Plant Science 217–218 (2014) 8–17

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Plant Science journal homepage: www.elsevier.com/locate/plantsci

Review

Facultative hyperaccumulation of heavy metals and metalloids A. Joseph Pollard a,∗ , Roger D. Reeves b , Alan J.M. Baker c a

Department of Biology, Furman University, Greenville SC 29613, USA Palmerston North, New Zealand c School of Botany, The University of Melbourne and Centre for Mined Land Rehabilitation, University of Queensland, Australia b

a r t i c l e

i n f o

Article history: Received 16 August 2013 Received in revised form 15 November 2013 Accepted 16 November 2013 Available online 23 November 2013 Keywords: Hyperaccumulation Facultative Constitutive Pseudometallophyte Serpentine Metal tolerance

a b s t r a c t Approximately 500 species of plants are known to hyperaccumulate heavy metals and metalloids. The majority are obligate metallophytes, species that are restricted to metalliferous soils. However, a smaller but increasing list of plants are “facultative hyperaccumulators” that hyperaccumulate heavy metals when occurring on metalliferous soils, yet also occur commonly on normal, non-metalliferous soils. This paper reviews the biology of facultative hyperaccumulators and the opportunities they provide for ecological and evolutionary research. The existence of facultative hyperaccumulator populations across a wide edaphic range allows intraspecific comparisons of tolerance and uptake physiology. This approach has been used to study zinc and cadmium hyperaccumulation by Noccaea (Thlaspi) caerulescens and Arabidopsis halleri, and it will be instructive to make similar comparisons on species that are distributed even more abundantly on normal soil. Over 90% of known hyperaccumulators occur on serpentine (ultramafic) soil and accumulate nickel, yet there have paradoxically been few experimental studies of facultative nickel hyperaccumulation. Several hypotheses suggested to explain the evolution of hyperaccumulation seem unlikely when most populations of a species occur on normal soil, where plants cannot hyperaccumulate due to low metal availability. In such species, it may be that hyperaccumulation is an ancestral phylogenetic trait or an anomalous manifestation of physiological mechanisms evolved on normal soils, and may or may not have direct adaptive benefits. © 2013 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

3. 4.

5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Defining and describing hyperaccumulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Biogeographic patterns: obligate and facultative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. How common is facultative hyperaccumulation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of research on facultative hyperaccumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ecophysiology of facultative hyperaccumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Is hyperaccumulator physiology population-specific or a species-wide trait? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Patterns of variation and environmental correlates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Facultative hyperaccumulators as physiological and genetic models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution of facultative hyperaccumulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +1 864 294 3244; fax: +1 864 294 2058. E-mail address: [email protected] (A.J. Pollard). 0168-9452/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2013.11.011

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A.J. Pollard et al. / Plant Science 217–218 (2014) 8–17

1. Introduction The interaction of plants with toxic metal and metalloid elements in soils has been used as a productive model for physiological, ecological, genetic and evolutionary research for over half a century. Although many such elements are essential micronutrients, most are toxic at high concentrations. The early studies in this field focused on tolerance mechanisms that allow some plants to grow in metal-contaminated soils where most species cannot survive [1]. More recent interest has centered on a small subset of these plants that not only tolerate metals, but also concentrate them to exceptional concentrations in their leaves, the phenomenon of hyperaccumulation [2,3]. Both tolerance and hyperaccumulation may have commercial applications, whether for revegetation of contaminated soils (phytostabilization), for extraction of metals for their intrinsic value (phytomining), or for plant-based remediation of polluted soils (phytoextraction) [4,5]. This review focuses on metal hyperaccumulator species that occur naturally on both metalliferous and non-metalliferous soils. Such species may broadly be described as facultative hyperaccumulators, a classification which will be explained more fully in Section 2.1. Facultative hyperaccumulators make up a minority of the known hyperaccumulator species and in general have been poorly studied, yet they also include a few of the most thoroughly investigated research models for metal tolerance and uptake. This paper does not attempt to comprehensively describe the biology of hyperaccumulation or its underlying genetics and physiology, which have been surveyed in other recent reviews [e.g. 2,3,6,7]. Instead, we will concentrate on the unique features of facultative hyperaccumulators. We will argue that ecological and evolutionary hypotheses regarding hyperaccumulating plants are expected to depend strongly on the biogeographic patterns of hyperaccumulator distribution, whether obligately restricted to metalliferous soils, primarily on metalliferous soils and occasionally on other substrates, or primarily on non-metalliferous soils but occasionally on metalliferous ones where they hyperaccumulate.

2. Defining and describing hyperaccumulators A recent critique [7] has attempted to clarify, refine and update the definition of metal hyperaccumulation. To paraphrase its conclusions, a hyperaccumulator can be defined as a plant whose leaves contain a metallic element at a concentration exceeding a specified threshold, when growing in nature (not in experimental cultivation). The threshold concentration should be 2–3 orders of magnitude higher than in leaves of most species on normal soils, and at least one order of magnitude greater than the usual range found in plants from metalliferous soils. Based on these concepts, the proposed nominal threshold criteria (all in units of ␮g metal per g of dry leaf tissue) are: 100 for Cd, Se and Tl; 300 for Co, Cr and Cu; 1000 for As, Ni and Pb; 3000 for Zn and 10,000 for Mn [7]. High concentrations of Al have also been reported in several species [8] but it is not clear that accumulation of major soil elements is a comparable phenomenon to hyperaccumulation of trace elements [7], so it will not be considered further in this review. The stipulation that these threshold concentrations should occur in plants growing in nature does not exclude weeds of anthropogenic habitats, but does imply that hyperaccumulators must possess sufficient metal tolerance to maintain a self-sustaining population in metalliferous soils. Based on the above criteria, approximately 500 taxa have been proposed as hyperaccumulators of one or more metals; thus, hyperaccumulation is a rare phenomenon known in less than 0.2% of the world’s inventory of vascular plants [7]. Of the known hyperaccumulators, a huge majority (>450 taxa) are hyperaccumulators

9

of nickel, generally occurring on serpentine (ultramafic) soils. Documented cases of hyperaccumulation of other metals are much rarer. This rarity, combined with their potential for practical application and commercial value, is a strong justification for basic biological studies. 2.1. Biogeographic patterns: obligate and facultative Hyperaccumulators make up a subset of the category known as metallophytes, plants that occur on metal-enriched soils. Early studies on such plants generated a confusing, redundant and overlapping terminology to describe the biogeographic distribution of metallophytes [1,9,10]. Species that occur exclusively on metalliferous soils have been variously described by the terms indicator, endemic, bodenstet, eumetallophye, absolute metallophyte, strict metallophyte, or obligate metallophyte. Species known to occur on both metalliferous and non-metalliferous soils have been termed bodenvag (meaning “soil-wandering”; alternative spellings include bodenwag and bodenwaag) species, pseudometallophytes and facultative metallophytes. We will consistently employ the adjectives obligate and facultative when describing whether or not a species is restricted to metalliferous soils. For the sake of simplicity, we will use the phrase “normal soils” to imply those which are not unusually enriched in metallic elements, recognizing that this is a gross oversimplification of potential variations in soil structure, texture, chemistry and hydration unrelated to metal concentration. One early attempt at ecological classification of metallophytes further subdivided the facultative species into three categories: (a) “elective pseudometallophytes”, occurring primarily and with greatest vigor on metalliferous soils; (b) “indifferent pseudometallophytes”, growing indiscriminately and equally well on metalliferous and normal soils; and (c) “accidental pseudometallophytes”, represented by weeds and ruderals appearing sporadically and with reduced vigor on metal-contaminated soils [1,8]. Although this classification has not been widely adopted, it serves as a reminder that the single term “facultative” encompasses a range of biogeographic patterns, ecological relationships and physiological responses. Most plants that survive on metalliferous soils do so by excluding metals from their shoots [2,7]. In contrast, hyperaccumulators make up only a small subset of metallophytes. Nonetheless, the terms obligate and facultative can be applied to hyperaccumulators to describe their fidelity to metalliferous soils. Because the phrase “hyperaccumulators that are facultative metallophytes” is long and unwieldy, we propose the more compact label “facultative hyperaccumulators”, the full meaning of which will be explored throughout this review. One of the first publications to describe facultative hyperaccumulation was a study of Rinorea bengalensis from Southeast Asia [11], and the first to systematically categorize obligate and facultative hyperaccumulators across a whole flora appears to have been a series of botanical surveys on the extensive serpentine soils of Cuba [12,13, and references therein]. As with facultative metallophytes in general, the distributions of facultative hyperaccumulators are expected to vary, from species that occur primarily on metalliferous soils, to species that rarely do. A somewhat different type of variation is occasionally found, in which a species growing on metalliferous soils includes some populations or genotypes that hyperaccumulate metals, and others that do not. Possible examples include Senecio coronatus [14] and Pimelea leptospermoides [15]. This phenomenon might be termed “erratic hyperaccumulation”. Because the plants involved are all obligate metallophytes, we regard it as fundamentally different from facultative hyperaccumulation and will not discuss it further in this review. Little is known about why it occurs, in any case. Some studies have claimed to identify hyperaccumulator species that are not metallophytes at all, in that they are not

A.J. Pollard et al. / Plant Science 217–218 (2014) 8–17

Foliar Zn (ug/g)

0.09

8000 0.06

6000 4000

0.03

2000 0 40000

Foliar Zn (µg/g)

Shoot Mass (g)

0

25

50

75

100

0 150 0.05

125

0.04

30000

0.03

20000

0.02

10000 0

0.01 0

200

400

600

800

Shoot Mass (g)

Foliar Zn (µg/g)

10000

Shoot Mass (g)

10

0 1000

Solution Zn Concentration (µM) Fig. 1. Comparison of metal “breakthrough” in Lactuca sativa with genuine hyperaccumulation in Noccaea caerulescens. The upper panel shows responses of L. sativa to hydroponic zinc concentrations ranging from 0.2 to 150 ␮M in 10% Hoagland’s solution. Foliar zinc concentrations reached the hyperaccumulation threshold of 10,000 ␮g g−1 in the 150 ␮M treatment, but shoot mass was drastically reduced and symptoms of toxicity were observed in the plants. The lower panel shows responses of N. caerulescens under identical conditions but with solution concentrations up to 1000 ␮M. Hyperaccumulation was observed in zinc treatments where shoot mass was equal to or greater than the 0.2 ␮M control. In the highest zinc treatment there was some reduction in shoot mass compared to the control, but the difference was not statistically significant. (Pollard, previously unpublished data).

documented to occur on metalliferous soil or to hyperaccumulate in nature. This appears to be the case for cadmium hyperaccumulation in species of Allium, Amaranthus, Iris, Lonicera, Rorippa, Salsola and Solanum to give just a few examples. Given the spectrum of biogeographic distribution patterns discussed earlier, with metalliferous populations ranging from common to rare, it might seem that these examples are merely at the extreme “rare” end of the spectrum, so much so that the species in fact never occurs naturally on metalliferous soils. However, it has been forcefully argued that plants that take up high concentrations of metals only when cultivated in artificial hydroponic solutions or “metal-spiked” soils should not be considered hyperaccumulators if they do not exhibit hyperaccumulation in natural populations [7]. Many, perhaps most, plants can be forced take up high concentrations of metals under laboratory conditions, albeit with the consequence of strongly reduced growth (Fig. 1), potentially resulting in reproductive failure or mortality. This does not represent genuine hyperaccumulation but is instead a non-specific “breakthrough” of metals into the shoot system, as the plant’s mechanisms of metal exclusion and homeostasis are compromised by metal toxicity [6,7]. Even if a non-metallophyte species is reported to possess high levels of both metal tolerance and metal accumulation, these traits could not have evolved in response to selection pressures imposed by high metal concentrations if the species does not grow on metalliferous soils. Although these plants may have interesting physiology and even some potential for phytoremediation, they are not necessarily the physiological, ecological, or evolutionary equivalents to plants that hyperaccumulate in the field.

on both metalliferous and normal soil. These estimates are at best rough approximations for reasons discussed in the next paragraph. Precise counts of facultative versus obligate hyperaccumulation are problematic because of several issues. First, there may be uncertainties related to basic taxonomy whenever new hyperaccumulators are discovered. A recent survey of serpentine plants in Brazil [16] found at least 40 previously unrecognized hyperaccumulators; however, only 13 of them could be confidently identified to species. The others may represent species new to science, not yet described or named, much less characterized in terms of geographic and edaphic distribution. Secondly, even for species whose classification and nomenclature are generally accepted, there are surely many cases where the full distribution is not known, including metallophytes whose existence on normal soils has not yet been recorded, and conversely species thought to be non-accumulators that may eventually be found somewhere to hyperaccumulate. Another taxonomic issue arises in cases where there is disagreement regarding the classification and nomenclature of local metallophyte populations, which will directly affect their categorization as facultative or obligate. For example, the standard taxonomic reference for continental Europe [19] regards the Iberian crucifer Alyssum serpyllifolium as widespread on both limestone and serpentine soils and recognizes no sub-specific taxa. Serpentine populations are known to hyperaccumulate Ni [20], so the species would thus be considered a facultative hyperaccumulator. However, in other taxonomic works the serpentine plants have been segregated as subspecies [21] or distinct species [22,23], under which treatment they would be regarded as obligate hyperaccumulators. In addition to these genuinely problematic issues of taxonomy and biogeography, there are simple cases of errors in identification or locality, especially on herbarium specimens. Finally, chemical analyses of leaves are subject to contamination and error [7,24]. Much basic, descriptive research is still ongoing, as documented by the ever-increasing list of proposed hyperaccumulators [7]. Despite the limitations described in the previous paragraph, it is revealing to compare the frequency of obligate and facultative hyperaccumulators in different regional floras. This comparison can meaningfully be made only in cases where large numbers of hyperaccumulators have been surveyed, as in studies of Ni hyperaccumulators of the serpentine soils in New Caledonia [25], Cuba [12,13], Turkey [17] and Brazil [16]. Collectively these four regions contain 281 hyperaccumulating taxa, or over half of all the hyperaccumulators currently known in the world. The relative numbers of obligate and facultative hyperaccumulators in each region are shown in Fig. 2. In all regions, obligate hyperaccumulators outnumbered facultative ones; however, there were large differences from one region to another. The data suggest a trend for a higher proportion of obligate hyperaccumulators in island floras (especially a small, remote island like New Caledonia), and a higher occurrence of facultative hyperaccumulation in mainland floras. It would be interesting to test the hypothesis that facultative hyperaccumulation is more common in mainland floras than in isolated islands. At present there are not sufficient data to draw conclusions with confidence, but hopefully more areas will be explored and more uncertainties resolved in the future. A recent study examining the general phenomenon of serpentine endemism (with no reference to hyperaccumulation) did not observe clear patterns of differential endemism in this regard, instead finding both endemic-rich and endemic-poor examples on islands and mainlands [26].

2.2. How common is facultative hyperaccumulation? Published estimates suggest that 85–90% of hyperaccumulator species are obligate endemics to metalliferous soils [16–18], especially soils derived from serpentine (ultramafic) rocks. Accordingly, as few as 10–15% of hyperaccumulators are facultative and occur

3. Analysis of research on facultative hyperaccumulation A summary of published research on facultative hyperaccumulators is presented in Table 1. We have attempted to be as

A.J. Pollard et al. / Plant Science 217–218 (2014) 8–17

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Table 1 Facultative hyperaccumulators, grouped by the metals known to be hyperaccumulated, with an analysis of the types of research that have been published on each. Type I studies are those that experimentally compare populations from metalliferous and normal soils, through cultivation in a common environment or comparisons of genetics, biochemistry, physiology, etc. Type II studies are those that report nickel concentrations in field-collected or herbarium specimens from both metalliferous and normal soils. Type III studies include other publications that focus on a facultative hyperaccumulator without making comparisons between metalliferous and non-metalliferous populations. Species ranges are based on the cited publications with additional information from standard regional floras and online searches of herbarium records from the Missouri Botanical Garden (MO), New York Botanical Garden (NYBG), Kew Gardens (K), and the Australian Virtual Herbarium (AVH). Metal

Species

Family

Range

I

II

III

Zn/Cd

Brassicaceae

[88]

>80 studies

Crassulaceae Pteridaceae

Mn/Cd

Phytolacca americana

Phytolaccaceae

Co

Nyssa sylvatica

Nyssaceae

Tl

Biscutella laevigata

Brassicaceae

Ni/Zn

Brassicaceae

Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni

Noccaea goesingense (=Thlaspi goesingense) Noccaea ochroleuca (=Thlaspi ochroleucum) Alyssum bracteatum Alyssum inflatum Alyssum longistylum Alyssum murale s.l. Alyssum peltarioides Alyssum penjwinensis Alyssum serpyllifolium s.l. Alyssum sibiricum Centaurea spicata Commelina ensifolia Chionanthus domingensis

Mts. of C. Europe to S.W. Alps S, W, and C Europe E to Poland and Balkans SE China, Korea, Japan Widespread in old-world subtropics & tropics E. United States and invasive worldwide E. United States and Mexico C. and S. Europe and N. to Belgium Balkan Peninsula and E.C. Europe E. Europe, Turkey

[34,35,58,70,85,86]

Zn/Cd As

Arabidopsis halleri (=Cardaminopsis halleri) Noccaea caerulescens (=Thlaspi caerulescens) Sedum alfredii Pteris vittata

Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae Asteraceae Commelinaceae Oleaceae

[94]

Ni

Evolvulus alsinoides

Convolvulaceae

Ni

Heliotropium salicoides

Boraginaceae

Ni

Hybanthus enneaspermus

Violaceae

Ni Ni

Acanthaceae Brassicaceae

Ni

Justicia lanstyakii Noccaea fendleri ssp. glauca (=Thlaspi montanum var. montanum) Ouratea nitida

Iran and Caucasus Iran and Caspian region Iran, Turkey, and Caucasus Balkans, Turkey, Caucasus Turkey Iran, Iraq, Turkey Portugal, Spain, S. France Turkey to Siberia Turkey India, Sri Lanka, Australia S. Mexico to Panama and E. to Puerto Rico Widespread: Americas, Africa, Asia, Australia Brazil, Paraguay, Bolivia, Colombia Trop. Africa, Asia, Australia South America North America from Pacific to Rocky Mts.

Ni Ni Ni

Ouratea striata Phyllanthus incrustatus Psychotria costivenia

Ochnaceae Euphorbiaceae Rubiaceae

Ni

Psychotria grandis

Rubiaceae

Ni

Rubiaceae

Ni Ni

Psychotria viridis (=P. glomerata) Rostellularia adscendens Rinorea bengalensis

Ni

Ruellia geminiflora

Acanthaceae

Ni

Sida linifolia

Malvaceae

Ni

Turnera subnuda

Turneraceae

Zn/Cd/Ni

Ni/Zn

Brassicaceae

Brassicaceae

Ochnaceae

Acanthaceae Violaceae

Publications by type

S. Mexico to Costa Rica and E. to Cuba Cuba and Puerto Rico Cuba S. Mexico to Costa Rica and E. to Cuba Guatemala to Ecuador and E. to Puerto Rico Nicaragua to Bolivia and E. to Cuba Northern 2/3 of Australia S.E. Asia from Sri Lanka to Solomon Is. C. Mexico to Paraguay and Brazil Mexico to Paraguay, Caribbean and C. Africa Brazil

comprehensive and accurate as possible, given the uncertainties in distinguishing between facultative and obligate hyperaccumulators discussed in the previous section. The table is not simply a catalog of publications, but is intended to permit analysis of how research has been distributed across taxonomic groups, geographic areas, and metals. In order to evaluate the amount of research focused on the pheomenon of facultative hyperaccumulation itself, we have classified the published literature into three categories:

[31–33,51–53,55–57,71,89,90]

>200 studies

[40–48] [38,43]

>70 studies >130 studies

[36,37,80]

>15 studies [91]

[92]

[49] [29]

>15 studies [93] [95] [96] [96] [17,97] [17] [20]

[20,21,39]

>30 studies

[22,23] [17] [17] [15] [13,15] [62] [16] [62]

[30]

[98]

[16] [99]

[13] [13,15] [12] [13] [15,77] [13] [15] [11] [16] [16] [16]

• Type I studies represent controlled experimental research focused on the biology of facultative hyperaccumulation. Examples include measurement of metal uptake and tolerance in conspecific plants from metalliferous and normal soils when grown in a common environment, comparisons of the biochemical or physiological properties of such populations, or genetic analysis of controlled crosses between plants from metalliferous and normal soils.

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A.J. Pollard et al. / Plant Science 217–218 (2014) 8–17

Number of hyperaccumulator species

140 120 Facultave 100

Uncertain Obligate

80 60 40 20 0 New Caledonia

Cuba

Turkey

Brazil

Fig. 2. Relative frequency of obligate and facultative hyperaccumulators of nickel, in extensive surveys of four serpentine regions. Facultative hyperaccumulators are known to hyperaccumulate nickel when occurring on serpentine soil, but are also known to occur on non-metalliferous soils. Classification as facultative or obligate is based on information in the original reports, supplemented by examination of species ranges in online herbarium databases of the Missouri Botanical Garden (MO), New York Botanical Garden (NYBG) and Royal Botanical Garden at Kew (K). The large uncertainty in categorization of Brazilian species results from unclear taxonomy, including tentative identifications, unknown species ranges, and discovery of previously un-named species of hyperaccumulators, as discussed in the text. References for data: New Caledonia [25]; Cuba [12,13]; Turkey [17]; Brazil [16].

• Type II papers are generally descriptive studies based on fieldcollected samples. Typically they report metal concentrations in plant materials collected in the field from populations on metalliferous and normal soils. Such studies can document the existence of facultative hyperaccumulation and the range of phenotypes it produces, but cannot reveal whether differences in metal concentrations result from environmental causes or biological differences among plants. Ideally such studies also include analyses of soil chemistry (metal concentration, pH, etc.) but in many cases they do not. Some are based on analysis of material removed from herbarium specimens. • Type III studies examine some aspect of hyperaccumulation but do not make any comparisons between plants originating from metalliferous and normal soils. We include them in the table only if the hyperaccumulator species is known to occur on normal soils in addition to metalliferous ones, or can reasonably be inferred to do so based on its geographic range. For species that have been extensively researched as physiological models or potential phytoremediation crops, an approximate number of Type III studies (based on database searches) is listed without citing specific sources. The great majority of publications in Table 1 (over 550 across all species) are Type III studies. This indicates that most of the research using facultative hyperaccumulators is not actually focused on their facultative nature, per se. It is immediately apparent from Table 1 that research focused on the biology of facultative hyperaccumulation has concentrated disproportionately on a few species and metals. Type I studies making experimental comparisons of tolerance and accumulation in facultative hyperaccumulators grown under uniform conditions have only been published for 10 of the 36 species, and 7 of those 10 belong to one family, the Brassicaceae. Although approximately 90% of all cases of hyperaccumulation involve Ni, very few of the Type I and Type II studies have examined Ni hyperaccumulators, focusing instead on hyperaccumulation of Zn, Cd and to a lesser

extent As. There are several understandable reasons for this emphasis. For any research in molecular biology, there are advantages to studying members of the Brassicaceae because of genetic similarity to Arabidopsis thaliana, for which a complete genome sequence is available, along with many other laboratory and bioinformatic tools [27]; this is especially true for the congeneric hyperaccumulator A. halleri. Small, herbaceous plants that are easily cultivated and cross-pollinated are practical subjects for genetic and physiological research; many Brassicaceae have these characteristics. Cadmium and arsenic are widespread and toxic pollutants, thus generating strong interest in their phytoremediation. All these factors, along with the pragmatism of proximity to major population centers, combine to create a strong attraction toward studies of temperate-zone hyperaccumulators of Zn and Cd, especially in the family Brassicaceae. However, as indicated in Table 1 and pointed out elsewhere [15], hyperaccumulation is in fact most common in tropical plants growing on serpentine soils, in a wide range of families. Moving toward a general understanding of the phenomenon of facultative hyperaccumulation will require expanding the geographic and phylogenetic foundation on which inferences are based. 4. Ecophysiology of facultative hyperaccumulation Facultative hyperaccumulators afford opportunities for research on questions that cannot be addressed in the majority of hyperaccumulators, which are obligate metallophytes. Perhaps the most fundamental question is whether the physiological ability to tolerate and accumulate metals occurs only in plants growing on metalliferous soils, or whether it is a universal property of the species. The latter has often been described as constitutive hyperaccumulation; however, this phrase may potentially cause confusion because the term constitutive is also commonly used in a biochemical sense in the literature on hyperaccumulation, to indicate continuous or non-inducible expression of a gene [e.g. 28]. Therefore, we prefer to describe hyperaccumulation in these cases as a “species-wide” trait [6]. 4.1. Is hyperaccumulator physiology population-specific or a species-wide trait? Pioneering studies with two facultative hyperaccumulators, Noccaea (Thlaspi) goesingense [29] and Noccaea fendleri ssp. glauca (Thlaspi montanum var. montanum) [30], indicated that Ni hyperaccumulation was a species-wide trait, whether the plants were collected from serpentine soils or normal soils. This finding sparked considerable interest because it stood in distinct contrast to the situation for metal tolerance in non-accumulators, which are the more common type of metallophytes. For non-accumulator, facultative metallophytes (e.g. metal-tolerant populations on mines), the paradigm had been that tolerance evolves at the population level in response to local edaphic conditions. In fact, the seminal review on this topic stated bluntly that “there is no evidence that a species has constitutional tolerance to heavy metals: evolution has always occurred when mine habitats are colonized” [1]. For hyperaccumulators, however, several more recent studies, covering a wide range of species and metals, have given further support to the hypothesis of species-wide tolerance and hyperaccumulation [31–38]. Conversely, in a few cases it has been suggested that the physiology for hyperaccumulation and tolerance is restricted to populations on metalliferous soils. This was the conclusion of the earliest publications on this topic, which reported that A. serpyllifolium (sensu lato, including Alyssum pintodasilvae and Alyssum malacitanum) from the Iberian Peninsula could hyperaccumulate

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Ni when growing on serpentine soil, but that populations originating from limestone-derived soils completely lacked Ni-tolerance and hyperaccumulation ability [20,21,39]. However, these findings must be regarded with some caution because all three papers were exceedingly vague about the provenance of seeds supposedly from limestone-derived soils. Preliminary results from a geographically extensive study [Pollard and Smith, unpublished data] suggest that A. serpyllifolium plants from some limestone-derived soils are only slightly less able to tolerate and hyperaccumulate Ni than those from some serpentine soils. The differences seem more clear-cut for Zn and Cd in Sedum alfredii [40–48] and for Tl in Biscutella laevigata [49]; in both species experimental studies indicate that plants from non-metalliferous soils lack the ability to tolerate and hyperaccumulate metals, which exists in populations from contaminated soils. These findings merit further study. The inconsistent findings regarding species-wide versus population-specific tolerance and hyperaccumulation may in part be related to the definitions of hyperaccumulation and tolerance. As mentioned earlier, hyperaccumulation is typically defined in relation to a specified threshold metal concentration. Although this is arguably somewhat arbitrary [7], it can at least be applied uniformly. There is no similar criterion that can be applied to define tolerance. Instead, tolerance is a relative concept, and the choice of a non-tolerant reference species may strongly influence whether a particular plant or population is perceived as being tolerant or not. Disagreement among studies may also result from an overly simplistic dichotomy between species-wide and populationspecific. Several of the most thorough studies, cited below, indicate a pattern that is more complex and nuanced. Tolerance and hyperaccumulation ability are at least partly under independent genetic control [50,51], and both may vary significantly among and within populations [31–35]. Results may differ depending on the particular set of populations considered in a given study. In species that can hyperaccumulate more than one metal, responses may depend on the metal under investigation. In N. caerulescens it appears that the properties of Zn tolerance and hyperaccumulation occur specieswide (albeit with some degree of variability), whereas Cd and Ni tolerance and hyperaccumulation are much more populationspecific [52,53]. Finally, neither hyperaccumulation nor tolerance is a one-step physiological process; both result from multiple processes, which may include root foraging, rhizosphere interactions, membrane transport, chelation or complexation of metals and compartmentation into specific organelles, cells, or tissues, and are under the control of multiple genetic factors [2,3]. This in turn can result in complex patterns of variation; for example, in N. caerulescens, it has been reported that strong translocation from root to shoot is a species-wide trait for Zn, Ni, and Cd [52] as well as Mn and Co [54], but enhanced total accumulation of Ni and Cd is a population-specific trait occurring only in plants from areas contaminated with those metals [52], and with some variability within those populations. 4.2. Patterns of variation and environmental correlates Rather than debating whether hyperaccumulation and tolerance are species-wide properties, it has been more fruitful to describe and analyze patterns of intraspecific variation. Even when facultative hyperaccumulators are reported to possess specieswide metal tolerance, intraspecific genetic variation in the degree of tolerance has been found in most studies specifically designed to look for it [38,52,53,55–58]. When a study finds no variation in metal tolerance, it may turn out to have been based on screening at a single concentration [e.g. 33]. Detection of differences in metal tolerance depends critically upon the metal concentrations employed in testing; too low a concentration may fail to

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suppress the growth of any populations, while too high a concentration may be uniformly toxic. Measurement of tolerance across a range of concentrations is most reliable. When variation is found, the highest levels of tolerance have consistently been found in populations growing on the most strongly metalliferous soils [38,52,53,55–58], in agreement with the long-held principle (first formulated for non-accumulating plants) that heavy metals in soils exert strong local selective pressures for the evolution of tolerance [1]. Hyperaccumulation (as measured in a standardized soil or other medium) also has intraspecific variation in most facultative hyperaccumulators, but the correlation between this character and metal concentration in the soil of origin has been much less predictable. Different results have been reported depending on the species, populations and metals used in the study. In some cases, the ability to accumulate metals is greatest in populations from soils enriched in that metal, especially for Cd [59–61]. Because such populations also tend to have the highest metal tolerance, this may represent a positive correlation between tolerance and hyperaccumulation. Correlation may be suggestive of some form of physiological dependence between the processes, but cannot on its own explain the nature of that dependence. Conversely, there have been several studies in which the strongest physiological ability to accumulate a metal occurs in populations on soils with low concentrations of that metal [32–34,38,51,57]. This is an intriguing finding in light of the adaptive explanations for hyperaccumulation discussed in Section 5. It is tempting to speculate that if possessing high metal concentrations confers some advantage on hyperaccumulators, then populations from low-metal soils may have been selected for the greatest ability to take up, concentrate and sequester metals in the face of a strong concentration gradient. It has also been hypothesized that N. caerulescens populations on some high-Zn (calamine) soils may have been selected for reduced metal uptake, to avoid internal Zn concentrations that might exceed even the extremely high tolerance of this species [51]. Although these adaptive explanations remain speculative, the overall conclusion that populations from normal soils have the highest capacity for hyperaccumulation seems well supported, at least in N. caerulescens. However, it is worth cautioning that a few studies [e.g. 32,38] report high metal concentrations in plants that appear to be under significant stress from metal toxicity in the experimental growth media, as indicated by reduced root or shoot growth. That finding could easily represent a case of non-specific “breakthrough” of metals into the shoot system, as discussed in Section 2.1. When attempting to measure hyperaccumulation under uniform conditions in the laboratory, it is important that the protocol be designed so that none of the treatments exceed the limits of tolerance in the populations under study. Despite attempts to understand patterns of intraspecific variation in a general framework, there are still some species whose behavior is paradoxical. In particular, some of the facultative hyperaccumulators in Table 1 possess high metal concentrations at only some of their metalliferous localities and not others (in addition to the low metal concentrations they always have on non-metalliferous soils). Examples include Alyssum sibiricum [17], Commelina ensifolia [15], Rostellularia adscendens [15], Evolvulus alsinoides [62] and Hybanthus enneaspermus [62]. These five species have wide geographic ranges (Table 1) including both normal and serpentine soils; however, hyperaccumulation of Ni has only been recorded in a small number of sites for each of them. It is unclear why they do not hyperaccumulate in other, apparently suitable, serpentine sites. Possibilities include both environmental characteristics of the sites (especially variations in soil pH and metal availability) and genetic characteristics of the populations, but none of them have been investigated in detail, with no Type I studies on

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these species (Table 1). This is another promising opportunity for further study. 4.3. Facultative hyperaccumulators as physiological and genetic models Recent years have seen a huge research enterprise dedicated to understanding the physiology, biochemistry, molecular biology and genetics of hyperaccumulation, fueled in part by potential commercial applications in biotechnology including phytoremediation and phytomining. The two species which have most often been used as model organisms for such research are the facultative hyperaccumulators N. caerulescens and A. halleri, as suggested by the vast preponderance of research on these species shown in Table 1. We will not attempt to summarize all this literature, for two reasons. First, most of it has been covered in other recent reviews [2,3,6,27,63,64]. But secondly and more importantly, although these species are indeed facultative hyperaccumulators, most of the research on them consists of Type III studies (Table 1), and does not actually relate to facultative hyperaccumulation per se. In the majority of cases, the same studies could have been conducted whether or not these two species ever occurred on normal soil. Nonetheless, there are at least two general areas of research in which comparisons between populations from metalliferous soils and normal soils have been productive and show promise for the future. Researchers attempting to identify mechanisms or genes responsible for hyperaccumulation frequently employ a comparative method, focusing on differences between hyperaccumulating and non-accumulating plants in order to identify factors that might underlie hyperaccumulation. Many studies compare hyperaccumulators with other species chosen because they are closely related to the hyperaccumulator or because they are well-characterized model plants. However, when making interspecific comparisons, there certainly must be many differences between the species, beyond whether or not they hyperaccumulate. The comparative method works best when comparisons are made across a uniform background, i.e. when the organisms being compared are as similar as possible except for the feature of interest. Because facultative hyperaccumulator species occur on both metalliferous and normal soil, and because of the documented intraspecific variation in hyperaccumulation and tolerance that exists among populations, they can represent excellent models for comparative physiological research. For the most part these comparisons have focused on describing the metal tolerance and uptake characteristics of populations on metalliferous and normal soils, as described in the previous section on environmental correlates [33,52,58,61,65]. A few researchers have attempted to use this comparative method to look for intraspecific differences in molecular physiology between plants from populations on different soil types. Results have suggested that the vacuolar transporter ZTP1 may contribute to increased Zn tolerance in populations of N. caerulescens from high-Zn soils compared to normal or serpentine soils [66]. In contrast, comparisons of N. caerulescens from contrasting soils have shown that differential expression of HMA4, the gene for a metal-transporting ATPase strongly implicated in hyperaccumulation based on interspecific comparisons, does not explain the differences among intraspecific variants [67]. In S. alfredii, another vacuolar transporter, MTP1, may be responsible for enhanced Zn translocation and tolerance in populations of from metalliferous soils [48]. This is a promising approach for any facultative hyperaccumulators found to have variation in metal uptake or tolerance. Facultative hyperaccumulators also provide the opportunity to conduct crosses between populations originating from different soil types (“inter-ecotypic crosses”), to further understand the genetic basis of hyperaccumulation. Most studies to date have

examined segregation ratios in order to determine whether tolerance and uptake are independent processes, whether there is genetic correlation in the uptake of different metals, or to estimate the number of loci involved in hyperaccumulation [51,53,56,68]. Application of quantitative trait locus (QTL) analysis has also been productive [57,69] and may eventually yield sufficiently fine-scale results to reveal the molecular basis of phenotypic differences between populations [70]. 5. Evolution of facultative hyperaccumulators It has widely been assumed that hyperaccumulation of heavy metals must incur some cost to the plant, related to transport, concentration, storage and detoxification of potentially toxic elements [6,71,72], although quantification of these costs has been problematic. In the face of such costs, many researchers have hypothesized that there must also be some compensatory benefit selecting for the evolution of hyperaccumulation. An influential paper on this topic [73] divided these hypotheses into five categories: (a) hyperaccumulation as a mechanism of metal tolerance, including sequestration and disposal of metals; (b) hyperaccumulation as a mechanism of allelopathic interference with competing plants; (c) hyperaccumulation as a mechanism of drought tolerance; (d) hyperaccumulation as an inadvertent consequence of other processes such as efficient nutrient uptake; (e) hyperaccumulation as a mechanism of defense against herbivores and pathogens. In over 20 years since the publication of this framework, there has been little research and even less support for the first four hypotheses [74], but a considerable body of mostly supportive findings for the defense hypothesis [75,76]. There are challenges in applying most of these hypotheses to facultative hyperaccumulators. As discussed earlier in this review, research has generally found that facultative hyperaccumulators growing on normal soil possess at least some physiological ability to tolerate and hyperaccumulate metals but do not usually achieve hyperaccumulation because of low metal availability in the soil. The difficulty in these situations is explaining the adaptive benefit of a species-wide physiological trait that is not fully manifested in the phenotype of the plants in many, perhaps a majority, of the sites where the species occurs. We suggest three hypotheses that may apply in this situation, two of which differ from those suggested in the preceding paragraph. As we will discuss, the three hypotheses are not mutually exclusive. The first, which we will label the “phylogenetic conservation” hypothesis, is that hyperaccumulation evolved originally in plants on metalliferous substrates, in response to selection pressures such as those outlined above. This implies that facultative hyperaccumulators may have descended from obligate hyperaccumulators, in the process of expanding their range to include non-metalliferous soils. The presence of hyperaccumulator physiology on normal soils would thus have little or no adaptive benefit, but merely be conserved from the ancestral populations. This hypothesis would be most likely in facultative hyperaccumulators that are closely related to obligate hyperaccumulators, and there are several potential examples. Table 1 includes eight species of facultative hyperaccumulators in the genus Alyssum, and as many as seven others may exist, though the evidence is not as clear [20; Reeves, unpublished data]. All are members of sect. Odontarrhena, which also includes at least 33 species of obligate hyperaccumulators from serpentine soils [17,20], suggesting a clear phylogenetic trend toward species-wide hyperaccumulation in this genus. As another

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example, three facultative hyperaccumulators in the genus Psychotria that are widespread across the Caribbean region are listed in Table 1 [15,77], but several obligate hyperaccumulators are also known in this genus from serpentine soils in Cuba [13] and New Caledonia [78]. In what may represent a parallel situation, a recent study of endemism in the California flora [79] found a relatively high frequency of evolutionary reversals, in which an endemic species gave rise to a non-endemic species, although the absolute number of such reversals was low because of the relative rarity of endemics. Although the study in question was not concerned with hyperaccumulation, a similar pattern could apply in hyperaccumulators, among which obligate (endemic) hyperaccumulators are rare, but nonetheless much more common than facultative hyperaccumulators. The second hypothesis, which we will call “incremental advantage”, postulates that there is some selective benefit of possessing the physiology associated with hyperaccumulation, even for plants growing on normal soil. Several studies have shown that facultative hyperaccumulators on normal soil have remarkably high concentrations of trace elements, even if those concentrations fall short of hyperaccumulation [52,60,80]. It is not known whether these concentrations benefit the plants, but there is some evidence that metal concentrations below the hyperaccumulation threshold might reduce feeding by herbivores [81]. This argument is thus related to the defensive enhancement hypothesis proposed as a possible explanation for the evolution of hyperaccumulation [76], but it differs in two respects. First, we are not limiting the incremental advantage on non-metalliferous soils to defense; there could also be incremental benefits in terms of nutrient acquisition, osmotic balance, or a multitude of other factors. Second, the defensive enhancement hypothesis assumes that higher concentrations of metals are generally more effective as defenses, and thus natural selection will favor continued increases in metal uptake ability [76]. In proposing that perhaps the physiology associated with hyperaccumulation has selective advantages for plants growing on normal soil, we also recognize the possibility that when such plants encounter metalliferous soils, they may be predisposed to hyperaccumulate metals to concentrations that are non-adaptive, if not maladaptive. Finally, the third hypothesis is the “inadvertent uptake” concept previously proposed by other researchers [73], suggesting that hyperaccumulation may result coincidentally as a side-effect of an especially efficient mechanism evolved for acquiring nutrients other than the heavy metal in question, or for heavy-metal homeostasis in general. Such a mechanism would be a species-wide trait, but only manifest itself as hyperaccumulation when members of that species occurred on metalliferous soils. Again, the hyperaccumulation itself might be non-adaptive or maladaptive. This explanation was proposed to explain Ni hyperaccumulation in N. fendleri ssp. glauca (T. montanum var. montanum), a species ranging over a huge area of western North America but hyperaccumulating only on isolated serpentine outcrops [30], but there is little direct evidence of what the coincidental benefit might be, nor what physiological processes might link it to hyperaccumulation. Given the relatively close relationship of this taxon to the well-known European hyperaccumulator species now placed in the strictly-defined genus Noccaea [82], it seems parsimonious that phylogenetic conservation may also contribute to hyperaccumulation in N. fendleri ssp. glauca. Evaluation of these hypotheses will be aided enormously by studies of the molecular phylogeny of facultative hyperaccumulators and their obligate relatives. In particular, it will be important to resolve the polarity of ancestral-descendent relationships, i.e. whether facultative hyperaccumulators have evolved from obligate hyperaccumulators or vice versa. Existing large-scale phylogenies for clades including non-accumulators, facultative

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hyperaccumulators, and obligate hyperaccumulators [e.g. 82–84] generally do not include enough species (especially rare endemics) and do not possess the resolution to answer these questions. Several detailed studies have recently attempted to use molecular and genomic data to elucidate the origin of the facultative hyperaccumulator A. halleri [58,85–87]. Evidence suggests that duplication of the HMA4 gene occurred concurrent with speciation, leading to species-wide ability to hyperaccumulate long before human industrial activities made metalliferous environments widespread [87]. Overall genetic structure and differentiation of populations is predicted by geographic factors rather than metal availability in the soil [85,86]. Although tolerance occurs species-wide, enhanced tolerance in populations on metalliferous is inferred to have arisen recently, as plants colonized polluted areas; however, there are also cases that appear to involve populations on low-metal soils that were founded from those on metalliferous soils and simply inherited elevated tolerance [58], a potential parallel to our phylogenetic conservation hypothesis. The three hypotheses we have proposed are not intended to be competing ideas; any of them might be accurate in a given circumstance. We suggest that the most important factor determining which hypothesis applies in a particular case is the distribution of the species in relation to metalliferous and normal soils. In many facultative hyperaccumulators, the species occurs mostly on metalliferous substrates and only occasionally on normal soil. Under these circumstances it is reasonable that the genetic traits leading to hyperaccumulation may have evolved in response to some selective advantage on metalliferous soil where hyperaccumulation does actually occur, and phylogenetically conserved in the less common situations where the species or its relatives occupy normal soil. There is a possibility that pollen or seed flow could transfer genes for tolerance and hyperaccumulation to populations on normal soil, but this could be tested in a relatively straightforward manner by studying the relationship between the characteristics of a population and its degree of isolation. In addition, there is a need to clarify the metabolic cost of possessing hyperaccumulator physiology on non-metalliferous soils, which is also not well understood. At the other extreme, some facultative hyperaccumulators are widespread and common on normal soils and only sporadically occur on metalliferous soils. An example is Phytolacca americana, a ruderal species native to the southeastern United States but found as an invasive weed worldwide. It has been identified as a hyperaccumulator of Mn and Cd on mine-spoil in China [49,50], and also observed to hyperaccumulate Mn on outcrops of manganiferous schist within its native range in North America [Pollard, unpublished data]. There is evidence that the physiological potential for Mn hyperaccumulation is a species-wide trait [36,80], even though Mn hyperaccumulation has only been reported to occur in natural populations at two localities in the world. It is highly unlikely that the ability to hyperaccumulate in this case evolved in the exceedingly rare metalliferous populations and somehow spread throughout the species. The much more likely explanation is that the species was predisposed to hyperaccumulation, either by some incremental advantage of elevated but sub-hyperaccumulation concentrations of Mn in plants on normal soil [80,81], or by inadvertent uptake related to some other element. A similar argument may apply to the facultative hyperaccumulation of As by the widespread fern Pteris vittata [38]. In such cases, it would quite possibly be fruitless to look for an ecological advantage caused by hyperaccumulation in the small subset of these plants growing on contaminated soils. As stated earlier, the various hypotheses discussed here need not be mutually exclusive. Hyperaccumulation could arise by inadvertent uptake and yet be found in several related species through phylogenetic conservation. It is also possible that

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hyperaccumulator physiology that originated through inadvertent uptake or incremental advantage could, under appropriate circumstances, be the starting point for directional selection as a defense against herbivores or pathogens, as has been explicitly modeled in discussions of the elemental defense hypothesis [76]. 6. Conclusions The majority of plants that hyperaccumulate heavy metals are obligately endemic to metalliferous soils, but a significant minority are facultative metallophytes, including many of the best-known research models. Facultative hyperaccumulators can be used as promising model systems for physiological and genetic research. However, previously-published evolutionary models attempting to explain the adaptive significance and selective advantage of hyperaccumulation do not apply well to facultative hyperaccumulators, because their physiological potential to hyperaccumulate metals is generally a species-wide trait, yet hyperaccumulation cannot usually occur on non-metalliferous soils. We have put forward several hypotheses to explain the evolution of facultative hyperaccumulation, and proposed that the explanation which applies to a particular species is likely to depend strongly on whether that species occurs commonly on metalliferous soils, or conversely occurs primarily on normal soils and only occasionally on metal-enriched soils. Some data are available to evaluate these hypotheses; however, the literature is strongly focused on only a few species and a few metals, with remarkably few detailed experimental studies of facultative hyperaccumulators of nickel, especially those from tropical regions. A broader base of research is required in order to progress toward a general understanding of the biology of facultative hyperaccumulation. Acknowledgements We thank Jonathan Gressel and eight anonymous reviewers of an earlier version of this paper for their helpful comments. References [1] J. Antonovics, A.D. Bradshaw, R.G. Turner, Heavy metal tolerance in plants, Adv. Ecol. Res. 7 (1971) 1–85. [2] U. Krämer, Metal hyperaccumulation in plants, Ann. Rev. Plant Biol. 61 (2010) 517–534. [3] E. Fasani, Plants that hyperaccumulate heavy metals, in: A. Furini (Ed.), Plants and Heavy Metals, SpringerBriefs in Biometals, Springer, 2012, pp. 55–74. [4] H. Ali, E. Khan, M.A. Sajad, Phytoremediation of heavy metals – concepts and applications, Chemosphere 91 (2013) 869–881. [5] V. Sheoran, A.S. Sheoran, P. Poonia, Phytomining: a review, Miner. Eng. 22 (2009) 1007–1019. [6] A.J. Pollard, K.D. Powell, F.A. Harper, J.A.C. Smith, The genetic basis of hyperaccumulation in plants, Crit. Rev. Plant Sci. 21 (2002) 539–566. [7] A. van der Ent, A.J.M. Baker, R.D. Reeves, A.J. Pollard, H. Schat, Hyperaccumulators of metal and metalloid trace elements: facts and fiction, Plant Soil 362 (2013) 319–334. [8] F. Metali, K.A. Salim, D.F.R.P. Burslem, Evidence of foliar aluminium accumulation in local, regional and global datasets of wild plants, New Phytol. 193 (2012) 637–649. [9] J.P. Lambinon, P. Auquier, La flore et la vegetation de terrains calaminaires de la Wallonie septentrionale et de la Rhenanie aixoise: types chorologiques et groups écologiques, Natura Mosana 16 (1963) 113–131. [10] A.R. Kruckeberg, Ecotypic response to ultramafic soils by some plant species of northwestern United States, Brittonia 19 (1967) 133–151. [11] R.R. Brooks, E.D. Wither, Nickel accumulation by Rinorea bengalensis (Wall.) O.K, J. Geochem. Explor. 7 (1977) 295–300. [12] R.D. Reeves, A.J.M. Baker, A. Borhidi, R. Berazaín, Nickel-accumulating plants from the ancient serpentine soils of Cuba, New Phytol. 133 (1996) 217–224. [13] R.D. Reeves, A.J.M. Baker, A. Borhidi, R. Berazaín, Nickel hyperaccumulation in the serpentine flora of Cuba, Ann. Bot. 83 (1999) 29–38. [14] R.S. Boyd, M.A. Davis, K. Balkwill, Elemental patterns in Ni hyperaccumulating and non-hyperaccumulating ultramafic soil populations of Senecio coronatus, S. Afr. J. Bot. 74 (2008) 158–162. [15] R.D. Reeves, Tropical hyperaccumulators of metals and their potential for phytoextraction, Plant Soil 249 (2003) 57–65.

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