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Phylogenetic variation in heavy metal accumulation

Blackwell Science, Ltd

in angiosperms
Martin R. Broadley1, Neil J. Willey 2, Janine C. Wilkins2, Alan J. M. Baker3, Andrew Mead1 and Philip J. White1
1
Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK; 2University of the West of England, Frenchay, Bristol BS16 1QY, UK;
3
University of Melbourne, Victoria 3010, Australia

Summary

Author for correspondence: • The influence of phylogeny on shoot heavy metal content in plants was
Martin R. Broadley investigated and the hypothesis tested that traits impacting on the accumu-
Tel: +44 (0) 1789 470382
lation of cadmium, chromium, copper, nickel, lead and zinc in plant shoots are
Fax: +44 (0) 1789 470552
Email: martin.broadley@hri.ac.uk associated.
• Data suitable for comparative analyses were generated from a literature survey,
Received: 13 March 2001
using a residual maximum likelihood (REML) procedure. Both pair-wise regressions
Accepted: 5 June 2001
and principal components analyses (PCA) were performed on independent contrasts
of shoot metal content.
• Significant variation in shoot metal content occurred at the classification level of
order and above, suggesting an ancient evolution of traits. Traits impacting on the
accumulation of metals in plant shoots were associated.
• This information can be used to improve predictions of soil-to-plant metal transfer,
to formulate hypotheses on the origins of metal-accumulating phenotypes and to
inform the exploitation of plant genetic resources for nutritional improvement and
phytoremediation.

Key words: Cd, Cr, Cu, heavy metals, hyperaccumulation, Ni, Pb, Zn.

© New Phytologist (2001) 152: 9–27

independently at a taxonomic level below the genus many

Introduction
Plants require at least 17 elements to complete their life cycles,
including the heavy metals Cu, Zn and Ni (Marschner,
1995). Plants also accumulate nonessential metals, such as
Pb and Cd, when they are present in the environment (Reeves
& Baker, 2000). Most metals contained within plant tissues
are acquired from the soil solution, and plants have evolved
elaborate rooting systems (Robinson, 1994; Whiting et al.,
2000) and transport mechanisms (White, 1997; Fox &
Guerinot, 1998; Morsomme & Boutry, 2000; Williams et al.,
2000) to mediate this process.
Plants display a wide range of adaptations to soils with
contrasting metal contents. Such adaptations have occurred
throughout evolution. For example, the phenotype of shoot
metal hyperaccumulation is an extreme plant response to soils
with elevated metal content. Often there is a single species
exhibiting hyperaccumulation within a genus (Reeves & Baker,
2000), suggesting that the trait of hyperaccumulation has evolved
times. Similarly, the calciotrophic phenotype, which also
impacts on the shoot content of other metals, has evolved
independently many times (Kinzel, 1982; Lee, 1999). Such evid-
ence indicates that some differences in shoot metal content
can be attributed to recent evolutionary processes. By contrast,
hyperaccumulation appears to have evolved most frequently
in certain plant groups (Reeves & Baker, 2000) (Fig. 1) and the
shoot Ca and Mg contents of dicotyledons (eudicots) are much
greater than those of monocotyledons (magnoliids) (Thompson
et al., 1997). This implies that differences in shoot metal
content can also be attributed to ancient evolutionary
processes. The influence of phylogeny on shoot metal content
has not previously been quantified. In this paper the variation
in shoot metal content has been partitioned into different
classification levels. In addition, the hypothesis was tested
that, independent of phylogeny, traits impacting on the
accumulation of Cd, Cr, Cu, Ni, Pb and Zn in plant shoots
are associated.

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10 Research

Fig. 1 Mean relative shoot metal contents of angiosperm orders. Data were obtained from loge-transformed data from 44 studies (Appendix 7).
These data were subject to residual maximum likelihood (REML) analyses and hierarchical ANOVA. Mean values are normalised for each metal
with mean and standard deviation values for each metal presented prior to normalisation. The phylogeny is based on multi-gene sequence data
(Soltis et al., 1999). The number of hyperaccumulator taxa in these orders are those listed at 1st January 2000 (Reeves & Baker, 2000; RD Reeves
& AJ Baker, unpublished).

between-study variances and means, respectively, to generate

Materials and Methods
Selecting primary data
A recent angiosperm phylogeny (Soltis et al., 1999) was used
to partition variation in shoot metal content of six heavy
metals: Cd, Cr, Cu, Ni, Pb and Zn. The primary data were
obtained from 44 studies (Appendix 7) in which direct
comparisons of shoot metal content were made in two or more
angiosperm species. These studies did not include herbarium
specimens or studies in which plants were grown in undefined
substrates or under noncomparative conditions. For a particular
metal, the studies conformed to the largest set in which at least
one plant species was contained within another study, and in
which no subsets of unlinked data arose. Different treatments
within these studies were treated as independent studies.
Generating comparative data using residual maximum
likelihood analyses
For each metal, the original data from the 44 studies were loge-
transformed and a residual maximum likelihood (REML)
analysis was performed on these transformed data (Broadley
et al., 1999). These procedures adjusted for differences in
the mean relative shoot metal content for each plant species
from studies performed under different experimental conditions
(Table 1). Hierarchical, nested ANOVA was used to partition
variation in relative shoot metal content between the
classification levels of species, genus, family and order for each
metal. All statistical analyses were performed using Genstat 5
(Genstat 5 Committee, 1997).
Testing comparative hypotheses
Hypotheses were tested that, independent of phylogeny,
traits impacting on Cd, Cr, Cu, Ni, Pb and Zn contents of
plant shoots are associated. Comparative data were obtained
by deriving independent contrasts for relative shoot metal
content, using the method of comparative analyses of
independent contrasts (CAIC) (Purvis & Rambaut, 1995).
This approach is based on the logic of comparing pairs of
species (or higher nodes) within a phylogeny that share an
immediate common ancestor. For each such pair of nodes,
the difference, or contrast, between the relative shoot metal
contents is calculated, providing an observation that is
independent of the position of the nodes within the
phylogeny, and independent of any evolutionary events

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© New Phytologist (2001) 152: 9 – 27 www.newphytologist.com
Table 1 Mean relative shoot metal contents of angiosperm species. Data were obtained from loge-transformed data from 44 studies (Appendix I). These data were subject to REML analyses
and hierarchical ANOVA. Mean values are normalized for each metal. Mean and standard deviation values for each metal are presented prior to normalization

Order Species Cd Cr Cu Ni Pb Zn Order Species Cd Cr Cu Ni Pb Zn

Fagales Myrica gale - - - − 1.32 - - Ericales Anagalis tenella - - - - - −1.13

Cucurbitales Cucumis melo − 0.02 - - - - - Chamaedaphne calyculata - - - − 1.78 - -
Cucumis sativus − 0.34 - - - 1.45 - Solanales Capsicum annuum 0.03 - - - - -
Cucurbita maxima − 0.51 - - - - - Ipomoea purpurea −1.86 - - - - -
Cucurbita vulgaris − 0.12 - - - - - Lycopersicon esculentum − 0.10 - - - - -
Rosales Sanguisorba minor - − 0.41 - − 0.51 - - Nicotiana tabacum 2.27 - - - - -
Fabales Astragalus glyciphyllus - - - - - − 1.39 Solanum lycopersicum − 0.67 - - - - -
Glycine max − 0.91 - - - − 0.02 - Solanum melongena − 0.18 - - - - -
Lotus ornithopodioides 2.44 1.53 0.22 0.86 1.16 1.71 Lamiales Calamintha nepeta 1.84 1.00 0.13 0.42 0.35 − 0.38
Medicago sativa − 1.27 - - - - - Linaria peloponnesiaca − 0.10 − 0.94 − 2.09 − 1.29 − 1.93 − 1.53
Onobrychis viciifolia − 1.62 - - - - - Marrubium peregrinum 0.93 0.91 − 0.26 − 0.06 0.55 − 0.70
Ononis spinosa 1.36 1.13 − 0.07 0.61 0.79 − 0.98 Marrubium vulgare 1.36 1.30 0.30 0.38 − 0.03 − 0.98
Phaseolus vulgaris − 1.19 - - 0.47 0.79 - Plantago holosteum 0.54 1.12 − 0.18 0.11 0.05 − 0.47
Pisum sativum − 1.24 - - - 0.23 - Plantago lanceolata - 1.93 - 0.50 - -
Trifolium arvense − 0.10 − 0.17 − 1.08 0.03 − 0.97 − 0.51 Plantago major - - 2.33 - 0.68 − 0.64
Trifolium palescens - - - - - − 0.48 Satureja montana - - - - - − 0.27
Trifolium pratense − 0.11 - - - - - Scrophularia canina − 0.10 − 0.39 −1.09 − 0.90 − 1.32 − 1.34
Trifolium repens − 0.62 - - 0.27 - - Stachys germanica - 0.28 −1.13 - - − 0.53
Vicia faba − 0.91 - - - - - Stachys serpentini - − 0.85 - − 0.84 - -
Vicia villosa − 1.22 - - - - - Teucrium polium 1.84 1.27 – 0.31 0.61 0.45 − 0.98
Malpighiales Euphorbia cyparissias - 0.23 - 0.69 - - Thymus ophioliticus - − 0.58 - − 0.65 - -
Euphorbia myrsinites 0.74 − 0.16 − 0.72 − 0.39 0.06 − 1.10 Verbascum blattaria 0.72 1.32 0.33 0.15 − 0.83 0.20
Euphorbia prostrata - − 1.29 - 0.00 - - Verbascum glandulosum − 0.17 0.17 - - − 0.13
Euphorbia taurinensis - - 1.99 - 0.54 − 0.12 Gentianales Galium degenii 0.54 0.08 0.04 − 0.07 − 0.11 − 0.76
Linum usitatissimum 0.67 - - - - - Caryophyllales Armeria denticulata - − 0.92 - − 0.93 - -
Malvales Cistus incanus - - 1.91 - 0.78 2.21 Beta vulgaris 0.29 −1.98 − 0.58 0.36 − 0.47 0.77
Cistus salviaefolius - - 0.88 - 1.00 1.81 Cerastium diffusum - - 1.85 - - 0.39
Brassicales Alyssum bertolonii - 0.16 - 3.04 - - Cerastium ligusticum - 1.25 - − 0.04 - -
Alyssum bertolonioides - 0.76 - 3.84 - - Ceterach officinarum - 0.02 - 0.03 - -
Alyssum murale 1.66 − 0.12 − 0.36 3.09 0.10 1.11 Dianthus sylvestris - − 0.96 - − 0.45 - -
Biscutella laevigata - - - - − 0.94 - Herniaria hirsuta 2.99 3.16 0.76 1.80 - 0.32
Brassica carinata − 0.22 - - - - - Minuartia ophiolitica - − 0.60 - − 0.66 - -
Brassica juncea − 0.18 - - - − 0.39 - Petrorhagia velutina - - 1.10 - 0.50 − 0.45
Brassica napus 0.51 - - - 0.77 - Rumex acetosa − 0.33 - - - - − 0.81
Brassica nigra − 0.20 - - - - - Rumex scutatus - 0.20 −1.13 - - 0.29
Brassica oleracea 0.16 − 0.93 − 0.73 0.68 0.09 2.36 Silene fabarioides - − 0.49 0.00 - - − 0.38
Brassica pekinensis − 0.39 - - - - - Silene gallica 0.54 0.14 1.81 − 0.42 0.03 0.20
Brassica rapa 0.41 - - - - - Silene italica 1.36 − 0.22 − 0.07 − 0.34 – 0.11 – 0.76

Research
Erysimum repandum 0.74 – 0.01 – 0.45 – 0.65 0.46 – 0.91 Silene vulgaris 0.34 – 0.17 0.07 - 0.07 – 0.66

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Table 1 continued

Order Species Cd Cr Cu Ni Pb Zn Order Species Cd Cr Cu Ni Pb Zn

Brassicales Raphanus sativus 0.26 - – 0.96 1.85 1.22 – 0.04 Caryophyllales Spinacea oleracea 0.80 – 1.00 0.38 – 0.07 – 0.59 1.24
(cont.) (cont.)
Rorippa nasturium-aquaticu 0.98 - - - - - Ranunculales Consolida orientalis – 0.10 0.60 – 0.34 0.00 – 0.77 1.28
Sinapis alba 0.37 - - - - - Delphinium peregrinum 0.93 – 0.57 – 0.45 – 1.13 – 0.11 0.52
Thlaspi caerulescens 0.82 - - - – 0.95 3.49 Nigella arvensis 0.54 – 0.40 – 0.07 – 1.22 – 0.43 0.32
Thlaspi ochroleucum –0.17 - - - – 0.57 1.24 Papaver somniferum 0.40 - - - - -
Thlaspi rotundifolium - - - - – 0.26 - Poales Aegilops geniculata 0.54 0.46 – 0.60 0.10 2.56 – 0.38
Saxifragales Sedum spp. - – 1.87 - – 1.27 - - Aegilops uniaristata 0.54 0.63 – 0.60 0.01 1.08 – 0.65
Apiales Apium graveolens 0.45 – 0.91 0.02 – 0.02 – 0.15 1.68 Agrostis capillaris – 1.37 - - 0.99 0.91 -
Coriandrum sativum - – 1.72 0.28 – 1.04 – 2.11 - Avena sativa – 1.45 - - 0.64 0.65 -
Daucus carota –0.64 - - - - - Bromus squarrosus 0.63 – 0.13 – 1.26 – 0.12 – 0.54 – 0.96
Pastinaca sativa –1.52 - - - - - Carex stricta - - - – 1.65 - -
Petroselinum crispum –1.07 – 1.17 – 1.32 – 0.35 – 1.94 – 0.41 Dactylis glomerata – 0.73 - - - - -
Dipsacales Sambucus ebulus –0.10 0.77 – 0.54 – 0.51 – 0.66 – 0.11 Festuca ovina - - - 0.26 – 0.44 -
Asterales Achillea coarctata 0.54 – 0.62 – 0.18 – 0.69 – 0.72 – 1.25 Festuca pratensis – 0.53 - - - - -
Achillea millefolium - - - - - – 1.24 Festuca rubra – 0.82 - - 0.23 – 0.17 -
Ambrosia artemisiifolia - - - - – 0.67 - Holcus lanatus – 0.98 - - - - -
Anthemis tinctoria - - 1.65 - 0.72 0.06 Hordeum distichum – 0.73 - - - - -
Arctium lappa –0.91 - - - - - Hordeum sativum – 1.48 - - - - -
Campanula ligulata - - 1.71 - 0.85 0.23 Hordeum vulgare – 0.99 - - 0.07 – 0.10 -
Centaurea rupestris - –1.63 - – 0.92 - - Koeleria eriostachya 0.54 – 0.51 – 0.45 – 0.67 4.70 0.10
Cichorium intybus –0.24 – 0.28 – 0.69 – 0.39 – 0.17 0.50 Lolium multiflorum – 0.47 - - - - -
Chrysanthemum coronarium 0.05 - - - - - Lolium perenne – 0.36 - - 0.46 0.20 -
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Ditrichia viscosa –0.10 0.98 – 0.17 – 0.03 – 1.04 – 0.81 Oryza sativa – 2.37 - - 0.21 - -
Erigeron canadensis –0.57 – 0.44 - – 0.67 - - Phleum pratense – 0.66 - - 0.53 0.05 -
Eupatorium capillifolium –1.20 – 0.04 - – 0.70 - - Poa trivialis – 0.10 – 0.17 – 2.57 0.07 – 1.22 – 0.11
Filago eriocephala 1.36 0.88 1.24 1.38 0.24 0.99 Secale cereale - - - - 0.82 -
Filago vulgaris –0.10 0.28 – 0.66 – 0.32 – 1.04 – 0.93 Sorghum vulgare – 0.05 - - - - -
Helianthus annuus –0.72 - - - - - Triticum aestivum – 0.67 - 0.60 –1.43 – 0.29 – 1.19
Inula germanica 2.44 1.91 0.64 0.79 0.65 0.04 Triticum durum 1.82 - - - - -
Jasione heldreichii - - 2.18 - - 0.37 Triticum sativum – 1.57 - - - - -
Lactuca sativa 0.55 – 1.82 0.30 – 0.91 – 1.20 0.00 Triticum turgidum – 1.60 - - - - -
Lactuca viminea - 0.24 – 0.57 - - – 0.13 Triticum vulgare - - - - – 1.58 -
Leontodon hispidus - - - - – 0.57 - Zea mays – 0.01 - - – 0.55 0.30 -
Picris hieracioides 1.84 1.25 0.64 0.41 0.90 0.37 Liliales Allium ampeloprasum – 0.43 – 1.37 – 0.17 – 0.60 – 0.59 0.76
Senecio erucifolius - - - 0.74 - - Allium cepa – 0.30 – 0.06 – 0.15 – 0.90 – 0.01 1.14
Taraxacum officinale 0.59 - – 0.51 - - 0.90 Allium fistulosum – 0.62 - - - - -
Xeranthemum annuum –0.10 0.28 – 0.92 0.88 – 1.32 0.02 Allium tuberosum – 0.95 - - - - -
Xeranthemum inapertum 0.54 – 0.62 – 0.07 – 0.62 0.30 – 1.41 Asparagus officinalis – 0.61 - - - - -
Mean prior to normalisation 1.94 2.12 2.21 2.06 3.22 3.88
Standard deviation prior to 0.85 0.90 0.84 1.32 1.14 0.68
normalisation
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prior to that common ancestor. It is these independent con- Table 2 Percentage of the total sum-of-squares, at each level within
trasts that are then compared to assess whether evolutionary the hierarchically nested ANOVA, for relative shoot concentrations of
six heavy metals
changes impacting on the shoot content of different metals are
correlated. Analyses were performed on the loge-transformed
Cd Cr Cu Ni Pb Zn
means, derived from the REML analyses, and a punctuated
evolutionary model was assumed by setting equal branch Order and above 27.3 23.6 24.0 46.1 20.4 44.5
lengths within the phylogeny (Purvis & Rambaut, 1995). Family 8.7 13.3 22.9 6.3 11.5 4.1
Two approaches were used to test comparative hypotheses. Genus 52.8 51.7 32.9 42.6 61.6 40.9
First, separate null hypotheses of independent evolution were Species 11.2 11.3 20.2 5.0 7.5 10.5
tested for each pair-wise combination of metals. For each No. of studies/ 90 16 27 45 28 35
metal, the independent contrasts, as calculated above, were treatments
No. of species 108 69 64 79 81 70
regressed onto similar contrasts derived for each other metal
in turn (the independent variable). The contrast values for
the independent variable were always calculated to give
positive differences, with those for the dependent variable
calculated using the same algebraic comparison of nodes.
Results
Fitted regression lines were constrained to pass through the
Influence of phylogeny on shoot metal
origin (Harvey & Pagel, 1991), with a fitted slope significantly
accumulation
different from zero indicating a correlation between contrast
values for the two metals. Regressions were performed Hierarchical, nested ANOVA was used to quantify the
twice for each pair of metals, allowing each metal in turn to be variation in relative shoot metal content between the
considered as the independent variable. classification levels of species, genus, family and order (Table 2).
In the second approach the associations between all six Study-to-study variability within species can be estimated
heavy metals were tested simultaneously. Contrasts were from the residual variance in each REML analysis. This is
obtained for the shoot metal contents of 42 plant species expressed as the standard error of the predicted mean (SE)
yielding data for all six metals, with calculations performed for each metal (Appendices 1–6). A small SE indicates a lack
with each metal considered as the independent variable. As of variation and allows confidence in the data used for
the CAIC procedure considers the true phylogeny to bifur- subsequent analyses. The hierarchical ANOVA indicates that
cate, and thus splits daughter taxa of multiple nodes into two phylogeny influences the trait of metal accumulation in
monophyletic groups according to the trait value (Purvis & angiosperms. A large proportion of the variation in shoot
Rambaut, 1995), each metal may define a slightly different Ni (46%) and Zn (45%) content occurred at the level of the
phylogeny, hence a slightly different set of contrasts. A prin- order or above.
cipal components analysis (PCA) was performed on the sets of The mean relative shoot contents of different plant orders
contrasts calculated for each of these six defined phylogenies. for the six heavy metals (Cd, Cr, Cu, Ni, Pb and Zn) are pre-
The PCA summarizes the joint variation between the con- sented in Fig. 1. The widest range of relative shoot Ni con-
trasts for the six metals. The first principal component is the tents between different orders occurred between Brassicales
linear combination of these six variables that accounts for the (high) and Ericales (low) and for Zn between Malvales/Brassi-
greatest proportion of the total variation, with the second cales (high) and Ericales (low). Using the phylogeny of Soltis
principal component being a further linear combination that et al. (1999), it is noteworthy that hyperaccumulation is more
accounts for the greatest proportion of the remaining vari- prevalent in certain orders. For example, Ni hyperaccu-
ation, and so on. The loadings for the six variables on these mulators are prevalent in the Brassicales, Malphigiales and
principal components are the contribution of each to these Asterales (Fig. 1).
linear combinations, and the correlations between the direc-
tions defined by these loadings indicate the degree of associ-
Association between traits, independent
ation between the contrast values for the six metals. Plotting
of phylogeny
the loadings against the first two dimensions provides a visual
impression of this association. Correlations between the The hypotheses that, independent of phylogeny, traits
contrasts for different metals were calculated as cosines of impacting on the Cd, Cr, Cu, Ni, Pb and Zn contents of plant
the mean angles between the loadings, averaged across the shoots are associated were tested by two approaches (pair-wise
configurations produced from the six analyses. To obtain regression and PCA) on data obtained using a CAIC
95% confidence limits for these correlations, the 95% procedure. The null hypothesis that traits are not associated
confidence limits were calculated for the mean angles could be rejected for 21 of the 30 pair-wise regressions of
across the six configurations, and cosines of these limits then contrasts between metals (P < 0.05; Box 1). This implies
calculated. that traits have evolved which impact on the shoot content of

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Box 1 Significance of pair-wise regressions of contrasts of relative shoot metal concentration. The explanatory metal defines the
phylogeny. Regressions were constrained through the origin to test the null hypotheses of no association between pairs of metals. Values
shown are F-probabilities from the fitted regressions

Response

Cd – < 0.001 < 0.001 0.017 < 0.001 0.002

Cr < 0.001 – 0.043 < 0.001 0.051 0.185
Cu < 0.001 0.036 – 0.182 < 0.001 0.002
Ni 0.006 < 0.001 0.141 – 0.827 0.004
Pb < 0.001 0.046 < 0.001 0.816 – 0.175
Zn 0.016 0.295 < 0.001 0.002 0.237 –
Explanatory Cd Cr Cu Ni Pb Zn

Fig. 2 Biplots from principal components

analyses of contrasts, calculated with each
element in turn defining the phylogeny.
Loadings for each element are plotted against
the first two principal components (PC1 on
horizontal axis, PC2 on vertical axis).
Correlations between elements, calculated as
cosines of the mean angles between the
loadings across the six configurations, are
shown in Box 2.

more than one metal. For example, the evolution of traits
influencing the shoot content of Cr, Cu, Ni, Pb and Zn also
impacts on shoot Cd content, and the evolution of traits
influencing shoot content of Cd, Cr and Cu impacts on shoot
Pb content.
The PCA allows the hypothesis that traits impacting on
heavy metal accumulation in plant shoots are associated to
be tested for all six metals simultaneously. As the first
two principal components accounted for between 73%
and 80% of the variation in each of the six analyses, the
correlations between the loadings in these first two
dimensions were assumed to estimate the true correlations,
the remaining dimensions merely describing noise. The
loadings on these first two dimensions are presented
graphically for each of the six analyses in Fig. 2. Correla-
tions between metals, calculated from these loadings, are pre-
sented in Box 2. These correlations indicate strong positive
associations between Ni and Cr, Cu and Cd, Zn and Cd, and
Zn and Cr and a lack of association between Pb and Ni, Cr
or Zn.

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Box 2 Correlations between contrasts of shoot metal concentration of six heavy metals. Values calculated as cosines of the angles
between the loadings from the six principal components analysis configurations (Fig. 2), with each element in turn defining the
phylogeny. Mean values shown in bold with upper and lower 95% confidence limits

Cr 0.90
0.81
0.69
Cu 0.98 0.79
0.94 0.64
0.90 0.49
Ni 0.78 1.00 0.60
0.65 0.97 0.44
0.49 0.90 0.26
Pb 0.74 0.27 0.92 − 0.12
0.62 0.04 0.80 − 0.19
0.48 − 0.19 0.64 − 0.26
Zn 0.99 0.99 0.94 0.96 0.47
0.92 0.94 0.81 0.88 0.30
0.78 0.82 0.64 0.77 0.11
Cd Cr Cu Ni Pb

content is for the Brassicales, which supports an association

Discussion
Discussion of the method and recommendations for
future analyses
Here, a hierarchical ANOVA has been used to quantify the
influence of phylogeny on relative shoot metal content. This
method attempted to integrate data from a number of
sources, growth conditions and experimental designs.
However, since the total number of species represented is
small and the sampling biased, the precise conclusions may
not be definitive. Nevertheless, this paper provides a suitable
model analysis, which can be revisited as more data becomes
available.
The plants included in the present study were mostly crop
species. However, a number of species that naturally hyper-
accumulate heavy metals were also included. Such hyper-
accumulators represent only a small proportion of the species
within an order. For example, of the approximately 23 000
species within the Asterales, only 38 have been reported to
hyperaccumulate heavy metals. The proportion of hyper-
accumulator species included in this study was greater than their
natural occurrence, and it is possible that the very high shoot
Ni and Zn contents reported here for the Brassicales (Fig. 1)
are dominated by the inclusion of hyperaccumulator species.
There is an apparent discrepancy between the mean relative
shoot metal content and the number of hyperaccumulator
species within an order (Fig. 1). Part of this might be attrib-
uted to the relative sizes of the various orders, but it is also pos-
sible that the trait of metal hyperaccumulation is unrelated to
the trait of shoot metal content studied here. For the case of
Ni, although the highest value for mean relative shoot Ni
between Ni content and hyperaccumulation, other orders in
which Ni hyperaccumulation is common have similar mean
relative shoot Ni content to orders in which Ni hyperaccumu-
lators are absent or rare. Likewise, for the case of Zn, the mean
relative shoot Zn content of the Brassicales, which contains
several Zn hyperaccumulator species, is high, but not as high
as in the Malvales in which Zn hyperaccumulators are
unknown. Thus, it appears that the trait of shoot metal con-
tent investigated here is largely independent of hyperaccu-
mulation. Indeed, since hyperaccumulators represent such a
small proportion of plant species, with sufficient sampling
one would not expect such species with abnormally large
metal content to dominate the analysis.
Ideally, an analysis of the effects of phylogeny on shoot
metal content would involve sampling all species within the
phylogeny of interest. In reality, this would be impractical for
the phylogeny studied here and, to avoid bias in the results, the
sampling strategy should be carefully designed. One impor-
tant consideration is to ensure that the whole phylogeny is
fairly represented within the study, which could be achieved
by sampling proportionally from all parts of the phylogeny.
So, for example, if the Asterales contains approx. 10% of the
species in the phylogeny, then approx. 10% of species sampled
should be from this order. A second important consideration
is that species should be selected at random, with all species
within a particular order/family/genus having an equal prob-
ability of selection.
In some circumstances it may not be possible to include
observations from some part of the phylogeny. Where the
omission is a complete branch of the phylogeny, then this part
should be removed. Although this should have little impact

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16 Research
on the analysis, conclusions must obviously be restricted to
the remaining phylogeny and no extrapolation of conclusions
to missing branches should be attempted. Another problem
occurs where the sampling strategy cannot follow the ‘equal
proportions’ approach described above, as was the case in this
study. Here, there are three options. The first is to remove
the branch(es) containing too few observations, with the
obvious loss of information. A second approach is to reduce
the total number of observations so that the ‘equal proportion’
requirement can be satisfied, again with a consequent loss of
information. The third approach, as adopted in this paper, is to
work with the available data. In this case, the conclusions are
only applicable to the observed subset of species. However,
hypotheses can be developed from these data which can then
be tested using an appropriate sampling strategy.
An overall strategy to exploring associations between traits
within a complex phylogeny could follow a sequential design
approach. A first study might concentrate on the associations
at the level of order or above, collecting a random sample from
each order, in proportion to the relative number of species in
that order. Results from such a study would identify orders of
interest. Further studies would then concentrate on the asso-
ciations within a particular order, or on a more complete
phylogeny but with orders of little interest removed. Further
resolution of any phylogeny would be undertaken at the level
of family or genus. Eventually, species–environment interac-
tions could be tested within a phylogeny of interest.
A further uncertainty in the method described here arises
from the combination of studies employing contrasting
experimental conditions and, in particular, the inclusion of
studies utilizing plants grown on substrates with contrasting
metal availability. This assumes that the shoot metal content
of different species respond in the same way to elevated metals
in the environment. However, this assumption may be chal-
lenged. For example, the difference between a metal hyper-
accumulator and a nonaccumulator is likely to be greater in
a high metal environment than in a low metal environment.
Nevertheless, although the method does not address variation
in the responses of species to elevated metals at present, this
information could be incorporated at a later date.
The predictive utility of partitioning variation in shoot
metal content
The distribution of variation in shoot metal content (Table 2)
demonstrates a significant phylogenetic influence on this
parameter. Similar conclusions were reached by Thompson
et al. (1997) for the nutritional elements Ca and Mg, by
Broadley et al. (1999) for Cs, and by Jansen et al. (2000) for
Al. Indeed, the trait of shoot Al content is used as a character
trait in plant systematics (Chenery & Sporne, 1976).
Thompson et al. (1997) noted that leaf-metal content was
strongly influenced by phylogeny and habitat, which are
themselves highly correlated. In the present study, any effect
of environmental variables was removed by the REML
analysis of loge-transformed data.
By quantifying the influence of phylogeny on shoot metal
accumulation, the information reported here can be used to
improve predictions of soil-to-plant metal transfer. For
example, many soil-to-plant contaminant transfer models rely on
transfer coefficients or transfer factors to relate the concentra-
tion of a contaminant in the soil to that in the shoot of a plant
(Alloway, 1995). Often, data are unavailable for particular
habitats or species. Since hierarchical, nested ANOVA can
generate explicit intraclass correlations (i.e. trait values for
species where data are lacking can be predicted from related
species) (Harvey & Pagel, 1991), the data reported here may
be useful in estimating transfer coefficients where experi-
mental data are unavailable and in improving predictions of rela-
tive soil-to-plant metal transfer in general. The interpretation
of an intraclass correlation is the correlation expected between
any two data points (species) selected at random from the
same group. If sufficient sampling is undertaken, and 52% of
the variation in shoot Ni content in angiosperms is accounted
for by orders plus families within orders, the correlation
between Ni content of randomly chosen species from the
same family will be 0.52. Thus, estimates of the shoot Ni con-
tent of unassayed species could be made from species whose
content is known.
Traits have evolved that impact on the shoot
accumulation of several heavy metals
Quantifying phylogenetic influences on metal accumulation
allows hypotheses on the origins of metal-accumulating
phenotypes to be investigated. The CAIC analysis, in
combination with pair wise regression of independent
contrasts (Box 1) and PCA (Fig. 2; Box 2), has tested
whether traits impacting on the shoot content of metals in
plant shoots are associated. Standard pair-wise regression
techniques revealed that, in most comparisons of pairs of
metals, shoot metal contents are correlated. Results obtained
from PCA, which yielded analyses of associations between
all metals simultaneously, were generally consistent with those
obtained from pair-wise regressions. These results can be
interpreted as indicating that traits have evolved which impact
on the shoot content of more than one metal.
Notable correlations between certain elements have also
been reported for plant samples collected from the field. In
particular, a high correlation between K, Ca and Mg is often
observed (Garten, 1976; Markert, 1994; Grime et al., 1997;
Thompson et al., 1997). Kinzel (1982), on the basis of field
ecology, postulated the existence of three distinct patterns of
cation content: ‘Oxalate plants’ that precipitate Ca as the
oxalate, as exemplified by certain families of the Caryophyl-
lales and Malpighiales; ‘calciotrophes’ that contain high con-
centrations of free Ca2+, as exemplified by certain families of
the Brassicales and Fabales; and ‘potassium plants’ that have
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NPH_238.fm Page 17 Wednesday, August 29, 2001 5:13 PM
a high K : Ca ratio, as exemplified by families in the Apiales
and Asterales.
The observation that the shoot contents of most metals
(except Pb) are correlated, could result from phylogenetic
influences on a nonspecific physiological trait such as the
ratios of organic/inorganic matter or tissue fresh weight/ dry
weight. In future analyses it would be worthwhile therefore to
include data relating to potentially unlinked elements such as
N (for dry weight) and K (for fresh weight) to address whether
the appearance of linkage is related to some general phen-
omenon, or to a specific, selective transport or sequestration
process.
Shoot metal content is determined by root uptake and
sequestration within root vacuoles, translocation in the xylem
and phloem, and dilution within the shoot through growth.
Mechanisms for the transport of metals across cell membranes
(White, 1997; Fox & Guerinot, 1998; Morsomme & Boutry,
2000; Williams et al., 2000) and the chelation of metals in cell
saps (Krämer et al., 1996; Rauser, 1999; Cobbett, 2000) can
be both specific and generic. Thus, differential expression
of transport or metal-chelating proteins may account for
phylogenetic differences in shoot metal content between
angiosperm clades, and also for associations between subsets
of metals, independent of phylogeny. Structural charact-
eristics such as cell wall cation-exchange capacity (CEC) and
transpiration rates will influence movement to the shoot of
those metals following an apoplastic pathway to the xylem
(White, 2001). Thus, genotypical differences in the shoot Ca
requirements of monocotyledons and dicotyledons, which are
attributed to differences in the CEC of cell walls (Marschner,
1995), may similarly apply to other metals. In the absence of
the redistribution of specific metals within the plant, shoot
growth and development will affect the accumulation of all
metals similarly. Since phylogeny is intimately related to
shoot growth and development, associations between metals
and differences between clades are perhaps inevitable.
In summary, this study demonstrates the use of a recent
molecular phylogeny as a comparative tool. It reveals that
traits impacting on heavy metal accumulation in angiosperms
have arisen throughout evolution and that traits could have
evolved which impact on the shoot content of more than one
metal. Although causal mechanisms cannot be inferred from
our study, traits impacting on metal accumulation are likely
to be controlled by quantitative gene effects, which can be
mapped onto chromosomal loci using analogous methodo-
logy (Alonso-Blanco & Koorneef, 2000). There is considerable
interest in exploiting allelic variation or molecular approaches
to generate plants suitable for phytoremediation (Salt et al.,
1998), either to extract metals from substrates or to prevent
incorporation into the food chain. Equally, there is interest
in the nutritional enhancement of crops (Guerinot & Salt,
2001). Since phylogeny influences the ability of plants to
accumulate metals in their shoot, it may be appropriate to
identify clades that are desirably constrained when embarking
© New Phytologist (2001) 152: 9 – 27 www.newphytologist.com
Research 17
upon crop improvement strategies. For example, the Caryo-
phyllales show a general ability to accumulate metals in their
shoot. This order includes the families Chenopodiaceae,
Polygonaceae and Amaranthaceae, which contain many
rapidly growing species. Conversely, crop plants that have low
shoot metal content include members of the magnoliids, such
as cereals and alliums.
Acknowledgements
We thank Ian Burns and John Fenlon (HRI), Kathy Fawcett
(UWE), Steve Whiting (University of Melbourne) and the
anonymous referees for their useful comments on earlier
manuscripts.
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Appendix 1
Table A1 Mean relative shoot metal content, sample size and primary references for Cd data

Species Shoot Cd content n Reference

Achillea coarctata 2.40 1 36

Aegilops geniculata 2.40 1 36
Aegilops uniaristata 2.40 1 36
Agrostis capillaris 0.78 1 9
Allium ampeloprasum 1.57 2 38
Allium cepa 1.68 2 38, 42
Allium fistulosum 1.42 2 21
Allium tuberosum 1.14 2 21
Alyssum murale 3.35 9 3, 36
Apium graveolens 2.33 2 38
Arctium lappa 1.17 2 21
Asparagus officinalis 1.43 2 21
Avena sativa 0.71 3 19, 32, 33
Beta vulgaris 2.19 10 19, 21, 31, 38, 39
Brassica carinata 1.75 1 9
Brassica juncea 1.79 2 9
Brassica napus 2.37 4 9, 32, 33
Brassica nigra 1.77 2 9
Brassica oleracea 2.08 13 9, 13, 18, 19, 20, 31, 33, 38, 40, 42
Brassica pekinensis 1.61 10 15, 21, 22
Brassica rapa 2.29 5 9, 21, 31
Bromus squarrosus 2.48 2 36
Capsicum annuum 1.97 4 21, 31
Cichorium intybus 1.74 10 24, 36, 37, 38
Chrysanthemum coronarium 1.99 2 21
Consolida orientalis 1.86 1 36
Cucumis melo 1.93 2 21
Cucumis sativus 1.66 10 21, 22, 30, 32, 33
Cucurbita maxima 1.51 8 21, 22, 30
Cucurbita vulgaris 1.84 2 21
Dactylis glomerata 1.32 1 19
Daucus carota 1.39 12 15, 19, 21, 33, 39
Delphinium peregrinum 2.73 1 36
Ditrichia viscosa 1.86 1 36
Erigeron canadensis 1.46 4 24
Erysimum repandum 2.57 2 36
Eupatorium capillifolium 0.92 4 24

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Table A1 continued

Species Shoot Cd content n Reference

Euphorbia myrsinites 2.57 2 36

Festuca pratensis 1.49 1 19
Festuca rubra 1.24 5 6, 9
Filago eriocephala 3.09 1 36
Filago vulgaris 1.86 1 36
Calamintha nepeta 3.50 1 36
Galium degenii 2.40 1 36
Glycine max 1.16 10 4, 21, 22
Helianthus annuus 1.33 3 19, 33, 42
Herniaria hirsuta 4.48 1 36
Holcus lanatus 1.11 1 42
Hordeum distichum 1.32 2 21
Hordeum sativum 0.69 1 19
Hordeum vulgare 1.10 4 31, 40
Inula germanica 4.01 1 36
Ipomoea purpurea 0.36 1 9
Koeleria eriostachya 2.40 1 36
Lactuca sativa 2.41 23 5, 18, 19, 21, 26, 28, 31, 33, 38, 39
Linaria peloponnesiaca 1.86 1 36
Linum usitatissimum 2.51 2 8
Lolium multiflorum 1.54 2 21
Lolium perenne 1.63 17 6, 13, 19, 27, 42
Lotus ornithopodioides 4.01 1 36
Lycopersicon esculentum 1.86 10 19, 21, 22, 31, 42
Marrubium peregrinum 2.73 1 36
Marrubium vulgare 3.09 1 36
Medicago sativa 0.86 1 19
Nicotiana tabacum 3.87 10 27
Nigella arvensis 2.40 1 36
Onobrychis viciifolia 0.56 1 19
Ononis spinosa 3.09 1 36
Oryza sativa –0.08 9 4, 13, 21, 30
Papaver somniferum 2.28 1 7
Pastinaca sativa 0.65 1 19
Petroselinum crispum 1.03 4 21, 38
Phaseolus vulgaris 0.93 19 4, 7, 13, 14, 21, 22, 30, 31, 33
Phleum pratense 1.38 1 19
Picris hieracioides 3.50 1 36
Pisum sativum 0.89 9 20, 21, 23, 30, 33
Plantago holosteum 2.40 1 36
Poa trivialis 1.86 1 36
Raphanus sativus 2.16 20 3, 19, 20, 21, 22, 33, 39
Rorippa nasturium-aquaticum 2.78 1 19
Rumex acetosa 1.66 1 33
Sambucus ebulus 1.86 1 36
Scrophularia canina 1.86 1 36
Silene gallica 2.40 1 36
Silene italica 3.09 1 36
Silene vulgaris 2.23 8 5
Sinapis alba 2.26 1 33
Solanum lycopersicum 1.37 1 33
Solanum melongena 1.79 6 21, 22
Sorghum vulgare 1.90 3 19, 21
Spinacea oleracea 2.62 6 13, 20, 26, 38,42
Taraxacum officinale 2.44 4 37
Teucrium polium 3.50 1 36
Thlaspi caerulescens 2.64 11 5, 9, 25
Thlaspi ochroleucum 1.79 2 25
Trifolium arvense 1.86 1 36
Trifolium pratense 1.85 1 19
Trifolium repens 1.41 3 19, 21

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Table A1 continued

Species Shoot Cd content n Reference

Triticum aestivum 1.37 17 4, 16, 19, 21, 22, 28, 42

Triticum durum 3.49 1 7
Triticum sativum 0.60 2 32, 33
Triticum turgidum 0.58 4 8, 16
Verbascum blattaria 2.55 1 36
Vicia faba 1.17 1 19
Vicia villosa 0.90 1 9
Xeranthemum annuum 1.86 1 36
Xeranthemum inapertum 2.40 1 36
Zea mays 1.93 21 4, 13, 14, 19, 21, 22, 23, 30, 31, 33
Standard Error range for all taxa 0.31−1.04

Appendix 2
Table A2 Mean relative shoot metal content, sample size and primary references for Cr data

Species Shoot Cr content n Reference

Achillea coarctata 1.56 1 36

Aegilops geniculata 2.54 1 36
Aegilops uniaristata 2.69 1 36
Allium ampeloprasum 0.88 2 38
Allium cepa 2.06 1 38
Alyssum bertolonii 2.26 2 12
Alyssum bertolonioides 2.80 1 12
Alyssum murale 2.01 4 1, 36
Apium graveolens 1.30 2 38
Armeria denticulata 1.29 3 12
Beta vulgaris 0.33 1 38
Brassica oleracea 1.28 1 38
Bromus squarrosus 2.00 2 36
Centaurea rupestris 0.65 2 12
Cerastium ligusticum 3.25 3 12
Ceterach officinarum 2.14 1 12
Cichorium intybus 1.86 6 24, 36, 38
Consolida orientalis 2.66 1 36
Coriandrum sativum 0.57 3 29
Delphinium peregrinum 1.61 1 36
Dianthus sylvestris 1.26 2 12
Ditrichia viscosa 3.00 1 36
Erigeron canadensis 1.72 4 24
Erysimum repandum 2.11 2 36
Eupatorium capillifolium 2.08 4 24
Euphorbia cyparissias 2.32 1 12
Euphorbia myrsinites 1.98 2 36
Euphorbia prostrata 0.96 2 12
Filago eriocephala 2.91 1 36
Filago vulgaris 2.37 1 36
Calamintha nepeta 3.02 1 36
Galium degenii 2.19 1 36
Herniaria hirsuta 4.96 1 36
Inula germanica 3.84 1 36
Koeleria eriostachya 1.66 1 36
Lactuca sativa 0.48 4 29, 38
Lactuca viminea 2.34 1 1
Linaria peloponnesiaca 1.28 1 36
Lotus ornithopodioides 3.50 1 36
Marrubium peregrinum 2.94 1 36
Marrubium vulgare 3.29 1 36

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Table A2 continued

Species Shoot Cr content n Reference

Minuartia ophiolitica 1.58 2 12

Nigella arvensis 1.76 1 36
Ononis spinosa 3.14 1 36
Petroselinum crispum 1.07 2 38
Picris hieracioides 3.25 1 36
Plantago holosteum 3.13 2 12, 36
Plantago lanceolata 3.86 1 12
Poa trivialis 1.97 1 36
Rumex scutatus 2.30 1 1
Sambucus ebulus 2.81 1 36
Scrophularia canina 1.77 2 1, 36
Sedum spp. 0.44 2 12
Silene fabarioides 1.68 1 1
Silene gallica 2.25 1 36
Silene italica 1.92 4 12, 36
Silene vulgaris 1.97 1 1
Spinacea oleracea 1.22 4 29, 38
Stachys germanica 2.37 1 1
Stachys serpentini 1.35 1 12
Teucrium polium 3.27 1 36
Thymus ophioliticus 1.60 2 12
Trifolium arvense 1.97 1 36
Verbascum blattaria 3.30 1 36
Verbascum glandulosum 1.97 1 1
Xeranthemum annuum 2.37 1 36
Xeranthemum inapertum 1.56 1 36
Standard Error range for all taxa 0.53 − 0.90

Appendix 3
Table A3 Mean relative shoot metal content, sample size and primary references for Cu data

Species Shoot Cu content n Reference

Achillea coarctata 2.06 1 36

Aegilops geniculata 1.7 1 36
Aegilops uniaristata 1.7 1 36
Allium ampeloprasum 2.07 2 38
Allium cepa 2.08 1 38
Alyssum murale 1.9 10 1, 3, 36
Anthemis tinctoria 3.6 3 2
Apium graveolens 2.23 2 38
Beta vulgaris 1.72 1 38
Brassica oleracea 1.6 1 38
Bromus squarrosus 1.15 2 36
Campanula ligulata 3.64 3 2
Cerastium diffusum 3.76 2 2
Cichorium intybus 1.63 6 36, 37, 38
Cistus incanus 3.81 1 2
Cistus salviaefolius 2.95 1 2
Consolida orientalis 1.92 1 36
Coriandrum sativum 2.44 3 29
Delphinium peregrinum 1.83 1 36
Ditrichia viscosa 2.06 1 36
Erysimum repandum 1.84 2 36
Euphorbia myrsinites 1.6 2 36
Euphorbia taurinensis 3.88 1 2
Filago eriocephala 3.25 1 36
Filago vulgaris 1.66 1 36

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Table A3 continued

Species Shoot Cu content n Reference

Calamintha nepeta 2.32 1 36

Galium degenii 2.24 1 36
Herniaria hirsuta 2.85 1 36
Inula germanica 2.75 1 36
Jasione heldreichi 4.04 1 2
Koeleria eriostachya 1.84 1 36
Lactuca sativa 2.47 7 28, 29, 38
Lactuca viminea 1.73 1 1
Linaria peloponnesiaca 0.46 1 36
Lotus ornithopodioides 2.39 1 36
Marrubium peregrinum 1.99 1 36
Marrubium vulgare 2.46 1 36
Nigella arvensis 2.15 1 36
Ononis spinosa 2.15 1 36
Petrorhagia velutina 3.13 2 2
Petroselinum crispum 1.1 2 38
Picris hieracioides 2.75 1 36
Plantago holosteum 2.06 1 36
Plantago major 4.16 1 2
Poa trivialis 0.05 1 36
Raphanus sativus 1.41 6 3
Rumex scutatus 1.26 1 1
Sambucus ebulus 1.76 1 36
Scrophularia canina 1.3 2 1, 36
Silene fabarioides 2.21 1 1
Silene gallica 3.73 2 2, 36
Silene italica 2.15 1 36
Silene vulgaris 2.27 1 1
Spinacea oleracea 2.53 4 29, 38
Stachys germanica 1.26 1 1
Taraxacum officinale 1.78 4 37
Teucrium polium 1.95 1 36
Trifolium arvense 1.3 1 36
Triticum aestivum 2.71 3 28
Verbascum blattaria 2.49 1 36
Verbascum glandulosum 2.35 1 1
Xeranthemum annuum 1.44 1 36
Xeranthemum inapertum 2.15 1 36
Standard Error range for all taxa 0.36 − 0.95

Appendix 4
Table A4 Mean relative shoot metal content, sample size and primary references for Ni data

Species Shoot Ni content n Reference

Achillea coarctata 1.14 1 36

Aegilops geniculata 2.20 1 36
Aegilops uniaristata 2.07 1 36
Agrostis capillaris 3.37 4 34
Allium ampeloprasum 1.27 2 38
Allium cepa 0.88 1 38
Alyssum bertolonii 6.08 4 11, 12
Alyssum bertolonioides 7.13 1 12
Alyssum murale 6.14 9 3, 36
Apium graveolens 2.03 2 38
Armeria denticulata 0.84 3 12
Avena sativa 2.90 3 10
Beta vulgaris 2.53 1 38
Brassica oleracea 2.96 6 13, 38, 44
Bromus squarrosus 1.90 2 36
Carex stricta − 0.11 2 35

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Table A4 continued

Species Shoot Ni content n Reference

Centaurea rupestris 0.84 2 12

Cerastium ligusticum 2.01 3 12
Ceterach officinarum 2.10 1 12
Chamaedaphne calyculata − 0.29 2 35
Cichorium intybus 1.54 6 24, 36, 38
Consolida orientalis 2.06 1 36
Coriandrum sativum 0.69 3 29
Delphinium peregrinum 0.57 1 36
Dianthus sylvestris 1.47 2 12
Ditrichia viscosa 2.02 1 36
Erigeron canadensis 1.17 4 24
Erysimum repandum 1.21 2 36
Eupatorium capillifolium 1.14 4 24
Euphorbia cyparissias 2.97 1 12
Euphorbia myrsinites 1.54 2 36
Euphorbia prostrata 2.06 2 12
Festuca ovina 2.40 4 34
Festuca rubra 2.36 4 34
Filago eriocephala 3.88 1 36
Filago vulgaris 1.63 1 36
Calamintha nepeta 2.61 1 36
Galium degenii 1.96 1 36
Herniaria hirsuta 4.44 1 36
Hordeum vulgare 2.15 3 10
Inula germanica 3.10 1 36
Koeleria eriostachya 1.18 1 36
Lactuca sativa 0.86 7 28, 29, 38
Linaria peloponnesiaca 0.36 1 36
Lolium perenne 2.67 15 10, 13, 34, 43, 44
Lotus ornithopodioides 3.19 1 36
Marrubium peregrinum 1.99 1 36
Marrubium vulgare 2.56 1 36
Minuartia ophiolitica 1.19 2 12
Myrica gale 0.32 2 35
Nigella arvensis 0.45 1 36
Ononis spinosa 2.86 1 36
Oryza sativa 2.34 1 13
Petroselinum crispum 1.60 2 38
Phaseolus vulgaris 2.68 3 13, 14
Phleum pratense 2.76 4 34
Picris hieracioides 2.60 1 36
Plantago holosteum 2.20 2 12, 36
Plantago lanceolata 2.72 1 12
Poa trivialis 2.16 1 36
Raphanus sativus 4.50 6 3
Sambucus ebulus 1.39 1 36
Sanguisorba minor 1.39 2 12
Scrophularia canina 0.88 1 36
Sedum spp. 0.39 2 12
Senecio erucifolius 3.04 1 12
Silene gallica 1.50 1 36
Silene italica 1.61 6 11, 12, 36
Spinacea oleracea 1.97 5 13, 29, 38
Stachys serpentini 0.95 1 12
Teucrium polium 2.86 1 36
Thymus ophioliticus 1.21 2 12
Trifolium arvense 2.10 1 36
Trifolium repens 2.41 4 43
Triticum aestivum 0.18 3 28
Verbascum blattaria 2.26 1 36
Xeranthemum annuum 3.23 1 36
Xeranthemum inapertum 1.24 1 36
Zea mays 1.33 10 13, 14, 43, 44
Standard Error range for all taxa 0.45 − 1.59

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Appendix 5

Table A5 Mean relative shoot metal content, sample size and primary references for Pb data

Species Shoot Pb content n Reference

Achillea coarctata 2.40 1 36

Aegilops geniculata 6.13 1 36
Aegilops uniaristata 4.45 1 36
Agrostis capillaris 4.26 4 34
Allium ampeloprasum 2.55 2 38
Allium cepa 3.21 2 38, 41
Alyssum murale 3.33 3 36
Ambrosia artemisiifolia 2.46 2 17
Anthemis tinctoria 4.04 3 2
Apium graveolens 3.05 2 38
Avena sativa 3.96 3 10
Beta vulgaris 2.68 1 38
Biscutella laevigata 2.15 1 41
Brassica juncea 2.78 2 17
Brassica napus 4.10 1 41
Brassica oleracea 3.32 1 38
Bromus squarrosus 2.60 2 36
Campanula ligulata 4.19 3 2
Cichorium intybus 3.02 2 36, 38
Cistus incanus 4.11 1 2
Cistus salviaefolius 4.36 1 2
Consolida orientalis 2.34 1 36
Coriandrum sativum 0.81 3 29
Cucumis sativus 4.87 1 41
Delphinium peregrinum 3.10 1 36
Ditrichia viscosa 2.03 1 36
Erysimum repandum 3.75 2 36
Euphorbia myrsinites 3.28 2 36
Euphorbia taurinensis 3.83 1 2
Festuca ovina 2.72 4 34
Festuca rubra 3.02 8 6, 34
Filago eriocephala 3.50 1 36
Filago vulgaris 2.03 1 36
Calamintha nepeta 3.62 1 36
Galium degenii 3.09 1 36
Glycine max 3.20 1 41
Hordeum vulgare 3.10 4 10, 41
Inula germanica 3.96 1 36
Koeleria eriostachya 8.58 1 36
Lactuca sativa 1.85 4 29, 38
Leontodon hispidus 2.57 1 41
Linaria peloponnesiaca 1.02 1 36
Lolium perenne 3.45 11 6, 10, 34
Lotus ornithopodioides 4.55 1 36
Marrubium peregrinum 3.85 1 36
Marrubium vulgare 3.19 1 36
Nigella arvensis 2.74 1 36
Ononis spinosa 4.12 1 36
Petrorhagia velutina 3.79 2 2
Petroselinum crispum 1.00 2 38
Phaseolus vulgaris 4.12 1 41
Phleum pratense 3.28 4 34
Picris hieracioides 4.25 1 36
Pisum sativum 3.48 1 41
Plantago holosteum 3.27 1 36
Plantago major 4.00 1 2
Poa trivialis 1.83 1 36

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Research 25

Table A5 continued

Species Shoot Pb content n Reference

Raphanus sativus 4.61 1 41

Sambucus ebulus 2.47 1 36
Scrophularia canina 1.71 1 36
Secale cereale 4.15 1 41
Silene gallica 3.25 2 2, 36
Silene italica 3.09 1 36
Silene vulgaris 3.30 1 41
Spinacea oleracea 2.55 4 29, 38
Teucrium polium 3.73 1 36
Thlaspi caerulescens 2.13 3 17, 25
Thlaspi ochroleucum 2.57 1 25
Thlaspi rotundifolium 2.92 2 17
Trifolium arvense 2.12 1 36
Triticum aestivum 2.88 2 17
Triticum vulgare 1.42 1 41
Verbascum blattaria 2.27 1 36
Xeranthemum annuum 1.71 1 36
Xeranthemum inapertum 3.56 1 36
Zea mays 3.57 3 17, 41
Standard Error range for all taxa 0.63–1.20

Appendix 6
Table A6 Mean relative shoot metal content, sample size and primary references for Zn data

Species Shoot Zn content n Reference

Achillea coarctata 3.03 1 36

Achillea millefolium 3.04 1 2
Aegilops geniculata 3.62 1 36
Aegilops uniaristata 3.44 1 36
Allium ampeloprasum 4.40 2 38
Allium cepa 4.65 1 38
Alyssum murale 4.64 10 36, 3, 1
Anagalis tenella 3.11 1 2
Anthemis tinctoria 3.92 3 2
Apium graveolens 5.03 2 38
Astragalus glyciphyllus 2.93 1 2
Beta vulgaris 4.41 1 38
Brassica oleracea 5.48 1 38
Bromus squarrosus 3.23 2 36
Campanula ligulata 4.04 3 2
Cerastium diffusum 4.15 2 2
Cichorium intybus 4.22 6 36, 37, 38
Cistus incanus 5.38 1 2
Cistus salviaefolius 5.11 1 2
Consolida orientalis 4.75 1 36
Delphinium peregrinum 4.24 1 36
Ditrichia viscosa 3.33 1 36
Erysimum repandum 3.26 2 36
Euphorbia myrsinites 3.13 2 36
Euphorbia taurinensis 3.80 1 2
Filago eriocephala 4.56 1 36
Filago vulgaris 3.25 1 36
Calamintha nepeta 3.62 1 36
Galium degenii 3.37 1 36
Herniaria hirsuta 4.09 1 36
Inula germanica 3.91 1 36

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Table A6 continued

Species Shoot Zn content n Reference

Jasione heldreichii 4.13 1 2

Koeleria eriostachya 3.95 1 36
Lactuca sativa 3.88 10 5, 28, 38
Lactuca viminea 3.79 1 1
Linaria peloponnesiaca 2.84 1 36
Lotus ornithopodioides 5.05 1 36
Marrubium peregrinum 3.40 1 36
Marrubium vulgare 3.21 1 36
Nigella arvensis 4.09 1 36
Ononis spinosa 3.21 1 36
Petrorhagia velutina 3.57 3 2
Petroselinum crispum 3.60 2 38
Picris hieracioides 4.13 1 36
Plantago holosteum 3.56 1 36
Plantago major 3.45 1 2
Poa trivialis 3.81 1 36
Raphanus sativus 3.85 6 3
Rumex acetosa 3.33 2 2
Rumex scutatus 4.08 1 1
Sambucus ebulus 3.81 1 36
Satureja montana 3.70 2 2
Scrophularia canina 2.97 2 1, 36
Silene fabarioides 3.62 1 1
Silene gallica 4.02 2 2, 36
Silene italica 3.37 1 36
Silene vulgaris 3.43 7 1, 5
Spinacea oleracea 4.72 1 38
Stachys germanica 3.52 1 1
Taraxacum officinale 4.49 4 37
Teucrium polium 3.21 1 36
Thlaspi caerulescens 6.25 9 5, 25
Thlaspi ochroleucum 4.72 3 25
Trifolium arvense 3.53 1 36
Trifolium palescens 3.55 1 2
Triticum aestivum 3.07 3 28
Verbascum blattaria 4.02 1 36
Verbascum glandulosum 3.79 1 1
Xeranthemum annuum 3.90 1 36
Xeranthemum inapertum 2.92 1 36
Standard Error range for all taxa 0.44 − 0.88

Appendix 7

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