Evidence of foliar aluminium accumulation in local, regional and global datasets of wild plants

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1 Research Evidence of foliar aluminium accumulation in local, regional and global datasets of wild plants Faizah Metali 1, Kamariah A. Salim 1 and David F. R. P. Burslem 2 1 Biology Programme, Faculty of Science, University of Brunei Darussalam, Jalan Tungku Link, Bandar Seri Begawan, BE1410, Brunei Darussalam; 2 School of Biological Sciences, Cruickshank Building, University of Aberdeen, St Machar Drive, Aberdeen, AB24 3UU, UK Author for correspondence: Faizah Metali Tel: faizah.metali@ubd.edu.bn Received: 30 August 2011 Accepted: 10 October 2011 doi: /j x Key words: Al accumulation, Al accumulator, foliar nutrient concentrations, phylogenetic signal, phylogeny. Summary High foliar concentrations of aluminium (Al) have been reported in numerous plant species, but progress on the understanding of the functional significance of this trait is constrained by the absence of a quantitative analysis of its distribution among plant lineages and across biomes. We constructed a global dataset of foliar Al and nutrient concentrations for 1044 plant species from literature sources and new data collections in Brunei Darussalam. Our results provide statistical support for the existence of Al accumulators and non-al accumulators in global, regional and local floras based on foliar Al concentrations. A value of 1 mg Al g )1 leaf dry mass is a suitable threshold to distinguish between these two groups in a sample of species that lacks any geographical reference. However, a higher threshold foliar Al concentration is required to distinguish between Al accumulators in tropical ( mg Al g )1 leaf dry mass) than in temperate (1.1 mg Al g )1 leaf dry mass) floras. There was a phylogenetic signal in the foliar concentrations of Al, but phylogeny did not explain the difference in the mean foliar Al concentration between tropical and temperate floras in a phylogenetically controlled analysis. Phylogeny and soil chemistry are potential factors driving Al accumulation in certain groups of plants. Introduction High aluminium (Al) concentrations in the tissues of some plants were first recognized > 300 yr ago (Hutchinson, 1943). In Herbarium Amboinense, published posthumously in 1743, Rumphius described a tree (Arbor aluminosa) whose leaf and bark extracts were used as a mordant instead of alum (cited in Hutchinson, 1943). This tree was identified as a species of Symplocos (Symplocaceae), which is now known to have high tissue Al concentrations (Merrill, 1917; Hutchinson, 1943; Jansen et al., 2002a). Since then, general surveys of foliar Al concentrations have been conducted (Chenery, 1948a,b, 1949; Webb, 1954), and several authors have asserted that the presence of high Al concentrations in some plants points to the existence of a qualitatively distinct group of species that can be classified as Al accumulators (Hutchinson, 1945; Chenery, 1948a). In the most extensive of these surveys, Chenery (1948b, 1949) determined Al concentrations in the leaves of herbarium specimens and classified 1779 dicot species (62% of 2859 species examined), 33 monocots (13% of 269 species) and 476 pteridophytes (46% of 1044 species) as Al accumulators. Al accumulators were defined by Hutchinson (1945) and Chenery (1948a) as plants with Al concentrations of > 1 mg Al g )1 dry mass of their leaf tissues, whereas non-al accumulators, such as most herbaceous plants, have leaf Al concentrations < 0.2 mg Al g )1 dry mass (Hutchinson, 1943). The term Al hyper-accumulator was introduced by Jansen et al. (2002a) to unify terminology with other metal hyper-accumulators (Baker et al., 2000), and is now used interchangeably with Al accumulator to refer to plants with >1 mg Al g )1 dry mass of leaf tissues. However, none of these studies has provided statistical evidence that Al accumulators form a qualitatively distinct group of species, or whether they are simply taxa drawn arbitrarily from the upper tail of a normal distribution of foliar Al concentration. Similarly, the thresholds presented for distinguishing between groups have not been defined on the basis of statistical criteria, and the phylogenetic and geographical variations in these threshold values are unknown. Studies of metal concentrations in plants must allow for the possibility of soil-borne contamination, such as residual dust adhering to leaf epidermal surfaces (Cook et al., 2009). To account for contamination, Berrow (1988) proposed that titanium (Ti) concentrations > 10 mg kg )1 in samples of wild plants would be an effective indicator of soil contamination because Ti concentrations are much higher in soils than in plants (Berrow, 1988; Cook et al., 2009). We present evidence to evaluate the hypothesis that soil contamination contributes to the identification of high foliar Al concentrations in wild plants. 637

2 638 Research Phytologist Foliar Al concentration is a phylogenetically constrained trait (Jansen et al., 2002a; Watanabe et al., 2007). For example, species and genera in the evolutionarily advanced families Anisophylleaceae, Melastomataceae, Pentaphylacaceae, Polygalaceae, Rubiaceae and Symplocaceae are consistently well represented in surveys recording taxa with high foliar Al concentrations (Chenery & Sporne, 1976; Jansen et al., 2000a), and the Rubiaceae (Gentianales) and Melastomataceae (Myrtales) are thought to have the highest number of Al-accumulating species (Jansen et al., 2000a,b, 2002b, 2003). High foliar Al concentrations have now been discovered in 60 angiosperm families (Jansen et al., 2002a). These families are distributed broadly within a major group of dicotyledons (the Eudicots), as well as monocotyledons, which suggests that a capacity to accumulate a high foliar Al concentration has arisen many times during angiosperm evolution. Nonflowering plant families, including ferns and some mosses, have also been shown to contain Al-accumulating species (Chenery, 1949; Webb, 1954; Olivares et al., 2009), suggesting that Al accumulators are also represented among ancient taxa. However, there is no evidence of Al accumulation in gymnosperms (Chenery, 1949; Webb, 1954). Studies analysing the distribution and functional significance of Al accumulation must take into account the lack of phylogenetic independence among the species included within the sample (Haridasan, 1982; Harvey & Pagel, 1991; Masunaga et al., 1998a). For example, Al accumulators are thought to be more frequent among tropical woody plants than in plants of other life-forms and in different regions (Hutchinson, 1943; Jansen et al., 2002a). The association between Al accumulation and tropical ecosystems may arise because tropical forest soils are often leached and strongly acidic, which gives rise to soils that contain a significant pool of soluble Al ions (Jansen et al., 2002a). This perspective is supported by a lower frequency of Al accumulators in tropical vegetation types in drier environments or less acidic soils (Hutchinson, 1943). However, an alternative possibility is that the association between Al accumulation and tropical woody plants arises because the species in the sample are phylogenetically biased, and this bias has not been accounted for in the analytical techniques. In this study, we compiled a global dataset of published values of foliar Al concentration for 986 species and supplemented this with new data on 58 species of tropical woody plants from three sites in Brunei Darussalam, northwest Borneo. We used these datasets to address the following questions: Is there evidence of bimodality in the distribution of foliar Al concentration within plants, which would be indicative of a distinct group of species with high foliar Al concentrations? Does the shape of this distribution differ for foliar concentrations of Al and the major nutrient elements (nitrogen (N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg))? What is the threshold foliar Al concentration that defines the Al accumulator group? Does this threshold value differ between tropical and temperate floras? Is there a phylogenetic signal in foliar Al and other major nutrient element concentrations (N, P, K, Ca and Mg) for angiosperms based on recent updates in our understanding of the angiosperm phylogeny? Taking phylogeny into account, are foliar Al concentrations from tropical floras higher than those from temperate floras? Materials and Methods Data for this study were obtained from two sources: a thorough bibliographical search to collate a database of foliar Al concentrations in plants, and new data collection from three study sites in Brunei Darussalam. Papers were selected if they reported datasets of foliar Al concentrations of wild plants. The sample of species derived from the literature search is not drawn randomly from the global flora, and therefore contains a geographical bias and unknown biases in terms of life-form representation and leaf age and phenology (Supporting Information, Table S1). Foliar nutrient concentrations in plants from the published literature Data on foliar Al concentrations were collated from Chenery (1948a, 1949), Haridasan (1982, 1987), Cuenca & Herrera (1987), Mazorra et al. (1987), Masunaga et al. (1998a), Osaki et al. (1998, 2003), Jansen et al. (2003) and Watanabe et al. (2007). These sources yielded data for a total of 986 plant species drawn from 493 genera in 193 families. In cases in which species were duplicated between datasets, the mean value across all studies was computed and used in the analyses. The dataset comprised 757 angiosperms and 229 nonflowering plants, including ferns (151 species), mosses (27 species), a lichen (one species), club mosses (seven species) and gymnosperms (43 species). The plant material for this dataset was collected from wild plants growing in Brazil (36 species), Hawaii (one species), Indonesia (123 species), Japan (506 species), Madagascar (two species), Malaysia (one species), Zealand (56 species), Philippines (one species), Thailand (51 species) and Venezuela (25 species). The origin of the remaining 184 species was not mentioned in the original papers, but they could be classified as either tropical (144 species) or temperate (40 species) on the basis of a literature search. In this article, we have divided the dataset into species from temperate climatic zones (those from Japan and Zealand and the 40 temperate species with an unrecorded origin) and the tropics (the rest). There were 349 and 466 angiosperms, and 93 and 136 nonflowering plants, in the tropical and temperate species lists, respectively. Data on the foliar concentrations of N, P, K, Ca and Mg were extracted from the same sources, although this yielded lower sample sizes for these elements because the papers were focused primarily on foliar Al concentrations (N, 51 species; P, 63 species; K, 671 species; Ca, 684 species; Mg, 656 species). The methods used to measure Al and other major nutrient concentrations varied among studies, but, in all cases, leaf samples were acid digested and concentrations of Al, K, Ca and Mg were mostly determined using atomic absorption spectrophotometry (Haridasan, 1982, 1987; Cuenca & Herrera, 1987; Mazorra et al., 1987; Osaki et al., 1998, 2003; Jansen et al., 2003); the exceptions were Chenery (1948a, 1949),

3 Phytologist Research 639 Masunaga et al. (1998a) and Watanabe et al. (2007), who measured Al using an aluminon colorimetric method, inductively coupled plasma atomic emission spectrometry and neutronactivation analysis, respectively. Total N and P concentrations were determined using a semi-micro Kjeldahl method (Osaki et al., 1998, 2003) and a vanado-molybdate colorimetric method (Haridasan, 1982; Osaki et al., 1998, 2003), respectively. Elemental concentrations were converted to mg g )1 from the units reported in the original papers. data collection in Brunei Darussalam Study sites To sample the variation in foliar Al and nutrient concentrations within a local tropical tree flora, three sites representing two distinct forest types were selected for study in Brunei Darussalam on the island of Borneo. Sampling was conducted on 0.96-ha permanent plots in mixed dipterocarp forest (MDF) at Andulau Forest Reserve (4 39 N, E, elevation m) and in heath forests (HFs, locally referred to as kerangas ) in the Badas (4 34 N, E, elevation m) and Bukit Sawat (4 34 N, E, elevation m) Forest Reserves. These sites are representative of two of the most important variants of lowland tropical rain forest on Borneo (Ashton, 1964; Whitmore, 1984). Their contrasting floristic composition and structural characteristics are associated with different underlying geologies, soil conditions and biogeochemical cycling (Brünig, 1974; Whitmore, 1984; Dent et al., 2006). Brunei Darussalam had a mean annual rainfall of c mm in with dry periods in February April and July September (records from Sungai Liang Agricultural Station; Department of Agriculture and Agrifood, Brunei Darussalam, unpublished data). Andulau, Badas and Sawat are located 4 km southeast, 14 km southwest and 11 km south of Sungai Liang, respectively. The mean monthly temperature ranges from 24 to 35 C (Brunei Meteorological Service, unpublished data). The soils at Andulau range from yellow podsols (sandy-textured haplic acrisols) to hydromorphic alluvium overlying alternate clay and sandstone layers of the Liang Formation (Ashton, 1964). The soils at Badas and Sawat are deep white sand albic arenosols and humic podsols overlying a Pleistocene marine terrace, respectively (Brünig, 1974). The plots are divided into 24 subplots of m 2 and were established and enumerated by the Biology Department, University of Brunei Darussalam. A detailed account of the floristic composition and community structure of all three forests is provided by Davies & Becker (1996). Andulau has the highest basal area, and the HF at Sawat has the highest stem density (Table S2). Dryobalanops aromatica Gaertn. f. (Dipterocarpaceae), Agathis borneensis V. Sl. (Araucariaceae) and Gluta beccarii (Engl.) Ding Hou (Anacardiaceae) contribute most to the basal area of trees 5 cm diameter at breast height (dbh) at Andulau (MDF), Badas (HF) and Sawat (HF), respectively. Mallotus wrayi King. Ex Hook. F. (Myrtaceae), Eugenia bankensis (Myrtaceae) and Dipterocarpus borneensis V. Sl. (Dipterocarpaceae) had the highest stem density at Andulau (MDF), Badas (HF) and Sawat (HF), respectively. Sampling A total of 322 leaf samples from 58 species (31 genera in 18 families) was collected from individuals 5 cm dbh growing on the three plots (Table S3). Tree species selected for sampling were common, ideally represented in more than one site and drawn from a wide range of families from different angiosperm clades (Table S4). The number of individual trees sampled per species ranged from one to 15 across all sites, with a mean of four trees. Where possible, fully expanded sun leaves with minimal or no damage were collected by tree climbing, although shade leaves were sampled in some cases because of the difficulty in accessing sun leaves from tall trees. In a paired t-test, foliar Al concentrations of sun (0.93 ± 1.48 mg Al g )1 dry mass; mean ± SE) and shade (1.45 ± 1.35 mg Al g )1 dry mass) leaves were not significantly different (t = 2.05, df = 28, P = 0.07). On the day of collection, the fresh leaf samples were oven dried at 60 C for 48 h in a laboratory at the University of Brunei Darussalam, and were then transported to Aberdeen University, UK, for chemical analysis. Chemical analysis Dried leaf samples were finely milled using a ball mill and analyses were conducted after digesting 0.1-g samples using a mixture of acid reagents (4.8% H 2 SO 4 and H 2 O 2 ) and an LiSO 4 catalyst at 365 C (Allen et al., 1989). Foliar N and P concentrations were determined colorimetrically using flow injection auto-analysers (Skalar FIA, Breda, the Netherlands and Tecator FIAstar 5010 Analyser, Croydon, Surrey, UK, respectively). Foliar K concentrations were determined by flame emission spectrophotometry (Perkin Elmer AAnalyst 100, Norwalk, CT, USA), and foliar Al, Ca, Mg and Ti concentrations were measured using atomic absorption spectrophotometry after diluting the acid-digested samples with LaCl 3 (H 2 SO 4 : LaCl 3 in the ratio of 1 : 1). Foliar Ti concentrations were measured to validate that foliar Al concentrations were not derived from soil-borne Al contamination or dust that had adhered to the leaf surfaces of samples of wild plants. In these analyses, foliar Ti concentrations were below the detectable limit in all samples. Therefore, we conclude that the measured Al concentrations in these samples were derived from plant material and not from contamination by soil or dust. Statistical analysis All statistical analyses were conducted using R version (R Development Core Team, 2009). During model development, all data were explored for the normality of residuals and homogeneity of variances and, where necessary, data were log 10 -transformed to fulfil model assumptions. distributions of foliar Al and macro-nutrient element concentrations The global dataset of foliar Al concentrations comprised values from 1044 plant species drawn from 513 genera in 195 families. This total included the published values for 986 plant species and the additional 58 tropical tree species from Brunei Darussalam (hereafter these data are referred to as the global dataset). The global dataset comprised 815 angiosperms and 229 nonflowering plants, and species were approximately equally distributed between tropical (442 species) and temperate (602 species) biomes. The global dataset of foliar concentrations of N,

4 640 Research Phytologist P, K, Ca and Mg comprised values from 109, 121, 729, 742 and 714 plant species, respectively, derived from the same sources. Hartigan s dip test (Hartigan & Hartigan, 1985) was used to determine whether or not the distributions of the mean foliar Al and nutrient concentrations of the species were unimodal. P values for the dip test statistic (d) were obtained from the dip test package (Maechler, 2009) in R version (R Development Core Team, 2009). The distributions of foliar element concentrations were also analysed for the presence of distinct clusters of values, which might be indicative of discrete groups of Al-accumulating and non-al-accumulating species. These analyses were conducted using the EMclust function from the mclust package in R (Fraley & Raftery, 2009). The best-fit model defining the number of clusters was selected by maximizing the Bayesian Information Criterion (BIC) value (Fraley & Raftery, 1999). The cluster models from this analysis also generated means, variances and number of species for the definition of the lower and upper limits of foliar element concentrations for the allocation of species to each cluster. In the case of Al concentrations, we used these values to define clusters indicative of Al-accumulating and non-al-accumulating species, and identified the threshold value that distinguished between these groups. Phylogenetic tree of angiosperms For all phylogenetic analyses involving foliar nutrient element concentrations, we constructed hypothesized phylogenetic trees using the software Phylomatic (Webb & Donoghue, 2005). Estimates of branch lengths (in millions of yr) were assigned on the basis of the age of known divergence events using the bladj function in Phylocom version 4.1 (Webb et al., 2008). The branch lengths are estimated from the known node ages based on fossil records (Wikström et al., 2001). Phylogenetic signal of foliar Al and macro-nutrient element concentrations in angiosperms We tested for a phylogenetic signal in foliar Al and macro-nutrient element (N, P, K, Ca and Mg) concentrations using a randomization test for continuous variables (Blomberg et al., 2003), which compares the observed variances of phylogenetically independent contrasts (hereafter referred to as contrasts) in species traits with a null distribution of randomly generated variances of these contrasts. We assessed the significance of the K statistic by randomly shuffling the observed variances of contrasts among species 999 times, and the distribution of randomly generated variances of contrasts was compared with the observed variances of contrasts calculated from the variance covariance matrix using the actual phylogeny. A significant phylogenetic signal was inferred if at least 95% of randomly generated variances of contrasts were greater than the actual trait variance of contrasts (P 0.05). We report the K statistic, which is the ratio of the amount of phylogenetic signal observed to the amount expected under a Brownian motion null model of trait evolution (Blomberg et al., 2003). The Brownian motion model assumes that a continuous trait evolves randomly in any direction. A K statistic close to zero indicates a lack of a phylogenetic signal, whereas values close to or more than unity demonstrate a higher degree of phylogenetic signal than predicted by a Brownian motion model of trait evolution, that is, a tendency for close relatives to be similar. P and K statistic values were obtained from the Picante package (Kembel et al., 2009) in R version (R Development Core Team, 2009). Foliar Al concentrations and angiosperm distribution We used a phylogenetic comparative approach to determine whether foliar Al concentrations of angiosperms differed between tropical and temperate biomes, between tropical regions (South America vs South-East Asia and Brazil vs South-East Asia) and within South- East Asia (Brunei Darussalam vs Indonesia vs Thailand). A phylogenetic generalized least-squares (gls) method based on an Ornstein Uhlenbeck (OU) model of trait evolution was used to account for the phylogenetic structure of the dataset (Martins & Hansen, 1997; Paradis, 2006). The OU model assumes that the trait is constrained to evolve under stabilizing selection and this was implemented using the cormartins phylogenetic correlation structure available in the ape package (Paradis et al., 2004) of R version (R Development Core Team, 2009). A pair-wise multiple comparison of means was conducted using Tukey s honestly significant difference (Tukey s HSD) test. Results distributions of foliar Al and macro-nutrient element concentrations The frequency distribution of foliar Al concentrations in the global dataset of 1044 species deviated significantly from unimodality (Fig. 1a, Table 1). Similarly, the cluster model with the highest BIC value split the dataset into two clusters of species with contrasting Al concentrations (Table 2). By contrast, there was no evidence of a departure from unimodality in the frequency distributions of foliar concentrations of N, P, K, Ca or Mg in the global dataset (Fig. 2, Table 3), and the cluster model with the highest BIC value retained all species in the same group in all five cases (Table 3). When the global foliar Al concentration dataset was partitioned, evidence of deviation from unimodality (Table 1) was obtained for the frequency distributions of foliar Al concentrations for nonflowering plants (229 species, Fig. 1c), South- East Asian angiosperms (228 species, Fig. 1d) and all tropical plants (442 species, Fig. 1e), but not for angiosperms in general (815 species, Fig. 1b) or temperate plants (602 species, Fig. 1f). For all five datasets, the cluster model that partitioned species into two clusters had a higher BIC value than models with one or more than two clusters (Table 2). The subset of 58 tropical tree species from Brunei Darussalam also showed significant departure from unimodality for foliar concentrations of Al, but not N, P, K, Ca or Mg (Fig. 3, Table 4), and the cluster model with the highest BIC value defined two clusters of species in the case of foliar Al concentration, but only one cluster of species for the five nutrient elements (Tables 2, 4). The threshold foliar Al concentration for distinguishing between the two clusters varied from 0.91 to 3.86 mg g )1 for comparisons between biomes, localities and taxonomic groups (Table 2). For the global dataset of all plant species, and for all

5 Phytologist Research 641 (a) (b) (c) (d) Fig. 1 distributions of log 10 -transformed mean foliar aluminium (Al) concentrations of species: (a) all plant species (1044 species); (b) angiosperms (815 species); (c) nonflowering plants (229 species); (d) South- East Asian (SEA) angiosperms (228 species); (e) tropical plants (442 species); (f) temperate plants (602 species). The data were derived from the global dataset and subsets of that dataset. Before transformation, foliar Al concentrations were expressed in mg Al g )1 dry mass. (e) (f) Table 1 Dip test statistics (d) and probability values (P) testing for deviations from unimodality in the frequency distributions of foliar aluminium (Al) concentrations for plant species from the global dataset (1044 species) and for subsets of that dataset Dataset No. of species Dip test statistic (d) (a) All plant species (global dataset) (b) Angiosperms (c) Nonflowering plants (d) Angiosperms from South-East Asia (e) Tropical plants < (f) Temperate plants Foliar Al concentrations were log 10 transformed before analysis. P values in bold are significant (P 0.05). angiosperms and nonflowering plants considered separately, a foliar Al concentration of 1.0 mg g )1 was an appropriate P threshold for distinguishing between the two clusters of species. However, when only tropical plants were considered, the threshold foliar Al concentration for distinguishing between clusters of species with low and high foliar Al concentration ranged from 2.3 to 3.9 mg g )1. The lowest threshold foliar Al concentration for distinguishing between clusters was for nonflowering plants and plants from temperate ecosystems (values of c. 1.0 mg Al g )1 in both cases). In the global dataset and all subsets examined here, there was a higher proportion of species in the low foliar Al concentration cluster (range 52 85%) than in the high foliar Al concentration cluster (15 48%, Table 2). The mean foliar Al concentrations ranged from 0.13 to 0.41 and 3.05 to mg g )1 in the two clusters, respectively (Table 2). Mean (± SEM) foliar Al concentrations were lowest in both clusters for temperate plants (0.13 ± 1.04 vs 3.05 ± 1.11 mg g )1, respectively), whereas the highest values for the low foliar Al cluster occurred among trees

6 642 Research Phytologist Table 2 Mean (± SEM) and 95% confidence intervals (CI) of foliar aluminium (Al) concentrations (mg Al g )1 dry mass), and number and proportion (%) of species in clusters defined from analysis of the global dataset and subsets of that dataset Cluster 1 Cluster 2 Dataset Parameters Low foliar Al concentration High foliar Al concentration (a) All plant species Mean foliar Al ± SEM 0.16 ± ± 1.07 (global dataset) 95% CI 0.15, , 6.18 n = 1044 No. of species (proportion of species) 786 (75.3%) 258 (24.7%) Best-fit number of clusters (BIC) 2 () ) Range (lower upper) Estimate of threshold foliar Al concentration 1.28 mg Al g )1 (b) Angiosperms Mean foliar Al ± SEM 0.15 ± ± 1.09 n = % CI 0.14, , 8.00 No. of species (proportion of species) 667 (81.8%) 148 (18.2%) Best-fit number of clusters (BIC) 2 () ) Range (lower upper) Estimate of threshold foliar Al concentration 1.53 mg Al g )1 (c) Nonflowering plants Mean foliar Al ± SEM 0.20 ± ± 1.10 n = % CI 0.17, , 4.97 No. of species (proportion of species) 119 (52.0%) 110 (48.0%) Best-fit number of clusters (BIC) 2 () ) Range (lower upper) Estimate of threshold foliar Al concentration 0.91 mg Al g )1 (d) South-East Asian Mean foliar Al ± SEM 0.25 ± ± 1.11 angiosperms 95% CI 0.20, , n = 228 No. of species (proportion of species) 187 (82.0%) 41 (18.0%) Best-fit number of clusters (BIC) 2 () ) Range (lower upper) Estimate of threshold foliar Al concentration 3.86 mg Al g )1 (e) Tropical plants Mean foliar Al ± SEM 0.27 ± ± 1.06 n = % CI 0.23, , No. of species (proportion of species) 289 (65.4%) 153 (34.6%) Best-fit number of clusters (BIC) 2 () ) Range (lower upper) Estimate of threshold foliar Al concentration 2.68 mg Al g )1 (f) Temperate plants Mean foliar Al ± SEM 0.13 ± ± 1.11 n = % CI 0.12, , 3.72 No. of species (proportion of species) 513 (85.2%) 89 (14.8%) Best-fit number of clusters (BIC) 2 () ) Range (lower upper) Estimate of threshold foliar Al concentration 1.05 mg Al g )1 (g) Tropical trees from Brunei Mean foliar Al ± SEM 0.41 ± ± 1.21 Darussalam 95% CI 0.33, , n = 58 No. of species (proportion of species) 42 (72.4%) 16 (27.6%) Best-fit number of clusters (BIC) 2 () ) Range (lower upper) Estimate of threshold foliar Al concentration 2.32 mg Al g )1 The total number of species in each analysis is given by n. The number of clusters in the model with the highest Bayesian Information Criterion (BIC) was two in all cases. Foliar Al concentrations were log 10 transformed before analysis, but the values of the mean, SEM and CI were back-transformed for presentation. Ranges of foliar Al concentration values for each cluster are also presented. An estimate of the threshold foliar Al concentration for distinguishing between clusters was calculated as the mean of the upper range for cluster 1 (low foliar Al concentration) and the lower range for cluster 2 (high foliar Al concentration). from Brunei Darussalam (0.41 ± 1.12 mg g )1 ) and for the high foliar Al cluster among South-East Asian angiosperms (11.08 ± 1.11 mg g )1 ). The tropical tree species sampled in Brunei Darussalam, allocated to each cluster using these threshold values, are presented in Table S5. Phylogenetic signal of foliar Al and macro-nutrient concentrations in angiosperms A significant phylogenetic signal was detected in the foliar Al concentration of 58 tropical tree species from Brunei

7 Phytologist Research 643 (a) (b) Log 10 foliar N concentration (c) (d) Log 10 foliar P concentration Log 10 foliar K concentration Fig. 2 distributions of log 10 -transformed mean concentrations of species: (a) foliar aluminium (Al) (1044 species); (b) foliar nitrogen (N) (109 species); (c) foliar phosphorus (P) (121 species); (d) foliar potassium (K) (729 species); (e) foliar calcium (Ca) (742 species); (f) foliar magnesium (Mg) (714 species). The data were derived from the global dataset. Before transformation, foliar nutrient concentrations were expressed in mg g )1 dry mass. (e) Log 10 foliar Ca concentration (f) Log 10 foliar Mg concentration Table 3 Dip test statistics (d) and probability values (P) testing for deviation from unimodality in the frequency distributions of foliar nitrogen (N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) concentrations in the global dataset Foliar element No. of species tested Dip test statistic (d) P Best-fit number of clusters (BIC) Mean foliar nutrient concentrations ± SEM (mg g )1 ) N (93.09) ± 1.03 P () 77.09) 0.70 ± 1.07 K () ) ± 1.17 Ca () ) ± 1.37 Mg () 21.11) 3.23 ± 1.15 The mean (± SEM) foliar nutrient concentrations (mg g )1 dry mass) and the number of clusters in the model with the highest Bayesian Information Criterion (BIC) are also presented. Foliar nutrient concentrations were log 10 transformed before analysis, but the values of the mean and SEM were back-transformed for presentation. Darussalam, but not for foliar concentrations of N, P, K, Ca or Mg in the same sample of species. For foliar Al concentration in Brunei trees, the K statistic suggested that the signal was more than half of that expected under Brownian motion (K = 0.60, P < 0.05, Table 5), and this was the highest amongst other K values (Tables 5, 6). For angiosperms in the global dataset, there was also evidence of a phylogenetic signal for foliar Al concentrations (K = 0.22, P = 0.001, Table 5) and for foliar concentrations of N, P, K, Ca and Mg (Table 5). Similarly, a phylogenetic signal was observed in foliar Al concentrations for the subsets of angiosperms from the global dataset (tropical, temperate, South-East Asian and tropical South American angiosperms, Table 6). Foliar Al concentrations and angiosperm distribution Taking phylogeny into account, the mean foliar Al concentration of angiosperm species from the tropical vegetation zone

8 644 Research Phytologist (a) (b) Log 10 foliar N concentration (c) (d) Log 10 foliar P concentration Log 10 foliar K concentration (e) Log 10 foliar Ca concentration (f) Log 10 foliar Mg concentration Fig. 3 distributions of log 10 -transformed mean concentrations of species: (a) foliar aluminium (Al); (b) foliar nitrogen (N); (c) foliar phosphorus (P); (d) foliar potassium (K); (e) foliar calcium (Ca); (f) foliar magnesium (Mg). The data were derived from 58 tropical woody plants from Brunei Darussalam. Before transformation, foliar nutrient concentrations were expressed in mg g )1 dry mass. Our conclusions are unaffected by the inclusion of the outlier value of foliar P concentration in the analyses. (0.73 ± 1.16 mg g )1 ) was significantly higher than that of angiosperms from the temperate zone (0.16 ± 1.07 mg g )1 ) (F 1,813 = 95.52, P < 0.001, Fig. 4). When we analysed foliar Al concentrations between tropical regions separately, whilst accounting for phylogeny, the mean foliar Al concentration of angiosperm species from South-East Asia (Brunei Darussalam, Indonesia, Malaysia, Philippines and Thailand; 0.48 ± 1.14 mg g )1 ) was significantly lower than that from tropical South America (Brazil and Venezuela; 0.86 ± 1.23 mg g )1 ) (F 1,286 = 13.36, P < 0.001, Fig. 5a) or from Brazil alone (0.90 ± 1.34 mg g )1 ; F 1,262 = 10.12, P < 0.01, Fig. 5b). Within South-East Asia, the mean foliar Al concentration of angiosperms, after accounting for phylogeny, was significantly different between species from Brunei Darussalam, Indonesia and Thailand (F 1,223 = 28.03, P < 0.001, Fig. 6). The sample of species from Thailand (0.10 ± 1.18 mg g )1 ) had significantly lower mean foliar Al concentrations than species from Brunei (0.96 ± 1.23 mg g )1, t = ) 0.98, df = 102, P < 0.001, Fig. 6) or Indonesia (0.59 ± 1.20 mg g )1, t = ) 0.77, df = 167, P < 0.001), which were not significantly different (t = ) 0.21, df = 180, P = 0.18). Discussion Foliar Al and macro-nutrient concentrations This study presents an analysis of the variation in foliar Al and macro-nutrient concentrations in a global dataset of plant species within a phylogenetic framework. The frequency distribution of foliar Al concentrations in this dataset was clearly bimodal and, similarly, a cluster analysis supported the existence of two groups of species in the dataset. This conclusion supports the hypothesis that Al accumulators and non-al accumulators exist as distinct, but overlapping, groups of species. Bimodality in the frequency distributions of foliar Al concentrations has been reported previously (Masunaga et al., 1997; Watanabe et al., 2007), but these studies have not provided statistical support for their inference of

9 Phytologist Research 645 Table 4 Dip test statistics (d) and probability values (P) testing for deviation from unimodality in the frequency distributions of foliar aluminium (Al), nitrogen (N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) concentrations in 58 tropical tree species from Brunei Darussalam Foliar element Table 5 Tests for a phylogenetic signal in the foliar concentrations of aluminium (Al), nitrogen (N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) for 58 tropical tree species from Brunei Darussalam and angiosperms from the global dataset Dataset Tropical tree species in Brunei Angiosperms from the global dataset Dip test statistic (d) Nutrient element P n Best-fit number of clusters (BIC) K statistic Observed mean variance of contrasts Mean foliar nutrient concentrations ± SEM (mg g )1 ) Al () ) 0.41 ± 1.12, 8.71 ± 1.21 N (51.70) ± 1.04 P () 2.06) 0.46 ± 1.07 K (35.21) 9.62 ± 1.17 Ca () 50.74) 3.03 ± 1.11 Mg () 29.64) 3.12 ± 1.09 The mean (± SEM) foliar nutrient concentrations (mg g )1 dry mass) and the number of clusters in models with the highest Bayesian Information Criterion (BIC) from the cluster analysis are also presented. Foliar nutrient concentrations were log 10 transformed before analysis, but were backtransformed for presentation. P values in bold are significant (P 0.05). Random mean variance of contrasts Al N P K Ca Mg Al N P K Ca Mg Bloomberg s K statistic is the ratio of the observed mean variances of contrasts to the expected mean variances of contrasts; P is the proportion of randomized mean variance of contrast values that were smaller than the observed mean variance of contrasts; n is the number of plant species analysed in each test. P values in bold suggest significant (P 0.05) evidence of a phylogenetic signal. bimodality. The two statistical approaches adopted here provide independent sources of evidence that species with high foliar Al concentrations are not simply representative of the upper tail of a normal distribution of foliar Al concentration among species. This supports the suggestion that Al accumulation represents a distinct dichotomous trait possessed by some plants. By contrast, there was no evidence of bimodality in the frequency distributions of the foliar concentrations of N, P, K, Ca or Mg, which suggests that these traits vary continuously in plants. These findings are comparable with those of previous studies, which also P showed either normal or skewed frequency distributions of foliar concentrations for these macro-nutrient elements (Masunaga et al., 1997; Watanabe et al., 2007). Bimodality in the frequency distribution of foliar nutrient concentrations is suggestive of a trait being controlled by a simple genetic basis, whereas unimodality implies a multigenetic basis (Meharg, 2003). The estimated threshold value of foliar Al concentration that distinguishes Al accumulators from non-al accumulators varies geographically. For all plants in the global dataset, our estimated threshold value of 1.28 mg Al g )1 is essentially identical to the value of 1 mg Al g )1 proposed by Hutchinson (1943) and Chenery (1948a,b), although they did not state how they arrived at this value. In addition, our results suggest that the threshold value is lower for temperate (1.1 mg Al g )1 ) than tropical (range of mg Al g )1 ) plants. This geographical lability in the threshold value of foliar Al concentration for species to be considered as Al accumulators helps to explain the discrepancies among studies in proposed threshold values (e.g. Chenery, 1948a,b; Masunaga et al., 1998a). The foliar Al concentration threshold of tropical plants might be greater than that of temperate plants because of differences in the chemistry of tropical and temperate soils, and these habitat differences might be a selective factor for both high foliar Al concentrations (Jansen et al., 2002b) and the higher foliar Al concentration threshold. The threshold foliar Al concentration for distinguishing between Al accumulators and non-al accumulators did not vary between angiosperms and nonflowering plants (values of c. 1mgAlg )1 in both cases), despite the divergence of these major taxa c. 120 million yr ago (APG, 1998). However, a much higher proportion of nonflowering plants (45% of 229 species examined) than angiosperms (19% of 815 species) were classified as Al accumulators in our global dataset. Similarly, Webb (1954) has highlighted the prevalence of Al accumulators amongst nonflowering plants (11% of 101 nonflowering plant species vs 6% of 1223 angiosperm species). Previous studies have also reported Al accumulators among some ferns (e.g. in Cyatheaceae and Lycopodiaceae) and mosses (Chenery, 1949; Olivares et al., 2009). Meharg (2002) proposed that arsenic (As) hyper-accumulation amongst ferns and their allies could have evolved in As-rich habitats, or the trait could have been inherited from their ancestors. Likewise, the prevalence of Al accumulators within nonflowering plants suggests that Al accumulation evolved early in the evolution of land plants and might be a primitive character associated with survival in ancestral Al-rich environments (Hutchinson, 1943, 1945; Jansen et al., 2002a). The retention of the trait in some relatively advanced families of angiosperms (e.g. in Anisophylleaceae, Melastomataceae, Phyllanthaceae and Polygalaceae) implies that it confers a selective advantage to some species in these groups, although the basis of this selective advantage is currently unknown. Although some authors have shown that plants that hyper-accumulate metals, such as nickel (Ni) and selenium (Se), are able to resist invertebrate and vertebrate herbivores, and pathogens (Boyd & Martens, 1994; Boyd et al., 1994, 2002; Freeman et al., 2009), we are unaware of any equivalent evidence for Al accumulators. The frequency of Al accumulators in the studies reviewed above is much greater than the frequency of

10 646 Research Phytologist Table 6 Tests for a phylogenetic signal in the foliar concentrations of aluminium (Al) for subsets of angiosperms from the global dataset Dataset n Kstatistic Observed mean variance of contrasts Random mean variance of contrasts P Tropical angiosperms Temperate angiosperms South-East Asian angiosperms South American angiosperms Bloomberg s K statistic is the ratio of the observed mean variance of contrasts to the expected mean variance of contrasts; P is the proportion of randomized mean variance of contrast values that were smaller than the observed mean variance of contrasts; n is the number of plant species analysed in each test. South-East Asian angiosperms comprised data from Brunei Darussalam, Indonesia, Thailand, Malaysia and the Philippines, and South American angiosperms comprised data from Brazil and Venezuela. P values in bold suggest significant (P 0.05) evidence of a phylogenetic signal. (a) Log10 foliar Al concentration Temperate Tropical Fig. 4 Box and whisker plots displaying median (horizontal line), interquartile range (boxes), maximum and minimum values (whiskers) and outliers (dots) of log 10 foliar aluminium (Al) concentrations in angiosperms from tropical (349 species) and temperate (466 species) zones before phylogenetic correction. Foliar Al concentrations (mg Al g )1 dry mass) were log 10 transformed before analysis and back-transformed values are presented in the text. Red horizontal lines display the fitted mean values based on phylogenetic generalized least-squares models, as described in the text. accumulators of transition metals or As (Baker et al., 2000; Reeves & Baker, 2000; Meharg, 2002, 2003), which suggests that the Al accumulator trait conveys a selective advantage in a wider range of ecological contexts. Differences in foliar Al concentrations among angiosperms This study has shown for the first time that there is a significant phylogenetic signal in foliar Al concentrations in angiosperms based on a synthesis of molecular phylogenetic research (APG III, 2009). This conclusion confirms previous research highlighting the greater prevalence of Al accumulators in some families than others (Chenery, 1948a,b, 1949; Webb, 1954; Jansen et al., 2000a, 2002a,b, 2003, 2004), and the observation that phylogenetic variation in foliar Al concentrations occurs above the species level (Jansen et al., 2002a,b, 2004; Watanabe et al., 2007). Previous research has uncovered taxonomic variation in shoot element concentrations (N, P, carbon (C), Ca, K, Mg, Ni and zinc (Zn)) in plants, especially angiosperms (Broadley et al., 2001, 2003, 2004; Kerkhoff et al., 2006; Watanabe et al., 2007). Our analyses of the phylogenetic signal for a global dataset of species of angiosperms further confirms the importance of phylogeny in influencing the variation in foliar concentrations of N, P, K, Ca, Mg and Al. The absence of a phylogenetic signal in (b) SA Brazil SEA SEA Fig. 5 Box and whisker plots (defined in Fig. 4) of log 10 foliar aluminium (Al) concentrations in angiosperms from: (a) the tropical South American (SA) and South-East Asian (SEA) regions of the global dataset (288 species: SA, 60 species; SEA, 228 species) and (b) Brazil and SEA regions of the global dataset (264 species; Brazil, 36 species; SEA, 228 species). Foliar Al concentrations (mg Al g )1 dry mass) were log 10 transformed before analysis and back-transformed values are presented in the text. Red horizontal lines display the fitted mean values based on phylogenetic generalized least-squares models, as described in the text. foliar nutrient concentrations among the 58 species of tree sampled in Brunei Darussalam reflects the fact that these species represented a phylogenetically restricted sample (drawn from 31 genera in 18 families). The existence of a phylogenetic signal implies that more closely related organisms tend to resemble each other with respect to trait values. For plant nutrient concentrations, a phylogenetic signal may arise because trait variation is phylogenetically conserved, or because related species occupy relatively similar habitats that differentially influence nutrient uptake and accumulation (Thompson et al., 1997). For example, Schreeg et al. (2010)

11 Phytologist Research a a b conveys differential fitness in response to local environmental conditions (Webb, 1954; Masunaga et al., 1998b,c; Jansen et al., 2002b, 2003; Watanabe et al., 2007). The ecological significance of the Al accumulation trait has not yet been established, although one hypothesis is that it confers tolerance to high Al availability (Webb, 1954; Cuenca et al., 1990; Watanabe et al., 2005, 2006). Future research should address the fitness costs and benefits of Al accumulation and the mechanisms of the local coexistence of plants possessing this trait alongside those that do not. Brunei Indonesia Thailand Fig. 6 Box and whisker plots (defined in Fig. 4) of log 10 foliar aluminium (Al) concentrations in angiosperms within South-East Asia from the global dataset (226 species; Brunei, 58 species; Thailand, 45 species; Indonesia, 123 species). Foliar Al concentrations (mg Al g )1 dry mass) were log 10 transformed before analysis and back-transformed for presentation in the text. Red horizontal lines display the fitted mean values based on phylogenetic generalized least-squares models, as described in the text. Different letters represent significantly different means (P < 0.05). Species from Malaysia and the Philippines in the global dataset were not included in this analysis as each country was represented by only one species. have provided evidence for significant associations between high soil exchangeable Al and manganese (Mn) concentrations and the distributions of trees in the Vochysiaceae and Myrtaceae on a 50- ha plot in lowland moist tropical forest in Panama. Previous authors have commented that species with high Al concentrations (including Al accumulators) are more frequent in tropical than temperate plants (Hutchinson, 1943; Jansen et al., 2002a), but these conclusions have not been validated by correcting for the phylogenetic structure of the sample of species examined. Our analyses support the conclusion that mean foliar Al concentration is higher in tropical than temperate angiosperms even after correcting for phylogeny. Foliar Al concentrations may be higher in tropical than temperate angiosperms because of differences in soil chemistry, in particular soil ph and Al concentrations, between typical soils of these climate zones (Webb, 1954; Jansen et al., 2002a). The Al solubility in soil solution increases in response to declining soil ph, which leads to high Al concentrations in old and intensively leached tropical soils (Webb, 1954; von Uexküll & Mutert, 1995; Jansen et al., 2002a). By contrast, temperate soils are typically younger, more weakly weathered and less acidic than many tropical soils (Robertson & Grandy, 2006). As a result, c. 60% of the world s acid soils are located in the tropical and subtropical regions (von Uexküll & Mutert, 1995). An association between high foliar Al concentrations and low soil ph is supported by the low frequency of Al accumulators in tropical dry forests with neutral soils and low rainfall (Hutchinson, 1943; Webb, 1954). In addition to highlighting contrasts in mean foliar Al concentration between plants of different biomes, our synthesis emphasizes that Al accumulators coexist with non-al accumulators in all terrestrial communities in which large-scale surveys have been conducted (Haridasan, 1982; Cuenca & Herrera, 1987; Masunaga et al., 1998a; Osaki et al., 1998, 2003). The coexistence of species possessing divergent values of a trait suggests that this trait Acknowledgements We thank the University of Brunei Darussalam and the Department of Forestry, Brunei Darussalam, for providing access to work in the permanent tropical rain forest plots in the forest reserves of Brunei Darussalam. This research study was funded by the Brunei Government and the University of Aberdeen, and F.M. s student scholarship was fully funded by the Brunei Government. References Allen SE, Grimshaw HM, Parkinson JA, Quarmby C Chemical analysis of ecological materials. Oxford, UK: Blackwell Scientific Publications. APG (Angiosperm Phylogeny Group) An ordinal classification for the families of flowering plants. Annals of the Missouri Botanical Garden 85: APG III (Angiosperm Phylogeny Group) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. 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