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  1. 1. 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
  2. 2. 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
  3. 3. 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.
  4. 4. 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
  5. 5. 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,
  6. 6. 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 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
  7. 7. 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’,
  8. 8. 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).
  9. 9. 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
  10. 10. 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.
  11. 11. 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.
  12. 12. 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.
  13. 13. ARE RED ALGAE PLANTS? 93Iorarachnlon 1242 NMChtorarachnlon 1-57 NM-AtexandrtumCrypthecodlnlumTetrahymenaErythrocladla’ErythrotrlchlaPyrenomonas NMCryptomonas NMPorphyra mlniataPorphyra leucostlctaPorphyra purpureaPorphyra umblllcalls- PhycodrysGraclllariaHlldenbrandiaDixonlellaAcanthopleuraPtacooectenChlamydomonasPyre;omonas NUCryptomonas NU- GonlomonasEmlllanaOchromonasPtasmoatumDlctyostellumEntamoebaFigure 4(B).
  14. 14. 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 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 (, andfrom it removed prokaryote sequences other than that of Escherichiu coli(Migula) Cast. & Chal m., multiple sequences from individual species, sequences
  15. 15. 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.
  16. 16. 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
  17. 17. 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 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
  18. 18. 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
  19. 19. 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.
  20. 20. 100 M. A. RAGAN AND R. R. GUTELLApt RI?, Grossman AR. 1993. Characterizaton and transcript analysis of the major phycobiliproteinsubunit genes from Aglaothamnion neglecturn (Rhodophyta). Plant Molecular Biology 21: 27-38.Apt ICE, Hoffian NE, Grossman AR 1992. The y subunit of R-phycoerythrin and its possible modeof transport into the plastid of red algae. Journal of Biological Chemistry 268: 16208-16215.Ariztia EV, Andersen RA, Sogln ML. 1991. A new phylogeny for chromophyte algae using l&S-likerRNA sequences from Mallomonas papillosa (Synurophyceae) and Tribonema aequale (Xanthophyceae).Journal of Phycology 27: 428-436.Baldauf SL, Palmer JD. 1993. Animals and fungi are each other’s closest relatives: congruent evidencefrom multiple proteins. &,oceedings of the National Academy of Sciences of the United States of America90: 11558-I 1562.B-JR, Jenkins MC, Danforth I-ID. 1991. Evolutionary relationships of avain Eimeria species amongother apicomplexan protozoa: monophyly of the Apicomplexa is supported. Molecular Biology andEvolution 8: 345-355.Bartling FrG. 1830. Ordines naturales plantarum, eorumque charactcres et afinitater, adjectii generumenumeratione auctore Fr. Th Bartling. Gottingen: Dieterich.Berbee ML, Taylor JW. 1992. Convergence in ascospore discharge mechanism among pyrenomycetefungi based on 18s ribosomal RNA gene sequence. Molecular Phylogenetics and Evolution 1: 59-71.Berkeley IQ. 1857. Introduction to Cryptogamic Botany. London: Bailhere.Bernard C, Thomas JC, Maze1 D, Mousseau A, Castets AM, Tandeau de Marsac N, Dubacq JP.1992. Characterization of the genes encoding phycoerythrin in the red alga RhodelZa viohzcea: evidencefor a splitting of the rpeB gene by an i&on. Proceedings of the National Academy of Sciences of theUnited States if America *89: 9564-9568..Berthold G. 1882. Die Banaiaceen des Golfes von Neanel und der anmenzenden Meeres-Abschnitte.Eine Monographie. Faunl und Flora des Go@ von Nea,bel 8: l-28+1 PI. Leipzig: Engelmann.Bhattacharya D, Elwood HJ, Goff LJ, Sogin ML. 1990. Phylogeny of Gracilaria lemaneifkmis(Rhodophyta) based on sequence analysis of its small subunit ribosomal RNA coding region. Journalof Phycology 26: 181-186.Bird CJ, Rice EL,, Murphy CA, Ragan MA. 1992. Phylogenetic relationships in the Gracilariales(Rhodophyta) as determined by 18s rDNA sequences. Phycologia 31: 510-522.Bory de Saint Vincent JBGM. 1824. Psycodiaire (Regne). In: LamourouxJVF, Bory de Saint VincentJBGM, Deslongchamps E, eds. Encyclojridie mithodique. Histoire naturelle des zoophytes, ou animauxrayonnkr, faisant suite a l’hirtoire naturelle des vers, de Bruguike. Paris: Veuve Agasse, 657-663.Bory de Saint Vincent JBGM. 1825. Une nouvelle distribution des corps naturels en cinq regnes.From Dictionnaire cbxique d’histoire nature&, reprinted in Revue enncyclopedique (82’ C&.-T. XXVIII),Septieme annee, seconde serie, Oct. 1825. Paris: Rignow, l-25.Boyen C, Leblanc C, Bonuard G, Grienenberger J-M, KIoareg B. 1994. Nucleotide sequence ofthe ~0x3 gene from Chondrus crisper: evidence that UGA encodes hyptophan and evolutionaryimplications. Nucleic Aciak Research 22: 1400-1403.Bremer K. 1989. The hierarchy of life (Preface, Fig. 1). In: Femholm B, Bremer K, Jomvall H, eds.The Hierarchy of Life. Molecules and Morphology in Phylogenetic Analysis (Nobel Symp. 70). Amsterdam:Exerpta Medica, 1.Brinkmann H, Martinez P, Quigley F, Martin W, Cerff R. 1987. Endosymhiotic origin and codonbias of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize.Journal of Molecular Evolution 26: 320-328.Cavalier-Smith T. 1981. Eukaryote kingdoms: seven or nine? BioSystems 14: 461-481.Cavalier-Smith T. 1986. The kingdom Chromista: origin and systematics. Progress in Phycological Research4: 309-347.Cavalier-Smith T. 1987. Glaucophyceae and the origin of plants. Evolutionary Trena!r in Plants 2: 75-78.Cavalier-Smith T. 1993. Kingdom Protozoa and its 18 phyla. Microbiological Reviews 57: 953-994.Cavender JA, Felsenstein J. 1987. Invariants of phylogenies in a simple case with discrete states.Journal of Chrssttcation 4: 51-7 1.Cedergren R, Gray MLW, Abel Y, Sankoff D. 1988. The evolutionary relationships among knownlie forms. Journal of Molecular Evolution 28: 98-112.Chapman DJ. 1974. Taxonomic status of Cyanidium caldarium, the Porphylidialev and t&niotru$&s.Nova Hedwigin 25: 673-682.Chapman RI.+ Buchheim MA. 1991. Ribosomal RNA gene sequences: analysis and significance inthe phylogeny and taxonomy of green algae. Critical Reviews in Plant Sciences 10: 343-368.Chase Mw, Soltis DE, Olmstead RG, Morgan D, Les DH, Mishler BD, Duvall MB, Price RA,Hills HG, Qiu Y-L Kron KA, Rettig JH, Conti J3, Palmer JD, Manhart JR, Sytsma KJ,Michaels I-&J,Kress WJ, Karol KG, Clark WD, Hedren M, Gaut BS, Jansen BK, Kim K-J,Wipee CF, Smith JF, Furnier GR, Strauss SH, Xiang Q, Plunkett GM, Soltis PS, SwensenSW Wi&ms SE, Gadek PA, Quinn CJ, Eguiarte LE, Golenberg F, Learn GH Jr, GrahamSW, Barrett SCH, Dayanandan S, Albert VA. 1993. DNA sequence phylogenetics of seed plants:an analysis of the plastid gene rbcL. Annals of the Missouri Botanical Garden 80: 528-580.
  21. 21. ARE RED ALGAE PLANTS? 101Christensen T. 1964. The gross classification of algae. In: Jackson DF, ed. A&aae and Man. New York:Plenum, 59-64.Cohn F. 1867. Beitrage zur Physiologie der Phycochromaceen und Florideen. Archiv fbr MikroskopischeAnatomie 3: l-60.Copeland HF. 1938. The kingdoms of organisms. Quarterly Review of Biology 13: 383-420.Copeland HF. 1947. Progress report on basic classification. American Naturalisl 8: 340-361.Copeland HF. 1956. The class$cation of lower organisms. Palo Alto CA: Pacific Books.Corliss JO. 1994. An interim utilitarian (‘user-friendly’) hierarchical classification and characterizationof the protists: Acta Protozoologica 33: l-5 1.Cronquist A. 1960. The divisions and classes of plants. Botanical Review 26: 425-482.Dayhoff MO, Hunt LT, McLaughlin PJ, J ones DD. 1972. Gene duplication in evolution: the globins.In: Dayhoff MO, ed. Atlas of Protein Sequence and Structure 7972, Vol. .5. Silver Spring MD: NationalBiomedical Research Foundation, Candolle Al?. 1813. lheorie elementaire de la botanique, ou exposition des principles de la classificationnaturelle et de Part de decrire et dltudier ku vegetaux. Paris: Jussieu AL. 1789. Genera plantarum secundum ordines naturales disposita, juxta methodum in horto regioparisiensi exaratum anno M.D.CC.LxxN. Paris: Herissant et Barrois.Dixon MT, HilBs DM. 1993. Ribosomal RNA secondary structure: compensatory mutations andimplications for phylogenetic analysis. Molecular Biology and Evolution 10: 256-267.Dobell CC. 1911. The principles of protistology. Archiv. fiir Protistenkunde 23: 269-3lO+Taf. 13.Dodson EO. 1971. The kingdoms of organisms. Systematic Zoology 20: 265-281.Dougherty EC, Allen MB. 1958. The words ‘protist’ and ‘Protista’. Experientia 14: 78.Douglas SE. 1994. Chloroplast origins and evolution. In: Bryant DA, ed. The molecular biology of thecyanobacteria. Dordrecht: Kluwer, 91-l 18.Douglas SE, Murphy CA, Spencer DF, Gray MW. 1991. Molecular evidence that cryptomonadalgae are evolutionary chimaeras of two phylogenetically distinct unicellular eukaryotes. Nature(London) 350: 148-151.Douglas SE, Turner S. 1991. Molecular evidence for the origin of plastids from a cyanobacterium-like ancestor. Journal of Molecular Evolution 33: 267-273.Drew KM. 1950. Rhodophyta. In: Smith GM, ed. Manual of phycology. An introduction to the algae andtheir biology. New York: Ronald Press, 167-191.Eichler AW. 1886. Syllabus der Vorlesungen tiber specielle und medicinisch-pharmaceutische Botanik. Berlin:Borntraeger, 4th edn.Em&her S. 1836-1840. Genera phntarum secundum ordines naturales disposita. Vienna: Beck.End&her S, Unger F. 1843. Grum&ige der Botanik. Vienna.Feldmarm J. 1967. Sur une Bangiophycees endozoique (Neevea repens Batters) et ses affinites (Rhodophyta).Blumea 15: 25-29.Felsenstein J. 1985. Confidence limits on phylogenies with a molecular clock. Systematic Zoology 34:152-161.Felsenstein J. 1989. PHYLIP-phylogeny inference package (version 3.2). Cladirks 5: 164-166.Fredericq S, Norris JN. 1995. A new order (Rhodogorgonales) and family (Rhodogorgonaceae) of redalgae composed of hvo tropical calciferous genera, Renoztxia gen. nov. and Rhodogorgon. CryptogamicBotany 5, in press.Freshwater DW, Fredericq S, Butler BS, Hommersand MI-I, Chase MW. 1994. Phylogenetic studiesof plastid rbcL sequences from red algae (Rhodophyta). Proceedings of the National Academy of Sciencesof the United States of America 91: 7281-7285.Gagnon S, Levesque RC, Sogin ML, Gajadbar AA. 1993. Molecular cloning, complete sequence ofthe small subunit ribosomal RNA coding region and phylogeny of Toxoplasma gondii. Molecular andBiochemicnl Parasitology 60: 145-148.Gajadhar AA, Marquardt WC, Hall R, Gunderson J, Ariatia-Carmona EV, Sogin ML. 1991.Ribosomal RNA sequences of SarcoLyslis muris, Theileria annulata and Crypthecodinium cohnii revealevolutionary relationships among apicomplexans, dinoflagellates, and ciliates. MoLecubzr and BiochemicalParasitology 45: 147-154.Garbary DJ, Gabrielson PW. 1990. Taxonomy and evolution. In: Cole KM, Sheath RG, eds. Biologyof the red algae. Cambridge: Cambridge University Press, 477-498.Garbary DJ, Hansen GI, Scagel RF. 1980. A revised classification of the Bangiophyceae (Rhodophyta).Nova Hedwtgin 33: 145-166.Gardner MJ, Feagin JE, Moore DJ, Rangacharl K, Williamson DH, Wilson RJM. 1993. Sequenceand organization of large subunit rRNA genes from ths extrachromosomal 35 kb circular DNA .ofthe malaria parasite Pbzsmodium falctparum. Nucleic A&B Research 21: 1067-1971.Gibbs SP. 1981. The chloroplasts of some algal groups may have evolved from endosymbioticeukaryotic algae. Annals of the New York Academy of Sciences 361: 193-208.Gogarten JP, Kibak H, D&rich P, Taiz L, Bowman EJ, Bowman, BJ, Manolson MF, Poole RJ,Date T, Oshima T, Konishi J, Denda K, Yosbida M. 1989. Evolution of the vacuolar H+-ATPase:implications for the origin of eukaryotes. Proceedings of the National Academy of Sciences of the UnitedStates of America 86: 6661-6665.
  22. 22. 102 M. A. RAGAN AND R. R. GUTELLGoggln CL, Barker SC. 1992. Phylogenetic position of the genus Perkinsus (Protista, Apicomplexa)based on small subunit ribosomal RNA. Molecular and Biochemical Parasitology 60: 65-70.Gray SF. 1821. A natural arrangement of British plants. London: Baldwin, Craddock & Joy, 2 vols.Grossman AR, Schaefer MB, Chlang.GG, Collier JL. 1993. The phycobilisome, a light-harvestingcomplex responsive to environmental conditions. Microbiological Reviews 57: 725-749.Gutell RB. 1993a. Collection of small subunit (16% and 16Slike) ribosomal RNA structures. NucleicAcids Research 21: 3051-3054.GutelI RR. 1993b. The simplicity behind the elucidation of complex structure in ribosomal RNA. In:Nierhaus KH. Franceschi F. Subramanian AR Erdmann VA, eds. The Translational Apparatus. NewYork: Plenum, 477-488. ’ ’._Gutell RR, Larsen N, Woese CR 1994. Lessons from an evolving rRNA: 16s and 23s rRNAstructures from a comparative perspective. Microbiological Reviews 58: 10-26.Haeckel E. 1866. Generelle Morphologic der Organismen: Allgemeine Grun&itge der organischen Formen-Wienschaj, mechanisch begriindet durch die von Charh Darwin rgormirte Descendenr-Theorie. Berlin:Reimer, 2 vols.Haeckel E. 1878. Dar Protislenreich. Eine pop&ire Uebersicht iiber das Formengebiet der niedersten Lebewesen.Mit einem wissensc~jlichen Anhange: System der Protisten. Leipzig: Gunther.Haeckel E. 1904. The wonders of life. A popular study of biological philosophy. New York: Harper.Halanych KM. 1991. 5S ribosomal RNA sequences inappropriate for phylogenetic reconstruction.Molecular Biology and Evolution 8: 249-253.Harvey WH. 1836. Algae. In: Mackay JT, ed. Flora Hibernico, vol. 2: 157-256. Dublin: Curry.Harvey WH. 1841. A manual of the British algae: containing generic and specific descriptions of all the knownBritish species of sea-weeds, and of Conferoae, both marine and jizsh-water. London: van Voorst.Hendriks I., De Baere B, Van de Peer Y, Needs J, Goris A, De Wachter R 1991. The evolutionaryposition of the rhodophyte Porphyra umbilicalis and the basidiomycete Leucosporidium scottii amongother eukaryotes as deduced from complete sequences of small ribosomal subunit RNA. Journal ofMolecular Evolution 32: 167-177.Hendy MD, Penny D. 1989. A framework for the quantitative study of evolutionary trees. SystematicZoology 38: 297-309.Hinkle G, Sogln ML. 1993. The evolution of the Vahlkamphidae as deduced from 16S-like ribosomalRNA analysis. Journal of Eukaryotic Microbiology 40: 599-603.Hogg J. 1860. On the distinctions of a plant and an animal, and on a fourth kingdom of nature.Edinburgh New Philosophical Journal, New Series 12: 216-225+P1. III.Horl H, Osawa S. 1986. Evolutionary change in 5S rRNA secondary structure and a phylogenetictree of 352 5S rRNA species. BioSystems 19: 163-172.Hori H, Osawa S. 1987. Origin and evolution of organisms as deduced from 5S ribosomal RNAsequences. Molecular Biology and Evolution 4: 445-472.Howe Cl. 1992. Plastid origin of an extrachromosomal DNA molecule from Plasmodium, the causativeagent-of malaria. Journal-of Theoretical Biology 158: 199-205.Iwabe N, Kuma K-I, Hasegawa M, Osawa S, Miyata T. 1989. Evolutionary relationship ofarchaebacteria, eubacteria, and eukaryotes inferred from phylogenetic trees of duplicated genes.Proceedings of the National Academy of Sciences of the United States of America 86: 9355-9359.Kelly-Borges M, Bergquist PR, Bergquist PL. 1991. Phylogenetic relationships within the orderHadromerida (Porifera, Demospongiae, Tetractinomorpha) as indicated by ribosomal RNA sequencecomparisons. Biochemical and Systematic Ecology 19: 117-125.Kishlno H. Hasepawa M. 1989. Evaluation of the maximum likelihood estimate of the evolutionarvtree topologies” from DNA sequence data, and the branching order in Hominoidea. Journal ifMolecular Evolution 29: 170-179.Knoll AH. 1992. The early evolution of eukaryotes: a geological perspective. Science 256: 622-627.Kraft GT. 1981. Rhodophyta: morphology and classification. In: Lobban CS, Wynne MT, eds. Thebiology of seaweedr. Oxford: Blackwell, (i-51.Krebber I-I, W&harm C, Bakker-Grunwald T. 1994. Evidence for the existence of a single ubiquitingene in Giardia lamblia. FEBS Lelters 343: 234-236.Kiitzing FT. 1843. Phycologia generalis, oder Anatomic,Brockhaus.Physiologie und Systematik der Tange. Leipzig:Kugrens P, Lee RE. 1991. Organization of cryptomonads. In: Patterson DJ, Larsen J, eds. The biologyof free-living heterotrophic flagellates. Oxford: Clarendon Press, 2 19-233.Lake JA. 1987. A rate-independent technique for analysis of nucleic acid sequences: evolutionaryparsimony. Molecular Biology and Evolution 4: 167-191.Lamouroux JVF. 1805. Dissertations sur plusieurs especes des Fucus, peu connues ou nouvelles; avec leurdescription en latin et cn frawais. Agen: Noubel.Lamourou~ JVF. 1813. Essai sur les genres de la famille de Thalassiophytes non articulees. Annalesdu Museum (National) d’Histoire Naturelle (Parts) 20: 21-47, 115-139, 267-293+7 pl.Leipe DD, Wainright PO, Gunderson JH, Porter D, Patterson DJ, VaIois I?, Himmerich S, SoginML. 1994. The stramenopiles from a molecular perspective: 16%like rRNA sequences fromLabyrinthuloides minuta and Cajteria roenbergensis. Phycologia 33: 369-377.
  23. 23. ARE RED ALGAE PLANTS? 103Liaud M-F, Zhang DK, Cerff R 1990. Differential intron loss and endosymbiotic transfer of chloroplastglyceraldehyde-3-phosphate dehydrogenase genes to the nucleus. Proceedings of the National Academyof Sciences of the United States of America 87: 8918-8922.Liaud M-F, Valentin C, Brandt U, Bouget F-Y, Kloareg B, Cerff R 1993. The GAPDH genesystem of the red alga Chondrus crr$ur promoter structures, intron/exon organization, genomiccomplexity and differential expression of genes. Plant Molecular Biology 23: 981-994.Liaud M-F, Valentin C, Martin W, Bouget F-Y, KIoareg B, Cerff R 1994. The evolutionary originof red algae as deduced from the nuclear genes encoding cytosolic and chloroplast glyceraldehyde-d-phosphate dehydrogenases from Chondrus crispus. Journal of Molecular Evolution 38: 319-327.Lim B-L, Kawai H, Hori I-I, Osawa S. 1986. Molecular evolution of 5S ribosomal RNA from redand brown algae. Japanese Journal of Genetics 61: 169-176.Lindey J. 1853. The vegetable kingdom; or, the structure, classtfication, and uses of plants, illustrated upon thenatural system. London: Bradbury & Evans, 3rd edn.Linnaeus C. 1753. Species plantarum, exhibentes plantas rite cognitas, ad genera relatas, cum dr~erentilspectjicis, nominibus tribialibus, synonymis selectis, locis natalibus, secundum systema sexuale dig&as. Stockholm:Laurentii Salvi, 2 vols.Lipscombe DL. 1989. Relationships among the eukaryotes. In: Femholm B, Bremer K, Jomvall H,eds. The hierarchy of life. Molecules and morphology in phylogenetic anaijsis (Nobel Symp. 70). Amsterdam:Exerpta Medica, 161-178.Lluisma AO, Ragan MA. Relationships among Eucheuma denticulatum, Eucheuma iriforme, and Kappaphycusalvare& (Gigartinales, Rhodophyta) based on nuclear ssu-rDNA gene sequences. Journal of AppliedPhycology, in press.McFadden GI, G&on PR, Douglas SE. 1994. The photosynthetic endosymbiont in cryptomonad cellsproduces both chloroplast and cytoplasmic-type ribosomes. Journal of Cell Science 107: 649-657.McFadden GL Gilson PB. Hill DBA. 1994. Gonimomonar: rRNA sequences indicate that thisphagotrophic flagellate is a close relative of the host component of cryp;omonads. European Journalof Phycology 29: 29-32.Magne F. 1989. Classification et phylogenie des Rhodophycees. Cryptogamie Algologie. 10: 101-115.Maier U-G, Hofmanu CJB, Eschbach S, Wolters J, Igloi G. 1991. Demonstration of nucleomorph-encoded eukaryotic small subunit ribosomal RNA in cryptomonads. Molecular and General Genetics230: 155-160.Martin W, Brinkmann H, Savona C, Cerff R 1993. Evidence for a chimeric nature of nucleargenomes: eubacterial origin of eukaryotic glyceraldehyde-3-phosphate dehydrogenase genes. Proceedingsof the National Academy pfSciences of the United States of America 90: 8692-8696.Mereschkowsky C. 1905. Uber Natur und Ursprung der Chromatophoren im Pflanzenreiche. BiologischesCentralblatt 25: 593-604, 689-691.Mereschkowsky C. 1910. Theorie der zwei Plasmaarten als Grundlage der Symbiogenesis, einer neienLehre von der Entstehung der Organismen. Biologisches Centralblatt 30: 278-288, 289-303, 321-347,354-367.Michener CD, SokaI RR. 1957. A quantitative approach to a problem in classification. Evolution 11:130-162.Morden CW, Delwiche CF, Kuhsel M, Palmer JD. 1992. Gene phylogenies and the endosymbioticorigin of p&ids. BioSystems 28: 75-90.NggeB C. 1847. Die Neuem Algensysteme und versuch zur Begriingung eines eigenen Systems derAlgen und Florideen. Zurich: Schulthess.O’Kelly CJ. 1992. Flagellar apparatus architecture and the phylogeny of ‘green’ algae: chlorophytes,euglenoids, glaucophytes. In: Menzel D, ed. nhe cytoskeleton of the algae. Boca Raton FL: CRC Press,315-346.OBveira MC, Kurniawan J, Bird CJ, Rice EL, Murphy CA, Singh ,I& Gutell RR, Bagan MA.1995. A preliminary investigation of the order Bangiales (Bangiophycidae, Rhodophyta) based onsequences of nuclear small-subunit ribosomal RNA genes. Phycological Research 1: 71-79.OIiveira MC, Ragan MA. 1994. Variant forms of a group I intron in nuclear small-subunit rRNAgenes of the marine red alga Porphyra spiralis var. amplif0lia. Molecular Biology and Evolution 11: 195-207.Olsen GJ, Matsuda H, Hagstrom II, Overbeek R 1994. faslDNAm1: A tool for construction ofphylogenetic trees of DNA sequences using maximum likelihood. Computer Applications in theBiosciences 10: 41-48.Owen R 1860. Palaeontoloa or a systematic summary of extinct animals and their geological relations.Edinburgh: A & C. Black.Owen R 1861. Paleontology or a systematic summary of extinct animals and their geological relulions.Edinburgh: A & C. Black, 2nd edn.Patterson DJ. 1989. Stramenopiles: chromophytes from a protistan perspective. In: Leadbeater BSC,ed. The chromophyte algae. Oxford: Clarendon Press, 357-379.Pearson RCM, Burns RG. 1993. Cyanidium caldarium, a thermophilic eucaryote: the cells, the tubulin,and the a-tub&n gene. Molecular Biology of the Cell. 4: 262a.
  24. 24. 104 M. A. RAGAN AND R. R. GUTELLpuppi RA. 1837. Beweis, doss die Nulliporen F’tlunzen sind. Archiv j?ir Naturgexhichte 3: CM. 1990. Cell structure. In: Cole KM, Sheath RG, eds. Biology of the red algae. Cambridge:Cambridge University Press, 7-41.Sagan MA, Chapman DJ. 1978. A biochemical phylogeny of the prolists. New York: Academic Press.Ragan MA, Bird CJ, Rice EL, GuteB RR, Murphy CA, Singh RR. 1994; A molecular phylogenyof the marine red algae (Rhodophyta) based on the nuclear small-subunit rRNA gene. Proceedingsof the National Academy of Sciences of the United States of America 91: 7276-7280.Reith ME, Muuholland J. 1993. A high-resolution gene map of the chloroplast genome of the reda&p Porphyra purpurea. l7te Plant Cell 5: 465-475.RoelI MK, Morse DE. 1993. Organization, expression and nucleotide sequences of the operon encodingR-phycocyanin a and P subunits from the red alga Polysiphonia boldii. Phznt Molecular Biology 21: 47-58.Rothschild LJ. 1985. Assessment of evolutionary relationships among protistan phyla and a blue-greenprokaryote by comparison of the enzyme ribulose-1,5-bisphosphate carboxylase. Ph.D. Thesis, BrownUniversity, Providence, R.I. xi+113 pp.Ruprecht FJ. 1851. Phycologia Ochotiensis, Tange des Ochotskischen Meeres. In: von MiddendorffAT, ed. Sibirische Reise, Botanih l(2): 1(193)-243(435). St Petersburg: Kais. Akademie Wissenschaften.Saunders GW, Druehl LD. 1992. Nucleotide sequences of the small-subunit ribosomal RNA genesfrom selected Laminariales (Phaeophyta): implications for kelp evolution. Journal of Phycology 28:544-549.Saunders GW, Kraft GT. 1994. Small sub-unit rRNA gene sequences from representatives of selectedfamilies of the Gigartinales and Rhodymeniales (Rhodophyta). 1. Evidence for the Plocamiales.Canadian Journal of Botany 72: 1250-1263.Saunders GW, Bird CJ, Rice EL, Ragan MA. 1995. Phylogeny of the red algal orders Acrochaetialesand Palmariales based on sequences of nuclear small-subunit ribosomal RNA genes. Journal ofPhycology 31(4), in press.Scherer S, Lechuer S, Biiger P. 1993. psbD sequences of Bumilleriopsir jliformis (Heterokontophyta,Xanthophyceae) and Porphyridium purpureum (Rhodophyta, Bangiophycidae): evidence for polyphyleticorigins of plastids. Current Genetics 24: 437-442.Schlegel M. 1991. Protist evolution and phylogeny as discerned from small subunit ribosomal RNAsequence comparisons. European Journal of Protistology 27: 207-219.Schmitz F. 1892. Florideae. In: Engler A, ed. Syllabus der Pflaruenfamilien. Berlin: Bomtraeger, 16-23.Schmitz F, Haupttleisch P. 1897. Rhodophyceae. In: Engler A, Prantl K, eds. Die naturlichenPflanznfmilien nebst ihren Gattungen und wichtigeren Arten insbesondere den Nut@fla~ I(2): 298-544.Leipzig: Engelmann.Schweigger AF. 1819. Beobachtungen auf naturhistorischen Reisen, anatomisch-physiologische Untersuchungeniiber Coralbsn. Berlin: Reimer. viii+128 pp+12 charts+8 pl.Silva PC. 1984. Extrinsic factors in gTeen algal systematics. In: Irvine DEG, John DM, eds. Systematicsof the green algae. London: Academic Press, 419-433.Skuja H. 1939. Versuch einer systematischen Einteilung der Bangioideen oder Protoflorideen. ActaHorti Botanici Universitatis Latviensis 11112: 23-40.Sogin ML. 1989. Evolution of eukaryotic microorganisms and their small subunit ribosomal RNAs.American Zoologist 29: 487-499.Sogin ML. 1990. Amplification of ribosomal RNA genes for molecular evolution studies. In: InnisMA, Gelfund DH, Sninsky B, White TJ, eds. PCR protocols: Q guide to methodr and applications. SanDiego: Academic Press, 307-324.Sogin ML, Edman U, PIwood HJ. 1989. A single kingdom of eukaryotes. In: Femholm B, BremerY J8mvaB H, eds. The hierarchy of life. Molecules and morphology in phylogenetic analysis (Nobel Symp.70). Amsterdam: Exerpta Medica, 133-143.Steele w, Holsmger mi Jamen RR, Taylor DW. 1991. Assessing the reliability of 5S rRNAsequence data for phylogenetic analysis in green plants. Molecular Biology and Evolution 8: 240-248.Steinkiitter J, Bhattacharya D, Semmelroth I, Bibeau C, Melhonian M. 1994. Prasinophytes formindependent lineages within the Chlorophytaz evidence from ribosomal RNA sequence comparisons.Journal of Phycology 30: 340-345.Swofford DL 1993. PAUP: Phylogenetic analysis using parsimony, Version 3.1. Computer programand manual distributed by the Illinois Natural History Survey, Champaign, Illinois, vi+257 pp.Swofford DL, Olsen GJ. 1990. Phylogeny reconstruction.Jystematics. Sunderland MA: Sinauer. 4 1l-50 1.In: Hillis DM, Moritz C, eds. MolecularTayior FJR. 1978. Problems in the development of an explicit hypothetical phylogeny of the lowerorganisms. BioSvstemc 10: 67-89.Temaeton AR. i983. Phylogenetic inference from restriction endonuclease cleavage site maps withparticular reference to the evolution of humans and the apes. Evolution 37: 221-244.Thuret G. 1855. Note sur un nouveau genre da&es, de la fumille des Floridies. Cherbourg: Feuardent.Reprinted as Mbmoires de la Societe des S&rues Nature&s de Cherbourg 3: 155-160+2pLTilden JE. 1933. A classification of algae based on evolutionary development, with special referenceto pigmentation. Botanical Gwtte 95: 59-77.
  25. 25. ARE RED ALGAE PLANTS? 105Tflden JE. 193.5. The algae and their life relations. Fundamentals of phycology. Minneapolis: University ofMinnesota.Tomas RN, Cox F,R. 1973. Observations on the symbiosis of Peridinium balticurn and its intracellularalgae. Journal of Phycology 9: 304-323.Turner D. 1898. Fun’ sive plantarum fitcorum pm’ a botanicis arcripturum icones descn~tiones et historia.London: M’Creery, 4 ~01s.Van de Peer Y, Neefs J-M, de Rijk P, De Wachter R 1993. Evolution of eukaryotes as deducedfrom small ribosomal subunit RNA sequences. Biochemical and Systematic Ecology 21: 43-55.van Keulen H, Gutell RR, Campbell SR, Erlandsen SI., JarroB EL. 1992. The nucleotide sequenceof the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris.Journal of Molecular Evolution 35: 318-328.VeUey T. 1795. Coloured figures of marine plants, found on the southern coast of England; illustrated withdescripions and observations; accompanied with a jgun of the Arabis stricta from St. Vincent’s Rock Towhich ir prefixed an inquiry into the mode of jrropagation peculiar to sea plants. Bath: Hazard.Wainright PO, Hinlcle G, Sogin MI, Stickel SA. 1993. Monophyletic origins of the metazoa: anevolutionary link with fungi. Science 260: 340342.West GS, F&s& FE. 1927. A treatise on the British freshwater algae in which are included all the pigmentedprotophyta hitherto found in British fieshwaters. Cambridge: Cambridge University Press.West L, Powers D. 1993. Molecular phylogenetic position of hexactinelhd sponges in relation to theProtista and Demospongiae. Molecular Marine Biology and Biotechnology 2: 71-75.Wettstein R von. 1901. Handbuch der syslematischen Botanik, 1. Band. Deuticke, Leipzig.Whadey JM. 1981. Chloroplast evolution-ancient and modem. Annals of the New York Academy ofSciences 361: 154-165.WhatIey JM, John P, Whatley FR. 1979. From extracellular to intracellular: the establishment ofmitochondria and chloroplasts. Proceedings of the Royal Society of London B204: 165-187.Whittaker RI-I. 1969. New concepts of kingdoms of organisms. Science 163: 150-160.Wilson TB, Cassin J. 1863. On a third kingdom of organized beings. Proceedings of the Academy ofNatural Sciences of Philadephia 15: 113-121.Woelkerling WJ. 1990. An introduction. In: Cole KM, Sheath RG, eds. Biology of the red algae.Cambridge: Cambridge University Press, 1-6.Woese C. 1987. Bacterial evolution. Microbiological Reviews 51: 221-271.Wolfe GFt, Cunnin gham FX, Durnford D, Green BR, Gantt E. 1994. Evidence for a commonorigin of chloroplasts with light-harvesting complexes of different pigmentation. Nature (London) 367:566-568.Zhou Y-H, Ragan MA. 1993. cDNA cloning and characterization of the nuclear gene encoding thechloroplast glyceraldehyde-3-phosphate dehydrogenase from the marine red alga Gracilaria verrucosa.Current Genetics 23: 483-489.Zhou Y-H., Ragan MA. 1994. Cloning and characterization of the nuclear gene encoding plastidglyceraldehyde-3-phosphate dehydrogenase from the marine red alga Gracilana verrucosa. CurrentGenetics 26: 79-86.Zhou Y-H, Ragan MA 1995a. Characterization of the polyubiquitin gene in the marine red algaGracilaria verrucosa. Biochimica et Biophysics Acta: 1261: 215-222.Zhou Y-H, Ragan MA 1995b. The nuclear gene and cDNAs encoding cytosolic glyceraldehyde-3-phosphate dehydrogenase from the marine red alga Gracilaria verrucosa: cloning, characterization andphylogenetic analysis. Current Genetics: in press.Zhou Y-H, Ragan MA. 1995c. Cloning and characterization of the nuclear gene and cDNAs fortriosephosphate isomerase of the marine red alga Gracilaria verrucosa. Current Genetics: in press.Z~IOU Y-H, Ragan MA. 19951. Characterization of the nuclear gene encoding mitochondrial aconitasein the marine red alga Gracilan’a venucosa. Plant Molecular Biolom, in press.Zhou Y-H, Ragan IviA: in press. Nuclear-encoded protein-coding genes of the agarophyte Gracilariaverrmosa (Hudson) Papenfuss. In: Lindstrom SC, Chapman DJ, eds. Proceedings of the Xv InternationalSeaweed Symposium. Dordrecht: Kluwer Academic Publishers, in press.