# Copper and cobalt accumulation in plants: a critical assessment of the current status of knowledge

Bastien Lange, Antony van Der Ent, Alan John Martin Baker, Guillaume

Echevarria, Grégory Mahy, François Malaisse, Pierre Meerts, Olivier Pourret,

Nathalie Verbruggen, Michel-Pierre Faucon

► To cite this version:

Bastien Lange, Antony van Der Ent, Alan John Martin Baker, Guillaume Echevarria, Grégory Mahy, et al.. Copper and cobalt accumulation in plants: a critical assessment of the current status of knowledge. A critical assessment of the current state of knowledge. New Phytologist, 2017, 213 (2), pp.537-551. 10.1111/nph.14175. hal-02265598

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Copper and cobalt accumulation in plants: a critical assessment of
the current status of knowledge

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Bastien Lange¹,²,*, Antony van der Ent³,⁴, Alan John Martin Baker³,⁴,⁵, Guillaume

Echevarria⁴, Grégory Mahy G⁶, François Malaisse⁶, Pierre Meerts², Olivier Pourret¹, Nathalie

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Verbruggen⁷§, Michel-Pierre Faucon¹§

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¹Hydrogeochemistry and Soil-Environment Interactions (HydrISE), UP.2012.10.102, Institut Polytechnique LaSalle Beauvais, Beauvais, France.

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²Laboratory of Plant Ecology and Biogeochemistry, Université Libre de Bruxelles, 1050 Brussels, Belgium.

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³Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, Australia.

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⁴Université de Lorraine – INRA, Laboratoire Sols et Environnement, UMR 1120, France.

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⁵School of BioSciences, The University of Melbourne, Victoria, Australia.

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⁶Departement of Forest, Nature and Landscape, Biodiversity and Landscape Unit, Gembloux
Agro-Bio Tech, University of Liège, Gembloux, Belgium.

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⁷Laboratory of Plant Physiology and Molecular Genetics, Université Libre de Bruxelles, Brussels, Belgium.

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*Author for correspondence: Bastien Lange

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Tel: +33 (0)6 89 83 73 17; Email: bastien.lange2@lasalle-beauvais.fr

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§ Co-last author

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- Total word count: 3 967

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- Number of words per section:

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I. Introduction : 517

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II. Ecophysiology of tolerance and accumulation of Cu and Co : 1 314

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III. Functional and evolutionary ecology of Cu and Co accumulation : 357

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IV. Inter- and intra-specific variations of shoot/foliar Cu and Co concentrations : 1 072

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V. Phytomining technology using Cu and Co accumulator plants : 321

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VI. Conclusions and Outlook : 324

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VII. Acknowledgement : 62

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- Number of Figures: 4 (Fig. 1 and Fig. 4 in color)
35 **Summary**

37 This review synthesizes contemporary understanding of Cu-Co tolerance and accumulation in
38 plants. Accumulation of foliar Cu and Co to >300 µg g⁻¹ is exceptionally rare globally, and
39 known principally from the Copperbelt of Central Africa. Cobalt accumulation is also
40 observed in a limited number of Ni hyperaccumulator plants occurring on ultramafic soils
41 around the world. None of the putative Cu or Co hyperaccumulator plants appears to comply
42 with the fundamental principle of hyperaccumulation as foliar Cu-Co accumulation is strongly
43 dose-dependent. Abnormally high plant-tissue Cu concentration occurs only when plants are
44 exposed to high soil Cu concentrations with a low shoot translocation factor. Most Cu tolerant
45 plants are Excluders *sensu* Baker and therefore setting threshold values for Cu
46 hyperaccumulation is not informative. Abnormal accumulation of Co occurs under similar
47 circumstances in the Copperbelt of the DR Congo, however, Co tolerant plants behave
48 physiologically as Indicators *sensu* Baker and sporadically coincides with Ni
49 hyperaccumulation on ultramafic soils. Practical application of Cu-Co accumulator plants in
50 phytomining is limited due to their dose-dependent accumulation characteristics, although for
51 Co trials may be warranted on highly Co-contaminated minerals wastes because it its high
52 metal value.

54 **Keywords** Accumulation, Cobalt, Copper, Hyperaccumulation, Metallophyte, Metal-
tolerance, Phytomining, Toxicity.
**Introduction**

Plant species that can grow and reproduce on soil strongly enriched in trace elements (TE), i.e. metalliferous soils of natural or anthropogenic origin, have proved to be powerful model systems for studying the ecophysiology and evolution of adaptation to toxicity stresses (Baker, 1987). Metallophytes are species able to tolerate concentrations of such TE (e.g. Ni, Cu or Co) in soil that are toxic for most other species (Baker, 1981). These metallophytes can be restricted to metalliferous soils (i.e. obligate metallophytes) or occur also on 'normal' soils (i.e. facultative metallophytes) (Pollard et al., 2014). Among these, some have the ability to actively accumulate certain TE in leaves, from either low or high available concentrations in soil without toxicity symptoms or growth inhibition, and are known as hyperaccumulators plants (Jaffré et al., 1976; van der Ent et al., 2013).

By far the greatest number of hyperaccumulators are known for Ni (>1000 µg g⁻¹ foliar Ni) and found on ultramafic soils (Reeves, 2003). (Hyper)accumulation of Cu and Co was first defined as >1000 µg g⁻¹ foliar Cu/Co (Malaisse et al., 1978), but later revised downwards to >300 µg g⁻¹ (van der Ent et al., 2013). More than 95% of the putative Cu-Co hyperaccumulators are found in the southeastern Democratic Republic of Congo (D. R. Congo) (see Brooks et al., 1980), where there are a large number of natural Cu-Co outcrops; the so-called 'Copper Hills' of the 'Katangan Copperbelt'. However, putative Cu-Co hyperaccumulators have also been reported from other countries on ultramafic soils enriched with Cu and Co. An overview of putative Cu and/or Co hyperaccumulator plants is provided in Fig. 1. Apart from the Katangan Copperbelt, high Co concentrations are usually found in ultramafic soils worldwide. Copper can also be present at high concentrations in some ultramafic soils. Putative Co hyperaccumulation on ultramafic soils has been reported for some individuals of two Ni-hyperaccumulating *Rinorea* species (Violaceae) (Brooks et al., 1977). A few cases of putative Cu hyperaccumulation have also been reported on Cu-enriched ultramafic soils in Sri Lanka, Brazil and Malaysia (Rajakaruna & Bohm, 2002; van der Ent & Reeves, 2015). Concluding, putative Cu and/or Co hyperaccumulation may occur in: Cu-Co outcrops (principally the Katangan Copperbelt), Cu-Co-enriched ultramafic soils (e.g. Goiás state, Brazil) and Co-enriched ultramafic soils (e.g. Barberton, South Africa).

High inter- and intra-specific variations in foliar/shoot Cu and/or Co have been reported among Cu-Co accumulators (Faucon et al., 2009; Lange et al., 2014). Some of the early
reports of Cu-Co (hyper)accumulation have been based upon analysis of herbarium materials (see e.g. Baker & Brooks, 1989; Reeves & Baker, 2000) and there is now substantial experimental data to suggest that many of these samples were contaminated with soil dust, so giving spurious high values to foliar dry matter analyses (Faucon *et al.*, 2007).

This review synthesizes recent advances and contemporary research on the understanding of Cu-Co tolerance and (hyper)accumulation, by (i) reviewing current state of knowledge about Cu-Co tolerance and (hyper)accumulation by physiological, ecological and biogeochemical approaches; (ii) presenting an updated global record of unusually high Cu-Co accumulation in plant species; (iii) evaluating possible applications for Cu-Co accumulating plants.

## Ecophysiology for tolerance and accumulation of Cu and Co

### Strategies of tolerance

Plants that can grow and reproduce on metalliferous soils can be classified based upon their TE uptake response when exposed to increasing soil concentrations of the element under consideration (Antonovics *et al.*, 1971). The three main strategies of tolerance are: ‘Excluder’, ‘Bioindicator’ and ‘Hyper accumulator’ (Baker, 1981). Tolerance to certain TE and their accumulation patterns have been widely studied over the past decades, especially for Zn, Ni and Cd (Verbruggen *et al.*, 2009). Copper- and Co-tolerant populations have been reported in the literature for a large number of species collected from nature, but the capacity to grow under elevated Cu and/or Co concentrations without toxicity symptoms or growth inhibition has rarely been demonstrated experimentally (Table 1).

Most Cu tolerant plants respond ecophysiologically as Excluders sensu Baker with a very limited Cu translocation to the leaves (Macnair, 1981; Schat *et al.*, 1993; Song *et al.*, 2004; Chipeng *et al.*, 2010; Lange *et al.*, 2016). *Crassula helmsii* could accumulate more 9000 µg g⁻¹ in its shoot at low Cu concentration in the nutrient solution would be the exception of Cu (hyper)accumulation (Küpper *et al.*, 2009). Cobalt accumulation exists in *Anisopappus chinensis* as foliar Co concentration increases in relation to Co soil concentration without a decrease of biomass or toxicity symptoms (Lange, 2016). None of the putative Cu or Co hyperaccumulator plants, however, appear to comply with the fundamental principle of hyperaccumulation that high shoot concentrations are maintained over a wide range, from low to high, of Cu or Co in the soil or growth medium (Van der Ent *et al.*, 2013).
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125 *Physiology of Cu tolerance and accumulation*

126 Copper is an essential transition metal with two oxidation states under physiologically relevant conditions. Due to its ability to cycle between the oxidized Cu(II) and reduced Cu(I) states, it is involved in biological processes such as photosynthesis, respiration, oxygen superoxide scavenging, ethylene sensing, cell wall metabolism and lignification (Burkhead *et al.*, 2009). For the very reason that it is essential, Cu can also be highly toxic (Fernandes & Henriques, 1991). Free Cu catalyzes Fenton reactions that generate hydroxyl radicals causing damage to lipids, proteins and DNA (Cohu & Pilon, 2010). Copper has also been reported to interfere with iron homeostasis (Bernal *et al.*, 2012). An overall reduction of plant biomass, inhibition of root growth, chlorosis, bronzing and necrosis are the usual reported symptoms of a Cu excess due to increased production of Reactive Oxygen Species and harmful interactions at the cellular level.

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138 Copper tolerance in metallophytes is thought to arise through adaptation of components of basic metallic homeostasis. Cellular Cu levels are controlled by interplay between the ATPase exporters and the Ctr family of Cu permeases (Leary & Winge, 2007). Most Cu-ions in cells are either compartmentalized or bound to proteins or metabolites. Copper is delivered to enzymes by specific chaperones while excess can be chelated by thiol-rich compounds such as metallothioneins which are low molecular weight cysteine-rich proteins, or phytochelatins, which are synthesized from glutathione. Long distance transport of Cu apparently involves nicotianamine, a ubiquitous metal chelator in higher plants, synthesized from three molecules of S-adenosyl-methionine (Zheng *et al.*, 2012).

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148 As previously stated, most of Cu-tolerant plants behave as Excluders. The genetic control of Cu tolerance in *S. vulgaris* and *M. guttatus* seems to be governed by a single major gene while tolerance level seems to be controlled by a number of modifiers (Macnair, 1983; Schat & Ten Bookum, 1992). Major determinants of Cu tolerance in Cu-tolerant plants are not known or genetically confirmed. In *S. vulgaris*, Cu tolerance is associated with efficient ATP-dependent Cu efflux through the root plasma membrane (van Hoof *et al.*, 2001a). In *Arabidopsis thaliana*, which is sensitive to Cu, *HMA5* (HEAVY METAL ATPase 5) was shown to co-localize with a major QTL explaining 52% of the difference in Cu tolerance between two ecotypes (Kobayashi *et al.*, 2008). *HMA5* is a Cu ATPase with a role in Cu compartmentation and detoxification in roots by moving Cu from the symplasm to apoplasm (Andrés-Colás *et al.*, 2012).
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al., 2006; del Pozo *et al.*, 2010). *HMA5* activity could correspond to the ATP-dependent Cu efflux highlighted in the study of van Hoof *et al.* (2001a) with *S. vulgaris*. Some authors have suggested a role for *HMA5* in xylem loading (Kobayashi *et al.*, 2008), which has been debatable since this transport was mostly maintained in the *HMA5* loss of function *Arabidopsis* mutant (Andrés-Colás *et al.*, 2006). Enhanced tolerance in *S. vulgaris* has also been associated with increased transcript levels of metallothionein 2B (van Hoof *et al.*, 2001b).

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Furthermore, Cu tolerance was found to be inversely correlated with long-term K⁺ efflux in several species, sensitive or tolerant, including *M. guttatus* (Strange & Macnair, 1991; Murphy *et al.*, 1999). K⁺ serves as a counter-ion for citrate that is released upon exposure to Cu excess, thus reducing Cu availability in the medium (Murphy *et al.*, 1999).

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No molecular mechanism of (hyper)accumulation has been described yet in any of putative Cu hyperaccumulator plants. Similar to the hyperaccumulation of other TE, root sequestration is limited to allow mobility of Cu to the shoot. Due to its toxicity, Cu excess is released in shoot and immobilized by efficient chelation and sequestration mechanisms. In the aquatic plant *C. helmsii*, Cu was found to be almost exclusively bound by oxygen ligands, likely organic acids, and not sulphur ligands (Küpper *et al.*, 2009). The concentration of organic acids in CAM plants may be so high that it is not limiting for Cu complexation. These authors suggested that main storage sites of Cu were vacuoles and cell walls. Similar investigations have not yet been undertaken on land C3 Cu accumulator plants.

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## Physiology of Co tolerance and accumulation

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Cobalt is a transition metal not essential for plants with seven possible oxidation states. In physiological conditions, the oxidation states of Co are mainly II and III, which makes Co a possible catalyzer of Fenton reactions. Beneficial effects of Co supply have been associated with symbiotic rhizobia that inhabit in the nodules of leguminous plants, and need vitamin B₁₂ (cobalamin) for the activity of several enzymes involved in nitrogen fixation. Other reported beneficial effects include retardation of leaf senescence through inhibition of ethylene biosynthesis, and increased drought resistance in seeds (reviewed by Pilon-Smits *et al.*, 2009). Toxicity of Co excess is linked to oxidative stress, inhibition of photosynthesis and iron deficiency (Palit *et al.*, 1994; Morrissey *et al.*, 2009). Cobalt has been reported to disrupt iron homeostasis and compete with iron for access to transporters in many organisms

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including plants (Barras & Fontecave, 2011). Cobalt can be transported into root epidermal cells of *A. thaliana* by IRT1 (Korshunova *et al.*, 1999). Once inside cells, ferroportins IRON REGULATED1 (IREG1/FPN1) and IREG2/FPN2 play a role in Co detoxification. IREG2/FPN2 can transport Co²⁺ inside vacuoles of root epidermal and cortical cells thereby sequestrating Co in the outer most layers of roots. Mobile Co can be loaded into the xylem by FPN1 and translocated to the shoot (Morrissey *et al.*, 2009). Additionally, it is believed that *HMA3*, which is mainly expressed in roots, also transports Co²⁺ inside vacuoles in addition to other metallic ions (Cd²⁺, Zn²⁺, Pb²⁺) (Morel *et al.*, 2009).

Mechanisms of Co tolerance and accumulation are still poorly understood. Since some of Co accumulators seem also to accumulate Cu (Faucon *et al.*, 2007), some tolerance and/or accumulation mechanisms are believed to be shared. Considering the targets of Co toxicity, plants that (hyper)accumulate Co have most probably evolved adjustments of Fe homeostatic mechanisms. Such adaptation has already been observed in Zn and Cd hyperaccumulators showing modified regulation of genes involved in iron homeostasis (Krämer, 2010; Hanikenne & Nouet, 2011; Shanmugam *et al.*, 2011). Another critical aspect of Co (hyper)accumulation is expected to be an efficient Co detoxification system to avoid the accumulation of free ions that can induce oxidative stress. Cellular Co tolerance of *C. cobalticola* was associated with an increase in citric acid as well as in cysteine, without any further increase in phytochelatin levels (Oven *et al.*, 2002).

## Functional and evolutionary ecology of Cu and Co accumulation

### Trace element (hyper)accumulation as a functional trait

Functional traits are defined as “morpho-physio-phenological traits which impact fitness indirectly via their effects on growth, reproduction and survival” (Violle *et al.*, 2007). Under that definition, TE concentrations in leaf tissues qualify as functional traits. Hypotheses related to the ecological significance of TE accumulation should be reformulated in the broader framework of functional plant ecology (Delhaye *et al.*, 2016). Due to trade-offs between different traits, some trait combinations are more likely to occur than others. The range of possible traits has become known as the ‘Leaf Economics Spectrum’ (Wright *et al.*, 2004). How (hyper)accumulation relates to the Leaf Economics Spectrum is still an open question, but the concept is being extended to elements other than carbon and nitrogen (Reich, 2014). Is (hyper)accumulation more likely to evolve in association with traits favouring fast
resource capture or with traits favouring nutrient conservation? Trade-offs between the
capacity to accumulate TE and the uptake of major nutrients might strongly constrain the
evolution of TE accumulation. Recent findings confirm that TE accumulation is correlated to
the capture/use of major nutrients (Lambers et al., 2015), thus supporting the so-called
hypothesis of ‘inadvertent uptake’ (Boyd & Martens, 1992). However, this hypothesis should
be confirmed contrary to the hypothesis that TE (hyper)accumulation may serve as a defense
against herbivores and pathogens (demonstrated for Ni, Zn and Cd hyperaccumulation)
(Boyd, 2013; Cappa & Pilon-Smits, 2014). Functional leaf and root traits involved in nutrient
acquisition and conservation should be studied to understand Cu-Co accumulation.

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236 *Evolution of trace element accumulation*

237 Does evolution of increased TE accumulation capacity involve changes in the niche
238 parameters? Circumstantial evidence suggests that TE exclusion is more frequently observed
239 in broad-niched metallophytes compared to narrow-niched (Delhaye et al., 2016). This
240 observation is thought to be the consequence of the cost of tolerance and/or
241 (hyper)accumulation (Maestri et al., 2010). Future work should test if evolutionary transitions
242 to (hyper)accumulation are linked to changes in competitive ability and niche metrics.
243 Another hypothesis posits that lower occurrence of pathogenic fungi and herbivores in Cu-
244 enriched soils may conceivably relax selection pressure of defences in Cu-tolerant populations
245 (Chipeng et al., 2010). Copper-tolerant populations would then be more susceptible to
246 pathogenic fungi compared to non-tolerant populations.
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248 Inter- and intra-specific variations of shoot/foliar Cu and Co concentrations

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250 *Phylogeny of variations in Cu and Co accumulation*

251 The absence of a suitable field test for Cu-Co in plant materials as exists for Ni has so far
252 limited the widespread screening for significant accumulation of these elements. Furthermore,
253 foliar Cu-Co concentrations are not routinely measured in many ecological studies around the
254 world. Many of the records for abnormal Cu-Co concentrations in plants, therefore, result
255 from targeted testing of plants from the Katangan Copperbelt, and for most part by atomic
256 absorption spectrometry (and hence not obtaining multi-element data – but see Brooks et al.,
257 1986). As a result, systematic analysis of the Cu-Co concentrations in plant species is limited.
258 Only recently has systematic multi-element mass-screening of herbarium collections using
259 non-destructive techniques (X-ray Fluorescence Spectroscopy) commenced (van der Ent,
260 2016). Therefore, at present it is difficult to objectively observe phylogenetic patterns of Cu and Co accumulation. Nevertheless, in Central Africa, substantial Cu-Co accumulation is mostly recorded in the Lamiaceae (Table 1). On ultramafic soils, accumulation of Co is a feature in a number of Ni hyperaccumulators, especially in the Brassicaceae, Violaceae and Phyllanthaceae (Table 1).
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266 Genetic variability of Cu and Co accumulation

267 The substantial phenotypic variation of Cu-Co accumulation recorded in Cu-Co metallophytes
268 is expressed between species, populations, and individuals within a population (Faucon et al.,
269 2007). For Cu, these variations can be of genetic origin. Peng et al. (2012) demonstrated the
270 foliar Cu variability for six distinct metallicolous populations of Haumaniastrum katangense.
271 Six times as much Cu was obtained in non-metallicolous compared to metallicolous plants of
272 Crepidorhopalon tenuis (approx. 115 µg g⁻¹) (Faucon et al., 2012a). For a given concentration
273 in the growth medium, Cu accumulation variation could be related to variations in the degree
274 of tolerance, with the less Cu-accumulating populations the most tolerant (Ouzounidou et al.,
275 1994; Gonnelli et al., 2001; Weng et al., 2005).

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277 In the case of Co, accumulation variation have been demonstrated between three closely
278 related *Silene* taxa (Baker *et al.*, 1983). The most tolerant taxon was the less accumulating for
279 a given soil Co concentration. The intra-specific variation of Co accumulation by
280 metallophytes from natural Co-enriched soils has not yet been tested. For the facultative
281 metallophyte *Anisopappus chinensis*, the strong variation in the degree of Co tolerance
282 between populations suggests intra-specific variation of accumulation (Lange, 2016). It may
283 be hypothesized that a lower accumulation variation within metallicolous populations than
284 within non-metallicolous populations exists, as a result of a long-term directional selection in
285 metallicolous populations (Dechamps *et al.*, 2007).
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287 Copper and Co chemical speciation in metalliferous soils and accumulation by plants

288 The Cu-Co outcrops have high total Cu-Co soil concentrations (up to 27793 µg g⁻¹ and 3451 µg g⁻¹, respectively; Pourret *et al.* 2016) from which a fraction is mobile (1145–5225 µg g⁻¹ and 39–2146 µg g⁻¹, respectively; Fig. 2a, 3a). This permits high Cu-Co accumulation by *A. chinensis* (Fig. 2b, 3b; up to 736 µg g⁻¹ foliar Cu and 2822 µg g⁻¹ foliar Co). Moreover, Cu accumulation variations (by *C. perennis* and *A. chinensis*) were mostly controlled by Cu adsorbed onto Mn and Fe oxides (Lange *et al.*, 2014). Mobility is higher in soils impacted by
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mining. Overall, the strong affinity of Mn oxides for Co may explain the lower Co mobility in
Mn-rich soils (Collins & Kinsela, 2011). Cobalt accumulation (in *C. perennis* and *A. chinensis*) was strongly influenced by free Co and by Co adsorbed onto the OM and Fe oxide
fractions (Lange *et al.*, 2014).

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Copper and Co concentrations in soils from Goiás (513–2015 µg g⁻¹ and 430–1230 µg g⁻¹, respectively) only have a small mobile fraction (0.05–4.25 µg g⁻¹ and 7–134 µg g⁻¹, respectively; Fig. 2a, 3a). The Ni-hyperaccumulator *Pfaffia sarcophylla* (Amaranthaceae) displays high foliar Cu concentrations (105–298 µg g⁻¹) on Niquelândia 7 soil but far lower concentrations (8–26 µg g⁻¹) on Niquelândia 5 soil (van der Ent & Reeves, 2015; Fig. 2b). *Piriqueta sp.* (Turneraceae) can accumulate up to 149 µg g⁻¹ foliar Co (van der Ent & Reeves, 2015; Fig. 3b).

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Soils from Barberton have lower Cu-Co concentrations (50–150 µg g⁻¹ and 95–272 µg g⁻¹, respectively) with a very low mobile fraction (0.01–1.86 µg g⁻¹ and 2–13 µg g⁻¹, respectively; Fig. 2a, 3a). No Cu accumulation has been found. When soils are waterlogged (i.e. as at Groenvaly, Fig. 3), Co is mainly associated with amorphous Fe oxides. Soluble Co/Ni ratio becomes then higher (i.e. 1/5) than in typical ultramafic soils (1/20–1/100) and *Berkheya coddii* may thus accumulate >600 µg g⁻¹. Cobalt is preferred over Ni by *B. coddii* when both are supplied at isomolar ratios as for *Alyssum* species (Homer *et al.*, 1991). Cobalt (hyper)accumulation in ultramafic soils is triggered by the available Co/Ni ratio.

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*Influence of the rhizosphere biota on trace element uptake by plants*

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Soil-plant-microbe interactions are widely investigated for the study of soil-plant processes (Huang *et al.*, 2014; Dessaux *et al.*, 2016). Soil microbial community has the capacity to mobilize/immobilize trace element, and thus, to affect the soil TE speciation (reviewed by Gadd, 2004) and availability (Cuypers *et al.*, 2013). However, the rhizosphere biota influence on Cu-Co accumulation remains poorly understood. It should be emphasised that natural soil properties are difficult to replicate synthetically and directly influence accumulation patterns in plants. The most appropriate method to study strategies of tolerance, therefore, is using natural soils from the habitat of the species under investigation (van der Ent *et al.*, 2015a).

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A diversity of metal-tolerant bacteria and fungi are naturally present in metalliferous soils (including plant growth-promoting bacteria, endophytic bacteria and Arbuscular Mycorrhizal

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Fungi (AMF)), having in some cases a beneficial effect on plant establishment, growth and
TE tolerance and accumulation (Sessitsch et al., 2013; Lenoir et al., 2016; Ma et al., 2016).
However, the majority of studies have focused on Ni- and Zn-accumulating plants. For Cu,
Chen et al. (2005) demonstrated that Cu-tolerant bacteria strains from the rhizosphere of E.
splendens had a positive effect on both the Cu mobility in soil and uptake by the plant. In the
same species, the presence and diversity of AMF had a positive effect on the uptake of Cu
from a Cu-contaminated soil (±15%) (Wang et al., 2005; 2007).

Endophytic bacteria associated with *H. katangense* and *C. tenuis* (Katangan Copperbelt) have been characterized (Kabagale *et al.*, 2010). However, no studies have yet been investigating the relationships between Cu-Co accumulation by these plants and their rhizosphere biota diversity/activity. Hypothetical influence of rhizosphere biotic processes on Cu-Co speciation, availability and uptake is displayed in Fig. 4. Further studies should be performed, especially *in situ* characterization of the rhizosphere microbial community from a Cu-Co metallophyte in relationship to Cu-Co uptake and accumulation.

## Phytomining technology using Cu-Co (hyper)accumulator plants

Phytomining involves extracting target elements from the soil by accumulation in the plant biomass, which are then harvested and processed for recovering the target elements from the “bio-ore” (Chaney *et al.*, 1998; van der Ent *et al.*, 2015b). The feasibility of phytomining has been demonstrated at field scale (Brooks & Robinson, 1998; Angle *et al.*, 2001; Chaney *et al.*, 2007; van der Ent *et al.*, 2015b, Bani *et al.*, 2015), but is limited to Ni.

Copper accumulation was tested using *A. chinensis* from southeastern D. R. Congo, and some individuals could accumulate up to 5 g Cu plant⁻¹ DW and 1 335 µg Cu g⁻¹ DW, i.e. a yield of 437 g Cu ha⁻¹ year⁻¹ (Grison *et al.*, in press). The amount of Cu phytoextracted would be of the same magnitude as other species such as *E. splendens* (Yang *et al.*, 2005). Given the low economic value of Cu, combined with very limited achievable yields, it is unlikely that Cu phytomining will be commercially viable.

Cobalt phytomining was first proposed in the early 1990s as a possibility using Ni-hyperaccumulator species on ultramafic soils (Homer *et al.*, 1991), but it was noted that the presence of Ni limits the uptake of Co in most Ni-hyperaccumulator plants (Malik *et al.*,
2000). *Alyssum* species can grow with shoot concentrations >1000 µg g⁻¹ in Co-contaminated soils (Malik *et al.*, 2000) and *B. coddii* can have foliar Co concentrations of >2000 µg g⁻¹ (Keeling *et al.*, 2003). In the Copper Flora, *Haumaniastrum robertii*, with leaf Co concentration can >4000 µg g⁻¹ (Morrison *et al.*, 1981), might be a good candidate for Co phytomining. *B. coddii* could also be a candidate in Co-enriched tailings due to its high biomass and foliar Co (up to 5000 µg g⁻¹, unpublished results). Cobalt phytomining may, therefore, be considered for Co-contaminated lands because of the possible attainable yields and the high metal product value (LME is USD $24,000/tonne in March 2016).

## Conclusions and Outlook

Accumulation of Cu and/or Co in leaves is exceptionally rare globally and principally known from the Cu-Co outcrops of the Copperbelt of Central Africa. None of the putative Cu or Co hyperaccumulator plants appears to comply with the fundamental principle of hyperaccumulation that uptake and accumulation characteristics are not dose-dependent. In the case of plants growing on ultramafic soils, we postulate that Co accumulation coincides with Ni accumulation operating on similar physiological pathways. True Cu hyperaccumulation does not appear to exist as Cu tolerant plants are essentially "Excluders" sensu Baker and there is no physiological mechanism of Cu hyperaccumulation known in these species. The occasional observations of high foliar Cu concentrations can be explained by stress and the breakdown of tolerance mechanisms reducing Cu storage capacity in the roots, leading to a release of Cu in the plant shoot, which highly Cu-tolerant genotypes can survive up to a certain level.

The ecophysiology of Co tolerance and accumulation is still poorly understood compared to the state of knowledge on Cu regulation in plants, and investigations of the interactions with simultaneous tolerance/accumulation of Co and Cu makes a strong science case. Application of next-generation sequencing technologies to research in non-model organisms as Cu- and Co-tolerant and Co accumulating plants is expected to uncover novel adaptation mechanisms (Verbruggen *et al.*, 2013). The ecological significance of Cu-Co accumulation should be investigated in relation to the “leaf economics spectrum”, and what the adaptive significance of Cu-Co accumulation might be. Adaptation to local soil, by characterizing the soil microbial communities and compare the response of Cu-Co-tolerant *versus* non-tolerant populations to the pathogen pressure by reciprocal transplantation should be investigated.
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Author contributions

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The application of Co accumulator plants in phytomining is limited because their accumulation characteristics mean that only ‘ore-grade soils’ may yield sufficient accumulation in the shoots to develop this technology. Cobalt phytomining may be considered for strongly Co-contaminated soils and tailings, because of the high metal product value compared to Cu.

The Belgian Fund for Scientific Research (FRS-FNRS, Belgium) is acknowledged for financial support to B.L. as research fellow of the “Fonds pour la Recherche dans l'Industrie et l'Agriculture”. A.V.D.E. was the recipient of a post-doctoral scholarship from the French National Research Agency through the national “Investissements d'avenir” program (ANR-10-LABX-21 - LABEX RESSOURCES21), and currently an Australian Research Council Post Doctoral Fellowship (DE160100429).

B.L., A.V.D.E. and M.P.F. conceived the manuscript. B.L., A.V.D.E., A.J.M.B., G.E., G.M., F.M., P.M., O.P., N.V. and M.P.F. wrote the manuscript with B.L. coordinating the writing process.
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724

*Biology* **36**: 409–430.

725

Zheng L, Yamaji N, Yokosho K, Ma JF. 2012. YSL16 is a phloem-localized transporter of

726

the copper-nicotianamine complex that is responsible for copper distribution in rice. *The Plant*

727

*Cell* **24**: 3767–3782.
728 **Table captions**

729

730 **Table 1.** Species for which the capacity of population(s) to grow under elevated concentrations of Cu and/or Co has been experimentally demonstrated.

731
732 The reported concentrations correspond to the higher experimental treatment, or correspond to
733 the concentration above which plants' growth was inhibited. CB = Katangan Copperbelt.

734

735 **Table 2.** Updated list of species recorded with at least one healthy specimen with a foliar
736 concentration >300 mg kg⁻¹ DW.

737 Were considered in this list plants that have received a washing protocol only (clearly
738 mentioned in the publication). When available, a range of variation is given. Otherwise,
739 values are single measurements, or mean values with standard deviation. *NA* = Not Available
740 data. *Ex situ* = measurement on plants growing under experimental conditions. CB =
741 Katangan Copperbelt.

742
743 **Figure captions**

744

745 **Figure 1.** Visual appearance of selected Cu- and/or Co-accumulator plants.

746 **(a)** *Haumaniastrum robertii* (Lamiaceae) (D. R. Congo). Photo credit: copperflora.org; **(b)**

747 *Hibiscus rhodanthus* (Malvaceae) (D. R. Congo). Photo credit: copperflora.org; **(c)**

748 *Anisopappus chinensis* (Asteraceae) (D. R. Congo). Photo credit: B. Lange; **(d)** *Rinorea*

749 *bengalensis* (Violaceae) (S-E Asia). Photo credit: A. van der Ent; **(e)** *Crepidorhopalon tenuis*

750 (Linderniaceae) (D. R. Congo). Photo credit: B. Lange; **(f)** *Berkheya codii* (Asteraceae)

751 (South-Africa). Photo credit: A. van der Ent; **(g)** *Alyssum murale* (Brassicaceae) (USA). Photo

752 credit: A. van der Ent.

753

754 **Figure 2.** Copper speciation in soils (modeled after Pourret *et al.*, 2015) and accumulation in

755 metallophytes from various origins.

756 **(a)** Proportion of Cu species in considered soils; **(b)** Inorganic Cu concentrations (mobile

757 concentrations considered as available) in soils. Accumulated concentrations in plants are

758 added when available. F5: Fungurume 5; G2: Goma 2; Et: Etoile; Niq: Niquelândia; Gr:

759 Groenvaly; So: Songimvelo; AM: Agnes Mine.

760

761 **Figure 3.** Cobalt speciation in soils (modeled after Pourret *et al.*, 2015) and accumulation in

762 metallophytes from various origins.

763 **(a)** Proportion of Co species in considered soils; **(b)** Inorganic Co concentrations in soils

764 (mobile concentrations considered as available). Accumulated concentrations in plants are

765 added when available. F5: Fungurume 5; G2: Goma 2; Et: Etoile; Niq: Niquelândia; Gr:

766 Groenvaly; So: Songimvelo; AM: Agnes Mine.

767

768 **Figure 4.** Hypothetical rhizosphere processes influencing Cu and Co availability and uptake

769 by metallophytes originating from naturally Cu- and Co-enriched soils.

770 LOA = Low-molecular-weight Organic Acids. The proportions of the different Cu and Co

771 fractions in the soil are related to the font size (Lange *et al.*, 2014; Pourret *et al.*, 2015).
<table><caption>Table 1</caption><thead><tr><th></th><th>Origin</th><th>Experimental demonstration of Cu tolerance, or of the capacity to grow under elevated concentration of Cu</th><th>Experimental demonstration of Co tolerance or of the capacity to grow under elevated concentration of Co</th><th>Growth medium</th><th>References</th></tr></thead><tbody><tr><td><strong>Asteraceae</strong><br>Anisopappus chinensis Hook.f. & Arn.</td><td>CB (D.R. Congo)</td><td>up to 500 µg g<sup>-1</sup><br>(total Cu)</td><td>up to 50 µg g<sup>-1</sup><br>(total Cu)</td><td>Soil</td><td>Lange et al., 2014</td></tr><tr><td><strong>Caryophyllaceae</strong><br>Minuartia hirsuta (M.Bieb.) Hand.-Mazz.</td><td>Kilkis (Greece)</td><td>up to 80 µM CuSO<sub>4</sub></td><td>-</td><td>Nutrient solution</td><td>Ouzounidou et al., 1994</td></tr><tr><td>Silene burchelli Otth ex DC. (Ecotype)</td><td>Luitia (D. R. Congo)</td><td>&gt;1 000 µg g<sup>-1</sup><br>(total Cu)</td><td>up to 800 µg g<sup>-1</sup><br>(total Co)</td><td>Soil</td><td>Baker et al., 1983</td></tr><tr><td>Silene burchelli var. angustifolia Sond.</td><td>Kundelungu Plateau (D. R. Congo)</td><td>up to 500 µg g<sup>-1</sup><br>(total Cu)</td><td>up to 100 µg g<sup>-1</sup><br>(total Co)</td><td>Soil</td><td>Baker et al., 1983</td></tr><tr><td>Silene cobalticola P.A. Duvign. & Plancke</td><td>CB (D.R. Congo)</td><td>&gt;1 000 µg g<sup>-1</sup><br>(total Cu)</td><td>&gt;1 000 µg g<sup>-1</sup><br>(total Co)</td><td>Soil</td><td>Baker et al., 1983</td></tr><tr><td>Silene vulgaris (Moench) Garcke</td><td>Ontario (Canada)</td><td>-</td><td>up to 5 mg l<sup>-1</sup></td><td>Nutrient solution</td><td>Paliouris & Hutchinson, 1991</td></tr><tr><td></td><td>Marsberg - Imsbach - Harlingerode<br>(Germany)</td><td>up to 8 µM</td><td>-</td><td>Nutrient solution</td><td>Schat & Ten Bookum, 1992</td></tr><tr><td></td><td>Imsbach (Germany)</td><td>19 to 8 645 µg g<sup>-1</sup><br>(total Cu)</td><td>-</td><td>Soil</td><td>Song et al., 2004</td></tr></tbody></table>
<table><tbody><tr><td><strong>Crassulaceae</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>Crassula helmsii</em> (Kirk) Cockayne</td><td>Oer-Erkenschwick (Germany)</td><td>up to 20 µM</td><td>-</td><td>Nutrient solution</td><td>Küpper et al., 1996</td></tr><tr><td><strong>Lamiaceae</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>Aeolanthus biformifolius</em> De Wild.</td><td>CB (D.R. Congo)</td><td>up to 9 000 µg g<sup>-1</sup><br>(total Cu)</td><td>up to 670 µg g<sup>-1</sup><br>(total Co)</td><td>Soil</td><td>Morrison et al., 1979</td></tr><tr><td><em>Elsholtzia haichowensis</em> Sun.</td><td>Yangtze River (China)</td><td>0.32 to 500 µM</td><td>-</td><td>Nutrient solution</td><td>Lou et al., 2004</td></tr><tr><td><em>Elsholtzia splendens</em> Nakai ex F.Maek.</td><td>Zhejiang (China)</td><td>up to 1 000 µg g<sup>-1</sup><br>(total Cu)</td><td>-</td><td>Soil</td><td>Jiang et al., 2004</td></tr><tr><td></td><td>Zhejiang (China)</td><td>up to 5 146 µg g<sup>-1</sup><br>(total Cu)</td><td>-</td><td>Soil</td><td>Song et al., 2004</td></tr><tr><td><em>Haumaniastrum katangense</em> (S. Moore)<br>Duvign. & Plancke</td><td>CB (D.R. Congo)</td><td>up to 100 µM</td><td>-</td><td>Nutrient solution</td><td>Chipeng et al., 2010; Peng et al., 2012</td></tr><tr><td></td><td></td><td>up to 1 400 µg g<sup>-1</sup></td><td>up to 600 µg g<sup>-1</sup></td><td>Soil</td><td>Morrison et al., 1979</td></tr><tr><td><em>Haumaniastrum robertii</em> (Robyns) P.A.<br>Duvign. & Plancke</td><td>CB (D.R. Congo)</td><td>up to 8 500 µg g<sup>-1</sup><br>(total Cu)</td><td>up to 4 000 µg g<sup>-1</sup><br>(total Co)</td><td>Soil</td><td>Morrison et al., 1979</td></tr><tr><td><strong>Linderniaceae</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>Crepidorhopalon tenuis</em> (S. Moore) Eb.<br>Fisch.</td><td>CB (D.R. Congo)</td><td>up to 60 µM</td><td>-</td><td>MS medium</td><td>Faucon et al., 2012a</td></tr><tr><td><em>Crepidorhopalon perennis</em> (P.A. Duvign.)<br>Eb. Fisch.</td><td>CB (D.R. Congo)</td><td>up to 60 µM</td><td>-</td><td>MS medium</td><td>Faucon et al., 2012a</td></tr></tbody></table>
<table><tbody><tr><td><strong>Phrymaceae</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>Mimulus guttatus</em> Fischer ex DC.</td><td>California (USA)</td><td>1.0 parts/10<sup>6</sup></td><td>-</td><td>Nutrient solution</td><td>Allen &amp; Sheppard, 1971; Macnair, 1983</td></tr><tr><td><strong>Poaceae</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>Agrostis capillaris</em> L.</td><td>Parys Mountain, Anglesey (UK)<br>Black Forest (Germany)</td><td>up to 15 µM</td><td>-</td><td>Nutrient solution</td><td>Jowett, 1958</td></tr><tr><td><em>Agrostis gigantea</em> Roth. (one clone)</td><td>Ontario (Canada)</td><td>up to 20 mM</td><td>up to 170 mM</td><td>Nutrient solution</td><td>Hogan &amp; Rauser, 1979</td></tr><tr><td><em>Agrostis stolonifera</em> L.</td><td>Piesky (Slovakia)</td><td>up to 15 µM</td><td>-</td><td>Nutrient solution</td><td>Jowett, 1958</td></tr><tr><td><em>Calamagrostis epigejos</em> (L.) Roth</td><td>Legnica (Poland)</td><td>up to 2 mM</td><td>-</td><td>Nutrient solution</td><td>Lehmann &amp; Rebele, 2004</td></tr><tr><td><em>Cynodon dactylon</em> (L.) Pers.</td><td>Guangdong (China)</td><td>up to 0.5 mg l<sup>-1</sup></td><td>-</td><td>Nutrient solution</td><td>Shu et al., 2002</td></tr><tr><td><em>Deschampsia cespitosa</em> (L.) Beauv.</td><td>Coniston smelter population, Ontario<br>(Canada)</td><td>0.3 µg cm<sup>-3</sup></td><td>-</td><td>Nutrient solution</td><td>Cox &amp; Hutchinson, 1980</td></tr><tr><td><em>Paspalum distichum</em> L.</td><td>Guangdong (China)</td><td>up to 1 mg l<sup>-1</sup></td><td>-</td><td>Nutrient solution</td><td>Shu et al., 2002</td></tr></tbody></table>
<table><caption>Table 2</caption><thead><tr><th></th><th>Foliar Cu<br>(µg g<sup>-1</sup> DW)</th><th>Foliar Co<br>(µg g<sup>-1</sup> DW)</th><th>Origin<br>of<br>samples</th><th>Site</th><th>Restriction to<br>metalliferous<br>soils</th><th>Life-cycle</th><th>References</th></tr></thead><tbody><tr><td colspan="8"><strong>MAGNOLIOPSIDA</strong></td></tr><tr><td colspan="8"><strong>Amaranthaceae</strong></td></tr><tr><td><em>Celosia trigyna</em> L.</td><td>345 to 603</td><td>75 to 501</td><td>In situ</td><td>CB (D.R. Congo)</td><td>Facultative</td><td>Annual</td><td>Faucon et al., 2007</td></tr><tr><td rowspan="3"><em>Pandiaka carsonii</em> Baker (C.B. Clarke)</td><td>273 to 445</td><td>6 to 7</td><td>In situ</td><td>CB (D.R. Congo)</td><td rowspan="3">Facultative</td><td rowspan="3">Perennial</td><td rowspan="3">Lefèbvre et al., unpublished ;<br>Duvigneaud & Denaeyer-De Smet, 1963<br>Delhaye et al., 2016</td></tr><tr><td>740</td><td>NA</td><td>In situ</td><td>CB (D.R. Congo)</td></tr><tr><td>1 870</td><td>NA</td><td>In situ</td><td>CB (D.R. Congo)</td></tr><tr><td colspan="8"><strong>Apocynaceae</strong></td></tr><tr><td><em>Carissa spinarum</em> L.</td><td>702 (leaf+stem)</td><td>1.6 (leaf+stem)</td><td>In situ</td><td>Ginigalpelessa (Sri<br>Lanka)</td><td>Facultative</td><td>Perennial</td><td>Rajakaruna & Bohm, 2002</td></tr><tr><td colspan="8"><strong>Asclepiadaceae</strong></td></tr><tr><td><em>Calotropis gigantea</em> L. R. Br.</td><td>582 (leaf+stem)</td><td>0.8 (leaf+stem)</td><td>In situ</td><td>Ginigalpelessa (Sri<br>Lanka)</td><td>Facultative</td><td>Perennial</td><td>Rajakaruna & Bohm, 2002</td></tr><tr><td colspan="8"><strong>Asteraceae</strong></td></tr><tr><td><em>Anisopappus chinensis</em> Hook.f. & Arn.</td><td>4 to 2800</td><td>3 to 1300</td><td>In situ</td><td>CB (D.R. Congo)</td><td>Facultative</td><td>Perennial</td><td>Lange et al., 2014</td></tr><tr><td><em>Berkheya coddii</em> Roessler</td><td>NA</td><td>40 to 2116</td><td>Ex situ</td><td>South-Africa</td><td>Obligate</td><td>Perennial</td><td>Keeling et al., 2003</td></tr><tr><td><em>Vernoniastrum latifolium</em> (Steetz) H. Rob.</td><td>269 to 1942</td><td>82 to 549</td><td>In situ</td><td>CB (D.R. Congo)</td><td>Facultative</td><td>Annual</td><td>Faucon et al., 2007</td></tr><tr><td colspan="8"><strong>Boraginaceae</strong></td></tr><tr><td><em>Onosma stenosiphon</em> Boiss.</td><td>300 to 657</td><td>NA</td><td>In situ</td><td>Sarcheshmeh (Iran)</td><td>Facultative</td><td>Biennial to<br>perennial</td><td>Ghaderian & Ravandi, 2012</td></tr></tbody></table>
<table>
  <tbody>
    <tr>
      <td><strong>Brassicaceae</strong></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>Alyssum corsicum Duby</td>
      <td>NA</td>
      <td>1 080 ± 260 (shoot)</td>
      <td>Ex situ</td>
      <td>Greece</td>
      <td>Facultative</td>
      <td>Perennial</td>
      <td>Malik et al., 2000</td>
    </tr>
    <tr>
      <td>Alyssum heldreichii Hausskn.</td>
      <td>NA</td>
      <td>&lt;5 to &gt;1 000</td>
      <td>Ex situ</td>
      <td>NA</td>
      <td>Obligate</td>
      <td>Perennial</td>
      <td>Homer et al., 1991</td>
    </tr>
    <tr>
      <td>Alyssum murale Waldst. & Kit.</td>
      <td>NA</td>
      <td>2 070 ± 252 (shoot)</td>
      <td>Ex situ</td>
      <td>NA</td>
      <td>Facultative</td>
      <td>Perennial</td>
      <td>Tappero et al., 2007</td>
    </tr>
    <tr>
      <td></td>
      <td>NA</td>
      <td>1 320 ± 300 (shoot)</td>
      <td>Ex situ</td>
      <td>Greece</td>
      <td></td>
      <td></td>
      <td>Malik et al., 2000</td>
    </tr>
    <tr>
      <td>Alyssum serpyllifolium Desf.</td>
      <td>NA</td>
      <td>&lt;5 to &gt;1 000</td>
      <td>Ex situ</td>
      <td>NA</td>
      <td>Facultative</td>
      <td>Perennial</td>
      <td>Homer et al., 1991</td>
    </tr>
    <tr>
      <td>Alyssum tenium Halácsy</td>
      <td>NA</td>
      <td>&lt;5 to &gt;1 000</td>
      <td>Ex situ</td>
      <td>NA</td>
      <td>Obligate</td>
      <td>Perennial</td>
      <td>Homer et al., 1991</td>
    </tr>
    <tr>
      <td>Alyssum troodii Boiss.</td>
      <td>71</td>
      <td>10 to 2 325</td>
      <td>Ex situ</td>
      <td>Republic of Cyprus</td>
      <td>Obligate</td>
      <td>Perennial</td>
      <td>Homer et al., 1991</td>
    </tr>
    <tr>
      <td><strong>Caryophyllaceae</strong></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>Silene cobalticola P.A. Duvign. & Plancke</td>
      <td>1660</td>
      <td>NA</td>
      <td>In situ</td>
      <td>CB (D.R. Congo)</td>
      <td>Obligate</td>
      <td>Perennial</td>
      <td>Duvigneaud & Denaeyer-De Smet, 1963</td>
    </tr>
    <tr>
      <td><strong>Cornaceae</strong></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>Nyssa sylvatica var. biflora (Walt.) Sarg.</td>
      <td>NA</td>
      <td>0 to 438</td>
      <td>Ex situ</td>
      <td>Southeastern USA</td>
      <td>Facultative</td>
      <td>Perennial</td>
      <td>McLeod & Ciravolo, 2007</td>
    </tr>
    <tr>
      <td></td>
      <td>NA</td>
      <td>300</td>
      <td>Ex situ</td>
      <td>Florida (USA)</td>
      <td>Facultative</td>
      <td>Perennial</td>
      <td>Malik et al., 2000</td>
    </tr>
    <tr>
      <td>Nyssa sylvatica var. sylvatica Marsh.</td>
      <td>NA</td>
      <td>800</td>
      <td>Ex situ</td>
      <td>Florida (USA)</td>
      <td>Facultative</td>
      <td>Perennial</td>
      <td>Malik et al., 2000</td>
    </tr>
    <tr>
      <td><strong>Crassulaceae</strong></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>Crassula helmsii (Kirk) Cockayne</td>
      <td>9200 ±1500</td>
      <td>NA</td>
      <td>Ex situ</td>
      <td>Oer-Erkenschwick (Germany)</td>
      <td>Facultative</td>
      <td>Perennial</td>
      <td>Küpper et al., 2009</td>
    </tr>
    <tr>
      <td><strong>Euphorbiaceae</strong></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>Acalypha cupricola Robyns</td>
      <td>390 to 2890</td>
      <td>207 to 904</td>
      <td>In situ</td>
      <td>CB (D.R. Congo)</td>
      <td>Obligate</td>
      <td>Perennial</td>
      <td>Faucon et al., 2007</td>
    </tr>
    <tr>
      <td>Croton bonplandianus Baill.</td>
      <td>2 163 (leaf+stem)</td>
      <td>2 (leaf+stem)</td>
      <td>In situ</td>
      <td>Ginigalpelessa (Sri)</td>
      <td>Facultative</td>
      <td>Perennial</td>
      <td>Rajakaruna & Bohm, 2002</td>
    </tr>
  </tbody>
</table>
Lanka)

Fabaceae

<table><tr><td>Senna auriculata L.</td><td>885 (leaf+stem)</td><td>1 (leaf+stem)</td><td>In situ</td><td>Ussangoda (Sri Lanka)</td><td>Facultative</td><td>Perennial</td><td>Rajakaruna &amp; Bohm, 2002</td></tr><tr><td>Tephrosia villosa Pers.</td><td>1 858 (shoot)</td><td>0.2</td><td>In situ</td><td>Indikolapelessa (Sri Lanka)</td><td>Facultative</td><td>Annual to perennial</td><td>Rajakaruna &amp; Bohm, 2002</td></tr><tr><td>Vigna dolomitica R. Wilczek</td><td>222 to 1000</td><td>241 to 540</td><td>In situ</td><td>CB (D.R. Congo)</td><td>Obligate</td><td>Perennial</td><td>Faucon et al., 2007</td></tr></table>

Lamiaceae

<table><tr><td>Aeolanthus biformifolius De Wild.</td><td>0 to 78</td><td>0 to 1 900</td><td>Ex situ</td><td>CB (D.R. Congo)</td><td>Obligate</td><td>Perennial</td><td>Morrison et al., 1978</td></tr><tr><td>Elsholtzia haichowensis Sun.</td><td>NA</td><td>17 to 391</td><td>In situ</td><td>Yangtze River (China)</td><td>Obligate</td><td>Annual</td><td>Tang et al., 1999</td></tr><tr><td>Elsholtzia splendens Nakai ex F.Maek.</td><td>&lt;10 to &gt;500</td><td>NA</td><td>Ex situ</td><td>Zhuji (China)</td><td>Facultative</td><td>Annual</td><td>Yang et al., 2002</td></tr><tr><td>Ocimum tenuiflorum L.</td><td>2 265 (leaf+stem)</td><td>11 (leaf+stem)</td><td>In situ</td><td>Yodhagannawa (Sri Lanka)</td><td>Facultative</td><td>Perennial</td><td>Rajakaruna &amp; Bohm, 2002</td></tr><tr><td>Haumaniastrum katangense (S. Moore) Duvign. &amp; Plancke</td><td>187 to 317</td><td>224 to 772</td><td>In situ</td><td>CB (D.R. Congo)</td><td>Facultative</td><td>Annual</td><td>Faucon et al., 2007</td></tr><tr><td>Haumaniastrum robertii (Robyns) P.A. Duvign. &amp; Plancke</td><td>62 to 6159</td><td>62 to 326</td><td>In situ</td><td>CB (D.R. Congo)</td><td>Obligate</td><td>Annual</td><td>Duvigneaud &amp; Denaeyer-De Smet, 1963</td></tr><tr><td></td><td>28</td><td>&gt;7 200</td><td>Ex situ</td><td>CB (D.R. Congo)</td><td></td><td></td><td>Morrison et al., 1979</td></tr></table>

Linderniaceae

<table><tr><td>Crepidorhopalon tenuis (S. Moore) Eb. Fisch.</td><td>0 to 2524</td><td>8 to 605</td><td>In situ</td><td>CB (D.R. Congo)</td><td>Facultative</td><td>Annual</td><td>Faucon et al., 2007</td></tr><tr><td>Crepidorhopalon perennis (P.A. Duvign.) Eb. Fisch.</td><td>80 to 1384</td><td>61 to 1105</td><td>In situ</td><td>CB (D.R. Congo)</td><td>Obligate</td><td>Short-lived perennial</td><td>Faucon et al., 2007</td></tr></table>

Malvaceae
<table>
  <tr>
    <td>Abutilon indicum Sweet</td>
    <td>915 (leaf+stem)</td>
    <td>1 (leaf+stem)</td>
    <td>In situ</td>
    <td>Indikolapelessa (Sri Lanka)</td>
    <td>Facultative</td>
    <td>Perennial</td>
    <td>Rajakaruna &amp; Bohm, 2002</td>
  </tr>
  <tr>
    <td>Hibiscus rhodanthus Gürke</td>
    <td>102 to 515</td>
    <td>42 to 274</td>
    <td>In situ</td>
    <td>CB (D.R. Congo)</td>
    <td>Facultative</td>
    <td>Perennial</td>
    <td>Faucon et al., 2007</td>
  </tr>
  <tr>
    <td>Triumfetta welwitschii Mast.</td>
    <td>111 to 704</td>
    <td>162 to 1971</td>
    <td>In situ</td>
    <td>CB (D.R. Congo)</td>
    <td>Facultative</td>
    <td>Perennial</td>
    <td>Faucon et al., 2007</td>
  </tr>
  <tr>
    <td><strong>Onagraceae</strong></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>Epilobium hirsutum L.</td>
    <td>1300 to 1581</td>
    <td>NA</td>
    <td>In situ</td>
    <td>Sarcheshmeh (Iran)</td>
    <td>Facultative</td>
    <td>Perennial</td>
    <td>Ghaderian &amp; Ravandi, 2012</td>
  </tr>
  <tr>
    <td><strong>Orobanchaceae</strong></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>Alectra sessiliflora (Vahl) O. Ktze.</td>
    <td>45 to 769</td>
    <td>21 to 866</td>
    <td>In situ</td>
    <td>CB (D.R. Congo)</td>
    <td>Facultative</td>
    <td>Annual</td>
    <td>Faucon et al., 2007</td>
  </tr>
  <tr>
    <td>Buchnera henriquesii Engl.</td>
    <td>142 to 242</td>
    <td>404 to 930</td>
    <td>In situ</td>
    <td>CB (D.R. Congo)</td>
    <td>Facultative</td>
    <td>Annual</td>
    <td>Faucon et al., 2007</td>
  </tr>
  <tr>
    <td><strong>Phrymaceae</strong></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>Mimulus guttatus Fischer ex DC.</td>
    <td>120 to 780</td>
    <td>NA</td>
    <td>Ex situ</td>
    <td>USA</td>
    <td>Facultative</td>
    <td>Annual and perennial</td>
    <td>Tilstone &amp; Macnair, 1997</td>
  </tr>
  <tr>
    <td><strong>Phyllanthaceae</strong></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>Aporosa chalarocarpa Airy Shaw</td>
    <td>NA</td>
    <td>468</td>
    <td>In situ</td>
    <td>Sabah (Malaysia)</td>
    <td>Facultative</td>
    <td>Perennial</td>
    <td>Van der Ent et al., 2015c</td>
  </tr>
  <tr>
    <td>Glochidion cf. sericeum (Blume) Zoll. &amp; Moritzi</td>
    <td>NA</td>
    <td>442 to 1310</td>
    <td>In situ</td>
    <td>Sabah (Malaysia)</td>
    <td>Facultative</td>
    <td>Perennial</td>
    <td>Van der Ent et al., 2015c</td>
  </tr>
  <tr>
    <td><strong>Poaceae</strong></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>Agrostis stolonifera L.</td>
    <td>100 to 1000</td>
    <td>NA</td>
    <td>In situ</td>
    <td>Prescot (UK)</td>
    <td>Facultative</td>
    <td>Perennial</td>
    <td>Wu et al., 1974</td>
  </tr>
  <tr>
    <td>Polypogon fugax Nees ex steud.</td>
    <td>1550 to 4012</td>
    <td>NA</td>
    <td>In situ</td>
    <td>Sarcheshmeh (Iran)</td>
    <td>Facultative</td>
    <td>Annual</td>
    <td>Ghaderian &amp; Ravandi, 2012</td>
  </tr>
  <tr>
    <td><strong>Polygonaceae</strong></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>Rumex acetosa Linn.</td>
    <td>340 to 1102</td>
    <td>NA</td>
    <td>In situ</td>
    <td>Yangtze River (China)</td>
    <td>Obligate</td>
    <td>Perennial</td>
    <td>Tang et al., 1999</td>
  </tr>
</table>
<table><tbody><tr><td><strong>Solanaceae</strong><br><i>Hyoscyamus senecionis</i> Willd.</td><td>87 to 331</td><td>NA</td><td>In situ</td><td>Sarcheshmeh (Iran)</td><td>Facultative</td><td>Perennial</td><td>Ghaderian & Ravandi, 2012</td></tr><tr><td><strong>Sterculiaceae</strong><br><i>Waltheria indica</i> L.</td><td>1 504 (leaf+stem)</td><td>1.3 (leaf+stem)</td><td>In situ</td><td>Ginigalpelessa (Sri Lanka)</td><td>Facultative</td><td>Short-lived perennial</td><td>Rajakaruna & Bohm, 2002</td></tr><tr><td><strong>Verbenaceae</strong><br><i>Clerodendrum infortunatum</i> L.</td><td>2 280 (leaf+stem)</td><td>0.6 (leaf+stem)</td><td>In situ</td><td>Ginigalpelessa (Sri Lanka)</td><td>Facultative</td><td>Perennial</td><td>Rajakaruna & Bohm, 2002</td></tr><tr><td><strong>Violaceae</strong><br><i>Rinorea bengalensis</i> (Wall.) O.Ktze.</td><td>NA</td><td>0.5 to 545</td><td>In situ</td><td>S.E. Asia</td><td>Facultative</td><td>Perennial</td><td>Brooks et al., 1977</td></tr><tr><td><i>Rinorea javanica</i> (Bl.) O.Ktze.</td><td>NA</td><td>3 to 670</td><td>In situ</td><td>S.E. Asia</td><td>Facultative</td><td>Perennial</td><td>Brooks et al., 1977</td></tr></tbody></table>

## LILIOPSIDA

<table><tbody><tr><td><strong>Commelinaceae</strong><br><i>Commelina communis</i> Linn.</td><td>19 to 587</td><td>NA</td><td>In situ</td><td>Yangtze River (China)</td><td>Facultative</td><td>Annual</td><td>Tang et al., 1999</td></tr><tr><td><i>Commelina zigzag</i> P.A. Duvign. & Dewit</td><td>1 210</td><td>NA</td><td>In situ</td><td>CB (D.R. Congo)</td><td>Obligate</td><td>Perennial</td><td>Duvigneaud & Denaeyer-De Smet, 1963</td></tr><tr><td><i>Cyanotis longifolia</i> Benth. var. longifolia</td><td>7.5 to 286</td><td>2.3 to 1 697</td><td>In situ</td><td>CB (D.R. Congo)</td><td>Obligate</td><td>Perennial</td><td>Delhaye et al., unpublished</td></tr><tr><td><strong>Cyperaceae</strong><br><i>Ascolepis metallorum</i> P.A. Duvign. & G. Léonard</td><td>1200</td><td>NA</td><td>In situ</td><td>CB (D.R. Congo)</td><td>Obligate</td><td>Perennial</td><td>Duvigneaud & Denaeyer-De Smet, 1963</td></tr></tbody></table>