Evolutionary dynamics of nickel ... - Wiley Online Library

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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 Dipartimento di Biologia Animale e Genetica, Università di Firenze, via Romana 17–19, I−50125 Firenze, Italy; 2School of Botany, The University of

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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: [email protected] 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 &

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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

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Hyperaccumulator (max. Ni, mg kg–1) 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) Yes (24300) No (1) No (47) Yes (7080) Yes (7080) No (1) Yes (7290) No (2) Yes (7600) Yes (9330) Yes (9000) Yes (22200) No (1) No (10) No (2) Yes (8810) Yes (3420) Yes (6320) No (1) No No n.d.

Calcareous rocks Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Calcareous rocks Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Calcareous soils Serpentine Serpentine Serpentine Calcareous rocks Serpentine Not serpentine (areas near to serpentine outcrops) Serpentine Serpentine Serpentine Serpentine Calcareous rocks Lead/zinc mine waste Serpentine Serpentine Serpentine Serpentine Calcareous rocks Serpentine Calcareous rocks n.d

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)

Alyssum murale ssp. murale var. murale (2)

Alyssum nebrodense Alyssum oxycarpum Alyssum peltarioides (1)

© New Phytologist (2003) 159: 691– 699 www.newphytologist.com Antalya (Turkey) Malaucène (France) Anduze (France) Ankara (Turkey) Bursa (Turkey) Tinos (Greece) Kütahya (Turkey) Kosche (Austria) Tuscany (Italy) Tuscany (Italy) n.d.

West of Yesilova, Burdur (Turkey) Çanakkale (Turkey) Bragança (Portugal)

Sicily (Italy) Seyhan (Turkey) Gevas, Van (Turkey)

Ankara (Turkey)

Mugla (Turkey) Tuscany (Italy) Tuscany (Italy) Panórama, Thessaloniki (Greece)

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)

Location

Turkey France, Spain France, Spain South-east Europe, Turkey South-east Europe, Turkey Tinos Turkey Friuli (Italy), Austria Balkans, Italy, France South Europe Europe

Turkey Turkey North Portugal

Turkey South Europe, Turkey South Europe Balkans, Turkey, Transcaucasia Balkans, Turkey, Transcaucasia Sicily Turkey Turkey

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

Distribution

AY237931 AY237923 AY237922 AY237928 AY237927 AY237926 AY237925 AY237924 AY237921 AY237920 AF401114

AY237932 AY240871 AY237929

AY237935 AY237934 AY237933

AY237937

AY237940 AY237939 AY237938 AY237936

AY237957 AY237956 AY237955 AY237954 AY237930 AY237953 AY237952 AY237951 AY237950 AY237949 AY237948 AY237947 AY237946 AY237945 AY237944 AY237943 AY237942 AY237941

GenBank accession no.

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.

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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

Samples

Substrate/ habitat

Table 1 Samples examined in this study1

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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.


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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


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


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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