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Leaf phenotypic variation and developmental instability in relation to different light regimes

ABSTRACT

For pioneer plants, shaded habitats represent a stressful condition, where sunlight exposure is below the optimum level and so leaves expand in order to intercept a greater amount of light. We investigated changes in both phenotypic variation and stress of Bauhinia brevipes in sunny and shaded microhabitats. Leaf area was used as a measure of phenotypic variation, whereas leaf asymmetry (difference between right and left sides of leaves), was used as a measure of stress. We hypothesized an increase in leaf area and stress in shaded locations, which might indicate that B. brevipes was compensating for low light absorption, and elevated levels of stress, respectively. Plants in the sun fitted a fluctuating asymmetry pattern (normal distribution of right minus left sides), while shaded plants were clearly antisymmetric (bimodal distribution of leaf side differences). Leaf asymmetry and area were 5% and 26.8% higher in plants in the shade compared to plants in the sun, respectively. These results were expected since B. brevipes is found predominantly in open areas; so sunlight exposure is important for its development. The presence of antisymmetry is rare in studies of developmental instability, and here it might indicate higher stress compared to plants with fluctuating asymmetry.

Keywords
antisymmetry; Bauhinia brevipes; fluctuating asymmetry; leaf morphometry; sunlight exposure

Introduction

Organisms in nature can be subjected to unfavourable developmental conditions that affect their growth and persistence (Jan et al. 2012Jan AT, Singhal P, Haq QMR. 2012. Plant abiotic stress: deciphering remedial strategies for emerging problem. Journal of Plant Interactions 8: 97-108.). For instance, plants living in salty, shaded or poor nutrient habitats usually present increased stress levels in comparison to plants in favourable environments (Palmer & Strobeck 1986Palmer AR, Strobeck C. 1986. Fluctuating asymmetry: measurement, analysis, patterns. Annual Review of Ecology and Systematics 17: 391-421.; Møller & Dongen 2003Møller AP, Dongen S. 2003. Ontogeny of asymmetry and compensational growth in elm Ulmus glabra leaves under different environmental conditions. International Journal of Plant Sciences 164: 519-526.; Cornelissen & Stiling 2011Cornelissen T, Stiling P. 2011. Similar responses of insect herbivores to leaf fluctuating asymmetry. Arthropod-Plant Interactions 5: 59-69.; Alves-Silva 2012Alves-Silva E. 2012. The influence of Ditylenchus (Nematoda) galls and shade on the fluctuating asymmetry of Miconia fallax (Melastomataceae). Ecología Austral 22: 53-61.). To persist in stressing habitats, plants have evolved, by natural selection, the ability to respond through morphological and physiological modifications, which is reflected in their phenotypic variation (Cardoso & Lomônaco 2003Cardoso GL, Lomônaco C. 2003. Variações fenotípicas e potencial plástico de Eugenia calycina Cambess. (Myrtaceae) em uma área de transição cerrado-vereda. Revista Brasileira de Botânica 26: 131-140.; Miner et al. 2005Miner BG, Sultan SE, Morgan SG, Padilla DK, Relyea RA. 2005. Ecological consequences of phenotypic plasticity. Trends in Ecology and Evolution 20: 685-692.; Rozendaal et al. 2006Rozendaal DMA, Hurtado VH, Poorter L. 2006. Plasticity in leaf traits of 38 tropical tree species in response to light; relationships with light demand and adult stature. Functional Ecology 20: 207-216.). These modifications may occur rapidly and in several structures (e.g., leaves) as a response to adverse conditions (Alpert & Simms 2002Alpert P, Simms EL. 2002. The relative advantages of plasticity and fixity in different environments: when is it good for a plant to adjust? Evolutionary Ecology 16: 285-297.).

Sunlight is an essential resource for the growth and performance of plants; however, plants can sometimes occur in places where sunlight exposure is below the optimum level (Niinemets 2010Niinemets Ü. 2010. A review of light interception in plant stands from leaf to canopy in different plant functional types and in species with varying shade tolerance. Ecological Research 25: 693-714.). For instance, many species in the Cerrado (Brazilian savanna) occur predominantly in open areas (e.g., Melastomataceae; Myrtaceae; Malpighiaceae; Solanaceae - EA Silva unpubl. res.), where they are exposed to high amounts of solar exposure. In fact, the germination and performance of some Cerrado plants can depend largely on sunny habitats (Lima et al. 2014Lima YBC, Durigan G, Souza FM. 2014. Germinação de 15 espécies vegetais do cerrado sob diferentes condições de luz. Bioscience Journal 30: 1864-1872.; see also Toledo-Aceves & Swaine 2008Toledo-Aceves T, Swaine MD. 2008. Biomass allocation and photosynthetic responses of lianas and pioneer tree seedlings to light. Acta Oecologica 34: 38-49.). Therefore, when light is minimum (i.e., shady conditions), plants can respond accordingly (Marques et al. 1999Marques AR, Garcia QS, Fernandes GW. 1999. Effects of sun and shade on leaf structure and sclerophylly of Sebastiania myrtilloides (Euphorbiaceae) from Serra do Cipó, Minas Gerais, Brazil. Boletim de Botânica da Universidade de São Paulo 18: 21-27.; Rossatto et al. 2010Rossatto DR, Takahashi FSC, Silva LCR, Franco AC. 2010. Características funcionais de folhas de sol e sombra de espécies arbóreas em uma mata de galeria no Distrito Federal, Brasil. Acta Botanica Brasilica 24: 640-647.; Ronquim et al. 2013Ronquim CC, Prado CHBA, Souza JP. 2013. Estabelecimento e crescimento de plantas jovens de Vochysia tucanorum Mart. (Vochysiaceae) em área aberta e sombreada sob dossel florestal. Scientia Forestalis 41: 121-130.), and approaches such as the assessment of phenotypic variation have been used to evaluate the performance of plants in different microhabitats (Marques et al. 2000Marques AR, Garcia QS, Rezende JLP, Fernandes GW. 2000. Variations in leaf characteristics of two species of Miconia in the Brazilian cerrado under different light intensities. Tropical Ecology 41: 47-60.; Ackerly et al. 2002Ackerly DD, Knight CA, Weiss SB, Barton K, Starmer KP 2002. Leaf size, specific leaf area and microhabitat distribution of chaparral woody plants: Contrasting patterns in species level and community level analyses. Oecologia 130: 449-457.; Niinemets 2010Niinemets Ü. 2010. A review of light interception in plant stands from leaf to canopy in different plant functional types and in species with varying shade tolerance. Ecological Research 25: 693-714.). Usually, plants enhance the leaf area/length in order to intercept higher amounts of light (Valladares et al. 2007Valladares F, Gialoni E, Gómez JM. 2007. Ecological limits to phenotypic plasticity. New Phytologist 176: 749-763.). In this context, it is expected that plants present intraspecific variation when individuals are exposed to sun or shade (Marques et al. 2000Marques AR, Garcia QS, Rezende JLP, Fernandes GW. 2000. Variations in leaf characteristics of two species of Miconia in the Brazilian cerrado under different light intensities. Tropical Ecology 41: 47-60.; Valladares & Niinemets 2008Valladares F, Niinemets U. 2008. Shade tolerance, a key plant feature of complex nature and consequences. Annual Review of Ecology, Evolution, and Systematics 39: 237-257.).

Intense responses to environments such as sun or shade can generate a phenomenon called developmental instability (DI) (Alves-Silva & Del-Claro 2013Alves-Silva E, Del-Claro K. 2013. Effect of post-fire resprouting on leaf fluctuating asymmetry, extrafloral nectar quality, and ant-plant-herbivore interactions. Naturwissenschaften 100: 525-532.; Venâncio et al. 2016Venâncio HS, Alves-Silva E, Santos JC. 2016. On the relationship between fluctuating asymmetry, sunlight exposure, leaf damage and flower set in Miconia fallax (Melastomataceae). Tropical Ecology 57: 419-427.), which is the inability of organisms to cope with stressing conditions (Alpert & Simms 2002Alpert P, Simms EL. 2002. The relative advantages of plasticity and fixity in different environments: when is it good for a plant to adjust? Evolutionary Ecology 16: 285-297.; Alves-Silva 2012Alves-Silva E. 2012. The influence of Ditylenchus (Nematoda) galls and shade on the fluctuating asymmetry of Miconia fallax (Melastomataceae). Ecología Austral 22: 53-61.). Developmental instability is commonly assessed through the fluctuating asymmetry (FA) analysis, which measures the imperfect growth of supposedly bilateral structures, such as leaves (Graham et al. 2010Graham JH, Raz S, Hel-Or H, Nevo E. 2010. Fluctuating asymmetry: methods, theory, and applications. Symmetry 2: 466-540.). Large deviations from perfect symmetry are evidence of high stress, and so this technique is widely used to compare stress levels in several habitats (reviewed by Møller & Shykoff 1999Møller AP, Shykoff JA. 1999. Morphological developmental stability in plants: patterns and causes. International Journal of Plant Sciences 160: 135-146.).

According to the literature, FA is the most common estimate of developmental instability (Graham et al. 2010Graham JH, Raz S, Hel-Or H, Nevo E. 2010. Fluctuating asymmetry: methods, theory, and applications. Symmetry 2: 466-540.; Santos et al. 2013Santos JC, Alves-Silva E, Cornelissen TG, Fernandes GW. 2013. The effect of fluctuating asymmetry and leaf nutrients on gall abundance and survivorship. Basic and Applied Ecology 14: 489-495.). Nonetheless, there is some evidence that other types of asymmetry, such antisymmetry (bimodal distribution of leaf sides, i.e., the population can have both increased left and right sides) and directional asymmetry (one leaf side is invariably greater than the other) also depict developmental instability (Graham et al. 1998Graham JH, Emlen JM, Freeman DC, Leamy LJ, Kieser JA. 1998. Directional asymmetry and the measurement of developmental instability. Biological Journal of the Linnean Society 64: 1-16.). However, so far only a few studies have examined this issue (Lens & Dongen 2000Lens L, Dongen S. 2000. Fluctuating and directional asymmetry in natural bird populations exposed to different levels of habitat disturbance, as revealed by mixture analysis. Ecology Letters 3: 516-522.; Silva et al. 2015Silva MHM, Almeida-Cortez JS, Silva EA, Santos JC. 2015. Relationship between gall-midge parasitism, plant vigor, and developmental instability in Ouratea polygyna Engl (Ochnaceae) in a patch of a Brazilian Atlantic Forest. Acta Botanica Brasilica 29: 274-277.). Therefore, any new relationship between the other types of asymmetry and a given stressing factor might promote advances for the studies of DI (McKenzie & O'Farrell 1993McKenzie JA, O'Farrell K. 1993. Modification of developmental instability and fitness: malathion-resistance in the Australian sheep blowfly, Lucilia cuprina. Genetica 89: 67-76.; Telhado et al. 2016Telhado C, Silveira FAO, Fernandes GW, Cornelissen T. 2016. Fluctuating asymmetry in leaves and flowers of sympatric species in a tropical montane environment. Plant Species Biology (in press). doi: 10.1111/1442-1984.12122
https://doi.org/10.1111/1442-1984.12122...
).

In the Brazilian savanna, the legume tree Bauhinia brevipes Vogel (Fabaceae) is a good model to investigate the relationship between phenotypic variation and DI according to different sunlight conditions. This species occurs in several phytophysiognomies within the savanna (Vaz & Tozzi 2003Vaz AMSF, Tozzi AMGA. 2003. Bauhinia ser. Cansenia (Leguminosae: Caesalpinioideae) no Brasil. Rodriguésia 54: 55-143.) and thus can be expected to occur in habitats with a wide variety of sunlight conditions. Bauhinia brevipes presents phenotypic variation and FA according to herbivory levels (Cornelissen & Fernandes 2001Cornelissen T, Fernandes GW. 2001. Induced defences in the neotropical tree Bauhinia brevipes (Vog.) to herbivory: effects of damage-induced changes on leaf quality and insect attack. Trees 15: 236-241.; Santos et al. 2013Santos JC, Alves-Silva E, Cornelissen TG, Fernandes GW. 2013. The effect of fluctuating asymmetry and leaf nutrients on gall abundance and survivorship. Basic and Applied Ecology 14: 489-495.), but environmental factors as agents that promote phenotypic variation and DI have not yet been examined. Thus, in this study, we investigated changes in both phenotypic variation and DI of B. brevipes in sites with different levels of sunlight exposure. Our measure of phenotypic variation was leaf area and DI was assessed through the differences between the leaf right and left sides (following Santos et al. 2013Santos JC, Alves-Silva E, Cornelissen TG, Fernandes GW. 2013. The effect of fluctuating asymmetry and leaf nutrients on gall abundance and survivorship. Basic and Applied Ecology 14: 489-495.). We hypothesized that increased leaf area and DI would occur in shady locations, which might indicate B. brevipes compensation for light absorption and elevated levels of stress, respectively.

Materials and methods

Study area

Fieldwork was conducted in November 2015 in a rupestrian field at the Parque Estadual da Serra de Caldas Novas, located in Caldas Novas city, Brazil (17°46'S - 48°40'W). The park has 123 km² of area, and sustains several phytophysiognomies distinctive of the Brazilian savanna (Cerrado biome), especially the Cerrado strictu sensu (see Felfili & Silva Jr. 1993Felfili JM, Silva Jr MC. 1993. A comparative study of cerrado (sensu stricto) vegetation in Central Brazil. Journal of Tropical Ecology 9: 277-289.; Castro et al. 1999Castro AAJF, Martins FR, Tamashiro JY, Shepherd GJ. 1999. How rich is the flora of Brazilian Cerrados? Annals of the Missouri Botanical Garden 86: 192-224. for detailed information about the Cerrados). The park has a tropical climate with two well-defined seasons, a hot-wet summer (October-March) and a (less hot) dry winter (April-September) (Lima et al. 2010Lima TA, Pinto JRR, Lenza E, Pinto ADS. 2010. Florística e estrutura da vegetação arbustivo-arbórea em uma área de cerrado rupestre no parque estadual da Serra de Caldas Novas, Goiás. Biota Neotropica 10: 159-166.). The sites chosen for the study supported sparse vegetation with a layer of herbs and grasses and a predominance of shrubs (<1 m tall) and medium trees (2-3 m tall).

Plant species

Bauhinia brevipes Vogel (Fabaceae) is a semi-deciduous tree that is widely distributed in the Brazilian savanna (Santos et al. 2013Santos JC, Alves-Silva E, Cornelissen TG, Fernandes GW. 2013. The effect of fluctuating asymmetry and leaf nutrients on gall abundance and survivorship. Basic and Applied Ecology 14: 489-495.), especially in sites where taller trees in the vicinity possess narrow canopies (Gonçalves-Alvim & Fernandes 2001Gonçalves-Alvim SJ, Fernandes GW. 2001. Comunidades de insetos galhadores (Insecta) em diferentes fisionomias do cerrado em Minas Gerais, Brasil. Revista Brasileira de Zoologia 18: 289-305.; Vaz & Tozzi 2003Vaz AMSF, Tozzi AMGA. 2003. Bauhinia ser. Cansenia (Leguminosae: Caesalpinioideae) no Brasil. Rodriguésia 54: 55-143.). Adult plants can reach up to 3 m in height, but many individuals in the Cerrado occur as shrubs (1-1.5 m tall), principally in areas where fires are common, such as at the park where the study was carried out (Lopes et al. 2009Lopes SF, Vale VS, Schiavini I. 2009. Efeito de queimadas sobre a estrutura e composição da comunidade vegetal lenhosa do cerrado sentido restrito em Caldas Novas, GO. Revista Árvore 33: 695-704.; Frizzo et al. 2012Frizzo TLM, Campos RI, Vasconcelos HL. 2012. Contrasting effects of fire on arboreal and ground-dwelling ant communities of a neotropical savanna. Biotropica 44: 254-261.). Bauhinia brevipes has green and smooth margined subcoriaceous leaves that may reach up to 9 cm in length and 7 cm in width. Small trichomes occur throughout the adaxial leaf surface (Vaz & Tozzi 2003Vaz AMSF, Tozzi AMGA. 2003. Bauhinia ser. Cansenia (Leguminosae: Caesalpinioideae) no Brasil. Rodriguésia 54: 55-143.). Leaf flush takes place in October and November at the onset of the rainy season (Santos et al. 2013Santos JC, Alves-Silva E, Cornelissen TG, Fernandes GW. 2013. The effect of fluctuating asymmetry and leaf nutrients on gall abundance and survivorship. Basic and Applied Ecology 14: 489-495.). At the time of sampling some individuals within the population of B. brevipes presented different phenological conditions, so we narrowed our search to those plants that had similar patterns of leaf and maturation and growth.

Study design

All B. brevipes selected for this study were located in a 5-ha radius, and we tagged individual plants that were at least 10 m distant from each other. In addition, only plants in the same phenological conditions (with mature leaves) and minimum presence of herbivory (i.e., galls and chewers) were chosen for the study design. Plants were chosen to permit the assessment of different sunlight conditions (e.g., shade and sunlight exposure). Following these criteria, we were able to select 26 B. brevipes shrubs (< 1.5 m tall) that fulfilled our requirements. Individual plants were equally assigned as 'shade' (plants that occurred near tall trees with large canopies that prevented B. brevipes shrubs from receiving direct sunlight) and 'sun' (plants that were not totally shaded by the canopy of any trees nearby), as adapted from Clark & Clark (1992)Clark DA, Clark DB. 1992. Life history diversity of canopy and emergent trees in a neotropical rain forest. Ecological monographs 62: 315-344. and Venâncio et al. (2016)Venâncio HS, Alves-Silva E, Santos JC. 2016. On the relationship between fluctuating asymmetry, sunlight exposure, leaf damage and flower set in Miconia fallax (Melastomataceae). Tropical Ecology 57: 419-427. (Fig. 1A-B).

From each individual plant, five branches located at approximately the same distance from the soil were chosen and one apical leaf was collected from each branch (n = 65 leaves from each plant group). Special care was given to this sampling and we chose only leaves without herbivory marks or hypersensitive reactions to galls (Santos et al. 2008Santos J, Silveira F, Fernandes G. 2008. Long term oviposition preference and larval performance of Schizomyia macrocapillata (Diptera: Cecidomyiidae) on larger shoots of its host plant Bauhinia brevipes (Fabaceae). Evolutionary Ecology 22: 123-137.). Leaves were photographed under a layer of transparent glass, and then we proceeded to the assessment of leaf area and sides (developmental instability) following Cornelissen & Stiling (2005)Cornelissen T, Stiling P. 2005. Perfect is best: low leaf fluctuating asymmetry reduces herbivory by leaf miners. Oecologia 142: 46-56..

Figure 1
Study design and leaf measurements of Bauhinia brevipes in a neotropical savanna. (A) plants labelled as 'sun', as they received sunlight most part of day; (B) shaded plants under canopy of tall trees; (C) B. brevipes leaf: Ls - left side, Rs - right side. Scale bar = 25 mm.

Leaf measurements

To investigate whether the leaf size varied according to microhabitat conditions (sun/shade), the area (mm²) of all leaves collected was estimated using Image J software from digital images of leaves. In addition, all leaves of B. brevipes were measured on both the right and left sides (Rs and Ls, in mm), and the horizontal middle portion of the leaves was used as the reference in order to account for the widest part of leaves (Fig. 1C). Individual leaf asymmetry was assessed as d = Rs - Ls. Trait size for leaf sides was calculated as (Rs + Ls)/2 and it was used to verify whether |d| (i.e. unsigned right minus left sides) varied according to leaf size. In order to verify whether our measurements were conducted with enough accuracy to eliminate errors, we used the Index of Repeatability of Falconer, which takes into account the individual versus side variance to estimate the measurement accuracy (Yezerinac et al. 1992Yezerinac SM, Lougheed SC, Handford P. 1992. Measurement error and morphometric studies: statistical power and observer experience. Systematic Biology 41: 471-482; Cornelissen & Stiling 2005Cornelissen T, Stiling P. 2005. Perfect is best: low leaf fluctuating asymmetry reduces herbivory by leaf miners. Oecologia 142: 46-56.). A subsample of 50 leaves was re-measured and compared with the original measurements of Rs and Ls.

Statistical analyses

A two-way ANOVA using leaf sides, individuals and measurements was conducted and the variance within and between the samples was used to calculate the Index of Repeatability (Yezerinac et al. 1992Yezerinac SM, Lougheed SC, Handford P. 1992. Measurement error and morphometric studies: statistical power and observer experience. Systematic Biology 41: 471-482), which varies from 0 (maximum error) to 1 (absence of measurement error). Before any inference on the role of environmental stress on the developmental instability levels, it is necessary to verify whether the leaf asymmetry measurements fit the pattern of fluctuating asymmetry (FA; i.e., small and random deviations from perfect symmetry), directional asymmetry (DA; i.e., one particular side is significantly larger than the other) or antisymmetry (AS; bimodal distribution of the Rs - Ls measurements). The presence of DA was tested with the Student's t test with the mean being equal to zero. Following Cowart & Graham (1999)Cowart NM, Graham JH. 1999. Within- and among-individual variation in fluctuating asymmetry of leaves in the fig (Ficus carica L.). International Journal of Plant Sciences 160: 116-121., we examined measures of skew (γ1) and kurtosis (γ2) to see whether the data deviated from normality (indication of antisymmetry). According to the tests, normal data should have a skewness value around zero and a kurtosis value around 3. These analyses were conducted with the package 'moments' in R statistical software 3.2.3. In addition, Shapiro-Wilk normality tests were also performed to check the normality of the data. We also conducted the Hartigans' Dip test for unimodality (package 'diptest' in R), which indicates potential bimodality in our data (evidence of AS). Measures of relative (coefficient of variation) and absolute (mean, standard deviation and median) were estimated for |d| and leaf area (mm²) in both plant groups.

The relationship between |d|, and trait size (both leaf width ((Ls+Rs)/2) and leaf area (mm²)) was examined with linear regressions. A significant relationship between these variables requires the use of a specific formula to assess developmental instability. Since leaf area was related to |d| (see Results section), it indicated that our data set fitted a multiplicative error according to Cowart & Graham (1999)Cowart NM, Graham JH. 1999. Within- and among-individual variation in fluctuating asymmetry of leaves in the fig (Ficus carica L.). International Journal of Plant Sciences 160: 116-121.. In this scenario, the values of |d| incur transformations before subsequent analyses. First, a log transformation was performed (|d| = log Ls - log Rs) and then data were Box Cox transformed (d* = (|d| + 0.00005)0.33). These series of transformations remove any size scaling from multiplicative error (when leaf asymmetry and trait size are related) and normalize the data (Cowart & Graham 1999Cowart NM, Graham JH. 1999. Within- and among-individual variation in fluctuating asymmetry of leaves in the fig (Ficus carica L.). International Journal of Plant Sciences 160: 116-121.). In addition, as we found antisymmetry in our data set (see Results section), the Box-coxed transformed data is more appropriate for statistical tests and comparisons between plant groups.

The DI index in plants was then assessed as DI = Ʃ(|d*|)/n, where 'n' is the number of leaves measured. The mean DI (i.e. leaf asymmetry) per plant was used for the subsequent statistical analyses. Comparisons of DI levels and leaf area (mm²) between plants in the sun and shade were made with Student's t tests.

Results

The Index of Repeatability showed that our measurements were conducted with enough accuracy, thus eliminating possible errors during the process of leaf morphometry evaluation (IR = 0.98). Our data indicated that plants in the sun presented purely FA, as both DA and AS were discarded, whereas data from shaded plants did not show DA or FA, but fit into AS. Neither kurtosis nor skewness were statistically significant in any plant group (Tab. 1). Density plots showed a clear bimodality in the leaf asymmetry data for plants in the shade (Fig. 2).

Figure 2
Density plots showing the distribution of leaf sides (right and left sides) according to plant location: (A) in the sun and (B) in the shade. Complete lines - adjusted distribution; dashed lines - predicted distribution.

Table 1
Statistical analyses to check for directional asymmetry (DA, one-sample Student's t test), antisymmetry (AS, Shapiro-Wilk and Hartigan's Dip test), skewness and kurtosis in two groups of Bauhinia brevipes in a Brazilian savanna. n.s. - non-significant; * P < 0.05 and ** P < 0.001. Values in bold indicate statistically significant differences.

Data were skewed to the right for both plant groups (skew > 0 in both plant groups). For the kurtosis, plants in the sun presented a platykurtic distribution. Shaded plants had a bimodal distribution, and so kurtosis interpretation was impractical. Leaf width was not related to leaf asymmetry |d| (sun: F11 = 0.0260; = 0.0023; P > 0.05; shade: F11 = 2.8230; = 0.2042; P > 0.05), but was positively and significantly related to leaf area from shaded plants (F11 = 6.0859; = 0.3562; P < 0.05; sun: F11 = 1.2464; = 0.1017; P > 0.05) (Fig. 3).

Figure 3
Relationship between leaf asymmetry |d|, leaf width and leaf area. Results were positive in all cases, but significant only for the relation between leaf asymmetry and area in shaded plants.

Developmental instability (d*) was roughly 5% (average values) higher in plants in the shade compared to plants in the sun, but results were not significant (t24 = 0.9413, P = 0.1779) (Fig. 4A). Leaf area, however, was statistically greater in shaded plants by 26.8% (t24 = 2.1500, P < 0.05) (Fig. 4B). Variation of leaf DI was higher for plants in the sun, while leaf area varied more for shaded plants. The range of DI was also higher for sunny plants and the opposite occurred for leaf area, as shaded plants presented larger variations compared to sunny plants. Values of median indicated that shaded leaves were 14% more asymmetrical and 22% larger than their counterparts in the sun (Tab. 2).

Figure 4
Leaf asymmetry as a measure of developmental instability, and phenotypic variation in Bauhinia brevipes according to location, i.e. sun or shade in a Brazilian savanna. (A) Leaf asymmetry; (B) leaf area. n.s. - non-significant.

Table 2
Measures of relative and absolute variability of developmental instability and leaf area in leaves of Bauhinia brevipes according to their occurrence in a Brazilian savanna. C.V. - coefficient of variation.

Discussion

Several plant species have the ability to modify their structures through phenotypic variation to maintain their performance in the face of stressing conditions (Cardoso & Lomônaco 2003Cardoso GL, Lomônaco C. 2003. Variações fenotípicas e potencial plástico de Eugenia calycina Cambess. (Myrtaceae) em uma área de transição cerrado-vereda. Revista Brasileira de Botânica 26: 131-140.). This modification is fairly common in light-dependent plant species growing in shaded habitats (Markesteijn et al. 2007Markesteijn L, Poorter L, Bongers F. 2007. Light-dependent leaf trait variation in 43 tropical dry forest tree species. American Journal of Botany 94: 515-25.). In our study, the legume shrub B. brevipes presented marked differences in leaf area in response to the microhabitat, as shaded plants had significantly larger leaves in comparison to plants in sunny sites. The modification of leaves is implemented by some plant species in order to increase the absorption of sunlight (Valladares et al. 2000Valladares F, Wright SJ, Lasso E, Kitajima K, Pearcy RW. 2000. Plastic phenotypic response to light of 16 congeneric shrubs from a Panamanian rainforest. Ecology 81: 1925-1936.; Baesse et al. 2014Baesse CQ, Tolentino VCM, Melo C. 2014. Phenotypic plasticity in Miconia albicans (sw.) Triana (Melastomataceae) between forest and cerrado areas at the Parque Estadual da Serra De Caldas Novas, in Goiás. International Journal of Science Commerce and Humanities 2: 119-129.). We have no data to show that B. brevipes is a pioneer species, but this plant is observed to resprout rapidly on the edges and in areas subjected to disturbances such as fire (EA Silva unpubl. res.), and so we are inclined to believe that it is fully adapted to open/sunny sites. Therefore, increased leaf area in shaded plants, as found in our study, can be a countermeasure of plants to persist in the late stages of the succession processes (Valladares et al. 2000Valladares F, Wright SJ, Lasso E, Kitajima K, Pearcy RW. 2000. Plastic phenotypic response to light of 16 congeneric shrubs from a Panamanian rainforest. Ecology 81: 1925-1936.; Valladares et al. 2002Valladares F, Chico JM, Aranda I, Balaguer L, Dizengremel P, Manrique E. 2002. The greater seedling high-light tolerance of Quercus robur over Fagus sylvatica is linked to a greater physiological plasticity. Trees 16: 395-403.; Rozendaal et al. 2006Rozendaal DMA, Hurtado VH, Poorter L. 2006. Plasticity in leaf traits of 38 tropical tree species in response to light; relationships with light demand and adult stature. Functional Ecology 20: 207-216.). Within the area where the study was carried out, fires are frequent and produce clearances for the colonisation of pioneer plants (Lopes et al. 2009Lopes SF, Vale VS, Schiavini I. 2009. Efeito de queimadas sobre a estrutura e composição da comunidade vegetal lenhosa do cerrado sentido restrito em Caldas Novas, GO. Revista Árvore 33: 695-704.). However, as the succession process advances, pioneer plants are subjected to high levels of light heterogeneity created by the canopy of large trees (Valladares et al. 2000Valladares F, Wright SJ, Lasso E, Kitajima K, Pearcy RW. 2000. Plastic phenotypic response to light of 16 congeneric shrubs from a Panamanian rainforest. Ecology 81: 1925-1936.; Poorter et al. 2004Poorter L, Plassche M, Willems S, Boot RGA. 2004. Leaf traits and herbivory rates of tropical tree species differing in successional status. Plant Biology 6: 746-754.). This microhabitat heterogeneity might have been responsible for the increased levels of variability in leaf area from shaded plants, which might allow for a better exposure to sunlight and postpone the persistence of B. brevipes in shaded habitats.

As we hypothesized, leaves in the shade presented increased levels of stress (but not statistically significant), as measured by the difference between the leaf sides. However, these differences were of only 5% (mean values; median = 14% differences). Leaf asymmetry was positively related to leaf area, but the data were significant only for shaded plants. This result may indicate that as the leaves grow larger to intercept more sunlight, they tend to become more asymmetric. A significant relationship between leaf asymmetry and area was not found for plants in the sun, indicating that leaf growth is not followed by developmental instability.

Some studies have found a relationship between sunlight and leaf developmental instability (Puerta-Piñero et al. 2008Puerta-Piñero C, Gómez JM, Hódar JA. 2008. Shade and herbivory induce fluctuating asymmetry in a mediterranean oak. International Journal of Plant Sciences 169: 631-635.; Alves-Silva & Del-Claro 2013Alves-Silva E, Del-Claro K. 2013. Effect of post-fire resprouting on leaf fluctuating asymmetry, extrafloral nectar quality, and ant-plant-herbivore interactions. Naturwissenschaften 100: 525-532.; Venâncio et al. 2016Venâncio HS, Alves-Silva E, Santos JC. 2016. On the relationship between fluctuating asymmetry, sunlight exposure, leaf damage and flower set in Miconia fallax (Melastomataceae). Tropical Ecology 57: 419-427.). For instance, developmental instability in leaves of the pioneer shrub Miconia fallax (Melastomataceae) was 25% higher in shaded plants than their co-specifics growing on the edges and exposed to sunlight (Alves-Silva 2012Alves-Silva E. 2012. The influence of Ditylenchus (Nematoda) galls and shade on the fluctuating asymmetry of Miconia fallax (Melastomataceae). Ecología Austral 22: 53-61.). All these studies found that leaves presented fluctuating asymmetry, and in fact, in a recent study, Santos et al. (2013)Santos JC, Alves-Silva E, Cornelissen TG, Fernandes GW. 2013. The effect of fluctuating asymmetry and leaf nutrients on gall abundance and survivorship. Basic and Applied Ecology 14: 489-495. found true patterns of fluctuating asymmetry in B. brevipes. Fluctuating asymmetry has long been considered as a phenotypic indicator of developmental instability and a biomarker of plant stress (Kozlov et al. 2001Kozlov MV, Zvereva EL, Niemelä P. 2001. Shoot fluctuating asymmetry: a new and objective stress index in Norway spruce (Picea abies). Canadian Journal of Forest Research 31: 1289-1291.), and it is related to several biotic (e.g., herbivory, competition, parasitism) and abiotic factors (e.g., pollution, climate, nutrients) (Rettig et al. 1997Rettig JE, Fuller RC, Corbett AL, Getty T. 1997. Fluctuating asymmetry indicates levels of competition in an even-aged poplar clone. Oikos 80: 123-127.; Cuevas-Reyes et al. 2011Cuevas-Reyes P, Oyama K, González-Rodríguez A, Fernandes GW, Mendoza-Cuenca L. 2011. Contrasting herbivory patterns and leaf fluctuating asymmetry in Heliocarpus pallidus between different habitat types within a Mexican tropical dry forest. Journal of Tropical Ecology 27: 383-391.; Beasley et al. 2013Beasley DAE, Bonisoli-Alquati A, Mousseau TA. 2013. The use of fluctuating asymmetry as a measure of environmentally induced developmental instability: a meta-analysis. Ecological Indicators 30: 218-226.; Uhl 2014Uhl D. 2014. Variability of selected leaf traits in European beech (Fagus sylvatica) in relation to climatic factors - some implications for palaeoenvironmental studies. Phytologia Balcanica 20: 145-153.; Ivanov et al. 2015Ivanov VP, Ivanov YV, Marchenko SI, Kuznetsov VV. 2015. Application of fluctuating asymmetry indexes of silver birch leaves for diagnostics of plant communities under technogenic pollution. Russian Journal of Plant Physiology 62: 340-348.; Alves-Silva & Del-Claro 2016Alves-Silva E, Del-Claro K. 2016. Herbivory-induced stress: leaf developmental instability is caused by herbivore damage in early stages of leaf development. Ecological Indicators 61: 359-365.). For instance, B. brevipes with high parasitism by galls have increased fluctuating asymmetry (Santos et al. 2013Santos JC, Alves-Silva E, Cornelissen TG, Fernandes GW. 2013. The effect of fluctuating asymmetry and leaf nutrients on gall abundance and survivorship. Basic and Applied Ecology 14: 489-495.). Other types of leaf asymmetry (e.g., directional asymmetry and antisymmetry) in relation to plant stress have rarely been examined, both because they do not seem to be common in nature and because researchers are still unsure whether they depict developmental instability (Graham et al. 1993Graham JH, Freeman DC, Emlen JM. 1993. Antisymmetry, directional asymmetry, and dynamic morphogenesis. Genetica 89: 121-137.; Silva et al. 2015Silva MHM, Almeida-Cortez JS, Silva EA, Santos JC. 2015. Relationship between gall-midge parasitism, plant vigor, and developmental instability in Ouratea polygyna Engl (Ochnaceae) in a patch of a Brazilian Atlantic Forest. Acta Botanica Brasilica 29: 274-277.; Telhado et al. 2016Telhado C, Silveira FAO, Fernandes GW, Cornelissen T. 2016. Fluctuating asymmetry in leaves and flowers of sympatric species in a tropical montane environment. Plant Species Biology (in press). doi: 10.1111/1442-1984.12122
https://doi.org/10.1111/1442-1984.12122...
).

In the present study, we found two patterns of asymmetry in B. brevipes. Plants in the sun, despite the high variability in bilateral morphometry, presented true patterns of fluctuating asymmetry, whereas leaves from shaded plants were rigorously antisymmetric. Leaf asymmetry was 5% higher in shaded plants (although this was not significant), revealing that these plants were, presumably, in a more stressed habitat compared to plants in the sun (see Puerta-Piñero et al. 2008Puerta-Piñero C, Gómez JM, Hódar JA. 2008. Shade and herbivory induce fluctuating asymmetry in a mediterranean oak. International Journal of Plant Sciences 169: 631-635.; Alves-Silva & Del-Claro 2013Alves-Silva E, Del-Claro K. 2013. Effect of post-fire resprouting on leaf fluctuating asymmetry, extrafloral nectar quality, and ant-plant-herbivore interactions. Naturwissenschaften 100: 525-532.; Venâncio et al. 2016Venâncio HS, Alves-Silva E, Santos JC. 2016. On the relationship between fluctuating asymmetry, sunlight exposure, leaf damage and flower set in Miconia fallax (Melastomataceae). Tropical Ecology 57: 419-427.). This makes us inclined to believe that antisymmetry can also depict stress, because if we assume that leaf asymmetry is related to stress levels, shaded plants were unable to buffer the developmental processes, thus giving rise to increased leaf asymmetry. Nonetheless, as differences in leaf asymmetry were low (5%) between plant groups, we cannot rule out the fact that leaf asymmetry may rather reflect the acclimation potential of the species over a wide host range with a diverse array of abiotic conditions (i.e., sun, shade, wind, edaphic conditions, relief, etc). In order to conclude that shade environment was stressful, additional investigations should be performed.

According to Graham et al. (1993Graham JH, Freeman DC, Emlen JM. 1993. Antisymmetry, directional asymmetry, and dynamic morphogenesis. Genetica 89: 121-137. and references therein), antisymmetry can arise from 'symmetry-breaking phase transitions', which occur when organisms are under severe stress, resulting in nonlinear processes of development (see also Lens & Dongen 2000Lens L, Dongen S. 2000. Fluctuating and directional asymmetry in natural bird populations exposed to different levels of habitat disturbance, as revealed by mixture analysis. Ecology Letters 3: 516-522., and Graham et al. 1998Graham JH, Emlen JM, Freeman DC, Leamy LJ, Kieser JA. 1998. Directional asymmetry and the measurement of developmental instability. Biological Journal of the Linnean Society 64: 1-16., for similar considerations on directional asymmetry). However, most studies on antisymmetry have been conducted with animal populations (Pratt & McLain 2002Pratt AE, McLain DK. 2002. Antisymmetry in male fiddler crabs and the decision to feed or breed. Functional Ecology 16: 89-98.; Will & Liebherr 2015Will KW, Liebherr JK. 2015. Antisymmetric male genitalia in Western Australian populations of Mecyclothorax punctipennis (Coleoptera: Carabidae: Moriomorphini). Insect Systematics & Evolution 46: 393-409.), and so the stress-antisymmetry relationship is lacking for plants. According to Sakai & Shimamoto (1965Sakai K-I, Shimamoto Y. 1965. Developmental instability in leaves and flowers of Nicotiana tabacum. Genetics and Molecular Research 51: 801-813.), both fluctuating asymmetry and antisymmetry can manifest in the same plant species. In B. brevipes, we found both fluctuating asymmetry and antisymmetry in the same population, and Santos et al. (2013)Santos JC, Alves-Silva E, Cornelissen TG, Fernandes GW. 2013. The effect of fluctuating asymmetry and leaf nutrients on gall abundance and survivorship. Basic and Applied Ecology 14: 489-495. found only fluctuating asymmetry in another distant population. This indicates that in some cases, different types of leaf asymmetry might depend on both spatial scales and stressing factors.

Here we showed changes in the phenotypic variation of B. brevipes in response to different microhabitats, and we also provide evidence that a plant outside its supposedly optimum habitat presents increased levels of leaf asymmetry, which fits the antisymmetry pattern. Whether other types of asymmetry (directional asymmetry and antisymmetry) reflect developmental instability still remains to be discussed (Graham et al. 1993Graham JH, Freeman DC, Emlen JM. 1993. Antisymmetry, directional asymmetry, and dynamic morphogenesis. Genetica 89: 121-137.; Graham et al. 2010Graham JH, Raz S, Hel-Or H, Nevo E. 2010. Fluctuating asymmetry: methods, theory, and applications. Symmetry 2: 466-540.; Telhado et al. 2016Telhado C, Silveira FAO, Fernandes GW, Cornelissen T. 2016. Fluctuating asymmetry in leaves and flowers of sympatric species in a tropical montane environment. Plant Species Biology (in press). doi: 10.1111/1442-1984.12122
https://doi.org/10.1111/1442-1984.12122...
) and more studies may clarify the relationship between plant stress and leaf asymmetry.

Acknowledgements

We thank the UFU for the discipline 'Field Ecology' and the Parque Estadual da Serra de Caldas Novas for providing logistical support for it. We appreciate the suggestions of two reviewers which increased the quality of the manuscript. We also thank Capes (Coordination for the Improvement of Higher Education Personnel), CNPq (National Counsel of Technological and Scientific Development, JCS grant nº 486742/2012-1) and Fapemig (Foundation of Support Research of the State of Minas Gerais) for funding.

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Publication Dates

  • Publication in this collection
    Apr-Jun 2016

History

  • Received
    14 Mar 2016
  • Accepted
    27 Apr 2016
Sociedade Botânica do Brasil SCLN 307 - Bloco B - Sala 218 - Ed. Constrol Center Asa Norte CEP: 70746-520 Brasília/DF. - Alta Floresta - MT - Brazil
E-mail: acta@botanica.org.br