Drivers of C4 evolution in the grasses

Biologists have long been fascinated by the biological complexity that exists today, with organisms
displaying a remarkable diversity of adaptations that have enabled them to thrive in almost every
environment. Many of the most impressive adaptations would be classified as complex traits, with
structural and metabolic elements working in synergy. To evolve, multiple elements need to be
rewired, which can be achieved by incremental modifications over successive generations.
Understanding evolutionary steps leading to the emergence of a complex trait can help solve the
evolutionary complexity puzzle. The C4 photosynthetic pathway is a complex adaptation involving
multiple biochemical and anatomical changes that have allowed certain plant lineages to succeed in
specific environmental conditions, including warm and arid climates. Here, we aimed to investigate
the evolutionary modifications required to construct the C4 cycle, a complex trait. I initially focused
on examining the remarkable photosynthetic variation in the grass Alloteropsis semialata, a species
unique in having both C4 and non-C4 photosynthetic genotypes. In my research, I conducted a
comparative analysis to identify leaf traits associated with the proportion of carbon fixed using the
C4 cycle. The findings revealed that plants with higher C4 activity generally have a greater ratio of
photosynthetically active bundle sheath tissue. Subsequently, I employed a genome-wide
association study (GWAS) to identify several candidate genes associated with the strength of the C4
cycle and enhancing the proportion of bundle sheath tissue. Finally, in the final chapter of my
research, I adopted a more comprehensive perspective to investigate the genetic precursors that may
have enabled the repeated evolution of C4 photosynthesis in the PACMAD grasses. I found that
genes associated with cell wall modifications and stomatal aperture were duplicated at the base of
the PACMAD clade, potentially facilitating the repeated convergent evolution of Kranz anatomy.
Collectively, all three data chapters highlight the importance of anatomical modifications in the
emergence of the C4 cycle. Overall my thesis identifies several key changes required for C4 evolution, and the results may be relevant to engineering C4 in C3 species, such as rice.