BotanicalJournal ofthe Linnean Society (1995), 178: 81-105. With 5 figuresAre red algae plants?MARK A. RAGANCanadian Imtitute for Advanced Research Program in Evolutionary Biology, and NRCInstitute for Marine Biosciences, 1417 Oxford Street, Hal+?, Nova Scotia,Canada B3H 32 7ANDROBIN R. GUTELLDepartment of Molecular, Cell and Developmental Biology, and Department ofChemistry and Biochemistry, University of Colorado, Boulder, CO 80309-02 15,U.S.A.Received September 7994, acceptedfor@blication June 1995For 200 years prior to the 1938 publication of H. F. Copeland, all authorities (with oneexception) classified red algae (Rhodophyta) within Kingdom Plantae or its equivalent.Copeland’s reclassification of red algae within Kingdom Protista or Protoctista drew from analternative tradition, dating to Cohn in 1867, in which red algae were viewed as the earliestor simplest eukaryotes. Analyses of ribosomal RNA (rRNA) sequence data initially favouredCopeland’s reclassification. Many more rRNA gene (rDNA) sequences are now available fromthe eukaryote lineages most closely related to red algae, and based on these data, thehypothesis that red algae and green plants are sister groups cannot be rejected. An increasingbody of sequence, intron-location and functional data from nuclear- and mitochondriallyencoded proteins likewise supports a sister-group relationship between red algae and greenplants. Submerging Kingdoms Plantae, Animalia and Fungi into Eukarya would provide amore natural framework for the eventual resolution of whether red algae are plants orprotists.0 199.5 The Linnean Socicly of LondonADDITIONAL KEY WORDS:-Eukarya - Kishino-Hasegawa test L molecular-sequencephylogeny - Plantae - Protista - Rhodophyta - 5s rRNA gene - 18s rRNA gene -Templeton-Felsenstein test.CONTENTSRhodophytaas amonophyletic group .Rhodophyta as plants us Rhodophyta as pro&. . . . . 82. . . 87A second tradition: red algae as ‘the deepest eukaryotes’ . . 88Resolution: both traditions appear incorrect . . . . . 89Are red algae plants after all? A new generation of rDNA trees . . 89The eukatyote 5S rRNA tree revisited . . . . . 94Evidence from nuclear genes . . . . . . . . 95Evidence from mitochondrial and plastid genes . . . . 970024-4074/95/060081+25 $08.00/O81Q 1995 The Linnean Society of London
82 M. A. RAGAN AND R. R. GUTELLEvidence from nonsequence data .............. 97Indirect evidence from studies of plastid origin ........... 98Acknowledgements ................. 99References .................... 99RHODOPHYTA AS A MONOPHYLETIC GROUPThe Rhodophyta encompasses 2500-6000 species (Woelkerling, 1990) ofeukaryotes sharing the character states listed in Table 1. None of thesecharacter states is a synapomorphy unique to red algae. Zygomycetes,ascomycetes and basidiomycetes also lack flagella and store a-1,4 glucans intheir cytosol; glaucophytes and green plants have flattened mitochondrialcristae and two plastid-envelope membranes; cyanobacteria lack accessorychlorophylls and typically possess unstacked thylakoids with superficialphycobilisomes. Red algal pit connections may not be homologous with thesuperficially similar structures of some vascular plants and cyanobacteria, andpit phigs (Pueschel, 1990) are uniquely red algal, but neither pit connectionsnor pit plugs occur in all red algae. Red algae are, however, unique inexhibiting the combination of character states listed in Table 1, and inpractice there is little difficulty in distinguishing what is or is not a red alga.Linnaeus (1753) did not establish a separate taxon for the red algae thenknown, but instead distributed them among at least three genera within theCryptogamia Algae subdivision of Class XXIV (Cryptogamia). Thus red algaenow assigned to the genera Chondrus and Furcellaria were placed in Linnaeangenus Fucus (thalloid cryptogams); Batrachospermum and Lemanea were includedin the Linnaean Conferua (filamentous cryptogams); and Porphyra was consideredwithin the Linnaean UZua (membranous cryptogams). In the early decades ofthe nineteenth century these artificial genera were broken apart, and newtaxa were established based on the colour of the thallus and spores. Onlythen did the morphologically more-complex red algae begin to be groupedtogether as FlorideCs (Lamouroux,Rhodospermeae (Harvey, 1836),1813), Purpureae (C. A. Agardh, 1824),or Rhodophyceae (Ruprecht, 1851). At thesame time, coralline red algae were distinguished from zoocorals (Schweigger,1819; Gray, 1821; Philippi, 1837). Thuret (1855) was the first to group bothbangiophycidean and florideophycidean red algae into a single taxon(Rhodophyceae), although only after the careful work of Berthold (1882) didthis concept gain widespread acceptance.Since early in the present century (von Wettstein, 1901) red algae havebeen afforded their own Division Rhodophyta, containing class I&odophyceaeTARL~ 1. Distinguishing features of red algaeEukaryoticNo centrioles, flagellar basal bodies, flagella, or other 9+2 structuresFlattened mitochondrial cristaePlastid envelope composed of two membranes (i.e. no external plastid endoplasmicreticulum-derived membrane)Chlorophyll u as only chlorophyllUnstacked thylakoidsPhycqbiliproteins in stalked phycobilisomes on the thylakoid surfaceStorage of a-1,4 glucans (starches) in the cytosol
ARE BED ALGAE PLANTS? 83T~ar.e 2. Characters differentiating members of subclasses Bangiophycidae andFlorideophycidaeBangiophycidae FlorideophycidaeNuclear conditionPlastidPlastid locationCell divisionThallus complexityPit connectionsSexual reproductionFilamentous gonimoblastTetrasporangiauninucleateusually single stellaletypically axileintercalaryuni- or multicellulausually absentabsent, rare, orcontroversial, exceptin genus Porphyraabsentabsentusually multinucleateusually multiple discoidalusually peripheralmostly apicalmulticellularpresentwidespreadusually presentusually presentand usually two subclasses, Bangiophycidae and Florideophycidae. Charactersdistinguishing bangiophytes from florideophytes are presented in Table 2.Again the paucity of clear-cut, positive defining synapomorphies is notable;but again, in practice there is little difficulty in assigning a red alga to oneor the other subclass.The modern classification of red algae follows Schmitz (1892) and Schmitz& Hauptfleisch (1897), who established one bangiophycidean and fourflorideophycidean orders (Figure 1). Schmitz based his delineation offlorideophyte orders on characters of female reproductive morphology andpost-fertilization development: structures of the carpogonium and thecarpogonial branch, presence and fate of auxiliary cell(s), orientation of theplanes of cytokinesis, and pattern of zygote amplification. As the ultrastructure,SANGIALESCDMPSOPDGON4LESRHODDCHAETALESPDRPHYR/D/ALESGONlOTRKX4LESCRYPTDNEMIALESI ‘-NEMALMLESSATR4CHDSPERhbUESACRDCMUlALESSONNEMAlSONlALESGELIDIALESGlGARTlNALESAHNFELTIALESGR4CURMESCRYPTCNEMMLESHILDENSRANDIALESRHODYMENIALESPALMAFTlALES1 I I I I I I I I I I I I I I I I I I I I I I1Eso 1w 1910 1920 1530 1s‘l4 1960 lsso 1970 ls80 1980 2mt0 GONlOTRCiALES llSS9l. ABANDONEDSCHMllZ, CklAETANGlAL!3 llS63~. NOT ACCEPTED1992* NEMASTOMALES WZSI, ABANDONED. SPHAEROCOCCALES l19261, ABANDONEDFigure 1. History or recognition of orders within Bhodophyta, from Schmitz (1892) to thepresent. The newly proposed Bhodogorgonales (1994) contains recently discovered genera(Renouxia, Rhodogorgon) which had not previously been formally placed in any other order.We thank Carolyn J. Bird and Bob Richards for assistance with this figure.
84 M. A. RAGAN AND R. R. GUTELLNEIWLIALES::2_______..__ _.---.-. ___.--. -..=LACRtKHAETIALES2Ig.lcystes monospermesl.f-----.--.------.I------- --_--_.--__ ---_-___- - _- - -:IBANGIALE:I 1; __-_-- _- --_---.-_-lcystes plurispermes!---.I[EUR~~~DOP~~YCIDAEI -1(cystes d production__ -.-. _-_-.-rRtlOC’X-l4ETALES----JLIERYTHROPELTIDALEScmPsoPOGONA’-=PORPHYRIDIALES(pas de vrais cysteslFigure 2. Phylogeny of red algae according to Magne (1989), based on morphology andmode of fertile cell formation within gametocysts and sporocysts. Reproduced with permission.reproduction and life histories of red algae became better understood duringthe twentieth century, additional characters were introduced, and existingones reinterpreted @raft, 1981; Garbary & Gabrielson, 1990: 479-481); as aconsequence, additional florideophycidean orders were erected (Fig. 1). Mostauthorities now recognize 11-13 orders of florideophytes (two new orders,Rhodogorgonales (Fredericq & Morris, 1994) and Plocamiales (Saunders &Kraft, 1994), were recently proposed); at least two of these orders,Acrochaetiales and Gigartinales, are widely suspected not to be monophyletic.Delineation of bangiophyte orders has received somewhat less attention.Skuja (1939) recognized four or five orders based on macromorphologicalcharacters. As the ultrastructure and reproductive morphology of theseorganisms became better known, orders were merged, a new order erected,and families reassigned (Feldmann, 1967; Chapman, 1974; Garbary, Hansen& Scagel, 1980). Most authorities now recognize four orders of bangiophycideanred algae, at least one of which (Porphyridiales) may not be monophyletic.Classification of red algae, as indeed of most algae, has historically beencarried out without reference to phylogeny. Few if any delineations of neworders mention character synapomorphy, and various subjective, aesthetic,and didactic factors have played important roles in algal taxonomy (Silva,1984). Garbary and Gabrielson were the first to apply phylogenetic principlesin classifying red algae (reviewed by Garbary & Gabrielson, 1990). Mag-rre(1989; Fig. 2) proposed that two lineages of multicellular red algae exist, one
ARE RED ALGAE PLANTS? a5composed of multicellular bangiophytes other than order Bangiales, the otherof Bangiales plus a monophyletic Florideophycidae.The first red algal phylogenies based on molecular sequences were putforward by Lim et aZ. (1986) and Hori & Osawa (1986, 1987). Theseauthors sequenced nuclear-encoded 5s ribosomal RNAs from six genera offlorideophytes and one bangiophyte, and inferred trees by cluster analysis(UPGMA). Their trees show red algal 5s rRNAs to form a single cluster,and the 5s r-RNA of Por@yra to diverge most deeply within this cluster. As5s rRNAs are small, highly constrained molecules with few informative sites(Halanych, 1991; Steele et aZ., 1991), the value of these trees in revealingred algal phylogeny has been doubted.Sequences of nuclear-encoded small-subunit rRNA genes (SSU rDNAs)have proven useful in investigating phylogenetic relationships within or amongmany problematic taxa (e.g. Woese, 1987; Cedergren et al, 1988; Sogin,Edman & Elwood, 1989; Ariztia, Andersen & Sogin, 1991; Berbee & Taylor,1992; Bird et al, 1992; Saunders & Druehl, 1992; van Keulen et al, 1992;Andersen et aZ., 1993; Gagnon et aC, 1993; Goggin & Barker, 1993; Hinkle& Sogin, 1993). SSU rDNA s are relatively large and information-rich, andthe presence of highly conserved regions as their immediate 5’ and 3’ endsfacilitates their amplification via the polymerase chain reaction (Sogin, 1990).The first detailed trees of red algal SSU rDNAs showed Rhodophyceae andFlorideophycidae, but not Bangiophycidae, to be monophyletic (Figure 3 andothers from Ragan et aZ., 1994 and unpublished). In these trees, bangiophyteSSU rDNAs constitute the three or four most basal branches withinRhodophyceae. All 71 tested alternative topologies consistent with amonophyletic (holophyletic) Bangiophycidae could be rejected using theKishino-Hasegawa test under maximum likelihood, and 68 of these couldalso be rejected using the Templeton-Felsenstein test under parsimony (Raganet al, 1994); thus the SSU rDNA sequences strongly suggest thatBangiophycidae is paraphyletic. Further SSU rDNA sequences are availablefor the monophyletic orders (Gracilariales (Bird et al, 1992) and Bangiales(Oliveira et al, 1995), for the large and possibly heterogeneous orderGigartinales (Saunders & Kraft, 1994; Lluisma & Ragan, in press), and forthe monophyletic Nemaliales-Acrochaetiales-Palmariales complex (Saunders etal, 1995).Remarkably (given the difficulties inherent in phylogenetic inference basedon 5s rRNAs, cited above), the 5s rRNA UPGMA tree of Hori C Osawa(1987) is congruent with the SSU rDNA tree not only in showing theBangiales to branch basally within Rhodophyceae, but also in showingPalmariales to branch more basally within Florideophycidae than membersof Gigartinales, Gelidiales and Gracilariales.The monophyly of rhodophytan SSU rDNAs apparent from Figure 3 isnot an artifact of outgroup selection. Replacement of the cryptomonad nuclearrDNA sequences with small numbers of other SSU rDNAs (unpublisheddata) or with a wide selection of eukaryote rDNAs (below) likewise yields amonophyletic Rhodophyta. There is, however, one complication. SSU rDNAsfrom the nucleomorph genomes of the cryptomonads Cryptomonas sp. phiand Qrenomonas salina S&tore group among red algal rDNAs (Douglas etal, 1991; Maier et al, 1991), specifically with those of genera Erythrocludia,
86 M. A. RAGAN AND R R. GUTELLI 4.05 unitsICPalmarialesAcrochaedalesNemalialesGigarQnalesGigarttnalesGigartinalesCeramialesGelidia esIRhodymenta esGrgartmalesBonnemarsonialesCeramialesCeramialesCeramialesCeramialesGracilarialesGracilariales- Gracilaria cornea Gracilariales1 ’ Gracilaria verrucosa Gracilariales4 IO- Gracilariopsis lemaneifotis Gracilariales-7 CurdieaGracilarialesMelanthalia . GracilarialesL Gracilariopsts sp. Gracilariales-Ahnfehia AhnfeltialesCorallinaHildenbrandiaCorallinalesHi@p$!;~PorphyridialesBangialesPalmarialesPalmarialesCompsopogonalesPorphyridialesFigure 3. Bootstrapped maximum-likelihood tree (DNAML) of red algal SSU rDNA sequences,based on most-conservative sequence regions (1566 nucleotide positions). With permissionfrom Ragan et al (1994). Authorities for taxa in Figs 3-5 are as follows:Anabaena variabilis Kiitz. Nicotiana tabacum L.Aspergillus nidulans (Eidam) Winter Physcomitrella patens (Hedw.) B.S.G.Bacillus megaterium de Bary Pisum sativum L.Bacillus stearothermophilus Donk Porphyra acanthophora Oliveira & CoilCaenorhabditis elegans (Maupas) Dough. Porphyra leucosticfa Tltur. in Le Jol.Chlamydomonas reinhardtii Dang. Porphyra miniata (C. Ag.) C. Ag.Chondrus crispus Stackh. Porphyra purpurea (Roth) C. Ag.Cochliobob heteroskophus Drech. Porphyra spiralis Oliveira & CoilDrosophila melanogaster Meig. Porphyra umbilicalis (L.) J. Ag.Escherichia coli (Migula) Cast. & Chalm. Saccharomyces cerevisiae HansenGallus gallus L. Schistosoma mansoni SambonGracilaria chilensis Bird et al Sinapis alba L.Gracilan’a cornea J. Ag. Spinacia oleracea L.Gracihrria Uvahiae McLachlanGracilaria venucosa (Huds.) Papenf.Tiypanosoma brucei Plimm. & Bradf.U&ago maydb (DeCand.) CordaGracilariopti lemaneiformis (Bory) Dawson Zea mays L.et al Zygosaccharomyces rouxii (Boutr.) YarrowHomo sapiens L. Zymomonas mobilis (Lindner) Kluyver &Magnolia lilh$ora Desr. in Lam. van NeilErythrotrichirz, and Stylonema (Fig. 3 and below). As cryptomonad nucleomorphshave never been considered to be red algae, Rhodophyceae might technicallybe paraphyletic: similar problems arise with viruses, which may have arisenon multiple occasions from various taxa. Various solutions are possible, the
ARE RED ALGAE PLANTS? 87easiest being simply to acknowledge that the cryptomonad nucleomorph hasapparently arisen from a red alga, and formally or informally cross-referencethis compartment and its genes to Rhodophyta.A large sequence database is also available for the plastid-encoded geneencoding the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase(LS-RuBisCO) (Ch ase et al, 1993). Freshwater et al. (1994) have recentlysequenced 81 red algal LS-RuBisCO genes and inferred a parsimony tree.As their tree is not rooted outside the red algae, no conclusions can bedrawn about monophyly of Rhodophyta or of its two subclasses; but if theLS-RuBisCO gene tree were rooted as in Figure 3 (placing Compsopogonalesbut not Bangiales on the deepest branch), Florideophycidae would again bemonophyletic and Bangiophycidae again not monophyletic. Although sometopological details differ, both the LS-RuBisCO and SSU rDNA trees indicatethat Hildenbrandiales, Corallinales, Acrochaetiales, Palmariales, Nemalialesand Ahnfeltiales are the earliest-diverging lineages within Florideophycidae.RHODOPHYTA AS PLANTS KS RHODOPHYTA AS PROTISTSAll available molecular data support the decision of the early phycologiststo recognize a monophyletic grouping for red algae. At which point did thislineage diverge from the other eukaryotes. 3 What are the closest relatives ofred algae? From West & Fritsch (1927: 416) and Drew (1950: 190) toGarbary & Gabrielson (1990: 493), Woelkerling (1990: 2) and Corliss (1994:25), answers to these questions have been ‘unknown’ and ‘problematical’.Historically, red algae were considered to be plants. From the days ofPliny the Elder if not earlier, all organisms had been considered to be eitherplant or animal; not surprisingly, de Jussieu (1789), Velley (1795), Lamouroux(1805, 1813), T umer (1808), de Candolle (1813), C. A. Agardh (1824),Bartling (1830), Harvey (1836, 1841), Endlicher (1836-1840), J. G. Agardh(1842-1901), Endlicher & Unger (1843), Ktitzing (1843), Nageli (1847), Lindley(1853) and Berkeley (1857) followed Linnaeus in treating red algae as plants.Nonetheless “. . . there are numerous expressions in the works of naturalistsof all times, which show a suspicion that organisms exist which are not tobe regarded properly as either animal or vegetable in their structure andnature” (Wilson & Cassin, 1863: 120). The latter authors cite Buffon,Daubenton and Pliny himself to this effect (pp. 119-121), while Bory deSaint V%rcent (1824: 660 and 1825: 6) reads these sentiments into Linnaeus’sZoo~hytu and Lamar&s Animaux u~at/ziques.Bory de Saint Vincent was apparently the first to propose a third kingdomof life (Psycho&are, or two-souled organisms) encompassing “les Arthrodiees,les Spongiaires, [et] la plupart des Polypiers”; his Regne Vegetal remainedcompositionally similar to the classical concept of plants “minus some oftheir cryptogams” (1824: 659 and 1825: 8). H. F. Copeland (1947: 351) laterdenied priority to Bory’s Regne Psychodiare on grounds that it was notLatinate.Without citing Bory, Owen (1860: 4) recognized that “. . . there are numerousbeings, mostly of minute size and retaining the form of nucleated cells,which manifest the common organic characters, but without the distinctivesuperadditions of true plants or animals. Such organisms are called ‘Protozoa’,
88 M. A. RAGAN AND R. R. GUTELLand include the sponges or Amorphozoa, the Foraminifera or Rhizopods,the Polycystineae, the Diatomaceae, Desmidiae, Gregarinae, and most of theso-called Polygastria of Ehrenberg, or infusorial animalcules of older authors”.Owen did not mention red algae, but from his delineation of Protozoa it isclear that he too considered red algae to be plants. Hogg (1860) acceptedOwen’s grouping but challenged his choice of the term Protozoa for it,preferring instead Regnum Primigenum. This kingdom comprised Protoctista(“all the lower creatu&s, or the primary organic beings”) and Amorphoctista(sponges). Owen later (1861: 6) proposed an alternative name, Acrita.Wilson & Cassin (1863) erected an alternative third kingdom of life,Primalia, with five subkingdoms, the first of which was Algae. These authorsdid not mentioned red (or brown or green) algae, but made it clear (pp118-119) that Primalia was to be much more inclusive than Owen’sProtozoa. As by the 1860s scholarly treatments of algae usually included theflorideophytes, Wilson & Cassin appear to be the first authorities to haveremoved red algae from among the plants. The influential Haeckel (1866,1878, 1904), however, expressly included Florideae among Plantae, not amonghis own third kingdom, Protista.Haeckel’s placement of red algae was not seriously challenged in thebotanical (Eichler, 1886) or protistological (Dobell, 1911) literature until H.F. Copeland (1938) grouped red algae with Protista-with disclaimers thatrhodophytes are “a highly evolved group of unknown origin” (Copeland,1938: 402, 409) and a diagram (p. 410) hs owing the red algal lineage closelyappressed to Plantae. Copeland later (1947, 1956) substituted Hogg’s termProtoctista on the basis of priority. Since Copeland, most authorities haveconsidered red algae among the Protista or Protoctista, although Whittaker(1969) and D odson (1971) continued treating them as plants.A SECOND TRADITION: RED ALGAE AS ‘THE DEEPEST EUKARYOTES’As early as 1867, Cohn concluded that “. . .in a natural system, thefilamentous cyanobacteria must be separated from the unicellular green algae,with which they are usually arranged, and moreover placed as the bottommoststep of a different organizational ranking immediately before the red algae”(1867: 36; translation by M. R.). This idea of red algae constituting theoldest, deepest-branching, or most ‘primitive’ group of eukaryotes permeatesthe subsequent phycological and botanical literature (Tilden, 1933, 1935;Copeland, 1947, 1956; Cronquist, 1960; Christensen, 1964; Whittaker, 1969Figure 3; Ragan & Chapman, 1978; Taylor, 1978; Lipscomb, 1989; Bremer,1989). Dougherty & Allen (1958) formalizedas ‘mesoprotists’.the idea by classifying red algaeIt is not hard to understand the roots of this idea, given the characterstates defining red algae (Table 1). In pigmentation and ultrastructure, redalgal plastids most closely resemble cyanobacteria; and even if this wereascribed to a particular course of endosymbiosis, red algae lack flagellarbasal bodies and centrioles. Moreover, the UPGMA tree of Hoi-i & Osawa(1987) showed red algal 5s rRNAs as the most basally diverging lineagewithin the eukaryotes (but see below).
ARE RED ALGAE PLANTS?RESOLUTION: BOTH TRADITIONS APPEAR INCORRECT89Primarily through the efforts of Sogin and colleagues, a unified view ofeukaryote phylogeny, based on analysis of SSU rDNA sequences, came intofocus in the late 1980s (Sogin, 1989; Sogin et al, 1989; Schlegel, 1991; Vande Peer et al, 1993). Two features of the emerging SSU rDNA treeimmediately illuminated the debate on classification of red algae. Red algalSSU rDNA sequences clearly grouped not at the base of the eukaryote tree,but just basally to the divergence of morphologically complex taxa includinganimals, plants, fungi, and straminipiles (Bhattacharya et al, 1990; Hendrikset al, 1991). [Organisms with tubular mitochondrial cristae and tripartitetubular flagellar hairs (bicosoecids, oomycetes, hyphochytrids, opanlinids,proteromonads, raphidiophytes, eustigmatophytes, chrysophytes, xantophytes,synurophytes, phaeophytes, bacillariophytes, labyrinthulids, andthraustochytrids) are straminipiles (see Patterson (1989) for origin of the term‘stramenopiles’). Haptophytes, cryptomonads, and dinoflagellates, which havesometimes been included among the Chromophyta, are excluded fromstraminipiles.] Moreover, the earliest-diverging rDNA lineages were those ofamitochondriate, not aflagellate, eukaryotes. Thus the cyanobacteria-likepigmentation of red algae did in fact reflect a primary endosymbiotic event,and the absence of flagella was clearly reductive. In these initial trees,however, red algal SSU rDNAs did not appear to be specifically related toSSUs of green plants; thus neither of the two historical traditions wasconfirmed by the early SSU rDNA data.ARE RED ALGAE PLANTS AFTER ALL? A NEW GENERATION OF rDNA TREESAlthough the shape and major features of the eukaryote SSU rDNA treewere well-established by the end of the 198Os, resolution of the relationshipsamong red algae, plants, fungi, animals and other closely related lineageshas not become stabilized. Both for analytical reasons (Cavender & Felsenstein,1987; Lake, 1987; Hendy & Penny, 1989) and from practical experience,several researchers realized that resolution would require determination ofSSU rDNA sequences of many more early-diverging animals, fungi, redalgae, cryptomonads, apicomplexans, and ciliates. The SSU rDNAs of manysuch organisms were subsequently sequenced (Barta, Jenkins & Danforth,1991; Chapman & Buchheim 1991; Douglas et ah, 1991; Gajadhar et al,1991; Kelly-Borges, Bergquist & Bergquist, 1991; Wainright et ah, 1993; West& Powers, 1993; Oliveira & Ragan, 1994; Ragan et al, 1994; Steinkotter etal, 1994). It also became apparent that the 8rst ‘PO@@ SSU rDNA(Hendriks et cd, 1991) actually belonged to a member of Pahnariales(Florideophycidae).Our alignment of red algal with other eukaryote SSU rDNAs has proceededin stages over more than three years. An initial group of red algal rDNAswas integrated into the existing database of aligned eukaryote SSU rDNAs,with alignment based on conservation of secondary- and higher-order structuresas revealed by covariation of nucleotides (Gutell, 1993a,b; Gutell, Larsen &Woese, 1994); folded structures of these red algal rRNAs were simultaneouslyconstructed (unpublished). T rees were inferred from the most conservative
90 M. A. RAGAN AND R. R. GUTELL(i.e. most credibly aligned) regions of this initial alignment matrix, and, basedon the results, sequences were reordered within the database to reflect betterthe phylogenetic relationships. More red algal and other sequences wereadded as they became available, alignments in the less-structured regionswere refined, trees were again inferred from the (now more extensive)credibly aligned regions, and the database was again reordered. Five completeiterations of this pro$ess, and numerous local modifications, have yielded astructure-based alignment of 62 red algal and approximately 650 othereukaryote SSU rDNa sequences. From this alignment we have selected 87aligned SSU rDNAs: two eubacterial and three archaeal rRNAs as outgroups,and 19 red algal and 63 other eukaryote sequences. The latter were selectedto overrepresent lineages which in our analyses diverge closest to red algae,and within those lineages to include earlier-diverging representatives (i.e. tominimize the lengths of internal edges, and thereby to try to avoid topologicalartifacts arising from differential apparent rates of acceptance of mutations).As documented elsewhere (Ragan et al, 1994), we inferred trees from twovariants of the sequence matrix: the ‘essentially complete’ matrix (i.e. afterremoving only the primer regions and the most sparsely populated variableregions: 1815 nucleotide positions), and the securely aligned ‘conservativecore’ (1098 nucleotide positions). Distances were calculated under Felsenstein’s‘maximum likelihood’ (generalized Kimura two-parameter) model of sequencechange. Bootstrapped (rz = 500 to 1000) neighbour-joining trees were inferredon a Sun lo/30 workstation using SEQBOOT, DNADIST, NEIGHBOR andCONSENSE from PHYLIP version 3.53c, and bootstrapped (n = 400 to 763)parsimony trees on an AVX series 2 computer (64 parallel Intel 860 boardslinked through an Intel 805 transponder) using SEQBOOT, DNAPARS andCONSENSE from PHYLIP 3.53~ (Felsenstein, 1989). Parsimony trees weresometimes ‘refit by least squares’ by appending a user tree (the consensustopology) to the distance matrix for analysis using FITCH and PHYLIP3.53~; when parsimony and distance trees are topologically similar, this yieldsrough estimates of the numbers of steps separating nodes. Consistent withthe experience of Olsen et al. (1994) for matrices of this size, we did notobtain stable results using fastDNAml.Not surprisingly, in all our trees, the greatest distances (or numbers ofsteps) separated prokaryote from eukaryote SSU rDNAs. The five mostbasally branching eukaryote rDNA lineages were respectively those ofEncephalitozoon and Vairimorpha; Hexamita and three Giardia species; Physarum;Euglena, Bodo, Trypanosoma, Leishmania and Crithidia; and Entamoeba, sometimestogether with Naegleriu. To make the relationships among red algal andnearby lineages more easily visible, branches more basal than Entumoebahave been removed from Figure 4A and B.Results from bootstrapped parsimony and neighbour-joining trees inferredfrom the conservative core regions are shown in Figure 4A and B, respectively.Red algal rDNAs appear monophyletic in both analyses, and the twocryptomonad nucleomorph rDNAs group among the bangiophycidean lineages.In agreement with Baldauf & Palmer (1993) and Wainright et al. (1993),both trees show a sister-group relationship between animal and fungal rDNAs;nuclear rDNAs of two cryptomonads plus Goniomonas and Glaucocystis likewisegroup together (cf. McFadden, Gilson & Hill, 1994). In both analyses (Fig.
ARE RED ALGAE PLANTS? 914A,B), rDNAs of four groups (red algae; green plants including green algaesensu ho; cryptomonads, Goniomonas and Gluucocystk and animals plus fungi)form an exclusive monophyletic group, with red algal rDNAs isolated; butdistances or numbers of steps are small, and bootstrap support is less than50%. We refer to these four groups, perhaps anthropocentrically, as the‘crown taxa’ (after Knoll, 1992).In an attempt to find the sister group of red algae, we inferred bootstrappedparsimony and neighbour-joining trees (results not shown) from the essentiallycomplete sequence matrix. Although the addition of less-securely alignedregions and more gaps might be expected to degrade the phylogenetic signal,these effects should be most severe near the base of the tree (where sequencesare more divergent), while resolution could improve among closely relatedgroups such as the crown taxa. However, possible improvements in resolutionmust be balanced against the unpredictable consequences of including in theanalyses numerous vacant (gapped) nucleotide positions.Upon neighbour-joining analysis, bootstrap support was significantlyimproved (to 92%) only for the animal-fungal clade; support for a monophyleticRhodophyceae actually decreased, while the cryptomonad nucleomorphrDNAs moved outside those of the (other?) rhodophytes. The volatile EmdianarDNA appeared among those of plants and cryptomonads, attracting theGlaucocystti but not the Goniomonas rDNA; and the straminipiles (SJ.VZU~,Ochromonas, NuuicuZa, Fucus, AchZp) moved into the crown of the tree.Bootstrapped parsimony analysis presented a rather different picture. Theanimal-fungal grouping was preserved (but did not gain additional bootstrapsupport) and associated somewhat more strongly with plant rDNAs. Aclassical Rhodophyta remained weakly intact and isolated from other majorcrown taxa. Most notably, the cryptomonad nucleomorphs were securelyattracted to the corresponding cryptomonad nuclear sequences, as were thehitherto separate chlorarachniophyte nucleomorphs to the chlorarachniophytenuclear rDNAs. The determinants of these attraction effects are not obviousfrom analysis of the cryptomonad (or chlorarachniophyte) rDNA sequences;there are no stretches of pan-wise-identical nucleotides in nonconservativeregions sufficiently long to be taken as evidence for example of post-endosymbiotic gene conversion. We interpret these results as artefacts arisingfrom the inclusion of numerous gapped positions.Thus the sister-group of red algae cannot readily be identified by inferenceof trees and bootstrapping. As an alternative we sought at least to rule outsome alternatives by use of nonparametric tests. Kishinb & Hasegawa (1989)have described a test of alternative topologies under maximum likelihood; acorresponding test under parsimony was introduced by Templeton (1983)and modified by Felsenstein (1985). Any four independent lineages (such asthe crown taxa identified above) can be related in only 15 ways via a rootedbifurcating tree, and we forced each of these topologies in turn, as a usertree, on the data using the DNAML and DNAPARS programs with PHYLIP3.53~; topologies within each of the four lineages appeared relatively robust(Fig. 4A,B and unpublished data) and for computational reasons were notallowed to vary. Alternative topologies were rejected if they decreased thelikelihood, or increased the number of steps, by more than 1.96 standarddeviations.
92 M. A. RAGAN AND R. R. GUTELLbdrlumPlasmc- Tetrahymena1rdlumnas... - - -..-< Cryptomonas NMPvrenomonas NMFigure 4. A (above), Detail from bootstrapped (n = 763) parsimony (DNAPARS) tree of SSUrDNA sequences, based on the most-conservative 1098 nucleotide positions. Tree refit byleast squares to show approximate numbers of nucleotide changes. B, Detail from bootstrapped(n = 1000) neighbour-joining (NEIGHBOR) tree of SSU rDNA sequences, based on the most-conservative 1098 nucleotide positions. Width of line indicates bootstrap support: widest, 90-100%; second widest, 70-89%, third widest, 50-690/o; narrowest, less than 50%.The 15 alternative topologies may be classified into five groups accordingto the taxa they require to be the sister group to red algae (Table 3).Interestingly, the only sister-group relationship for red algae which couldnever be rejected was that between red algae and green plants. However,as only seven of the 15 topologies could be rejected at all (and only twobased on the core matrix), these results should not be taken as strong supportfor, or strong rejection of, any of these five possibilities.
94 M. A. RAGAN AND R. R. GUTELLTABLE 3. Acceptability of alternative topologies of crown taxa undernonparametric testsFull matrix Core matrixT-F K-H Tree T-F K-H+++++++NoiLO+++++++ -WWF,R))+ -CO-W,R))+ -WW,R)-WW’,W+WC(P,R)),-LWW’,R)-mw-GR))-AF(W,R))-W,P)(C,R)+ ++ ++ ++ No+ ++ +++ +-I-+ ++ +Red algaeand plants aresister groupsRed algaeand cryptos aresister groupsRed algae andanima.ls+fungi aresister groupsNo++No+No+ -P(RW,C)) + + Red algae are++ -C(NM,P)) No + sister group to+ -“wwm + + two crown taxa+ -RV’LQ,C)) + + Red algae aref -W(~,P)) + + more basal than+ -WW,P)) + + three crown taxaAF = animals+fungi C = cryptomonads P = green plants R = red algae ++best treeWe are forced to conclude that the existing SSU rDNA sequence datahave been ‘squeezed’ as much as possible using standard inferential techniques,without revealing the precise evolutionary origin or nearest relatives of thered algae. It is possible, although not likely, that future data may resolvethese features to produce a stable classification. Only two further lines ofanalysis appear promising with the existing rDNA matrices: structure-correctedinference, and signature analysis. In the former, covarying nucleotidepositions are downweighted to minimize effects of the violation of positionalindependence. Both theoretical and practical difficulties remain, and to ourknowledge this approach has never been attempted with a data set asextensive and varied as ours. Moreover, simplified analyses (Dixon & Hillis,1993) suggest that improvements using this approach may be modest. Thelatter approach involves identifying features of folded rRNAs characteristicof each lineage and of putative common ancestors, and deciding amongthem, e.g. by energy minimization. We hope to be able to report on suchan analysis in due course.THE EUKARYOTE 5S rRNA TREE REVISITEDThe very different phylogenetic position of red algae in our SSU rDNAtrees (Fig. 4A,B) ve7sus the 5S rRNA tree of Hori & Osawa (1986, 1987)bears comment. As noted above, the 5S rBNA molecule presents problemsfor phylogenetic inference, but the extreme difference between the two treesinvites further analysis. To this end we downloaded an aligned set of 2925S rRNA sequences from the EMBL server (ftpembl-heidelberg.de), andfrom it removed prokaryote sequences other than that of Escherichiu coli(Migula) Cast. & Chal m., multiple sequences from individual species, sequences
ARE RED ALGAE PLANTS? 95from very closely related species, and pseudogenes. From the resulting matrixof 229 5s rRNAs we inferred trees by UPGMA and neighbour-joining,bootstrapping each analysis 100 times.The results (not shown) are striking. In the UPGMA tree, the red algal5s rRNAs cluster near the base of the tree, separated from the E. cob 5Sr-RNA by only five internal nodes. In the neighbour-joining tree, the redalgal 5s rRNAs are 13 nodes removed from E. co& firmly in the crown ofthe tree. (These numbers must be considered approximate, as bootstrapsupport is < 15% for most nodes relating phyla or divisions). The red algal5s rRNAs exhibit long branches; 5s rRNAs of some other eukaryotes areas long or longer, but most are much shorter. Thus the set of 5s rRNAsequences is in egregious violation of ultrametricity (clock-like behaviour onall branches). Like other clustering methods, UPGMA requires, and is verysensitive to violations of, ultrametric behaviour (Michener & Sokal, 1957;Swofford & Olsen, 1990 pp. 440-441). UPGMA is misapplied in analysis ofthese data, and this is a major reason why the global 5s rRNA tree of Hori& Osawa (1987) is so seriously misleading.EVIDENCE FROM NUCLEAR GENESThe non-rRNA molecular biology of red algae is in its infancy, and thatof red algal nuclear genes even more so. Sequences are known for P-tubulingenes of Porphyra purpurea (R. M. MacKay and J. W. Gallant, unpublished)and the cc-tubulin gene of Cyanidium culdarium (Roth) C.Ag. (Pearson &Burns, 1993), while those of the p- and y-tub&n genes of C. culdarium arenearing completion (B. R. Oakley & R. G. Burns, pers. comm. 1994). Apolyubiquitin cDNA of Aghothmnion neglecturn Feldmann-Mazoyer (Apt &Grossman, 1992) and the polyubiquitin gene of Gruciluriu uerrucosu (Huds.)Papenf. (Zhou & Ragan, 1995a) have also been sequenced. Although thehighly conserved tubulin and ubiquitin sequences provide too few informativecharacters for use in inference of global phylogenies, the presence of apolyubiquitin gene suggests that red algae are not among the oldest eukaryotelineages (Krebber, Wostmann & Bakker-Grunwald, 1994). Gene sequencesare available for mitochondrial aconitase (Zhou & Ragan, 1995d) and acalmodulin-like protein from G. uerrucosu (Y.-H. Zhou & M. A. Ragan,unpublished), but the former is only the fourth eukaryote aconitase socharacteri?ed, while paralogy complicates inference of phylogenetic trees fromthe latter.Sequences of the nuclear-encoded NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase genes (gupc and gupA respectively) of Chondruscrispus Stackh. (Liaud et al, 1993, 1994) and GruciZuriu uerrucosa (Zhou &Ragan, 1993, 1994, 1995b) are known. We inferred six most-parsimoniousGAPDH protein trees using PROTPARS (100 independent addition series),and calculated a consensus using CONSENSE in PHYLIP version 3.53. AsgupA and gupC have apparently arisen via duplication of a single ancestralgene, we rooted the consensus tree (Fig. 5) on the point of duplication(Dayhoff et a& 1972; Gogarten et uk, 1989; Iwabe et al, 1989). As withother GAPDH protein trees, some features of Figure 5 are not readilyexplicable, and conclusions must be drawn cautiously.
96 M. A. RAGAN AND R. R. GUTELL-IAspergillus nidulansCochliobolus heterostrophus-Gracilaria verrucosa GapCChondrus crispus GapC- Escherichia coli gap1Trypanosoma brucei cytosolic- Chlamydomonas reinhardtii GapCTtypanosoma brucei glycosomalAnabaena variabilis gap1Nicotiana tabacumGapAChondrus crispus GapA- Anabaena variabilis gap2Bacillus stearothermophilus gapBacillus megaterium gapEscherichia coli gap2Zymomonas mobilis gapAnabaena variabilis gap3Figure 5. Protein parsimony tree of glyceraldehyde3phosphate dehydrogenase sequences.Majority-rule consensus (CONSENSE) of the six most-parsimonious trees (2728 steps each)inferred using PROTPARS with 500 independent random addition orders. All positions wereweighted equally. When positions were weighted inversely (i.e. by (n-I)-’ where n = numberof different amino acids at that position among fully sequenced GAPDHs), no changes wereobserved in the topology of the GAPA subtree, while support for the red alga-green plantclade was weakened (Y.-H. Zhou & M. A. Ragan, unpublished)The gupA gene, whose mature product is plastid-localized, appears to haveentered the eukaryote lineage endosymbiotically (Brinkmann et al, 1987;Liaud, Zhang & Cerff, 1990; Martin et al, 1993) before the divergence ofred algae and green plants (Zhou & Ragan, 1993), and subsequently to havebeen transferred to the nucleus and duplicated within the green plant lineage.In agreement with these results, the position of the Anabaena gap2 gene inFigure 5 supports a cyanobacterial origin for gupA.~The ga..C branch of Figure 5 clearly supports sistergroup relationshipsbetween red algae and green plants, and between animals and fungi (Zhou& Ragan, 1995b). Six equally parsimonious trees were found (each 2728
ARE RED ALGAE PLANTS? 97steps in length), and both sister-group relationships appear in all six trees.The same result is obtained with inverse weighting (i.e. when the relativeweight is (n-l)-‘, where n is the number of different amino acids at thatsite). When the GAPDH protein trees is bootstrapped (n = loo), however,support for the red alga-green plant clade is found to be weak (52%).The triosephosphate isomerase gene of Grucilaria uerruco.ru has recentlybeen sequenced (Zhou & Ragan, 199%). The G. verruc~~a TPI tends tobranch deeply among eukaryote TPIs, but because the protein is relativelysmall, bootstrap support for many topological features is poor. Inverseweighting resolves the G. verrucosa and green plant TPIs as sister groups,although with modest bootstrap support (Zhou & Ragan, 1995c).The red alga Por$$~ru purpureu contains at least two nuclear genes encodingelongation factor (EF) la, one expressed in both the conchocelis (diploidsporophyte) and leafy-thallus (haploid gametophyte) phases, the other expressedonly in the conchoecelis (Q Y. Liu, S. L. Baldauf & M. E. Reith, unpublished).The protein parsimony tree (inferred using PAUP 3.1: Swofford, 1993) showsthe Porphyra EF- la sequences to branch near that of Dictyostelium, just basallyto EF-la’s of animals and fungi but less basally than those of green plants.This result tends to support red algae being a crown taxon; but cautionmust again be exercised, because the ~rypanosoma EF-lcr behaves anomalouslyin this tree.EVIDENCE FROM MITOCHONDRIAL AND PLASTID GENESNeighbour-joining analysis of the mitochondrial gene encoding subunit 3of cytochrome oxidase (~0x3) from the red alga Chondrzls crispus suggests thatred algae are the sister group to green plants (Boyen et al, 1994). Data onother mitochondrial genes from other red algae are becoming available (B.F. Lang & G. Burger, pers. comm.) but have not yet been published.Sequences are also available for genes encoding several phycobilisomesubunits (Bernard et al, 1992; Apt & Grossman, 1993; Grossman et aL, 1993;Roe11 & Morse, 1993), and the entire plastid genome of Par-hyra purpureahas recently been completed (Reith & Munholland, 1993; M. E. Reith, pers.comm.). Like gapA sequences, however, these data are of limited utility inglobal phylogenies because the corresponding genes are typically absent fromnonphotosynthetic eukaryotes.EVIDENCE FROM NONSEQUENCE DATARothschild (1985: 93) noted a much stronger immunological crossreactivitybetween ribulose 1,5-bisphosphate carboxylase large subunits of pea and twored algae than between those of pea and chromophytes. Apt, Hoffman &Grossman (1993) h ave shown that in transgenic constructs the peptidetranslated from the plastid-transit-encoding region of the Aglaothamnion neglecturngene encoding small subunit of ribulose 1,5-bisphosphate carboxylase isfunctional in targeting the corresponding peptide of pea across the peachloroplast membrane. Zhou and Ragan (in press) have recently demonstratedthat the polyadenylation in the red alga Gracilaria verrucosa more closelyresembles that of green plants than that of fungi or animals; multiple
98 M. A. RAGAN AND R. R. GUTELLpolyadenylated transcripts can be produced from individual nuclear genes,the AATAAA polyadenylation signal is not strictly conserved, and GT-richclusters downstream from the poly(A) addition site are absent from somegenes.INDIRECT EVIDENCE FROM STUDIES OF PLASTID ORIGINMost molecular-s;quence evidence indicates that plastids aroseendosymbiotically from cyanobacterial ancestors (Douglas & Turner, 199 1;Douglas, 1994). Mereschkowsky (1905: 602 and 1910) initially proposed thatplastids of red, brown and green algae arose independently from differentgroups of cyanobacteria, and some molecular-sequence data do appear tosupport this idea (Scherer, Lechner & Boger, 1993). Most molecular data,however, are more consistent with plastids having arisen from a single pointwithin the cyanobacteria (Morden et al, 1992; Douglas, 1994; Wolfe et al,1994). The location of intron 1 in the, transit-peptide-encoding region of gu&4in green plants and G. verrucosa (Zhou & Ragan, 1994) may likewise suggestthat gupA entered the eukaryote lineage only once, hence that red algae andgreen plants share a common photosynthetic eukaryotic ancestor.The plastids of cryptomonads and photoautotrophic straminipiles are nowwidely thought to have arisen via secondary associations between eukaryotichosts and eukaryotic symbionts (Douglas, 1994). In the case of cryptomonads,this symbiont appears to have been a red alga (Douglas et al 1991) or aclose relative thereof, and the eukaryotic host a Goniomonas-like flagellate(Kugrens & Lee, 1991; McFadden, Gilson & Hill, 1994). Numerousobservations appear consistent with this scenario, including the numbers ofextraplastidal membranes in cryptomonads and photoautotrophic straminipiles(Tomas & Cox, 1973; Gibbs, 1981; Whatley, John & Whatley, 1979; Whatley,1981), the presence of and biosynthetic capability within the nucleomorphcompartment of cryptomonads (McFadden, Gilson & Douglas, 1994), and thenonphotosynthetic nature of the most basal lineages within the straminipiles(Leipe et al, 1994). This scenario is further supported by the specificrelationship observed between rDNAs of Goniomonas and cryptomonad nuclei(Fig. 4A,B, and McFadden, Gilson & Hill, 1994).Evidence is also accumulating that secondarily nonphotosynthetic eukaryotescan retain molecular evidence of the lost plastid genome for evolutionarilysignificant periods of time (e.g. Plasmodium: Howe, 1992; Gardner et al,1993). TO our knowledge there is no evidence that animals or fungi descendfrom a photosynthetic ancestor. If red algae, green plants, fungi and animalsare the crown taxa in the sense argued above, and if red algae and greenplants do in fact descend from a common photosynthetic eukaryote, and ifanimals and fungi were never photosynthetic, then red algae and green plantsmust be sister groups.As a consequence of this result, the presence of two membranes surroundingrhodoplasts and chloroplasts can be interpreted not only as evidence of theorigin of these plastids in a primary endosymbiotic event involving acyanobacterium but also, most parsimoniously, as a synapomorphy. Storageof starch in the cytoplasm and the absence of phagotrophy might likewisebe synapomorphies in red algae and green plants, and a Kingdom Plantae
ARE RED ALGAE PLANTS? 99TABLE 4. Plantae sensu Cavalier-Smith(1981)Kingdom PlantaeSubkingdom ViridiplantaeDivision ChlorophytaDivision CharophytaDivision EmbryophytaSubkingdom BiliphytaDivision GlaucophytaDivision Rhodophytasel2su Cavalier-Smith (1981) (Table 4) might in fact be founded onsynapomorphic character states (see also Cavalier-Smith, 1993: 956-957).This classification implies a relationship between glaucophytes (e.g.Cyanophora, Glaucocystk, Glaeochaete) and red algae, and between glaucophytesand green plants. Cavalier-Smith (1987: 75-76) interprets ultrastructuralfeatures as indicating that “(t)he transition between Glaucophyceae andRhodophyceae is so gradual that they might almost be treated as a singleclass”, and both Cavalier-Smith (1986: 323) and O’Kelly (1992) commentupon ultrastructural features linking glaucophytes with green plants. The SSUrDNA data do not resolve these issues; other types of analyses (above) andsequence data from more-variable genes or gene regions will probably berequired.ACKNOWLEDGEMENTSIt is a pleasure to acknowledge the contribution our colleagues Carolyn J.Bird, Ellen Rice Kenchington, Colleen A. Murphy and Rama K. Singh insequencing red algal SSU rDNAs. Sandie L. Baldauf, Debashish Bhattacharya,Roy G. Bums, Detlef D. Leipe, Qng Yan Liu, Ron M. MacKay, Geoff I.McFadden, Michael E. Reith and Mitchell L. Sogin graciously allowed usaccess to unpublished data and(or) to manuscripts prior to publication. Wethank Tom Cavalier-Smith, Georges E. Merinfeld and Lynn J. Rothschild forhelpful comments. Joe Felsenstein and Gary J. Olsen supplied sourcecode for PHYLIP and fastDNAm1 respectively; Burkhard Plache compiledDNAPARS and fast DNAml for the AVX-2, and Optimax Software (Halifax,N.S.) donated computer time. R.R.G. acknowledges support from U.S.National Institutes of Health Grant GM 48207 and from the W. M. KeckFoundation.REFERENCESAgardh CA. 1824. Systema algurum Lund: Berlingianis.Agardh JG. 1842-1901. Species pera et ordines Fucoidearum, seu desm’ptiones succinctae specierum generumet ordinum, guibus jkoidearum clussis constituitur. Lund: Gleerup.Andersen RA, Saunders GW, Paskind q, Sexton JP. 1993. Ultrastructure and 18s rRNA genesequences for Pefagomonas calceoiuta gen. et sp. nov. and the description of a new algal class, thePelagophyceae classis nov. Journal of Phycology 29: 701-715.Apt KE, Grossman AR. 1993. A polyubiquitin cDNA from a red alga. Plnnt Physiology 99: 1732-1733.
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