Title Molecular Clocks and Inferring Evolutionary Milestones and Biogeography in the Microalgae Author(s) Medlin, Linda K. Edited by Hisatake Okada, Shunsuke F. Mawatari, Noriyuki Suzuki, Pitambar Gautam. ISBN: 978-4-9903990-0-9, 31- Citation 42 Issue Date 2008 Doc URL http://hdl.handle.net/2115/38433 Type proceedings Note International Symposium, "The Origin and Evolution of Natural Diversity". 1‒5 October 2007. Sapporo, Japan. File Information p31-42-origin-08.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Molecular Clocks and Inferring Evolutionary Milestones and Biogeography in the Microalgae Linda K. Medlin* Alfred Wegener Institute for Polar and Marine Research, Department of Biological Oceanography, Bremerhaven, Germany 27570 ABSTRACT Molecular clocks are proving to be very useful tools for unravelling the evolution of protistan taxa relative to geological change. Molecular clocks have been used to reconstruct the biogeographic history of non-fossilized microalgae from calibration of trees/clocks based on taxa with a fossil re- cord. They have also been used to extrapolate back to the origin of microalgal lineages, to docu- ment the appearance of new morphotypes in the fossil record that should be recognised as new species, to estimate the timing of major evolutionary events, viz., endosymbioses and to infer pos- sible explanations for selective survival during global change extinction events. Keywords: Molecular clocks, Phylogeny, Historical biogeography tive and qualitative shifts set the stage for evolution- INTRODUCTION ary recoveries [1]. Molecular clocks are proving to be very useful Before constructing a molecular clock, calibrated tools for unravelling the evolution of protistan taxa. studies must be performed to determine the reliabili- Molecular clocks have been used to reconstruct bio- ty/test intrinsic error margins of clocks and to pro- geographic histories, divergence times of many pro- vide baseline estimates of molecular evolution rates. tists ranging from their origins to the divergence of Calibration of the molecular tree is possible in two cryptic species. Microalgae, such as diatoms, dino- ways: First, a calibration point can be established flagellates and coccolithophores, have mineralised where a clade in the tree has a derived character walls that preserve well. These microalgal groups that is unique to that clade and that can be traced in have better preserved fossil records than their the fossil record. In this case one can infer that (a) metaphyton and metazoan counterparts and molecu- the character must have evolved on the branch of lar clocks made using calibrations from microalgae the tree leading to the base of the clade and (b) that are better calibrated. Many microalgal groups suf- the character must have evolved prior to its first oc- fered greatly at mass extinctions only to reradiate currence in the fossil record. One can refer to these after the event, whereas others pass through the as character- based constraints. Alternatively, a cali- events relatively unscathed. Mass extinctions are bration point can be established where sister taxa in important to macroevolution not only because they the tree have good fossil records, then the first oc- involve a sharp increase in extinction intensity over currences (FOs) of the taxa can be used as a mini- “background” levels, but also because they bring a mum age constraint for the node in the tree at which change in extinction selectivity and these quantita- the two taxa diverge. If both taxa have sufficient de- *e-mail: [email protected] Information for use in citing this article: Okada, H., Mawatari, S.F., Suzuki, N. and Gautam, P. (eds.), Origin and Evolution of Natural Diversity, Proceedings of International Symposium “The Origin and Evolution of Natural Di- versity”, 1–5 October 2007, Sapporo, pp. 31–42. 32 L.K. Medlin rived characters to be certain they are monophyletic, euglenoid or chlororachniophyte alga. If the en- then the FO of the older taxon can be used for the gulfed alga was a red alga, then the resultant new constraint, and a range extension for the younger eukaryotic cell was a cryptomonad, haptophyte, het- taxon inferred. One can refer to these as divergence- erokont or dinoflagellate alga. Each of these new based constraints. eukaryote cells has 3–4 membranes around the plas- In constructing a molecular clock, one must take tid, each one representing a different membrane in account of the rates of evolution that will likely the serial endosymbiosis (Fig. 1). vary across the tree of interest. There are basically The number and timing of these events has been two methods that can be used to correct for this controversial. Early single gene phylogenies showed variation. One can average the rate of evolution that there were multiple events and the timing of across the tree, e.g. in a linearised tree [2], after these events was placed at a recent date [9]. Multi- eliminating all taxa from the tree that vary in their ple gene phylogenies of the plastid clearly showed rate of evolution outside a poisson distribution. This that the plastid were monophyletic [6] and molecu- is a strict molecular clock. Ranch lengths are re- lar clocks using calibration dates from the outgroup gressed against fossil dates and displayed in the taxa and not any ingroup taxa estimated the origin model provided by Ref 3. Nodes without dates can of the red algae plastid to be soon after the primary be inferred from the regression line. Alternatively, endosymbiotic event. Host gene phylogenies have one can allow the rate of evolution to vary across proven very difficult to recover that supported a the tree [4]. This is a relaxed molecular clock and is monophyletic origin of the hosts [10]. Even now a preferred method because it helps to remove one that both primary and the red linage secondary en- of the biases of constructing a molecular clock. dosymbioses appear to have happened only once, it In this paper I will review the use of molecular is clear that an early timing of this event does not clocks in the microalgae to estimate their evolution- match the fossil record of the phytoplankton who ary milestones and to infer the biogeography of se- are the modern components of the red algal second- lected genera. ary endosymbiotic event [11]. Clearly, the host lin- eages did not take immediate advantage of their The Origin of the Plastids newly acquired organelle and photosynthetic func- Because photosynthesis has play such a funda- tion. All of the early divergences in the heterokont mental role in shaping the biosphere, the origins of tree are heterotrophic and they appear to have lost the plastids have remained one of the most intriging the plastid from their secondary endosymbiosis. and well researched topics in biology [see reviews There is a final divergence in this lineage of all of in 5, 6, 7]. The origin of the initial photosynthetic the autotrophic golden brown and brown algae (Fig. eukaryote likely involved the phagotrophic uptake 2A). In the haptophytes, there is a long branch from of a cyanobacterium by a heterotrophic host cell. the origin before the divergence of the two classes This endosymbiosis resulted in formation of three and after that there is another long branch in each primary algal lineages: the green algae, the red al- of the classes until the classes diverge into orders gae and the glaucophytes. Each of these algal and families (Fig. 3A). The same appears to be true groups contains plastids that are surrounded by two for the dinoflagellates, whose basal lineages are het- membranes, which constitute the two membranes of erotrophic or parasitic and there is a later divergence the original cell membrane of the cyanobacterium of the photosynthetic lineage with many multiple (Fig. 1). losses of their plastids and serial replacement of the Although many early single gene phylogenetic original plastid with other algal groups [12] (Fig. 4). analyses suggested that this may have happened Using a molecular clock we are able to estimate the multiple times, the analysis of multiple gene phy- divergence of the autotrophic heterokonts and the logenies of the host plant and the plastid have indi- dinoflagellates and the divergences of the modern cated that the red, green and glaucophytes host and diversity in the haptophyte lineage. Each of these plastid lineages are monophyletic [8] and there is a major groups of secondary endosymbiotic algae ap- single endosymbiotic event giving rise to these al- pear to have radiated after 250 Ma. In other words, gae. Following this endosymbiotic event, a second- the plastid did not really confer any adaptive advan- ary event took place, which one of the primary tage before this time because all earlier lineages do endosymbiotic algae was engulfed by a second het- not retain their plastids and are heterotropic in the erotrophic host. If the engulfed alga was green al- case of the pigmented heterokonts of which the dia- gae, then the resultant new eukaryote cell was a toms are a member and the dinoflagellates or are all Molecular Clocks, Evolution and Microalgae 33 Fig. 1 Scenario showing the serial endosymbiosis leading to the algae in the primary endosymbiosis (top row) and the red algae in the secondary endosymbiosis (bottom row). Drawn by Wiebe Kooistra. extinct in the case of the haptophytes. At 250 Ma, fied by differences in cell shape and in the geometry the world suffered a major mass extinction with ap- of the apical pore complex (APC), by the presence proximately 98% of all life in the ocean going ex- or absence of a ventral pore on the apical plate (1’), tinct. It is at this time that the red algal plastid and by the tendency to form chains or not. Phyloge- containing lineages began to proliferate and diversi- netic studies of the Alexandrium tamarense species fy.