X-ray fluorescence elemental mapping of roots, stems and leaves of the nickel hyperaccumulators Rinorea cf. bengalensis and Rinorea cf. javanica (Violaceae) from Sabah (Malaysia), Borneo Antony van der Ent, Martin D. de Jonge & Rachel Mak & Jolanta Mesjasz-Przybyłowicz & Wojciech J. Przybyłowicz & Alban D. Barnabas & Hugh H. Harris Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, Brisbane, Australia Australian Synchrotron, ANSTO, Melbourne, Australia School of Chemistry, University of Sydney, Sydney, Australia Department of Botany and Zoology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa Faculty of Physics & Applied Computer Science, AGH University of Science and Technology, al. Mickiewicza 30, 30-059 Kraków, Poland Materials Research Department, iThemba LABS, National Research Foundation, P.O. Box 722, Somerset West 7129, South Africa Department of Chemistry, The University of Adelaide, Adelaide, Australia 1 ABSTRACT Aims There are major knowledge gaps in understanding the translocation leading from nickel uptake in the root to accumulation in other tissues in tropical nickel hyperaccumulator plant species. This study focuses on two species, Rinorea cf. bengalensis and Rinorea cf. javanica and aims to elucidate the similarities and differences in the distribution of nickel and physiologically relevant elements (potassium, calcium, manganese and zinc) in various organs and tissues. Methods High-resolution X-ray fluorescence microscopy (XFM) of frozen-hydrated and fresh- hydrated tissue samples and nuclear microprobe (micro-PIXE) analysis of freeze-dried samples were used to provide insights into the in situ elemental distribution in these plant species. Results This study has shown that the distribution pattern of nickel hyperaccumulation is typified by very high levels of accumulation in the phloem bundles of roots and stems. In the leaves, nickel is preferentially located in epidermal cell region, whereas manganese is located mainly in the lower epidermis and zinc in the upper epidermis and palisade mesophyll. The abundant formation of calcium-oxalate crystals, lining the collenchyma, is a prominent feature of both Rinorea cF. bengalensis and Rinorea cF. javanica. Conclusions Future investigations on Rinorea cf. bengalensis and Rinorea cf. javanica should focus on unravelling the mechanisms of nickel uptake in the root, specifically targeting the identification of nickel specific membrane transporters. Keywords Agromining, Elemental mapping, Facultative hyperaccumulation, Micro-PIXE, nuclear microprobe, XFM 2 INTRODUCTION Hyperaccumulators are unusual plants that accumulate trace elements to extreme concentrations in their living tissues (Baker and Brooks 1989; van der Ent et al. 2013a). Some of these plants can reach up to 7.6 Wt% nickel in leaves (Mesjasz-Przybyłowicz et al. 2004) and up to 25 Wt% in phloem sap (Jaffré et al. 1976). Hyperaccumulator plants can achieve such extreme levels of foliar sequestration due to enhanced uptake and translocation mechanisms (Baker 1981, 1987). The hyperaccumulation phenomenon is extremely rare (exhibited by <0.2% of angiosperms) with ~70% of the known 700 hyperaccumulator species recorded for Ni (Reeves 2003; Reeves et al. 2017). Hyperaccumulator plants are found on all continents except Antarctica, in temperate and tropical biomass, with the greatest numbers found in New Caledonia, Cuba and the Mediterranean Region (van der Ent et al. 2013a). Nickel hyperaccumulators have been recorded from at least 40 different plant families (Reeves 2006). On a global scale, Ni hyperaccumulation is most prevalent in the order Brassicales (Brassicaceae, genera Alyssum, Arabidopsis, Noccaea, etc.) in temperate regions and in the Asterales (Berkheya, Pentacalia, Senecio), the Buxales (Buxaceae; Buxus) and the supraordinal COM clade (Celastrales, Oxalidales, Malpighiales, mainly Euphorbiaceae, Phyllanthaceae and Violaceae families) in tropical regions (Jaffré et al. 2013; Jaffré et al. 2018; Reeves 2003). Limited systematic screening across phylogenetic lineages means that at present there is no comprehensive understanding of the phenomenon, although such efforts are currently underway using handheld X-ray Fluorescence (XRF) devices (Gei et al. 2017; van der Ent et al. 2019a, b). Such an understanding is important for elucidating whether Ni hyperaccumulator species evolved from nonaccumulating ancestors, and whether the ability to grow on ultramafic soils is a prerequisite for this character to appear (Jaffré et al. 2013). Nevertheless, phylogenetic studies have been undertaken for a number of families and genera, which have shown different patterns. In some genera, Ni hyperaccumulating taxa occur as singularities in their genus, such as Stackhousia tryonii from Australia out of 33 taxa in this genus (Burge and Barker 2010), Streptanthus polygaloides from the USA out of 35 taxa in this genus (Reeves et al. 1981), and Psychotria gabriellae out of 60 taxa in this genus in New Caledonia (Baker et al. 1985). This contrasts with a number of genera that have numerous Ni hyperaccumulators, such as Alyssum sect. Odontarrhena (Mediterranean Region), Buxus (Cuba), Geissois (New Caledonia) and Phyllanthus (New Caledonia and elsewhere) (Jaffré et al. 1979; Kersten et al. 1979; Reeves et al. 1996; Cecchi et al. 2010; Jestrow et al. 2012). Hyperaccumulating plant species can be further subdivided in either ‘strict (or obligate) species and ‘facultative’ species (Baker et al. 2010). Obligate 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). One unusual ‘facultative’ species is Senecio coronatus (Asteraceae) which has genotypes that either hyperaccumulate or do not hyperaccumulate on ultramafic soils (Mesjasz-Przybyłowicz et al. 2007; 3 Meier et al. 2018). Another example of a ‘facultative’ hyperaccumulator is Rinorea bengalensis (Violaceae), which can accumulate up to 1.78 Wt% foliar Ni when growing on ultramafic soils, but < 0.03 Wt% foliar Ni when growing on ‘normal’ soils (Reeves 2003; van der Ent et al. 2013a). Approximately 85–90% of hyperaccumulator species are obligate to metalliferous (ultramafic) soils, and only 10–15% are facultative and occur on metalliferous and normal soils (Pollard et al. 2014). The diversity of Ni hyperaccumulator plants is widely distributed across phylogenies, suggestive that physiological processes of hyperaccumulation have evolved independently, and may therefore differ between species (Meier et al. 2018). Hence, the results reported from model plants in the Brassicaceae (Noccaea caerulescens, Alyssum murale) could essentially differ from tropical plants which remain understudied. The mechanisms that lead from root uptake to sequestration in the shoots (leaves) are incompletely understood, especially in tropical taxa. After the discovery of Ni hyperaccumulation in the ligneous shrub Hybanthus Floribundus (Violaceae) from Western Australia that accumulates up to 1.35 Wt% Ni (Severne and Brooks 1972; Cole 1973), two more hyperaccumulating Hybanthus species were discovered in New Caledonia, H. austrocaledonicus (accumulating up to 2.55 Wt% Ni) and H. caledonicus (accumulating up to 1.75 Wt% Ni) (Brooks et al. 1974; Kelly et al. 1975). Hybanthus enneaspermus from Sri Lanka accumulated up to 1860 μg g-1 Ni (Rajakaruna and Bohm 2002). Another Violaceae in New Caledonia, Agatea longipedicellata accumulates up to 2500 μg g-1 Ni (Jaffré 1980; Boyd and Jaffré 2009). Brooks and coworkers then set out to test other Violaceae for nickel accumulation and discovered that Rinorea bengalensis accumulated up to 1.75 Wt% Ni (Brooks and Wither 1977). It was noted that this species accumulates relatively high Ni concentrations even from non-ultramafic soils (e.g., 500 μg g-1 foliar Ni when growing on limestone). In addition, they observed that Rinorea is unusual in tending to accumulate more cobalt than nickel. Further analysis of Ni and Co in 181 herbarium specimens covering 70 Rinorea species also revealed another species, Rinorea javanica (Bl.) Kuntze, as a Ni hyperaccumulator, with up to 2170 μg g-1 in the leaves and, in addition, up to 670 μg g-1 Co (Brooks et al. 1977a, b). It is noteworthy that none of the other numerous species of Rinorea occurring around the world, other than the Southeast Asian species, were hyperaccumulators. Another, as yet unnamed Ni hyperaccumulating Rinorea species has been reported from Mt Piapi on Karakelong Island, northeast of Sulawesi in Indonesia with up to 1830 μg g-1 Ni (Proctor et al. 1994). More recently, R. niccoliFera has been described from Luzon in the Philippines where it accumulates up to 1.8 Wt% Ni (Fernando et al. 2014). Most recently, the elemental distribution and chemical form of Ni was determined in R. bengalensis from Sabah, Malaysia (van der Ent et al. 2017). The Violaceae occur in temperate and tropical regions, with Viola (~400 spp.) the largest genus, followed by Rinorea (~200 spp.), Hybanthus (~70 spp.) and ~50 spp. in other genera (Jacobs and Moore 1971). Rinorea is a pan-tropical genus of forest shrubs and trees. It is the second most species-rich genus in the family after Viola, with an estimated total of 225–275 species throughout the tropics. In the Malesian region, 11 species occur with