Introduction

Ultramafic soils represent a category of substrates derived from ultramafic bedrock and are sparsely distributed around the world. Such soils are known for relatively high concentrations of potentially phytotoxic trace elements, including nickel (Ni), cobalt (Co) and chromium (Cr) while concomitantly having cation imbalances and general nutrient deficiencies (Brooks 1987; Proctor 2003). Of the trace elements enriched in ultramafic soils, Ni is a micronutrient that is essential for some plant species, and influences plant senescence, nitrogen metabolism, germination and plant disease resistance (Brown et al. 1987; Welch 1995). On ultramafic soils, however, this trace element can be phytotoxic and symptoms indicating excess can include leaf chlorosis and reduced growth (Brune and Dietz 1995; Kukier and Chaney 2001; Weng et al. 2003). Some plants restricted to ultramafic soils have evolved ecophysiological mechanisms to tolerate and accumulate Ni, and are termed Ni hyperaccumulators when having in excess of 1000 μg g−1 Ni in the foliage (Reeves 1992; Van der Ent et al. 2013a). The phenomenon is exceptionally rare and known in approximately 400 species worldwide in a range of different plant families. The Salicaceae, Buxaceae, Phyllanthaceae and Rubiaceae are the most common families for Ni hyperaccumulators in tropical regions (Reeves 2006). Nickel hyperaccumulators can be categorised into ‘strict’ and ‘facultative’ hyperaccumulators. Strict hyperaccumulators are exclusively confined to ultramafic soil and all populations of the particular species are hyperaccumulators. However, species that are ‘facultative’ hyperaccumulators have populations on ultramafic soils that are hyperaccumulators, and populations on other soils that are not (Pollard et al. 2014). An example of a facultative Ni hyperaccumulator which occurs in Sabah is Rinorea bengalensis (Violaceae), that has Ni concentrations of 1000–17,750 μg g−1 in the foliage of specimens of this species growing on ultramafic soils, and 1–300 μg g−1 in foliage of specimens of this species growing on non-ultramafic soils (Reeves 2003; Van der Ent et al. 2013a).

Hyperaccumulation is hypothesised to have evolved to interfere with other competing plant species (‘elemental allelopathy’), or to protect against insect herbivores (‘elemental herbivory defense’), although a variety of other explanations have also been suggested (Martens and Boyd 1994; Boyd and Jaffré 2001). The first hypothesis suggests that hyperaccumulators increase Ni concentrations in the soil area around the plant base (‘phyto-enrichment’) via leaf litter deposition as a result of leaf senescence and abscission which, as a result of toxicity effects, might reduce growth performance and germination of competing plant species (Boyd and Martens 1998). Given the inherent Ni-tolerance of hyperaccumulators, this might also provide advantages in survival of seedlings of the same hyperaccumulator species (or indeed, of different Ni hyperaccumulator species if these occur in the same habitat). The second hypothesis suggests that high foliar Ni concentrations protect against insect herbivores. As a consequence, Ni hyperaccumulators suffer less damage as a result of insect herbivory, and hence have competitive advantages. Further refinements of this model led to the formulation of the ‘Defensive Enhancement Hypothesis’, which proposes that after an initial defensive benefit resulting from relatively low initial foliar Ni concentrations, increased concentrations provided increased plant fitness, and led to a step-wise increase in foliar Ni accumulation (Boyd 2012). Furthermore, the ‘Joint Effects Hypothesis’ proposes that Ni accumulation in combination with organic chemicals (such as alkaloids) could have synergistic effects (Boyd 2012). In the context of ‘elemental herbivory defense’, foliar Ni accumulation has the distinct benefit of requiring limited energetic resources (although the uptake and transport physiology requires Ni complexing ligands such as citrate) because Ni is not produced but rather translocated from the soil and, contrary to organic molecules, Ni cannot be broken down or metabolised to avoid toxicity (Martens and Boyd, 1994; Boyd and Martens 1998). However, whereas the synthesis of organic toxic molecules by plants is relatively flexible in evolutionary terms, Ni accumulation is not (Cheruiyot et al. 2013).

The ultramafic soils of the Malaysian state of Sabah on the Island of Borneo are extensive, occupying a total area of about 3500 km2 (Proctor et al. 1988) and is renowned for high species richness (Van der Ent et al. 2014). The flora of Sabah has an estimated 8000 vascular plant species (Wong 1992) with over 5000 plant species in the <1200 km2 Kinabalu Park (Beaman 2005). Prior to this study, the following Ni hyperaccumulators were known to occur in Sabah: Rinorea bengalensis and R. javanica (Violaceae) (Brooks and Wither 1977; Brooks et al. 1977), Phyllanthus balgooyi (Phyllanthaceae) (Baker et al. 1992; Hoffmann et al. 2003), Dichapetalum gelonioides (Dichapetalaceae) (Baker et al. 1992), Psychotria cf. gracilis (Rubiaceae) (Reeves 2003) and Shorea tenuiramulosa (Dipterocarpaceae) (Proctor et al. 1989). The objective of this study was to screen the flora of ultramafic outcrops in Sabah, mainly Kinabalu Park, for the occurrence of (more) Ni hyperaccumulators. Further aims were to elucidate general phylogenetic patterns of Ni hyperaccumulation, habitat characteristics, overall plant-soil relationships and potential ecological interactions relating to herbivory and allelopathy.

Materials and methods

Study area and field collection

As part of a larger study on the relationships between plant diversity and soil chemistry of ultramafic outcrops at Mount Kinabalu and Mount Tambuyukon in Sabah, Malaysia, plants were screened for Ni hyperaccumulators (Van der Ent et al. 2013c). During the fieldwork on the ultramafic soils in Kinabalu Park (January 2011–September 2012), leaf samples were collected from all plants in plots (there were a total of 101 plots, ranging is size from 2000 to 250 m2 and as many different plants as possible in the surrounding vegetation). In addition, plants were screened during fieldwork in the following Forest Reserves elsewhere in Sabah: Mount Tavai, Bidu-Bidu Hills, Mount Silam and Bukit Hampuan. Figure 1 shows a map of Sabah (Malaysia) with the study localities. In the field, the leaf samples were pressed against white test paper impregnated with the Ni-specific colorimetric reagent, dimethylglyoxime (‘DMG’), which changes colour, to purple, upon contact with Ni. Approximately 5000 plant samples have been tested using this method. All samples that tested visually positive were re-collected (fully grown sun leaves, at least 2 m above the soil surface) by hand. Fresh plant leaves were put in paper bags to prevent decomposition before transport to the field station. Leaves were thoroughly washed with demineralised water to remove potential dust contamination and then dried at 70 °C for 5 days in a dehydrating oven, packed for transport to Australia and gamma irradiated at Steritech Pty. Ltd. in Brisbane following Australian quarantine regulations. Soil samples (20–30 cm from base of the Ni hyperaccumulator plant, 10–20 cm deep) were also collected for analysis. Leaf litter samples were collected under Ni hyperaccumulator plants by carefully collecting partly decomposed leaves from a 1-m2 area, ensuring that no mineral constituents were adhered to the collected material. In addition to new collections of potential Ni hyperaccumulators in the family Phyllanthaceae (specifically in the genera Phyllanthus, Glochidion and Breynia) collected from the field, examples from existing herbarium specimens held at the Sabah Parks Herbarium (SNP) Herbarium were also sourced. For each species of interest (220 specimens across 41 species), 50–100 mg samples were obtained and the original collection data associated with the specimens were recorded.

Fig. 1
figure 1

Map of Sabah (Malaysia) with study localities. The insert shows the location of Sabah on the Island of Borneo. Satellite imagery is Landsat, from Google Earth with insert from ArcGIS (esri)

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Chemical analyses of plant tissue samples

Foliar (and leaf litter) samples were crushed and ground, and a 300-mg subsample was digested in 4 mL concentrated nitric acid (70 %) and 1 mL hydrogen peroxide (30 %) in a microwave oven. The digest was diluted to 40 mL with TDI water before analysis with ICP-AES (Varian Vista Pro II). Elements included in the analysis were Al, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, S and  Zn. The potential for foliar contamination with soil particulates is a major risk for accurate analysis of foliar elemental composition. This risk is highest in samples of ground-herbs, and lesser so for trees, but it cannot be entirely avoided. Concomitantly high foliar concentrations of Fe (>2500 μg g−1) and Cr (>50 μg g−1) are an indication for soil contamination as these elements are major constituents of ultramafic soils.

Chemical analyses of soil samples

Soil samples (300 mg subsample) were digested using freshly prepared ‘reverse’ Aqua Regia (9 mL 70 % nitric acid and 3 mL 37 % hydrochloric acid per sample) in a digestion microwave for a 2-h programme and diluted with TDI water to 45 mL before analysis. The method followed Rayment and Higginson (1992) method 17B1 and results in ‘pseudo-total’ elemental concentrations. Soil pH and electrical conductivity (EC) were measured in a 1:2.5 soil:water mixture. Exchangeable Ni, Co, Cr and Mn were extracted in 0.1 M Sr(NO3)2 at a soil:solution ratio of 1:4 (10 g : 40 mL) and 2-h shaking time. Phytoavailable Ni, Co, Cr and Mn were extracted with diethylene triamine pentaacetic acid (DTPA) according to Lindsay and Norvell 1978, but with modifications from Bequer et al. (1995) (excluding TEA, buffered at pH 5.3). Another method for phytoavailable Ni, Co, Cr and Mn was also used, namely extraction with organic acids (acetic, malic and citrate acid in molar ratio of 1:2:2 at 0.01 M) at a soil:solution ratio of 1:4 (10 g : 40 mL) and 2-h shaking time. Exchangeable cations were extracted with silver-thiourea (Dohrmann 2006) over 16 h. All soil extractions were undertaken in 50-mL PP centrifuge tubes. Soil samples were weighed using a four-decimal balance and weights recorded for correction of the precise weights in the mass balance calculations. Samples were agitated for method-specific times using an end-over-end shaker at 60 rpm and subsequently centrifuged (10 min at 4000 rpm). The resultant supernatant was collected in 10-mL PP tubes. All soil samples were analysed with ICP-AES (Varian Vista Pro II) for Al, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, S and  Zn. The ICP-AES instrument was calibrated using a six-point multi-element standard prepared in each extraction solution.

Statistical analysis

The ranges and means of the foliar and soil elemental concentrations were calculated. Correlation coefficients between the soil and plant chemistry data were also calculated. These analyses were undertaken using the software packages STATISTICA Version 9.0 (StatSoft) and Excel for Mac version 2011 (Microsoft).

Results

Confirmation of Ni hyperaccumulator status and new discoveries

All previously known Ni hyperaccumulators from Sabah were located during the current field explorations of this study and chemical analysis of foliar samples re-confirmed their Ni hyperaccumulation status. Of these, Phyllanthus balgooyi is a widespread shrub often dominating open habitats on mountain ridges and along riverbanks, for example on Bukit Hampuan and in the Mount Tawai Forest Reserve. The climber Psychotria cf. gracilis (here identified as P. sarmentosa complex) is a common understory creeper of open forests. In contrast, the medium-sized trees Rinorea bengalensis and R. javanica (Violaceae) are comparatively rare. Another widespread, but rare, species, Dichapetalum gelonioides subsp. sumatranum and D. gelonioides subsp. pilosum, is a strong hyperaccumulator of zinc (Zn), but not of Ni on non-ultramafic soils, and vice versa on ultramafic soils. Finally, the tree Shorea tenuiramulosa (Dipterocarpaceae) is restricted to Mount Silam and the Meliau Range, and the only dipterocarp known to hyperaccumulate Ni (Proctor et al. 1989). In Table 1, newly discovered and confirmed Ni hyperaccumulators from Sabah are listed with their highest foliar Ni values. The strongest Ni hyperaccumulators are Phyllanthus cf. securinegoides (23,300 μg g−1) and Psychotria sarmentosa complex (24,200 μg g−1), whereas Ptyssiglottis cf. fusca (Acanthaceae) (1160 μg g−1) and Baccaurea lanceolata (Phyllanthaceae) (1450 μg g−1) are weak Ni hyperaccumulators, only just reaching the threshold foliar concentration that defines a Ni hyperaccumulator. Of some potential concern is the knowledge that fruits from Bacaurea species are frequently harvested and eaten. This is disconcerting in view of its Ni hyperaccumulation characteristic when growing on ultramafic soils, although fruits have not been analysed.

Table 1 New and confirmed nickel hyperaccumulators from Sabah, Malaysia
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Habitats of nickel hyperaccumulators

Nearly all Ni hyperaccumulators recorded in Sabah grow in successional or open habitats, such as on natural landslides, burnt areas, along rivers and on the top of ridges. Furthermore, all Ni hyperaccumulators have been recorded from lowland areas up to the lower montane forest boundary, and no Ni hyperaccumulators have been recorded at altitudes >1200 m a.s.l. (despite extensive screening at higher altitudes). The growth forms of all known Ni hyperaccumulators in Sabah are small trees (Rinorea bengalensis up to a height of 23 m with a bole diameter of 58 cm is the largest) with Psychotria sarmentosa complex being the only exception (a creeper). The five main habitat types in which nickel hyperaccumulators have been recorded in Sabah are: (1) open shrub on hill ridges; (2) riparian zone and open forest along rivers; (3) landslide areas on serpentinite soils; (4) disturbed areas in lowland forest; and (5) seepage zones below peridotite rock cliffs. Figure 2 shows some of the Ni hyperaccumulator species from Sabah studied here. Their habitats are described in more detail below.

Fig. 2
figure 2

Nickel hyperaccumulator species from Sabah studied in the present study

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Open shrub on hill ridges

The main site that falls in this category is located in Nalumad. Here, a lowland (400 m a.s.l.) hill near the Mekadou River has been burnt as a result of an uncontrolled forest fire in 2008. Prior to burning, the site had been logged and most major trees were removed. At present, the site has a short and open shrub community (1–3 m) dominated by pioneer non-accumulating species such as Macaranga kinabaluensis (Euphorbiaceae). In this habitat type, the Ni hyperaccumulator Phyllanthus cf. securinegoides is common with others including Mischocarpus sundaicus (Sapindaceae), Rinorea javanica, Psychotria sarmentosa complex, Glochidion cf. brunneum (Phyllanthaceae) and Xylosma luzonensis (Salicaceae) also present. The site is also the only known site for Actephila sp. nov. (Phyllanthaceae). The shrub Phyllanthus balgooyi, characteristically colonising rock crevices on the bare ridges, occurs in this habitat type at Nalumad and many other similar sites. Unusually, at nearby Bukit Hampuan, Phyllanthus balgooyi grows as a tree up to 9 m tall (and a bole diameter of 20 cm) in an open lower montane forest. A not dissimilar habitat at Mount Silam is a site where Shorea tenuiramulosa occurs.

Riparian zone and open forest along rivers

The main sites in this category are those along the Bambangan River, the Wuluh River, the Bangau-Bangau River and the Panataran River. Several Ni hyperaccumulators are associated with the riparian habitats at these sites: the tree Flacourtia kinabaluensis (Salicaceae) grows on the streambed, the shrub Phyllanthus balgooyi (Phyllanthaceae) grows inside the riverbed forming a fringe, and Glochidion rubrum (Phyllanthaceae) grows as a large tree on the alluvial clays. Besides the riparian habitat, a small valley near Bukit Hampuan has an open shrub community dominated by Ni hyperaccumulators, including the trees Kibara coriacea (Monimiaceae), Walsura cf. pinnata (Meliaceae), Mischocarpus sundaicus, the shrubs Phyllanthus cf. securinegoides, Phyllanthus balgooyi and Xylosma luzonensis, and the creeper Psychotria sarmentosa complex.

Landslide areas on serpentinite soils

The vegetation on serpentinite rocks is characterised by the (non-accumulating) trees Ceuthostoma terminale or Gymnostoma sumatranum (Casuarinaceae). Serpentinite outcrops are prone to landslides, and the pioneer vegetation in such habitats hosts Xylosma luzonensis, Glochidion species and Psychotria sarmentosa complex. The main sites in this category are at the Wuluh and Panataran Rivers in Kinabalu Park.

Disturbed areas in lowland forest

Open areas in lowland forest, such as at Serinsim, form the habitats of the trees Glochidion cf. rubrum and G. cf. sericeum and the herb Ptyssiglottis cf. fusca. These species, together with Baccaurea lanceolata, are the only Ni hyperaccumulators known to occur to date in tall lowland forest.

Seepage zones below peridotite rock cliffs

Seepages zones below rock cliffs near Serinsim and at Bambangan form the habitat of several Ni hyperaccumulators. At Serinsim, peridotite cliff faces are up to 70–80 m, Phyllanthus balgooyi is a common shrub growing on the rock face, whereas at the foot of these cliffs Rinorea bengalensis, R. javanica, Mischocarpus sundaicus (Sapindaceae) and Flacourtia kinabaluensis occur where seepage water drains out. A similar habitat is found at Bambangan (on Mount Kinabalu) where Glochidion rubrum grows below the rock face in the seepage zone.

Taxonomy and phylogeny of Ni hyperaccumulators

The main families in which Ni hyperaccumulators occur in Sabah are the Phyllanthaceae, Rubiaceae, Salicaceae and the Violaceae. This conforms to the global trends for Ni hyperaccumulators, with these families also being the most important in other tropical hotspots for Ni hyperaccumulators, such as in New Caledonia and Cuba (Reeves 2003). These families, except the Rubiaceae, are all in the Order Malpighiales (Angiosperm Clade Rosids). The records for Monimiaceae and Sapindaceae are the believed to be the first records for Ni hyperaccumulators in these families globally. The genera Phyllanthus and Glochidion, in the large family Phyllanthaceae (over 2000 species in 60 genera), have the highest diversity in the Malesian and Australian regions (Govaerts et al. 2000; Kawakita, 2010). The genus Phyllanthus has over 800 species globally, with major centres of diversity in New Caledonia (113 spp.), Madagascar (63 spp.), Cuba (50 spp.) and Venezuela (58 spp.) (Govaerts et al. 2000). Of the New Caledonian species, 14 are Ni hyperaccumulators (Kersten et al. 1979; Reeves 2003) and of the Cuban species, 19 are Ni hyperaccumulators (Reeves 2003). Approximately 12 species of this genus are known in Sabah and it is noteworthy that a number of the newly discovered Ni hyperaccumulators also represent undescribed plant species. This mainly reflects the complex taxonomy of Phyllanthaceae, which to date has not been revised in the ‘Tree Flora of Sabah and Sarawak’ or ‘Flora Malesiana’ (taxonomical revisions for the region). It is further worth noting that the majority of Ni-hyperaccumulating Phyllanthus and Glochidion species seem restricted to a single or a few ultramafic outcrops and are hence rare. The exception is Phyllanthus balgooyi, which is widespread and locally common, but the P. balgooyi species complex is taxonomically not completely understood (Hoffmann et al. 2003), and there might be several variants that warrant subspecies status, based on their morphologies and the distinct ecological niches they occupy (e.g. riparian zones versus rock ridges). This species was first discovered as a Ni hyperaccumulator in the Philippines by Baker et al. (1992) as P. ‘palawanensis’ and subsequently described as a new species (P. balgooyi). Maximum foliar Ni was 16,230 μg g−1 in the Philippine material (Baker et al. 1992), whereas in this Sabah study, the maximum Ni concentration recorded was 8606 μg g−1. Extremely high phloem tissue Ni concentrations (up to 88,580 μg g−1) are another characteristic of this species (Hoffmann et al. 2003). Taxonomical difficulties also pertain to Phyllanthus cf. securinegoides, which is also part of a difficult section in the genus. This species was first recorded as a Ni hyperaccumulator from historic collections made in Palawan and Mindanao in the Philippines from which herbarium samples were analysed by Baker et al. (1992). This species was not known from Sabah or the island of Borneo, until collections were made as part of this study (if indeed it belongs to the same taxon). This species is rare and appears to be restricted in Sabah to a narrow band of ultramafic outcrops stretching between Bukit Hampuan and Nalumad. The Philippine material accumulated up to 34,750 μg g−1 Ni in the foliage (Baker et al. 1992) whereas the maximum found in Sabah in this study was 23,300 μg g−1.

Since several new Ni hyperaccumulators in the family Phyllanthaceae were discovered during the field sampling, it seemed possible that this family could contain more Ni hyperaccumulators. Therefore, 220 specimens covering 41 species were sampled from existing herbarium specimens held at the SNP Herbarium. Analytical results of the elemental analysis of plant species in the Phyllanthaceae occurring in Kinabalu Park are given in Table 2. Several more species of Phyllanthus and Glochidion were found to be Ni hyperaccumulators. It should be noted that phylogenetic studies have shown that Phyllanthus is paraphyletic over Glochidion and Breynia and that the species in these genera be will transferred to Phyllanthus in a future revision for the region (Hoffmann et al. 2006; Kathriarachchi et al. 2006; Wagner and Lorence 2011). The collection data confirmed that all of specimens that hyperaccumulated Ni had been collected from ultramafic soils. In addition, there was a propensity for the Phyllanthaceae to accumulate not only Ni, but also other trace elements such as Co, Cr, Mn and Zn. Cobalt was accumulated to a significant level in Aporosa chalarocarpa (468 μg g−1), while the Glochidion cf. sericeum specimens contained 442–1310 μg g−1 (and is therefore clearly a hyperaccumulator of this element). One specimen of Baccaurea lanceolata was unusual in containing 179 μg g−1 Co, 143 μg g−1 Cr, 1451 μg g−1 Ni and 1450 μg g−1Zn. Manganese concentrations were also relatively high, but varied widely. One species (Aporosa falcifera) appeared to be a Mn hyperaccumulator with 13,500 μg g−1 Mn, as the foliar threshold for Mn hyperaccumulation is >10,000 μg g−1 (Van der Ent et al. 2013a). Aporosa chalarocarpa, Baccaurea lanceolata, Cleistanthus sp. 1, Glochidion arborescens, Glochidion sp. ‘bambangan’, G. cf. brunneum, G. lanceilimbum, G. mindorense, Glochidion sp. ‘nalumad’, G. cf. sericeum, Phyllanthus balgooyi, and P. cf. securinegoides are Ni hyperaccumulators, but almost all species collected from ultramafic soils have higher than normal Ni concentrations in their tissues. As Glochidion sp. ‘panantaran’ probably represents the same species as Glochidion sp. ‘nalumad’ we do not consider this a new record (the infertile specimens were of poor quality making identification impossible). Some specimens collected from non-ultramafic soils have unusually high Zn concentrations, such as Glochidion glomeratum (185 μg g−1 Zn) and Phyllanthus amarus (571 μg g−1 Zn). The new Ni hyperaccumulation record for Actephila sp. nov. also represents a new genus holding a Ni hyperaccumulator in the Phyllanthaceae family, with up to 11,520 μg g−1 Ni. The relatively small family Dichapetalaceae, which is mainly distributed in Africa, has a few species in Southeast Asia of which subspecies of D. gelonioides are of particular interest with regard to metal accumulation. D. gelonioides subsp. tuberculatum and subsp. pilosum are strong Ni hyperaccumulators when occurring on ultramafic soils (up to 26,650 μg g−1 Ni), and strong Zn hyperaccumulators (up to 30,000 μg g−1 Zn) when occurring on non-ultramafic soils, whereas D. gelonioides subsp. sumatranum is a Zn hyperaccumulator (up to 15,660 μg g−1), and does not accumulate Ni (Baker et al. 1992). In this research, only D. gelonioides subsp. pilosum was located and analysed. The specimens accumulated little Ni, but contained up to 4922 μg g−1 Zn, confirming the results of earlier studies.

Table 2 Elemental concentrations (Co, Cr, Mn, Ni and Zn) in Phyllanthaceae
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The Rubiaceae, and the genus Psychotria in particular, is well represented globally with Ni hyperaccumulators including Psychotria gabriellae (formerly P. douarrei) from New Caledonia, P. grandis from Puerto Rico, P. clementis, P. costivenia, P. glomerata, P. osseana and P. vanhermanii from Cuba (Reeves et al. 1999; Reeves 2003). In Sabah, Psychotria cf. gracilis (here named as P. sarmentosa complex) has previously been recorded by Reeves (2003). Samples of this species were collected from various locations during the current study and confirmed its status as one of the strongest known Ni hyperaccumulators in the region. The taxonomy of this species is, however, difficult and probably comprises several closely related species and subspecies. High, though sub-hyperaccumulator, levels of Ni also occur in other members of the Rubiaceae, including Timonius cf. eskerianus (500 μg g−1 Ni).

The record for Ptyssiglottis cf. fusca (Ni up to 1160 μg g−1) in the Acanthaceae is interesting because another member of that family, Rostellularia adscendens var. hispida, was recorded in Queensland, Australia, with concentrations up to 2190 μg g−1 (Reeves 2003), and Phidiasia lindavii from Cuba with concentrations up to 1853 μg g−1 (Reeves et al. 1999). The only Buxus species in Sabah, Buxus rolfei (Buxaceae) from a family with a large number of Ni hyperaccumulators in Cuba (17 Ni hyperaccumulators out of 37 species that occur in the country; Reeves et al. 1996), does not accumulate Ni. The Sapotaceae, which contains the famous Ni hyperaccumulator Pycnandra acuminata (formerly Sebertia acuminata) (from New Caledonia and renowned for its green nickel-rich sap; Jaffré et al. 1976), and the Ni hyperaccumulator Planchonella oxyedra from Indonesia (Wither and Brooks 1977), did not contain Ni hyperaccumulators in Sabah in the 18 species across five genera tested, with Planchonella sp. nov. from Nalumad being the highest with a Ni concentration of 160 μg g−1. The record for Dalbergia beccarii recorded in this study with 2620 μg g−1 Ni is interesting, because another species in this genus (D. melanoxylon) and another member in this family (Pearsonia metallifera) are (hyper)accumulators of Ni in Zimbabwe (Cole 1971; Reeves 2003). The Ochnaceae has a Ni hyperaccumulator in Brackenridgea palustris (several subspecies) from Palawan and Sulawesi (Baker et al. 1992), and this species has also been recorded from Sabah. The Clusiaceae contains Ni hyperaccumulators in Garcinia bakeriana, G. revoluta and G. ruscifolia from Cuba (Reeves et al. 1999), but analyses in this study reveal no hyperaccumulators (29 species across five genera tested), the highest Ni concentrations being found in Mesua paniculata with 146 μg g−1, Kayea macrantha with 125 μg g−1, Calophyllum soulattri with 123 μg g−1 and Garcinia bancana with 105 μg g−1 Ni. Although Ni hyperaccumulators have been recorded in the Myrtaceae in Cuba and New Caledonia, no Ni hyperaccumulators were recorded in this family during this study, with the highest Ni concentrations found in a specimen of Leptospermum recurvum with 140 μg g−1, Syzygium cf. pterophera with 115 μg g−1 and Rhodamnia cinerea with 108 μg g−1 Ni. Finally, although the genus Chionanthus (Oleaceae) has yielded a Ni hyperaccumulator species (C. domingensis) from Cuba, the two species in that genus tested in this study contained <6 μg g−1 Ni.

Soil chemistry and foliar concentrations in Ni hyperaccumulators

The results of the analysis of the soil chemistry in the rhizosphere of 12 Ni hyperaccumulators are given in Tables 3 and 4. These soils are characterised by circumneutral pH (mean pH 6.7), relatively high total (mean 2985 μg g−1) and exchangeable Ca (mean 1417 μg g−1), extremely high total Mg (mean 43.8 mg g−1) and low exchangeable K (Table 3). Total Co (mean 387 μg g−1), Cr (mean 3205 μg g−1) and Mn (mean 3946 μg g−1) are also high (Table 3). However, phytoavailable concentrations of these elements are low. Mean total soil Ni is high (1890 μg g−1) and potentially phytoavailable Ni (mean 99, 127, 128 μg g−1 for Mehlich, DTPA and organic acid-extractable Ni, respectively) is also high. Despite the fact that the soils originate from different localities and have wide ranging properties, extractable Ni concentrations are similar (Table 4).

Table 3 Main soil chemistry parameters in the rooting zone (major cations)
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Table 4 Ni concentrations in soil in the rooting zone
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Foliar elemental concentrations in hyperaccumulators were compared with soil elemental concentrations (total and extractable/exchangeable) of the hyperaccumulator soils (of matched pairs). Table 4 shows summarised elemental concentrations in foliage and associated soils in a range of hyperaccumulators. On average, foliar Ni is three times higher than soil Ni (but reaches a factor of sixfold in Phyllanthus cf. securinegoides). While such levels of biomagnification are impressive, it should be noted that hyperaccumulator plants (particularly trees) are long lived and hence slowly accumulate Ni from the soil into their biomass over an extended period of time. Working with native hyperaccumulators in New Caledonia, Lee et al. (1977) found a positive relationship between foliar Ni and extractable Mn and Ni in the soil. However, in this study only a weak (but significant) correlation was found between foliar Ni and total soil Ni (r = 0.20), and correlations between foliar and extractable soil Ni were weaker. There are also (weak) correlations between foliar Ni and pH (r = − 0.28) and foliar Ni and soil P (r = 0.47). Further correlations were found between foliar S and P and soil Mg (r = − 0.40 and −0.27), foliar K and exchangeable Al, Ca and K (r = 0.32, 0.28 and 0.30, respectively) and foliar Al and pH (r = −0.28).

High-Ni leaf shedding and potential allelopathic effects

The field survey showed that most Ni hyperaccumulators were locally abundant in their habitats, both in terms of numbers of individuals and relative numbers of (hyperaccumulator) species. This suggests preferential habitats on certain soil types, but the high relative density of hyperaccumulators could also potentially locally induce allelopathic effects. It has been shown that leaves shed by the New Caledonian Ni hyperaccumulator tree (Pycnandra acuminata) increases the concentrations of Ni in the top soil directly under the canopy substantially (Boyd and Jaffré 2001) potentially constituting an allelopathic system that could give the hyperaccumulator competitive advantages. Recent work with Se hyperaccumulators have shown that such plants may reduce the growth of sensitive neighbouring plants while facilitating tolerant plants with the latter experiencing less herbivory (El Mehdawi et al. 2011a; El Mehdawi and Pilon-Smits 2011). These effects were linked to ‘phyto-enrichment’ (increase of Se in the surrounding soil) and hence causing elemental allelopathy (El Mehdawi et al. 2011b). In contrast, experimental work with a temperate Ni hyperaccumulator demonstrated that leaf shedding did not inhibit germination or growth of competing plants (Zhang et al. 2006).

Leaf litter samples collected at Nalumad under the canopies of Phyllanthus cf. securinegoides and Rinorea bengalensis contained 665 and 2128 μg g−1 Ni, respectively, and exceed the concentrations in the sub-surface soil. Compared to the mean Ni concentration (74 μg g−1) in leaf litter samples from 84 plots in Kinabalu Park (Van der Ent et al. unpublished), the leaf litter under these hyperaccumulators is extremely Ni rich. It is to be expected, however, that co-occurring plant species are also Ni tolerant. Nevertheless, the high leaf litter Ni could exert a selective force on some (generalist) plant species that may outcompete Ni hyperaccumulators. Furthermore, high Ni in the organic topsoil could have mutualistic effects on seedlings of both the Ni hyperaccumulator species that is the source of the leaf litter, and co-occurring Ni hyperaccumulator species. As such, Ni loading in the topsoil could benefit hyperaccumulator seedlings by having a ready supply of Ni to their shallow roots to aid herbivory protection in emerging leaves. Indeed, high densities of seedlings of Rinorea bengalensis at Nalumad, and Phyllanthus balgooyi at Bukit Hampuan, have been observed. These interactions might not explain why Ni hyperaccumulation has evolved, but are rather consequences of the immediate Ni hyperaccumulator environment. Leaf shedding could also act as a tolerance mechanism by translocation of Ni in abscised leaves (Martens and Boyd 1994). Although leaf litter Ni concentrations were high as outlined earlier, they were still lower than samples from living (attached) leaves on the plants, hence it would be an inefficient process. It should be noted that leaf litter decomposes very rapidly under the conditions of intense rainfall and the tropical humid climate, and any contained Ni probably leaches rapidly.

Interaction of Ni hyperaccumulators with epiphytes

The ecological relationships between epiphytes and hyperaccumulators is poorly understood, although Boyd et al. (2009) found that epiphytes (bryophytes) growing on Ni hyperaccumulator hosts contained greater levels of Ni than those growing on non-hyperaccumulator hosts in New Caledonia. Even more so, they concluded that bryophyte Ni concentrations often exceeded Ni hyperaccumulation thresholds (Boyd et al. 2009). Although it should be noted that bryophytes have no active vascular system for Ni uptake and translation and hence the definition of ‘hyperaccumulation’ should probably not apply to such non-vascular plants. Nevertheless, this seems to indicate that the suitability for epiphytes to grow on hyperaccumulators as hosts may depend on their capability to tolerate high Ni concentrations and this may in turn define the epiphyte community composition between Ni hyperaccumulator and non-hyperaccumulator hosts (Boyd et al. 2009). Most Ni hyperaccumulators in Sabah are small shrubs and hence do not carry epiphytes, but an exception are some particularly large specimens of Phyllanthus balgooyi on Bukit Hampuan, where ferns have colonised branches of these trees. Foliage of these ferns contained 251 μg g−1 Ni, whereas the bark of the host plant contained 358 μg g−1 Ni. This shows that the bark of some Ni hyperaccumulators is a high-Ni environment and that epiphytes can indeed take up Ni from the living or decomposing bark (or decomposing dropped leaves) on which they grow. As such, there is some selective force for Ni-tolerance in epiphytes growing on Phyllanthus balgooyi hosts, but it is unknown whether this might have any potential beneficial effects for the epiphyte (‘enhanced herbivory protection’).

Interaction of Ni hyperaccumulators with insect herbivores

The ‘Elemental Herbivory Defense’ hypothesis (Boyd and Martens 1998; Boyd 2009) suggests that high Ni loading in the leaves of Ni hyperaccumulators could result in reduced (insect) herbivory attack. However, field observations on Mount Bloomfield, Palawan (Philippines), showed that Ni hyperaccumulators did not experience less herbivory (Proctor et al. 2000), and the Ni hyperaccumulator Shorea tenuiramulosa suffered as much foliar herbivory damage as ‘normal’ plant species growing in the area on Mount Silam in Sabah (Proctor et al. 1989). It should be noted, however, that this species achieves only relatively low Ni accumulation (351–1787 μg g−1 recorded in this study), and the extremely high concentrations in, for example Psychotria sarmentosa complex (up to 24,200 μg g−1) and Phyllanthus cf. securinegoides (up to 23,300 μg g−1) will undoubtedly exert a stronger toxicity effect on insect herbivores. Nevertheless, extensive insect herbivory damage to all Ni hyperaccumulator species in Sabah was observed during this study. Specialist insect herbivores may feed on Ni hyperaccumulators without negative effects (Boyd 2009), and one geometric moth species (Erebidae: Erebinae tribe Poaphilini) has been found in this study to feed exclusively on the Ni hyperaccumulator Phyllanthus balgooyi (illustrated in Fig. 3, top). Such Ni-tolerant insects preferentially feed on Ni hyperaccumulators to increase their own body Ni content to deter their predators (Boyd 2009). Aphids have also been found feeding on Phyllanthus cf. securinegoides at Nalumad (also see Fig. 3, bottom). The extremely high Ni concentrations in the tissues of these hyperaccumulators necessitate exceptional Ni-tolerance by these insects. If not tolerant, other strategies that could be employed by herbivore insects to avoid Ni toxicity include diet dilution (feeding on low-Ni plant parts in addition to high-Ni plant parts), or avoidance (by selective feeding) (Martens and Boyd 1994; Boyd and Martens 1998; Boyd 2009).

Fig. 3
figure 3

Geometric moth (Erebidae: Erebinae tribe Poaphilini) feeding on the leaves of the Ni hyperaccumulator Phyllanthus balgooyi (top) and aphids, with ants, feeding on Phyllanthus cf. securinegoides (bottom)

Full size image

Discussion

Laboratory analysis with ICP-AES confirmed the indicative results achieved from the initial testing in the field with dimethylglyoxime (DMG). Field testing with DMG paper therefore remains a reliable and quick method for Ni hyperaccumulator reconnaissance. The method is, however, fairly insensitive, and depends on Ni being present in the aqueous phase. Some plants, for example Shorea tenuiramulosa, did not react with DMG in the field, and only laboratory analysis showed significant Ni accumulation. This intriguing example is suggestive of the various biochemical forms in which Ni may be present in plants. Recent progress has been made with understanding Ni chemical forms in Alyssum ssp. (Tappero et al. 2007; McNear et al. 2010), and in some hyperaccumulators from New Caledonia, most notably Pycnandra acuminata (formerly Sebertia acuminata) (Perrier et al. 2004; Callahan et al. 2008), providing evidence for the important role of carboxylic acids such as citrate in Ni complexation. The wide range of plant families and life forms involved in Ni hyperaccumulation suggests that various different physiologies, and hence Ni chemical forms, might be present in hyperaccumulator plants. Such differences in chemical bonding of Ni in plant tissues might also contribute to lower or higher toxicity to insect herbivores.

Nickel hyperaccumulators in Sabah occur mainly in open, successional habitats, mainly on ridges, below rock faces and along rivers. Recent studies on soils associated with these hyperaccumulators have shown that the occurrence is correlated with soils with highly available Ni concentrations (Van der Ent et al. unpublished). These open, often bare ultramafic soils are also difficult for most plants to colonise and make plants colonising these habitats potentially more susceptible to insect herbivores. The local abundance of Ni hyperaccumulators might indicate possible advantages, including highly efficient nutrient sequestration, or foliar toxicity to reduce insect herbivory (‘elemental herbivory defense’) or competitive advantages over other plant species (‘elemental allelopathy’). Step-wise evolution of the Ni hyperaccumulator trait, starting with relatively low foliar metal concentrations and associated competitive advantages over other plants with normal foliar concentrations has been hypothesised to lead to hyperaccumulation (Boyd 2012; Cheruiyot et al. 2013). The question arising from this hypothesis is why some Ni hyperaccumulators reach extremely high Ni concentrations (for example, 23,300 μg g−1 in Phyllanthus cf. securinegoides or >100-fold higher than MLC for the generalist insect herbivore in Cheruiyot et al. 2013), which presumably comes at an energetic cost. The answer could lie in a biogeochemical ‘arms race’ with Ni-tolerant insects (Boyd 2004, 2009).

Several specialist Ni-tolerant insects have been discovered that feed on Ni hyperaccumulators: for example, the insects Melanotrichus boydi (Schwartz and Wall 2001) and Chrysolina pardalina (Mesjasz-Przybylowicz and Przybylowicz 2001). Such specialised insects have co-evolved with Ni hyperaccumulators and adapted to increasing foliar Ni concentrations that are lethal for generalist insect herbivores. The field observation of widespread herbivory damage on Ni hyperaccumulators in Sabah, and the local discovery of specialist herbivores suggest that Ni-tolerant insects are perhaps a relatively common feature in the ultramafic flora of Sabah. Though in order to test this, controlled experiments are needed in which herbivory damage between two closely related species (a Ni hyperaccumulator and a non-hyperaccumulator) is carefully compared. Apart from scientific interest in the co-evolution of Ni hyperaccumulator plants and specialist insects, this could have economic consequences, because as indicated by Boyd (1998), potential future sites for phytomining might already harbour specialist Ni-tolerant herbivores that could pose a threat to the phytomining crop. Moreover, metal transfer over trophic levels could have ecosystem health implications. As such either mammals (wild animals such as deer or orang-utan, or domestic animals such as cattle) or invertebrates (such as insects) feeding on high-nickel plants could be exposed to Ni (Peterson et al. 2003; Miranda et al. 2009).

On a global scale, Ni hyperaccumulation occurs in approximately 400 species (about 40 families). This widespread phylogenetic occurrence of Ni hyperaccumulation indicates that the trait has independently evolved multiple times (Pollard 2000; Krämer 2010). Globally, 30 % of the Ni hyperaccumulators (and 83 % of those in New Caledonia) belong to the COM clade (Orders Celastrales, Oxalidales, Malpighiales) of the Rosids (Jaffré et al. 2013). In Sabah, Ni hyperaccumulation occurs most frequently in the Order Malpighiales (mainly the families Dichapetalaceae, Phyllanthaceae, Salicaceae, Violaceae), and is particularly common in the Phyllanthaceae (genera Phyllanthus, Glochidion). The genus Phyllanthus has most Ni hyperaccumulators worldwide, and is well represented in Sabah. In this genus, hybridization and introgression could be responsible for the frequent occurrence of the Ni hyperaccumulation trait, as virtually all species in this genus occurring on ultramafic soils in Sabah are Ni hyperaccumulators. Not infrequently, several Phyllanthus spp. co-occur in the same habitat. In this respect, the observation that certain Phyllanthus hybrids, for example P. x pallidus (P. discolor x orbicularis) from Cuba (Reeves et al. 1996) reach Ni hyperaccumulation far in excess of the parental species, is of particular interest.

Conclusion

With only approximately 10 % of the ultramafic flora of Sabah screened, it is expected that more Ni hyperaccumulators will be recorded in the near future, particularly in the order Malpighiales. The restriction of most hyperaccumulators species to ultramafic-derived soils, which can be mining targets, means that these species are both rare and threatened (Baker et al. 2010). It is therefore necessary that systematic screening and cataloguing of Ni hyperaccumulator plant species and other plants native to ultramafic ecosystems take place prior to any land clearing or Ni mining activities. Screening also supports strategies aimed at the preservation of hyperaccumulator germplasm, which is especially critical for local ecotype populations possessing enhanced hyperaccumulation traits. Unfortunately such screening is not part of any environmental impact assessment (EIA) undertaken on Ni mining leases in the region.

Although there are no current Ni mining activities in Sabah, there has been substantial loss of forest cover with >7000 km2 lost between 1990 and 2010 (Reynolds et al. 2011). Protected areas amount to approximately 8 % of the land surface in Sabah (Bryan et al. 2013). The recent establishment of the ‘Hyperaccumulator Botanical Garden’ in Sabah, under the auspices of Sabah Parks, serves as an example of ex situ preservation of hyperaccumulator germ plasm. Such preservation is essential if Ni hyperaccumulator species (and ecotype populations) are to be utilised in potential future Ni phytomining operations, aimed at growing such plants at agricultural scale to harvest Ni bio-ore (Van der Ent et al. 2013b). In the ‘Hyperaccumulator Botanical Garden’, living plants of all currently known Ni hyperaccumulator species from Sabah have been planted (10–20 plants for each species) in naturally occurring ultramafic soil.

Despite the richness of the ultramafic flora, very few studies have focussed on the occurrence of Ni hyperaccumulators in this region. As such, many important questions remain unanswered: for example, how Ni hyperaccumulators have evolved and whether this trait can be induced in facultative Ni hyperaccumulators such as Rinorea bengalensis by exposure to ultramafic soil? The richness not only in Ni hyperaccumulator species, but also in Ni-tolerant insects, indicates potentially rewarding avenues for future scientific research in Sabah.