857 J. Phycol. 34, 857–864 (1998) SEQUENCES OF THE LARGEST SUBUNIT OF RNA POLYMERASE II FROM TWO RED ALGAE AND THEIR IMPLICATIONS FOR RHODOPHYTE EVOLUTION 1 John W. Stiller 2 and Benjamin D. Hall Department of Botany, University of Washington, Box 355325, Seattle, Washington 98195 ABSTRACT The Rhodophyta are a widespread group of multicellular eukaryotes that occupy a range of habitats and exhibit a broad variety of morphologies and life histories. Unlike green plants, animals, and even brown algae, the red algae have attained this diversity without having evolved true tissue differentiation. Moreover, the relationship of red al- gae to other multicellular lineages remains controversial, and the molecular and biochemical mechanisms of their development are largely unexplored. We have isolated the 3' end of the gene encoding the largest subunit (RPB1) of RNA polymerase II (Pol II) from two divergent red algae, Porphyra yezoensis Ueda and Bonnemaisonia hami- fera Heriot. The Pol II largest subunit lies at the catalytic center of the enzyme responsible for mRNA transcription in eukaryotic cells. In plants, animals, and fungi, the 3' dis- tal region of the RPB1 gene encodes a carboxyl-terminal domain (CTD) that is the organizing center for assembly of the RNA Pol II holoenzyme essential for developmentally regulated transcription. Examination of rhodophyte RPB1 C-terminal regions as well as phylogenetic analyses based on conserved gene sequences suggest that the red algae emerged in the course of eukaryotic evolution before the CTD had been codified in the remaining crown taxa. The possibility that a causal relationship underlies the corre- lated lack of a CTD and absence of tissue differentiation in red algae is proposed as an intriguing subject for further study. Key index words: Bonnemaisonia hamifera; carboxyl- terminal domain; evolution; molecular phylogeny; mRNA transcription; multicellular development; Porphyra ye- zoensis; Rhodophyta; RNA polymerase II; RPB1 Abbreviations: CTD, C-terminal domain; RPB1, RNA polymerase II largest subunit; RPC1, RNA polymerase III largest subunit Red algae have been recognized as a group of related organisms, separate and distinct from other macroalgae, since the mid-19th century; this conclu- sion is supported strongly by extensive molecular analyses (see Ragan and Gutell 1995 for review). Based on both fossil and molecular evidence (But- terfield et al. 1990, Ragan et al. 1994), the Rho- dophyta represent an ancient evolutionary lineage that has proliferated widely in form, ecological hab- itat, and life history. Red algal taxa occupy marine, freshwater, terrestrial, and even geothermal environ- ments. One puzzling aspect of the group is that, un- 1 Received 16 December 1997. Accepted 5 May 1998. 2 Author for reprint requests; e-mail stiller@u.washington.edu. like green algae, their green plant descendants, and even the chromophytic algae, red algae have achieved this broad diversity without the differenti- ation of complex tissues (Coomans and Hommer- sand 1990). The place of the Rhodophyta in the eukaryotic world has been debated for more than a century (Ragan and Gutell 1995). The oldest fossil that is clearly recognizable as a relative of an extant, mul- ticellular eukaryotic taxon is a red alga (Butterfield et al. 1990). Much older bladelike fossils (at least 1.7 billion years old) from the Tuanshanzi forma- tion in China have ‘‘hyphae filamentous structure’’ and appear to be macroalgae (Zhu and Chen 1995). They were interpreted by the original authors to be members of the Phaeophyta because of a superficial morphological similarity to kelp; however, a large body of molecular, biogeographical, and fossil evi- dence indicates that kelp originated within the last 30 million years (Lu ¨ning 1990, Saunders and Druehl 1992). Their filamentous structure suggests that, if they are related to any extant group of mul- ticellular eukaryotes, the Tuanshanzi fossils more likely are the remains of rhodophytic taxa similar in morphology to modern bladelike genera in the or- der Gigartinales. In addition to fossil evidence, molecular analyses suggest that the Rhodophyta may be the most an- cient group of multicellular eukaryotes. The se- quence divergence among nuclear small-subunit rRNA genes (ssu rDNA) within red algae is greater than that found among green plants or fungi and even between plants and an amoeboid protist (Ra- gan et al. 1994). This may result from a faster rate of substitution in the Rhodophyta, but it is equally plausible that the increased sequence divergence re- flects their greater antiquity. Moreover, extensive phylogenetic analyses of ssu rDNA (Kumar and Rzhetsky 1996, Van de Peer et al. 1996) consistently place the emergence of rhodophytes before the common ancestor of most other so-called ‘‘crown’’ eukaryotic taxa (Knoll 1992) including green plants, animals, and fungi. Recently, we reported phylogenetic analyses (Still- er and Hall 1997) of the gene encoding the largest subunit (RPB1) of RNA polymerase II (Pol II), the enzyme responsible for transcription of messenger RNA (mRNA) in the eukaryotic nucleus. This study provided the first strong statistical support from mo- lecular data for an early emergence of the red algae, before the common ancestor of the other multicel- lular taxa included in our data set. Our analyses in-