Striking between-population floral divergences in a habitat specialized plant.

When the habitat occupied by a specialist species is patchily distributed, limited gene flow between the fragmented populations may allow population differentiation and eventual speciation. 'Sky islands'-montane habitats that form terrestrial islands-have been shown to promote diversification in many taxa through this mechanism. We investigate floral variation in Impatiens lawii, a plant specialized on laterite rich rocky plateaus that form sky islands in the northern Western Ghats mountains of India. We focus on three plateaus separated from each other by ca. 7 to 17 km, and show that floral traits have diverged strongly between these populations. In contrast, floral traits have not diverged in the congeneric I. oppositifolia, which co-occurs with I. lawii in the plateaus, but is a habitat generalist that is also found in the intervening valleys. We conducted common garden experiments to test whether the differences in I. lawii are due to genetic differentiation or phenotypic plasticity. There were strong differences in floral morphology between experimental plants sourced from the three populations, and the relative divergences between population pairs mirrored that seen in the wild, indicating that the populations are genetically differentiated. Common garden experiments confirmed that there was no differentiation in I. oppositifolia. Field floral visitation surveys indicated that the observed differences in floral traits have consequences for I. lawii populations, by reducing the number of visitors and changing the relative abundance of different floral visitor groups. Our results highlight the role of habitat specialization in diversification, and corroborates the importance of sky islands as centres of diversification.

Leaf thermotolerance in dry tropical forest tree species: relationships with leaf traits and effects of drought.

Understanding how tropical trees will respond to extreme temperatures and drought is essential to predict how future increases in the severity, frequency and duration of extreme climatic events will affect tropical systems. In this study, we investigated leaf thermotolerance by quantifying the temperatures that resulted in a 50 % decrease in photosystem II function (T50) in experimentally grown saplings of 12 tree species from a seasonally dry tropical forest. We examined the relationship of thermotolerance with leaf functional traits and photosynthetic rates. Additionally, we tested how water limitation altered thermotolerance within species, and examined the relationship between thermotolerance and drought tolerance among species. Thermotolerance ranged from 44.5 to 48.1 °C in the least and most thermotolerant species, respectively. The observed variation in thermotolerance indicates that the upper limits of leaf function are critically close to maximum temperatures in this region, and that these species will be vulnerable to, and differentially affected by, future warming. Drought increased temperature tolerance, and species that were more drought tolerant were also more thermotolerant. Importantly, thermotolerance was positively related to the key leaf functional trait-leaf mass per area (LMA), and congruent with this, negatively related to photosynthetic rates. These results indicate that more productive species with lower LMA and higher photosynthetic rates may be more vulnerable to heat and drought stress, and more likely to be negatively affected by future increases in extreme climatic events.

Leaf thermotolerance in tropical trees from a seasonally dry climate varies along the slow-fast resource acquisition spectrum.

Knowledge of the upper limits of temperature tolerance is essential to understand how tropical trees will respond to global warming. We quantified leaf thermotolerance in 41 tree species growing in a seasonally dry tropical region of the Indian subcontinent to examine: (1) differences between evergreen and deciduous species; (2) relationships with leaf mass per area (LMA) and leaf size; and, (3) seasonal variation in thermotolerance. Thermotolerance ranged from 45.5 °C to 50.5 °C among species, was higher for evergreen than deciduous species, and was negatively related to a continuous estimate of deciduousness. Species with higher LMA had higher thermotolerance, but we did not detect any relationship between leaf size and thermotolerance. Seasonal changes in thermotolerance varied among species implying that species' capacity to acclimate may differ. Thermal safety margins, the difference between thermotolerance and maximum habitat temperatures indicate that most species may be highly vulnerable to future warming. Overall our results show that deciduous, and fast growing species with low LMA are likely to be more negatively affected by global warming. This differential vulnerability may lead to directional changes in composition in dry tropical forests, and such changes could alter vegetation-atmosphere feedbacks and further exacerbate global warming.

Pleiotropy in the wild: the dormancy gene DOG1 exerts cascading control on life cycles.

In the wild, organismal life cycles occur within seasonal cycles, so shifts in the timing of developmental transitions can alter the seasonal environment experienced subsequently. Effects of genes that control the timing of prior developmental events can therefore be magnified in the wild because they determine seasonal conditions experienced by subsequent life stages, which can influence subsequent phenotypic expression. We examined such environmentally induced pleiotropy of developmental-timing genes in a field experiment with Arabidopsis thaliana. When studied in the field under natural seasonal variation, an A. thaliana seed-dormancy gene, Delay Of Germination 1 (DOG1), was found to influence not only germination, but also flowering time, overall life history, and fitness. Flowering time of the previous generation, in turn, imposed maternal effects that altered germination, the effects of DOG1 alleles, and the direction of natural selection on these alleles. Thus under natural conditions, germination genes act as flowering genes and potentially vice versa. These results illustrate how seasonal environmental variation can alter pleiotropic effects of developmental-timing genes, such that effects of genes that regulate prior life stages ramify to influence subsequent life stages. In this case, one gene acting at the seed stage impacted the entire life cycle.

Seed after-ripening and dormancy determine adult life history independently of germination timing.

• Seed dormancy can affect life history through its effects on germination time. Here, we investigate its influence on life history beyond the timing of germination. • We used the response of Arabidopsis thaliana to chilling at the germination and flowering stages to test the following: how seed dormancy affects germination responses to the environment; whether variation in dormancy affects adult phenology independently of germination time; and whether environmental cues experienced by dormant seeds have an effect on adult life history. • Dormancy conditioned the germination response to low temperatures, such that prolonged periods of chilling induced dormancy in nondormant seeds, but stimulated germination in dormant seeds. The alleviation of dormancy through after-ripening was associated with earlier flowering, independent of germination date. Experimental dormancy manipulations showed that prolonged chilling at the seed stage always induced earlier flowering, regardless of seed dormancy. Surprisingly, this effect of seed chilling on flowering time was observed even when low temperatures did not induce germination. • In summary, seed dormancy influences flowering time and hence life history independent of its effects on germination timing. We conclude that the seed stage has a pronounced effect on life history, the influence of which goes well beyond the timing of germination.

Natural variation in germination responses of Arabidopsis to seasonal cues and their associated physiological mechanisms.

BACKGROUND AND AIMS: Despite the intense interest in phenological adaptation to environmental change, the fundamental character of natural variation in germination is almost entirely unknown. Specifically, it is not known whether different genotypes within a species are germination specialists to particular conditions, nor is it known what physiological mechanisms of germination regulation vary in natural populations and how they are associated with responses to particular environmental factors. METHODS: We used a set of recombinant inbred genotypes of Arabidopsis thaliana, in which linkage disequilibrium has been disrupted over seven generations, to test for genetic variation and covariation in germination responses to distinct environmental factors. We then examined physiological mechanisms associated with those responses, including seed-coat permeability and sensitivity to the phytohormones gibberellic acid (GA) and abscisic acid (ABA). KEY RESULTS: Genetic variation for germination was environment-dependent, but no evidence for specialization of germination to different conditions was found. Hormonal sensitivities also exhibited significant genetic variation, but seed-coat properties did not. GA sensitivity was associated with germination responses to multiple environmental factors, but seed-coat permeability and ABA sensitivity were associated with specific germination responses, suggesting that an evolutionary change in GA sensitivity could affect germination in multiple environments, but that of ABA sensitivity may affect germination under more restricted conditions. CONCLUSIONS: The physiological mechanisms of germination responses to specific environmental factors therefore can influence the ability to adapt to diverse seasonal environments encountered during colonization of new habitats or with future predicted climate change.

DOG1 expression is predicted by the seed-maturation environment and contributes to geographical variation in germination in Arabidopsis thaliana.

Seasonal germination timing of Arabidopsis thaliana strongly influences overall life history expression and is the target of intense natural selection. This seasonal germination timing depends strongly on the interaction between genetics and seasonal environments both before and after seed dispersal. DELAY OF GERMINATION 1 (DOG1) is the first gene that has been identified to be associated with natural variation in primary dormancy in A. thaliana. Here, we report interaccession variation in DOG1 expression and document that DOG1 expression is associated with seed-maturation temperature effects on germination; DOG1 expression increased when seeds were matured at low temperature, and this increased expression was associated with increased dormancy of those seeds. Variation in DOG1 expression suggests a geographical structure such that southern accessions, which are more dormant, tend to initiate DOG1 expression earlier during seed maturation and achieved higher expression levels at the end of silique development than did northern accessions. Although elimination of the synthesis of phytohormone abscisic acid (ABA) results in the elimination of maternal temperature effects on dormancy, DOG1 expression predicted dormancy better than expression of genes involved in ABA metabolism.

Major flowering time gene, flowering locus C, regulates seed germination in Arabidopsis thaliana.

Flowering locus C (FLC) is a major regulator of flowering responses to seasonal environmental factors. Here, we document that FLC also regulates another major life-history transition-seed germination, and that natural variation at the FLC locus and in FLC expression is associated with natural variation in temperature-dependent germination. FLC-mediated germination acts through additional genes in the flowering pathway (FT, SOC1, and AP1) before involving the abscisic acid catabolic pathway (via CYP707A2) and gibberellins biosynthetic pathway (via GA20ox1) in seeds. Also, FLC regulation of germination is largely maternally controlled, with FLC peaking and FT, SOC1, and AP1 levels declining at late stages of seed maturation. High FLC expression during seed maturation is associated with altered expression of hormonal genes (CYP707A2 and GA20ox1) in germinating seeds, indicating that gene expression before the physiological independence of seeds can influence gene expression well after any physical connection between maternal plants and seeds exists. The major role of FLC in temperature-dependent germination documented here reveals a much broader adaptive significance of natural variation in FLC. Therefore, pleiotropy between these major life stages likely influences patterns of natural selection on this important gene, making FLC a promising case for examining how pleiotropy influences adaptive evolution.

Interactive effects of elevated CO2 and growth temperature on the tolerance of photosynthesis to acute heat stress in C3 and C4 species.

Determining effects of elevated CO2 on the tolerance of photosynthesis to acute heat-stress (heat wave) is necessary for predicting plant responses to global warming, as photosynthesis is thermolabile and acute heat-stress and atmospheric CO2 will increase in the future. Few studies have examined this, and past results are variable, which may be due to methodological variation. To address this, we grew two C3 and two C4 species at current or elevated CO2 and three different growth temperatures (GT). We assessed photosynthetic thermotolerance in both unacclimated (basal tolerance) and pre-heat-stressed (preHS = acclimated) plants. In C3 species, basal thermotolerance of net photosynthesis (P(n)) was increased in high CO2, but in C4 species, P(n) thermotlerance was decreased by high CO2 (except Zea mays at low GT); CO2 effects in preHS plants were mostly small or absent, though high CO2 was detrimental in one C3 and one C4 species at warmer GT. Though high CO2 generally decreased stomatal conductance, decreases in P(n) during heat stress were mostly due to non-stomatal effects. Photosystem II (PSII) efficiency was often decreased by high CO2 during heat stress, especially at high GT; CO2 effects on post-PSII electron transport were variable. Thus, high CO2 often affected photosynthetic theromotolerance, and the effects varied with photosynthetic pathway, growth temperature, and acclimation state. Most importantly, in heat-stressed plants at normal or warmer growth temperatures, high CO2 may often decrease, or not benefit as expected, tolerance of photosynthesis to acute heat stress. Therefore, interactive effects of elevated CO2 and warmer growth temperatures on acute heat tolerance may contribute to future changes in plant productivity, distribution, and diversity.

Interactive effects of elevated CO2 and ozone on leaf thermotolerance in field-grown Glycine max.

Humans are increasing atmospheric CO2, ground-level ozone (O3), and mean and acute high temperatures. Laboratory studies show that elevated CO2 can increase thermotolerance of photosynthesis in C3 plants. O3-related oxidative stress may offset benefits of elevated CO2 during heat-waves. We determined effects of elevated CO2 and O3 on leaf thermotolerance of field-grown Glycine max (soybean, C3). Photosynthetic electron transport (et) was measured in attached leaves heated in situ and detached leaves heated under ambient CO2 and O3. Heating decreased et, which O3 exacerbated. Elevated CO2 prevented O3-related decreases during heating, but only increased et under ambient O3 in the field. Heating decreased chlorophyll and carotenoids, especially under elevated CO2. Neither CO2 nor O3 affected heat-shock proteins. Heating increased catalase (except in high O3) and Cu/Zn-superoxide dismutase (SOD), but not Mn-SOD; CO2 and O3 decreased catalase but neither SOD. Soluble carbohydrates were unaffected by heating, but increased in elevated CO2. Thus, protection of photosynthesis during heat stress by elevated CO2 occurs in field-grown soybean under ambient O3, as in the lab, and high CO2 limits heat damage under elevated O3, but this protection is likely from decreased photorespiration and stomatal conductance rather than production of heat-stress adaptations.

Variation in heat-shock proteins and photosynthetic thermotolerance among natural populations of Chenopodium album L. from contrasting thermal environments: implications for plant responses to global warming.

Production of heat-shock proteins (Hsps) is a key adaptation to acute heat stress and will be important in determining plant responses to climate change. Further, intraspecifc variation in Hsps, which will influence species-level response to global warming, has rarely been examined in naturally occurring plants. To understand intraspecific variation in plant Hsps and its relevance to global warming, we examined Hsp content and thermotolerance in five naturally occurring populations of Chenopodium album L. from contrasting thermal environments grown at low and high temperatures. As expected, Hsp accumulation varied between populations, but this was related more to habitat variability than to mean temperature. Unexpectedly, Hsp accumulation decreased with increasing variability of habitat temperatures. Hsp accumulation also decreased with increased experimental growth temperatures. Physiological thermotolerance was partitioned into basal and induced components. As with Hsps, induced thermotolerance decreased with increasing temperature variability. Thus, populations native to the more stressful habitats, or grown at higher temperatures, had lower Hsp levels and induced thermotolerance, suggesting a greater reliance on basal mechanisms for thermotolerance. These results suggest that future global climate change will differentially impact ecotypes within species, possibly by selecting for increased basal versus inducible thermotolerance.

Effects of elevated CO2 on the tolerance of photosynthesis to acute heat stress in C3, C4, and CAM species.

Determining the effect of elevated CO(2) on the tolerance of photosynthesis to acute heat stress (AHS) is necessary for predicting plant responses to global warming because photosynthesis is heat sensitive and AHS and atmospheric CO(2) will increase in the future. Few studies have examined this effect, and past results were variable, which may be related to methodological variation among studies. In this study, we grew 11 species that included cool and warm season and C(3), C(4), and CAM species at current or elevated (370 or 700 ppm) CO(2) and at species-specific optimal growth temperatures and at 30°C (if optimal ≠ 30°C). We then assessed thermotolerance of net photosynthesis (P(n)), stomatal conductance (g(st)), leaf internal [CO(2)], and photosystem II (PSII) and post-PSII electron transport during AHS. Thermotolerance of P(n) in elevated (vs. ambient) CO(2) increased in C(3), but decreased in C(4) (especially) and CAM (high growth temperature only), species. In contrast, elevated CO(2) decreased electron transport in 10 of 11 species. High CO(2) decreased g(st) in five of nine species, but stomatal limitations to P(n) increased during AHS in only two cool-season C(3) species. Thus, benefits of elevated CO(2) to photosynthesis at normal temperatures may be partly offset by negative effects during AHS, especially for C(4) species, so effects of elevated CO(2) on acute heat tolerance may contribute to future changes in plant productivity, distribution, and diversity.

Diversification of phytochrome contributions to germination as a function of seed-maturation environment.

Environmental conditions during seed maturation influence germination, but the genetic basis of maternal environmental effects on germination is virtually unknown. Using single and multiple mutants of phytochromes, it is shown here that different phytochromes contributed to germination differently, depending on seed-maturation conditions. Arabidopsis thaliana wild-type seeds that were matured under cool temperatures were intensely dormant compared with seeds matured at warmer temperature, and this dormancy was broken only after warm seed-stratification followed by cold seed-stratification. The warm-cold stratification broke dormancy in fresh seeds but not in dry after-ripened seeds. Functional PHYB and PHYD were necessary to break cool-induced dormancy, which indicates a previously unknown and ecologically important function for PHYD. Disruption of PHYA in combination with PHYD (but not PHYB) restored germination to near wild-type levels, indicating that PHYA contributes to the maintenance of cool-induced dormancy on a phyD background. Effects of seed-maturation temperature were much stronger than effects of seed-maturation photoperiod. PHYB contributed to germination somewhat more strongly in seeds matured under short days, whereas PHYD contributed to germination somewhat more strongly in seeds matured under long days. The variable contributions of different phytochromes to germination as a function of seed-maturation conditions reveal further functional diversification of the phytochromes during the process of germination. This study identifies among the first genes to be associated with maternal environmental effects on germination.

Stress responses and metal tolerance of Chlamydomonas acidophila in metal-enriched lake water and artificial medium.

Chlamydomonas acidophila faces high heavy-metal concentrations in acidic mining lakes, where it is a dominant phytoplankton species. To investigate the importance of metals to C. acidophila in these lakes, we examined the response of growth, photosynthesis, cell structure, heat-shock protein (Hsp) accumulation, and metal adsorption after incubation in metal-rich lake water and artificial growth medium enriched with metals (Fe, Zn). Incubation in both metal-rich lake water and medium caused large decreases in photosystem II function (though no differences among lakes), but no decrease in growth rate (except for medium + Fe). Concentrations of small Hsps were higher in algae incubated in metal-rich lake-water than in metal-enriched medium, whereas Hsp60 and Hsp70A were either less or equally expressed. Cellular Zn and Fe contents were lower, and metals adsorbed to the cell surface were higher, in lake-water-incubated algae than in medium-grown cells. The results indicate that high Zn or Fe levels are likely not the main or only contributor to the low primary production in mining lakes, and multiple adaptations of C. acidophila (e.g., high Hsp levels, decreased metal accumulation) increase its tolerance to metals and permit survival under such adverse environmental conditions. Supposedly, the main stress factor present in the lake water is an interaction between low P and high Fe concentrations.

Phytochrome mediates germination responses to multiple seasonal cues.

We identified a new role of phytochrome in mediating germination responses to seasonal cues and thereby identified for the first time a gene involved in maternal environmental effects on germination. We examined the germination responses of a mutant, hy2-1, which is deficient in the phytochrome chromophore. The background genotype, Landsberg erecta (Ler), lacked dormancy in most treatments, while hy2-1 required cold stratification for germination in a manner that resembled a more dormant ecotype, Columbia (Col). Unlike Col, hy2-1 was not induced into dormancy by warm stratification. Therefore, the down-regulation of phytochrome-mediated germination pathways results in sensitivity to cold, but we found no evidence that reduced phytochrome activity enables the warm-induction of dormancy. Cool temperatures during seed maturation induced dormancy. The hy2-1 mutants did not overcome this dormancy, indicating that phytochrome-mediated pathways are required to break cold-induced dormancy. Ler did not respond to post-stratification temperature, but hy2-1 did respond, suggesting phytochrome pathways are involved in germination responses to temperature. In summary, phytochromes mediate dormancy and germination responses to seasonal cues experienced both during seed maturation and after dispersal. Phytochromes therefore appear to be involved in mediating seasonal germination timing, a trait of great ecological importance and one that is under strong natural selection.

Temperature- and pH-dependent accumulation of heat-shock proteins in the acidophilic green alga Chlamydomonas acidophila.

Chlamydomonas acidophila, a unicellular green alga, is a dominant phytoplankton species in acidic water bodies, facing severe environmental conditions such as low pH and high heavy metal concentrations. We examined the pH-, and temperature-dependent accumulation of heat-shock proteins in this alga to determine whether heat-shock proteins play a role in adaptation to their environment. Our results show increased heat-shock proteins accumulation at suboptimal pHs, which were not connected with any change in intracellular pH. In comparison to the mesophilic Chlamydomonas reinhardtii, the acidophilic species exhibited significantly higher accumulations of heat-shock proteins under control conditions, indicating an environmental adaptation of increased basal levels of heat-shock proteins. The results suggest that heat-shock proteins might play a role in the adaptation of C. acidophila, and possibly other acidophilic algae, to their extreme environment.

Variation in chloroplast small heat-shock protein function is a major determinant of variation in thermotolerance of photosynthetic electron transport among ecotypes of Chenopodium album.

Chloroplast small (low-molecular-weight) heat-shock proteins (csHsps) can protect photosynthetic electron transport (Pet), and quantitative variation in csHsps is correlated with thermotolerance of net photosynthesis and Photosystem II. However, the functional (i.e. protective) consequence of natural variation in csHsps is unknown. To investigate this, we used an in vitro assay to determine the contribution of csHsps to the tolerance of Pet to high temperatures in five ecotypes of Chenopodium album collected from habitats ranging from cool to warm, and we partitioned total Pet thermotolerance into basal and induced Pet components (without and with a pre-heat treatment, respectively, to induce csHsps). The ecotypes varied in total Pet thermotolerance and this was correlated with habitat temperature. Variation in total Pet thermotolerance was associated primarily with variation in induced Pet thermotolerance, and not with basal Pet thermotolerance. Variation in induced Pet was highly correlated with csHsp protection of Pet. Variation in csHsp function was associated with variation in csHsp content among ecotypes. These results are the first to demonstrate the direct functional consequences for natural variation in Hsps in plants, and show that functional variation is associated with evolutionary adaptation to specific habitats among ecotypes.