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Ecology |
Department of Biological, Geological and Environmental Sciences, Cleveland State University, 2121 Euclid Avenue, Cleveland, Ohio 44115 USA; 3Department of Biological Sciences, University of Windsor, Windsor, Ontario, N9B 3P4, Canada
Received for publication June 1, 2004. Accepted for publication January 24, 2005.
ABSTRACT
In colonizing species, high phenotypic plasticity can contribute to survival and propagation in heterogenous adventive environments, and it has been suggested as a predictor of invasiveness. Observation of natural populations of an invasive species, Lythrum salicaria salicaria, indicated extensive variation in its growth and reproductive traits. Phenotypic plasticity of different life history traits of L. salicaria was investigated using vegetative clones of each of 12 genotypes from one population in Ontario, Canada. We chose soil moisture as the treatment factor because of its importance in wetland species and raised all 12 genotypes in each of four soil moisture treatments. We examined an array of vegetative and reproductive traits, including root and shoot mass, shoot and inflorescence length, total seed set, floral mass, and morphometric variables. All observed vegetative as well as reproductive traits demonstrated significant phenotypic plasticity in response to soil moisture treatment. Even the stigma–anther separation involved significant genotype by environment interactions, suggesting that soil moisture may modify the relative positions of anthers and stigma. Compared to vegetative traits, most reproductive traits demonstrated crossing reaction norms, implying that the average differences in those traits among genotypes vary with the environment maintaining the genetic variation in a population.
Key Words: colonizing weed • norm of reaction • phenotypic plasticity • purple loosestrife • soil moisture • tristyly
Colonizing weeds have been considered to be ecological generalists, able to inhabit diverse new sites without undergoing local genetic adaptation (Sultan, 2000
). Plasticity in ecologically important traits can contribute to survival and propagation of such species in sites having heterogenous environments. This has been suggested as an important predictor of invasiveness (Baker, 1974
; Via, 1993
; Rejmanek and Richardson, 1996; Meekins and McCarthy, 2000
; Claridge and Franklin, 2002
). Although some plastic responses are likely to be inevitable effects of environmental limits on growth and physiology, adaptive plastic responses in colonizing weeds may often help them maintain function and fitness across a range of environments and influence their ecological breadth and response to natural selection (Sultan, 2000
; Daehler, 2003
).
Adaptive plasticity can help outcrossing colonizing weeds overcome population bottlenecks. Although population growth in self-compatible and hermaphroditic colonizing weeds is facilitated by self-fertilization (Baker, 1965
), the population growth of many outcrossing and dioecious species is limited by mating systems that do not function optimally at low population densities during initial colonizing episodes. Despite this constraint, some outcrossing colonizing weeds are extremely successful (Price and Jain, 1981
; Mal et al., 1992
). Moreover, plasticity in mating systems of outcrossing colonizing weeds can help them override effects of long-distance colonization and population bottlenecks, by assuring reproduction in low population density and producing new and better-adapted genotypes. Yet relatively few studies exist dealing with the phenotypic plasticity of plant reproductive traits (but see Vogler et al., 1999
; Dorken and Barrett, 2004
).
Although the majority of flowering plant species are hermaphroditic, outcrossing is facilitated in many of them through spatiotemporal and physiological mechanisms. In heterostylous hermaphrodites, the reciprocal positioning of male and female organs leads to two, or three, kinds of style morphs in a population (Darwin, 1877
). Unlike self-compatible hermaphrodites, a self-incompatible heterostylous species requires an individual of another morph for successful pollination and faces different kinds of challenges during colonizing episodes when "legitimate" mating partners are not present. Here we report phenotypic plasticity in vegetative and reproductive traits in Lythrum salicaria (purple loosestrife; family Lythraceae), a heterostylous species having three morphs in a typical population (tristyly): long (stigma above the two staminal whorls), mid (stigma between the two staminal whorls), and short (stigma below the two staminal whorls) (Darwin, 1877
).
Lythrum salicaria is an invasive weed of North American wetlands that tends to form extensive monocultures, often excluding native species (Mal et al., 1992
, 1997a
; Mal and Lovett-Doust, 1997
). It is native to Eurasia and was reported first in North America in 1814 (Stuckey, 1980
). The infestation of L. salicaria is most severe in eastern Canada (Mal et al., 1992
) and the northeastern United States, including the Great Lakes region. The species was relatively sparse until 1930, and then a significant increase in population growth was documented (Cutright, 1986
). The reason for its sudden increase in population growth is not known, although evolution of breeding systems, introgressive hybridization between L. salicaria and Lythrum alatum, and a lack of native herbivores have been suggested for its success and spread in North America (Strefeler et al., 1996
; Mal and Lovett-Doust, 1997
).
Results of an extensive survey of adventive populations of L. salicaria indicated significant effects of site on growth and reproduction, and experimental results demonstrated significant effects of soil moisture and fertilizer application on these traits (Mal et al., 1997b
; Mal and Lovett-Doust, 1997
). Because phenotypic plasticity is considered as an important predictor of invasiveness, we investigated the amplitude of plasticity in vegetative and reproductive traits with respect to soil moisture in controlled environmental conditions. We chose soil moisture as the treatment factor because this is one of the most important environmental factors for a wetland species such as L. salicaria. In the present paper, we report patterns of phenotypic plasticity in growth and reproductive traits of L. salicaria, in response to changes in the moisture content of soil. We hypothesize that (1) the availability of soil moisture will affect phenotypic plasticity of vegetative and reproductive traits of L. salicaria and (2) individual genotypes will respond to the available soil moisture gradient by producing different phenotypes.
MATERIALS AND METHODS
Four large genets of each of the three style morphs of L. salicaria were collected from a wetland population at LaSalle, Essex County, Ontario, Canada and grown at the greenhouse of the University of Windsor, Windsor, Ontario, Canada. Each of the 12 genotypes (genotypes 1–4 belong to the long morph, genotypes 5–8 belong to the mid, and genotypes 9–12 belong to the short morph) was cloned from the root buds to generate 28 plants, which were then individually potted in plastic pots (20 cm diameter, 25 cm deep) containing topsoil. All together, 336 plants were grown in one room of the greenhouse. Each plant started from a root bud and therefore had only one ramet. Seven randomly selected cloned individuals of each genet were then placed in each of four water treatments: (1) standing water treatment (SW), individuals were totally flooded, i.e., throughout the growth period there was at least 2.5 cm of water on top of the soil; (2) high water treatment (HW), 750 mL of water per day per pot; (3) medium water treatment (MW), 500 mL of water per day per pot; (4) low water treatment (LW), 250 mL of water per day per pot. The water treatments were selected to represent a gradient of soil moisture found in L. salicaria populations in North America (Mal et al., 1992
, 1997b
; Mal and Lovett-Doust, 1997
; T. K. Mal, personal observation). All pots and treatments were rotated each week to counter any positional effect of pots within treatments. We recorded the following growth and reproductive parameters.
Vegetative growth
Total length of all branches was recorded at the beginning of the first treatment and again near the end of the experiment. Net vegetative growth was estimated by subtracting total initial length from the total final length; this value was used in our statistical analysis.
Root and shoot biomass
Both above- and belowground parts were harvested separately for each plant. Belowground parts were carefully cleaned of soil to minimize loss of root tissue. Plant parts were then dried in convection ovens at 60 C for a week and weighed individually to obtain dry biomass (hereafter dry mass).
Floral morphometry
Five flowers were sampled from each plant for morphometric measures on 29 July 1996. The flowers were dissected open and laminated on Scotch tape. It was confirmed that no detectable shrinkage occurred in flowers subjected to this treatment (Mal, 1995
; Mal and Lovett-Doust, 1997
). Lengths of calyx, pistil, and all available stamens were measured using a dissecting microscope and ocular micrometer, and stigma–anther separation was calculated (see Mal and Lovett-Doust, 1997
).
Floral biomass
Five flowers were sampled from each plant for floral biomass estimates on 13 August. Floral parts were partitioned into perianth or attractive structures (calyx and corolla together), pistil, and stamens, and they were dried separately in a convection oven at 60°C for a week. Dry mass was obtained by weighing individual floral parts in a microbalance.
Reproductive effort and fertility level
Reproductive effort was investigated by measuring the total length and dry mass of the infructescence for each plant at harvest. We investigated fertility level by introducing bumblebees into the greenhouse. One hive (Koppert Biological Systems, Kingsville, Ontario, Canada) was kept at a central location of the greenhouse for a week. Each inflorescence was marked by color-coded wire, at the beginning and end of bee introduction. The number of capsules formed following bee introduction was determined on 10 cm of the marked section of infructescence. After 4 wk, five mature capsules were collected from each plant randomly from the marked section. We counted number of seeds formed in each of these capsules and obtained mean seed set per capsule. The total number of seeds was calculated as the number of capsules multiplied by the average number of seeds per capsule.
Statistical analysis
Data were transformed to improve normality when necessary. Only mean values of each trait from individual plants were used in the analysis (Hurlbert, 1984
). We used factorial analysis of variance. We considered soil moisture treatment as a fixed factor and genotype as a random factor. Significant differences among groups were obtained by Tukey post-hoc analyses. We assessed significance of crossing reaction norms by significant interaction terms between soil moisture treatment and genotypes. Data were analyzed using SAS (SAS, 1989
) and SYSTAT (Wilkinson, 1998
) statistical packages.
RESULTS
Vegetative characters such as shoot length and shoot dry mass differed among soil moisture treatments and genotypes, and there were no significant interactions between them (Table 1; Figs. 1– 2). Shoot length in the high water treatment was significantly greater than in the medium and low water treatments, followed by the standing water treatment (Fig. 1A). Shoot dry mass was also significantly greater in the high water treatment, followed by the standing and medium water treatments. Individuals in the driest treatment (low water treatment) had lowest dry mass (Fig. 1B). Clearly, genotype 8 had the shortest shoot (Fig. 2A), and genotypes 6 and 8 had significantly lower shoot mass compared to several others, while genotypes 1 and 3 had significantly greater shoot mass than many (Fig. 2B). Root dry mass was significantly different among treatments and genotypes. There were also significant interactions between them, indicating that the different genotypes interacted with available soil moisture differently (Table 1, Fig. 3).
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All floral traits (mass of flower, perianth, stamens and pistil, and stigma–anther separation) were significantly affected by soil moisture and genotype, and all (except calyx length) demonstrated significant interactions between soil moisture treatments and genotypes (Table 2). Calyx was significantly larger in the standing water treatment than in other soil moisture treatments (Fig. 1). Plasticity in total number of seeds produced by individuals after the pollinator introduction also differed significantly among genotypes (Table 2, Fig. 3).
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DISCUSSION
In the present study, we observed significant phenotypic plasticity not only in vegetative traits, but also in all the measured reproductive traits. Intuitively, characters formed over a long period of meristematic activity are likely to be subjected to greater cumulative environmental influences. Plant size is such a character, and thus it is expected to be more plastic (Stebbins, 1950
). In contrast, flowers are modified shoots that are determinate in their growth, and their meristematic tissues remain active for a relatively short period. Thus the environment has relatively little time to act on the flower, so floral traits are generally regarded as canalized and nonplastic (Bradshaw, 1965
), and their plasticity has been relatively unexplored (Dorken and Barrett, 2004
).
Although all floral traits demonstrated significant variation due to variation in soil moisture availability, floral biomass and morphometry observed here were typical of this species and agree with our previous results (Mal, 1995
, 1998
; Mal and Lovett-Doust, 1997
; Mal et al., 1997b
) (Table 2). It is interesting, however, that the stigma–anther separation is significantly affected by the soil moisture treatment. The distance between the anther and stigma may produce a separation that helps a heterostylous species reduce self-pollination. The stigma–anther separation was significantly affected by both water treatment as well as by genotypes and also demonstrated significant interactions between treatment and genotype. A Tukey post-hoc test of the main factor, soil moisture treatment, indicated a significant reduction in stigma–anther distance in the driest two treatments. The occurrence of semi-homostylous flowers with smaller stigma–anther separation in drier habitats has been reported previously in other tristylous species (Barrett, 1992
).
Phenotypic plasticity in stigma–anther separation in response to soil moisture can play vital roles in the evolution of the breeding system of tristylous species. Plasticity in stigma– anther separation can be adaptive in some colonizing heterostylous species in which reduced stigma–anther separation can provide reproductive assurance. Such evolutionary changes related to long-distance colonization, founder events, and genetic bottlenecks have been documented in tristylous species, such as Eichhornia paniculata, E. crassipes (Barrett, 1992
), and Oxalis alpina (Weller, 1976
). Even in L. salicaria, in which self-incompatibility is considered to be strong, some degree of weakness or leakiness has been observed. Any seed set that results from illegitimate pollinations represents leakiness in the incompatibility system (Mal et al., 1999
). Mal and Lovett-Doust (1997)
reported variant flowers with stigma–anther separation values of zero (or very close to zero) and documented significant site effects on floral morphometry in L. salicaria. However, how such variant flowers and leaky self-incompatibility can provide reproductive assurance in this species is unclear.
We observed significant effects of soil moisture upon all vegetative traits. Maximum growth occurred in the treatment with intermediate soil moisture content (Fig. 1). Sultan and Bazzaz (1993a
, b
, c
) studied phenotypic plasticity in Polygonum persicaria in response to light, moisture, and nutrient content of soil, and they found marked morphological plasticity in leaf, stem, root, and fruit, and in structures related to reproductive support following changes in soil moisture (Sultan and Bazzaz, 1993b
). Under drought conditions, overall plant growth was reduced as a result of both biochemical disruptions and reduced cell enlargement, which in turn led to reduced leaf expansion and total leaf area and therefore reduced whole-plant photosynthesis. Although growth was affected in the drier treatments, the total number of seeds produced in sampled 10-cm inflorescence fragments was significantly greater in medium and low water treatments. Previously, environmental stressors such as salinity have been demonstrated to reduce vegetative growth in Iris hexagona, but they led to either increased or neutral effects on sexual reproduction (van Zandt et al., 2003). Such environmental stressors also have been documented to increase evolutionary potential in wild mustard, Sinapis arvensis (Stanton et al., 2000
).
Genotypes differed significantly in all vegetative and reproductive traits across the gradient of available soil moisture. The traits (shoot length and mass, inflorescence length, calyx length) that exhibited no interactions between treatment and genotypes may be readily subject to selection. For example, some genotypes of mid morph consistently have a smaller inflorescence in all environments (Fig. 2), and those genotypes may be selected against. On the other hand, some genotypes of the mid morph had flowers with significantly smaller stigma–anther separation across the environmental gradient, and those genotypes may be selected during founder events (Fig. 3). When we used a partially hierarchical factorial analysis of variance using morph and soil moisture treatment as the main factors and genotypes were nested within morph, we found that morphs varied significantly in reproductive effort and in phenotypic plasticity of ecologically important traits such as reproductive effort and stigma–anther separation. However, the detailed results of such analyses have not been reported here because of inadequate number of genotypes within each morph. Smaller stigma–anther separation in some genotypes of mid morph may provide an opportunity for reproductive assurance when "legitimate" mating partners (or pollinators) are not available.
In the present study, all but one of the reproductive traits (inflorescence mass; mass of flower, perianth, stamens, and pistil; stigma–anther separation; total number of seeds) had significant interactions between genotypes and soil moisture treatments (Tables 1, 2). Norms of reaction of individual genotypes crossed to differing degrees in these traits that demonstrated significant interactions (Fig. 3). In such a situation, the underlying genetic differences are not likely to be available to natural selection (Gupta and Lewontin, 1982
; Schlichting and Pigliucci, 1998
). Sultan and Bazzaz (1993b)
proposed that crossing reaction norms, combined with environmental variation, will act to prevent elimination of any particular genotype. Environmentally specialized populations may evolve if individual genotypes fare differently, altering the phenotypic ranks in contrasting environments (Wade, 1990
). In this situation, selection will favor different genotypes in different environments.
Sultan and Bazzaz (1993a
, b
) found that no two genotypes of Polygonum persicaria differed significantly from one another in any trait across an environmental gradient. Thus, consistent superiority or inferiority has not been found in any of the genotypes, although nonsignificant interactions between genotype and environment have been documented. Sultan and Bazzaz (1993a
, b
) argued that existence of average differences among genotypes, and correlation between environments, can operate to obstruct selection for particular norms of reaction. Such a pattern will also enable the population to maintain genetic structure in heterogenous environments (Thompson, 1991
). Significant interactions between genotype and environment in most traits observed in this study may influence the colonization success of L. salicaria by maintaining its population genetic structure in adventive environments.
Phenotypic plasticity in life history traits of invasive species is an important characteristic that can be used in predicting the ability of a species to invade new habitats. The present paper demonstrates significant phenotypic plasticity in many vegetative and reproductive traits that are important ecologically and can facilitate successful colonization of new habitats by L. salicaria. The results also suggest that L. salicaria can maximize growth and reproduction in habitats with a wide range of soil moisture availability and such information can be crucial for developing management strategies and predictive models of its spread.
FOOTNOTES
1 The authors thank Drs. S. C. H. Barrett, C. D. Schlichting, S. Scheiner, C. Eckert, L. Lovett-Doust, A. Corbett, R. Krebs, and two anonymous reviewers for their suggestions and critiques on the manuscript and many undergraduate students for their help in the experiment. T. K. M. acknowledges research support from the George Gund Foundation, Research Challenge Grant, EFFRD Program, and the Center for Excellence in Risk Analysis at Cleveland State University. J. L. D. thanks the Natural Sciences and Engineering Research Council of Canada and the Environmental Youth Corps Program of the Ontario Ministry of Natural Resources for financial support. 
2 E-mail: t.mal{at}csuohio.edu
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