Pascal-Antoine Christin

The Origins of C4 Grasslands: Integrating Evolutionary and Ecosystem Science

Grassland Emergence The evolution of the C4 photosynthetic pathway from the ancestral C3 pathway in grasses led to the establishment of grasslands in warm climates during the Late Miocene (8 to 3 million years ago). This was a major event in plant evolutionary history, and their high rates of foliage production sustained high levels of herbivore consumption. The past decade has seen significant advances in understanding C4 grassland ecosystem ecology, and now a wealth of data on the geological history of these ecosystems has accumulated and the phylogeny of grasses is much better known. Edwards et al. (p. 587) review this multidisciplinary research area and attempt to synthesize emerging knowledge about the evolution of grass species within the context of plant and ecosystem ecology. The evolution of grasses using C4 photosynthesis and their sudden rise to ecological dominance 3 to 8 million years ago is among the most dramatic examples of biome assembly in the geological record. A growing body of work suggests that the patterns and drivers of C4 grassland expansion were considerably more complex than originally assumed. Previous research has benefited substantially from dialog between geologists and ecologists, but current research must now integrate fully with phylogenetics. A synthesis of grass evolutionary biology with grassland ecosystem science will further our knowledge of the evolution of traits that promote dominance in grassland systems and will provide a new context in which to evaluate the relative importance of C4 photosynthesis in transforming ecosystems across large regions of Earth.

The C4 plant lineages of planet Earth

Using isotopic screens, phylogenetic assessments, and 45 years of physiological data, it is now possible to identify most of the evolutionary lineages expressing the C(4) photosynthetic pathway. Here, 62 recognizable lineages of C(4) photosynthesis are listed. Thirty-six lineages (60%) occur in the eudicots. Monocots account for 26 lineages, with a minimum of 18 lineages being present in the grass family and six in the sedge family. Species exhibiting the C(3)-C(4) intermediate type of photosynthesis correspond to 21 lineages. Of these, 9 are not immediately associated with any C(4) lineage, indicating that they did not share common C(3)-C(4) ancestors with C(4) species and are instead an independent line. The geographic centre of origin for 47 of the lineages could be estimated. These centres tend to cluster in areas corresponding to what are now arid to semi-arid regions of southwestern North America, south-central South America, central Asia, northeastern and southern Africa, and inland Australia. With 62 independent lineages, C(4) photosynthesis has to be considered one of the most convergent of the complex evolutionary phenomena on planet Earth, and is thus an outstanding system to study the mechanisms of evolutionary adaptation.

Oligocene CO2 Decline Promoted C4 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.

Contemporaneous and recent radiations of the world's major succulent plant lineages.

The cacti are one of the most celebrated radiations of succulent plants. There has been much speculation about their age, but progress in dating cactus origins has been hindered by the lack of fossil data for cacti or their close relatives. Using a hybrid phylogenomic approach, we estimated that the cactus lineage diverged from its closest relatives ≈35 million years ago (Ma). However, major diversification events in cacti were more recent, with most species-rich clades originating in the late Miocene, ≈10–5 Ma. Diversification rates of several cactus lineages rival other estimates of extremely rapid speciation in plants. Major cactus radiations were contemporaneous with those of South African ice plants and North American agaves, revealing a simultaneous diversification of several of the worlds major succulent plant lineages across multiple continents. This short geological time period also harbored the majority of origins of C4 photosynthesis and the global rise of C4 grasslands. A global expansion of arid environments during this time could have provided new ecological opportunity for both succulent and C4 plant syndromes. Alternatively, recent work has identified a substantial decline in atmospheric CO2 ≈15–8 Ma, which would have strongly favored C4 evolution and expansion of C4-dominated grasslands. Lowered atmospheric CO2 would also substantially exacerbate plant water stress in marginally arid environments, providing preadapted succulent plants with a sharp advantage in a broader set of ecological conditions and promoting their rapid diversification across the landscape.

C4 Photosynthesis evolved in grasses via parallel adaptive genetic changes.

Phenotypic convergence is a widespread and well-recognized evolutionary phenomenon. However, the responsible molecular mechanisms remain often unknown mainly because the genes involved are not identified. A well-known example of physiological convergence is the C4 photosynthetic pathway, which evolved independently more than 45 times [1]. Here, we address the question of the molecular bases of the C4 convergent phenotypes in grasses (Poaceae) by reconstructing the evolutionary history of genes encoding a C4 key enzyme, the phosphoenolpyruvate carboxylase (PEPC). PEPC genes belong to a multigene family encoding distinct isoforms of which only one is involved in C4 photosynthesis [2]. By using phylogenetic analyses, we showed that grass C4 PEPCs appeared at least eight times independently from the same non-C4 PEPC. Twenty-one amino acids evolved under positive selection and converged to similar or identical amino acids in most of the grass C4 PEPC lineages. This is the first record of such a high level of molecular convergent evolution, illustrating the repeatability of evolution. These amino acids were responsible for a strong phylogenetic bias grouping all C4 PEPCs together. The C4-specific amino acids detected must be essential for C4 PEPC enzymatic characteristics, and their identification opens new avenues for the engineering of the C4 pathway in crops.

Anatomical enablers and the evolution of C4 photosynthesis in grasses

C4 photosynthesis is a series of anatomical and biochemical modifications to the typical C3 pathway that increases the productivity of plants in warm, sunny, and dry conditions. Despite its complexity, it evolved more than 62 times independently in flowering plants. However, C4 origins are absent from most plant lineages and clustered in others, suggesting that some characteristics increase C4 evolvability in certain phylogenetic groups. The C4 trait has evolved 22–24 times in grasses, and all origins occurred within the PACMAD clade, whereas the similarly sized BEP clade contains only C3 taxa. Here, multiple foliar anatomy traits of 157 species from both BEP and PACMAD clades are quantified and analyzed in a phylogenetic framework. Statistical modeling indicates that C4 evolvability strongly increases when the proportion of vascular bundle sheath (BS) tissue is higher than 15%, which results from a combination of short distance between BS and large BS cells. A reduction in the distance between BS occurred before the split of the BEP and PACMAD clades, but a decrease in BS cell size later occurred in BEP taxa. Therefore, when environmental changes promoted C4 evolution, suitable anatomy was present only in members of the PACMAD clade, explaining the clustering of C4 origins in this lineage. These results show that key alterations of foliar anatomy occurring in a C3 context and preceding the emergence of the C4 syndrome by millions of years facilitated the repeated evolution of one of the most successful physiological innovations in angiosperm history.

Causes and evolutionary significance of genetic convergence

Convergent phenotypes provide extremely valuable systems for studying the genetics of new adaptations. Accumulating studies on this topic have reported surprising cases of convergent evolution at the molecular level, ranging from gene families being recurrently recruited to identical amino acid replacements in distant lineages. Together, these different examples of genetic convergence suggest that molecular evolution is in some cases strongly constrained by a combination of limited genetic material suitable for new functions and a restricted number of substitutions that can confer specific enzymatic properties. We discuss approaches for gaining further insights into the causes of genetic convergence and their potential contribution to our understanding of how the genetic background determines the evolvability of complex organismal traits.

Phylogenomics of C4 Photosynthesis in Sedges (Cyperaceae): Multiple Appearances and Genetic Convergence

C(4) photosynthesis is an adaptive trait conferring an advantage in warm and open habitats. It originated multiple times and is currently reported in 18 plant families. It has been recently shown that phosphoenolpyruvate carboxylase (PEPC), a key enzyme of the C(4) pathway, evolved through numerous independent but convergent genetic changes in grasses (Poaceae). To compare the genetics of multiple C(4) origins on a broader scale, we reconstructed the evolutionary history of the C(4) pathway in sedges (Cyperaceae), the second most species-rich C(4) family. A sedge phylogeny based on two plastome genes (rbcL and ndhF) has previously identified six fully C(4) clades. Here, a relaxed molecular clock was used to calibrate this tree and showed that the first C(4) acquisition occurred in this family between 19.6 and 10.1 Ma. According to analyses of PEPC-encoding genes (ppc), at least five distinct C(4) origins are present in sedges. Two C(4) Eleocharis species, which were unrelated in the plastid phylogeny, acquired their C(4)-specific PEPC genes from a single source, probably through reticulate evolution or a horizontal transfer event. Acquisitions of C(4) PEPC in sedges have been driven by positive selection on at least 16 codons (3.5% of the studied gene segment). These sites underwent parallel genetic changes across the five sedge C(4) origins. Five of these sites underwent identical changes also in grass and eudicot C(4) lineages, indicating that genetic convergence is most important within families but that identical genetic changes occurred even among distantly related taxa. These lines of evidence give new insights into the constraints that govern molecular evolution.

Evolutionary Switch and Genetic Convergence on rbcL following the Evolution of C4 Photosynthesis

Rubisco is responsible for the fixation of CO2 into organic compounds through photosynthesis and thus has a great agronomic importance. It is well established that this enzyme suffers from a slow catalysis, and its low specificity results into photorespiration, which is considered as an energy waste for the plant. However, natural variations exist, and some Rubisco lineages, such as in C4 plants, exhibit higher catalytic efficiencies coupled to lower specificities. These C4 kinetics could have evolved as an adaptation to the higher CO2 concentration present in C4 photosynthetic cells. In this study, using phylogenetic analyses on a large data set of C3 and C4 monocots, we showed that the rbcL gene, which encodes the large subunit of Rubisco, evolved under positive selection in independent C4 lineages. This confirms that selective pressures on Rubisco have been switched in C4 plants by the high CO2 environment prevailing in their photosynthetic cells. Eight rbcL codons evolving under positive selection in C4 clades were involved in parallel changes among the 23 independent monocot C4 lineages included in this study. These amino acids are potentially responsible for the C4 kinetics, and their identification opens new roads for human-directed Rubisco engineering. The introgression of C4-like high-efficiency Rubisco would strongly enhance C3 crop yields in the future CO2-enriched atmosphere.

COMPLEX EVOLUTIONARY TRANSITIONS AND THE SIGNIFICANCE OF C3–C4 INTERMEDIATE FORMS OF PHOTOSYNTHESIS IN MOLLUGINACEAE

C4 photosynthesis is a series of biochemical and structural modifications to C3 photosynthesis that has evolved numerous times in flowering plants, despite requiring modification of up to hundreds of genes. To study the origin of C4 photosynthesis, we reconstructed and dated the phylogeny of Molluginaceae, and identified C4 taxa in the family. Two C4 species, and three clades with traits intermediate between C3 and C4 plants were observed in Molluginaceae. C3–C4 intermediacy evolved at least twice, and in at least one lineage was maintained for several million years. Analyses of the genes for phosphoenolpyruvate carboxylase, a key C4 enzyme, indicate two independent origins of fully developed C4 photosynthesis in the past 10 million years, both within what was previously classified as a single species, Mollugo cerviana. The propensity of Molluginaceae to evolve C3–C4 and C4 photosynthesis is likely due to several traits that acted as developmental enablers. Enlarged bundle sheath cells predisposed some lineages for the evolution of C3–C4 intermediacy and the C4 biochemistry emerged via co‐option of photorespiratory recycling in C3–C4 intermediates. These evolutionarily stable transitional stages likely increased the evolvability of C4 photosynthesis under selection environments brought on by climate and atmospheric change in recent geological time.