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Department of Integrative Biology, University of California, Berkeley, California 94720-3140
Received for publication March 17, 1998. Accepted for publication February 1, 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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13C) ranged from -28 to -22
and became less negative at higher salinities, indicating an improved water use efficiency in the seedlings at high salt concentrations. In addition, 13C values were generally less negative at high than at low nutrients. Differences among taxa were generally small. The results show that salinity affects both establishment and growth of hybridizing Carpobrotus. The overall weak species differences in salt tolerance indicate that the exotic C. edulis can occupy the same sites as C. chilensis in terms of salinity. The similarity of hybrids in their response to salinity suggests that they may contribute to the invasion by Carpobrotus.
Key Words: Aizoaceae • Carpobrotus; • germination • invasion • salinity tolerance • water use efficiency
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Vince and Snow, 1984
). Coastal substrate salinity can vary considerably, ranging from 0.1 to 3% (Barbour, de Jong, and Pavlik, 1985
), and depends on the time of year and distance from the sea. Plant species of coastal environments are mostly halophytes (Ungar, 1991
), adapted to cope with saline environments. The growth of many halophytes is stimulated by some levels of salinity, but at higher levels salinity causes stress, reducing growth and fitness (Waisel, 1972
; Wainwright, 1980
; Goldstein et al., 1996
). In addition, nitrogen and phosphorus availability can be important factors determining plant growth in saline environments. Nitrogen uptake is often reduced in the presence of salts (Jefferies, 1977
; Loveland and Ungar, 1983
; Drake and Ungar, 1989
), and studies have demonstrated that fertilization with nitrogen increased biomass production and water uptake in the case of salt marsh plants (Ungar, 1991
).
Coastal environments are often invaded by non-native (exotic) plant species (Drake et al., 1989
). The spread of exotic plant species into coastal plant communities depends, besides other factors, on the salinity tolerance of the invading species. Physiological and morphological plasticity as an adaptation to saline environments may determine the extent of establishment and distribution along the salt gradient. Since the colonization of a new area by an exotic species depends on recruitment, the most crucial stages for establishment are germination and subsequent seedling growth. Studies have shown that salinity tolerance varies at different developmental stages of a plant, seedlings being often the most sensitive one (Ungar, 1991
; Shumway and Bertness, 1992
).
The rate of spread of an exotic species and its ecological impact may be altered if hybridization between the exotic species and a congener occurs. In such a situation, differences in salinity tolerance of the species involved and the relative performance of hybrids determine whether the ecological niche of the exotic invader can be enlarged. The salt tolerance of hybrids relative to parental species may determine the extent to which hybrids can invade the sites occupied by parental species. Also, hybridization may increase phenotypic plasticity in the direction of increased salt tolerance and may thus lead to a greater invasiveness of hybrids.
The purpose of this study was to assess morphological and physiological responses of seeds and seedlings of hybridizing Carpobrotus species to soil salinity. Carpobrotus edulis, a succulent perennial introduced from South Africa, is an aggressive invader of coastal plant communities throughout California (Zedler and Scheid, 1988
; D'Antonio, 1990
; D'Antonio, Dennis, and Tyler, 1993
). Carpobrotus edulis has a wide ecological amplitude, occurring in coastal habitats but also in nonsaline environments such as road edges, grass slopes, and forest edges. Carpobrotus chilensis is of smaller habit and distinguished from C. edulis mainly by smaller leaves and flowers. The species is much less abundant than C. edulis and confined to coastal habitats. It grows in sites directly exposed to salt spray and storm waves, e.g, coastal bluffs and rocks near the sea, but can also be found in backdunes. The origin of this species is not known, but it has been present in California since at least the early 1600s (Bicknell and Mackey, 1988
). Both species produce fleshy fruits with many small seeds and are obligately animal dispersed. Once established, Carpobrotus spreads mainly by means of clonal growth.
Previous studies have shown that hybrid-appearing phenotypes are found in many sites along the coast, leading to a complete phenotypic range between the two species (Albert, D'Antonio, and Schierenbeck, 1997
). Morphological and genetic studies have confirmed the presence of introgressive hybridization between C. edulis and C. chilensis (Albert, D'Antonio, and Schierenbeck, 1997
; Gallagher, Schierenbeck, and D'Antonio, 1997
). Analysis of isozyme markers has shown that introgression is occurring mainly in the direction of C. edulis (Gallagher, Schierenbeck, and D'Antonio, 1997
). Hybrids can be fully fertile and reach high local abundances. For the purpose of this study, we compared the two parental species and intermediate plants ("hybrids").
In this study, we asked the following questions: (1) To what extent are seeds and seedlings produced by C. edulis, C. chilensis, and hybrids different in their responses to salt? (2) How are these differences affected by nutrient availability? (3) How do hybrid offspring differ from parental species in salt tolerance?
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Leaf length and shape are important characters for distinguishing species and hybrids in Carpobrotus because of strong species differences and low phenotypic plasticity of these characters (Weber and D'Antonio, 1997
). Seeds were removed from the fruits and stored at room temperature. A random sample of seeds of each taxon was used for the experiments. No attempts were made to identify and select seed genotypes prior to experiments. Previous studies have shown that both species are selfing and that seedlings from the parental species resemble them in leaf shape and size. Thus, we assume that seeds from the three taxa contain mainly genotypes from either of the species and hybrids, respectively.
Germination
Germination tests for each taxon were carried out at 0, 10, 20, and 50% seawater. We used artificial seawater with a 3% salt content (Bio Sea, San Carlos, California). Two experiments were carried out. For experiment A, forty seeds of each taxon were placed on Whatman's Grade 182 filter paper in 50 mm diameter petri dishes on 11 February 1996. Each dish was watered with 5 mL of test solution. Each salt treatment was replicated ten times, and the dishes were randomly arranged on a bench in a glasshouse without artificial light. All dishes were kept inside covered plastic trays and misted regularly with tap water to reduce evaporation. Seeds were considered as germinated when both cotyledons were visible. Germination was scored for a total of 15 wk. In this experiment, we investigated the pattern of germination in the presence of salt.
For experiment B, seeds that did not germinate in experiment A were transferred to new dishes with fresh water only, in order to test for germination recovery after removal of salt stress. Germination was scored for an additional 3 wk.
Growth and physiology of seedlings
Seeds were germinated on moist vermiculite. Seedlings were transferred to 8 cm diameter pots when the first leaves were emerging, at a size of 
10 mm. A mixture of one part sand and one part soil (U. C. Davis mix) was used as substrate. Plants of each of the three taxa (parental species and hybrids) were randomly allocated to eight treatments, resulting from the combination of four levels of substrate salinity (irrigation with 0, 10, 20, and 50% seawater) and two levels of nutrient availability (fertilized and not fertilized). We used a 0.5% solution of a commercial 20:20:20 NPK fertilizer and added 20 mL to each pot of the high-nutrient treatment once per month (equal to 20 mg N per plant and month). Low-nutrient pots received the same amount of water but no fertilizer. In total, 432 plants were used, 18 replicates for each salinity treatment and taxon.
Pots were placed in trays randomly arranged on benches outside, and trays were re-arranged twice a month. All plants were watered with seawater and tap water, respectively. To avoid increase in salt concentration in the soil, all plants were watered with tap water 1 wk after irrigation with seawater to simulate rain. Seawater and tap water were given in excess, and pots were allowed to drain.
All plants were harvested in November 1996 and the following measurements were taken: aboveground biomass, fresh mass and dry mass of the largest leaf, and length of the same leaf. Leaf water content was calculated as the ratio:
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13C), dried leaves were finely ground and 3–4 mg samples were analyzed on a Europa (Franklin, Ohio) mass spectrometer by the stable isotope laboratory at U. C. Berkeley. Stable carbon isotope composition was expressed as the 13C/12C ratio relative to that of peach leaves standard (NIST number 1547, U.S. Department of Commerce).
Data analyses
Germination was expressed as percentage germinated seeds (germination rate) and germination rate relative to control, e.g., relative to germination rate at 0% seawater (percentage of control). Both measures were determined for experiment A (germination in presence of salt) and for experiment B (germination recovery). A two-way analysis of variance (ANOVA) with taxon (fixed effect) and salt concentration (random effect) was performed on germination rate to test for differences among taxa and effects of seawater.
Growth data were analyzed by a mixed-model analysis of variance (ANOVA) for each character. Nutrient treatment and taxon were considered as fixed effects and seawater concentration as a random effect. A significant seawater effect indicates plasticity, whereas a significant taxon effect indicates differences among taxa. Significant interactions suggest that taxa differ in their responses. If appropriate, data were log transformed to achieve normal distribution and homogeneity of variances. Differences among taxa within environments were tested by one-way ANOVAs. For all analyses, we used general linear models (procedure GLM; SAS, 1990
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In addition to these general patterns in germination behavior, we found significant differences among taxa as well as significant interactions with seawater in both germination rate after salt exposure and final germination rate (P < 0.001 for all effects). Parental species and hybrids differed in the magnitude of germination rates: Carpobrotus chilensis had the highest percentage germination both after salt exposure and after transfer to fresh water (Fig. 1). Germination rates of hybrids were intermediate. (Fig. 2). However, if germination is expressed as percentage of control, hybrids had higher relative germination rates at moderate seawater concentrations, whereas C. chilensis and C. edulis were similar (Fig. 2). Thus, the effect of seawater on germination was less pronounced in the case of hybrids compared to parental species.
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13C values were generally less negative at high than at low nutrients. The shift in 13C from 0 to 50% seawater was 1.92 at low and 3.38 at high nutrients, respectively (averaged over all three taxa). Water content of leaves increased with increasing salinity, and differences among taxa were apparent at high salinities (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) after transferring to fresh water, e.g., seeds that were exposed to salt had a higher final germination rate than control. The inhibition of germination by salt is thought to be an osmotically enforced dormancy (Ungar, 1978
), preventing the establishment of seedlings under unfavorable conditions. The inhibition was rather strong in our study: half-strength seawater caused a nearly 100% inhibition in Carpobrotus, whereas seeds of other halophytic species can have higher salt tolerances (Ungar, 1991
). However, germination responses of halophytes are variable and species specific (Ungar, 1982, 1991;
Woodell, 1985
). Carpobrotus edulis grows primarily in grassland and dunes, but can occasionally be found in wet places near the coast. It has been suggested that salt-induced dormancy and the ability to germinate better after immersion in seawater is an advantage for rapid establishment (Woodell, 1985
). Carpobrotus fruits ripen in late summer and seeds get exposed to soil in fall. They germinate during winter rains, thus salt stimulation might promote establishment in Carpobrotus.
Varying germination responses among a set of species in saline habitats can determine which species will successfully colonize a new area. We found differences in germination behavior among the two species and hybrids in several aspects. Carpobrotus chilensis is distinct from C. edulis in the overall higher germination in fresh water and at low salinities, which suggests that salt does not induce dormancy as strongly as in C. edulis. Previous studies also have shown that C. chilensis germinated more easily in fresh water and under field conditions than C. edulis (Vilà and D'Antonio, 1998a
). The low germination of C. edulis may be compensated by a higher number of fruits and seeds per clone than C. chilensis (Vilà and D'Antonio, 1998a
). In addition, fruits of C. edulis and hybrids are removed more quickly by mammals than those of C. chilensis (Vilà and D'Antonio, 1998b
), and plants of C. edulis are more resistant to mammal herbivory than C. chilensis (Vilà and D'Antonio, 1998c
).
Hybrids were intermediate with respect to percentage germinated seeds. We do not know the identity of seed genotypes, but assume that hybrid offspring contains more hybrid genotypes than offspring of parental species. We found, however, that hybrids had higher germination relative to control, e.g., at low and moderate salinity, more seeds germinated than in the case of parental species. This means that the inhibitory effects of seawater on germination are not as strong in hybrids as in parental species and suggests that hybrids are more salt tolerant than the other taxa with respect to germination. In saline habitats, early stages of a plant's life cycle, e.g., germination and establishment, are thought to be important in determining which species will be present in an unoccupied area. Thus, any small differences in germination and seedling growth may translate into a pre-emption of available space. If seeds of hybrids do germinate faster and are likely to germinate better at low salinities than those of parental species, this may well be an advantage for hybrids to colonize new areas. To support this hypothesis, field studies are necessary.
Growth and physiological responses
Seedlings of the two species and hybrids showed varying responses in growth and physiology to increased levels of salinity. Overall, plants of all three taxa exhibited strong plasticities in the characters measured, and differences among taxa generally were low.
Despite ambiguous responses among the taxa, we found some clear patterns. Low and moderate salinity caused an enhancement in biomass production compared to control in all three taxa at low nutrients, but the relative increase in biomass was rather low. Salt-stimulated growth has been described for many halophytes (Ungar, 1991
). At high nutrients, no salt stimulation was apparent, although the plants at 20% seawater produced more biomass than control in the case of hybrids. The interaction between nutrient availability and salt in halophytes is not yet fully understood (Ungar, 1991
). Several authors have demonstrated that halophytes at high salinities grow better if sufficient nitrogen is supplied (Smart and Barko, 1980
; Drake and Ungar, 1989
). In our experiment, seedlings at low nutrients could have been limited by nitrogen and thus limited in photosynthesis.
The 13C values we obtained are in the range found for other members of the Aizoaceae. For example, Carpobrotus acinaciformis had a carbon isotope content ranging from -27.7 to -21.6 at natural sites (Ziegler, 1996
). The 13C values in our study plants ranged from about -28 to -22, becoming less negative at higher salinities. This certainly indicates an increase in water use efficiency, however, we do not know whether a shift to Crassulacean acid metabolism (CAM) occurred. Typical CAM plants have 13C values ranging from -29 to -11 (Ziegler, 1995
), and many halophytes have the ability to change to CAM under stress conditions. Mesembryanthemum crystallinum, a succulent annual, rapidly shifts from C3 to CAM carbon fixation as water stress increases (Bloom and Troughton, 1979
), and Schmitt and Piepenbrock (1992)
found that transient reductions in leaf water content leads to the expression of CAM. Winter and von Willert (1972)
could induce a shift to the CAM pathway in Carpobrotus edulis when the salinity level was raised to 400 mmol/L NaCl. Interestingly, we found a strong effect of nutrient levels on carbon isotope composition: the 13C values were less negative at high nutrients compared to low nutrients. Similar results were obtained for Crassula ovata (Ehleringer, Hall, and Farquhar, 1993
). In this species, the 13C discrimination was increased (13C values becoming more negative) with low nitrogen supply. In our study, plants grown in the high nutrient treatment produced more biomass than those in the low nutrient treatment even though they were exposed to a similar salinity level and had similar osmotic adjustments. However, different developmental stages of a plant can show different responses to salinity (Ungar, 1991
).
Differences among taxa
Parental species were undistinguishable in their physiologial adjustments, e.g., osmolarity and carbon isotope composition. Hybrids exhibited less negative carbon isotope values at low and moderate salinity, but only at low nutrients. Carpobrotus chilensis had less negative 13C values than C. edulis, as has been shown for cuttings from these species grown in a garden (Weber and D'Antonio, 1997
). At low nutrients, the two species were more different from each other than at high nutrients. Carpobrotus chilensis may thus cope better with more stressful environments, e.g., those that are nutrient poor and saline.
Hybrids were mostly intermediate in their growth responses. Exceptions were biomass and leaf length at low nutrients, where hybrids exceeded parental species at low salinity. At high nutrients, hybrids became intermediate. These results likely are the result of different ontogenic phases and allometric relationships: plants at low nutrients were much smaller and their leaves probably were not fully expanded. The findings confirm that different stages within a plant's life cycle can exhibit different plastic reactions.
Conclusions
The germination patterns suggest that seeds are stimulated to germinate if inundated with seawater and washed by rain afterwards. Hybrids were more strongly stimulated than both parental species, which may indicate that hybrids have a higher probability of establishing seedlings. It is clear that field studies are necessary to elucidate survival in different microhabitats.
Our results from the growth experiment suggest that Carpobrotus chilensis, C. edulis, and hybrids are very similar in their ability to adjust to saline environments. Two conclusions for the invasion process can be drawn: (1) hybrids can colonize the same sites as parental species along a salt gradient, (2) the absence of profound species differences in salt tolerance may explain why both species, together with hybrids, occur intermixed without recognizable zonation. Hybrids may, however, have an advantage over parental species in nutrient-poor soils.
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FOOTNOTES |
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2
Author for correspondence, current address: Swiss Research Station for Fruitgrowing, Viti- and Horticulture, CH-8820 Wädenswil, Switzerland.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Barbour, T. M. De Jong, and B. M. Pavlik. 1985 Marine beach and dune plant communities. In B. F. Chabot and H. A. Mooney [eds.], Physiological ecology of North American plant communities, 296–322. Chapman and Hall, New York, NY.
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