Integrated global analysis in spider flowers illuminates features underlying the evolution and maintenance of C₄...

The carbon concentrating mechanism-C₄ photosynthesis-represents a classic example of convergent evolution, but how this important trait originated and evolved remains largely enigmatic. The spider flower Gynandropsis gynandra is a valuable leafy vegetable crop and medicinal plant that has also been recognized as a C₄ model species. Here we present a high-quality chromosome-scale annotated genome assembly of G. gynandra through a combination of Oxford Nanopore Technology (ONT), HiFi and Hi-C technology. The 17 super-scaffolds cover 98.66% of the estimated genome (997.61 Mb), with a contig N50 of 11.43 Mb and a scaffold N50 of 51.02 Mb. Repetitive elements occupy up to 71.91% of its genome, and over half are long terminal repeat retrotransposons (LTR-RTs) derived from recent bursts, contributing to genome size expansion. Strikingly, LTR-RT explosion also played a critical role in C₄ evolution by altering expression features of photosynthesis-associated genes via preferential insertion in promoters. Integrated multiomics analyses of G. gynandra and the ornamental horticulture C₃ relative Tarenaya hassleriana reveal that species-specific whole-genome duplication, gene family expansion, recent LTR-RT amplification, and more recent tandem duplication events have all facilitated the evolution of C₄ photosynthesis, revealing uniqueness of C₄ evolution in the Cleome genus. Moreover, high leaf vein density and heat stress resilience are associated with shifted gene expression patterns. The mode of C₃-to-C₄ transition found here yields new insights into evolutionary convergence of a complex plant trait. The availability of this reference-grade genomic resource makes G. gynandra an ideal model system facilitating efforts toward C₄-aimed crop engineering.