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.