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Botanical Journal of the Linnean Society, 2023, XX, 1–13. With 3 figures.
Discolobium and Riedeliella (Fabaceae – Faboideae –
Dalbergieae clade): leaflet anatomy, secretory structures
and their systematic implications
JOÃO PEDRO SILVÉRIO PENABENTO1, MARCUS PAULO GONÇALVESROSA2, and
ÂNGELA LÚCIA BAGNATORISARTORI2,3,*
1Programa de Pós-Graduação em Biologia Vegetal, Instituto de Biologia, Universidade Estadual de
Campinas, Barão Geraldo, Campinas, SP 13083-862, Brazil
2Programa de Pós-Graduação em Biologia Vegetal, Instituto de Biociências, Universidade Federal de
Mato Grosso do Sul, Avenida Costa e Silva, s/nº, Campo Grande, Mato Grosso do Sul, 79070-900, Brazil
3Universidade Federal de Mato Grosso do Sul, Instituto de Biociências, Laboratório de Sistemática
Vegetal, Avenida Costa e Silva, s/nº Campo Grande, Mato Grosso do Sul, 79070-900, Brazil
Received 14 April 2022; revised 11 August 2022; accepted for publication 20 October 2022
Discolobium and Riedeliella, exclusively South American genera, have been shown in phylogenetic analyses to be the
sister group to the Pterocarpus clade. The species of the two genera can be confused when reproductive structures
are absent, resulting in taxonomic misunderstandings in botanical collections. This study aimed to investigate the
anatomical characters of leaflets of representatives of Discolobium and Riedeliella in terms of their diagnostic value
at the genus and species levels and to provide information about the type, location and histochemistry of their
secretory structures, and their usefulness for the systematics of the genera. The taxa were analysed using light
microscopy and scanning electron microscopy. Unistratified epidermis, epidermal cell contour and fibre bundles of
the main vein are features shared by the two genera. The position of the stomata and of the tector trichomes and the
shape of the epidermal cells and of the mucilaginous idioblasts are diagnostic anatomical features at the genus level.
Epidermal mucilaginous idioblasts are confirmed for all species studied. Phenolic idioblasts are present in Riedeliella
and Discolobium, and secretory trichomes are found only in Discolobium. The anatomical characters of the leaflets
are useful for the taxonomy of all species studied. Furthermore, our dataset shows novelties for the systematics of
Fabaceae.
ADDITIONAL KEYWORDS: bulbous-based trichomes – leaflet anatomy – mucilaginous idioblasts – Pterocarpus
clade.
INTRODUCTION
Discolobium Benth. and Riedeliella Harms are
exclusively South American genera belonging
to Fabaceae subfamily Faboideae. In molecular
phylogenetic studies, the genera emerged as the sister
group of the Pterocarpus clade in the dalbergioid clade
(Cardoso et al., 2013). This clade has members well
represented in seasonally dry tropical forests, savannas
and temperate biomes, with > 60% of the genera and
species endemic to the New World (Schrire, Lewis
& Lavin, 2005). Discolobium comprises six species
occurring preferably in the seasonally or permanently
flooded areas in the vegetation formations of Caatinga,
Chaco and Pantanal (Sartori et al., 2017). The species
in this genus usually occur in areas practically without
woody vegetation along river banks and are distributed
in Brazil, Argentina and Paraguay (Rudd, 1981; Lavin
et al., 2001; Sartori et al., 2017). More than 50% of
the species occur in Pantanal and Chaco, one species
occurs in Caatinga and one is found in the Amazon
Forest (Sartori et al., 2017). Riedeliella occurs in south-
eastern South America, with three species distributed
in Brazil and Paraguay (Lima, Studart & Vaz, 1984).
The species occur in seasonally dry tropical forest,
Cerrado formations, thorn shrubland (caatinga) and
‘Mata de Tabuleiro’. Discolobium spp. have affinities
*Corresponding author. E-mail: albsartori@gmail.com
© The Author(s) 2023. Published by Oxford University Press on behalf of The Linnean Society of London.
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2 J. P. S. P. BENTO ET AL.
© 2023 The Linnean Society of London, Botanical Journal of the Linnean Society, 2023, XX, 1–13
with more humid areas of the Pantanal (Sartori et al.,
2017), whereas Riedeliella spp. occur in dry areas in
the Cerrado, mainly in central-western Brazil (Lima et
al., 1984). Despite the beauty of the flowers and fruits,
the species are little used as ornamentals.
The taxonomic circumscription of both genera has
been well established on the basis of reproductive
characters. Jointed loment fruits are present in
Discolobium (Sartori et al., 2017) and samara fruits are
present in Riedeliella (Lima et al., 1984). Papilionaceous
flowers, a tetradelphous androecium and a folded ovary
are floral characters of Discolobium (Sartori et al., 2017;
Bento, Kochanovski & Sartori, 2021), whereas radial
flowers, a monadelphous androecium and an unfolded
ovary occur in Riedeliella (Lima et al., 1984; Bento
et al., 2021). However, individuals from these genera
lacking reproductive structures can be confused due to
the external morphological similarity of their branches
and leaves, resulting in misguided identifications at
the species level (Lima et al., 1984; Sartori et al., 2017).
Furthermore, some of the vegetative characters overlap,
such as leaflet shape, prevalence of discolourous
leaflets, usually brochidodromous venation and the
type of indumentum, and these make it impossible to
distinguish between Riedeliella sessiliflora Kuhlmann
and Riedeliella graciliflora Harms, for example.
According to leaflet number and shape, it has been
possible to identify only Riedeliella magalhaesii (Rizz.)
Lima & Vaz from the other species (Lima et al., 1984).
Morphoanatomical studies of leaflets in Fabaceae have
been promising in the identification of new characters
used in the taxonomy of genera and species. Therefore,
morphoanatomical studies of vegetative organs should
contribute additional characters and corroborate
the taxonomical circumscription of Discolobium and
Riedeliella.
Studies of leaflet anatomy have shown promising
results in groups that have emerged in phylogenetic
analyses (Palermo et al., 2017; Silva et al., 2018; Bento
et al., 2020) of tribal taxonomy (Freire & Freyre, 1984;
Rashid et al., 2019), genera and species (Rao & Rao,
1994; Sartori & Tozzi, 2002; Coutinho, Francino &
Meira, 2013; Silva et al., 2018; Flores, Costa & Dias,
2019; Mendes, Fortuna-Perez & Rodrigues, 2019; Bento
et al., 2020) of Faboideae. Leaflet characters such
as the positioning of stomata, location of trichomes,
epidermal cell shape, presence of sclerenchyma
tissue, vascular bundle shape, margin shape and
ornamentation and information regarding secretory
structures have been used for the separation of genera
and species (Freire & Freyre, 1984; Rao & Rao, 1994;
Sartori & Tozzi, 2002; Coutinho et al., 2013; Palermo et
al., 2017; Silva et al., 2018; Flores et al., 2019; Mendes
et al., 2019; Rashid et al., 2019; Bento et al., 2020).
Among the anatomical characters of leaflets, papillose,
subpapillose and mucilaginous epidermal cells and
spherical crystals have been reported for Discolobium
(Metcalfe & Chalk, 1950). However, information about
leaf anatomy is lacking in the literature for Riedeliella.
Secretory structures are common and diverse in
leaflets of Faboideae. Such structures can occur in
the epidermis or in the mesophyll of the leaflets, with
variations in structure types, shape and location and
in the exudate type found (Evert, 2013). Characters
obtained from analyses of secretory structures such
as location, type of structure and secretion chemical
composition are of great importance in Faboideae
both for the taxonomy of tribes/clades (Lackey,
1978; Silva et al., 2018; Vargas et al., 2018; Bento et
al., 2020) and the separation of genera and species
(Marquiafável, Ferreira & Teixeira, 2009; Coutinho et
al., 2013; Vargas, Sartori & Dias, 2015; Flores et al.,
2019; Mendes et al., 2019; Bento et al., 2020) and for
the confirmation of certain structures seen with the
naked eye such as the presence of translucent spots,
which may be glands or intercellular spaces (Teixeira,
Castro & Tozzi, 2000). Among the secretory structures
reported for Discolobium are different morphotypes
of glandular trichomes present in various organs
including the leaflets, suggesting more than one type/
form of glandular trichome among the species of
the genus (Sartori et al., 2017). In the floral organs
and inflorescence parts, bulbous-based glandular
trichomes and mucilage idioblasts have been reported
for Discolobium pulchellum Benth. and uniseriate
glandular trichomes and phenolic compound idioblasts
have been reported for R. graciliflora (Bento et al.,
2021), suggesting a possible relationship of secretory
structures in reproductive and vegetative organs
according to recent studies (Leite et al., 2022).
This study aimed to investigate the anatomical
characters of Discolobium and Riedeliella leaflets of
diagnostic value at the generic and species levels and
to provide information about the type, location and
histochemistry of secretory structures and usefulness
in the systematics of the genera.
MATERIAL AND METHODS
Subterminal leaflets of five of six accepted Discolobium
spp. (Sartori et al., 2017) and two of three Riedeliella
spp. (Lima et al., 1984) were sampled. The samples
were obtained from an herbarium or collected in
the field and analysed in three replicates whenever
possible (Table 1).
The collected leaflets were fixed in NBF (neutral
buffered formalin) (Clark, 1981), washed in distilled
water, gradually dehydrated in an ethanol series up to
70% and preserved in the same solution. Herborized
leaflets were rehydrated in distilled water at 80
°C until fully immersed, and two drops of 2% KOH
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ANATOMY OF DISCOLOBIUM AND RIEDELIELLA 3
© 2023 The Linnean Society of London, Botanical Journal of the Linnean Society, 2023, XX, 1–13
solution were added (modified from Smith & Smith,
1942). After 24h, the leaflets were washed in distilled
water, fixed in NBF, dehydrated and stored in 70%
ethanol.
Samples from the median region of the leaflets
consisting of the main vein, intercostal region and
margin were dehydrated in an ethyl alcohol series up
to 95%, embedded in methacrylate (Meira & Martins,
2003) and sectioned transversely (5–7 µm thick) with
a Leica RM 2145 rotary microtome. Sections were
stained with 0.5% toluidine blue in sodium phosphate
buffer, pH 6.8 (modified from O’Brien, Feder & McCully,
1964). Additionally, some samples were dehydrated in
an ethyl and butyl acetate series, embedded in paraffin
(Johansen, 1940) and later transversely sectioned
(7–10 µm thick) with a rotary microtome. Sections
were stained with Alcian blue and safranin (Luque,
Sousa & Kraus, 1996), and mounted on slides with
synthetic resin (Gerlach, 1969). Leaflet fragments were
dissociated in a heated hydrogen peroxide and glacial
acetic acid solution (1:1), stained with basic fuchsin
in 50% ethanol, mounted in 50% glycerin and sealed
with colourless nail polish (Franklin, 1945, modified
by Kraus & Arduin, 1997).
Histochemical tests were carried out using lugol
for the identification of starch (Johansen, 1940),
ruthenium red for pectins (Johansen, 1940), toluidine
blue for mucilage (modified from O’Brien et al., 1964),
ferric chloride for non-structural phenolic compounds
(Johansen, 1940), sodium phosphate buffer solution
for total lipids (Kirk, 1970) and xylidine Ponceau
for proteins (Vidal, 1970). Controls were performed
according to the author of each technique, also
using reference material (Ventrella et al., 2013). The
classification of mucilaginous idioblasts followed
the terminology of Matthews & Endress (2006).
Photomicrographs were obtained with a Nikon Eclipse
Ci light microscope (LM) equipped with a Motic
Moticam Pro 252B digital camera. An identification
key based on anatomical data is presented for the
studied species.
To study epidermis microstructure, leaflets
fragments of previously herborized materials, c. 1cm2,
were analysed using scanning electronic microscopy
(SEM). Fragments were mounted on metal stubs with
carbon tape, and covered with a layer of gold c. 10nm
thick with a sputter Denton Vacuum Desk III and
Balzers SCD-050. Electron micrographs were obtained
with Jeol JSM 6380LV and Jeol JSM 5800LV SEMs.
RESULTS
In frontal view, epidermal cell walls showed a straight
to undulated contour (Fig. 1A–K, Table 2) in all
species evaluated, under LM and SEM. Epicuticular
waxes are absent in Riedeliella (Fig. 1E, G, H). All
Discolobium spp. had crystalloid epicuticular waxes
in a rosette pattern on the epidermis (Fig. 1B, I, J).
Unstratified epidermis was observed in all species
(Fig. 2A–C). Papillose epidermis, in cross-section, was
recorded on both sides (Fig. 2A, B) in the main vein,
intercostal and margin regions of Discolobium. In
cross-section, globose or quadrangular epidermal cells
may occur on the adaxial side in the main vein region,
papillary epidermal cells may occur on the abaxial
side and globose, quadrangular, papillose or tabular
cells may occur in the intercostal region on both sides
of the epidermis in Riedeliella. Tabular epidermal
cells, in cross-section, were recorded in R. sessiliflora,
Table 1. Information about the studied taxa
Taxa Voucher Herbaria
Discolobium hirtum Benth. de Queiroz, L.P. 5757 HUEFS
Guedes, M.L. 7853 HUEFS
Fernandes s/n HUEFS
Discolobium junceum Micheli Guerrero, L; Zardini, E.M. 37566 INTA
Discolobium leptophyllum Benth. Souza, V.C. 14595 UEC
Anderson, W.R. 11336 MBM
Discolobium psoraleaefolium Benth. Miotto, 2022 ICN
Discolobium pulchellum Benth. *Bento, J.P.S.P. 84 CGMS
Meyer, T; Schulz, A.G.S. 18509 LIL
Sartori, A.L.B. 1114 CGMS
Silva, R.H. 28394 CGMS
Riedeliella graciliflora Harms *Rosa, M.P.G. 78885 CGMS
de Queiroz, L.P. 12745 HUEFS
Hoehne, W. s/n UEC
Riedeliella sessiliflora Kuhlmann Andrade-Lima, D. 75-6516 ASE
*Specimens collected in this study.
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4 J. P. S. P. BENTO ET AL.
© 2023 The Linnean Society of London, Botanical Journal of the Linnean Society, 2023, XX, 1–13
Figure 1. Front view of the epidermis of the leaflets of species of Discolobium and Riedeliella. A, Straight and ondulate
contour of the anticlinal wall of epidermal cells on the abaxial side in D. leptophyllum, revealing the stomata (black
arrowhead). B, Detail of the stomata of D. leptophyllum in SEM, showing the presence of crystalloid epicuticular wax in
rosettes. C, Straight and undulate contour of the anticlinal wall of epidermal cells on the adaxial side of D. leptophyllum,
showing the stomata (arrowhead) and tector trichomes (white arrow). D, Straight and ondulate contour of the anticlinal
wall of epidermal cells on the abaxial side of R. graciliflora, showing the stomata (arrowhead) and tector trichomes (white
arrow). E, Trichome of R. graciliflora in SEM, showing the rugose surface of the trichome. F, Straight and undulate contour
of the anticlinal wall of epidermal cells on the adaxial side of R. graciliflora, showing the tector trichomes (white arrow).
G, Epidermis of R. graciliflora in SEM, showing absence of epicuticular wax and tectors trichomes (white arrowheads). H,
Epidermis of R. sessiliflora, showing absence of epicuticular wax and tectors trichomes (white arrowheads). I, Detail of the
stomata of Discolobium pulchellum, showing presence of crystalloid epicuticular wax in rosettes in the epidermal cells. J,
Epidermis of D. pulchellum in SEM, demonstrating the presence of crystalloid epicuticular wax in rosettes throughout its
extension. K, Detail of the tector trichomes of D. pulchellum in SEM, demonstrating the presence of ornamentation in the
apical cell of the trichome. Scale bars: 5 µm (B), 10 µm (A, C, E, I, J, K), 100 µm (D, F) and 50 µm (G, H).
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ANATOMY OF DISCOLOBIUM AND RIEDELIELLA 5
© 2023 The Linnean Society of London, Botanical Journal of the Linnean Society, 2023, XX, 1–13
globose, quadrangular or papillose cells occurred in
R. graciliflora, and only globose cells occurred in R.
sessiliflora.
Stomata were recorded on both sides of the
epidermis (amphistomatic leaflet) (Figs 1A–C, 2A) in
Discolobium and on the abaxial side (hypoestomatic
leaflet) in Riedeliella (Figs 1D, F, 2C, Table 2). Paracytic
and anisocytic stomata occurred in all Discolobium
spp. and staurocytic stomata occurred only in
Discolobium leptophyllum Benth. Paracytic stomata
were seen in both Riedeliella spp., and cyclocytic and
anisocytic stomata occurred only in R. graciliflora.
Uniseriate tector trichomes were observed on the
abaxial side (Fig. 1B) of the main vein and intercostal
region of Discolobium, and only on the margin of D.
leptophyllum (Table 2). These trichomes were observed
on both sides of the epidermis (Fig. 1C, D) and in all
regions of the leaflet in Riedeliella. When analysed
with SEM, R. graciliflora (Fig. 1E) and D. pulchellum
(Fig. 1K) possess ornamented trichomes with rugous
walls along its extension.
A dorsiventral mesophyll (Fig. 2A–C) was recorded
in all species of Discolobium and Riedeliella (Table
3). Collateral vascular bundles were observed in all
studied species (Fig. 2A–C). Bundles surrounded by
sheath parenchymatic cells (Fig. 2A) were detected
in Discolobium hirtum Benth. and D. pulchellum, and
bundles consisting of a fibre cap facing the abaxial
face (Fig. 2B) occurred in D. leptophyllum, Discolobium
junceum Micheli and Discolobium psoraleaefolium
Benth. (Table 3). Bundles with fibre caps facing both
faces of the epidermis were observed in the vascular
bundles of Riedeliella (Fig. 2C). Isolated prismatic
crystals were observed in the bundle sheath cells
and sometimes in the parenchyma near the fibres of
the vascular bundles in Discolobium. Isolated and
grouped prismatic crystals were present in some
cells of the abaxial epidermis, palisade parenchyma,
spongy parenchyma and vascular bundle sheath cells
of Riedeliella.
The relief of the region of the main vein was adaxially
convex (Fig. 2D) in D. leptophyllum and R. sessiliflora,
straight (Fig. 2E) to concave (Fig. 2F) in D. hirtum, D.
junceum, D. psoraleaefolium and D. pulchellum and
only straight in R. graciliflora. Layers of collenchyma
were recorded below the epidermis on the adaxial
side, and cells with secondary wall deposition were
observed in all studied Discolobium spp. (Fig. 2F).
Regular parenchyma cells extending to the vascular
bundle were detected on both sides of the epidermis
in Riedeliella (Fig. 2G). The main vascular bundle
was collateral in all species and was surrounded
by a fibre cap facing the abaxial side (Fig. 2D, F) in
Discolobium, whereas D. junceum had fibre caps facing
both sides of the main vascular bundle (Fig. 2E). The
main vascular bundle of Riedeliella was composed of
Table 2. Epidermic characterization of the leaflets of Discolobium Benth. and Riedeliella Harms species
Taxa Epidermal cell shape Trichome
location (aba:
ada)
Epidermic relief in the main
vein region on the adaxial side
Crystals Classification of leaflets con-
cerning the position of stomata
Frontal view Transverse view (aba: ada)
aba ada NP RI M NP RI M
D. hirtum O/R O/R P: P P: P P+: - +: - + straight/ slig. concave - amphistomatic
D. junceum * * P: P P: P P+: - +: - + straight - amphistomatic
D. leptophyllum O/R O/R P: P P: P P+: - +: - - slig. convex - amphistomatic
D. psoraleaefolium * * P: P P: P P+: - +: - + straight - amphistomatic
D. pulchellum * * P: P P: P P+: - +: - + straight/ slig. concave - amphistomatic
R. graciliflora O/R O/R G/Q: P G/Q/P: G/Q/P G/P/Q +: + +: + + straight + (aba) hypostomatic
R. sessiliflora O/R O/R G/Q: P G/Q/T: G/Q/T G +: + +: + + concave + hypostomatic
aba, abaxial side; ada, adaxial side; NP, main vein; RI, intercostal region; M, margin; G, globose; slig., slightly; O, ondulate; P, papillose; Q, quadrangular; R, straight; *, could not determine; +, present;
-, absent.
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6 J. P. S. P. BENTO ET AL.
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Figure 2. Transverse section of the intercostal region, leaflet margin and main vein of the species of Discolobium and
Riedeliella. A, Dorsiventral mesophyll of D. pulchellum showing elongated mucilaginous idioblasts (arrowhead) and
vascular bundles surrounded by sheath cells. B, Dorsiventral mesophyll of D. leptophyllum showing elongated mucilaginous
idioblasts (arrowhead), phenolic compounds of idioblasts (white arrow) and bundles with the presence of a fibre cap facing
the abaxial side of the epidermis. C, Dorsiventral mesophyll of R. graciliflora showing vascular bundles with fibre caps
on both sides of the epidermis and special mucilaginous idioblasts in the epidermis (arrowhead). D, Convex relief of the
main vein region of the adaxial side, reniform vascular system with a fibre cap on the abaxial side in D. leptophyllum. E,
Straight relief of the main vein region of the adaxial side, reniform vascular system with a fibre cap on the abaxial side, and
sclerenchyma tissue on the adaxial side in D. junceum. F, Concave relief of the main vein region on the adaxial side, elliptical
vascular system with a fibre cap on the abaxial side in D. hirtum showing elongated mucilaginous idioblasts (arrowhead). G,
Straight relief of the main vein region of the adaxial side, elliptical vascular system with a bundle showing two gelatinous
fibre caps (black arrowheads) in R. sessiliflora. H, Obtuse margin showing a bundle with a fibre cap in D. leptophyllum
with elongated mucilaginous idioblasts (arrowhead) and phenolic idioblast compounds (white arrow). I, Acute margin in D.
hirtum showing elongated mucilaginous idioblasts (arrowhead). J, Curved margin in R. graciliflora. K, Obtuse margin in R.
sessiliflora showing special mucilaginous idioblasts in the epidermis (arrowhead). Scale bars: 100 µm (A–K).
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ANATOMY OF DISCOLOBIUM AND RIEDELIELLA 7
© 2023 The Linnean Society of London, Botanical Journal of the Linnean Society, 2023, XX, 1–13
two fibre caps, one on each side (Fig. 2G). The main
vascular bundle contour was oval (Fig. 2F), circular or
elliptical in D. hirtum, D. pulchellum and Riedeliella
graciliflora, reniform (Fig. 2D) in D. leptophyllum, D.
psoraleaefolium and D. junceum, and elliptical (Fig.
2G) in R. sessiliflora. Gelatinous fibres were only found
in R. sessiliflora (Fig. 2G). Layers of collenchyma were
located between the vascular bundle and the epidermis
of the abaxial surface (Fig. 2F) in Discolobium spp.
Layers of regular parenchyma were recorded between
the vascular bundle and the abaxial epidermis of
Riedeliella (Fig. 2G). Isolated prismatic crystals were
observed in some cells of the phloem and fibres in
both genera, in the collenchyma of Discolobium and
in the regular parenchyma of Riedeliella. Idioblasts
with phenolic compounds (Fig. 2D) were observed in
some cells of the collenchyma in D. leptophyllum and
D. hirtum.
The leaflet margin was rounded (Fig. 2H) to acute
(Fig. 2I) in D. hirtum and D. pulchellum, and rounded
in D. leptophyllum and D. psoraleaefolium. The
margin was rounded to acute, slightly curved on the
abaxial side (Fig. 2J) in R. graciliflora, and rounded
without curvature on one side of the epidermis in
R. sessiliflora (Table 3). Vascular bundles were
observed near the margin region in all species (Fig. 2K),
except for D. leptophyllum, which had a vascular
bundle at the edge of the margin and an evident fibre
cap (Fig. 2H). Collenchyma was seen below the leaf
margin epidermis only in Riedeliella (Fig. 2J). Isolated
crystals, if present, were always observed associated
with the vascular bundle.
Secretory StructureS
Secretory trichomes and idioblasts were observed in
Discolobium and only idioblasts in Riedeliella (Table 3).
The secretory idioblasts varied regarding the shape
and nature of the exudate produced (Table 4).
Secretory trichomes with a bulbous base (Fig. 3A–D)
were detected only in D. hirtum, with two regions: a
basal one consisting of a complex of cells with dense
cytoplasm without established organization and
with a variable number of cells, and an apical one
corresponding to more elongated cells. Trichomes with
a bulbous base (Fig. 3A) had a cavity delimited by
non-turgid cells in the basal portion and mucilage was
detected in the intracellular region (Fig. 3A, B).
Idioblasts containing phenolic compounds were
observed in all species of Riedeliella, and in D. hirtum
and D. leptophyllum. Idioblasts occurred below both
epidermal surfaces and in parenchymatic cells of the
mesophyll in Riedeliella (Fig. 3E), between the sheath
cells of the vascular bundles of the intercostal region
in D. hirtum, below the epidermis of the abaxial face
of the intercostal region and below the epidermis of
Table 3. Anatomical features of the species of Discolobium Benth. and Riedeliella Harms
Taxa Mesophyll Vascular bundle contour Fibre caps on
vascular bundles in
the intercostal region
Margin with
vascular
bundles
Margin: contour/shape Secretory structures
TG IM ICF
D. hirtum dorsiventral oval/circular/elliptical - - straight/obtuse, acute + + +
D. junceum dorsiventral reniform + - straight/obtuse, acute - + -
D. leptophyllum dorsiventral reniform + + curved, abaxial side/ obtuse - + +
D. psoraleaefolium dorsiventral reniform + - straight/obtuse, acute - + -
D. pulchellum dorsiventral oval/circular/elliptical - +/ - straight/obtuse, acute - + -
R. graciliflora dorsiventral oval/circular/elliptical + - curved, abaxial side/ obtuse,
acute
- + +
R. sessiliflora dorsiventral elliptical + - straight/obtuse - + +
IM, mucilaginous idioblasts; ICF, phenolic compound idioblasts; TG, glandular trichomes; +, present; -, absent.
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8 J. P. S. P. BENTO ET AL.
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the abaxial surface in the region of the main vascular
bundle in D. leptophyllum. The phenolic idioblasts
reacted positively to the ferric chloride test in the
species analysed (Fig. 3E, Table 4).
Mucilaginous idioblasts in the epidermis were
classified into three types according to their shape
and to the pattern of mucilage deposition. Unspecified
mucilaginous idioblasts (Fig. 3F) were characterized
by an intracellular region occupied by mucilage and
slightly larger than that of the other epidermal cells.
These idioblasts occurred in all species analysed, on
both sides of the epidermis in the intercostal region
and margin of Riedeliella spp. and on the abaxial side of
the epidermis in the intercostal region of Discolobium
spp. In elongated mucilaginous idioblasts (Fig. 3G), the
entire intracellular region was occupied by mucilage
with the cell projecting towards the central region of
the mesophyll. These idioblasts were observed only
on the adaxial side of all Discolobium spp., except
for D. hirtum that had mucilaginous idioblasts on
both sides of the epidermis. Special mucilaginous
idioblasts (Fig. 3H) showed the deposition of mucilage
on one side of the wall, with the remaining cytoplasmic
contents being present at the other end of the cell.
These types were seen on both sides of the epidermis
only in R. graciliflora. Mucilage can extravasate into
the intercellular space of the mesophyll (Fig. 3I), as
observed in D. pulchellum. The presence of mucilage
and pectins was detected in all mucilaginous idioblasts
(Table 4).
DISCUSSION
Amphistomatic and hypostomatic leaves are shared
by species of Discolobium and Riedeliella, respectively,
representing a diagnostic character for each genus.
In Faboideae, the location of stomata in the leaflets
may vary within the same genus (Metcalfe & Chalk,
1979). However, the cited authors mention that
Arachis L. and Dalbergia L.f. have stomata on both
sides of the leaflets and that Aeschynomene L. and
Lonchocarpus Kunth have stomata only on the abaxial
surface, with the location of the stomata thus being
variable for these representatives of Dalbergieae.
Possibly, the sub-shrubby habit and the preference
for open areas practically devoid of woody vegetation
are conditions that may have favoured the presence
of stomata on both sides in the leaves of Discolobium
spp. Otherwise, Riedeliella spp. with a shrubby habit
and occurring mainly in closed-canopy vegetation
should have contributed to the hypostomatic leaves.
Amphistomatic leaves have been correlated with
both high light environments (Jordan, Carpenter
& Brodribb, 2014) and rapid growth plants or
herbaceousness (Muir, 2018). Hypostomatic leaves
are considered to be advantageous in light-limited
conditions (Mott & O´Leary, 1984; Jordan et al., 2014),
although these leaves are found also throughout a
range of light environments (Mott & Michaelson, 1991;
Jordan et al., 2014). The stomatal distribution between
leaf surfaces have been correlated with a number of
environmental factors (Mott & O´Leary, 1984; Mott
& Michaelson, 1991; Jordan et al., 2014; Muir, 2018).
Further investigations are needed regarding the
distribution of stomata, as they should elucidate their
application to the ecology of these genera. Paracytic
and anisocytic types of stomata were detected in both
Discolobium and Riedeliella, so that this character
could not be used as a diagnostic feature, as recorded
in some genera of Faboideae (Metcalfe & Chalk, 1950;
Silva et al., 2018).
The crystalloids in a rosette pattern are confirmed
only in Discolobium spp., and their presence thus has
taxonomic value at the generic level. Arrangement
of platelets into multiple more or less radially
assembled clusters, evenly spread over an epidermal
surface was recognized for Fabales, e.g. in Calliandra
haematomma (Bertero ex DC) Benth. (Barthlott et al.,
1998). This pattern has been also observed in other
families, including Connaraceae, Malpighiaceae and
Erythroxylaceae (Ditsch & Barthlott, 1997). Functions
Table 4. Histochemical tests performed on the leaflets of species of Discolobium Benth. and Riedeliella Harms
Taxa Ferric chloride—
phenolic compounds
Lugol
reagent—
starch
Neutral
red—lipids
Ruthenium
red—pectin
Toluidine
blue—mucilage
Xylidine
Ponceau—
proteins
D. hirtum + + - + + -
D. junceum * * * * * *
D. leptophyllum + + - + + -
D. psoraleaefolium * * * * * *
D. pulchellum + + - + + -
R. graciliflora + - - - + -
R. sessiliflora ++-++ -
+, positive; -, negative; *, could not determine.
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ANATOMY OF DISCOLOBIUM AND RIEDELIELLA 9
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Figure 3. Cross-section of a leaflet illustrating secretory structures in species of Discolobium and Riedeliella. A, Bulbous
base trichome; B, detail of the base with the formation of the cavity (arrowhead) containing mucilage (asterisk) in D.
hirtum, in LM; C, bulbous base trichome in D. hirtum, in SEM. D, SEM micrographs of bulbous base trichome (white arrow)
and tector trichomes (white arrowhead) in the margin of D. hirtum. E, Ferric chloride test result identifying phenolic
compounds idioblasts in R. graciliflora. F, Unspecified mucilaginous idioblasts (arrowhead) in D. pulchellum. G, Elongated
mucilaginous idioblasts in D. pulchellum. H, Special mucilaginous idioblasts with the cytoplasm distinct (asterisk) from
mucilage (arrowhead) in R. graciliflora. I, Mesophyll with intercellular spaces (asterisk) filled with mucilage extravasated
from the idioblasts in D. pulchellum. Scale bars: 10 µm (F, H), 20 µm (B, E), 50 µm (C, D) and 100 µm (A, G, I).
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10 J. P. S. P. BENTO ET AL.
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of the epicuticular wax on leaves have been attributed
to a photoprotective layer (Barthlott et al., 1998;
Burchard, Bilger & Weissenböck, 2000), the reduction
of the water loss through transpiration and the
protection to the plant against pathogens (Müller &
Riederer, 2005).
Non-glandular, multicellular, elongate trichomes
have been reported for Discolobium (Metcalfe &
Chalk, 1950) and Riedeliella (Lima et al., 1984). In
this study, we found that the occurrence of tector
trichomes on one or both sides and their localization in
each leaflet region (main vein, intercostal region and
margin) are relevant for taxonomic distinction. The
occurrence and location of trichomes on leaflets have
also been important at the genus and species levels in
members of the Amburaneae and Dipterygeae clades,
both belonging to Faboideae (Silva et al., 2018; Bento
et al., 2020).
Our results show that most species have trichomes
with smooth walls. However, trichomes with ruffled
walls occurs in D. pulchellum and R. graciliflora. The
trichome wall ornamentation has been taxonomically
useful for several groups of angiosperms (Martin &
Juniper, 1970), at the generic and species levels. In
Fabaceae, the trichomes with ornamented walls have
been used in systematics of Phaseoleae (Lackey, 1978),
Taralea Aubl. and Monopteryx Spruce ex Benth. (Silva
et al., 2018), both genera of the Dipterygeae clade.
Prismatic crystals were detected in both genera,
being reported here for the first time for Riedeliella.
Spherical crystals are mentioned for leaflets of
Discolobium (Metcalfe & Chalk, 1950), but only
prismatic crystals were observed in the present study.
The relief of the adaxial surface of the epidermis
in the region of the main vein, in cross-section, was
important in distinguishing Riedeliella spp., being
rectilinear in R. graciliflora and concave relief in R.
sessiliflora. These species are easily confused when in
the vegetative state, however, the recorded anatomical
structures contribute to their recognition and
differentiation. In Discolobium, this characteristic is
variable in most species, except for the slightly convex
relief in D. leptophyllum. The relief of the adaxial side
of the epidermis in the region of the main vein has been
considered a relevant feature for species separation in
taxa of Faboideae (Metcalfe & Chalk, 1950; Sartori
& Tozzi, 2002; Silva et al., 2018; Mendes et al., 2019;
Bento et al., 2020).
The main vascular bundle consisting of two
sclerenchyma regions facing both faces of the
epidermis differentiates D. junceum from other species,
with only one fibre cap extending to one surface of
the epidermis. The presence of sclerenchyma tissue
in the region of the main vein has the potential to
differentiate species and genera of Faboideae (Sartori
& Tozzi, 2002; Mendes et al., 2019; Bento et al., 2020).
IdentIfIcatIon key accordIng to the anatomIcal characterS of leafletS of SpecIeS of
Discolobium and RieDeliella
1a. Hypostomatic leaflets; tector trichomes on both sides of the epidermis; quadrangular, globose, tabular and
papillose cells in transverse view; absence of elongated mucilaginous idioblasts and of epicuticular wax..
............................................................................................ .............................................................................. 2
1b. Amphistomatic leaflets; tector trichomes restricted to the abaxial side, papillose epidermic cells in trans-
verse view; presence of elongated mucilaginous idioblasts and of epicuticular wax.....................................3
2a. Straight relief on the adaxial side of the main vein region; epidermic crystals on cells of the abaxial side;
margin slightly curved on the abaxial side; presence of special and unspecified mucilaginous idioblasts
........................................................................................ ............................................................ R. graciliflora
2b. Concave relief on the adaxial side of the main vein region; epidermic crystals on both sides; straight
margin; presence of only unspecified mucilaginous idioblasts.................................................. R. sessiliflora
3a. Main vascular bundle with two fibre caps............................................................ ........................ D. junceum
3b. Main vascular bundle with one fibre cap............................................................................ ........................... 4
4a. Margin with a fibre cap on the vascular bundle..................................... .............................. D. leptophyllum
4b. Margin without a fibre cap on the vascular bundle or vascular bundle absent.............. ............................ 5
5a. Vascular bundles with a fibre cap on the intercostal region........... ................................. D. psoraleaefolium
5b. Vascular bundles without fibre caps on the intercostal region.................................... ................................ 6
6a. Glandular trichomes present; elongated mucilaginous idioblasts on both sides of the epider-
mis........................................................................................................... ...........................................D. hirtum
6b. Glandular trichomes absent; elongated mucilaginous idioblast restricted to the adaxial side of the
epidermis................................................................................... ................................................. D. pulchellum
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ANATOMY OF DISCOLOBIUM AND RIEDELIELLA 11
© 2023 The Linnean Society of London, Botanical Journal of the Linnean Society, 2023, XX, 1–13
The diagnostic potential of the organization of the
main vascular bundle can be extended to the bundles
of the intercostal regions associated with the type of
secretory structure (D. hirtum and D. pulchellum) and
margin (D. leptophyllum).
The contour of the leaflet margin is a feature that
proved effective in distinguishing R. graciliflora and R.
sessiliflora and D. leptophyllum. Contour and shape of
the margin have been applied to early diverging clades
of Faboideae (Silva et al., 2018; Bento et al., 2020)
at the generic and species levels. In the margin, the
presence of a vascular bundle with a fibre cap occurs
only in D. leptophyllum, thus being of diagnostic value
for the species. A vascular bundle near the margin can
occur in D. pulchellum, but without a fibre cap. The
margin with a vascular bundle at the extremity is a
character of diagnostic value at the generic level for
the Amburaneae clade (Bento et al., 2020), and the
present study demonstrated that the character can
also be of value at the species level depending on the
group, as in Discolobium.
Secretory StructureS
Secretory trichomes confirmed only in D. hirtum
reveal the diagnostic value of this structure for species
differentiation. Similar glandular trichomes have been
recorded for D. pulchellum on the inflorescence axis,
bract, bracteole, calyx and ovary (Bento et al., 2021). The
occurrence of glandular trichomes has been useful for
the taxonomy of genera of Faboideae including Dussia
Krug & Urb. ex Taub. (Bento et al., 2020), Indigofera L.
(Marquiafável et al., 2009), Pterodon Vogel (Silva et al.,
2018), Eriosema (DC.) G.Don (Seixas, Fortuna-Perez &
Rodrigues, 2019) and Rhynchosia Lour. (Vargas et al.,
2015; Flores et al., 2019).
Glandular trichomes are considered synapomorphies
for the dalbergioid clade (Lavin et al., 2001). However,
the definition of the term is broad, encompassing
several morphologies, including bulbous base
trichomes. The occurrence of a cavity at the base of
bulbous trichomes is uncommon in Fabaceae, with
cavitated spherical trichomes having been reported
thus far for Bauhinia L. (Duarte-Almeida et al., 2015;
Marinho, Oliveira & Teixeira, 2016) and Indigofera
L. (Kumar et al., 1986). Further investigations are
needed regarding the presence of cavities in the basal
region of bulbous trichomes, as they should elucidate
the types of trichome and broaden their application to
the systematics of the group.
Phenolic compound idioblasts are present in
Riedeliella and in two Discolobium spp. The presence of
phenolic idioblasts can vary depending on the group of
Faboideae. In the Dipterygeae clade, these structures
are confirmed in the leaflets (Silva et al., 2018), but
variable in the anthers, where they occur only in
Monopteryx Spruce ex Benth. (Leite et al., 2022). In
the Amburaneae clade, they occur in only a few genera
(Bento et al., 2020), whereas in the Cajaninae tribe
(Vargas et al., 2018) they are present in all genera
but not in all species. Thus, variation in the presence
of phenolic idioblasts assists in the taxonomy of
Discolobium and Riedeliella, as demonstrated in the
Amburaneae clade (Bento et al., 2020) and Cajaninae
(Vargas et al., 2018). Phenolic compounds are associated
with the function of protecting the organs where
they are present and may confer protection against
excessive exposure to ultraviolet light, pathogens and,
especially, against herbivory (Beckman, 2000; Haslam,
2007; Barros et al., 2017).
Elongated mucilaginous idioblasts are of taxonomic
value at the generic level for Discolobium and special
mucilaginous idioblasts are useful at the species
level for Riedeliella. Mucilaginous idioblasts have
been used in the taxonomic delimitation of species of
the Hymenaeae clade (Pinto et al., 2018), of Zornia
J.F.Gmel (Fortuna-Perez, Castro & Tozzi, 2012),
Poiretia Vent. (Mendes et al., 2019) and Amburana
Schwacke & Taub. (Bento et al., 2020). The presence of
mucilaginous idioblasts in Discolobium and Riedeliella
suggests that this type of secretory structure must
have appeared more than once in Dalbergieae. The
presence of mucilaginous idioblasts was also confirmed
in the Adesmia clade (Fortuna-Perez et al., 2021) and
in the genera of the Pterocarpus clade (J. Gonzalez,
unpubl. data).
CONCLUSIONS
Leaflet characters (mainly on the epidermis) and the
type of secretory structures are useful for the recognition
of the genera. Characters such as the relief of the
adaxial side of the main vein region, composition of the
vascular system of the main vein and lateral veins of
the intercostal region, presence of crystals in epidermal
cells, the shape of the leaflet margin and the type and
shape of the secretory structures are of taxonomic
value for species of Discolobium and Riedeliella. The
confirmed secretory structures, i.e. mucilaginous
idioblasts, phenolic idioblasts and secretory trichomes,
are useful anatomical features for the taxonomy of both
genera. In addition, elongated mucilaginous idioblasts
are novelties for Discolobium, as are mucilaginous and
phenolic idioblasts for Riedeliella.
The anatomical structures have proved to be useful
for the taxonomic distinction of the genera and species
evaluated. The anatomical characters of leaflets may
be potential synapomorphies for future phylogenetic
studies involving the Pterocarpus clade, requiring
morphological and molecular studies.
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12 J. P. S. P. BENTO ET AL.
© 2023 The Linnean Society of London, Botanical Journal of the Linnean Society, 2023, XX, 1–13
ACKNOWLEDGEMENTS
The authors wish to thank Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) for a scholarship granted to JPSPB, Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) for a scholarship granted to MPGR, and the
Laboratório de Anatomia Vegetal—LAVe (UFMS)
and the Fundação Universidade Federal de Mato
Grosso do Sul (UFMS/MEC) Brasil, for support. The
authors are also grateful for access to the equipment
and assistance provided by the Electron Microscope
Laboratory (LME/UNICAMP) and the Multi-User
Material Analysis Laboratory (MULTILAM/UFMS).
DATA AVAILABILITY
The data presented in this paper are available from
the corresponding author upon reasonable request.
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