Genome biogeography reveals the intraspecific spread of adaptive mutations for a complex trait

Physiological novelties are often studied at macro‐evolutionary scales such that their micro‐evolutionary origins remain poorly understood. Here, we test the hypothesis that key components of a complex trait can evolve in isolation and later be combined by gene flow. We use C₄ photosynthesis as a study system, a derived physiology that increases plant productivity in warm, dry conditions. The grass Alloteropsis semialata includes C₄ and non‐C₄ genotypes, with some populations using laterally acquired C₄‐adaptive loci, providing an outstanding system to track the spread of novel adaptive mutations. Using genome data from C₄ and non‐C₄ A. semialata individuals spanning the species’ range, we infer and date past migrations of different parts of the genome. Our results show that photosynthetic types initially diverged in isolated populations, where key C₄ components were acquired. However, rare but recurrent subsequent gene flow allowed the spread of adaptive loci across genetic pools. Indeed, laterally acquired genes for key C₄ functions were rapidly passed between populations with otherwise distinct genomic backgrounds. Thus, our intraspecific study of C₄‐related genomic variation indicates that components of adaptive traits can evolve separately and later be combined through secondary gene flow, leading to the assembly and optimization of evolutionary innovations.     

C4 anatomy can evolve via a single developmental change

C₄ photosynthesis is a complex trait that boosts productivity in warm environments. Paradoxically, it evolved independently in numerous plant lineages, despite requiring specialised leaf anatomy. The anatomical modifications underlying C₄ evolution have previously been evaluated through interspecific comparisons, which capture numerous changes besides those needed for C₄ functionality. Here, we quantify the anatomical changes accompanying the transition between non‐C₄ and C₄ phenotypes by sampling widely across the continuum of leaf anatomical traits in the grass Alloteropsis semialata. Within this species, the only trait that is shared among and specific to C₄ individuals is an increase in vein density, driven specifically by minor vein development that yields multiple secondary effects facilitating C₄ function. For species with the necessary anatomical preconditions, developmental proliferation of veins can therefore be sufficient to produce a functional C₄ leaf anatomy, creating an evolutionary entry point to complex C₄ syndromes that can become more specialised.     

Evolution of the C4 photosynthetic pathway: events at the cellular and molecular levels

The biochemistry and leaf anatomy of plants using C₄ photosynthesis promote the concentration of atmospheric CO₂ in leaf tissue that leads to improvements in growth and yield of C₄ plants over C₃ species in hot, dry, high light, and/or saline environments. C₄ plants like maize and sugarcane are significant food, fodder, and bioenergy crops. The C₄ photosynthetic pathway is an excellent example of convergent evolution, having evolved in multiple independent lineages of land plants from ancestors employing C₃ photosynthesis. In addition to C₃ and C₄ species, some plant lineages contain closely related C₃–C₄ intermediate species that demonstrate leaf anatomical, biochemical, and physiological characteristics between those of C₃ plants and species using C₄ photosynthesis. These groups of plants have been extremely useful in dissecting the modifications to leaf anatomy and molecular biology, which led to the evolution of C₄ photosynthesis. It is now clear that great variation exists in C₄ leaf anatomy, and diverse molecular mechanisms underlie C₄ biochemistry and physiology. However, all these different paths have led to the same destination—the expression of a C₄ CO₂ concentrating mechanism. Further identification of C₄ leaf anatomical traits and molecular biological components, and understanding how they are controlled and assembled will not only allow for additional insights into evolutionary convergence, but also contribute to sustainable food and bioenergy production strategies.     

Photosynthetic innovation broadens the niche within a single species

Adaptation to changing environments often requires novel traits, but how such traits directly affect the ecological niche remains poorly understood. Multiple plant lineages have evolved C₄ photosynthesis, a combination of anatomical and biochemical novelties predicted to increase productivity in warm and arid conditions. Here, we infer the dispersal history across geographical and environmental space in the only known species with both C₄ and non‐C₄ genotypes, the grass Alloteropsis semialata. While non‐C₄ individuals remained confined to a limited geographic area and restricted ecological conditions, C₄ individuals dispersed across three continents and into an expanded range of environments, encompassing the ancestral one. This first intraspecific investigation of C₄ evolutionary ecology shows that, in otherwise similar plants, C₄ photosynthesis does not shift the ecological niche, but broadens it, allowing dispersal into diverse conditions and over long distances. Over macroevolutionary timescales, this immediate effect can be blurred by subsequent specialisation towards more extreme niches.     

Environmental and Evolutionary Preconditionsfor the Origin and Diversification of the C4 PhotosyntheticSyndrome

Abstract: C₄ photosynthesis is an evolutionary solution to high rates of photorespiration and low kinetic efficiency of Rubisco in CO₂‐depleted atmospheres of recent geologic time. About 7500 plant species are C₄, in contrast to 30 000 CAM and 250 000 C₃ species. All C₄ plants occur in approximately 90 genera from 18 angiosperm families. In all of these families, the C₄ pathway evolved independently. In many, multiple independent origins have occurred, such that over 30 distinct evolutionary origins of the C₄ pathway are recognized. Fossil and carbon isotope evidence show that the C₄ syndrome is at least 12 to 15 million years old, although estimates based on molecular sequence comparisons indicate it is over 20 million years old. The evolutionary radiation of herbaceous angiosperms may have been required for C₄ plant evolution. All C₄ species occur in advanced angiosperm families that appeared in the fossil record in the past 70 million years. Most of these families diversified in terms of genera and species numbers between 20 to 40 million years ago, during a period of global cooling, atmospheric CO₂ reduction and aridification. During the period of diversification, numerous traits arose in the C₃ flora that enhanced their performance in arid environments and atmospheres of reduced CO₂. Some of these traits may have predisposed certain taxa to develop the C₄ pathway once atmospheric CO₂ levels declined to a point where the ability to concentrate CO₂ had a selective advantage. Leading traits in C₃ plants that may have facilitated the initial transition to C₄ photosynthesis include close vein spacing and an enlargement of the bundle sheath cell layer to form a Kranz‐like anatomy. Ecological factors not directly connected with photosynthesis probably also played a role. For example, extensive ecological disturbance may have been needed to convert C₃‐dominated woodlands into open, high‐light habitats where herbaceous C₄ plants could succeed. Disturbances in the form of fire, and browsing by large mammals, increase during the time of C₄ plant evolution and diversification. Fire increased because of the drying climate, while browsing increased with the evolutionary diversification of the mammalian megafauna in the Oligocene and Miocene epochs. In summary, the origin of C₄ plants is hypothesized to have resulted from a novel combination of environmental and phylogenetic developments that, for the first time, established the preconditions required for C₄ plant evolution.     

Two forward genetic screens for vein density mutants in sorghum converge on a cytochrome P450 gene in the brassinosteroid pathway

The specification of vascular patterning in plants has interested plant biologists for many years. In the last decade a new context has emerged for this interest. Specifically, recent proposals to engineer C₄ traits into C₃ plants such as rice require an understanding of how the distinctive venation pattern in the leaves of C₄ plants is determined. High vein density with Kranz anatomy, whereby photosynthetic cells are arranged in encircling layers around vascular bundles, is one of the major traits that differentiate C₄ species from C₃ species. To identify genetic factors that specify C₄ leaf anatomy, we generated ethyl methanesulfonate‐ and γ‐ray‐mutagenized populations of the C₄ species sorghum (Sorghum bicolor), and screened for lines with reduced vein density. Two mutations were identified that conferred low vein density. Both mutations segregated in backcrossed F₂ populations as homozygous recessive alleles. Bulk segregant analysis using next‐generation sequencing revealed that, in both cases, the mutant phenotype was associated with mutations in the CYP90D2 gene, which encodes an enzyme in the brassinosteroid biosynthesis pathway. Lack of complementation in allelism tests confirmed this result. These data indicate that the brassinosteroid pathway promotes high vein density in the sorghum leaf, and suggest that differences between C₄ and C₃ leaf anatomy may arise in part through differential activity of this pathway in the two leaf types.     

C4 photosynthesis evolved in warm climates but promoted migration to cooler ones

C₄ photosynthesis is considered an adaptation to warm climates, where its functional benefits are greatest and C₄ plants achieve their highest diversity and dominance. However, whether inherent physiological barriers impede the persistence of C₄ species in cool environments remains debated. Here, we use large grass phylogenetic and geographical distribution data sets to test whether (1) temperature influences the rate of C₄ origins, (2) photosynthetic types affect the rate of migration among climatic zones, and (3) C₄ evolution changes the breadth of the temperature niche. Our analyses show that C₄ photosynthesis in grasses originated in tropical climates, and that C₃ grasses were more likely to colonise cold climates. However, migration rates among tropical and temperate climates were higher in C₄ grasses. Therefore, while the origins of C₄ photosynthesis were concentrated in tropical climates, its physiological benefits across a broad temperature range expanded the niche into warmer climates and enabled diversification into cooler environments.     

C4 grasses adapted to low precipitation habitats show traits related to greater mesophyll conductance and lower leaf hydraulic conductance

In habitats with low water availability, a fundamental challenge for plants will be to maximize photosynthetic C-gain while minimizing transpirational water-loss. This trade-off between C-gain and water-loss can in part be achieved through the coordination of leaf-level photosynthetic and hydraulic traits. To test the relationship of photosynthetic C-gain and transpirational water-loss, we grew, under common growth conditions, 18 C₄ grasses adapted to habitats with different mean annual precipitation (MAP) and measured leaf-level structural and anatomical traits associated with mesophyll conductance (g_m) and leaf hydraulic conductance (K_leaf). The C₄ grasses adapted to lower MAP showed greater mesophyll surface area exposed to intercellular air spaces (S_mes) and adaxial stomatal density (SD_ada) which supported greater g_m. These grasses also showed greater leaf thickness and vein-to-epidermis distance, which may lead to lower K_leaf. Additionally, grasses with greater g_m and lower K_leaf also showed greater photosynthetic rates (A_net) and leaf-level water-use efficiency (WUE). In summary, we identify a suite of leaf-level traits that appear important for adaptation of C₄ grasses to habitats with low MAP and may be useful to identify C₄ species showing greater A_net and WUE in drier conditions.     

Ecological selection pressures for C4 photosynthesis in the grasses

Grasses using the C₄ photosynthetic pathway dominate grasslands and savannahs of warm regions, and account for half of the species in this ecologically and economically important plant family. The C₄ pathway increases the potential for high rates of photosynthesis, particularly at high irradiance, and raises water-use efficiency compared with the C₃ type. It is therefore classically viewed as an adaptation to open, arid conditions. Here, we test this adaptive hypothesis using the comparative method, analysing habitat data for 117 genera of grasses, representing 15 C₄ lineages. The evidence from our three complementary analyses is consistent with the hypothesis that evolutionary selection for C₄ photosynthesis requires open environments, but we find an equal likelihood of C₄ evolutionary origins in mesic, arid and saline habitats. However, once the pathway has arisen, evolutionary transitions into arid habitats occur at higher rates in C₄ than C₃ clades. Extant C₄ genera therefore occupy a wider range of drier habitats than their C₃ counterparts because the C₄ pathway represents a pre-adaptation to arid conditions. Our analyses warn against evolutionary inferences based solely upon the high occurrence of extant C₄ species in dry habitats, and provide a novel interpretation of this classic ecological association.     

Evolutionary implications of C3–C4 intermediates in the grass Alloteropsis semialata

C₄ photosynthesis is a complex trait resulting from a series of anatomical and biochemical modifications to the ancestral C₃ pathway. It is thought to evolve in a stepwise manner, creating intermediates with different combinations of C₄‐like components. Determining the adaptive value of these components is key to understanding how C₄ photosynthesis can gradually assemble through natural selection. Here, we decompose the photosynthetic phenotypes of numerous individuals of the grass Alloteropsis semialata, the only species known to include both C₃ and C₄ genotypes. Analyses of δ¹³C, physiology and leaf anatomy demonstrate for the first time the existence of physiological C₃–C₄ intermediate individuals in the species. Based on previous phylogenetic analyses, the C₃–C₄ individuals are not hybrids between the C₃ and C₄ genotypes analysed, but instead belong to a distinct genetic lineage, and might have given rise to C₄ descendants. C₃ A. semialata, present in colder climates, likely represents a reversal from a C₃–C₄ intermediate state, indicating that, unlike C₄ photosynthesis, evolution of the C₃–C₄ phenotype is not irreversible.     

Lateral Gene Transfer Acts As an Evolutionary Shortcut to Efficient C4 Biochemistry

The adaptation of proteins for novel functions often requires changes in their kinetics via amino acid replacement. This process can require multiple mutations, and therefore extended periods of selection. The transfer of genes among distinct species might speed up the process, by providing proteins already adapted for the novel function. However, this hypothesis remains untested in multicellular eukaryotes. The grass Alloteropsis is an ideal system to test this hypothesis due to its diversity of genes encoding phosphoenolpyruvate carboxylase, an enzyme that catalyzes one of the key reactions in the C₄ pathway. Different accessions of Alloteropsis either use native isoforms relatively recently co-opted from other functions or isoforms that were laterally acquired from distantly related species that evolved the C₄ trait much earlier. By comparing the enzyme kinetics, we show that native isoforms with few amino acid replacements have substrate K_M values similar to the non-C₄ ancestral form, but exhibit marked increases in catalytic efficiency. The co-option of native isoforms was therefore followed by rapid catalytic improvements, which appear to rely on standing genetic variation observed within one species. Native C₄ isoforms with more amino acid replacements exhibit additional changes in affinities, suggesting that the initial catalytic improvements are followed by gradual modifications. Finally, laterally acquired genes show both strong increases in catalytic efficiency and important changes in substrate handling. We conclude that the transfer of genes among distant species sharing the same physiological novelty creates an evolutionary shortcut toward more efficient enzymes, effectively accelerating evolution.     

Photosynthesis, Morphology, Leaf Anatomy, and Cytogenetics of Hybrids between C(3) and C(3)/C(4)Panicum Species

The Laxa group of the Panicum genus contains species which have CO₂ exchange and anatomical characteristics intermediate to C₃ and C₄ photosynthetic types (C₃/C₄), and also species characterized as C₃. Hybrids were made between two of the C₃/C₄ species and two C₃ species. Carbon dioxide exchange and morphological, leaf anatomical, and cytogenetic characteristics of F₁ hybrids between Panicum milioides Nees. ex Trin (C₃/C₄) and P. laxum Mez. (C₃), P. spathellosum Doell (C₃/C₄) and P. boliviense Hack. (C₃), and P. spathellosum and P. laxum were studied. There were no consistent differences in apparent photosynthesis, although two of the three hybrids had higher net CO₂ uptake than the C₃ parent. Values of inhibition of apparent photosynthesis by 21% O₂, CO₂ loss in the light, and CO₂ compensation concentration for the hybrids were between those of the parents. All three hybrids showed leaf anatomical traits, especially organelle quantities in the bundle sheath cells, between those of their respective parents. Linear regression of CO₂ compensation concentration on the percentage of mitochondria and chloroplasts in vascular bundle sheaths of the parents and hybrids gave correlation coefficients of −0.94. This suggests that the reduction in CO₂ loss in the C₃/C₄ species, and to a lesser degree in the F₁ hybrids, was due to development of organelles and perhaps a higher proportion of leaf photorespiration in bundle sheaths. The overall morphology of the hybrids was so different from the parents that they could be described as new taxonomic forms. The chromosomes in the hybrids were mainly unpaired or paired as bivalents indicating possible homology between some parental genomes.     

The age of the grasses and clusters of origins of C4 photosynthesis

At high temperatures and relatively low CO₂ concentrations, plants can most efficiently fix carbon to form carbohydrates through C₄ photosynthesis rather than through the ancestral and more widespread C₃ pathway. Because most C₄ plants are grasses, studies of the origin of C₄ are intimately tied to studies of the origin of the grasses. We present here a phylogeny of the grass family, based on nuclear and chloroplast genes, and calibrated with six fossils. We find that the earliest origins of C₄ likely occurred about 32 million years ago (Ma) in the Oligocene, coinciding with a reduction in global CO₂ levels. After the initial appearance of C₄ species, photosynthetic pathway changed at least 15 more times; we estimate nine total origins of C₄ from C₃ ancestors, at least two changes of C₄ subtype, and five reversals to C₃. We find a cluster of C₄ to C₃ reversals in the Early Miocene correlating with a drop in global temperatures, and a subsequent cluster of C₄ origins in the Mid‐Miocene, correlating with the rise in temperature at the Mid‐Miocene climatic optimum. In the process of dating the origins of C₄, we were also able to provide estimated times for other major events in grass evolution. We find that the common ancestor of the grasses (the crown node) originated in the upper Cretaceous. The common ancestor of maize and rice lived at 52 ± 8 Ma.     

Contrasted histories of organelle and nuclear genomes underlying physiological diversification in a grass species

C₄ photosynthesis evolved multiple times independently in angiosperms, but most origins are relatively old so that the early events linked to photosynthetic diversification are blurred. The grass Alloteropsis semialata is an exception, as this species encompasses C₄ and non-C₄ populations. Using phylogenomics and population genomics, we infer the history of dispersal and secondary gene flow before, during and after photosynthetic divergence in A. semialata. We further analyse the genome composition of individuals with varied ploidy levels to establish the origins of polyploids in this species. Detailed organelle phylogenies indicate limited seed dispersal within the mountainous region of origin and the emergence of a C₄ lineage after dispersal to warmer areas of lower elevation. Nuclear genome analyses highlight repeated secondary gene flow. In particular, the nuclear genome associated with the C₄ phenotype was swept into a distantly related maternal lineage probably via unidirectional pollen flow. Multiple intraspecific allopolyploidy events mediated additional secondary genetic exchanges between photosynthetic types. Overall, our results show that limited dispersal and isolation allowed lineage divergence, with photosynthetic innovation happening after migration to new environments, and pollen-mediated gene flow led to the rapid spread of the derived C₄ physiology away from its region of origin.     

Transition from C3 to proto-Kranz to C3–C4 intermediate type in the genus Chenopodium (Chenopodiaceae)

The Chenopodiaceae is one of the families including C₄ species among eudicots. In this family, the genus Chenopodium is considered to include only C₃ species. However, we report here a transition from C₃ photosynthesis to proto-Kranz to C₃–C₄ intermediate type in Chenopodium. We investigated leaf anatomical and photosynthetic traits of 15 species, of which 8 species showed non-Kranz anatomy and a CO₂ compensation point (Γ) typical of C₃ plants. However, 5 species showed proto-Kranz anatomy and a C₃-like Γ, whereas C. strictum showed leaf anatomy and a Γ typical of C₃–C₄ intermediates. Chenopodium album accessions examined included both proto-Kranz and C₃–C₄ intermediate types, depending on locality. Glycine decarboxylase, a key photorespiratory enzyme that is involved in the decarboxylation of glycine, was located predominantly in the mesophyll (M) cells of C₃ species, in both M and bundle-sheath (BS) cells in proto-Kranz species, and exclusively in BS cells in C₃–C₄ intermediate species. The M/BS tissue area ratio, number of chloroplasts and mitochondria per BS cell, distribution of these organelles to the centripetal region of BS cells, the degree of inner positioning (vacuolar side of chloroplasts) of mitochondria in M cells, and the size of BS mitochondria also changed with the change in glycine decarboxylase localization. All Chenopodium species examined were C₃-like regarding activities and amounts of C₃ and C₄ photosynthetic enzymes and δ¹³C values, suggesting that these species perform photosynthesis without contribution of the C₄ cycle. This study demonstrates that Chenopodium is not a C₃ genus and is valuable for studying evolution of C₃–C₄ intermediates.

     

Root vascular anatomy predicts maximum growth rates in savanna trees and grasses

Root-based functional traits are relatively overlooked as drivers of savanna plant community dynamics, an important gap in water-limited ecosystems. Recent work has shed light on patterns of trait coordination in roots, but less is known about the relationship between root functional traits, water acquisition, and plant demographic rates. Here, we investigated how fine-root vascular and morphological traits are related in two dominant PFTs (C₃ trees and C₄ grasses from the savanna biome), whether root traits can predict plant relative growth rate (RGR), and whether root trait multivariate relationships differ in trees and grasses. We used root data from 21 tree and 18 grass species grown under greenhouse conditions, and quantified a suite of vascular and morphological root traits. We used a principal components analysis (PCA) to identify common axes of trait variation, compared trait correlation matrices between the two PFTs, and investigated the relationship between PCA axes and individual traits and RGR. We found that there was no clear single axis integrating vascular and morphological traits, but found that vascular anatomy predicted RGR in both trees and grasses. Trait correlation matrices differed in trees and grasses, suggesting potentially divergent patterns of trait coordination between the two functional types. Our results suggested that, despite differences in trait relationships between trees and grasses, root conductivity may constrain maximum growth rate in both PFTs, highlighting the critical role that water relations play in savanna vegetation dynamics and suggesting that root water transport capacity is an important predictor of plant performance in the savanna biome.