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Patterns of herbivory and leaf morphology in two Mexican hybrid oak complexes: Importance of fluctuating asymmetry as indicator of environmental stress in hybrid plants
Ecological Indicators 90 (2018) 164–170
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Original Articles
Patterns of herbivory and leaf morphology in two Mexican hybrid oak complexes: Importance of fluctuating asymmetry as indicator of environmental stress in hybrid plants
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Pablo Cuevas-Reyesa, Armando Canché-Delgadoa, Yurixhi Maldonado-Lópezb, , G. Wilson Fernandesc, Ken Oyamad, Antonio González-Rodrígueze a
Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo, Ciudad Universitaria, Morelia, 58030 Michoacán, Mexico CONACYT-Instituto de Investigaciones sobre los Recursos Naturales, Universidad Michoacana de San Nicolás de Hidalgo, Avenida San Juanito Itzícuaro SN, Nueva Esperanza, 58330 Michoacán, Mexico c Ecologia Evolutiva & Biodiversidade/DBG, C P 486, ICB/Universidade Federal de Minas Gerais, 31270 901 Belo Horizonte, MG, Brazil d Escuela Nacional de Estudios Superiores, Unidad Morelia, Universidad Nacional Autónoma de México, Morelia, Michoacán, Mexico e Instituto de Investigaciones en Ecosistemas y Sustentabilidad, Universidad Nacional Autónoma de México, Antigua Carretera a Pátzcuaro No. 8701, Col. Ex-Hacienda de San José de la Huerta, Morelia, 58190 Michoacán, Mexico b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hybrid complexes Quercus Leaf morphology Herbivory Fluctuating asymmetry Environmental stress
Interspecific hybridization is a prevalent process in plant species that may have different ecological and evolutionary consequences. Interactions with herbivorous insects may be altered because of hybridization among host plants. These changes result from the morphological, physiological and chemical traits expressed in hybrid individuals. Therefore, it is of interest to document the changes in traits such as leaf morphology and their consequences on patterns of herbivory by insects in hybrid complexes of plants. Another useful indicator that may serve to evaluate developmental instability resulting from genetic or environmental stress in hybrid plants is fluctuating asymmetry. In this study, we used two previously genetically characterized complexes of hybridizing Mexican oaks as models to compare and understand the relationships between leaf morphology, fluctuating asymmetry and herbivory levels in parental and hybrid individuals. Results indicated that in the Quercus affinis × Q. laurina complex, hybrid individuals show a distinct morphology in relation to the parental species, while in the Q. magnoliifolia × Q. resinosa complex, hybrids were similar to Q. resinosa. In both hybrid complexes, our results show that hybrid individuals have higher levels of fluctuating asymmetry and herbivory levels, which may reflect higher levels of genetic or environmental stress in comparison to the parental species. These results might help explain why oak species usually remain distinct despite the high frequency of hybridization characteristic of the genus.
1. Introduction Fluctuating asymmetry (FA) describes random morphological differences between the two sides of a bilateral character in organisms, and it is considered a good indicator of developmental instability caused by genetic and environmental stress (e. g., Palmer and Strobeck, 1986; Møller and Shykoff, 1999; Cornelissen and Stiling, 2005; CuevasReyes et al., 2011b). These morphological changes in plants (Palmer and Strobeck, 2003; Cornelissen and Stiling, 2010; Cuevas-Reyes et al., 2011a) can be caused by abiotic factors such as pollution (Zvereva et al., 1997; Cornelissen et al., 2003), nutrient or water stress (Freeman et al., 2004), and biotic factors as hybridization (Albarrán-Lara et al.,
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2010) and the incidence of pathogens and herbivores (Zvereva et al., 1997). Because the degree of FA reflects the inability of individuals to maintain homeostasis during development under stressful conditions (Møller and Swaddle, 1997), high levels of FA are usually correlated with low performance (i.e., growth, survival and fecundity) (Díaz et al., 2004; Møller, 1999; but see Clarke 1998). In this way, plants with high levels of FA could also be more susceptible to herbivores (e.g., Lempa et al., 2000; Cornelissen and Stiling, 2005; Cuevas-Reyes et al., 2011a), because of underlying differences in leaf nutritional quality and chemical defenses (Cornelissen and Stiling, 2005, 2011). However, other authors have proposed that herbivory can directly act as a plant
Corresponding author. E-mail address: [email protected] (Y. Maldonado-López).
https://doi.org/10.1016/j.ecolind.2018.03.009 Received 10 October 2016; Received in revised form 10 February 2018; Accepted 5 March 2018 1470-160X/ © 2018 Elsevier Ltd. All rights reserved.
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complexes, individuals analyzed here were the same used in previous genetic characterizations of the hybrid zone structure (Pérez-López et al., 2016; Ramos-Ortiz et al., 2016). The first hybrid complex is formed by the red oaks Quercus affinis × Quercus laurina. Quercus affinis Scheidweiler occurs along the Sierra Madre Oriental and is distributed along an altitudinal gradient from about 1600 to 2800 m (GonzálezRodríguez et al., 2004). This tree can reach 30 m in height, and its leaves are oblong lanceolate, 4.5–9 cm long, 1.5–2 cm wide, smooth on both sides, with toothed margin from the mid region to the apex (Rzedowski, 1978). Quercus laurina Humboldt et Bonpland is distributed along the Sierra Madre del Sur and in the Trans-Mexican Volcanic Belt. It is a tree growing up to 25 m with deciduous leaves, broadly lanceolate or oblanceolate, 7–10 cm long, 2.5–3 cm wide, smooth and shiny on the adaxial surface, undersides with woolly pubescence (Rzedowski, 1978). The hybrid zone between these two species is located in the eastern portion of the Trans-Mexican Volcanic Belt and northern Oaxaca (González-Rodríguez et al., 2004; Ramos-Ortiz et al., 2016). The second hybrid complex is composed by the white oaks Quercus magnoliifolia × Q. resinosa and is located at the Tequila volcano, Jalisco state, Mexico (20°50′ N, 103°5′ W). At this site, both plant species are distributed along an altitudinal gradient from about 1400 to 2100 m. Quercus magnoliifolia Née occurs between 1400 and 1800 m. It is a tree, growing up 25 m, with obovate leaves from 7.5 to 23 cm long and 3.5 to 13 cm wide. The adaxial surface is lustrous and almost glabrous, tomentose on the abaxial surface and with glabrescent petioles (Arizaga et al., 2009). Quercus resinosa Liebm occurs from 1700 to 2100 m. This tree can grow up to 20 m in height, it has large leaves 15–25 cm long and 5–16 cm wide. The leaves are obovate, rugose on the adaxial surface and pale-green or yellowish, tomentose on the abaxial surface and with densely tomentose petioles (Arizaga et al., 2009). The hybridization zone between the two species is located between 1600 and 1800 m at the Tequila volcano (Albarrán-Lara et al., 2010; Pérez-López et al., 2016). Herbivorous insect species attacking the plant species studied here have not been previously documented in the literature. However, during the study, we observed at least nine free-feeders insect species of different families such as Lepidoptera (Geometridae), Orthoptera (Acrididae) and Coleoptera (Chrysomelidae) responsible for most of the apparent damage in the Q. affinis × Q. laurina hybrid complex. In the case of the Q. resinosa × Q. magnoliifolia hybrid complex, we detected the presence of some free-feeders species of Lepidoptera (Geometridae, Noctuidae, Pyralidae, Phaloniidae, Gelechiidae, Lymantridae, Thyridiidae, Limacodidae, Arctiidae), Coleptera (Chrysomelidae), Hymenoptera (Formicidae, Pergidae) and Orthoptera (Gryllidae).
stressor, modifying the patterns of leaf symmetry and increasing FA levels (Díaz et al., 2004; Santos et al., 2013; Alves-Silva and Del-Claro, 2016). Fluctuating asymmetry may also increase as a result of hybridization because of the disruption of coadapted gene combinations (Leary and Allendorf, 1989). Natural hybridization is an important process in plant evolution that results in new genetic combinations by the introduction of semicompatible genes into another genome as consequence of interbreeding between two or more different species, which can produce fertile or infertile individuals called hybrids (Rieseberg, 1997; Martinsen et al., 2001). It has been proposed that insect herbivory on hybrid plants can potentially affect plant evolution modifying gene flow between hybrids (Pearse and Baty, 2012). In turn, other studies suggest that herbivore communities and herbivory levels can be affected by high genetic variation of hybrid plants that show a mosaic of phenotypic traits in comparison with their parental species (Rieseberg and Ellstrand, 1993; Arnold, 1997; Whitham et al., 2003; Floate et al., 2016). Therefore, differences in genetic, morphological and chemical traits among parental species and hybrids can influence insect herbivore communities, insect incidence and herbivory patterns in the hybrid plant complex (Driebe and Whitham, 2000; Cattell and Stiling, 2004; Bangert et al., 2006). Despite the high frequency of hybridization among Quercus species (González-Rodríguez et al., 2004; Tovar-Sánchez and Oyama, 2004; Pérez-López et al., 2016; Ramos-Ortiz et al., 2016) these usually remain distinct, suggesting that selection may act to decrease the number of hybrids that survive to reproduction (Howard et al., 1997; William et al., 2001). Because higher levels of herbivory on hybrids have been reported in many plant systems (Strauss, 1994; Whitham et al., 1999), herbivory may select against hybrid genotypes to maintain species integrity. However, several studies in sympatric oaks species indicated high levels of hybridization with viable and fertile offspring (Dodd and Afzal-Rafi, 2004; Hipp and Weber, 2008). A longer-term observation of the success of hybrids considering environmental filters as well as their interactions with natural enemies, such as herbivores, is crucial. Hence, to understand the ecology and evolution of hybrid complexes it is necessary to consider the negative effects of herbivory on plant growth and fitness (Maldonado-López et al., 2015) and, therefore their plant stressors. The formation of hybrid zones between Quercus species in Mexico is very common (González-Rodríguez et al., 2004; Tovar-Sánchez and Oyama, 2004; Albarrán-Lara et al., 2010; Pérez-López et al., 2016; Ramos-Ortiz et al. 2016). Depending on the introgression levels, hybrids may present a continuum of leaf shapes (Whitham, 1989); hence we postulate that it would also lead to different levels of FA on such areas. Therefore, hybrid oak complexes represent an ideal system to understand the relationship between plant-herbivore interactions and FA. In our study, we determined the patterns of herbivory, leaf morphology and fluctuating asymmetry in two previously genetically characterized hybrid oak complexes (Quercus affinis × Quercus laurina, and Quercus magnoliifolia × Q. resinosa) that occur in different regions of Mexico. We addressed the following questions: (1) Does leaf morphology differ between parental and hybrid plants in each hybrid complex? (2) Are hybrid plants more susceptible to herbivory in both hybrid complexes, and (3) Is leaf FA associated with the levels of herbivory?
2.2. Sample collection The studied individuals of the Quercus affinis × Quercus laurina complex were the same analyzed genetically by Ramos-Ortiz et al. (2016) in seven different localities: Tizapán, Hidalgo state (20.62° N, 98.6° W); Zacatlán, Puebla state (19.9° N, 97.95° W); Tonayán, Veracruz state (19.72° N, 96.9° W); Zoquitlán, Puebla state (18.28° N, 97.08° W); Pápalo, Oaxaca state (17.85° N, 96.8° W); Lachao, Oaxaca state (16.22° N, 97.13° W) and Suchixtepec, Oaxaca state (16.06° N, 96.48° W). In each population, 15 trees were studied. After the previous genetic analysis, individuals in the Tizapán and Zacatán populations were assigned as Q. affinis except for one and two individuals, respectively, that were assigned as hybrids. In Tonayán population there were six Q. affinis individuals, four Q. laurina individuals and five hybrids, while in population Zoquitlán there were 12 Q. laurina individuals and three hybrids. In the three southernmost populations all individuals were assigned as Q. laurina (see Ramos-Ortiz et al., 2016 for more details). In the second oak hybrid complex, from the individuals analyzed genetically by Pérez-López et al. (2016), we randomly selected a set of 30 trees at each of three different altitudes, 1400–1500, 1600–1800 and
2. Materials and methods 2.1. Study system This study was conducted in different regions of Mexico where the two hybrid oak complexes are located. Both regions are characterized for the presence of deciduous or brevideciduous oak species that occur in seasonal temperate forests (Hernández-Calderón et al., 2013; PérezLópez et al., 2016; Ramos-Ortiz et al., 2016). For both hybrid 165
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configuration of all leaves as reference and then, we calculated the shape variables (Procrustes distances) based on superimposition coordinates to eliminate the effect of leaf size (Cuevas-Reyes et al., 2011a). Finally, in each oak hybrid complex, we applied a principal components analysis to determine the differences in leaf morphology between the three groups of plants considering the configuration of all leaves. This type of analysis has been used to classify species, distinguish hybrid groups, locating the most likely parent of a known hybrid with several possible parents (Hardig et al. 2000; Viscosi 2015).
1900–2100 m. The previous genetic characterization of these individuals indicated that at the lower altitude, 25 individuals were Q. magnoliifolia and five were hybrids, while at the intermediate altitude there were eight Q. magnoliifolia individuals, 17 hybrids and four Q. resinosa individuals, and at the higher altitude there were nine Q. resinosa and 21 hybrids. Sample collections were conducted during 2014, at the end of the rainy season after the peak of herbivore activity (October–November). For each individual the leaf sampling was random. Because the studied species are deciduous or brevideciduous, all collected leaves correspond to the same cohort. Three branches were collected from the top, intermediate, and bottom strata of the canopy of each tree, sampling a minimum of 100 fully developed leaves per individual (Cuevas-Reyes et al., 2004a,b). To control for plant size, the stem diameter at breast height (dbh) was measured for each individual sampled (Cuevas-Reyes et al., 2006). In all cases, leaves from each individual were separated into two categories: a random set of leaves was used to estimate the herbivory level on each tree; and a set of undamaged leaves, which was used for morphometric and fluctuating asymmetry analysis (CuevasReyes et al., 2011a).
2.4. Fluctuating asymmetry measurements Fluctuating asymmetry was calculated in approximately 50 fully expanded intact mature leaves of each tree sampled in the two hybrid oak complexes. A digital image was taken for each leaf and then we measured the distance from the right side and left side from the leaf edge to the midvein at the midpoint of the leaf corresponding to its widest part (Fig. 1b). FA was estimated as the absolute value of the difference between the distances from the midvein to the left and right margins of the leaf (|Ai − Bi|) divided by the average distance (Ai + Bi/ 2) to correct for the fact that asymmetry may be size-dependent (Cornelissen and Stiling, 2005; Cuevas-Reyes et al., 2011a). We obtained an individual FA value of each tree from the average of the 50 leaves analyzed. A subsample of 25 leaves combined across all trees sampled within a hybrid complex was re-measured to control the measurement error in FA. Then, we checked the significance of FA relative to measurement error using a two-way mixed-model ANOVA. This model considers as factor individual, leaf (random) and side (right or left). Both measurements were considered as replicates (Palmer and Strobeck, 2003; Cuevas-Reyes et al., 2011a). The significance of the interaction (individual × leaf × side) indicated that variation in FA was greater than expected by measurements error (F24, 50 = 24.9; P < 0.0001). According to Palmer and Strobeck (2003), it is necessary to discriminate FA from other kinds of asymmetry. Fluctuating asymmetry, directional asymmetry, and antisymmetry represent the three types of deviation from the perfect bilateral symmetry of the organisms (Albarrán-Lara et al., 2010; Cuevas-Reyes et al., 2011a). FA represents the variance in left–right (L-R) differences distributed with a mean value of zero, whereas in directional asymmetry, the L-R differences are distributed about a mean that is significantly greater or less than zero, reflecting a consistent bias of a character towards greater development of one side than the other. Finally, antisymmetry is the lack of symmetry represented by bimodal or platykurtic distribution of L-R differences about a mean of zero (Van Valen, 1962). Hence, FA reflects random variation from the perfect symmetry of the bilateral trait, whereas, directional asymmetry and antisymmetry are considered as inappropriate descriptors of developmental instability, because they are developmentally controlled and are probably adaptive (Palmer and Strobeck, 1986). To confirm that our data reflected only FA and no other types of asymmetry, we checked for the directional asymmetry using a Student’s t test considering whether the average L-R difference value differed from zero. In the same way, to test for antisymmetry the Lilliefors’ normality test was performed considering the distribution of L-R differences (Alves-Silva and Del-Claro, 2016). Because in our data L-R differences followed a normal distribution and the mean value of L-R differences did not deviate significantly from zero (t = 2.3 P > 0.05), the values were considered to reflect FA exclusively.
2.3. Morphometric analysis of leaves We obtained digital images of approximately 50 intact leaves with no visible damage along the margins per tree to determine the differences in leaf morphology and size between the three groups of plants (i.e., the two parental species and the hybrids) in each oak complex. In each digital image, 32 landmarks and two additional landmarks were placed as reference of size (see Fig. 1). All landmarks correspond to homologous loci, which are unambiguous and repeatable marks in all the leaves, representing their shape (sensu Bookstein, 1991; CuevasReyes et al., 2011a). We used the TpsDig program (Rohlf, 2015) to record the coordinates (x, y) of the 32 landmarks in each leaf image. A Procrustes superimposition analysis was used for the configuration of the coordinates of each landmark using the CoordGen6 program, in the Integrated Morphometrics Package (IMP series: http://www.canisius. edu/~sheets/morphsoft.html). We considered the average
2.5. Measurement of herbivory To estimate the leaf area consumed by folivores, we examined approximately 50 leaves from each individual the two hybrid oak complexes. First, we took a digital image of each leaf sampled and estimated the total leaf area and the area consumed by folivorous insects using the Image analysis software for plant disease quantification (Assess: Image
Fig. 1. Morphometric measurements of leaves. Digital image of a leaf of Q. laurina showing the 32 morphological landmarks along the leaf margin. Two additional landmarks (33 and 34) were added on a reference ruler as a scale (a). Representation of the measurements used to estimate leaf FA in Q. affinis. RW right width, LW left width (b).
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Analysis Software for Plant Disease Quantification, APS Press, Saint Pail, MN). In all cases, the normality was tested after suitable transformations. Herbivory data were transformed as arc-sine square root (Pascual-Alvarado et al., 2008). 2.6. Statistical analyses Because in both oak hybrid complexes there were several sampling sites (i. e. seven sites across a latitudinal gradient in the Q. affinis × Q. laurina complex and three different altitudes in the Q. resinosa × Q. magnoliifolia complex), we used two-way ANOVA tests to assess the effects of site and plant group (two parental species and hybrids) on FA and herbivory levels. Because not all plant groups were present at all sites in the two hybrid oak complexes, the interaction between FA and herbivory was not considered. Finally, we used Spearman’s rank correlation analyses to determine the relationships between FA, herbivory, foliar area and DBH, separately within each plant group (i.e., two parental species and hybrids), for each hybrid oak complex. 3. Results The degree of leaf shape variation based on mean configuration of superimposition coordinates, showed that leaves of Q. laurina were more elongated and wider than leaves of the hybrids and Q. affinis
Fig. 3. Differences in leaf shape morphology between parental species and hybrid plants in two Mexican hybrids oak complex according to principal components analysis. Gray circles: Q. affinis. Black crosses: Hybrids. Gray stars: Q. laurina (a). Gray circles: Q. resinosa. Black crosses: Hybrids. Gray stars: Q. magnoliifolia: (b).
(Fig. 2a). In the second hybrid complex, leaves of hybrids were more elongated and wider than Q. resinosa and Q. magnoliifolia (parental species) (Fig. 2b). In addition, the scatter plot of PC1 and PC2 scores of Q. affinis × Q. laurina complex accounting for 44.0% and 27.1% of total variance respectively; and separates three distinct groups: Q. laurina with a minimum overlap from Q. affinis, and hybrids totally separated from these two species (Fig. 3a). In the second oak hybrid complex, the scatter plot of PC1 accounted for 96.4% of total variance and PC2 for only 0.1%. Q. resinosa was separated, with a small overlap from hybrids and Q. magnoliifolia was a different group as shown by the principal components analysis (Fig. 3b). In the Q. affinis x Q. laurina complex, both sites (F = 11.02; d. f. = 6, P < 0.0001) and plant group (F = 3.57; d. f. = 2; P = 0.03) had a significant effect on FA levels. Mean ( ± standard error) FA in Q. laurina individuals was 0.17 ± 0.006, which did not differ from the mean value in Q. affinis individuals (0.17 ± 0.009), but which was significantly lower than the mean value in hybrid individuals (0.26 ± 0.017). Similarly, in the Q. resinosa × Q. magnoliifolia complex, both site (altitude) (F = 55.37; d. f. = 2; P < 0.0001), and plant group (F = 3.97; d. f. = 2; P = 0.022) had a highly significant effect on FA levels. FA was lower in Q. magnoliifolia (0.11 ± 0.01) than in Q. resinosa (0.15 ± 0.01), the hybrids (0.17 ± 0.008), which did not differ between each other. Leaf area consumed by folivorous insects differed significantly among sites in the Q. affinis × Q. laurina complex (F = 46.98; d. f. = 6; P < 0.0001) and among plant groups (F = 3.8; d. f. = 2; P = 0.026).
Fig. 2. Leaf morphological variation between the three groups of plants in each hybrid oak complex: mean shape of coordinates of landmark configuration of leaves. Q. affinis × Q. laurina (a) and Q. resinosa x Q. magnoliifolia (b).
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species (Whitham et al., 2003; González-Rodríguez et al., 2004). In the Q. resinosa × Q. magnoliifolia complex, we found variation in leaf morphology, but hybrid plants had a similar leaf shape to the parental species Q. resinosa. Resemblance of hybrids to one of the parental species is a common observation that can be explained by dominance (Rieseberg et al., 2003). Herbivore preference, performance, and distribution in hybrid complexes (Tovar-Sánchez et al., 2013; Pérez-López et al., 2016), are influenced by plant attributes modified in plant hybridization conditions, such as morphology, growth, physiology and chemical defense (Rehill et al., 2005; Bangert et al., 2006; Travis et al., 2008; Cheng et al., 2011; Schweitzer et al., 2004). In the same way, taxonomic relationships between parental species, age of the hybrid complex, structure and assemblages of insect herbivores may affect the herbivory patterns in hybrid complexes (Boecklen and Spellenberg, 1990; TovarSánchez and Oyama, 2006; Pearse and Hipp, 2009). In our study, despite the effect of sampling at different sites, we found that leaf FA was significantly greater in leaves of hybrids than in leaves of parental species (or at least one parental species), in both hybrid oak complexes. Because the degree of divergence among parental species and hybrids influences hybrid performance (Hochwender and Fritz, 1999), high values of fluctuating asymmetry can be associated to a disruption of coadapted gene complexes and by the interaction among genes that functionally diverged between species, resulting in hybrids with higher levels of fluctuating asymmetry (Albarrán-Lara et al., 2010). In parallel, we found that hybrid individuals received in general a higher level of damage by herbivorous insects than parental species individuals, even though this difference was only significant in the Q. affinis × Q. laurina complex. However, when we analyzed the correlation between the percentage of leaf area removed and FA separately within plant groups (Table 1) we found a consistent positive correlation between the two variables within the hybrid class in the two oak hybrid complexes. This correlation was also observed for parental individuals in the Q. magnoliifolia × Q. resinosa complex, but not in the Q. affinis × Q. laurina complex. Therefore, in general we found evidence that plants with more asymmetric leaves experience greater herbivore consumption. Differences in leaf morphology, leaf size and shape between parental species and hybrid plants have explained the variation in the levels of consumption by herbivores and insect distribution (Driebe and Whitham, 2000; Cattell and Stiling, 2004). It is possible that leaves with higher FA also have a higher nutritional quality and lower concentrations of secondary metabolites compared with more symmetric leaves, as has been demonstrated in other species of Quercus (Cornelissen and Stiling, 2005). Also, plant hybridization can disrupt the chemical resistance of plants against herbivore insects in different ways: no differences between hybrids and parental species, additive or intermediate resistance in hybrids, dominance (similar resistance of hybrids to one of the parent species) and hybrid susceptibility (Fritz et al., 1999). Different hypotheses have been proposed to understand the significance of plant interspecific hybridization on plant-herbivore interactions (“Additive hypothesis”, Boecklen and Spellenberg 1990; “Dominance hypothesis”, Fritz et al., 1994; “Hybrid resistance hypothesis”, Boecklen and Spellenberg, 1990 and “Hybrid depression hypothesis”, Arnold and Hodges, 1995). Our results support the “hybrid susceptibility hypothesis” also called the “hybrids-as-sinks hypothesis” (Whitham, 1989), which states that hybrid plants are more susceptible to herbivores or parasites than their parental plants, and act as sinks for herbivore populations (Whitham, 1989). We found that folivory levels were higher on hybrid plants in comparison with their parental species in both hybrid oak complexes studied. Interestingly, in a previous study, conducted in the same individuals of the Q. magnoliifolia × Q. resinosa complex, we found higher diversity of gall morphospecies in hybrids than in both parental species (Pérez-López et al., 2016). Our results are also consistent with the “hybrid depression hypothesis” (Arnold and Hodges, 1995) that proposes higher levels of
Table 1 Spearman’s correlation coefficients of total leaf area, DBH, herbivory and fluctuating asymmetry (**P < 0.05). Q. affinis × Q. laurina (a) and Q. resinosa × Q. magnoliifolia (b). (a) Plant species Q. affinis
Hybrids
Q. laurina
Leaf area removed
FA
FA Total leaf area (cm2) DBH (cm)
0.17 0.51**
0.66**
0.11
0.08
FA Total leaf area (cm2) DBH (cm)
0.91** 0.08
0.14
0.15
0.11
FA Total leaf area (cm2) DBH (cm)
0.13 0.48**
0.71**
0.14
0.13
Total leaf area
0.46**
0.22
0.17
(b) Plant species
Q. resinosa
Hybrids
Q. magnoliifolia
Leaf area removed
FA
FA Total leaf area (cm2) DBH (cm)
0.63** 0.22
0.21
0.07
0.11
FA Total leaf area (cm2) DBH (cm)
**
FA Total leaf area (cm2) DBH (cm)
0.76 0.12
0.15
0.15
0.15
Total leaf area
0.36**
0.47**
**
0.54 0.18
0.21
0.11
0.13
0.37**
Percentage herbivory did not differ between the two parental species (16.2 ± 1.5 in Q. laurina, 19% ± 2.3 in Q. affinis), but was significantly higher (34.8 ± 4.3) in the hybrids. In contrast, in the Q. resinosa × Q. magnoliifolia complex, percentage herbivory differed significantly among altitudes (F = 36.07; d. f. = 2; P < 0.0001) but the effect of plant groups was not significant (F = 2.47; d. f. = 2; P = 0.09). In this case, herbivory was higher at the intermediate altitude (18.5 ± 1.1) than in the higher and lower altitudes (7.6 ± 1.15 and 8.3 ± 1.4, respectively). A positive relationship was found between leaf area consumed and FA in the hybrid group of the Q. affinis × Q. laurina complex, but not in the parental species individuals (Table 1). Instead, leaf area consumed was positively correlated with total leaf area in both Q. affinis and Q. laurina. In contrast, in the Q. magnoliifolia × Q. resinosa hybrid complex, leaf area consumed and FA levels were positively correlated in all three plant groups and there was no correlation with total leaf area. 4. Discussion The presence of hybrid zones in the genus Quercus is common (Spellenberg, 1995; González-Rodríguez et al., 2004; Pérez-López et al., 2016). Hybridization in oaks has been detected using molecular markers and morphological characters of leaves (González-Rodríguez et al., 2004; Tovar-Sánchez and Oyama, 2004; Albarrán-Lara et al., 2010; Pérez-López et al., 2016). In our study, we found different patterns of leaf morphology in the two hybrid oak complexes using geometric morphometrics methods. In the Q. affinis × Q. laurina complex, leaf morphology of hybrid plants was statistically different to their parental species. Different phenomena can account for these differences, such as transgressive segregation or gene-environment interactions, resulting in the differentiation of leaf shape between hybrids and their parental 168
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herbivory on hybrids than parental species as a selection mechanism that promotes maintenance or even speciation events. In this case, herbivore adaptation to new or alternative host plant species may lead to reproductive isolation mechanisms and genetic differentiation (hostassociated differentiation) (Dickey and Medina, 2010). For example, if hybrids are sufficiently different from parental species, is possible to expect adaptation, reproductive isolation and host associated differentiation in herbivorous insects, and therefore, more herbivory levels on hybrids than on parental species (Evans et al., 2008, 2012). 5. Conclusions Our results show that hybridization in both complexes of Mexican oaks has important consequences on plant insect interactions, increasing both herbivory and FA levels in hybrid plants. This emphasizes the role of hybrid individuals as potential sinks or bridges for the colonization of host plants by herbivorous insects. Therefore, we provide additional information showing the importance of preserving the genetic diversity of plants as a way of conserving dependent animal species. Finally, our study shows the importance of fluctuating asymmetry as indicator of genetic and environmental stress. Acknowledgments This project was supported by CONACYT project CB105755 and DGAPA-PAPIIT-UNAM project RV201015. Cuevas-Reyes P thanks Coordinación de la Investigación Científica UMSNH for their generous support, and GWF thanks CNPq and FAPEMIG for support. We also thank Fidel Anguiano for editing figures. Declaration of interest All authors declare that we have any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, our work. References Albarrán-Lara, A.L., Mendoza-Cuenca, L., Valencia-Avalos, S., González-Rodríguez, A., Oyama, K., 2010. Leaf fluctuating asymmetry increases with hybridization and introgression between Quercus magnoliifolia and Quercus resinosa (Fagaceae) through an altitudinal gradient in Mexico. Int. J. Plant Sci. 171, 310–322. http://dx.doi.org/10. 1086/650317. Alves-Silva, E., Del-Claro, K., 2016. Herbivory-induced stress: Leaf developmental instability is caused by herbivore damage in early stages of leaf development. Ecol. Indic. 61, 359–365. Arizaga, S., Martínez-Cruz, J., Salcedo-Cabrales, M., Bello-González, M.A., 2009. Aspectos generales de los encinos. In: Arizaga, S., Cruz, J.M., Cabrales, M.S., González, M.A.B. (Eds.), Manual de la biodiversidad de encinos michoacanos. Secretaría de Medio Ambiente y Recursos Naturales (Semarnat), Instituto Nacional de Ecología (INESemarnat), México, D., pp. 12–141. Arnold, M.L., 1997. Natural Hybridization and Evolution. Oxford University Press, New York. Arnold, M.L., Hodges, S.A., 1995. Are natural hybrids fit or unfit relative to their parents. Trends Ecol. Evol. 10, 67–71. Bangert, R.K., Turek, R.J., Rehill, B., Wimp, G.M., Schweitzer, J.A., Allan, G.J., Bailey, J.K., Martinsen, G.D., Leim, P., Lindroth, R.L., Whitham, T.G., 2006. A genetic similarity rule determines arthropod community structure. Mol. Ecol. 15, 1379–2139. Boecklen, W.J., Spellenberg, R., 1990. Structure of herbivore communities in two oak (Quercus spp.) hybrid zones. Oecologia 85, 2–100. Bookstein, F.L., 1991. Morphometric Tools for Landmark Data: Geometry and Biology. Cambridge University Press, New York. Cattell, M.V., Stilling, P., 2004. Tritrophic interactions and trade-offs in herbivore fecundity on hybridising host plants. Ecol. Entomol. 29, 255–263. Cheng, D., Vrieling, K., Klinkhamer, P.G.L., 2011. The effect of hybridization on secondary metabolites and herbivore resistance: implications for the evolution of chemical diversity in plants. Phytochem. Rev. 10, 107–117. Clarke, G.M., 1998. Developmental stability and fitness: the evidence is not quite so clear. Am. Nat. 152, 732–766. Cornelissen, T., Stiling, P., 2005. Perfect is best: low leaf fluctuating asymmetry reduces herbivory by leaf miners. Oecologia 142, 46–56.
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Original Articles
Patterns of herbivory and leaf morphology in two Mexican hybrid oak complexes: Importance of fluctuating asymmetry as indicator of environmental stress in hybrid plants
T
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Pablo Cuevas-Reyesa, Armando Canché-Delgadoa, Yurixhi Maldonado-Lópezb, , G. Wilson Fernandesc, Ken Oyamad, Antonio González-Rodrígueze a
Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo, Ciudad Universitaria, Morelia, 58030 Michoacán, Mexico CONACYT-Instituto de Investigaciones sobre los Recursos Naturales, Universidad Michoacana de San Nicolás de Hidalgo, Avenida San Juanito Itzícuaro SN, Nueva Esperanza, 58330 Michoacán, Mexico c Ecologia Evolutiva & Biodiversidade/DBG, C P 486, ICB/Universidade Federal de Minas Gerais, 31270 901 Belo Horizonte, MG, Brazil d Escuela Nacional de Estudios Superiores, Unidad Morelia, Universidad Nacional Autónoma de México, Morelia, Michoacán, Mexico e Instituto de Investigaciones en Ecosistemas y Sustentabilidad, Universidad Nacional Autónoma de México, Antigua Carretera a Pátzcuaro No. 8701, Col. Ex-Hacienda de San José de la Huerta, Morelia, 58190 Michoacán, Mexico b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hybrid complexes Quercus Leaf morphology Herbivory Fluctuating asymmetry Environmental stress
Interspecific hybridization is a prevalent process in plant species that may have different ecological and evolutionary consequences. Interactions with herbivorous insects may be altered because of hybridization among host plants. These changes result from the morphological, physiological and chemical traits expressed in hybrid individuals. Therefore, it is of interest to document the changes in traits such as leaf morphology and their consequences on patterns of herbivory by insects in hybrid complexes of plants. Another useful indicator that may serve to evaluate developmental instability resulting from genetic or environmental stress in hybrid plants is fluctuating asymmetry. In this study, we used two previously genetically characterized complexes of hybridizing Mexican oaks as models to compare and understand the relationships between leaf morphology, fluctuating asymmetry and herbivory levels in parental and hybrid individuals. Results indicated that in the Quercus affinis × Q. laurina complex, hybrid individuals show a distinct morphology in relation to the parental species, while in the Q. magnoliifolia × Q. resinosa complex, hybrids were similar to Q. resinosa. In both hybrid complexes, our results show that hybrid individuals have higher levels of fluctuating asymmetry and herbivory levels, which may reflect higher levels of genetic or environmental stress in comparison to the parental species. These results might help explain why oak species usually remain distinct despite the high frequency of hybridization characteristic of the genus.
1. Introduction Fluctuating asymmetry (FA) describes random morphological differences between the two sides of a bilateral character in organisms, and it is considered a good indicator of developmental instability caused by genetic and environmental stress (e. g., Palmer and Strobeck, 1986; Møller and Shykoff, 1999; Cornelissen and Stiling, 2005; CuevasReyes et al., 2011b). These morphological changes in plants (Palmer and Strobeck, 2003; Cornelissen and Stiling, 2010; Cuevas-Reyes et al., 2011a) can be caused by abiotic factors such as pollution (Zvereva et al., 1997; Cornelissen et al., 2003), nutrient or water stress (Freeman et al., 2004), and biotic factors as hybridization (Albarrán-Lara et al.,
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2010) and the incidence of pathogens and herbivores (Zvereva et al., 1997). Because the degree of FA reflects the inability of individuals to maintain homeostasis during development under stressful conditions (Møller and Swaddle, 1997), high levels of FA are usually correlated with low performance (i.e., growth, survival and fecundity) (Díaz et al., 2004; Møller, 1999; but see Clarke 1998). In this way, plants with high levels of FA could also be more susceptible to herbivores (e.g., Lempa et al., 2000; Cornelissen and Stiling, 2005; Cuevas-Reyes et al., 2011a), because of underlying differences in leaf nutritional quality and chemical defenses (Cornelissen and Stiling, 2005, 2011). However, other authors have proposed that herbivory can directly act as a plant
Corresponding author. E-mail address: [email protected] (Y. Maldonado-López).
https://doi.org/10.1016/j.ecolind.2018.03.009 Received 10 October 2016; Received in revised form 10 February 2018; Accepted 5 March 2018 1470-160X/ © 2018 Elsevier Ltd. All rights reserved.
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complexes, individuals analyzed here were the same used in previous genetic characterizations of the hybrid zone structure (Pérez-López et al., 2016; Ramos-Ortiz et al., 2016). The first hybrid complex is formed by the red oaks Quercus affinis × Quercus laurina. Quercus affinis Scheidweiler occurs along the Sierra Madre Oriental and is distributed along an altitudinal gradient from about 1600 to 2800 m (GonzálezRodríguez et al., 2004). This tree can reach 30 m in height, and its leaves are oblong lanceolate, 4.5–9 cm long, 1.5–2 cm wide, smooth on both sides, with toothed margin from the mid region to the apex (Rzedowski, 1978). Quercus laurina Humboldt et Bonpland is distributed along the Sierra Madre del Sur and in the Trans-Mexican Volcanic Belt. It is a tree growing up to 25 m with deciduous leaves, broadly lanceolate or oblanceolate, 7–10 cm long, 2.5–3 cm wide, smooth and shiny on the adaxial surface, undersides with woolly pubescence (Rzedowski, 1978). The hybrid zone between these two species is located in the eastern portion of the Trans-Mexican Volcanic Belt and northern Oaxaca (González-Rodríguez et al., 2004; Ramos-Ortiz et al., 2016). The second hybrid complex is composed by the white oaks Quercus magnoliifolia × Q. resinosa and is located at the Tequila volcano, Jalisco state, Mexico (20°50′ N, 103°5′ W). At this site, both plant species are distributed along an altitudinal gradient from about 1400 to 2100 m. Quercus magnoliifolia Née occurs between 1400 and 1800 m. It is a tree, growing up 25 m, with obovate leaves from 7.5 to 23 cm long and 3.5 to 13 cm wide. The adaxial surface is lustrous and almost glabrous, tomentose on the abaxial surface and with glabrescent petioles (Arizaga et al., 2009). Quercus resinosa Liebm occurs from 1700 to 2100 m. This tree can grow up to 20 m in height, it has large leaves 15–25 cm long and 5–16 cm wide. The leaves are obovate, rugose on the adaxial surface and pale-green or yellowish, tomentose on the abaxial surface and with densely tomentose petioles (Arizaga et al., 2009). The hybridization zone between the two species is located between 1600 and 1800 m at the Tequila volcano (Albarrán-Lara et al., 2010; Pérez-López et al., 2016). Herbivorous insect species attacking the plant species studied here have not been previously documented in the literature. However, during the study, we observed at least nine free-feeders insect species of different families such as Lepidoptera (Geometridae), Orthoptera (Acrididae) and Coleoptera (Chrysomelidae) responsible for most of the apparent damage in the Q. affinis × Q. laurina hybrid complex. In the case of the Q. resinosa × Q. magnoliifolia hybrid complex, we detected the presence of some free-feeders species of Lepidoptera (Geometridae, Noctuidae, Pyralidae, Phaloniidae, Gelechiidae, Lymantridae, Thyridiidae, Limacodidae, Arctiidae), Coleptera (Chrysomelidae), Hymenoptera (Formicidae, Pergidae) and Orthoptera (Gryllidae).
stressor, modifying the patterns of leaf symmetry and increasing FA levels (Díaz et al., 2004; Santos et al., 2013; Alves-Silva and Del-Claro, 2016). Fluctuating asymmetry may also increase as a result of hybridization because of the disruption of coadapted gene combinations (Leary and Allendorf, 1989). Natural hybridization is an important process in plant evolution that results in new genetic combinations by the introduction of semicompatible genes into another genome as consequence of interbreeding between two or more different species, which can produce fertile or infertile individuals called hybrids (Rieseberg, 1997; Martinsen et al., 2001). It has been proposed that insect herbivory on hybrid plants can potentially affect plant evolution modifying gene flow between hybrids (Pearse and Baty, 2012). In turn, other studies suggest that herbivore communities and herbivory levels can be affected by high genetic variation of hybrid plants that show a mosaic of phenotypic traits in comparison with their parental species (Rieseberg and Ellstrand, 1993; Arnold, 1997; Whitham et al., 2003; Floate et al., 2016). Therefore, differences in genetic, morphological and chemical traits among parental species and hybrids can influence insect herbivore communities, insect incidence and herbivory patterns in the hybrid plant complex (Driebe and Whitham, 2000; Cattell and Stiling, 2004; Bangert et al., 2006). Despite the high frequency of hybridization among Quercus species (González-Rodríguez et al., 2004; Tovar-Sánchez and Oyama, 2004; Pérez-López et al., 2016; Ramos-Ortiz et al., 2016) these usually remain distinct, suggesting that selection may act to decrease the number of hybrids that survive to reproduction (Howard et al., 1997; William et al., 2001). Because higher levels of herbivory on hybrids have been reported in many plant systems (Strauss, 1994; Whitham et al., 1999), herbivory may select against hybrid genotypes to maintain species integrity. However, several studies in sympatric oaks species indicated high levels of hybridization with viable and fertile offspring (Dodd and Afzal-Rafi, 2004; Hipp and Weber, 2008). A longer-term observation of the success of hybrids considering environmental filters as well as their interactions with natural enemies, such as herbivores, is crucial. Hence, to understand the ecology and evolution of hybrid complexes it is necessary to consider the negative effects of herbivory on plant growth and fitness (Maldonado-López et al., 2015) and, therefore their plant stressors. The formation of hybrid zones between Quercus species in Mexico is very common (González-Rodríguez et al., 2004; Tovar-Sánchez and Oyama, 2004; Albarrán-Lara et al., 2010; Pérez-López et al., 2016; Ramos-Ortiz et al. 2016). Depending on the introgression levels, hybrids may present a continuum of leaf shapes (Whitham, 1989); hence we postulate that it would also lead to different levels of FA on such areas. Therefore, hybrid oak complexes represent an ideal system to understand the relationship between plant-herbivore interactions and FA. In our study, we determined the patterns of herbivory, leaf morphology and fluctuating asymmetry in two previously genetically characterized hybrid oak complexes (Quercus affinis × Quercus laurina, and Quercus magnoliifolia × Q. resinosa) that occur in different regions of Mexico. We addressed the following questions: (1) Does leaf morphology differ between parental and hybrid plants in each hybrid complex? (2) Are hybrid plants more susceptible to herbivory in both hybrid complexes, and (3) Is leaf FA associated with the levels of herbivory?
2.2. Sample collection The studied individuals of the Quercus affinis × Quercus laurina complex were the same analyzed genetically by Ramos-Ortiz et al. (2016) in seven different localities: Tizapán, Hidalgo state (20.62° N, 98.6° W); Zacatlán, Puebla state (19.9° N, 97.95° W); Tonayán, Veracruz state (19.72° N, 96.9° W); Zoquitlán, Puebla state (18.28° N, 97.08° W); Pápalo, Oaxaca state (17.85° N, 96.8° W); Lachao, Oaxaca state (16.22° N, 97.13° W) and Suchixtepec, Oaxaca state (16.06° N, 96.48° W). In each population, 15 trees were studied. After the previous genetic analysis, individuals in the Tizapán and Zacatán populations were assigned as Q. affinis except for one and two individuals, respectively, that were assigned as hybrids. In Tonayán population there were six Q. affinis individuals, four Q. laurina individuals and five hybrids, while in population Zoquitlán there were 12 Q. laurina individuals and three hybrids. In the three southernmost populations all individuals were assigned as Q. laurina (see Ramos-Ortiz et al., 2016 for more details). In the second oak hybrid complex, from the individuals analyzed genetically by Pérez-López et al. (2016), we randomly selected a set of 30 trees at each of three different altitudes, 1400–1500, 1600–1800 and
2. Materials and methods 2.1. Study system This study was conducted in different regions of Mexico where the two hybrid oak complexes are located. Both regions are characterized for the presence of deciduous or brevideciduous oak species that occur in seasonal temperate forests (Hernández-Calderón et al., 2013; PérezLópez et al., 2016; Ramos-Ortiz et al., 2016). For both hybrid 165
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configuration of all leaves as reference and then, we calculated the shape variables (Procrustes distances) based on superimposition coordinates to eliminate the effect of leaf size (Cuevas-Reyes et al., 2011a). Finally, in each oak hybrid complex, we applied a principal components analysis to determine the differences in leaf morphology between the three groups of plants considering the configuration of all leaves. This type of analysis has been used to classify species, distinguish hybrid groups, locating the most likely parent of a known hybrid with several possible parents (Hardig et al. 2000; Viscosi 2015).
1900–2100 m. The previous genetic characterization of these individuals indicated that at the lower altitude, 25 individuals were Q. magnoliifolia and five were hybrids, while at the intermediate altitude there were eight Q. magnoliifolia individuals, 17 hybrids and four Q. resinosa individuals, and at the higher altitude there were nine Q. resinosa and 21 hybrids. Sample collections were conducted during 2014, at the end of the rainy season after the peak of herbivore activity (October–November). For each individual the leaf sampling was random. Because the studied species are deciduous or brevideciduous, all collected leaves correspond to the same cohort. Three branches were collected from the top, intermediate, and bottom strata of the canopy of each tree, sampling a minimum of 100 fully developed leaves per individual (Cuevas-Reyes et al., 2004a,b). To control for plant size, the stem diameter at breast height (dbh) was measured for each individual sampled (Cuevas-Reyes et al., 2006). In all cases, leaves from each individual were separated into two categories: a random set of leaves was used to estimate the herbivory level on each tree; and a set of undamaged leaves, which was used for morphometric and fluctuating asymmetry analysis (CuevasReyes et al., 2011a).
2.4. Fluctuating asymmetry measurements Fluctuating asymmetry was calculated in approximately 50 fully expanded intact mature leaves of each tree sampled in the two hybrid oak complexes. A digital image was taken for each leaf and then we measured the distance from the right side and left side from the leaf edge to the midvein at the midpoint of the leaf corresponding to its widest part (Fig. 1b). FA was estimated as the absolute value of the difference between the distances from the midvein to the left and right margins of the leaf (|Ai − Bi|) divided by the average distance (Ai + Bi/ 2) to correct for the fact that asymmetry may be size-dependent (Cornelissen and Stiling, 2005; Cuevas-Reyes et al., 2011a). We obtained an individual FA value of each tree from the average of the 50 leaves analyzed. A subsample of 25 leaves combined across all trees sampled within a hybrid complex was re-measured to control the measurement error in FA. Then, we checked the significance of FA relative to measurement error using a two-way mixed-model ANOVA. This model considers as factor individual, leaf (random) and side (right or left). Both measurements were considered as replicates (Palmer and Strobeck, 2003; Cuevas-Reyes et al., 2011a). The significance of the interaction (individual × leaf × side) indicated that variation in FA was greater than expected by measurements error (F24, 50 = 24.9; P < 0.0001). According to Palmer and Strobeck (2003), it is necessary to discriminate FA from other kinds of asymmetry. Fluctuating asymmetry, directional asymmetry, and antisymmetry represent the three types of deviation from the perfect bilateral symmetry of the organisms (Albarrán-Lara et al., 2010; Cuevas-Reyes et al., 2011a). FA represents the variance in left–right (L-R) differences distributed with a mean value of zero, whereas in directional asymmetry, the L-R differences are distributed about a mean that is significantly greater or less than zero, reflecting a consistent bias of a character towards greater development of one side than the other. Finally, antisymmetry is the lack of symmetry represented by bimodal or platykurtic distribution of L-R differences about a mean of zero (Van Valen, 1962). Hence, FA reflects random variation from the perfect symmetry of the bilateral trait, whereas, directional asymmetry and antisymmetry are considered as inappropriate descriptors of developmental instability, because they are developmentally controlled and are probably adaptive (Palmer and Strobeck, 1986). To confirm that our data reflected only FA and no other types of asymmetry, we checked for the directional asymmetry using a Student’s t test considering whether the average L-R difference value differed from zero. In the same way, to test for antisymmetry the Lilliefors’ normality test was performed considering the distribution of L-R differences (Alves-Silva and Del-Claro, 2016). Because in our data L-R differences followed a normal distribution and the mean value of L-R differences did not deviate significantly from zero (t = 2.3 P > 0.05), the values were considered to reflect FA exclusively.
2.3. Morphometric analysis of leaves We obtained digital images of approximately 50 intact leaves with no visible damage along the margins per tree to determine the differences in leaf morphology and size between the three groups of plants (i.e., the two parental species and the hybrids) in each oak complex. In each digital image, 32 landmarks and two additional landmarks were placed as reference of size (see Fig. 1). All landmarks correspond to homologous loci, which are unambiguous and repeatable marks in all the leaves, representing their shape (sensu Bookstein, 1991; CuevasReyes et al., 2011a). We used the TpsDig program (Rohlf, 2015) to record the coordinates (x, y) of the 32 landmarks in each leaf image. A Procrustes superimposition analysis was used for the configuration of the coordinates of each landmark using the CoordGen6 program, in the Integrated Morphometrics Package (IMP series: http://www.canisius. edu/~sheets/morphsoft.html). We considered the average
2.5. Measurement of herbivory To estimate the leaf area consumed by folivores, we examined approximately 50 leaves from each individual the two hybrid oak complexes. First, we took a digital image of each leaf sampled and estimated the total leaf area and the area consumed by folivorous insects using the Image analysis software for plant disease quantification (Assess: Image
Fig. 1. Morphometric measurements of leaves. Digital image of a leaf of Q. laurina showing the 32 morphological landmarks along the leaf margin. Two additional landmarks (33 and 34) were added on a reference ruler as a scale (a). Representation of the measurements used to estimate leaf FA in Q. affinis. RW right width, LW left width (b).
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Analysis Software for Plant Disease Quantification, APS Press, Saint Pail, MN). In all cases, the normality was tested after suitable transformations. Herbivory data were transformed as arc-sine square root (Pascual-Alvarado et al., 2008). 2.6. Statistical analyses Because in both oak hybrid complexes there were several sampling sites (i. e. seven sites across a latitudinal gradient in the Q. affinis × Q. laurina complex and three different altitudes in the Q. resinosa × Q. magnoliifolia complex), we used two-way ANOVA tests to assess the effects of site and plant group (two parental species and hybrids) on FA and herbivory levels. Because not all plant groups were present at all sites in the two hybrid oak complexes, the interaction between FA and herbivory was not considered. Finally, we used Spearman’s rank correlation analyses to determine the relationships between FA, herbivory, foliar area and DBH, separately within each plant group (i.e., two parental species and hybrids), for each hybrid oak complex. 3. Results The degree of leaf shape variation based on mean configuration of superimposition coordinates, showed that leaves of Q. laurina were more elongated and wider than leaves of the hybrids and Q. affinis
Fig. 3. Differences in leaf shape morphology between parental species and hybrid plants in two Mexican hybrids oak complex according to principal components analysis. Gray circles: Q. affinis. Black crosses: Hybrids. Gray stars: Q. laurina (a). Gray circles: Q. resinosa. Black crosses: Hybrids. Gray stars: Q. magnoliifolia: (b).
(Fig. 2a). In the second hybrid complex, leaves of hybrids were more elongated and wider than Q. resinosa and Q. magnoliifolia (parental species) (Fig. 2b). In addition, the scatter plot of PC1 and PC2 scores of Q. affinis × Q. laurina complex accounting for 44.0% and 27.1% of total variance respectively; and separates three distinct groups: Q. laurina with a minimum overlap from Q. affinis, and hybrids totally separated from these two species (Fig. 3a). In the second oak hybrid complex, the scatter plot of PC1 accounted for 96.4% of total variance and PC2 for only 0.1%. Q. resinosa was separated, with a small overlap from hybrids and Q. magnoliifolia was a different group as shown by the principal components analysis (Fig. 3b). In the Q. affinis x Q. laurina complex, both sites (F = 11.02; d. f. = 6, P < 0.0001) and plant group (F = 3.57; d. f. = 2; P = 0.03) had a significant effect on FA levels. Mean ( ± standard error) FA in Q. laurina individuals was 0.17 ± 0.006, which did not differ from the mean value in Q. affinis individuals (0.17 ± 0.009), but which was significantly lower than the mean value in hybrid individuals (0.26 ± 0.017). Similarly, in the Q. resinosa × Q. magnoliifolia complex, both site (altitude) (F = 55.37; d. f. = 2; P < 0.0001), and plant group (F = 3.97; d. f. = 2; P = 0.022) had a highly significant effect on FA levels. FA was lower in Q. magnoliifolia (0.11 ± 0.01) than in Q. resinosa (0.15 ± 0.01), the hybrids (0.17 ± 0.008), which did not differ between each other. Leaf area consumed by folivorous insects differed significantly among sites in the Q. affinis × Q. laurina complex (F = 46.98; d. f. = 6; P < 0.0001) and among plant groups (F = 3.8; d. f. = 2; P = 0.026).
Fig. 2. Leaf morphological variation between the three groups of plants in each hybrid oak complex: mean shape of coordinates of landmark configuration of leaves. Q. affinis × Q. laurina (a) and Q. resinosa x Q. magnoliifolia (b).
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species (Whitham et al., 2003; González-Rodríguez et al., 2004). In the Q. resinosa × Q. magnoliifolia complex, we found variation in leaf morphology, but hybrid plants had a similar leaf shape to the parental species Q. resinosa. Resemblance of hybrids to one of the parental species is a common observation that can be explained by dominance (Rieseberg et al., 2003). Herbivore preference, performance, and distribution in hybrid complexes (Tovar-Sánchez et al., 2013; Pérez-López et al., 2016), are influenced by plant attributes modified in plant hybridization conditions, such as morphology, growth, physiology and chemical defense (Rehill et al., 2005; Bangert et al., 2006; Travis et al., 2008; Cheng et al., 2011; Schweitzer et al., 2004). In the same way, taxonomic relationships between parental species, age of the hybrid complex, structure and assemblages of insect herbivores may affect the herbivory patterns in hybrid complexes (Boecklen and Spellenberg, 1990; TovarSánchez and Oyama, 2006; Pearse and Hipp, 2009). In our study, despite the effect of sampling at different sites, we found that leaf FA was significantly greater in leaves of hybrids than in leaves of parental species (or at least one parental species), in both hybrid oak complexes. Because the degree of divergence among parental species and hybrids influences hybrid performance (Hochwender and Fritz, 1999), high values of fluctuating asymmetry can be associated to a disruption of coadapted gene complexes and by the interaction among genes that functionally diverged between species, resulting in hybrids with higher levels of fluctuating asymmetry (Albarrán-Lara et al., 2010). In parallel, we found that hybrid individuals received in general a higher level of damage by herbivorous insects than parental species individuals, even though this difference was only significant in the Q. affinis × Q. laurina complex. However, when we analyzed the correlation between the percentage of leaf area removed and FA separately within plant groups (Table 1) we found a consistent positive correlation between the two variables within the hybrid class in the two oak hybrid complexes. This correlation was also observed for parental individuals in the Q. magnoliifolia × Q. resinosa complex, but not in the Q. affinis × Q. laurina complex. Therefore, in general we found evidence that plants with more asymmetric leaves experience greater herbivore consumption. Differences in leaf morphology, leaf size and shape between parental species and hybrid plants have explained the variation in the levels of consumption by herbivores and insect distribution (Driebe and Whitham, 2000; Cattell and Stiling, 2004). It is possible that leaves with higher FA also have a higher nutritional quality and lower concentrations of secondary metabolites compared with more symmetric leaves, as has been demonstrated in other species of Quercus (Cornelissen and Stiling, 2005). Also, plant hybridization can disrupt the chemical resistance of plants against herbivore insects in different ways: no differences between hybrids and parental species, additive or intermediate resistance in hybrids, dominance (similar resistance of hybrids to one of the parent species) and hybrid susceptibility (Fritz et al., 1999). Different hypotheses have been proposed to understand the significance of plant interspecific hybridization on plant-herbivore interactions (“Additive hypothesis”, Boecklen and Spellenberg 1990; “Dominance hypothesis”, Fritz et al., 1994; “Hybrid resistance hypothesis”, Boecklen and Spellenberg, 1990 and “Hybrid depression hypothesis”, Arnold and Hodges, 1995). Our results support the “hybrid susceptibility hypothesis” also called the “hybrids-as-sinks hypothesis” (Whitham, 1989), which states that hybrid plants are more susceptible to herbivores or parasites than their parental plants, and act as sinks for herbivore populations (Whitham, 1989). We found that folivory levels were higher on hybrid plants in comparison with their parental species in both hybrid oak complexes studied. Interestingly, in a previous study, conducted in the same individuals of the Q. magnoliifolia × Q. resinosa complex, we found higher diversity of gall morphospecies in hybrids than in both parental species (Pérez-López et al., 2016). Our results are also consistent with the “hybrid depression hypothesis” (Arnold and Hodges, 1995) that proposes higher levels of
Table 1 Spearman’s correlation coefficients of total leaf area, DBH, herbivory and fluctuating asymmetry (**P < 0.05). Q. affinis × Q. laurina (a) and Q. resinosa × Q. magnoliifolia (b). (a) Plant species Q. affinis
Hybrids
Q. laurina
Leaf area removed
FA
FA Total leaf area (cm2) DBH (cm)
0.17 0.51**
0.66**
0.11
0.08
FA Total leaf area (cm2) DBH (cm)
0.91** 0.08
0.14
0.15
0.11
FA Total leaf area (cm2) DBH (cm)
0.13 0.48**
0.71**
0.14
0.13
Total leaf area
0.46**
0.22
0.17
(b) Plant species
Q. resinosa
Hybrids
Q. magnoliifolia
Leaf area removed
FA
FA Total leaf area (cm2) DBH (cm)
0.63** 0.22
0.21
0.07
0.11
FA Total leaf area (cm2) DBH (cm)
**
FA Total leaf area (cm2) DBH (cm)
0.76 0.12
0.15
0.15
0.15
Total leaf area
0.36**
0.47**
**
0.54 0.18
0.21
0.11
0.13
0.37**
Percentage herbivory did not differ between the two parental species (16.2 ± 1.5 in Q. laurina, 19% ± 2.3 in Q. affinis), but was significantly higher (34.8 ± 4.3) in the hybrids. In contrast, in the Q. resinosa × Q. magnoliifolia complex, percentage herbivory differed significantly among altitudes (F = 36.07; d. f. = 2; P < 0.0001) but the effect of plant groups was not significant (F = 2.47; d. f. = 2; P = 0.09). In this case, herbivory was higher at the intermediate altitude (18.5 ± 1.1) than in the higher and lower altitudes (7.6 ± 1.15 and 8.3 ± 1.4, respectively). A positive relationship was found between leaf area consumed and FA in the hybrid group of the Q. affinis × Q. laurina complex, but not in the parental species individuals (Table 1). Instead, leaf area consumed was positively correlated with total leaf area in both Q. affinis and Q. laurina. In contrast, in the Q. magnoliifolia × Q. resinosa hybrid complex, leaf area consumed and FA levels were positively correlated in all three plant groups and there was no correlation with total leaf area. 4. Discussion The presence of hybrid zones in the genus Quercus is common (Spellenberg, 1995; González-Rodríguez et al., 2004; Pérez-López et al., 2016). Hybridization in oaks has been detected using molecular markers and morphological characters of leaves (González-Rodríguez et al., 2004; Tovar-Sánchez and Oyama, 2004; Albarrán-Lara et al., 2010; Pérez-López et al., 2016). In our study, we found different patterns of leaf morphology in the two hybrid oak complexes using geometric morphometrics methods. In the Q. affinis × Q. laurina complex, leaf morphology of hybrid plants was statistically different to their parental species. Different phenomena can account for these differences, such as transgressive segregation or gene-environment interactions, resulting in the differentiation of leaf shape between hybrids and their parental 168
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herbivory on hybrids than parental species as a selection mechanism that promotes maintenance or even speciation events. In this case, herbivore adaptation to new or alternative host plant species may lead to reproductive isolation mechanisms and genetic differentiation (hostassociated differentiation) (Dickey and Medina, 2010). For example, if hybrids are sufficiently different from parental species, is possible to expect adaptation, reproductive isolation and host associated differentiation in herbivorous insects, and therefore, more herbivory levels on hybrids than on parental species (Evans et al., 2008, 2012). 5. Conclusions Our results show that hybridization in both complexes of Mexican oaks has important consequences on plant insect interactions, increasing both herbivory and FA levels in hybrid plants. This emphasizes the role of hybrid individuals as potential sinks or bridges for the colonization of host plants by herbivorous insects. Therefore, we provide additional information showing the importance of preserving the genetic diversity of plants as a way of conserving dependent animal species. Finally, our study shows the importance of fluctuating asymmetry as indicator of genetic and environmental stress. Acknowledgments This project was supported by CONACYT project CB105755 and DGAPA-PAPIIT-UNAM project RV201015. Cuevas-Reyes P thanks Coordinación de la Investigación Científica UMSNH for their generous support, and GWF thanks CNPq and FAPEMIG for support. We also thank Fidel Anguiano for editing figures. Declaration of interest All authors declare that we have any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, our work. References Albarrán-Lara, A.L., Mendoza-Cuenca, L., Valencia-Avalos, S., González-Rodríguez, A., Oyama, K., 2010. Leaf fluctuating asymmetry increases with hybridization and introgression between Quercus magnoliifolia and Quercus resinosa (Fagaceae) through an altitudinal gradient in Mexico. Int. J. Plant Sci. 171, 310–322. http://dx.doi.org/10. 1086/650317. Alves-Silva, E., Del-Claro, K., 2016. Herbivory-induced stress: Leaf developmental instability is caused by herbivore damage in early stages of leaf development. Ecol. Indic. 61, 359–365. Arizaga, S., Martínez-Cruz, J., Salcedo-Cabrales, M., Bello-González, M.A., 2009. Aspectos generales de los encinos. In: Arizaga, S., Cruz, J.M., Cabrales, M.S., González, M.A.B. (Eds.), Manual de la biodiversidad de encinos michoacanos. Secretaría de Medio Ambiente y Recursos Naturales (Semarnat), Instituto Nacional de Ecología (INESemarnat), México, D., pp. 12–141. Arnold, M.L., 1997. Natural Hybridization and Evolution. Oxford University Press, New York. Arnold, M.L., Hodges, S.A., 1995. Are natural hybrids fit or unfit relative to their parents. Trends Ecol. Evol. 10, 67–71. Bangert, R.K., Turek, R.J., Rehill, B., Wimp, G.M., Schweitzer, J.A., Allan, G.J., Bailey, J.K., Martinsen, G.D., Leim, P., Lindroth, R.L., Whitham, T.G., 2006. A genetic similarity rule determines arthropod community structure. Mol. Ecol. 15, 1379–2139. Boecklen, W.J., Spellenberg, R., 1990. Structure of herbivore communities in two oak (Quercus spp.) hybrid zones. Oecologia 85, 2–100. Bookstein, F.L., 1991. Morphometric Tools for Landmark Data: Geometry and Biology. Cambridge University Press, New York. Cattell, M.V., Stilling, P., 2004. Tritrophic interactions and trade-offs in herbivore fecundity on hybridising host plants. Ecol. Entomol. 29, 255–263. Cheng, D., Vrieling, K., Klinkhamer, P.G.L., 2011. The effect of hybridization on secondary metabolites and herbivore resistance: implications for the evolution of chemical diversity in plants. Phytochem. Rev. 10, 107–117. Clarke, G.M., 1998. Developmental stability and fitness: the evidence is not quite so clear. Am. Nat. 152, 732–766. Cornelissen, T., Stiling, P., 2005. Perfect is best: low leaf fluctuating asymmetry reduces herbivory by leaf miners. Oecologia 142, 46–56.
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