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.     

Differences in drought sensitivities and photosynthetic limitations between co-occurring C3 and C4 (NADP-ME) Panicoid grasses

Background and Aims

The success of C₄ plants lies in their ability to attain greater efficiencies of light, water and nitrogen use under high temperature, providing an advantage in arid, hot environments. However, C₄ grasses are not necessarily less sensitive to drought than C₃ grasses and are proposed to respond with greater metabolic limitations, while the C₃ response is predominantly stomatal. The aims of this study were to compare the drought and recovery responses of co-occurring C₃ and C₄ NADP-ME grasses from the subfamily Panicoideae and to determine stomatal and metabolic contributions to the observed response.

Methods

Six species of locally co-occurring grasses, C₃ species Alloteropsis semialata subsp. eckloniana, Panicum aequinerve and Panicum ecklonii, and C₄ (NADP-ME) species Heteropogon contortus, Themeda triandra and Tristachya leucothrix, were established in pots then subjected to a controlled drought followed by re-watering. Water potentials, leaf gas exchange and the response of photosynthetic rate to internal CO₂ concentrations were determined on selected occasions during the drought and re-watering treatments and compared between species and photosynthetic types.

Key Results

Leaves of C₄ species of grasses maintained their photosynthetic advantage until water deficits became severe, but lost their water-use advantage even under conditions of mild drought. Declining C₄ photosynthesis with water deficit was mainly a consequence of metabolic limitations to CO₂ assimilation, whereas, in the C₃ species, stomatal limitations had a prevailing role in the drought-induced decrease in photosynthesis. The drought-sensitive metabolism of the C₄ plants could explain the observed slower recovery of photosynthesis on re-watering, in comparison with C₃ plants which recovered a greater proportion of photosynthesis through increased stomatal conductance.

Conclusions

Within the Panicoid grasses, C₄ (NADP-ME) species are metabolically more sensitive to drought than C₃ species and recover more slowly from drought.     

Parallelism and diversity in multiple origins of c4 photosynthesis in the grass family

C₄ photosynthesis is thought to be an adaptation to warm environments, involving complex changes in the expression of genes governing photosynthesis, intermediary metabolism, and leaf anatomy and histology. Such complexity should be difficult to evolve, yet the pathway has arisen multiple times in the history of the flowering plants and at least four times in the grass family alone. We have used immunolocalization techniques to compare photosynthetic gene expression across all four origins, to determine which genetic changes occur in parallel and which are unique to a particular lineage. The only gene expression patterns common to all origins of the pathway are up-regulation of PEP carboxylase and down-regulation of RuBisCO in mesophyll cells. Both NAD-malic enzyme and NADP-malic enzyme are expressed in bundle sheaths. Expression patterns of light-harvesting chlorophyll a/b binding proteins and pyruvate orthophosphate dikinase appear to be lineage specific, and may be localized to bundle sheaths or to mesophyll or expressed throughout the photosynthetic tissue of the leaf. We suggest that future studies of parallel origin of the C₄ pathway concentrate on regulation of the two carboxylases, as well as the increased density of vascular tissue, which is the only histological characteristic common to all origins of the pathway.     

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.     

Endosymbiotic ratchet accelerates divergence after organelle origin

We hypothesize that as one of the most consequential events in evolution, primary endosymbiosis accelerates lineage divergence, a process we refer to as the endosymbiotic ratchet. Our proposal is supported by recent work on the photosynthetic amoeba, Paulinella, that underwent primary plastid endosymbiosis about 124 Mya. This amoeba model allows us to explore the early impacts of photosynthetic organelle (plastid) origin on the host lineage. The current data point to a central role for effective population size (N_e) in accelerating divergence post-endosymbiosis due to limits to dispersal and reproductive isolation that reduce N_e, leading to local adaptation. We posit that isolated populations exploit different strategies and behaviors and assort themselves in non-overlapping niches to minimize competition during the early, rapid evolutionary phase of organelle integration. The endosymbiotic ratchet provides a general framework for interpreting post-endosymbiosis lineage evolution that is driven by disruptive selection and demographic and population shifts. Also see the video abstract here: https://youtu.be/gYXrFM6Zz6Q     

C3 and C4 Photosynthetic and Anatomical forms of Alloteropsis semialata (R.Br.) Hitchcock: I. Variability in Photosynthetic Characteristics, Water Utilization Efficiency and Leaf Anatomy

The grass Alloteropsis semialata (R.Br.) Hitchcock is uniquein that both Kranz and non-Kranz leaf anatomy has been reportedin this species. The present study investigates Kranz formsof A. semialata collected from a single ecological niche. Theseplants exhibit morphological and anatomical differences withrespect to leaf area, stomatal size and stomatal distribution.Carbon dioxide and water exchange measurements in the two formsshow the expected pattern of higher photosynthetic rate andhigher water utilization efficiency associated with Kranz anatomy.No intermediate physiological response or anatomical form wasobserved in this sample. Alloteropsis semialata (R.Br.) Hitchcock, C₃ photosynthetic, C₄ photosynthesis, water utilization, leaf anatomy, Kranz anatomy     

A molecular phylogeny of the genus Alloteropsis (Panicoideae, Poaceae) suggests an evolutionary reversion from C4 to C3 photosynthesis

Background and Aims: The grass Alloteropsis semialata is the only plant species withboth C₃ and C₄ subspecies. It therefore offers excellent potentialas a model system for investigating the genetics, physiologyand ecological significance of the C₄ photosynthetic pathway.Here, a molecular phylogeny of the genus Alloteropsis is constructedto: (a) confirm the close relationship between the C₃ and C₄subspecies of A. semialata; and (b) infer evolutionary relationshipsbetween species within the Alloteropsis genus. Methods: The chloroplast gene ndhF was sequenced from 12 individuals,representing both subspecies of A. semialata and all four ofthe other species in the genus. ndhF sequences were added tothose previously sequenced from the Panicoideae, and used toconstruct a phylogenetic tree. Key Results: The phylogeny confirms that the two subspecies of A. semialataare among the most recently diverging lineages of C₃ and C₄taxa currently recognized within the Panicoideae. Furthermore,the position of the C₃ subspecies of A. semialata within theAlloteropsis genus is consistent with the hypothesis that itsphysiology represents a reversion from C₄ photosynthesis. Thedata point to a similar evolutionary event in the Panicum stenodes–P.caricoides–P. mertensii clade. The Alloteropsis genusis monophyletic and occurs in a clade with remarkable diversityof photosynthetic biochemistry and leaf anatomy. Conclusions: These results confirm the utility of A. semialata as a modelsystem for investigating C₃ and C₄ physiology, and provide moleculardata that are consistent with reversions from C₄ to C₃ photosynthesisin two separate clades. It is suggested that further phylogeneticand functional investigations of the Alloteropsis genus andclosely related taxa are likely to shed new light on the mechanismsand intermediate stages underlying photosynthetic pathway evolution.     

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.     

Occurrence of the suberized lamella in leaves of grasses of different photosynthetic types. I. In parenchymatous bundle sheaths and PCR (“Kranz”) sheaths

Summary Leaf blades of 42 grasses (Poaceae) have been examined ultrastructurally for the occurrence of a suberized lamella in walls of parenchymatous bundle sheaths and PCR (= Kranz) sheaths in both large and small vascular bundles. The sample includes species from a range of major grass taxa, and represents all photosynthetic types found in the grasses. Three grasses with unusual C₄ leaf anatomy were also included:Alloteropsis semialata, Aristida biglandulosa, Arundinella nepalensis. The presence of a suberized lamella in PCR cell walls was perfectly correlated with photosynthetic type. All PEP-carboxykinase type and NADP-malic enzyme type C₄ species examined possessed a suberized lamella in outer tangential and radial walls, but with variable presence in inner tangential walls. PCR cells of bothAlloteropsis semialata andArundinella nepalensis also possessed a suberized lamella. A lamella was totally absent from parenchymatous bundle sheath cells of the C₃ species examined (5 spp.) and ofPanicum milioides, a C₃-C₄ intermediate. It was also absent from PCR cells of NAD-malic enzyme type C₄ species (14 spp.) andAristida biglandulosa. The results are discussed in relation to the leakage of CO₂ from PCR cells, and to differences between C₄ types in ¹³C values, chloroplast position in PCR cells, and other anatomical characteristics.     

Geographic distributions and physiological characteristics of co-existing Flaveria species in south-central Mexico

The genus Flaveria consists of 23 species with significant variation in photosynthetic physiologies. We tested whether photosynthetic pathway variation in seven co-existing Flaveria species corresponds to geographic distributions or physiological performance in C₃, C₄, and intermediate species growing under natural conditions in south-central Mexico. We found that Flaveria pringlei (C₃) was the most widely distributed species with multiple growth habits. Numerous populations of Flaveria kochiana (C₄), a recently described species with a previously unknown distribution, were located in the Mixtec region of Oaxaca. Flaveria cronquistii (C₃) and Flaveria ramosissima (C₃-C₄) were only located in the Tehuacán Valley region while Flaveria trinervia (C₄) was widely distributed. Only one population of Flaveria angustifolia (C₃-C₄) and Flaveria vaginata (C₄-like) were located near Izúcar de Matamoros. Midday leaf water potential differed significantly between Flaveria species, but did not vary according to growth habit or photosynthetic pathway. The quantum yield of photosystem II did not vary between species, despite large differences in leaf nitrogen content, leaf shape, plant size and life histories. We did not find a direct relationship between increasing C₄ cycle characteristics and physiological performance in the Flaveria populations examined. Furthermore, C₃ species were not found at higher elevation than C₄ species as expected. Our observations indicate that life history traits and disturbance regime may be the primary controllers of Flaveria distributions in south-central Mexico.     

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.     

C3 and C4 Photosynthetic and Anatomical Forms of Alloteropsis semialata (R.Br.) Hitchcock: 2. A Comparative Investigation of Leaf Ultrastructure and Distribution of Chlorenchyma in the Two Forms

Alloteropsis semialata (R.Br.) Hitchcock is a widely distributedgrass species known to show marked morphological, anatomicaland physiological variation. An unusual feature of this grassis that it shows both C₃, and C₄ photosynthetic pathways withthe respective anatomy related to these pathways in one speciesand within a single environment. This study investigates ultrastructuraldifferences between C₃ and C₄ forms of A. semialata, and showsdistribution of chlorenchyma in the two forms, at light microscopelevel. Quantitative assessment shows marked differences in mesophylland Kranz sheath cells with regard to size and organelle content;also in distribution of starch grains, lipid droplets and organellesin the mesophyll cells of both forms. These differences arediscussed in relation to the C₃/C₄ syndrome. Measurements were made using a Digiplan electronic planimeteron transmission electron micrographs of mature leaves sectionedtransversely at mid-lamina, and on drawings of leaf sectionsshowing the distribution of chlorenchymatous tissue. Alloteropsis semialata(R.Br.) Hitchcock, leaf structure, chlorenchyma, ultrastructure, C₃-photosynthesis, C₄-photosynthesis     

Diversity in forms of C4 in the genus Cleome (Cleomaceae)

Background and Aims

Cleomaceae is one of 19 angiosperm families in which C₄ photosynthesis has been reported. The aim of the study was to determine the type, and diversity, of structural and functional forms of C₄ in genus Cleome.

Methods

Plants of Cleome species were grown from seeds, and leaves were subjected to carbon isotope analysis, light and scanning electron microscopy, western blot analysis of proteins, and in situ immunolocalization for ribulose bisphosphate carboxylase oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC).

Key Results

Three species with C₄-type carbon isotope values occurring in separate lineages in the genus (Cleome angustifolia, C. gynandra and C. oxalidea) were shown to have features of C₄ photosynthesis in leaves and cotyledons. Immunolocalization studies show that PEPC is localized in mesophyll (M) cells and Rubisco is selectively localized in bundle sheath (BS) cells in leaves and cotyledons, characteristic of species with Kranz anatomy. Analyses of leaves for key photosynthetic enzymes show they have high expression of markers for the C₄ cycle (compared with the C₃–C₄ intermediate C. paradoxa and the C₃ species C. africana). All three are biochemically NAD-malic enzyme sub-type, with higher granal development in BS than in M chloroplasts, characteristic of this biochemical sub-type. Cleome gynandra and C. oxalidea have atriplicoid-type Kranz anatomy with multiple simple Kranz units around individual veins. However, C. angustifolia anatomy is represented by a double layer of concentric chlorenchyma forming a single compound Kranz unit by surrounding all the vascular bundles and water storage cells.

Conclusions

NAD-malic enzyme-type C₄ photosynthesis evolved multiple times in the family Cleomaceae, twice with atriplicoid-type anatomy in compound leaves having flat, broad leaflets in the pantropical species C. gynandra and the Australian species C. oxalidea, and once by forming a single Kranz unit in compound leaves with semi-terete leaflets in the African species C. angustifolia. The leaf morphology of C. angustifolia, which is similar to that of the sister, C₃–C₄ intermediate African species C. paradoxa, suggests adaptation of this lineage to arid environments, which is supported by biogeographical information.     

The Historical Speciation of Mauremys Sensu Lato: Ancestral Area Reconstruction and Interspecific Gene Flow Level Assessment Provide New Insights

Mauremys sensu lato was divided into Mauremys, Chinemys, Ocadia, and Annamemys based on earlier research on morphology. Phylogenetic research on this group has been controversial because of disagreements regarding taxonomy, and the historical speciation is still poorly understood. In this study, 32 individuals of eight species that are widely distributed in Eurasia were collected. The complete mitochondrial (mt) sequences of 14 individuals of eight species were sequenced. Phylogenetic relationships, interspecific divergence times, and ancestral area reconstructions were explored using mt genome data (10,854 bp). Subsequent interspecific gene flow level assessment was performed using five unlinked polymorphic microsatellite loci. The Bayesian and maximum likelihood analyses revealed a paraphyletic relationship among four old genera (Mauremys, Annamemys, Chinemys, and Ocadia) and suggested the four old genera should be merged into the genus (Mauremys). Ancestral area reconstruction and divergence time estimation suggested Southeast Asia may be the area of origin for the common ancestral species of this genus and genetic drift may have played a decisive role in species divergence due to the isolated event of a glacial age. However, M. japonica may have been speciated due to the creation of the island of Japan. The detection of extensive gene flow suggested no vicariance occurred between Asia and Southeast Asia. Inconsistent results between gene flow assessment and phylogenetic analysis revealed the hybrid origin of M. mutica (Southeast Asian). Here ancestral area reconstruction and interspecific gene flow level assessment were first used to explore species origins and evolution of Mauremys sensu lato, which provided new insights on this genus.     

C4 Photosynthesis Promoted Species Diversification during the Miocene Grassland Expansion

Identifying how organismal attributes and environmental change affect lineage diversification is essential to our understanding of biodiversity. With the largest phylogeny yet compiled for grasses, we present an example of a key physiological innovation that promoted high diversification rates. C₄ photosynthesis, a complex suite of traits that improves photosynthetic efficiency under conditions of drought, high temperatures, and low atmospheric CO₂, has evolved repeatedly in one lineage of grasses and was consistently associated with elevated diversification rates. In most cases there was a significant lag time between the origin of the pathway and subsequent radiations, suggesting that the ‘C₄ effect’ is complex and derives from the interplay of the C₄ syndrome with other factors. We also identified comparable radiations occurring during the same time period in C₃ Pooid grasses, a diverse, cold-adapted grassland lineage that has never evolved C₄ photosynthesis. The mid to late Miocene was an especially important period of both C₃ and C₄ grass diversification, coincident with the global development of extensive, open biomes in both warm and cool climates. As is likely true for most “key innovations”, the C₄ effect is context dependent and only relevant within a particular organismal background and when particular ecological opportunities became available.