Introduction

Mixed temperate forests are characterized by the co-occurrence of two or more tree species that are immediate neighbors and maintain inter- and intraspecific interactions between them (Bravo-Oviedo et al. 2014). These forests represent an important source of ecological services due to their high primary productivity (Chamagne et al. 2017; Jactel et al. 2018), stability against environmental and anthropogenic changes (Morin et al. 2014; Van der Plas et al. 2016) and resistance to abiotic (i.e. drought and fire) and biotic stressors (i.e. insect herbivores and pathogens) (Jactel et al. 2017). Consequently, it has been suggested that tree diversity is an important factor that confers resistance and resilience to forest in relation to the incidence of insect herbivores (Bauhus et al. 2017). For example, mixed species forests have low herbivory levels by insects compared to monospecific forests, according to a meta-analysis of planted and natural forests and biodiversity-ecosystem functioning experiments (Jactel and Brockerhoff 2007; Castagneyrol et al. 2014). Additionally, it has been corroborated that trees are generally more prone to suffering higher levels of damage by insect herbivores when that grow in monospecific stands than when are associated with other tree species (Vehviläinen et al. 2007; Castagneyrol et al. 2014). Conversely, other studies have shown neutral effects of plant diversity on the incidence of generalist insect herbivores that cause foliar damage (Rosado-Sánchez et al. 2018) and even an increase in foliar damage as tree diversity increases (Novotny et al. 2010) in the case of host plant species of the same genus that tend to support similar insect herbivore communities compared to insect communities that feed on host plant species of different taxa (Ødegaard et al. 2005). Thus, plant diversity can reduce or increase the insect herbivore diversity and the herbivory levels on their host plants (Barbosa et al. 2009; Muiruri et al. 2019). For example, the plant species richness hypothesis has been proposed to explain differences in species richness of insect herbivores among plant communities. This hypothesis proposes that the floristic diversity of habitats may be responsible for the variation in the local patterns of insect herbivore species richness because more plant species represent more potential sites to colonize (Fernandes and Price 1988).

In this way, the vegetation complexity and characteristics of neighboring plants can greatly influence the interactions among insect herbivores and the focal host plant, affecting host plant selection and plant susceptibility (Plath et al. 2012; Kostenko et al. 2017). This fact is known as associational effects (Root 1973; Barbosa et al. 2009) which involve direct and indirect interactions between nearby plants according to the following two distinct hypotheses: (i) “The associational resistance hypothesis” (ARH) that proposes that the increase of plant diversity generates heterespecific neighborhoods of plant species that can reduce the damage by specialist insect herbivores on a particular plant species (Field et al. 2020). This prediction is based in which a focal plant and its morphological, nutritional quality, and chemical defense characteristics may experience associational resistance against insect herbivores when growing in a neighborhood in close proximity to other plant species (Coley and Barone 1996; Plath et al. 2012), and (ii) “The associational susceptibility hypothesis” (ASH) that state that an increase in plant diversity increases the damage by generalist insect herbivores due to a wider range of food resource availability to insects in more diverse plant communities (Unsicker et al. 2008; Plath et al. 2011). Associational susceptibility can also occur in the case when a focal plant is less preferred by insect herbivores, but growing in close proximity and surrounded by an extremely preferred host plants, which favors the incidence of generalist insect herbivores after depletion of the preferred host plant (White and Whitham 2000).

Under natural conditions, plant nutritional quality and chemical defenses may be influenced by the diversity of the neighboring plant community as result of competition between the host plant and its neighbors for the availability of resources (i.e. light, soil nutrients, water) (Abbas et al. 2013; Kos et al. 2015; Moreira et al. 2014; Kostenko et al. 2017). For example, Mraja et al. (2011) documented that the catalpol concentrations (a iridoid glycoside associated with herbivory defense) increased as the plant species richness was higher in the experimental grassland plots of Plantago lanceolata. Some theories predict that the concentration of plant secondary metabolites can be influenced by the diversity of the plant community. For example, “The growth-defense trade-off hypothesis” (Coley et al. 1985; Endara and Coley 2010; Eisenhauer et al. 2009) that states that plants will allocate more resources for defense in more diverse plant communities, where a greater plant diversity increases the competition for nutrients, water and light, reducing growth of the focal plants and the levels of herbivore attack. Contrarily, the specialist-generalist hypothesis (Van der Meijden 1996) suggests that the concentration of secondary metabolites associated with plant defense is influenced by the proportion of generalist and specialist herbivorous insects present in the community, where specialists (e.g. gall-inducing insects) have a preference for plants with high concentrations of defensive compounds, while generalists (e.g. leaf-chewing insects) prefer plants with low defenses.

In the Northern Hemisphere, oak (Quercus spp.) forests host rich communities of canopy arthropods compared to other kind of forests (Valencia-Cuevas et al. 2015; Maldonado-López et al. 2018). High arthropod diversity often results in a complex community structure of herbivores, predators and parasites (Mitchell et al. 2019; Sierzega and Eichholz, 2019). Mexico is an important center of oak diversification and endemism (Hipp et al. 2018) with a total number of species close to 170 and 100 endemics (Hipp et al. 2018). Furthermore, a wide range of oak communities with different species diversity and composition can be found (Torres-Miranda et al. 2013; Rodríguez-Correa et al. 2015). Some areas in central-western and southern Mexico are particularly rich in oak species, where between two and up to six species can co-occur at the local level. These diversity gradients are an excellent model to evaluate different ecological aspects of oak communities such as canopy arthropod composition. Our main objective was to evaluate whether more diverse oak species communities harbor a greater canopy insect herbivore diversity and produce higher rates of herbivory on the focal plant Quercus laurina along the oak diversity gradient. The specific questions addressed were as follows: (1) Does the oak diversity gradient increase the diversity of the canopy insect herbivore community in Q. laurina? (2) Are Q. laurina chemical defense compounds and herbivory levels affected by oak diversity? and (3) Is there a relationship between leaf chemical defense, herbivory levels and the insect herbivore diversity of Q. laurina along oak diversity gradient?

Material and methods

In all study sites, oak trees were highly dominant in the plant community; representing at least 90% of the trees (See supplementary materials). Overall, we found that of the five oak species, three belonged to the Lobatae section (Q. laurina, Q. crassifolia and Q. calophylla) and two to the Quercus section (Q. obtusata and Q. rugosa). Our focal species (Quercus laurina) co-occurs with these oak species, which are not phylogenetically close as they are located in different clades according with the North American oak’s phylogeny (Hipp et al. 2020).

Study system

Quercus laurina Humb et Bonpl is an endemic oak species from México which belongs to the section Lobatae (red oaks) of the genus Quercus (Nixon 1993). It is a tree with a height between 10 and 30 m that occurs in mixed oak and pine forests in the Sierra Madre del Sur and the Trans-Mexican Volcanic Belt (TMBV) (Valencia 1994). The leaves are coriaceous, lanceolate or elliptic-oblanceolate, with a green and lustrous surface and its fruit is an ovoid acorn, with an average size of 15–20 mm long and 15–17 mm in diameter (Arizaga et al. 2009). Based on data from a previous study where we analyzed the canopy arthropod diversity in five sites with different oak species diversity (Vaca-Sánchez et al. 2021a, b), we selected a data subset that included insect herbivores which were separated based on their feeding group and thus, analyze both the diversity and changes in the assemblages of insect herbivore feeding group along the oak diversity gradient. The study sites are located in the central-western portion of the Trans-Mexican Volcanic Belt: (1) Tequila Volcano, (2) Los Azufres, (3) Indaparapeo, (4) Carindapaz and (5) Cerro Burro (Table 1).

Table 1 Geographical characteristics and community composition of oak species along the diversity gradient
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In each site, we sampled three independent transects of 100 × 40 m with at least 500 m of separation from each other. Within each transect, we recorded the number of individuals with a diameter at breast height (DBH) ≥ 10 cm of each of the oak species present. From these data, we calculated the oak species richness (OSR), the total density of oak individuals (TDOI; trees per hectare, including all species), the density of individuals belonging to the Lobatae section (DILS; trees per hectare including only the oaks trees of this section) and the density of individuals belonging to the Quercus section (DIQS; trees per hectare including only the oaks trees of this section). A full characterization of oak community composition is given in Supplementary Materials (Appendix 1).

Canopy insect herbivores sampling

Sampling of canopy insect herbivores was performed at the end of the rainy season (September) of 2014. In each study site, five mature medium-sized trees (DBH 30–40 cm) of Q. laurina were randomly selected to collect canopy insect herbivores using fogging techniques (Erwin and Geraci 2009). We choose these tree sizes because larger trees are harder to sample with this technique. Fogging was applied in all trees during the morning, between 05:00 and 6:00 A.M. Each tree was nebulized for a period of 10 min using a Swingfog SN-50 Thermal Fogger to disperse a mix of synergized pyrethrins (30 g/l) and piperonyl butoxide (150 g/l). Under each tree canopy, we place 8 funnel-shaped trays of 1m2 each one under tree canopy, and after 50 min, we performed the first insect collection (Barringer et al. 2019). The remaining insects were captured using ropes to shake the canopy after 2 h from the first insect collection (Marques et al. 2006). The insect herbivores collected were stored in 70% ethanol and transported to the Agroecology Laboratory of Universidad Michoacana de San Nicolás de Hidalgo, for taxonomic identification. Insect herbivores were separated and identified to the family level and were assigned to feeding group: (i) exophagous (i.e. leaf-chewing and sap-sucking) and endophagous insects (i.e. leaf-miners and gall-inducing insects) (Triplehorn et al. 2005; Ubick et al. 2017). Abundance and species richness were estimated using morphospecies criteria (recognizable taxonomic units) (Majer et al. 2000; Stiegel and Mantilla-Contreras, 2018).

Foliar chemical analysis

For the quantification of the secondary metabolites, in the same adult trees of Q. laurina selected to analyze the canopy insect herbivores, we collected 30 intact leaves and immediately stored in liquid nitrogen. The chemical metabolites quantified were as follows: total soluble phenols, total flavonoids, proanthocyanidins and galotannines. The total content of soluble phenolics was estimated using a modification of the Folin–Ciocalteau method (Torres et al. 1987; Maldonado-López et al. 2015). In the case of flavonoids, the samples were extracted with 80% ethanol. 0.1 ml of supernatant was diluted in 0.9 ml 80% ethanol. A 0.5-ml aliquot was placed in a tube, and 0.3 ml NaNO2 (1:20) were added. After 5 min, 3 ml AlCl3 (1:10) was added. Six min later, the solution was mixed and the absorbance was measured at 510 nm (Zhuang et al. 1992; Maldonado-López et al. 2015). The content of total flavonoids was calculated from a standard curve of quercitin.

For tannin quantification, samples were extracted with 70% aqueous acetone, allowed to stand for 1 h at room temperature with continuous stirring and centrifuged for 10 min at 3000 rpm. The acetone extract was reduced to the aqueous phase and then frozen and lyophilized. 0.1% ascorbic acid was used as conservative (Hagerman 1987; Maldonado-López et al. 2015). To quantify gallotannins, a modification of the method by Inoue and Hagerman (1988) was used. One ml of the samples suspended in 0.2 N H2SO4 was placed in a 2-ml tube and dissolved in 1 ml 1 M H2SO4. The samples were hydrolyzed for 4 h at 100 °C. A 0.1-ml aliquot of the hydrolysate was mixed with 0.3 ml of 0.667% methanlicrhodanine solution and 0.2 ml of 0.5 M KOH solution. After 2.5 min, the mixture was diluted to 5 ml with distilled water. The absorbance at 520 nm was read. The rhodanine assay was standardized with gallic acid (Cuevas-Reyes et al. 2017). Soluble proanthocyanidins (PAS) were measured using a modification of the method of Watterson and Butler (1983). Samples were resuspended in 50% methanol. 0.1 ml of extract and 0.6 ml of water were added to 6 ml 1-butanol/concentrated HCl (95:5, v/v). After mixing, the tubes were placed in a bath for 50 min at 95 C. The absorbance at 555 nm was measured. The standard curve was prepared with known concentrations of (+) catechin hydrate (Maldonado-López et al. 2015).

Herbivory measurements

To determine the herbivory levels by leaf-chewing insects, ten adult trees of Q. laurina were selected in each study site (five trees not previously sampled and the same five used for the analysis of the canopy insect herbivores). We used a stratified sampling in the canopy of each tree, collecting three branches at each canopy stratum (lower, medium and high). From each branch, 10 leaves were randomly selected (N = 30 leaves per individual and N = 300 leaves per site). We took a digital image of each leaf to calculate the total leaf area and the leaf area removed by insect herbivores using the program ImageJ 1.51j87 (https://imagej.nih.gov/ij/) (Aguilar-Peralta et al. 2020). The percentage of leaf area removed by leaf-chewing insects was calculated for each leaf by (leaf area consumed/total leaf area) × 100. Herbivory data were transformed using the logit transformation to meet normality (Warton and Hui 2011).

Statistical analysis

Canopy insect herbivores’ composition analysis

Canopy insect herbivore abundance and species richness were compared between study sites using a GLM analysis with a Poisson error distribution and a log link function. The study sites were used as the independent variable and insect herbivore abundance and species richness was considered as the response variables.

To evaluate the differences in the frequency of the insect herbivore orders between study sites, we performed a logistic regression analysis using the CATMOD procedure (SAS 2008) that is a general procedure for modelling categorical data. To obtain the frequency of each order of herbivorous insects and build this model, the abundance of herbivorous insects of each order per tree was added and divided by the total abundance per site and multiplied by 100. Because the distribution of the dependent variable data did not follow a normal distribution, a Poisson distribution with a logarithmic link function was used (Stokes et al. 2000).

A Pielou-evenness index was used at the insect herbivore order level to estimate insect herbivore diversity in each study site. This measure of equitability compares the observed Shannon–Wiener index against the distribution of individuals between the observed species which would maximize diversity (Wan et al. 2014). Additionally, a mixed-effect model was performed to determine the effects of the parameters of the oak community on the Shannon diversity index and the Pielou-evenness index. The values of the Shannon diversity index and the Pielou-evenness index were considered as response variables and the parameters of the oak community as explanatory variables. These analyses were performed using SAS ver. 11 (SAS 2000).

The richness of the canopy insect herbivore families of each study site was obtained by rarefaction curves using the program EstimateS 9.1.0 (Colwell 2013). The scale of the independent variable (X axis) was represented by the number of estimated individuals of insect herbivores in the canopy. For the application of the rarefaction method, a data set was standardized and compared using the number of individuals as the sampling effort (Gotelli and Colwell, 2001). A 95% confidence interval (CI) was used in each rarefaction curve to determine if the differences in richness between localities was a result of true richness or due to abundance. Also, rarefaction curves were compared among each study site using an ANOVA analysis (King and Tschinkel 2008). This analyses were performed in SAS ver. 11 (SAS 2000).

A GLM analysis was applied to determine the differences in canopy insect herbivore abundance and species richness between study sites for each feeding guild groups of insect herbivores. A Poisson error distribution and a log link function were used (Stokes et al. 2000). To evaluate the differences in insect herbivores’ composition between the five study sites, a Multivariate analysis was used. The five communities of insect herbivores were ordered by non-metric multidimensional scaling (NMDS) using an abundance similarity matrix and the Bray–Curtis index as a distance metric. After, a non-parametric permutation procedure (ANOSIM) was used to check whether phytophagous insect species’ composition differed among the five study sites. The ANOSIM was based on a binary matrix insect species occurrence in each study site, using the Bray–Curtis index as a distance metric and 5000 permutations (Hammer et al. 2001). Pairwise ANOSIMs were performed between all pairs of sites as a post-hoc test.

Foliar secondary metabolites and herbivory levels analysis

The following oak community parameters were calculated in all the following study sites: (i) the oak species richness (OSR), (ii) Shannon diversity index (H’), (iii) the total density of oak individuals (TDOI), (iv) the density of individuals belonging to the Lobatae section (DILS) and (v) the density of individuals belonging to the Quercus section (DIQS). A mixed-effect model was carried out to test the effects of the oak community parameters on the concentration of foliar secondary metabolites and herbivory. The model considers the oak community parameters as the independent variable and secondary metabolites’ concentration and the herbivory as the response variables. The study sites and trees were considered as a random factor. A LSMeans test was used for a posteriori comparisons of secondary metabolites concentration and the herbivory (SAS 2000).

We performed Spearman's correlation analyses to determine the relationship between the herbivory with the total phenols, flavonoids, proanthocyanidins and gallotanins. All these analyses were performed in SAS ver. 11 (SAS 2000). In addition, to evaluate the effect of the study site (oak diversity) on herbivory considering its covariance of secondary compounds, a series of analyses of covariance (ANCOVA tests) were performed (Maldonado-López et al. 2014). In the model, study sites were considered as explanatory variable, herbivory as response variable and total phenols, flavonoids, PAS and gallotanins as covariates (JMP statistical software version 15.1.0) (SAS Institute Inc., Cary, NC, USA).

Relationship between the oak community parameters and the insect herbivore diversity

To determine the effects of the parameters of the oak community on the canopy herbivore insect diversity, as well on the diversity of each insect herbivores feeding guild, we performed two mixed-effect models separately. In the first analysis, the abundance and richness of canopy insect herbivores were considered as response variables, while the explanatory variables were the parameters of the oak community. In the second mixed-effects model, the abundance and richness of each insect herbivore feeding guild (i.e. leaf-chewing, sap-sucking, leaf-miners and gall-inducing insects) were considered as the response variables, and the parameters of the oak community were used as independent variables. The study sites and trees were considered as a random factor in both analyses. In addition, Shannon's diversity index was excluded from the models and did not show significant effects for any of the mixed-effect models performed (Stokes et al. 2000).

Results

Insect herbivore community along the oak diversity gradient

We registered a total of 3,693 individuals in the canopy of Q. laurina individuals in all study sites: 1,909 in Tequila Volcano, 223 in Los Azufres, 544 in Indaparapeo, 488 in Carindapaz and 529 in Cerro Burro. These were separated into 485 different morphospecies grouped in 7 orders (Fig. 1) (See Appendix 1). The frequency of insect herbivore orders was different between study sites (χ2 = 928.38; d.f. = 4; P < 0.0001).Tequila Volcano, which was the site with higher oak species richness had a higher frequency of the Coleoptera (58.87%), Lepidoptera (16.34%), Hymenoptera (6.75%) and Thysanoptera (5.76%), while the site with lowest richness of oaks (i.e. Cerro Burro) had 36.95% of Coleoptera, 8.55% of Lepidoptera and (2.92%) of Hymenoptera (Fig. 1).

Fig. 1
figure 1

Differences in the frequency of insect herbivore orders associated with the canopy of Q. laurina along oak diversity gradient

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Shannon’s diversity index values per site at the order level were for Tequila Volcano = 2.41, Los Azufres = 2.19, Indaparapeo = 2.21, Carindapaz = 2.32 and Cerro Burro = 2.03. All study sites differed in the evenness of the abundance of orders according to Pielou-evenness index, which ranged from J = 0.62 in Tequila Volcano to J = 0.93 in Cerro Burro (Table 2). According to the mixed-effects model, the parameters of the oak community showed significant effects on the Shannon’s diversity index and the Pielou-evenness index. Only the OSR (F = 21.07; d.f. = 3; P < 0.0001) and DOLS (F = 14.34; d.f. = 3; P < 0.007) on the Shannon index and OSR (F = 8.23; d.f. = 3; P < 0.045) and DOLS (F = 14.3; d.f. = 3; P < 0.041) on the Pielou index showed significant effects (Table 3).

Table 2 Shannon’s diversity Index and Pielou-evenness index values in each study site
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Table 3 Results of mixed effects model for the effects of parameters of oak community (Oak species richness: OSR, Total density of all oak species: TDOS, Density of oaks Lobatae section: DOLS and Density of oaks Quercus section: DOQS) on the Shannon diversity index and the Pielou-evenness index
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The richness of the insect herbivore families was significantly higher in the Volcano Tequila, followed by Indaparapeo, Carindapaz, Los Azufres and Cerro Burro according the rarefaction analysis (Fig. 2). The ANOVA analysis of the rarefaction curves showed significant differences between study sites (F = 133.58, d.f. = 4, P < 0.0001), indicating a higher species richness of insect herbivore families at the Tequila Volcano site.

Fig. 2
figure 2

Rarefaction curves of canopy arthropods of Q. laurina for each of the study sites along the oak diversity gradient. The data were rarefied considering the number of individuals observed (white circles) to allow a valid comparison of arthropod family richness between the study sites

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Canopy insect herbivore abundance of Q. laurina (measured as the mean number of insect herbivores per tree) was different along the oak diversity gradient (χ2 = 799.03; d.f. = 4, P < 0.0001). Trees of Q. laurina in the Tequila Volcano harbored higher insect herbivore abundance (381.8 ± 1.73) than trees growing in Los Azufres (55.75 ± 0.30), Indaparapeo (108.8 ± 0.63), Carindapaz (97.7 ± 1.40) and Cerro Burro (105.8 ± 1.22). Similarly, the richness of insect herbivores per tree was greater in the Tequila Volcano (60.8 ± 2.62) than in Los Azufres (32 ± 1.03) and Indaparapeo (43.6 ± 5.67 SE) and lower in Carindapaz (27.4 ± 3.8) and Cerro Burro (29.2 ± 1.3) (χ2 = 104.5; d.f. = 4, P < 0.0001).

Overall, the insect herbivore feeding guilds were represented as follows: leaf-chewing (69.72%), sap-sucking (14.02%), gall-inducing (9.72%) and leaf-miners (6.52%) (For more details, see Appendix 2). The abundance of insect herbivore feeding guilds differed significantly between the study sites (χ2 = 460.04; d.f. = 4, P < 0.0001). Leaf-chewing insects had the higher abundance along the oak diversity gradient in the Tequila Volcano (311.2 ± 2.33) followed by Indaparapeo (85.8 ± 0.89) while the lowest abundance was detected in Los Azufres (28.25 ± 0.38). Gall-inducing insects were the second most abundant feeding guild in Tequila Volcano (38.6 ± 2.24). Contrary, Cerro Burro had the higher abundance of the sap-sucking insects in comparison with the other study sites (χ2 = 6.84; d.f. = 4, P < 0.018) (Fig. 3a).

Fig. 3
figure 3

Diversity patterns of feeding guilds of insect herbivores associated with canopy of Q. laurina. A Insect herbivore abundance guilds. B Species richness of insect herbivore guilds. Common letters identify means that were similar according to LSMeans test (P < 0.05)

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The species richness of each insect herbivore feeding guild also varied among study sites. The Tequila volcano had the highest species richness of leaf-chewing insects in comparison with the other study sites (χ2 = 102.78; d.f. = 4, P < 0.0001). Significant differences were also observed for leaf-miner insects (χ2 = 8.95; d.f. = 4, P < 0.008), gall-inducing insects (χ2 = 6.71; d.f. = 4, P < 0.0009) and sap-sucking insects (χ2 = 20.13; d.f. = 4, P < 0.0001) (Fig. 3b).

The similarity of insect herbivores community showed that NMDS ordination explained 90.5% of the variance between sampling points (axis 1 = 90% and axis 2 = 0.5%), where each point is a two-dimensional representation of the composition of insect herbivore species in a single tree based on global NMDS. The composition of insect herbivores associated to Q. laurina varied between the study sites (ANOSIM r = 0.28, n = 24, P < 0.002) (Fig. 4). The post-hoc pairwise ANOSIM tests showed different composition of insect herbivore species between all study sites. Particularly, the Tequila Volcano and Carindapaz had totally different assemblages in comparison with the other three study sites (P < 0.05).

Fig. 4
figure 4

Non-metric dimensional scaling (NMDS) ordinations illustrating similarity of insect herbivores’ taxonomic composition between the study sites along oak diversity gradient. Each point is a two-dimensional (axis 1 and axis 2) representation of insect herbivore species composition on an individual tree based on global, non-metric multidimensional scaling (NMDS)

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Chemical composition and foliar herbivory levels

The mixed-effects model showed significant effects of the parameters of the oak community on the herbivory percentage, as well on the secondary metabolites’ concentration. Particularly, TDOS (F = 12.2; d.f. = 21; P < 0.03) and DOLS (F = 11.4; d.f. = 20; P < 0.04) had significant effects on the percentage of herbivory (Table 4). In addition, we detected significant effects of all oak parameters on total phenols, as well as some effects of these parameters on the proanthocyanidins and gallotanins’ concentration (see Table 4). Particularly, OSR (F = 12.3; d.f. = 22; P < 0.03), TDOS (F = 9.8; d.f. = 21; P < 0.01) and DOLS (F = 19.8; d.f. = 20; P < 0.0001) had significant effects on the PAS. In the same way, the concentration of gallotanins was affected by OSR (F = 9.7; d.f. = 22; P < 0.04), TDOS (F = 22.5; d.f. = 21; P < 0.034) and DOLS (F = 19.5; d.f. = 20; P < 0.039) (Table 4).

Table 4 Results of mixed effects model for the effects of parameters of oak community (Oak species richness: OSR, Total density of all oak species: TDOS, Density of oaks Lobatae section: DOLS and Density of oaks Quercus section: DOQS) on the percentage of herbivory and secondary metabolites’ concentration
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Differences in the leaf secondary metabolites’ concentration between individuals of Q. laurina along the oak diversity gradient were detected according to LSMeans test (P < 0.05) (Table 5). Q. laurina trees growing in the site with the greatest oak species richness (i.e. Tequila volcano) had higher total phenols concentration, flavonoids, proanthocyanidins (PAS) and gallotanins’ concentration in comparison with Q. laurina individuals occurring in sites with less oak diversity (see Table 5).

Table 5 Differences in the mean foliar secondary metabolites concentration, as well the herbivory percentage between Q. laurina individuals growing along the oak diversity gradient according to LSMeans test (P < 0.05)
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The LSMeans test (P < 0.05) for herbivory showed that the amount of leaf area consumed by leaf-chewing insects differed significantly between the five study sites (see Table 5). Along the oak diversity gradient, Q. laurina trees growing in the Tequila volcano had higher herbivory levels in comparison to the other study sites.

In general, significant relationships were detected between the secondary metabolites’ concentration and herbivory levels according the Spearman's correlation analyzes. Particularly, a negative correlation was observed between herbivory and the total phenols (P =  – 0.41, P < 0.04), PAS (P =  – 0.4, P < 0.02) and gallotanins (P =  – 0.51, P < 0.0001). We did not find correlation among the herbivory and flavonoids (ρ =  – 0.2, P < 0.16). Additionally, the results of ANCOVA analyses showed that only gallotanins and PAS concentration covaried negatively with herbivory along oak diversity gradient (Table 6). No significant effects of total phenols and flavonoids on herbivory was found (Table 6).

Table 6 Results of analyses of covariance (ANCOVA tests) that show the effects of site, herbivory and secondary metabolites
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Oak community parameters and the insect herbivore diversity

The mixed-effects models showed that the oak community parameters had significant effects on the canopy insect herbivore diversity, as well on the diversity of each insect herbivores feeding guild. Particularly, OSR (F = 13.3; d.f. = 22; P < 0.0001), TDOS (F = 34.2; d.f. = 21; P < 0.0001) and DOLS (F = 568.3; d.f. = 20; P < 0.0001) showed significant effects on the insect herbivore abundance and richness (Table 7). The abundance of leaf-chewing was affected by three different parameters of the oak community: OSR (F = 14.3; d.f. = 22; P < 0.0001), TDOS (F = 45.3; d.f. = 21; P < 0.0001) and DOLS (F = 110.48; d.f. = 20; P < 0.0001). The richness of leaf-chewing insects was influenced for OSR (F = 10.3; d.f. = 22; P < 0.0001), TDOS (F = 12.16; d.f. = 21; P < 0.0001) and DOLS (F = 10.5; d.f. = 19; P < 0.0001) (Table 7). Similarly, gall-inducing insect abundance was significantly influenced for two parameters of the oak community: TDOS (F = 40.4; d.f. = 22; P < 0.0001) and DOLS (F = 44.67; d.f. = 20; P < 0.0001). The abundance of sap-sucking insects was influenced by DOQS (F = 7.36; d.f. = 20; P < 0.045). Finally, we did not find significant effects on leaf-miner insect abundance and richness (Table 7).

Table 7 Results of mixed effects models for the relationships of the parameters of oak community on the abundance and species richness of insect herbivores, as well on each herbivore insect feeding guild associated with the canopy of Q. laurina along the oak diversity gradient
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Leaf secondary metabolites and the insect herbivore community

The Canonical correspondence analyses (CCA) between foliar chemical defense (i.e. secondary metabolites) and the insect herbivore community along the oak diversity gradient showed significant correlations according to the Permutation test of all canonical axes (Trace = 0.11, P = 0.01). The first two axes explained 89.84% (axis 1 = 79.13% and axis 2 = 10.71%) of the spatial variation of the insect herbivore community relative to secondary metabolites, where total phenols, flavonoids, proanthocyanidins (PAS) and gallotannis had a strong correlation with the insect herbivore composition. Particularly, the insect herbivore community of the Tequila Volcano was more influenced by the flavonoids, proanthocyanidins (PAS) and gallotannis; being the leaf-chewing insects the most influenced by the presence of these secondary metabolites (Fig. 5).

Fig. 5
figure 5

Canonical correspondence analyses (CCA) between foliar chemical defense (secondary metabolites) and the insect herbivore community along the oak diversity gradient. Each point is a two-dimensional (axis 1 and axis 2) representation of insect herbivores’ composition on an individual tree

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Discussion

Effects of the oak diversity on insect herbivore community

Plant diversity effects on chemical defense composition and its potential links with insect herbivore community might be regulating plant–insect interactions along the plant communities (Koricheva et al. 2017, Ebeling et al. 2019). Some studies have evaluated the effects of the insect herbivore community on the composition of the chemical defense (Valencia-Cuevas et al. 2015; Visakorpi et al. 2019; Field et al. 2020). Our study analyzed the effects of the oak diversity on both leaf chemical defenses and the assembling of insect herbivore community. Overall, we found 485 different morphospecies of insect herbivores grouped into 7 orders that were associated with the canopy of Q. laurina along oak diversity gradient. Additionally, we detected that insect–herbivore species composition was different along the oak diversity gradient, as well as a greater insect herbivore species diversity on trees growing in sites with higher oak diversity such as the Volcano Tequila. These results can be explained by “The plant species richness hypothesis” proposed by Fernandes and Price (1988) that suggests that communities with greater plant diversity represent potential resources and niches available to be colonized by a greater number of insect herbivore species (“bottom-up effects”) (Ebeling et al.2018). Our results agree with others studies at the local scale that have shown a positive relationship between plant species richness and herbivore insect richness (Novotny et al. 2002; Cuevas-Reyes et al. 2004). Similarly, a greater diversity of insect herbivores present in highly diverse plant communities can increase the higher trophic levels as predators, affecting the community structure of arthropods (Haddad et al. 2011; Moreira et al. 2016; Randlkofer et al. 2018). Therefore, plant diversity can be a key factor that determines the arthropod diversity along diversity gradients (Knops et al. 1999; Haddad et al. 2011). We also found that the abundance and richness of insect herbivores of the canopy of Q. laurina were significantly correlated to some parameters of the oak community such as the abundance and the oak species richness, Shannon diversity index, as well as with the total density of oaks and the density of oak individuals of the Lobatae section. These results confirm the fact that oak communities with higher species richness represent a higher resources and niches availability to be colonized by a greater number of insect herbivores, where species richness of insect herbivores from the canopy of Q. laurina increases as more oak species are available. Our results accord with a meta-analysis involving 52 independent comparison (i.e. 18 correlations derived from gall-inducing insects and 34 from non-galling herbivores) (Araújo 2013), confirming that insect herbivore diversity increases as the diversity of plants increases.

Effects of the oak diversity on herbivory levels

In the same way, our results showed that the herbivory levels by leaf-chewing insects were higher in the Tequila volcano, which was the site with highest diversity of oak species. This result can be explained by the higher abundance of leaf-chewing insects registered in sites with highest oak diversity and that belonging to families of generalist insects (i.e. Pyrallidae, Lymantridae, Geometridae, Arctiidae). This fact is consistent with the idea that plant diversity is a key factor that may increase the plant susceptibility to insect herbivore attack according to the associational susceptibility hypothesis (ASH) (Brown and Ewel 1987; Barbosa et al. 2009) that proposes that an increase in plant diversity increases the damage by generalist insect herbivores due to a wider range of food resource availability to insects in more diverse plant communities (Unsicker et al. 2008; Plath et al. 2011). An alternative explanation is that Q. laurina trees were less preferred by this generalist insect herbivores, but growing in close proximity and surrounded by an extremely preferred as host plants (other oak species), which favors the incidence of generalist insect herbivores after depletion of the preferred host plant (Unsicker et al. 2008; Karban et al. 2010; Castagneyrol et al. 2014; Welti et al. 2017). For example, Castagneyrol et al. (2014) found that phylogenetic distance between the focal plant and alternative host plant species that occur in the same neighborhood is an important factor that determines the percentages of foliar damage caused by generalist insect herbivores, where more related host plants are more likely to share functional traits involved in host recognition and exploitation by insects (Gómez et al. 2010; Wiens et al. 2010) and, therefore, are more prone to share the same herbivores (Ødegaard et al. 2005; Weiblen et al. 2006).

Effects of the oak diversity on chemical foliar composition

It has been also proposed that the changes in foliar chemical composition affects the insect herbivore community to be the direct link between plants and insect herbivores (Zunjarrao et al. 2020). In our study, the foliar secondary metabolites’ concentration varied along the oak diversity gradient, where Q. laurina trees growing in sites with the highest diversity of oaks (i.e. Tequila Volcano and Los Azufres) had higher concentration of total phenols, flavonoids, PAS and gallotannins in comparison with less diverse oak sites such as Cerro Burro and Carindapaz.

According to the mixed models, we detected effects of the oak community parameters on foliar secondary metabolites such as total phenols, proanthocyanidins and gallotanins concentration. These results can be partially explained by “The growth-defense trade-off hypothesis” (Eisenhauer et al. 2009),which states that plants will allocate more resources for chemical defense in more diverse plant communities, due the competition for nutrients such as water and light increase in greater plant diversity communities, reducing plant growth. In our case, it is possible that some compounds such as total phenols and flavonoids may be more associated with other physiological functions such as leaf photoprotection and not act as a effective defenses against generalist herbivores because individuals of Q. laurina that occurred in sites of greater oak diversity had higher levels of herbivory and higher concentration of this secondary metabolites (Barbehenn and Constabel 2011). For example, Rosado-Sánchez et al. (2018) showed a negative effect of the tree diversity on total phenols and phenolic compounds in Swietenia macrophylla and Tabebuia rosea between two types of conditions (i.e. monoculture vs polyculture).

Another important result is the relationship detected between secondary metabolites’ concentration and the insect herbivory community along the oak diversity gradient. We detected that total phenols, flavonoids, PAS and Gallotannis concentration had a strong correlation with the insect herbivore composition, particularly with the leaf-chewing insect guild. This relationship could be explained by “The hypothesis of the specialist-generalist dilemma” (Van der Meijden 1996) that proposes that the concentration of secondary plant compounds in a plant depends on the proportion of generalist and specialist herbivores. In our case, the different proportion of generalist phytophagous (i.e. leaf-chewing insects) between the study sites may be indirectly driven by changes in concentration of chemical compounds associated with the defense against generalist herbivores, as a consequence of changes in the diversity of oaks along the diversity gradient. This result matches with Moreira et al. (2014), which reported increased production of anti-herbivores, such as phenolic compounds (i.e. total phenols) chemical defense and changes in herbivory levels on Swietenia macrophylla in sites with highest plant diversity.

Conclusions

Our study showed changes in insect herbivore diversity of the canopy of Q. laurina along the oak diversity gradient. The general pattern showed higher insect herbivore diversity in the Tequila volcano, which represented the site with the greatest diversity of oaks. Also, we detected an increment of the herbivory levels in more diverse oak communities, as well as changes in the composition of the insect herbivore community. Our findings confirm that at the local scale, oak species richness is the main factor that determines the insect herbivore diversity associated with canopy of Q. laurina, as well the herbivory levels and foliar chemical composition of Q. laurina. We highlight the relevance of conserving oak species because they are key elements of temperate forest in the North Hemisphere that harbor high diversity of insect herbivores and are important for the maintenance of biotic interactions in this ecosystem.

Author contribution statement

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by MSV-S, YM-L, JSA-P, MAZB, MLF, MF and PC-R. The first draft of the manuscript was written by MSV-S, YM-L, KO, GD, PC-R and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.