Foliar elemental profiles in the ultramafic flora of Kinabalu Park (Sabah, Malaysia)

Antony van der Ent, David Robert Mulligan, Rimi Repin, Peter Damian Erskine

Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, Brisbane, Australia

Laboratoire Sols et Environnement, Universite´ de Lorraine-INRA, UMR-1120, Vandœuvre-Le` s-Nancy, France

Sabah Parks, Sabah, Malaysia

Abstract The foliar elemental profile of most plants reflects that of the soil on which plants grow in their natural habitat. The aim of this study was to elucidate foliar elemental profiles in the ultramafic flora of Kinabalu Park in Sabah, Malaysia. Kinabalu Park is ideal for studying plant elemental profiles because of its exceptionally rich flora and diversity in soil types. Foliar elemental profiles of 594 plant species in 99 families were analysed (totalling-1710 samples). This included 495 species (90 families) from ultramafic soils, and 120 species (45 families) from non-ultramafic soils (used as a comparison dataset). In general, the foliar elemental uptake ranges from exclusion of phytotoxic elements such as Ni and Mn, to a limited number of plant species characterised by extreme accumulation of specific elements, including trace elements (Co, Mn, Ni, Zn,), or non-essential elements (Al). This research identified hyperaccumulator behaviour (as defined by exceedances of established threshold foliar concentrations) in numerous plant species: Al >1000 µg g -1 (38 spp.), Mn >10 mg g -1 (7 spp.), Co >300 µg g -1 (3 spp.), Ni >1000 µg g -1 (24 spp.) and Zn 3000 µg g -1 (2 spp.). Distinct phylogenetic patterns emerged for Ni in which 18 of the identified 24 Ni hyperaccumulators were in the order Malpighiales, predominantly in the families Phyllanthaceae, Salicaceae and Violaceae.

Introduction Plants require at least-16 mineral elements for completion of their life cycle and these can be subdivided in macronutrients (Ca, K, Mg, P, S), micronutrients (Fe, B, Cl, Cu, Mn, Mo, Ni, Zn) and elements that are beneficial to some plants (Na, Co, Si) (Marschner 1995; Hopkins 1999), and the foliar elemental abundance generally decreases as a function of atomic mass (Markert 1992). The essential elements can further be subdivided in three groups based on their functions: (i) P, Cu, S and Fe which are associated with the ‘nucleic acid-protein set’, (ii) Mg, Ca, K, Zn and Mn which are associated with the ‘structural and photosynthetic set’, and (iii) Mn, K and Mg which are associated with the ‘enzymatic set’ (Garten 1978; Wright et al. 2005; Zhang et al. 2011). Plants also take up non-essential elements, such as Al, Sr, Ba and As (Willey 2016). The supply of the essential elements to a plant ranges from deficiency to optimum and eventual phytotoxicity (Chapin- 1980; Ernst 2006), and differs greatly between elements, being particularly narrow for micronutrients such as Zn and Cu (Clemens et al. 2002). The sequestration of essential elements is governed by highly specific mechanisms that enable uptake in the rhizosphere interface and transport and regulation inside the plant (Rengel and Marschner 2005). The necessity of different elements for ecophysiological functioning results in an overall proportionality (stoichiometry) between elements in foliar matter (Epstein 1972; Ågren 2008). Under similar environmental conditions and on the same soil, systematic differences in elemental accumulation between plant species results from differences in the uptake (and selectivity) as a function of species-specific physiology as well as associated ecological interactions including mycorrhizal symbioses (Marschner 1995; van der Heijden et al. 2015). Systematic differences in the foliar elemental profiles (e.g. the accumulation patterns of elements) are a reflection of evolutionary processes and hence plant phylogeny (Broadley et al. 2004; Watanabe et al. 2007), and thus also provide a potentially useful taxonomic tool (i.e. ‘chemotaxonomy’ viz. Kersten et al. 1979). Plant species that accumulate foliar elements to extreme levels (including Ni, Co and Mn) are known as hyper- accumulators (Jaffre et al. 1976; Reeves 2003; Reeves et al. 2017), a phenotypic character that has evolved independently in multiple orders and families (Pollard et al. 2000; Macnair 2003; Kramer 2010).

There have been efforts to define reference concentrations for elements in plants to serve as a ‘baseline’ (Markert 1996), but such generalizations disregard the importance of soil chemistry and genotypic and phenotypic variation of elemental uptake (Ernst 1998). Furthermore, the large variability in elemental concentrations within and between plants makes the establishment of a standard reference a difficult task, as a study comparing tropical rainforest plants illustrates (Breulmann et al.1998,1999). Most studies on foliar elemental concentrations have focused on ‘‘normal’’ or agricultural soils, but studying trace element-rich environments (such as ultramafic soils), however, has also provided a better understanding of foliar elemental concentrations in the presence of an excess of specific trace elements in the soil (van der Ent and Lambers 2016; Rajakaruna 2017). Of the naturally trace element-enriched environments, ultramafic soils are by far the most widespread and extensive, especially in Cuba, New Caledonia, the Philippines, Indonesia and Malaysia (van der Ent et al. 2015a; Galey et al. 2017). Ultramafic soils have high concentrations of Mg, Fe, Ni, Mn, Cr and Co, but are low in Ca, K and P (Proctor et al. 2000; Proctor 2003). Analyses of foliar plant samples from Mount Silam (ultramafic mountain on the east coast of Sabah, Malaysia) (Proctor et al. 1988), Mount Bloomfield (ultramafic mountain on the island of Palawan, Philippines) (Proctor et al. 2000) and Mount Giting-Giting (ultramafic mountain on Sibuyan Island, Philippines) (Proctor et al. 1998) showed that the elemental profile of plants on ultramafic soils is a reflection of the unusual soil chemistry, but important differences exist between the elements accumulated, but comprehensive phylogenetic information on such patterns is scant. Hyperaccumulation thresholds for trace elements have been defined at 300 µg g -1 for Cr, Co and Cu, 1000 µg g -1 for Ni, 3000 µg g -1 for Zn and 10 mg g -1 for Mn (Reeves 2003; van der Ent et al. 2013). The threshold for Al accumulators was defined at 1000 µg g -1 (Hutchinson 1945; Chenery-1948), but such

plants have also now been termed ‘Al hyperaccumulators’ (Jansen et al. 2002) to unify the terminology with trace element hyperaccumulators.

Kinabalu Park in the Malaysian state of Sabah has large surface exposures of ultramafic soils and is renowned for its biodiversity with at least 5000 species recorded in an area 1200 km2 (Beaman and Beaman 1990; Beaman 2005). In total 2854 plant species in 742 genera and-188 families were recorded from ultramafic soils in Kinabalu Park (van der Ent et al. 2014, 2015b, 2016). The extremely high levels of plant diversity combined with the high diversity in soil types (van der Ent et al. 2018) in a small surface area make this site an ideal location for studying patterns in the foliar elemental profiles of a wide range of plant species, which was the aim of this study.

Methods

Collection of plant material During 2010–2014 a large ecological research project was conducted in Kinabalu Park, and in the nearby Bidu-Bidu Hills and Trus Madi Forest Reserves, all in the Malaysian state of Sabah on the Island of Borneo. The research project focussed on the plant-soil relationships of the vegetation on ultramafic soils at these localities (van der Ent and Mulligan 2015; van der Ent et al. 2015a, b, c, 2016). The foliar elemental frequency distributions and links between foliar and soil chemistry were the subjects of a separate study that uses the same foliar dataset, in addition to soil samples from the plots (van der Ent and Erskine, unpublished). Non- permanent vegetation plots were established in Kinabalu Park (n = 88 plots and 1211 foliar samples) and plant foliar samples were collected from composite samples of fully expanded leaves from each of the 3–5 most dominant tree species (in terms of largest combined basal area), or the most dominant herb or fern (in terms of ground- cover) from the plots. Foliar samples from the ultra- mafic Bidu-Bidu Hills (n = 322 foliar samples) were also included in the analysis, and details on sample collection are provided in van der Ent and Reeves (2015). Similarly, foliar samples were collected from plots on non-ultramafic soils in the Trus Madi Forest Reserve (n = 13 plots and 99 foliar samples) and from around Park HQ in Kinabalu Park (78 foliar samples) to serve as a comparison dataset. This resulted in a total of-1710 foliar samples (n = 1533 foliar samples from ultramafic soils and n = 177 foliar samples from non- ultramafic soils). The foliar samples were washed with water after collection to remove potential soil contamination and dried at 70 °C for 5 days in a drying oven.

Identification of plant species Plant specimens from the plots were identified at the Sabah Parks Herbarium (SNP) and the Herbarium of the Sabah Forestry Department (SAN) and, as required, taxonomic specialists from the Kew (K), Leiden (L) and Singapore (SING) herbaria were consulted. As it was not possible to identify some plants to species level without floral parts present, collection numbers and vegetative morphology (i.e. ‘morpho-species’) were used as the identifier in some cases.

Laboratory analyses: foliar concentrations All foliar samples were crushed and ground, and a 300 mg representative subsample was digested using 5 ml concentrated HNO3 (70%) and 1 mL H2O2 (30%) in a microwave oven (Milestone, Italy). The digests were diluted to 30 ml with demineralised water before analysis with Inductively coupled plasma atomic emission spectroscopy (ICP-AES), which included measurements of the following elements: Ni, Co, Cr, Cu, Zn, Mn, Fe, Mg, Ca, Na, K, S and P. The microwave acid digestion and ICP-AES analysis included standard reference material (National Institute of Standards and Technology, NIST 1515, USA) to determine the accuracy and repeatability of the method for determining elemental concentrations in plant leaves (see ESM-1). The

potential for contamination with soil dust adhered to plant leaves is a problem for accurate analyses of the foliar elemental composition, even with sample washing. This risk is highest for ground-herbs, and lesser so for trees, but cannot be entirely avoided. Concomitantly high foliar concentrations of Cr (> 60 µg g-1) and Fe (>2500 µg g-1) are a useful indication for soil contamination as these elements are major constituents of ultramafic soils (Cary and Kubota 1990). By means of comparison, the mean values of a large dataset (608 samples) from a (non-ultramafic) rainforest in Sumatra, Indonesia from Masunaga et al. (1998) are used in this study as a reference: Al (2.04 mg g-1), Ca (16.9 mg g-1), Fe (0.16 mg g-1), K (9.44 mg g-1), Mg (2.62 mg g-1), Mn (0.5 mg g-1), Na (0.15 mg g-1), P (1.0 mg g-1) and S (2.78 mg g-1). To provide a comparison of leaf elemental content from ultramafic soils, the mean values from Proctor et al. (1988), plots at 610 m asl (52 species) on Mount Silam (Sabah, Malaysia) are also used: Ca (12.3 mg g-1), K (5.4 mg g-1), Na (2.5 mg g-1), Fe (0.04 mg g-1), Mg (3.7 mg g-1), Mn (0.16 mg g-1), and P (0.47 mg g-1).

Statistical analyses The foliar chemistry data was analysed using the software packages STATISTICA Version 9.0 (StatSoft, USA) and Excel for Mac version 2011 (Microsoft, USA).

Macronutrients and major ions (Al, Ca, Mg, P, K, Na, S)

Results and discussion In total, the elemental data of 594 plant species in 99 families were analysed (total number of foliar samples = 1710). These included 495 species (90 families) from ultramafic soils and 120 species (45 families) from non-ultramafic soils (comparison dataset). This large dataset allowed for detailed analysis of elemental profiles and phylogenetic patterns of elemental accumulation. Overall, plants from ultramafic soils had higher concentrations of Fe, Mg, Co, Cr, and Ni, and lower concentrations of Ca, K and P. Hyperaccumulators of any element were absent from the non-ultramafic soils, but for Ni numbered 24 species in nine families from the ultramafic soils. Furthermore, 38 Al accumulators, two Zn hyperaccumulators, seven Mn hyperaccumulators, and three Co hyperaccumulators were found. Unusually high foliar accumulations, with the highest values for each species per element, are shown in Table 1. Edaphically limited (ultramafic) foliar elemental profiles. In the following sections, we discuss the foliar concentrations of each element in the dataset and single out cases of unusual accumulation and hyperaccumulation behaviour. We start with the macronutrients and major ions (Al, Ca, Mg, P, K, Na, S) followed by the trace elements (Cu, Co, Cr, Fe, Mn, Ni, Zn).

Macronutrients and major ions (Al, Ca, Mg, P, K, Na, S)

Aluminium Al accumulation in foliage (>1000 µg g -1 or > 1 mg g-1) occurred in 38 species (17 families). The highest Al accumulation was found in the members of the family Symplocaceae: Symplocos buxifolia with 50 mg g-1, S. deflexa with 38.4 mg g-1, S. gambliana with 37.5 mg g-1, S. adenophylla with 29.4 mg g-1. The first known Al hyperaccumulator was also a species in the genus Symplocos, now known as Symplocos cochinchinensis var. philippinensis, and was called ‘Arbor aluminosa’ or ‘Aluyn-Boom’ (Jansen et al. 2002). Species in other families with high Al concentrations include Urophyllum longideus (Rubiaceae) with 36 mg g -1 and Aporosa chalarocarpa (Phyllanthaceae) with 25.9 mg g-1. In acid soils, Al toxicity is one of the most growth-limiting 3+ 3+ factors for plants, and at pH 5 Al is mostly present in the form of Al(H2O)6 or Al which are the most phytotoxic forms (Miyasaka et al. 2007). At pH 5.0–6.2, Al is mainly present in the form of Al(OH)2+,

Al(OH)2 + and Al(OH)4 which are non-toxic (Kidd and Proctor 2000). Al toxicity inhibits the uptake of

nutrients and affects the functioning of the plasma membrane (Huang et al. 1992; Ishikawa and Wagatsuma- 1998), which results in inhibition of root growth and elongation (Pilon-Smits et al. 2009). The high total soil Al concentrations (mean pseudo-total Al 19 mg g-1) combined with acidic conditions (pH 5.1–5.5) of most montane ultramafic soils (see Dystric Folic Cambisols in van der Ent et al. 2018) means there is high potential for Al uptake and toxicity. The relatively high occurrence of Al accumulators on leached tropical soils is consistent with the lower reported frequency of Al accumulators in drier environments or less acidic soils (Hutchinson 1943). In some adapted plant species, excess soil Al enhances growth (Jansen et al. 2002) and one such example recorded in this study was Melastoma malabathricum (Melastomataceae) with 3670 µg g-1 foliar Al. This species has increased root and shoot growth under high Al conditions, a strategy that has been attributed to amelioration of Fe phytotoxicity (Watanabe et al. 2005; 2006).

Calcium Foliar concentrations of Ca were generally low in the ultramafic plants (mean 6364 ± 163 µg g1), but 58 species (34 families) had >10 mg g-1 and 16 species (12 families) had > 20 mg g-1. Of these two groups, seven samples were of Podocarpus brevifolius (Podocarpaceae), and three other samples were Ni hyperaccumulators (these were in the Phyllanthaceae). The highest Ca concentrations were in Symplocos buxifolia (Symplocaceae) with 53.9 mg g-1, Schefflera foetida (Araliaceae) with 51.8 mg g -1 and Semnostachya galeopsis (Acanthaceae) with 49.5 mg g-1. Species in the Symplocaceae, Podocarpaceae and Phyllanthaceae generally had high Ca concentrations. The mean Mg/Ca quotient across the foliar samples analysed was 0.97 (range 0.02–31.9).

Magnesium Foliar concentrations of Mg were much lower (mean 3025 ± 68 µg g-1) than those of Ca, with 25 species (12 families) containing >10 mg g-1. Among these were three Ni hyperaccumulators: Ptyssiglottis cf. fusca (Acanthaceae) with 35.4 mg g-1, Baccaurea lanceolata (Phyllanthaceae) with 26.7 mg g -1 and Kibara coriacea (Monimiaceae) with 22.8 mg g-1. High levels of Mg accumulation are pronounced in the families Acanthaceae, Euphorbiaceae, Phyllanthaceae and Rubiaceae.

Phosphorus P concentrations in most plants was relatively low (mean of 413 ± 8.6 µg g-1), but there were 10 specimens of Pityrogramma calomelanos (Hemionitidaceae) that had unusually high foliar P concentrations. This species is a known As hyperaccumulator when it occurs on As-rich mining soils (Francesconi et al. 2002), possibly the result of its high P uptake (and physiological inability of differentiating between As and P). Most Glochidion spp. (Phyllanthaceae) also had very high foliar P concentrations (>-1000 µg g-1).

Potassium The majority of plants had extremely low foliar K concentrations (mean 3854 ± 83 µg g-1), but 26 species in- 18 families) had >10,000 µg g -1 (>10 mg g-1). The Ni hyperaccumulator Actephila sp. nov. (now named A. alanbakeri—Phyllanthaceae) had consistently extremely high foliar K concentrations (>12 mg g-1). The highest foliar K concentrations were in Drypetes crassipes (Putranjivaceae) with 32.7 mg g-1, an unidentified Glochidion sp. (Phyllanthaceae) with 29 mg g-1, Rinorea bengalensis (Violaceae) with 28 mg g -1 and Casearia velutinosa (Salicaceae) with 22.9 mg g-1. These species are all in the order Malpighiales. Substantial K accumulation appears to be frequent among Ni hyperaccumulators. Such extreme levels of foliar K accumulation are remarkable given the very low soil K concentrations. Potassium fulfils an important role in plant water conservation via regulation of the stomata in the leaves; hence, increased foliar K concentrations could be viewed as an indicator of increased drought tolerance (Shabala 2003; Wang et al. 2013).

Sodium Seven species (five families) have > 5000 µg g -1 foliar Na, of which Croton oblongifolius (Euphorbiaceae) and an unidentified Syzygium sp. (Myrtaceae) had 14.1 and 13 mg g-1, respectively. Given the low soil Na concentrations, a consequence of the highly leached tropical soils, such levels of foliar accumulation were unexpected.

Sulphur Foliar S concentrations were particularly high in plants of the ‘primitive’ families Equisetaceae and Polypodiaceae (11.7–13.2 mg g-1), but also in Dichapetalum gelonioides (Dichapetalaceae). The specimens of the latter species with high foliar S did not hyperaccumulate Ni or Zn, whereas the specimens that did, had relatively low foliar S. This suggests that S is not involved with complexation of either Ni or Zn in this species.

Trace elements (Cu, Co, Cr, Fe, Mn, Ni, Zn)

Copper Foliar Cu is controlled over a very narrow range, with a mean of 6.6 ± 0.4 µg g-1. Only 10 species (nine families) had > 50 µg g -1 Cu and these were also Ni hyperaccumulators. The highest foliar Cu concentrations were found in Mischocarpus sundaicus (Sapindaceae) with 369 µg g-1, in Agrostemma cf. hameliifolium (Rubiaceae) with 228 µg g-1, and in Gluta wallichii (Anacardiaceae) with 171 µg g-1. Only Mischocarpus sundaicus might be listed as a hyperaccumulator of this metal, but given that only one sample exceeded the nominal threshold of > 300 µg g -1 (out of eight samples analysed for this species, and the mean of 25 µg g -1 Cu excluding the single high value), it is highly doubtful whether this species is a true Cu hyperaccumulator.

Cobalt Co is not generally considered an essential trace element for plants, but it has been shown to be beneficial to the growth of legumes, probably because Co is essential for symbiotic rhizobia that live in nitrogen-fixing nodules associated with the roots of legumes (Pilon-Smits et al.2009). All plants with Co > 50 µg g -1 were also Ni hyperaccumulators, or were facultative Ni hyperaccumulators in which populations of the same species hyperaccumulate Ni, but Ni non-hyperaccumulating populations also had unusually high Co. In total, there were 27 species (eight families) with Co > 50 µg g -1 and 16 species (four families) with Co >-100 µg g-1, predominantly in the families Phyllanthaceae and Euphorbiaceae. In the Ni hyperaccumulators, the Ni/Co quotient ranged from-1.5 to-18,590 (mean was 475). The highest Ni/Co quotients are in strong Ni hyperaccumulators, for example Psychotria sarmentosa (n = 8), which had mean Ni and Co concentrations of- 12,825 and 3.6 µg g-1, respectively. The specificity of Ni uptake over Co is thus remarkable. The highest concentrations were found in the Co hyperaccumulator Glochidion cf. sericeum (Phyllanthaceae) with 909– 1310 µg g-1. This species is unusual in having a very low Ni/Co ratio (ranging from 1.7 to 2) and would be considered a moderate Ni hyperaccumulator (accumulating up to 2192 µg g -1 Ni in the foliage). Lower Co concentrations were found in Mischocarpus sundaicus (Sapindaceae) with-1139 µg g-1, Aporosa chalarocarpa (Euphorbiaceae) with 468 µg g-1, and Glochidion arborescens (Phyllanthaceae) with 315 µg g-1. All of these records are above the defined hyperaccumulation threshold of 300 µg g -1 Co (van der Ent et al. 2013).

Chromium Cr is a non-essential element for plants, and Cr phytoavailability in soils is generally extremely low, even in] ultramafic soils which can contain up to 2% Cr. As a consequence, foliar Cr concentrations are generally low (mean 13 ± 0.8 µg g-1). Unusually high Cr concentration, combined with high Fe, is often an indication of contamination of leaf samples by soil particulates. If such suspect samples are excluded from our dataset,

there are still four species (from three families) with Cr >300 µg g-1 i.e. exceeding the nominal hyperaccumulation threshold for this element (van der Ent et al. 2013). Almost all of these species were Ni hyperaccumulators mostly from the Phyllanthaceae, Monimiaceae and Violaceae. Examples of plants with high foliar Cr include Anisophyllea disticha (Anisophylleaceae) with 482 µg g -1 and Psychotria sarmentosa (Rubiaceae) with 426 µg g-1. The fact that these records occur as erratic outliers with only single specimens within a species having high Cr values, strongly suggest that this is not true hyperaccumulator behaviour but results from contamination (see also comments about Cu above).

Iron The mean foliar Fe concentration was 96 ± 8.4 µg g-1, but 17 species (13 families) had >-1000 µg g-1, of which the highest were Agrostemma cf. hameliifolium (Rubiaceae) with 6534 µg g-1, Taenitis blechnoides (Adiantaceae) with 4249 µg g-1, Phyllanthus cf. reticulatus (Phyllanthaceae) with 3292 µg g -1 and Thottea triserialis (Aristolochiacaea) with 2694 µg g-1.

Manganese Foliar Mn concentrations vary widely between plants (range 0.01–23,100 µg g -1 with a mean of 588 ± 35 µg g-1) with 51 species (24 families) with > 2000 µg g -1 foliar Mn and-19 species (12 families) with >5000 µg g -1 foliar Mn. The highest values were recorded for Hedyotis sp. (Rubiaceae) with 23.1 mg g-1, Antidesma cf. coriaceum (Phyllanthaceae) with 16.9 mg g-1, and Ilex oppositifolia (Aquifoliaceae) with 13.4 mg g-1. Foliar Mn accumulation was particularly notable in the Phyllanthaceae with six species of Glochidion having >2000 µg g-1. In total, seven species (five families) were hyperaccumulators of Mn (>10 mg g-1). Relatively few (about species 20) Mn hyperaccumulators are known globally, mainly from Australia and New Caledonia. In Australia Mn hyperaccumulators are from the Myrtaceae with various species in the genus Gossia and in the Proteaceae in the genus Macadamia (Bidwell et al. 2002; Fernando et al. 2006, 2009). In New Caledonia Mn hyperaccumulators are known from the Proteaceae genus Virotia, and other species such as Garcinia amplexicaulis (Clusiaceae), Alyxia poyaensis (Apocynaceae) and Denhamia fournieri (Celastraceae) (Jaffre 1977; 1979; Brooks et al. 1981; van der Ent et al. 2015c). Manganese is an essential plant element and abundant in ultramafic soils, however, at low soil pH, Mn can be phytotoxic. Proctor (2003) noted that 21% of the species tested in New Caledonia had foliar Mn concentrations >1000 µg g-1, suggesting that Mn accumulation is a relatively common phenomenon on ultramafic soils.

Nickel Ni was the most recently discovered trace element to be essential to (most) plants (Brown et al. 1987) and, along with Mo, critical concentrations for Ni are the lowest (0.1 µg g-1) of all micronutrients (Epstein 1972). Nickel is effectively excluded from uptake in most plants to avoid potential toxic effects (Seregin and Kozhevnikova 2006) With Ni hyperaccumulators excluded (delimited at >-1000 µg g-1), the mean foliar Ni concentration across the samples collected was 55 µg g-1. In total, 24 species (12 families) were Ni hyperaccumulators (>1000 µg g-1), predominantly in the Phyllanthaceae, Rubiaceae and Violaceae. The highest concentrations were in Psychotria sarmentosa (Rubiaceae) with 24.2 mg g-1, Phyllanthus cf. securinegoides (Phyllanthaceae) with 23.3 mg g-1, Glochidion cf. ‘bambangan’ (Phyllanthaceae) with-16.7 mg g-1, Rinorea bengalensis (Violaceae) with 12.8 mg g -1 and Actephila sp. nov. (A. alanbakeri) with-11.5 mg g-1. In total, six species accumulate more than-10 mg g -1 (1%) and such plants have been termed ‘hypernickelophores’ by Jaffre and Schmid (1974). Although hyperaccumulators of trace elements are generally extremely specific in the uptake and accumulation of one metal, and rarely two, it appears from our dataset that co-accumulation of a range of metals does occur sometimes; for example, Aporosa chalarocarpa (Phyllanthaceae) with Al 25.9 mg g-1, Mn 1443 µg g-1, and Ni 1558 µg g-1, Baccaurea lanceolata

(Phyllanthaceae) with Al 18.4 mg g-1, Ca 21.9 mg g-1, Mg 26.7 mg g-1, Ni 1450 µg g -1 and Zn 202 µg g-1, and Urophyllum cf. marcophyllum (Rubiaceae) with Al 27.1 mg g-1, Mg 3259 µg g-1, and Mn 10.5 mg g-1. It is unlikely these cases result from contamination of the samples with soil particulates, as Fe and Cr concentrations are low. However, from a physiological point of view it is hard to explain how and why such co-uptake could occur, particularly because these ions are of different charges and ionic radii.

Zinc Zn is an essential micronutrient with critical concentrations of approximately 20 µg g -1 (Epstein 1972). Two species, Dichapetalum gelonioides subsp. pilosum (Dichapetalaceae) and an unidentified Glochidion sp. (Phyllanthaceae), with Zn concentrations of 4922 and 4275 µg g-1, respectively, exceed the hyperaccumulation threshold of > 3000 µg g -1 (van der Ent et al. 2013). The case of Dichapetalum gelonioides is particularly interesting because a subspecies (D. gelonioides subsp. tuberculatum) this taxon is also a strong Ni hyperaccumulator on ultramafic soils. Baker et al. (1992) reported Zn hyperaccumulation up to-15.6 mg g -1 from an herbarium specimen collected from near Lahad Datu in Sabah (from non-ultramafic soils) and 34.4 mg g -1 from a specimen in the Philippines. Phyllanthus amarus, a common weed growing mainly on non- ultramafic soils, had a relatively high Zn concentration (1475 µg g-1).

Elemental accumulation patterns across families and genera Overall, the foliar concentrations of Ca and K are lower in plants growing on ultramafic soils than in plants growing on non-ultramafic soils. In contrast, Mg, Ni, Cr and Co are mostly higher in plants growing on ultra- mafic soils (compare Figs.1 with 2 and 3 with 4). Comparison of elemental profiles of 90 families from ultramafic soils reveals differences between elemental accumulation behaviour. For example, the Acanthaceae, Moraceae, Pittosporaceae and Rosaceae accumulate relatively high levels of Ca, K and Mg, whereas Dracenaceae, Polygalaceae and Putranjivaceae are lower in these elements (Fig. 1). The Zn concentration is constrained over a narrow range (mean 70 ± 6.0 µg g-1), but Mn over a wider range (mean 588 ± 35 µg g-1). Families with consistently high Zn include the Dichapetalaceae, Meliaceae, Symplocaceae and Tiliaceae. In plants from non-ultramafic soils, these overall patterns are also apparent, but absolute concentrations of all these elements are lower (Figs. 2, 4). Lower overall foliar elemental concentrations are also apparent when comparing Ca, Mg and K concentrations in seven common families (Theaceae, Podocarpaceae, Myrtaceae, Lauraceae, Fagaceae, Fabaceae and Ericaceae, none contains any hyperaccumulators) from ultramafic and non-ultramafic soils (Fig. 5a, b). On the non-ultramafic soils the foliar ranges for these elements are wider ranging than on ultramafic soils, but the general proportionality between elements within families is similar between ultramafic and non-ultramafic soils.

There are differences between families, for example, the family Myrtaceae (genera: Leptospermum, Xanthomyrthus, Syzygium) and Theaceae (genera: Schima, Adinandra) have high concentrations of Al and Mg and low concentrations of Ca and K. This relates to the high- altitude acid soils on which these families predominantly occur. Further, higher order phylogenetic patterns occur in the foliar accumulation of P, S and K. The association of Al accumulation in the families Symplocaceae, Polygalaceae, Rubiaceae, Melastomataceae and Anisophylleaceae is well-known in the literature (Chenery and Sporne 1976; Jansen et al. 2002) and in this study these families were unequivocally the most important Al accumulators (Table 2). In addition, the family Phyllanthaceae (genus: Aporosa) also has high Al accumulation, and this has also been reported elsewhere (Masunaga et al. 1998; Jansen et al. 2002). Al accumulation is a widespread phenomenon, known from at least 60 angiosperm families (Jansen et al. 2002), suggesting that Al accumulation has evolved independently numerous times (Metali et al. 2011). In contrast, phylogenetic patterns for Ni accumulation are more distinct, and dominate in the Order Malpighiales (Pillon et al. 2010; Jaffre et al. 2013). The Malpighiales

is one of the largest orders of flowering plants, containing approximately 16,000 species in 42 families globally, accounting for approximately 7.8% of Eudicots (Wurdack and Davis 2009; Stevens 2013). Of those, 21 families and 292 species have been recorded in Kinabalu Park of which 115 species occur predominantly or exclusively on ultramafic soils (Beaman 2013). The families Phyllanthaceae (genera: Phyllanthus, Glochidion) and Violaceae (genus: Rinorea) have the most Ni hyperaccumulator species. A study on rainforest plants (on non-ultramafic soils) in Sarawak found that the Euphorbiaceae accumulated more Co compared to the Dipterocarpaceae (Breulmann et al. 1999). This re-iterates the propensity for this family to accumulate transition elements, not just Ni. This is also evident from the frequency of species in the Phyllanthaceae represented as having the highest accumulation values for Cu, Co, Cr, Fe, Mn and Zn (Table 2). Above- average Co and Cr accumulation

Conclusion The approach in this study limits biogeographical effects, although site-specific (e.g. habitat) variation is still important. Specifically, plant assemblages from high- altitude scrub and lowland tall rainforest have vastly different elemental profiles. This exemplifies that the foliar elemental profiles are a result of both present-day ecophysiology and the association of a plant species to particular edaphic niches as a result of its evolution (Watanabe et al. 2007). As such, phylogenetic patterns of elemental accumulation represent the sum of past selection pressures, and may be independent of present day soil habitat and soil requirements. For example, in the case of Al accumulators, higher-level phylogenetic patterns have been shown to be independent of present day soil preferences (Jansen et al. 2002). However, as opposed to Al accumulators, the restriction of Ni and Co hyperaccumulators to ultramafic soils is sine qua non to the presence of these elements, as the soil concentrations in non-ultramafic soils are too low to enable any substantial accumulation. In Sabah, a correlation was found between the occurrence of Ni hyperaccumulator plant species and soil Ni concentrations (van der Ent et al. 2016). Some Ni hyperaccumulators such as sub-species of Dichapetalum gelonioides occur also on non- ultramafic soils (as such they are facultative hyperaccumulators) but hyperaccumulate Zn, which is a more abundant ion in non-ultramafic soils than Ni. This raises important questions about the evolution of Ni and Zn hyperaccumulation in this species. Such ‘inter-element’ hyperaccumulator behaviour is not known from any other plant species in the region, although Phyllanthus spp. occurring on non-ultramafic soils also have abnormally high Zn (and several Phyllanthus spp. on ultramafic soils hyperaccumulate Ni). Overall, however, the great majority of hyperaccumulator species are ‘obligate’ and are physiologically linked to ultramafic soils as a result of their evolution. On a global scale, hyperaccumulation appears to have evolved most frequently in the Brassicaceae, Euphorbiaceae (including Phyllanthaceae) and Asteraceae families (Cappa and Pilon-Smits 2013), whereas in New Caledonia 83% of the Ni hyperaccumulator species are in the COM clade (Celastrales, Oxalidales, and Malpighiales) (Pillon et al. 2010; Jaffre et al. 2013). This study shows that this holds true for Sabah where the majority of Ni, Co and Zn hyperaccumulators are from the Malpighiales.

Acknowledgements We wish to thank Sabah Parks for their support and the SaBC for granting permission for conducting research in Sabah, and extend our gratitude to Sukaibin Sumail and Rositti Karim (Sabah Parks) for assistance in Kinabalu Park. The French National Research Agency through the national ‘‘Investissements d’avenir’’ program (ANR-10-LABX-21, LABEX RESSOURCES21) and through the ANR-14-CE04-0005 Project ‘‘Agromine’’ is acknowledged for funding support. A. van der Ent is the recipient of a Discovery Early Career Researcher Award (DE160100429) from the Australian Research Council.

References Ågren GI (2008) Stoichiometry and nutrition of plant growth in natural communities. Annu Rev Ecol Evol Syst 39:153–170

Baker AJM, Proctor J, van Balgooy MMJ, Reeves RD (1992) Hyperaccumulation of nickel by the flora of the ultramafics of Palawan, Republic of the Philippines. In: Baker AJM, Proctor J, Reeves RD (eds) The vegetation of ultramafic (serpentine) soils. Intercept, Andover, pp 291–304

Beaman JH (2005) Mount Kinabalu: hotspot of plant diversity in Borneo. Biol Skr 55:103–127

Beaman RS (2013) Database of Mount Kinabalu collections made available to Antony van der Ent in digital format. The database entries are also available online in a website from: http://141.211.83.135:591/kinabalu/index.html

Beaman JH, Beaman RS (1990) Diversity and distribution patterns in the flora of Mount Kinabalu. In: Baas P, Kalkman K, Geesink R (eds) The plant diversity of Malesia. Kluwer Acacemic Publishers, pp-147–160

Bidwell SD, Woodrow IE, Batianoff GN, Sommer-Knusden J (2002) Hyperaccumulation of manganese in the rainforest tree Austromyrtus bidwillii (Myrtaceae) from Queensland, Australia. Funct Plant Biol 29:899–905

Breulmann G, Ogino K, Ninomiya I, Ashton PS, La Frankie IV, Leffler US, Weckert V, Lieth H, Konschak R, Markert B (1998) Chemical characterisation of Dipterocarpaceae by use of chemical fingerprinting—a multielement approach at Sarawak, Malaysia. Sci Total Environ 215:85–100

Breulmann G, Ogino K, Markert B, Leffler US, Herpin U, Weckert V, Konschak R, Kikugawa Y, Ohkubo T (1999) Comparison of chemical elements in Dipterocarpaceae and Euphorbiaceae from a tropical rain forest in Sarawak, Malaysia. Sci Total Environ 225:231–240

Broadley MR, Bowen HC, Cotterill HL, Hammond JP, Meacham MC, Mead A, White PJ (2004) Phylogenetic variation in the shoot mineral concentration of angiosperms. J Exp Bot 55:321– 336

Brooks RR, Trow J, Veillon J, Jaffre T (1981) Studies on manganese-accumulating Alyxia species from New Caledonia. Taxon 30:420–423

Brown P, Welch R, Cary E (1987) Nickel: a micronutrient essential for higher plants. Plant Physiol 85:801– 893

Cappa JJ, Pilon-Smits EAH (2013) Evolutionary aspects of elemental hyperaccumulation. Planta 239:267– 275

Cary EE, Kubota J (1990) Chromium concentration plants: effects of soil chromium concentration and tissue contamination by soil. J Agric Food Chem 38:108–114

Chapin FS (1980) The mineral nutrition of wild plants. Annu Rev Ecol Evol Syst 11:233–260

Chenery EM (1948) Aluminium in plants and its relation to plant pigments. Ann Bot 12:121–136

Chenery EM, Sporne KR (1976) A note on the evolutionary status of aluminium accumulators among dicotyledons. New Phytol 76:551–554

Clemens S, Palmgren MG, Kra¨ mer U (2002) A long way ahead: understanding and engineering plant metal accumulation. Trends Plant Sci 7:309–315

Epstein E (1972) Mineral nutrition of plants: principles and perspectives. Wiley, New York

Ernst WHO (1998) Instrumental element and multi-element analysis of plant samples. Methods and applications—review. Plant Ecol-136:250–251

Ernst WHO (2006) Evolution of metal tolerance in higher plants. For Snow Landsc Res 80:251–274

Fernando DR, Bakkaus EJ, Perrier N, Baker AJM, Woodrow IE, Batianoff GN, Collins RN (2006) Manganese accumulation in the leaf mesophyll of four tree species: a PIXE/EDAX localization study. New Phytol 171:751–758

Fernando DR, Guymer G, Reeves RD, Woodrow IE, Baker AJM, Batianoff GN (2009) Foliar Mn accumulation in eastern Australian herbarium specimens: prospecting for ‘new’ Mn hyper- accumulators and potential applications in taxonomy. Ann Bot 103:931–939

Francesconi K, Visoottiviseth P, Sridokchan W, Goessler W (2002) Arsenic species in an arsenic hyperaccumulating fern, Pityrogramma calomelanos: a potential phytoremediator of arsenic- contaminated soils. Sci Total Environ 284:27–35

Galey MC, van der Ent A, Iqbal MCM, Rajakaruna N (2017) Ultramafic geoecology of South and Southeast Asia. Bot Stud 58:18. https://doi.org/10.1186/s40529-017-0167-9

Garten CT Jr (1978) Multivariate perspectives on the ecology of plant mineral element composition. Am Nat- 112:533–544

Hopkins WG (1999) Introduction to plant physiology, 2nd edn. Wiley, New York, pp 61–76

Huang JW, Shaff JE, Grunes DL, Kochian LV (1992) Aluminum effects on calcium fluxes at the root apex of aluminium-tolerant and aluminium-sensitive wheat cultivars. Plant Physiol 98:230– 237

Hutchinson GE (1943) The biogeochemistry of Al and of certain related elements. Q Rev Biol-18:1–29

Hutchinson GE (1945) Aluminum in soils, plants and animals. Soil Sci 60:29–40

Ishikawa S, Wagatsuma T (1998) Plasma membrane permeability of root-tip cells following temporary exposure to Al ions is a rapid measure of Al tolerance among plant species. Plant Cell Physiol 39:516–525

Jaffre T (1977) Accumulation du manganese par des especes associees aux terrains ultrabasiques de Nouvelle- Cale´ donie. C R Acad Sci (Paris) Ser D 284:1573–1575

Jaffre T (1979) Accumulation du manganese par les proteacees de Nouvelle-Caledonie. Comptes Rendus Academie des Science Paris Serie D 289:425–428

Jaffre T, Schmid M (1974) Accumulation du nickel par une Ru- biace´ e de Nouvelle Cale´ donie, Psychotria douarrei (G. Beau- visage) Daniker. C R Acad Sci (Paris) Ser D 278:1727–1730

Jaffre T, Brooks RR, Lee J, Reeves RD (1976) Sebertia acuminata: a hyperaccumulator of nickel from New Caledonia. Science-193:579–580

Jaffre T, Pillon Y, Thomine S, Merlot S (2013) The metal hyper- accumulators from New Caledonia can broaden our understanding of nickel accumulation in plants. Front Plant Sci. https://doi.org/10.3389/fpls.2013.00279

Jansen S, Broadley M, Robbrecht E, Smets E (2002) Aluminum hyperaccumulation in angiosperms: a review of its phylogenetic significance. Bot Rev 68:235–269

Kersten W, Brooks RD, Reeves RD, Jaffre´ T (1979) Nickel uptake by New Caledonian species of Phyllanthus. Taxon 28:529–534

Kidd PSP, Proctor J (2000) Effects of aluminium on the growth and mineral composition of Betula pendula Roth. J Exp Bot 51:1057–1066

Kramer U (2010) Metal hyperaccumulation in plants. Plant Biol 61:517–534

Macnair M (2003) The hyperaccumulation of metals by plants. Adv Ecol Res 40:63–105

Markert B (1992) Presence and significance of naturally occurring chemical elements of the periodic system in the plant organism and consequences for future investigations on inorganic environmental chemistry in ecosystems. Vegetatio 103:1–30

Markert B (1996) Instrumental element and multi-element analysis of plant samples. Methods and applications. Wiley, New York

Marschner H (1995) Mineral nutrition of higher plants, 2nd edn. Academic, London

Masunaga T, Kubota D, Hotta M, Wakatsuki T (1998) Nutritional characteristics of mineral elements in leaves of tree species in tropical rain forest, west Sumatra, Indonesia. Soil Sci Plant Nutr 44:315–329

Metali F, Salim KA, Burslem DFRP (2011) Evidence of foliar aluminium accumulation in local, regional and global datasets of wild plants. New Phytol 193:637–649

Miyasaka SC, Hue NV, Dunn MA (2007) Aluminum. In: Barker AV, Pilbeam DJ (eds) Handbook of plant nutrition. Tayler and Francis Group, Boca Raton, pp 439–497

Pillon Y, Munzinger J, Amir H, Lebrun M (2010) Ultramafic soils and species sorting in the flora of New Caledonia. J Ecol 98:1108–1116

Pilon-Smits EA, Quinn CF, Tapken W, Malagoli M, Schiavon M (2009) Physiological functions of beneficial elements. Curr Opin Plant Biol 12:267–274

Pollard AJ, Dandridge KL, Jhee EM (2000) Ecological genetics and the evolution of trace element hyperaccumulation in plants. In: Terry N, Banuelos GS (eds) Phytoremediation of contaminated soil and water. CRC Press, Boca Raton, pp 251–264

Proctor J (2003) Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East. Perspect Plant Ecol 6:105–124

Proctor J, Lee YF, Langley AM, Munro W, Nelson T (1988) Ecological studies on Gunung Silam, a small ultrabasic mountain in Sabah, Malaysia. I. Environment, forest structure and floristics. J Ecol 76:320–340

Proctor J, Argent G, Madulid D (1998) Forests of the ultramafic mount Giting-Giting, Sibuyan Island, the Philippines. Edinb J Bot 55:295–316

Proctor J, Baker AJM, Bruijnzeel LA, Van Baµgooy MMMJ, Fairweather GM, Madulid DA (2000) Foliar chemistry and leaf herbivory on Mount Bloomfield, Palawan, Philippines. Bot J Scotl 52:79–89

Rajakaruna N (2017) Lessons on evolution from the study of edaphic specialization. Bot Rev. https://doi.org/10.1007/s12229-017-9193-2 (in press)

Reeves RD (2003) Tropical hyperaccumulators of metals and their potential for phytoextraction. Plant Soil 249:57–65

Reeves RD, Baker AJM, Jaffre T, Erskine PD, Echevarria G, van der Ent A (2017) A global database for hyperaccumulator plants of metal and metalloid trace elements. New Phytol. https://doi.org/10.1111/nph.14907 (in press)

Rengel Z, Marschner P (2005) Nutrient availability and management in the rhizosphere: exploiting genotypic differences. New Phytol-168:305–312

Seregin IV, Kozhevnikova AD (2006) Physiological role of nickel and its toxic effects on higher plants. Russ J Plant Physiol 53:257–277

Shabala S (2003) Regulation of potassium transport in leaves: from molecular to tissue level. Ann Bot 92:627– 634

Stevens PF (2013) Malpighiales: angiosperm phylogeny Website on Missouri Botanical Garden Website. http://www.mobot. org/MOBOT/research/APweb/. Accessed-12 Oct 2013 van der Ent A, Lambers H (2016) Plant-soil interactions in global biodiversity hotspots. Plant Soil 403:1–5 van der Ent A, Mulligan D (2015) Multi-element concentrations in plant parts and fluids of Malaysian nickel hyperaccumulator plants and some economic and ecological considerations. J Chem Ecol 41:396–408

van der Ent A, Reeves RD (2015) Foliar metal accumulation in plants from copper-rich ultramafic outcrops: case studies from Malaysia and Brazil. Plant Soil 389:401–418 van der Ent A, Reeves RD, Baker AJM, Pollard J, Schat H (2013) Hyperaccumulators of metal and metalloid trace elements: facts and fiction. Plant Soil 362:319–334 van der Ent A, Repin R, Sugau J, Wong KM (2014) The ultramafic flora of Sabah: an introduction to the plant diversity on ultra- mafic soils. Natural History Publications (Borneo), Kota Kinabalu. ISBN 9789838121521 van der Ent A, Wong KM, Sugau J, Repin R (2015a) Plant diversity of ultramafic outcrops in Sabah (Malaysia). Aust J Bot 63:204–215 van der Ent A, Erskine PD, Sumail S (2015b) Ecology of nickel hyperaccumulator plants from ultramafic soils in Sabah (Malaysia). Chemoecology 25:243–259 van der Ent A, Jaffre´ T, L’Huillier L, Gibson N, Reeves RD (2015c) The flora of ultramafic soils in the Australia-Pacific Region: state of knowledge and research priorities. Aust J Bot 63:173–190 van der Ent A, Erskine PD, Mulligan DR, Repin R, Karim R (2016) Vegetation on ultramafic edaphic islands in Kinabalu Park (Sabah, Malaysia) in relation to soil chemistry and altitude. Plant Soil 403:77–101 van der Ent A, Cardace D, Tibbett M, Echevarria G (2018) Eco- logical implications of pedogenesis and geochemistry of ultra- mafic soils in Kinabalu Park (Malaysia). Catena 160:154–169 van der Heijden MGA, Martin FM, Selosse M-A, Sanders IR (2015) Mycorrhizal ecology and evolution: the past, the present, and the future. New Phytol 205:1406–1423

Wang M, Zheng Q, Shen Q, Guo S (2013) The critical role of potassium in plant stress response. Int J Mol Sci 14:7370–7390

Watanabe T, Jansen S, Osaki M (2005) The beneficial effect and the role of citrate in Al accumulation in Melastoma malabathricum. New Phytol 165:773–780

Watanabe T, Jansen S, Osaki M (2006) Al-Fe interactions and growth enhancement in Melastoma malabathricum and Mis- canthus sinensis dominating acid sulphate soils. Plant Cell Environ 29:2124–2132

Watanabe T, Broadley MR, Jansen S, White PJ, Takada J, Satake K, Takamatsu T, Tuah SJ, Osaki M (2007) Evolutionary con- trol of leaf element composition in plants. New Phytol-174:516– 523

Willey N (2016) Environmental plant physiology. Garland Publishing Inc., Taylor & Francis Inc, p 320

Wright IJ, Reich PB, Cornelissen JHC, Falster DS, Garnier E, Hikosaka K, Lamont BB, Lee W, Oleksyn J, Osada N, Poorter H, Villar R, Warton DI, Westoby M (2005) Assessing the generality of global leaf trait relationships. New Phytol 166:485–496

Wurdack KJ, Davis CC (2009) Malpighiales phylogenetics: gaining ground on one of the most recalcitrant clades in the angiosperm tree of life. Am J Bot 96:1551–1570

Zhang SB, Zhang JL, Slik JWF, Cao KF (2011) Leaf element concentrations of terrestrial plants across China are influenced by taxonomy and the environment. Glob Ecol Biogeogr 21:809– 818

FIGURES AND TABLES

Fig. 1 Boxplots of foliar accumulation (Ca, K, Mg) of ultramafic soils (n = 1352), with concentrations in µg g-1. Key to symbols: open squares are the ± mean, whiskers are ± standard deviation, circles are outliers and asterisks are extreme outliers.

Fig. 2 Boxplots of foliar accumulation (Ca, K, Mg) of non-ultramafic soils (n = 177), with concentrations in µg g-1. Key to symbols: open squares are the ± mean, whiskers are ± standard deviation, circles are outliers and asterisks are extreme outliers.

Fig. 3 Boxplots of foliar accumulation (Ni, Mn, Zn) of ultramafic soils (n = 1352), with concentrations in µg g1. Key to symbols: open squares are the ± mean, whiskers are ± standard deviation, circles are outliers and asterisks are extreme outliers.

Fig. 4 Boxplots of foliar accumulation (Ni, Mn, Zn) of non-ultramafic soils (n = 177), with concentrations in µg g-1. Key to symbols: open squares are the ± mean, whiskers are ± standard deviation, circles are outliers and asterisks are extreme outliers.

Fig. 5 Elemental profiles of common families on ultramafic (a) and non-ultramafic soils (b), with concentrations in µg g-1. Key to symbols: open squares are the ± mean, whiskers are ± standard deviation, circles are outliers and asterisks are extreme outliers.

Table -1 Extreme foliar accumulation for a range of elements.

Table 2 Patterns of metal accumulation among classes, clades, orders and families.

FIGURE 1

FIGURE 2

FIGURE 3

FIGURE 4

FIGURE 5

TABLE 1

TABLE 2