C4 Evolution Research Articles

The origins and diversification of C4 grasses and savanna‐adapted ungulates

AbstractC4 grasses constitute the main component of savannas and are pervasive in other dry tropical ecosystems where they serve as the main diet for grazing animals. Among potential factors driving C4 evolution of grasses, the interaction between grasses and grazers has not been investigated. To evaluate if increased grazing pressure may have selected for higher leaf silica production as the grasses diverged, we reconstructed the phylogeny of all 800 genera of the grass family with both molecular (combined multiplastid DNA regions) and morphological characters. Using molecular clocks, we also calculated the age and number of origins of C4 clades and found that shifts from C3 to C4 photosynthesis occurred at least 12 times starting 30.9 million years ago and found evidence that the most severe drop in atmospheric carbon dioxide in the late Oligocene (between 33 and 30 million years ago) matches the first origin of C4 photosynthesis in Chloridoideae. By combining fossil and phylogenetic data for ungulates and implementing a randomization procedure, our results showed that the appearance of C4 grass clades and ungulate adaptations to C4‐dominated habitats match significantly in time. An increase of leaf epidermal density of silica bodies was found to correspond to postulated shifts in diversification rates in the late Miocene [24 significant shifts in diversification (P<0.05) were detected between 23 and 3.7 million years ago]. For aristidoid and chloridoid grasses, increased grazing pressure may have selected for a higher leaf epidermal silica production in the late Miocene.

Oligocene CO 2 Decline Promoted C 4 Photosynthesis in Grasses

C4 photosynthesis is an adaptation derived from the more common C3 photosynthetic pathway that confers a higher productivity under warm temperature and low atmospheric CO2 concentration [1, 2]. C4 evolution has been seen as a consequence of past atmospheric CO2 decline, such as the abrupt CO2 fall 32-25 million years ago (Mya) [3-6]. This relationship has never been tested rigorously, mainly because of a lack of accurate estimates of divergence times for the different C4 lineages [3]. In this study, we inferred a large phylogenetic tree for the grass family and estimated, through Bayesian molecular dating, the ages of the 17 to 18 independent grass C4 lineages. The first transition from C3 to C4 photosynthesis occurred in the Chloridoideae subfamily, 32.0-25.0 Mya. The link between CO2 decrease and transition to C4 photosynthesis was tested by a novel maximum likelihood approach. We showed that the model incorporating the atmospheric CO2 levels was significantly better than the null model, supporting the importance of CO2 decline on C4 photosynthesis evolvability. This finding is relevant for understanding the origin of C4 photosynthesis in grasses, which is one of the most successful ecological and evolutionary innovations in plant history.

The evolution of C4 photosynthesis.

C4 photosynthesis is a series of anatomical and biochemical modifications that concentrate CO2 around the carboxylating enzyme Rubisco, thereby increasing photosynthetic efficiency in conditions promoting high rates of photorespiration. The C4 pathway independently evolved over 45 times in 19 families of angiosperms, and thus represents one of the most convergent of evolutionary phenomena. Most origins of C4 photosynthesis occurred in the dicots, with at least 30 lineages. C4 photosynthesis first arose in grasses, probably during the Oligocene epoch (24-35million yr ago). The earliest C4 dicots are likely members of the Chenopodiaceae dating back 15-21million yr; however, most C4 dicot lineages are estimated to have appeared relatively recently, perhaps less than 5million yr ago. C4 photosynthesis in the dicots originated in arid regions of low latitude, implicating combined effects of heat, drought and/or salinity as important conditions promoting C4 evolution. Low atmospheric CO2 is a significant contributing factor, because it is required for high rates of photorespiration. Consistently, the appearance of C4 plants in the evolutionary record coincides with periods of increasing global aridification and declining atmospheric CO2 . Gene duplication followed by neo- and nonfunctionalization are the leading mechanisms for creating C4 genomes, with selection for carbon conservation traits under conditions promoting high photorespiration being the ultimate factor behind the origin of C4 photosynthesis. Contents Summary 341 I. Introduction 342 II. What is C4 photosynthesis? 343 III. Why did C4 photosynthesis evolve? 347 IV. Evolutionary lineages of C4 photosynthesis 348 V. Where did C4 photosynthesis evolve? 350 VI. How did C4 photosynthesis evolve? 352 VII. Molecular evolution of C4 photosynthesis 361 VIII. When did C4 photosynthesis evolve 362 IX. The rise of C4 photosynthesis in relation to climate and CO2 363 X. Final thoughts: the future evolution of C4 photosynthesis 365 Acknowledgements 365 References 365.

Gene Duplication, Neofunctionalization, and the Evolution of C4Photosynthesis

The evolution of C4 photosynthesis provides one of the most interesting examples of evolutionary novelty in plants. As an adaptation that enhances plant carbon gain in warm climates with high light and relatively low atmospheric CO2 concentration, the complex interactions between C4 anatomy and biochemistry appear to have evolved over thirty times independently within the angiosperms. Past theories have explained the multiple appearances of C4 photosynthesis solely on the basis of global decreases in atmospheric CO2 concentration during the past 50 million years. The premise of such theories is that the C4 pathway provides selective advantages in terms of plant carbon gain in an atmosphere of low CO2 concentration. These “carbon balance” theories, however, are limited in their ability to explain why or how C4 photosynthesis evolved so many times independently and why certain patterns in the taxonomic distribution of C4 photosynthesis exist; e.g., the absence of C4 photosynthesis in canopy‐forming forest tree species and the paucity of C4 species within eudicots compared to monocots. In this review, I present the case that one of the most often overlooked aspects of C4 evolution is the potential for genetic limitation, specifically that associated with gene duplication and subsequent modification, which is crucial to the evolution of C4 biochemistry. I describe the research to date that provides insight into the origins of C4 genes, and I derive the conclusion that the evolution of C4 photosynthesis is largely a story of gene duplication while plants are still in the ancestral, C3 state. Once a reservoir of key, duplicated, and preserved C3 genes is present, a small amount of subsequent modification within gene promoter regions is all that is necessary to transform certain C3 patterns of gene expression to C4 patterns. Quantitative theory predicts that the most likely factors to be associated with the accumulation of a reservoir of duplicated C3 genes are large population size, short generation time, and frequent recruitment of sexually produced individuals. When combined with the selective pressures of reduced atmospheric CO2 concentration, consideration of population and life history factors, and the genetic constraints that they impose, could help explain certain patterns of C4 distribution.

Evolution of CAM and C4Carbon‐Concentrating Mechanisms

Mechanisms for concentrating carbon around the Rubisco enzyme, which drives the carbon‐reducing steps in photosynthesis, are widespread in plants; in vascular plants they are known as crassulacean acid metabolism (CAM) and C4 photosynthesis. CAM is common in desert succulents, tropical epiphytes, and aquatic plants and is characterized by nighttime fixation of CO2. The proximal selective factor driving the evolution of this CO2‐concentrating pathway is low daytime CO2, which results from the unusual reverse stomatal behavior of terrestrial CAM species or from patterns of ambient CO2 availability for aquatic CAM species. In terrestrials the ultimate selective factor is water stress that has selected for increased water use efficiency. In aquatics the ultimate selective factor is diel fluctuations in CO2 availability for palustrine species and extreme oligotrophic conditions for lacustrine species. C4 photosynthesis is based on similar biochemistry but carboxylation steps are spatially separated in the leaf rather than temporally as in CAM. This biochemical pathway is most commonly associated with a specialized leaf anatomy known as Kranz anatomy; however, there are exceptions. The ultimate selective factor driving the evolution of this pathway is excessively high photorespiration that inhibits normal C3 photosynthesis under high light and high temperature in both terrestrial and aquatic habitats. CAM is an ancient pathway that likely has been present since the Paleozoic era in aquatic species from shallow‐water palustrine habitats. While atmospheric CO2 levels have undoubtedly affected the evolution of terrestrial plant carbon‐concentrating mechanisms, there is reason to believe that past atmospheric changes have not played as important a selective role in the aquatic milieu since palustrine habitats today are not generally carbon sinks, and the selective factors driving aquatic CAM are autogenic. Terrestrial CAM, in contrast, is of increasing selective value under extreme water deficits, and undoubtedly, high Mesozoic CO2 levels reduced the amount of landscape perceived by plants as water limited. Late Tertiary and Quaternary reductions in atmospheric CO2, coupled with increasing seasonality, were probably times of substantial species radiation and ecological expansion for CAM plants. C4 photosynthesis occurs in only about half as many families as CAM, and three‐fourths of C4 species are either grasses or sedges. Molecular phylogenies indicate C4 is a more recent innovation than CAM and that it originated in the mid‐Tertiary, 20–30 Ma, although some data support an earlier origin. While the timing of the origin of C4 remains controversial, the nearly explosive increase in C4 species is clearly documented in the late Miocene, 4–7 Ma. Increasing seasonality has been widely suggested as an important climatic stimulus for this C4 expansion. Alternatively, based on models of photosynthetic quantum yield at different temperatures and CO2 concentration, it has been hypothesized that the late Miocene C4 expansion resulted from declining atmospheric CO2 levels. This model is most appropriate for explaining the transition from C3 grasslands to C4 grasslands but by itself may not be sufficient to explain the more likely scenario of a late Miocene transition from C3 woodland/savanna to C4 grasslands. A largely unexplored hypothesis is that climatic changes in late Miocene altered disturbance regimes, in particular the incidence of fires, which today are often associated with maintenance of C4 grasslands. Oceanic charcoal sediments that appear to represent Aeolian deposits from continental wildfires follow a strikingly similar pattern of explosive increase in late Miocene. Climate, CO2, and disturbance are not mutually exclusive explanations and probably all acted in concert to promote the expansion of C4 grasslands. More recently, late Quaternary changes in CO2 may have been responsible for driving major changes in the landscape distribution of C4 species. The theory is sound; however, many of the studies cited in support of this model are open to alternative interpretations, and none has eliminated climatic factors as important selective agents. CAM and C4 evolution required coupling of biochemical pathways with structural changes in photosynthetic tissues, succulence in CAM and Kranz in C4. This was apparently accomplished by piecemeal evolution beginning with mechanisms for recapturing respiratory CO2, although this need not have been so in aquatic CAM species. It has been proposed that the extreme rarity of both pathways in the same plant results from biochemical and structural incompatibilities (Sage 2002). Equally important is the fact that the selective environments are quite different, with CAM evolution thriving on stressful sites inhospitable to C3 species whereas C4 evolution has selected for rapid growth capable of outcompeting associated C3 plants.

Are crassulacean acid metabolism and C4 photosynthesis incompatible?

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. Despite sharing a similar metabolism, crassulacean acid metabolism (CAM) and C4 photosynthesis are not known to occur in identical species, with the exception of Portulaca spp. In Portulaca, C4 and weak CAM photosynthesis occur in distinct regions of the leaf, rather than in the same cells. This is in marked contrast to the situation in most CAM species where C3 and CAM photosynthesis are active in the same cell over the course of a day and growing season. The lack of CAM and C4 photosynthesis in identical cells of a plant indicates these photosynthetic pathways are incompatible. Incompatibilities between CAM and C4 photosynthesis could have a number of biochemical, anatomical and evolutionary explanations. Biochemical incompatibilities could result from the requirement for spatial separation of C3 and C4 phases in C4 plants versus temporal separation in CAM plants. In C4 plants, regulatory systems coordinate mesophyll and bundle sheath metabolism, with light intensity being the major environmental signal. In CAM plants, a circadian oscillator coordinates day and night phases of CAM. The requirement for rapid intercellular transport in C4 plants may be incompatible with the intracellular transport and storage needs of CAM. For example, the large vacuole required for malate storage in CAM could impede metabolite diffusion between mesophyll and bundle sheath cells in C4 plants. Anatomical barriers could also exist because both CAM and the C4 pathway require distinct leaf anatomies for efficient function. Efficient function of the C4 pathway generally requires an outer layer of cells specialized for phosphoenolpyruvate (PEP) carboxylation and regeneration and an inner layer for CO2 accumulation and refixation, while CAM species require enlarged vacuoles and tight cell packing. In evolutionary terms, barriers preventing CAM and C4 photosynthesis in the same species may be the initial steps in the respective evolutionary pathways from C3 ancestors. The first steps in C4 photosynthesis are related to scavenging photorespiratory CO2 via localization of glycine decarboxylase in the bundle sheath cells. The initial steps in CAM evolution are associated with the scavenging of respiratory CO2 at night by PEP carboxylation. In each, simplified versions of the specialized anatomy may need to be present for the evolutionary sequence to begin. For C4 evolution, enhanced bundle sheath size may be required in C3 ancestors; for CAM evolution, succulence may be required. Thus, before CAM or C4 photosynthesis began to evolve, the outcome of the evolutionary experiment may have been predetermined.

Molecular evolution of a host-range gene in geminiviruses infecting asexual populations of Eupatorium makinoi.

Asexual plants of Eupatorium makinoi is frequently infected with tobacco leaf curl geminivirus (TLCV). The host range of TLCV is narrow, and ORF C4 is considered to function as a host range determinant. Using this TLCV-Eupatorium system, we tested the expectation that the rate of amino acid replacements will be accelerated in ORF C4 if resistant genes of the host plants drive molecular evolution in ORF C4. ORF C4 is entirely contained within a longer ORF C1 encoding a replication protein. We analyzed 21 sequences containing ORF C4 and a part of ORF C1. While per-site number of synonymous substitutions exceeded that of replacements in ORF C1, per-site number of replacements exceeded that of synonymous substitutions in ORF C4. However, this excess of per-site replacement in ORF C4 was mostly explained by the overlap gene nature, because most synonymous substitutions in ORF C1 change amino acid of ORF C4. In conclusion, not positive but negative selection is a predominant mode characterizing molecular evolution of ORF C4.

Ｃ３植物からＣ４植物への進化 イネの光合成回路改良の可能性

Ｃ３植物からＣ４植物への進化 イネの光合成回路改良の可能性

Acclimation of Photosynthetic Phenotype to Environmental Heterogeneity

Inducible C4—like photosynthetic metabolism in Hydrilla verticillata leaf tissue elicits variability in photosynthetic phenotype, expressed as CO2 compensation point (Τ). We conducted a field and laboratory study to investigate the ecological and adaptive significance of this physiological phenomenon. Spatial horizontal environmental heterogeneity was observed within clonal populations of H. verticillata in Florida, USA. Measured at midday, the edge habitat at the expanding periphery of the clone exhibited a dissolved inorganic carbon (DIC) concentration of 0.7 mmol/L, pH 7.1, a dissolved oxygen (DO) level of 0.13 mmol/L, and biomass of 0.2 kg/m2. The mat habitat, located 200 cm towards the interior of the surface mat, exhibited DIC 0.1 mmol/L, pH 10.2, DO 0.48 mmol/L, and biomass 0.8 kg/m2. DIC depletion and DO supersaturation characterized the mat habitat for most of the day and much of the growing season. Furthermore, net photosynthesis, daily carbon gain, and relative growth rate (RGR) of H. verticillata were reduced 80% by mat conditions compared to edge conditions. Τs of H. verticillata were positively correlated with CO2 and bicarbonate concentration, and negatively correlated with pH, DO, and biomass. Low and high Τ photosynthetic phenotypes were associated with the mat and edge habitats, respectively. Photosynthetic phenotype of H. verticillata appears to acclimate to environmental heterogeneity within a clone in the field. Net photosynthesis and daily carbon gain of low Τphenotype H. verticillata was 128% and 40% greater than the high Τ phenotype when measured in the mat habitat, but was 21% lower than the high Τphotosynthetic phenotype when measured in the edge habitat under low quantum flux. Laboratory experiments showed a negative curvilinear relationship between the Τ of H. verticillata and plant density. The data demonstrate that plasticity in photosynthetic phenotype of H. verticillata is a density—dependent, physiological response that optimizes carbon gain within a stressful heterogeneous environment. Evolution of facultative C4—like photosynthetic metabolism in H. verticillata may have been an adaptation to the contraints imposed upon carbon gain by DIC and quantum flux limitation in the mat habitat.

Helical Motion and Non-Adiabatic Expansion in the Jet of 3C345

We developed a kinematic jet model for the motion and flux density evolution of the high frequency VLBI jet components C4 and C5 in the quasar 3C345 (Zensus et al. 1994) assuming the conservation of three basic quantities: the Lorentz factor, the angular momentum, and the opening angle of the jet. This model is a simplified description of the helical motion in a conical jet expected from the magnetodynamical model of Camenzind (1986) which is based on a black hole surrounded by a magnetized accretion disc. Our best fit yields Lorentz factors of 5.7 and 5.0 for components C4 and C5, respectively, and an angle between the jet axis and the observer's line of sight of 7.5°. These values are very close to those obtained by Unwin & Wehrle (1992) from component motion further out. An intrinsic bending of the jet axis is necessary to account for the common bent path of all jet components at core separations larger than about 4 mas. We found that differential Doppler boosting alone is not able to explain the flux density variations of component C4. A non-adiabatic expansion model of an inhomogeneous plasma cloud combined with differential Doppler boosting on a helical path fits the flux density evolution (Steffen et al. 1994). We find that the expansion in the decreasing part of the lightcurve is slower than expected from adiabatic expansion.

Control of Phenotypic Plasticity Via Regulatory Genes

Control of Phenotypic Plasticity Via Regulatory Genes

Organization and evolution of C4 and CYP21 genes in primates: importance of genomic segments

The evolutionary relationship between two central major histocompatibility complex (MHC) genes, C4 and CYP21, was investigated by employing pulsed field gel electrophoresis (PFGE) and conventional restriction fragment length polymorphism (RFLP) analyses in human and nonhuman primates. Using Taq I in conjunction with C4 and CYP21 probes, it has been found that there are four major types of C4 genes [defined by 7.0, 6.4, 6.0, and 5.4 kilobases (kb) Taq I fragments] and two major types of CYP21 genes (3.7 and 3.2 kb fragments) in human and nonhuman primates including chimpanzee, gorilla, and orangutan. All of the eight possible combinations of C4 and CYP21 genes can be identified on one or more human ancestral haplotypes (AH). It is concluded that each of the major types of C4 and CYP21 (and each of the combinations between these) predated human speciation. PFGE analysis with Mlu I and Pvu I suggested that each C4+CYP21 segment has a specific length of 30-50 kb and that each AH carries one, two, three, or even more segments. In the case of C4, it is important to note that there is no simple relationship between the RFLP and the protein classifications. Thus, at least some of the expressed polymorphisms could be relatively recent in that they are carried by the same or different gene types. These findings are consistent with the hypothesis that MHC AHs have been formed from a large pool of specific genomic segments and that further haplospecific polymorphism has developed subsequently.

Serum sickness‐like syndrome associated with minocycline therapy

A 19 year-old youth was taking oral minocycline and after 8 days he presented all four cardinal symptoms of serum sickness (urticaria, fever, lymphadenopathy and joint symptoms). C3, C4 and CH50 evolution imitate experimental serum sickness complement evolution. We exclude other causes of this syndrome. Although other hypersensitivity reactions have occurred with minocycline usage, to our knowledge serum sickness-like syndrome has not been previously reported with this drug.

GRASS LEAF ULTRASTRUCTURAL VARIATIONS

The results of an ultrastructural examination of leaf anatomy and cytology of plastids of both non‐Kranz and Kranz grasses are presented. Variations of Kranz anatomy examined include agranal and two granal conditions of kranz‐cell chloroplasts, details of the two typical parenchyma sheaths of Aristida, and the “distinctive cells” of species of some small panicoid tribes. These conditions are discussed in relation to C4 photosynthesis, evolution, and classification.