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2Department of Botany, University of Hawai'i M
anoa, 3190 Maile Way, Honolulu, Hawaii 96822;
3Bodega Marine Laboratory, P.O. Box 247, Bodega Bay, California 94923; and
4Biology Department, University of Leicester, Leicester, LE1 7RH, UK
Received for publication June 4, 1998. Accepted for publication December 10, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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21 cm), tenfold the tiller density (4000 tillers/m2), and is restricted to growth in the upper intertidal zone. Chromosome counts of the dwarfs are identical to typical smooth cordgrass (2n = 62), and smooth cordgrass-specific random amplified DNA markers confirm the species identity of the dwarf. Field-collected clonal fragments of the dwarf grown for 2 yr under high-nutrient conditions maintained the dwarf syndrome, as did plants grown from the seed of a dwarf. The dwarf condition is not caused by endophytic fungi. The first dwarf smooth cordgrass patch was discovered in 1991, and by 1996 five separate dwarf patches had appeared within 200 m of the original. Since 1991, total area covered by the dwarf ecotype has increased sixfold to 140 m2. The ecological range of the dwarf smooth cordgrass ecotype is similar to that of S. patens, a competitor on the Atlantic coast. We suggest that the absence of S. patens from most of San Francisco Bay has allowed the dwarf ecotype of smooth cordgrass to survive and spread.
Key Words: competition • dwarf • founding population • invasion • Poaceae • smooth cordgrasss • Spartina • random amplified polymorphic DNA
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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; Hiesey and Milner, 1965
). Ecotypic differentiation is common within plant species and has been reported between subpopulations separated by as little as a few metres (Blits and Gallagher, 1991
; Masuda and Washitani, 1992
). Recent theory indicates that changes in fitness functions, often associated with population introductions into a new environments, can lead to peak shifts in a theoretical fitness landscape, resulting in substantial morphological evolution (Whitlock, 1997
). Earlier theories also predicted rapid genetic differentiation of small, founding populations in association with genetic drift (Wright, 1931
; Carson and Templeton, 1984
). Thus, several theories have suggested that new ecotypes can quickly evolve in small, introduced populations.
Here we report the discovery and spread of a distinctly dwarf ecotype of introduced smooth cordgrass, Spartina alterniflora Loisel., in San Francisco Bay, California, USA. This dwarf ecotype has evolved since the introduction of S. alterniflora to San Francisco Bay from Maryland in the 1970s (Daehler and Strong, 1994
), and as far as we know, a similar dwarf ecotype has not been reported from within the native range of S. alterniflora. The new ecotype differs ecologically from typical or "wild-type" S. alterniflora and also differs significantly from the previously reported "short-form" S. alterniflora (Gallagher et al., 1988
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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. In August 1996, measurements of stem height and stem density were taken in the field on five patches of the dwarf ecotype and five adjacent patches (within 1 m) of wild-type S. alterniflora.
Greenhouse growth studies
Four replicate clonal fragments were dug from each of five separate patches of the dwarf ecotype and five wild-type S. alterniflora patches at San Bruno in April 1994. The fragments were propagated in a greenhouse at Bodega Bay, California for 2 yr in 2.8-L pots, in a mixture of 50% Bodega Bay intertidal mud and 50% vermiculite (by volume). During the summer, plants were fertilized once per week with 50 mL of a solution of 5% Plantex 20-20-20 (dissolved in water). High-nutrient common growing conditions were used to determine whether the short stature of the dwarf plants in the field was due to nutrient limitation. Under these high-nutrient greenhouse conditions S. alterniflora grows vigorously (Daehler, personal observations). Stem heights and stem diameters of the dwarf ecotype and wild-type S. alterniflora were compared after 2 yr in the common environment.
Field experiments
To compare the growth range potential of the dwarf ecotype and wild-type S. alterniflora, field transplants were made along an intertidal gradient ranging from +0.6 to +1.7 m above mean low water (MLW). In June 1996, seven common gardens were established along the tidal gradient, with each garden consisting of five replicates of the dwarf ecotype containing at least ten live stems each (dug directly from the field) and two replicates of wild-type S. alterniflora. Survival of the transplants along the tidal gradient was recorded in December 1996. Plants classified as dead in December 1996 were confirmed to be dead in spring 1997. To examine competitive interactions between the dwarf ecotype and wild type, we recorded lateral spread at points where the two ecotypes came into direct contact. In June 1996, wires were buried in the mud, tracing the border between patches of the two ecotypes (seven contact zones in total, each averaging 1 m in length). In September 1996, new growth of each ecotype at these borders was assessed by counting the number of new tillers of each ecotype that had spread into the patch of the opposing ecotype.
Heritability of dwarf growth characters
Three open-pollinated seeds of the dwarf ecotype and ten seeds from neighboring wild-type S. alterniflora were stored over winter in 50% seawater at 5°C (Daehler and Strong, 1994
) and germinated in spring 1992. These seedlings were grown in a greenhouse at Bodega Bay for three summers, after which time tillers were measured.
Chromosome counts
Root tips of rapidly growing plants from two patches of the dwarf ecotype were collected in the late morning and pretreated in a saturated aqueous solution of alpha bromo napthalene for 22 h at 4°C. The root tips were then fixed in 3:1 ethanol : glacial acetic acid, hydrolyzed for 10 min in 5 mol/L hydrochloric acid, and placed into 70% ethanol. Meristems were dissected out in 2% aceto-orcein, squashed, and examined with a Zeiss Universal microscope at 1000X.
Confirmation of species using DNA markers
We used species-specific DNA markers to confirm that the patches of the dwarf ecotype were S. alterniflora. We also wanted to confirm that the patches of dwarf ecotype were not hybrids between S. alterniflora and the native S. foliosa (Daehler and Strong, 1997
). Five patches of the dwarf ecotype were screened for the presence of S. alternflora-specific and S. foloisa-specific DNA markers. Additional species-specific markers that have been identified since publication of Daehler and Strong (1997)
were used in this study (see Table 1).
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extraction buffer. After 1–2 h, the proteinase K was denatured by heating, and samples were centrifuged at 8000 x g for 10 min; 500 µL of supernatant was withdrawn and combined with 50 µL of 2 mol/L sodium acetate and 500 µL isopropanol. The solution was placed at -20°C for at least 1 h and then centrifuged as above. The pellet was recovered and suspended in 100 µL Tris-EDTA buffer; 5 µg of RNAase were added and the sample was incubated at 37°C for 20 min. The DNA was precipitated by adding 10 µL of 2 mol/L sodium acetate and 250 µL 100% ethanol and chilling at -20°C for at least 1 h. The pellet was recovered and resuspended as above. Centrifugation was repeated as above; 90 µL of supernatant were withdrawn and mixed with 200 µL Tris-EDTA buffer.
Amplification
Polymerase chain reaction was performed with a Perkins Elmer 9600 thermocycler (Norwalk, Connecticut) using the following protocol: 1 cycle, 94°C (1.5 min), 42°C (30 sec), 72°C (65 sec); 44 cycles, 94°C (15 sec), 42°C (30 sec), 72°C (65 sec); 1 cycle 72°C (4 min). Reaction volumes of 15 µL contained 10% by volume MgCl2-free 10x reaction buffer A (Promega, Madison, Wisconsin), 0.6 units Taq polymerase (Promega, Madison, Wisconsin), 360 picounits primer (Operon Technologies, Alameda, California), 3 mmol/L MgCl2, 200 µmol/L each dATP, dCTP, dGTP,and dTTP (Promega, Madison, Wisconsin), and 30 ng genomic DNA. All reactions were repeated at least twice to confirm consistency. Following electrophoresis on 1.5% agarose gels, DNA was stained with ethidium bromide and visualized under UV light.
Assaying for endophyte infection
To test the hypothesis that endophytic fungi were responsible for the dwarfism (Fisher and Holton, 1957
), we assayed the leaves of the dwarf and wild-type S. alterniflora using the culture techniques of Carroll and Carroll (1978)
. Each sample comprised two pieces from the same leaf, each 3 cm in length, cultured on sterile water agar in separate 150-mm petri plates. Of the dwarf, we tested four patches, two replicate leaves from each. Of the wild-type S. alterniflora, we cultured leaves from three clones growing near the dwarfs.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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), while the dwarf ecotype does not. The vegetative and reproductive morphologies of the dwarf ecotype do not match any other described Spartina species described by Mobberly (1956). Instead, the dwarf morphology is consistent with a miniaturized form of S. alterniflora.
Morphology of the dwarf ecotype
The most striking characteristic of the dwarf ecotype is its small size and extremely high tiller density, relative to the wild-type S. alterniflora (Figs. 1–2). These differences were maintained after 2 yr of growth in common greenhouse environment under high-nutrient conditions (Table 2). In addition, progeny grown from open-pollinated seed also maintained the dwarf morphology, demonstrating heritability of the dwarf condition (Table 2). These seed progeny were likely selfed because the cross-pollination rates in San Francisco Bay were low (Daehler, 1998
). A "short-form" of S. alterniflora has been previously reported from the species' native range, and there has been considerable controversy over whether this "short-form" is environmentally induced or has a genetic basis (Valiela, Teal, and Deuser, 1978
; Gallagher et al., 1988
). Some controversy remains because heritability studies based on seed progeny have never been published. Nevertheless, the dwarf ecotype we describe here differs substantially from the "short form" S. alterniflora found commonly on the Atlantic and Gulf coasts. Stem density of the dwarf ecotype is over five times higher than in the "short form" S. alterniflora (4000 vs. 700 tillers/m2), and mean stem diameter is less than half that of the "short form" (1.7 vs. 3.7 mm) (Gallagher et al., 1988
). Endophytic fungi can sometimes cause dwarfism in grasses (Fisher and Holton, 1957
), but they were not detected in any of our plants. Chromosome counts of the dwarf and wild type were identical (Table 1). In many cereal crops, dwarf genetic mutants have been identified that may be similar to the dwarf ecotype of S. alterniflora. In most of these cases, dwarfism is due to mutations that either reduced synthesis of gibberellins, or mutations involving the gibberellin response pathway (Devi et al., 1994
; Evans, Blundell, and King, 1995
; Ogawa et al., 1996
).
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). Because stems of the dwarf are inundated much sooner on the rising tide than wild-type S. alterniflora, the longer submergence times of the dwarf could account for its restriction to the high intertidal. When grown in the absence of competition, both ecotypes survived equally well in the upper intertidal (Table 2). However, where the dwarf ecotype and wild type came into contact in the upper intertidal, the dwarf was more successful at spreading (Table 2). The extremely high tiller density of the dwarf ecotype appears to prevent the wild-type S. alterniflora from invading. Patches of the dwarf ecotype have remained homogeneous and have continued to spread, despite coming into contact with neighboring wild-type plants. The much lower stem density in the wild-type S. alterniflora allows more light to reach the mud surface and apparently allows shoots of the dwarf to spread into established wild-type patches, especially in the spring when both ecotypes are more similar in height.
Distribution of the dwarf ecotype
Since discovery of the original dwarf patch in 1991, we have observed the development of four new patches, all within 200 m of the original. The new patches probably established from seeds because we did not observe erosion surrounding the original patch that would have led to vegetative fragmentation. All dwarf patches show very similar RAPD profiles, suggesting, but not proving that the original patch was the source of the new patches. The total area covered by the dwarf ecotype has grown from 25 m2 in 1991 to 140 m2 in 1996. Given the abundant open mud habitat and the dwarf ecotype's high growth rate and high competitive ability, the dwarf ecotype will probably continue to spread in San Francisco Bay. On the Atlantic and Gulf coasts of North America, S. patens, a much shorter species is competitively dominant over S. alterniflora in the upper intertidal (Bertness, 1991
). We speculate that the absence of S. patens in San Francisco Bay has contributed to the success of the dwarf S. alterniflora ecotype.
Conclusions
A similar dwarf form was reported for the hybrid species Spartina anglica during its invasion of mudflats in Britain (Chater and Jones, 1951
) and New Zealand (Bascand, 1970
). In contrast to the vigorous seedling growth we observed in the dwarf S. alterniflora ecotype, seeds of the S. anglica dwarf plants could not be germinated (Chater and Jones, 1951
), limiting its potential for spread. Chater and Jones (1951)
suggested that S. anglica dwarfs resulted from disintegration of a formerly stable hybrid polyploid. Since S. alterniflora is not a hybrid and the dwarf ecotypes is of the normal ploidy level, a new explanation for the dwarf ecotypes of both species is suggested based on consistencies between these cases. In both cases, invasion of new geographic regions led to the appearance and spread of a new ecotype. Natural variants that would be selected against in the native habitat may survive and flourish under novel selective regimes associated with invasions, particularly when an invader spreads over a heterogeneous habitat while experiencing little interspecific competition from native species.
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FOOTNOTES |
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5 Author for correspondence (FAX 808-956-3923; Tel. 808-956-3929; e-mail: daehler{at}hawaii.edu
).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Bertness, M. D. 1991 Zonation of Spartina patens and Spartina alterniflora in a New England salt marsh. Ecology 72: 138–148.[CrossRef][ISI]
Blits, K. C., and J. L. Gallagher. 1991 Morphological and physiological responses to increased salinity in marsh and dune ecotypes of Sporobolis virginicus (L.) Kunth. Oecologia 87: 330–335.[CrossRef][ISI]
Callaway, J. C., and M. N. Josselyn. 1992 The introduction and spread of smooth cordgrass (Spartina alterniflora) in South San Francisco Bay. Estuaries 15: 218–226.[CrossRef][ISI]
Carroll, G. C., and F. E. Carroll. 1978 Studies of the incidence of coniferous needle endophytes in the Pacific Northwest. Canadian Journal of Botany 56: 3034–3043.[CrossRef]
Carson, H. L., and A. R. Templeton. 1984 Genetic revolutions in relation to speciation phenomena: the founding of new populations. Annual Review of Ecology and Systematics 15: 97–131.
Chater, E. H., and H. Jones. 1951 New forms of Spartina townsendii (Groves). Nature 168: 126.
Clausen, J., and W. M. Hiesey. 1958 Experimental studies on the nature of species. IV, Genetic structure of ecological races. Carnegie Institute of Washington, Publication 615, Washington, D.C.
Daehler, C. C. 1998 Self-fertility variation and the reproductive advantage of self-fertility for an invading plant (Spartina alterniflora). Evolutionary Ecology 12: 553–568.[CrossRef][ISI]
Daehler, C. C., and D. R. Strong. 1994 Variable reproductive output among clones of Spartina alterniflora (Poaceae) invading San Francisco Bay, California: the influence of herbivory, pollination, and establishment site. American Journal of Botany 81: 307–313.[CrossRef][ISI]
———, and ———. 1996 Status, prediction, and prevention of introduced cordgrass (Spartina spp.) invasions in Pacific estuaries, USA. Biological Conservation 78: 51–58.[CrossRef][ISI]
———, and ———. 1997 Hybridization between introduced smooth cordgrass (Spartina alterniflora; Poaceae) and native California cordgrass (S. foliosa) in San Francisco Bay, California, USA. American Journal of Botany 84: 607–611.[Abstract]
Devi, K. U., M. K. Rao, S. J. Croker, P. Hedden, and A. Rao. 1994 Coleoptile length, gibberellin sensitivity and concentration in five non-allelic dwarf mutants of pearl millet—Pennisetum glaucum (L.) R. Br. Plant Growth Regulation 15: 215–221.
Evans, L. T., C. Blundell, and R. W. King. 1995 Developmental responses by tall and dwarf isogenic lines of spring wheat to applied gibberellins. Australian Journal of Plant Physiology 22: 365–371.[ISI]
Fisher, G. W., and C. S. Holton. 1957 Biology and control of the smut fungi. Ronald Press, New York, NY.
Gallagher, J. L., G. F. Sommers, D. M. Grant, and D. M. Seliskar. 1988 Persistent differences in two forms of Spartina alterniflora: a common garden experiment. Ecology 69: 1005–1008.[CrossRef][ISI]
Guidet, F. 1994 A powerful new technique to quickly prepare hundreds of plant extracts for PCR and RAPD analyses. Nucleic Acids Research 22: 1772–1773.
Hiesey, W. M., and H. W. Milner. 1965 Physiology of ecological races and species. Annual Review of Plant Physiology 16: 203–216.
Masuda, M., and I. Washitani. 1992 Differentiation of spring emerging and autumn emerging ecotypes in Galium spurium L. var. echinospermon. Oecologia 89: 42–46.[CrossRef][ISI]
Mobberley, D. G. 1956 Taxonomy and distribution of the genus Spartina. Iowa State College Journal of Science 30: 471–574.
Ogawa, S., T. Toyomasu, H. Yamane, N. Murofushi, R. Ikeda, Y. Morimoto, Y. Nishimura, and T. Omori. 1996 A step in the biosynthesis of gibberellins that is controlled by the mutation in the semi-dwarf rice cultivar Tan-Ginbozu. Plant Cell Physiology 37: 363–368.
Valiela, I., J. M. Teal, and W. G. Deuser. 1978 The nature of growth forms in the salt marsh grass Spartina alterniflora. American Naturalist 112: 461–470.
Whitlock, M. C. 1997 Founder effects and peak shifts without genetic drift: adaptive peak shifts occur easily when environments fluctuate. Evolution 51: 1044–1048.[CrossRef][ISI]
Wright, S. 1931 Statistical theory of evolution. Journal of the American Statistical Association 265: 201–208.
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