A systematic assessment of the occurrence of trace element hyperaccumulation in the flora of New Caledonia Vidiro Gei1, Sandrine Isnard2,3, Peter D. Erskine1, Guillaume Echevarria1,4, Bruno Fogliani5, Tanguy Jaffré2,3, Antony van der Ent1,4* 1Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, St Lucia, QLD 4072, Australia 2botAnique et Modelisation de l’Architecture des Plantes et des végétation (AMAP), Université Montpellier, IRD, CIRAD, CNRS, INRA, Montpellier, France 3botAnique et Modelisation de l’Architecture des Plantes et des végétation (AMAP), IRD, Herbier de Nouvelle-Calédonie, Nouméa, New Caledonia 4Laboratoire Sols et Environnement, Université de Lorraine – INRAE, F54000 Nancy, France 5Équipe ARBOREAL (AgricultuRe BiOdiveRsité Et vALorisation), Institut Agronomique néo-Calédonien (IAC), 98890 Païta, New Caledonia *Corresponding author. E-mail: [email protected] ABSTRACT New Caledonia is a global biodiversity hotspot known for its metal hyperaccumulator plants. X-ray fluorescence technology (XRF) has enabled non-destructive and quantitative determination of elemental concentrations in herbarium specimens from the ultramafic flora of the island. Specimens belonging to six major hyperaccumulator families (Cunoniaceae, Phyllanthaceae, Salicaceae, Sapotaceae, Oncothecaceae and Violaceae) and one to four specimens per species of the remaining ultramafic taxa in the herbarium were measured. XRF scanning included a total of c. 11 200 specimens from 35 orders, 96 families, 281 genera and 1484 species (1620 taxa) and covered 88.5% of the ultramafic flora. The study revealed the existence of 99 nickel hyperaccumulator taxa (65 known previously), 74 manganese hyperaccumulator taxa (11 known previously), eight cobalt hyperaccumulator taxa (two known previously) and four zinc hyperaccumulator taxa (none known previously). These results offer new insights into the phylogenetic diversity of hyperaccumulators in New Caledonia. The greatest diversity of nickel hyperaccumulators occur in a few major clades (Malphigiales and Oxalidales) and families (Phyllanthaceae, Salicaceae, Cunoniaceae). In contrast, manganese hyperaccumulation is phylogenetically scattered in the New Caledonian flora. KEYWORDS: cobalt – Cunoniaceae – manganese – nickel – Oncothecaceae – Phyllanthaceae – Salicaceae – Sapotaceae – ultramafic – Violaceae – zinc. INTRODUCTION Ultramafic soils and hyperaccumulator plants Ultramafic soils derive from mantle rock consisting largely of magnesium-iron-silicate minerals. These soils have relatively high concentrations of the trace elements nickel (Ni), cobalt (Co) and chromium (Cr), but at the same time have cation imbalances as a result of high magnesium (Mg) and low calcium (Ca) (Proctor, 2003; Echevarria, 2018). Ultramafic outcrops contribute disproportionately to regional plant diversity throughout the globe and especially in biodiversity hotspots including California, Cuba, Borneo and New Caledonia (Jaffré, 1992; Borhidi, 1992; Harrison et al., 2006; van der Ent, Erskine & Sumail, 2015a; van der Ent et al., 2015b, d, 2016; Isnard et al., 2016; Galey et al., 2017). The chemical and physical adversity of ultramafic soils is associated with major transformations and adaptations of some plants, such as accumulation of trace elements at extremely high concentrations in their leaves known as ‘hyperaccumulation’ (Brooks et al., 1977; van der Ent et al., 2012; Reeves, van der Ent & Baker, 2018b). Although ultramafic vegetation is often highly species-rich, plants that hyperaccumulate trace elements are comparatively rare. Current estimates suggest that hyperaccumulation occurs in < 0.2% of angiosperms (Baker & Brooks, 1989; Reeves, 2003) and in 1–2% of the ultramafic flora (van der Ent et al., 2015c). Worldwide there are at least 523 Ni, 53 copper (Cu), 42 Co, 20 zinc (Zn) and 42 manganese (Mn) hyperaccumulators known to date (Baker & Brooks, 1989; Reeves, 1992; 2003; van der Ent et al., 2012; Reeves et al., 2018a; Reeves, van der Ent & Baker, 2018b). Most known hyperaccumulator plants hyperaccumulate Ni; however, the disparity in the number of known Ni hyperaccumulator taxa in comparison to the numbers of hyperaccumulators that accumulate other elements is due to the availability of an initial field-testing method using dimethylglyoxime (DMG)-treated paper (Gambi, 1967; Gei et al., 2018) and because of the presence of ultramafic soils worldwide which are enriched in Ni (Brooks, 1987; Echevarria, 2018). A small portion of plants growing on ultramafic soil hyperaccumulate trace elements; however, most plants are metal excluders, i.e., they maintain a low and relatively constant concentration of trace elements in their tissues when compared to concentrations in the soil (Baker, 1981). Hyperaccumulation involves high metal concentration in plant tissues especially in the leaves (van der Ent et al., 2012), implying extremely high physiological tolerance to specific elements. As such, plant species growing on ultramafic soils show a gradient in physiological responses than can lead to the accumulation of variable concentrations of metal in their tissues. Threshold values for hyperaccumulation have been established as an order of magnitude higher than the “normal” concentration in plants (Jaffré & Schmid, 1974; Brooks et al., 1977). In a more recent review, van der Ent et al. (2012) used the criteria of: (i) two to three orders of magnitude higher than plants on normal soils, and (ii) one order of magnitude higher than in typical plants on metalliferous soils. This concept was applied to all elements, not just Ni. For the main metals that have been studied in plants, the hyperaccumulator thresholds are: 300 µg G-1 for Co, Cu and Cr, 1000 µg G-1 for Ni, 3000 µg G-1 for Zn and 10 mg G-1 for Mn (Reeves, 2003; Reeves et al., 2018a; van der Ent et al., 2012) (Table 1). Plants that accumulate >10 000 µg G-1 of Ni have been further referred to as ‘hypernickelophores’ (Jaffré & Schmid, 1974). The phylogenetic distribution of metal hyperaccumulation indicates repeated evolution of the trait during plant evolution (Krämer, 2010; Jaffré et al., 2013; Cappa & Pilon-Smits, 2014). Such repeated independent evolution is suggestive of an adaptive benefit of hyperaccumulation, although selective advantages are probably diverse and depend on the metal, species physiology, phylogeny and ecology (Boyd, 2004; Reeves et al., 2018a). In New Caledonia, hyperaccumulation is particularly common in a few clades, which are over- represented in the native flora (Pillon et al., 2010; Jaffré et al., 2013). As such, the disharmony of the New Caledonian flora, as observed in many island floras, has been suggested to be strongly driven by preadaptation to ultramafic soils (Pillon et al., 2010). As hyperaccumulation is a complex trait, a better understanding of the evolution of this phenomenon requires a broad knowledge on the phylogenetic diversity of hyperaccumulator plants. the Ultramafic flora of New Caledonia and hyperaccumulator plants New Caledonia is an archipelago located in the south- western Pacific and is a renowned global biodiversity hotspot (Myers et al., 2000; Mittermeier et al., 2004). The total land area of the island is approximately 19 000 km2 and it harbours an exceptional flora of c. 3300 vascular species with c. 75% endemism (Morat et al., 2012; Pillon, Barrabé & Buerki, 2017). In New Caledonia, ultramafic outcrops cover about one-third of the main island (Grande Terre), including the islands of Belep and des Pins (totalling c. 5600 km2) (Pelletier, 2006). New Caledonia is recognized as a global hotspot of Ni hyperaccumulator plants with 65 Ni and 11 Mn hyperaccumulator plant species currently recorded (Jaffré et al., 2013; Losfeld et al., 2015a). Approximately 70% of the currently known Ni hyperaccumulators in New Caledonia were discovered between 1974 and 1980. In 1976, Pycnandra acuminata (Pierre ex Baill.) Swenson & Munzinger [formerly Sebertia acuminata (Pierre ex Baill.) Engl.] was discovered to have the highest ever recorded concentration of Ni in a living organism (257.4 mg G-1 or 25.74 percent by weight (wt%) in the latex) (Jaffré et al., 1976, 2018). Considerable interest was generated in New Caledonia to discover more hyperaccumulators, most of which were discovered in 1979 in Cunoniaceae, Phyllanthaceae and Salicaceae (Jaffré, Brooks & Trow, 1979a; Jaffré et al., 1979b; Kersten et al., 1979) and in 1980 from Argophyllaceae, Celastraceae, Cunoniaceae, Phyllanthaceae and Violaceae (Jaffré, 1980). The remainder of the recorded Ni hyperaccumulators were discovered in 2007 and 2013 (Amir et al., 2007; Jaffré et al., 2013). Apart from the Ni hyperaccumulators, New Caledonia also hosts 11 Mn hyperaccumulators; the first discovered in 1977 was Denhamia fournieri (Pancher & Sebert) M.P.Simmons subsp. fournieri [formerly Maytenus bureaviana (Loes.) Loes.] in Celastraceae and Alyxia poyaensis (Boiteau) D.J.Middleton (formerly Alyxia rubricaulis (Baill.) Guillaumin subsp. poyaensis Boiteau) in Apocynaceae (Jaffré, 1977). These were followed by discoveries of three Mn hyperaccumulators in the Proteaceae (Jaffré, 1979), Garcinia amplexicaulis Vieill. ex Pierre in Clusiaceae, Gossia clusioides (Brongn. & Gris) N.Snow var. ploumensis (Däniker) N.Snow comb. ined. in Myrtaceae (Jaffré, 1980) and more recently Polyscias pancheri (Baill.) Harms in Araliaceae, Gossia diversifolia (Brongn. & Gris) N.Snow in Myrtaceae and Grevillea meisneri Montrouz. in Proteaceae (Losfeld et al., 2015a). Herbarium