Enzymes are adapted to perform optimally in different thermal regimes that would otherwise alter kinetics and stability. Whether adaptive evolution in the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) also compensates for thermal variation remains uncertain. We examined molecular evolution and modeled the change in free energy of protein folding (ΔΔG, where negative values indicate stabilization) of the rubisco large subunit (RbcL) in four phylogenetically distant plant genera: wood ferns (Dryopteris), sea lavenders (Limonium), pines (Pinus), and viburnums (Viburnum). Using codon evolutionary models in each genus, we observed widespread positive selection and parallel substitution in the catalytic α/β barrel domain. Species with warmer growing seasons had derived amino acids with stronger hydrogen bond contributions to ΔΔG. Protein structure-based modeling showed that the hydrogen bond contribution to stability tracked the growing season temperature of species carrying the derived amino acid. Stronger hydrogen bonds were offset by weaker contributions from hydrophobic solvation interactions, such that total ΔΔG showed no relationship with growing season temperature. In Viburnum, the strength of positive selection differed among biomes, with cold temperate and cloud forest clades showing stronger positive selection. These patterns are consistent with environmental tuning of non-covalent interactions within the enzyme. However, modest effect sizes indicate that other components of the rubisco holoenzyme likely also contribute to its thermal evolution.

BACKGROUND AND AIMS:To better understand C4 evolution in monocots, we characterized C3-C4 intermediate phenotypes in the grass genus Homolepis (subtribe Arthropogoninae). METHODS:Carbon isotope ratio (δ13C), leaf gas exchange, mesophyll (M) and bundle sheath (BS) tissue characteristics, organelle size and numbers in M and BS tissue, and tissue distribution of the P-subunit of glycine decarboxylase (GLDP) were determined for five Homolepis species and the C4 grass Mesosetum loliiforme from a phylogenetic sister clade. We generated a transcriptome-based phylogeny for Homolepis and Mesosetum species to interpret physiological and anatomical patterns in an evolutionary context, and to test for hybridization. KEY RESULTS:Homolepis contains two C3 species (H. glutinosa, H. villaricensis), one species with a weaker form of C2 termed sub-C2 (H. isocalycia), and two C2 species (H. longispicula, H. aturensis). Homolepis longispicula and H. aturensis express over 85 % of leaf glycine in centripetal mitochondria within the BS, and have increased fractions of leaf chloroplasts, mitochondria and peroxisomes within the BS relative to H. glutinosa. Analysis of leaf gas exchange, cell ultrastructure and transcript expression show M. loliiforme is a C4 plant of the NADP-malic enzyme subtype. Homolepis comprises two sister clades, one containing H. glutinosa and H. villaricensis and the second H. longispicula and H. aturensis. Homolepis isocalycia is of hybrid origin, its parents being H. aturensis and a common ancestor of the C3  Homolepis clade and H. longispicula. CONCLUSIONS:Photosynthetic activation of BS tissue in the sub-C2 and C2 species of Homolepis is similar to patterns observed in C3-C4 intermediate eudicots, indicating common evolutionary pathways from C3 to C4 photosynthesis in these disparate clades. Hybridization can diversify the C3-C4 intermediate character state and should be considered in reconstructing putative ancestral states using phylogenetic analyses.

With over 60 parallel origins representing evolutionary replicates, C4 photosynthesis is well-suited for studying complex trait evolution. However, lineages with diverse C3-C4 intermediate species are scarce, leaving uncertainty in models of C4 evolution. Phenotypic characterization of 28 living species of Blepharis (Acanthaceae) is presented, including photosynthetic gas exchange, enzyme activity assays, cell ultrastructure, and δ13C assays, the latter including 92 herbarium specimens from three species with phenotypic diversity. A well-resolved transcriptome-based phylogeny provides evolutionary context. C3, proto-Kranz, C2, C4-like, and C4 phenotypes occur in Blepharis sect. Acanthodium. The phylogeny supports a stepwise progression from C3 through C2 to C4 states and up to five distinct origins of the C4 cycle. Substantial intraspecific C2-C4 variation is demonstrated in Blepharis mitrata, Blepharis furcata, and Blepharis macra. Blepharis gazensis is a monospecific C4 lineage exhibiting an NADP malic enzyme C4 pathway with features of the NAD-ME subtype, extending the ways in which the C4 cycle is known to function. Substantial photosynthetic diversity exists in Blepharis that rivals or exceeds the range of character states present in other C3 to C4 transitional lineages. This diversity in Blepharis represents a robust new model for studying convergent evolution of C4 photosynthesis and complex traits in general.

C4 plants exhibit greater nitrogen use efficiency (NUE) than C3 plants, primarily due to lower ribulose-1,5-bisphosphate carboxylase/oxygenase requirements. However, plant NUE also varies among C4 species, suggesting that other factors, such as cell wall composition, contribute to nitrogen economy. To investigate the contribution of cell wall architecture to nitrogen economy, we compared plant species with distinct cell wall types: eudicots with type I cell walls (T1CW) and grasses with type II cell walls (T2CW), under four nitrogen regimes: deficit, low, medium, and normal. Species with different photosynthetic metabolisms were compared to confirm known differences in photosynthetic nitrogen use efficiency (pNUE), while species with similar metabolism but distinct cell wall types were compared assessing the influence of cell wall on nitrogen economy. The pNUE of C4 grasses was higher than that of C4 eudicots, increasing from +54 % in normal nitrogen to +81 % in nitrogen deficit. C4 grasses presented lower structural nitrogen (-26 %) in normal nitrogen supply, which decreased to -58 % in nitrogen deficit, in comparison to C4 eudicots. An exploratory parameter cwpNUE (photosynthetic rate/structural nitrogen) resulted in a much higher value in C4 grasses (∼1.1 μmol CO2 s-1 mmol-1 cell wall nitrogen) than in the other groups (∼0.5 μmol CO2 s-1 mmol-1 cell wall nitrogen). In turn, the ester-linked ferulic acid increased from +177 % in C4 grasses in normal nitrogen to +362 % in nitrogen deficit, when compared to C4 eudicots. Data supports the hypothesis that in hot, humid tropical environments, nitrogen became a major limiting nutrient for C4 plant growth and development. The overlap between extensins and FA-GAX in crosslinking CW polymers observed in T2CW suggest that nitrogen scarcity may have exerted a selection pressure for adaptations in C4 grasses to contribute to NUE.

Enzymes are thought to be tuned to perform similarly in different thermal regimes. Whether the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) follows similar rules, especially when considering evolutionary history, is uncertain. The molecular, structural, and ecological factors of the rubisco large subunit (RbcL) were examined in four plant clades: wood ferns, pines, sea lavenders, and viburnums. Using rbcL gene sequences, codon evolutionary models were used to test for positive and divergent selection and convergent evolution. Protein structure modeling was performed to predict side chain changes and protein stability. Phylogenetic comparative methods were used to examine the relationship between protein stability to growing season temperature. All four clades showed significant evidence of positive selection, with multiple convergent substitutions predicted to alter side chain polarity and interactions with the solvent. In viburnums, biome transition rates were dependent on amino acid substitution, with positive selection was concentrated in cold temperate and cloud forest clades. Rubiscos with higher stability occurred in species from warmer environments. However, this correlation was weaker after correcting for phylogeny. These analyses support a hypothesis that RbcL evolution is influenced by both environmental tuning and evolutionary history. ### Competing Interest Statement The authors have declared no competing interest. Natural Sciences and Engineering Research Council, https://ror.org/01h531d29

Our understanding of how photosynthetic capacity varies among C4 species and across growth and measurement conditions remains limited. We collated 1696 CO2 response curves of net CO2 assimilation rate (A/Ci curves) from C4 species grown and measured at various environmental conditions and used these data to estimate the apparent maximum carboxylation activity of phosphoenolpyruvate carboxylase (VpmaxA) and CO2-saturated net photosynthetic rate (Amax), two key parameters describing photosynthetic capacity. We examined how VpmaxA and Amax vary with species-specific traits, growth and measurement conditions. We found little systematic variation of VpmaxA and Amax across the classical C4 biochemical subtypes or growth forms, but showed that growth temperature and measurement conditions are major factors determining C4 photosynthetic capacity. We found no evidence that common C4 model species (e.g. maize, sorghum and Setaria viridis) differ in photosynthetic capacity from other C4 species when grown in controlled environments. However, C4 model species showed up to twice the photosynthetic capacity of other C4 species when grown in the field. Our multivariate model accounts for 47-51% of the variation reported in VpmaxA and Amax, and we argue that environmental conditions have a greater influence on C4 photosynthetic capacity than biochemical subtypes or growth forms.

It has been 60 years since the discovery of C4 photosynthesis, an event that rewrote our understanding of plant adaptation, ecosystem responses to global change, and global food security. Despite six decades of research, one aspect of C4 photosynthesis that remains poorly understood is how the pathway fits into the broader context of adaptive trait spectra, which form our modern view of functional trait ecology. The C4 CO2-concentrating mechanism supports a general C4 plant phenotype capable of fast growth and high resource-use efficiencies. The fast-efficient C4 phenotype has the potential to operate at high productivity rates, while allowing for less biomass allocation to root production and nutrient acquisition, thereby providing opportunities for the evolution of novel trait covariances and the exploitation of new ecological niches. We propose the placement of the C4 fast-efficient phenotype near the acquisitive pole of the world-wide leaf economic spectrum, but with a pathway-specific span of trait space, wherein selection shapes both acquisitive and conservative adaptive strategies. A trait-based perspective of C4 photosynthesis will open new paths to crop improvement, global biogeochemical modeling, the management of invasive species, and the restoration of disturbed ecosystems, particularly in grasslands.

C2 photosynthesis is a photosynthetic pathway in which photorespiratory CO2 release and refixation are enhanced in leaf bundle sheath (BS) tissues. The evolution of C2 photosynthesis has been hypothesized to be a major step in the origin of C4 photosynthesis, highlighting the importance of studying C2 evolution. In this study, physiological, anatomical, ultrastructural, and immunohistochemical properties of leaf photosynthetic tissues were investigated in six non-C4 Tribulus species and four C4 Tribulus species. At 42°C, T. cristatus exhibited a photosynthetic CO2 compensation point in the absence of respiration (C*) of 21 µmol mol-1, below the C3 mean C* of 73 µmol mol-1. Tribulus astrocarpus had a C* value at 42°C of 55 µmol mol-1, intermediate between the C3 species and the C2 T. cristatus. Glycine decarboxylase (GDC) allocation to BS tissues was associated with lower C*. Tribulus cristatus and T. astrocarpus allocated 86% and 30% of their GDC to the BS tissues, respectively, well above the C3 mean of 11%. Tribulus astrocarpus thus exhibits a weaker C2 (termed sub-C2) phenotype. Increased allocation of mitochondria to the BS and decreased length-to-width ratios of BS cells, were present in non-C4 species, indicating a potential role in C2 and C4 evolution.

The world's forests store large amounts of carbon (C), and growing forests can reduce atmospheric CO2 by storing C in their biomass. This has provided the impetus for world-wide tree planting initiatives to offset fossil-fuel emissions. However, forests interact with their environment in complex and multifaceted ways that must be considered for a balanced assessment of the value of planting trees. First, one needs to consider the potential reversibility of C sequestration in trees through either harvesting or tree death from natural factors. If carbon storage is only temporary, future temperatures will actually be higher than without tree plantings, but cumulative warming will be reduced, contributing both positively and negatively to future climate-change impacts. Alternatively, forests could be used for bioenergy or wood products to replace fossil-fuel use which would obviate the need to consider the possible reversibility of any benefits. Forests also affect the Earth's energy balance through either absorbing or reflecting incoming solar radiation. As forests generally absorb more incoming radiation than bare ground or grasslands, this constitutes an important warming effect that substantially reduces the benefit of C storage, especially in snow-covered regions. Forests also affect other local ecosystem services, such as conserving biodiversity, modifying water and nutrient cycles, and preventing erosion that could be either beneficial or harmful depending on specific circumstances. Considering all these factors, tree plantings may be beneficial or detrimental for mitigating climate-change impacts, but the range of possibilities makes generalisations difficult. Their net benefit depends on many factors that differ between specific circumstances. One can, therefore, neither uncritically endorse tree planting everywhere, nor condemn it as counter-productive. Our aim is to provide key information to enable appropriate assessments to be made under specific circumstances. We conclude our discussion by providing a step-by-step guide for assessing the merit of tree plantings under specific circumstances.

ABSTRACT As a complex trait, C 4 photosynthesis has multiple independent origins in evolution. Phylogenetic evidence and theoretical analysis suggest that C 2 photosynthesis, which is driven by glycine decarboxylation in the bundle sheath cell, may function as a bridge from C 3 towards C 4 photosynthesis. However, the exact molecular mechanism underlying the transition between C 2 photosynthesis towards C 4 photosynthesis remains elusive. Here, we provide multiple evidence suggesting a role of higher α-ketoglutarate (AKG) concentration during this transition. Metabolomic data of 12 Flaveria species, including multiple photosynthetic types, show that AKG concentration initially increases in the C 3 -C 4 intermediate with a further increase in C 4 species. Petiole feeding of AKG increased the concentrations of C 4 related metabolites in C 3 -C 4 and C 4 species but not the activity of C 4 related enzymes. Sequence analysis shows that glutamate synthase (Fd-GOGAT), which catalyzes the generation of glutamate using AKG, was under strong positive selection during the evolution of C 4 photosynthesis. Simulations with a constraint-based model for C 3 -C 4 intermediate further show that decreasing the activity of Fd-GOGAT facilitates the transition from a C 2 -dominant to a C 4 -dominant CO 2 concentrating mechanisms. All these provide an insight into the mechanistic switch from C 3 -C 4 intermediate to C 4 photosynthesis.

C4 NAD-malic enzyme (NAD-ME) species occurs in drier regions and exhibit different drought responses compared to C4 NADP-malic enzyme (NADP-ME) species. However, a physiological mechanism explaining the geographical discrepancies remains uncertain. This study examined gas exchange patterns that might explain different distributions observed between two subtypes of C4 photosynthesis. We measured the response of leaf gas exchange to vapour pressure deficit (VPD) and CO2 in plants from six distinct C4 clades having closely related NAD-ME and NADP-ME species using a Li-Cor 6400 gas exchange system. We found that NAD-ME species exhibited greater relative reductions in stomatal conductance with increases in VPD than NADP-ME species but observed no consistent subtype differences in C4 cycle activity as indicated by the initial slope of the A response to intercellular CO2 concentration. Based on these results, we hypothesise the greater response of gs to increasing VPD may enable NAD-ME plants to outperform NADP-ME plants in hot, dry environments where VPD is normally high.

Societal Impact StatementBiological samples and their associated information are an essential resource used by scientists, governments, policymakers, practitioners and communities to ensure that biodiversity can be appropriately protected and sustainably used. Yet, considering the enormous task of documenting the vast numbers of as‐yet‐unknown plant and fungal species, greater international coordination for biological collecting and recording is necessary, built on equitable collecting practices and standards. Here, we propose five commitments to accelerate and enhance scientific knowledge of plant and fungal diversity, while increasing collaboration, benefit sharing and efficiency.SummaryAlmost all life depends on plants and fungi, making knowledge of their diversity and distribution—primarily derived from biological collections—fundamental to national and international conservation, restoration and sustainable use commitments. However, it is estimated that some 15% of all plant species and over 90% of all fungal species have not yet been scientifically described, hampering our ability to assess and demonstrate the impact of efforts to halt biodiversity loss. In addition, organisations and researchers around the world lack a concerted strategy for increasing complementarity and avoiding overlap in botanical and mycological research, particularly in relation to the collection of specimens. We here present the 2030 Declaration on Scientific Plant and Fungal Collecting, summarising a commitment towards such a necessary strategy. Its components were identified from discussions during and after a series of four workshops and plenary discussions at the 2023 State of the World's Plants and Fungi symposium convened by the Royal Botanic Gardens, Kew, and were then consolidated into the present form by the authors. The Declaration was subsequently opened up for endorsement by signatories. Collectively, we agree on a set of five commitments for cataloguing the world's flora and funga, designed to maximise efficiency, facilitate knowledge exchange and promote equitable collaborations: (1) use evidence‐based collection strategies; (2) strengthen local capacity; (3) collaborate across taxa and disciplines; (4) collect for the future; and (5) share the benefits. This Declaration is a first step towards increased global and regional coordination of scientific collecting efforts.

Considering the prevalence of ever-changing conditions in the natural world, investigation of photosynthetic responses in C-4 plants under fluctuating light is needed. Here, we studied the effect of dynamic illumination on photosynthesis in totally 10 C-3, C-3-C-4 intermediate, C-4-like and C-4 dicots and monocots at CO2 concentrations of 400 and 800 mu mol mol(-1.) C-4 and C-4-like plants had faster photosynthetic induction and light-induced stomatal dynamics than C-3 plants at 400 mu mol mol(-1), but not at 800 mu mol mol(-1) CO2, at which the CO2 supply rarely limits photosynthesis. C-4 and C-4-like plants had a higher water use efficiency than C-3 plants at both CO2 concentrations. There were positive correlations between photosynthetic induction and light-induced stomatal response, together with CO2 compensation point, which was a parameter of the CO2-concentrating mechanism of C-4 photosynthesis. These results clearly show that C-4 photosynthesis in both monocots and dicots adapts to fluctuating light conditions more efficiently than C-3 photosynthesis. The rapid photosynthetic induction response in C-4 plants can be attributed to the rapid stomatal dynamics, the CO(2-)concentrating mechanism or both.

C4 photosynthesis compensates for photosynthetic limitations imposed by low atmospheric CO2. C4 plants concentrate CO2 into the bundle sheath (BS) cells where Rubisco is localized. This leads to more efficient photosynthesis in warm climates and thus facilitates domination of open landscapes of low-to-mid latitude. C4 photosynthesis also allows exotic C4 grasses to become aggressive weeds. Where humans facilitate grass establishment, invasive C4 grasses can initiate autocatalytic grass-fire cycles that destroy tropical forests. Although exotic C4 grasses are expanding, much of the Earth's native C4 diversity is being degraded due to overexploitation of their habitat for agricultural purposes.

Eleocharis vivipara, an amphibious sedge in the Cyperaceae family, has several remarkable properties, most notably its alternate use of C-3 photosynthesis underwater and C-4 photosynthesis on land. However, the absence of genomic data has hindered its utility for evolutionary and genetic research. Here, we present a high-quality genome for E. vivipara, representing the first chromosome-level genome for the Eleocharis genus, with an approximate size of 965.22 Mb mainly distributed across 10 chromosomes. Its Hi-C pattern, chromosome clustering results, and one-to-one genome synteny across two subgroups indicates a tetraploid structure with chromosome count 2n = 4x = 20. Phylogenetic analysis suggests that E. vivipara diverged from Cyperus esculentus approximately 32.96 million years ago (Mya), and underwent a whole-genome duplication (WGD) about 3.5 Mya. Numerous fusion and fission events were identified between the chromosomes of E. vivipara and its close relatives. We demonstrate that E. vivipara has holocentromeres, a chromosomal feature which can maintain the stability of such chromosomal rearrangements. Experimental transplantation and cross-section studies showed its terrestrial culms developed C-4 Kranz anatomy with increased number of chloroplasts in the bundle sheath (BS) cells. Gene expression and weighted gene co-expression network analysis (WGCNA) showed overall elevated expression of core genes associated with the C-4 pathway, and significant enrichment of genes related to modified culm anatomy and photosynthesis efficiency. We found evidence of mixed nicotinamide adenine dinucleotide - malic enzyme and phosphoenolpyruvate carboxykinase type C-4 photosynthesis in E. vivipara, and hypothesize that the evolution of C-4 photosynthesis predates the WGD event. The mixed type is dominated by subgenome A and supplemented by subgenome B. Collectively, our findings not only shed light on the evolution of E. vivipara and karyotype within the Cyperaceae family, but also provide valuable insights into the transition between C-3 and C-4 photosynthesis, offering promising avenues for crop improvement and breeding.

BACKGROUND AND AIMS:CAM photosynthesis is hypothesized to have evolved in atmospheres of low CO2 concentration in recent geological time because of its ability to concentrate CO2 around Rubisco and boost water use efficiency relative to C3 photosynthesis. We assess this hypothesis by compiling estimates of when CAM clades arose using phylogenetic chronograms for 73 CAM clades. We further consider evidence of how atmospheric CO2 affects CAM relative to C3 photosynthesis.RESULTS:Where CAM origins can be inferred, strong CAM is estimated to have appeared in the past 30 million years in 46 of 48 examined clades, after atmospheric CO2 had declined from high (near 800 ppm) to lower (<450 ppm) values. In turn, 21 of 25 clades containing CAM species (but where CAM origins are less certain) also arose in the past 30 million years. In these clades, CAM is probably younger than the clade origin. We found evidence for repeated weak CAM evolution during the higher CO2 conditions before 30 million years ago, and possible strong CAM origins in the Crassulaceae during the Cretaceous period prior to atmospheric CO2 decline. Most CAM-specific clades arose in the past 15 million years, in a similar pattern observed for origins of C4 clades.CONCLUSIONS:The evidence indicates strong CAM repeatedly evolved in reduced CO2 conditions of the past 30 million years. Weaker CAM can pre-date low CO2 and, in the Crassulaceae, strong CAM may also have arisen in water-limited microsites under relatively high CO2. Experimental evidence from extant CAM species demonstrates that elevated CO2 reduces the importance of nocturnal CO2 fixation by increasing the contribution of C3 photosynthesis to daily carbon gain. Thus, the advantage of strong CAM would be reduced in high CO2, such that its evolution appears less likely and restricted to more extreme environments than possible in low CO2.

center dot Background and Scope The growth of experimental studies of crassulacean acid metabolism (CAM) in diverse plant clades, coupled with recent advances in molecular systematics, presents an opportunity to re-assess the phylogenetic distribution and diversity of species capable of CAM. It has been more than two decades since the last comprehensive lists of CAM taxa were published, and an updated survey of the occurrence and distribution of CAM taxa is needed to facilitate and guide future CAM research. We aimed to survey the phylogenetic distribution of these taxa, their diverse morphology, physiology and ecology, and the likely number of evolutionary origins of CAM based on currently known lineages.center dot Results and Conclusions We found direct evidence (in the form of experimental or field observations of gas exchange, day-night fluctuations in organic acids, carbon isotope ratios and enzymatic activity) for CAM in 370 genera of vascular plants, representing 38 families. Further assumptions about the frequency of CAM species in CAM clades and the distribution of CAM in the Cactaceae and Crassulaceae bring the currently estimated number of CAM-capable species to nearly 7 % of all vascular plants. The phylogenetic distribution of these taxa suggests a minimum of 66 independent origins of CAM in vascular plants, possibly with dozens more. To achieve further insight into CAM origins, there is a need for more extensive and systematic surveys of previously unstudied lineages, particularly in living material to identify low-level CAM activity, and for denser sampling to increase phylogenetic resolution in CAM-evolving clades. This should allow further progress in understanding the functional significance of this pathway by integration with studies on the evolution and genomics of CAM in its many forms.