Aluminum accumulation and its relationship with mineral plant nutrients in 12 pteridophytes from Venezuela

Environmental and Experimental Botany 65 (2009) 132–141

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Aluminum accumulation and its relationship with mineral plant nutrients in 12 pteridophytes from Venezuela ˜ a , Eunice Marcano b , Julian ´ Mostacero c , Guillermina Aguiar a , Elizabeth Olivares a,∗ , Eder Pena a a Malfy Ben´ıtez , Elizabeth Rengifo a b c

Centro de Ecolog´ıa, Instituto Venezolano de Investigaciones Cient´ıficas (IVIC), Carretera Panamericana Km. 11, Sector Altos de Pipe, Estado Miranda, ZP 1204, Venezuela Centro de Qu´ımica, Instituto Venezolano de Investigaciones Cient´ıficas (IVIC), Carretera Panamericana Km. 11, Sector Altos de Pipe, Estado Miranda, ZP 1204, Venezuela Fundaci´ on Instituto Bot´ anico de Venezuela, Herbario Nacional de Venezuela, Universidad Central de Venezuela, Apartado 2156, Caracas 1010-A, Venezuela

a r t i c l e

i n f o

Article history: Received 10 March 2008 Accepted 2 April 2008 Keywords: Aluminum accumulation Metal excluders Neotropical Ferns Pteridophytes

a b s t r a c t The purpose of this study was to investigate the aluminum (Al) concentration in Lycopodium clavatum, Dicranopteris flexuosa, Sticherus nudus, Anemia villosa, Cyathea gibbosa, Pteridium arachnoideum, Pteris vittata, Thelypteris dentata, Blechnum occidentale, Elaphoglossum sporadolepis, Nephrolepis cordifolia and Polypodium pseudoaureum, species from 11 families with different phylogenetic position, found on soils with a high concentration of Al (up to 13 g kg−1 dry mass (DM)). When Al concentration and mineral nutrients in aerial organs were considered, pteridophytes were classified into three groups: group one included pteridophytes with Al concentrations over 1000 mg kg−1 DM in their aerial organs, a ratio between Al and essential plant nutrients such as Ca, Mg and P higher than one and a K/Al ratio between 0.68 and 2.56 mol mol−1 . In group 1 was the well known Al-accumulator L. clavatum (Lycophyte) as well as the Neotropical ferns D. flexuosa, S. nudus (both basal leptosporangiate ferns), and C. gibbosa (core leptosporangiate tree fern). Group 2, ferns which accumulate Al over 1000 mg kg−1 DM in their fronds, and had an Al/Ca and Al/Mg ratio <1. Species in this group included E. sporadolepis and N. cordifolia (derived polypod ferns). Group 3, ferns classified as Al-excluders, showing Al concentration <782 mg kg−1 DM in the fronds, had Al/Ca and Al/Mg ratios <1, Al/P ratio ≤1 and a K/Al ratio between 18.10 and 80.36 mol mol−1 . In group 3, were A. villosa (basal leptosporangiate fern) and the derived polypod ferns P. arachnoideum, P. vittata, T. dentata, B. occidentale and P. pseudoaureum. The translocation factor of Al from subterranean to aerial organs was up to 4 in S. nudus, and subterranean organs from E. sporadolepis showed the highest concentration of Al (12 g kg−1 DM). We coincide with early literature in that other criteria in addition to the Al concentration should be considered to define the Al accumulation, such as its relationship with macronutrients. For example, we propose the inclusion of K/Al ratio. We conclude that out of six Al-excluders five belonged to the derived polypods while two species from Polypodiales showed high Al concentrations. We reconfirm accumulation of Al in L. clavatum and C. gibbosa and discover two new Al-accumulating species in the more ancient ferns: S. nudus and D. flexuosa. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Chenery (1949) applied the “aluminon” (ammonium aurine tricarboxilate) test to 1044 pteridophyte species from the Kew Herbarium and defined 476 as aluminum (Al) plants. The pinkish to orange reagent turned to a distinctive dark red to crimson color when the Al content in the tissue tested exceeded 1000 mg kg−1 dry mass (DM). He reported 515 Al accumulating plants among 1178 pteridophyte species tested, including data from former literature sources published between 1851 and 1943, and concluded

∗ Corresponding author. Tel.: +58 212 5041363; fax: +58 212 5041088. E-mail address: [email protected] (E. Olivares). 0098-8472/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2008.04.002

that Al-accumulators are almost entirely confined to ancient families and genera. However, in 98 pteridophyte species, he found contrasting Al concentrations inside some genus after adopting a quantitative colorimetric method. For example, he reported for the marattioid ferns Angiopteris arborescens and A. brooksii Al concentrations of 580 and 9880 mg kg−1 DM, respectively. Furthermore in the leptosporangiate ferns Gleichenia elongata and G. revoluta (now Sticherus revolutus), he reported Al concentrations of 100 and 15,100 mg kg−1 DM, respectively, and in the Polypod ferns Blechnum serrulatum and B. diversifolium Al concentrations of 145 and 23,200 mg kg−1 DM, respectively. Webb (1954) using the “aluminon” test also concluded that the recorded Al-accumulators were mainly restricted to what are usually regarded as the more primitive groups of Filicales. He found 11 Al-accumulators among

E. Olivares et al. / Environmental and Experimental Botany 65 (2009) 132–141

87 pteridophyte species in the Australian-New Guinea flora distributed in the following way: one Lycopodium species accumulates Al out of seven species tested, one Angiopteris (Marattiaceae), one Selenodesmium (now Trichomanes, Hymenophyllaceae, a filmy fern), one Dipteris (Dipteridaceae, order Gleicheniales in Smith et al., 2006, denoted as Polypodiaceae in Webb, 1954), one Dicranopteris (Gleicheniaceae), two out of two Sticherus (Gleicheniaceae), two out of five Cyathea (Cyatheaceae, tree ferns), and two out of nine Lindsaea (Lindsaeaceae, order Polypodiales in Smith et al., 2006, denoted as Pteridaceae in Webb, 1954). According to Pryer et al. (2004) a phylogenetic dichotomy occurred in the early-mid Devonian, circa 400 million years ago (Myr), separating the modern lycophytes (less than 1% of extant vascular plants) from a group that contains all other living vascular plant lineages, the euphyllophytes: seed plants and monilophytes. In the ferns (monilophytes), the first dichotomy separates a clade consisting of whisk ferns and ophioglossoid ferns from a clade comprising horsetails, marattioid ferns and leptosporangiate ferns, which include osmundaceous ferns, filmy ferns, gleichenioid ferns, schizaeoid ferns and finally core leptosporangiate ferns that include heterosporous ferns, tree ferns and polypod ferns. The earliest known occurrence of leptosporangiate ferns is found in the early Carboniferous, 280 Myr (Schneider et al., 2004). The schizaeoid ferns take a sister position to the core leptosporangiate ferns. The more derived polypod ferns, which comprise more than 80% of living fern species diversified in the Cretaceous, 121 Myr (Schneider et al., 2004). Based on Chenery (1949) and Webb (1954) we expect to see a higher probability of Al-excluders in the derived polypod ferns. Chenery (1948,1949) determined the Al concentrations in the aerial plant tissues of thousands of plant species, and classified them as Al-accumulators (≥1000 mg kg−1 DM) or Al non-accumulators (<1000 mg kg−1 DM). Under these criteria most plants are Al non-accumulators, with levels below 300 mg kg−1 DM (Jansen et al., 2004). Masunaga et al. (1998) found that Alaccumulators with an Al concentration <3000 mg kg−1 DM showed a continuous distribution pattern with the non accumulators in their linear relationships between Al concentrations and five other elements (Ca, Mg, P, S and Si), while Al-accumulators with an Al concentration in leaves ≥3000 mg kg−1 DM conformed a different group in this relationship. The correlation coefficients (r) between Ca, Mg, P, S and Si with Al for the Al-accumulators with an Al concentration in leaves ≥3000 mg kg−1 DM were 0.14, 0.25, 0.09, 0.44, 0.29, respectively, not significant for Ca and P, however if the Al-accumulators (≥1000 mg kg−1 DM) with an Al concentration <3000 mg kg−1 DM were included, the coefficients were −0.10, −0.03, −0.13, 0.43, 0.04, with only S significant. Authors proposed criterion of 3000 mg kg−1 DM or to use the Al/Ca ratio in order to define Al accumulators, in addition to the criterion of Chenery (>1000 mg kg−1 DM) and reported an Al/Ca ratio of 0.04 g g−1 (=0.06 mol mol−1 ) in nonaccumulators, 0.05 g g−1 (=0.07 mol mol−1 ) in Al accumulators <3000 mg kg−1 DM and 0.72 g g−1 (=1.07 mol mol−1 ) in Al accumulators ≥3000 mg kg−1 DM. In the present study, a field survey was conducted to explore Al-accumulating pteridophyte species of northern Venezuela. The area chosen consisted of tropical montane cloud forest that has been changed in some areas to secondary savannas due to recurrent human-related fires in the last two centuries. Soils investigated were typically acidic and contained high concentrations of Al. Furthermore, several studies have reported Al-accumulator species other than pteridophytes (Cuenca and Herrera, 1987; Cuenca and Medina, 1990; Izaguirre-Mayoral and Flores, 1995). However, to our knowledge, in this study site Al-accumulation in pteridophytes has not been reported with the exception of high Al concentrations

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in the subterranean organs of Pteridium aquilinum (Olivares et al., 2007). In the literature the majority of screening studies that investigate pteridophytes have examined arsenic (As) (Meharg, 2003; Wang et al., 2007) as well as cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb), zinc (Zn) (Kachenko et al., 2007), and iron (Fe) (Cornara et al., 2007) and failed to address Al. Therefore, the aims of present study were to (1) screen twelve pteridophyte species for Al-accumulation in their natural environment in the Neotropics of Venezuela using in addition to the Al concentration criterion, the relationships of Al with nutrients, including the K/Al ratio which had not been previously reported and (2) relate the degree of Al-accumulation with taxonomy because the pteridophytes under study belong to 11 families which have been described in early literature as primitive or recent. 2. Materials and methods 2.1. Sample collection from the field Sampling was done within the grounds of the “Instituto Venezolano de Investigaciones Cient´ıficas” (IVIC, Venezuelan Institute of Scientific Research), located in “Altos de Pipe” (Venezuela), where the predominant vegetation is a primary cloud forest, surrounded by secondary forests and a secondary savanna community (Table 1). The species were chosen based on their abundance and accessibility along the roads at IVIC (Table 2). The soils referred to in Tables 1 and 3 were collected with plants at each site at a depth of 0–10 cm. Fronds from all fern species growing at Site A and C were collected in June 2005, excepting Anemia villosa which was sampled in site C on May 2006 when adult individuals were found. As two species from site C seemed to be Al-accumulators they were collected again in the same site from 2005 and in other sites at IVIC (sites B, E, F) where other ferns were also sampled, such as the known Al-accumulators Lycopodium clavatum and the tree fern Cyathea gibbosa. Lycophyles (leaves with an intercalary meristem, Pryer et al., 2004) from the Lycophyte and fronds from the 11 ferns were harvested from the field during rainy season (dates in Table 4) from adult pteridophytes. Subterranean organs (rhizome and/or roots) were only sampled in Al-accumulators at least in one site. Roots in adults of the tree fern C. gibbosa were not sampled. 2.2. Sample pretreatment and chemical analysis The soil pH was determined on air-dried samples sieved to <2 mm with a pH-meter (Jones, 2001). Plant material was thoroughly washed at first with tap water and afterwards with deionised water. Tubers of Nephrolepis cordifolia were detached from roots and excluded from the elemental analysis. Aerial and underground organs were dried to constant weight in a ventilated oven for approximately 78 h at 60 ◦ C and then ground with a Wiley mill (3383-L10, Thomas Scientific, USA). The concentrations of Al, K, Ca, Mg, Fe, Mn, Zn, Cu and Ni in pteridophytes and soils were determined on a dry mass basis following digestions in nitric-perchloric acid (Miller, 1998) using an atomic absorption spectrometer (SpectrAA 55B, Varian Techtron, Victoria, Australia). The method by Miller does not allow a complete digestion of soil samples and soil reference material was not used to state recovery, nevertheless those analyses allow a comparison of the elemental soil composition between two sites with contrasting pH and vegetation. A blank sample and a duplicate certified reference of peach leaves (1547, National Institute of Standard and Technology, Gaithersburg, USA) were included for quality control in each batch of 40 digestions, the recovery in above ground organs analyses was greater

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E. Olivares et al. / Environmental and Experimental Botany 65 (2009) 132–141

Table 1 Sampling sites characterization in “Altos de Pipe” (Venezuela) Site A. Check point (entrance)

Altitude (m)

Localization

1240

N10◦ 23 53

Soil pH in KCl 0–10 cm deep 4.7

ab

Vegetation

Pteridophyte species sampleda

Secondary forest

Pv, Pp

W66◦ 58 15 B. Main road, km 3.5

1479

N10◦ 23 53 W66◦ 58 32

3.7 b

Secondary forest

Df, Sn

C1. Deposit-south

1619

N10◦ 24 12 W66◦ 58 27

4.1 b

Secondary savanna

Df, Pv

C2. Deposit-east

1621

N10◦ 24 12 W66◦ 58 27

4.9 a

Secondary savanna

Pa, Pv, Td

C3. Deposit-north

1624

N10◦ 24 13 W66◦ 58 27

4.2 b

Secondary savanna

Sn, Av, Bo

D. Cloud forest

1625

N10◦ 24 05 W66◦ 58 53

3.4 b

Primary cloud forest

Cg, Es

E. Chemistry center

1716

N10◦ 24 02 W66◦ 58 53

3.5 b

Secondary forest

Lc, Sn, Td, Nc

F. Physics center

1724

N10◦ 23 53 W66◦ 59 01

3.7 b

Secondary forest

Lc, Df, Sn

a The “Pteridophyte species sampled” column refer to species shown in Table 2: Pv, P. vitatta; Pp, P. pseudoaureum; Df, D. flexuosa; Sn, S. nudus; Pa, P. arachnoideum; Td, T. dentata; Av, A. villosa; Bo, B. occidentale; Cg, C. gibbosa; Es, E. sporadolepis; Lc, L. clavatum, Nc, N. cordifolia. b Means followed by the same letter are not significantly different at P ≤ 0.05, n = 4–12.

Table 2 Plant material analyzed Order

Family

Species

Lycophyte (clubmosses) Lycopodiales

Lycopodiaceae

Lycopodium clavatum L.

Euphyllophyte, Class Polypodiopsida = Filicopsida = Monilophyte (ferns) clade leptosporangiates Gleicheniales Gleicheneaceae Gleicheniales Gleicheniaceae Schizaeales Anemiaceae

Dicranopteris flexuosa (Schrader) Underw. Sticherus nudus (Moritz ex.Reichardt) Nakai Anemia villosa Humb. and Bonpl. ex Willd.

Clade core leptosporangiates Cyatheales

Cyatheaceae

Cyathea gibbosa (Klotzsch) Domin.

Clade polypods Polypodiales Polypodiales

Dennstaedtiaceae Pteridaceae

Pteridium arachnoideum (Kaulf.) Maxon Pteris vittata L.

Clade eupolypods II Polypodiales Polypodiales

Thelypteridaceae Blechnaceae

Thelypteris dentata (Forssk.) E. St. John Blechnum occidentale L.

Clade eupolypods I Polypodiales Polypodiales Polypodiales

Dryopteridaceae Lomariopsidaceae Polypodiaceae

Elaphoglossum sporadolepis (Kunze ex Kuhn) T. Moore Nephrolepis cordifolia (L.) C. Presl. Polypodium pseudoaureum Cav.

The ferns classification is according to Smith et al. (2006).

Table 3 Mineral concentrations of metals in soils at Sites A and D from Table 1 having contrasting pH and located at different altitude Element

Concentration (mg kg−1 dry mass) Site A

Al K Ca Mg Fe Mn Zn Cu Ni

8,202 12,705 710 3,013 1,865 204 29 9 15

Significance levela

Site D ± ± ± ± ± ± ± ± ±

233 502 70 114 86 16 1 1 1

13,193 10,615 296 2,365 3,538 18 30 7 29

± ± ± ± ± ± ± ± ±

504 268 36 111 216 1 1 0 2

*** ** *** *** *** *** ns *** ***

a Significance levels: ***P < 0.001, **P < 0.01, ns = no significance. Mann–Whitney Rank Sum test was performed. Values are means of 20 replicates ± S.E.

than 96% for investigated elements. Phosphorus was measured colorimetrically (Murphy and Riley, 1962) in the digested material of pteridophytes by UV/visible spectrophotometer (Ultrospec 2000, Amersham Pharmacia, Cambridge, England). Molar ratios (mol mol−1 ) instead of mass ratios (g g−1 ) for Al/Ca, Al/Mg, Al/P, K/Al, K/Ca, Ca/Mg, Al/Fe, Al/Mn, Al/Zn were calculated in the present work because they are more common in physiological research, as they reflect the actual stoichiometric relationships (Britez et al., 2002; Jansen et al., 2003). For comparative purposes mass ratios, common in the literature, were recalculated to molar ratios.

2.3. Statistical analysis A statistical comparison of soil pH means or Al concentration in the pteridophytes was examined with the all pair wise multiple

E. Olivares et al. / Environmental and Experimental Botany 65 (2009) 132–141

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Table 4 Aluminum concentration (mean ± S.E.) and molar ratios with macronutrients in the aerial green organs of the pteridophyte species sampled in the sites indicated in Table 1 Species

Date

Group 1 (Al > 1000 mg/kg, Al/Ca, Al/Mg and Al/P > 1, K/Al < 3) Lycopodium clavatum June 10/06 July 7/06 Mean

Site

n

Al (mg kg−1 DM)

Al/Ca

Al/Mg (mol mol−1 )

F E

4 10 14

2237 ± 285 aa 2921 ± 83 b 2725 ± 127

2.08 4.82 3.68

4.24 3.75 3.85

7.17 2.27 2.70

1.13 2.13 1.89

Al/P

K/Al

Dicranopteris flexuosa

July 1/05 January 19/06 January 19/06 April 4/06 Mean

C1 C1 B F

8 10 8 8 34

1013 1538 2407 3658 2118

± ± ± ± ±

122 a 203 b 74 c 259 d 192

1.75 1.20 2.33 1.96 1.77

2.70 2.90 4.07 4.22 3.61

4.67 9.07 9.75 9.79 8.58

1.38 2.56 1.27 1.15 1.51

Sticherus nudus

July 1/05 January 19/06 January 19/06 April 4/06 July 7/06 Mean

C3 C3 B F E

2 6 4 4 10 26

3319 2129 2842 3280 5733 3894

± ± ± ± ± ±

356 a 96 a 452 a 568 a 199 b 327

3.91 2.46 3.11 3.26 3.91 3.46

6.49 2.57 3.41 3.30 3.46 3.39

10.75 9.37 15.21 13.01 7.50 8.90

0.68 1.35 0.83 0.81 1.03 0.99

Cyathea gibbosa adult Juvenile

July 7/06 March /07

D D

10 2

3815 ± 597 3616 ± 20

3.44 2.00

1.52 1.53

3.61 8.80

2.47 1.85

Group 2 (Al > 1000 mg/kg, Al/Ca and Al/Mg < 1, Al/P > 1, 8 < K/Al > 4) Elaphoglossum sporadolepis July 7/06 D Nephrolepis cordifolia July 7/06 E

10 10

1507 ± 176 1632 ± 79

0.30 0.28

0.31 0.31

1.71 1.54

7.40 4.83

Group 3 (Al < 1000 mg/kg, Al/Ca and Al/Mg < 1, Al/P ≤ 1, K/Al > 18) Anemia villosa May 23/06 C3 Pteridium arachnoideum July 1/05 C2 Pteris vittata July 1/05 A Jan 19/06 C1 July 1/05 C2 Jan 19/06 C2 Mean

4 4 2 6 6 6 20

0.08 0.03 0.11 0.12 0.06 0.05 0.08

0.10 0.06 0.16 0.24 0.10 0.08 0.14

0.40 0.15 0.29 1.03 0.43 0.24 0.47

18.10 80.36 33.33 20.77 39.50 29.38 27.01

215 81 140 261 88 100 149

± ± ± ± ± ± ±

1 2 3b 42 b 8a 11 a 21

Thelypteris dentata

July 1/05 July 7/06 Mean

C2 E

2 4 6

291 ± 32 a 782 ± 162 a 619 ± 146

0.03 0.20 0.11

0.07 0.24 0.17

0.33 0.32 0.32

26.15 24.13 24.45

Blechnum occidentale Polypodium pseudoaureum

July 1/05 July 1/05

C3 A

4 4

481 ± 212 357 ± 10

0.19 0.04

0.25 0.08

0.79 0.22

33.86 52.10

The date of collection and the number of samples (n) are shown. a Different letters within the same species indicate a significant difference at P < 0.05.

comparison procedure (Dunn’s Method), available in the statistical package SigmaStat 3.1 (2004). A cluster analysis was performed with Statistical 6.0 (StatSoft Inc., 2001) after transforming the variables (Al concentration, and the ratios Al/Ca, Al/Mg, Al/P and K/Al) with the formula y = log10 (x + 1) following Legendre and Legendre (1998). 3. Results 3.1. Elemental analysis in two contrasting soils The elemental composition of soil in site A, with pH 4.7 and located at 1240 m altitude, was contrasting in mineral composition to soil in site D, with pH 3.4 and located at 1625 m (Table 3). In site A, lower concentrations of Al, Fe and Ni, but higher K, Ca, Mg, Mn and Cu concentrations were found compared to site D and Zn levels were similar in both sites. 3.2. Aluminum concentration in aerial green organs Pteridophytes were classified into three groups when Al concentration in harvestable biomass and their relationship with mineral nutrients were considered (Table 4). In group 1 L. clavatum, Dicranopteris flexuosa, Sticherus nudus, and C. gibbosa, accumulated Al > 1000 mg kg−1 DM in their lycophylls or fronds, had a ratio >1 between Al and essential plant minerals such as Ca, Mg and P, and a relatively low K/Al ratio, ranging from 0.68 to 2.56 mol mol−1 . In

group 2 Elaphoglossum sporadolepis and N. cordifolia also accumulated Al over 1000 mg kg−1 DM in their fronds, but had Al/Ca and Al/Mg ratios <1. In group 3 A. villosa, Pteridium arachnoideum, Pteris vittata, Thelypteris dentata, Blechnum occidentale and Polypodium pseudoaureum showed Al concentration <782 mg kg−1 DM in the fronds, had Al/Ca and Al/Mg ratios <1, a Al/P ratio ≤1 and a high K/Al ratio, between 18.10 and 80.36 mol mol−1 . Species collected in more than one site showed significant differences in Al concentrations (Table 4). For example in site E, L. clavatum showed higher Al concentrations in their aboveground organs than in site F. Moreover, S. nudus and D. flexuosa showed differences between sites and dates and in site F, where both ferns coexisted, showed similar Al concentrations (Student’s t-test P = 0.497). A cluster analysis (Fig. 1) was performed to separate the sample species. In a first dichotomy, pteridophytes with Al/Ca and Al/Mg ratios >1 (group 1) were separated and in the second dichotomy, group 3 (Al < 1000 mg kg−1 DM and K/Al > 18), species were separated from group 2 (Al > 1000 mg kg−1 DM and K/Al < 7). Pteridophytes from group 1 included the Lycophyte, two basal and one core leptosporangiate ferns, while ferns from groups 2 and 3 included eupolypods or polypods, with one exception A. villosa which is a basal leptosporangiate. 3.3. Nutrient concentrations in aerial green organs The concentrations of nutrients K, Ca, Mg, P, Fe, Mn, Zn, Cu and Ni are presented in Table 5. The highest K concentration occurred in

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E. Olivares et al. / Environmental and Experimental Botany 65 (2009) 132–141

Fig. 1. Cluster analysis for 12 pteridophyte species sampled in Altos de Pipe. The variables Al concentration, Al/Ca, Al/Mg, Al/P and K/Al shown in Table 4 were transformed with the formula y = log10 (x + 1). The clade (Table 2) is indicated: Lycophyte (L), core leptosporangiate (CL), basal leptosporangiate (L), polypods (P), eupolypods II and I (EPII, EPI).

P. pseudoaureum, followed by B. occidentale and T. dentata, all from group 3. High K concentration also occurred in E. sporadolepis from group 2. Conversely the lowest concentration of K was observed in fronds of D. flexuosa, followed by S. nudus, both from group 1. Low values similar to those in group 1 were also observed in members of group 3, such as A. villosa and P. vittata. The concentration of Ca of the four pteridophyte species from group 1 and P. vittata from group 3 was lower than those of other species studied. The highest concentration of Ca in the studied pteridophytes was found in P. pseudoaureum and T. dentata from group 3 comparable to the two species from group 2. Dicranopteris flexuosa presented the lowest concentrations of Mg and P while the highest concentration of Mg was obtained in E. sporadolepis and N. cordifolia, from group 2. High Mg concentration was observed relative to E. sporadolepis and N. cordifolia in T. dentata and P. pseudoaureum. Highest Fe and Mn concentrations occurred in E. sporadolepis and N. cordifolia (Table 5). Ferns from group 3 showed lower concen-

trations of Mn than pteridophytes from other groups. Nephrolepis cordifolia, A. villosa, P. vittata and T. dentata showed high Zn concentrations and were located in different sites (Table 1). C. gibbosa showed the highest mean concentrations of Cu and Ni. The intraspecific variance was higher for Al than for Mn, Zn, Cu and Ni in 10 out of 12 species from 11 families and only in A. villosa and P. arachnoideum Fe and Zn had higher variance than Al (data not shown). The differences between sites in Al concentration shown in Table 4 were also found for other metals, such as Fe, Mn and Zn (Fig. 2). The highest values of Fe in above ground organs from the study pteridophytes present in more than one site were observed in site E in L. clavatum, S. nudus and T. dentata. High Mn concentrations were observed not only in E. sporadolepis from site D or N. cordifolia from site E (Table 5), but in D. flexuosa from sites B and F (Fig. 2). The highest values of Zn were observed at site C in D. flexuosa and T. dentata, but high Zn concentrations were also observed in ferns sampled on other sites, such as N. cordifolia in site E (Table 5).

Table 5 Nutrients concentrations on a dry mass basis (mean ± S.E.) in the same aerial organs of pteridophyte species from Table 4 Species Group 1 Lycopodium clavatum, n = 14 Dicranopteris flexuosa, n = 34 Sticherus nudus, n = 26 Cyathea gibbosa Adult, n = 10 Cyathea gibbosa Juvenile, n = 2 Group 2 Elaphoglossum sporadolepis, n = 10 Nephrolepis cordifolia, n = 10 Group 3 Anemia villosa, n = 4 Pteridium arachnoideum, n = 4 Pteris vittata, n = 20 Thelypteris dentate, n=6 Blechnum occidentale, n=4 Polypodium pseudoaureum, n=4

K (g kg−1 )

Ca (g kg−1 )

Mg (g kg−1 )

P (g kg−1 )

Fe (mg kg−1 )

Mn (mg kg−1 )

Zn (mg kg−1 )

Cu (mg kg−1 )

Ni (mg kg−1 )

7.48 ± 0.90

1.10 ± 0.21

0.64 ± 0.04

1.16 ± 0.24

288.32 ± 36.19

159.21 ± 20.98

39.74 ± 2.84

6.15 ± 0.22

2.32 ± 0.53

4.63 ± 0.35

1.77 ± 0.14

0.53 ± 0.03

0.28 ± 0.02

80.26 ± 9.98

466.53 ± 54.43

48.87 ± 10.56

5.15 ± 0.63

3.18 ± 0.69

5.60 ± 0.50 13.66 ± 0.98

1.67 ± 0.13 1.65 ± 0.20

1.03 ± 0.08 2.25 ± 0.08

0.50 ± 0.07 1.21 ± 0.19

223.04 ± 43.42 247.75 ± 24.88

295.00 ± 23.70 53.15 ± 3.55

24.60 ± 2.28 45.41 ± 2.26

8.15 ± 0.17 24.99 ± 2.92

3.94 ± 0.62 9.63 ± 4.86

9.68 ± 0.19

2.69 ± 0.03

2.13 ± 0.00

0.47 ± 0.00

107.19 ± 4.34

147.99 ± 0.01

27.30 ± 4.08

31.76 ± 1.26

10.12 ± 0.38

16.17 ± 1.41

7.41 ± 0.82

4.33 ± 0.13

1.01 ± 0.05

638.25 ± 75.56

638.63 ± 33.96

77.91 ± 2.95

5.40 ± 0.36

4.90 ± 0.90

11.43 ± 0.56

8.68 ± 0.77

4.70 ± 0.14

1.21 ± 0.04

724.36 ± 58.84

660.54 ± 31.69

124.63 ± 5.55

9.67 ± 0.32

5.75 ± 0.71

5.65 ± 0.51 9.43 ± 0.05

3.84 ± 0.05 4.47 ± 0.34

1.85 ± 0.04 1.14 ± 0.03

0.62 ± 0.01 0.62 ± 0.02

205.74 ± 17.19 128.50 ± 2.72

29.06 ± 0.65 50.16 ± 0.16

158.97 ± 1.51 79.09 ± 2.92

5.17 ± 0.79 4.39 ± 0.78

5.56 ± 0.17 3.39 ± 0.45

5.83 ± 0.47 21.91 ± 3.46

2.81 ± 0.19 8.69 ± 2.03

0.95 ± 0.05 3.24 ± 0.23

0.36 ± 0.03 2.21 ± 0.49

130.44 ± 12.37 179.89 ± 39.51

14.26 ± 0.89 8.17 ± 1.06

94.08 ± 16.40 82.92 ± 44.32

8.41 ± 0.42 11.38 ± 0.99

4.26 ± 1.28 3.92 ± 0.40

23.58 ± 1.25

3.85 ± 0.23

1.73 ± 0.02

0.70 ± 0.19

82.42 ± 33.26

11.69 ± 2.85

31.12 ± 5.63

4.61 ± 0.35

2.71 ± 0.76

26.92 ± 1.25

12.06 ± 0.16

3.79 ± 0.00

1.83 ± 0.12

82.17 ± 3.49

38.87 ± 1.12

39.72 ± 0.30

6.55 ± 0.18

6.06 ± 0.08

E. Olivares et al. / Environmental and Experimental Botany 65 (2009) 132–141

Fig. 2. Concentration of Fe, Mn and Zn (mean ± S.E.) on a dry mass (DM) basis in the aerial green organs of the pteridophyte species sampled in more than two sites: Lc, L. clavatum, sites F and E; Df, D. flexuosa, sites C1, B, and F; Sn, S. nudus, sites C3, B, F and E; Pv, P. vitatta, sites A, C1 and C2 and Td, T. dentata, sites C2 and E. In samples from the same species and site the date is indicated (denoted with 5 for 2005 or 6 for 2006).

The ratios of K/Ca, Ca/Mg, Al/Fe, Al/Mn and Al/Zn are presented in Fig. 3. Among all species the K/Ca ratio was >1 and <4 mol mol−1 , excepting in L. clavatum and T. dentata in site E, as well as in adult individuals of C. gibbosa and B. occidentale. Only L. clavatum and S. nudus from site E, and C. gibbosa had a Ca/Mg ratio <1 and the

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Fig. 3. Molar ratios of K/Ca, Ca/Mg, Al/Fe, Al/Mn and Al/Zn in the aerial green organs of the pteridophyte species sampled in “Altos de Pipe”: Lc, L. clavatum; Df, D. flexuosa; Sn, S. nudus; Cg, C. gibbosa; Cg J, C. gibbosa juvenil; Es, E. sporadolepis; Nc, N. cordifolia; Av, A. villosa; Pa, P. arachnoideum; Pv, P. vitatta; Td, T. dentata; Bo, B. occidentale and Pp, P. pseudoaureum in the sites indicated in Table 1 (A–F). In samples from the same species and site the date is indicated (denoted with 5 for 2005 or 6 for 2006). The means from Tables 4 and 5 transformed to molar units were used in ratio calculations.

highest values were observed in P. arachnoideum and D. flexuosa from site C. The Al/Fe, Al/Mn and Al/Zn ratios in all species under study were >1. In general group 1 showed higher Al/Fe and Al/Zn ratios than groups 2 and 3, but with contrasting values between members of the same species, for example in S. nudus. For Al/Mn was observed that species from different groups, may reach high values, such as C. gibbosa (group 1) and T. dentata (group 3) from

Table 6 Aluminum concentration (mean ± S.E.) and molar ratios for mineral elements in the subterranean organs of Al-accumulator pteridophyte species from “Altos de Pipe” Al (mg kg−1 DM)

Al/Ca (mol mol−1 )

Al/Mg (mol mol−1 )

Al/P (mol mol−1 )

K/Al (mol mol−1 )

Site

Organ

F E Mean

Roots Roots Roots

1,917 ± 86 5,208 ± 346 4,660 ± 468

1.38 8.70 6.38

2.62 5.26 4.92

2.66 5.15 4.84

1.18 1.19 1.19

Dicranopteris flexuosa

F

Roots Rhizomes

4,146 ± 164 8,748 ± 526

2.47 3.27

3.37 3.43

20.60 79.70

0.60 1.92

Sticherus nudus

F

Roots Rhizomes

3,057 ± 1,556 763 ± 133

3.25 2.72

7.36 1.71

14.04 4.02

1.06 6.99

Cyathea gibbosa (Juvenile)a

D

Roots

3,852 ± 163

2.42

4.01

9.41

1.00

Group 2 Elaphoglossum sporadolepis Nephrolepis cordifolia

D E

Rhizomes + rootsa Roots

12,176 ± 1,584 1,943 ± 150

14.84 0.99

17.70 1.58

18.49 4.50

0.17 1.23

Group 1 Lycopodium clavatum

Groups were defined in Table 4. a Roots from adult tree-ferns were not sampled.

Sticherus nudus

79.39 197.17 87.97 86.61 129.69 27.76 87.90 11.13 3.09 1.68 1.19 1.60 2.45 2.57 1.22 11.70 ± 0.19 14.62 ± 1.40 14.84 ± 4.52 30.25 ± 2.91 16.84 ± 1.55 7.15 ± 0.09 117.58 ± 4.56 149.66 ± 71.06 53.53 ± 2.31 90.56 ± 5.55 191.16 ± 31.30 142.53 ± 14.43 1008.99 ± 40.76 2264.21 ± 69.40 1302.63 ± 73.49 0.48 ± 0.04 0.76 ± 0.07 0.50 ± 0.02 0.87 ± 0.00 0.62 ± 0.24 1.11 ± 0.02 2.40 ± 0.35 1.22 ± 0.77 2.92 ± 0.14 5.56 ± 0.34 3.06 ± 0.26 3.47 ± 0.14 Cyathea gibbosa Juvenile Roots D Elaphoglossum sporadolepis Rhizomes + roots D Nephrolepis cordifolia Roots E

152.24 275.33 740.85 142.24 93.80 92.29 85.27 67.55 22.18 210.55 20.98 100.40 1.36 1.05 2.26 0.63 1.49 6.28 3.43 19.00 4.89 0.23 3.01 0.67 ± ± ± ± 10.32 3.51 13.67 1.63 1.37 0.46 4.31 0.49 ± ± ± ± 9.69 3.60 13.46 5.84 12.00 10.00 5.00 1.00 ± ± ± ± 66.00 77.00 10.00 13.00 19.00 45.00 40.00 2.00 ± ± ± ± 90.00 193.00 73.00 23.00 47.00 14.00 88.65 6.65 ± ± ± ± 387.00 86.00 301.54 15.73 0.04 0.03 0.08 0.02 ± ± ± ± 0.23 0.13 0.25 0.22 0.09 0.41 0.08 0.02 ± ± ± ± Roots Rhizomes Roots Rhizomes Dicranopteris flexuosa

F F F F

3.62 24.36 4.67 7.73

0.41 3.58 0.29 0.63

2.49 3.98 1.40 0.42

0.19 0.95 0.24 0.04 ± ± ± ± ± ± ± ±

1.11 2.30 0.37 0.40

72.30 302.56 248.32 53.56 273.37 213.33 26.79 5.56 5.88 1.90 0.61 0.77 1.63 10.33 7.57 5.18 ± 0.01 4.77 ± 0.62 4.81 ± 1.43 7.78 ± 0.00 7.50 ± 0.21 7.53 ± 0.20 64.26 ± 3.86 41.71 ± 3.05 45.47 ± 3.60 72.87 ± 4.28 38.79 ± 3.21 44.47 ± 4.69 148.13 ± 9.15 1938.39 ± 134.83 1640.02 ± 229.92 0.83 ± 0.03 1.16 ± 0.07 1.10 ± 0.07 0.66 ± 0.04 0.89 ± 0.04 0.85 ± 0.04 2.07 ± 0.06 0.89 ± 0.07 1.09 ± 0.14 3.29 ± 0.15 8.96 ± 0.82 8.02 ± 0.93 F E

Ni (mg kg−1 ) Cu (mg kg−1 ) Zn (mg kg−1 ) Mn (mg kg−1 ) Fe (mg kg−1 )

Roots Roots Mean

The pH from soils in sites A and C2 were significantly higher than values obtained from the remaining locations (Table 1), however all soils were acidic and therefore Al is considered the major exchangeable cation in acid soils, being the solubility of Al (Al3+ ion) in water 0.3 ppm at pH 4.5 and 76.4 ppm at pH 3.11 (Osaki et al., 2003). At a pH <5, mononuclear Al3+ is dominant, which is known to inhibit root elongation (Schulze et al., 2005). The pteridophyte species studied (Table 2) appeared to resist high concentrations of Al (Fig. 1), accumulating (groups 1 and 2) or excluding (group 3) the metal. In site A, where P. vittata and P. pseudoaureum were present (Table 1), the concentration of Al in the soil was 8 g kg−1 DM (Table 3); and at site D, where C. gibbosa and E. sporadolepis were present, the concentration was 13 g kg−1 DM. These concentrations are higher than those we found in the pteridophytes (Tables 4 and 6). An Al concentration of 12 g kg−1 DM was observed in subterranean organs from E. sporadolepis (Table 6), however plants with root accumulation should not be interpreted as accumulators (Jansen et al., 2002). Differences in the chemical properties in soils at IVIC with different vegetation had been previously reported (Marulanda, 1998), for example at 0–5 cm depth (horizont A1-1 ) differences in pH KCl ranging from 3.5 in the cloud forest to 4.0 in the savanna, carbon from 5.95 to 2.34%, cation-exchange capacity from 10.71 to

Lycopodium clavatum

4. Discussion

Mg (g kg−1 ) P (g kg−1 )

Rhizomes of D. flexuosa showed high concentrations of K (Table 7) and very low Ca concentrations were observed in S. nudus rhizomes and L. clavatum roots, consequently the K/Ca ratio was higher than those found in other species analyzed. In site F roots of L. clavatum had lower concentration of K, Mg, P and Fe than in site E. The concentration of Fe in subterranean organs of L. clavatum and N. cordifolia in site E, as well as E. sporadolepis and C. gibbosa juvenile in site D was higher than in other species studied while the concentration of Fe and Mn in S. nudus rhizomes was lower. Roots had higher concentrations of Cu and Ni than rhizomes, but this was not observed for Zn. Translocation factor (TF) of Al was 1.17 and 1.07 when the translocation was calculated from the roots of L. clavatum and S. nudus from site F, respectively, and was <1 for other Al-accumulator species studied (Table 8) but was 4.30 for S. nudus when the translocation was calculated from the rhizomes. E. sporadolepis presented very high TF values for K, Ca and Mg. Nephrolepis cordifolia presented high translocation factors for K, Ca, Mg, Mn, Zn and Cu. In L. clavatum from site E the TF for P was higher than in site F, contrary to Fe. L. clavatum and D. flexuosa showed a TF <1 for Mg. The highest values of TF for Mn were observed in rhizomes from S. nudus.

Ca (g kg−1 )

3.5. Nutrients concentration in subterranean organs and translocation factor

Site K (g kg−1 )

Highest Al concentrations were observed in D. flexuosa rhizomes and lowest in S. nudus rhizomes (Table 6). Subterranean organs (rhizomes and roots) from E. sporadolepis showed the highest concentration of Al. In site E L. clavatum showed higher Al concentration than in site F in roots as in above ground organs (Table 4). The Al/Ca ratio ranged from 0.99 in N. cordifolia to 14.84 mol mol−1 in E. sporadolepis, and Al/Mg and Al/P ratios were higher than one in all the species. The highest value of Al/P ratio was observed in D. flexuosa, which along with N. cordifolia showed a K/Al ratio lower than one.

Table 7 Nutrients concentrations on a dry mass basis (mean ± S.E.) and molar ratios for mineral elements in the same subterranean organs of Al-accumulator pteridophyte species from Table 6

3.4. Aluminum concentration in subterranean organs

Organ

site E, or show low values, for example D. flexuosa (group 1) from site B and P. arachnoideum (group 3).

K/Ca Ca/Mg Al/Fe Al/Mn Al/Zn (mol mol−1 ) (mol mol−1 ) (mol mol−1 ) (mol mol−1 ) (mol mol−1 )

E. Olivares et al. / Environmental and Experimental Botany 65 (2009) 132–141

Species

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E. Olivares et al. / Environmental and Experimental Botany 65 (2009) 132–141

139

Table 8 Translocation factor (TF), ratio of the metal concentration in aerial green organs divided by that in subterranean organs Species

Site

Organ

Al

K

Ca

Mg

P

Fe

Lycopodium clavatum

F E

Roots Roots

1.17 0.56

1.11 1.12

0.77 0.83

0.73 0.82

0.43 1.35

0.84 0.22

Dicranopteris flexuosa

F

Roots Rhizomes

0.88 0.42

1.69 0.25

1.11 0.70

0.70 0.34

1.87 3.31

Sticherus nudus

F

Roots Rhizomes

1.07 4.30

0.83 0.70

0.50 3.57

2.43 2.25

Cyathea gibbosa Juvenile Elaphoglossum sporadolepis Nephrolepis cordifolia

D D E

Roots Rhizomes + roots Roots

0.94 0.12 0.84

1.74 5.28 3.29

1.12 6.07 2.97

2.45 6.98 4.23

Mn

Zn

Cu

Ni

3.16 2.94

0.44 0.97

0.41 0.82

1.28 0.49

0.40 1.80

6.88 3.21

0.61 0.52

0.67 1.82

0.52 1.52

1.20 1.36

0.22 4.18

4.59 14.57

1.50 1.15

0.38 0.88

1.00 8.39

0.98 1.33 2.42

0.11 0.28 0.56

1.63 3.34 4.63

0.23 0.52 2.33

1.05 0.32 1.35

0.86 0.34 0.39

The subterranean organ from the metal is translocated is indicated.

5.33 Cmolc /kg, and exchangeable Al from 9.03 to 2.55 Cmolc /kg (7.29–2.06 g kg−1 DM). However, the Al-accumulators D. flexuosa and S. nudus sampled in the savanna (Site C1 and C3) showed up to 1538 and 3319 mg kg−1 DM, respectively (Table 4) while S. nudus statistically similar values in the secondary forest (B and F) where soil Al concentration was higher. This supports the idea suggested by Osaki et al. (2003) and Watanabe et al. (2007) that the Al concentration in leaves is not determined by the soil type in which plants grow but by the specific characteristics of plants for Al accumulation or tolerance. In this study, two new Al-accumulator Neotropical ferns are reported which belong to the more primitive groups: S. nudus and D. flexuosa (Fig. 1; Table 4). We also report Al accumulation in the tree fern C. gibbosa. This fact had been reported previously by Chenery (1949) but the fern was identified as Alsophila gibbosa. He reported concentrations of Al 2990–22,900 mg kg−1 DM in the Cyatheaceae and found that this family showed the largest number of Al-accumulators (289 species out of 426). Anemia villosa (Schizaeaceae) is a basal leptosporangiate and was not found in group 1 (Table 3) and similarly Chenery (1949) and Webb (1954) investigated 10 and 3 species respectively of Schizaeaceae (the taxonomic identification was at family level) and did not detect Al accumulation. In the Gleichenaceae family, Chenery (1949) did not analyzed Dicranopteris species, however, in the genus Gleichenia 65 out of 67 species analyzed using the “aluminon” test were Al-accumulators. Webb (1954) reported Al accumulation using the same colorimetric test in one Dicranopteris and two Sticherus, identified to genus level. To our knowledge Al has not been reported in the Neotropical fern D. flexuosa, however Chau and Lo (1980) reported up to 3900 mg kg−1 DM in Dicranopteris linearis. The authors reported Ca, Mg and K, but not P concentrations. From their study, we can calculate Al/Ca, Al/Mg and K/Al ratios of 19.39, 0.59 and 2.48 mol mol−1 , respectively. Dicranopteris dichotoma, from Japan accumulates lanthanum (Koyama et al., 1987) and other REEs (Ichihashi et al., 1992). There are reports about accumulation of barium in D. linearis in Japan (Ozaki et al., 1997) and rare earth elements (REEs), mainly lanthanum, cerium, praseodymium, neodymium, samarium, gadolinium and yttrium in D. linearis in China (Zhenggui et al., 2001). This species is one of the most widely distributed ferns throughout Old World tropics and subtropics, a pioneer in mountain ridges, precipices and taluses, and is credited with preventing erosion on steep slopes where few other species can become established (Russell et al., 1998). Dicranopteris flexuosa from the Neotropics grows in similar habitats. In Table 5 low concentrations of K (<9 g kg−1 DM) were observed in the Al-accumulators (group 1) and similarly in the Al-excluders (group 3). Broadley et al. (2004) found a range of K from 9.8 g kg−1 DM in Gladiolus blandus (Iridaceae) to 91.5 g kg−1 DM in Borago officinalis (Boraginaceae) when they studied 117 species of

hydroponically grown angiosperms representing 24 orders. The concentration of Ca in pterydophytes from group 1 and P. vittata was lower than 2.69 g kg−1 DM, however values as low as 1.1 g kg−1 DM have been reported in monocots, for example in Phoenix canariensis (Aracaceae) (e.g. Broadley et al., 2004). The concentration of Mg can be as high as 7.6 g kg−1 DM in Caryophyllales (Broadley et al., 2004), in this study we report highest Ca concentration of 4.7 g kg−1 DM in N. cordifolia. Broadley et al. (2004) found that shoot Ca and Mg concentration regressed significantly across the angiosperms when Caryophyllales were excluded, and reported a Ca/Mg ratio of 7.7 mol mol−1 , however in the present study Ca/Mg ratio ranged from 0.44 to 2.38 mol mol−1 . Among all species investigated, Al-accumulators and nonaccumulators, showed low concentrations of P in the aerial green organs (Table 5) and subterranean organs (Table 7), compared to the lowest concentration (3.7 g kg−1 DM) reported in angiosperms by Broadley et al. (2004). Our study concurs with values reported by Liao et al. (2004), who showed a concentration of P of 1.98 g kg−1 DM in pinnae of P. vittata sampled in China. Similarly Russell et al. (1998) reported a concentration of P ranging from 0.27 to 0.47 g kg−1 DM in fronds of Dicranopteris linearis in Hawai, and indicated that this species had an exceptionally high phosphorus use efficiency (amount of biomass produced per unit nutrient taken up from the environment). Lycopodium, Sticherus, Dicranopteris, Pteridium, Nephrolepis, Elaphoglossum, Blechnum and Polypodium were among the 85 genera of pteridophytes identified by Page (2004) as having species tolerant to low-nutrient substrates. In the present study those pterydophytes showed low concentrations of P and were found in soils with low pH and high concentrations of Al, as well as Cyathea, Anemia, Pteris and Thelypteris. In sites E and D, pH were 3.4 and 3.5, respectively, and when pH decreased below 3.5, ferric iron is soluble and toxicity would be expected (Osaki et al., 2003), however pteridophytes did not show visible symptoms of toxicity, such as bronzing, necrosis, etc. but high concentration of Fe in aerial organs (Table 5; Fig. 2) and subterranean organs (Table 7). Osaki et al. (2003) reported that the Fe concentration in leaves was not always related to pH when 166 species from different phylogeny grown in various soil types in the temperate region were analyzed. Concentrations of Fe in fronds varied among species C. gibbosa and E. sporadolepis located at the same site D (Table 5). Similarly results were observed with Mn and Zn accumulation in both species. The background concentration of Fe in leaves ranges from 50 to 250 mg kg−1 DM (Schulze et al., 2005) and the normal concentration of Mn ranged from 20 to 400 mg kg−1 DM (Lindberg and Greger, 2002). The high concentrations of Fe and Mn found in E. sporadolepis and N. cordifolia were not observed in the aerial green organs from other species, but high concentrations of Fe were also found in roots of L. clavatum and C. gibbosa (Table 7). In P. vittata, Liao et al. (2004) reported Fe concentrations up to

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893 mg kg−1 DM in pinnae, up to 2509 mg kg−1 DM in rhizomes, and up to 1812 mg kg−1 DM in roots. Jansen et al. (2003) reported a range of Fe concentrations of 746–651 mg kg−1 DM in the foliar tissues of the Al-accumulators Coccocypselum canescens (Rubiaceae) and Melastoma malabathricum (Melastomataceae). In the present study, the species N. cordifolia, A. villosa, P. vittata and T. dentata showed concentrations of Zn higher than 83 mg kg−1 DM. The normal concentration in plants ranged from 20 to 400 mg kg−1 DM for Zn, 5 to 25 mg kg−1 DM for Cu and 1 to 10 mg kg−1 DM for Ni (Lindberg and Greger, 2002). C. gibbosa had higher concentrations of Cu than normal values and the concentration of Ni was in the upper limit of the range. The translocation of Al from subterranean to aerial organs only was slightly higher than one in L. clavatum from site E and in S. nudus was 1.07 from roots and 4.30 from rhizomes (Table 8). Cuenca and Herrera (1987) reported in the same cloud forest of Altos de Pipe a translocation factor of Al from 1.06 to 5.17 in the Rubiaceae Palicourea fendleri and P. angustifolia, respectively, in a total of four Al-accumulators species, contrasting to 0.05–0.5 in the nonaccumulators Vismia ferruginosa (Clusiaceae) and Didymopanax glabratus (Araliaceae), out of a total of 12 non Al-accumulators species. They reported a maximum of 11,582 mg kg−1 DM Al in leaves from Richeria grandis (Euphorbiaceae) with a translocation factor of 1.86. Roots in Dioclea guyanensis (Fabaceae), sampled also at the same site, presented 2750 mg kg−1 DM Al in roots besides 1250 in leaves (Izaguirre-Mayoral and Flores, 1995), which produces a translocation factor of 0.45. Pteridium caudatum, sampled also at Altos de Pipe, has a translocation factor of 0.02 in roots and 0.09 in rhizomes and shows high concentrations of Al in the subterranean organs (Olivares et al., 2007). Mazorra et al. (1987) also found low translocation from roots to leaves in five Alaccumulator plants in Venezuela, with a foliar concentration of Al up to 6899 mg kg−1 DM in Miconia stephantera (Melastomataceae). The recalculated translocation factor in those plants ranged from 0.45 to 1.02. L. clavatum, which is nearly cosmopolitan, but absent in Australia (Berry et al., 1995), has been reported as Al-accumulator (Krupitz, 1969), however Osaki et al. (2003) reported Al concentration of 6 mg kg−1 DM in L. clavatum L. var. nipponicum Nakai. European specimens of L. clavatum tend to contain more Al than American specimens (Webb, 1954) and in tropical conditions at Altos de Pipe behaved as an Al-accumulator (Table 4), but with low TF (Table 8). In the present study, the concentration of Mn was higher in fronds than in the subterranean organs in all the species, but Fe and Ni were sequestered in roots and only rhizomes of S. nudus showed translocation factors higher than one for these elements. In N. cordifolia (Table 8) higher TF were found than those reported by Sridokchan et al. (2005) in ferns grown with 0.01 As for Al (0.54), K (1.36), Ca (1.25), Mg (1.25), P (1.14), Fe (0.25), Zn (0.39) and Cu (0.34) and than those of Kachenko et al. (2007) for Zn (0.86) and Cu (0.20), but we found lower translocation for Ni (0.51). Other TF higher than one were found for Zn in S. nudus as well as for Cu in rhizomes of D. flexuosa and juvenile individuals of C. gibbosa. Further investigation is required to explore the relationship between Al availability and its uptake. The study of ferns grown in hydroponics with controlled concentrations of mineral elements would help to understand if they are sensitive to Al and therefore reduce their biomass when exposed to high Al concentrations or if they are tolerant and growth is not affected by this element. 5. Conclusion The results presented here suggested that the K/Al ratio is useful to define Al-accumulator pteridophytes, when used together with

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