REGULAR ARTICLE. J. Escarré & C. Lefèbvre & H. Frérot & S. Mahieu & N. Noret

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1 Plnt Soil DOI 1.17/s z REGULAR ARTICLE Metl concentrtion nd metl mss of metllicolous, non metllicolous nd serpentine Nocce cerulescens popultions, cultivted in different growth medi J. Escrré & C. Lefèvre & H. Frérot & S. Mhieu & N. Noret Received: 27 August 212 / Accepted: 25 Jnury 213 # Springer Science+Business Medi Dordrecht 213 Astrct Aims Evlute the genetic nd environmentl vriility of metl concentrtion nd metl mss of Nocce cerulescens, from metlliferous (MET), non metlliferous (NMET) nd serpentine (SERP) soils. Methods 18 popultions were cultivted in 18 different growth conditions, such s soil mine tiling, soils mended with zinc (Zn), cdmium (Cd) nd nickel (Ni) slts (in mixtures or in monometllic slts) nd hydroponic solution with two Zn concentrtions. Results MET popultions hd Zn concentrtions lower thn NMET nd SERP in the different soils ut higher Cd mss (the product of eril iomss nd folir metl concentrtion). SERP hd the highest Responsile Editor: Fngjie Zho. J. Escrré (*) : S. Mhieu Centre d Ecologie Fonctionnelle et Evolutive (CNRS) UMR 5175, Route de Mende 1919, Montpellier Cedex 5, Frnce e-mil: jose.escrre@cefe.cnrs.fr C. Lefèvre : N. Noret Lortoire d Ecologie végétle et Biogéochimie, Université Lire de Bruxelles, ULB-Cmpus de l Pline, CP244. Boulevrd du Triomphe, 15 Bruxelles, Belgium H. Frérot Lortoire de Génétique et Evolution des Popultions Végétles, UMR CNRS 8198, Université Lille 1, Bâtiment SN2, Villeneuve d Ascq Cedex, Frnce Ni concentrtion nd Ni mss vlues. The ddition of Cd or Ni to Zn-contminted soil significntly decreses Zn concentrtion. In hydroponics, MET nd NMET hd equivlent Zn concentrtions ut these were three times higher thn those otined in soil experiments. Zn mss of NMET ws significntly lower thn MET with the ltter hving Zn mss vlues lrgely ove those otined in mine soil. Conclusions Results showed lrge heterogeneity of responses mong popultions depending on the sustrte used, nd it ws not possile to correctly ssign single popultion to its ccurte origin with only one experiment. Finlly, dt on metl concentrtion otined in culture soils re closer to those in field soils thn those from hydroponics so tht they could give more ccurte informtion on the ccumulting cpcity of Nocce cerulescens nd its use in phytoextrction of metls in field conditions. Keywords Thlspi cerulescens. Mine soil. Plnt popultions. Phytoremedition. Zn/Cd/Ni hyperccumultion. Metl tolernce Introduction Nturl soils with high metl concentrtions, such s nickel-rich ultrmfic soils or nthropogenic metlcontminted soils from the mining industry, re the thetre of evolutionry processes resulting in genetic nd physiologicl dpttions tht enle orgnisms to

2 Plnt Soil colonise these extreme environments (Antonovics et l. 1971). Most plnt species growing on these sites show tolernce to high metl concentrtions due to restriction of metl trnsport to the eril tissues; however limited numer of these species, clled hyperccumultors, re le to ccumulte metls t very high concentrtions (e.g. 1 % of Zn) in eril dry prts (Bker nd Wlker 199; vn der Ent et l. 212). Among these species, Nocce (formerly Thlspi) cerulescens, Brssiccee tht hyperccumultes Zn, Cd nd Ni, hs een widely studied (Assunção et l. 23). The species shows lrge climtic nd geogrphicl rnge in Europe. Popultions re found from the lowlnds up to 2 m (Alpine climte) nd from the North of Spin to Scndinvi (Tutin et l ). N. cerulescens grows on soils contminted y hevy metls (Zn, Cd, P), on serpentine soils (Ni) nd is lso present on non-contminted soils (Reeves et l. 21; Meyer 26). Studies compring plnts from metl-contminted soils (metllicolous popultions, MET) nd non-contminted soils (nonmetllicolous popultions, NMET) showed tht NMET plnts often hve lower iomss thn those in metl-contminted soils. Interestingly, NMET individuls usully ccumulte more Zn thn MET plnts when grown in the sme soil (e.g. Meerts nd Vn Iscker 1997; Escrré et l. 2; Meerts et l. 23; Dechmps et l. 27). Serpentine popultions (SERP), tht hve een less studied, cn lso ccumulte Zn even with low extrctle soil Zn concentrtions (Peer et l. 23). Despite the high numer of ppers concerning N. cerulescens, most experimentl studies investigting metl concentrtion ptterns hve compred very low numer (usully 1 or 2) of MET popultions (with the exception of Peer et l. 23 nd Roosens et l. 23). Most experiments were performed with nutrient solutions (e.g. Brown et l. 1995; Assunção et l. 23) or with non-contminted soils supplemented with metl slts (e.g. Meerts et l. 23). Only very few numer included soils from contminted sites (e.g. Meerts nd Vn Iscker 1997; Escrré et l. 2; Lomi et l. 21). In ddition, experimentl studies compring MET with NMET popultions simultneously re scrce nd were mostly conducted y Belgin nd French tems (Meerts nd Vn Iscker 1997; Escrré et l. 2; Meerts et l. 23; Noret et l. 25, 27; Dechmps et l. 27, 28), nd even less common re studies tht compred popultions from metl-contminted nd serpentine soils (Peer et l. 23; Roosens et l. 23). Only two studies (Assunção et l. 23; 28) compred one popultion of ech of the three origins. However, when only one popultion per origin is used, generlistion is hzrdous s popultion replictes re missing. To our knowledge there re no pulished experiments compring severl popultions of plnts from ech origin, i.e. non-contminted, metlcontminted nd serpentine soils. If N. cerulescens must e used for the phytoremedition of moderte contminted soils (Zho et l. 23), it would e necessry to conduct lrge screening process to evlute the mong-popultion vriility of metl ccumultion with different metl concentrtions nd growth medi, including contminted soils from the field. Here, severl experiments re presented tht compre lef metl concentrtions nd metl msses (lef metl concentrtion times eril iomss, i.e. the mount of metl in the eril prts) of N. cerulescens plnts from the three origins tht were cultivted in different sustrt. The following questions were ddressed: 1) Do metllicolous, non-metllicolous nd serpentine popultions exhiit contrsting ptterns of iomss, metl concentrtion nd metl mss? 2) Do these differences vry ccording the chrcteristics of culture medi, of metllic slts nd of the composition of their mixtures? 3) Is there evidence of interctions mong metls, prticulrly etween Zn nd Cd or etween Zn nd Ni? Mteril nd methods Plnt nd soil mteril N. cerulescens seeds were collected from 7 metlliferous sites, from 2 serpentine soils nd from 9 nonmetlliferous soils in Belgium, the Grnd Duchy of Luxemourg nd Frnce (Tle 1). In ech popultion, seeds from 2 3 flowering plnts were collected. To nlyse the extrctle Zn, Cd, P nd Ni concentrtions of soil, ulk smples were ir-dried, ground nd the <2 mm frctions were seprted from corse rock

3 Plnt Soil Tle 1 Chrcteristics of metllicolous, non-metllicolous nd serpentine sites of Nocce cerulescens popultions. Soil concentrtions re expressed s mgkg -1 mmonium cette-edta extrctle element. ABBR: Arevitions of loclities. Arevitions of tretments: AS (Les Avinières soil), BM (Binry metllic slt mixtures), MS (Monometllic slts), HY (Hydroponics) Loclities ABBR. Tretments Altitude (m) Ltitude (N) Longitude (E) Soil Zn Soil Ni Soil Cd Environment Metllicolous Avinières mine AV AS-BM-MS-HY Clcreous wste grsslnd Durfort mine DU AS-BM <1 69 clcreous wste grsslnd Pryon smelter PR AS-BM (2) <1 429 limestone nd shle grsslnd St Felix mine SF AS-BM-MS-HY <1 36 clcreous wste grsslnd Treves mine TR AS-BM-MS " 6887 <1 41 clcreous wste grsslnd Vernissiere mine VE AS-BM <1 28 clcreous wste shrulnd Viviez smelter VI AS-BM c c micschist grsslnd Non metllicolous Brquette BA AS-BM-MS-HY <1 <1 clcreous shrulnd Buege BU AS-BM clcreous shrulnd Nvcelles NA AS-BM <1 <1 clcreous shrulnd Rnqus RA AS-BM clcreous shrulnd St Budille SB AS-BM <1 <1 clcreous shrulnd St Come SC AS-BM-MS <1 <1 clcreous shrulnd St Michel SM AS-BM <1 <1 clcreous shrulnd Sernne SE AS-BM-MS-HY <1 <1 <1 clcreous shrulnd Wilwerwitz WI AS-BM (2) <1 <1 shle grsslnd Serpentine Bergench BE AS-BM-MS d <1 serpentine grsslnd Puy wolf e PW AS-BM-MS e <1 serpentine shrulnd (Frérot et l. 26) Totl Zn ±1762 mg/kg; totl Cd 899±2 n=3 (Escrré et l. 211) (Dechmps et l. 28,; Molitor et l. 25) c Totl Zn concentrtion (Reeves et l. 21) d Totl Ni concentrtion: 146 to 25 mg/kg (Chrdot et l. 27) e Totl Ni concentrtion: mg/kg (Reeves et l. 21)

4 Plnt Soil frgments y sieving. All chemicl nlyses were performed on the <2 mm soil frctions. Minerl elements were extrcted with mmonium cette-edta 1 N (ph4.65) for 3 min (1 g dry soil in 5 ml of extrctnt) (Cottenie et l. 1982). The superntnt ws filtered nd nlysed y inductively-coupled plsm opticl emission spectroscopy (ICP-OES; Vrin Vist MPX). This method is known to extrct the lile nd less lile pools of trce elements (Fngueiro et l. 25; Lnowski et l. 28), i.e. the minerl frction potentilly ville to plnts vi root sorption. In previous experiments, this frction showed good correltion with the concentrtions of Zn nd Cd in the eril tissues of Nocce cerulescens (Roinson et l. 1998). Extrctle Ni concentrtions were nlysed only for the ultrmfic soils from Bergench nd Puy de Wolf. A smpling of the other soils from Southern Frnce ws lso nlysed ut the Ni EDTA concentrtions were ll lower thn 1 mg/kg. Plnt culture in soils Germinted seedlings were trnsferred into.5 L continers nd ssigned to different tretments for 3 months (from My to July) in glsshouse with nturl dy: night regime nd wtered with distilled wter t the CEFE CNRS experimentl field Sttion in Montpellier (Frnce). Continers were completely rndomised in the glsshouse. The following tretments were used: 1) Contminted soil. This ws collected in tiling pond t Les Avinières mine. The soil hd high metl content (Tle 1), low orgnic mtter nd shortge of mjor plnt nutrients. It ws mixed with commercil compost (9 % soil-1 % compost) to fcilitte plnt growth. Initilly, 1 plnts per popultion were used, ut the mortlity in some popultions reduced the numer of surviving plnts t the end of the experiment. Only plnt Zn nd Cd concentrtions nd mss were nlysed ecuse the Ni concentrtion vlues in this soil re very low. We used Les Avinières soil ecuse in previous experiments (Escrré et l. 2) it llowed to discriminte MET nd NMET origins with low mortlity. In ddition it is true soil used s control to compre with the other tretments. 2) Grden soil with metllic (Zn, Cd, Ni) slts dded. ) BINARY MIXTURES. The soil ws mixture of commercil grden compost nd soil from the CEFE-CNRS experimentl sttion with very low orgnic C content (3.8 %) nd low C:N rtio (16) (see Escrré et l. 2 for other chrcteristics of this soil). Zn ws supplied to soil s ZnSO 4.7H 2 O nd mixed with two slts (CdSO 4 or NiCl 2.6H 2 O) in six tretments: 1) 25 mg/kg Zn 1 mg/kg Cd (herefter 25Zn1Cd; id for the following tretments); 2) 1 mg/kg Zn 25 mg/kg Cd; 3) 25 mg/kg Zn 25 mg/kg Cd; 4) 1 mg/kg Zn 25 mg/kg Ni; 5) 25 mg/kg Zn 25 mg/kg Ni; 6) 25 mg/kg Zn 1 mg/kg Ni. ) MONOMETALLIC ZN, CD AND NI SALTS. Different Zn, Cd nd Ni slts with contrsted soluility in wter (2 C) were used: 25 nd 15 mg/kg ZnSO 4.7H 2 O (soluility 96.5 g/1 ml); 15 mg/kg ZnO (soluility.16 g/1 ml); 25 nd 5 mg/kg CdSO 4 (soluility 76.4 g/1 ml); 5 mg/kg CdCl 2 (soluility 135 g/1 ml); 25 nd 1 mg/kg NiCl 2.6H 2 O (soluility 254 g/1 ml); 1 mg/kg NiSO 4.6H 2 O (soluility 65 g/1 ml). Five plnts per popultion nd per tretment were used in the experiments with metllic slts. Soil metl concentrtions of the mixtures were reltively low compred to those of soils from Les Avinières mine (Tle 1) nd in the rnge of ultrmfic soils for Ni (Tle 1). Our gol ws to llow survivl nd optiml growth for popultions from the three origins. Nevertheless, six of nine tretments with monometllic slts (Zn, Cd nd Ni) hd very high concentrtions of metls with the purpose to test the tolernce of popultions nd the eril metl concentrtion of the plnts from three origins in extreme soil conditions. Plnt culture in hydroponics Individuls of four popultions (2 MET: AV, SF; 2 NMET: BA, SE) were rndomly plced into 3.9 L continers with cpcity for 16 plnts, in growth room with rtificil light, s detiled in Grnier (1992), nd then filled in with nutritive solution

5 Plnt Soil descried y Koch et l. (1987). The photoperiod ws set t 16:8 h (dy:night), the ir temperture ws 22:18 C nd the reltive humidity ws mintined ove 6 %. The Photosyntheticlly Active Rdition flux (PAR) t seedling height ws 515±8 μmol photons m -2 s -1 (n=1). The solution ws renewed twice week, the ph ws djusted to 5.5 with HCl every 2 dys, nd the continers were periodiclly moved to homogenize light distriution. Nine continers received 1.5 μm of zinc, nd nine received 2 μm (ZnSO 4.7H 2 O form). Plnts were hrvested t 7, 2, 4, 6, 8, 1 nd 125 dys fter trnsplnting. The numer of plnts t ech hrvest ws vrile depending on the mortlity. Two or three plnts were collected per popultion nd tretment t the first hrvest, nd five for the other hrvests, while for the lst hrvest ll survivors were collected (4 7 individuls) for totl of 265 plnts. At ech hrvest, only one plnt per popultion nd per continer ws tken t rndom nd the continers were lso rndomly selected. Plnt nlysis Aeril iomss of ll plnts grown in soil ws hrvested fter 3 months, rinsed in deionised wter nd dried t 6 C for 3d. Due to the very lrge numer of elementl nlyses, only suset of MET nd NMET popultions were nlysed for Cd nd Ni in the experiments with slt mixtures nd monometllic slts, wheres the two SERP were consistently nlysed (Tle 1). Some plnts with too low iomss were excluded from Cd/Ni nlyses. Dried smples were ground nd suset ws minerlised in mixture of nitric nd perchloric cid with Tector digestor, nd their individul cdmium nd nickel contents determined y ICP-OES (Vrin Vist MPX). The rest of the ground mteril ws used to mesure the individul Zn concentrtion y the zincon method (see herefter). The totlity of individuls grown in soil nd in hydroponics ws nlysed for Zn concentrtions with the Zincon method developed for Aridopsis hlleri (Mcnir nd Smirnoff 1999). This method is sed on UV-visile spectrophotometry using zincon s coloured Zn-chelting gent nd is less expensive thn ICP. The Zincon method llows the nlysis of individuls with low iomss. This method hs een previously vlidted with Nocce cerulescens (Frérot et l. 25). Metl mss ws clculted s the product of metl concentrtion, times the ove-ground iomss. Despite the fct tht metl mss is concentrtiondependent, unlike iomss, this mesure ws lso selected ecuse it provides good ide of the potentil of ech Nocce cerulescens popultion to extrct metls from soil. Sttisticl methods First, dry eril metl concentrtions nd metl msses were nlysed for ll soil tretments y 6 three-wy prtilly nested ANOVAs (SAS 24) with the following fctors: origins (MET, NMET nd SERP), tretments (1 for Zn, 7 for Cd nd 6 for Ni) nd popultions nested within origins nd tretments. The popultion fctor ws considered s rndom. Type III sums of squres nd the Stterthwite pproximtion were used. Mens were compred using lest squre mens tests. These nlyses were mde to compre oth the men vlues in the different tretments nd the performnces of the three origins. Secondly, ecuse of the complex interctions etween popultions nd tretments (see results), ech tretment ws lso nlysed independently y nested ANOVA with the three origins nd the popultions nested within the origins. Popultion ws considered s rndom. The ANOVAs for metl concentrtions nd msses were s follows: i) one for the experiment with the mine sustrte, ii) 6 for ech of the tretments with Zn inry slt mixtures (3 with Cd nd 3 with Ni), nd iii) 9 for the monometllic slt tretments (3 for ech metl). Lstly, differences in metl concentrtions or metl msses mong slt mixtures nd monometllic slts for popultion were performed y one-wy ANOVAs followed y posterioricontrsts. Dt from hydroponics were nlysed independently ecuse the experimentl conditions were not the sme s for the other experiments. Zn concentrtion nd mss were nlysed y prtilly-nested ANOVAs with the following fctors nd their interctions: dtes (7), origins (3), tretments (2) nd popultions (4) nested within origins. Vlues were log-trnsformed prior to sttisticl nlyses. Therefore, the results of the ANOVAs re given for log-trnsformed vlues, ut mens (±SE) re in rithmeticl vlues for the ske of clrity. Survivl differences etween origins nd tretments were tested using Fisher's exct test (Sttistix 23).

6 Plnt Soil Results Comprisons mong soil tretments nd origins (popultions nested within origin nd tretment) ) Biomss nd survivl. Plnt cultivted only with Cd or Ni monometllic slts hd lower iomss (Fig. 1) thn those grown with mixtures (ANOVA s Tretment effect significnt). In most tretments MET hd higher iomss thn NMET nd SERP (ANOVA s Origin effect significnt). The ltter hd on verge the lowest iomss (see the three histogrms t the right end of the grph). The only exception ws in the 25ZnSO 4 tretment nd in the tretments with Ni monometllic slts where SERP iomss ws equivlent to tht of MET individuls. As for iomss, survivl vlues (Tle 2) were lowest for plnts grown without Zn ut with Cd slts (including 5 CdCl 2 : 43 % survivl) nd to lesser extent with Ni slts of (1 NiSO 4 :63% survivl). Overll survivl of plnts growing with Zn slts (in mixtures or with monometllic slts) ws significntly higher (97.6 % 645/66) thn plnts without Zn slts (69.6 % 167/24). There ws no difference in survivl etween origins for mixtures. On the other hnd NM survivl (66.6 % 9/135) ws significntly lower thn tht of MET (79.2 % 9/135) nd tht of the SERP (82.2 % 74/9) when plnts were cultivted in Zn, Cd nd Ni monometllic slts. Survivls of individuls grown in Les Avinières soil ws 76 % (137/18) similr to tht of monometllic slts ut significntly lower thn tht of mixtures. There were no significnt survivl differences mong origins in this soil. ) Zinc. Plnts cultivted in monometllic slts or in Les Avinières soil hd the highest Zn concentrtion vlues per plnt (significnt tretment effect, cpitl letters ove rs in Fig. 2) nd plnts from Zn-Cd mixtures hd the lowest Zn concentrtion vlues (Fig. 2). Overll, plnts from MET origin hd the lowest men Zn concentrtion (MEAN, lst three rs t the right of ech grph) ut high Zn men mss (with NMET) ecuse the high iomss of MET individuls compensted for the low Zn concentrtion. SERP individuls hd overll the highest Zn concentrtion nd the Aeril iomss (g) AB AB AB A A AB AB BC C AB CD C D A C C 1Zn25Cd 25Zn1Cd Ori F (2,165) = 13.2*** 25Zn25Cd Trit F (15,16) = 6.3*** 1Zn25Ni OrixTrit F (3,16) =1.2 25Zn1Ni 25Zn25Ni Pop (OrixTrit) F (154,787) = 5.7*** 25 ZnSO 4 15 OZn 15 ZnSO 4 MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS ORIGIN c AVINIÈRES 25 CdSO 4 5 CdSO 4 5 CdCl 2 25 NiCl 2 1 NiCl 2 1 NiSO 4 MEAN Fig. 1 Men (±SE) eril iomss vlue per individul of metllicolous (M), non-metllicolous (N) nd serpentine (S) Nocce cerulescens plnts cultivted in grden soil contminted with different metl concentrtions nd in Les Avinières mine soil. In inry mixtures, Zn ws provided s ZnSO 4,Cd s CdSO 4 nd Ni s NiCl 2. In monometllic slts, Zn ws provided s ZnSO 4 nd ZnO, Cd s CdSO 4 nd CdCl 2 nd Ni s NiCl 2 nd NiSO 4. For ech tretment, rs topped with the sme lower letter re not significntly different with lest squres mens test (SAS). Results of the mixed nested ANOVA including ll tretments re shown. Tretments with the sme cpitl letter re not significntly different with Tukey test (SAS). Mens for origins cross tretments re shown s the lst three rs

7 Plnt Soil Tle 2 Survivl (percentge numer of living individuls/totl numer) of metllicolous, non-metllicolous nd serpentine popultions of Nocce cerulescens cultivted in 3 Zn/Cd nd 3 Zn/Ni slt mixtures, in Zn, Cd nd Ni monometllic slts nd in contminted soil from the tiling sins of Les Avinières mine. For slt mixtures nd monometllic slt tretments, survivl vlues with the sme letter re not significntly different with Fisher s exct test Survivl (%) Slt mixtures 1Zn25Cd 25Zn1Cd 25Zn25Cd 1Zn25Ni 25Zn1Ni 25Zn25Ni AVINIÈRES SOIL Metllicolous 97 (34/35) 1 (35/35) 97 (34/35) 1 (35/35) 1 (35/35) 97 (34/35) 73 (51/7) Non-metllicolous 97 (44/45) 97 (44/45) 1 (45/45) 1 (45/45) 97 (44/45) 95 (43/45) 8 (71/9) Serpentine 1 (1/1) 1 (1/1) 1 (1/1) 1 (1/1) 1 (1/1) 1 (1/1) 75 (15/2) Totl 98 (88/9) 99 (89/9) 99 (89/9) 1 (9/9) 99 (89/9) 97 (87/9) 76 (137/18) Monometllic slts 25ZnSO 4 15ZnSO 4 15ZnO 25CdSO 4 5CdSO 4 5CdCl 2 25NiCl 2 1NiCl 2 1NiSO 4 Metllicolous 93 (14/15) 93 (14/15) 1 (15/15) 6 (9/15) 1 (15/15) 53 (8/15) 1 (15/15) 53 (8/15) 6 (9/15) Non-metllicolous 86 (13/15) 86 (13/15) 1 (15/15) 4 (6/15) 93 (14/15) 26 (4/15) 1 (15/15) 73 (11/15) 53 (8/15) Serpentine 1 (1/1) 9 (9/1) 1 (1/1) 5 (5/1) 9 (9/1) 5 (5/1) 1 (1/1) 8 (8/1) 8 (8/1) Totl 93 (37/4) 9 (36/4) 1 (4/4) 5 (2/4) c 95 (38/4) 43 (17/4) c 1 (4/4) 68 (27/4) 63 (25/4) c lowest Zn mss due to their smll iomss, prticulrly in some Zn-Cd tretments nd Cd monometllic slts, proly due to the low Cd tolernce of these popultions. NMET individuls hd intermedite Zn men concentrtion. In the 15 ZnSO 4 tretment (the most solule slt) plnts from SERP hd vlues ove 1, mg Zn/kg (the dmitted threshold for Zn hyperccumultion). Zn concentrtions were significntly lower in metllic slt mixtures (Zn-Cd or Zn-Ni) thn in monometllic slts (25ZnSO 4 ) t equivlent Zn concentrtion (25 mg/kg) (Fig. 2). Zn mss ws significntly lower for Zn-Cd (ut not for Zn-Ni) mixtures (25Zn1Cd nd 25Zn25Cd), thn in monometllic slts (Fig. 2). c) Cdmium. The highest Cd concentrtions were mesured in plnts grown with monometllic Cd slts (Fig. 2c), wheres plnts grown in Les Avinières soil hd the lowest Cd concentrtions nd Cd mss (Fig. 2c, d). No differences mong the origins were found for Cd concentrtion, ut MET popultions hd significntly higher Cd mss overll thn the others. In ll tretments nd origins Cd concentrtions were ove the Cd hyperccumultion threshold. Cd concentrtions (ut not Cd mss) were significntly lower in slt mixtures (1Zn25Cd nd 25Zn25Cd) thn in monometllic slt (25 CdSO 4 ) t equivlent Cd concentrtions (25 mg/kg) (Fig. 2c, d). Even t high Cd concentrtions (especilly in 5 mg/kg CdCl 2 ) Cd mss ws very low ecuse Cd (prticulrly in the most solule slt CdCl 2 ) decresed iomss nd therefore Cd mss. d) Nickel. Plnts cultivted with the highest concentrtion of Ni monometllic slts (1, NiCl 2 or 1 NiSO 4 ) hd the highest Ni concentrtions, nd those with Zn-Ni inry mixtures hd the lowest (Fig. 2e). Overll, SERP popultions hd significntly higher men Ni concentrtions nd msss; while MET hd the lowest concentrtions ut similr msses s NMET (Fig. 2e, f). In 1 mg Ni/kg tretments, SERP popultions hd men Ni concentrtion vlues ove the Ni hyperccumultion threshold (1 mg Ni/kg plnt dry mss).

8 Plnt Soil ) ) Zn concentrtion (mg/kg) D C D Ori F (2,129) = 28.9*** Trit F (9,128) =3.*** OrixTrit F (18,128) =1.4 Pop (OrixTrit) F (123,638) =2.4*** B C MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS C AB AB B A c 1Zn25Cd 25Zn1Cd 25Zn25Cd 1Zn25Ni 25Zn1Ni 25Zn25Ni 25 ZnSO 4 15 ZnSO 4 15 ZnO AVINIERES MEAN c) d) Zn mss (mg/plnt) DE CD E ABC AB BCD A DE CDE Ori F (2,129) = 4.5* Trit F (9,127) =5.4*** OrixTrit F (18,127) =1.3 Pop(OrixTrit) F (123,638) =3.*** MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS MNS A Cd concentrtion (mg/kg) B B BC A A Ori F (2,78) =.5 Trit F (6,7) =1.9*** OrixTrit F (12,7) =.6 Pop (OrixTrit) F (53,194) =3.9*** MNS MNS MNS MNS MNS MNS MNS MNS A C 1Zn25Cd 25Zn1Cd Zn25Cd25 25 CdSO4 5 CdSO4 5 CdCl2 AVINIERES MEAN Cd mss (mg/plnt) Ori F (2,74) = 8.6*** Trit F (6,67) =4.9*** OrixTrit F (12,66) =.7 Pop (OrixTrit) F (53,194) =4.7*** A A AB AB AB B B MNS MNS MNS MNS MNS MNS MNS MNS e) f) D BC CD B A A Ni concentrtion (mg/kg) Ori F (2,49) = 14.4*** Trit F (5,47) =14.6*** OrixTrit F (1,46) =.4 Pop (OrixTrit) F (43,12) =2.9*** c c MNS MNS MNS MNS MNS MNS MNS ORIGIN 1Zn25Ni 25Zn1Ni 25Zn25Ni 25 NiCl 2 1 NiCl 2 1 NiSO 4 MEAN Ni mss (mg/plnt) B A AB A Ori F (2,49) = 5.6** Trit F (5,47) =2.6* OrixTrit F (1,46) =1.2 Pop (OrixTrit) F (43,12) =3.*** MN S MN S MN S MN S MN S MN S MN S ORIGIN A A Fig. 2 Men (±SE) Zn (), Cd (c) nd Ni (e) concentrtions (mg/kg) nd men (±SE) Zn (), Cd (d) ndni(f) msses (mg/plnt) per individul in Nocce cerulescens eril prts of metllicolous (M), non-metllicolous (N) nd serpentine (S) Nocce cerulescens plnts cultivted in grden soil contminted with different metl concentrtions nd in Les Avinières mine soil. In inry mixtures, Zn ws provided s ZnSO 4, Cd s CdSO 4 nd Ni s NiCl 2. In monometllic slts, Zn ws provided s ZnSO 4 nd ZnO, Cd s CdSO 4 nd CdCl 2 nd Ni s NiCl 2 nd NiSO 4.For ech tretment, rs topped with the sme lower letter re not significntly different with lest squres mens test (SAS). Tretments with the sme cpitl letter re not significntly different with lest squres mens test (SAS). Mens for origins cross tretments re shown s the lst three rs in ech grph. Hyperccumultion thresholds for Zn (), Cd (c) nd Ni (e) re indicted with dotted horizontl line. Results of the mixed nested ANOVA including ll tretments re shown The comprison of Ni concentrtion nd mss etween Zn-Ni mixtures nd monometllic slts (25NiCl 2 ) t the sme soil Ni concentrtion (25 mg/kg) did show significnt decrese in Ni concentrtion ut not in Ni mss (Fig. 2e, f). In ll nlyses there ws significnt interction effect for Pop (Ori x Trit), indicting tht metl concentrtion nd metl mss mong popultions within origins nd tretments were heterogeneous. For this reson, dt were renlysed tretment y tretment. Comprisons for ech soil tretment (popultions nested within origin) Biomss, survivl, Zn nd Cd concentrtion nd mss in Les Avinières soil (Fig. 3) Overll, MET popultions (ut Pryon) hd high men iomss vlues (Fig. 3e). NM nd SERP hd lower ut similr vlues. The two SERP popultions hd very different iomss with BE hving high nd PW hving low vlues (similr to PR nd WI). Survivl ws very

9 f ) 25 ANOVA Ori F (2,16) = 8.5* Pop(Ori) F (15,12) =4.5*** ) 1 ANOVA Ori F (2,16) = 1.7 Pop(Ori) F (15,12) =3.2*** Zn concentrtion (mg/kg) ef def cdef cde cd cd c cde Zn mss (mg/plnt) e cde f Plnt Soil de e de c e de de cde cde cd e de e VEAV TRPR VI DUSF SMSE SBRASCBABUNA WI BEPW M N S VE AV TRPR VI DUSF SMSE SBRASCBABUNA WI BEPW M N S c) ANOVA d) 15 Ori F (2,2) = 1.2 Pop(Ori) F (15,112) =2.1* 2. ANOVA Ori F (2,2) = 3.9* Pop(Ori) F (15,112) = 4.4*** e) Cd concentrtion (mg/kg) Aeril iomss (g) 1 5 VE AVTRPR VI DUSF SMSESBRASCBABUNAWI BEPW M N S ANOVA Ori F (2,16) = Pop(Ori) F (15,12) =5.7*** VE AV TRPR VI DUSF SMSE SBRASCBABUNA WI BEPW M N S c c cd c ef cd c c de c c cd c c cd c de cd c ef Cd mss (mg/plnt) c e cd de cde cd e cd de. VEAV TRPR VI DUSF SMSESBRASCBABUNAWI BEPW M N S POPULATION MEAN POPULATION MEAN Fig. 3 Men (±SE) Zn () nd Cd () concentrtion (mg/kg), men (±SE) Zn (c) nd Cd (d) mss (mg/plnt) nd men (±SE) iomss (g) (e) per individul of metllicolous (lck rs), non-metllicolous (white rs) nd serpentine (htched rs) popultions of Nocce cerulescens cultivted in Les Avinières soil. For popultion nmes plese see Tle 1. Popultion mens with the sme letter re not significntly different with lest squres mens test (SAS). M metllicolous, N non-metllicolous, S serpentine. Results of the nested ANOVA re shown low for PR (1/1), SC (4/1) nd PW (6/1). Surprisingly, ll WI individuls were live despite their low iomss. MET popultions hd Zn concentrtions lower thn NMET nd SERP (significnt differences mong origins). There ws high heterogeneity within origins: individuls from WI hd two times higher zinc concentrtions thn the other NMET popultions nd the PW (SERP) ccumulted 8 % more Zn thn BE (SERP). There were no differences mong the origins for Zn mss (Fig. 3c), ut the northern popultions PR (MET), WI (NMET) nd PW (SERP) hd the lowest zinc mss (due to low iomss), nd the southern popultions DU nd SF (MET), NA nd BA (NMET) hd the highest [significnt popultion (origin)]. There were no significnt differences mong origins for Cd concentrtion (Fig. 3), ut gret heterogeneity occurred, prticulrly mong MET popultions [significnt popultion (origin)]. Overll, MET popultions hd higher Cd msses thn NMET or SERP (Fig. 3d). However, there ws high heterogeneity within MET, with SF showing the highest vlues nd the northern PR the lowest.

10 Plnt Soil Biomss, survivl, Zn concentrtion nd Zn mss in inry mixtures nd in Zn monometllic slts (Tle 3) In 4 of 9 tretments there were significnt differences mong origins for plnt iomss with MET hving higher vlues thn NMET nd SERP. In 5 of 6 mixture tretments, NM popultions hd the highest iomss (exception: MET PR in the 1Zn25Cd). This highlights the high tolernce of NM to moderte levels of metls. In 6 of 9 tretments, there were significnt differences mong popultions. This shows tht within origins popultion respond differently to metls. Survivl of the different popultions ws very high (see lso Tle 2) in mixtures s in tretments with monometllic slts (two of which hd very high concentrtions of Zn). Despite tht SERP hd low iomss there ws no mortlity in ny tretment. Zn slt mixtures (Zn-Cd nd Zn-Ni) generted contrsting responses mong popultions for Zn concentrtion nd mss (Tle 3). Thus, for the three Zn-Cd tretments there ws only one significnt difference (p<.1) in the Zn concentrtion mong origins nd mong popultions (25Zn1Cd). The mixture with 25Zn25Cd showed significnt differences for zinc mss mong origins (SERP hd the lowest vlues, see lso Fig. 2) nd mong popultions, with the northern popultions WI (NMET) nd PW (SERP) hving the lowest Zn mss vlues (Tle 3). Out of the three Zn-Ni tretments, two (1Zn25Ni nd 25Zn1Ni) showed significnt Zn concentrtion differences mong origins (Tle 3) withserpshowing high vlues (Tle 3 &Fig.2). However, ll of the Zn-Ni tretments hd significnt popultion (origin) effect for Zn concentrtion nd Zn mss. Unexpectedly, some popultions hd contrsted differences in Zn mss for the sme soil Zn concentrtion (25 mg/kg). For instnce, WI (NMET) hd the highest Zn mss vlues in the 25Zn25Ni tretment (7.3 mg/plnt) nd the lowest in the 25Zn1Ni tretment (.7 mg/plnt). The two zinc sulphte tretments (monometllic slts) showed significnt differences for Zn concentrtion mong the origins (Tle 3) with the two SERP popultions hving the highest vlues (Tle 3). There were lrge vritions for some popultions mong the three tretments with monometllic slts (e.g. Zn concentrtion vlues mong BE (SERP) individuls did vry from 4, to nerly 2 mg/kg), nd the tretment with the lowest Zn concentrtion in the soil (25 mg/kg) in other popultions (BA, TR, SF) gve the highest vlues of Zn concentrtion in plnts. Both 15 Zn tretments showed significnt differences mong popultions, prticulrly for the two SERP popultions. Thus, PW hd higher Zn concentrtion thn BE for zinc oxide, ut hd the lowest zinc mss. However, for zinc sulphte t 15 mg/kg, BE hd the highest Zn concentrtion. Previously (see Fig. 2), it ws shown tht Zn concentrtions were significntly lower in metllic slt mixtures (Zn-Cd or Zn-Ni) thn in monometllic slts (25ZnSO 4 ) t equivlent Zn concentrtions (25 mg/kg). We checked whether this occurred in the 8 popultions tested or if only some popultions showed such decrese. The one-wy ANOVAs followed y posteriori contrsts showed tht the three MET popultions (AV, SF nd TR), two NMET (BA nd SC) nd the two SERP (BE nd PW) popultions hd significntly (p<.5) lower Zn concentrtions vlues in the Zn-Cd mixtures compred to monometllic slts. Only one NMET (SE) showed nonsignificnt Zn concentrtion differences (p>.5). For the Zn-Ni TR (MET), BA nd SC (NMET) nd PW (SERP) showed significnt (p<.5) decrese of Zn concentrtion vlues in mixtures, wheres the other popultions AV, SF (MET), SE (NMET) nd BE (SERP) did not show ny significnt differences. The results of the tretments with Zn slts clerly showed tht the highest Zn concentrtions nd msses were chieved y different popultions in different tretments (Tle 3). Thus, the mximum Zn concentrtion in ech tretment ws chieved y 6 different popultions (4 NMET nd the 2 SERP), nd the mximum Zn mss in ech tretment ws chieved y 8 different popultions (2 MET, 4 NMET, 1 SERP) (Tle 3). Biomss, survivl, Cd concentrtion nd Cd mss in inry mixtures nd monometllic slts (Tle 4) There were no significnt iomss differences mong origins for ny of the tretments, ut for ll tretments, there were significnt differences mong popultions within origin. Some popultions hd n importnt mortlity t high Cd concentrtions. This ws the cse of SF (MET), BA nd SC (NMET) nd PW (SERP). The ltter popultion hd only one survivor in the 5 CdCl 2 tretment.

11 Plnt Soil Tle 3 Results of nested ANOVAs for eril iomss, Zn concentrtion nd Zn mss () nd mens (±SE) for eril iomss (g) (old), Zn concentrtion (mg/kg) (norml) nd Zn shoot mss (mg/plnt) (itlics) per individul () of metllicolous, non-metllicolous (old) nd serpentine (itlics) popultions of Nocce cerulescens cultivted in six Zn slt mixtures (ZnSO 4 ) nd in three Zn monometllic slts (ZnSO 4, ZnO). For ech slt tretment, popultion mens with the sme letter (old for iomss, norml for Zn concentrtion nd itlics for Zn mss) re not significntly different with lest squres mens test (SAS). Mximl iomss, Zn concentrtion nd mss vlues for ech tretment re underlined. Smpling numer is enclosed in prentheses fter the men eril iomss Zinc slt mixtures Zinc monometllic slts 1Zn 25Cd 25Zn 1Cd 25Zn 25Cd 1Zn 25Ni 25Zn 1Ni 25Zn 25Ni 25ZnSO4 15ZnSO4 15ZnO ANOVAS Shoot iomss Ori F 2,15 =3.1 Ori F 2,15 =1.6 ns Ori F 2,15 =4.* Ori F 2,15 =4.2* Ori F 2,15 =.1 ns Ori F 2,15 =5.7* Ori F 2,5 =.8 ns Ori F 2,5 =1. ns Ori F 2,5 =1.2 ns Pop(Ori) F 15,7 =1.6 ns Pop(Ori) F 5,7 =4.6*** Pop(Ori) F 15,7 =4.7*** Pop(Ori) F 15,72 =3.2** Pop(Ori) F 15,68 =6.6** Pop(Ori) F 15,67 =2.4** Pop(Ori) F 5,28 =1.7 ns Pop(Ori) F 5,28 =8.9 *** Pop(Ori) F 5,32 =16.9*** Shoot zinc concentrtion Ori F2,15=1.2 ns Ori F2,15=2.9 Ori F2,15=1.6 ns Ori F2,15=3.7* Ori F2,15=3.1 Ori F2,15=2. ns Ori F2,5=6.8* Ori F2,5=7.4* Ori F2,5=1.1 ns Pop(Ori) F5,7=1.3 ns Pop(Ori) F15,7=1.7 Pop(Ori) F15,7=1. ns Pop(Ori) F15,72=3.5** Pop(Ori) F15,68=5.2** Pop(Ori) F15,67=2.* Pop(Ori) F5,28=.7 ns Pop(Ori) F5,28=2.2 Pop(Ori) F5,32 =6.5*** Shoot zinc mss Ori F 2,15 =.9 ns Ori F 2,15 =1.3 ns Ori F 2,15 =3.8* Ori F 2,15 =1.7 ns Ori F 2,15 =.7 ns Ori F 2,15 =2.6 Ori F 2,5 =3.2 Ori F 2,5 =.5 ns Ori F 2,5 =.8 ns Pop(Ori) F 15,7 =1.4 ns Pop(Ori) F 15,7 =3.1*** Pop(Ori) F 15,7 =2.5** Pop(Ori) F 15,72 =1.9* Pop(Ori) F 15,68 =5.1*** Pop(Ori) F 15,67 =1.6 Pop(Ori) F 5,28 =.7 ns Pop(Ori) F 5,28 =8.3*** Pop(Ori) F 5,32 =9.2*** Aeril iomss (g)/zn Concentrtion (mg/kg)/zn MASS (mg/plnt) Avinières (AV).9±.34 (5) 1.34±.14 (5).61±.23 c (4) 1.8±.24 (5).78±.11 (5).38±.21 cd (4).76±.9 (5) 1.58±.23 (5) 1.29±.26 (5) 1289± ±3 1763± ±926 defg 3428±122 cdef 3987±224 cd 4429± ±299 cd 471±756 c 1.6±.7 3.7±.4 c 1.6±1 c 5.3±1.4 3.±.2 c 2.1± ±.7 7.2±.9 6.3±1.6 Durfort (DU) 1.4±.42 (5) 1.19±.26 c (5).71±.15 c (5).81±.17 (5) 1.1±.16 (5).71±.22 cd (5) 1455± ± ± ±659 cd 3725±289 cde 352±851 cde 1.8±.8 3.8±.9 c 1.7±.5 c 5.3± ±.9 2.9±1.2 Pryon (PR) 1.37±.24 (5).37±.22 cd (5).97±.32 c (5).71±.17 (5).28±.7 c (5).63±.22 cd (5) 2456± ± ± ±591 efgh 231±447 efg 281±992 cde 3.3±.6.7±.5 de 3.3±.7 2.6±.8.8±.3 cd7 1.8±.7 St Felix (SF) 1.16±.36 (5).99±.22 c (5).99±.38 c (5) 1.14±.29 (5) 1.44±.22 (5) 1.5±.26 c (5).35±.13 (4) 1.4±.33 (5) 1.33±.24 (5) 192± ± ± ±834 defg 3718±59 cde 4443±453 cd 629±21 326±1875 cd 3938±558 c 2.4±.8 4.6± ± ± ± ± ± ± ±1.5 Treves (TR) 1.8±.57 (4).62±.28 cd (5).21±.8 cde (5) 1.34±.34 (5) 1.7±.43 (5).62±.29 cd (5).58±.17 (5).34±.3 c (4).48±.2 (5) 2222± ± ± ±759 defg 1594±232 g 2121±383 e 5129± ±296 d 3575±444 c 2.5± ±.8 cd.5±2 c 4.6±.7 1.9±.8 cd 1.6±.8 2.9±1. 1.7±1.1 c 1.9±.9 c Vernissiere (VE).87±.3 (5).34±.14 cd (5) 1.18±.3 (5).62±.21 (5).95±.35 (5) 1.12±.14 (5) 1343± ± ± ±344 h 1475±138 g 3113±796 cde 1.6±.7 1.2±.5 de 2.3±.6 c 1.3±.5 1.4±.6 cd 3.8±1.5 Viviez (VI) 1.3±.22 (5) 1.12±.16 (5).6±.18 c (5).79±.12 (5) 1.18±.8 (5).7±.4 cd (5) 1912± ± ± ±45 efgh 2128±22 fg 283±782 e 2.1±.6 3.6±.8 c 1.2±.5 c 2.2± ±.2 c 1.5±.6 Brquette (BA).84±.18 (5) 1.54±.13 (5) 1.81±.16 (5) 1.67±.2 (5) 1.18±.17 (5) 1.6±.21 (5).52±.14 (5).5±.1 c (5).13±.7 c (5) 2541± ± ± ±612 gh 3179±266 defg 3676±66 cde 7539± ±2563 cd 451±1328

12 Plnt Soil Tle 3 (continued) Zinc slt mixtures Zinc monometllic slts 1Zn 25Cd 25Zn 1Cd 25Zn 25Cd 1Zn 25Ni 25Zn 1Ni 25Zn 25Ni 25ZnSO 4 15ZnSO 4 15ZnO 2.±.6 4.±.5 c 3.6±.8 4.5±.9 3.6±.4 5.6±.6 4.3±1.6.1±.1 c.6±.4 c Buege (BU).65±.16 (5) 1.1±.19 c (5).85±.13 c (5).93±.23 (5) 1.13±.11 (5).87±.14 cd (5) 227± ± ± ±756 cde 4195±537 cd 3458±26 cde 1.6±.6 4.6±.9 2.4±.5 5.± ±.6 3.±.6 Nvcelles (NA).61±.21 (5).72±.21 c (5).48±.17 cd (5).92±.1 (5) 1.17±.17 (5).53±.13 cd (5) 2338± ± ± ±748 defg 466±492 c 4961±833 c 1.2±.3 2.8±.7 cd 1.4±.6 c 4.7±1 5.3±.8 2.9±.7 Rnqus (RA).15±.3 (4).47±.13 cd (5).48±.13 cd (5).56±.1 (5) 1.1±.5 (5).26±.7 cd (5) 1482± ± ± ±49 cd 2911±287 defg 2649±78 de.2±.1 1.5±.5 de.7±.7 c 3.5±.8 3.±.3 c.8±.3 Sernne (SE).74±.27 (5).66±.23 c (5).84±.15 c (5) 1.78±.12 (5) 1.1±.13 (5) 1.15±.27 (5).87±.8 (3).74±.17 (5) 1.13±.9 (5) 1862± ± ± ±544 defg 2849±47 defg 2821±667 de 4116± ±1875 cd 7392±742 c 1.4±.7 2.5±1 cde 2.2±.5 8.±.9 3.1±.4 c 3.5± ±.6 3.7± ±.3 St Budille (SB).61±.12 (5) 1.3±.31 (5).99±.15 (5).94±.2 (5).92±.6 (4).91±.17 cd (5) 1787± ± ± ±958 defg 3478±41 cde 3947±863 cde 1.1±.2 4.7± ±.7 5.± ±.4 c 3.9±.9 St Come (SC).7±.29 (5) 1.11±.37 c (5).57±.17 c (5) 1.12±.19 (5) 1.57±.2 (5).69±.2 cd (5).48±.3 (5).63±.4 c (3).12±.9 cd (5) 198± ± ± ±516 defg 2941±336 defg 4871±77 c 6477± ±4639 c 12286± ±.7 3.7±1 c 1.6±.7 c 5.±.7 4.4±.2 3.7± ±.3 1.1±1. c 1.3±.9 c St Michel (SM).41±.1 (5).55±.9 c (5).49±.6 c (5).69±.2 (5).92±.23 (5).29±.3 cd (5) 1921± ± ± ±1245 defg 437±6 cd 3631±1259 cde.8±.3 2.3±.4 cde 1.2±.1 c 3.2± ±.6 c 1.2±.5 Wilwerwiltz (WI).23±.16 (5).8±.3 d (4).6±.1 e (5).17±.6 (5).12±.4 c (5).58±.14 cd (3) 2351±6 3723± ± ± ± ±568.9±.7.3±.2 e.1±.4 c 2.1±.9.7±.3 d 7.3±1.9 Bergench (BE).43±.16 (5).28±.15 cd (5).18±.6 cde (5).35±.15 (5).91±.5 (5).17±.6 d (5).56±.1 (5).36±.14 c (5).65±.19 (5) 2 349± ± ± ± ±653 efg 6579± ± ± ±65 c.8±.2 1.4±.8 cde.5±.3 c 3.2± ±.7 cd 1.4±.5 4.4± ± ±.8 Puy de Wolf (PW).45±.5 (5).22±.6 cd (5).1±.4 de (5).16±.5 (5).55±.8 (5).24±.12 cd (5).35±.8 (5).3±.5 c (4).1±.3 d (5) 1647± ± ± ±893 c 6696± ±1589 cd 1368± ± ±2428.8±.2 1.±.4 de.2±.1 c 1.3±.4 3.5±.6 c 1.8± ±1.5.4±.1 c.2±.1 c *** p<.1; ** p<.1; * p<.5; p<.1; ns p>.1

13 Plnt Soil Tle 4 Results of nested ANOVAs for eril iomss, Cd concentrtion nd mss () nd men (±SE) eril iomss (g) (old), Cd concentrtion (mg/kg) (norml) nd men (±SE) Cd shoot mss (mg/plnt) (itlics) per individul () from metllicolous (norml), non-metllicolous (old) nd serpentine (itlics) popultions of Nocce cerulescens cultivted in three Cd slt mixtures (CdSO 4 ) nd in three Cd monometllic slts (CdSO 4, CdCl 2 ). For ech slt tretment, popultion mens with the sme letter (old for iomss, norml for Cd concentrtion nd itlics for Cd mss) re not significntly different with lest squres mens test (SAS). Mximl Cd concentrtion nd mss vlues for ech tretment re underlined. Smpling numer is enclosed in prentheses fter the men eril iomss. Degrees of freedom differ mong iomss nd Cd nlyses ecuse smpling numer ws not the sme Cdmium slt mixtures Cdmium monometllic slts 1Zn 25Cd 25Zn 1Cd 25Zn 25Cd 25CdSO 4 5CdSO 4 5CdCl 2 ANOVAS Shoot iomss Ori F2,9=1.9 ns Ori F2,9=1.5 ns Ori F2,9=2.3 ns Ori F2,5=.1 ns Ori F2,5=.14 ns Ori F2,2=1. ns Pop(Ori) F9,47=2. Pop(Ori) F9,47=7.*** Pop(Ori) F9,47=6.3*** Pop(Ori) F5,12=5.6** Pop(Ori) F5,3=28.5*** Pop(Ori) F2,12=15.8*** Shoot cdmium concentrtion Ori F2,9=.2 ns Ori F2,9=.3 ns Ori F2,9=1.4 ns Ori F2,3=.2 ns Ori F2,4=.9 ns Ori F2,3=3.2 ns Pop(Ori) F9,23=3.6** Pop(Ori)F9,21=6.6*** Pop(Ori) F9,19=4.2** Pop(Ori) F3,4=15.8** Pop(Ori) F4,7=.7 ns Pop(Ori) F1,3=.1 ns Shoot cdmium mss Ori F2,9=.7 ns Ori F2,9=.8 ns Ori F2,9=3.2 ns Ori F2,3=.4 ns Ori F2,4=.2 ns Ori F2,3=8.7 ns Pop(Ori) F9,23=2.2 Pop(Ori) F9,21=3.6** Pop(Ori) F9,19=4.8** Pop(Ori) F3,4=13.6** Pop(Ori) F4,7=7.5* Pop(Ori) F1,3=1.4 ns Aeril iomss (g)/cd Concentrtion (mg/kg)/cd MASS (mg/plnt) Avinières (AV).9±.34 (5) 1.34±.14 (5).61±.23 c (4).34±.24 (4).78±.3 (5).13±.1 (4) 575±94 cd 1131±353 cd 83±92 cd 1991±199 c 3547± ±115.8±.1 1.5±.6.8±.3 cd 1.4±.9 1.3±.1.5±.2 Pryon (PR) 1.37±.24 (5).37±.22 cd (5).97±.32 c (5) 635±312 cd 3±56 d 673±92 cd 1.±.5.2±.2 d.9±.2 cd St Felix (SF) 1.16±.36 (5).99±.22 c (5).99±.38 c (5).12 (1).67±.22 (5) ded 3152± ± ± ± ±.6 2.1±.5 3.6±.8 3.5±.7 Treves (TR) 1.8±.57 (4).62±.28 cd (5).21±.8 cde (5).18±.4 (4).3±.1 (5).7±.2 (4) 3417± ± ± ± ± ± ±.4.8±.6 cd.7±.1.1 c.3±.1 Viviez (VI) 1.3±.22 (5) 1.12±.16 (5).6±.18 c (5) 432±37 d 1447± ±32.6±.1 1.9±.3 1.7±.4 c Brquette (BA).84±.18 (5) 1.54±.13 (5) 1.81±.16 (5).2 (1).7±.4 (5).3±.1 (4) 89±41 cd 566±16 cd 479±1 cd 1844± ±.4.9±.3 c.8±.1 cd.3±.2 c.1 Buege (BU).65±.16 (5) 1.1±.19 c (5).85±.13 c (5) 629±116 cd 42±49 d 55±95 cd.6±.2.5±.1 cd.6±.2 cd

14 Plnt Soil Tle 4 (continued) Cdmium slt mixtures Cdmium monometllic slts 1Zn 25Cd 25Zn 1Cd 25Zn 25Cd 25CdSO4 5CdSO4 5CdCl2 Sernne (SE).74±.27 (5).66±.23 c (5).84±.15 c (5).5±.1 (3) 1.1±.2 (5) ded 1567±514 c 581±91 cd 543±7 cd 915 c 262± ±.3.6±.2 cd.6±.1 cd.1 2.8±1.1 St Come (SC).7±.29 (5) 1.11±.37 c (5).57±.17 c (5).16±.13 (2).2±.4 (4) ded 4759± ±475 c 3921± ± ±.8 2.9± c Wilwerwiltz (WI).23±.16 (5).8±.3 d (4).6±.1 e (5) 25±1796 cd 1969± d 1.6±1.6.2±.1 cd.1 d Bergench (BE).43±.16 (5).28±.15 cd (5).18±.6 cde (5).7±.18 (2).4±.16 (4).8±.2 (4) 461±29 cd 576±122 cd 151±633 c 2823± ±632 37±441.3±.1.3±.1 cd.4±.2 cd 2.±.6.7±.2 c.4±.1 Puy de Wolf (PW).45±.5 (5).22±.6 cd (5).1±.4 de (5).1±.2 (3).25±.8 (5).2 c (1) 1847±231 c 2312± ±659 cd ±.2.8±.3 c.2±.1 d.2 n=1 *** p<.1; ** p<.1; * p<.5; p<.1; ns p>.1

15 Plnt Soil We did not found sttisticl differences in Cd concentrtions mong origins when plnts were cultivted in mixtures of Zn-Cd, ut lrge significnt differences were found mong popultions of the sme origin (Tle 4). For instnce, SC individuls (NMET) hd high Cd concentrtion vlues (3921 mg/kg) in the tretment 25Zn25Cd, wheres the other NMET popultions hd vlues elow 55 mg/kg (Tle 4). Similr contrsting results were otined etween this SC popultion nd the other NMET in the tretment 1Zn25Cd. Likewise, the MET popultions SF nd TR hd vlues ove 3 mg/kg in the 1Zn25Cd tretment, wheres the other MET popultions hd vlues lower thn 65 mg/kg. For Cd mss in the sme tretment, SC (NMET), SF nd TR (MET) showed vlues of round 5 mg/plnt wheres SERP individuls hd Cd vlues lower thn 1 mg/plnt (Tle 4). In monometllic slts, there ws high mortlity (8-1 %) for SF (MET), BA, SE nd SC (NMET), nd PW (SERP). Plnts in 5CdCl 2 soil showed low Cd mss despite high concentrtion vlues, proly ecuse of the high soluility of this slt decresed iomss (4 of 8 popultions died in this tretment). Physiologicl studies investigting Cd nd Zn influx in the root poplst suggested tht the Cd trnsport from root to shoot ws not Zn suppressile nd medited y the sme trnsporter with higher ffinity for Cd thn for Zn in the MET AV (clled Gnges ecotype ; Lomi et l. 21; Zhoetl.22). Roosens et l. (23) further showed tht the ddition of Zn (1 or 1 μm) in hydroponic solutions contining Cd (3 μm) did not decrese lef Cd concentrtion in SF popultion (lso clled Gnges ecotype ), confirming the ide tht Cd is ccumulted independently of Zn in these popultions. We tested this hypothesis for the AV popultion nd two others y compring the Cd concentrtion etween the two slt mixtures (1Zn25Cd nd 25Zn25Cd) nd the monometllic slt 25 CdSO 4 t the sme Cd concentrtion (25 mg Cd/kg). Cd concentrtions nd mss differences were tested with one-wy ANOVAs followed y posteriori contrsts for ech popultion (it ws not possile to test SF, BA, SE, SC nd PW ecuse of high mortlity ( 8 %) in the 25 CdSO 4 tretment. In contrst to precedent studies we showed significnt decrese of lef Cd concentrtions in MET AV (p<.5) nd in SERP BE (p<.1) in the two slt mixtures compred with monometllic slts. For TR (MET), the men Cd concentrtions nd mss vlues were not significntly different etween monometllic slts nd slt mixtures (see Tle 4 for men vlues). Compred to Zn, only reduced numer of popultions chieved the highest Cd concentrtions nd mss vlues in the different tretments (Tle 4). Thus, SC (NMET) hd the highest concentrtion vlues in 4 tretments nd BA (NMET) nd PW (SERP) in the other two tretments. The SF (MET) popultion showed the highest Cd mss vlues for 4 tretments nd AV (MET) nd BE (SERP) for the other two tretments. Biomss, survivl, Ni concentrtion nd Ni mss in inry mixtures nd Ni monometllic slts (Tle 5) Only 2 of the 6 tretments showed significnt differences mong origins. None of them involved monometllic slts. On the other hnd, 5 of 6 tretments showed significnt differences mong popultions within n origin. Popultion survivl ws higher in mixtures since ll plnts hd survived until the end of the experiment. Agin plnts cultivted in monometllic slts with high concentrtions of Ni (1 NiCl 2 nd 1 NiSO 4 ), hd high mortlity e.g. MET (SF, TR), NM(SE,SC)ndtolesserextentintheSERP (PW). BE ws the only popultion which showed no mortlity. There were significnt differences mong origins for shoot Ni concentrtions in tretments with 25 mg Ni/kg only (inry mixtures nd monometllic slts) (Tle 5). In ddition, significnt differences mong popultions were found in ll tretments for Ni concentrtions nd msses, with the northern PR (MET) hving less Ni thn the others (Tle 5). Unexpectedly, the highest Ni concentrtion in mixtures ws otined for PW (SERP) t the lowest soil Ni concentrtion (25Zn1Ni). Similr results were otined with TR (MET), SE nd WI (NMET). The PW popultion hd the highest Ni concentrtion with monometllic slts (25451 mg Ni/kg with 1 NiSO 4 ). Previous results (see Fig. 2e) showed tht the Ni concentrtions were significntly lower in metllic slt mixtures (Zn-Ni) thn in monometllic slts (25NiCl 2 ) t equivlent Ni concentrtions (25 mg/kg). As for Zn nd Cd, we checked whether the 8 popultions tested ll showed such decrese. One of the three MET

16 Plnt Soil Tle 5 Results of nested ANOVAs for eril iomss, Ni concentrtion nd mss () nd men (±SE) eril iomss (g) (old), Ni concentrtion (mg/kg) (norml) nd men (±SE) Ni shoot mss (itlics) (mg/plnt) per individul () from metllicolous (norml), non-metllicolous (old) nd serpentine (itlics) popultions of Nocce cerulescens cultivted in three Ni slt mixtures (NiCl 2 ) nd in three Ni monometllic slts (NiCl 2, NiSO 4 ). For ech slt tretment, popultion mens with the sme letter (old for iomss, norml for Ni concentrtion nd itlics for Ni mss) re not significntly different with lest squres mens test (SAS). Mximl iomss, Ni concentrtion nd mss vlues for ech tretment re underlined. Smpling numer is enclosed in prentheses fter the men eril iomss. Degrees of freedom differ mong iomss nd Ni nlyses ecuse smpling numer ws not the sme Nickel slt mixtures Nickel monometllic slts 1Zn 25Ni 25Zn 1Ni 25Zn 25Ni 25NiCl2 1NiCl2 1NiSO4 ANOVAS Shoot iomss Ori F2,9=3.4 Ori F2,9=.1 ns Ori F2,9=12.5*** Ori F2,5=.4 ns Ori F2,5=.3 ns Ori F2,3=1.4 ns Pop(Ori) F9,48=5.2*** Pop(Ori) F9,48=8.*** Pop(Ori) F9,45=1.3 ns Pop(Ori) F5,32=6.4*** Pop(Ori) F5,8=3.8* Pop(Ori) F3,19=8.7*** Shoot nickel concentrtion Ori F 2,9 =3.6 Ori F 2,9 =1.2 ns Ori F 2,9 =3.2 Ori F 2,8 =1.6** Ori F 2,5 =2.9 ns Ori F 2,3 =2.8 ns Pop(Ori) F9,22=7.4*** Pop(Ori) F9,23=2.3* Pop(Ori) F9,21=3.2* Pop(Ori) F8,22=2.2 Pop(Ori) F5,8=1.4** Pop(Ori) F3,6=8.1* Shoot nickel mss Ori F2,9=.4 ns Ori F2,9=.6 ns Ori F2,9=3.5 Ori F2,8=6.* Ori F2,5=3. ns Ori F2,3=2.4 ns Pop(Ori) F 9,22 =7.3*** Pop(Ori) F 9,23 =2.7* Pop(Ori) F 9,21 =3.2* Pop(Ori) F 8,22 =1.3 ns Pop(Ori) F 5,8 =3.1 Pop(Ori) F 3,6 =2.3 ns Aeril iomss (g)/ni Concentrtion (mg/g)/ni MASS (mg/plnt) Avinières (AV) 1.8±.24 (5).78±.11 (5).38±.21 cd (4).99±.14 (5).9±.4 c (3).64±.9 (5) 33±78 417±158 c 345±28 c 432± ±369 d 1994±51 c.3±.1.3±.1 cd.2±.1 cd.4±.1.1±.1 d 1.1±.4 Pryon (PR).71±.17 (5).28±.7 c (5).63±.22 cd (5) 25±9 d 76±18 c 124±28 c.2±.5 c.3±.4 d.1±.7 d St Felix (SF) 1.14±.29 (5) 1.44±.22 (5) 1.5±.26 c (5).91±.9 (5).65±.33 (3) ded 124±37 c 638±122 c 621±89 c 428± ±379 cd.2±.1 c 1.1±.2 c.8±.1.4±.2 1.8±.9 Treves (TR) 1.34±.34 (5) 1.7±.43 (5).62±.29 cd (5).66±.11 (5).7±.5 (2).12±.7 c (4) 11±19 c 433±3 c 157±24 c 573± cd 2738±177 c.2±.1 c.8±.1 cd.1±.3 d.4±.1.1 cd.5±.4 Viviez (VI).79±.12 (5) 1.18±.8 (5).7±.4 cd (5) 132±28 c 255±23 c 161±15 c.1±.3 c.3±.5 cd.1±.1 d Brquette (BA) 1.67±.2 (5) 1.18±.17 (5) 1.6±.21 (5).33±.9 c (5).5±.4 c (3).4±.7 c (5) 157±4 c 612±74 c 524±83 c 963± c 235 c.3±.1.9±.2 c.9±.2.3±.4.7 cd.2 Buege (BU).93±.23 (5) 1.13±.11 (5).87±.14 cd (5) 364±76 521±9 c 563±8 c

17 Plnt Soil Tle 5 (continued) Nickel slt mixtures Nickel monometllic slts 1Zn 25Ni 25Zn 1Ni 25Zn 25Ni 25NiCl2 1NiCl2 1NiSO4.5±.1.7±.1 cd.5±.1 c Sernne (SE) 1.78±.12 (5) 1.1±.13 (5) 1.15±.27 (5).74±.6 (5).31±.6 (5) ded 15±1 cd 197±87 c 449±116 c 748± ±227 d.2±.1 c 1.4±.1.7±.1.6±.2.3±.1 cd St Come (SC) 1.15±.19 (5) 1.57±.2 (5).69±.2 cd (5).87±.6 (5).16±.11 c (3).8±.6 c (3) 222±23 c 619±394 c 866±7 12± ± c.3±.5 1.2±.7.8±.2.8±.1.9±.6 c.9 Wilwerwiltz (WI).17±.6 (5).12±.4 c (5).58±.14 cd (3) 115±3 c 1333± ±34 c.3±.1 c.3±.5 cd.4±.1 cd Bergench (BE).35±.15 (5).91±.5 (5).17±.6 d (5) 1.1±.12 (5).38±.13 (5).63±.22 (5) 522± ±81 c 474±446 c 1486± ± ±117.2±.1.3±.1 cd.2±.1 cd 1.4±.7 2.5±.7 4.7±2 Puy de Wolf (PW).16±.5 (5).55±.8 (5).24±.12 cd (5).46±.12 c (5).15±.7 c (3).9±.3 c (3) 789± ± ± ± ± ±1797.2±.2 2.3±.3.7±.2 1.7±.5 2.7± ±1 n=1 ***p<.1; **p<.1; *p<.5; p<.1; ns p>.1