Evolution of C4 photosynthesis in Neurachne

As a carbon-concentration mechanism, C4 photosynthesis enables plants to be highly productive even in carbon-deficient, hot and dry conditions. From the ancestral C3 pathway, C4 photosynthesis has evolved more than 60 times, and approximately half of C4 origins have occurred in monocots. Uniquely for monocots, the Australian genus Neurachne R.Br. consists of eight closely related species that conduct different photosynthetic types (i.e. C3, C4, and so-called C3-C4 intermediate photosynthesis). Furthermore, the two C4 species of Neurachne represent two independent origins of C4 photosynthesis opening up the possibility to determine if the mechanisms of establishing C4 photosynthesis differ between the species.By means of mRNA sequencing of leaves of seven of the eight Neurachne species (four C3 species, one C3-C4 intermediate species, and two C4 species) we firstly revisit the phylogenetic relationships of Neurachne. To increase our understanding of differential gene expression between closely related species with differing photosynthetic types, we then conduct a comparative transcriptomic analysis between these seven Neurachne species. By using the newly inferred phylogeny of Neurachne and transcriptome sequence information, we finally test whether the key proteins of C4 photosynthesis are under positive selection.Our phylogenetic analysis shows slightly differing relationships in Neurachne compared to previously inferred relationships, but affirm two independent C4 origins within the genus. The gene expression pattern mostly concurs with results observed in comparative transcriptome analyses between C3 and C4 species of other plant lineages. For example, transcriptional investment of genes known to be involved in C4 photosynthesis is very high in leaves of the two C4 species while insignificant in C3 and C3-C4 intermediate species. Interestingly, the number of differentially expressed genes is highest between the two C4 species. Positive selection is absent in most of the C4 proteins when labelling the two branches where C4 is hypothesised to have evolved. However, several amino acids in the primary C4 carboxylase, phosphoenolpyruvate carboxylase, are detected to be under positive selection; one of these in the N terminus—a region underrepresented in all positive selection analyses up to now. This particular amino acid change not only occurs in the two C4 species in Neurachne, but also is present in other C4 grasses.