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(American Journal of Botany. 2008;95:931-942.) doi: 10.3732/ajb.2007364 © 2008 Botanical Society of America, Inc.

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Plasticity in salt tolerance traits allows for invasion of novel habitat by Japanese knotweed s. l. (Fallopia japonica and F.xbohemica, Polygonaceae)¹

² Department of Ecology and Evolution, Stony Brook University, Stony Brook, New York 11794 ³ Department of Biology and Center for Genomics and Systems Biology, New York University, New York, New York 10003 ⁴ Biology Department, University of Leicester, Leicester, LE1 7RH, UK

Received for publication 8 November 2007. Accepted for publication 28 May 2008.

ABSTRACT

Japanese knotweeds are among the most invasive organisms in the world. Their recent expansion into salt marsh habitat provides a unique opportunity to investigate how invasives establish in new environments. We used morphology, cytology, and AFLP genotyping to identify taxa and clonal diversity in roadside and salt marsh populations. We conducted a greenhouse study to determine the ability to tolerate salt and whether salt marsh populations are more salt tolerant than roadside populations as measured by the efficiency of PSII, leaf area, succulence, height, root-to-shoot ratio, and total biomass. Clonal diversity was extremely low with one F. japonica clone and five F. xbohemica genotypes. The two taxa were significantly different in several traits, but did not vary in biomass or plasticity of any trait. All traits were highly plastic in response to salinity, but differed significantly among genets. Despite this variation, plants from the salt marsh habitats did not perform better in the salt treatment, suggesting that they are not better adapted to tolerate salt. Instead, our data support the hypothesis that plasticity in salt tolerance traits may allow these taxa to live in saline habitats without specific adaptation to tolerate salt.

Key Words: adaptive evolution • clonal plant • Fallopia japonica (Polygonaceae) • Fallopia xbohemica • Japanese knotweed • natural hybridization • salt tolerance • succulence

Concern over the ecological and economic impacts of biological invasions has generated tremendous interest in the factors determining invasion success (Wilcove et al., 1998; Mack et al., 2000; Pimentel et al., 2000; Pysek et al., 2003; Minchinton et al., 2006). Although all plants have at least a limited ability to respond to the environment, several studies have found that some introduced species have evolved rapidly in response to novel conditions (Lee, 2002; Leger and Rice, 2003; Maron et al., 2004; Wolfe et al., 2004). While invasive species should be subject to genetic bottlenecks, rapid evolution may be facilitated by the infusion of genetic variation and the generation of novel genotypes through inter- or intraspecific hybridization (Daehler and Strong, 1997; Ellstrand and Schierenbeck, 2000; Bleeker, 2003; Pysek et al., 2003; Bímová et al., 2004; Mandák et al., 2004). These novel hybrid genotypes may also be able to occupy a wider range of habitats than the original invaders had in their native range (Lavergne and Molofsky, 2007). On the other hand, phenotypic plasticity has frequently been suggested as a potentially important mechanism in plant invasions (Baker, 1965; Rice and Mack, 1991; Sexton et al., 2002; Sultan, 2004). Recently, several studies have shown that plasticity in ecologically relevant traits may contribute to the success of invasive plant species by allowing increased fitness across a range of habitats (Cheplick, 2006; Pan et al., 2006; Richards et al., 2006; Muth and Pigliucci, 2007). Tolerating novel conditions, through phenotypic plasticity or rapid differentiation of trait mean or trait plasticity, is likely an important component of invasion ecology. Therefore, studies that reveal the underpinnings of adaptation in the particular circumstances of invasive populations are greatly needed.

The coastal communities of North America have been dramatically changed by invasive species (Sayce, 1988; Daehler and Strong, 1997; Ayres et al., 1999; Saltonstall, 2002; Silliman and Bertness, 2004; Freeman and Byers, 2006; Minchinton et al., 2006). One striking example is on the west coast, where Spartina alterniflora and the hybrids produced between S. alterniflora and the native S. foliosa have extensively invaded the once open mudflats. In the process, many soft-sediment organisms as well as migratory birds, have been displaced and the structure of the community dramatically altered (Sayce, 1988; Daehler and Strong, 1997; Ayres et al., 1999). Another conspicuous example is on the east coast, where the extremely successful invader Phragmites australis has spread at the expense of other wetland plants (Chambers et al., 1999; Amesbury et al., 2000; Saltonstall, 2002; Minchinton and Bertness, 2003; Silliman and Bertness, 2004). Similarly, Fallopia species and hybrids have been historically associated with roadside or freshwater wetlands (Pysek et al., 2001; Mandák et al., 2004), but have the potential to become a serious problem in coastal areas in the future.

Fallopia japonica (Houtt.) Ronse Decraene and F. sachalinensis (F. Schmidt) Ronse Decraene are considered among the world’s most invasive plants (Bailey and Conolly, 2000; Pysek et al., 2001; Bailey, 2003). These species can spread extensively by rhizome growth, but recent studies have shown that hybridization between the two has created even more highly invasive genotypes of the hybrid F. xbohemica (Chrtek and Chrtkova) J. Bailey that establish by seed (Pysek et al., 2003; Bímová et al., 2004; Mandák et al., 2004, Tiébré et al., 2007a, b). Invasive populations of this Fallopia species complex, referred to as Japanese knotweed sensu lato, occupy a wide range of habitats, including natural and manmade freshwater waterways and relatively undisturbed grassland, forest, and scrublands, as well as roadsides and urban habitats (Pysek et al., 2001; Mandák et al., 2004). On Long Island, New York, Japanese knotweeds have colonized an even broader array of habitats than previously reported. In addition to freshwater riparian areas, the invaded habitat on Long Island includes salt marshes, where Japanese knotweed is often growing next to or on the terrestrial border of marshes that are inundated with Phragmites australis. These populations are remarkable because even though Japanese knotweed rhizomes or stems are known to survive immersion in salt water, which has aided in transport between islands in Great Britain (Beerling et al., 1994), the species has not previously been found in salt marshes (J. P. Bailey, unpublished data). It is not known whether phenotypic novelty created by hybridization, response to selection or plasticity allows Fallopia species to colonize these novel habitats because we have no information on the genetic makeup of these populations. An important component of this study therefore was to use morphology, cytology, and AFLP markers to identify the taxonomic status and clonal diversity of roadside and salt marsh populations to determine whether hybrid genotypes are more prevalent in the salt marsh habitat.

Whatever the taxonomic makeup of the Long Island salt marsh populations and whether they consist of F. japonica, F. sachalinensis, F. xbohemica, or backcross hybrids, these populations may be expected to have undergone evolutionary change because of the physiological challenges of living in salt marsh habitats and therefore to be quite different from Japanese knotweed populations found elsewhere. Plants that are able to tolerate highly saline areas are characterized by traits that specifically ameliorate the toxic and osmotic effects of substrate salinity to allow for growth under these conditions (Flowers et al., 1977; Yeo et al., 2000). One common response to salinity is to close stomata to limit transpirational water loss and uptake of excess ions. Closing the stomata also limits photosynthetic capacity by reduced CO₂ uptake. The reaction centers of photosystem II may then be exposed to excess energy, become over reduced, and lose efficiency (as measured by _PSII, Qiu et al., 2003; Redondo-Gomez et al., 2006). Plants that are able to tolerate higher salt uptake, through increased succulence or salt excretion, may be able to maintain higher transpiration rates and therefore higher PSII efficiency. Alternatively, plants may employ various mechanisms to maintain high photosynthetic rates at low internal CO₂ concentrations or to dissipate excess energy and maintain high PSII efficiency (Quick et al., 1991; Brugnoli and Björkman, 1992; Yordanov et al., 2000). Regardless of the mechanism, _PSII should be a good indicator of a plant’s ability to maintain photosynthetic capacity under saline conditions, and genets that are particularly tolerant would have less reduction in _PSII in response to salt.

Increased succulence and increased allocation of biomass to roots are two additional traits that are thought to be adaptive responses to salt (Flowers et al., 1977; Reimann and Breckle, 1995; Lexer et al., 2003; Inan et al., 2004; Maggio et al., 2007). For example, studies on Helianthus paradoxus suggest that the evolution of succulence may have been an important adaptation to allow H. paradoxus to survive salty habitats, which was novel for Helianthus (Rosenthal et al., 2002; Lexer et al., 2003; Karrenberg et al., 2006). Similarly, H. paradoxus increased allocation to roots in response to salt, while the ancestral, nontolerant parents H. annuus and H. petiolaris did not (Karrenberg et al., 2006).

Because Japanese knotweed s. l. has not been previously reported in a saline habitat, the individuals that have established there may have survived because of increased salt tolerance compared to those established in the more common roadside habitat. Increased salt tolerance could result if invasive Japanese knotweed populations had heritable phenotypic variation for traits that increase salt tolerance, like succulence, increased allocation to roots, or traits that allow for maintaining efficiency of photosystem II. Selection should act on these traits in salt marsh habitats, leading to differentiation of genotypes from those found along roadsides and therefore to locally adapted populations (Levene, 1953; Hedrick, 1976; Caisse and Antonovics, 1978; Feder et al., 1997). If these populations are adapted to habitats with different salt concentrations, we expect to see distinct phenotypic differences for plants from the two habitat types in response to controlled salt treatments. Although salt treatments will reduce growth (for example, leaf area and height) and overall performance (total biomass) for plants from all sites, the prediction of local adaptation would be supported if plants from salt marsh habitats performed better under salt treatment than plants from roadside habitats, with the rankings reversing under no salt conditions. Alternatively, these species may be very successful invasives because of high levels of phenotypic plasticity, and genotypes from both habitats may be equally able to adjust their phenotypes appropriately.

To test these hypotheses, we carried out a greenhouse experiment with replicate cuttings of genets from roadside and salt marsh habitats and asked three questions. (1) How do Japanese knotweed genets respond to treatment with salt? (2) Is there molecular or quantitative trait genetic variation within or among populations for the response to salt? (3) Do marsh genets or populations tolerate salt treatment better than those from the roadside?

MATERIALS AND METHODS

Fallopia species complex
The taxonomy of the Japanese knotweeds has been complicated historically (for full review, see Bailey and Conolly, 2000; Bailey and Wisskirchen, 2004). Originally from Japan, the two species, F. japonica and F. sachalinensis, were introduced to Europe in the 1830s and to the United States by the end of the 19th century (Townsend, 1997). These species have been referred to by three different generic names: Polygonum, Reynoutria, and Fallopia (Beerling et al., 1994). The most recent treatments of these species place them in the genus Fallopia (Ronse Decraene and Akeroyd, 1988; Beerling et al., 1994; Lamb Frye and Kron, 2003). In Japan, both tetraploid (2n = 44) and octoploid (2n = 88) F. japonica plants can be found in natural populations, although the tetraploid is more common (Bailey, 1999, 2003). The dwarf tetraploid montane plants, referred to as F. japonica var. compacta, are considered primary colonizers, important in the establishment of vegetation on newly formed, bare volcanic habitat like that of Mt. Fuji (Zhou et al., 2003). In this habitat, F. japonica var. compacta is thought to establish primarily by seed, then developing into patches by vegetative spread. These patches accumulate soil and provide safe haven for the establishment of other plant species (Zhou et al., 2003). Japanese populations of F. japonica are extremely variable in morphology and at the molecular level (Inamura et al., 2000; Bailey 2003; Zhou et al., 2003).

The distribution of the closely related species F. sachalinensis is much more limited, restricted to the Sakhalin Islands and northern Japan. This species is typically found along freshwater waterways (Bailey, 2003; Bímová et al., 2003). Although F. sachalinensis (2n = 44) is distinct morphologically from F. japonica, the two species are not differentiated in chloroplast DNA (cpDNA). From the sequencing of 68 individuals collected from 22 sites across Japan, five distinct groups of F. japonica corresponding to geographic distribution were identified. Three accessions of F. sachalinensis did not group together, but were dispersed among two of the F. japonica groups, implying that there has probably been gene flow between the two species in native populations (Inamura et al., 2000).

While hybridization between these two species appears to be rare in Japan, where the species are not usually sympatric, F. xbohemica is much more common in the invasive range of Europe (Bailey, 2003; Bailey and Wisskirchen, 2004), and has greater genetic diversity than either parent in the invasive range (Pysek et al., 2003; Bímová et al., 2004; Mandák et al., 2005, Tiébré et al., 2007a, b). Cytological studies of natural populations of F. xbohemica in the British Isles and across Europe, show that these hybrids are found with three different ploidy levels: tetraploid (2n = 44), hexaploid (2n = 66) and octoploid (2n = 88, Bailey, 1999; Bailey and Wisskirchen, 2004).

Claims that the first Fallopia in the United States were planted in Central Park in the late 1800s are unsubstantiated (Townsend, 1997), but the first voucher specimens in New York state are from Kings County on Long Island in 1893 and the Bronx in 1893 and 1900 (New York Flora Association, http://atlas.nyflora.org/; New York Botanical Garden herbarium specimen). In Massachusetts, the earliest herbarium record dates to 1877 (Forman and Kesseli, 2003). Little is known about the genetic makeup of the populations in the United States, but recent studies suggest that spread in the United States is through both vegetative and sexual reproduction and that the morphological variation present in the United States is much greater than that reported in Europe (Forman and Kesseli, 2003; Gammon et al., 2007; Grimsby et al., 2007).

Sampling sites
We collected Japanese knotweed s. l. plants from three marsh sites and three roadside sites across Suffolk County, Long Island, New York. Roads on Long Island are salted during winter storms; however, salt application is sporadic and infrequent because of the relatively mild winters on Long Island. In the winters of 2005–2006 and 2006–2007, for instance, roads in Suffolk County required salting for only three storms (Suffolk County Executive Levi’s Office, personal communication). Soil sampling with a refractomer in midsummer, when the plants are actually growing, confirmed that these sites have 0 ppt salt, while the marsh sites were typically in the range of 2–10 ppt (C. L. Richards, unpublished data). The marsh sites were separated by 14–34 km and were located in the Terrell River County Park in Center Moriches Marsh (CMM), Wertheim National Wildlife Refuge (WH), and behind a privately owned boathouse on the Peconic Bay in Riverhead (RHBH). The roadside sites were separated by 20–32 km and were located on Montauk Highway at Terrell River County Park in Center Moriches (CMR), Hagerman Landing Road in Rocky Point (HL), and Chauncey Road at Highway 24 in Riverhead (RHC, Table 1). In Center Moriches, the marsh and roadside sites were adjacent to each other (separated by 20 m). The close proximity of these two sites could provide insight into the ability of adjacent populations to adapt to different habitats, given the likeliness of pollen and seed transfer as well as vegetative spread over such a short distance. In Riverhead, the marsh and roadside sites were separated by 1 km. The final two sites, Wertheim and Hagerman Landing Road, were separated by 25 km.

We used the Qiagen DNeasy Plant Mini kit (Qiagen, Valencia, California, USA) to extract DNA in duplicate from each of the samples at each site. We followed the standard protocol suggested by Qiagen, but eluted the DNA with water instead of Tris EDTA buffer (TE) in the final step. We used an AFLP protocol based on standard methods (Vos et al., 1995; Liu et al., 2001), with some modifications. We digested 200 ng of genomic DNA at 37°C for 3 h. Each restriction reaction contained 10 units of EcoRI, 10 units of MseI and 1x EcoRI buffer (New England Biolabs, Beverly, Massachusetts, hereafter referred to as NEB) in a final volume of 20 µL. Immediately after 3 h of digestion, we added 20 µL of ligation mixture consisting of 75 µM of EcoRI adapters, 75 µM of MseI adaptors, 1x T4 DNA ligase buffer (NEB) and 20 units of T4 DNA ligase (NEB). Adapters were ligated overnight (16–20 h) at 4°C and then diluted with 160 µL of water.

For the preselective amplification, we used the single selective base A at the 3' end of the EcoRI primer and the single selective base C at the 3' end of the MseI primer. Each reaction contained 40 pmol of the EcoRI+A and the MseI+C primers, 200 µM dNTPs, 1.5 mM MgCl₂, 1x PCR buffer (NEB), 2.5 units Taq polymerase (Bioline USA, Randolph, Massachusetts, USA), and 10 µL dilute ligation reaction. We used the following PCR conditions: 75°C for 2 min; 20 cycles of 94°C for 30 s, 56°C for 30 s, 75°C for 2 min; final extension at 60°C for 30 min. We ran 5 µL of this PCR product on a 1% agarose gel to verify that the reactions had worked. Successful reactions produced a smear of DNA in the 100–1500 bp range.

We diluted the preselective PCR products with sterile water to 7% and used the dilute preselective products for selective amplification. For the selective amplification, we multiplexed two fluorescently labeled EcoRI primers. One EcoRI primer was labeled with 6-carboxy-fluorescein (6-FAM) and had the additional three selective bases AGC at the 3' end. The second EcoRI primer was labeled with 4,7,2',4',5',7'-hexachloro-6-carboxy-fluorescein (HEX) and had the additional three selective bases ACG at the 3' end. The MseI selective primers had the additional bases CAA at the 3' end. Each reaction contained 4 pmol of each of the labeled EcoRI+3 primers and 25 pmol of the MseI+CAA primer. In addition, each reaction contained 300 µM dNTPs, 1.5 mM MgCl2, 1x PCR buffer (NEB), 1 unit of Taq polymerase (Bioline), and 5 µL dilute preselective amplification reaction. We used the following PCR conditions: 94°C for 2 min; 10 touchdown cycles of 94°C for 30 s, 65°C for 30 s (reducing 1°C per cycle), 72°C for 2 min; 30 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 2 min; and a final extension at 60°C for 5 min. We ran 5 µL of this PCR product on a 1% agarose gel to verify that the reactions had worked. Successful reactions produced a smear of DNA in the 50–500 bp range. We submitted the selective amplification products to the DNA Facility at Iowa State University, where they were electrophoretically separated on an ABI 3700 DNA analyzer (Applied Biosystems, Foster City, California, USA). We visually inspected the AFLP fragments using the open source program Genographer (Benham, 2001) and manually categorized genotypes based on identical banding patterns across 150 total loci for the HEX and 6FAM primer sets combined. Repeatability of banding patterns was assessed across duplicates for each sample.

Experimental treatments
At each site, we collected approximately 1 m of rhizome from six to eight genets to allow us to examine the response of replicates of each genet to different salt treatments. Again, we collected rhizomes that were approximately 10 m apart to maximize the likelihood of collecting different genotypes, considering that several studies have reported that these plants are characterized by very large clones (Hollingsworth and Bailey, 2000; Pysek et al., 2003; Mandák et al., 2005; Grimsby et al., 2007). In the Stony Brook University greenhouse, we cut each rhizome into at least 12 pieces of 8–12 g fresh mass (6 sites x 6–8 rhizomes per site for a total of 44 genets x 12 replicates = 528 rhizome pieces). For each genet, we randomly assigned six rhizome pieces to each of two treatments in a split-plot completely randomized block design. We planted the rhizome pieces in individual 668 cm³ wells in 12-well flats with Promix potting medium (Promix Bx, Quakertown, Pennsylvania, USA), and 1 teaspoon of slow release fertilizer (15-9-12 Osmocote Plus 8–9 mo, Marysville, Ohio). The two treatments were (1) a 5 ppt salinity treatment made with pure salt (NaCl) and tap water, initiated five weeks after planting when shoots had fully emerged; and (2) water only (2 treatments x 44 genets x 6 replicates = 528 rhizome pieces). We chose 5 ppt because field conditions at the marsh sites were typically in the range of 2–10 ppt (C. L. Richards, unpublished data). Salinity treatments were administered twice weekly with enough water to completely flush the soil to prevent the buildup of salt through evapotranspiration. The flats were placed in a temperature-controlled greenhouse under conditions approximating midsummer in Suffolk County, Long Island. Day temperature was maintained at 30°C and night temperature at 25°C. We grew the plants for approximately eight months because response to salinity is thought to require long time spans, especially to detect variation in tolerance between genotypes (Yeo et al., 2000).

Traits measured
We measured six traits related to stress response, salt tolerance, and overall performance for each plant: _PSII (using a LI-6400 portable gas exchange and fluorescence system; Li-Cor, Lincoln, Nebraska, USA), succulence (g water in all leaves/cm² total leaf area), total leaf area (using a LI-3100 leaf area meter; Li-Cor), height, root-to-shoot based on dry biomass, and total biomass at final harvest. Light-adapted quantum efficiency of photosystem II (_PSII) was measured on the third uppermost fully expanded leaf. Light response curves on multiple field- and greenhouse-grown plants indicated a saturating response to light at 1500 µmol•m^–2•s^–1 (R. L. Walls, unpublished data). Standard conditions for measurements therefore were 350 µmol CO₂, saturating light level 1500 µmol•m^–2•s^–1 and leaf temperature 28.7 ± 0.2°C. Measurements were taken once leaf parameters had stabilized (<1% total system coefficient of variation, change of CO₂ and H₂O signals over time) after 3–5 min. Once stabilized, steady state fluorescence (F_s) was measured. Then, maximum fluorescence yield (F_m') was measured after a saturating light pulse of 15000 µmol•m^–2•s^–1 for 0.7 s temporarily inhibited PSII photochemistry (Redondo-Gomez et al., 2006). Quantum efficiency of PSII was calculated as: _PSII = (F_m' – F_s)/F_m' (Genty et al., 1989).

For each plant, all live leaf tissue at final harvest was weighed before and after drying to calculate succulence. Plants were dried in a forced air oven at 60°C for at least 72 h to determine total dry biomass. We evaluated the performance of genets in terms of height and leaf area but used biomass as our overall measure of fitness to assess degree of adaptive response. These taxa have extensive clonal growth and many individuals may not flower at all in the field, so biomass is probably the best indicator of fitness (McLellan et al., 1997; Watson et al., 1997; Johnston et al., 2004).

Data analysis
We used the SAS statistical package (version 9.1.3 for Windows; SAS Institute, Cary, North Carolina, USA) for all data analyses. An ANCOVA model was constructed that included the effects of salt treatment, taxon, habitat, site nested within taxon and habitat (3 salt marsh and 3 roadside sites), genet nested within site, taxon and habitat, block, covariate of initial rhizome weight and the interactions between salinity treatment and (1) taxon, (2) habitat (3) site (nested within taxon and habitat), and (4) genet (nested within site, taxon and habitat). We evaluated each response variable by including only plants that were alive at harvest, and we performed a separate univariate mixed model ANCOVA using the type III method of PROC MIXED (sensu Littell et al., 2006). For these univariate analyses, treatment, taxon and habitat were considered fixed effects and genet, block, and genet by treatment interaction were designated as random effects. In addition, our design was a split plot with salt treatment as the whole plot factor. This design requires designating block and the block by salt treatment interaction terms as random effects and using the block by salt treatment interaction term as the error term to test for the main effect of salt treatment (Snedecor and Cochran, 1989; Littell et al., 2004). Prior to the analysis, all variables, with the exception of _PSII, were box-cox transformed to meet the assumptions of normality and homoscedasticity. Finally, we analyzed plant mortality using a nominal logistic model with the effects of salt treatment, site, genet nested within site, and rhizome weight as a covariate.

RESULTS

Identification and genotyping of the accessions
Using diagnostic leaf characteristics (Bailey and Wisskirchen, 2004), along with chromosome counts (2n = 66), we identified the plants from four of the six sites studied as F. xbohemica, produced from hybridization between F. japonica and F. sachalinensis. Our data cannot determine whether these individuals were F1 hybrids or later generation hybrids that were produced by the crossing of F1s. These sites represented two roadside (HL and RHC) and two marsh (RHBH and WH) habitats. One roadside (CMR) and one marsh (CMM) population included here appear to consist of the parent F. japonica (2n = 88, Table 1), which has the same genotype as the single clone found across Europe (Hollingsworth and Bailey, 2000). We found that no plants were F. sachalinensis.

We identified six different AFLP genotypes, which we labeled A–F (Table 1). Only one site had more than one genotype: the salt marsh site WH (genotypes A–C). Of the other five, the sites that were very near each other had the same genotype: all samples from CMM and CMR were genotype D, and all samples from RHBH and RHC were genotype E.

Response to experimental treatments
The three-way ANCOVA with initial rhizome mass as a covariate indicated that the F. japonica and F. xbohemica differed significantly in leaf area (P < 0.001), succulence (P < 0.01), height (P < 0.001) and R:S, but they did not differ in _PSII, total biomass, or plasticity of any of the traits (Taxon and Salt x Taxon terms, Table 2).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Reaction norms (means ± 1 SE) of Fallopia japonicaand F.xbohemicaacross two salt treatments for each site. (A) PSII, (B) total leaf area of all leaves at final harvest, (C) succulence as measured on all leaves at final harvest, (D) final height, (E) ratio of dry root biomass to dry shoot biomass, and (F) total dry biomass at final harvest.

View this table: [in this window] [in a new window]   Table 3. Nominal logistic regression of mortality of Fallopia japonicaandF.xbohemica. Wald 2 and significance are presented for the main effects of salinity treatment, site and genet nested within site.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Reaction norms (means ± 1 SE) of Fallopia japonicaand F.xbohemicafor succulence across two salt treatments for surviving genets at each site. (A) Center Moriches Marsh (CMM), (B) Riverhead Boathouse (RHBH), (C) Worthheim (WH), (D) Center Moriches Road (CMR), (E) Riverhead Chauncey Road (RHC), and (F) Hagerman Landing Road (HL).

View larger version (22K): [in this window] [in a new window]   Fig. 3. Reaction norms (means ± 1 SE) across two salt treatments for surviving genets of Fallopia japonicaat the Center Moriches marsh (CMM) site. (A) Total dry biomass at final harvest, (B) total leaf area of all leaves at final harvest, (C) final height, (D) ratio of dry root biomass to dry shoot biomass, and (E) succulence as measured on all leaves at final harvest. Dark black lines in each plot indicate genets CMM1 and CMM6, which maintained biomass across the treatments.

In coastal areas of North America, invasive species have dramatically changed the community makeup of local ecosystems, and many studies suggest that the ability to tolerate novel conditions through plasticity or rapid differentiation of trait mean or plasticity is likely an important component of invasion ecology (Sayce, 1988; Daehler and Strong, 1997; Ayres et al., 1999; Saltonstall, 2002; Silliman and Bertness, 2004; Freeman and Byers, 2006; Richards et al., 2006; Muth and Pigliucci, 2007). In this study, we investigated how hybridization, adaptive evolution, and/or phenotypic plasticity may have contributed to increased salt tolerance in one of the world’s most invasive plants. We found that six Long Island Japanese knotweed populations were either F. japonica or F. xbohemica, with both taxa established in salt marsh habitat. Across the six sites, clonal diversity was low with only the same F. japonica genotype that has been found across Europe and in the United States (Hollingsworth and Bailey, 2000; Grimsby et al., 2007) and five F. xbohemica AFLP genotypes. The two taxa differed in several traits, but not in total biomass or response to salt treatments. Across the two taxa, plants from both roadside and marsh habitats were highly plastic in response to treatment with salt, and within sites, there were significant genet differences in biomass, as well as genet variation in plasticity for succulence, leaf area, R:S, and height. This trait variation is surprising because these populations have very little or no genetic variation as measured by AFLP markers. Despite this phenotypic variation across sites, plants from the salt marsh habitats did not perform better in the salt treatment, suggesting that selection by the salt content of the marsh habitat has not created genotypes adapted to high salinity. Instead, the results demonstrate a substantial degree of phenotypic plasticity in these species and that a few F. japonica genets (from marsh site CMM), not F. xbohemica, may be especially tolerant.

Contribution of hybridization
Although Fallopia taxa have not been reported in these habitats before, novel trait expression resulting from hybridization (transgressive traits) could allow for this type of expansion into novel habitat (Ellstrand and Schierenbeck, 2000; Johnston et al., 2001; Rosenthal et al., 2002; Lexer et al., 2003). Several recent studies have documented that transgressive trait expression has important ecological consequences (Weber and D’Antonio, 1999; Gaskin and Schaal, 2002; Rosenthal et al., 2002, 2005; Lexer et al., 2003; Gross et al., 2004; Johnston et al., 2004; Ludwig et al., 2004; Karrenberg et al., 2006). Johnston et al., (2004) found that high clonal fitness in F1 hybrids between Iris brevicaulis and I. fulva may allow for this genotypic class to perform well in many habitats. In particular, F1s had a greater allocation to shallow roots than did both parental species, which was thought to benefit these individuals by maintaining roots in more oxygenated layers of flooded soils. In addition, extremely high specific leaf area in the F1s could contribute to shade tolerance in these individuals. In another system, hybrids between the native Carpobrotus chilensis and native C. edulis had higher biomass in response to low salinity treatments under low nutrient conditions (Weber and D’Antonio, 1999), indicating that they may have an advantage in nutrient poor soils. These and other studies suggest that novel genome combinations may allow for rapid adaptation to new environments by recombining different genetically based adaptations (Anderson and Stebbins, 1954; Ellstrand and Schierenbeck, 2000; Facon et al., 2005; Gross and Rieseberg, 2005).

Hybridization between F. japonica and F. sachalinensis to form the hybrid F. xbohemica has similarly been highlighted as a mechanism capable of increasing the genetic diversity of the depauperate invasive F. japonica gene pool (Pysek et al., 2003; Bímová et al., 2004; Mandák et al., 2004). Such a combination brings together two taxa with different distributional ranges in Japan: F. japonica extends much farther south than F. sachalinensis. These taxa can also occur close to the sea in native habitat, so there may have been selection for salt tolerance in some populations. We therefore expected that the populations of Japanese knotweed s.l. invading novel salt marsh habitat might be composed of particularly aggressive F. xbohemica genotypes. Morphology and chromosome counts of these populations suggest that four of the six populations are made up of hybrids between the two species F. japonica and F. sachalinensis and that hybridization may be an important source of genetic variation in North American Fallopia populations. Considering we found only one genotype of F. japonica in this study and have not found any F. sachalinensis in this study or in the course of a more extended survey (C. Richards and M. Pigliucci, unpublished data), our findings corroborate studies in Europe concluding that F. xbohemica has greater genetic diversity than either parent in the invasive range (Pysek et al., 2003; Bímová et al., 2004; Mandák et al., 2005). Our data also concur with recent studies by Kesseli and colleagues that reveal sexual reproduction and seedling establishment in Massachusetts (Forman and Kesseli, 2003; Gammon et al., 2007; Grimsby et al., 2007).

However, even if transgressive segregation through hybridization might be a source for increased salt tolerance, we found that one of our robust salt marsh populations consisted only of the same single vigorous F. japonica clone that has been found throughout the invasive range. In addition, across all the sites, the genets that seemed to be most tolerant of our salt treatments were from the CMM marsh population and were most likely the same vigorous F. japonica clone. The presence of F. japonica at this marsh site means that at least one of the parent species is capable of invading salt marsh habitat and that this salt tolerance could be inherited by hybrids or back cross hybrids. Although we did not find direct evidence for the presence of F. sachalinensis, the existence of several hybrid genotypes indicates that either there is F. sachalinensis available somewhere on Long Island or that these hybrids are advanced generation hybrids. The fact that one F. japonica population exists in the salt marsh habitat may be significant in the future because any seed produced in the salt marsh will be immediately subject to selection for salt tolerance. The formation of any hybrid or backcross seed with grossly rearranged genotypes may provide a natural laboratory for the production of plants that are even more salt tolerant. Such an increase in salt tolerance may ultimately allow Japanese knotweed to move into higher saline areas of the salt marsh and displace the more salt tolerant salt marsh flora.

Response to salt treatment
Another successful invader, Phragmites australis, is historically found in freshwater marshes, but recently has been aggressively invading marshes exposed to full strength seawater and displacing native marsh plants (Amsberry et al., 2000; Minchinton and Bertness, 2003; Silliman and Bertness, 2004). The latest findings suggest that the Phragmites invasion of salt marshes could be the result of introduction of a particularly aggressive, nonnative genotype in areas where habitats have been altered by human activity (Saltonstall, 2002; Silliman and Bertness, 2004). This invasive, nonnative genotype has increased salt tolerance compared to the native, as measured by a greater rate of new shoot initiation, higher growth rates across the salinity range, and the ability to tolerate salinities up to three times that tolerated by the less salt-tolerant native Phragmites (Vasquez et al., 2005). Because Japanese knotweed s. l. has not been previously reported in saline habitat, we suspected that there may be a similar increased salt tolerance in the populations that are invading salt marshes compared to those established in the more common roadside habitat. This type of adaptive response could be evolving in Japanese knotweed s. l. regardless of whether the Long Island populations consist of F. japonica, F. sachalinensis, F. xbohemica or mixed populations. We expected salt treatments to reduce growth and overall performance for plants from all sites. This reduction was true in general, although there was variation within and among sites for most traits. However, we expected plants from salt marsh populations to suffer less from the effects of salinity, but they did not. Instead, plants from both marsh and roadside sites may tolerate or avoid salt stress equally well on average.

Because PSII efficiency integrates multiple functional traits, we anticipated that this trait would be a good indicator of tolerance to salt conditions and that genets that were particularly tolerant would have less reduction in _PSII in response to salt treatment. Although we did not measure stomatal conductance or transpiration rate, _PSII was decreased by half in the salt treatment, suggesting that the salt treatment did lead to reduced stomatal conductance and reduced efficiency of PSII. However, the lack of differences among plants within and among sites indicates that this trait may not be important in the differential success of genets.

The differences in succulence in response to salt among sites suggest that succulence is one trait that could aid in the adaptation to salt marsh habitats in Fallopia. This trait could be important for invasion of salty habitats because ions taken up from the salt water are typically stored in the vacuole of the cell. Therefore, the ability to become succulent and dilute the toxic effect of concentrated salt ions is essential (Flowers et al., 1977). Reimann and Breckle (1995) found dramatic intraspecific variation in succulence in Salsola kali originating from different sources and concluded that the salt tolerant subspecies S. kali traga was able to increase succulence more than the nonsalt tolerant S. kali ruthenica. In our Japanese knotweed plants, we found a high degree of variation in succulence in response to salt treatment within and among roadside and marsh populations. Many genets across all six sites had no change or even a slight reduction in succulence. Such a seemingly maladaptive response is consistent with an historical lack of selection for salt tolerance. However, despite the fact that our AFLP data suggest no genotypic diversity at five of the six sites, every site had at least one genet that more than doubled in succulence, indicating that at least some genets had the potential to tolerate higher salt concentrations through increased succulence.

The dramatically different response from what should be the same genotype is surprising and under other circumstances could reflect the importance of maternal effects or provisioning. However, in our study we were careful to start plants with similar initial rhizome masses (8–12 g) and the covariate term for rhizome mass was not significant for most traits, including succulence. One possibility is that these are rare genotypes that established by seed and we somehow missed them in our AFLP sampling. However, each rhizome we collected was at least a meter in length, suggesting that the individual was substantial in size. It seems unlikely therefore, that we would have missed them in our sampling.

Another possibility is that other fine scale effects, like persistent epigenetic effects, may have been induced on a microenvironmental scale and may be effecting the expression of important phenotypes like succulence. There is now mounting evidence that heritable variation in ecologically relevant traits can be generated through a suite of epigenetic mechanisms, even in the absence of genetic variation (Grant-Downton and Dickinson, 2005, 2006; Jablonka and Lamb, 2005; Rapp and Wendel, 2005; Richards, 2006). Recent studies indicate that in some cases environmentally induced epigenetic changes may be inherited by future generations (Richards, 2006; Whitelaw and Whitelaw, 2006; Bossdorf et al., 2008). More importantly, there is increasing evidence that epigenetic processes are an important component of hybridization events and may therefore play a key role in the biology of many invasive species (Rapp and Wendel, 2005; Salmon et al., 2005; Bossdorf et al., 2008). Our ongoing survey of methylation-sensitive AFLP (msAFLP) will shed light on the level of epigenetic variation in these populations (e.g., Cervera et al., 2002; Salmon et al., 2005; Keyte et al., 2006; C. L. Richards and M. Pigliucci, unpublished data).

Although the adaptive value of plasticity in succulence seems obvious, our study emphasizes the importance of dissecting the response of traits at the genet level and relating those responses to the effect on fitness (Richards et al., 2006; Muth and Pigliucci, 2007). Even though the comparison among sites suggests that increased succulence could be adaptive in this system, there was no relationship between succulence and biomass within the salt treated individuals (P = 0.5207), and a few genets that were able to maintain biomass across the two treatments actually had little response in succulence to salt. There may be a variety of strategies, even within this species complex, for tolerating salinity and a dramatic response in succulence may be important for only some genets. Further studies investigating other mechanisms of salt tolerance, as well as the extent and rate of spread of those few genets that maintained biomass but did not increase succulence would elucidate which other traits or combinations of traits are important for salt tolerance in natural populations of Japanese knotweed.

Whatever the mechanism of divergence, which could include drift, selection, transgressive segregation, or epigenetic effects, this study demonstrates that there is persistent phenotypic variation present in these Japanese knotweed populations and that the parental F. japonica genotype on Long Island is as tolerant as the hybrid F. xbohemica to our salt treatments. This variation in ecologically important traits provides the potential for future adaptation that could increase its already high rate of spread in North America and in salt marsh habitats in particular.

FOOTNOTES

¹ The authors thank J. Wendel, R. Percifield, and J. Hawkins for generous assistance with developing and interpreting the AFLP genotyping and Stony Brook University greenhouse staff M. Axelrod and J. Clumpp, as well as D. Arcuri, O. Bossdorf, and J. Banta for assistance in the greenhouse and with harvesting. J. Banta, O. Bossdorf, and N. Muth provided valuable comments on the manuscript. The authors thank J. Funk and an anonymous reviewer for thoughtful review of the manuscript. This work was partially supported by the Research Foundation of the State University of New York and a New York SEA grant.

⁵ Author for correspondence (e-mail: clr3{at}nyu.edu), phone: 212-998-8465, fax: 212-995-4015

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