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Genetic variation in zinc-tolerant populations of Glyceria fluitans
Aquatic Botany 82 (2005) 157–167 www.elsevier.com/locate/aquabot
Genetic variation in zinc-tolerant populations of Glyceria fluitans David J. Matthews *, Thomas F. Gallagher, Marinus L. Otte Department of Botany, Wetland Ecology Research Group, University College Dublin, Belfield, Dublin 4, Ireland Received 6 August 2004; received in revised form 19 March 2005; accepted 13 April 2005
Abstract Amplified fragment length polymorphism (AFLP) was used to conduct a study of the genetic diversity of zinc-tolerant populations of Glyceria fluitans from 10 sites from across Europe. Six different primer combinations were used on five to nine plants from each of the 10 sites to generate a total of 796 bands, of which 670 were polymorphic. These data were then used to calculate a dendrogram by agglomerative clustering using the unweighed pair group method with average linkage (UPGMA). The dendrogram contained two distinct clusters, with little overlap between populations. Genetic diversity between populations of G. fluitans did not always correlate with geographical distances, for example, plants from the Navan population from Ireland were more genetically similar to populations from Poland than other populations from Ireland. In other instances, geographical origin was significant, for example, all Polish populations were genetically similar to each other. Populations from two English sites only 160 km apart, showed such a high degree of genetic diversity that they were placed in different clusters in the dendrogram. They were more closely related to Irish and Polish populations than to each other. Plants from different zinccontaminated sites were found not to cluster together. The conclusion was that the cluster groupings were not related to exposure to zinc at the sites of origin, and that the drive to generate distinct metaltolerant populations may not occur in this species due to the existence of a constitutive tolerance to metals. # 2005 Elsevier B.V. All rights reserved. Keywords: AFLP; Floating sweetgrass; Innate zinc tolerance
* Corresponding author. Tel.: +353 171 62019. E-mail address: [email protected] (D.J. Matthews). 0304-3770/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2005.04.002
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1. Introduction Glyceria fluitans (L.) R. Br. (floating sweetgrass) is a wetland grass found in habitats, such as ponds, canals, streams and marshes (Stace, 1997). It is found throughout Europe and extends into West Asia and North Africa (Preston and Croft, 1997) and has been introduced into America and Australia. It is diploid (2n = 40) (Borill, 1955). It possesses innate zinc tolerance (Matthews et al., 2004a). Metal tolerance can be defined as the ability of a plant to survive and reproduce on sites that are toxic to most other plants, because the soil contains elevated concentrations of one or more metals (Macnair and Baker, 1994). These metal-tolerant populations are genetically distinct from non-tolerant populations (Antonovics et al., 1971). Such plants do not possess an innate or constitutive tolerance to metals, but tolerant populations evolved after exposure to a contaminated environment (Antonovics et al., 1971). However, the basis of metal tolerance in wetland plants seems to be different to that of dryland plants. Comparisons of populations of wetland plants from metal enriched sites with those from non-polluted sites have shown that they were equally tolerant of metals. This led to the theory that wetland plants have an innate tolerance to heavy metals (McCabe et al., 2001). Innate tolerance to metals, such as zinc has been found in several other species of wetland plants, including Typha latifolia (McNaughton et al., 1974; Ye et al., 1997a), Phragmites australis (Ye et al., 1997b), Eriophorum angustifolium (Matthews et al., 2004b) and Carex rostrata (Matthews et al., 2005). Previous work on zinc tolerance in G. fluitans populations from across Europe (Ireland, England, Denmark and Poland), collected from both metal-contaminated and non-metal-contaminated sites found that plants were equally tolerant to elevated levels of zinc, regardless of prior exposure (Moran and Otte, 2004; Matthews et al., 2004a). Zinc uptake differed between populations, but this did not affect the tolerance of individual populations to elevated zinc concentrations. Some populations sequestered more zinc in their roots, while other populations sequestered more zinc in their leaves. The aim of this investigation was to use amplified fragment length polymorphism (AFLP) to determine if populations of G. fluitans from zinc-contaminated sites were genetically distinct compared to populations from non-contaminated sites. Also investigated was whether Irish populations were genetically closer to each other than to populations in Europe. Populations were also investigated to assess if different zinc sequestering methods reflected markedly different genetic profiles. AFLP analysis (Vos et al., 1995) was chosen for this study.
2. Materials and methods 2.1. Plant material G. fluitans populations were collected from 10 locations in the spring of 2002. Populations were collected from four locations in Ireland, two locations in England, three locations in Poland and a single location in Denmark (Table 1). The plants from Ireland and England were collected directly from the field, while the plants from Denmark and Poland were germinated from seed. Plant and soil samples collected from
Site location
Country
Coordinates
Approximate site size (m)
Soil (mmol g1)
Porewater (mmol L1) (P = 0.001)
Roots (mmol g1) (P = 0.006)
Live leaves (mmol g1) (P = 0.002)
Navan Lough Dan Glendalough Djouce Woods Camborne Sommerset Thisted Radostowo Bartniki Chomotowo
Ireland Ireland Ireland Ireland England England Denmark Poland Poland Poland
068430 W, 538420 N 068180 W, 538050 N 068230 W, 538000 N 068150 W, 538100 N 058190 W, 508150 N 028550 W, 518100 N 088420 E, 568570 N 208370 E, 538580 N 208450 E, 548040 N 158110 E, 548020 N
5 40 10 10 45 55 10 10 10 10 100 100 50 50 10 10 14 14 32 32
6.1 3.8b 1.5 0.2a 175 96c 0.8 0.1a 0.8 0.2a 0.8 0.2a – – – –
4.7 0.7 b 1.1 0.4 a 105 15 c 2.9 0.4 a 0.7 0.2 a 0.7 0.2 a – – – –
6.9 2.2 b 0.6 0.2 a 50 24 c 1.1 0.1 a 1.2 0.4 a 1.2 0.4 a – – – –
0.8 0.1a 0.2 0.1a 24 2.0c 0.4 0.1a 0.8 0.1a 0.8 0.1a – – – –
The degree of statistical significance, probability P, as determined by one-way ANOVA and Tukey’s comparisons of means tests is shown. Different superscripted letters indicate significant differences between values in columns at P < 0.05; (–) indicates data not available).
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Table 1 Information on site, country, coordinates and mean zinc concentrations standard deviations of soil, porewater, roots and live leaves of G. fluitans
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the field were placed in plastic bags and transported back to the Thornfield Horticultural Unit Glasshouse Complex in University College Dublin, Ireland. Samples of soil, porewater and plants were also transferred to the laboratory and analyzed for zinc content. Soil samples were collected from soil around the plants. Porewater samples were taken to determine the total amount of zinc available to the plant. Ten dialysis vials, consisting of 20-mL plastic scintillation vials covered with 20-mm millipore mesh, were buried at each site at depths of between 5 and 10 cm (Doyle and Otte, 1997). These were left for 3 weeks in the soil to equilibrate, retrieved, filtered using 0.45-mm membrane filters and analyzed for zinc. The Danish and Polish plants were collected from areas with no known contamination with heavy metals (Hansen and Zukev, personal communications, 2002). 2.2. Chemical analysis of soil, porewater and plant samples Samples of soil and plants were dried in an oven at 60 8C until constant weight was obtained. Zinc concentrations were determined following digestion of 100 mg of dried homogenized soil or 50 mg of dried homogenized plant material in Teflon bombs containing 2 mL of HNO3:HCl in a (4:1) mix (Otte et al., 1995). The samples were cooled, diluted and filtered using 0.45-mm membrane filters. Acid digests of soil and plant samples were analyzed for zinc using a Unicam 929 flame atomic absorption spectrometer. Porewater samples were acidified with HNO3 before analysis. Plant standard reference samples supplied by Glen Spectra Reference Materials, England, of oriental tobacco leaves (CTA–OTL–1) were also analyzed for zinc content and deviated on average by 2% from certified values for zinc. 2.3. DNA extraction DNA was extracted using a method based on that of Pich and Schubert (1993), using approximately 0.2 g of leaf material. Extracted DNA was ethanol precipitated and resuspended in 200 mL of sterile distilled water. DNA was re-precipitated by adding 100 mL of 7.5 mol L1 ammonium acetate and 600 mL of 100% ethanol. DNA was then pelleted at 10,000 g for 10 min, and the pellet washed five times in 300 mL of 70% ethanol. The dried pellet was resuspended in 200 mL of TE at pH 7.5. DNA quality and quantity was estimated by separating 5 mL aliquots on 0.8% agarose TBE gels. Only samples that showed non-degraded DNA were used in subsequent AFLP analysis. The DNA amounts were estimated by comparison with known molecular weight standards. 2.4. AFLP protocol The AFPL protocol was carried out as described by Liscum and Oeller (1995). 5 mL (500–600 ng) of DNA was digested with five units of EcoR1 and five units of Mse1 for 3 h at 37 8C. The digestion was carried out in 1 Pharmacia ‘‘One-PHOR-All+’’ buffer containing 50 ng mL1 BSA. EcoR1 and Mse1 adapters were ligated to the digestion DNA at 37 8C. The adapted ligated DNA was pre-amplified in a reaction containing 0.5 mL of both EcoR1-oligo 1 (50 -CTCGTAGACTGCGTACCAATTC-30 ) and Mse1-oligo 1
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(50 -GACGATGAGTCCTGAGTAA-30 ) (50 ng mL1) to 1 mL of ligated DNA, along with 2 mL of 5 mmol L1 dNTPs, 2 mL of Promega Taq buffer, 12 mL of 25 mmol L1 MgCl2, 0.08 mL of 0.4 U Taq Polymerase and 12.72 mL of sterile distilled water. This mixture was then amplified using a PCR thermocyclic profile: (1) 94 8C for 2 min; (2) 94 8C for 30 s; (3) 50 8C for 30 s; (4) 72 8C for 1 min; steps 2–4 repeated 34 times. The amplification product was then diluted by 10 times in TE (10 mmol L1 Tris–HCl, 0.1 mmol L1 EDTA). A selected PCR reaction was performed in which 1 mL of this amplified diluted mixture was added to 0.25 mL of 33P-labelled EcoR1 primer, 0.3 mL of Mse1 primer (six different combinations:s EcoR1–AAG and Mse1–CAC, EcoR1–AAG and Mse1–CAC, EcoR1– AAG and Mse1–CTC, EcoR1–ACC and Mse1–CAC, EcoR1–ACC and Mse1–CAC, EcoR1–ACC and Mse1–CTC), 1 mL of 5 mmol L1 dNTPs, 1 mL of 10 Promega Taq buffer, 0.6 mL of 25 mmol L1 MgCl2, 0.04 mL of 0.4 U Taq Polymerase and 6.31 mL of sterile distilled water. The PCR reaction was performed with the following profile: (1) 94 8C for 2 min; (2) 94 8C for 30 s; (3) 65 8C for 30 s; (4) 72 8C for 1 min; (5) steps 1–4 repeated 11 times; (6) 94 8C for 30 s; (7) 56 8C for 30 s; (8) 72 8C for 1 min; (9) steps 6–8 repeated 25 times; (10) 72 8C for 2 min. The 33P-labelled products were then separated by electrophoresis on a 6% polyacrymide gel in 1 TBE buffer at 50 W for about 3.5 h. The gels were vacuum-dried with a gel dryer (model 583, Bio-Rad) for 55 min at 80 8C. Then the gels were exposed to Kodak Scientific Imaging Film (X-OMATTM) for 48–76 h, depending on the amount of radiation, which was determined with a Geiger–Mu¨ ller counter and developed. 2.5. Data analysis The resulting autoradiograms were scanned using a HP ScanJet 6100C highresolution scanner. The resulting images were saved as TIFF files, and were processed using GelCompare II (Version 3.0) software (Applied Maths, Sint-Martens-Latem, Belgium). The automatic band searching facility was used. Any band that represented more than 1% of the total surface under the densitometric curve was scored. The scoring of the bands was then edited manually. Bands smaller than 100 bp were eliminated, as they could not be scored consistently. Bands on the gel were scored as present (1) or absent (0). Gels were normalized by the alignment of 25 bp molecular weight markers (Promega), which were included at regular intervals in each gel. This was to ensure that all bands were uniformly distributed on the gels. The data were transformed into band frequencies and diversity values based on phenotype frequency (phenotypes being the band patterns produced by individual primer pairs). A matrix of genetic distances between populations based on the number of shared amplification products was calculated using the metric of Nei and Li (1979). These data were then used to calculate a dendrogram by agglomerative clustering using the unweighed pair group method with average linkage (UPGMA) (Sneath and Sokal, 1973). The dendrogram was calculated using a composite of all six-primer combinations so that the contribution of each primer pair was equal, irrespective of the number of bands that had been generated by each primer combination. The support values for the degree of confidence at the nodes of the dendrogram were evaluated by bootstrap analysis (Felsenstein, 1985). One thousand bootstrap datasets were calculated.
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3. Results 3.1. Zinc in soil, porewater and plants from the field The populations analyzed in this study were collected from sites with low and high zinc levels in Ireland and England (Table 1). The Camborne site is in a known copper mining area and so soils were analyzed for copper: an average soil concentration of 2.8 mmol g1 of copper was found. No original soil or plant material was available for analysis from Denmark and Poland, but seeds were collected from areas with no known contamination with metals (Hansen and Zukev, personal communications, 2002). The Glendalough site had significantly higher concentrations of zinc in the soil, porewater and plant parts than the other two contaminated sites at Navan and Camborne. The concentration of zinc at the contaminated sites was higher than the concentration of zinc at the non-contaminated sites at Djouce and Lough Dan. The soil, porewater and plant parts from Djouce and Lough Dan contained the least amount of zinc from the five sites analyzed. 3.2. AFLP polymorphism Fifty-three individuals from the 10 locations were assayed using six selective primer combinations. In total, 796 bands (mean 132 bands per primer combination) were generated (Table 2) across all the G. fluitans populations. The number of monomorphic generated bands was low, with many bands being polymorphic. In total, 670 polymoprhic bands were scored, which gave a total of 84% polymorphism. Primer combination 5 generated the greatest number of bands (172) and primer combination 2 revealed the highest levels of polymorphism (93%). The genetic distance matrix (Nei and Li, 1979) was used to establish the level of genetic divergence between the populations. 3.3. Cluster analysis Cluster analysis divided the 53 populations into two large groups (Fig. 1). Cluster A contained the three Polish populations, the populations from Sommerset (England) and Navan (Ireland). Cluster B contained the remaining Irish populations (Glendalough, Lough Dan and Djouce), and the populations from Thisted (Denmark) and Camborne (England). The cluster analysis shows that populations from any given location are more closely Table 2 AFLPs generated among 65 individuals of G. fluitans representing 10 populations across Europe Primer pair
Polymorphic bands
Total bands
Polymorphism rate (%)
1 2 3 4 5 6
110 109 88 125 129 109
137 117 96 136 172 139
80 93 91 91 75 78
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Fig. 1. UPGMA dendrogram of the different individuals of 10 different G. fluitans populations.
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related to themselves than to populations from other locations. In each case, populations from a particular location form distinct clusters (Fig. 1). There were two exceptions, the first is an individual from Radostowo that is located at the top of cluster A and secondly, an individual from Lough Dan, which is grouped with the Glendalough population. This indicates that there was greater variation between populations from different locations than within the populations from a single location. Clusters A and B were different in their patterns of similarity between populations, with Group A exhibiting a higher level of genetic diversity. The populations of cluster A were 59% similar, and individuals within the populations were from 62% to 85% similar. The populations of cluster B were 62% similar and individuals within the population were from 67% to 90% similar. There was a tendency for plants from the same location to cluster together with little overlap between populations from different locations. The dendrogram constructed with all individuals was generally supported by high bootstrap values, but the two large clusters have different degrees of support. For cluster A, 16 out of 26 bootstrap values were above 50%, suggesting a limited reliability of the pattern found compared with cluster B, which exhibited high bootstrap values, 25 out of 26 values were above 50%. The individual populations within cluster A were supported by high bootstrap value of 91 on the first node. The populations within cluster B were supported by high bootstrap value of 100 on the first node.
4. Discussion Cluster analysis of the AFLP profiles divided the 53 individuals into two groups. Populations did not segregate with respect to the metal status of their sites, as the populations from metal-contaminated sites (Glendalough, Camborne and Navan) did not cluster together. Cluster A contained populations collected from across a large geographic range, whereas populations in cluster B were mainly restricted to Ireland and England. The highest level of genetic diversity was found in the Radostowo group, followed by the other groups from cluster A. G. fluitans plants from the same location tended to cluster together, with minimal overlap between populations. This trend is in agreement with the findings for Lolium multiflorum (Cresswell et al., 2001), Moringa oleifera (Muluvi et al., 1999) and Sticherus flabellatus (Keiper and McConchie, 2000). The partitioning of genetic diversity between populations rather than within populations can be attributed to genetic drift, inbreeding and the homogenizing effects of restricted gene flow (Loveless and Hamrick, 1984). Most of the plants were collected from small sites. Plants collected from isolated sites can display lower diversity, as was demonstrated for Coreopsis integrifolia (Cosner and Crawford, 1994). In contrast, much higher degrees of diversity are observed in populations that cover large geographic areas (Frankham, 1996; Frankham and Ralls, 1998). The plants from Glendalough in cluster B display high similarity between individuals. This could be because currently this site is more isolated than the other sites. The Glendalough site is a tailings pond from a small lead/zinc mine abandoned in the 1950s. The populations from Djouce, Camborne and Lough Dan were also collected from relatively small sites, but within each group do not display as high a degree of similarity as the Glendalough
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population. However, there is some overlap between the Glendalough and Lough Dan populations, signifying some genetic similarity. These populations are geographically close together, at a distance of approximately 20 km apart. Genetic similarity of plants from these two sites may be accounted for by clonal reproduction. G. fluitans can reproduce by clonal means, as detached shoots may be washed away and readily re-root in new localities (Preston and Croft, 1997). This may have happened in Wicklow in the recent past and could account for the overlapping and genetic similarities between these two populations. The findings indicated that in some cases genetic distances was dependent on geographical distance, for example, the close genetic relationship between the Glendalough and Lough Dan populations. However, other populations did not cluster together based on geographical proximity. For example, the Sommerset and Camborne populations in England are only about 160 km apart, but are in separate clusters. This lack of geographical grouping has been found in other species, such as Calycophyllum spruceannum (Russell et al., 1999). The Camborne population is genetically closer to Irish populations, and the Sommerset population is genetically closer to the Polish populations. A possible reason for the unexpected similarities between geographically distant populations may be the role of migratory birds, which can spread seeds across wide distances and account for genetic similarities between distant populations (Stankiewicz et al., 2001). The populations from sites with a history of metal exposure (Glendalough, Navan and Camborne) were not genetically similar to each other. Thus, genetic similarity appears not to be related to the metal status of the sites. It is possible that the drive to generate distinct metal-tolerant populations may not occur in G. fluitans due to the existence of a constitutive metal tolerance. However, there are significant differences in the levels of zinc in these three sites. The Glendalough site has zinc levels that are 175-fold above levels from non-contaminated sites, while the other two sites have much lower levels of zinc. The manner in which the different populations clustered is also not related to the manner in which they partition zinc. The Glendalough and Camborne populations had been shown to sequester more zinc in their roots than their dead leaves, while the Thisted, Navan and Radostowo populations sequestered more zinc in their dead leaves than their roots (Matthews et al., 2004a). The Glendalough and Camborne populations do not form a group in cluster B, while the Thisted, Navan and Radostowo geneotypes are dispersed throughout both clusters A and B. Innate metal tolerance occurs in some other wetland plants, including T. latifolia, P. australis, E. angustifolium and C. rostrata (Ye et al., 1997a,b; Matthews et al., 2004b, 2005). Based on the findings presented here, it is possible that the drive to generate distinct metal-tolerant populations in other wetland plant species may not occur due to the existence of innate tolerance, as genetic similarity appears not to be related to the contamination status of sites. In a separate study, using inter-simple sequence repeat (ISSR) amplification, populations of the wetland species E. angustifolium from contaminated and non-contaminated locations from around Ireland did not segregate with respect to the metal status of their sites (Wilson, personal communication, 2004). This species has also been found to possess innate tolerance (Matthews et al., 2004b). It is possible that innate metal tolerance is a general feature of wetland plants, and consequently these plants do not evolve distinct metal-tolerant populations.
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Acknowledgements This work was funded by Enterprise Ireland (Project No: SC/01/361). We thank the staff of Wicklow Mountains National Park and Outokumpu-Zinc Tara Mines Ltd. for allowing access to the tailings ponds in Glendalough and Navan. Special thanks to Dr. Loveday Jenkin from the Camborne School of Mining, England, Dr. Hans Vilhelm Hansen of the Botanic Gardens, University of Copenhagen, Denmark and Dr. Grzegorz Zurek of the Bydgoszcz Botanic Garden of Plant Breeding and Acclimatization, Poland for help in obtaining the populations from England, Denmark and Poland.
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Genetic variation in zinc-tolerant populations of Glyceria fluitans David J. Matthews *, Thomas F. Gallagher, Marinus L. Otte Department of Botany, Wetland Ecology Research Group, University College Dublin, Belfield, Dublin 4, Ireland Received 6 August 2004; received in revised form 19 March 2005; accepted 13 April 2005
Abstract Amplified fragment length polymorphism (AFLP) was used to conduct a study of the genetic diversity of zinc-tolerant populations of Glyceria fluitans from 10 sites from across Europe. Six different primer combinations were used on five to nine plants from each of the 10 sites to generate a total of 796 bands, of which 670 were polymorphic. These data were then used to calculate a dendrogram by agglomerative clustering using the unweighed pair group method with average linkage (UPGMA). The dendrogram contained two distinct clusters, with little overlap between populations. Genetic diversity between populations of G. fluitans did not always correlate with geographical distances, for example, plants from the Navan population from Ireland were more genetically similar to populations from Poland than other populations from Ireland. In other instances, geographical origin was significant, for example, all Polish populations were genetically similar to each other. Populations from two English sites only 160 km apart, showed such a high degree of genetic diversity that they were placed in different clusters in the dendrogram. They were more closely related to Irish and Polish populations than to each other. Plants from different zinccontaminated sites were found not to cluster together. The conclusion was that the cluster groupings were not related to exposure to zinc at the sites of origin, and that the drive to generate distinct metaltolerant populations may not occur in this species due to the existence of a constitutive tolerance to metals. # 2005 Elsevier B.V. All rights reserved. Keywords: AFLP; Floating sweetgrass; Innate zinc tolerance
* Corresponding author. Tel.: +353 171 62019. E-mail address: [email protected] (D.J. Matthews). 0304-3770/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2005.04.002
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1. Introduction Glyceria fluitans (L.) R. Br. (floating sweetgrass) is a wetland grass found in habitats, such as ponds, canals, streams and marshes (Stace, 1997). It is found throughout Europe and extends into West Asia and North Africa (Preston and Croft, 1997) and has been introduced into America and Australia. It is diploid (2n = 40) (Borill, 1955). It possesses innate zinc tolerance (Matthews et al., 2004a). Metal tolerance can be defined as the ability of a plant to survive and reproduce on sites that are toxic to most other plants, because the soil contains elevated concentrations of one or more metals (Macnair and Baker, 1994). These metal-tolerant populations are genetically distinct from non-tolerant populations (Antonovics et al., 1971). Such plants do not possess an innate or constitutive tolerance to metals, but tolerant populations evolved after exposure to a contaminated environment (Antonovics et al., 1971). However, the basis of metal tolerance in wetland plants seems to be different to that of dryland plants. Comparisons of populations of wetland plants from metal enriched sites with those from non-polluted sites have shown that they were equally tolerant of metals. This led to the theory that wetland plants have an innate tolerance to heavy metals (McCabe et al., 2001). Innate tolerance to metals, such as zinc has been found in several other species of wetland plants, including Typha latifolia (McNaughton et al., 1974; Ye et al., 1997a), Phragmites australis (Ye et al., 1997b), Eriophorum angustifolium (Matthews et al., 2004b) and Carex rostrata (Matthews et al., 2005). Previous work on zinc tolerance in G. fluitans populations from across Europe (Ireland, England, Denmark and Poland), collected from both metal-contaminated and non-metal-contaminated sites found that plants were equally tolerant to elevated levels of zinc, regardless of prior exposure (Moran and Otte, 2004; Matthews et al., 2004a). Zinc uptake differed between populations, but this did not affect the tolerance of individual populations to elevated zinc concentrations. Some populations sequestered more zinc in their roots, while other populations sequestered more zinc in their leaves. The aim of this investigation was to use amplified fragment length polymorphism (AFLP) to determine if populations of G. fluitans from zinc-contaminated sites were genetically distinct compared to populations from non-contaminated sites. Also investigated was whether Irish populations were genetically closer to each other than to populations in Europe. Populations were also investigated to assess if different zinc sequestering methods reflected markedly different genetic profiles. AFLP analysis (Vos et al., 1995) was chosen for this study.
2. Materials and methods 2.1. Plant material G. fluitans populations were collected from 10 locations in the spring of 2002. Populations were collected from four locations in Ireland, two locations in England, three locations in Poland and a single location in Denmark (Table 1). The plants from Ireland and England were collected directly from the field, while the plants from Denmark and Poland were germinated from seed. Plant and soil samples collected from
Site location
Country
Coordinates
Approximate site size (m)
Soil (mmol g1)
Porewater (mmol L1) (P = 0.001)
Roots (mmol g1) (P = 0.006)
Live leaves (mmol g1) (P = 0.002)
Navan Lough Dan Glendalough Djouce Woods Camborne Sommerset Thisted Radostowo Bartniki Chomotowo
Ireland Ireland Ireland Ireland England England Denmark Poland Poland Poland
068430 W, 538420 N 068180 W, 538050 N 068230 W, 538000 N 068150 W, 538100 N 058190 W, 508150 N 028550 W, 518100 N 088420 E, 568570 N 208370 E, 538580 N 208450 E, 548040 N 158110 E, 548020 N
5 40 10 10 45 55 10 10 10 10 100 100 50 50 10 10 14 14 32 32
6.1 3.8b 1.5 0.2a 175 96c 0.8 0.1a 0.8 0.2a 0.8 0.2a – – – –
4.7 0.7 b 1.1 0.4 a 105 15 c 2.9 0.4 a 0.7 0.2 a 0.7 0.2 a – – – –
6.9 2.2 b 0.6 0.2 a 50 24 c 1.1 0.1 a 1.2 0.4 a 1.2 0.4 a – – – –
0.8 0.1a 0.2 0.1a 24 2.0c 0.4 0.1a 0.8 0.1a 0.8 0.1a – – – –
The degree of statistical significance, probability P, as determined by one-way ANOVA and Tukey’s comparisons of means tests is shown. Different superscripted letters indicate significant differences between values in columns at P < 0.05; (–) indicates data not available).
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Table 1 Information on site, country, coordinates and mean zinc concentrations standard deviations of soil, porewater, roots and live leaves of G. fluitans
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the field were placed in plastic bags and transported back to the Thornfield Horticultural Unit Glasshouse Complex in University College Dublin, Ireland. Samples of soil, porewater and plants were also transferred to the laboratory and analyzed for zinc content. Soil samples were collected from soil around the plants. Porewater samples were taken to determine the total amount of zinc available to the plant. Ten dialysis vials, consisting of 20-mL plastic scintillation vials covered with 20-mm millipore mesh, were buried at each site at depths of between 5 and 10 cm (Doyle and Otte, 1997). These were left for 3 weeks in the soil to equilibrate, retrieved, filtered using 0.45-mm membrane filters and analyzed for zinc. The Danish and Polish plants were collected from areas with no known contamination with heavy metals (Hansen and Zukev, personal communications, 2002). 2.2. Chemical analysis of soil, porewater and plant samples Samples of soil and plants were dried in an oven at 60 8C until constant weight was obtained. Zinc concentrations were determined following digestion of 100 mg of dried homogenized soil or 50 mg of dried homogenized plant material in Teflon bombs containing 2 mL of HNO3:HCl in a (4:1) mix (Otte et al., 1995). The samples were cooled, diluted and filtered using 0.45-mm membrane filters. Acid digests of soil and plant samples were analyzed for zinc using a Unicam 929 flame atomic absorption spectrometer. Porewater samples were acidified with HNO3 before analysis. Plant standard reference samples supplied by Glen Spectra Reference Materials, England, of oriental tobacco leaves (CTA–OTL–1) were also analyzed for zinc content and deviated on average by 2% from certified values for zinc. 2.3. DNA extraction DNA was extracted using a method based on that of Pich and Schubert (1993), using approximately 0.2 g of leaf material. Extracted DNA was ethanol precipitated and resuspended in 200 mL of sterile distilled water. DNA was re-precipitated by adding 100 mL of 7.5 mol L1 ammonium acetate and 600 mL of 100% ethanol. DNA was then pelleted at 10,000 g for 10 min, and the pellet washed five times in 300 mL of 70% ethanol. The dried pellet was resuspended in 200 mL of TE at pH 7.5. DNA quality and quantity was estimated by separating 5 mL aliquots on 0.8% agarose TBE gels. Only samples that showed non-degraded DNA were used in subsequent AFLP analysis. The DNA amounts were estimated by comparison with known molecular weight standards. 2.4. AFLP protocol The AFPL protocol was carried out as described by Liscum and Oeller (1995). 5 mL (500–600 ng) of DNA was digested with five units of EcoR1 and five units of Mse1 for 3 h at 37 8C. The digestion was carried out in 1 Pharmacia ‘‘One-PHOR-All+’’ buffer containing 50 ng mL1 BSA. EcoR1 and Mse1 adapters were ligated to the digestion DNA at 37 8C. The adapted ligated DNA was pre-amplified in a reaction containing 0.5 mL of both EcoR1-oligo 1 (50 -CTCGTAGACTGCGTACCAATTC-30 ) and Mse1-oligo 1
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(50 -GACGATGAGTCCTGAGTAA-30 ) (50 ng mL1) to 1 mL of ligated DNA, along with 2 mL of 5 mmol L1 dNTPs, 2 mL of Promega Taq buffer, 12 mL of 25 mmol L1 MgCl2, 0.08 mL of 0.4 U Taq Polymerase and 12.72 mL of sterile distilled water. This mixture was then amplified using a PCR thermocyclic profile: (1) 94 8C for 2 min; (2) 94 8C for 30 s; (3) 50 8C for 30 s; (4) 72 8C for 1 min; steps 2–4 repeated 34 times. The amplification product was then diluted by 10 times in TE (10 mmol L1 Tris–HCl, 0.1 mmol L1 EDTA). A selected PCR reaction was performed in which 1 mL of this amplified diluted mixture was added to 0.25 mL of 33P-labelled EcoR1 primer, 0.3 mL of Mse1 primer (six different combinations:s EcoR1–AAG and Mse1–CAC, EcoR1–AAG and Mse1–CAC, EcoR1– AAG and Mse1–CTC, EcoR1–ACC and Mse1–CAC, EcoR1–ACC and Mse1–CAC, EcoR1–ACC and Mse1–CTC), 1 mL of 5 mmol L1 dNTPs, 1 mL of 10 Promega Taq buffer, 0.6 mL of 25 mmol L1 MgCl2, 0.04 mL of 0.4 U Taq Polymerase and 6.31 mL of sterile distilled water. The PCR reaction was performed with the following profile: (1) 94 8C for 2 min; (2) 94 8C for 30 s; (3) 65 8C for 30 s; (4) 72 8C for 1 min; (5) steps 1–4 repeated 11 times; (6) 94 8C for 30 s; (7) 56 8C for 30 s; (8) 72 8C for 1 min; (9) steps 6–8 repeated 25 times; (10) 72 8C for 2 min. The 33P-labelled products were then separated by electrophoresis on a 6% polyacrymide gel in 1 TBE buffer at 50 W for about 3.5 h. The gels were vacuum-dried with a gel dryer (model 583, Bio-Rad) for 55 min at 80 8C. Then the gels were exposed to Kodak Scientific Imaging Film (X-OMATTM) for 48–76 h, depending on the amount of radiation, which was determined with a Geiger–Mu¨ ller counter and developed. 2.5. Data analysis The resulting autoradiograms were scanned using a HP ScanJet 6100C highresolution scanner. The resulting images were saved as TIFF files, and were processed using GelCompare II (Version 3.0) software (Applied Maths, Sint-Martens-Latem, Belgium). The automatic band searching facility was used. Any band that represented more than 1% of the total surface under the densitometric curve was scored. The scoring of the bands was then edited manually. Bands smaller than 100 bp were eliminated, as they could not be scored consistently. Bands on the gel were scored as present (1) or absent (0). Gels were normalized by the alignment of 25 bp molecular weight markers (Promega), which were included at regular intervals in each gel. This was to ensure that all bands were uniformly distributed on the gels. The data were transformed into band frequencies and diversity values based on phenotype frequency (phenotypes being the band patterns produced by individual primer pairs). A matrix of genetic distances between populations based on the number of shared amplification products was calculated using the metric of Nei and Li (1979). These data were then used to calculate a dendrogram by agglomerative clustering using the unweighed pair group method with average linkage (UPGMA) (Sneath and Sokal, 1973). The dendrogram was calculated using a composite of all six-primer combinations so that the contribution of each primer pair was equal, irrespective of the number of bands that had been generated by each primer combination. The support values for the degree of confidence at the nodes of the dendrogram were evaluated by bootstrap analysis (Felsenstein, 1985). One thousand bootstrap datasets were calculated.
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3. Results 3.1. Zinc in soil, porewater and plants from the field The populations analyzed in this study were collected from sites with low and high zinc levels in Ireland and England (Table 1). The Camborne site is in a known copper mining area and so soils were analyzed for copper: an average soil concentration of 2.8 mmol g1 of copper was found. No original soil or plant material was available for analysis from Denmark and Poland, but seeds were collected from areas with no known contamination with metals (Hansen and Zukev, personal communications, 2002). The Glendalough site had significantly higher concentrations of zinc in the soil, porewater and plant parts than the other two contaminated sites at Navan and Camborne. The concentration of zinc at the contaminated sites was higher than the concentration of zinc at the non-contaminated sites at Djouce and Lough Dan. The soil, porewater and plant parts from Djouce and Lough Dan contained the least amount of zinc from the five sites analyzed. 3.2. AFLP polymorphism Fifty-three individuals from the 10 locations were assayed using six selective primer combinations. In total, 796 bands (mean 132 bands per primer combination) were generated (Table 2) across all the G. fluitans populations. The number of monomorphic generated bands was low, with many bands being polymorphic. In total, 670 polymoprhic bands were scored, which gave a total of 84% polymorphism. Primer combination 5 generated the greatest number of bands (172) and primer combination 2 revealed the highest levels of polymorphism (93%). The genetic distance matrix (Nei and Li, 1979) was used to establish the level of genetic divergence between the populations. 3.3. Cluster analysis Cluster analysis divided the 53 populations into two large groups (Fig. 1). Cluster A contained the three Polish populations, the populations from Sommerset (England) and Navan (Ireland). Cluster B contained the remaining Irish populations (Glendalough, Lough Dan and Djouce), and the populations from Thisted (Denmark) and Camborne (England). The cluster analysis shows that populations from any given location are more closely Table 2 AFLPs generated among 65 individuals of G. fluitans representing 10 populations across Europe Primer pair
Polymorphic bands
Total bands
Polymorphism rate (%)
1 2 3 4 5 6
110 109 88 125 129 109
137 117 96 136 172 139
80 93 91 91 75 78
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Fig. 1. UPGMA dendrogram of the different individuals of 10 different G. fluitans populations.
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related to themselves than to populations from other locations. In each case, populations from a particular location form distinct clusters (Fig. 1). There were two exceptions, the first is an individual from Radostowo that is located at the top of cluster A and secondly, an individual from Lough Dan, which is grouped with the Glendalough population. This indicates that there was greater variation between populations from different locations than within the populations from a single location. Clusters A and B were different in their patterns of similarity between populations, with Group A exhibiting a higher level of genetic diversity. The populations of cluster A were 59% similar, and individuals within the populations were from 62% to 85% similar. The populations of cluster B were 62% similar and individuals within the population were from 67% to 90% similar. There was a tendency for plants from the same location to cluster together with little overlap between populations from different locations. The dendrogram constructed with all individuals was generally supported by high bootstrap values, but the two large clusters have different degrees of support. For cluster A, 16 out of 26 bootstrap values were above 50%, suggesting a limited reliability of the pattern found compared with cluster B, which exhibited high bootstrap values, 25 out of 26 values were above 50%. The individual populations within cluster A were supported by high bootstrap value of 91 on the first node. The populations within cluster B were supported by high bootstrap value of 100 on the first node.
4. Discussion Cluster analysis of the AFLP profiles divided the 53 individuals into two groups. Populations did not segregate with respect to the metal status of their sites, as the populations from metal-contaminated sites (Glendalough, Camborne and Navan) did not cluster together. Cluster A contained populations collected from across a large geographic range, whereas populations in cluster B were mainly restricted to Ireland and England. The highest level of genetic diversity was found in the Radostowo group, followed by the other groups from cluster A. G. fluitans plants from the same location tended to cluster together, with minimal overlap between populations. This trend is in agreement with the findings for Lolium multiflorum (Cresswell et al., 2001), Moringa oleifera (Muluvi et al., 1999) and Sticherus flabellatus (Keiper and McConchie, 2000). The partitioning of genetic diversity between populations rather than within populations can be attributed to genetic drift, inbreeding and the homogenizing effects of restricted gene flow (Loveless and Hamrick, 1984). Most of the plants were collected from small sites. Plants collected from isolated sites can display lower diversity, as was demonstrated for Coreopsis integrifolia (Cosner and Crawford, 1994). In contrast, much higher degrees of diversity are observed in populations that cover large geographic areas (Frankham, 1996; Frankham and Ralls, 1998). The plants from Glendalough in cluster B display high similarity between individuals. This could be because currently this site is more isolated than the other sites. The Glendalough site is a tailings pond from a small lead/zinc mine abandoned in the 1950s. The populations from Djouce, Camborne and Lough Dan were also collected from relatively small sites, but within each group do not display as high a degree of similarity as the Glendalough
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population. However, there is some overlap between the Glendalough and Lough Dan populations, signifying some genetic similarity. These populations are geographically close together, at a distance of approximately 20 km apart. Genetic similarity of plants from these two sites may be accounted for by clonal reproduction. G. fluitans can reproduce by clonal means, as detached shoots may be washed away and readily re-root in new localities (Preston and Croft, 1997). This may have happened in Wicklow in the recent past and could account for the overlapping and genetic similarities between these two populations. The findings indicated that in some cases genetic distances was dependent on geographical distance, for example, the close genetic relationship between the Glendalough and Lough Dan populations. However, other populations did not cluster together based on geographical proximity. For example, the Sommerset and Camborne populations in England are only about 160 km apart, but are in separate clusters. This lack of geographical grouping has been found in other species, such as Calycophyllum spruceannum (Russell et al., 1999). The Camborne population is genetically closer to Irish populations, and the Sommerset population is genetically closer to the Polish populations. A possible reason for the unexpected similarities between geographically distant populations may be the role of migratory birds, which can spread seeds across wide distances and account for genetic similarities between distant populations (Stankiewicz et al., 2001). The populations from sites with a history of metal exposure (Glendalough, Navan and Camborne) were not genetically similar to each other. Thus, genetic similarity appears not to be related to the metal status of the sites. It is possible that the drive to generate distinct metal-tolerant populations may not occur in G. fluitans due to the existence of a constitutive metal tolerance. However, there are significant differences in the levels of zinc in these three sites. The Glendalough site has zinc levels that are 175-fold above levels from non-contaminated sites, while the other two sites have much lower levels of zinc. The manner in which the different populations clustered is also not related to the manner in which they partition zinc. The Glendalough and Camborne populations had been shown to sequester more zinc in their roots than their dead leaves, while the Thisted, Navan and Radostowo populations sequestered more zinc in their dead leaves than their roots (Matthews et al., 2004a). The Glendalough and Camborne populations do not form a group in cluster B, while the Thisted, Navan and Radostowo geneotypes are dispersed throughout both clusters A and B. Innate metal tolerance occurs in some other wetland plants, including T. latifolia, P. australis, E. angustifolium and C. rostrata (Ye et al., 1997a,b; Matthews et al., 2004b, 2005). Based on the findings presented here, it is possible that the drive to generate distinct metal-tolerant populations in other wetland plant species may not occur due to the existence of innate tolerance, as genetic similarity appears not to be related to the contamination status of sites. In a separate study, using inter-simple sequence repeat (ISSR) amplification, populations of the wetland species E. angustifolium from contaminated and non-contaminated locations from around Ireland did not segregate with respect to the metal status of their sites (Wilson, personal communication, 2004). This species has also been found to possess innate tolerance (Matthews et al., 2004b). It is possible that innate metal tolerance is a general feature of wetland plants, and consequently these plants do not evolve distinct metal-tolerant populations.
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Acknowledgements This work was funded by Enterprise Ireland (Project No: SC/01/361). We thank the staff of Wicklow Mountains National Park and Outokumpu-Zinc Tara Mines Ltd. for allowing access to the tailings ponds in Glendalough and Navan. Special thanks to Dr. Loveday Jenkin from the Camborne School of Mining, England, Dr. Hans Vilhelm Hansen of the Botanic Gardens, University of Copenhagen, Denmark and Dr. Grzegorz Zurek of the Bydgoszcz Botanic Garden of Plant Breeding and Acclimatization, Poland for help in obtaining the populations from England, Denmark and Poland.
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