Research
Evolutionary dynamics of nickel hyperaccumulation in
Alyssum revealed by ITS nrDNA analysis
Blackwell Publishing Ltd.
A. Mengoni1, A. J. M. Baker2, M. Bazzicalupo1, R. D. Reeves3, N. Adigüzel4, E. Chianni5, F. Galardi5,
R. Gabbrielli5 and C. Gonnelli5
1
Dipartimento di Biologia Animale e Genetica, Università di Firenze, via Romana 17–19, I−50125 Firenze, Italy; 2School of Botany, The University of
Melbourne, Melbourne, VIC 3010, Australia; 3Institute of Fundamental Sciences, Massey University, Palmerston North 5301, New Zealand; 4Gazi University,
Faculty of Science and Arts, Department of Biology, T−06500, Ankara, Turkey; 5Dipartimento di Biologia Vegetale, Laboratorio di Fisiologia Vegetale,
Università di Firenze, via Micheli 1, I−50121 Firenze, Italy
Summary
Author for correspondence:
A. Mengoni
Fax: +39 0552288 250
Email: alessiome@unifi.it
Received: 14 April 2003
Accepted: 21 May 2003
doi: 10.1046/j.1469-8137.2003.00837.x
• Molecular phylogeny based on ribosomal internal transcribed spacer (ITS) sequences was studied to investigate the phyletic relationships among some nickel (Ni)hyperaccumulating and nonhyperaccumulating species of the genus Alyssum in
relation to their geographic distribution and Ni-hyperaccumulating phenotype.
• Thirty-seven samples belonging to 32 taxa were analysed by sequencing the
polymerase chain reaction-amplified ITS region and performing neighbor joining,
maximum parsimony and maximum likelihood phylogenetic analyses.
• The ITS region in the sampled species varied from 221 to 307 bp of ITS1 and from
194 to 251 bp of ITS2. A total of 765 characters was used to infer the phylogeny
and the average nucleotide variation detected was 15.15%.
• Nickel-hyperaccumulation could have been lost or acquired independently more
than once during the speciation of the genus. The geographical location of species
could not be related to phylogenetic affinities.
Key words: Alyssum, heavy metals, nickel, hyperaccumulation, evolution, molecular
phylogeny, internal transcribed spacers.
© New Phytologist (2003) 159: 691– 699
Introduction
Serpentine soils derived from a wide range of ultramafic rock
types are widely distributed around the world. They are
characterized by high levels of nickel (Ni), cobalt (Co) and
chromium, low levels of nitrogen (N), phosphorus (P),
potassium (K), calcium (Ca), and a high Mg/Ca quotient
(Brooks, 1987). These extreme chemical properties render
serpentine soils uninhabitable for most plant species but also
comprise a major selective force in the evolution of endemic
serpentine taxa ( Pichi Sermolli, 1948; Wild & Bradshaw,
1977; Kruckeberg & Kruckeberg, 1990). Areas of serpentine
soil can be considered as ‘ecological islands’ (Lefèbvre &
Vernet, 1990) and the occurrence of plant taxa restricted to
serpentine substrates has been documented since the sixteenth
century ( Vergnano Gambi, 1992).
Among the plants adapted to survive in these soils are a
small number of Ni-hyperaccumulating plants (Baker &
© New Phytologist (2003) 159: 691– 699 www.newphytologist.com
Brooks, 1989; Reeves & Baker, 2000), able to concentrate Ni
in their aboveground parts to concentrations greater than
0.1% on a dry weight basis, generally in excess of the substrate
concentration. Most of these species are virtually restricted to
serpentine soils. There is considerable debate over an evolutionary explanation of the hyperaccumulation trait. One of
the most favored hypotheses is defense, which states that
metal hyperaccumulation has evolved as a mechanism to
reduce damage by parasitism, herbivory and disease (Boyd &
Martens, 1992). Several lines of experimental evidence support this hypothesis (Pollard, 2000). The genetic background
of metal hyperaccumulation is still poorly understood
(Pollard et al., 2002) but there is evidence, at the molecular
level, for the involvement of specific metal transporters (Pence
et al., 2000; Assunção et al., 2001).
In temperate latitudes the Ni-hyperaccumulation trait is
mainly found in members of the family Brassicaceae (especially in the genera Alyssum and Thlaspi ). The first record of a
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hyperaccumulator of Ni was for Alyssum bertolonii, in which
up to 1.22% Ni was found in the leaves (Minguzzi &
Vergnano, 1948). Many taxa in this genus have subsequently
been shown to accumulate Ni in their aerial parts (Doksopulo,
1961; Menezes de Sequeira, 1969; Brooks & Radford 1978;
Brooks et al., 1979). Alyssum is a genus of about 175 species,
mainly found in Mediterranean Europe and Turkey, with a
few species in North Africa, the Near East (Iran, Iraq and
Transcaucasia) and scattered across the Ukraine and Siberia
into the north-west of the American continent (Alaska,
Yukon). In Europe, it is confined to the southern half of the
continent and may well be a preglacial relic since its distribution is to the south of areas formerly covered by the ice sheet
during the Ice Ages. Alyssum is presently subdivided into six
sections: Meniocus (Desv.) Hook. f.; Psilonema (C.A. Meyer)
Hook. f.; Alyssum; Gamosepalum (Hausskn.) Dudley;
Tetradenia (Spach) Dudley; Odontarrhena (C.A. Meyer)
Koch. The largest sections are Alyssum and Odontarrhena,
each with about 73–75 species. All the Ni hyperaccumulators
occur in section Odontarrhena, which consists of yellowflowered perennials with ovules and seeds solitary in each
loculus; the seeds are mucilage-producing. Nothing is known
about the evolution of the Ni hyperaccumulation trait in the
section and in particular whether or not hyperaccumulation
is monophyletic or polyphyletic. Interest is growing concerning the evolutionary origins of metal accumulation (e.g.
recent papers dealing with arsenic accumulation in fern spp.
(Zhao et al., 2002) and zinc (Zn)/cadmium (Cd) accumulation in Arabidopsis halleri ( Bert et al., 2002; Macnair, 2002)).
Recently, Pepper & Norwood (2001) showed that serpentine
taxa in Streptanthus, Caulanthus and Guillenia complexes were
all nonmonophyletic, suggesting that tolerance to serpentine
may be gained or lost through relatively few genetic changes.
Nickel accumulation certainly implies serpentine tolerance,
but the HIGH number of tolerant, nonaccumulating species,
suggests that accumulation and tolerance are under different
genetic control (Macnair et al., 1999).
The nucleotide sequences of the internal transcribed spacers 1 and 2 (ITS1 and ITS2) from the nuclear 18S−26S ribosomal DNA (nrDNA) region are widely used in molecular
phylogenetics at the genus level and provide a valuable source
of variable characters that can be used in phylogenetic analyses
(Baldwin, 1992; Baldwin et al., 1994). Here we apply these
molecular markers to an analysis of phylogenetic relationships
among Alyssum spp., mainly in section Odontarrhena, aiming
to shed light on the possible evolutionary scenarios of the
Ni-hyperaccumulation phenotype in this genus.
montanum, Alyssum minus and Alyssum wulfenianum) were
analyzed for ITS sequence variation (Table 1). For some
widespread taxa more than one sample was analysed (Alyssum
corsicum, Alyssum sibiricum, Alyssum murale, Alyssum peltarioides,
Alyssum serpyllifolium spp. serpyllifolium). Moreover, samples
for the species Iberis umbellata and Lobularia maritima were
included as well as the sequence, available in GenBank, of
Alyssum alyssoides. In Table 1 the locality of origin of the
sample and the Ni concentration in the leaves, if determined,
is also shown. The choice of materials was dictated by
the intention to cover the Mediterranean basin (from Turkey
to Portugal) and to have both Ni-hyperaccumulating and
nonaccumulating species. More than one sample was included
for those species that appeared to be widely dispersed in
the basin.
DNA preparation
DNA was extracted from dried leaf tissues or seeds with a
cetyltrimethylammonium bromide (CTAB) protocol as described
in Mengoni et al. 2000. The extracted DNA was quantified
after agarose gel electrophoresis of the samples (0.6% w : v) in
TAE buffer (1 m ethylenediaminetetraacetic acid (EDTA),
40 m Tris-acetate) containing 1 µg ml−1 (w : v) of ethidium
bromide by comparison with a known mass standard.
Primers and polymerase chain reaction
The primers ITS4 and ITS5 (Baldwin et al., 1994) were
used for the amplification of the internal transcribed spacers
ITS1 and ITS2 and of the 5.8S subunit of nuclear rDNA
intron. Polymerase chain reaction (PCR) amplifications were
performed in a total volume of 50 µl containing 5 µl of 10×
reaction buffer (Dynazyme II; Finnzyme, Espoo, Finland),
1.5 m MgCl2, 20 pmol of each primer, 200 µ of each
dNTP, 1 U of Taq DNA polymerase (Dynazyme II; Finnzyme)
and 10 ng of template DNA. Reactions were performed in a
Perkin-Elmer 9600 thermocycler (Perkin Elmer, Norwalk,
CT, USA) programmed for an initial melting at 95°C for
5 min followed by 40 cycles at 95°C for 30 min, 52°C for
55 min, 72°C for 1 min, and a final extension step at 72°C
for 10 min. Then, 5 µl of each amplification mixture was
analysed by agarose gel (1.5% w : v) electrophoresis in TAE
buffer containing 1 µg ml−1 (w : v) of ethidium bromide. The
PCR reactions were purified from excess salts and primer with
the PCR Purification Kit (Roche, Mannheim, Germany).
DNA sequencing and phylogenetic analysis
Materials and Methods
Plant samples
Thirty-seven Alyssum samples including 29 taxa from section
Odontarrhena and three taxa from section Alyssum (Alyssum
Automated DNA sequencing was performed on both strands
directly from the ITS4 and ITS5 primers on the purified PCR
products using BigDye Terminator v.2 chemistry and an
ABI310 sequencer (PE-Applied Biosystems, Norwalk, CT,
USA) according to the manufacturer’s recommendations. The
www.newphytologist.com © New Phytologist (2003) 159: 691–699
Table 1 Samples examined in this study1
© New Phytologist (2003) 159: 691– 699 www.newphytologist.com
Samples
Substrate/
habitat
Hyperaccumulator
(max. Ni, mg kg–1)
Location
Distribution
GenBank
accession no.
Alyssum alpestre
Alyssum anatolicum
Alyssum argenteum
Alyssum bertolonii
Alyssum bertolonii ssp. scutarinum
Alyssum biovulatum
Alyssum caricum
Alyssum condensatum ssp. flexibile
Alyssum corsicum (1)
Alyssum corsicum (2)
Alyssum cypricum
Alyssum davisianum
Alyssum fallacinum
Alyssum floribundum
Alyssum guitianii2
Alyssum huber-morathii
Alyssum lesbiacum
Alyssum malacitanum (Alyssum
serpyllifolium ssp. malacitanum)
Alyssum masmenaeum
Alyssum minus
Alyssum montanum
Alyssum murale ssp. murale var. murale (1)
Calcareous rocks
Serpentine
Serpentine
Serpentine
Serpentine
Serpentine
Serpentine
Calcareous rocks
Serpentine
Serpentine
Serpentine
Serpentine
Serpentine
Serpentine
Serpentine
Serpentine
Serpentine
Serpentine
No (4)
No (153)
Yes (29400)
Yes (13400)
Yes (10200)
No (5)
Yes (16500)
No (14)
Yes (13500)
Yes (13500)
Yes (23600)
Yes (19600)
Yes (3960)
Yes (7700)
Yes (9000)
Yes (13500)
Yes (22400)
Yes (10000)
Col du Lautaret (France)
Near Gumusane (Turkey)
Val d’Aosta (Italy)
Tuscany (Italy)
East of Prizren (Yugoslavia)
Chorn-Aksy (Russia)
Köycegiz (Turkey)
East Turkey
Kütahya (Turkey)
Bastia, Corsica (France)
Burdur (Turkey)
Kütahya (Turkey)
Crete (Greece)
Içel (Turkey)
Puente Basadre, Galicia (Spain)
Burdur (Turkey)
Lesvos (Greece)
Sierra Berméja, Andalucia (Spain)
South Europe
Turkey
North-west Italy
Central Italy
Italy, Albania, Balkans
South Siberia
Turkey
Turkey, Syria, Iraq
Turkey
Corsica (France)
Cyprus, Turkey
Turkey
Crete
Turkey
Spain
Turkey
Lesvos
Spain
AY237957
AY237956
AY237955
AY237954
AY237930
AY237953
AY237952
AY237951
AY237950
AY237949
AY237948
AY237947
AY237946
AY237945
AY237944
AY237943
AY237942
AY237941
Serpentine
Calcareous soils
Serpentine
Serpentine
Yes (24300)
No (1)
No (47)
Yes (7080)
Mugla (Turkey)
Tuscany (Italy)
Tuscany (Italy)
Panórama, Thessaloniki (Greece)
AY237940
AY237939
AY237938
AY237936
Alyssum murale ssp. murale var. murale (2)
Serpentine
Yes (7080)
Ankara (Turkey)
Alyssum nebrodense
Alyssum oxycarpum
Alyssum peltarioides (1)
Calcareous rocks
Serpentine
Not serpentine (areas
near to serpentine outcrops)
Serpentine
Serpentine
Serpentine
No (1)
Yes (7290)
No (2)
Sicily (Italy)
Seyhan (Turkey)
Gevas, Van (Turkey)
Turkey
South Europe, Turkey
South Europe
Balkans, Turkey,
Transcaucasia
Balkans, Turkey,
Transcaucasia
Sicily
Turkey
Turkey
Yes (7600)
Yes (9330)
Yes (9000)
West of Yesilova, Burdur (Turkey)
Çanakkale (Turkey)
Bragança (Portugal)
Turkey
Turkey
North Portugal
AY237932
AY240871
AY237929
Serpentine
Calcareous rocks
Lead/zinc mine waste
Serpentine
Serpentine
Serpentine
Serpentine
Calcareous rocks
Serpentine
Calcareous rocks
n.d
Yes (22200)
No (1)
No (10)
No (2)
Yes (8810)
Yes (3420)
Yes (6320)
No (1)
No
No
n.d.
Antalya (Turkey)
Malaucène (France)
Anduze (France)
Ankara (Turkey)
Bursa (Turkey)
Tinos (Greece)
Kütahya (Turkey)
Kosche (Austria)
Tuscany (Italy)
Tuscany (Italy)
n.d.
Turkey
France, Spain
France, Spain
South-east Europe, Turkey
South-east Europe, Turkey
Tinos
Turkey
Friuli (Italy), Austria
Balkans, Italy, France
South Europe
Europe
AY237931
AY237923
AY237922
AY237928
AY237927
AY237926
AY237925
AY237924
AY237921
AY237920
AF401114
1
AY237935
AY237934
AY237933
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The taxon name, the substrate and location of collection with the nickel (Ni) concentration (where determined), the distribution of the species (after Ball & Dudley, 1993) and the GenBank
accession number of the internal transcribed spacer (ITS) sequence are shown for each sample. Alyssum alyssoides sequence was retrieved from GenBank database. The criterion used to classify
the species as hyperaccumulator was a Ni content above 1000 mg kg−1 dry weight. n.d., not determined. 2Alyssum guitianii is a name locally applied to the Galician serpentine A. serpyllifolium
species, and is not at present validly published.
Research
Alyssum peltarioides (2)
Alyssum pinifolium
Alyssum pintodasilvae (Alyssum
serpyllifolium ssp. lusitanicum)
Alyssum pterocarpum
Alyssum serpyllifolium ssp. serpyllifolium (sample 1)
Alyssum serpyllifolium ssp. serpyllifolium (sample 2)
Alyssum sibiricum (1)
Alyssum sibiricum (2)
Alyssum tenium
Alyssum virgatum
Alyssum wulfenianum
Iberis umbellata
Lobularia maritima
Alyssum alyssoides
AY237937
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nucleotide sequences obtained were checked for orthology
to the sequence of I. umbellata, which was then used as the
outgroup for dendrogram construction. Multiple alignment
with hierarchical clustering was performed with the program
(Corpet, 1988, http://protein.toulouse.inra.fr/
multalin.html) using the Alternate DNA symbol comparison
table. The alignment was further examined and slightly
modified manually. Neighbor-joining (NJ), maximum
parsimony (MP) and maximum likelihood (ML) methods
were used to analyse the aligned sequences. Dendrograms
were constructed on the basis of the total sequence of the ITS.
Neighbor-joining trees were made by the NJ method (Saitou
& Nei, 1987) performed on a Kimura-2 parameter distance
matrix (Kimura, 1980). Maximum parsimony trees were
constructed by the MP method with a heuristic search adding
sequences at random. Maximum likelihood was based on the
MP tree with a heuristic search adding sequences at random
using Tree-Bisection Reconnection branch swapping. Phylogenetic trees were calculated with (ver 2.1; Kumar et al.,
1993) for MP and NJ and * (ver 4.0b; Swofford, 2000)
for ML. Bootstrap analyses were carried out with 64238
random seed and 1000 or 500 replicates (for NJ or MP and
ML, respectively) (Felsenstein, 1985).
Phylogenetic analysis
Phylogenetic analyses were performed considering I. umbellata,
L. maritima or A. alyssoides as outgroups. All analyses gave
identical results and that with I. umbellata showed the highest
resolution within the Alyssum species analysed and is hence
presented. Neighbor joining, MP and ML analyses of Alyssum
ITS sequences rendered similar topologies (Figs 1–3). The
MP analysis resulted in trees of 814 steps whose strict
consensus had a consistency index (CI) of 0.773 and a
retention index (RI) of 0.667. Sorted ML parameters were:
–Ln likelihood = 3582.81, gamma-shape = 0.4415, ti : tv
ratio = 0.922 (K = 1.882). Bootstrap values were in general
higher for MP than for ML and NJ trees. From the
comparison of MP, ML and NJ dendrograms four groups, each
comprising both hyperaccumulator and nonhyperaccumulator
species, can be recognized: (1) a group with A. murale specimens,
A. wulfenianum, A. minus and A. montanum; (2) a group with
A. corsicum, A. lesbiacum and A. sibiricum specimens; (3) a
group with A. peltarioides specimens, A. pterocarpum and
A. bertolonii; (4) a large group comprising A. argenteum, A.
bertolonii ssp. scutarinum, A. tenium, A. fallacinum and A.
floribundum, A. serpyllifolium ssp. serpyllifolium specimens,
A. malacitanum and A. guitianii.
Results
Discussion
DNA sequences
Reliable DNA sequence data for the specimens were obtained
by manually cross-checking the complementary sequences of
both DNA strands of the PCR products for base-calling
ambiguities. The DNA sequences have been submitted
to the GenBank–EMBL–DDBJ database and can be
retrieved using the accession numbers indicated in
Table 1. The DNA sequences obtained were then aligned to
the I. umbellata, L. maritima or A. alyssoides sequences. The
alignment can be retrieved from EMBL database (ftp://
ftp.ebi.ac.uk/pub/databases/embl/align/) under the accession
number ALIGN_000514. The alignment with I. umbellata
ITS sequence (629 bp long) showed the greatest similarity
with the Alyssum species analyzed. The ITS region (comprising all of the 5.8S ribosomal RNA gene, but not including
nucleotides from the 18S and 26S ribosomal RNA genes)
varied in its overall length from 614 bp (A. anatolicum) to
691 bp (A. malacitanum). The average G + C content was
56.46%. The ITS1 sequences were 221–307 nucleotides in
long and the ITS2 sequences were 194–251 nucleotides long.
The outgroup I. umbellata had ITS1 253 bp long and ITS2
219 bp long. Sequence comparisons of the ITS region
revealed a total of 765 characters, 373 of which were constant,
180 variable uninformative sites and 212 parsimony
informative sites. The average number of pairwise nucleotide
differences, without I. umbellata, was 15.15 (2.3% on the
average length of ITS).
This is the first study reporting an analysis of the relationships
between Ni-hyperaccumulator and nonhyperaccumulator
species in the genus Alyssum using molecular markers.
The variability of the ITS sequences observed was able to
cluster unambiguously many of the species investigated. In
particular, all the analyses showed that at least four main
groups within the genus Alyssum may be recognized, supported by high bootstrap levels (especially in MP analysis).
Each of the groups comprises both Ni-hyperaccumulator and
nonhyperaccumulator species.
In the first group, species are present from the section
Alyssum (A. minus, A. montanum and A. wulfenianum) and
the hyperaccumulator A. murale. Alyssum murale is a very
variable, polymorphic species and geographically it includes
Ni-hyperaccumulators from the Balkans to Greece, Turkey,
Armenia and Crimea; a relationship between the large distribution of this species and the fact that this group contains
species from all over the Mediterranean basin could be
hypothesized.
The second group includes A. sibiricum, A. lesbiacum and
A. corsicum. Our results show that the A. corsicum population
from Corsica is the same as the one from Turkey, supporting
the theory of a ‘human factor’ in its transport from Turkey
(where it is a widespread serpentine-endemic) to Corsica
(where it is localized on serpentine near the seaport of Bastia).
The third group recognized includes A. peltarioides, A.
pterocarpum and A. bertolonii.
www.newphytologist.com © New Phytologist (2003) 159: 691–699
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Fig. 1 Neighbour-joining tree of Alyssum
species based on ITS1 + ITS2 sequence
alignment. Numbers at nodes show the
bootstrap values obtained for the 1000replicate analysis. Values lower than 50 are
not shown. Nickel-hyperaccumulators are
shown in bold type. Bar, the Kimura 2
parameter distance.
Finally, a fourth large group can be recognized. This
includes Ni-hyperaccumulator and nonhyperaccumulator
species distributed along the northern part of the Mediterranean basin from Turkey to Spain. In this group, two main subgroups could be identified, which were strongly separated in
the ML tree, while in NJ and MP trees they were connected
to the same node: the first one comprised only hyperaccumulating species distributed from Turkey to the Balkans (A.
floribundum, A. bertolonii ssp. scutarinum, A. argenteum, A.
tenium and A. fallacinum) and A. pintodasilvae supported by
high bootstrap levels in MP analysis. However, for this subgroup, NJ analysis clustered A. pintodasilvae in a different
group with A. lesbiacum. These species could have evolved
from the same hyperaccumulating progenitor populating that
area. In this group, A. bertolonii ssp. scutarinum appeared to
be a different species from A. bertolonii (they were not even
grouped in the same cluster). The second subgroup contained
© New Phytologist (2003) 159: 691– 699 www.newphytologist.com
Iberian species (A. serpyllifolium ssp. serpyllifolium and the
hyperaccumulators A. guitianii and A. malacitanum). There
has been a history of changing opinions about the A. serpyllifolium complex, with the Ni accumulators at various times
having been raised to subspecific or even specific rank
(Dudley, 1986a,b) and then reduced to the status of variants
of A. serpyllifolium ‘possibly divisible into a number of species’
(Ball & Dudley, 1993). On the basis of the dendrograms
presented, we can speculate that the A. serpyllifolium variants
actually belong to different species. However, more detailed
molecular analyses using different molecular markers could be
helpful in elucidating the taxonomic and phylogenetic status
within the A. serpyllifolium group. In this group, the ability to
hyperaccumulate Ni could have been lost during the evolution of the group (e.g. A. serpyllifolium ssp. serpyllifolium).
Other possible clusters were formed, but their interpretation
remains uncertain. The ML analysis showed a cluster with
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Fig. 2 Consensus maximum parsimony tree
of Alyssum species based on ITS1 + ITS2
sequence alignment. Numbers at nodes
show the bootstrap values obtained for the
500-replicate analysis. Values lower than 50
are not shown. Nickel-hyperaccumulators
are shown in bold type.
A. caricum, A. anatolicum, A. condensatum and A. biovulatum,
but the NJ and MP analyses did not recognize these species
as members of the same group. In particular, A. caricum was
clustered with only A. anatolicum and A. condensatum was
clustered with only A. biovulatum. The MP and ML analyses
showed A. nebrodense and A. virgatum as members of the same
clusters but NJ analysis did not confirm this grouping.
Looking at the patterns depicted by the dendrograms, the
nonhyperaccumulating trait was interspersed within the dendrograms in most of the nodes. In each of the main groups it
was possible to recognize both Ni-hyperaccumulator and
nonhyperaccumulator species. Moreover, for A. sibiricum and
A. peltarioides there are many nonaccumulating populations
that do not appear on serpentine, several nonaccumulating
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Fig. 3 Maximum likelihood tree derived
from of Alyssum species based on
ITS1 + ITS2 sequence alignment. Numbers at
nodes show the bootstrap values obtained
for the 500-replicate analysis. Values lower
than 50 are not shown. Nickelhyperaccumulators are shown in bold type.
populations on serpentine, and a range of Ni-accumulating
populations on serpentine, ranging from a few hundred to
many thousand p.p.m. of Ni. With our analysis, hyperaccumulators and nonaccumulators samples were shown to belong
to the same taxon. This could indicate a polymorphism for this
trait even at the species level, although analyses in controlled
conditions (e.g. hydroponic cultures) are necessary to establish
the real hyperaccumulation capacities of these species/populations. Indeed, it is possible that some nonhyperaccumulators
living in nonserpentine soils could accumulate Ni when
grown on serpentine soil. This was recently shown for arsenate
hyperaccumulation in the fern Pteris vittata, where accessions
from arsenic (As)-contaminated soils hyperaccumulated
As similarly to accessions from uncontaminated soils (Zhao
© New Phytologist (2003) 159: 691– 699 www.newphytologist.com
et al., 2002), as well as for metallicolous and nonmetallicolous
populations of Arabidopsis halleri which were similarly able to
hyperaccumulate Zn and Cd (Bert et al., 2002).
The reported data could lead to a hypothesis that the
Ni-hyperaccumulating or the nonaccumulating phenotypes
are polyphyletic. The polyphyletic origin of the serpentine
endemics has been shown for the species of the Streptanthus
cluster (Pepper & Norwood, 2001) and polyphyletic origins
for metallophyte taxa has also been suggested for the zinc
hyperaccumulator Thlaspi caerulescens (Koch et al., 1998).
From the data presented here, it is possible to speculate that
Ni-hyperaccumulation evolved ancestrally (and possibly
independently in A. murale) in the section Odontarrhena.
The hyperaccumulating phenotype could then have been
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lost in a number of species. There is evidence that A. nebrodense is unable to hyperaccumulate Ni when grown under
high Ni concentrations in hydroponics (Gabbrielli et al.,
1982). Moreover, at least for A. anatolicum and A. sibiricum
1, which are not serpentine-endemic species, our specimens
collected on serpentine soils did not contain high levels of
Ni. However, without prior molecular knowledge of the
molecular basis for Ni accumulation or ad hoc experiments
it is premature to formulate any conclusions about the
polyphyletic/monophyletic origins of Ni-hyperaccumulation
in Alyssum.
In the absence of a previous phylogenetic analysis of section
Odontarrhena based on other independent characters, our
results can be discussed only in relation to the current classification of groups, which is based mainly on morphological
characters. This is an important limitation, especially in case
of disagreements (Doyle & Doyle, 1993; Palacios et al.,
2000).
The phylogenetic relationships depicted by the dendrograms did not match well with the geographical proximity of
the hyperaccumulating species, apparently in contrast with
the hypothesis of Brooks (1987) about a spreading of these
species from a center of origin located in Turkey, since in this
region there is the highest number of hyperaccumulators,
along with the highest levels of Ni hyperaccumulation. This
fact could imply that the present area of distribution of the
species may reflect historical phenomena such as spreading
immediately after the Ice Ages for the more cold-sensitive
species. Moreover, considerable divergence exists between
clusters based on the data presented, obtained using DNA
sequences, and the traditional, morphologically based taxonomy. These included the perennial A. murale which is the
most widespread member of the section Odontarrhena in
close DNA similarities to the (annual) members of section
Alyssum and the various subspecies of A. serpyllifolium (which
are morphologically almost identical) which appeared to be
distributed in different subclusters. For the position of A.
murale within the dendrogram data were consistent and the
different methods gave similar results. More uncertain was the
case of A. serpyllifolium for which NJ and MP reconstructions
gave slightly different results. This could result from the discriminatory power of the marker used, which could not support the phylogenetic reconstruction well at that taxonomic
level. To obtain a better resolution of phylogenetic relationships, it may be necessary to sequence more DNA regions,
such as the nuclear gene Adh (Small et al., 1998) or the chloroplast DNA of matK ( Johnson & Soltis, 1994), or to score
for more variable genetic markers, such as amplified fragment
length polymorphisms, which have been used to elucidate
relationships among cogeneric species (Roa et al., 1997).
More in-depth analyses on the other species of the section
Odontarrhena using also other molecular markers need to be
performed to better elucidate the phylogenetic dynamics of
Ni-hyperaccumulation in the genus Alyssum.
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