Mineral nutrition of campos rupestres plant species on contrasting...

Mineral nutrition of campos rupestres plant species oncontrasting nutrient-impoverished soil types

Rafael S. Oliveira1,2, Hugo C. Galv~ao1, Mariana C. R. de Campos2, Cleiton B. Eller1, Stuart J. Pearse2 and

Hans Lambers2

1Departamento de Biologia Vegetal, Universidade Estadual de Campinas, Rua Monteiro Lobato 255, Campinas 13083-862, Brazil; 2School of Plant Biology, University of Western Australia,

35 Stirling Highway, Crawley, Perth, WA 6009, Australia

Authors for correspondence:Hans Lambers

Tel: +55 1935216177Email: [email protected]

Rafael S. OliveiraTel: +55 19 3521 6177

Email: [email protected]

Received: 25 August 2014Accepted: 13 October 2014

New Phytologist (2014)doi: 10.1111/nph.13175

Key words: cerrado, dauciform roots, leafmanganese (Mn) concentration, leaf nutrientconcentrations, mycorrhizas,nitrogen : phosphorus (N : P) ratio,phosphorus nutrition, sand-binding roots.

Summary

� In Brazil, the campos rupestres occur over the Brazilian shield, and are characterized by

acidic nutrient-impoverished soils, which are particularly low in phosphorus (P). Despite rec-

ognition of the campos rupestres as a global biodiversity hotspot, little is known about the

diversity of P-acquisition strategies and other aspects of plant mineral nutrition in this region.� To explore nutrient-acquisition strategies and assess aspects of plant P nutrition, we mea-

sured leaf P and nitrogen (N) concentrations, characterized root morphology and determined

the percentage arbuscular mycorrhizal (AM) colonization of 50 dominant species in six com-

munities, representing a gradient of soil P availability. Leaf manganese (Mn) concentration

was measured as a proxy for carboxylate-releasing strategies.� Communities on the most P-impoverished soils had the highest proportion of nonmycorrhi-

zal (NM) species, the lowest percentage of mycorrhizal colonization, and the greatest diversity

of root specializations. The large spectrum of leaf P concentration and variation in root mor-

phologies show high functional diversity for nutritional strategies. Higher leaf Mn concentra-

tions were observed in NM compared with AM species, indicating that carboxylate-releasing

P-mobilizing strategies are likely to be present in NM species.� The soils of the campos rupestres are similar to the most P-impoverished soils in the world.

The prevalence of NM strategies indicates a strong global functional convergence in plant

mineral nutrition strategies among severely P-impoverished ecosystems.

Introduction

Landscapes characterized by very low soil phosphorus (P) concen-trations, such as those of south-western Australia (Holford,1997) and the Cape Floristic Region in South Africa (Witkowski& Mitchell, 1987), are frequently hotspots of biodiversity (Myerset al., 2000). The species native to these regions are adapted tolow concentrations of soil P through several mechanisms. Aseverely limiting P availability is associated with leaf scleromor-phy (Specht & Rundel, 1990; Wright et al., 2002) and also withslow growth and longer leaf longevity (Lambers & Poorter, 1992;Wright & Westoby, 2003). Species from P-impoverished soilsoften present low leaf P concentration ([P]) and high photosyn-thetic P-use efficiency (Wright et al., 2004; Lambers et al., 2012),and can exhibit high levels of P resorption from senescing leaves(Kobe et al., 2005; Denton et al., 2007; De Campos et al., 2013).These nutrient-conserving mechanisms may co-occur with P-acquisition specializations, which include morphological traitssuch as a root architecture configured for exploration of thesuperficial layers of the soil, increased specific root length,increased root : shoot ratio or increased proliferation of root hairs(Lambers et al., 2006). Other specializations are the development

of cluster roots, which increase the root surface area and exudecompounds that release P from fractions sorbed onto soil parti-cles or from organic fractions (Shane & Lambers, 2005), andassociations with mycorrhizal fungi, which extend the volume ofsoil explored and allow access to nutrient-rich patches (Tibbett,2000).

While mycorrhizal associations are very common in landplants (Brundrett, 2009), they are proportionally less common inextremely P-impoverished landscapes where cluster roots are amajor P-acquisition specialization (Lambers et al., 2008, 2010).Cluster roots are a morphological and physiological specializationthat increases plant P availability through the exudation of carb-oxylates that release P from forms that are strongly bound to thesoil (Dinkelaker et al., 1989; Veneklaas et al., 2003). Clusterroots are abundant on the most severely P-impoverished soils,but on less impoverished soils cluster roots may be less advanta-geous than mycorrhizas, probably because of the higher carboncost of cluster roots to the plant (Lambers et al., 2006).

In addition to releasing P from soil particles, carboxylatesmobilize a range of micronutrients, including manganese (Mn)(Jauregui & Reisenauer, 1982; Wang & Stone, 2006). Therefore,leaf Mn accumulation would be expected in plants with a

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carboxylate-releasing strategy in response to P deficiency. In arecent study across a chronosequence in south-western Australia,Hayes et al. (2014) showed that nonmycorrhizal (NM) specieswith root specializations (cluster roots, dauciform roots, andsand-binding roots) have higher leaf [Mn] compared with mycor-rhizal species. These results suggest that leaf Mn accumulation isa simple proxy for carboxylate release (Lambers et al., 2014b).

The Brazilian campos rupestres, which harbor lineages originat-ing from Gondwanaland (Mello-Silva et al., 2011), similar toSouth Africa and Australia, also exhibit very low concentrationsof soil P (Benites et al., 2007). These similarities, as well as a highbiodiversity in all three regions (Giulietti et al., 1997; Myerset al., 2000), have led us to propose that there are equivalentfunctional groups of species in the campos rupestres, the fynbos inSouth Africa and the kwongan in south-western Australia (Lam-bers et al., 2014a). We aimed to explore P-acquisition strategiesand assess aspects of plant P nutrition in six campos rupestres com-munities with contrasting soil nutrient availability, in comparisonwith those in similar nutrient-impoverished landscapes in south-western Australia and the Cape Floristic Region in South Africa.Little is known about root structures and the ecophysiology ofspecies with regard to plant P nutrition in the campos rupestres.The present study is the first explorative investigation of rootadaptations in P-impoverished habitats of the campos rupestres.

We hypothesized that soil [P] plays a major role in determin-ing root specializations and leaf nutrient concentrations in plantcommunities, similar to what has been observed in other severelynutrient-impoverished regions such as the kwongan in south-western Australia and fynbos in the Cape Floristic Region inSouth Africa (Lambers et al., 2014a). We expect plant communi-ties with lower soil [P] to have lower mycorrhizal root coloniza-tion and a higher proportion of NM species with rootspecializations that enhance P acquisition, such as a high densityof root hairs and evidence of root carboxylate exudation. We alsoexpect that plant communities with lower soil [P] will showhigher leaf [Mn], especially NM species, as evidence of release ofcarboxylates to increase soil P availability (Lambers et al., 2014b).Leaf [P] would decrease and the leaf N : P ratio would increasewith decreasing soil [P].

Materials and Methods

Study site

Campos rupestres (rocky fields) are characterized by the occurrenceof rock outcrops and sandy fields with shallow skeletal soils (Fur-ley & Ratter, 1988). They generally originate from nutrient-poorparent rocks such as quartzites and less commonly granites orsandstones (Alves & Kolbek, 1994; Benites et al., 2007) and thisparent material is believed to have been formed in the Pre-Cam-brian era (Conceic�~ao et al., 2007).

Our study was performed on six field sites (Table 1) located inthe State of Minas Gerais, central Brazil. These sites are locatedin the Espinhac�o Range and were chosen because, despite beinggenerally nutrient impoverished, they form a natural gradient ofsoil P availability. The vegetation of the field sites varied from

very sparse herbaceous vegetation over white sandy soils (WS1and WS2), to savanna shrublands over gravel-rich soil (GC1 andGC2), to grasslands over peat-bog soils (PB1 and PB2) (Fig. 1).The climate is continental and, for the region surveyed, mini-mum daily temperatures range between 10 and 15°C and max-ima between 20 and 28°C, and rainfall ranges between 1250 and1550 mm (INMET, 1911–2010). We measured leaf nutrientconcentrations, characterized root morphology and determinedthe percentage of arbuscular mycorrhizal (AM) colonization onthree to six individuals from each of the 10 most abundantspecies at each site.

Plant analyses

We collected fully expanded mature green leaves with no appar-ent damage for leaf nutrient analyses. The leaves were dried in alamp oven in the field and in a convection oven at 60°C for 3 dupon return from the field. Dried leaves were ground with amortar and pestle, digested with nitric and perchloric acid andanalyzed for [P], following the molybdate and malachite greenmethod (Motomizu et al., 1983). Leaf [Mn] was analyzed byatomic absorption spectrometry (Perkin Elmer 5300 DV ICP-OES; Perkin Elmer, Waltham, MA, USA). Leaf N results wereobtained through combustion under continuous helium flux inan elemental analyzer (model CHN-1110; Carlo Erba, Milan,Italy).

The roots of the same individuals sampled for leaves were exca-vated during the wet season and field observations of visibleradicular specializations were recorded. Root samples were col-lected and kept in 70% (v/v) ethanol. The entire root system wascollected when possible, and when this was not possible, asubsample of the terminal portion of the roots was taken instead.Root samples kept in ethanol were prepared following the proto-col used by Brundrett et al. (1996). Roots were cleared (diaphan-ized) with 10% (w/v) KOH in an autoclave for 20 min at 121°Cand then stained with 0.05% (w/v) Trypan Blue and preserved inlactoglycerol. Arbuscular mycorrhizal colonization counts wereperformed under a stereomicroscope and recorded as a percent-age of colonization. Arbuscules, vesicles and hyphae were consid-ered in the counts. During this process, any structural rootmodifications were also recorded. Species that presented < 1%AM colonization in roots of all collected individuals were consid-ered NM (Brundrett, 2009). In addition to the 50 most abun-dant species, we excavated the root systems of 12 species ofimportant campos rupestres families (Velloziaceae, Xyridaceae andEriocaulaceae) outside our study plots to evaluate their rootmorphology and mycorrhizal status.

Soil sampling and nutrient analyses

Three soil samples that represented the substrate of each commu-nity were collected at each study site. Samples were taken at 0–20 cm depth in each area and sieved (< 2 mm) to remove rootsand organic debris. All soil samples collected were taken back tothe Department of Soil Science at the University of S~ao Paulo.The samples were homogenized, air-dried and analyzed following

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the standardized methods of the Institute of Agriculture ofCampinas (IAC). In order to measure readily available P in thesoil, several methods are available, such as the resin-extractablemethod, the Colwell method, the Bray I and II methods, and theOlsen method (Bray & Kurtz, 1945; Olsen et al., 1954; Ameret al., 1955; Colwell, 1963). The applicability of such methodol-ogies depends on soil characteristics such as texture, mineralogyand pH. We opted to measure readily available P by the ion-exchange resin method because it is suitable for a wide range ofsoils (Amer et al., 1955), with a detection limit (DL) of1 mg kg�1 (set by the calibration curve). Therefore, we used amixture of cationic and anionic resins saturated with NaHCO3

to extract P, potassium (K), calcium (Ca), and magnesium (Mg)from soil samples (Van Raij et al., 1986). We used colorimetry toquantify the resin-extracted P, atomic emission spectrophotome-try to quantify Ca and Mg, and flame photometry to quantify K.We used the diethylene triamine pentaacetic acid (DTPA)method to extract soil iron (Fe), Mn, copper (Cu), and zinc (Zn),and atomic absorption spectrophotometry to quantify them (DL,respectively, 0.1, 0.1, 0.2 and 0.7 mg kg�1). The methods andDLs for the analyses performed for other soil parameters were:aluminum (Al) by colorimetry with potassium chlorideextraction, DL 0.8 mmol kg�1; sulfur (S) by turbidimetry withcalcium phosphate extraction, DL 1 mg kg�1; boron (B) by col-orimetry with warm water extraction, DL 0.14 mg kg�1. Soil pHwas measured in 10 mM CaCl2.

We determined soil organic matter (OM) using dichromateoxidation and colorimetry (Walkley & Black, 1934) and soil Nusing a mass spectrophotometer (Finnigan Delta +; FinniganMAT, Bremen, Germany) and a CN analyzer (Carlo-Erba). Soilsamples were also analyzed for texture, which was analyzed bydensity and split between sand (soil particle diameters between0.05 and 2 mm), silt (0.002–0.05 mm) and clay (< 0.002 mm).

Statistical analyses

Soil nutrient data were subjected to analyses of variance (ANO-VAs) and honestly significant difference (HSD) Tukey’s post hoctests. The data were log-transformed (natural base) when necessaryto satisfy the assumptions of residual homoscedasticity and nor-mality. Leaf nutrient differences between sites with different soil[P] were analyzed using linear mixed-effects models. We estimatedrandom intercepts per species, as we collected three to six

individuals from the same species at each site. When necessary, welog-transformed (natural base) the response variable to decreaseresidual heteroscedasticity. We investigated differences betweeneach site using least-squares means and 95% confidence intervals(CIs). We used simple linear and polynomial regressions todescribe the relationship between site mean values of mycorrhizalcolonization, leaf [Mn], [P], [N] and N : P and soil [P]. Soil [P]was log-transformed (natural base) in these analyses to satisfy theassumptions of residual homoscedasticity and normality. All sta-tistical analyses were performed using the software R (version3.0.2; R Development Core Team, 2012) and the packages LME4(Bates et al., 2014) and LMERTEST (Kuznetsova et al., 2012).

Results

Soil analyses

The soil pHCaCl2 of all sites was very acidic: between 3.1 and 3.9,and only PB2 had a significantly lower pHCaCl2 than the othersites (Fig. 2c). WS1 andWS2 had the most nutrient-impoverishedsoils, showing a lower soil concentration of every nutrient exceptZn, S and Ca (Fig. 2). There was a clear gradient of increase in soil[P] from WS1 to PB2, which ranged from 1mg kg�1 in whitesands to 7.3 mg P kg�1 in peat bogs (Fig. 2a). A similar patternwas found for OM, which ranged from 3 g OM kg�1 soil in whitesands to 18.8 g OM kg�1 soil in peat bogs (Fig. 1b). The gravelcerrado sites (GC1 and GC2) generally presented higher soil con-centrations of Al, N, Fe, Ca, K, and Mn. Total N was extremelylow in white sand soils (WS1 and WS2) and in one of the peat-bog soils (PB2). Extremely low values of B and Cu were found atall sites, with only two locations (PB1 and PB2) showing concen-trations above the detection limit for Cu (Supporting InformationTable S1). Sulfur concentration ranged from 2.3 mg S kg�1 soil inwhite sands to 3.6 g S kg�1 soil in peat bogs (Table S1). Analysisof the texture of the soil showed low percentages of clay (3%) forfour of the sites. GC1 and GC2 were the exceptions, with 15%clay. The two areas in the Cip�o reserve (GC1 and GC2) were theones with the highest silt percentages (6%).

Leaf nutrient analyses

Leaf [N] ranged from 5.6 to 27.9 mg N g�1 leaf dry weight(DW), with an average of 12.4 mg N g�1 leaf DW. Although

Table 1 Study sites with common names and site codes assigned in this study

Common site name Reserve Site code GPS location Soil type

Arei~ao Cabral Cabral WS1 S17°420 W44°110 White sandCabral’s swamp grassland Cabral PB1 S17°420 W44°110 Peat bogCerrado Cip�o I Cip�o GC1 S19°160 W43°390 Oxisol with gravelCerrado Cip�o II Cip�o GC2 S19°160 W43°400 Oxisol with gravelArei~ao do Deco Rio Preto WS2 S18°050 W43°200 White sandRio Preto’s swamp grassland Rio Preto PB2 S18°050 W43°200 Peat bog

The reserve names refer to the conservation units in which the sites are located (Parque Estadual da Serra do Cabral; Parque Nacional da Serra do Cip�o;Parque Estadual do Rio Preto). All sites are located in the State of Minas Gerais, Brazil. In all landscapes in Cabral, Cip�o, and Rio Preto, the parent material isquartzite and arenite.


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plants in all vegetation types showed relatively similar leaf [N],there was a weak tendency for plant communities to have lowerleaf [N] at sites more enriched in soil [P] (Fig. 3a,b). Leaf [P]ranged from 0.07 to 1.2 mg P g�1 leaf DW, with an average of0.3 mg P g�1 leaf DW for all vegetation types. The sites with thelowest soil [P] (WS1 and WS2) showed higher values of leaf [P]than other sites, while sites with > 2 mg P kg�1 soil had relativelysimilar values to each other, generating a curvilinear relationshipbetween leaf [P] and soil [P] (Fig. 3c,d). The leaf N : P ratio of allvegetation types was high, with a species minimum of 16, a

maximum of 116 and an average of 43. These values are withinthe range expected for plants on P-limited soils, and were gener-ally similar between vegetation types, except for plants at GC1,which showed significantly higher values than those at the othersites (Fig. 3e). There was no clear relationship between soil [P]and leaf N : P among plant communities (Fig. 3f).

Roots and nutrient-acquisition strategies

We found differences in the proportions of species with NM andAM root strategies between vegetation types (Fig. 4). The whitesand sites were dominated by NM species: 75% and 83% atWS1 and WS2, respectively; the GC2 site also presented a highproportion of NM species (60%). Considering all studied spe-cies, we observed AM in only 38% of individuals analyzed, andin at least one individual of 42% of the species. No mycorrhizaswere found in any specimens of Cyperaceae and Cactaceae, andin most species of Velloziaceae. In Eriocaulaceae, both mycorrhi-zal and NM species were found. Grasses in the white sands wereeither NM or had low mycorrhizal colonization. It should be

(a)

(b)

(c)

Fig. 1 Overview of the different vegetation types at the study sites incentral Brazil. (a) Campos rupestres over white quartzite sands in ParqueEstadual da Serra do Cabral. (b) Scrubland over oxisol with gravels in Serrado Cip�o. (c) Peat-bog community in Parque Estadual do Rio Preto.

(a) (b)

(c) (d)

(e) (f)

(g) (h)

(i) (j)

Fig. 2 Comparison of the concentrations of (a, e–j) soil nutrients, (d)aluminum and (b) organic matter (OM) and (c) of soil pH (measured in10mM CaCl2) among the different vegetation types. Bars are mean� SE.Different letters indicate P < 0.05 in the Tukey post hoc test. WS1, whitesands 1; WS2, white sands 2; GC1, gravel cerrado; GC2, gravel cerrado 2;PB1, peat bog 1; PB2, peat bog 2.



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borne in mind that a sample with no mycorrhizal structures doesnot mean that the species from which that sample was collected isinvariably NM (Brundrett, 2009).

Most of the NM species possessed some alternative root spe-cializations (Fig. 5). These root specializations included ‘sand-binding roots’, that is, roots covered by a layer of sand aggluti-nated by an exudate released from the root, in Actinocephaluscabralensis, Syngonanthus sp., Xyris sp. (Xyridaceae), andDiscocactus placentiformis (Cactaceae) (Fig. 5). These sand-binding structures covered approximately three-quarters of theroots, from the tip to the base. Other species presented much lessextensive sand-binding (Fig. 5). Some roots, such as those ofSyngonanthus sp. (Fig. 5), were covered in very fine root hairs,not only at the tip of the root, but over its entire length. In fact,more than half of the collected species (32 out of 50) were cov-ered, not only at the root cap, but also all over the roots, with fineclear hairs, most of them in the Eriocaulaceae and Xyridaceae,but also in Asteraceae. These often had sand granules firmlyattached to them which did not come loose when rinsed withwater, suggesting that these hairs could be releasing exudates.

Some species (e.g. Paepalanthus sp.) had both sand-binding rootsand mycorrhizas. We found dauciform roots in the genusLagenocarpus sp. (Cyperaceae). We did not find any ‘true clusterroots’ in any species; however, we found a number of roots withspecializations morphologically similar to cluster roots.

The proportion of root colonization of plants by AM fungiwas positively related to soil [P] across vegetation types (Fig. 6).Among the species sampled, 30% presented some AM coloniza-tion; most of these species were in peat-bog communities (PB1and PB2). However, these plants (56.5%) generally had a level ofcolonization under 5%. The extent to which all of the roots werecolonized varied between 0% and 27% (Table S2), with a largevariation between samples of the same species, and some varia-tion between the sites.

We found that NM plants had higher values of leaf [Mn] thanplants with arbuscular mycorrhizas (Fig. 7a). However, this dif-ference was not observed within sites (Supporting InformationFig. S1). There was also variation in leaf [Mn] among sites, pro-ducing a weak trend for plants to have lower leaf [Mn] at siteswith higher soil [P] (Fig. 7b,c).

(a) (b)

(c) (d)

(e) (f)

Fig. 3 (a, b) Leaf nitrogen (N)concentrations, (c, d) phosphorus (P)concentrations and (e, f)nitrogen : phosphorus ratio (N : P) across thesoil [P] gradient in different vegetation types.The central vertical bar in each boxrepresents the median, the box representsthe interquartile range and the whiskersrepresent the most extreme data points thatare still within 1.5 of the upper or lowerquartiles. The circles outside the whiskers arevalues that are > 1.5 from the upper andlower quartiles. The notches representconfidence intervals around the median.Different letters indicate significantdifferences based on 95% confidenceintervals estimated from the linear mixed-effects model. The dashed lines in (a) and (c)indicate the global averages for N and P,respectively (Vergutz et al., 2012). Thedashed line in (e) represents the threshold forP limitation following G€usewell (2004). Theslope of the linear relationships betweenmean site values and soil [P] was notsignificantly different from zero in (b) and (f)(P = 0.23 and 0.78, respectively) despite therelatively high R2 in (b). The slope of thesecond-order polynomial regression in (d)was marginally significant at P = 0.09. WS1,white sands 1; WS2, white sands 2; GC1,gravel cerrado; GC2, gravel cerrado 2; PB1,peat bog 1; PB2, peat bog 2.





Discussion

We demonstrate that the soils of campos rupestres communitiespresent very low P availability, similar to the most P-impover-ished soils in the world (Lambers et al., 2010, 2014a), andthis low soil [P] might select for plant root specializationsand, to some extent, low leaf nutrient concentrations. Plantproductivity in campos rupestres is probably limited by P,rather than N, as evidenced by the community-level low leaf[P] and high leaf N : P ratio. However, total [N] was also verylow in white-sand communities and in one peat-bog commu-nity (PB2), so further studies based on alternative methods toassess nutrient limitation (e.g. phytometers) in campos rupestrescommunities are needed. Our results support the model pro-posed by Lambers et al. (2008, 2010) by showing that mycor-rhizal colonization was less common on the more severely P-impoverished soils. We also show a great diversity of root spe-cializations in the most P-impoverished soils. The ‘cluster-like’morphology of several species in the studied area suggests thatthese roots have a role in ‘mining’ sorbed P from soil particlesby exuding carboxylates (Lambers et al., 2006, 2008). Thehigher leaf [Mn] in NM species and on sites with low soil[P] corroborates this hypothesis (Lambers et al., 2014b). Thisis the first survey of campos rupestres plants focusing on min-eral nutrition strategies and it shows a strong functional con-vergence in plant mineral nutrition strategies among severelynutrient-impoverished ecosystems around the world.

Soil analyses

Readily available soil [P] was measured with the resin method,which is considered the most appropriate method when soil Pvalues are very low (Van Raij et al., 1994). Our results showthat values for white sand campos rupestres soil [P] are similarto those of older soils on south-western Australian sandplains(< 0.5 mg total P kg�1 soil; Lalibert�e et al., 2012; Hayes et al.,2014). Cape Floristic Region soils are also impoverished in P,with [P] lying somewhere between the values found in south-

western Australia and in some campos rupestres communities inBrazil. In the Cape, soil [P] ranges between 0.4 and 2.5 mgresin-extractable P kg�1 soil (Mitchell et al., 1984) andbetween 0.1 and 3.2 mg resin-extractable P kg�1 soil for fyn-bos areas (Witkowski & Mitchell, 1987). A recent reviewshowed an average of 87 mg total P kg�1 soil for the Cape(Stock & Verboom, 2012). These values suggest that thekwongan in south-western Australia, the fynbos in SouthAfrica and the campos rupestres over white sands in Brazil aresimilar environments in terms of P availability.

The low P availability we measured agrees with the predictionthat white sandy soils, resulting from in situ weathering of quartz-ite in an ancient geological formation dating between 950 and650 million yr ago (Alves & Kolbek, 1994), are severely P impov-erished. The low P availability can be partly explained by the lowpH, between 3.1 and 4.0 (Marschner, 1991).

Leaf N and P concentrations

In a global comparison, the leaf [N] of most campos rupestres spe-cies in this study was below the world average of 17.25 mg N g�1

leaf DW (Vergutz et al., 2012), and higher than values for thesouth-western Australian kwongan and Cape fynbos flora (Lam-bers et al., 2010; Hayes et al., 2014). The weak associationbetween soil [P] and leaf [N] might indicate that plant N-usestrategies are, to some extent, determined by plant P-use strate-gies, as shown for Proteaceae in south-western Australia (Sulpiceet al., 2014). As plants in lower soil [P] sites presented a widerange of root specializations to enhance P uptake, it is possiblethat some of these specializations also enhance the acquisition ofother soil nutrients, such as N.

The mature leaf [P] results from our survey match those ofplants in other severely P-limited environments such as south-western Australia (range from 0.11 to 3 mg P g�1 leaf DW; aver-age of 0.26 mg P g�1 leaf DW for communities on the oldestsoils) and the Cape floristic region of South Africa (0.34 mgP g�1 leaf DW), and are much lower than the world average of1.02 mg P g�1 leaf DW and values obtained in adjacent forestsand savannas (Lambers et al., 2010; Viani et al., 2011, 2014;Hayes et al., 2014). The white-sand communities, which are thesites with the lowest soil P availability and the highest abundanceof NM plants, showed the highest values of mature leaf [P]. Thisresulted in a relationship between leaf [P] and soil P availabilitydifferent from what we expected and what is normally observedin other systems (Vitousek et al., 1995; Aerts & Chapin, 2000;Hayes et al., 2014). This pattern could be attributed to species(especially from the white-sand communities) accessing P frac-tions other than readily available resin P or facilitation in P acqui-sition among coexisting species with contrasting nutrient-acquisition strategies (Muler et al., 2014).

Leaf N : P ratio

Despite the large variation in leaf N : P between species (16–116), genera (e.g. Eriocaulaceae and Xyridaceae), and communi-ties, all species had an N : P ratio of 16 or above, indicating that

Fig. 4 Proportion of nutrient-acquisition strategies (AM, arbuscularmycorrhizal (mid gray bars); LAM, low-arbuscular mycorrhizal (light graybars); NM, nonmycorrhizal (dark gray bars)) for the 10 most abundantspecies of each vegetation type (WS1, white sands 1; WS2, white sands 2;GC1, gravel cerrado; GC2, gravel cerrado 2; PB1, peat bog 1; PB2, peatbog 2).



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(a) (b) (c)

(d)

(e) (f) (g)

(h) (i)

(j) (k) (l)

Fig. 5 Root and foliar specializations assessed in the field in plant species of the campos rupestres of central Brazil. (a) Sand-binding roots of Paepalanthussp. (Eriocaulaceae); (b) sand-binding roots of Actinocephalus cabralensis (Ericaulaceae); (c) abundance of fine root hairs in Actinocephalus cabralensis; (d)fine roots along the central root axis of Syngonanthus sp.; (e) abundance of root hairs over the entire length of the main roots in Xyris obcordata; (f) sand-binding in Xyris sp.1; (g) sand-binding roots of Lagenocarpus rigidus (Cyperaceae); (h) sand-binding roots of Discocactus placentiformis; (i) dauciform rootof Lagenocarpus sp.1 (Cyperaceae); (j) nodules inMimosa misera (Fabaceae) roots; (k) rosette in Paepalanthus bromelioides (protocarnivorous plant); (l)nematodes over the underground leaf of Philcoxia minensis (carnivorous plant). Bars: (b, h, j) 2 cm; 1 cm (a, c, d, e, f, g, i); 100 lm (l). Photo credits: photo(a–k) Rafael Oliveira; photo (l) Caio Pereira.





P was the key limiting macronutrient for plant productivity inthe campos rupestres (Koerselman & Meuleman, 1996). Thisagrees with the low soil and leaf [P] data.

From a global perspective, we note that the average N : P ratiofor the species in this study was higher than the average values inother P-impoverished environments. For south-western Australia,the calculated average N : P ratios were 24 (Lambers et al., 2010)and 17, with a range of 8–82 (Stock & Verboom, 2012). For theCape floristic region, the calculated values were 25 (Lambers et al.,2010) and 22, with a range of 10–80 (Stock & Verboom, 2012).

Leaf Mn concentration

Most species in all communities showed leaf [Mn] higher thanadequate for crop growth (50 lg Mn g�1 leaf DW; Epstein &Bloom, 2005). However, sites with more NM species showedgenerally higher values of leaf [Mn], and species with low or nomycorrhizal colonization also had higher values of leaf [Mn],independent of site. Considering the relationship between leaf[Mn] and release of carboxylates in NM plants (Shane & Lam-bers, 2005; Abrah~ao et al., 2014; Hayes et al., 2014; Lamberset al., 2014b), this observation supports our hypothesis that theroot specializations in NM plants are functional adaptations tomobilize P in P-impoverished soils, through exudation ofcarboxylates. We did not find a correlation between [P] and [Mn]in leaves, and neither did Hayes et al. (2014) along the JurienBay dune chronosequence. This lack of correlation does not indi-cate that leaf [Mn] is not associated with plant P-acquisitionstrategy; just that leaf [P] is not a good surrogate for a ‘successful’P-acquisition strategy. The most efficient species also have a veryhigh P-use efficiency, showing rapid rates of photosynthesis atlow leaf [P] (Lambers et al., 2012).

Root specializations

Mycorrhizas No extensive research on mycorrhizal colonizationhas been published on plant species of the campos rupestres. As we

Fig. 6 Linear relationship (P < 0.05) between percentage of mycorrhizalcolonization and soil phosphorus (P) concentration among differentvegetation types. The circles are the mean values of each vegetation type.WS1, white sands 1; WS2, white sands 2; GC1, gravel cerrado; GC2,gravel cerrado 2; PB1, peat bog 1; PB2, peat bog 2.

(a)

(b)

(c)

Fig. 7 (a) Leaf manganese (Mn) concentrations in plants with differentnutrient-acquisition strategies (AM, arbuscular mycorrhizal; LAM, lowarbuscular mycorrhizal; NM, nonmycorrhizal) and (b) across vegetationtypes. The central vertical bar in each box represents the median, the boxrepresents the interquartile range and the whiskers represent the mostextreme data points that are still within 1.5 of the upper or lower quartile.The circles outside the whiskers are values that are > 1.5 from the upperand lower quartiles. The notches represent confidence intervals around themedian. Bracketed numbers on the x-axis refer to soil resin-P values.Different letters indicate significant differences based on 95% confidenceintervals estimated from the linear mixed-effects model. The dashed linedindicates the leaf Mn concentration considered adequate for crop growth(Epstein & Bloom, 2005). (c) Linear relationship between soil [P] and leafMn; the slope was not significantly different from zero (P = 0.39). WS1,white sands 1; WS2, white sands 2; GC1, gravel cerrado; GC2, gravelcerrado 2; PB1, peat bog 1; PB2, peat bog 2.



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hypothesized, the sites where there was low resin P (white sands)were also the sites with the lowest occurrence of AM. Conversely,in the peat-bog communities, where our resin-P results showmoderate values of readily available P, our counts showed muchhigher levels of mycorrhizal colonization. These observations,and the strong positive relationship between the proportion ofmycorrhizal colonization and soil P availability, reinforce previ-ous observations that AM are favored at intermediate [P] (Red-dell et al., 1997; Siqueira & Saggin-J�unior, 2001) and lendsupport to Lambers et al.’s (2008, 2010) model of prevalence ofnutrient-acquisition strategies along gradients of soil nutrientavailability.

Mycorrhizas are a prevalent symbiosis, occurring world-widein 82% of all vascular plants, and AM are the most common,occurring in 73% of all taxa (Brundrett, 2009). However, inparticular environments, the dominance of a certain group ofspecies or soil conditions may affect this proportion. NM spe-cies are particularly abundant in severely P-impoverished soils.For south-western Australia and the Cape region, the propor-tion of plants with AM is smaller and the NM fraction isgreater (Brundrett, 2009; Lambers et al., 2010; Hayes et al.,2014), notably NM species of the ‘Proteaceae type’ sensu Lam-bers & Teste (2013), on the most severely P-impoverished soils(Lambers et al., 2014a). Our observations show that the camposrupestres also have a smaller proportion of mycorrhizal speciesand a wide range of root specializations, indicating a functionalconvergence in roots between the campos rupestres, kwongan,and fynbos. Several root types that we found in campos rupestresspecies are morphologically very similar to root specializationsthat enhance P uptake in nutrient-poor habitats in south-western Australia and the Cape region (such as a high density ofroot hairs and sand-binding roots). These roots often possessspecializations that increase P acquisition (Lambers et al., 2006;Abrah~ao et al., 2014).

We recognize that the species pool we have sampled might notrepresent the nutrient-acquisition strategies of the entire commu-nity, because of the very high species diversity in these environ-ments (Giulietti et al., 1997). However, when all species within acommunity are considered and the classification scheme ofBrundrett (2009) is used, NM species are still relativelyabundant, representing up to 50% of the local flora for thecommunity (F. Silveira et al., unpublished).

Sand-binding roots Sand-binding roots, with a very similarstructure to that detailed for south-western Australian Lyginiabarbata (Anarthriaceae) (Shane et al., 2009) and several Haem-odoraceae species (Smith et al., 2011), were found in Eriocaul-aceae species in the campos rupestres. Four out of nineEriocaulaceae species possessed sand-binding roots, with largesheaths of sand grains. These were most pronounced forActinocephalus sp. and Paepalanthus sp. Based on the high leaf[Mn] in Eriocaulaceae, similar to concentrations in Australianspecies that have sand-binding roots (Hayes et al., 2014), wesurmise that the roots of these plants have a similar role inplant mineral nutrition (Shane et al., 2009; Smith et al.,2011).

In a number of species, such as Xyris obcordata, roots werefound with sand firmly attached to them that resisted washingand even scraping. However, these roots did not form a sheath,and are probably the result of local exudation. We also observedsand-binding roots in Discocactus placentiformis (Cactaceae). Thisspecies had the highest shoot [Mn], and exudate analysis showedthat these roots increase their release of carboxylates in responseto P deficiency (Abrah~ao et al., 2014).

Dauciform roots This survey showed the first record of dauci-form roots in South America. These root specializations werefound in all individuals of Lagenocarpus sp. in white sands. It isessential to note that Cyperaceae is one of the most prominentfamilies in the campos rupestres and accounts for a substantial per-centage of the aboveground biomass in some communities. TheCyperaceae species we sampled presented very high leaf [Mn],and dauciform roots are known to release carboxylates, facilitat-ing access to sorbed P in soils of low fertility (Playsted et al.,2006; Shane et al., 2006).

Carnivory The carnivory cost/benefit model proposed byGivnish et al. (1984) predicts that the nutrient-impoverished,well-lit and seasonally dry regime of the campos rupestresmakes carnivory a likely nutrient-acquisition strategy in theseenvironments. In fact, carnivory is a common nutrient-acquisi-tion strategy, with species showing contrasting prey-capturemechanisms (Drosera, Genlisea, and Utricularia). The recentlydescribed carnivorous genus, Philcoxia (Plantaginaceae),showed the highest leaf [N] and [P] within its community,indicating the importance of alternative nutrient sources forplant nutrition (Pereira et al., 2012). In addition, protocarni-vory is a common strategy in rosette species of Eriocaulaceaeand Bromeliaceae (Nishi et al., 2012).

Concluding comments

The prevalence of NM strategies in the severely P-impoverishedcampos rupestres corroborates the model proposed by Lamberset al. (2008, 2010), predicting how plant nutrition strategieschange with soil P availability. As the first investigation of thiskind in the campos rupestres, our study not only stresses the gener-ality of this model, but has also produced many novel findings,such as sand-binding structures in Eriocaulaceae and ‘cluster-likeroots’, yet to be investigated.

The low levels of soil P availability in the campos rupestres arereflected not only in the root specializations, but also in the spe-cies’ very high leaf N : P ratios. We propose that the similarity inpatterns of nutrient-acquisition strategies among the camposrupestres, kwongan, and fynbos, which are all known for their hy-perdiversity, provides a fascinating example of convergent evolu-tion of root specializations involved in P acquisition. However,while most NM families differ from those in Australia and SouthAfrica, they function in a remarkably similar manner. We explainthis similarity by referring to Hopper’s OCBIL (old, climatically-buffered, infertile landscapes) theory (Hopper, 2009). Thecampos rupestres feature several very old Gondwanan lineages, for





example, Eriocaulaceae and Velloziaceae (Mello-Silva et al.,2011; Trovo et al., 2013), that evolved (in situ) a wide range ofroot specializations to survive with P limitation.

Acknowledgements

Thanks to the Department of Plant Biology and the GraduateProgram on Ecology at Unicamp for the funding and infra-struc-ture provided, CAPES for the scholarship awarded to H.C.G.,FAPESP for the PhD scholarship awarded to C.B.E. (2013/19555-2) and Instituto Estadual de Florestas-MG for logisticalsupport. Thanks to the University of Western Australia for theIPRS/SIRF PhD scholarship awarded to M.C.R.d.C, and to theSchool of Plant Biology for the funding and infra-structure pro-vided. This research was supported by the Brazilian NationalCouncil for Scientific Research (CNPq 474670/2008-2 toR.S.O.), FAPESP (2010/17204-0 to R.S.O.), the AustralianResearch Council (ARC; DP0985685 and DP110101120 toH.L.) and the ANZ Holsworth Wildlife Foundation and MaryJanet Lindsay of Yanchep Memorial Fund who provided addi-tional grants. R.S.O. received productivity scholarships fromCNPq. We thank the volunteers who have helped with fieldworkand sample analyses: Ana L. Muler, Andre V. Scatigna, Maria C.Penteado, Caio G. Pereira and Patricia Brito. We thank the twoanonymous reviewers for their insightful comments that helpedus to improve this manuscript.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Leaf [Mn] least square means estimated from a linearmixed effects model.

Table S1 Soil copper (Cu), boron (B) and sulfur (S) for the sixstudied campos rupestres communities





Table S2 List of species, families and habits, and average resultsfor percentage mycorrhizal colonization (% Col), leaf nitrogen(N) and phosphorus (P) concentration, leaf N : P ratio, and leafmass per unit leaf area (z) for the six studied campos rupestrescommunities

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