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(American Journal of Botany. 2002;89:472-478.)
© 2002 Botanical Society of America, Inc.

Ecology

Habitat divergence between a homoploid hybrid sunflower species, Helianthus paradoxus (Asteraceae), and its progenitors1

Mark E. Welch2 and Loren H. Rieseberg

Indiana University, Department of Biology, Bloomington, Indiana 47405 USA

Received for publication June 5, 2001. Accepted for publication September 7, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The diploid hybrid species Helianthus paradoxus is restricted to salt marshes with sodium concentrations that exceed those found in the habitats of its progenitors, H. annuus and H. petiolaris. The observed association with saline habitats has led to the hypothesis that H. paradoxus is more salt tolerant than its progenitors. This hypothesis was tested by growing all three species in three NaCl treatments (0 mmol/L, 100 mmol/L, and 200 mmol/L). Helianthus paradoxus treated with NaCl was found to be more than five times as fit, in terms of biomass and survivorship, than its progenitors. Selection for salt tolerance in early generation hybrids may have contributed to the formation of H. paradoxus because theory predicts that homoploid hybrid speciation is feasible even when selection favoring hybrid genotypes is much weaker. Additionally, we show that H. paradoxus is significantly different from its parental species for several traits that often distinguish salt-tolerant species and suggest a mechanistic basis for the elevated salt tolerance expressed by H. paradoxus.

Key Words: Asteraceae • Helianthus paradoxus • hybridization • salt tolerance • speciation • sunflowers • transgressive segregation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Many authors believe that habitat divergence plays a crucial role in speciation, particularly in sessile organisms such as plants (Templeton, 1981 ; Levin, 2000 ). In the classic allopatric model of speciation, geographically isolated populations diverge through selection associated with local environments and through genetic drift (Mayr, 1963 ). If significant habitat divergence accrues between these populations, then competition will be reduced upon secondary contact. Prezygotic reproductive isolation is likely because individuals with divergent habitat requirements will be less likely to meet and mate (Templeton, 1981 ; Schluter, 1998 ). Postzygotic isolation is probable as well, because hybrids between the divergent parental populations are likely to be less fit in parental habitats.

Habitat divergence may be even more important when speciation is sympatric or parapatric, because the evolution of reproductive barriers must somehow take place in the presence of gene flow. This process occurs most readily if the reproductive barrier arises as a correlated by-product of divergent selection (Antonovics, 1968 ; Felsenstein, 1981 ; Rice and Hostert, 1993 ). As noted above, habitat divergence provides a particularly plausible means by which this might occur, because both prezygotic and postzygotic reproductive isolation are likely by-products of adaptation to local environments.

One form of sympatric or parapatric speciation that has received increased attention recently is diploid or homoploid hybrid speciation (Abbott, 1992 ; Arnold, 1997 ; Rieseberg, 1997 ). This mode of speciation requires that a fertile hybrid genotype that is at least partially reproductively isolated from its parents becomes locally fixed (Stebbins, 1957 ; Rieseberg, 1997 ; Brochmann, Borgen, and Stabbetorp, 2000 ). Theory indicates that this process is most likely to be driven by habitat divergence (McCarthy, Asmussen, and Anderson, 1995 ; Buerkle et al., 2000 ), and in fact, all well-documented examples of homoploid hybrid plant species occur in habitats that are different from those of their parental species (reviewed in Rieseberg, 1997 ). Hybrid animal taxa appear to follow this same trend (Taylor and Hebert, 1992 ; Taylor, Hebert, and Colbourne, 1996 ; Dowling and Secor, 1997 ).

So how do new hybrid lineages come to occupy different habitats? The leading hypothesis is based on the observation that segregating hybrids frequently exhibit traits that are extreme relative to those of either parental species (Anderson and Stebbins, 1954 ; Lewontin and Birch, 1966 ; Rieseberg, Archer, and Wayne, 1999 ). Referred to as transgressive segregation, this phenomenon appears to be a consequence of parental species being fixed for sets of genes that have opposing effects within species (de Vicente and Tanksley, 1993 ; Rieseberg, Archer, and Wayne, 1999 ). In this complementary gene model, recombination may generate hybrid genotypes with gene effects in the same direction, producing extreme phenotypes. If the habitat occupied by a hybrid population is extreme relative to that of either parental species, then selection for a transgressive genotype/phenotype could readily lead to its fixation.

While this scenario is appealing, we know little about the specific traits that facilitate habitat divergence in hybrid lineages. However, specific hypotheses have been suggested. For example, Wang and Szmidt (1994) suggest that Pinus densata, a high-altitude, homoploid hybrid pine species, may be more tolerant of colder temperatures than either of its parental species, which are associated with lower altitude temperate and semitropical forests. Likewise, 18 of 50 morphological and ecophysiological traits in a hybrid sunflower species, Helianthus anomalus, were significantly transgressive when compared to those of its parental species (Schwarzbach, Donovan, and Rieseberg, 2001) . Many of these traits could be interpreted as adaptations for the sand dune habitat occupied by H. anomalus, but their fitness effects were not tested. The authors note that it is difficult to distinguish among traits required for habitat divergence, correlated traits, and traits arising as incidental by-products of hybridization.

Here, we consider one case where habitat divergence may have played a pivotal role in hybrid speciation. Helianthus paradoxus, the puzzle sunflower, appears to have arisen through hybridization between H. annuus and H. petiolaris (Rieseberg, Carter, and Zona, 1990 ; Rieseberg, 1991 ). It grows exclusively in brackish marshes that have soil sodium concentrations ranging from 2000 to 20 000 ppm (Rogers, Thompson, and Seiler, 1982 ; C. Lexer, Indiana University, unpublished data), typically in habitats associated with Distichlis spicata, saltgrass (Bush and Van Auken, 1997 ; Van Auken and Bush, 1998 ). Plant species found in habitats with soil salt concentrations greater than 100 mmol/L, such as H. paradoxus, are thought to require special adaptations and are classified as halophytes (Flowers, Hajibagheri, and Clipson, 1986 ). The progenitors of H. paradoxus occur in more xeric and less halogenous soils; soil sodium concentrations average 24 ± 10 ppm (± SE) for five distinct H. annuus habitats, and 120 ± 27 ppm for six distinct H. petiolaris habitats (M. Welch, Indiana University, unpublished data). This habitat segregation may be due to differences in salt tolerances among these three species, or soil sodium concentrations may be coincidental with other ecological parameters. Previous reports suggest that H. paradoxus can survive at higher salt concentrations than cultivated lines of H. annuus, supporting the salt tolerance hypothesis (Miller, 1993, 1995 ). However, the fitness of the three taxa when challenged with salt concentrations comparable to those found in the wild has not been measured, and nothing is known about salt tolerance in H. petiolaris. This study tests the hypothesis that H. paradoxus is more salt tolerant than both parental species.

We tested the salt tolerance hypothesis by measuring two indicators of fitness, survivorship and biomass, in H. paradoxus and its progenitors under varying degrees of salinity in a laboratory environment. We then considered whether habitat-dependent fitness differences among species are strong enough to promote speciation. Finally, we determined whether H. paradoxus is transgressive for specific traits that typically distinguish halophytes from salt sensitive species (glycophytes), including succulence, degree of salt-induced root growth inhibition, levels of sodium bioaccumulation, and water-use efficiency. These data suggest a possible mechanistic basis for salt tolerance differences among these species and provide ecological insight into the genesis of H. paradoxus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant material
Achenes from the following four accessions were used in these experiments: wild H. annuus from the Hopi Indian Reservation ~400 m east of Second Mesa, Arizona, USA (Rieseberg no. 1294); H. paradoxus from Grants, New Mexico, USA (courtesy of C. MacDonald, United States Fish and Wildlife Service, Albuquerque, New Mexico, USA); H. petiolaris from 8 km southeast of Moenkopi, Arizona, USA (Rieseberg no. 1283); and cultivated H. annuus, cms HA89 (cytoplasmic male sterile HA89, courtesy of G. Seiler, USDA-ARS, Fargo, North Dakota, USA). The latter cultivar was included in this experiment because it was used in a separate study designed to map quantitative trait loci (QTL) for salt tolerance in sunflower (M. E. Welch and L. H. Rieseberg, unpublished data).

Germination
To synchronize germination across species, the peduncular ends of ~60 achenes from each accession were removed with a razor blade. Seeds were then allowed to imbibe one of the three following treatment solutions: 0 mmol/L, 100 mmol/L, or 200 mmol/L NaCl. These salt concentrations are within the range reported for natural populations of H. paradoxus (Rogers, Thompson, and Seiler, 1982 ; C. Lexer, Indiana University, unpublished data). After 72 h, 12 germinating seeds from each accession and treatment were selected at random for planting (3 treatments x 4 accessions x 12 individuals = 144).

Growth conditions
Selected seeds were planted in 13-cm plastic pots with two parts sand and one part soil. Pots were spaced in plastic trays and subirrigated with 6 L of treatment solution and 6 g of Peter's 15-16-17 (N-P-K) fertilizer (Scotts-Sierra Horticultural Products Company, Marysville, Ohio, USA). Treatment solutions were the same as those used for germination. Four blocks of each treatment consisting of three plants from each accession were implemented (3 treatments x 4 accessions x 4 blocks x 3 individuals = 144). Blocks, which we defined as individual subirrigation trays, were arranged in four rows in a Conviron PGW 36 growth chamber (Controlled Environments, Winnipeg, Manitoba, Canada) so that each treatment was represented only once per row. Solution levels in trays were monitored daily and maintained with deionized water. The growth chamber was set to provide a 16 h photoperiod and maintain a constant temperature of 25°C. Photosynthetically active radiation (PAR) was approximated at 400 µmol·m–2·s–1 at soil level using a Licor LI-250 (Licor, Lincoln, Nebraska, USA). Because of the standing water in the subirrigation trays, the relative humidity in the growth chamber was generally quite high (~80%).

Data collection
Plant survivorship was assessed 21 d after planting. Plants that were erect and green were considered to be survivors.

Plants were harvested 23 d after planting. Roots were rinsed clean of soil and sand and plants were separated into roots, shoots, and the second set of true leaves. Fresh mass of these parts were measured immediately after harvesting. Dry mass of plant sections were measured after being oven dried at 60°C for 3 d.

Sodium concentrations (in parts per thousand; ppt) in second true leaves and roots were measured by means of inductively coupled argon plasma (ICAP) spectrometry (Midwest Laboratories, Omaha, Nebraska, USA).

Stomatal conductance (H2O loss per unit leaf area; in moles of H2O per square meter per second) and photosynthetic rates (CO2 uptake per unit leaf area; in micromoles of CO2 per square meter per second), were measured using a Licor 6400 portable photosynthesis system at PAR of ~400 µmol·m–2·s–1, light conditions similar to those in the growth chamber. Measurements were taken from a single leaf on each plant between 1000 and 1600 over a 3-d period beginning 21 d after planting. Variation associated with time of day was assumed to be negligible because of the constant light conditions in the growth chamber. Further, plants of a block were measured consecutively so that any such variation should appear as block effects (see Data analysis below).

Data analysis
Multiplicative fitness estimates for individual plants were calculated by combining two proxies of fitness; survivorship and total dry biomass. Individuals that failed to survive the experiment were scored as having zero fitness, and total dry biomass was scored as fitness for the survivors. Individual multiplicative fitness values were then divided by the mean multiplicative fitness of the accession under the 0 mmol/L NaCl treatment to calculate a species relative fitness; this is the fitness value that is analyzed and discussed throughout this paper. Fitness was computed in this manner to account for inherent differences between species with respect to growth rate and size. Leaf succulence (percentage H2O) was calculated by subtracting leaf dry biomass from leaf fresh biomass, dividing by leaf fresh biomass, and multiplying by 100. Root to shoot ratio is root dry biomass divided by shoot dry biomass. Water-use efficiency (WUE) was calculated by dividing photosynthetic rate by stomatal conductance (in micromoles CO2 to moles H2O).

We analyzed fitness, leaf succulence, root to shoot ratio, leaf sodium concentrations, and WUE using mixed model ANOVAs. In the basic ANOVA design, NaCl treatment, accession, NaCl treatment by accession interaction, and block effects were tested for significance. Blocks were nested within NaCl treatments to account for variation attributable to differences among blocks. Because of low survivorship in the high salt treatment, it was necessary to omit this treatment from ANOVA for all traits except fitness. Tukey-Kramer honestly significant difference (HSD) tests were performed to determine which accession means within all three treatments were significantly different.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Fitness
Survivorship varied dramatically across accessions and treatments. All 48 plants (4 accessions x 12 individuals) survived the 0 mmol/L NaCl treatment, and only 19 individuals survived the 200 mmol/L NaCl treatment. Helianthus paradoxus had the highest rate of survival in both the 100 mmol/L and 200 mmol/L treatments (Table 1). Total dry biomass also varied greatly across accessions and treatments. In the 0 mmol/L treatment, the two H. annuus accessions acquired more biomass than H. paradoxus, which in turn acquired more biomass than H. petiolaris (Table 1). However, in the 100 mmol/L treatment there was little difference among mean biomasses of H. paradoxus and the two H. annuus accessions. Helianthus paradoxus had the highest mean biomass in the 200 mmol/L treatment.


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  		Table 1. Plant measurements (mean ± SE and ranges for continuous traits) from all accessions and all NaCl treatments; N is 12 unless otherwise noted in parentheses. Dashes indicate that no measurements could be taken for a specific accession at a given treatment level

 
Fitness was significantly affected by accession, treatment, and block effects, as well as accession by treatment interactions (Fig. 1; Table 2). Because species means are set equal to 1.0 in the 0 mmol/L treatment, fitness variation attributable to accession and accession by treatment interaction effects must result from differences among accessions in the 100 mmol/L and 200 mmol/L treatments. The Tukey-Kramer HSD test results indicate that the mean fitness of H. paradoxus is significantly higher than that of all other accessions in the 100 mmol/L and 200 mmol/L treatments and that no significant fitness differences exist among the remaining accessions in these treatments (Fig. 1). Thus, significant accession and accession by treatment interaction effects largely result from higher fitness of H. paradoxus at 100 mmol/L and 200 mmol/L NaCl.



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  		Fig. 1. Mean fitnesses of all accessions of Helianthus across NaCl treatments. All individual fitness values were adjusted by accession mean fitness in the 0 mmol/L NaCl treatment (see text). Bars represent SE, and accession means from the same treatment sharing a letter (a–c) were not significantly different using the Tukey-Kramer HSD test. N for all accessions in all treatments is 12

 

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  		Table 2. P values from ANOVA of experimental effects on plant measurements. Note that block effects were nested within treatments

 
Leaf succulence
Among individuals in the 0 mmol/L and 100 mmol/L treatments, significant variation in leaf succulence could be attributed to accession effects (Table 2). Treatment effects were only marginally significant. Tukey-Kramer HSD test results demonstrate that leaf succulence in the second true leaves of H. paradoxus is greater than that of all other accessions in all three treatments (Fig. 2). Thus, leaves of H. paradoxus are significantly more succulent than those of its parental species.



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  		Fig. 2. Mean leaf succulence (percentage H2O by mass) of all accessions across NaCl treatments. Bars represent SE, and accession means from the same treatment sharing a letter (a–c) were not found to be significantly different using the Tukey-Kramer HSD test. N for all accessions in the 0 mmol/L NaCl treatment and for H. paradoxus in the 100 mmol/L treatment is 12. N in the 100 mmol/L treatment is 9 for both H. annuus accessions and 4 for H. petiolaris. In the 200 mmol/L treatment, N is 4, 3, and 10 for wild H. annuus, cms HA89, and H. paradoxus, respectively. The one H. petiolaris individual surviving the 200 mmol/L treatment was not large enough for leaf succulence to be measured

 
Root to shoot ratios
All experimental factors had highly significant effects on root to shoot ratios among individuals in the 0 mmol/L and 100 mmol/L treatments (Table 2). The mean root to shoot ratio of H. paradoxus was found to be lower than that for both H. annuus accessions at 0 mmol/L NaCl, but greater than all accessions in both the 100 mmol/L and 200 mmol/L treatments. This caused a significant accession by treatment interaction effect (Fig. 3).



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  		Fig. 3. Mean root to shoot ratios for all accessions across NaCl treatments. Bars represent SE, and accession means from the same treatment sharing a letter (a–c) were not found to be significantly different using the Tukey-Kramer HSD test. N for all accessions in the 0 mmol/L NaCl treatment and for H. paradoxus in the 100 mmol/L treatment is 12. N in the 100 mmol/L treatment is 9 for both H. annuus accessions and 4 for H. petiolaris. In the 200 mmol/L treatment, N is 4, 3, and 10 for wild H. annuus, cms HA89, and H. paradoxus, respectively. The one H. petiolaris individual surviving the 200 mmol/L treatment was not large enough for root to shoot ratio to be measured

 
Sodium concentrations
Variation in leaf sodium concentration among individuals in the 0 mmol/L and 100 mmol/L NaCl treatments was significantly affected by accession, treatment, and accession by treatment interactions, but not by block (Table 2). Helianthus paradoxus had significantly higher leaf sodium concentrations than all other accessions in all treatments (Fig. 4). Further, mean sodium concentrations in H. paradoxus have a greater response to increasing NaCl than any other accession accounting for the significant interaction effect.



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  		Fig. 4. Mean sodium content by mass (ppt) for all accessions across NaCl treatments. Bars represent SE, and accession means from the same treatment sharing a letter (a–c) were not found to be significantly different using the Tukey-Kramer HSD test. N for all accessions in the 0 mmol/L NaCl treatment and for H. paradoxus in the 100 mmol/L treatment is 12. N in the 100 mmol/L treatment is 9 for both H. annuus accessions and 4 for H. petiolaris. In the 200 mmol/L treatment, N is 4, 3, and 10 for wild H. annuus, cms HA89, and H. paradoxus, respectively. The one H. petiolaris individual surviving the 200 mmol/L treatment was not large enough for leaf sodium concentration to be measured

 
Root sodium concentrations are different from the leaf sodium concentrations (Table 1). Roots of H. paradoxus had the highest sodium concentration in the 0 mmol/L treatment, but no significant difference between H. paradoxus and either H. annuus accession was detected at 100 mmol/L or 200 mmol/L NaCl. Comparisons with H. petiolaris were not feasible in the 100 mmol/L and 200 mmol/L treatments because insufficient root tissue was available for analysis.

Water-use efficiency
Accession, treatment, and block effects were all significant with regard to WUE (Table 2). Tukey-Kramer HSD test results indicated that WUE values from H. petiolaris were significantly different from those for wild H. annuus in the 0 mmol/L treatment and significantly different from those of all accessions in the 100 mmol/L treatment. No other pairwise difference among means from the same treatment was found to be significant (Fig. 5). Note that physiological measurements for three cms HA89 individuals in the 0 mmol/L treatment were not collected because of obvious signs of drought stress; plants were removed from subirrigation trays 24 h prior to measurement and the pots of these three individuals dried out.



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  		Fig. 5. Mean water-use efficiency (WUE, in micromoles of CO2 per moles of H2O) for all accessions across NaCl treatments. Bars represent SE, and accession means from the same treatment sharing a letter (a–c) were not found to be significantly different using the Tukey-Kramer HSD test. N for all accessions in all treatments are the same as for stomatal conductance and photosynthetic rate and are shown in Table 1

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Fitness
Helianthus paradoxus is significantly more tolerant of salt than either parental species, regardless of the fitness component measured. It had higher survivorship, maintained more of its biomass, and exhibited greater relative root growth when treated with NaCl than did either of its parental species (Table 1; Figs. 1 and 3). These findings are consistent with previous reports that showed H. paradoxus to be more resilient than H. annuus when treated with NaCl (Miller, 1993, 1995 ) and demonstrate that H. petiolaris may be even more susceptible to NaCl than H. annuus. Thus, H. paradoxus can be viewed as significantly and positively transgressive for salt tolerance relative to its parental species. The low fitness of H. annuus and H. petiolaris in a saline environment suggest that this may be an important ecological parameter in defining habitat boundaries among these species.

The fitness differences between H. paradoxus and its parental species in a saline environment appear to be strong enough to account for its origin. When treated with NaCl, Helianthus paradoxus is 5–14 times more fit, in terms of biomass and survivorship, than H. annuus and 15–47 times more fit than H. petiolaris. Theory indicates that hybrid speciation is feasible even when hybrids have less than a twofold habitat-dependent fitness advantage over their progenitors (McCarthy, Asmussen, and Anderson, 1995 ; Buerkle et al., 2000 ). Thus, if early generation hybrids had only a fraction of the selective advantage documented for H. paradoxus, then it should have been strong enough to drive homoploid hybrid speciation. It is noteworthy that neither theoretical study considered scenarios in which hybrid viability was greater than twice that of the parental species. However, both the rate of origin and persistence of hybrid neospecies were shown to increase with greater hybrid fitness, presumably indicating that a selective advantage as large as that reported here should further increase the probability of establishing and maintaining a hybrid neospecies. Note that both of these simulation studies are based on the model of recombinational speciation, in which chromosomal rearrangements contribute to reproductive isolation between parental species and their hybrid derivative (Grant, 1981 ).

The mechanistic basis for salt tolerance
High soil sodium concentrations stress plants through both ionic and osmotic effects (Hasegawa et al., 2000) . When sodium reaches high cytoplasmic concentrations certain crucial metabolic enzymes exhibit a reduction or a complete loss of function in vitro (Yeo, 1998 ). To avoid the toxic effects sodium may have in the cytoplasm, numerous species (including cultivated H. annuus) actively exclude it (Flowers, Troke, and Yeo, 1977 ; Flowers, Hajibagheri, and Clipson, 1986 ; Ashraf and O'Leary, 1995 ; Francois, 1996 ). However, this salt-tolerance strategy may only be effective at relatively low salt concentrations, because an increasing difference between soil and cytoplasm solute concentrations should result in increasing osmotic stress on plants (Hasegawa et al., 2000) . Thus, sodium exclusion may be metabolically expensive, because plants using this strategy must pump ions against an osmotic gradient and may be forced to invest resources in osmotic adjustment (Hasegawa et al., 2000) .

An alternative salt tolerance strategy involves the internal accumulation and sequestration of sodium. This strategy allows plants to avoid much of the osmotic stress experienced by sodium excluders, because it reduces the difference between the osmotic potentials of the soil and the plant (Flowers, Hajibagheri, and Clipson, 1986 ). Relative to total dry biomass, H. paradoxus has significantly higher sodium concentrations in its leaves than either of its parental species (Fig. 4). Because the cytosolic enzymes of halophytes and glycophytes are not thought to differ in their resistance to the toxic effects of sodium, accumulators must be able to sequester sodium outside of the cytoplasm or produce metabolites that ameliorate the adverse effects of high cytoplasmic sodium concentrations (Flowers, Hajibagheri, and Clipson, 1986 ). Many halophytes have been shown to sequester sodium in the vacuoles by means of sodium pumps (Hasegawa et al., 2000) , and strong evidence indicates that even glycophytic H. annuus cultivars can compartmentalize sodium in their roots (Ballesteros et al., 1997 ). We found leaf sodium concentrations in wild sunflower accessions to be higher than that for cultivated H. annuus treated with NaCl (Fig. 4). Of the accessions studied in this paper, H. paradoxus had a significantly higher concentration of sodium in the leaves and the greatest fitness. This suggests that salt tolerance in H. paradoxus may be dependent upon its capacity to sequester sodium in leaves.

Plants may also be more salt tolerant due to better water maintenance strategies. Rates of ion uptake are correlated with rates of transpiration, and hence a reduction in stomatal conductance may reduce salt stress on plants (Yeo, 1998 ). However, rates of carbon fixation are also correlated with transpiration rates, and photosynthate is necessary for plant biomass accumulation. Because plants can transpire copious amounts of water during gas exchange, they can lessen salt stress through increasing the efficiency of photosynthesis. Helianthus paradoxus does appear to improve its water-use efficiency under moderate salt stress (100 mmol/L NaCl, Fig. 4), and this improvement results almost entirely from a decrease in stomatal conductance; cms HA89 appears to react similarly (Table 1). The species in this experiment with the highest rate of carbon fixation, H. petiolaris, also had the lowest water-use efficiency, because its rate of stomatal conductance was also the highest (Table 1). This high rate may account for some of the sodium buildup in the leaves of this species as an alternative to sequestering sodium in the vacuoles and may in turn explain the salt-sensitive nature of H. petiolaris.

The physiological measures of photosynthesis can also be used to address the issue of ionic stresses in the cytoplasm, because certain enzymes in the photosynthetic pathway are known to be highly salt sensitive. Photosynthetic rates among these species do not decrease significantly with the addition of NaCl. The rate of carbon fixation in wild H. annuus actually increases, although not significantly, under moderate salt stress (Table 1). This suggests that differences in biomass accumulation among these species across salt treatments reflect the metabolic efficiency of their different salt tolerance mechanisms and not their capacity to photosynthesize.

With the exception of leaf sodium concentrations, the most striking difference recorded here is in degree of succulence. Helianthus paradoxus has significantly higher leaf succulence than either of its parental species in all treatments, and H. petiolaris is more succulent than H. annuus (Fig. 2). In fact, H. paradoxus is considerably more succulent than other halophytic members of the Asteraceae (see Flowers, Hajibagheri, and Clipson, 1986 ). The benefits of succulence to halophytes may be twofold. First, as mentioned previously, increasing water-use efficiency may reduce the rate of sodium ion uptake. Succulence may reflect an increased thickness in the palisade layer of the mesophyll where gas exchange actually occurs, increasing the potential rate of gas exchange without necessarily increasing the water loss that would occur by increasing the number of stomates. Second, a greater water volume may lead to lower cytoplasmic ion concentrations. This aspect of succulence is particularly intriguing given that H. paradoxus has the greatest sodium concentrations in the leaves relative to total dry biomass (Fig. 4), while the leaves of wild H. annuus under treatment with NaCl actually have slightly higher sodium concentrations relative to total fresh biomass.

Does the expression of traits correlated with salt tolerance in these three species match the expectations of the complementary gene action model for transgressive segregation? In other words, can we account for transgressive salt tolerance in H. paradoxus through combinations of traits found in H. annuus and H. petiolaris? While highly speculative, such a scenario does seem feasible. For example, it is possible that H. paradoxus acquired the genetic machinery necessary to sequester sodium in the leaves from H. annuus, whereas greater succulence and reduced root growth inhibition is derived from H. petiolaris.

Summary and conclusions
We have demonstrated that H. paradoxus, a homoploid, hybrid species, is more salt tolerant than its parental species. This finding supports the hypothesis that selection favoring extreme or transgressive traits in hybrids was instrumental in speciation. The selective advantage H. paradoxus has over its progenitors in a saline environment is much greater than viability advantages included in previous simulation studies, in which hybrid speciation was rare. Our findings reduce the odds against this speciational mode, at least for extreme habitats such as that characteristic of H. paradoxus.

Additionally, when compared to its progenitors, H. paradoxus was found to be extreme for several traits that are commonly associated with salt tolerance in plants. Most notable and statistically supported among these traits are leaf sodium concentration and leaf succulence, which suggest specific mechanisms confer greater salt tolerance in H. paradoxus. High leaf sodium concentrations are evidence of sodium sequestration, and greater leaf succulence may also increase salt tolerance. Because H. paradoxus is more similar to H. annuus in its leaf sodium concentrations and more similar to H. petiolaris in its leaf succulence, it's possible that complementation of genes associated with different salt tolerance mechanisms in the hybrid species may have led to its extreme salt tolerance. In the future, we hope to test this hypothesis by mapping quantitative trait loci (QTL) associated with leaf sodium concentrations and leaf succulence in H. paradoxus and determining the parental origin of these QTL. Our complementation hypothesis would be supported if QTL associated with greater leaf sodium concentration are predominantly derived from H. annuus and those associated with greater leaf succulence are largely derived from H. petiolaris.


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  		Table 1. Extended

 

   FOOTNOTES
 
1 The authors thank Nicole Welch and Ella Ingram for assistance in setting up the experiment; the Indiana University Greenhouse Staff for assistance and advice in plant care; Lynda Delph for loaning us her Licor 6400 portable photosynthesis system and instructing us in its use; and Troy Wood, Alex Widmer, Michele Arntz, John Burke, Eva Allen, Roger Hangarter, and Rhonda Rieseberg for comments on the manuscript. This study was supported by NIH grants R01 GM59065 and T32 GM07757-21. Back

2 Author for reprint requests (marwelch{at}indiana.edu ) Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Abbott R. J. 1992 Plant invasions, interspecific hybridization and the evolution of new plant taxa. Trends in Ecology and Evolution 7: 401-405[CrossRef]

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Antonovics J. 1968 Evolution in closely adjacent plant populations. V. Evolution of self-fertility. Heredity 23: 219-238

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Ashraf M. J. W. O'leary 1995 Distribution of cations in leaves of salt-tolerant and salt-sensitive lines of sunflower under saline conditions. Journal of Plant Nutrition 18: 2379-2388[ISI]

Ballesteros E. E. Blumwald J. P. Donaire A. Belver 1997 Na+/H+ antiport activity in tonoplast vesicles isolated from sunflower roots induced by NaCl stress. Physiologia Plantarum 99: 328-334[CrossRef]

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