3.4 Analysis of anatomical traits of intact and damaged flowers
The thickness of the adaxial and abaxial cuticle of intact and damaged
flowers did not differ statistically. In turn, the median thickness of
the adaxial and abaxial epidermis and median thickness of the mesophyll
differed significantly between intact and damaged flowers according to
the non-parametric Kruskau-Wallis test. Thus, intact flowers had thicker
tissues than damaged flowers (p = 0.05) (Table 2).
The structural organization of the mesophyll did not differ between
intact and damaged flowers, except for the intercellular spaces
described above. However, we observed that while the mesophyll of intact
flowers remained expanded and linear (Figure 5F), damaged flowers had
constrictions with undulations on the epidermis, forming depressions
along the mesophyll (Figure 5G-H). These differences in the conformation
of the mesophyll can be seen in Figure 5. Mesophyll thickness also
differed statistically between intact and damaged flowers: the median
thickness of the mesophyll of intact flowers was significantly larger
than that of damaged flowers.
3.5 Histochemistry of intact and damaged flowers
Based on a control floral section (Figure 8A), all intact and damaged
flowers analyzed showed a positive reaction for the presence of
alkaloids (Figure 8B-C). However, the petals of intact flowers showed a
negative reaction, while all floral whorls of the petals of damaged
flowers showed a positive reaction for the presence of these compounds.
Only damaged flowers had phenolic compounds, exclusively in the anthers
damaged by florivores (Figure 8 D). Intact and damaged flowers showed a
positive reaction for proteins (Figure 8E-F) in all floral whorls and in
epidermal appendages, such as glandular trichomes. Starch was detected
in the petals and anthers of damaged flowers (Figure 8G), but only in
the petals of intact flowers. Pectins were identified in the cell walls
of the sepals (Figure 8H), petals, anthers, and ovaries of damaged
flowers (Figure 8I), but only in the sepals and anthers of intact
flowers. Tannins were detected in the anthers of damaged flowers, but
not identified in any part of intact flowers. Lipid substances occurred
in the sepals and anthers of both intact and damaged flowers (Figure
8J-L), but not in other whorls. Reducing sugars (glucose and fructose)
were found in intact and damaged flowers (Figure 8M-O). The tests for
essential oils and oleoresins were negative.
3.5.1 Percentage of chemical compounds in intact and damaged flowers
The percentages of nutritional substances were higher than those of
defense substances in both intact and damaged flowers (Figure 9). The
petals of damaged flowers presented approximately 100% of nutritional
substances, which include pectins, proteins, lipids and sugars. The
anthers of damaged flowers also presented a high percentage of these
compounds, with 60% in relation to the defense compounds. Low detection
of alkaloids and phenolic compounds was observed in all plant organs of
intact and damaged flowers. Higher percentages of defense compounds were
found only in the anthers in relation to the other whorls: 28% in
intact flowers and 38% in damaged flowers.
3.6 Resource production in intact and damaged flowers
Intact flowers produced a significantly higher amount of pollen compared
to damaged flowers (p = 0.01) (Figure 10a). The amount of pollen did not
differ significantly between the initial, intermediate, and pre-anthesis
stages of floral development (p = 0.8280) (Figure 10b).
4. DISCUSSION
4.1 Floral anatomy
The diversity of epicuticular waxes found in S. aversiflora is
important because waxes are considered taxonomically valuable characters
(Barthlott et al., 1998), especially helpful in the classification ofSenna species of the series Bacillares (Souto et al.,
2022). Furthermore, waxes play an important ecological role in plants,
protecting them against biotic and abiotic stresses, including
radiation, water loss, microorganisms, and insects (Ahmad et al., 2015;
Lewandowska et al., 2020). Waxes are also associated with insect-plant
interactions. Crystalloids, for example, help to prevent insects to
attach to plant organs (Gorb & Gorb, 2003), as demonstrated by Abbas et
al. (2023) in a study with Citrus L, in which cultivars with a
higher amount of waxes had lower densities of Lepidoptera larvae.
However, despite the presence and diversity of waxes in S.
aversiflora , they do not seem to hinder florivory because all floral
whorls of this species are widely consumed.
The ornamentation of epidermal surfaces can be phylogenetically useful
for the delimitation of groups and has an important functional role in
ecological interactions. Conical and/or papillose and lobular epidermal
surfaces are commonly found in Fabaceae groups such as Papilionoideae,
Mimosoideae, and Caesalpinioideae, which includes Cassia L andSenna Mill. species (Odeja et al., 2009). The patterns observed
are a rugose tabular epidermis with striations in Cassia
emarginata L. and a papillose conical epidermis in Senna alata (L.) Roxb (Odeja et al., 2009).
The sinuous anticlinal walls observed in the epidermis of S. aversiflora are similar to those observed in species with
different pollination syndromes (Kraaij & Van de Kooi, 2019). The
straight conformation of the anticlinal walls at the base of the concave
petal in S. aversiflora suggests a functional role in
pollination, by ricochet or looping. Pollen is released after the
vibration of the flowers by the bees and ejected towards the concave
petal, where it collides (ricochets) or runs (makes a loop) through its
entire surface to the apex, being then deposited on the back of the
pollinator (Westerkamp, 2004; Almeida et al., 2013; Amorim et al.,
2017). Considering that straight walls configure a more leveled
epidermis, we believe that this is an attribute that facilitates the
path of pollen grains when bees visiting S. aversiflora flowers
vibrated and pollen is released.
Flowers usually have petals with papillose epidermal cells, a
characteristic also associated with the attraction and interaction with
pollinators (Odeja et al., 2009; Costa et al., 2016; Bailes & Glover,
2018; Cavallini-Speisser et al., 2021). According to Costa et al.
(2016), several bee-pollinated flowers have a papillose epidermis that
can facilitate the landing and movement of bees in flowers. However,
even though S. aversiflora is pollinated by bees,
papillose epidermal cells were not observed in the species. The absence
of papillae may be related to the aforementioned ricochet or looping
pollination system. A papillose epidermis would probably make it
difficult for pollen to slide on the petals.
Regarding the striated cuticle observed in the petals by scanning and
light microscopy in S. aversiflora , it is known that this
character also has a role in the interaction between flowers and
pollinators. According to Whitney et al. (2009), regular folds in the
waxy cuticle of the epidermis of the petals may cause the diffraction of
light and serial propagation of bright reflections, working as a clue in
the petals perceptible to pollinators.
The presence of few trichomes in the petals may be related to the
distribution of florivores in S. aversiflora , since pubescence is
a character that promotes herbivore deterrence (Hnaley et al., 2007).
The high density of simple trichomes in plant organs creates an obstacle
for insect movement and feeding (Dai et al., 2010; Kaur & Kariyat,
2023). The mesophyll of flowers, including those of several species of
Fabaceae, is commonly homogeneous with braciform cells (Costa et al.,
2016), as observed in S. aversiflora . The organization of
braciform cells in the mesophyll form large air cavities. These
cavities, according to Van der Kooi et al. (2019), create differences in
the refractive indices of petal tissues and strong reflection and
scattering of light in relation to other flowers. This dynamic has an
effect on the appearance of petals by reflecting or diffusing light
(Cavallini-Speisser et al., 2021).
We associate the papillae found on the epidermis of the anthers to a
better adherence of the pollinators. Senna aversiflora has
flowers that are buzz pollinated by pollen-collecting bees that cling to
the anthers (Pritchard & Vallejo-Marín, 2020), and it has been proposed
that papillae facilitate the landing and movement of pollinators on
flowers (Aronne et al., 2012; Costa et al., 2016). The fibrous
endothelium gives rigidity to the anthers and consequently protection to
the pollen grains inside. The fibrous anthers of S. aversiflora may also contribute to pollination by the mechanical support provided,
being this one of the most important functional roles of lignin in
plants (Yadav, 2018). The bees that pollinate S. aversiflora are
large and, according to Dulberger (1981), the need to protect the
gynoecium from the action of pollinators is a possible explanation for
the existence of enantiostyly, a floral polymorphism observed inSenna species. In this sense, the existence of a lignified
endothelium in the gynoecium, observed in our study, may provide
resistance against the weight of the bees and their vibration during
visits.
4.2 Influence of florivory on anatomical traits
Our results corroborated the proposed hypothesis on the thickness of
anatomical traits, as we found that damaged flowers had less thick
traits, except for the cuticle. Studies on leaf herbivory indicate that
cuticle thickness generally functions as a mechanical barrier that
hinders the penetration of the oral tract of herbivores into the leaf
(Petters, 2002. Corrêa et al., 2008; Mostafa et al., 2022; Demis, 2024).
The epidermis of damaged flowers was less thick on both surfaces than
that of intact flowers, probably due to the stress caused by florivory,
including loss of biomass and water, consequently triggering a
retraction in cell volume.
The mesophyll of the petals of damaged flowers was also less thick than
that of intact flowers. In the case of leaves, the number of mesophyll
layers in conjunction with other structural traits such as the presence
of a lignified epidermis, a thickened hypodermis, and sclerenchyma are
considered potential barriers against insects (Correa et al., 2008;
Caldwell et al., 2016). The absence or reduction of these traits are
opportune for florivory. It has been observed that higher densities of
external chewers are associated with the absence of a thickened
hypodermis in flowering plants, including species of Acacia Mill., and Pultenaea Sm. of the family Fabaceae (Peeters, 2002).
Further, it has been demonstrated that the proportion of damage caused
by chewers decrease with increasing leaf thickness in four tree species
(Martini et al., 2022).
Deformations in the petals of damaged flower also include collapsed
mesophyll tissue and invaginations in the epidermis, unlike intact
flowers whose mesophylls are expanded and rectilinear. Changes similar
to the ones observed in the petals of S. aversiflora are
commonly seen in leaf galls (Nobrega et al., 2023). The present results
are the first record of these changes in floral whorls.
4.3 Histochemistry
The hypothesis that damaged flowers have lower concentration of defense
compounds was not confirmed, since both intact and damaged flowers
showed high percentages of nutritional compounds in relation to defense
compounds. Phenolic compounds and alkaloids, although present in the
flowers of S. aversiflora , occurred in low concentrations.
Phenolic compounds are especially involved in the defense and resistance
of plants against pathogens, including bacteria, viruses and fungi. They
also play a protective role against insect herbivory because they cause
oxidative damage to the herbivores and affect their growth, development,
and reproduction (Kumar et al., 2020). Alkaloids, in turn, are
considered toxic to herbivores (see Wari et al., 2021). The studies by
Adler et al. (2011) on the direct and indirect effects of alkaloids on
plant suitability for herbivory and pollination showed that alkaloids
directly reduced bud herbivory, but did not have strong effects on
pollination. The fact that S. aversiflora had low levels of
alkaloids means that the flowers of this species are vulnerable to
herbivore attacks.
As for attraction and nutritional compounds, the flowers and buds ofS. aversiflora are rich in proteins, sugars, lipids, and
carbohydrates. According to Erb et al. (2021), the primary metabolites
produced by plants influence and play a positive role on herbivory by
increasing the essential nutrients transferred across trophic levels.
Host signals, including the primary and secondary metabolites and the
nutritional value of the plant part consumed, determine the feeding
preferences of herbivores. Sugars, for example, can inhibit the aversive
taste of secondary metabolites and make tissues palatable (Machado et
al., 2021), while proteins and carbohydrates favor the growth of
herbivores (Le Gall et al., 2014). Le Gall et al. (2014) found that
higher concentration of carbohydrates and proteins had significant
positive effects on the body mass gain of nymphs of the grasshopperMelanoplus differentialis (Thomas). The availability of
macronutrients in S. aversiflora makes it an attractive source of
food, which possibly explains this strong antagonistic interaction in
the species.