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Citation: Chitchak, N.; Stewart, A.B.;
Traiperm, P. Functional Ecology of
External Secretory Structures in Rivea
ornata (Roxb.) Choisy
(Convolvulaceae). Plants 2022,11,
2068. https://doi.org/10.3390/
plants11152068
Academic Editor: Agnes Farkas
Received: 8 July 2022
Accepted: 5 August 2022
Published: 8 August 2022
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plants
Article
Functional Ecology of External Secretory Structures in Rivea
ornata (Roxb.) Choisy (Convolvulaceae)
Natthaphong Chitchak , Alyssa B. Stewart and Paweena Traiperm *
Department of Plant Science, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
*Correspondence: paweena.tra@mahidol.edu
Abstract:
Plants have evolved numerous secretory structures that fulfill diverse roles and shape their
interactions with other organisms. Rivea ornata (Roxb.) Choisy (Convolvulaceae) is one species that
possesses various external secretory organs hypothesized to be ecologically important. This study,
therefore, aimed to investigate five secretory structures (nectary disc, petiolar nectaries, calycinal
glands, staminal hairs, and foliar glands) using micromorphology, anatomy, histochemistry, and
field observations of plant–animal interactions in order to assess the functional contributions of
these structures. Results show that the nectary disc and petiolar nectaries are complex working
units consisting of at least epidermis and ground tissue, while the other structures are glandular
trichomes. Various groups of metabolites (lipids, phenolic compounds, polysaccharides, terpenoids,
flavonoids, and alkaloids) were detected in all structures, while starch grains were only found in the
nectary disc, petiolar nectaries, and their adjacent tissues. Integrating preliminary observation of
animal visitors with micromorphological, anatomical, and histochemical results, two hypotheses are
proposed: (I) nectary disc and staminal hairs are important for pollination as they potentially attract
and reward floral visitors, and (II) petiolar nectaries, calycinal glands, and foliar glands contribute to
plant defense. Specifically, petiolar nectaries and calycinal glands provide protection from herbivores
via guard ants, while calycinal and foliar glands may use plant metabolites to help prevent tissue
damage from dehydration and insolation.
Keywords:
plant–animal interaction; micromorphology; anatomy; histochemistry; plant
defense; pollination
1. Introduction
Plants have evolved remarkable adaptations that allow them to tolerate harsh environ-
mental conditions [
1
,
2
] and to shape their interactions with other organisms, whether it
be communicating with other plants in the community [
3
], attracting mutualistic partners
such as pollinators [
4
–
6
], or deterring antagonistic individuals such as herbivores [
7
,
8
].
Many of these adaptations involve metabolites that are delivered through diverse secretory
structures, such as nectaries, stinging hairs, and osmophores [
9
–
12
]. In general, secretion
refers to the release of substances from the protoplast [
13
] and sometimes includes local-
ization of substances within the cell [
14
]. Plants differentiate their organs both internally
and externally to form secretory structures, which are involved in various activities such
as excess ion elimination, metabolic waste compartmentalization, pollinator attraction,
and defense against herbivory [
15
]. Secretory structures are diverse in form, ranging from
simple to complex working units, based upon their functions, secreted substances, and
locations on the plant [13,14,16].
Convolvulaceae is a diverse family distributed mainly in tropical and warm temper-
ate climates [
17
], and secretory structures have been examined in several species within
the family. In particular, two internal secretory organs have been reported, laticifers and
crystal idioblasts, which possibly contribute to chemical defense and detoxification, re-
spectively [
18
–
21
], and several other external secretory structures with different forms and
Plants 2022,11, 2068. https://doi.org/10.3390/plants11152068 https://www.mdpi.com/journal/plants
Plants 2022,11, 2068 2 of 27
functions, found on various plant parts, have also been reported. For example, peltate
glands are ubiquitous on the epidermis of vegetative organs, with most reports coming
from leaf investigation, and they are believed to contribute to protection from unsuit-
able environmental conditions and herbivory [
18
,
20
,
22
–
26
]. Moreover, glandular hairs on
staminal filament bases have been found in several species within Convolvulaceae and
are of taxonomic value to the family [
27
,
28
]. A recent study examining the pollination of
Argyreia siamensis (Craib) Staples suggested that chemicals that accumulate in the hairs
might contribute to pollinator attraction [
29
]. Nectaries are another common external
secretory structure and can be found in three locations: flower, receptacle (or sometimes
defined as part of the calyx or pedicel), and petiole. The role of the floral nectary (nectary
disc) in rewarding pollinators has been well-established, and most species in the family
are partially or completely dependent on pollinators [
29
–
32
]. While the floral nectary is
conserved throughout the family, nectaries on petioles and/or receptacles predominantly
occur in Ipomoea L., Rivea Choisy, and a few species from Decalobanthus Ooststr. and Cuscuta
L. Extrafloral nectaries are generally considered to contribute to plant defense by attracting
guard insects, which have mostly been reported to be ants inhabiting the surrounding
areas [33–38].
One species in the Convolvulaceae family with unique secretory structures, such as
prominent petiolar nectaries, is Rivea ornata (Roxb.) Choisy. Rivea ornata is one out of just
three species from the genus Rivea Choisy, and its distribution ranges from the Indian
subcontinent across the Eastern Himalaya to Indochina, but it is only rarely found in
Thailand [
39
]. Most descriptions of this species mention the occurrence of a nectary disc
and petiolar nectaries [
17
,
28
,
40
,
41
]. Additional observations of dried herbaria specimens
and living plants in field surveys revealed the noticeably more prominent petiolar nectaries
of R. ornata, as compared to related species, making it taxonomically significant and
also leading to questions about the functional significance of such prominent extrafloral
nectaries. Examination of the entire plant revealed that this species possesses numerous
types of external secretory organs, in addition to petiolar nectaries. However, there is
limited micromorphological, anatomical, and histochemical evidence to explain or provide
support for their structures and functions.
Therefore, the aim of this study was to examine five external secretory structures in
R. ornata, i.e., the nectary disc, petiolar nectaries, calycinal glands, staminal hairs, and
foliar glands, in order to (I) identify and describe their micromorphological and anatomical
features, (II) detect the main classes of chemicals in the accumulated substances and tissues
by histochemical methods, and (III) conduct preliminary observations of animals visiting
these secretory structures. We predicted that the diverse external secretory structures of
R. ornata help shape their interactions with various, mainly insect, species (ranging from
mutualistic to antagonistic), and use micromorphological, anatomical, histochemical, and
ecological data to formulate hypotheses about these interactions.
2. Materials and Methods
2.1. Study Species and Sample Collection
Rivea ornata (Roxb.) Choisy is a perennial shrub with woody rootstock predominantly
found in the understory of deciduous dipterocarp forests and mixed forests [
28
]. Flowers
are fragrant, white, salverform, and night-blooming [
28
]. Anthesis usually starts around
1800 h, shortly before sunset (1830–1900 h), and lasts throughout the night until sunrise
(0600 h) (NC, pers. obs.). The total accumulated nectar of a single flower ranges between
14–64
µ
L, and nectar sugar concentration tested using a handheld refractometer (Atago N1,
0–32%) ranges between 12–20% sucrose wt/wt (NC, pers. obs.). Flowering usually occurs
during the rainy season, from around August to October [
28
] (NC, pers. obs.). Leaves are
cordate and possess a pair of prominent glands at the apex of the petiole, widely known
among convolvulaceous plants as “petiolar nectaries” [
28
,
33
,
35
,
42
,
43
]. Fruits are dry and
horizontally dehiscent when mature, and contain four seeds embedded in the spongy
matrix [
28
,
39
]. In Thailand, the current number of known R. ornata populations appears to
Plants 2022,11, 2068 3 of 27
be lower than the number of populations observed in herbarium records, which are mostly
from the last 30–60 years, possibly due to deforestation and climate change. Plant materials
used in this study were collected from the two largest known populations. The first study
population (>30 individuals) was in Mae Hong Son province in northern Thailand, at an
altitude of 590 m a.s.l. A second study population (ca. 30 individuals) was located in Sakon
Nakhon province in northeastern Thailand, at an altitude of ca. 300 m a.s.l. Interestingly,
there was a striking morphological difference found in plants between the two sites. Plants
from the northern population displayed dark purple petiolar nectaries, while petiolar
nectaries of plants in the northeastern population were green (Figure 1). Voucher specimens
(northern population: NC and PT 45, 63; northeastern population: NC and PT 60) were
prepared following the standard method for plant taxonomy [
44
] and deposited at the Forest
Herbarium in Bangkok, Thailand (BKF). Fresh materials (including fully opened flowers
and mature leaves) kept at 4
◦
C were used for histochemical examination, and materials
fixed in 70% ethanol with a few drops of glycerol were used for micromorphological and
anatomical study.
Plants 2022, 11, x FOR PEER REVIEW 3 of 28
known among convolvulaceous plants as “petiolar nectaries” [28,33,35,42,43]. Fruits are
dry and horizontally dehiscent when mature, and contain four seeds embedded in the
spongy matrix [28,39]. In Thailand, the current number of known R. ornata populations
appears to be lower than the number of populations observed in herbarium records,
which are mostly from the last 30–60 years, possibly due to deforestation and climate
change. Plant materials used in this study were collected from the two largest known pop-
ulations. The first study population (>30 individuals) was in Mae Hong Son province in
northern Thailand, at an altitude of 590 m a.s.l. A second study population (ca. 30 indi-
viduals) was located in Sakon Nakhon province in northeastern Thailand, at an altitude
of ca. 300 m a.s.l. Interestingly, there was a striking morphological difference found in
plants between the two sites. Plants from the northern population displayed dark purple
petiolar nectaries, while petiolar nectaries of plants in the northeastern population were
green (Figure 1). Voucher specimens (northern population: NC and PT 45, 63; northeastern
population: NC and PT 60) were prepared following the standard method for plant taxon-
omy [44] and deposited at the Forest Herbarium in Bangkok, Thailand (BKF). Fresh ma-
terials (including fully opened flowers and mature leaves) kept at 4 °C were used for his-
tochemical examination, and materials fixed in 70% ethanol with a few drops of glycerol
were used for micromorphological and anatomical study.
Figure 1. Photos of Rivea ornata from the two study populations. (A) A Rivea ornata plant during the
flowering season. (B) A dark purple petiolar nectary at the Mae Hong Son study site surrounded by
Camponotus rufoglaucus ants foraging on the droplet of nectar observed in the center of the nectary.
(C) A green petiolar nectary at the Sakon Nakhon study site visited by Crematogaster sp. ants forag-
ing on nectar.
2.2. Micromorphology and Anatomy
Anatomical investigation by paraffin technique modified from Johansen [45] was car-
ried out to examine the nectary disc, petiolar nectaries, calycinal glands, and foliar glands,
Figure 1.
Photos of Rivea ornata from the two study populations. (
A
) A Rivea ornata plant during the
flowering season. (
B
) A dark purple petiolar nectary at the Mae Hong Son study site surrounded by
Camponotus rufoglaucus ants foraging on the droplet of nectar observed in the center of the nectary.
(
C
) A green petiolar nectary at the Sakon Nakhon study site visited by Crematogaster sp. ants foraging
on nectar.
2.2. Micromorphology and Anatomy
Anatomical investigation by paraffin technique modified from Johansen [
45
] was
carried out to examine the nectary disc, petiolar nectaries, calycinal glands, and foliar
glands, using three to six flowers (nectary disc and calycinal glands) or leaves (petiolar
nectaries and foliar glands) from different individuals from each population. Staminal hairs
were excluded from anatomical investigation as it was difficult to obtain clear sections due
Plants 2022,11, 2068 4 of 27
to the disorganized nature of the hairs. For the four external secretory structures that could
be sectioned, samples were dehydrated through a series of ethanol and butanol, embedded
in paraffin, and cut using a sliding microtome (Leica SM2000R, Nussloch, Germany). The
thin sections were affixed on glass slides with gelatin; deparaffinized by a series of xylene
and ethanol; stained with a mixture of alcian blue, basic fuchsin, and acriflavine (AFA);
and permanently mounted with DPX (distyrene, plasticizer, and xylene). The slides were
examined and photographed using an Olympus CX21 light microscope equipped with a
Sony α6400 digital camera.
To examine the surface of the secretory structures, peeled epidermal layers with AFA
staining were prepared to observe both sides of the leaf lamina (to examine foliar glands),
the petiolar nectaries, and the adaxial sepal surfaces (to examine calycinal glands). To
examine staminal hairs, we performed a clearing technique using a potassium hydroxide so-
lution and Clorox with toluidine blue O staining. These samples were temporally mounted
in water and observed using the same microscope setup as described in the previous para-
graph. Additionally, nectary discs were dehydrated using an acetone series, critical-point
dried (Hitachi HCP-2, Macquarie Park, NSW, Australia), coated with platinum–palladium
using an ion sputter (Hitachi E-102), and observed under a scanning electron microscope
(SEM) (Hitachi S-2500). External secretory structures were described using anatomical
terminology following Metcalfe and Chalk [18], Werker [46], and Evert [13].
2.3. Histochemistry
Thin sections of fresh materials were obtained from nectary discs, petiolar nectaries,
sepals for calycinal glands, and leaves for foliar glands using a sliding microtome (Leica
SM2000R), and staminal hairs were pulled out from the filaments. Five flowers and leaves
from each population were used for each test, with three to five repetitions per flower or per
leaf to confirm the results. The samples were treated with histochemical assays as follows:
Sudan black B [
47
] and neutral red [
48
] for total lipids, ferric chloride [
45
] and potassium
dichromate [
49
] for general phenolic compounds, Lugol’s iodine [
45
] for starch, periodic
acid–Schiffs’s reagent (PAS) [
50
,
51
] for neutral polysaccharides, ruthenium red [
45
] for
acidic polysaccharides, mercuric bromophenol blue [
52
] for proteins, Nadi reagent [
53
]
for terpenoids, Naturstoff reagent A [
48
] for flavonoids, and Dragendorff [
54
] and Wag-
ner’s [
55
] reagents for alkaloids. Neutral red and Naturstoff treatments were observed
using a fluorescence microscope (Olympus BX53 with a DP73 camera set, Waltham, MA,
USA) with 365 nm and 436 nm exciter filters, respectively, while the remaining treatments
were observed under a light microscope (Olympus CX21 equipped with a Sony
α
6400 digi-
tal camera, Tokyo, Japan). Additionally, UV autofluorescence of the tissues were observed
in each structure in general.
2.4. Preliminary Observation of Rivea ornata Visitors
Opportunistic observations were conducted to examine the overall species diversity of
animal visitors (i.e., we did not quantify visitation frequency). Only visitors that appeared
to interact with the examined secretory structures were recorded, and we categorized
their potential role based on their behavior with R. ornata. The categories were: herbivore
(consumes plant organs, such as leaves or corolla lobes), potential protector (consumes
exudates and may protect plant from herbivores), exudate consumer (consumes exudates
but likely does not offer protection from herbivores), and potential pollinator (visits flowers
and possibly pollinates them). An animal visitor could be classified under more than one
category, with the exception of the potential protector and exudate consumer categories,
which were mutually exclusive. The Mae Hong Son population was examined during
14–16 September 2019 and 15–19 September 2020, and the Sakon Nakhon population was
examined during 21–22 September 2019 and 7–11 September 2020. Direct observations
were performed between 600–2200 h to cover both diurnal and nocturnal visitors as much
as possible given field constraints, accounting for 18 h in 2019 and 35 h in 2020 for the Mae
Hong Son study site, and for 8 h in 2019 and 30 h in 2020 for the Sakon Nakhon study site.
Plants 2022,11, 2068 5 of 27
Animal visitors were photographed, and some were collected and anesthetized in ethyl
acetate vapor. Identification was made to the lowest taxonomic rank possible using various
identification guides, e.g., Ants of Thailand [
56
] and Thailand butterfly guide [
57
], as well
as consulting with entomologists (see Acknowledgements). Additionally, in 2020, action
cameras (Yi Lite, Xiaomi) were set up to continuously record crepuscular and nocturnal
visitor behavior between 600–1800 h, accounting for 60 and 48 h at the Mae Hong Son
and Sakon Nakhon study sites, respectively. Since the action cameras were not equipped
with night vision, plants were dimly illuminated with red light. Due to the relatively low
resolution of the video footage, animal visitors were classified to the level of order.
3. Results
3.1. Micromorphology and Anatomy
Sectional and epidermal examination revealed that the nectary disc and petiolar
nectaries function as complex working units formed by at least epidermal tissue and
ground tissue (and vascular tissue is also found in nectary discs), while the calycinal glands,
staminal hairs, and foliar glands are glandular trichomes formed from modified epidermal
tissue only (Figures 2–4).
The nectary disc is a rounded, pentagonal, bulging ring that is slightly concave planar
along the sides, embracing around a quarter of the height of the ovary from where the
nectary disc attaches to the receptacle. Scanning electron microscopy revealed that surfaces
are glabrous (Figure 2A–C). Permanently opened nectarostomata were found on the apical
and adaxial regions of the nectary disc (Figure 2B,C,G,H). Transversal and longitudinal
sections revealed that the nectary disc is composed of three regions (epidermis, nectarif-
erous parenchyma, and subnectariferous parenchyma) and is vascularized (Figure 2D–F).
The nectary epidermal cells are anticlinally arranged in a single-cell layer. The nectarif-
erous parenchyma consists of unorganized isodiametric cells with thin walls and dense
cytoplasmic content, located underneath the epidermis. Vascular bundles pass through the
middle region of the nectariferous parenchyma parallel to the longitudinal section outline,
connecting to the vascular system in the receptacle and ovary (Figure 2D,E). Two types of
subnectariferous parenchyma cells were found. The first type consists of elongated cells with
intensely stained cytoplasm that are smaller in diameter than the nectariferous parenchyma
cells and are arranged in parallel surrounding the vascular tissue (Figure 2D–F). The second
type consists of cells that, compared to the nectariferous parenchyma cells, are larger in
size, have looser cytoplasmic components, and are located in the area where the nectary
disc attaches to the receptacle (Figure 2E).
The petiolar nectaries, in transversal view, appeared as a working unit comprising
peltate trichomes on the surface and nectariferous tissue beneath (Figure 3A,D). The single-
cell layer of the petiolar nectary epidermis is different from the adjacent areas by having
anticlinal elongated epidermal cells. When viewed from the top, peltate trichomes are
distributed solitarily and evenly across the petiolar nectary epidermis (Figure 3B,C). The
trichomes are composed of three parts: an asymmetrical, radially divided, multicellular
head; a unicellular stalk; and a uni- to multicellular base (Figure 3C,D). Only the basal cells
penetrate into the nectariferous parenchyma; the head and stalk cells are level with the
epidermal cells, or slightly sunken in the epidermal layer (Figure 3D). The nectariferous
parenchyma cells of petiolar nectaries are polyhedral with dense cytoplasm (Figure 3A,D).
Subnectariferous parenchyma cells are larger in size and have less cytoplasmic content
than the nectariferous parenchyma cells. They span from the nectariferous parenchyma to
the cortex zone, with several areas connecting to vascular bundles (Figure 3A). In the fresh
sample sections, druse crystals were observed in nectariferous tissue cells but they vanished
during the process of making permanent slides, thus leaving only large parenchyma cells
lacking cytoplasmic content (Figure 3D).
Plants 2022,11, 2068 6 of 27
Plants 2022, 11, x FOR PEER REVIEW 6 of 28
Figure 2. Micromorphology and anatomy of the nectary disc of Rivea ornata. (A) Abaxial surface
without nectarostomata. (B) Adaxial surface showing the distribution of nectarostomata (high-
lighted in red). (C) Top view of the nectary disc showing nectarostomata (highlighted in red) located
on the apical region and down along the adaxial side (double asterisk) but absent from the abaxial
side (single asterisk), with trace amounts of exudate (arrowhead). (D) Transversal and (E) longitu-
dinal views showing the epidermis, nectariferous parenchyma, subnectariferous parenchyma, and
vascular tissues. (F) Close-up view of (D) from an area near the adaxial surface. (G) Surface image
of nectarostoma. (H) Transversal section of nectarostoma. Note: (A–C,G) were taken with a scan-
ning electron microscope, while (D–F,H) were taken with a light microscope. Abbreviations: e, epi-
dermal cell; gc, guard cell; np, nectariferous parenchyma; o, ovary; ov, ovule; p, parenchyma; ph,
phloem; sp, subnectariferous parenchyma; st, stoma; v, vascular tissue; xy, xylem. Scale bars: (A–C)
500 μm; (D,E) 1000 μm; (F) 250 μm; (G,H) 50 μm.
The petiolar nectaries, in transversal view, appeared as a working unit comprising
peltate trichomes on the surface and nectariferous tissue beneath (Figure 3A,D). The sin-
gle-cell layer of the petiolar nectary epidermis is different from the adjacent areas by hav-
ing anticlinal elongated epidermal cells. When viewed from the top, peltate trichomes are
distributed solitarily and evenly across the petiolar nectary epidermis (Figure 3B,C). The
trichomes are composed of three parts: an asymmetrical, radially divided, multicellular
Figure 2.
Micromorphology and anatomy of the nectary disc of Rivea ornata. (
A
) Abaxial surface
without nectarostomata. (
B
) Adaxial surface showing the distribution of nectarostomata (highlighted
in red). (
C
) Top view of the nectary disc showing nectarostomata (highlighted in red) located on
the apical region and down along the adaxial side (double asterisk) but absent from the abaxial side
(single asterisk), with trace amounts of exudate (arrowhead). (
D
) Transversal and (
E
) longitudinal
views showing the epidermis, nectariferous parenchyma, subnectariferous parenchyma, and vascular
tissues. (
F
) Close-up view of (
D
) from an area near the adaxial surface. (
G
) Surface image of
nectarostoma. (
H
) Transversal section of nectarostoma. Note: (
A
–
C
,
G
) were taken with a scanning
electron microscope, while (
D
–
F
,
H)
were taken with a light microscope. Abbreviations: e, epidermal
cell; gc, guard cell; np, nectariferous parenchyma; o, ovary; ov, ovule; p, parenchyma; ph, phloem;
sp, subnectariferous parenchyma; st, stoma; v, vascular tissue; xy, xylem. Scale bars: (
A
–
C
) 500
µ
m;
(D,E) 1000 µm; (F) 250 µm; (G,H) 50 µm.
Plants 2022,11, 2068 7 of 27
Plants 2022, 11, x FOR PEER REVIEW 7 of 28
head; a unicellular stalk; and a uni- to multicellular base (Figure 3C,D). Only the basal
cells penetrate into the nectariferous parenchyma; the head and stalk cells are level with
the epidermal cells, or slightly sunken in the epidermal layer (Figure 3D). The nectarifer-
ous parenchyma cells of petiolar nectaries are polyhedral with dense cytoplasm (Figure
3A,D). Subnectariferous parenchyma cells are larger in size and have less cytoplasmic
content than the nectariferous parenchyma cells. They span from the nectariferous paren-
chyma to the cortex zone, with several areas connecting to vascular bundles (Figure 3A).
In the fresh sample sections, druse crystals were observed in nectariferous tissue cells but
they vanished during the process of making permanent slides, thus leaving only large
parenchyma cells lacking cytoplasmic content (Figure 3D).
Staminal hairs are distributed densely at the base of each filament (where they attach
to the corolla), as well as along the adjacent areas between each filament base on the inner
side of the corolla (Figure 3E). Two parts of the staminal hairs are noticeably distinct, i.e.,
head and stalk, but basal cells could not be differentiated from the other epidermal cells
(Figure 3E,F). The head is unicellular and obovoid or pyriform. The stalk is cylindrical and
formed by rows of long cells (Figure 3E,F). Peltate glandular trichomes were also sparsely
detected among the hairs located on the inner corolla (Figure 3E).
Figure 3. Micromorphology and anatomy of petiolar nectaries and staminal hairs of Rivea ornata via
light microscopy. (A) Transversal view of petiole showing a petiolar nectary formed by epidermis,
Figure 3.
Micromorphology and anatomy of petiolar nectaries and staminal hairs of Rivea ornata via
light microscopy. (
A
) Transversal view of petiole showing a petiolar nectary formed by epidermis,
peltate glandular trichomes, nectariferous parenchyma, and subnectariferous parenchyma, as well as
regions of petiole vascular tissues and cortex. (
B
) Surface of a petiolar nectary with peltate glandular
trichomes distributed solitarily and evenly across the epidermis. (
C
) Close-up view of (
B
) showing
details of the multicellular head of the peltate trichomes. (
D
) Close-up view of (
A
) along the epidermis
showing details of the peltate trichomes, epidermal cells, nectariferous parenchyma, and a cell used to
store druse crystals (asterisk). (
E
) The base of a stamen where it connects to the corolla tube covered in
staminal hairs. (
F
) Details of staminal hairs showing apical glands and stalks. Abbreviations: b, base;
c, corolla; e, epidermal cell; f, filament; gt, glandular trichome; h, head; np, nectariferous parenchyma;
p, parenchyma; ph, phloem; s, stalk; sh, staminal hair; sp, subnectariferous parenchyma; v, vascular
tissue; xy, xylem. Scale bars: (A,E) 1000 µm; (B) 500 µm; (C,D) 100 µm; (F) 200 µm.
Plants 2022,11, 2068 8 of 27
Plants 2022, 11, x FOR PEER REVIEW 8 of 28
peltate glandular trichomes, nectariferous parenchyma, and subnectariferous parenchyma, as well
as regions of petiole vascular tissues and cortex. (B) Surface of a petiolar nectary with peltate glan-
dular trichomes distributed solitarily and evenly across the epidermis. (C) Close-up view of (B)
showing details of the multicellular head of the peltate trichomes. (D) Close-up view of (A) along
the epidermis showing details of the peltate trichomes, epidermal cells, nectariferous parenchyma,
and a cell used to store druse crystals (asterisk). (E) The base of a stamen where it connects to the
corolla tube covered in staminal hairs. (F) Details of staminal hairs showing apical glands and stalks.
Abbreviations: b, base; c, corolla; e, epidermal cell; f, filament; gt, glandular trichome; h, head; np,
nectariferous parenchyma; p, parenchyma; ph, phloem; s, stalk; sh, staminal hair; sp, subnectarifer-
ous parenchyma; v, vascular tissue; xy, xylem. Scale bars: (A,E) 1000 μm; (B) 500 μm; (C,D) 100 μm;
(F) 200 μm.
Calycinal glands are dispersed across the epidermis of the adaxial surface of sepals,
in the form of solitary peltate trichomes, or in clusters of up to 30, and nested in a shallow
pit on the surface (Figure 4A–C). The trichomes contain three parts: a radially divided,
eight-cell head; a unicellular stalk; and a one- to few-cell base (Figure 4B,C). Tissues under
the glands were found to be collenchyma with sparse cell content (Figure 4C).
Foliar glands were found only on the adaxial surface of leaf blades, scattered solitar-
ily and evenly on the epidermis among the stomata, while all trichomes on the opposite
side of the leaf surface were non-glandular (Figure 4D,E). Similar to the structure of calyc-
inal glands, the glandular trichomes of foliar glands are peltate (Figure 4E,F), containing
three parts: a radially divided, eight-cell head; a unicellular stalk; and a unicellular base
(Figure 4F,G). Transversal view showed that the peltate trichomes are slightly sunken, so
as to have the same height as the surrounding epidermal cells (Figure 4G). A part of the
basal cell extends into the palisade mesophyll (Figure 4G).
Figure 4. Micromorphology and anatomy of calycinal (A–C) and foliar (D–G) glands of Rivea ornata
via light microscopy. (A) Adaxial surface of a sepal with clusters of calycinal glands. (B) Close-up
view of (A) showing details of the multicellular head of the calycinal glands. (C) Transversal view
Figure 4.
Micromorphology and anatomy of calycinal (
A
–
C
) and foliar (
D
–
G
) glands of Rivea ornata
via light microscopy. (
A
) Adaxial surface of a sepal with clusters of calycinal glands. (
B
) Close-up view
of (
A
) showing details of the multicellular head of the calycinal glands. (
C
) Transversal view of the
adaxial side of a sepal showing a cluster of calycinal glands in the shallow pit with traces of exudate
(double asterisk). (
D
) Abaxial leaf surface possessing only non-glandular trichomes. (
E
) Adaxial leaf
surface possessing only glandular trichomes distributed solitarily on the epidermis. (
F
) Close-up
view of (
E
) showing details of the multicellular head of the glandular trichomes. (
G
) Transversal
view of a leaf showing details of the glandular trichomes. Abbreviations: b, base; cl, collenchyma; e,
epidermal cell; f, filament; gt, glandular trichome; h, head; nt, non-glandular trichome; pp, palisade
parenchyma; s, stalk; spp, spongy parenchyma; st, stoma. Scale bars: (
A
) 500
µ
m; (
B
,
F
) 100
µ
m;
(D,E) 200 µm; (G) 50 µm.
Staminal hairs are distributed densely at the base of each filament (where they attach
to the corolla), as well as along the adjacent areas between each filament base on the inner
side of the corolla (Figure 3E). Two parts of the staminal hairs are noticeably distinct, i.e.,
head and stalk, but basal cells could not be differentiated from the other epidermal cells
(Figure 3E,F). The head is unicellular and obovoid or pyriform. The stalk is cylindrical and
formed by rows of long cells (Figure 3E,F). Peltate glandular trichomes were also sparsely
detected among the hairs located on the inner corolla (Figure 3E).
Calycinal glands are dispersed across the epidermis of the adaxial surface of sepals, in
the form of solitary peltate trichomes, or in clusters of up to 30, and nested in a shallow
pit on the surface (Figure 4A–C). The trichomes contain three parts: a radially divided,
eight-cell head; a unicellular stalk; and a one- to few-cell base (Figure 4B,C). Tissues under
the glands were found to be collenchyma with sparse cell content (Figure 4C).
Foliar glands were found only on the adaxial surface of leaf blades, scattered solitarily
and evenly on the epidermis among the stomata, while all trichomes on the opposite side
of the leaf surface were non-glandular (Figure 4D,E). Similar to the structure of calycinal
Plants 2022,11, 2068 9 of 27
glands, the glandular trichomes of foliar glands are peltate (Figure 4E,F), containing three
parts: a radially divided, eight-cell head; a unicellular stalk; and a unicellular base
(Figure 4F,G). Transversal view showed that the peltate trichomes are slightly sunken, so as
to have the same height as the surrounding epidermal cells (Figure 4G). A part of the basal
cell extends into the palisade mesophyll (Figure 4G).
3.2. Histochemistry
The histochemical assays revealed that the secretory structures in R. ornata produce var-
ious groups of metabolites, as all examined compounds tested positive, with the exception
of proteins (Table 1and Figures 5–9). Lipids appear to be restricted to structural layers such
as cell walls, while phenolic compounds, terpenoids, flavonoids, and alkaloids are mainly
found in substances that accumulate in cytoplasmic components. Polysaccharides, in gen-
eral, occur in both cell walls and cell contents (Table 1and Figures 5–9). Additionally, starch
grains were found only in the nectary disc, and only sparsely (Table 1and Figure 5L,M), but
they were also detected in abundance in parenchyma cells of the receptacle (Figure 5L,M)
and in cells near petiolar nectaries (Figure 6H,I).
The nectary disc contained all classes of chemicals tested in this study except proteins.
It was the only structure that produced starch in its tissues. The histochemical results
from the nectary epidermis and the nectariferous tissue of the nectary disc were congruent
(Table 1and Figure 5). However, the positive results for most of the detected chemicals
were more pronounced in the nectariferous parenchyma near the adaxial epidermis (e.g.,
Figure 5F,N,P,T,V,X). Lipids appeared concentrated in the subnectariferous parenchyma
cells surrounding vascular bundles (Figure 5C,E). Blue-green autofluorescence under UV
wavelengths was also mainly found in cells in the abaxial regions (Figure 5BB,CC).
The unstained petiolar nectary sections revealed that the purple color observed in
the Mae Hong Son population was caused by the accumulation of anthocyanin in the
nectariferous parenchyma of the petiolar nectaries; anthocyanin was absent from the Sakon
Nakhon population, resulting in green petiolar nectaries due to chloroplasts (Figure 6A,B).
Histochemical results were not different between the two morphotypes. Substances with
positive histochemical results were principally stored in the head of the glandular trichome
and in the elongated epidermal cells of the petiolar nectaries, but they were sometimes
also found in the stalk and basal cells of the glandular trichomes, or even in nectariferous
cells (Table 1and Figure 6). Starch grains were not found in the glandular trichomes, but
accumulated in a strand of parenchyma cells located next to the outer edge of vascular
bundles in petiolar nectaries (Figure 6H,I); some of the starch grains were located in the
subnectariferous region, while others were found in the cortex, which was not a part of the
secretory working unit in petiolar nectaries. The cuticle layer on glandular trichomes and
epidermal cells exhibited blue autofluorescence under UV wavelengths (Figure 6Q).
Calycinal and foliar glands not only shared homologous structures but also presented
similar histochemical results (Table 1; Figures 7and 8). Positive reactions were mainly
found at the head and stalk cells of the trichomes. While lipids detected by Sudan black
B were generally found in both head and stalk regions, the results of the neutral red assay
revealed that lipids predominantly occurred in stalk cells (Figure 7B,C and Figure 8B,C). Ter-
penoids and flavonoids, as well as blue-green autofluorescence under UV wavelengths, were
more pronounced in the stalk cells rather than the head cells (Figure 7J,K,N and Figure 8J,K,N).
In addition to the vibrant blue-green autofluorescence, faint orangish-brown autofluores-
cence was observed for substances accumulated in the head of calycinal and foliar glands
(Figures 7N and 8N).
Plants 2022,11, 2068 10 of 27
Table 1. Metabolites identified in secretory structures of Rivea ornata using histochemical assays.
Metabolite
Group Test
Positive
Chromatic
Reaction
Nectary Disc
Petiolar Nectaries
Glandular Trichome Epidermis Nectariferous
Parenchyma
Head Stalk Base
Total lipids Sudan black B Dark blue
to black +, s +, s +, s - +, s -
Neutral red Yellow + +, s +, s - +, s -
General phenolic
compounds
Ferric chloride Brown or black +, c +, c - - +, c +, c
Potassium
dichromate Brown +, c +, c - - - -
Starch Lugol’s iodine Dark
blue to black +, c - - - - -
Neutral
polysaccharides
Periodic
acid–Schiff’s
reagent (PAS)
Pink +, c, s +, c, s +, s +, s +, c, s +, c, s
Acidic
polysaccharides Ruthenium red Pink to red +, c, s +, c, s +, s +, s +, c, s +, c, s
Proteins
Mercuric
bromophenol
blue
Blue - - - - - -
Terpenoids Nadi reagent Dark blue or
violet +, c +, c - - +, c +, c
Flavonoids Naturstoff
reagent A Yellow +, c +, s - - +, s -
Alkaloids
Dragendorff
reagent Reddish brown +, c +, c - - - -
Wagner’s reagent
Reddish brown +, c +, c - - +, c +, c
Metabolite
Group Test
Positive
Chromatic
Reaction
Calycinal Glands Foliar Glands Staminal
Hairs
Head Stalk Base Head Stalk Base Head Stalk
Total lipids Sudan black B Dark blue to
black +, s +, s - + + - + +
Neutral red Yellow - +, s - - +, s - + +
General phenolic
compounds
Ferric chloride Brown or black +, c +, c - +, c + - - +, c
Potassium
dichromate Brown +, c +, c - +, c - - - +, c
Starch Lugol’s iodine Dark
blue to black - - - - - - - -
Neutral
polysaccharides
Periodic
acid–Schiff’s
reagent (PAS)
Pink + + + +, c, s +, s +, s +, s +, s
Acidic
polysaccharides Ruthenium red Pink to red +, c, s +, c, s +, s +, s +, s +, s +, s +, s
Proteins
Mercuric
bromophenol
blue
Blue - - - - - - - -
Terpenoids Nadi reagent Dark blue
or violet + + +, c + + - +, c +, c
Flavonoids Naturstoff
reagent A Yellow +, c, s +, c, s - + + - +, c +
Alkaloids
Dragendorff
reagent Reddish brown +, c +, c - +, c +, s - +, c +
Wagner’s reagent
Reddish brown +, c +, c - +, c - - +, c +, c
Note: -, negative result; +, positive result; c, metabolite clearly detected in cytoplasmic components; s, metabolite
clearly detected in structural layers, such as cell walls and/or cell membranes; positive results (+) without c or s
labels indicate that the metabolite was detected, but it was unclear whether they were in cytoplasmic components
or structural layers.
Plants 2022,11, 2068 11 of 27
Plants 2022, 11, x FOR PEER REVIEW 10 of 28
results (+) without c or s labels indicate that the metabolite was detected, but it was unclear whether
they were in cytoplasmic components or structural layers.
The nectary disc contained all classes of chemicals tested in this study except pro-
teins. It was the only structure that produced starch in its tissues. The histochemical re-
sults from the nectary epidermis and the nectariferous tissue of the nectary disc were con-
gruent (Table 1 and Figure 5). However, the positive results for most of the detected chem-
icals were more pronounced in the nectariferous parenchyma near the adaxial epidermis
(e.g., Figure 5F,N,P,T,V,X). Lipids appeared concentrated in the subnectariferous paren-
chyma cells surrounding vascular bundles (Figure 5C,E). Blue-green autofluorescence un-
der UV wavelengths was also mainly found in cells in the abaxial regions (Figure 5BB,CC).
Figure 5. Histochemical results obtained from the nectary disc of Rivea ornata. (A) Transversal view
and (B) close-up view of an unstained nectary disc. (C–E) Sudan black B test for total lipids; note
Figure 5.
Histochemical results obtained from the nectary disc of Rivea ornata. (
A
) Transversal
view and (
B
) close-up view of an unstained nectary disc. (
C
–
E
) Sudan black B test for total lipids;
note cells in the subnectariferous parenchyma presenting intense positive results in accumulated
substances (arrowhead). (
F
,
G
) Neutral red fluorochrome test for total lipids. (
H
,
I
) Ferric chloride test
for phenolic compounds. (
J
,
K
) Potassium dichromate test for phenolic compounds. (
L
,
M
) Lugol’s
iodine test for starch; note the positive results also present in parenchyma tissue of the receptacle.
(
N
,
O
) Periodic acid–Schiff’s reagent (PAS) test for neutral polysaccharides. (
P
,
Q
) Ruthenium red
test for acidic polysaccharides. (
R
,
S
) Mercuric bromophenol blue test for proteins. (
T
,
U
) Nadi
reagent test for terpenoids. (
V
,
W
) Naturstoff reagent A under fluorescent microscopy detecting
flavonoids. (
X
,
Y
) Dragendorff reagent test for alkaloids. (
Z
,
AA
) Wagner ’s reagent test for alkaloids.
(
BB
,
CC
) UV autofluorescence of the nectary disc. Note: (
C
–
Q
,
T
–
AA
) show positive reactions, while
(
R
,
S)
show negative reactions. Abbreviations: e, epidermal cell; np, nectariferous parenchyma; o,
ovary; r, receptacle; sg; starch grain; sp, subnectariferous parenchyma; v, vascular tissue. Scale
bars: (A,C,F,H,J,L,N,P,R,T,V,X,Z,BB) 500 µm; (B,D,E,G,I,K,M,O,Q,S,U,W,Y,AA,CC) 50 µm.
Plants 2022,11, 2068 12 of 27
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Figure 6. Histochemical results obtained from petiolar nectaries of Rivea ornata. (A) Unstained peti-
olar nectary from the Mae Hong Son study site showing anthocyanins in the nectariferous paren-
chyma. (B) Unstained petiolar nectary from the Sakon Nakhon study site without anthocyanins in
the nectariferous parenchyma. (C) Sudan black B test for total lipids. (D) Neutral red fluorochrome
test for total lipids. (E) Ferric chloride test for phenolic compounds. (F) Potassium dichromate test
for phenolic compounds. (G–I) Lugol’s iodine test for starch; note that positive results are only pre-
sent in a strand of parenchyma cells surrounding the vascular bundle. (J) Periodic acid–Schiff’s re-
agent (PAS) test for neutral polysaccharides. (K) Ruthenium red test for acidic polysaccharides. (L)
Mercuric bromophenol blue test for proteins. (M) Nadi reagent test for terpenoids. (N) Naturstoff
reagent A under fluorescent microscopy detecting flavonoids. (O) Dragendorff reagent test for al-
kaloids. (P) Wagner’s reagent test for alkaloids. (Q) UV autofluorescence of petiolar nectaries. Note:
(C–F,H–K,M–P) show positive reactions, while (G,L) show negative reactions. Abbreviations: b,
base; d, druse crystal; e, epidermal cell; h, head; s, stalk; sg, starch grain; v, vascular tissue. Scale
bars: (A–H,J–Q) 50 μm; (I) 500 μm.
Figure 6.
Histochemical results obtained from petiolar nectaries of Rivea ornata. (
A
) Unstained
petiolar nectary from the Mae Hong Son study site showing anthocyanins in the nectariferous
parenchyma. (
B
) Unstained petiolar nectary from the Sakon Nakhon study site without anthocyanins
in the nectariferous parenchyma. (
C
) Sudan black B test for total lipids. (
D
) Neutral red fluorochrome
test for total lipids. (
E
) Ferric chloride test for phenolic compounds. (
F
) Potassium dichromate test
for phenolic compounds. (
G
–
I
) Lugol’s iodine test for starch; note that positive results are only
present in a strand of parenchyma cells surrounding the vascular bundle. (
J
) Periodic acid–Schiff’s
reagent (PAS) test for neutral polysaccharides. (
K
) Ruthenium red test for acidic polysaccharides.
(
L
) Mercuric bromophenol blue test for proteins. (
M
) Nadi reagent test for terpenoids. (
N
) Naturstoff
reagent A under fluorescent microscopy detecting flavonoids. (
O
) Dragendorff reagent test for
alkaloids. (
P
) Wagner ’s reagent test for alkaloids. (
Q
) UV autofluorescence of petiolar nectaries.
Note: (
C
–
F
,
H
–
K
,
M
–
P
) show positive reactions, while (
G
,
L
) show negative reactions. Abbreviations:
b, base; d, druse crystal; e, epidermal cell; h, head; s, stalk; sg, starch grain; v, vascular tissue. Scale
bars: (A–H,J–Q) 50 µm; (I) 500 µm.
Plants 2022,11, 2068 13 of 27
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Figure 7. Histochemical results obtained from calycinal glands of Rivea ornata. (A) Unstained calyc-
inal glands. (B) Sudan black B test for total lipids. (C) Neutral red fluorochrome test for total lipids.
(D) Ferric chloride test for phenolic compounds. (E) Potassium dichromate test for phenolic com-
pounds. (F) Lugol’s iodine test for starch. (G) Periodic acid–Schiff’s reagent (PAS) test for neutral
polysaccharides. (H) Ruthenium red test for acidic polysaccharides. (I) Mercuric bromophenol blue
test for proteins. (J) Nadi reagent test for terpenoids. (K) Naturstoff reagent A under fluorescent
microscopy detecting flavonoids. (L) Dragendorff reagent test for alkaloids. (M) Wagner’s reagent
test for alkaloids. (N) UV autofluorescence of calycinal glands. Note: (B–E,G,H,J–M) show positive
reactions, while (F,I) show negative reactions. Abbreviations: b, base; h, head; s, stalk. Scale bars 20
μm.
In the staminal hairs, positive histochemical results were found in both the head and
stalk regions, but they generally showed different degrees of chromatic reaction (Table 1
and Figure 9). Only phenolic compounds were absent from the head of the hairs (Table 1
and Figure 9D,E). For lipids, while Sudan black B gave relatively indistinct positive reac-
tions in both the head and stalk regions, neutral red fluorochrome results were noticeably
evident at the head (Figure 9B,C). For neutral polysaccharides, the head stained magenta
but the stalk stained red (Figure 9G). A similar result was observed for terpenoids, in
which substances stored in the head turned violet or blue, but the wall of the head and
the stalk were solely dark blue (Figure 9J). The test for acidic polysaccharides generally
showed patterns of positive staining in the head, but some heads were completely stained,
some were stained only at the area attaching to the stalk, and some were entirely un-
stained (Figure 9H). Both parts of the staminal hairs emitted dim blue autofluorescence
under UV wavelengths (Figure 9N).
Figure 7.
Histochemical results obtained from calycinal glands of Rivea ornata. (
A
) Unstained
calycinal glands. (
B
) Sudan black B test for total lipids. (
C
) Neutral red fluorochrome test for total
lipids. (
D
) Ferric chloride test for phenolic compounds. (
E
) Potassium dichromate test for phenolic
compounds. (
F
) Lugol’s iodine test for starch. (
G
) Periodic acid–Schiff’s reagent (PAS) test for neutral
polysaccharides. (
H
) Ruthenium red test for acidic polysaccharides. (
I
) Mercuric bromophenol blue
test for proteins. (
J
) Nadi reagent test for terpenoids. (
K
) Naturstoff reagent A under fluorescent
microscopy detecting flavonoids. (
L
) Dragendorff reagent test for alkaloids. (
M
) Wagner ’s reagent
test for alkaloids. (
N
) UV autofluorescence of calycinal glands. Note: (
B
–
E
,
G
,
H
,
J
–
M
) show positive
reactions, while (
F
,
I
) show negative reactions. Abbreviations: b, base; h, head; s, stalk. Scale bars
20 µm.
In the staminal hairs, positive histochemical results were found in both the head
and stalk regions, but they generally showed different degrees of chromatic reaction
(Table 1and Figure 9). Only phenolic compounds were absent from the head of the
hairs (Table 1and Figure 9D,E). For lipids, while Sudan black B gave relatively indistinct
positive reactions in both the head and stalk regions, neutral red fluorochrome results were
noticeably evident at the head (Figure 9B,C). For neutral polysaccharides, the head stained
magenta but the stalk stained red (Figure 9G). A similar result was observed for terpenoids,
in which substances stored in the head turned violet or blue, but the wall of the head and
the stalk were solely dark blue (Figure 9J). The test for acidic polysaccharides generally
showed patterns of positive staining in the head, but some heads were completely stained,
some were stained only at the area attaching to the stalk, and some were entirely unstained
(Figure 9H). Both parts of the staminal hairs emitted dim blue autofluorescence under UV
wavelengths (Figure 9N).
Plants 2022,11, 2068 14 of 27
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Figure 8. Histochemical results obtained from foliar glands of Rivea ornata. (A) Unstained foliar
gland. (B) Sudan black B test for total lipids. (C) Neutral red fluorochrome test for total lipids. (D)
Ferric chloride test for phenolic compounds. (E) Potassium dichromate test for phenolic compounds.
(F) Lugol’s iodine test for starch. (G) Periodic acid–Schiff’s reagent (PAS) test for neutral polysac-
charides. (H) Ruthenium red test for acidic polysaccharides. (I) Mercuric bromophenol blue test for
proteins. (J) Nadi reagent test for terpenoids. (K) Naturstoff reagent A under fluorescent microscopy
detecting flavonoids. (L) Dragendorff reagent test for alkaloids. (M) Wagner’s reagent test for alka-
loids. (N) UV autofluorescence of foliar gland. Note: (B–E,G,H,J–M) show positive reactions, while
(F,I) show negative reactions. Abbreviations: ch, chloroplast; b, base; e, epidermal cell; h, head; s,
stalk. Scale bars 20 μm.
Figure 8.
Histochemical results obtained from foliar glands of Rivea ornata. (
A
) Unstained foliar gland.
(
B
) Sudan black B test for total lipids. (
C
) Neutral red fluorochrome test for total lipids. (
D
) Ferric
chloride test for phenolic compounds. (
E
) Potassium dichromate test for phenolic compounds. (
F
) Lu-
gol’s iodine test for starch. (
G
) Periodic acid–Schiff’s reagent (PAS) test for neutral polysaccharides.
(
H
) Ruthenium red test for acidic polysaccharides. (
I
) Mercuric bromophenol blue test for proteins.
(
J
) Nadi reagent test for terpenoids. (
K
) Naturstoff reagent A under fluorescent microscopy detecting
flavonoids. (
L
) Dragendorff reagent test for alkaloids. (
M
) Wagner ’s reagent test for alkaloids. (
N
) UV
autofluorescence of foliar gland. Note: (
B
–
E
,
G
,
H
,
J
–
M
) show positive reactions, while (
F
,
I
) show
negative reactions. Abbreviations: ch, chloroplast; b, base; e, epidermal cell; h, head; s, stalk. Scale
bars 20 µm.
Plants 2022,11, 2068 15 of 27
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Figure 9. Histochemical results obtained from staminal hairs of Rivea ornata. (A) Unstained staminal
hairs. (B) Sudan black B test for total lipids. (C) Neutral red fluorochrome test for total lipids. (D)
Ferric chloride test for phenolic compounds. (E) Potassium dichromate test for phenolic compounds.
(F) Lugol’s iodine test for starch. (G) Periodic acid–Schiff’s reagent (PAS) test for neutral polysac-
charides. (H) Ruthenium red test for acidic polysaccharides. (I) Mercuric bromophenol blue test for
proteins. (J) Nadi reagent test for terpenoids. (K) Naturstoff reagent A under fluorescent microscopy
detecting flavonoids. (L) Dragendorff reagent test for alkaloids. (M) Wagner’s reagent test for alka-
loids. (N) UV autofluorescence of staminal hairs. Note: (B–E,G,H,J–M) show positive reactions,
while (F,I) show negative reactions. Abbreviations: h, head; p, pollen grain; s, stalk. Scale bars 200
μm.
3.3. Preliminary Observations of Rivea ornata Visitors
Thirty-five taxa of insects and one Helicinan snail interacted with at least one of the
four parts of the plant related to the studied secretory structures, i.e., calyx (calycinal
glands), petioles (petiolar nectaries), flowers (nectary disc and staminal hairs), and leaf
blades (foliar glands) (Figures 1B,C, 10 and 11). Among these visitors, 63% of all visitor
diversity (22 taxa) were observed only at the Mae Hong Son study site, while 23% (8 taxa)
were observed only at the Sakon Nakhon study site, and 14% (5 taxa) were found at both
sites.
Figure 9.
Histochemical results obtained from staminal hairs of Rivea ornata. (
A
) Unstained staminal
hairs. (
B
) Sudan black B test for total lipids. (
C
) Neutral red fluorochrome test for total lipids.
(
D
) Ferric chloride test for phenolic compounds. (
E
) Potassium dichromate test for phenolic com-
pounds. (
F
) Lugol’s iodine test for starch. (
G
) Periodic acid–Schiff’s reagent (PAS) test for neutral
polysaccharides. (
H
) Ruthenium red test for acidic polysaccharides. (
I
) Mercuric bromophenol blue
test for proteins. (
J
) Nadi reagent test for terpenoids. (
K
) Naturstoff reagent A under fluorescent
microscopy detecting flavonoids. (
L
) Dragendorff reagent test for alkaloids. (
M
) Wagner ’s reagent
test for alkaloids. (
N
) UV autofluorescence of staminal hairs. Note: (
B
–
E
,
G
,
H
,
J
–
M
) show positive
reactions, while (
F
,
I
) show negative reactions. Abbreviations: h, head; p, pollen grain; s, stalk. Scale
bars 200 µm.
3.3. Preliminary Observations of Rivea ornata Visitors
Thirty-five taxa of insects and one Helicinan snail interacted with at least one of the
four parts of the plant related to the studied secretory structures, i.e., calyx (calycinal
glands), petioles (petiolar nectaries), flowers (nectary disc and staminal hairs), and leaf
blades (foliar glands) (Figure 1B,C, Figures 10 and 11). Among these visitors, 63% of all
visitor diversity (22 taxa) were observed only at the Mae Hong Son study site, while 23%
(8 taxa) were observed only at the Sakon Nakhon study site, and 14% (5 taxa) were found
at both sites.
The majority of the visitors were hymenopterans, accounting for 57% of visitor
species diversity. Within this order, we observed numerous ant species (Formicidae)
(Figure 1B,C, Figures 10 and 11A–C,G,H) and one paper wasp species (Ropalidia sp.,
Vespidae) (Figures 10 and 11C), all of which visited either the petiolar nectaries alone or
visited both the petiolar nectaries and calyxes. The wasps were active during the day and
roamed in groups of up to approximately five individuals per plant. Ants were observed
interacting with R. ornata at all times of day and night, patrolling mainly around the stems,
Plants 2022,11, 2068 16 of 27
petiolar nectaries, and calyxes (of both flower buds and mature, open flowers), where
they made routes from the ground to find food, but they were not often observed on leaf
blades. They were more often observed on young and newly mature leaves than on aged
leaves. In general, ants occupying the same area of the plant (e.g., a branch containing
inflorescences and leaves), or the entire plant, belonged to the same species. We observed
one instance of territory protection, where Oecophylla smaragdina ants were aggressive
towards individuals of Camponotus sp. While ants were generally not aggressive towards
insects from other families (e.g., cockroaches and butterflies) found on areas of the plant
that were not patrolled by ants (Figure 11H), multiple ant species were observed to act
aggressively towards cockroaches on calyxes and nearby areas (e.g., the corolla tube surface
next to the sepal apexes). Such aggression was observed during both day and night, with
the ants attacking and chasing any cockroaches encountered, causing the cockroaches to
move to other, ant-free, areas. Herbivores on plant organs beyond the ant-patrolling areas
(e.g., flowers and leaf blades) were not observed to be chased or harmed by the ants.
Eight taxa of lepidopterans, accounting for 23% of total visitor species, were spotted
as visitors either at flowers, calyxes, or leaves (Figures 10 and 11D–G). Skippers (Gangara
thyrsis, Hesperiidae) visited flowers at dusk, as soon as the flowers began to bloom, and
sphinx moths (Sphingidae) visited flowers after it was dark (Figures 10 and 11D,E). All
butterfly and moth species inserted their proboscises through the long corolla tube (and
in doing so, through the clump of staminal hairs), to access nectar stored in the nectar
chamber. In doing so, their proboscises were certain to contact anthers and stigmas,
which were closely bundled together due to the narrow diameter of the corolla tube. We,
therefore, classified skippers and sphinx moths as potential pollinators because pollen
grains were observed on the proboscises of individuals visiting R. ornata flowers, which
were confirmed to be the pollen of R. ornata by examining captured specimens. Three
butterfly species (Lycaenidae and Nymphalidae) were also observed visiting calyxes in the
early morning, and they used their proboscises to forage on sepal exudate and leachate, so
we classified them as exudate consumers (Figures 10 and 11F). The larval stage of a species
of Homodes moth (Erebidae) was observed consuming the leaves (and, therefore, classified
as an herbivore), and they were associated with and protected by Oecophylla smaragdina
(Formicidae) (Figures 10 and 11G).
Three taxa of cockroaches (Ectobiidae, Blattodea), accounting for 9% of all visitor
species, were mainly observed as exudate consumers at calyxes (Figures 10 and 11H,I).
They were active beginning in the evening, throughout the night, and during the early
morning hours before sunlight directly illuminated the plants. Moreover, they were also
found to visit the corolla throat and contact stigmas and anthers, possibly to consume
stigma fluid or pollen. We, therefore, also classified them as potential pollinators, since
pollen grains were observed on their antennae, head, legs, and dorsal areas of the thorax
and abdomen.
A Helicinan snail (Stylommatophora, Helicoidea), kattydids (Orthoptera, Tettigoni-
idae), and tortoise beetles (Coleoptera, Chrysomelidae), together accounting for 11% of
the visitor species diversity, were classified as exudate consumers, floral herbivores, and
foliar herbivores, respectively (Figures 10 and 11J–L). The snail visited petiolar nectaries
where ants were not present (Figures 10 and 11J). The katydids were active from night until
the following morning; they only came during nights when the plants were in bloom, and
they were observed consuming the corolla lobes of flowers, so that just the corolla tube and
midpetaline bands remained (Figures 10 and 11K). Tortoise beetles were frequently spotted
consuming the leaf blades of young and newly mature leaves (Figures 10 and 11L). We did
not observe ants acting aggressively towards katydids and tortoise beetles.
Plants 2022,11, 2068 17 of 27
Plants 2022, 11, x FOR PEER REVIEW 16 of 28
Figure 10. Diagram of animal visitors observed visiting plant parts where the studied external se-
cretory structures are found (nectary disc and staminal hairs at flowers, calycinal glands at calyxes,
petiolar nectaries at petioles, and foliar glands at leaves). Visitors were classified as: herbivore (con-
sumes plant organs, such as leaves or flowers), potential protector (consumes exudates and may
protect plant from herbivores), exudate consumer (consumes exudates but likely does not offer pro-
tection from herbivores), or potential pollinator (visits flowers and possibly pollinates them). Most
visitors fell under a single category, with the exception of cockroaches (Blattodea), which were clas-
sified as both exudate consumers and potential pollinators. Note: Dashed lines connecting visitor
names to plant parts only indicate that visits were observed, they do not indicate visitation fre-
quency.
The majority of the visitors were hymenopterans, accounting for 57% of visitor spe-
cies diversity. Within this order, we observed numerous ant species (Formicidae) (Figures
1B,C, 10 and 11A–C,G,H) and one paper wasp species (Ropalidia sp., Vespidae) (Figures
10 and 11C), all of which visited either the petiolar nectaries alone or visited both the pet-
iolar nectaries and calyxes. The wasps were active during the day and roamed in groups
of up to approximately five individuals per plant. Ants were observed interacting with R.
ornata at all times of day and night, patrolling mainly around the stems, petiolar nectaries,
and calyxes (of both flower buds and mature, open flowers), where they made routes from
Figure 10.
Diagram of animal visitors observed visiting plant parts where the studied external
secretory structures are found (nectary disc and staminal hairs at flowers, calycinal glands at calyxes,
petiolar nectaries at petioles, and foliar glands at leaves). Visitors were classified as: herbivore
(consumes plant organs, such as leaves or flowers), potential protector (consumes exudates and
may protect plant from herbivores), exudate consumer (consumes exudates but likely does not offer
protection from herbivores), or potential pollinator (visits flowers and possibly pollinates them).
Most visitors fell under a single category, with the exception of cockroaches (Blattodea), which were
classified as both exudate consumers and potential pollinators. Note: Dashed lines connecting visitor
names to plant parts only indicate that visits were observed, they do not indicate visitation frequency.
Plants 2022,11, 2068 18 of 27
Plants 2022, 11, x FOR PEER REVIEW 18 of 28
Figure 11. Animal visitors of Rivea ornata (hv: herbivore, pr: potential protector, ec: exudate con-
sumer, po: potential pollinator). (A) Polyrhachis proxima (pr) on petiolar nectaries. (B) Oecophylla am-
aragdina (pr) on calyx and petiolar nectaries. (C) Ropalidia sp. (ec; blue arrowhead) and Crematogaster
sp. (pr; red arrowhead) visiting calyces. (D) Gangara thyrsis (po) visiting a flower. (E) Agrius convol-
vuli (po) visiting a flower. (F) Rapala dieneces dieneces (ec) visiting a calyx. (G) Caterpillar of Homodes
sp. (hv; red arrowhead) on a branch, undisturbed by Oecophylla amaragdina (pr; blue arrowhead).
(H) Balta sp. (ec; yellow arrowhead), Lobopterella dimidiatipes (ec; blue arrowhead), and Camponotus
sp. (pr; red arrowhead) on calyces. (I) Balta sp. (po) visiting a flower; note the pollen grains on its
antennae. (J) Unidentified Helicinan snail (ec) on a petiolar nectary. (K) Unidentified katydid (hv)
consuming the corolla of a flower. (L) Aspidimorpha sp. (hv) on a leaf.
4. Discussion
Our study revealed that diverse secretory organs can appear very similar micromor-
phologically and anatomically, such as calycinal and foliar glands, or have a completely
distinct structure, such as the nectary disc. The richness of chemicals produced by these
secretory structures, as evidenced by the histochemical tests performed, provide insight
into their ecological roles, especially when assessed in combination with the visitor obser-
vation results. From our micromorphological, anatomical, histochemical, and ecological
results, we propose two hypotheses, with support from the previous related literature,
Figure 11.
Animal visitors of Rivea ornata (hv: herbivore, pr: potential protector, ec: exudate consumer,
po: potential pollinator). (
A
)Polyrhachis proxima (pr) on petiolar nectaries. (
B
)Oecophylla amaragdina
(pr) on calyx and petiolar nectaries. (
C
)Ropalidia sp. (ec; blue arrowhead) and Crematogaster sp. (pr;
red arrowhead) visiting calyces. (
D
)Gangara thyrsis (po) visiting a flower. (
E
)Agrius convolvuli (po)
visiting a flower. (
F
)Rapala dieneces dieneces (ec) visiting a calyx. (
G
) Caterpillar of Homodes sp. (hv;
red arrowhead) on a branch, undisturbed by Oecophylla amaragdina (pr; blue arrowhead). (
H
)Balta
sp. (ec; yellow arrowhead), Lobopterella dimidiatipes (ec; blue arrowhead), and Camponotus sp. (pr;
red arrowhead) on calyces. (I)Balta sp. (po) visiting a flower; note the pollen grains on its antennae.
(
J
) Unidentified Helicinan snail (ec) on a petiolar nectary. (
K
) Unidentified katydid (hv) consuming
the corolla of a flower. (L)Aspidimorpha sp. (hv) on a leaf.
4. Discussion
Our study revealed that diverse secretory organs can appear very similar micromor-
phologically and anatomically, such as calycinal and foliar glands, or have a completely
distinct structure, such as the nectary disc. The richness of chemicals produced by these se-
cretory structures, as evidenced by the histochemical tests performed, provide insight into
their ecological roles, especially when assessed in combination with the visitor observation
results. From our micromorphological, anatomical, histochemical, and ecological results,
Plants 2022,11, 2068 19 of 27
we propose two hypotheses, with support from the previous related literature, about the
function of secretory organs in Rivea ornata: (I) the nectary disc and staminal hairs, which
are floral secretory organs, are important in pollinator attraction [
29
,
30
], and (II) petiolar
nectaries, calycinal glands, and foliar glands are associated with defense mechanisms.
Specifically, petiolar nectaries and calycinal glands appear to provide indirect defense
against herbivores by attracting guard ants [
33
–
37
], and the calycinal glands may also
protect flowers and fruit from abiotic stressors [
26
,
58
]. Foliar glands appear to be related to
a wide array of defensive mechanisms, e.g., reducing herbivory through secretion of plant
metabolites or by harboring beneficial microbes, as well as providing protection against
abiotic stress in hot and dry environments [19,25,36,59–64].
4.1. Floral Secretory Structures Potentially Important to Pollination
The micromorphological and anatomical features of the nectary disc found in R. ornata
were generally similar to those of other species in the family, as described in previous
studies, both in terms of nectary tissue composition and vascularization [
29
,
30
,
65
,
66
]. The
secretion of nectar through modified stomata (i.e., nectarostomata) is common among flow-
ering plants across diverse, taxonomically distant species [
67
]. Galetto and Bernardello [
30
]
reported three species-specific distribution patterns for nectarostomata on the nectary disc
epidermis in six species of Ipomoea L., i.e., homogeneously distributed over the surface,
restricted to only the apex and base, and restricted only to the apical area. The latter
distribution pattern was found in a related morning glory species, Argyreia siamensis (Craib)
Staples [
29
]. Nectarostomata in R. ornata, however, presented a different pattern, appearing
on the apical region and down along the adaxial surface of the epidermis, but absent from
the abaxial surface. Nectarostomata are the openings through which nectar are secreted [
68
],
and the function of the nectary disc in producing nectar as a reward for pollinators is widely
acknowledged [30,69,70].
Several groups of metabolites detected in the tissues and accumulated substances of
the nectary disc in R. ornata have also been found in the floral nectaries of other plant taxa,
such as from Anacardiaceae, Solanaceae, Zeyheria Mart. (Bignoniaceae), and Pedicularis
L. (Orobanchaceae) [
71
–
74
], and these studies often reported a lack of proteins in their
nectariferous tissue as well. Starch grains present in the nectary disc and receptacle of R.
ornata are likely the substrate that is subsequently hydrolyzed into sugars during the process
of nectar production [
75
,
76
]. Polysaccharides and lipids stored in the cells could serve as
sources of essential nectar nutrients for pollinators [
67
], and positive histochemical reactions
for these two chemical classes also indicate their presence in the cell wall or cuticle layer, as
they are primary components of these structures [
77
,
78
]. Moreover, the positive detection
for lipids in cells within in the subnectariferous parenchyma of the nectary disc could
indicate the presence of laticifers [
36
], however, they could not be identified with certainty
using our anatomical technique. Tissues that appeared to be bundles of subnectariferous
parenchyma and vascular tissue in R. ornata were also found and defined as secretory ducts
in the nectary disc of Argyreia siamensis [
29
]. Alkaloids and phenolic compounds present
in the nectary disc might serve to discourage visitors or herbivores that are susceptible
to such chemicals [
79
–
81
]. Flavonoids are typically thought to be responsible for floral
coloration to attract pollinators; in the case of R. ornata, since the nectary disc is hidden
from pollinator view due to the narrow corolla tube, these chemicals may serve other
functions, such as stress detoxification or defense against pathogens [
82
,
83
]. Terpenoids
are crucial scent compounds that play prevalent roles in pollinator attraction [
84
] and in
defense against herbivores [
83
]. The presence of terpenoids in the nectary disc tissues
suggests that they may infuse into the nectar as well [
85
], however, the nectary disc is not
the only organ producing scent in R. ornata flowers, since the staminal hairs also presented
a strong positive histochemical reaction for terpenoids. Indeed, almost all of the metabolites
found in the nectary disc were also found in the staminal hairs, which, given the fact that
both occur in R. ornata flowers, suggests that these two organs function similarly and
work synergistically.
Plants 2022,11, 2068 20 of 27
Flowers of R. ornata closely match the typical sphingophilous pollination syndrome by
having white, fragrant corollas with a long, narrow corolla tube and nocturnal anthesis [
4
].
As expected, sphingid moths visited R. ornata individuals with flowers and showed high
potential to contribute to pollination, given that R. ornata pollen was found on their bodies.
Pollinator observations made at Ipomoea alba L. flowers, a species with convergently evolved
moth flowers similar to those of R. ornata, revealed that sphingid moths were the only
pollinators of this species [
30
,
31
]. At night, white flowers exhibit the greatest contrast to
dark backgrounds, and are perceived by moths via photoreceptors in their large, sensitive
eyes [
86
], and, simultaneously, floral scents are detected by their antennae and help guide
them to the flower [
87
,
88
]. Among the floral scent components known to attract moths,
terpenoids, especially acyclic terpene alcohols such as linalool, nerolidol, and farnesol, are
frequently among the most common [
89
–
91
]. Therefore, it is highly likely that the scent of
R. ornata flowers stems from terpenoids produced in the nectary disc and staminal hairs.
In addition to sphingid moths, we also observed two other insect groups visiting and
potentially pollinating R. ornata flowers: skipper butterflies (Hesperiidae) and cockroaches
(Blattodea). Although skipper butterflies are active during the day [
92
], they were able
to visit R. ornata flowers during approximately the first hour of anthesis (1800–1900 h),
before sunset. Pollination by cockroaches is relatively rare, with only 11 flowering plant
species confirmed to date to have cockroaches as effective pollinators [
4
,
93
,
94
]. They
do not have the long appendages necessary to forage on the nectar of R. ornata, given
the long, narrow corolla tube, but the behaviors observed in this study correspond to
previous reports that cockroaches feed on pollen and stigmatic exudates, and, thus, may
help pollinate flowers [
4
,
93
]. We also observed pollen on the bodies of skipper butterflies
and cockroaches, so further research is necessary to compare the contributions of sphingid
moths, skipper butterflies, and cockroaches and to determine which groups are effective
pollinators of R. ornata.
4.2. Secretory Structures Potentially Important to Defense
Petiolar nectaries are the only extrafloral nectary found in the genus Rivea, while
several species of the sister genus Ipomoea exhibit either petiolar nectaries, receptacular
nectaries, or both [
33
–
38
], and another genus in the family, Cuscuta L., is unique for nectaries
located along the stem [
95
,
96
]. Based on the classification of petiolar nectaries studied in
Ipomoea by Keeler and Kaul [
35
], the petiolar nectaries of R. ornata fits the description of a
superficial nectary (secretory tissues located on the surface exposed to the environment),
rather than one of the other two types, i.e., a crypt nectary (secretory tissues located deep
within a cavity, with nectar transferred through a duct and released to the surface by a pore),
or a basin nectary (secretory tissues located in slightly recessed depressions). Superficial
nectaries are rare among Ipomoea, with the only other occurrence reported in Ipomoea
leptophylla Torr. [
42
]. This type of nectary, however, also seems to occur in Decalobanthus
peltatus (L.) A.R. Simões & Staples because its nectary appearance and tissue arrangement
is similar to our findings in R. ornata [
97
]. The petiolar nectaries of R. ornata and other
species in the family have similar peltate trichomes on the petiolar nectary epidermis and
potentially secrete nectar through these trichomes as the main pathway, while petiolar
nectaries in other plant lineages have been reported to secrete nectar through either a
modified epidermal layer (e.g., Sapium biglandulosum Müll. Arg., Euphorbiaceae; Smilax
polyantha Griseb., Smilacaceae; and Passiflora spp., Passifloraceae) or through various kinds
of glandular trichomes (e.g., Hibiscus forsteri F. D. Wilson and Eriotheca gracilipes (K. Schum.)
A. Robyns in the Malvaceae family) [36,43–102].
Our work revealed that tissues involved with the petiolar nectaries of R. ornata pro-
duced diverse groups of metabolites, similar to those found in the nectary disc, and they
probably function in similar ways as discussed for the nectary disc. In contrast, Mar-
tins et al. [
36
] reported histochemical tests of the crypt petiolar nectaries in Ipomoea asarifolia
Roem. & Schult. and found positive reactions only for polysaccharides. The presence of
chemicals involved in defense (such as alkaloids, phenolic compounds, flavonoids, and
Plants 2022,11, 2068 21 of 27
terpenoids) in R. ornata petiolar nectaries might be due to their exposed nature, which poses
a higher risk of getting damaged from pathogens, desiccation, and UV radiation, therefore
rendering protective chemicals essential, as opposed to I. asarifolia petiolar nectaries, which
are hidden [
36
,
43
]. Even though the boundary of the petiolar nectaries defined in this
study was limited to the secreting tissues near the surface, localizations of starch grains
near these secreting tissues (presumably as a precursor of nectar sugars) imply that the
areas where nectar production occurs are not limited to just the secreting tissues [
75
,
76
].
The petiolar nectaries of the northeast population lacked anthocyanins, resulting in green
petiolar nectaries, which possibly have a genetic basis, such as decreased expression of
genes related to anthocyanin biosynthesis [
103
,
104
]. Anthocyanins belong to a class of
flavonoids that are widely known to be involved in plant response to both biotic and abiotic
stressors [
105
,
106
]. Thus, further work is needed to assess how the lack of this protective
substance in some populations of R. ornata affects plant fitness.
The calycinal glands found in R. ornata were morphologically identical to the calycinal
glands of other species in the family [
26
,
58
]. Stictocardia tiliifolia (Desr.) Hallier f., Operculina
turpethum (L.) Silva Manso, and O. codonantha (Benth.) Hallier f. were reported to secrete
obvious quantities of slimy fluids, while exudates from other species, e.g., Ipomoea pes-caprae
(L.) R.Br., I. quamoclit L., and Argyreia mollis (Burm.f.) Choisy, were secreted in unnoticeable
amounts [
58
], and this latter case was also found for R. ornata. Nevertheless, the positive
reaction of the ruthenium red test showed that the calycinal glands of R. ornata likely
contain mucilaginous and hygroscopic substances, as was also found in Ipomoea cairica (L.)
Sweet [
26
], and were defined as colleters (any of the morphologically diverse secretory
organs functioning homologously in secreting mucilaginous or resinous fluids [
13
]). Based
on the properties of the secreted substances, calycinal glands are believed to protect flower
buds, from their development until fruiting, from desiccation and insolation [26,58,107].
Although the petiolar nectaries and calycinal glands of R. ornata are located on different
parts of the plant, they appear to have the same function in feeding visitors, especially
diverse species of ants. Ants are widely known to be the main nectar consumer of extrafloral
nectaries and are generally acknowledged as guards against herbivores [
9
,
108
,
109
], but
we have not found any reports about ants or other insects visiting secretory organs that
function as colleters. Therefore, our study may be the first to report another potential
role of calycinal glands, or calycinal colleters, beyond protection from abiotic factors. It is
possible that R. ornata gained the capability of attracting guard ants through its calycinal
colleters instead of developing receptacular nectaries, which are found in several Ipomoea
species [
35
]. Nevertheless, the effectiveness of protection by guard ants depends on nectar
quality and the natural aggressiveness of each ant species [
110
,
111
]. Our study further
suggests that the effectiveness of ant guards also depends on the density of external
secretory structures, the distribution of external secretory structures on the plant, and any
plant traits that exclude ants (e.g., corolla tubes too narrow for ants to enter), as these
traits influence the plant’s ability to attract ant guards to specific areas or prevent them
from accessing certain areas. Unlike other plant species that have ant-attracting secretory
structures distributed on the leaf blade and leaf margin or located on the exposed parts
of the flower, such as Passiflora spp., Miconia tococa (Desr.) Michelang., Turnera subulata
Sm., and Mallotus japonicus (L.f.) Müll.Arg. [
109
,
112
–
115
], petiolar nectaries and calycinal
glands in R. ornata appear to only attract ants on certain parts of the plant. Specifically,
in R. ornata, ants gather at the petiolar nectaries and calycinal glands and also traverse
stems, branches, petioles, and inflorescence axes, but other parts were left ant-free most of
the time (i.e., leaf blades and corolla tubes and lobes), leaving herbivores on these areas
(tortoise beetles and katydids) undisturbed by ants. In Ipomoea leptophylla Torr., the presence
of ants on flowers significantly reduced damage from grasshopper herbivores and also
decreased seed