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Does Domestication Affect Structural and Functional Leaf Epidermal Traits? A Comparison between Wild and Cultivated Mexican Chili Peppers (Capsicum annuum)

During domestication, lineages diverge phenotypically and genetically from wild relatives, particularly in preferred traits. In addition to evolutionary divergence in selected traits, other fitness-related traits that are unselected may change in concert. For instance, the selection of chili pepper fruits was not intended to change the structure and function of the leaf epidermis. Leaf stomata and trichome densities play a prominent role in regulating stomatal conductance and resistance to herbivores. Here, we assessed whether domestication affected leaf epidermis structure and function in Capsicum annuum. To do this, we compared leaf stomata and trichome densities in six cultivated varieties of Mexican Capsicum annuum and their wild relative. We measured stomatal conductance and resistance to herbivores. Resistance to (defense against) herbivores was measured as variation in the herbivory rate and larvae mortality of Spodoptera frugiperda fed with leaves of wild and cultivated plants. As expected, the different varieties displayed low divergence in stomatal density and conductance. Leaf trichome density was higher in the wild relative, but variation was not correlated with the herbivory rate. In contrast, a higher mortality rate of S. frugiperda larvae was recorded when fed with the wild relative and two varieties than larvae fed with four other varieties. Overall, although domestication did not aim at resistance to herbivores, this evolutionary process produced concerted changes in defensive traits.


Introduction
Domestication is an evolutionary process in which plant and animal species are consciously and unconsciously modified by humans through artificial selection [1][2][3]. An outcome of this process is that the selection of particular traits produces dramatic changes that clearly distinguish domesticated lineages from their wild relatives [4,5]. Domestication in plants is also accompanied by changes in the environment from wild to agricultural conditions, which may act as a selective factor over other phenotypic traits, favoring phenotypes that confer an advantage under new conditions [6][7][8]. However, some phenotypic traits could remain similar between wild and domesticated varieties because those phenotypic traits have physiological and ecological functions that are equally relevant for survival and reproduction in natural and in agronomic conditions [9][10][11]. A scenario such as this predicts that some traits will, and some will not, change under domestication, depending on their relevance for plant functioning. Although domestication produces ubiquitous divergence between cultivated and wild progenitors in selected traits, its effects on functional Two studies compared a single cultivated variety with its wild relative [23,33] but see [27], which limits our understanding of the broad spectrum of functional results expected in this group of plants [42].
Here, the aim of this study was to assess the impact of domestication on the epidermal traits implicated in water-use efficiency and resistance to herbivores in the wild-cultivated system Capsicum annuum. Stomatal density and conductance, as well as leaf trichome density and resistance to herbivores, were measured in six varieties of Mexican chili pepper and compared to its wild ancestor C. annuum var. glabriusculum. First, it was expected to find a higher density of stomata and higher water evapotranspiration in cultivated plants because cultivated plants have evolved under agronomic conditions, where they have been exposed to beneficial conditions of water availability in relation to wild relatives under natural conditions [43]. Likewise, a reduction in the density of leaf trichomes and a reduction in plant resistance to herbivores was expected in cultivated varieties as a consequence of plant domestication [27][28][29]. To assess the role of trichomes in resistance, variation in leaf trichome density was associated with the level of damage caused by a generalist herbivore Spodoptera frugiperda. This is the first study assessing how epidermal traits, critical to plant functioning, diverge between cultivated and wild varieties of C. annuum. These results show that plant resistance to herbivores has decreased in some cultivated varieties of Capsicum annuum highlighting the need to study differences in plant functioning and herbivore resistance in many varieties to better understand the outcomes and mechanisms of evolution under domestication in C. annum and other plants.

Variation between Upper and Lower Epidermal Surfaces
The results show significant differences between varieties in five out of six epidermal traits (Table 1). Moreover, the leaf-side effect was significant for five out of six traits of density or cell size, indicating that the upper and lower epidermies differed in some cell types (Table 1 and Figure 1). The lower epidermis had a higher mean density and mean cell size than the upper epidermis ( Figure 1). The covariate leaf area was only significant and positive for the number of pavement cells ( Table 1). The mean stomata width was the only trait that did not show significant differences between varieties and between the upper and lower epidermis.

Stomatal Density and Size of the Upper and Lower Epidermis
The stomatal density of the lower and upper epidermises was significantly different between the chili plants ( Table 2). The stomatal density of the lower epidermis differed between the wild population and two out of six cultivated varieties, YAHU and JAL. The mean stomatal densities of the YAHU and JAL varieties were 45% and 70% lower in comparison to the wild population (Tukey test lower epidermis : α = 0.05; Q = 3.304). In contrast, no differences in stomatal density were detected in the upper epidermis between the wild population and the cultivated varieties (Tukey test upper epidermis : α = 0.05, Q = 3.304).
The stomatal length was significantly different between the wild population and the cultivated varieties in the lower epidermis (Table 2 and Figure 1). In this case, the stomatal length of the wild population only differed significantly from that of the JAL variety, which had a 23% larger stomatal length than the wild variety ( Figure 1). The stomatal length of the upper epidermis and the stomatal width of the upper and lower epidermises were not significantly different.

Stomatal Density and Size of the Upper and Lower Epidermis
The stomatal density of the lower and upper epidermises was significantly different between the chili plants ( Table 2). The stomatal density of the lower epidermis differed between the wild population and two out of six cultivated varieties, YAHU and JAL. The mean stomatal densities of the YAHU and JAL varieties were 45% and 70% lower in comparison to the wild population (Tukey test lower epidermis: α = 0.05; Q = 3.304). In contrast, no differences in stomatal density were detected in the upper epidermis between the wild population and the cultivated varieties (Tukey test upper epidermis: α = 0.05, Q = 3.304).
The stomatal length was significantly different between the wild population and the cultivated varieties in the lower epidermis (Table 2 and Figure 1). In this case, the stomatal length of the wild population only differed significantly from that of the JAL variety, which had a 23% larger stomatal length than the wild variety ( Figure 1). The stomatal length of the upper epidermis and the stomatal width of the upper and lower epidermises were not significantly different.

Trichome Density in the Upper and Lower Epidermis
The foliar trichomes identified in all tested varieties were glandular and non-glandular trichomes. Glandular trichomes were observed in the wild and all cultivated varieties ( Figure 1C,G), whereas non-glandular trichomes were found in the wild population and in the cultivated JAL, SERR, and YAHU ( Figure 3D,E). Leaf trichome density in the lower epidermis was significantly different between chilis and the wild population (Table 2 and Figure 4). The mean trichome density of the wild population was significantly higher than that of the domesticated GÜE, SERR, and YAHU varieties (Tukey test: α = 0.05; Q = 3.41). The trichome density of the upper epidermis was not significantly different among any of the varieties ( Table 2). Univariate ANOVA was used to compare the the stomatal conductance of wild populations and cultivated varieties. Red bars indicate the mean and 1 ± SE for the wild variety. Gray bars indicate the mean and SE of the cultivated varieties. The asterisks indicate which cultivated variety significantly differs from the wild variety after Tukey's HSD test (α = 0.05).

Trichome Density in the Upper and Lower Epidermis
The foliar trichomes identified in all tested varieties were glandular and non-glandular trichomes. Glandular trichomes were observed in the wild and all cultivated varieties ( Figure 1C,G), whereas non-glandular trichomes were found in the wild population and in the cultivated JAL, SERR, and YAHU ( Figure 3D,E). Leaf trichome density in the lower epidermis was significantly different between chilis and the wild population (Table 2 and Figure 4). The mean trichome density of the wild population was significantly higher than that of the domesticated GÜE, SERR, and YAHU varieties (Tukey test: α = 0.05; Q = 3.41). The trichome density of the upper epidermis was not significantly different among any of the varieties ( Table 2).

Leaf Consumption Rate
The leaf area consumed by S. frugiperda was significantly different among varieties and wild plants (F1,6 = 4.94; p < 0.02; n = 180; R 2 = 0.08). Wild plants received the highest level of damage compared to cultivated varieties. The leaf area consumed by the wild variety was 43% higher than that of YAHU and 37% higher than that of the JAL varieties ( Figure 5). The correlation between the consumption rate and the trichome density of the lower epidermis was not significant (r = 0.42; p = 0.3).
The larval survival of S. frugiperda was significantly different between cultivars and between cultivars and wild plants (χ 2 L-R = 171. 22; d.f. = 6; p < 0.0001; R 2 = 0.65; n = 192). The lowest survival rate was found in larvae fed with leaf tissue of the wild population and

Leaf Consumption Rate
The leaf area consumed by S. frugiperda was significantly different among varieties and wild plants (F 1,6 = 4.94; p < 0.02; n = 180; R 2 = 0.08). Wild plants received the highest level of damage compared to cultivated varieties. The leaf area consumed by the wild variety was 43% higher than that of YAHU and 37% higher than that of the JAL varieties ( Figure 5). The correlation between the consumption rate and the trichome density of the lower epidermis was not significant (r = 0.42; p = 0.3).
The larval survival of S. frugiperda was significantly different between cultivars and between cultivars and wild plants (χ 2 L-R = 171. 22; d.f. = 6; p < 0.0001; R 2 = 0.65; n = 192). The lowest survival rate was found in larvae fed with leaf tissue of the wild population and two cultivated varieties (ARB and Yahu). In contrast, survival rates in larvae fed with tissue of other domesticated plants were higher, ranging from 100 to 95% for GÜE and PIM, and for the JAL and SERR varieties, survival was~75%.
Plants 2022, 11, x FOR PEER REVIEW two cultivated varieties (ARB and Yahu). In contrast, survival rates in larvae fed w sue of other domesticated plants were higher, ranging from 100 to 95% for GÜE and and for the JAL and SERR varieties, survival was ~75%.

Discussion
This study is the first multi-variety assessment of the consequences of domesti linking the morphology of the leaf epidermis and the function of Capsicum annuu one of the most important crop spices around the world. We included six cultivated eties of C. annuum that are known to have been derived from their wild ancestor, nuum var. glabriusculum, to interpret the results following an evolutionary framewor Domestication can produce abrupt changes, primarily increasing the size of selected [10]. However, this trend varies in magnitude, and in some crops, varieties sh changes in some traits even when they have relevant physiological and ecological r agricultural ecosystems.
This study found variation in the magnitude of the effect of domestication on e mal phenotypes and their functions between wild populations and cultivated varie Capsicum annuum. The results reveal partial support for the effect of domestication idermal phenotypic traits and their function in Capsicum annuum. Stomatal de showed great variation between varieties, but only two cultivated varieties differed their wild relative. At the functional level, ample variability was found in stomata ductance within all and the wild population, which is in line with the low differen in stomatal density and size. In relation to the effects of domestication on plant resis the wild C. annuum var. glabriusculum and two domesticated varieties had the str effects on herbivore survival, while four varieties caused lower or no herbivore mo We found higher trichome density in wild plants compared to cultivars. How changes in trichome density did not show an association either with consumption r S. frugiperda or with herbivore survival. Overall, the patterns observed in this study the complex effects of domestication on the leaf epidermis of C. annuum, suggestin domestication may be moderate in traits that are not consciously selected.
Stomatal density and size can increase during domestication because cult plants are exposed to new environmental conditions, such as high water availabilit

Discussion
This study is the first multi-variety assessment of the consequences of domestication, linking the morphology of the leaf epidermis and the function of Capsicum annuum (L.), one of the most important crop spices around the world. We included six cultivated varieties of C. annuum that are known to have been derived from their wild ancestor, C. annuum var. glabriusculum, to interpret the results following an evolutionary framework [34]. Domestication can produce abrupt changes, primarily increasing the size of selected traits [10]. However, this trend varies in magnitude, and in some crops, varieties show no changes in some traits even when they have relevant physiological and ecological roles in agricultural ecosystems.
This study found variation in the magnitude of the effect of domestication on epidermal phenotypes and their functions between wild populations and cultivated varieties of Capsicum annuum. The results reveal partial support for the effect of domestication in epidermal phenotypic traits and their function in Capsicum annuum. Stomatal densities showed great variation between varieties, but only two cultivated varieties differed from their wild relative. At the functional level, ample variability was found in stomatal conductance within all and the wild population, which is in line with the low differentiation in stomatal density and size. In relation to the effects of domestication on plant resistance, the wild C. annuum var. glabriusculum and two domesticated varieties had the strongest effects on herbivore survival, while four varieties caused lower or no herbivore mortality. We found higher trichome density in wild plants compared to cultivars. However, changes in trichome density did not show an association either with consumption rate by S. frugiperda or with herbivore survival. Overall, the patterns observed in this study show the complex effects of domestication on the leaf epidermis of C. annuum, suggesting that domestication may be moderate in traits that are not consciously selected.
Stomatal density and size can increase during domestication because cultivated plants are exposed to new environmental conditions, such as high water availability [44]. In particular, the densities and sizes of stomata are expected to increase during domestication if water losses are not traded off with the rate of CO 2 diffusion to the interior of the leaf [45]. We found significant variations between the wild population and varieties in terms of stomatal density and length. However, differences in stomatal density between the wild and cultivated varieties were found only in the lower epidermis. This low-level differentiation can explain the similarities in evapotranspiration rates found between the wild and cultivated plants in this study. Although the wild variety showed the lowest rate of evapotranspiration, it only had a significant difference from one cultivated variety (the YAHU variety).
The low level of differentiation observed in stomatal density and size (i.e., length) is not rare. In agreement with our results, previous studies have found that stomatal density and length do not show a high degree of divergence between wild and cultivated varieties in the common bean, Phaseolus vulgaris (Fabaceae) [24]. Moreover, an assessment of 24 species found that stomatal features showed low differentiation between wild and cultivated relatives [23]. These moderate differences in the density and size of stomata between the cultivated varieties and their wild ancestor explain the absence of a correlation between stomatal conductance and stomatal density [46]. Thus, the low degree of divergence in density and size between the cultivated varieties and their wild relative can explain the similitude in stomatal conductance observed in this study as well. In this case, this evidence suggests that the agronomic conditions of water availability did not promote an increase in stomatal traits implicated in ecological functions.
The effects of domestication were more evident in leaf trichomes than in the other epidermal cells (stomatal and pavemented cells). In this case, the wild plants had significantly more trichomes than the plants of three out of six cultivated varieties in the lower epidermis. As expected, the wild population had the highest density of trichomes, whereas the most dramatic change was that leaf trichomes were not detected in some cultivated varieties. Surprisingly, variation in trichome density did not correlate with the herbivory rate. Contrarily, the herbivory rates of S. frugiperda were higher in the leaf disks of wild plants in relation to domesticated plants. Given that wild plants had the highest trichome density, this study suggests that leaf trichomes did not effectively obstruct feeding by S. frugiperda larvae [47]. Previous studies have found similar evidence suggesting that changes in leaf trichome density associated with domestication did not explain the resistance to herbivores [33]. Thus, even when leaf trichomes seemed to not have an adaptive role as a component of resistance to herbivores, it remains to be tested whether trichomes provide defense for other herbivore species.
One objective of this study was to assess whether domestication reduces resistance to herbivores. Variation in mortality rate between wild and cultivated plants found in this study supports the hypothesis that domestication affects plant resistance to herbivores in Capsicum annuum. We found that S. frugiperda larvae exposed to a leaf-based diet of wild populations and two cultivars caused a dramatic survival reduction in S. frugiperda. In contrast, the leaf tissue of the other four cultivars had a lower impact on insect mortality. These results are comparable with evidence from a recent meta-analysis that found that insects fed with the leaf tissue of wild plants have higher mortality in relation to domesticated ones [48]. Specifically, in Capsicum annuum, previous studies have found similar results, supporting the notion that domestication reduces the level of resistance to herbivores as a function of insect mortality [49]. For instance, when resistance to herbivores in a wild population (i.e., Tapachula accession, not included in this study) and cultivated poblano and serrano varieties of C. annuum were compared, the mortality rates of S. frugiperda fed wild tissue were higher than those fed domesticated varieties [49]. Although resistance to herbivores decreased with domestication in four varieties analyzed in this study, wild and two cultivated chili varieties had a similar impact on insect mortality, suggesting that the cost of domestication is not equal in all species. Thus, domestication effects on plant defense largely depend on the variety and its evolutionary history, and they also need to be assessed on insects.
One caveat in this study is that we did not measure a biochemical component of resistance to herbivores. Other studies have found that leaf polyphenols affect Spodoptera performance [50,51], indicating the relevance of these types of compounds in resistance to herbivores. In our case, secondary metabolites, such as polyphenols, could explain resistance to herbivores, and domestication can affect chemical resistance to herbivores in Mexican varieties of Capsicum annuum. Further metabolomic studies are required to compare and identify the type and number of chemical compounds responsible for the mortality of the herbivore S. frugiperda in wild and cultivated varieties of C. annuum. This is among the few studies that have analyzed the changes in epidermal cells in different cultivated varieties as well as their functional implications. The lack of differences in the density of stomata and their conductance indicates that this trait has not been abruptly affected during the course of domestication. Second, wild plants produced significantly more trichomes than cultivars, but the trichomes did not obstruct herbivore feeding, as expected for a trait with a defensive role. In spite of this, the high survival rate of S. frugiperda fed with cultivated tissue indicated that domestication can have detrimental effects on plant resistance [48], but it seems that changes are occurring in secondary compounds. Traits related to water-use efficiency/CO 2 uptake and resistance to herbivores can show ample variation with no fitness cost for domesticated plants [23,43,52] possibly as a result of the relaxed selection occurring under agronomic conditions (i.e., irrigation and pesticide application). To examine the cost of domestication, further studies should expose cultivars to simulate the natural conditions of water stress and/or different levels of herbivores [53]. This will help clarify how cultivars depart from their ancestor and whether small phenotypic modifications [54], such as stomata and trichome density, associated with domestication in Capsicum annuum are relevant in natural environments [55].

Conclusions
Our results demonstrate for the first time that domestication of C. annuum is accompanied by changes in epidermal traits implicated in critical functional traits. We confirmed that domestication has detrimental effects on resistance to herbivores in some varieties, but the differences in leaf trichomes failed to explain variation in resistance to herbivores. Likewise, water evapotranspiration and stomata did not show dramatic changes. These findings thus have implications, which we discuss in more detail above, for understanding both the broad spectrum of functional responses expected under domestication in this group of plants and the variable consequences of domestication.

Plant Materials
For the experiment, we used seeds from six cultivated varieties and the wild relative Capsicum annumm var. glabriusculum. Seeds of the wild C. annuum var. glabriusculum were collected during 2016 and 2017 from different natural populations and stored in a germplasm collection. To examine epidermal features, we used wild seeds collected from San Javier, Sonora, located in northwestern Mexico. Furthermore, to measure water-use efficiency and plant resistance, we also included seeds collected from two more wild populations, namely Veracruz and Tapachula, which grow under more humid conditions. Those populations were included to cover a broad spectrum of variation in stomatal conductance, water-use efficiency, and resistance to herbivores ( Table 1). Seeds of the six cultivated varieties used were obtained from commercial stocks from a local market brand, Rancho Los Molinos (Mexico). The following cultivated varieties were used: Capsicum annuum, called "árbol" (ARB), "jalapeño" (JAL), "serrano" (SERR), "yahualica" (YAHU), "güero" (GÜE), and "morrón" (PIM). These varieties were randomly chosen, but we took care to include varieties for whom C. annuum var. glabriusculum is the common ancestor [54,55].

Plant Growth
We sowed 50 seeds per variety in germination trays filled with commercial soil (Berger BM2). All trays were placed in a germination room with a controlled photoperiod with a 14:10 h nightlight under a temperature of 25 ± 2 • C. Seedlings with four well-expanded leaves were transplanted to 4 L plastic pots filled with substrate (Berger BM2). A total of 15 seedlings per variety were transplanted, resulting in 105 plants from the San Javier wild variety and the six cultivated varieties. The plants were randomly distributed on six benches within a greenhouse to avoid shading between plants. During the experiment, fertilization consisted of a weekly application of 50 mL of solution containing 2 g × L −1 N-P-K (19-19-19) on the soil of each plant. All plants were watered ad libitum to avoid the potential influence of hydric stress on the leaf epidermis. In addition, the instructions of the seed producers indicated that the plants had to be very well irrigated.

Epidermal Impressions
To examine variations in the leaf epidermis, we collected leaves from a subsample of three plants per variety brought to the laboratory from the greenhouse. From each plant, we collected five mature leaves from the middle part of the stems of each individual plant. We obtained 400 epidermal impressions from the abaxial and adaxial surfaces of the sampled leaf. Epidermal impressions were made using the nail polish method. Briefly, we applied a film of clear nail polish to the leaf surface between the second and third secondary veins, avoiding the main and secondary veins. The nail polish was left to dry for 3 min, and then we placed a piece of transparent adhesive tape on the area covered with the nail polish. We pressed the adhesive tape evenly for 1 min using a piece of cotton rag. The adhesive tape was removed using fine scissors, and the sample was placed on a slide labeled with the name of the variety and the sample number.

Cell Counts and Stomatal Measurements
To obtain the number and measurements of epidermal cells, we digitized the upper and lower epidermises on a sample of 3-5 leaves per plant using a light optical microscope Primo star equipped with an ICc5 digital camera (Zeiss, Germany). A 40× magnification lens was used to obtain digital images, resulting in a visual area of 2.38 mm 2 for each sample. We counted all pavement cells, trichomes, and stomata observed in each digital image, excluding incomplete cells at the margins of images. Stomata length and width measurements were obtained from three closed stomata randomly chosen from the samples. Stoma length was measured as the distance from the upper to the lower tip of a guard cell. In turn, the stoma width was measured as the distance between the outer extremes of the two guard cells in the middle of each stoma. Cell counts and stomatal measurements were conducted using Zen lite software (Zeiss 2012).

Stomatal Conductance
Stomatal conductance was measured in a subsample of three plants per variety. Measurements were taken on a random sample of three mature leaves located in the middle part of the stem of each plant. Stomatal conductance was obtained on the upper side of the leaves using a leaf porometer (SC-1 model, Decagon Services) between 11:30 am and 12:00 pm. Plants from each variety were randomly distributed in the greenhouse. All plants were well irrigated for two days before measurements were conducted.

Insects
Spodoptera frugiperda eggs were obtained from the Laboratorio de Control Biológico, ECOSUR-Tapachula. The eggs were allowed to hatch in Petri dishes (90 mm × 15 mm) on moist paper in a growth room (L:D 14:10 h; 26 • C; 65% RH). Larvae were then individually moved to small plastic containers in the same growth room on a corn-based diet. Thirdinstar and fourth-instar larvae were used for both insect consumption rates and survival tests.

Insect Consumption Rate
To determine whether domestication influences insect consumption rates, third-and fourth-instar S. frugiperda larvae were given leaf disks from each of the six cultivated varieties and from each wild population. Plastic Petri dishes with a 3 cm diameter × 2 cm depth were used as arenas. Leaves were randomly removed from cultivated and wild plants in the greenhouse and brought to the laboratory, where a hole punch (1.96 cm 2 diameter) was used to cut the leaf disks. One leaf disk was placed into each Petri dish, and one larva was placed into a Petri dish and allowed to feed on leaf disks for 1 h in the growth room (see above). One hour was determined to be a reasonable length of time to assess consumption rates of herbivores but short enough to prevent insects from consuming all of the leaf disks from one treatment. A total of 24 larvae were assigned to be fed per domesticated variety or wild population, resulting in 216 larvae. When a larva did not start to eat within the first 20 min, it was excluded from the experiment. This resulted in 189 larvae. At the end of the experiment, the remaining leaf disks were removed from the Petri dish and digitized to obtain the remaining leaf disk areas for each cultivated variety and wild population. The remaining leaf areas were determined using the imaging program WinFolia (Regent Instruments, USA). The remaining leaf disk areas were subtracted from the original area of the leaf disks to determine the area consumed. We did not replace larvae because of the short time elapsed in this experiment.

Survival assay of Spodoptera frugiperda
To test whether domestication influenced insect survival, the larvae of Spodoptera frugiperda were allowed to feed on leaves from cultivated and wild plants from each of the six cultivated lines and the three wild accessions. The experiment began with a sample of 216 larvae, but larvae that did not start to eat after 4 h were excluded from the experiment, resulting in a sample size of 192. Each individual larva was placed in a Petri dish fed with a 1.5 cm 2 leaf tissue disk of only one variety until death or pupation in the growth room (see above). The leaf disks were changed every 12 h to maintain fresh tissue. This time was determined to be a reasonable length of time to maintain fresh tissue and was short enough to prevent insects from consuming all of the leaf disks and starving. Survival (1) and mortality (0) were registered daily until the last larva died or reached the pupa stage. All larvae fed the leaf tissue of wild chili died before reaching the pupa stage.

Statistical Analyses
First, an analysis of variance (ANOVA) of the leaf size was conducted, including the variety as a fixed effect to compare the leaf size between the wild populations and cultivated varieties. Second, a univariate analysis of covariance (ANCOVA) was conducted to compare the density and size of epidermal cells among the wild populations and cultivated varieties, including the following variables: (i) pavement cell density, (ii) density of stomata, (iii) stomatal length, (iv) stomatal width, and (v) trichome density. The ANCOVA included the effects of variety, leaf side (i.e., the upper and lower epidermises), and the variety × leaf size interaction. For these analyses, data on the upper and lower epidermises were pooled. When leaf size was not significant for a specific trait, it was removed from its respective ANCOVA. Furthermore, we performed Tukey's test to assess the significant differences between the wild population and cultivated varieties.
Because the number of cells differing between the upper and lower epidermises was statistically different, we conducted independent ANOVAs for the upper and lower epidermis for the same response variables mentioned above. This model only included the effect of variety as a fixed effect. We performed Tukey's test comparisons to assess the significant differences between the wild population and cultivated varieties.
To examine the differences in stomatal conductance and consumption rate by herbivores, we conducted an ANOVA for each variable, including variety as a fixed effect. Survival among larvae was analyzed with a logistic ANOVA, including variety as a fixed effect. Data of larvae fed wild chili plants were pooled because the consumption rate (F = 0.99; g.l. = 5; p = 0.4) and survival were not different between populations.
To examine the relationship between a trait and its functional role, individual Pearson's correlation analyses were used for pairs of variables. One correlation was estimated for stomatal conductance vs. stomatal density, and another correlation was conducted for consumption rate vs. trichome density. These correlations were performed using the mean values per variety calculated prior to the analysis. Data analysis was performed using JMP software v. 10

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.