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Species and population differences in plant ecophysiological traits related to carbon gain and resource use have long been observed and are often interpreted as potentially adaptive (Ackerly et al., 2000; Aerts & Chapin, 2000; Arntz & Delph, 2001). Phenotypic selection analysis provides a means of estimating the strength of direct and indirect selection on individual traits, and thus testing adaptive hypotheses for the evolution of these traits (Lande & Arnold, 1983; Brodie et al., 1995; Kingsolver et al., 2001). Detection of contrasting selection between habitats provides the strongest inference that a trait is adaptive in a particular habitat and that selection may drive population differentiation (Wade & Kalisz, 1990; Dudley, 1996a). Selection has been shown to vary spatially and temporally for morphological and life-history traits (Kalisz, 1986; Kelly, 1992; Bennington & McGraw, 1995; Maad & Alexandersson, 2004; Young, 2008). However, there have been relatively few tests of whether selection on plant ecophysiological traits differs by resource environment (Dudley, 1996a; Heschel & Riginos, 2005; Sherrard & Maherali, 2006; Saldana et al., 2007). Here we used a compilation of unpublished and published data for Helianthus to investigate the spatial and temporal variation in direct selection on plant ecophysiological traits.
Helianthus anomalus and Helianthus deserticola are annual sunflowers of recent homoploid hybrid origin (170 000–63 000 yr BP) from the same ancestral parent species: Helianthus annuus and Helianthus petiolaris (Schwarzbach & Rieseberg, 2002; Gross et al., 2003). The two hybrid species are endemic to desert southwest USA in habitats that differ from those of their ancestral parent species (Gross et al., 2003; Rosenthal et al., 2005a). Studies of naturally occurring plants in the hybrid species habitats indicate that the actively moving dune habitat of H. anomalus has lower N and higher water availability than the adjacent stabilized dune habitat of H. deserticola (Rosenthal et al., 2005a; Donovan et al., 2007). In addition, the growth of naturally occurring H. anomalus seedlings is enhanced by the addition of nitrogen (N) and water , but not by water alone (Ludwig et al., 2006). This raises the question of whether selection on ecophysiological traits differs between these two habitats and thus may have contributed to local adaptation and hybrid speciation. We address this question with a compilation of data for 10 Helianthus populations (Table 1), including artificial hybrids (BC2ann, BC2pet) planted into both H. anomalus and H. deserticola habitats, natural populations in both habitats, and H. anomalus planted into its own habitat. BC2ann and BC2pet are artificial hybrids of H. annuus and H. petiolaris (see the Materials and Methods section for details) that represent the ancestral hybrid populations from which the hybrid species arose and contain a range of values for individual traits and trait combinations, including ‘H. anomalus-like’ and ‘H. deserticola-like’ multi-trait phenotypes (Rieseberg et al., 2003; Rosenthal et al., 2005b). Hybrid speciation may have been facilitated by ‘escape’ of early generation hybrid phenotypes into the new habitats currently occupied by the H. anomalus and H. deserticola, and subsequent environmental selection (Rieseberg et al., 2003; Karrenberg et al., 2007).
Table 1. Summary of Helianthus populations analysed for phenotypic selection of leaf ecophysiological traits
|Population codes||Plant material||Habitat||Natural population or experimental plot||Year measured||Population sample size|
|BC2ann-ANO||BC2ann||H. anomalus||ANO plot1||2002||114|
|BC2pet-ANO||BC2pet||H. anomalus||ANO plot1||2002||104|
|BC2ann-DES||BC2ann||H. deserticola||DES plot2||2002||198|
|BC2pet-DES||BC2pet||H. deserticola||DES plot2||2002||192|
|BC2annF1-ANO-03||BC2ann-F1||H. anomalus||ANO plot||2003||175|
|H. anomalus-ANO||H. anomalus||H. anomalus||ANO plot1||2002||130|
|H. anomalus-ANO-03||H. anomalus||H. anomalus||ANO plot||2003||141|
|H. anomalus-wet||H. anomalus||H. anomalus||Natural population-wet3||2002||114|
|H. anomalus-dry||H. anomalus||H. anomalus||Natural population-dry3||2002||114|
|H. deserticola||H. deserticola||H. deserticola||Natural population3||2002||135|
For these Helianthus populations, we chose focal traits based on their potential relevance to the differing habitats and potential importance based on previous comparisons of these species in common environment experiments: individual leaf size (individual leaf area), leaf succulence, leaf N concentration, and time-integrated photosynthetic water-use efficiency (WUE, ratio of photosynthetic carbon gain to transpirational water loss) estimated from leaf carbon isotope ratio (δ13C). We expected water and N limitations in the H. anomalus and H. deserticola habitats to influence selection on these resource related traits (Ehleringer & Clark, 1988; Dudley, 1996a; Geber & Dawson, 1997; Heschel et al., 2002; Wright et al., 2003; Heschel & Riginos, 2005; Maron et al., 2007). Greater leaf size indicates more area for photosynthesis. In some species, leaf area is strongly correlated with plant size, although the causality is unclear (Dudley & Schmitt, 1996). Higher leaf succulence, as generally found in H. anomalus, may contribute to drought tolerance (Jennings, 1976; Brouillette et al., 2006). Leaf size, leaf N and leaf WUE are physiologically linked to each other and to carbon gain, water use and WUE. A smaller leaf size may increase WUE by reducing the leaf boundary layer and thus the leaf temperature and transpirational water loss for a given stomatal conductance (Jones, 1992; Nobel, 1999). Leaf N is a major determinant of the biochemical capacity of photosynthesis (Field & Mooney, 1986). Plants can increase leaf WUE by increasing leaf N invested in photosynthetic capacity and by decreasing stomatal conductance (Wright et al., 2001, 2003). However, only decreasing stomatal conductance will reduce water loss (Cohen, 1970; Ludlow, 1989). Smaller leaf size and greater WUE are generally expected to be favored in water-limited habitats, although there is debate about whether water limitation might also select for drought escape with lower WUE and short lifespan, depending on the timing of water availability (Geber & Dawson, 1990, 1997; Dudley, 1996a; Stanton et al., 2000; McKay et al., 2003; Heschel & Riginos, 2005; Sherrard & Maherali, 2006, Angert et al., 2007; Huxman et al., 2008).
Results from several of the study populations were previously published to address other objectives, as summarized here. Ludwig et al. (2004) compared the populations planted into the H. anomalus habitat (hereafter designated ‘ANO’ experimental plot) in 2002, including H. anomalus and artificial hybrids (BC2ann and BC2pet). There was selection on all of the leaf traits in at least one of the populations, and selection on some of the traits in the artificial hybrids was similar to, or could move the phenotype towards, the current H. anomalus phenotype. Gross et al. (2004) compared the artificial hybrids planted in to the H. deserticola habitat (hereafter designated ‘DES’ experimental plot) in 2002 for a different set of leaf traits and came to a similar conclusion that selection on some of the traits in the artificial hybrids could move the phenotype towards the current H. deserticola phenotype. Donovan et al. (2007) compared the wild populations in 2002 and found no evidence that the longer-lived H. anomalus is under selection for lower WUE characteristics consistent with drought tolerance, or that the shorter-lived H. deserticola is under direct selection on WUE consistent with drought escape.
By adding the previously unpublished 2003 ANO plot data and reanalysing the 2002 DES plot data for a comparable set of traits, we now have a compilation of data from 10 populations to ask several new questions. (1) Does selection differ spatially among habitats for artificial hybrids planted into both habitats? A spatial comparison of selection across habitats for the same traits and the same plant material provides the strongest inference of selective forces driving trait differentiation and local adaptation (Wade & Kalisz, 1990; see discussion in Dudley, 1996a; Geber & Griffen, 2003). To provide a context for habitat comparison, we ask two further questions: (2) Does direct selection differ spatially among planted and natural populations of H. anomalus within the H. anomalus habitat; and (3) Does direct selection differ temporally across years for planted populations within the H. anomalus habitat? Selection that varies between habitats, but not within a habitat or across years, is a likely candidate for driving population differentiation and speciation. Finally, we report the relationship of direct to total selection for each trait in order to address potential constraints on evolutionary response to selection.
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The contrast of the artificial hybrids (BC2ann, BC2pet) planted into the two habitats provides a direct test of the habitat differences. The hybrid plants grown in the ANO plot had less biomass than plants grown in the DES plot. Given that competition in both plots was minimized by the low plant cover characteristic of both habitats, and weeding of volunteers and planting design within the plots, the lower biomass in the ANO plot was most likely caused by abiotic factors such as N limitation and unstable sand surfaces (burial, excavation, abrasion from blowing sand). Although dunes look dry, they generally store more water than adjacent stabilized areas, provided that root growth is sufficient to access deeper soils (Pavlik, 1980; Rosenthal et al., 2005a). Lower biomass in the ANO plot was accompanied by lower leaf succulence, much lower leaf N and lower WUE compared with the DES plot, consistent with our expectation that the H. anomalus habitat has lower N availability and higher water availability than the H. deserticola habitat (Rosenthal et al., 2005a; Donovan et al., 2007). The two habitats differed for direct selection for leaf N and WUE in the BC2ann and BC2pet, respectively, but did not differ for direct selection on leaf size and succulence.
Selection on leaf N differed between habitats as expected on the basis of habitat resource availability. For BC2ann, direct selection tended to favor higher leaf N in the ANO plot with lower N availability, but tended to favor lower N in the DES plot with higher N availability. This habitat difference is consistent with previously reported habitat differences in selection on leaf N for naturally occurring populations: stronger selection for high leaf N in naturally occurring H. anomalus-wet than for H. deserticola (Donovan et al., 2007). The habitat differences in selection on leaf N are set against a background of relatively few differences within each habitat. Within each of the experimental plots, selection on N did not differ between BC2pet and BC2ann (Ludwig et al., 2004 for ANO plot; P > 0.05 for DES Plot). Within the ANO plot, the selection on leaf N did not differ between years (Table 4). The difference in selection on leaf N between habitats, but not within the H. anomalus habitat or across years, suggests that N limitation and selection on leaf N, or unmeasured traits related to achieving a higher leaf N, may be contributing to population differentiation and speciation.
Does direct selection on leaf N indicate that leaf N itself is the important trait, or could it be, instead, that underlying unmeasured traits allow the plant to gather and use N more efficiently? It is difficult to say because there are currently only a few studies that have investigated selection on leaf N and related traits. Maron et al. (2007) found total selection for higher leaf N, but not direct selection, for European Hypericum perforatum planted outside of its native range. Agrawal et al. (2008) found no direct selection for leaf C : N ratio for Asclepias incarnata planted into a common environment. However, for plants in the H. anomalus habitat, with such low fertility, it seems reasonable to hypothesize that the direct selection on leaf N reflects selection on an underlying suite of traits that enhance access and efficiency of uptake and subsequent use. For example, H. anomalus is capable of hydraulic lift (Howard et al., 2009), where water is transferred at night from wetter to drier soils via roots. This may allow plants to maintain root growth in dry soils and continue nutrient uptake by affecting root uptake capacity, nutrient mobility in the soil, and nutrient availability via microbial communities and decomposition (Caldwell et al., 1998; Bauerle et al., 2008). Also, plants in low nutrient habitats may use N more efficiently by prolonging leaf lifetime and reabsorbing more N before senescence (Aerts & Chapin, 2000). We are currently investigating whether H. anomalus differs from its ancestral parents for leaf lifetime and nutrient-use efficiency, and following nutrient-related traits in an artificial selection experiment under low soil N conditions.
Direct selection on WUE differed between habitats and within habitat. For BC2pet, higher WUE was favored in the ANO plot and lower WUE was favored in the DES plot. This was opposite to the expectation, based on water availability in the two habitats, that that greater water limitation generally favors more conservative water use (Dudley, 1996a; Heschel et al., 2002; Casper et al., 2005). One interpretation might be that selection on WUE and closely related parameters is dependent on the strength and timing of water deficits; for example, drought earlier in the growing season may favor a live fast, die young strategy (Geber & Dawson, 1990, 1997; Stanton et al., 2000; McKay et al., 2003; Heschel & Riginos, 2005; but see Sherrard & Maherali, 2006). This would be consistent with the earlier flowering of H. deserticola and selection for only lower WUE or no selection in the H. deserticola habitat. However, there was also substantial variation in selection on WUE within the sand dune habitat of H. anomalus. First, the H. anomalus planted in the ANO plot (H. anomalus-ANO) was only c. 20 m from the mesic natural population (H. anomalus-wet), and yet there was strong selection for higher and lower WUE, respectively, in these adjacent populations. Second, BC2ann and BC2pet in the ANO plot differed in selection on WUE (Ludwig et al., 2004). These differences may be caused by localized variation in water availability and plant rooting depth. The within-habitat variation in selection on WUE makes this less likely to be a candidate for driving population differentiation and speciation. In addition, among the focal traits in this study, the relationship between direct and total selection was weakest for WUE, indicating a larger contribution of indirect selection. This result fits into a larger context of a relative lack of indirect selection for most traits in wild populations, but the potential for greater indirect selection on plant physiological traits (Kingsolver et al., 2001; Geber & Griffen, 2003).
Larger leaf size was favored by direct and total selection in most of the planted and natural Helianthus populations, but direct selection did not differ by habitat for the BC2ann and BC2pet planted into both habitats. Other studies have varied as to whether drought results in direct selection for smaller leaf size (Bennington & McGraw, 1995; Dudley, 1996a; Heschel & Riginos, 2005; Caruso et al., 2006). For Helianthus species in these desert habitats, and within the context of these traits, differences in water availability did not result in differential selection on leaf size.
Direct selection on leaf succulence also did not differ between habitats. This was surprising because H. anomalus has a substantially higher leaf succulence than its ancestral parents and H. deserticola, suggesting that some factor in the H. anomalus habitat directly or indirectly favors greater leaf succulence (Schwarzbach et al., 2001; Rosenthal et al., 2002; Brouillette et al., 2006; Donovan et al., 2007). We found no evidence of strong indirect selection on succulence through other traits included in this study. In our current studies we have shifted the focus to related traits that that have also been suggested as potentially adaptive because of their correlation with plant functional types, such as leaf toughness, leaf thickness, leaf dry mass per area and leaf dry matter content (Garnier & Laurent, 1994; Cornelissen et al., 2003; Diaz et al., 2004; Ishida et al., 2008; but see Volaire, 2008). Leaf dry matter content is of particular interest because it can be correlated with other traits important for nutrient stress tolerance, such as relative growth rate and leaf life span (Cornelissen et al., 2003), but varies in the strength of correlation with succulence across the Helianthus study populations (Table S2).
Would we expect the habitat differences in phenotypic selection on leaf N to result in trait differentiation if we followed the artificial hybrids for several generations in these habitats? This requires consideration of the amount of standing genetic variation for selection to act on and the genetic correlations among traits. For these traits, genetic variation for the selection to act on is likely, as demonstrated by quantitative trait loci identified for the BC2pet Helianthus population in this study (Rieseberg et al., 2003; Brouillette et al., 2007) and heritability estimates reported for other species (Geber & Griffen, 2003; Caruso et al., 2005, Culley et al., 2006; Agrawal et al., 2008). However, heritability estimates vary by species, populations and environment, and are expected to be lower in natural populations where the environment is usually more variable than for common environment experiments. Unfortunately, we do not know whether the genetic correlations among traits would facilitate or constrain selection for these populations. We did not detect strong indirect selection for these traits (except for WUE). Although we did report phenotypic correlations among traits (for phenotypic trait correlations, see Ludwig et al. (2004) for ANO plot 2002, Donovan et al. (2007) for natural populations in 2002, and Tables S1 and S2 for DES plot 2002 and ANO plot 2003, respectively), this does not tell us the underlying genetic correlations for each population and whether these correlations are likely to constrain or facilitate trait evolution in Helianthus in these environments. Reports to date conflict on whether correlations among ecophysiological traits will likely constrain evolution (Dudley, 1996b; Geber & Griffen, 2003; Caruso et al., 2005; Agrawal et al., 2008).
Are the habitat differences in phenotypic selection reflected in differences in traits for the native Helianthus species endemic to these habitats? In the natural populations compared at the same time selection was estimated (June), H. anomalus did have higher leaf N than H. deserticola, despite the lower soil N in the H. anomalus habitat, consistent with the divergent phenotypic selection (Rosenthal et al., 2002; Donovan et al., 2007). Although species comparisons in glasshouse experiments under high- and low-nutrient treatments have not found a consistent species difference in leaf N relations (Schwarzbach et al., 2001; Rosenthal et al., 2002; Brouillette et al., 2006), the expression of the species differences may depend on environmental factors not replicated in glasshouse conditions.
There are several caveats that should be kept in mind when interpreting these results. One caveat is that in this study we were limited to using vegetative biomass as our proxy for fitness. We feel that it is a reasonable proxy for fitness when exploring traits expected to affect fitness through vegetative growth (Samson & Werk, 1986; Ehleringer & Clark, 1988; Ferris & Lechowicz, 1990; Dudley, 1996a). However, measures of reproductive biomass or seed production would have been preferred and may have changed the results if ecophysiological traits affect seed production more directly.
A second caveat is that, when estimating direct selection on traits, the choice of traits to be included can influence the results. As an example, our results for selection on leaf size differ from a previous analysis of leaf size for the artificial hybrids in the DES plot (Gross et al., 2004), where the total selection was still positive, but direct selection was negative. We attribute difference in results among studies to the choice of traits included in the selection analysis. Gross et al. (2004) included stem diameter, which appears to be a surrogate for plant size, and thus mixes both ‘morphology’ and ‘performance’ traits (sensu Arnold, 1983). Another example is that including leaf N in the analysis may affect the detection and interpretation of direct selection on WUE (Donovan et al., 2007). Plants can increase WUE either by increasing photosynthetic capacity (e.g. via increased leaf N) or by decreasing water loss. When both WUE and leaf N are incorporated in a selection analysis, selection for increased leaf N may include selection for increased photosynthetic capacity, and selection on WUE may only measure selection on reducing water loss. However, for studies that assess selection on WUE but not leaf N (Dudley, 1996a), selection on WUE should include selection for increasing photosynthetic capacity through increased leaf N as well as for reducing water loss, increasing the apparent adaptive value of the WUE. For a third example, leaf N concentration (mass basis) and leaf N per unit area are correlated for these populations (r2 = 0.47–0.86, P < 0.001 for all; Table S2) and both have received attention with regard to WUE, plant nutrient relations and leaf economics (Field & Mooney, 1986; Wright et al., 2001, 2003, 2004; Cornelissen et al., 2003; Huxman et al., 2008). We used leaf N concentration in all the analyses presented, but could have replaced it with leaf N on an area basis, and could also have included specific area in the analyses. We chose to use leaf N concentration and ignore specific leaf area because initial exploration of the data indicated that the combination of leaf area, N concentration, succulence and WUE gave us the greatest explanatory power for fitness for this study. However, this makes it less comparable to any studies that focus on leaf N on an area basis. While the choice of traits to be included in any given phenotypic selection study is usually influenced by the biology of the focal species and the question being asked, the growing number of studies of selection on ecophysiological traits will, it is hoped, lead to a consensus on traits worth exploring.
A third caveat is that our point measure of traits misses the complexity of how both traits and the environment change. While we did not find a difference in direct selection on traits when they were measured in June of 2002 and 2003, we did find within-season variation in selection on traits measured in June vs. July for the H. anomalus native populations (Donovan et al., 2007). In addition, selection on WUE may differ between juveniles and adults of long-lived perennials (Donovan & Ehleringer, 1994, Casper et al., 2005), differ across years for adults (Casper et al., 2005) and depend on when drought occurs during the growing season (Heschel & Riginos, 2005). Thus, shifting trait values as plants respond to their environment and shifting selection as the environment changes needs to be further explored to understand selection on, and evolutionary responses of, ecophysiological traits.
In summary, we detected substantial direct and total selection on the four-leaf ecophysiological traits for desert annual sunflowers. This adds to the growing evidence for selection on ecophysiological traits (Dudley, 1996a; Heschel et al., 2002; Casper et al., 2005; Heschel & Riginos, 2005; Caruso et al., 2006; Sherrard & Maherali, 2006; Maron et al., 2007; Saldana et al., 2007; Agrawal et al., 2008), although strengths and directions of natural selection vary. For the traits studied, except for WUE, direct and total selection were in good agreement, indicating little effect of indirect selection. The differences in selection on traits in the H. anomalus and H. deserticola habitat, combined with differences in plant biomass and trait means, suggest that nutrient limitation has been more important than water limitation as a driving force for adaptation of H. anomalus to the desert dune habitat. More studies assessing spatial and temporal variation in selection are needed to understand the evolution of ecophysiological traits.