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 role of polyploidy, particularly allopolyploidy, in plant diversification is a subject of debate. Whole-genome duplications precede the origins of many major clades (e.g., angiosperms, Brassicaceae, Poaceae), suggesting that polyploidy drives diversification. However, theoretical arguments and empirical studies suggest that polyploid lineages may actually have lower speciation rates and higher extinction rates than diploid lineages. We focus here on the grass tribe Andropogoneae, an economically and ecologically important group of C 4 species with a high frequency of polyploids. A phylogeny was constructed for ca. 10% of the species of the clade, based on sequences of four concatenated low-copy nuclear loci. Genetic allopolyploidy was documented using the characteristic pattern of double-labeled gene trees. At least 32% of the species sampled are the result of genetic allopolyploidy and result from 28 distinct tetraploidy events plus an additional six hexaploidy events. This number is a minimum, and the actual frequency could be considerably higher. The parental genomes of most Andropogoneae polyploids diverged in the Late Miocene coincident with the expansion of the major C 4 grasslands that dominate the earth today. The well-documented whole-genome duplication in Zea mays ssp. mays occurred after the divergence of Zea and Sorghum. We find no evidence that polyploidization is followed by an increase in net diversification rate; nonetheless, allopolyploidy itself is a major mode of speciation. P olyploidy (whole-genome duplication) is often linked with the acquisition of new traits and subsequent species diversification, particularly in plants (1, 2). Ancient polyploidy correlates with major land-plant radiations (3) and the origins of orders, large families, and major clades (4-7) although, in many cases, sharp changes in diversification rates are delayed for millions of years after the polyploidization event (1). This phylogenetic pattern has led to the hypothesis that polyploidy causes or promotes diversification. Good mechanistic reasons support such a hypothesis. Studies of naturally occurring and synthetic polyploids find changes in gene expression, gene loss, release of transposons, and changes in morphology and physiology immediately after polyploidy (4,(8)(9)(10)(11)(12)). This pattern is particularly true for allopolyploids, which originate from a cross between genetically distinct parents often representing different species; in such cases, the biological changes seen after polyploidization may not reflect the effects of genome doubling per se, but rather the effect of hybridization between distantly related progenitors (4).Despite the appeal of the hypothesis that polyploidy causes diversification, there is evidence to the contrary. As noted by Stebbins (13), "polyploidy has been important in the diversification of species and genera within families, but not in the origin of the families and orders themselves," implying that polyploidy is only a minor force in diversification (see also ref. 14). ...

Summary• Grasses rank among the world's most ecologically and economically important plants. Repeated evolution of the C 4 syndrome has made photosynthesis highly efficient in many grasses, inspiring intensive efforts to engineer the pathway into C 3 crops. However, comparative biology has been of limited use to this endeavor because of uncertainty in the number and phylogenetic placement of C 4 origins.• We built the most comprehensive and robust molecular phylogeny for grasses to date, expanding sampling efforts of a previous working group from 62 to 531 taxa, emphasizing the C 4 -rich PACMAD (Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae and Danthonioideae) clade. Our final matrix comprises c. 5700 bp and is > 93% complete.• For the first time, we present strong support for relationships among all the major grass lineages. Several new C 4 lineages are identified, and previously inferred origins confirmed. C 3 /C 4 evolutionary transitions have been highly asymmetrical, with 22-24 inferred origins of the C 4 pathway and only one potential reversal.• Our backbone tree clarifies major outstanding systematic questions and highlights C 3 and C 4 sister taxa for comparative studies. Two lineages have emerged as hotbeds of C 4 evolution. Future work in these lineages will be instrumental in understanding the evolution of this complex trait.

No abstract

C 4 photosynthesis is a series of anatomical and biochemical modifications to the typical C 3 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, C 4 origins are absent from most plant lineages and clustered in others, suggesting that some characteristics increase C 4 evolvability in certain phylogenetic groups. The C 4 trait has evolved 22-24 times in grasses, and all origins occurred within the PACMAD clade, whereas the similarly sized BEP clade contains only C 3 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 C 4 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 C 4 evolution, suitable anatomy was present only in members of the PACMAD clade, explaining the clustering of C 4 origins in this lineage. These results show that key alterations of foliar anatomy occurring in a C 3 context and preceding the emergence of the C 4 syndrome by millions of years facilitated the repeated evolution of one of the most successful physiological innovations in angiosperm history.precursor | preadaptation | phylogeny | Poaceae