Photosynthetic diversity in the genus Blepharis (Acanthaceae) and the evolutionary origins of C₄ photosynthesis

Such a comprehensive phenotyping effort revealed an unexpectedly high diversity in various key C4 components. C4 species are often nested within clades that also contain non-C4 plants that have adapted to efficiently refix CO2 derived from photorespiration. These so-called C2 plants have C4-like modifications to leaf structure and gene expression, which, together with their close relatedness with C4 lineages, suggest they represent evolutionary intermediates in the transition to a C4 physiology (Sage et al., 2012) (although it is important to note the evolutionary stability of 'C3–C4 intermediates' in several clades; see Lundgren & Christin, 2017). For this reason, such clades containing C4 and C2 species have been chosen as the main models for comparative studies on C4 evolution. These include the well-studied genus Flaveria (Asteraceae) (e.g. Adachi et al., 2023), and the grasses Alloteropsis (e.g. Pereira et al., 2023) and Neurachne (e.g. Khoshravesh et al., 2020), among others. Together, these studies have revealed a large diversity of C4-related traits, which have been fundamental to disentangling adaptive changes associated with the C4 mechanism from other lineage-specific traits. In an article recently published in New Phytologist, Stata et al. (2025; doi: 10.1111/nph.70426) contribute to this effort by revealing the diversity of photosynthetic traits in the eudicot genus Blepharis (Acanthaceae), establishing it as a new model system to understand the evolution of C4 photosynthesis. The genus Blepharis includes 128 species that occur in seasonally dry and arid areas across Africa, the Middle East, and southeastern Asia. The existence of different photosynthetic types in Blepharis had been previously reported (Fisher et al., 2015), but the diversity of intermediate phenotypes and their evolutionary significance remained unexplored. To address these gaps, Stata and colleagues conducted an extensive sampling of 29 Blepharis species, including several independent populations for some of them, and quantified biochemical, anatomical, and leaf-level photosynthesis parameters. Such a comprehensive phenotyping effort revealed an unexpectedly high diversity in various key C4 components. To shed light on general patterns across species, the authors used a multivariate statistical approach to discriminate the different samples across physiological space. This suggested that Blepharis species can be grouped into five main clusters, which the authors interpreted as different stages along the evolutionary transition from C3 to C4 physiology. The first cluster corresponded to typical C3 species with low activity of C4-related enzymes, such as phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase, and CO2 compensation point (CCP) above 50 μmol mol−1. This group is sister to the remaining C4 and intermediate lineages and likely retains the photosynthetic characteristics of the common ancestor of all Acanthaceae (Fig. 1). A second well-defined group corresponded to the C4 species, which exhibited the highest activity of C4-related enzymes, the lowest CCP values, and carbon isotope signatures (δ13C) that are diagnostic of C4 plants. Surprisingly, the phylogenetic distribution of these species suggested five independent C4 clades in Blepharis, which is the highest number of transitions to the C4 state so far reported for a single genus. The remaining species were assigned to three additional categories. The first was, to a large extent, similar to the C4 type, although with significantly lower PEPC activity and lower leaf-level carboxylation efficiency, which were associated with δ13C values at the upper limit of usual C4 values. The three species displaying this phenotype were classified as C4−, described by the authors as species with an incomplete expression of C4 characters, such as the absence of a full compartmentalization of Rubisco to the bundle sheath tissue. The two other categories included all species with key diagnostic features of C2 plants, including the restriction of the enzyme glycine decarboxylase (GDC) to the bundle sheath tissue, and intermediate CCP values between C3 and C4 plants. One of the C2 groups, named C2+, was composed of a single species, B. mitrata, which displayed higher activity of some C4-related enzymes and low CCP, although with C3-like δ13C values. One particular aspect of this study is the large intraspecific variation in δ13C observed in herbarium specimens of three species–B. furcata, B. macra, and B. mitrata–with values spanning typical C3 and C4 values. This suggests that the three species contain recently diverged C4 and non-C4 populations, a finding that has so far been limited to the grass Alloteropsis semialata. Although a phylogenetic assessment of the specimens analyzed is necessary to rule out taxonomic issues, this would not be surprising given the high diversity in C4-related traits in Blepharis. This highlights the importance of sampling multiple populations for trait evolution studies, particularly in cases of complex traits such as C4 photosynthesis. C4 research has diversified over the past 20 years from physiological and anatomical descriptions into phylogenetically controlled investigations, often accompanied by genetic insights from comparative genomic and transcriptomic analyses. This has created a broad understanding of the circumstances of repeated C4 evolution, and was fundamental for developing the initial strategies to engineer the C4 trait in C3 crops. However, two major gaps in our knowledge still remain. First, although some regulatory mutations linked to C4 anatomy and gene expression have been identified (e.g. Swift et al., 2024; Vlad et al., 2024), our knowledge of the genetic control of the C4 trait is largely limited to a few models, particularly maize. The second major gap is the lack of a consensus on the minimum set of components necessary for a full C4 physiology. The latter in particular is a difficult task because the phenotype we observe in living species is a result of lineage-specific constraints and increments that might be adaptive or not, and might have evolved after the trait was first put in place. The genus Blepharis provides a new opportunity to reassess these problems from a new perspective. Another important shift in C4 research regards the adoption of experimental strategies using controlled crosses and genetic transformation. These are powerful tools not only to dissect the genetic basis of C4 components (Simpson et al., 2022) but also to experimentally investigate long-standing hypotheses on C4 evolution from modelling studies (e.g. Mallmann et al., 2014), an effort that has long been missing. Nonetheless, progress has been slow due to the current lack of an ideal experimental model. In the grass A. semialata, for example, which includes closely related C3, C4, and intermediates, although F1 photosynthetic hybrids were obtained (Bianconi et al., 2022), the generation of segregating F2 hybrids has been proven to be challenging. Likewise, tissue culture protocols for the species have not advanced, which limits the implementation of genetic transformation approaches. Nonetheless, population-level genome sequencing across the species range allowed a genome-wide association study (GWAS) that identified several genomic regions underlying C4 features (Alenazi et al., 2024). The genus Blepharis, particularly if C2 and C4 phenotypes are confirmed within B. furcata, B. macra, and B. mitrata, provides a new opportunity for GWAS and genetic approaches to identify loci controlling C4 traits. Similarly, crossing between photosynthetic types could also be explored, although a different chromosome number among species has been reported in the genus (Ranganath & Krishnappa, 1982), which might complicate such efforts. Finally, the fast germination and growth rates as Stata et al. (2025) reported for some Blepharis species are desirable characteristics for a genetic model, and these might provide a promising alternative for establishing such a long-awaited model for experimental investigations. The authors would like to thank New Physiologist for the invitation to write this commentary. MEB is funded through a Marie Skłodowska-Curie Actions (MSCA) Postdoctoral Fellowship (101105838), and LTD is funded by a UK Natural Environment Research Council Independent Research Fellowship (NE/T011025/1). The New Phytologist Foundation remains neutral with regard to jurisdictional claims in maps and in any institutional affiliations.