Comparison of Tree Genotypic Diversity and Species Diversity Effects on Different Guilds of Insect Herbivores

Comparison of tree genotypic diversity and species diversity effects on different guilds of insect herbivores

Luis Abdala-Roberts , Kailen A. Mooney , Teresa Quijano-Medina , Mar í a Jos é Campos-Navarrete , Alejandra Gonz á lez-Moreno and V í ctor Parra-Tabla

L. Abdala-Roberts ([email protected]) and K. A. Mooney, Dept of Ecology and Evolutionary Biology, Univ. of California, Irvine, 321 Steinhaus Hall, Irvine, CA 92697-2525, USA. – LAR, M. J. Campos-Navarrete and V. Parra-Tabla, Depto de Ecolog í a Tropical, Campus de Ciencias Biol ó gicas y Agropecuarias, Univ. Aut ó noma de Yucat á n, Apartado Postal 4-116, Itzimn á , MX-97000 M é rida, Yucat á n, Mé xico. – T. Quijano-Medina, Wageningen Univ., Droevendaalsesteeg 2, NL-6708 PB Wageningen, the Netherlands. – A. Gonz á lez-Moreno, Inst. Tecnol ó gico de Conkal, Km. 16.3 Antigua Carretera M é rida-Motul, MX-97345 Conkal, Yucat á n, M é xico.

Although the eff ects of plant diversity on herbivores are contingent upon herbivore traits and the source of plant diversity (e.g. intra- and interspecifi c), most studies have analyzed these eff ects separately. We compared the eff ects of genotypic diversity of big-leaf mahogany Swietenia macrophylla with that of tree species diversity on two specialist caterpillars ( Hypsipyla grandella stem borers and Phyllocnistis meliacella leaf miners) and three generalist leafhoppers (Cicadellidae) feeding on mahogany in a large-scale (7.2 ha) forest diversity experiment in southern Mexico. T e experiment consisted of fi fty-nine 21 ϫ 21-m plots, with 64 tree saplings each (3-m spacing between plants). Plots were either mahogany monocultures or species polycultures of four species (including mahogany) and – within each of these two plot types – mahogany was represented by either one or four genotypes. T roughout a fi ve-month period, beginning six months after planting, we measured mahogany growth and monitored herbivore and predator (spider) abundance. We found no eff ect of mahogany genotypic diversity on either specialist caterpillars or generalist leafhoppers, and this result was consistent across levels of tree species diversity. In contrast, species diversity had signifi cant eff ects on both specialists but neither of the generalist herbivores. Specifi cally, species diversity lowered H. grandella attack at the middle of the sampling season, but increased attack at the end of the season, whereas P. meliacella abundance was consistently reduced. Such eff ects were not mediated by eff ects of species diversity on plant growth (of which there were none), but rather through resource heterogeneity. Diversity did not infl uence spider abundance. T is study is one of few to directly compare sources of plant diversity, and uniquely compares such eff ects among herbivores with contrasting life histories (e.g. diet breadths). Overall, we demonstrate that plant species diversity eff ects outweigh those of genotypes, and our results suggest that such eff ects are stronger on specialist than generalist herbivores.

Evidence has mounted for the eff ects of intra- (reviewed shelters or prey) resulting in stronger top – down suppression by Bailey et al. 2009) and inter-specifi c (Siemann 1998, of herbivore populations and thus lower herbivory (reviewed Haddad et al. 2009) plant diversity on higher trophic levels. by Russell 1989). Alternatively, the ‘ resource concentra- Numerous studies have found eff ects of plant diversity on tion hypothesis ’ (RCH) (Root 1973) holds that herbivore arthropod species richness and abundance (Koricheva et al. foraging is density-dependent and increasing plant species 2000, Crutsinger et al. 2006, Haddad et al. 2009) and on diversity at a constant plant density reduces the probabil- consumptive interactions at higher trophic levels (Moreira ity of fi nding a preferred host plant, which lowers herbivore and Mooney 2013, Abdala-Roberts and Mooney 2014), recruitment and damage on individual plants. While with the basis of such eff ects being variation in ecologically both hypotheses have received considerable attention, evi- important traits among plant species or genotypes within dence from natural systems has generated mixed support species (Hare 2002, Mooney and Singer 2012). Within this (Bommarco and Banks 2003, Underwood et al. 2014). context, a widely documented pattern is that greater plant T e inconsistent support for the EH and RCH could diversity frequently leads to reductions in herbivory (reviewed be due to unaccounted variation in herbivores traits by Andow 1991, Barbosa et al. 2009). Two hypotheses have (Vehvil ä inen et al. 2007, Plath et al. 2012). For example, been off ered to explain this phenomenon and invoke the infl u- the dynamics predicted by the EH should be more likely to ence of resource heterogeneity on consumers. According to occur for generalist herbivores because they are more sus- the ‘ enemies hypothesis ’ (hereafter EH; Root 1973), greater ceptible to natural enemies as they frequently lack defense habitat complexity at high plant diversity favors increased mechanisms found in specialists (e.g. crypsis, sequestration predator recruitment (e.g. because of greater availability of of plant toxic compounds; Mooney et al. 2012, Singer et al.

EV-1 2014). Similarly, the dynamics predicted by the RCH should enced by tree species diversity than generalists, as the former also depend on herbivore dietary specialization (Root 1973); are more sensitive to changes in density of a particular host in this case, however, specialist herbivores should be (i.e. mahogany) across levels of species diversity. Similarly, negatively infl uenced by plant diversity whereas general- the eff ects of mahogany genotypic diversity (albeit weaker ist herbivores should exhibit weak (or variable) responses overall) are expected to be stronger on specialists because since they are not limited to feeding on a specifi c host plant of reductions in the density of a preferred mahogany geno- (Jactel and Brockerhoff 2007, Castagneyrol et al. 2013, type at high genotypic diversity, whereas generalists are less Salazar et al. 2013). In addition, other traits such as mobility infl uenced by genotype variation (and thus diversity) within and feeding mode are also thought to be important predic- any one tree species. Alternatively, following the EH, gen- tors of plant diversity eff ects on herbivores (Koricheva et al. eralist herbivores are expected to be more strongly aff ected 2000, Bommarco and Banks 2003). by diversity than specialists because the former are more sus- Plant diversity eff ects on herbivores may also vary depend- ceptible to enhanced eff ects by predators at high diversity. ing on the magnitude of underlying plant trait variation. We Overall, this study builds towards a predictive understanding expect that because trait variation is typically greater among of plant diversity – herbivory relationships and more broadly species than among genotypes within a species (Albert et al. of consumer responses to resource heterogeneity. 2010), plant species diversity should lead to stronger eff ects due to greater resource heterogeneity infl uencing predator (as predicted by the EH) or herbivore (as predicted by Methods the RCH) foraging behaviors. However, only two studies have tested this prediction and found that genotypic diver- Study system sity eff ects were equally (Cook-Patton et al. 2011) or more (Crawford and Rudgers 2013) important than species diver- Big-leaf mahogany and its herbivores sity in structuring arthropod communities. Further research Big-leaf mahogany Swietenia macrophylla (Meliaceae), the comparing various sources of plant diversity is necessary in target tree species and the component of genotype variation order to assess the relative strength and combined action of evaluated in this study, is a self-compatible, long-lived plant intra- and interspecifi c diversity eff ects on consumers. perennial tree distributed from southern M é xico to Bolivia We report on the results of a large-scale (7.2-ha, 4780 (Pennington and Sarukh á n 2005). Adult tree density of plants) tree diversity experiment testing for the eff ects of mahogany is low in tropical forests, but seedlings occur at big-leaf mahogany Swietenia macrophylla genotypic diversity higher densities and can be found in mono-specifi c patches in and tree species diversity on two groups of insect herbivores: forest gaps (Grogan et al. 2003). T e main herbivores of this immobile, mandibulate specialists that feed exclusively on species in tropical forests are insect leaf chewers (Norghauer mahogany, Hypsipyla grandella s t e m b o r e r s a n d Phyllocnistis et al. 2010) and small mammals (Grogan et al. 2003), meliacella leaf miners (hereafter ‘ specialists ’ ), and mobile, whereas in managed systems the most common herbivores phloem-feeding generalists (leafhoppers, Cicadellidae; are Hypsipyla grandella (Lepidoptera: Pyralidae) stem-boring hereafter ‘ generalists ’ ). In addition, the specialists are caterpillars, Phyllocnistis meliacella (Lepidoptera: Gracillarii- concealed or internal feeders whereas the generalists are dae) leaf-mining caterpillars and phloem-feeding leafhoppers external feeders. T erefore, eff ects of plant diversity on each (Cicadellidae). T ese two specialist caterpillars as well as the group may refl ect diff erences not only in diet breadth but generalist phloem-feeders were the most abundant groups also in other traits such as mobility and feeding mode. A of herbivores in our system, and we thus chose to focus on side from herbivores and to evaluate the EH, we also tested them in this study. for diversity eff ects on spiders, a major predator group in the Hypsipyla grandella is a specialist herbivore that only system which could mediate diversity eff ects on herbivores. feeds on a handful of species of Meliaceae (Newton et al. Sampling was conducted on tree saplings at an early time 1993). Larvae carve tunnels through the terminal shoots of point in the experiment, prior to direct interactions among saplings and juvenile plants, resulting in deformation of the widely-spaced plants. T us, any eff ect of diversity on main stem and reduced growth, and a single caterpillar can consumers would be mostly due to plant-based habitat produce multiple damage sites per plant (Mo et al. 1997). heterogeneity infl uencing consumer recruitment, rather Likewise, P. meliacella is a caterpillar that also specializes on than eff ects of increased plant biomass as a result of resource a few species of Meliaceae (Becker 1976, Arguedas 2007). partitioning or facilitative interactions among plants. First, Leafminer larvae produce characteristic serpentine galleries we predicted that the strength of diversity eff ects corre- throughout the leaf surface (Becker 1976). Finally, leafhop- sponds to the magnitude of plant trait variation underly- pers (Hemiptera: Cicadellidae) are frequently found feeding ing each source of diversity, i.e. species diversity exerts a on mahogany in disturbed sites (S á nchez-Soto et al. 2009). stronger eff ect on herbivores and predators than genotypic Most of the leafhopper species found in our study system diversity (predicted by both the EH and RCH). Second, are dietary generalists (Maes and Godoy 1993), and at the we predicted that genotypic diversity eff ects on these con- study site the most common species belong to the genera sumer groups are weaker at high species diversity because Homalodisca , Oncometopia and Pseudophera . Abundance and the eff ects of genotype variation are overridden by increased damage by these generalist leafhoppers and by specialist variation at the tree species level. T ird, our predictions for caterpillars is greatest during the rainy season, which spans the response of the two herbivore groups diff er between the from June to October. EH and the RCH. Following the RCH, we predicted that In tropical forests of the Yucatan Peninsula, big-leaf specialist herbivores are more strongly (negatively) infl u- mahogany co-occurs with fi ve other tree species (CICY

EV-2 2010) that were used to manipulate tree species diversity stem borer attack: 3.8-fold) (Supplementary material Appen- in this experiment, namely: Tabebuia rosea (Bignonaceae), dix 1 Table A1), and chemical defenses (polyphenolics: fi ve- Ϫ1 Ϫ1 Ceiba pentandra (Malvaceae), Enterolobium cyclocarpum fold variation 10.44 mg g t o 5 0 . 3 9 m g g ; F 5,50 ϭ 6 . 3 0 , (Fabaceae), Piscidia piscipula ( F a b a c e a e ) a n d Cordia dodecandra p Ͻ 0.0001; data from Moreira et al. 2014). As such, these (Boraginaceae). T ese species are long-lived, deciduous, and genotype identity eff ects represent the basis of expected adult trees generally reach from 20 m (P. piscipula ) to 40 m genotypic diversity eff ects on herbivores. In December 2011, ( C. pentandra ) in maximum height, depending on the we established the diversity experiment by planting four species (Pennington and Sarukh á n 2005), and are distrib- month-old seedlings (40 – 60 cm in height) at a site owned uted from central M é xico to Central and South America by INIFAP near Muna, Yucat á n (100 km southwest of (Pennington and Sarukh á n 2005). Mococh á ; 20° 24 ′ 44 ′ ′ N, 89 ° 45 ′ 13 ′ ′ W). Plants were fertil- ized once in January 2012 with N, P, and K (20:30:10), and Experimental design irrigated with 2 l of water three times per week from January 2012 until June 2012. Seedlings were sampled from June Across 7.2 ha we planted 74 plots of 21 ϫ 21 m each at 2012 until November 2012. a density of 64 plants per plot, 3 m spacing among trees, for a total of 4780 plants. Aisles between plots were 6-m Measurements of plant growth and consumer wide. T e experiment was established on a recently cleared abundance site where vegetation was composed mostly of grasses and shrubs, and is currently surrounded by a matrix of secondary We measured the size of mahogany plants at the start (June tropical forest. For this study, we restricted our sampling to 2012; all mahogany plants n ϭ 2480) and end (October the 59 plots where mahogany was planted (ignoring 15 plots 2012: randomly chosen subset n ϭ 944) of the rainy sea- without mahogany). T ese plots were classifi ed into four son. Plant size was estimated by measuring canopy height, types, depending on the diversity treatment combination: width, and length, and calculating the product of these three 1) mahogany monocultures of a single genotype (12 plots, measures which represented a proxy of canopy volume (m3 ). two replicate plots/genotype), 2) mahogany monocultures Given the short time since the establishment of the planta- of four genotypes (20 plots), 3) polycultures of four species tion (six months), we found no eff ects of mahogany geno- within which all mahogany saplings planted were of one typic diversity or tree species diversity on mahogany canopy genotype (12 plots, two plots/genotype), and 4) polycultures volume at the end of the growing season, suggesting weak of four species within which mahogany plants were repre- eff ects of diversity on plant biomass via plant – plant interac- sented by four genotypes (15 plots) (Supplementary material tions (analyses based upon plot-level-means; see results in Appendix 5 Fig. A1). Treatments of both species and geno- Supplementary material Appendix 2 Table A2). Likewise, typic diversity included equal numbers of individuals of four analyses showed no initial diff erences in mahogany can- species and four mahogany genotypes drawn randomly from opy volume between diversity treatments (Supplementary pools of six species and six genotypes, respectively. All non- material Appendix 2 Table A2). mahogany species were equally represented across polycul- We conducted three surveys of H. grandella stem borer tures (each species present in six polyculture plots). Likewise, attack throughout the rainy season of 2012, approximately mahogany genotypes were represented in a similar number every 45 days, in late July (early season), mid-September of mahogany monocultures of four genotypes (8 – 9 plots per (mid-season), and late October (late season). During each genotype), and also in a similar number of species polycul- survey, we inspected the entire canopy of each mahogany tures where mahogany plants were of four genotypes (9 – sapling (n ϭ 2480 plants, 59 plots) and recorded the number 10 plots per genotype). Plots of each treatment combination of new attack sites (following Taveras et al. 2004). At the were randomly interspersed throughout the experimental start of the sampling season, mahogany height and canopy landscape. volume were 76.04 Ϯ 1.21 cm and 0.17 Ϯ 0.01 m 3 , respectively (mean Ϯ SE). For P. meliacella leaf miners, we ran- Seed sources and collection domly selected 16 mahogany plants from each plot (n ϭ 944 plants, 59 plots), examined the entire canopy of each plant, From January 2011 to March 2011, we collected seeds of and counted the number of leaves with mines (in Ͼ 90% all species from adult plants located in southern Quintana of cases we observed one mine per leaf). We conducted this Roo (M é xico), and germinated at the INIFAP (Instituto survey only once at the end of the rainy season of 2012 (mid- Nacional de Investigaciones Forestales Agr í colas y Pecuar- October). Because mahogany leaves are long-lived, both old ias) campus in Mococh á , Yucat á n (M é xico) (21° 6 ′ 40 ′ ′ N, and new mines were present on a given plant at the time 89 ° 26 ′ 35 ′ ′ W). For all species, we collected seed from six of sampling, and our survey thus represented an estimate mother trees (distance among trees ranged from 0.5 to of cumulative leaf miner abundance throughout the rainy 50 km, depending on the species; 3 to 50 km for mahogany). season. Adults of this herbivore were identifi ed following In the case of mahogany, these seed source distances fall within Becker (1976). the range used by previous studies to defi ne distinct popula- We used the same plants surveyed for leaf miner tions of this species (Gillies et al. 1999, Loveless and Gullison abundance to record leafhopper (Cicadellidae) and spider 2003). Accordingly, we found signifi cant variation among abundance by examining the entire canopy of each mahog- mahogany maternal families (mixture of full- and half-sibs; any plant three times throughout the rainy season of hereafter referred to broadly as ‘ genotypes ’ ) in growth-related 2012 (late July, late September, and late October). Across traits (e.g. canopy size: 2.5-fold), herbivore resistance (e.g. tree species, sap feeders represented 42.9% of all recorded

EV-3 specimens of generalist herbivores (Campos-Navarrete either source of diversity and survey date, we performed unpubl.), and they were the most abundant guild of general- component models testing for diversity eff ects separately ist herbivores found on mahogany during the sampling sea- within each survey date. In the case of P. meliacella leaf son (representing 88.2% of all generalist herbivore specimens miners, we performed a general linear model with PROC recorded for this tree species). Oncometopia sp., Homalodisca GLM in SAS to test for eff ects of genotypic diversity, hambletoni and Pseudophera atra accounted for 45.3% of all species diversity, and their interaction on the plot-level mean generalist sap-feeding specimens and were observed feed- number of leaves with mines (i.e. average of all sampled ing on all tree species (Supplementary material Appendix 3 mahogany plants within each plot). Table A3; Campos-Navarrete unpubl.). T ese three generalist herbivores were the subject of this study and exhibited up Generalist herbivores to 15-fold variation in abundance among tree species thus We performed a general linear model with PROC GLM in emphasizing the importance of tree species identity eff ects SAS using the same model previously described for leaf miner (Campos-Navarrete unpubl.). T e genus and species of abundance. We summed leafhopper counts across species and these leafhoppers were identifi ed following Young (1968). surveys for each plant and used the mean value across plants On the other hand, spiders were the most abundant per plot as response variable. We did not use a repeated mea- predator group on mahogany (87% of predatory arthropods sures model because preliminary analyses showed no tempo- surveyed, excluding ants; Abdala-Roberts unpubl.). Although ral variation in the strength or direction of diversity eff ects ants were more abundant than spiders, some ant species on leafhopper abundance (i.e. non-signifi cant survey date ϫ tended leafhoppers for their honeydew and thus their role as species diversity and survey date ϫ genotypic diversity inter- predators or mutualists is diffi cult to assess (Abdala-Roberts actions; F2,112 ϭ 0.14, p ϭ 0.86 and F2,112 ϭ 0.89, p ϭ 0.41, unpubl.). Based on this, we chose to focus only on spiders respectively). Analyses performed separately for each of the to test if natural enemies mediate plant diversity eff ects on three leafhopper species yielded qualitatively identical results herbivores. Spiders were frequently observed feeding on leaf- relative to a model based upon pooled data across species hoppers, as well as moths and butterfl ies (Abdala-Roberts (Supplementary material Appendix 4 Table A4). unpubl.), the latter suggesting that they likely fed upon adults of the studied specialist stem borer and leaf miner. It is Spiders important to note that by sampling leafhoppers and spiders We performed a general linear model with PROC GLM only on mahogany (rather than sampling all tree species), in SAS using the same model previously described for leaf our test of species diversity eff ects on these generalist con- miners and leafhoppers. We summed spider counts across sumers addresses the infl uence of tree species neighborhood surveys for each plant and used the mean value across plants on one of the component species in the system rather than per plot as response variable. As for leafhoppers, we did the overall eff ect of species diversity on these consumers (i.e. not fi nd temporal variation in the strength or direction of across all tree species). In addition, while this study focuses diversity eff ects on spider abundance (i.e. non-signifi cant on diversity eff ects on herbivore and predator abundance survey date ϫ species diversity and survey date ϫ geno- we acknowledge that measuring eff ects on herbivory (while typic diversity interactions; F2,111 ϭ 0.16, p ϭ 0.85 and diff erent in nature and depending on the questions being F2,111 ϭ 1.13, p ϭ 0.32, respectively). asked) is desirable to obtain a more complete understanding of diversity eff ects on plant – herbivore interactions. Testing for non-additive effects of diversity Whenever a genotypic or species diversity eff ect was signifi - Data analyses cant in the above models, we determined if such eff ect was additive, where diversity eff ects were due to sampling eff ects, Specialist herbivores or non-additive, where diversity results in higher or lower We used a repeated measures general linear model with values relative to monoculture (Houston 1997, Tilman the MIXED procedure in SAS ver. 9.2 using plot as sub- et al. 2002). Following Johnson et al. (2006), we calculated ject, to test for the eff ects of mahogany genotypic diversity mahogany genotype means for each consumer group at low (fi xed, two levels), tree species diversity (fi xed, two levels), diversity (i.e. expected values), and compared these values to survey (fi xed, three levels), all two-way interactions, and the the mean of each genotype at high diversity (i.e. observed three-way interaction on the proportion of plants with new values). For mahogany genotypic diversity, we compared the H. grandella attack sites per plot during each survey. We used mean of each genotype across mahogany monocultures with a repeated measures analysis (as opposed to cumulative or one genotype and species polycultures with one mahogany mean values across surveys) because we observed a signifi - genotype (expected values) to observed values of the corre- cant change through time in the eff ects of diversity on stem sponding genotype in mahogany monocultures of four geno- borer attack. Although a parallel repeated-measures model types and species polycultures with four genotypes (observed using the plot-level mean number of attacks per plant, per values). For species diversity, we compared the mean of each survey yielded qualitatively similar results (not shown), the genotype across mahogany monocultures of one genotype frequency of attack sites does not provide a reliable estimate and mahogany monocultures of four genotypes (expected of stem borer abundance as a single Hypsipyla larva can pro- values) to observed values of the corresponding genotype duce multiple damage sites on a plant. T erefore, we only in species polycultures with either one or four mahogany report results for the proportion of attacked plants which is genotypes (observed values). We performed genotypic and in keeping with our goal of measuring eff ects on herbivore species diversity tests separately by comparing observed and abundance. If we found a signifi cant interaction between expected values with one-way general linear models using

EV-4 the MIXED procedure in SAS. T ese models also included a temporal shift in the eff ect of tree species diversity (Fig. the eff ects of plot and genotype nested within plot which 1B). Specifi cally, we found that during the second survey made them similar to a paired test comparing observed date species polycultures exhibited a 55% lower proportion vs. expected values for each genotype (Johnson et al. 2006). of attacked plants relative to monocultures (F1,53 ϭ 5.35, A signifi cant diff erence between observed and expected p ϭ 0.02) (Fig. 1B), while for the third survey this pattern values is necessarily due to non-additivity as the compari- reversed as polycultures exhibited a 24% higher proportion son is performed by specifying the monoculture values of of attacked plants relative to monocultures (F1,53 ϭ 4.20, each genotype (i.e. sampling eff ects are accounted for p ϭ 0.04) (Fig. 1B). T e negative eff ect of species diversity by including genotype-specifi c expected values). For stem on stem borer attack during the second survey date was non- borer attack, we performed these tests separately for each additive (observed versus expected: F 1,67 ϭ 1 0 . 6 4 , p ϭ 0.002), survey whenever a diversity eff ect was signifi cant on a whereas for the third survey the eff ect was additive (F 1,65 ϭ 2.66, given date. p ϭ 0 . 1 1 ) . T ere was no overall eff ect of genotypic diversity (genotype monocultures vs. plots with four genotypes) on General considerations stem borer attack as well as no evidence of a genotypic diver- For all models we used plot-level data and included plot- sity ϫ species diversity interaction (Table 1, Fig. 1A). level mean canopy volume (mean across plants per plot) as a T ere was a signifi cant eff ect of species diversity on the covariate to account for residual variation in mahogany size plot-level mean number of Phyllocnistis meliacella leaf infl uencing herbivore and spider abundance. If the genotypic mines per plant (Table 1), with species polycultures show- diversity ϫ species diversity interaction was signifi cant for ing a 48% lower mean value relative to mahogany monocul- the herbivore abundance or non-additivity models, we per- tures (Fig. 2A). Such reduction in leaf miner abundance formed component models testing for an eff ect of each source for species polycultures was greater than expected based of diversity separately within each level of the other source of upon leaf miner abundance in mahogany monocultures diversity. For all models, we used a normal distribution with (F 1,66 ϭ 7 . 5 1 , p ϭ 0.008), indicating non-additive eff ects the identity as link function. T e assumption of normality of species diversity on this specialist herbivore. In con- was met in all cases following verifi cation of residuals and trast, there was no eff ect of mahogany genotypic diversity Kolmogorov – Smirnov tests. In addition, although we found (Fig. 2A). Likewise, we found no evidence of a genotypic evidence of heteroscedasticity for the models testing for non- diversity ϫ species diversity interaction on leaf miner additivity of diversity eff ects, non-parametric Wilcoxon tests abundance (Table 1, Fig. 2A). showed qualitatively similar results (not shown). T us, we report results for parametric models only. We present least- Effects of plant diversity on generalist herbivores square means and standard errors as descriptive statistics and report results for type III sums of squares. Oncometopia sp. was by far the most abundant of the three focal leafhopper species feeding on mahogany (1470 individuals, 82.5% of specimens recorded), followed by Results H. hambletoni (177 individuals, 9.2%), and P. atra (161 specimens, 8.3%). Based upon pooled abundances across Effects of plant diversity on specialist herbivores species and contrary to the results for the two specialist caterpillars, there was no evidence of tree species diversity on T e mean percent of mahogany plants with new attack sites the abundance of generalist leafhoppers (Table 1, Fig. 2B). by Hypsipula grandella stem borers per plot was 18.5 Ϯ 2.0% Likewise, there was no eff ect of genotypic diversity, as well (mean and SE across surveys). Although there was no over- as no evidence of a genotypic diversity ϫ species diversity all eff ect of tree species diversity (mahogany monocultures interaction on leafhopper abundance (Table 1, Fig. 2B). As versus species polycultures) on the proportion of plants with mentioned previously, models conducted separately for each new attacks per plot (Table 1, Fig. 1A), we found signifi cant leafhopper species yielded equivalent results (Supplementary species diversity ϫ survey interaction (Table 1), indicating material Appendix 4 Table A4).

Table 1 . Effects of tree species diversity (SD) and big-leaf mahogany genotypic diversity (GD) on attack by specialists (S) stem-boring caterpillar and leaf-mining caterpillars, generalist (G) sap-feeding herbivores (leafhoppers), and spiders. Shown are F- (with numerator and denominator degrees of freedom) and p-values (in parenthesis). Signiﬁ cant (p Ͻ 0.05) results are in bold. Plant size ϭ canopy volume (measured in m3 ).

Response variable Predictor Stem borers (S) Leaf miners (S) Leafhoppers (G) Spiders

SD F1,53 ϭ 0.89 (0.35) F1,54 ϭ 8.26 (0.006 ) F1,54 ϭ 0.17 (0.69) F1,54 ϭ 0.73 (0.39) GD F 1,53 ϭ 0.22 (0.63) F1,54 ϭ 1.13 (0.29) F1,54 ϭ 0.01 (0.99) F1,54 ϭ 1.89 (0.17) SD ϫ GD F 1,53 ϭ 0.15 (0.69) F1,54 ϭ 0.006 (0.99) F1,54 ϭ 2.58 (0.11) F1,54 ϭ 0.45 (0.50) Time F2,105 ϭ 64.61 ( Ͻ 0.0001 ) ––– Time ϫ SD F2,105 ϭ 6.08 ( 0.003 ) ––– Time ϫ GD F2,105 ϭ 0.22 (0.80) ––– Time ϫ SD ϫ GD F 2,105 ϭ 1.42 (0.24) ––– Plant size F 1,53 ϭ 9.18 ( 0.003 ) F1,54 ϭ 22.17 ( Ͻ 0.0001 ) F1,54 ϭ 1.67 (0.20) F1,54 ϭ 7.84 ( 0.007 )

EV-5 Figure 1. (A) Eff ects of big-leaf mahogany Swietenia macrophylla Figure 2. (A) Eff ects of tree species diversity and big-leaf mahogany genotypic diversity and tree species diversity on the proportion of ( Swietenia macrophylla ) genotypic diversity on the abundance of mahogany sapling attacked by the specialist stem-boring caterpillar the specialist leaf-mining caterpillar Phyllocnistis meliacella attack- Hypsipyla grandella . Grand least-square means Ϯ SE for each level ing mahogany. (B) Eff ects of tree species diversity and big-leaf of species diversity (i.e. mahogany monocultures and species poly- mahogany (Swietenia macrophylla ) genotypic diversity on the abun- cultures) are shown on each side using a diff erent color code to dance of generalist leafhoppers (Cicadellidae) feeding on mahogany diff erentiate from levels of genotypic diversity (black ϭ low species (pooled data across three leafhopper species). Circles are plot-level diversity; white ϭ h i g h s p e c i e s d i v e r s i t y ; l i g h t g r e y ϭ l o w g e n o t y p i c least-square means Ϯ SE from a general linear model accounting diversity; dark grey ϭ high genotypic diversity). Symbols are off set for plant size. Symbols are off set for purposes of visual clarity. for purposes of visual clarity. (B) Eff ects of tree species diversity on Grand least-square means Ϯ SE for each level of species diversity the proportion of plants attacked by H. grandella s h o w n s e p a r a t e l y (mahogany monocultures and species polycultures) are shown on for each of three surveys conducted in July, September, and each side using a diff erent color code to diff erentiate from levels October of 2012. For both panels, circles represent plot-level least- of genotypic diversity (black ϭ low species diversity; white ϭ high square means Ϯ SE from a repeated measures analysis accounting species diversity; light grey ϭ low genotypic diversity; dark for plant size. For panel B, the asterisk indicates signifi cant diff er- grey ϭ high genotypic diversity). ences (p Ͻ 0.05) between levels of species diversity (mahogany monocultures and species polycultures) during a particular survey date. intra- and interspecifi c). Because of presumably weak plant – Effects of plant diversity on predators plant interactions at the time of insect monitoring, diversity eff ects on the specialist herbivores likely occurred not through T ere was no evidence of either tree species diversity (mon- increased mahogany growth via plant resource partition- ocultures ϭ 0.70 Ϯ 0.05 spiders; polycultures ϭ 0.76 Ϯ 0.06) ing or facilitation, but rather through the eff ects of greater or mahogany genotypic diversity (plots with one geno- habitat heterogeneity. We found that specialist herbivores type ϭ 0.78 Ϯ 0.06 spiders; four genotypes ϭ 0.68 Ϯ 0.05) (i.e. immobile stem borers and leaf miners) were respon- on spider abundance (Table 1). Likewise, the genotypic sive to tree species but not to mahogany genotypic diversity, diversity ϫ species diversity interaction on spider abundance whereas generalist herbivores (i.e. mobile leafhoppers) were was not signifi cant (Table 1). not infl uenced by either form of diversity. Because diversity eff ects were observed for specialists only, they likely operated Discussion directly on herbivore foraging behaviors (as predicted by the RCH) rather than indirectly through suppression by pred- Our study provides insight into how herbivore species vary ators (as predicted by the EH). T e lack of diversity eff ects on in their response to diff erent sources of plant diversity (i.e. spider abundance further supports this interpretation. EV-6 Effects of tree species diversity on herbivores caterpillars have relatively immobile larval stages whereas the generalist leafhoppers have highly mobile nymphs (in Had the eff ects of diversity operated via natural enemies addition to adults). Dispersal ability can infl uence herbivore (according to the EH), we would expect an increase in responses to plant diversity, with mobile herbivores respond- predation or parasitism at high diversity which would have ing more than sedentary herbivores because they can more in turn led to stronger reductions in the abundance of readily disperse and choose among plant patches (Bommarco generalist herbivores (relative to specialists) because these are and Banks 2003). While we cannot separate the eff ects of generally more susceptible to natural enemies (Bernays 1998, diet breadth and mobility, had the latter predominated then Mooney et al. 2012, Singer et al. 2014). However, we found this would have presumably led to a stronger eff ect of tree the opposite pattern, as only the specialists were infl uenced species diversity on (mobile) generalist leafhoppers which by tree species diversity. In addition, tree species diversity runs counter to our fi ndings. T is suggests that diet breadth did not infl uence spider abundance suggesting that diver- (and not dispersal ability) was responsible for the reduction sity eff ects on the specialist herbivores were not mediated in damage by stem borers and leaf miners at high diversity. by this predator functional group. Recent work conducted However, a robust evaluation of the linkage between plant at the study plantation has similarly shown no eff ect of tree diversity and herbivore diet breadth requires testing for eff ects species diversity on parasitism associated with P. meliacella on generalist and specialist species within the same taxon, as (mahogany monocultures: 49.1 Ϯ 11.2%; species polycul- well as controlling for other factors such as feeding guild or tures: 42.9 Ϯ 9.2%; Abdala-Roberts unpubl.), and previous feeding mode (Ali and Agrawal 2012, Plath et al. 2012). studies conducted in mahogany and Mexican cedar Cedrela odorata plantations located near our study site (Chan-Basto Effects of plant genotypic diversity on herbivores 2006) have found low parasitism rates for H. grandella (2 to 6%). Although these fi ndings suggest that predators Despite using a mahogany parental tree sampling scheme and parasitoids probably did not mediate the eff ect of tree that provided equal or greater variation than that found at species diversity on the specialist herbivores, we cannot the population level, and despite observing detectable varia- entirely rule out their infl uence. Further work is necessary tion among mahogany genotypes in growth and chemical to assess whether natural enemies become a predominant defenses (Moreira et al. 2014; data from this study), we force controlling herbivore populations in subsequent suc- found no evidence of mahogany genotypic diversity eff ects cessional stages as observed in previous forest studies (Jactel on herbivores. T is fi nding runs counter to previous work and Brockerhoff 2007). showing strong eff ects of plant genotypic diversity on arthro- T at species diversity aff ected specialist but not generalist pods (Crutsinger et al. 2006, Parker et al. 2010, McArt and herbivores is in keeping with the RCH, which predicts that T aler 2013). Such discordant results are perhaps not sur- specialists are more strongly infl uenced by plant diversity. prising given that we did not fi nd signifi cant variation among For the two specialist herbivores, we found species diver- mahogany genotypes for leafhopper or leaf miner abundance sity reduced P. meliacella leaf miner abundance (48%) and (only for stem borers). In addition, we speculate that geno- H. grandella stem borer abundance for the middle survey typic diversity eff ects on consumers were weak because of the (55%). With respect to stem borers, we speculate that attack spatial scale of our experiment (Bommarco and Banks 2003) occurred in accordance to the mechanisms predicted by the and how scale relates to the magnitude of plant trait variation. RCH at the middle of the sampling season, with greater In particular, the magnitude of plant trait variation needed to attack occurring at low diversity because of easier host loca- manifest a diversity eff ect must increase with increasing spa- tion under higher densities of mahogany. T is eff ect was non- tial scale (relative to herbivore movement). Because heteroge- additive supporting the idea that such diversity eff ect was neity in forest systems occurs at a greater spatial scale relative mediated by emergent patch-level properties (i.e. resource to past experiments with herbaceous species, our design may heterogeneity). T e subsequent reversal of this pattern straddle this threshold such that the eff ects of tree species may have been due to reduced host availability in the low- diversity were strong enough to matter at the studied scale diversity plots (which exhibited greater attack rates earlier). whereas genotypic diversity eff ects were not. At the same time, our fi nding of generalist leafhoppers Mahogany genotypic diversity eff ects may have also been being insensitive to species diversity runs counter to results weak because our study was conducted prior to genotypic from several previous studies showing positive eff ects of diversity aff ecting producer biomass via plant-plant interac- plant species diversity on generalist herbivores (Loranger tions (Crutsinger et al. 2006, Cook-Patton et al. 2011). T is et al. 2014). T ese eff ects have been attributed to diet mix- could be a particularly important factor in early successional ing (Singer et al. 2004, Unsicker et al. 2008), increased forests dominated by widely-spaced tree saplings (Schuldt encounter rates with preferred hosts (Salazar et al. 2013), or et al. 2011), where the eff ects of resource partitioning of greater plant biomass (Crutsinger et al. 2006, Cook-Patton plant biomass and of habitat heterogeneity on herbivores et al. 2011). Our results are unsupportive of this last mecha- and predators take longer to emerge relative to systems dom- nism in particular because there were no eff ects of diversity inated by herbaceous plants (Siemann 1998, Haddad et al. on tree sapling growth. Accordingly, if plant diversity eff ects 2009). At the same time, however, herbivore eff ects during on generalist herbivores typically operate via increased plant initial plant developmental stages can strongly infl uence sub- biomass, this might explain or failure to detect such eff ects sequent plant growth and survival, and examinations such as for leafhoppers. ours of plant – herbivore interactions early in the establish- T e studied herbivore groups varied not only in diet ment of forest systems are important from both a basic and breadth, but also in their degree of mobility; the specialist an applied (forestry) perspective.

EV-7 Concluding remarks Becker, O. B. 1976. Microlepid ó pteros asociados con Carapa , Cedrela y Swietenia en Costa Rica. – In: Whitmore, L. (ed.), T e eff ects of plant diversity in this study, particularly in the Studies on the shootborer Hypsipyla grandella (Zeller) Lep. case of specialist herbivores, were likely caused by eff ects of Pyralidae. Volume II. CATIE, Costa Rica, pp. 75 – 101. habitat (plant-based) heterogeneity and not due to either Bernays, E. A. 1998. Evolution of feeding behavior in insect positive eff ects of diversity on plant biomass or stronger herbivores: success seen as diff erent ways to eat without being eaten. – Bioscience 48: 35 – 44. top– down eff ects of predators. Within this context, our Bommarco, R. and Banks, J. E. 2003. Scale as modifi er in vegeta- fi ndings indicate that herbivore traits are important prediction diversity experiments: eff ects on herbivores and predators. tors of the eff ects of plant diversity on herbivore foraging – Oikos 102: 440 – 448. behavior. Accordingly, we speculate that the eff ects of plant Castagneyrol, B. et al. 2013. Plant apparency, and overlooked diversity on higher trophic levels may vary among systems driver of associational resistance to insect herbivory. – J. Ecol. depending on the degree of dietary specialization and mobil- 101: 418 – 429. ity within key life stages of the dominant herbivores in each CICY (Centro de Investigaci ó n Cient í fi ca de Yucat á n) 2010. Flora community. In addition, whereas plant species diversity was de la Pen í nsula de Yucat á n. – Cat á logo de Flora, Yucat á n, M é xico. Ͻ www.cicy.mx/sitios/fl ora%20digital/index.phpϾ , expected to have stronger eff ects on herbivores relative to accessed 8 April 2014. plant genotypic diversity, comparisons of these sources of Chan-Basto, F. 2006. Parasitismo de Hypsipyla grandella Zeller diversity might depend upon the spatial scale of analysis. (Lepidoptera: Pyralidae), Barrenador de las Meli á ceas en Plant- Overall, this study shows that resource heterogeneity eff ects aciones de Cedro (Cedrela odorata L.) en Yucat á n. – Masters on consumer behaviors represent a prevalent mechanism of thesis, Univ. Aut ó noma de Yucat á n, M é xico. plant diversity eff ects on herbivores, and that our ability to Cook-Patton, S. C. et al. 2011. A direct comparison of the conse- predict plant diversity– herbivory relationships depends on quences of plant genotypic and species diversity on communi- linkages between the magnitude of plant trait variation and ties and ecosystem function. – Ecology 92: 915 – 923. Crawford, K. 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Supplementary material (available online as Appendix oik.02033 at Ͻ www.oikosjournal.org/readers/appendixϾ ). Appendix 1 – 5.

EV-9