ABSTRACT

Previous ecological studies of dioecious plant species have found that female plants preferentially occur at lower altitudes where there are typically better nutritional conditions, while male plants often occur in less favorable sites. We compared the ecophysiological performance of male and female plants in three populations of the dioecious Baccharis concinna, an endemic species of rupestrian grasslands of Serra do Cipó, in southeastern Brazil. We hypothesized that physiological differences between the sexes would explain the distribution patterns of the populations. Analyses of the tissue content of phosphorus (P), calcium (Ca), potassium (K) and sodium (Na), and carbon and nitrogen stable isotopes, were used to assess nutritional status and water use efficiency (WUE) in plant leaves, stems and roots of male and female plants in three populations located along an elevational gradient. Differences among populations were related to decreased nutrient levels and WUE at higher elevations, but an effect of sex was found only for %C, with male plants having slightly higher values. In conclusion, the sex ratios in the studied populations of B. concinna could not be attributed to differences in nutrient acquisition and WUE.

Keywords
Cerrado; campos rupestres; dioecy; plant sex; resource allocation; rupestrian grasslands; Serra do Cipó

Introduction

Many aspects of the natural history, ecology, and physiology of dioecious plants have been studied in an attempt to unravel the evolutionary processes and mechanisms involved in the differential distribution of the sexes among habitats of differing quality ( Lloyd 1973Lloyd DG. 1973. Sex ratios in sexually dimorphic Umbelliferae. Heredity 31: 239-249.; 1974Lloyd DG. 1974. Female-predominant sex ratios in angiosperms. Heredity 32: 35-44.; Freeman et al. 1976Freeman DC, Klikoff LG, Harper KT. 1976. Differential resource utilization by the sexes of dioecious plants. Science 193: 597-599.; Varga & Kytöviita 2011Varga S, Kytöviita M. 2011. Sex ratio and spatial distribution of male and female Antennaria dioica (Asteraceae) plants. Acta Oecologica 37: 433-440.; Castilla et al. 2012Castilla AR, Wiegandb T, Alonso C, Herrera CM. 2012. Disturbance-dependent spatial distribution of sexes in a gynodioecious understory shrub. Basic and Applied Ecology 13: 405-413.). Females have been reported to be relatively more abundant in habitats at lower elevations with higher humidity, lower salinity and less light exposure, and also in populations of low-density. Males in contrast, exhibit the opposite trend relative to these conditions (e.g., Pavón & Ramírez 2008Pavón NP, Ramírez IL. 2008. Sex ratio, size distribution and nitrogen reabsorption in the dioecious tree species Bursera morelensis (Burseraceae). Journal of Tropical Ecology 24: 463-466.; but see Vega-Fruits et al. 2013Vega-Frutis R, Munguía-Rosas MA, Varga S, Kytöviitaa M. 2013. Sex-specific patterns of antagonistic and mutualistic biotic interactions in dioecious and gynodioecious plants. Perspectives in Plant Ecology Evolution and Systematics 15: 45-55.; Riba-Hernandez et al. 2014Riba-Hernández P, Segura JB, Fuchs EJ, Moreira J. 2014. Population and genetic structure of two dioecious timber species Virola surinamensis and Virola koschnyi (Myristicaceae) in southwestern Costa Rica. Forest Ecology and Management 323: 168-176.). The spatial segregation of males and females of dioecious plants among habitats has been mostly attributed to sex-dependent differential resource requirements (e.g., Cox 1981Cox PA. 1981. Niche partitioning between sexes of dioecious plants. The American Naturalist 117: 295-307.; Meagher 1984Meagher TR. 1984. Sexual dimorphism and ecological differentiation of male and female plants. Annals of the Missouri Botanical Garden 71: 254-269.), in which female plants allocate more resources to reproduction, and preferentially occupy relatively more favorable habitats (e.g., Bierzychudek & Eckhart 1988Bierzychudek P, Eckhart V. 1988. Spatial segregation of the sexes of dioecious plants. The American Naturalist 152: 34-43.; Krischik & Denno 1990Krischik VA, Denno RF. 1990. Differences in environmental response between the sexes of the dioecious shrub, Baccharis halimifolia (Compositae). Oecologia 6: 176-181.; Herms & Mattson 1992Herms DA, Mattson WJ. 1992. The dilemma of plants: to grow or defend. The Quarterly Review of Biology 67: 283-335).

Analyses of δ¹³C and δ¹⁵N stable isotope composition have been successfully used to assess differences in carbon metabolism, water use and the magnitude of different N sources available to plants both at ecosystem ( Pate & Arthur 1998Pate J, Arthur D. 1998. δ13C analysis of phloem sap carbon: novel means of evaluating seasonal water stress and interpreting carbon isotope signatures of foliage and trunk wood of Eucalyptus globulus. Oecologia 117: 301-311.; Ehleringer et al. 2000Ehleringer JR, Evans RD, Williams D. 2000. Assessing sensitivity to change in desert ecosystems - a stable isotope approach. In: Griffts H. (ed.) Stable isotopes: integration of biological, ecological and geochemical processes. Oxford, Bios Scientific Publishers. p. 223-237.), and individual levels ( Robinson et al. 2000Robinson D, Handley LL, Scrimgeour CM, Gordon DC, Forster BP, Ellis RP. 2000. Using stable isotope natural abundances ((15N and δ13C) to integrate the stress responses of wild barley ( Hordeum spontaneaum C. Koch.) genotypes. Journal of Experimental Botany 51: 41-50.). (¹³C-ratios in organic material provide an estimate of the extent to which different components of gas exchange affect productivity ( Farquhar et al. 1989aFarquhar GD, Ehleringer JR, Hubick KT. 1989a. Carbon isotope discrimination and photosyntheis. Annual Review of Plant Physiology and Plant Molecular Biology 40: 503-537.), as well as screen C₃ genotypes for potential water-use efficiency ( Ehleringer et al. 1993Ehleringer JR, Hall AE, Farquhar GD. 1993. Stable isotopes and plant carbon/water relations. San Diego, Academic Press.). (¹⁵N-ratios, on the other hand, provide a quantitative estimate of the balance between nitrogen inputs and losses from soil, and species-specific patterns of nitrogen use ( Evans & Ehleringer 1993Evans RD, Ehleringer JR. 1993. A break in the nitrogen cycles in aridlands? evidence from (15N of soils. Oecologia 94: 314-317.). Environmental stresses, such as drought, modify δ¹³C in a largely predictable way ( Guy et al. 1998Guy RD, Wane PG, Reid DM. 1988. Stable carbon isotope ratio as an index of water-use efficiency in C3 halophytes - Possible relationship to strategies for osmotic adjustment. In: Rundel PW, Ehleringer JR, Nagy KA. (eds.) Stable isotopes in ecological research. Heidelberg, Springer . p. 55-75.), which is another useful tool for assessing the physiological mechanisms that influence the differential distribution of male and female dioecious plants.

The dioecious shrub Baccharis concinna (Asteraceae) is a threatened, endemic and locally rare species found in patches of rupestrian grasslands of the Serra do Cipó in southeastern Brazil ( Gomes et al. 2004Gomes V, Collevatti RG, Silveira FAO, Fernandes GW. 2004. The distribution of genetic variability in Baccharis concinna (Asteraceae), an endemic, dioecious and threatened shrub of rupestrian fields of Brazil. Conservation Genetics 5: 157-165.; Marques & Fernandes 2016Marques ESA, Fernandes GW. 2016. The gall inducing insect community on Baccharis concinna (Asteraceae): the role of shoot growth rates and seasonal variations. Lundiana 12: 17-26.). A higher proportion of female plants has been observed at lower elevations, while male plants are at higher relative proportions at higher elevations ( Marques et al. 2002Marques AR, Fernandes GW, Reis IA, Assunção RM. 2002. Distribution of adult male and female Baccharis concinna (Asteraceae) in the rupestrian fields of Serra do Cipó, Brazil. Plant Biology 4: 94-103.). These authors have reported that soils at higher elevations were more nutrient-impoverished than those at lower elevations. They also demonstrated that there is a trend towards the aggregation of plants of the same sex within habitats, but that the distribution of the sexes within habitats was random, perhaps owing to nutrient homogeneity of the soils within habitats as well as the absence of antagonism between the sexes. Therefore, this scenario presents an interesting opportunity to investigate the nutritional content of plants along an elevational gradient in order to make inferences about the functional responses of sex along an environmental gradient.

Here, we examined dried leaf, stem, and root samples from male and female plants from three populations of B. concinna along an elevational gradient. Both δ¹³C and (¹⁵N ratios were determined, aiming to assess C and N acquisition and water-use efficiency, and levels of C, N, C/N, P, Ca, K and Na were determined in order to assess the nutritional content of the samples analyzed. We hypothesize a strong sex effect, with female plants exhibiting a greater accumulation of nutrients, higher C and N acquisition and higher water-use efficiency than male plants. We also expect physiological differences along the elevational gradient because resource availability decreases with increasing elevation.

Materials and methods

Species, population sites and sample collection

The dioecious shrub Baccharis concinna G.M. Barroso (Asteraceae) is narrowly distributed in the Espinhaço Mountain Range in southeastern Brazil ( Barroso 1976Barroso GM. 1976. Compositae - subtribo Baccharidinae Hoffman: estudo das espécies ocorrentes no Brasil. Rodriguésia 40: 3-273.). The plant is an ericoid shrub reaching between 0.5 and 1.7 meters in height, and is endemic to the rupestrian grasslands of Serra do Cipó and Diamantina. Despite its local rarity, it supports one of the richest fauna of galling insects in rupestrian grasslands ( Fernandes et al. 1996Fernandes GW, Carneiro MAA, Lara ACF, et al. 1996. Galling insects on Neotropical species of Baccharis (Asteraceae). Tropical Zoology 9: 315-332.; 2014aFernandes GW, Silva JO, Espírito-Santo MM, Fagundes M, Oki Y, Carneiro MAA. 2014a. Baccharis: a Neotropical model system to study insect plant interactions. In: Fernandes GW, Santos JC. (eds.) Neotropical Insect Galls. New York, Springer. p. 193-219.; Marques & Fernandes 2016Marques ESA, Fernandes GW. 2016. The gall inducing insect community on Baccharis concinna (Asteraceae): the role of shoot growth rates and seasonal variations. Lundiana 12: 17-26.).

Rupestrian grasslands vegetation is a physiognomic formation of the Cerrado biome known for its high daily variation in temperature and humidity, and intense solar irradiance ( Fernandes et al. 2014bFernandes GW, Barbosa NPU, Negreiros D, Paglia AP. 2014b. Challenges for the conservation of vanishing megadiverse rupestrian grasslands. Natureza & Conservação 12: 162-165.; Fernandes 2016aFernandes GW. 2016a. The megadiverse rupestrian grassland. In: Fernandes GW. (ed.) Ecology and Conservation of Mountaintop grasslands in Brazil. Cham, Springer. p. 3-14.; Silveira et al. 2016Silveira FAO, Negreiros D, Barbosa NPU, et al. 2016. Ecology and evolution of plant diversity in the endangered campo rupestre: a neglected conservation priority. Plant Soil 403: 129-152). The soils are shallow, sandy, rocky (quartzite-derived) and with variable water retention capacity and organic matter content ( Negreiros et al. 2009Negreiros D, Fernandes GW, Silveira FAO, Chalub C. 2009. Seedling growth and biomass allocation of endemic and threatened shrubs of rupestrian fields. Acta Oecologica 35: 301-310.; Oliveira et al. 2015Oliveira RS, Galvão HC, de Campos MCR, Eller CB, Pearse SJ, Lambers H. 2015. Mineral nutrition of campos rupestres plant species on contrasting nutrient-impoverished soil types. New Phytologist 205: 1183-1194.). In tropical mountains, soil nutrient concentration generally decreases with increasing elevation ( Harrison et al. 1991Harrison AF, Taylor K, Hatton JC, Dighton J, Howard DM. 1991. Potential of root bioassay for determining P-deficiency in high altitude grassland. Journal of Applied Ecology 28: 277-289; Morecroft et al. 1992Morecroft MD, Woodward FI, Marrs RH. 1992. Altitudinal trends in leaf nutrient contents, leaf size and δ13C of Alchemilla alpina. Functional Ecology 6: 730-740.). Aluminum concentration also exhibits the same negative relationship with elevation ( Marques et al. 2002Marques AR, Fernandes GW, Reis IA, Assunção RM. 2002. Distribution of adult male and female Baccharis concinna (Asteraceae) in the rupestrian fields of Serra do Cipó, Brazil. Plant Biology 4: 94-103.), and has an important influence on nitrogen availability in the soil ( Goodland & Pollard 1972Goodland R, Pollard R. 1972. The Brazilian cerrado vegetation: a fertility gradient. Ecology 61: 219-225.). The climate of Serra do Cipó has four distinct seasons: a rainy season from November to January, a post-rainy season from February to April, a dry season from May to September, and a post-dry transition in October. Mean annual precipitation is around 1350 mm, low temperatures can be below 8 ^oC in the winter while high temperatures may rise above 35 ^oC in the summer ( Madeira & Fernandes 1999Madeira JA, GW Fernandes. 1999. Reproductive phenology of sympatric taxa od Chamaecrista (Leguminosae) in Serra do Cipó, Brazil. Journal of Tropical Ecology 15: 463-479.)

We studied three large patches (hereafter assumed to be distinct populations) of B. concinna at different elevations. The first population was located at km 101 of highway MG-010 at 900 m a.s.l. (19(18’S, 43(36’W), hereafter named Population 101. This population is in an area undergoing natural regeneration and contains numerous other shrub, grass and tree species. The second population was located at km 107 of highway MG-010 on privately owned property (Reserva Vellozia) with a relatively flat slope. This population is in a degraded area of rocky outcrop terrain at 1,150 m a.s.l. (19(17’S, 43(35’W) that is undergoing natural regeneration; hereafter named Population 107. The third population was in an eroded area outside Parque Nacional da Serra do Cipó at 1,500 m a.s.l. (km 123 highway MG-010, 19(14’S, 43(30’W); hereafter named Population 123. At each population, we randomly selected five male and five female individual plants and removed them in their entirety from the soil, taking care to completely remove the root system. Because (¹³C signatures may vary considerably between individuals in shaded and non-shaded habitats (see Damesin et al. 1997Damesin C, Rambal S, Joffre R. 1997. Between-tree variations in leaf δ13C of Quercus pubescens and Quercus ilex among Mediterranean habitats with different water availability. Oecologia 111: 26-35.; Pate & Arthur 1998Pate J, Arthur D. 1998. δ13C analysis of phloem sap carbon: novel means of evaluating seasonal water stress and interpreting carbon isotope signatures of foliage and trunk wood of Eucalyptus globulus. Oecologia 117: 301-311.), samples were taken only from individuals exposed to full sunlight. Plants were then placed into plastic bags, deposited in an icebox and taken to the laboratory where they were separated into roots, stems, and leaves, and dried in an oven at 50 (C for 72 h. The plants were then powdered in a mortar for analyses.

Nutrient analyses

To determine the concentration of sodium, calcium, and potassium in the tissues of B. concinna, 50 mg of dry powdered plant material was extracted with 5 ml distilled water for 30 min and centrifuged at 4000 x for 10 min. The supernatant was used to measure ion content by flame photometry (Eppendorf, Hamburg, Germany). To determine total phosphate content, 50 mg of dry powdered plant material was digested by boiling five times with 1 ml H₂SO₄ (95 %), HCl (70 %) and 3 droplets of H₂O₂ (30 %). Phosphate was determined colorimetrically according to Strickland & Parsons (1965Strickland JDH, Parsons TR. 1965. A manual of seawater analysis. Journal of the Fisheries Research Board of Canada 125: 203.).

Stable isotope analyses

Isotope ratio mass spectrometry (IRMS) was used to determine ( ¹⁵N, ( ¹³C, nitrogen and carbon content. Aliquots of dried plant material were weighed, placed in tin capsules (CE instruments, Milan, Italy) and injected into an elemental analyzer (EA) NC 2500 (CE instruments, Milan, Italy). Samples were combusted and oxidized at 1000 °C in an oxidation oven (HE 46820999 Hekatech, Wegberg, Germany). From the resulting gases (H₂O, CO₂, N₂, NO_x), nitrogen-oxides were reduced at 750 °C in a copper reduction oven (HE 46820899 Hekatech, Wegberg, Germany) to final N₂. H₂O was fixed with magnesium perchlorate. N₂ and CO₂ in the helium carrier gas flow were separated in a gas chromatographic column. Flow was split and N₂, which was diluted first from the column, was conducted without dilution into the Con Flow II interface (Finnigan MAT GmbH, Bremen, Germany), which connected the EA with the Delta^plus isotope ratio mass spectrometer (Finnigan MAT GmbH, Bremen, Germany). CO₂ was diluted with helium when passing through the interface. In IRMS, N₂ molecules (mass 28: 14N-14N, mass 29: 14N-15N, and mass 30: 15N-15N) and CO₂ molecules (mass 44: ¹²C-¹⁶O₂, mass 45: ¹³C-¹⁶O₂, ¹²C-¹⁷O-¹⁶O, ¹²C-¹⁶O-¹⁷O, and mass 46: ¹²C-¹⁸O-¹⁶O, ¹²C-¹⁶O-¹⁸O, ¹³C-¹⁷O-¹⁶O, ¹³C-¹⁶O-¹⁷O, ¹²C-¹⁷O₂, ¹⁴C-¹⁶O₂) were fractionated and detected by mass. Results were corrected with standard L-glutamate samples, calibrated against Pee Dee Be (CO₂) and N₂ in air (N₂), which were measured. The δ¹³C values were calculated as:

where R_Sample and R_PDB are ¹³C/¹²C ratios of the sample and the standard Pee Dee belemnite (PDB), respectively.

The δ¹⁵N values were calculated as:

where R_Sample and R_{N2 in air} are ¹⁵N/¹⁴N ratios of the sample and N₂ in air, respectively.

Total N content and total C content of the samples were calculated as sum of the single isotopes and expressed as proportion (%) of sample mass. From the measurements of carbon isotope ratio (δ¹³C), ¹³C discrimination (∆¹³C) was calculated as follows:

where δ_a and δ_p (in ‰) are carbon isotope ratios of the ambient air (assumed here to be - 8 ‰) and dried plant material, respectively ( Farquhar et al. 1989aFarquhar GD, Ehleringer JR, Hubick KT. 1989a. Carbon isotope discrimination and photosyntheis. Annual Review of Plant Physiology and Plant Molecular Biology 40: 503-537.; b; Broadmeadow et al. 1992Broadmeadow MSJ, Griffiths H, Maxwell C, Borland A. 1992. The carbon isotope ratio of plant organic material reflects temporal and spatial variations in CO2 within tropical forest formation in Trinidad. Oecologia 89: 435-441.).

Statistical analyses

For statistical analyses of ion, nutrient, and isotope concentrations in tissues we used two-way analyses of variance, which allowed us to compare the effects of the independent factors of plant sex and population, plus their interaction. Analyses were done separately for each plant organ. When significant differences were detected we performed post-hoc tests using the Tukey HSD test ( Zar 1996Zar JH. 1996. Biostatistical analysis. 3rd. edn. New Jersey, Prentice Hall.). For all analyses, we assessed the statistical assumptions and considered an α value of ≤ 0.05 as significant.

Results

Tables 1, 2 and 3 show the effects of the variables plant sex and population, and their interaction, for all measured parameters in leaf, stem and root tissue, respectively. The subsequent figures illustrate the differences found whenever there were significant effects of plant sex, population, or their interaction, on sex differences within populations.

The effect of plant sex

The effect of plant sex was restricted to the leaves of B. concinna, and only for %C ( Tab. 1), with the average value for males of all three populations being higher than the average for female plants (male = 47.4±4.1, female = 42.9±5.4, Fig. 1). No other significant difference was observed within populations. All the other parameters from stable isotope analysis (δ¹³C, Δ¹³C, δ¹⁵N and C/N), as well as tissue concentration of P, K⁺, Na⁺ and Ca⁺⁺, varied independently of plant sex.

The effect of population, and its interactions with plant sex

The variable population had a much clearer effect on a variety of parameters and appeared to be an important factor to the physiological behavior of B. concinna. Among the parameters of the isotope analysis, δ¹³C-values exhibited the clearest effect of population, which was observed across all studied organs ( Tabs. 1 - 3). Population 123 clearly possessed a less negative isotope ratio (in foliar, stem and root tissue) while Population 101 and Population 107 had either more negative or intermediately negative values (stems and roots) ( Fig. 2). A similar pattern was also observed with ¹³C discrimination (Δ¹³C) in leaf and stem tissue ( Tabs. 1, 2; Figs. 2, 3).

The %C and C/N ratio experienced an effect from the variable population only in foliar tissue ( Tab. 1 and Fig. 1). The population average %C was similar in Population 101 and Population 123, while it was lower in Population 107 than Population 123 at higher elevations. The C/N ratio was also lower in Population 107, but higher in Population 123 and Population 101, which did not differ significantly.

With regard to P concentration ( Fig. 4), the effect of population was found in foliar and root tissues, with the overall plant average of Population 107 having the highest values. For root tissue, Population 123 had the lowest average value. Population 101 had intermediate values, being similar to Population 123 with regard to leaves, and similar to Population 107 with regard to roots.

Among the ions analyzed, the effect of population on Ca⁺⁺ concentration was evident in root and stem tissue, with lower values for Population 123 at the highest elevation ( Fig. 5), while effects on K⁺ concentration were restricted to stem tissue ( Fig. 6), where a decreasing gradient was found with increasing elevation from Population 101 to Population 123.

Significant interaction between plant sex and population was rare among the parameters studied, and restricted to P concentration in stem and root tissue ( Fig. 4), and K⁺ concentration in leaf tissue ( Fig. 6). Stem P concentration in female plants was significantly higher than that in male plants in Population 101. However, the inverse pattern was observed in Population 107, and no difference was observed in Population 123. In roots, significant differences between sexes regarding P concentration exhibited an inverse pattern to that of stems: female plants had lower P-values in Population 101 and higher values in Population 107, while Population 123 exhibited no difference.

Lastly, K⁺ concentration only differed between the sexes in leaves of Population 101, where male plants had greater values than female plants. The other two populations did not exhibit any clear differences. Overall, the patterns observed for P and K⁺ concentrations regarding sex were irregular, with no absolute sex effect being observed in all cases.

Discussion

Male and female plants of dioecious species may differ in traits related to plant phenology and defense against herbivory, as well as in physiological traits such as photosynthetic performance and water use ( Dawson & Geber 1999Dawson TE, Geber MA. 1999. Sexual dimorphism in physiology and morphology. In: Geber MA, Dawson TE, Delph LF. (eds.) Gender and dimorphism of flowering plants. Berlin, Springer. p. 175-216.; Rowland & Johnson 2001Rowland DL, Johnson NC. 2001. Sexual demographics of riparian populations of Populus deltoides: can mortality be predicted from change in reproductive status? Canadian Journal of Botany 79: 702-710.; Cornelissen & Stiling 2005Cornelissen T, Stiling P. 2005. Sex-biased herbivory: a meta-analysis of the effects of gender on plant-herbivore interactions. Oikos 111: 488-500.; Vega-Fruits et al. 2013Vega-Frutis R, Munguía-Rosas MA, Varga S, Kytöviitaa M. 2013. Sex-specific patterns of antagonistic and mutualistic biotic interactions in dioecious and gynodioecious plants. Perspectives in Plant Ecology Evolution and Systematics 15: 45-55.). Male and female plants of many species have adapted to different ecological niches ( Shine 1989Shine R. 1989. Ecological causes for the evolution of sexual dimorphism: a review of the evidence. The Quarterly Review of Biology 64: 419-461.; Obeso 2002Obeso JR. 2002 The cost of reproduction in plants. New Phytologist 155: 321-348.; Vega-Fruits et al. 2013Vega-Frutis R, Munguía-Rosas MA, Varga S, Kytöviitaa M. 2013. Sex-specific patterns of antagonistic and mutualistic biotic interactions in dioecious and gynodioecious plants. Perspectives in Plant Ecology Evolution and Systematics 15: 45-55.), and many authors have argued that female individuals are more common in habitats of better quality (e.g., Freeman et al. 1976Freeman DC, Klikoff LG, Harper KT. 1976. Differential resource utilization by the sexes of dioecious plants. Science 193: 597-599.; Cox 1981Cox PA. 1981. Niche partitioning between sexes of dioecious plants. The American Naturalist 117: 295-307.; Wef & Berg 1995Wef HMG, Berg W. 1995. Nitrogen fertilization and sex expression effect size variability of fiber hemp ( Cannabis sativa L.) Oecologia 103: 462-470.; see review by Vega-Fruits et al. 2013Vega-Frutis R, Munguía-Rosas MA, Varga S, Kytöviitaa M. 2013. Sex-specific patterns of antagonistic and mutualistic biotic interactions in dioecious and gynodioecious plants. Perspectives in Plant Ecology Evolution and Systematics 15: 45-55.). Marques et al. (2002Marques AR, Fernandes GW, Reis IA, Assunção RM. 2002. Distribution of adult male and female Baccharis concinna (Asteraceae) in the rupestrian fields of Serra do Cipó, Brazil. Plant Biology 4: 94-103.) corroborated the hypothesis of sex segregation between habitats in an investigation of B. concinna, by finding a greater proportion of males at higher elevations and greater proportion of females at lower elevations. In the present study, we found minor differences in the physiological behavior of male and female plants of B. concinna, and argue that the unbalanced distribution of the sexes could have been influenced by soil quality, since the dominance of female plants at lower elevations coincided with greater soil fertility, in terms of P/N and Ca/Al ratios, at lower elevations ( Marques et al. 2002Marques AR, Fernandes GW, Reis IA, Assunção RM. 2002. Distribution of adult male and female Baccharis concinna (Asteraceae) in the rupestrian fields of Serra do Cipó, Brazil. Plant Biology 4: 94-103.).

Plant communities of rupestrian grasslands exist on extremely nutrient-impoverished soils ( Oliveira et al. 2015Oliveira RS, Galvão HC, de Campos MCR, Eller CB, Pearse SJ, Lambers H. 2015. Mineral nutrition of campos rupestres plant species on contrasting nutrient-impoverished soil types. New Phytologist 205: 1183-1194.; Fernandes 2016aFernandes GW. 2016a. The megadiverse rupestrian grassland. In: Fernandes GW. (ed.) Ecology and Conservation of Mountaintop grasslands in Brazil. Cham, Springer. p. 3-14.; bFernandes GW. 2016b. Ecology and Conservation of Mountaintop Grasslands in Brazil. Cham, Springer .). These soils possess exceptionally low levels of P, which is an essential element for seed production ( Fujita et al. 2014Fujita Y, Venterink HO, Bodegom PM, et al. 2014. Low investment in sexual reproduction threatens plants adapted to phosphorus limitation. Nature 505: 82-86.). Therefore, we anticipated to find pronounced differences in resource acquisition strategies between male and female plants. However, we found only minor differences in tissue nutrient accumulation between male and female plants, with the most significant differences occurring between populations at different elevations. Plants of B. concinna at higher elevations had the lowest nutrient concentrations, irrespective of their sex. At the study site, soil fertility decreased with elevation, and so individuals of B. concinna at higher elevations experienced the most extreme conditions of resource scarcity ( Fernandes et al. 2007Fernandes GW, Rodarte LHO, Negreiros D, Franco AC. 2007. Aspectos nutricionais em Baccharis concinna (Asteraceae), espécie endemic e ameaçada da Serra do Espinhaço, Brasil. Lundiana 8: 83-88.). Therefore, sexual reproduction in populations at the highest elevations can be severely constrained by resource limitation.

Values of Δ¹³C are known to be correlated with long-term average stomatal opening (g_H2O), and show a negative correlation with plant overall water-use-efficiency (WUE). Thus, this parameter is a good indicator of the overall WUE throughout the lifetime of a plant ( Farquhar et al. 1989 aFarquhar GD, Ehleringer JR, Hubick KT. 1989a. Carbon isotope discrimination and photosyntheis. Annual Review of Plant Physiology and Plant Molecular Biology 40: 503-537.; bFarquhar GD, Hubick KT, Coudon AG, Richards RA. 1989b. Carbon isotope fractionation and plant water-use efficiency. In: Rundel PW, Ehleringer JR, Nagy KA. (eds.) Stable isotopes in ecological research. Heidelberg, Springer. p. 21-40.; Broadmeadow et al. 1992Broadmeadow MSJ, Griffiths H, Maxwell C, Borland A. 1992. The carbon isotope ratio of plant organic material reflects temporal and spatial variations in CO2 within tropical forest formation in Trinidad. Oecologia 89: 435-441.; Ehleringer et al. 1993Ehleringer JR, Hall AE, Farquhar GD. 1993. Stable isotopes and plant carbon/water relations. San Diego, Academic Press.; Guehl et al. 2004Guehl J-M, Bonal D, Ferhi A, Barigah TS, Farquhar G, Granier A. 2004. Community-level diversity of carbon-water relations in rainforest trees. In: Gourlet-Fleury S, Guehl J-M, Laroussinie O. (eds.) Ecology and management of a neotropical rainforest. Amsterdam, Elsevier. p. 75-94.). This made it possible to infer that the population at the highest elevation had a higher WUE than other populations.

We had postulated that the differential occupation of habitats by male and female plants would result in different concentrations of ions in plant organs. Our study showed differences in performance among populations of B. concinna in traits related to water use efficiency (Δ¹³C) and nutrient acquisition (%C, C/N, P, Ca⁺⁺and K⁺). Among them, a minor effect of sex was suggested by the interaction of sex and population with regard to P and K⁺ concentrations, although the patterns were not enough to establish a clear difference in behavior between male and female plants. Foliar %C, however, was always higher in male plants independent of intra-population variation. This result could indicate that male plants of B. concinna are more efficient at fixing carbon than female plants, although the latter are more abundant at more nutritional sites ( Marques et al. 2002Marques AR, Fernandes GW, Reis IA, Assunção RM. 2002. Distribution of adult male and female Baccharis concinna (Asteraceae) in the rupestrian fields of Serra do Cipó, Brazil. Plant Biology 4: 94-103.). Nevertheless, this difference was very subtle since no significant differences were found within populations, and only in pooled averages from all sites. Zhao et al. (2012 Zhao H, Li Y, Zhang X, Korpelainen H, Li C. 2012. Sex-related and stage-dependent source-to-sink transition in Populus cathayana grown at elevated CO2 and elevated temperature. Tree Physiololgy 32: 1325-1338.) showed that adult and fully expanded leaves of male plants of the riparian species Populus cathayana exhibited improved photosynthesis-dependent traits related to source and sink differences compared to female plants. Conversely, Liebig et al. (2001Liebig M, F Scarano, E Mattos, H Zaluar, U Lüttge. 2001. Ecophysiological and floristic implications of sex expression in the dioecious neotropical CAM tree Clusia hilariana Schltdl. Trees 15: 278-288.) found that sex expression has no important general consequences for the physiology of the photosynthetic light reactions of Clusia hilariana, a dioecious and obligate CAM plant that grows on oligotrophic sandy coastal planes ( restingas) in Brazil

In conclusion, our study found minor differences in tissue nutrient content between male and female plants of Baccharis concinna, although a consistent decrease in nutrient content was detected with increasing elevation. It remains unclear whether the higher %C in males of B. concinna are a result of resource partitioning between male and female plants, and studies should be conducted to attempt to explain this finding. Such studies will serve to advance our understanding of the ecology of dioecious plants, and improve our ability to predict the response of such plants to changing climate conditions in mountains.

Acknowledgements

We thank F. R. Scarano, B. Rubia and two anonymous reviewers for their comments and criticisms, which improved the manuscript. This study was funded by MCTIC/CNPq, FAPEMIG, the International Foundation for Science (C-2487/1), and CAPES/DAAD. The logistical support provided by Reserva Vellozia and Planta Tecnologia Ambiental is also acknowledged. GWF and FAOS acknowledge a CNPq fellowship for research productivity.

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