RESEARCH ARTICLE
Functional role and evolutionary
contributions of floral gland morphoanatomy
in the Paleotropical genus Acridocarpus
(Malpighiaceae)
Isabel Reis Guesdon1¤, André Márcio Amorim2,3, Renata Maria Strozi Alves Meira ID1*
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1 Universidade Federal de Viçosa, UFV, Departamento de Biologia Vegetal, Viçosa, Minas Gerais, Brazil,
2 Universidade Estadual de Santa Cruz, UESC, Departamento de Ciências Biológicas, Ilhéus, Bahia, Brazil,
3 Herbário Centro de Pesquisas do Cacau, CEPEC, Itabuna, Bahia, Brazil
¤ Current address: Universidade Federal do Amazonas, UFAM, Instituto de Ciências Exatas e Tecnologia,
Itacoatiara, Amazonas, Brazil
* rmeira@ufv.br
Abstract
OPEN ACCESS
Citation: Guesdon IR, Amorim AM, Meira RMSA
(2019) Functional role and evolutionary
contributions of floral gland morphoanatomy in the
Paleotropical genus Acridocarpus (Malpighiaceae).
PLoS ONE 14(9): e0222561. https://doi.org/
10.1371/journal.pone.0222561
Editor: Juliana Hipólito, Instituto de Investigaciones
en Recursos Naturales, Agroecologia y Desarrollo
Rural (IRNAD), Universidad Nacional de Rio Negro
- Sede Andina, ARGENTINA
Received: March 22, 2019
Accepted: September 3, 2019
Published: September 17, 2019
Copyright: © 2019 Guesdon et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript.
The stereotyped floral morphology of Neotropical Malpighiaceae species—zygomorphic
and calyx with oil glands—is correlated with oil-bee pollination. In contrast, the floral trends
of the Paleotropical lineages are actinomorphy and lack of calyx glands, probably due to the
absence of oil-collecting bees. The Paleotropical genus Acridocarpus is an exception
because of its zygomorphic, gland-bearing flowers. Glands throughout Acridocarpus inflorescences were morphoanatomically evaluated to verify the occurrence of patterns related
to species and geographic distribution. Herbarium (25 species) and fresh samples of A.
longifolius were processed according to standard anatomical techniques. To verify the presence of glucose and protein, strip tests were applied to the exudate. Fresh samples were
fixed and submitted to histochemical tests. Based on the occurrence, number and placement of glands, three distribution patterns were recognized on the bracteole and ten on the
calyx. Bract, bracteole and sepal glands have a typical nectary structure with a palisade-like
epidermis and vascularized parenchyma. Glands were classified as short-stalked, sessile or
immersed. Histochemical tests performed on bract and sepal glands of A. longifolius were
positive for proteins, polysaccharides and phenolic compounds, and negative for oil compounds. Glucose and protein were detected in the exudate. These results allow us to recognize the glands in Acridocarpus inflorescences as nectaries. This comprehensive
morphoanatomical study helps to clarify the correlation between patterns of floral morphology and secretion consumers, as well as to better understand floral evolution in Malpighiaceae across their dispersal events.
Funding: This study was financed in part by the
Coordenação de Aperfeiçoamento de Pessoal de
Nı́vel Superior - Brasil (CAPES) - Finance Code
001. The authors thank the Coordenação de
Aperfeiçoamento de Pessoal de Nı́vel Superior
(CAPES) and Fundação de Amparo à Pesquisa do
Estado do Amazonas (FAPEAM/PROGRAMA RH-
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
INTERIORIZAÇÃO) for awarding the doctoral
scholarships to IRG; the Programa de PósGraduação em Botânica at the Universidade
Federal de Viçosa (UFV) for providing logistical
support; the Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico (CNPq)
for awarding research productivity fellowships to
AMA (grant #310717/2015-9) and RMSAM (grant
#307762/2016-5); and the Fundação de Amparo à
Pesquisa do Estado de Minas Gerais (FAPEMIG)
for the financial resources (UNIVERSAL APQ00383-16). We are also grateful to the MNHN
Herbarium and Jardin Botanique Meise for kindly
loaning samples. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Introduction
The family Malpighiaceae comprises approximately 1300 species of trees, shrubs, vines climbing and rarely herbs, distributed in the Neo- and Paleotropics [1, 2, 3]. Most species usually
have the following: 2-branched malpighiaceous trichomes; simple opposites leaves, with intraor interpetiolar stipules; pentamerous bisexual flowers; androecium with 10 stamens; gynoecium superior, tricarpellate, 1-ovulate; and fleshy or dry fruits [4, 5].
Although the pantropical distribution of Malpighiaceae has been explained over the past by
Gondwanan vicariance [1], fossil and phylogenetic evidence suggest a post-Gondwanan origin
in the Neotropics [6], which is in agreement with Anderson’s American hypothesis [2]. This
evidence combined with divergence time estimates indicate repeated migration events from
the Neo- to the Paleotropics [6, 7]. In addition, the last phylogeny of Malpighiaceae identified
nine Paleotropical clades [3], and most of them are placed within Neotropical lineages as sister
groups [8].
Secretory structures are well documented in Malpighiaceae species. They include nectaries
located throughout the leaf, which attract nectar consumers that may provide protection
against herbivory [1], as well as glands on the calyx, which play an important role in pollinator
attraction in the Neotropics, acting as oil-producing elaiophores [1, 2, 9]. Since these sepal
glands are typical of the Neotropical species, they are considered a synaphomorphy for Malpighiaceae with multiple loss events [1], which support the American origin of the family [1, 2,
9, 10]. On the other hand, although morphoanatomical and exudate analyses of Paleotropical
genera are scarce, Vogel [1] postulated that the sepal glands in these Paleotropical lineages of
Malpighiaceae have become modified and seem to behave as nectaries [1, 2, 11, 12]. Therefore,
Malpighiaceae constitute an interesting group to test hypotheses about floral evolution and to
examine the maintenance of morphological traits, since they exhibit a typical oil-flower pollination syndrome in the Neotropics, and the dissociation with this syndrome is predominantly
related to Paleotropical species [1, 13, 3, 8].
The floral morphology of Neotropical Malpighiaceae is highly conserved and typically zygomorphic, with clawed petals, one uppermost posterior petal and calyx glands on the abaxial
surface of sepals [1, 4, 5, 9, 14] (Fig 1A). The posterior petal is strongly correlated with the pollinator position to access the sepal glands [1, 2, 9, 11, 8, 15–20]. The pollinators are bees of
tribes Centridini, Tapinotaspidini and Tetrapediini, which scratch their specialized legs on
these glands to collect the fatty oil that is used as a larval food resource and nest coat [1, 2, 17,
18]. Interactions between oil-collecting bees and oil-flowers are a very specialized mutualism.
This specialized pollination system has driven the floral evolution of Malpighiaceae in the Neotropics [2, 8], where floral traits evolved under the selective pressure of oil-bees [1, 8].
Specialist oil-collecting bees are absent in the Paleotropics [1], resulting in the loss of the
selective pressure to maintain the typical Neotropical floral morphology [2]. The floral morphology of Paleotropical species presents instead actinomorphic flowers, weakly clawed petals,
posterior petal absence and eglandular calyx [1, 21, 22]. The records of heteranthery, poricidal
anthers and dry and dusty pollen in Paleotropical species [1, 3, 8, 20] are expected features in a
pollen syndrome flower [1]. These androecium changes suggest the loss of oil-bee pollination
in the majority of Paleotropical clades, and pollen is the only obvious pollinator reward [1, 3].
Acridocarpus Guill. & Perr. is one of the exceptional genera in the Paleotropics, as well as
Hiptage Gaertn. and Tristellateia Thouars, for having some floral morphology characteristics
found in Neotropical species, such as zygomorphic flowers, with posterior petals and glandular
calyx [1, 11, 21, 22]. This genus comprises about 30 species distributed in Africa, including
Madagascar, the Arabian Peninsula, and one species in New Caledonia [7, 8, 21, 22]. According to the phylogeny of Malpighiaceae, Acridocarpus composes the acridocarpoid clade with
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
Fig 1. Floral morphology of Neotropical (A) and Paleotropical Malpighiaceae (B). (A) Tetrapterys sp. with one
posterior petal (white arrow) and (B) Acridocarpus longifolius, showing two posterior petals (white arrows). Photos A
and B by I. R. Guesdon.
https://doi.org/10.1371/journal.pone.0222561.g001
Brachylophon Oliv. [3], which is a monospecific genus from the Malay Peninsula and Sumatra
[7]. Acridocarpus is characterized by the following: erect or climbing shrubs and rarely small
trees, with simple and alternate leaves; yellow flowers clustered in racemes or terminal panicles, sessile pedicels with floriferous bracts and two bracteoles at the base; bracteole sometimes
glandular; zygomorphic flowers; mostly with glandular calyx; weakly clawed petals, two posterior petals; poricide anthers; ovary 2-carpelate with curved styles; and fruits typically winged
[1, 20, 21, 22]. Interestingly, the zygomorphy of the Acridocarpus flower is reoriented, with
two posterior petals, two lateral petals and only one anterior petal, while the calyx presents
only one posterior sepal, two lateral sepals and two anterior sepals [1, 20] (Fig 1B). The occurrence of glands on the bracteoles and calyx is commonly documented in taxonomic studies of
Acridocarpus [21–23]. However, little attention has been given to the glands on the bracts at
the base of inflorescence peduncle. The functional role and structure of these glands are not
fully understood. In this work, we characterize the morphology and anatomy of these glands
in Acridocarpus species and evaluate the gland secretion with the aim of identifying patterns
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
across the genus, their association to species geographic distribution and their phylogenetic
framework. Finally, this study contributes to clarifying patterns of floral evolution within
Paleotropical Malpighiaceae.
Materials and methods
Floral samples were obtained from herbarium material of 25 Acridocarpus species, from the
Muséum National d’Histoire Naturelle (P), France (33 specimens in Table 1). The bracteole
and sepal glands were analyzed with a stereomicroscope (Stemi 2000-C Zeiss, Gottingen, Germany) equipped with a digital camera (AxioCam ERc; Zeiss, Gottingen, Germany). The bracts
at the base of inflorescence peduncle were not always well preserved and when these bracts
were present, the sampling from the exsiccate was discarded, in order to avoid injuries and to
preserve the herbarium collection.
Besides the herbarium material, samples of bracts at the base of the inflorescence, peduncle,
bracteoles, and calyx of Acridocarpus longifolius were obtained from a plant nursery, from collections at the Jardin Botanique de Meise, Belgium (19391489, 19700668). They were fixed in
FAA (formalin, acetic acid and 50% ethanol; 1:1:18 by volume) [24] for 48 h and then stored in
70% ethanol. The secretion exuded by all the glands on the inflorescence was submitted to glucose and protein concentration tests using urinalysis reagent strips (Insight, Acon Laboratories, San Diego, USA). A Sudan black test was also made to detect oil compounds on the
surface of the glands.
Table 1. List of material of Acridocarpus from the Muséum National d’Histoire Naturelle (MNHN) used in this
study, including collector and collection number.
Species
Acridocarpus adenophorus A. Juss.
Collector and number
Capuron 8883
Acridocarpus alopecurus Sprague
Haerdi 447, Sacleux 779
Acridocarpus alternifolius Nied.
Meikle 845, Breteler 5322
Acridocarpus austrocaledonicus Baill.
McPherson 3306
Acridocarpus camerunensis Nied.
Le Testu 7800
Acridocarpus chevalieri Sprague
Jaeger 3461
Acridocarpus chloropterus Oliv.
Schlieben 2427
Acridocarpus congolensis Sprague
Chevalier 28381
Acridocarpus excelsus A. Juss.
Schatz 2984
Acridocarpus humbertii Arènes
Phillipson 5989
Acridocarpus katangensis De Wild.
Gathy 1950
Acridocarpus longifolius Hook. F.
Bos 4924, Chevalier 26184
Acridocarpus macrocalyx Engl.
Letouzey 11775, Carvalho 3455
Acridocarpus monodii Arènes & Jaeger ex Birnbaum & J.Florence
Griaule 60, Birnbaum 615
Acridocarpus natalitius A. Juss.
Phillipson 3807
Acridocarpus orientalis A. Juss.
Popov 706
Acridocarpus perrieri Arènes
Rakotondrajaona 397
Acridocarpus plagiopterus Guill. & Perr.
Chevalier 14767; 20357
Acridocarpus prasinus Exell
Sita 3148
Acridocarpus smeathmannii Guill. & Perr.
Leeuwenberg 2409
Acridocarpus socrotanus Oliv.
Smith 204
Acridocarpus spectabilis (Nied.) Doorn-Hoekm.
Valenza 420, Birnbaum 751
Acridocarpus vanderystii R.Wilczek
Koechlin 6027, Chevalier 11097
Acridocarpus vivy Arènes
Schatz 4165
Acridocarpus zanzibaricus A. Juss.
Zhang 154
https://doi.org/10.1371/journal.pone.0222561.t001
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
Samples from herbarium material (Table 1) were subjected to a reversion process [25],
dehydrated and stored in 70% ethanol. Samples of both herbarium and fixed fresh material
were embedded in methacrylate (Historesin Leica; Heidelberg, Germany) following the manufacturer’s recommendations. Cross and longitudinal sections (5μm thickness) were made with
an automatic rotary microtome (Leica RM2155, Deerfield, USA). The sections were stained
with toluidine blue at pH 4.7 [26] and the slides were mounted in Permount (Fisher Scientific,
NJ, USA).
The following histochemical tests were performed on the fixed samples (Acridocarpus longifolius): for total proteins, xylidine ponceau [27] and Coomassie blue [28]; for total polysaccharides, periodic acid–Schiff reagent—PAS [29]; for mucilage and pectin, ruthenium red and for
starch, lugol [24]; for phenolic compounds, ferric chloride [24]; and for total lipids, Sudan red
[30].
Images were taken using a light microscope (Olympus AX70TRF) equipped with a digital
camera (AxioCam HRc; Zeiss, Gottingen, Germany) at the Laboratory of Plant Anatomy of
the Federal University of Viçosa (UFV), Brazil. Scanning electron microscopy (SEM) analyses
were conducted at the Center for Microscopy and Microanalysis (Viçosa, Brazil), with a LEO
1430VP (Zeiss, Cambridge, UK). The fixed samples were dehydrated, critically point dried
using CO2 (CPD 030, Bal-Tec, Balzers, Liechtenstein), fixed on stubs and sputter coated with
gold (SCD 050, Bal-tec, Balzers, Liechtenstein).
The morphological description was based mainly on Niedenzu [21, 22] and Anderson’s terminology [5, 14]. For anatomical descriptions, the glands were classified as stalked, when a
short non-secretory stalk was present, and immersed or sessile when the secretory tissues
were distributed above or below the level of the non-secretory epidermis surrounding the
gland, respectively. The types of gland and distribution patterns were classified according to
position in symmetrical plans. For bracts and bracteoles were observed the median or marginal
placement, while for calyx, beyond marginal or intersepalar position, was observed the distribution on the dorsal, lateral or anterior sepals.
Results
The number of sepal glands in sampled Acridocarpus species from different geographical distribution was confirmed (Table 2). The ranges of variation and the absence of glands reported
in taxonomical studies were also checked (see Table 2). The glands on the bract peduncle of
the inflorescence were described for the first time in the genus, and the bracteole glands in A.
austrocaledonicus was recorded for the first time.
Morphology and anatomy of bract and bracteole glands
The bract of the inflorescence peduncle was analyzed only in the fresh samples of Acridocarpus
longifolius. However, we believe that all Acridocarpus species bear such bracts, which are deciduous on the mature inflorescence. On the bracts examined from the fresh samples of Acridocarpus longifolius, two greenish glands were observed (Fig 2A and 2B), while one reddish
gland-like protrusion was recorded on the bracteoles (Fig 2A and 2C). The bract gland comprises a secretory epidermis and vascularized secretory parenchyma (Fig 2D and 2E). Although
a gland-like protrusion was evident on the bracteole of A. longifolius (Fig 2A and 2C), and curiously, no secretory features were observed (Fig 2F and 2G). The shape of the bract glands and
gland-like protrusion of A. longifolius, and the bracteole glands from herbarium samples, are
globose with an orbicular-oblong outline (Fig 2A, 2B, 2H and 2I).
Regarding the bracteole gland position, three groups were recognized: Group I, one basilaminar median gland (Fig 2H, 2J and 2L), in Acridocarpus alopecurus, A. austrocaledonicus, A.
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
Table 2. Geographic distribution of Acridocarpus species, occurrence and number variations of glands on the peduncle bract, bracteole and calyx, in the species analyzed in this study and in taxonomical previous studies. - indicates absence and x indicates unknown data.
Geographic distribution
Species
Glands on the Peduncle Bract / Bracteole / calyx
(this study)
Madagascar
x/2/-
x/1/-
[21, 22, 31]
A. excelsus
x/2/-
x / -; 1 / -
[21, 22, 31]
A. humbertii
x/2/-
x/1/-
[32]
A. perrieri
x/2/-
x/1/-
[31]
A. vivy
Continental
Africa
Arabian
Peninsula
New Caledonia
(previous studies)
A. adenophorus
x/2/-
x/1/-
A. alopecurus
x / 1 / 3(5)
x / 1 / 3(5); 2–3
A. alternifolius
x /—/ 5
x /—/ 2–4; 2
A. camerunensis
x /—/ 4
x / x / 3–4
A. chevalieri
x /—/ 3
x /—/ 2–3
A. chloropterus
x /—/ 3
x /—/ 3; 2–3
[31]
[21, 22, 33, 34]
[21, 22, 35]
[21, 22]
[35]
[21, 22, 33, 36, 37]
A. congolensis
x/1/2
x / 1 / 2–3
A. katangensis
x / 1 / 2(3)
x / 1 / 2; 2–3
[21, 22, 34, 37]
[21, 22, 34]
A. longifolius
2 / (relictual ?) / 1
x /—/ 1
[21, 22, 34, 35]
A. macrocalyx
x /—/ 2(4)
x / x / 2; 2–3
A. monodii
x /—/ 4
x/x/2
A. natalitius
x /—/ 3
x /—/ 2–3; 4
A. plagiopterus
x /—/ 2
x / x / 2; 3
A. prasinus
x/1/4
x / -; 1 / 2–3
A. smeathmanni
x/1/3
x / 1 / 3; 2 (3–4)
A. spectabilis
x /—/ 4
x / x / 5–10
[40]
A. vanderystii
x /—/ 5
x / 1 / 2–3
[41]
[21, 22, 34]
[38]
[21, 22, 37]
[21, 22, 36, 39]
[21, 22, 33, 34]
[21, 22, 33, 34, 36]
A. zanzibaricus
x /—/ 3
x /—/ 2; 3; 2–3
[21, 22, 33, 36]
A. orientalis
x /—/ 5
x / x / 2–3; 1–5
[21, 22, 42]
A. socotranus
x /—/ 2
x/x/2
[21, 22]
A. austrocaledonicus
x/1/-
x /—/ x
[21, 22]
https://doi.org/10.1371/journal.pone.0222561.t002
congolensis, A. katangensis, A. prasinus and A. smeathmanni; Group II, two glands medially
positioned (Fig 2I and 2K), in A. adenophorus, A. excelsus, A. humbertii, A. perrieri, and A.
vivy; and Group III, eglandular, in A. alternifolius, A. camerunensis, A. chevalieri, A. chloropterus, A. macrocalyx, A. monodii, A. natalitius, A. plagiopterus, A. spectabilis, A. vanderystii, A.
zanzibaricus, A. orientalis, and A. socotranus.
The anatomical constitution of the bracteole glands on samples from herbarium specimens
was the same observed for the bract glands on samples from fresh material of Acridocarpus
longifolius. The secretory epidermis cells are typically arranged in a palisade-like layer and
have densely stained cytoplasm with a conspicuous nucleus and thick cuticle, which develops a
subcuticular space (Fig 2M and 2O). No secretory pores or ruptured cuticles were observed
(Fig 2H and 2I). The secretory parenchyma comprises a few layers of cells (Fig 2N and 2O),
frequently filled with phenolic compounds (Fig 2O). Idioblasts containing druse crystals were
often observed scattered in the secretory parenchyma (Fig 2O).
The majority of species have sessile bracteole glands (Fig 2J and 2L), while stalked glands
(Fig 2M) were observed on the bracteoles of Acridocarpus adenophorus, A. excelsus, A. humbertii and A. perrieri. Stalked glands are usually associated with a flat secretory surface, while sessile bracteole glands possess a convex surface.
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
Fig 2. Morphoanatomy of bract and bracteole glands in Acridocarpus. (A-C) General view of inflorescence axis; showing glandular bract (B;
black arrowheads) and bracteole (Br; white arrowheads) on the base of the pedicels. (D) Paradermal section of bract in A. longifolius, note the bract
glands (asterisks). (E) Detail of the bract gland showing secretory epidermis (SE) and secretory parenchyma (SP), with voluminous nucleus and a
dense-staining cytoplasm. (F, G) Bracteole gland-like protrusion in A. longifolius, note a detail showing epidermis and non-secretory parenchyma
(G). (H, I) SEM image of bracteoles, one-glandular bracteole in A. smeathmanni (H) and two-glandular bracteole in A. perrieri (I). (J) Transversal
section of one-glandular bracteoles (asterisks) on the pedicel (Pe) of A. smeathmanni. (K) Transversal section of two-glandular bracteole
(asterisks) in A. vivy. (L) Sessile arrangement of bracteole gland in A. prasinus. (M) Stalked bracteole gland in A. humbertii, note the subcuticular
space (white arrowhead). (N, O) Detail of bracteole gland in A. prasinus (N) and A. humbertii (O). Highlighting the secretory epidermis (SE) and
the secretory epidermis (SP), note the presence of cystals (white arrow), phenolic idioblast (black arrow) and the vascularization (black
arrowhead). Photos A-C by I. R. Guesdon.
https://doi.org/10.1371/journal.pone.0222561.g002
Morphology and anatomy of sepal glands
One yellow gland was recorded on the calyx of fresh samples of Acridocarpus longifolius (Fig
3A and 3B). For the herbarium specimens analyzed, the sepal glands are marginal (Fig 3C) or
intersepalar (Fig 3B and 3D). These glands are globose with an oblong outline (Fig 3C) or, in
A. longifolius and A. zanzibaricus, impressed with a sagittate-acute outline (Fig 3B and 3D).
All sepal glands analyzed comprise a secretory epidermis with subcuticular space and vascularized secretory parenchyma with abundant phloem, and crystalliferous and phenolic idioblasts are common (Fig 3G and 3I). The anterior gland in Acridocarpus longifolius (Fig 3D and
3F) and A. zanzibaricus are immersed, while in the remaining species only sessile glands were
observed on the calyx (Fig 3E, 3I and 3N).
Regarding sepal gland distribution patterns, the glands were restricted to the posterior sepal
(Fig 3J and 3K), to the sepals of the ventral plane (Fig 3L) or distributed on all sepals (Fig 3M
and 3N); ten morphoanatomical types were established and are illustrated in Fig 4. Type I:
eglandular calyx, observed in in Acridocarpus adenophorus, A. austrocaledonicus, A. excelsus,
A. humbertii, A. perrieri, and A. vivy; Types II: two marginal glands in posterior sepal of A.
congolensis, A. katangensis, A. macrocalyx, A. plagiopterus, and A. socotranus; Type III: two
marginal glands in posterior sepal and one marginal gland on posterior side in each lateral
sepal in A. katangensis, A. macrocalyx, and A. prasinus; Type IV, each anterior sepal with two
glands in A. monodii and A. spectabilis; Type V: distinct from type IV due to the one anterior
gland in an intersepalar position of A. zanzibaricus; Type VI: one gland in the intersepalar portion of the anterior sepal pair in A. longifolius; Type VII: two marginal glands in one of the
anterior sepals and one marginal gland in the other anterior sepal, founded in A. alopecurus,
A. chevalieri, A. chloropterus, A. smeathmanni, and A. natalitius; Type VIII: two marginal
glands in the posterior sepal, two marginal glands in one of the anterior sepals and one marginal gland in the other anterior sepal in A. alopecurus, A. alternifolius, and A. orientalis; Type
IX: two marginal glands in the posterior sepal and one marginal gland on the posterior side of
each anterior sepal in A. camerunensis, and Type X: one gland in each intersepalar portion in
A. vanderystii.
In types II and III the glands are distributed only on the posterior plane of the calyx, while
in types IV–VII they are on the sepal on the ventral plane, and in types VIII–X on both planes.
However, intraspecific variations were recorded that point out different types in A. alopecurus
(Types VII and VIII), and in A. katangensis and A. macrocalyx (Types II and III).
The sepal glands in most Acridocarpus species are small and paired at least in one sepal (Fig
4); and even for the marginal glands of adjacent sepals a non-secretory intersepalar region was
observed (e.g., in Acridocarpus prasinus, Fig 3K). However, adjacent glands show different
degrees of fusion in some cases (Fig 5A–5C). In A. spectabilis, adjacent glands of the anterior
sepals are close to each other and the non-secretory intersepalar region is reduced (Fig 5D and
5G), while in A. monodii the intersepalar region between the adjacent glands is actually secretory, suggesting fusion, since they share the secretory epidermis and the secretory parenchyma
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
Fig 3. Morphoanatomy of sepal glands in Acridocarpus. (A, B) General view of inflorescence in A. longifolius (A) showing sepal gland of floral buds
exuding a nectar drop (black arrows), the magnification is displayed in (B). (C) SEM image of marginal sepal (Se) glands (white arrows) in A.
smeathmanni. (D) SEM image of the intersepalar gland of A. longifolius. (E, F) Transversal sections of a sessile sepal gland in A. smeathmanni (E) and
immersed sepal gland of A. longifolius (F). (G-I) Detail of secretory tissues of sepal gland note the voluminous nucleus and a dense-staining cytoplasm
of secretory epidermis (SE) and secretory parenchyma cells (SP) in A. longifolius (G), A. spectabilis (H) and A. vanderystii (I), note crystals (white
arrow), phenolic idioblast (black arrow) and subcuticular space (white arrowhead). (J-N) Sepal gland distribution patterns. (J-K) Glands (asterisks) on
posterior plane; glands on the posterior sepal of A. socrotanus (K), glands on posterior and lateral sepal of A. prasinus. (L) Glands (asterisks) on ventral
plane, on the anterior sepals of A. alopecurus. (M, N) Glands (asterisks) on both posterior and ventral plane in A. alternifolius (M) and A. vanderystii
(N). Photos A-C by I. R. Guesdon.
https://doi.org/10.1371/journal.pone.0222561.g003
(Fig 5E and 5H). Another degree of fusion was observed in A. longifolius and A. zanzibaricus
(Fig 5F and 5I), which have adjacent glands that share the secretory parenchyma, vascularization and the secretory epidermis, comprising a flat and homogeneous surface (Fig 5I). One
medial gland was observed in all intersepalar regions of A. vanderystii (Fig 3N); however, indications of fusion were unclear.
Floral gland secretion in Acridocarpus longifolius
Copious secretion was recorded on the sepal glands (Fig 3A and 3B), and nectar consumers
were observed visiting these structures (Fig 6A). Secretory activity begins during blooming
and remains active until fruiting (Fig 6B). The bract and sepal exudate reacted with the test
strips for the presence of glucose and protein. The concentration of glucose was higher in the
sepal glands (1000[60] mg/dL [mmol/L]) compared to the bract glands (250[15] mg/dL
[mmol/L]), while the concentration of proteins was the same in both glands (30[0.3] mg/dL[g/
L]).
In agreement with the anatomical analyses of Acridocarpus longifolius, the histochemical
test applied to the bract and sepal glands reacted positively for total polysaccharides (Fig 6C
and 6D), protein (Fig 6E–6G) and phenolic compounds (Fig 6J). Proteins and phenolic
Fig 4. Diagram in frontal view of the morphoanatomical patterns of calyx gland distribution. Type I, eglandular. Type II, two marginal
glands in posterior sepal. Type III, two marginal glands in posterior sepal and one marginal gland on posterior side in each lateral sepal. Type
IV, each anterior sepal with two glands. Type V, distinct from type IV due to the one anterior gland in an intersepalar position. Type VI, one
gland in the intersepalar portion of the anterior sepal pair. Type VII, two marginal glands in one of the anterior sepals and one marginal gland
in the other anterior sepal. Type VIII, two marginal glands in the posterior sepal, two marginal glands in one of the anterior sepals and one
marginal gland in the other anterior sepal. Type IX, two marginal glands in the posterior sepal and one marginal gland on the posterior side of
each anterior sepal. Type X, one gland in each intersepalar portion.
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
Fig 5. Morphoanatomy of sepal glands in Acridocarpus species. (A-C) SEM images of sepal glands (Se), showing two adjacent glands on the
anterior sepal pair in Acridocarpus spectabilis (A) and A. monodii (B), and a single gland shared by the anterior sepal pair in A. longifolius (C), with a
sagittate-acute outline; the white arrows are point sepal glands. (D-F) Transversal cross sections of the calyx, note the gland (asterisks) distribution in
A. spectabilis (D), A. monodii (E) and A. zanzibaricus (F). (G-I) Different degrees of glandular fusion: secretory epidermis of adjacent glands
juxtaposed, in A. spectabilis (G); adjacent glands sharing the epidermis and secretory parenchyma in A. monodii (H) and complete sharing of
secretory tissues in A. zanzibaricus (I).
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compounds were distributed in the epidermis and secretory parenchyma. A positive reaction
for lipid compounds was detected only on the cuticle (Fig 6H and 6I).
Discussion
Morphology, anatomy and composition of secretion: Functional insights
This is the first comprehensive study of morphoanatomy of Acridocarpus glands, and histochemical record of their secretion constitution. Based on morphoanatomy, histochemistry,
persistent secretory activity, detection of glucose and protein in the exudate and the absence of
lipid compounds in the exudate, we proved that the bract and sepal glands of Acridocarpus
longifolius are actually nectaries. Vogel [43] demonstrated oil production in the elaiophores of
three Neotropical species, which have been confirmed in morphoanatomical, histochemical
and ultrastructural studies in several Neotropical species [44– 47]. In contrast, after the old
finding of sugar in glandular secretions in two species of Paleotropical Malpighiaceae [11],
studies about any glands in Paleotropical species are scarce. Until now, morphoanatomical,
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
Fig 6. Aspects of secretion and histochemical results of sepal glands in Acridocarpus longifolius. (A) Nectar consumer on the sepal gland. (B)
Nectar available in sepal glands of pollinated flowers and during the development of fruit (white arrowhead). (C, D) Periodic acid/Schiff reagent
(PAS); magenta staining indicates neutral polysaccharides. (E-G) Protein detection by the blue stain from the Comassie blue test (E, F) and orange
stain from the Xylidine Ponceau test (G). (H, I) Sudan red test; reddish color indicates total lipids, note the negative reaction for the secretory tissues
(H, I) and positive reaction for the cuticle (black arrow) (I). (J) Phenolic compounds stained brown using the ferric chloride test. Photos A and B by I.
R. Guesdon.
https://doi.org/10.1371/journal.pone.0222561.g006
histochemical and ultrastructural data were restricted to only two species of Hiptage Gaertn.
[48–50].
Although the secretion of the bracteole glands was not tested, due to their placement and
similar morphoanatomy, the glands on the bracts, bracteole and sepals of Acridocarpus were
all recognized as nectaries. The morphoanatomical characters recorded in the glands of
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
Acridocarpus species analyzed have been commonly reported in nectaries of Malpighiaceae
species, such as a palisade-like epidermis, with epidermal cells well juxtaposed, subcuticular
space storing secretion, and idioblasts containing phenolic compounds or druse-type crystals
[45, 47, 51–54].
The floral glands in the majority of investigated species of Acridocarpus are sessile, with the
exception of stalked glands on the bracteole of A. adenophorus, A. excelsus, A. humbertii and
A. perrieri. In Malpighiaceae, stalked nectaries have been recorded on the leaf and bracteole of
Neotropical species [45, 51, 52] belonging to Banisteriopis C.B.Rob., Lophanthera Adr. Juss.,
and Mcvaughia W.R.Anderson [14, 45, 52]; while long stalked elaiophores were morphoanatomically described only in Dinemandra ericoides A. Juss., and were reported in some speciespoor genera like Dinemagonum A. Juss., Heladena A. Juss. and Henleophytum H. Karst. [55,
22]. Immersed and embedded nectaries were previously reported, mainly in Leguminosae leaf
[56, 57]; the sepal gland with secretory tissues immersed on the calyx in A. longifolius, called as
“magna” gland by Niedenzu [22], was the first record in Malpighiaceae.
The detection of sugar and protein found in the secretions of bract and sepal nectaries in
Acridocarpus longifolius could be related to nectar consumers, such as ants [1, 11]. This
increase in nutritional richness of the nectar by the presence of protein could strengthen the
ecological relations with these consumers, which prefer sugary solutions rich in amino acids
[58–60]. Although few observations in the field are available to A. longifolius, the aggressive
behavior of ant consumers of sepal nectar in the Paleotropical Malpighiaceae species Hiptage
benghalensis (L.) Kurz, was associated with a protection role against inflorescence herbivory
[12]. A similar interpretation is possible for A. longifolius, which the larger drops of nectar and
the higher glucose concentration in their sepal glands might be involved in protecting vulnerable reproductive organs by attracting protector consumers [61, 62], since nectar composition
may also vary according to plant demands [59, 63].
The presence of phenolic compounds detected in Acridocarpus glands, is a common feature
of secretory tissues [64], and has also been reported for leaf nectaries [47, 54, 65] and elaiophores of Neotropical species of Malpighiaceae [44, 46, 47]. These compounds may provide
chemical protection against herbivore attacks [63, 66].
In addition to the morphoanatomical similarity of nectaries and elaiophores reported for
Neotropical species, a mixture of hydro- and lipophilic compounds detected in both secretions
supports their homology [44–47, 53, 54], differing for a sugar or oil-rich secretion [1, 53]. The
oil production in elaiophores of Neotropical species [11, 43], was detected in species of Diplopterys A. Juss. [44], Banisteriopsis [45], Byrsonima Rich. ex. Kunth and Peixotoa A. Juss. [46],
Galphimia Cav. [53], Dinemandra A. Juss. [55], Mcvaughia, Burdachia Adr. Juss. and Glandonia Griseb. [47]. Unlike expected for elaiophores, the histochemical tests performed in the
bract and sepal glands of Acridocarpus longifolius, of both field and laboratory analysis, did not
detect lipids (Fig 5). Similar results were obtained for the sepal secretion of A. smeathmanii
[11] and Hiptage benghalensis [12], where most of the compounds were reducing sugars and
no lipids were found. Furthermore, the secretory activity of sepal glands in A. longifolius is premature and persistent during fruiting (Fig 5), as reported for sepal nectaries by Ren et al. [12].
In contrast, the elaiophore secretion starts to accumulate in the young bud stage and increases
until pollinator gathering in the anthesis stage [46].
An interesting similarity to note between nectaries and elaiophores, that the secretion is
accumulate in the subcuticular space [44–56]. In Neotropical species of Malpighiaceae, as a
consequence of a natural process of secretion release or because of the collection behavior of
oil-bees [46], the elaiophore secretion is often exposed because the cuticle rupture; this type of
damage is commonly reported [44, 46, 50, 53, 55]. However, no ruptured cuticles, pores, or
any damage caused by secretion consumers were observed in any glands of Acridocarpus
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
analyzed, even in the herbarium material. An intact cuticle was also observed in nectaries on
the bracteole of Burdachia, Glandonia and Mcvaughia, since the ants just slide their labrum on
the gland surface [47]. Other evidence of the non-nuptial function of sepal glands in Acridocarpus are the weakly clawed petals, since they might make the calyx glands difficult to be accessed
by pollinators, as recorded for Hiptage benghalensis [1]. In the latter, petals keep the calyx
gland enclosed during the whole anthesis period [12]. In summary, the reoriented zygomorphic flowers in Acridocarpus, petal morphology, and the poricide anthers (Fig 1B) probably
reflect a remarkable floral specialization to pollen-collecting pollinators, such as buzz-bees and
honey bees [11]; while morphoanatomy and histochemistry of sepal glands are probably associated to plant anti-herbivore defense.
Evolutionary trends from gland morphoanatomy and biogeography of
Acridocarpus
The mutualistic association of Neotropical Malpighiaceae with oil-bee pollinators is evident by
the floral morphology conservatism, including sepal oil-glands on the calyx. In the Neotropical
species, these ten glands are typically distributed in pairs on all five sepals, but may be absent
on the anterior sepal, on both anterior and lateral sepals or, rarely, the calyx is completely
eglandular [1, 9, 43]. According to Vogel [1], sepal glands are absent in most Paleotropical species or, when present, vary in number (one, three or five). However, here we identified for the
first time ten gland distribution patterns in the genus Acridocarpus.
The ventral absence of oil-glands on the calyx in the Neotropical species of Malpighiaceae
has been commonly attributed to an economic reduction, due to the inability of oil-bee pollinators to use ventral glands [1]; while the eglandular calyx is associated with the oil-bee syndrome loss [2, 67–69]. Despite the lack of studies about reproductive biology in Acridocarpus,
there is strong evidence of the loss of oil-bee syndrome, shown by the reduction/fusion or
absence of sepal glands (Fig 4) and the nectar secretion. Additionally, considering the morphoanatomical evidence of homology shared by nectaries and elaiophores [1, 45, 47, 55], and
the fact that nectar precedes oil secretion across the evolution [1], an expected outcome in
Acridocarpus species, given the loss of oil-bee pollination, is the reversal to non-nuptial nectar
secretion.
Although the sepal glands are taxonomically useful to distinguish Paleotropical species of
Hiptage [70], in Acridocarpus these characters should be interpreted cautiously due the variability in the number of glands in the same species (Table 2), as well as in the same specimens.
Intraspecific variation has been reported for some Neotropical species of Malpighiaceae, as
recorded in Byrsonima, Galphimia, and Stigmaphyllon A. Juss., having variable numbers of
glands on the anterior and lateral sepals [53, 67, 71, 72]. It is interesting to note that the high
variation in number and placement of sepal glands recorded in Acridocarpus (Fig 4) was not
described for any other Malpighiaceae genus, which may be due to a labile gene expression to
determine the presence of the glands on the calyx. According to Anderson [68] this range of
numerical variety is typically observed in nectaries. Additionally, Davis et al. [3, 8] proposed
that the Neotropical floral morphology, including the usual elaiophore distribution in pairs on
five or four sepals, are actually labile and were actively maintained by the mutualistic relation
with oil-bee pollinators. Our results in Acridocarpus suggest that in the Paleotropics, where the
species have pollen-flowers and are not under the oil-bees selective pressure, not only the
secretion of sepal glands was modified to nectar, but their distribution patterns on the calyx
have become highly variable (Fig 4).
Vogel [1] and Anderson [9] emphasized morphological trends of the sepal glands in Paleotropical species of Malpighiaceae, being smaller than elaiophores and typically intersepalar.
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Unexpectedly, in Acridocarpus this intersepalar placement was an exception, since a single
gland in the intersepalar position was only recorded in A. longifolius, A. zanzibaricus and A.
vanderystii (see Fig 4, types V, VI and X). This finding suggests that the absence of one gland
of the pair on the same sepal (see Fig 4, types III, VI-IX), may be due to the numerical variability expected in nectaries. Furthermore, the occurrence of small intersepalar glands on the calyx
is not exclusive to Paleotropical species, since this was reported for Neotropical species that
have lost the stereotyped malpighiaceus floral morphology [68].
This intersepalar gland position was considered by Vogel [1] as the result of fusion of a pair
of glands on adjacent sepals. However, Castro et al. [53] attribute the single gland in Galphimia
brasiliensis A. Juss. (Malpighiaceae) to loss and not fusion of such structures. Souto and Oliveira [73] suggest that the fusion of sepal glands in Mascagnia cordifolia (A. Juss.) Griseb (Malpighiaceae) is based mainly on shared vascular tissue. In Acridocarpus longifolius and A.
zanzibaricus, followed to the shared secretory tissues and vascular bundles, the sagittate-acute
outline may be strong evidence of the fusion of two glands on adjacent anterior sepals (see Fig
5C for A. longifolius). The anatomy of the sepal glands of A. longifolius, A. monodii, A. spectabilis and A. zanzibaricus suggests an evolutionary sequence from juxtaposed to partially connate to completely fused glands (Fig 5E–5J), reflecting fusion of tissues from the exterior to the
interior, which is in agreement with the fusion steps noted by Fahn [64].
According to phylogenetic evidence [3, 7], most Neotropical species of Malpighiaceae, like
those in the byrsonimoid clade, have sepal glands, which indicates an ancestral condition [9],
while their absence suggests a derived condition [73]. In the first dispersal of Neotropical
ancestors to the Paleotropics, the acridocarpoid clade gave rise to Asian and African lineages
(Brachylophon and Acridocarpus) [7]. Regarding that Brachylophon (sister group of Acridocarpus) is completely eglandular [22], the sepal glands in the acridocarpoid clade may be a reversal
to the ancestral condition, showing morphofunctional differences from the sepal glands of
most Neotropical species, probably due to the absence of oil-bee mutualism, as an adaption
against herbivores.
The reconstruction of the geographical history [7] suggest that Acridocarpus evolved from
migrations to continental Africa, Madagascar and New Caledonia, subsequently from east to
west of Africa and finally to the Arabian Peninsula. Davis et al. [7] proposed that Acridocarpus
species from Madasgascar and the only species from New Caledonia are probably a disjunction
by a long-distance dispersal event from Madasgascar to New Caledonia. Interesting, most
Madagascar species bear stalked bracteole glands, except for Acridocarpus vivy that has sessile
glands (Fig 2K), like in A. austrocaledonicus. Acridocarpus longifolius was phylogenetically
placed within African species with eglandular bracteoles [7], which suggests that the gland-like
protrusion of the bracteole can be a relictual signal. Additionally, an eglandular calyx associated to a glandular bracteole seems to be exclusive to Acridocarpus species from Madagascar
and New Caledonia (Fig 7) and support the phylogenetic proximity of Madagascar-New Caledonia species demonstrated by Anderson and Davis [3]. In comparison, most species from
continental Africa and Arabian Peninsula have a contrary condition, with eglandular bracteoles and glandular calyx (Fig 7).
Conclusions
Overall, the bract, bracteole and sepal glands in Acridocarpus are nectaries, which is probably
associated with attracting anti-herbivory protectors instead of pollinators. Due to the absence
of oil-collecting bees and oil-flower mutualism in Acridocarpus, the different types of gland
distribution patterns on the calyx identified in this study provide new evidence that such a feature is variable and labile. Our study also demonstrated the morphoanatomical similarity
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
Fig 7. Geographic distribution and glandular condition of bracteole and calyx. Brachylophon (black area) in Malay Peninsula and Sumatra: eglandular. Acridocarpus
(grey area) in Madagascar and New Caledonia: glandular bracteole and eglandular calyx; in continental Africa: eglandular bracteole (or glandular) and glandular calyx;
in Arabian Peninsula: eglandular bracteole and glandular calyx. Black circles: Localities of the specimens analyzed in this study.
https://doi.org/10.1371/journal.pone.0222561.g007
between nectaries and elaiophores of the Neotropical species and nectaries of the Acridocarpus
species, which can be interpreted as a sign of their homology and the secretion transformation
of sepal glands from oil to nectar, reinforcing Vogel and Anderson’s hypothesis that these
structures probably evolved following a secretion change. Considering the clues pointed out
by the phylogenetic framework, the glandular bracteole and eglandular calyx provide some
support for the sister-taxon relationships between Acridocarpus species from Madagascar and
New Caledonia. Finally, our findings suggest a relation between non-nuptial nectar consumers
and the floral glands in Acridocarpus, and contribute to a better understanding of how the dispersal events from the Neo- to the Paleotropics affected the floral morphology of Malpighiaceae. Further morphoanatomical studies in other Paleotropical glandular genera may also
improve the knowledge about floral evolution in Malpighiaceae.
Acknowledgments
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nı́vel
Superior—Brasil (CAPES)—Finance Code 001. The authors thank the following: the Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES) and Fundação de Amparo à
Pesquisa do Estado do Amazonas (FAPEAM/PROGRAMA RH-INTERIORIZAÇÃO) for
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Morphoanatomy of floral glands in Acridocarpus (Malpighiaceae)
awarding the doctoral scholarships to I.R.G; the Programa de Pós-Graduação em Botânica at
the Universidade Federal de Viçosa (UFV) for providing logistical support; the Conselho
Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq) for awarding research productivity fellowships to AMA (grant # 310717/2015-9) and RMSAM (grant # 307762/2016-5); and
the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the financial
resources (UNIVERSAL APQ-00383-16). We are also grateful to the MNHN Herbarium and
Jardin Botanique Meise for kindly loaning samples, and the anonymous reviewers as well as
Dr. Marcelo Pace for the comments and suggestions about this manuscript. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Author Contributions
Formal analysis: Renata Maria Strozi Alves Meira.
Funding acquisition: Renata Maria Strozi Alves Meira.
Investigation: Isabel Reis Guesdon, André Márcio Amorim, Renata Maria Strozi Alves Meira.
Methodology: Isabel Reis Guesdon.
Resources: Isabel Reis Guesdon.
Supervision: Renata Maria Strozi Alves Meira.
Writing – original draft: Isabel Reis Guesdon, André Márcio Amorim, Renata Maria Strozi
Alves Meira.
Writing – review & editing: Isabel Reis Guesdon, André Márcio Amorim, Renata Maria
Strozi Alves Meira.
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