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Ultramafic (serpentine) soils are adverse habitats for plants. The serpentine soils are characterized by low concentrations of the plant nutrients N, P, K and Ca and high, potentially toxic, concentrations of Mg and Ni (Brooks, 1987; Proctor, 1999). In addition, they are dry and exposed because of lack of organic material and their granular texture. Accordingly, the reason for the infertility of serpentine soils is a multifactorial phenomenon, sometimes referred to as the serpentine syndrome (Jenny, 1980; Kruckeberg, 1984). Although a range of both chemical and physical factors may influence plant growth in serpentine soils, high concentrations of Mg in relation to Ca, and elevated concentrations of Ni are considered to be important factors affecting plant growth and survival (Proctor, 1971; Marrs & Proctor, 1976; Johnston & Proctor, 1981; Gabbrielli & Pandolfini, 1984; Proctor & Nagy, 1992; Nagy & Proctor, 1997). High concentrations of Mg in relation to Ca may lead to diminished uptake of Ca. In addition, high concentration of Mg per se may have a toxic effect on plant growth (Proctor, 1970). Although Ni is an essential element, the required amounts are minor (Taiz & Zeiger, 1998). Elevated concentrations of Ni can per se effectively inhibit cell division at root meristems in non-tolerant plants (Robertson, 1985). In addition, Ni has a negative effect on photosynthesis, respiration and stomatal regulation of transpiration (Carlson et al., 1975). The role of Ni as a factor explaining infertility of serpentine soils is, however, unclear and contrasting results have been reported on its effect in combination with Mg (Johnston & Proctor, 1981; Gabbrielli & Pandolfini, 1984; Proctor & Baker, 1994). The plant available concentration of Ni also differs among serpentine soils (Proctor, 1971).
Some plants have the genetic resources to grow in serpentine soils and a distinctive flora has evolved on these soils (Rune, 1953; Brooks, 1987; Baker et al., 1992). In Scandinavia, the serpentine flora is characterized by several alpine plants of the family Caryophyllaceae (Rune, 1953; Rune & Westerbergh, 1992). One of the most common of these is a small perennial herb, Cerastium alpinum. This plant grows in serpentine soils in the Scandinavian Mountains, as well as in the boreal region in Finland. Outside serpentine soils, C. alpinum grows on open gravel rich land, alpine heaths, steep slopes and riverbanks (Hultén, 1956; Jonsell, 2001). C. alpinum is polyploid (2n = 9x = 72) and highly variable in morphology. It constitutes a complex with several subspecies and varieties (Hultén, 1956). These taxa have been distinguished on the basis of difference in hairiness, leaf morphology and the presence of subterranean shoots (Rune, 1953; Jonsell, 2001). C. alpinum is hermaphroditic and disperses with seeds formed through selfing or cross-fertilization, or vegetatively through runners.
Analyses of enzyme phenotypes by Nyberg Berglund & Westerbergh (2001) and Nyberg Berglund et al. (2001) suggest that C. alpinum has colonized Scandinavia through two independent immigration events resulting in an eastern and western lineage. These two genetic lineages seem to come into contact in a hybrid zone in northern Scandinavia. The C. alpinum populations on and off serpentine present an excellent opportunity to study the mode of serpentine tolerance in different genetic backgrounds.
Plant species have different abilities to evolve serpentine tolerant populations depending on their genetic resources. If all individuals of the two immigrating lineages of C. alpinum had the genetic prerequisites to colonize serpentine, serpentine and non-serpentine populations would respond to serpentine stress in a similar way. Alternatively, the immigrating populations may have had genetic variability in tolerance so that tolerant individuals would have been selected upon the colonization of each serpentine site. We have studied the response to serpentine stress in serpentine and non-serpentine populations in the two immigrating lineages, as well as in the presumed hybrid zone. Among the factors affecting plant growth in serpentine soils we have focused on Ni and Mg and investigated the individual and combined effects of these metals in nutrient solution culture experiments. The response to metal stress for a population is discussed in relation to the composition of the soil at each site. We address the following questions: Is there evidence for local adaptation in C. alpinum? If so, do the serpentine populations differ in the response to metal stress? Has the tolerance to Ni and Mg arisen in parallel in the two independent immigrating lineages?
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Plant adaptation to serpentine stress may occur only in those populations experiencing this stress or may be widespread among populations within a species (Antonovics et al., 1971; Wu, 1990). Whether or not the ability to tolerate serpentine stress is restricted to populations that grow on serpentine may have implications for questions dealing with the adaptation and evolution of stress tolerance, and its genetic control. The two lineages of C. alpinum in Scandinavia provide an excellent opportunity to study the mode of serpentine tolerance in different genetic backgrounds.
We have found significant differences between serpentine and non-serpentine populations in tolerance to Ni and Mg, two important factors for the infertility on serpentine. Plants from serpentine populations showed, in general, a higher tolerance against both Ni and Mg than plants from adjacent non-serpentine populations. This suggests that serpentine tolerance is an adaptive rather than a constitutive trait in C. alpinum. Previous enzymatic studies (Nyberg Berglund & Westerbergh, 2001) have shown that serpentine populations of C. alpinum are genetically more similar to non-serpentine populations within the same geographic region than with distant serpentine populations. This suggests that serpentine tolerant populations have repeatedly evolved within the species. Our findings in the present study of a higher tolerance to elevated concentrations of Ni and Mg in serpentine populations strengthen the evidence for multiple origins of serpentine populations. The serpentine populations differed, however, in metal response.
The serpentine population 1 S in the west and 5 S in the east showed similar responses to Ni and Mg stress, while 3 S in the hybrid zone differed in response. Population 3 S showed the highest tolerance to Ni, whereas the growth was strongly reduced in the other two serpentine populations (Fig. 3). Plants from 3 S responded negatively to Mg, whereas Mg did not have a negative effect on the root growth of plants from 1 S and 5 S compared to the control. The negative effect of Ni found in 1 S and 5 S was reduced in the presence of Mg, whereas Mg enhanced the negative effect of Ni in 3 S (Figs 4, 7b,c).
Why do the serpentine populations show different degrees of tolerance to Ni and Mg stress? The answer can be found in the different compositions of the serpentine soils, so that a higher degree of tolerance to Ni and Mg has evolved in C. alpinum populations from soils that contain higher effective concentrations of these metals. The effective concentration of a metal is a result of interactions between several factors. For example, pH has a crucial role in the concentration of Ni in the soil solution. In well-drained soils with pH higher than 6.0 Ni is unlikely to reach toxic concentrations (Jenne, 1968). The toxic effect of Mg is enhanced with relatively low concentration of Ca. The toxicity of Mg depends also on the actual concentrations of both elements (Proctor, 1971).
According to the known interactions among soil factors, we may describe the plant available Ni and Mg concentrations in the three serpentine soils in the following way: although the Ni concentration in the soil samples of 5 S is twice the concentration in the soil of 3 S the highest plant available concentration of Ni is most likely to be found in population 3 S. First, pH is > 1 unit lower in 3 S than in the other two serpentine populations. This will increase the solubility of the Ni present in the soil particles. Second, the average total cation concentration is only approx. two thirds of those in 1 S and 5 S, which may also increase the relative activity of Ni in the soil solution in 3 S. The relatively high pH in 1 S and 5 S reduces the toxic effect of Ni so that the plants are not adapted to severe Ni stress. According to the high Mg concentrations found in the soil of 1 S and 5 S these populations are exposed to severe Mg stress (Table 1). The Mg toxicity is less severe in 3 S because of the lower Mg concentrations per se and higher Ca concentrations, ameliorating the toxic effect of Mg. In the growth experiments, plants from 1 S and 5 S grew as well in solutions with elevated concentrations of Mg as in the control. However, plants from 3 S were clearly inhibited by the high Mg concentration. Correlation between metal tolerance in a population and the metal concentration of the soil has been shown in several classical studies on the grass Agrostis tenuis (e.g. McNeilly & Bradshaw, 1968).
The fact that Mg ameliorates the effect of Ni in two of the serpentine populations (1 S and 5 S), where the Ni tolerance is rather low, but not in the serpentine population in the hybrid zone (3 S), where the Ni tolerance is much higher, suggests that these C. alpinum populations achieve their tolerance to serpentine somewhat differently. Magnesium has been reported to enhance the toxicity of Ni in the wild grass, Festuca rubra (Johnston & Proctor, 1981), contrary to this, it has been shown to ameliorate the toxic effect of Ni in oat (Avena sativa; Proctor & MacGowan, 1976) and Alyssum bertolonii (Brassicaceae; Gabbrielli & Pandolfini, 1984).
As the difference in effective concentration of Ni and Mg in the serpentine soils is reflected in the different degrees of tolerance in the populations there seems to be a difference in intensities of selection among serpentine sites. However, the serpentine populations 1 S and 5 S in the two independent immigrating lineages grow in soils with similar intensities of selection and showed similar response to Ni and Mg stress. This suggests that Ni and Mg tolerance have evolved in parallel in the two genetic lineages of C. alpinum during the postglacial colonization of Scandinavia. We therefore propose that C. alpinum is a case for parallel evolution of multiple adaptive traits. Parallel evolution of metal tolerance has been suggested for Silene vulgaris (Caryophyllaceae; Schat et al., 1996) and parallel evolution of other physiological traits have recently been shown in Lasthenia californica (Asteraceae; Rajakaruna et al., 2003).
Repeated evolution of metal tolerance has occurred in several grasses such as Agrostis tenuis (Nichols & McNeilly, 1982) and A. capillaris (Al-Hiyaly et al., 1988) and in dicots such as Mimulus guttatus (Macnair, 1983) and Silene vulgaris (Schat et al., 1996). These examples suggest that the evolution of ecotypes from generalist-to-specialist is a common pathway. Plant species that are found in metalliferious soils possess, in general, low frequencies of tolerant individuals in non-metalliferious populations (Gartside & McNeilly, 1974; Ingram, 1988). In some other cases the tolerance has been shown to be a constitutive trait. No significant difference in Ni tolerance between populations on and off serpentine has been reported in a couple of Caryophyllaceae plants, Silene dioica in Sweden (Westerbergh, 1994) and Cerastium fontanum in Scotland (Nagy & Proctor, 1997). In addition, the Ni hyperaccumulator Thlaspi montanum var. montanum (Brassicaceae) was shown to be constitutively adapted (Boyd & Martens, 1998).
Studies of serpentine tolerance have often involved measurements of tolerance as a response on total root mass or growth of the longest root. However, the elongation of main roots, initiation and extension of lateral roots reacts differently to various environmental conditions (Waisel & Eshel, 2002). In the present study, C. alpinum plants from non-serpentine populations produced significantly more lateral roots than Mg tolerant serpentine plants in Mg stress. The higher production of lateral roots in non-serpentine plants could be a secondary effect when the growth of the longest root is inhibited. Alternatively, the serpentine and non-serpentine populations have evolved different growth strategies. We found evidence supporting the latter hypothesis. First, negative correlation between the growth of the longest root and the production of lateral roots as expected for non-serpentine plants under the secondary effect hypothesis was not found (data not shown). Second, the non-serpentine population 4 N-S, which grows in a soil with almost twice the Mg concentration of the two other non-serpentine sites (Table 1), produced less lateral roots under Mg stress than 2 N-S and 6 N-S even though the non-serpentine populations showed similar growth of the longest root (Fig. 3). Non-serpentine plants may have evolved a growth strategy where an increased formation of lateral roots is a result of a higher concurrence and limited resources (e.g. Mg) on non-serpentine soils. The serpentine populations may have evolved a strategy, where the formation of lateral roots is down regulated in high concentrations of Mg and more resources are allocated to longer and deep growing roots, which are important in dry serpentine soils.
Serpentine tolerance is easily evolved in Cerastium. In Europe, at least 15 Cerastium species have evolved serpentine populations (Brooks, 1987). This suggests that the ability to evolve serpentine tolerance has occurred in the early stages of the evolution of Cerastium and in the Caryophyllaceae, in general, as this phenomenon is found in several related genera in the plant family such as Silene, Lychnis and Arenaria (Rune, 1953; Rune & Westerbergh, 1992). What makes Cerastium plants such successful colonizers of serpentine? Besides Ni and Mg, drought tolerance is considered to be a major determinant for serpentine adaptation and evolution. In fact, drought tolerance is suggested to be the main driving force for serpentine tolerance in Mimulus (Gardner & Macnair, 2000; Hughes et al., 2001). Tolerance to drought is a common feature in the genus Cerastium as many species grow in dry and gravel rich soils in Europe (Jonsell, 2001). As serpentine tolerance is likely to depend on an interaction of many genes it is of great importance for the survival of the plant population to maintain blocks of adapted genes from one generation to another. The fact that the majority of the Cerastium species can spread vegetatively and produce seeds through selfing could be an additional explanation for the successful colonization of serpentine. All of the serpentine Cerastium species are polyploids with chromosome numbers ranging from 2n = 36 to 2n = 144 (Jalas & Suominen, 1988). Like most polyploids they are likely to have evolved through interspecific hybridization (allopolyploidization). For several alpine polyploid species such as Draba (Brassicaceae), repeated polyploidization events have been shown to occur within relatively small regions and short time scales (Brochmann et al., 1992). Possibly, the genetic effects of allopolyploidization as well as repeated polyploidization events within a species have increased the number of available pathways for the evolution of serpentine tolerance.