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Photosynthesis research on yellowtops: Macroevolution in progress
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Theory in Biosciences 125 (2007) 81–92 www.elsevier.de/thbio
Photosynthesis research on yellowtops: Macroevolution in progress U. Kutscheraa,, K.J. Niklasb a
Institut fu¨r Biologie, Universita¨t Kassel, Heinrich-Plett-Str. 40, 34109 Kassel, Germany Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA
b
Received 15 May 2006; accepted 1 June 2006
Abstract The vast majority of angiosperms, including most of the agronomically important crop plants (wheat, etc.), assimilate CO2 through the inefficient C3 pathway of photosynthesis. Under ambient conditions these organisms loose about 1/3 of fixed carbon via photorespiration, an energetically wasteful process. Plants with C4 photosynthesis (such as maize) eliminate photorespiration via a biochemical CO2-pump and thus have a larger rate of carbon gain. The genus Flaveria (yellowtops, Asteraceae) contains not only C3 and C4 species, but also many C3–C4 intermediates, which have been interpreted as evolving from C3 to fully expressed C4 metabolism. However, the evolutionary significance of C3–C4 Flaveria-intermediates has long been a matter of debate. A well-resolved phylogeny of nearly all Flaveria species has recently been published. Here, we review pertinent background information and combine this novel phylogeny with physiological data. We conclude that the Flaveria species complex provides a robust model system for the study of the transition from C3 to C4 photosynthesis, which is arguably a macroevolutionary event. We conclude with comments relevant to the current Intelligent Design debate. r 2006 Elsevier GmbH. All rights reserved. Keywords: Evolution; Flaveria; Intermediate forms; Macroevolution; Photosynthesis
Corresponding author.
E-mail addresses: [email protected] (U. Kutschera), [email protected] (K.J. Niklas). 1431-7613/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.thbio.2006.06.001
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Introduction Agronomists have long known that the average crop yield of maize (Zea mays) is much larger than that of many other domesticated grasses such as wheat (Triticum aestivum) or barley (Hordeum vulgare). According to Agricultural Statistics (USDA 1980), the average yields obtained for maize, wheat and barley are 3.06, 1.03 and 1.21 tons grain/acre, respectively. The reasons for these striking differences were debated when these data first became available 25 years ago. However, today we know that the approximately two-fold larger average crop yield of maize compared to wheat (and barley) is largely attributable to the more efficient photosynthetic mode of the maize plant (Sage and Monson, 1999; Sage, 2004). Like its relatives sugarcane and sorghum, Z. mays is characterized by a biochemical pump that concentrates the atmospheric carbon dioxide (CO2) around the key enzyme of photosynthesis, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco, localized in the stroma of chloroplasts) after diffusion via the stomata into leaf cells. This sophisticated two-step mechanism of CO2-assimilation was discovered in the 1960s, only a few years after Melvin Calvin (1911–1997) and his colleagues had resolved the details of photosynthetic CO2-fixation in suspensions of the green alga Chlorella (Hatch, 1992; Kutschera, 2002). In this contribution, we describe a model system that has been used to elucidate the phylogenetic development of the CO2-pump in higher plants such as maize. This (and other) arguably macroevolutionary transition has been addressed in previous publications (Kutschera and Niklas, 2004, 2005), where background information on the topic discussed here is summarized.
Photorespiration and the CO2-pump of the maize plant Over the past decades, it has become very apparent that more than 90% of all land plants, including most crop species (wheat, barley, etc.) assimilate CO2 via the onestep C3 ‘‘Chlorella-type’’ pathway of photosynthesis. In these green organisms, the five-carbon sugar RuBP is the primary CO2-acceptor and the first product of photosynthetic CO2-fixation is the three-carbon (C3) molecule 3-phosphoglycerate (PGA). However, Rubisco, the enzyme catalyzing the fixation of CO2, is bifunctional. Both CO2 and atmospheric oxygen (O2) compete with one another at the active site of Rubisco. Carboxylation results in the formation of 2 PGA, whereas oxygenation of RuBP leads to the production of 1 PGA and 1 glycollate-2-P (oxygenase reaction). Thus, the primary reaction of one-step C3photosynthesis occurring in chloroplasts can be summarized as follows: Rubisco
Mesophyll cell : RuBP þ CO2 ðO2 Þ ! 2 PGA ð1 PGA þ 1 glycolate2PÞ.
Importantly, only PGA can be converted in the stroma of chloroplasts into carbohydrates. The second product of the oxygenase reaction, glycolate-2-P, cannot
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be used for CO2-fixation in C3 plants; it is salvaged inefficiently in the photorespiratory pathway (Sage and Monson, 1999; Leegood, 2002). Photorespiration is the light-dependent uptake of O2 and concomitant release of CO2 (and NH3) associated with glycolate-2-P metabolism. Because the rate of CO2-assimilation in C3 plants such as wheat is inhibited by ca. 35% in normal air containing 21% O2 (compared with an artificial oxygen-poor atmosphere), photorespiration is a wasteful process and leads to a decrease in photosynthetic efficiency (De Veau and Burris, 1989; Monson et al., 1984). In other words, C3 plants suffer from a ‘‘leak’’ in their CO2-assimilatory pathway (Calvin-cycle). The photosynthetic ‘‘design’’ in these plants is sub-optimal or poor, because 1/3 of their photosynthetic potential is wasted (Mann, 1999). In contrast, the rate of photorespiration is close to zero in C4 plants and virtually no O2-dependent inhibition of photosynthesis occurs. This two-step C4 mode of CO2-fixation requires two subsequent carboxylations, which occur in separate cell types of the leaf. In the first step, atmospheric CO2 is fixed in mesophyll cells into a four-carbon (C4) compound by the enzyme phosphoenolpyruvate carboxylase (PEPC); carbon dioxide is attached to the three-carbon compound phosphoenolpyruvate (PEP, the primary CO2-acceptor) to produce oxaloacetate, and, in the second step, this four-carbon compound is converted into malate and transferred (pumped) into the bundle sheath cells where it is broken down into C3 compounds. During this reaction, CO2 is released and concentrated around the second carboxylating enzyme Rubisco, which is concentrated in the chloroplasts of bundle sheath cells where the final CO2-fixation occurs as in C3 plants: PEPC
Mesophyll cell : PEP þ CO2 ! oxaloacetate ! malate ðtransferÞ, Rubisco
Bundle sheath cell : malate ! CO2 þ RuBP ! 2 PGA ! carbohydrates:
As a result of this two-step CO2-pump mechanism, only PGA (but virtually no glycolate-2-P) is produced, i.e., photorespiration in C4 plants is almost entirely suppressed (Sage and Monson, 1999; Leegood, 2002).
The adaptive significance of C4 metabolism Rubisco evolved in ancient cyanobacteria about 3000 million years ago when the Earth’s atmosphere was warmer and substantially different in its gas composition than it is today (molecular oxygen was lacking; the CO2 concentration was higher than today; Schopf, 1999; Knoll, 2003). This enzyme may have been very efficient during the time in Earth’s history when it made its first appearance. But it is currently far from ‘‘optimal’’ photosynthetically because of the affinity of its active site for oxygen (O2). Molecular phylogenies and other data indicate that the chloroplasts of the land plants (embryophytes) and the ancestral group from which they descentded, the
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green algae (Chlorophyta), evolved as a result of ‘‘residency’’ with a heterotrophic prokaryote ‘‘host’’ cell and subsequently evolved into modern-day chloroplasts (Niklas, 1997; Kutschera and Niklas, 2004, 2005). Palaeochemical studies of Earth’s ancient atmosphere indicate that during the time this endosymbiotic event occurred, atmospheric CO2 levels were substantially higher than they are now. Likewise, studies of present-day cyanobacteria (and presumably the ancestral life-forms that eventually evolved into chloroplasts) demonstrate that they possess an inorganic CO2-pump located at the plasma membrane that delivers hydrogen carbonate (HCO–3) to the cytosol. Cyanobacteria also generate CO2 (by carbonic anhydrase activity) in carboxysomes, which nurture the majority of Rubisco activity in their cells (Kaplan et al., 1998; Price et al., 1998). In contrast, terrestrial C3 plants rely on passive diffusion for the entry of CO2 into their cells. It comes, therefore, as little surprise that the highest specific reaction rates for carboxylation, the lowest selectivity for CO2 over O2 and the lowest affinity for CO2 in the cyanobacteria Chlorophyta–embryophytes ‘‘Rubisco clade’’ are found in present-day cyanobacteria, nor that a higher selectivity for CO2 over O2 and a higher affinity for CO2 is reported for aquatic members of the Chlorophyta and embryophytes. In general, an even higher CO2/O2 selectivity and CO2 affinity is found in C4 plants (but with less the selectivity and affinity values for terrestrial C3 plants) (Badger et al., 1998; Bird et al., 1982; Palmqvist et al., 1995; Rintama¨ki and Aro, 1985). The significance of this phylogenetic trend in the context of CO2 and O2 concentrations at the active site of Rubisco in present-day cyanobacteria and ‘‘green’’ plants is that the CO2/O2 ratio around Rubisco in cyanobacteria is 10 or more times that in air-equilibrium solution as a result of the activity of inorganic carbon pump and carboxysomes, both of which saturate the carboxylase activity of cyanobacterial Rubisco (and thus minimizes the oxygenase activity of this enzyme). In contrast, because most bryophytes and all vascular plants, including gymnosperms and angiosperms (with the exception of C4 plants), depend on the passive diffusion of CO2 to Rubisco, the diffusive resistance to CO2 entry and O2 exit from photosynthetic cells results in the steady-state CO2/O2 ratio and CO2 concentration at the active site of Rubisco that yields only half the saturation of the Rubisco carboxylase activity. Hence, very significant rates of O2 uptake by Rubisco (i.e., photorespiration) occurs during photosynthesis at present-day atmospheric CO2 concentrations (De Veau and Burris, 1989; Kutschera, 2002). Based on numerous and extensive phylogenetic and palaeobotanical analyses, the oldest flowering plants are about 125 million years old (Archaefructus from the Cretaceous, see Niklas, 1997; Kutschera and Niklas, 2004); these ancient green organisms most likely possessed C3 metabolism. Similar analyses show that the CO2pump in C4 plants is a recent evolutionary innovation, occurring over the past 30–20 million years in 19 different monocotyledonous and dicotyledonous families (Sage, 2004). In terms of the distribution of C4 species, 75% are monocots (Poaceae and Cyperaceae). The remaining 25% of all known C4 species is found in the Brassicaceae, Cruciferae, Aizoaceae, Amaranthaceae, Chenopodiaceae and Asteraceae (Apel et al., 1997; Kadereit et al., 2003).
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The evolution of C4 species in so many phyletically diverse families inevitably raises the question, Why? Certainly, convergent evolution provides strong circumstantial evidence for adaptation by means of natural selection. Thus, the real question is, What is the adaptive value of C4 photosynthesis? One obvious answer is that C4 photosynthesis is an adaptive response to the fairly steady decrease in atmospheric CO2 over the last 600 million years (Sage, 2004). Another plausible answer is that C4 metabolism confers a competitive advantage in hot habitats. These two hypotheses are not mutually exclusive, because C4 metabolism under low CO2 partial pressure and high temperatures (which are generally, but not invariably associated with limited water availability) would significantly enhance the competitive ability of any photosynthetic plant (Hatch, 1992). In typical C3 plants (Fig. 1), the rate of CO2-assimilation increases to some limit and then decreases with both increasing ambient temperatures and with increasing atmospheric CO2 levels. At ambient CO2 levels, leaf photosynthesis is limited by the activity of the enzyme Rubisco, and the response reflects two competing processes: an increase in the carboxylation rate with increasing temperature and a decrease in the affinity of Rubisco for CO2 (Fig. 1). A comparison between the quantum yields of photosynthetic carbon fixation of C3 and C4 plants shows that across most species, the efficiency of C3 plants at lower ambient temperatures (15 1C) exceeds that of C4 plants (Fig. 2). However, at higher temperatures (35–40 1C), the efficiency of C4 leaves is larger than that of the corresponding organs in C3 plants (Ehleringer and Bjo¨rkman, 1977; Berry and Bjo¨rkman, 1980). For example, the CO2-pump in maize and other C4 plants allows these organisms to assimilate atmospheric CO2 at lower gas concentrations than their C3 congenitors. But this advantage is particularly evident in terms of leaf carbon gain in warm highlight environments (Sage, 2004). This feature of C4 metabolism is well shown by
Fig. 1. Changes in the rate of photosynthesis (mmol CO2/m2 s) as a function of temperature and ambient or saturating atmospheric CO2 concentrations in leaves of Nerium oleander, a typical C3 plant. Data taken from Berry and Bjo¨rkman (1980).
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Fig. 2. Quantum yield of photosynthetic carbon fixation (mol CO2 per absorbed quantum) in leaves of a C3 and C4 plant (Encelia californica and Atriplex rosea, respectively) plotted as a function of ambient temperature. The measurements were carried out at normal CO2 concentration. Data taken from Ehleringer and Bjo¨rkman (1977).
comparing the carbon dioxide compensation points (CP) of C3 and C4 species. This physiological parameter, which measures for the efficiency of the CO2-pump mechanism, is defined as the CO2 concentration at which light-dependent gas uptake (leaf assimilation) is just balanced by CO2 loss through photorespiration and mitochondrial (dark) respiration. For example, at comparable air temperatures, wheat and other C3 plants typically have CP-values of about 50 ml CO2/l air (at 21 vol% O2), whereas maize has a CP of 0–3 ml/l, depending on the developmental stage of the plant (Sage and Monson, 1999).
The model system Flaveria More detailed comparisons of CP-values can provide even deeper insights into the adaptive significance of C4 metabolism, particularly if these comparisons are drawn among species possessing C3, C3–C4 intermediate or C4 metabolism nested in the same genus. An excellent example is the genus Flaveria (common name: yellowtops), which is a member of the Asteraceae (the largest dicot family, with about 1300 genera and more than 21 000 species). This genus contains 23 species of small annual herbs and perennial shrubs, some of which are known to have C3, C3–C4 intermediate or C4 metabolism (Monson et al., 1984; Monson and Moore, 1989; Westhoff and Gowik, 2004; Brown et al., 2005; McKown et al., 2005). One of these is the C4 (and cosmopolitan invasive) species Flaveria trinervia (Syn. F. repanda), which can reach a height of 40–80 cm depending on environmental conditions (Fig. 3). Gas exchange measurements on the leaves of greenhouse-grown specimens of this species
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Fig. 3. Ontogeny of a model organism for the study of a phylogenetic (macroevolutionary) trend in higher plants. Flaveria trinervia (Sprengel) C. Mohr (Syn. F. repanda), common name: clustered yellowtops, is an annual dicot C4 plant (Asteraceae). Juvenile (A), flowering (B) and senescing sporophyte (C). Scale bar ¼ 4 cm.
indicate a CO2-CP at approximately 3.5 ml/l (Ku et al., 1991) (a CP-value of o1 has been reported previously, see Monson et al., 1984), a CO2-assimilation rate (A) of 32 mmol/m2 s, and inhibition of photosynthesis at 21 vol% O2 (I) occurs at roughly 1.5% (the corresponding data for maize is CP3 ml/l, A33 mmol CO2/m2 s and I1–2%, respectively, see De Veau and Burris, 1989). Two other widely distributed C4 Flaveria species (i.e., F. bidentis and F. australasica) display quantitatively similar leaf gas exchange characteristics. In contrast, three C3 Flaveria species (i.e., F. cronquistii, F. pringlei and F. robusta) have very different gas exchange characteristics, i.e., CP60–62 ml/l; A16–21 mmol CO2/m2 s; I30–35% (Ku et al., 1991). These values are similar to those measured on wheat (T. aestivum), a representative C3 monocot (i.e., CP50 ml/l, A22 mmol CO2/m2 s and inhibition of photosynthesis at 21 vol% O2 (I)30–40%) (De Veau and Burris, 1989). Note that the rate of CO2-assimilation (A) of the C3 dicot Nerium oleander under normal conditions (air, 25 1C) is about 20 mmol/m2 s (see Fig. 1). Data such as these show clearly that C3 and C4 Flaveria species (Fig. 3) display leaf photosynthetic characteristics similar to those of the classical model organisms for the study of macroevolutionary transitions in plants, i.e., wheat and maize (Kellogg, 2000). Perhaps far more relevant to understanding the evolution of C4 metabolism are the leaf photosynthetic characteristics of Flaveria species manifesting a C3–C4 intermediate or C4-like metabolisms. When these data are plotted on a recently published phylogenetic tree for the genus, it becomes immediately clear that any shift from the antecedent C3 condition toward the evolutionarily derived C4 condition is physiologically adaptive (Fig. 4). Certainly, a note of caution must be introduced whenever phenotypic trends are deduced from the distribution of character states ‘‘mapped’’ onto a cladogram such as the one shown in Fig. 4 because the polarity of these states has to be rigorously established. For example, without independent confirmation, it is just as reasonable
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Fig. 4. Phylogeny depicting the evolutionary history of 18 Flaveria species for which the carbon dioxide compensation point (CP) has been published (unit: ml CO2/l air at 21 vol% O2). Adapted from McKown et al. (2005). The CP-values are from Ku et al. (1991).
to argue that some or all of the Flaveria species possessing C3–C4 intermediate or C4-like photosynthesis reflect the loss of fully developed C4 metabolic features rather than natural plant populations in the process of evolving toward full C4 photosynthesis. Indeed, the ‘‘macroevolution-in-action-hypothesis’’ has been questioned by some workers (for a critical discussion, see Monson and Moore, 1989). However, the comprehensive analysis of 21 out of the 23 described Flaveria species published by McKown et al. (2005) has now unequivocally resolved this longstanding open question.
Phylogeny and photosynthetic features of yellowtops The data set and detailed analyses of McKown et al. (2005) show that C3 photosynthesis is the ancestral condition and that a latent genetic predisposition for the evolution of C4 photosynthesis exists in the genus Flaveria. Based on the biogeographic distribution and the ecological preferences of the species within this genus, it is also reasonably clear that the multiple origins of the C4 photosynthesis were the result of selection pressures for survival in hot, arid or saline conditions.
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The data set of McKown et al. (2005) establishes two well-supported clades, designated A and B, each of which is identifiable on the basis of morphological, reproductive or physiological character states (Fig. 4). For example, most of species nested in clade A are small to moderately sized annual plants with inflorescences possessing 3–4 phyllaries (Fig. 3). All self-compatible species in this clade have C4 photosynthesis. In contrast, most of the species in clade B are moderate sized perennial and self-incompatible plants possessing 5–6 phyllaries. The majority of species in this clade has C3–C4 intermediate photosynthesis, the exception being F. brownii (Fig. 4). The basal species in each clade are C3–C4 physiological intermediates. However, they differ in their CO2-CPs and O2 inhibition, and thus in the type of their C3–C4 intermediate metabolism. Specifically, the basal species F. ramosissima in clade A is type II, whereas the basal species F. angustifolia in clade B is type I. (type I/II C3–C4 intermediates are characterized by the absence/presence of a C4 cycle). The appearance of type II C3–C4 intermediate photosynthesis in clade A coincides with the evolution of an annual life cycle in F. ramosissima and suggests that the common ancestor of this species (and other C4-like or C4 species in the clade, which are also annual species, e.g., F. vaginata) possessed a type II C3–C4 intermediate metabolism. If true, then the capacity to fully express C4 metabolism evolved on three separate occasions in clade A (i.e., once each in F. campestris, a relative of F. palmeri, F. australasica/F. trinerva and F. bidentis). Each of these evolutionary events coincides with the appearance of self-incompatibility. Noting that the ancestral condition for both clade A and B is C3 photosynthesis, the appearance of type I C3–C4 intermediate photosynthesis at the base of clade B is likely a derived condition, which coincides with the appearance of perennial and selfincompatible Flaveria species (Fig. 4). Based on low genetic divergence values and genomic analyses of closely related species, the most evolutionarily derived species in each of the two Flaveria clades evolved rapidly (McKown et al., 2005). In turn, various lines of evidence support the hypothesis that Flaveria speciation has undergone comparatively recent and intense selection for C3–C4 intermediate, C4-like and C4 photosynthesis: prior work has shown that some type II C3–C4 intermediate species initially assimilate CO2 into C4 acids under low CO2 levels, a condition that promotes photorespiration (McKown et al., 2005). Although decreasing CO2 levels may have played a role in the evolution of C4 metabolism multiple times in Flaveria (as well as many other flowering plant lineages), a variety of other ecological factors that coincide with the habitats occupied by basal Flaveria species are likely to have played much more important roles (e.g., heat, soil–water deprivation, salinity). Recall that C4 metabolism confers an advantage to plants living in hot habitats, which are generally but not invariably associated with water-stressed conditions (see Fig. 2). In this context, the basal C3–C4 intermediate species F. ramosissima and F. angustifolia grow in the sandy soils of the warm and arid Puebla-Oaxaca region of Mexico. This environment fosters high rates of transpiration and arguably therefore favors the survival of species capable of compensating for water deprivation and eliminating or reducing photorespiration. Likewise, the only C4-like species in clade B (i.e., F. brownii)
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grows in warm saline sandy flats and brackish marshes, environments that can subject species to intense selection. Life-history features may have been equally important to the rapid physiological radiation reported for this genus. Consider that all known advanced C3–C4 intermediate, C4-like and C4 Flaveria species are annuals. Likewise, all C4 Flaveria species are self-compatible. As noted by McKown et al. (2005), the comparatively short generation times of annual species typically promotes gene recruitment processes and the rapid accumulation of genetic variation in plant populations. Likewise, self-compatible species that have evolutionarily survived the gauntlet of inbreeding depression, early in their history, are purged of maladaptive alleles during an initially intense episode of selection for adaptive genetic variants. As noted, the environments currently occupied by advanced C3–C4 intermediate, C4-like and C4 Flaveria species would have provided intense selection pressures for coping with arid as well as hot conditions.
Conclusions The phylogenetic development of the biochemical CO2-pump capable of suppressing photorespiration and thereby enhancing the rate of photosynthetic CO2-assimilation is arguably a macroevolutionary event (here defined as the evolutionary appearance of any morphological, physiological or biochemical novelty above the level of populations) (Eldredge, 1989; Kellogg, 2000; Futuyma, 1998; Kutschera, 2006a, b, c; Kutschera and Niklas, 2004, 2005; Simons, 2002; Mayr, 1982, 2001). The work of McKown et al. (2005) and others demonstrates that the yellowtop (Flaveria) species complex provides an exceptionally powerful living model system with which to explore a physiological and morphological macroevolutionary event as well as patterns of speciation and thus microevolution, particularly in terms of the roles played by environmental factors (such as heat as well as aridity and water deprivation) and reproductive syndromes (self-compatibility and -incompatibility). The molecular mechanisms responsible for the occurrence of the CO2-pump are also amenable to investigation (Kopriva et al., 1996; Sage, 2004). For example, preliminary results indicate that C4 gene evolution occurred via duplication of pre-existing C3 DNA-sequences, followed by advantageous mutations in one of the duplicates to yield a novel role (neofunctionalization) (Monson, 2003; Westhoff and Gowik, 2004). Such adaptive mutations may have been rapidly ‘‘locked’’ into the genomes of advanced C3–C4, C4-like and C4 Flaveria species as a consequence of the co-occurrence of self-compatible (inbreeding) reproductive systems functioning in arid and hot environments. The facts that Rubisco across present-day eukaryotic photoautotrophs is far from a ‘‘perfect enzyme’’ as well as the evidence for macroevolution in the Flaveria species complex (Fig. 4) serve as clear examples with which to counter the fallacies perpetuated by creationists and adherents of the Intelligent Design (ID)-movement. Although some creationists and ID adherents may admit that microevolution (variation or speciation) is unquestionable, they assert that there is no evidence for
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macroevolution in extant populations of organisms and that living beings are ‘‘perfectly designed’’ (Kutschera, 2003, 2006a, b, c). The evolution of C4 metabolism in the Flaveria species complex and the fact that roughly 90% of all extant angiosperms loose ca. 1/3 of the carbon fixed in the light reactions of C3 photosynthesis to photorespiration easily refute these assumptions. Rubisco evolved under conditions that made this enzyme reasonably efficient (anaerobic atmosphere; high CO2-level), particularly in the physiological context of the cyanobacteria-like organisms that ultimately evolved into present-day chloroplasts (Kutschera and Niklas, 2005). However, it is clear that once these cyanobacteria gained permanent residency in their host cells, the efficiency of photosynthesis was significantly reduced and only improved with subsequent physiological and morphological adaptive evolutionary events (i.e., the occurrence of C4 photosynthesis). In a very real sense, Rubisco and C3 metabolism are ‘‘phyletic legacies’’ that are imperfect in many ways. All available evidence indicates that photorespiration is a resounding waste of energy and has no obvious advantage to C3 plants (Mann, 1999; Leegood, 2002). Thus, most higher plants are poorly designed from an evolutionary point of view. Even taken in isolation, this simple fact should demonstrate to any reasonable person that organisms are not invariably ‘‘perfect’’. Finally, it cannot escape notice that numerous CO2-concentrating mechanisms have evolved multiple times in the algae, pteridophytes and gymnosperms, thus providing ample evidence for adaptive convergent macroevolution in members of the kingdoms Protoctista and Plantae. Acknowledgments We thank Mr. W. Kawollek (University of Kassel) for technical help. The cooperation of the authors was initiated by the Alexander von Humboldt-Stiftung (AvH), Bonn (Germany). References Apel, P., Horstmann, C., Pfeffer, M., 1997. The Moricandia syndrome in species of the Brassicaceae – evolutionary aspects. Photosynthetica 33, 205–215. Badger, M.R., Andrews, T.J., Whitney, S.M., Ludwig, M., Yellowlees, D.C., Leggat, W., Price, G.D., 1998. The diversity of coevolution of Rubisco, plastids, pyrenoids, and chloroplast-based CO2concentrating mechanisms in algae. Can. J. Bot. 76, 1052–1071. Berry, J., Bjo¨rkman, O., 1980. Photosynthetic response and adaptation to temperature in higher plants. Annu. Rev. Plant Physiol. 31, 491–543. Bird, I.F., Cornelius, M.J., Keys, A.J., 1982. Affinity of RuBP carboxylases for carbon dioxide and inhibition of the enzymes by oxygen. J. Exp. Bot. 33, 1004–1013. Brown, N.J., Parsley, K., Hibbert, J.M., 2005. The future of C4 research – maize, Flaveria or Cleome? Trends Plant Sci. 10, 215–221. De Veau, E.J., Burris, J.E., 1989. Photorespiratory rates in wheat and maize as determined by 18 O labeling. Plant Physiol. 90, 500–511. Ehleringer, J., Bjo¨rkman, O., 1977. Quantum yields for CO2 uptake in C3 and C4 plants. Dependence on temperature, CO2 and O2 concentration. Plant Physiol. 59, 86–90. Eldredge, N., 1989. Macroevolutionary Dynamics. McGraw-Hill Publishing Company, New York. Futuyma, D.J., 1998. Evolutionary Biology, third ed. Sinauer, Sunderland, MA. Hatch, M.D., 1992. C4 photosynthesis: an unlikely process full of surprises. Plant Cell Physiol. 33, 333–342.
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Theory in Biosciences 125 (2007) 81–92 www.elsevier.de/thbio
Photosynthesis research on yellowtops: Macroevolution in progress U. Kutscheraa,, K.J. Niklasb a
Institut fu¨r Biologie, Universita¨t Kassel, Heinrich-Plett-Str. 40, 34109 Kassel, Germany Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA
b
Received 15 May 2006; accepted 1 June 2006
Abstract The vast majority of angiosperms, including most of the agronomically important crop plants (wheat, etc.), assimilate CO2 through the inefficient C3 pathway of photosynthesis. Under ambient conditions these organisms loose about 1/3 of fixed carbon via photorespiration, an energetically wasteful process. Plants with C4 photosynthesis (such as maize) eliminate photorespiration via a biochemical CO2-pump and thus have a larger rate of carbon gain. The genus Flaveria (yellowtops, Asteraceae) contains not only C3 and C4 species, but also many C3–C4 intermediates, which have been interpreted as evolving from C3 to fully expressed C4 metabolism. However, the evolutionary significance of C3–C4 Flaveria-intermediates has long been a matter of debate. A well-resolved phylogeny of nearly all Flaveria species has recently been published. Here, we review pertinent background information and combine this novel phylogeny with physiological data. We conclude that the Flaveria species complex provides a robust model system for the study of the transition from C3 to C4 photosynthesis, which is arguably a macroevolutionary event. We conclude with comments relevant to the current Intelligent Design debate. r 2006 Elsevier GmbH. All rights reserved. Keywords: Evolution; Flaveria; Intermediate forms; Macroevolution; Photosynthesis
Corresponding author.
E-mail addresses: [email protected] (U. Kutschera), [email protected] (K.J. Niklas). 1431-7613/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.thbio.2006.06.001
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Introduction Agronomists have long known that the average crop yield of maize (Zea mays) is much larger than that of many other domesticated grasses such as wheat (Triticum aestivum) or barley (Hordeum vulgare). According to Agricultural Statistics (USDA 1980), the average yields obtained for maize, wheat and barley are 3.06, 1.03 and 1.21 tons grain/acre, respectively. The reasons for these striking differences were debated when these data first became available 25 years ago. However, today we know that the approximately two-fold larger average crop yield of maize compared to wheat (and barley) is largely attributable to the more efficient photosynthetic mode of the maize plant (Sage and Monson, 1999; Sage, 2004). Like its relatives sugarcane and sorghum, Z. mays is characterized by a biochemical pump that concentrates the atmospheric carbon dioxide (CO2) around the key enzyme of photosynthesis, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco, localized in the stroma of chloroplasts) after diffusion via the stomata into leaf cells. This sophisticated two-step mechanism of CO2-assimilation was discovered in the 1960s, only a few years after Melvin Calvin (1911–1997) and his colleagues had resolved the details of photosynthetic CO2-fixation in suspensions of the green alga Chlorella (Hatch, 1992; Kutschera, 2002). In this contribution, we describe a model system that has been used to elucidate the phylogenetic development of the CO2-pump in higher plants such as maize. This (and other) arguably macroevolutionary transition has been addressed in previous publications (Kutschera and Niklas, 2004, 2005), where background information on the topic discussed here is summarized.
Photorespiration and the CO2-pump of the maize plant Over the past decades, it has become very apparent that more than 90% of all land plants, including most crop species (wheat, barley, etc.) assimilate CO2 via the onestep C3 ‘‘Chlorella-type’’ pathway of photosynthesis. In these green organisms, the five-carbon sugar RuBP is the primary CO2-acceptor and the first product of photosynthetic CO2-fixation is the three-carbon (C3) molecule 3-phosphoglycerate (PGA). However, Rubisco, the enzyme catalyzing the fixation of CO2, is bifunctional. Both CO2 and atmospheric oxygen (O2) compete with one another at the active site of Rubisco. Carboxylation results in the formation of 2 PGA, whereas oxygenation of RuBP leads to the production of 1 PGA and 1 glycollate-2-P (oxygenase reaction). Thus, the primary reaction of one-step C3photosynthesis occurring in chloroplasts can be summarized as follows: Rubisco
Mesophyll cell : RuBP þ CO2 ðO2 Þ ! 2 PGA ð1 PGA þ 1 glycolate2PÞ.
Importantly, only PGA can be converted in the stroma of chloroplasts into carbohydrates. The second product of the oxygenase reaction, glycolate-2-P, cannot
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be used for CO2-fixation in C3 plants; it is salvaged inefficiently in the photorespiratory pathway (Sage and Monson, 1999; Leegood, 2002). Photorespiration is the light-dependent uptake of O2 and concomitant release of CO2 (and NH3) associated with glycolate-2-P metabolism. Because the rate of CO2-assimilation in C3 plants such as wheat is inhibited by ca. 35% in normal air containing 21% O2 (compared with an artificial oxygen-poor atmosphere), photorespiration is a wasteful process and leads to a decrease in photosynthetic efficiency (De Veau and Burris, 1989; Monson et al., 1984). In other words, C3 plants suffer from a ‘‘leak’’ in their CO2-assimilatory pathway (Calvin-cycle). The photosynthetic ‘‘design’’ in these plants is sub-optimal or poor, because 1/3 of their photosynthetic potential is wasted (Mann, 1999). In contrast, the rate of photorespiration is close to zero in C4 plants and virtually no O2-dependent inhibition of photosynthesis occurs. This two-step C4 mode of CO2-fixation requires two subsequent carboxylations, which occur in separate cell types of the leaf. In the first step, atmospheric CO2 is fixed in mesophyll cells into a four-carbon (C4) compound by the enzyme phosphoenolpyruvate carboxylase (PEPC); carbon dioxide is attached to the three-carbon compound phosphoenolpyruvate (PEP, the primary CO2-acceptor) to produce oxaloacetate, and, in the second step, this four-carbon compound is converted into malate and transferred (pumped) into the bundle sheath cells where it is broken down into C3 compounds. During this reaction, CO2 is released and concentrated around the second carboxylating enzyme Rubisco, which is concentrated in the chloroplasts of bundle sheath cells where the final CO2-fixation occurs as in C3 plants: PEPC
Mesophyll cell : PEP þ CO2 ! oxaloacetate ! malate ðtransferÞ, Rubisco
Bundle sheath cell : malate ! CO2 þ RuBP ! 2 PGA ! carbohydrates:
As a result of this two-step CO2-pump mechanism, only PGA (but virtually no glycolate-2-P) is produced, i.e., photorespiration in C4 plants is almost entirely suppressed (Sage and Monson, 1999; Leegood, 2002).
The adaptive significance of C4 metabolism Rubisco evolved in ancient cyanobacteria about 3000 million years ago when the Earth’s atmosphere was warmer and substantially different in its gas composition than it is today (molecular oxygen was lacking; the CO2 concentration was higher than today; Schopf, 1999; Knoll, 2003). This enzyme may have been very efficient during the time in Earth’s history when it made its first appearance. But it is currently far from ‘‘optimal’’ photosynthetically because of the affinity of its active site for oxygen (O2). Molecular phylogenies and other data indicate that the chloroplasts of the land plants (embryophytes) and the ancestral group from which they descentded, the
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green algae (Chlorophyta), evolved as a result of ‘‘residency’’ with a heterotrophic prokaryote ‘‘host’’ cell and subsequently evolved into modern-day chloroplasts (Niklas, 1997; Kutschera and Niklas, 2004, 2005). Palaeochemical studies of Earth’s ancient atmosphere indicate that during the time this endosymbiotic event occurred, atmospheric CO2 levels were substantially higher than they are now. Likewise, studies of present-day cyanobacteria (and presumably the ancestral life-forms that eventually evolved into chloroplasts) demonstrate that they possess an inorganic CO2-pump located at the plasma membrane that delivers hydrogen carbonate (HCO–3) to the cytosol. Cyanobacteria also generate CO2 (by carbonic anhydrase activity) in carboxysomes, which nurture the majority of Rubisco activity in their cells (Kaplan et al., 1998; Price et al., 1998). In contrast, terrestrial C3 plants rely on passive diffusion for the entry of CO2 into their cells. It comes, therefore, as little surprise that the highest specific reaction rates for carboxylation, the lowest selectivity for CO2 over O2 and the lowest affinity for CO2 in the cyanobacteria Chlorophyta–embryophytes ‘‘Rubisco clade’’ are found in present-day cyanobacteria, nor that a higher selectivity for CO2 over O2 and a higher affinity for CO2 is reported for aquatic members of the Chlorophyta and embryophytes. In general, an even higher CO2/O2 selectivity and CO2 affinity is found in C4 plants (but with less the selectivity and affinity values for terrestrial C3 plants) (Badger et al., 1998; Bird et al., 1982; Palmqvist et al., 1995; Rintama¨ki and Aro, 1985). The significance of this phylogenetic trend in the context of CO2 and O2 concentrations at the active site of Rubisco in present-day cyanobacteria and ‘‘green’’ plants is that the CO2/O2 ratio around Rubisco in cyanobacteria is 10 or more times that in air-equilibrium solution as a result of the activity of inorganic carbon pump and carboxysomes, both of which saturate the carboxylase activity of cyanobacterial Rubisco (and thus minimizes the oxygenase activity of this enzyme). In contrast, because most bryophytes and all vascular plants, including gymnosperms and angiosperms (with the exception of C4 plants), depend on the passive diffusion of CO2 to Rubisco, the diffusive resistance to CO2 entry and O2 exit from photosynthetic cells results in the steady-state CO2/O2 ratio and CO2 concentration at the active site of Rubisco that yields only half the saturation of the Rubisco carboxylase activity. Hence, very significant rates of O2 uptake by Rubisco (i.e., photorespiration) occurs during photosynthesis at present-day atmospheric CO2 concentrations (De Veau and Burris, 1989; Kutschera, 2002). Based on numerous and extensive phylogenetic and palaeobotanical analyses, the oldest flowering plants are about 125 million years old (Archaefructus from the Cretaceous, see Niklas, 1997; Kutschera and Niklas, 2004); these ancient green organisms most likely possessed C3 metabolism. Similar analyses show that the CO2pump in C4 plants is a recent evolutionary innovation, occurring over the past 30–20 million years in 19 different monocotyledonous and dicotyledonous families (Sage, 2004). In terms of the distribution of C4 species, 75% are monocots (Poaceae and Cyperaceae). The remaining 25% of all known C4 species is found in the Brassicaceae, Cruciferae, Aizoaceae, Amaranthaceae, Chenopodiaceae and Asteraceae (Apel et al., 1997; Kadereit et al., 2003).
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The evolution of C4 species in so many phyletically diverse families inevitably raises the question, Why? Certainly, convergent evolution provides strong circumstantial evidence for adaptation by means of natural selection. Thus, the real question is, What is the adaptive value of C4 photosynthesis? One obvious answer is that C4 photosynthesis is an adaptive response to the fairly steady decrease in atmospheric CO2 over the last 600 million years (Sage, 2004). Another plausible answer is that C4 metabolism confers a competitive advantage in hot habitats. These two hypotheses are not mutually exclusive, because C4 metabolism under low CO2 partial pressure and high temperatures (which are generally, but not invariably associated with limited water availability) would significantly enhance the competitive ability of any photosynthetic plant (Hatch, 1992). In typical C3 plants (Fig. 1), the rate of CO2-assimilation increases to some limit and then decreases with both increasing ambient temperatures and with increasing atmospheric CO2 levels. At ambient CO2 levels, leaf photosynthesis is limited by the activity of the enzyme Rubisco, and the response reflects two competing processes: an increase in the carboxylation rate with increasing temperature and a decrease in the affinity of Rubisco for CO2 (Fig. 1). A comparison between the quantum yields of photosynthetic carbon fixation of C3 and C4 plants shows that across most species, the efficiency of C3 plants at lower ambient temperatures (15 1C) exceeds that of C4 plants (Fig. 2). However, at higher temperatures (35–40 1C), the efficiency of C4 leaves is larger than that of the corresponding organs in C3 plants (Ehleringer and Bjo¨rkman, 1977; Berry and Bjo¨rkman, 1980). For example, the CO2-pump in maize and other C4 plants allows these organisms to assimilate atmospheric CO2 at lower gas concentrations than their C3 congenitors. But this advantage is particularly evident in terms of leaf carbon gain in warm highlight environments (Sage, 2004). This feature of C4 metabolism is well shown by
Fig. 1. Changes in the rate of photosynthesis (mmol CO2/m2 s) as a function of temperature and ambient or saturating atmospheric CO2 concentrations in leaves of Nerium oleander, a typical C3 plant. Data taken from Berry and Bjo¨rkman (1980).
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Fig. 2. Quantum yield of photosynthetic carbon fixation (mol CO2 per absorbed quantum) in leaves of a C3 and C4 plant (Encelia californica and Atriplex rosea, respectively) plotted as a function of ambient temperature. The measurements were carried out at normal CO2 concentration. Data taken from Ehleringer and Bjo¨rkman (1977).
comparing the carbon dioxide compensation points (CP) of C3 and C4 species. This physiological parameter, which measures for the efficiency of the CO2-pump mechanism, is defined as the CO2 concentration at which light-dependent gas uptake (leaf assimilation) is just balanced by CO2 loss through photorespiration and mitochondrial (dark) respiration. For example, at comparable air temperatures, wheat and other C3 plants typically have CP-values of about 50 ml CO2/l air (at 21 vol% O2), whereas maize has a CP of 0–3 ml/l, depending on the developmental stage of the plant (Sage and Monson, 1999).
The model system Flaveria More detailed comparisons of CP-values can provide even deeper insights into the adaptive significance of C4 metabolism, particularly if these comparisons are drawn among species possessing C3, C3–C4 intermediate or C4 metabolism nested in the same genus. An excellent example is the genus Flaveria (common name: yellowtops), which is a member of the Asteraceae (the largest dicot family, with about 1300 genera and more than 21 000 species). This genus contains 23 species of small annual herbs and perennial shrubs, some of which are known to have C3, C3–C4 intermediate or C4 metabolism (Monson et al., 1984; Monson and Moore, 1989; Westhoff and Gowik, 2004; Brown et al., 2005; McKown et al., 2005). One of these is the C4 (and cosmopolitan invasive) species Flaveria trinervia (Syn. F. repanda), which can reach a height of 40–80 cm depending on environmental conditions (Fig. 3). Gas exchange measurements on the leaves of greenhouse-grown specimens of this species
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Fig. 3. Ontogeny of a model organism for the study of a phylogenetic (macroevolutionary) trend in higher plants. Flaveria trinervia (Sprengel) C. Mohr (Syn. F. repanda), common name: clustered yellowtops, is an annual dicot C4 plant (Asteraceae). Juvenile (A), flowering (B) and senescing sporophyte (C). Scale bar ¼ 4 cm.
indicate a CO2-CP at approximately 3.5 ml/l (Ku et al., 1991) (a CP-value of o1 has been reported previously, see Monson et al., 1984), a CO2-assimilation rate (A) of 32 mmol/m2 s, and inhibition of photosynthesis at 21 vol% O2 (I) occurs at roughly 1.5% (the corresponding data for maize is CP3 ml/l, A33 mmol CO2/m2 s and I1–2%, respectively, see De Veau and Burris, 1989). Two other widely distributed C4 Flaveria species (i.e., F. bidentis and F. australasica) display quantitatively similar leaf gas exchange characteristics. In contrast, three C3 Flaveria species (i.e., F. cronquistii, F. pringlei and F. robusta) have very different gas exchange characteristics, i.e., CP60–62 ml/l; A16–21 mmol CO2/m2 s; I30–35% (Ku et al., 1991). These values are similar to those measured on wheat (T. aestivum), a representative C3 monocot (i.e., CP50 ml/l, A22 mmol CO2/m2 s and inhibition of photosynthesis at 21 vol% O2 (I)30–40%) (De Veau and Burris, 1989). Note that the rate of CO2-assimilation (A) of the C3 dicot Nerium oleander under normal conditions (air, 25 1C) is about 20 mmol/m2 s (see Fig. 1). Data such as these show clearly that C3 and C4 Flaveria species (Fig. 3) display leaf photosynthetic characteristics similar to those of the classical model organisms for the study of macroevolutionary transitions in plants, i.e., wheat and maize (Kellogg, 2000). Perhaps far more relevant to understanding the evolution of C4 metabolism are the leaf photosynthetic characteristics of Flaveria species manifesting a C3–C4 intermediate or C4-like metabolisms. When these data are plotted on a recently published phylogenetic tree for the genus, it becomes immediately clear that any shift from the antecedent C3 condition toward the evolutionarily derived C4 condition is physiologically adaptive (Fig. 4). Certainly, a note of caution must be introduced whenever phenotypic trends are deduced from the distribution of character states ‘‘mapped’’ onto a cladogram such as the one shown in Fig. 4 because the polarity of these states has to be rigorously established. For example, without independent confirmation, it is just as reasonable
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Fig. 4. Phylogeny depicting the evolutionary history of 18 Flaveria species for which the carbon dioxide compensation point (CP) has been published (unit: ml CO2/l air at 21 vol% O2). Adapted from McKown et al. (2005). The CP-values are from Ku et al. (1991).
to argue that some or all of the Flaveria species possessing C3–C4 intermediate or C4-like photosynthesis reflect the loss of fully developed C4 metabolic features rather than natural plant populations in the process of evolving toward full C4 photosynthesis. Indeed, the ‘‘macroevolution-in-action-hypothesis’’ has been questioned by some workers (for a critical discussion, see Monson and Moore, 1989). However, the comprehensive analysis of 21 out of the 23 described Flaveria species published by McKown et al. (2005) has now unequivocally resolved this longstanding open question.
Phylogeny and photosynthetic features of yellowtops The data set and detailed analyses of McKown et al. (2005) show that C3 photosynthesis is the ancestral condition and that a latent genetic predisposition for the evolution of C4 photosynthesis exists in the genus Flaveria. Based on the biogeographic distribution and the ecological preferences of the species within this genus, it is also reasonably clear that the multiple origins of the C4 photosynthesis were the result of selection pressures for survival in hot, arid or saline conditions.
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The data set of McKown et al. (2005) establishes two well-supported clades, designated A and B, each of which is identifiable on the basis of morphological, reproductive or physiological character states (Fig. 4). For example, most of species nested in clade A are small to moderately sized annual plants with inflorescences possessing 3–4 phyllaries (Fig. 3). All self-compatible species in this clade have C4 photosynthesis. In contrast, most of the species in clade B are moderate sized perennial and self-incompatible plants possessing 5–6 phyllaries. The majority of species in this clade has C3–C4 intermediate photosynthesis, the exception being F. brownii (Fig. 4). The basal species in each clade are C3–C4 physiological intermediates. However, they differ in their CO2-CPs and O2 inhibition, and thus in the type of their C3–C4 intermediate metabolism. Specifically, the basal species F. ramosissima in clade A is type II, whereas the basal species F. angustifolia in clade B is type I. (type I/II C3–C4 intermediates are characterized by the absence/presence of a C4 cycle). The appearance of type II C3–C4 intermediate photosynthesis in clade A coincides with the evolution of an annual life cycle in F. ramosissima and suggests that the common ancestor of this species (and other C4-like or C4 species in the clade, which are also annual species, e.g., F. vaginata) possessed a type II C3–C4 intermediate metabolism. If true, then the capacity to fully express C4 metabolism evolved on three separate occasions in clade A (i.e., once each in F. campestris, a relative of F. palmeri, F. australasica/F. trinerva and F. bidentis). Each of these evolutionary events coincides with the appearance of self-incompatibility. Noting that the ancestral condition for both clade A and B is C3 photosynthesis, the appearance of type I C3–C4 intermediate photosynthesis at the base of clade B is likely a derived condition, which coincides with the appearance of perennial and selfincompatible Flaveria species (Fig. 4). Based on low genetic divergence values and genomic analyses of closely related species, the most evolutionarily derived species in each of the two Flaveria clades evolved rapidly (McKown et al., 2005). In turn, various lines of evidence support the hypothesis that Flaveria speciation has undergone comparatively recent and intense selection for C3–C4 intermediate, C4-like and C4 photosynthesis: prior work has shown that some type II C3–C4 intermediate species initially assimilate CO2 into C4 acids under low CO2 levels, a condition that promotes photorespiration (McKown et al., 2005). Although decreasing CO2 levels may have played a role in the evolution of C4 metabolism multiple times in Flaveria (as well as many other flowering plant lineages), a variety of other ecological factors that coincide with the habitats occupied by basal Flaveria species are likely to have played much more important roles (e.g., heat, soil–water deprivation, salinity). Recall that C4 metabolism confers an advantage to plants living in hot habitats, which are generally but not invariably associated with water-stressed conditions (see Fig. 2). In this context, the basal C3–C4 intermediate species F. ramosissima and F. angustifolia grow in the sandy soils of the warm and arid Puebla-Oaxaca region of Mexico. This environment fosters high rates of transpiration and arguably therefore favors the survival of species capable of compensating for water deprivation and eliminating or reducing photorespiration. Likewise, the only C4-like species in clade B (i.e., F. brownii)
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grows in warm saline sandy flats and brackish marshes, environments that can subject species to intense selection. Life-history features may have been equally important to the rapid physiological radiation reported for this genus. Consider that all known advanced C3–C4 intermediate, C4-like and C4 Flaveria species are annuals. Likewise, all C4 Flaveria species are self-compatible. As noted by McKown et al. (2005), the comparatively short generation times of annual species typically promotes gene recruitment processes and the rapid accumulation of genetic variation in plant populations. Likewise, self-compatible species that have evolutionarily survived the gauntlet of inbreeding depression, early in their history, are purged of maladaptive alleles during an initially intense episode of selection for adaptive genetic variants. As noted, the environments currently occupied by advanced C3–C4 intermediate, C4-like and C4 Flaveria species would have provided intense selection pressures for coping with arid as well as hot conditions.
Conclusions The phylogenetic development of the biochemical CO2-pump capable of suppressing photorespiration and thereby enhancing the rate of photosynthetic CO2-assimilation is arguably a macroevolutionary event (here defined as the evolutionary appearance of any morphological, physiological or biochemical novelty above the level of populations) (Eldredge, 1989; Kellogg, 2000; Futuyma, 1998; Kutschera, 2006a, b, c; Kutschera and Niklas, 2004, 2005; Simons, 2002; Mayr, 1982, 2001). The work of McKown et al. (2005) and others demonstrates that the yellowtop (Flaveria) species complex provides an exceptionally powerful living model system with which to explore a physiological and morphological macroevolutionary event as well as patterns of speciation and thus microevolution, particularly in terms of the roles played by environmental factors (such as heat as well as aridity and water deprivation) and reproductive syndromes (self-compatibility and -incompatibility). The molecular mechanisms responsible for the occurrence of the CO2-pump are also amenable to investigation (Kopriva et al., 1996; Sage, 2004). For example, preliminary results indicate that C4 gene evolution occurred via duplication of pre-existing C3 DNA-sequences, followed by advantageous mutations in one of the duplicates to yield a novel role (neofunctionalization) (Monson, 2003; Westhoff and Gowik, 2004). Such adaptive mutations may have been rapidly ‘‘locked’’ into the genomes of advanced C3–C4, C4-like and C4 Flaveria species as a consequence of the co-occurrence of self-compatible (inbreeding) reproductive systems functioning in arid and hot environments. The facts that Rubisco across present-day eukaryotic photoautotrophs is far from a ‘‘perfect enzyme’’ as well as the evidence for macroevolution in the Flaveria species complex (Fig. 4) serve as clear examples with which to counter the fallacies perpetuated by creationists and adherents of the Intelligent Design (ID)-movement. Although some creationists and ID adherents may admit that microevolution (variation or speciation) is unquestionable, they assert that there is no evidence for
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macroevolution in extant populations of organisms and that living beings are ‘‘perfectly designed’’ (Kutschera, 2003, 2006a, b, c). The evolution of C4 metabolism in the Flaveria species complex and the fact that roughly 90% of all extant angiosperms loose ca. 1/3 of the carbon fixed in the light reactions of C3 photosynthesis to photorespiration easily refute these assumptions. Rubisco evolved under conditions that made this enzyme reasonably efficient (anaerobic atmosphere; high CO2-level), particularly in the physiological context of the cyanobacteria-like organisms that ultimately evolved into present-day chloroplasts (Kutschera and Niklas, 2005). However, it is clear that once these cyanobacteria gained permanent residency in their host cells, the efficiency of photosynthesis was significantly reduced and only improved with subsequent physiological and morphological adaptive evolutionary events (i.e., the occurrence of C4 photosynthesis). In a very real sense, Rubisco and C3 metabolism are ‘‘phyletic legacies’’ that are imperfect in many ways. All available evidence indicates that photorespiration is a resounding waste of energy and has no obvious advantage to C3 plants (Mann, 1999; Leegood, 2002). Thus, most higher plants are poorly designed from an evolutionary point of view. Even taken in isolation, this simple fact should demonstrate to any reasonable person that organisms are not invariably ‘‘perfect’’. Finally, it cannot escape notice that numerous CO2-concentrating mechanisms have evolved multiple times in the algae, pteridophytes and gymnosperms, thus providing ample evidence for adaptive convergent macroevolution in members of the kingdoms Protoctista and Plantae. Acknowledgments We thank Mr. W. Kawollek (University of Kassel) for technical help. The cooperation of the authors was initiated by the Alexander von Humboldt-Stiftung (AvH), Bonn (Germany). References Apel, P., Horstmann, C., Pfeffer, M., 1997. The Moricandia syndrome in species of the Brassicaceae – evolutionary aspects. Photosynthetica 33, 205–215. Badger, M.R., Andrews, T.J., Whitney, S.M., Ludwig, M., Yellowlees, D.C., Leggat, W., Price, G.D., 1998. The diversity of coevolution of Rubisco, plastids, pyrenoids, and chloroplast-based CO2concentrating mechanisms in algae. Can. J. Bot. 76, 1052–1071. Berry, J., Bjo¨rkman, O., 1980. Photosynthetic response and adaptation to temperature in higher plants. Annu. Rev. Plant Physiol. 31, 491–543. Bird, I.F., Cornelius, M.J., Keys, A.J., 1982. Affinity of RuBP carboxylases for carbon dioxide and inhibition of the enzymes by oxygen. J. Exp. Bot. 33, 1004–1013. Brown, N.J., Parsley, K., Hibbert, J.M., 2005. The future of C4 research – maize, Flaveria or Cleome? Trends Plant Sci. 10, 215–221. De Veau, E.J., Burris, J.E., 1989. Photorespiratory rates in wheat and maize as determined by 18 O labeling. Plant Physiol. 90, 500–511. Ehleringer, J., Bjo¨rkman, O., 1977. Quantum yields for CO2 uptake in C3 and C4 plants. Dependence on temperature, CO2 and O2 concentration. Plant Physiol. 59, 86–90. Eldredge, N., 1989. Macroevolutionary Dynamics. McGraw-Hill Publishing Company, New York. Futuyma, D.J., 1998. Evolutionary Biology, third ed. Sinauer, Sunderland, MA. Hatch, M.D., 1992. C4 photosynthesis: an unlikely process full of surprises. Plant Cell Physiol. 33, 333–342.
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