bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259127; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 A molecular timescale for the origin of red algal-derived plastids 2 Jürgen F. H. Strassert1,2,7, Iker Irisarri1,3,4,7, Tom A. Williams5, Fabien Burki1,6,* 3 1Department of Organismal Biology, Program in Systematic Biology, Uppsala University, 4 Norbyvägen 18D, 75 236 Uppsala, Sweden 5 2Department of Biology, Chemistry, Pharmacy, Institute of Biology, Free University of Berlin, 6 Königin-Luise-Straße 1–3, 14 195 Berlin, Germany 7 3Department of Biodiversity and Evolutionary Biology, Museo Nacional de Ciencias Naturales 8 (MNCN-CSIC), José Gutiérrez Abascal 2, 28 006 Madrid, Spain 9 4Current address: Department of Applied Bioinformatics, Institute for Microbiology and 10 Genetics, University of Göttingen, Goldschmidtstraße 1, 370 77 Göttingen, Germany 11 5School of Biological Sciences, University of Bristol, Life Sciences Building, Tyndall Avenue, 12 Bristol BS8 1TQ, UK 13 6Science for Life Laboratory, Uppsala University, Uppsala, Sweden 14 7These authors contributed equally 15 *Corresponding author: 16 Fabien Burki 17 Department of Organismal Biology, Program in Systematic Biology, Uppsala University, 18 Norbyvägen 18D, 752 36 Uppsala, Sweden 19 Phone: +46 (0)18 471 2779 20 E-mail: [email protected] 21 Jürgen F. H. Strassert: https://orcid.org/0000-0001-6786-7563 22 Iker Irisarri: https://orcid.org/0000-0002-3628-1137 23 Tom A. Williams: https://orcid.org/0000-0003-1072-0223 24 Fabien Burki: https://orcid.org/0000-0002-8248-8462 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259127; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 25 Abstract 26 In modern oceans, eukaryotic phytoplankton is dominated by lineages with red algal-derived 27 plastids such as diatoms, dinoflagellates, and coccolithophores. These lineages and countless 28 others representing a huge diversity of forms and lifestyles all belong to four algal groups: 29 cryptophytes, ochrophytes, haptophytes, and myzozoans. Despite the ecological importance of 30 these groups, we still lack a comprehensive understanding of their evolution and how they 31 obtained their plastids. Over the last years, new hypotheses have emerged to explain the 32 acquisition of red algal-derived plastids by serial endosymbiosis, but the chronology of these 33 putative independent plastid acquisitions remains untested. Here, we have established a 34 timeframe for the origin of red algal-derived plastids under scenarios of serial endosymbiosis, 35 using a taxon- and gene-rich phylogenomic dataset combined to Bayesian molecular clock 36 analyses. We find that the hypotheses of serial endosymbiosis are chronologically possible, as the 37 stem lineages of all red plastid-containing groups overlapped in time. This period in the Meso- 38 and Neoproterozoic Eras set the stage for the later expansion to dominance of red algal-derived 39 primary production in the contemporary oceans, which has profoundly altered the global 40 geochemical and ecological conditions of the Earth. 41 Introduction 42 Plastids (= chloroplasts) are organelles that allow eukaryotes to perform oxygenic photosynthesis. 43 Oxygenic photosynthesis (hereafter simply photosynthesis) evolved in cyanobacteria around 2.4 44 billion years ago (bya), leading to the Great Oxidation Event—a rise of oxygen that profoundly 45 transformed the Earth’s atmosphere and shallow ocean1,2. Eukaryotes later acquired the capacity 46 to photosynthesise with the establishment of plastids by endosymbiosis. Plastids originated from 47 primary endosymbiosis between a cyanobacterium and a heterotrophic eukaryotic host, leading to 48 primary plastids in the first photosynthetic eukaryotes. There are three main lineages with 49 primary plastids: red algae, green algae (including land plants), and glaucophytes—altogether 50 forming a large group known as Archaeplastida3,4. Subsequently to the primary endosymbiosis, 51 plastids spread to other eukaryote groups from green and red algae by eukaryote-to-eukaryote 52 endosymbioses, i.e., the uptake of primary plastid-containing algae by eukaryotic hosts. These 53 higher order endosymbioses resulted in complex plastids surrounded by additional membranes, 54 some even retaining the endosymbiont nucleus (the nucleomorph), and led to the diversification 55 of many photosynthetic lineages of global ecological importance, especially those with red algal- 56 derived plastids (e.g., diatoms, dinoflagellates, and apicomplexan parasites)5. 57 The evolution of complex red algal-derived plastids has been difficult to decipher, mainly 58 because the phylogeny of host lineages does not straightforwardly track the phylogeny of 59 plastids. From the plastid perspective, phylogenetic and cell biological evidence strongly support 60 a common origin of all complex red plastids6–11. This is at the centre of the chromalveolate 61 hypothesis12, which proposed that the series of events needed to establish a plastid are better 62 explained by a single secondary endosymbiosis in the common ancestor of alveolates, 63 stramenopiles, cryptophytes, and haptophytes: the four major groups known to harbour complex 64 red plastids. From the host side, however, the phylogenetic relationships of these four groups 65 have become increasingly difficult to reconcile with a single origin of all complex red algal- 66 derived plastids in a common ancestor. Indeed, over a decade of phylogenomic investigations bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259127; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 67 have consistently shown that all red plastid-containing lineages are most closely related to a 68 series of plastid-lacking lineages, often representing several paraphyletic taxa, which would 69 require extensive plastid losses under the chromalveolate hypothesis (at least ten)5. This situation 70 is further complicated by the fact that no cases of complete plastid loss have been demonstrated, 71 except in a few parasitic taxa13,14. 72 The current phylogeny of eukaryotes has given rise to a new framework for explaining the 73 distribution of complex red plastids. This framework, unified under the rhodoplex hypothesis, 74 invokes the process of serial endosymbiosis, specifically a single secondary endosymbiosis 75 between a red alga and a eukaryotic host, followed by successive higher-order—tertiary, 76 quaternary—endosymbioses spreading plastids to unrelated groups15. Several models compatible 77 with the rhodoplex hypothesis have been proposed, differing in the specifics of the plastid donor 78 and recipient lineages16–19 (Fig. 1). However, these models of serial endosymbiosis remain highly 79 speculative, in particular because we do not know if they are chronologically possible—did the 80 plastid donor and recipient lineages co-exist? Addressing this important issue requires a reliable 81 timeframe for eukaryote evolution, which has been challenging to obtain owing to a combination 82 of complicating factors, notably: 1) uncertain phylogenetic relationships among the major 83 eukaryote lineages; 2) the lack of genome-scale data for the few microbial groups with a robust 84 fossil record; and 3) a generally poor understanding of methodological choices on the dates 85 estimated for early eukaryote evolution. 86 Recent molecular clock analyses placed the origin of primary plastids in an ancestor of 87 Archaeplastida in the Paleoproterozoic Era, between 2.1–1.6 bya20. The origin of red algae has 88 been estimated to be in the late Mesoproterozoic to early Neoproterozoic (1.3–0.9 bya)20, after a 89 relatively long lag following the emergence of Archaeplastida. However, an earlier appearance in 90 the late Paleoproterozoic has also been proposed based on molecular analyses21,22. The earliest 91 widely accepted fossil for the crown-group Archaeplastida is the multicellular filamentous red 92 alga Bangiomorpha deposited ~1.2 bya23. Recently, older filamentous fossils (Rafatazmia and 93 Ramathallus) interpreted as crown-group red algae were recovered in the ~1.6 Ga old Vindhyan 94 formation in central India24. This taxonomic interpretation pushed back the oldest commonly 95 accepted red algal fossil record by 400 million years, and consequently the origin of red algae and 96 Archaeplastida well into the Paleoproterozoic Era. Evidence for the diversification of red algal 97 plastid-containing lineages comes much later in the fossil record, which is apparent in the well- 98 documented Phanerozoic continuous microfossil records starting from about 300 million years 99 ago (mya). This period marks the diversification of some of the most ecologically important algae 100 in modern oceans such as diatoms (ochrophytes), dinoflagellates (myzozoans), and 101 coccolithophorids (haptophytes). If the fossil record is taken at face value,