations
similar to rosary and stomata (ts).
(f) Abaxial face showing
expanded glandular trichome (gt),
stomata and trichome-like
emergence with long central cell

The leaf blade epidermis of C. bonplandianus and C. gracilipes con-
sists of common epidermal cells, stomata and subsidiary cells, unicellu-
lar and pluricellular non-glandular trichomes and colleters (Figures 2
and 3). C. bonplandianus possesses common epidermal cells with
straight to curved anticlinal cell wall (Figures 2a and 4a). Some epider-
mal cells are smaller than others, circular in shape, papillose, with
smooth external periclinal wall (Figure 2d,e). Around these cells, the
other epidermal cells are arranged radially (Figure 4a). In C. gracilipes,
common epidermal cells have slightly sinuous anticlinal cell walls on
the adaxial face, but more strongly sinuous on the abaxial face
(Figure 4a–d).
In the epidermis of C. bonplandianus, we found trichome-like
emergences (Figures 2a–b and 4e). These trichomes are formed by
elongated cells, enlarged, radially organized, and inserted at different
levels on an axis. The trichome cells have thin walls and smooth sur-
face. In the central region of this trichome, an elevated cell occurs
with a truncated apex and a smooth central cell (Figure 2c). The basal
region of this cell forms a smooth, uniform ring. These trichomes con-
sist of approximately 25 cells.
Only in C. gracilipes were unicellular simple trichomes found
(Figures 3a–c and 4f). These unicellular trichomes are simple, either
short or more elongated, pointed, and have an enlarged basal region
with smooth surface (Figure 3b). In this species, the trichome-like
emergences are multicellular, formed by a set of cells organized radi-
ally and located at the apex of a short peduncle. The cells in the region
that forms the apical portion are long with thin cell walls and a smooth
surface. The central region of this trichome is formed by a cell with a
pointed apex that can vary in length; in the basal portion, these cells
form elevations similar to a rosary (Figure 3c–e).
The two species evaluated have stomata on both sides of the leaf,
characterizing amphistomatic leaves (Figure 4a–d). The stomata are the
paracytic type (Figure 4a,b). C. bonplandianus has exposed stomata, while
in C. gracilipes, especially on the abaxial face, the stomata are protected
by numerous multicellular trichomes (Figures 2f, 3d, and 4e–f).
In the leaf margin, the epidermis of both species could be charac-
terized by the occurrence of colleters in the apices of the leaf teeth
(Figure 4c,g). These structures are pluricellular, approximately spheri-
cal, formed by long cells with thick cell walls and organized radially
ROSA ET AL. 5

F I G U R E 4 Epidermis in
frontal view and cross section of
Croton bonplandianus and Croton
gracilipes (Euphorbiaceae). (a,c,e,
g): C. bonplandianus; (b,d,f,h):
C. gracilipes. (a) Common
epidermal cells with straight and
curved cell walls, circular
epidermal cells (circles) and
stomata. (b) Common epidermal
cells with sinuous anticlinal cell
walls and stomata. (c) Abaxial side
of the epidermis on the leaf edge
showing colleters (coll). (d) Adaxial
face: unicellular non-glandular
trichomes (ts). (e) Abaxial face:
stomata and trichome-like
emergence (tle). (f) Abaxial face:
trichome like-emergence (tle).
(g) Epidermis on the edge of the
leaf showing a colleter in frontal
view (arrow). (h) Cross
section showing uniseriate
epidermis (*) and stomata (arrow)
[Color figure can be viewed at
wileyonlinelibrary.com]

(Figure 6f–g). The colleters presented positive reaction for neutral
lipids.
In cross section, both species present uniseriate epidermis
(Figure 5a,b). C. bonplandianus showed flattened epidermal cells with
thick periclinal cell walls. The stomata are located at the same level as
the other common epidermal cells (Figure 5a,c). C. gracilipes has epi-
dermal papillae on both sides of the leaf blade, and the stomata are
located at the same, or slightly above, the level of the other epidermal
cells (Figure 5b,d). In both species, the trichome-like emergences are
elevated through a set of cells with strongly thickened and cutinized
walls supporting the cells that form the radiated portion of the tri-
chome (Figures 5b and 6d,e). Phenolic compounds were detected in
the cells that make up the radiated region of the trichome-like emer-
gences (Figure 6c).
3.2 | Mesophyll
C. bonplandianus, in cross section, presents a heterogeneous isolateral
or isobilateral mesophyll composed of palisade and spongy
6 ROSA ET AL.

F I G U R E 5 Leaf blade of
Croton bonplandianus and Croton
gracilipes in cross
section (Euphorbiaceae). (a,c,e):
C. bonplandianus; (b,d,f,c):
C. gracilipes. (a) General aspect of
the leaf blade showing uniseriate
epidermis, palisade parenchyma
(pp), spongy parenchyma (sp) and
isolateral leaf structure.
(b) General aspect of the leaf
blade showing papillous
uniseriate epidermis, palisade
parenchyma (pp), spongy
parenchyma (sp) and dorsiventral
leaf structure. (c) Detail showing
stomata on both sides (st),
palisade parenchyma (pp), spongy
parenchyma (sp), vascular bundle
(in the circle), laticifers (lat) and
crystal idioblast in parenchyma
cell (arrow). (d) Detail showing
papillary epidermis (pe), stomata
(st), palisade parenchyma
(pp) with crystal idioblast (id),
spongy parenchyma (sp), vascular
bundle (in the circle), and
laticifers (lat). (e) Main vein:
epidermis, collenchyma (col),
regular parenchyma (rp), V-
shaped vascular system, laticifers
(arrows); note the concave region
on the adaxial face. (f) Main vein:
epidermis, collenchyma (col),
regular parenchyma (rp), arc-
shaped vascular system, laticifers
(arrows); note raised region on
the adaxial face [Color figure can
be viewed at
wileyonlinelibrary.com]

parenchyma (Figure 5a). The palisade parenchyma occurs in a single
layer of long cells under the upper face and another layer with shorter
cells near the lower face. The spongy parenchyma is restricted to the
central region of the mesophyll. It is formed by bulky cells with varied
shapes, and it is organized in a more compact way similar to aquiferous
parenchyma (Figure 5c). C. gracilipes showed dorsiventral mesophyll
containing a single layer of palisade parenchyma that occupies half of
the mesophyll. The spongy parenchyma has cells of varying shapes. It
is loosely organized with wide intercellular spaces (Figure 5d).
Throughout the mesophyll, we can observe the laticifers compos-
ing a set of cells that make up a secretory structure formed by a series
of cells joined by the ends. These structures can be observed parallel
to the cells of the palisade parenchyma, the spongy parenchyma, and
close to the epidermis, without a defined location (Figure 5a,c,d). His-
tochemical tests indicate that the laticifers contain neutral lipids, as
detected with Sudan IV; acidic lipids, as identified by the Nile Blue;
and phenolic compounds (Figure 6c). Idioblasts containing crystals can
be observed dispersed in the mesophyll of the two species evaluated.
Idioblasts are numerous and large in C. gracilipes (Figure 5d), and in
C. bonplandianus, crystal idioblasts are located in the median region of
mesophyll (Figure 5c, arrow).
The vascular system is composed of collateral-type vascular bun-
dles surrounded by a parenchymal sheath. C. bonplandianus has a
parenchyma sheath formed by bulky cells (Figure 5c,d).
3.3 | Midrib
Leaves in cross section showed a region of the central vein of
C. bonplandianus that presents a groove on the adaxial side (Figure 5e),
ROSA ET AL. 7

F I G U R E 6 Leaf histochemical
tests of Croton bonplandianus and
Croton gracilipes in cross
section (Euphorbiaceae). (a,b)
Laticifers with phenolic
compounds and acidic lipids (*) in
Croton bonplandianus (a and b)
and in C. gracilipes (c). (d,e) Lipids
in mesophyll (d) and in basal
region of non-glandular
pluricellular trichome (e, arrow).
(f) Croton bonplandianus with a
colleter in leaf margin, as
determined by Sudan IV reaction.
(g) Colleter in Croton gracilipes
with Sudan IV reaction [Color
figure can be viewed at
wileyonlinelibrary.com]

and in C. gracilipes, a projection is formed in this region (Figure 5f). The
epidermis is covered by a thick layer of cuticle, and common epidermal
cells have periclinal outer thick walls. C. bonplandianus has more volu-
minous epidermal cells on the upper surface (Figure 5a) and uniformly
rounded epidermal cells on the lower surface, while in C. gracilipes, the
cells are less voluminous and papillary (Figure 5d).
In the midvein of C. bonplandianus, we observed a concave region
in the adaxial surface (Figure 5e). Under the epidermis of the adaxial
surface, we observed a layer of parenchyma with elongated cells, simi-
lar to palisade parenchyma, and more internally, a fundamental paren-
chyma intersected by laticifers is present (Figure 5e, rp; arrows). Near
the abaxial side, 3–5 layers of angular collenchyma occur (Figure 5e,
col). In Croton gracilipes, the adaxial region forms a projection occupied
by angular collenchyma (Figure 5f). Internally, laticifers also pass
through the regular parenchyma (Figure 5f, rp; arrows). The vascular
system is formed by a single vascular bundle surrounded by a sheath
of non-lignified fibers in C. bonplandianus, but absent in C. gracilipes
(Figure 5e,f). The vascular system of C. bonplandianus is formed by pri-
mary xylem organized in a U-shape (Figure 5(e)) and surrounded by
primary phloem and fibers. In C. gracilipes, the vascular system is orga-
nized in an arc-shape (Figure 5(f)) without fibrous sheath.
3.4 | Petiole
In both species evaluated, we observed extrafloral nectaries located in
the distal region of the petiole, close to the insertion of the lamina
(Figure 7a). The extrafloral nectaries are circular in frontal view and
supported by a peduncle, as seen in cross section (Figure 7a,d). In
frontal view, the nectary epidermis is composed of papillary epidermal
cells with straight anticlinal walls (Figure 7b). In cross section, the epi-
dermal cells are elongated and translucent with thin cell walls
(Figure 7c). Under this layer, the nectary parenchyma has short and
polyhedral cells with thickened walls (Figure 7c); many idioblasts with
druses can be observed in this region (Figure 7d). Xylematic cells can
be observed in the basal region of nectary tissue. In nectary stalk, the
ground tissue is a compact parenchyma (Figure 7d).
Considering the anatomical organization, the petiole, on both spe-
cies studied, is similar. The epidermis is composed of a single layer of
cells covered by a thick cuticle (Figure 7e,f). Below the epidermis, con-
tinuous layers of collenchyma followed by ground parenchyma are
present (Figure 7f). Many laticifers and crystal idioblasts can be
observed in the cortical region, forming a very visible crystalliferous
sheath in C. gracilipes (Figure 7e).
8 ROSA ET AL.

F I G U R E 7 Nectary and petiole of Croton bonplandianus and Croton gracilipes (Euphorbiaceae). (a,b) Croton bonplandianus: frontal view of a
nectary in petiole (surface electronic microscopy). (b) Detail of nectary adaxial surface: epidermis. (c) Epidermis of nectary of Croton bonplandianus
in cross section: note the long palisade-like cells. (d) C. gracilipes: cross section showing nectary secretory epidermis, nectary parenchyma, xylem
cells, crystal idioblasts and the peduncle. (e,f) Cross section of petiole of C. bonplandianus (e) and C. gracilipes (f) showing epidermis, cortical region
and the arrangement of vascular bundles [Color figure can be viewed at wileyonlinelibrary.com]

The petiolar vascular system in both species is composed of col-
lateral vascular bundles with primary xylem organized in radial rows
surrounded abaxially by primary phloem. In C. bonplandianus, the
major vascular bundles are located abaxially, forming an open arc, and
in C. gracilipes, five vascular bundles are organized in a circle separated
by parenchyma cells with starch grains. Numerous laticifers and crys-
tal idioblasts can be seen in this region.
4 | DISCUSSION
Leaf anatomy reveals several taxonomic characters that can be used
in the delimitation of genera, tribes or species in various families of
Angiosperms (see Metcalfe & Chalk, 1950). Despite being one of the
largest genera of Euphorbiaceae, leaf anatomical studies aiming to
indicate specific diagnostic characters of Brazilian Croton species are
still scarce. Our study demonstrated that several anatomical charac-
teristics can be used to recognize the leaves of the two species evalu-
ated, especially those found in the epidermis.
The contour of the anticlinal cell wall can be considered a relevant
character in the distinction of the two species evaluated since it
proved to be straight for C. bonplandianus and sinuous in C. gracilipes,
the same type of contour observed in C. cajucara (Mendonça, Ilkiu-
Borges, & Souza, 2008), an important medicinal plant widely used in
the Brazilian Amazon region (Maciel et al., 2009). The variation
between these two formats is cited for the genus (Metcalfe &
Chalk, 1950).
The epidermis provides several relevant characters for the delimi-
tation of genera and species since the cells that compose it may
exhibit some cell types that do not change qualitatively, even when
the plants grow in different environments. Thus, for example, the
stoma and trichomes are highly conserved structural features, all-
owing their use in taxonomic and phylogenetic approaches (Silva,
Arruda, Alves, & Sartori, 2018; Vaz, Alves, & Arruda, 2019).
Considering Euphorbiaceae, for the Crotoneae tribe, the paracytic
stomata type is predominant (Metcalfe & Chalk, 1950; Riina
et al., 2015), a character which was corroborated in our study. Sto-
mata may occur on one or both sides of the leaf blade; in the same
species, the location of these structures may vary depending on envi-
ronmental conditions. The leaves of the two species of Croton individ-
uals evaluated here were classified as amphistomatic, regardless of
the location where they occur. In the Euphorbiaceae, the positioning
of stomata on the abaxial face can be considered as a relevant taxo-
nomic marker since the Croton species comprise the only group with
this characteristic (Metcalfe & Chalk, 1950).
Several types of trichomes are described for the Crotoneae tribe,
among which simple, single-celled trichomes, such as those observed
in Croton gracilipes, are considered extremely rare (Metcalfe &
Chalk, 1950) and thus may be relevant for the identification of this
species.
ROSA ET AL.
Various types of non-glandular pluricellular trichomes are fre-
quently mentioned and recognized as a relevant taxonomic character
for Croton constituting important elements in the recognition of the
genus and in the characterization of species (Metcalfe & Chalk, 1950;
Webster et al. 1996). In the plants analyzed here, we found that the
different morphologies of non-secreting trichomes can actually
applied in their recognition, corroborating the proposals of other
authors who also point out the use of these appendages in the delimi-
tation of clades or in phylogenetic approaches (Lucena & Sales, 2006;
Riina et al., 2009).
Trichome-like emergences were investigated by Vitarelli, Riina,
Cassino, and Meira (2016) concerning their ontogenetic development
and functions in three Croton species: C. erythroxyloides Baill.,
C. pygmaeus L. R. Lima and C. splendidus Mart. ex. Colla. The authors
established that the trichome-like emergences are actually produced by
protodermal and ground meristem cells capable of transporting water
from the atmosphere into the leaf vascular system. This feature can be
an important functional trace in the occupation of environments with
seasonal water deficit, such as the Pantanal and Brazilian Chaco where
the two species herein studied are found, since plants can get water
from the atmosphere (mist) through the leaves during dry periods.
Colleters are important secretory structures, especially for pro-
ducing secretions that protect young leaves. In the investigated spe-
cies, we found colleters only in the leaf margin, as also mentioned for
many Croton species, although they can be in various positions on the
leaf (Riina et al., 2015; Vitarelli et al., 2015).
Floral and extrafloral nectaries are probably the best-known
secretory structures in Crotoneae. This is especially true for Croton in
the context of mutualistic interactions, such as reward for pollinators
and recruitment of plant-protective insects (Vitarelli et al., 2015). Flo-
ral nectaries are located on floral parts, while extrafloral nectaries
(EFNs) are positioned on vegetative organs (Coutinho, Francino,
Azevedo, & Meira, 2012; Schmid, 1988). In both species evaluated,
petiolar or EFNs were identified. EFNs can be defined as structures
that occur on the surface of a plant's vegetative organs and produce a
secretion in which sugars predominate (Durkee, 1982; Roshchina &
Roshchina, 1993). EFN on leaves and stipules are typical of some Cro-
ton sections, but data on their structure are still scarce (Freitas,
Bernardello, Galetto, & Paoli, 2001). EFNs exhibit distinctive charac-
ters of diagnostic value (Webster, 1993) and are widely used in taxo-
nomic treatments for Croton; however, it is not an exclusive feature
and can be found in other genera of Euphorbiaceae (Bernhard, 1966;
Fiala & Maschwitz, 1991). The nectaries observed here can be classi-
fied as epidermal, differentiating among tissues. They generally display
a vascularized parenchyma bound by an epidermis covered by a cuti-
cle. Nectaries bear anatomical similarity among Croton species
(Vitarelli et al., 2015; Wilkinson, 1979).
The leaf structure results from the interaction with environmental
factors, such as incident light (Dickison, 2000). In leaves that receive
light energy on both sides, the isolateral pattern is common, as seen in
C. bonplandianus. Plants found in xeromorphic, arid and semi-arid
environments exhibit anatomical features that are potentially adaptive
9
to their habitat (Fahn & Cutler, 1992) and often have a dense leaf
induction, performing an adaptation to low water availability and high
temperatures (Sandquist & Ehleringer, 1997). Thick cuticle layers,
glandular and non-glandular trichomes were also observed, as well as
a dense distribution of crystalline idioblasts and the presence of phe-
nolic compounds.
The distribution of crystalline idioblasts in the mesophyll may also
be a relevant characteristic for the recognition of each species evalu-
ated, as also observed in other Croton species (Vitarelli et al., 2015).
The location and type of crystals can prove to be quite consistent and
useful in taxonomic classification (Evert, 2013); in addition, they may
be associated with the removal of excess calcium from the plant
(Francheschi & Nakata, 2005) since crystallization is the most effec-
tive way for plants to neutralize excess calcium transported by the
water flow, but also acting against herbivory and mechanical support
(Lersten & Horner, 2008).
Phenolic compounds observed can help to protect against herbiv-
ory, being considered food deterrents. These compounds are also
related to the reduction of photodamage and are often found in plants
subjected to intense sun exposure and high temperatures, suggesting
that these substances reduce the passage of light through epidermal
cells, avoiding exposure of the photosynthetic apparatus to high levels
of light, thus avoiding possible injuries (Jordan, Dillon, &
Weston, 2005).
Laticifers contain a complex solution including neutral lipids,
acidic lipids, and phenolic compounds essential for the defense of
plants against biotic and abiotic stresses (Naidoo, Naidoo, &
Dewir, 2020; Pott, Osorio, & Vallarino, 2019). According to Vitarelli
et al. (2015), laticifers in the Crotoneae subtribe are the articulated
type; they can be present in the subfamily Crotonoideae and consti-
tute a plesiomorphic character for Euphorbiaceae. A set of these sec-
ondary metabolites is produced in the laticifers which provides a more
efficient chemical barrier against herbivores and microorganisms
(Wink, 2010). These substances are highly valuable and widely used
to treat human diseases since phenolics have antioxidant, antip-
roliferative, anti-inflammatory, and antibacterial properties (Pott
et al., 2019). The presence of different types of chemical compounds
in the latex of C. bonplandianus and C. gracilipes serves to confirm the
popular use of these two species as medicinal plants.
5 | CONC LU SIONS
The two species investigated exhibit conserved characters to the
genus, such as the presence of glandular and non-glandular trichomes,
EFN, and laticifers. The mesophyll and petiole organization of both
species can offer additional elements for identification, proving the
value of leaf anatomical features as important support for the study of
medicinal plants. Besides these features, the two plants also show
some attributes that can be considered xeromorphic in relation to
water economy, such as the presence of trichomes, crystals in idio-
blasts and phenolic substances.
10
ACKNOWLEDGMENTS
We thank the Conselho Nacional de Desenvolvimento Cientifico e
Tecnológico (CNPq) (Project Proc. 143260/2013-8) for providing
financial support. A.C. Rosa thanks CNPq for the research grant (IC-
CNPq/UFMS); the authors also thank Luiz Felipe Plaça from the Phys-
ics Institute of the Universidade Federal do Mato Grosso do Sul (INFI/
UFMS) for assistance with SEM analysis.
AUTHOR CONTRIBUTIONS
We declare that all work performed in compiling this article was done
by the author(s), including Aline Camargo Rosa, Alexandre Ferraro,
Rosa Helena da Silva, Vali Joana Pott, Cristiane P. Victório and Rosani
do C.O. Arruda, and that all liabilities pertaining to claims relating to
the content of this article will be borne by the authors.
Aline Camargo Rosa: Collected plant material, anatomical and his-
tochemical test data. Alexandre Ferraro: Collected plant material, ana-
tomical and histochemical test data. Rosa Helena da Silva: Collected
and identified plant material. Rosa Helena da Silva: Collected and
identified plant material and drafted the manuscript. Rosa Helena da
Silva: Interpreted the results, translated and drafted the manuscript.
Rosani do C.O. Arruda: Designed the study, collected plant material,
anatomical and histochemical test data, interpreted the results and
drafted the manuscript.
ORCID
Cristiane Pimentel Victório https://orcid.org/0000-0002-2815-
3725
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