Photosynthetic eukaryotes have shaped the Earth's biosphere by producing oxygen and converting light into organic compounds in specialized organelles called plastids. Plastids evolved from free-living cyanobacteria ingested by heterotrophic unicellular eukaryotes. Two such independent engulfment processes, called cyanobacterial endosymbioses, have been reported so far. The first gave rise to primary plastids and three Archaeplastida lineages: glaucophytes, red algae and green algae with land plants, whereas the second resulted in chromatophores in the rhizarian amoeba Paulinella. Importantly, archaeplastidans donated their plastids to many protist groups, thereby further spreading photosynthesis across the tree of life. To reveal the complex plastid evolution, we performed comprehensive phylogenetic and multi-clock analyses based on new fossil calibration points and the greatest number yet of plastid-encoded proteins from 108 taxa, representing a large diversity of photosynthetic organisms. Our results indicate that primary plastids evolved prior to 2.1 - 1.8 Ba, i.e. before glaucophytes diverged from the other archaeplastidans, and Paulinella chromatophores most probably before 292 - 266 Ma. Red and green algae were engulfed by cryptophyte and chlorarachniophyte ancestors between 1.7 - 1.4 Ba, and 1.1 - 1.0 Ba, respectively; the former subsequently triggered plastid transfers to other eukaryotes. We also studied the impact of various molecular clocks and calibration sets on the age estimation and clearly indicate that the clocks are the source of greater differences.

Please see the README document ("README.txt ") and the SUPPLEMENTARY MATERIALS file ("Supplementary_Information.docx").Funding provided by: Narodowym Centrum NaukiCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100004442Award Number: 2017/26/D/NZ8/004444Funding provided by: Narodowym Centrum NaukiCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100004442Award Number: 2018/31/N/NZ2/01338Funding provided by: Narodowym Centrum NaukiCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100004442Award Number: 2019/35/N/MZ8/03366

Please see the README document ("README.txt ") and the SUPPLEMENTARY MATERIALS file ("Supplementary_Information.docx").