American Journal of Botany 84(8): 966–972. 1997.
RELATIONSHIPS OF THE
FROM NUCLEOTIDE SEQUENCES OF THE RIBOSOMAL
DANIEL POTTER,2,4 GARY W. SAUNDERS,3,5 ROBERT A. ANDERSEN2,6 2
Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine 04575; and 3Botany School, University of Melbourne, Parkville, Victoria 3052, Australia
Some earlier studies suggested an evolutionary relationship between the Raphidophyceae (chloromonads) and Xantho¨ omycete phyceae (yellow-green algae), whereas other studies suggested relationships with different algal classes or the O fungi. To evaluate the relationships, we determined the complete nucleotide sequences of the 18S ribosomal RNA gene from the raphidophytes Vacuolaria virescens, Chattonella subsalsa, and Heterosigma carterae, and the xanthophytes Vaucheria bursata, Botrydium stoloniferum, Botrydiopsis intercedens, and Xanthonema debile. The results showed that the Xanthophyceae were most closely related to the Phaeophyceae. A cladistic analysis of combined data sets (nucleotide sequences, ultrastructure, and pigments) suggested the Raphidophyceae are the sister taxon to the Phaeophyceae–Xanthophyceae clade, but the bootstrap value was low (40%). The raphidophyte genera were united with high (100%) bootstrap values, supporting a hypothesis based upon ultrastructural features that marine and freshwater raphidophytes form a monophyletic group. We ¨ omycetes, and we confirmed that examined the relationship between Vaucheria, a siphoneous xanthophyte alga, and the O Vaucheria is a member of the class Xanthophyceae. Partial nucleotide sequences of the 18S rRNA gene from eight xanthophytes (including Bumillariopsis filiformis, Heterococcus caespitiosus, and Mischococcus sphaerocephalus) produce a phylogeny that is not congruent with the current morphology-based classification scheme. Key words: phyceae.
¨ omycetes; phylogeny; Raphidophyceae; ribosomal RNA (18S); Vaucheria; Xanthoalgae; biodiversity; O
The evolutionary relationships of various chromophyte algal classes were never well established (or even attempted) based upon light microscopic information (e.g., Fritsch, 1935, Smith, 1950). Ultrastructural observations provided data valuable for phylogenetic studies, but quantitative data analysis such as cladistic analysis was uncommon (e.g., Andersen, 1991; Williams, 1991). Ultrastructural data were used to revise taxonomic classes, and these studies led to the description of several new classes such as the Haptophyceae (Christensen, 1962), Eustigmatophyceae (Hibberd and Leedale, 1971), Dictyochophyceae (Silva, 1980), Synurophyceae (Andersen, 1987), and Pelagophyceae (Andersen et al., 1993). The accumulation of gene sequence data, however, has led to numerous evolutionary studies of the chromophyte algae (e.g., Gunderson et al., 1987; Pe´rasso et al., 1989; Ariztia, Andersen, and Sogin, 1991; Bhattacharya et al., 1992; Saunders and Druehl, 1992; Andersen et al., 1993; Leipe
et al., 1994; Saunders et al., 1995; Cavalier-Smith, Chao, and Allsopp, 1995; Potter et al., 1997), and there has been one attempt to combine molecular and traditional (ultrastructure, biochemical) data sets in the same analysis (Saunders et al., 1995). This paper provides the 18S rRNA gene sequence data for representatives of the Raphidophyceae and the Xanthophyceae and addresses several questions related to them. The algal class Raphidophyceae, known as the Chloromonadophyceae in older literature, has both freshwater and marine representatives (Heywood, 1990). The chloroplast pigments of the freshwater species are remarkably different from those of the marine representatives. The pigments of freshwater raphidophytes resemble those of the class Xanthophyceae, i.e., both groups contain diadinoxanthin, heteroxanthin, and vaucheriaxanthin (or derivatives) and both lack fucoxanthin and violaxanthin (Bjørnland and Liaaen-Jensen, 1989). Scagel et al. (1966) and Christensen (1980, 1994) emphasized these pigment similarities when they included the freshwater raphidophytes in the Xanthophyta and Xanthophyceae, respectively. Other authors have also followed these classifications (e.g., Chapman and Haxo, 1966; Stewart, 1974; Bold and Wynne, 1978; Heywood, 1978). On the other hand, the marine raphidophytes have carotenoids that resemble those of the Chrysophyceae, Eustigmatophyceae, Phaeophyceae, and Synurophyceae, i.e., they contain fucoxanthin and violaxanthin but lack diadinoxanthin, heteroxanthin, and vaucheriaxanthin (Bjørnland and LiaaenJensen, 1989). Ultrastructural observations have also been interpreted to suggest an evolutionary relationship
1 Manuscript received 9 August 1996; revision accepted 12 December 1996. The authors thank Drs. Richard Moe and Paul Silva at the University of California-Berkeley Herbarium for taxonomic and nomenclatural assistance. We also thank Dr. Peter Heywood and Dr. Donald Ott for critically reviewing the manuscript. This work was supported by National Science Foundation Grants EHR-9108766, BRS-94-19498 and Office of Naval Research Grant N00014-92-J-1717 to RAA. 4 Current address: Department of Pomology, University of California, Davis, CA 95616. 5 Current address: Department of Biology, University of New Brunswick, Fredericton, New Brunswick, E3B 6E1, Canada. 6 Author for correspondence: telephone: (207)-633-9600; FAX: (207)-633-9715; email: [email protected]
ET AL.—PHYLOGENY FOR THE
between the marine Raphidophyceae and the Chrysophyceae (Leadbeater, 1969; Gibbs, Chu, and Magnussen, 1980; Gibbs, 1981). However, the splitting of the Raphidophyceae was not restricted to these two groups. Hovasse (1945) proposed that some raphidophyte taxa were related to the Cryptophyceae and others to the Dinophyceae. Some additional support for this proposal may be inferred from fatty acid and sterol data from the marine raphidophytes Heterosigma and Chattonella, which resemble those from the Dinophyceae and Haptophyceae (Nichols, Volkman, and Johns, 1983; Nichols et al., 1987). In contrast to studies that advocated splitting the Raphidophyceae, other studies argued for the retention of the Raphidophyceae as a distinct class or division (e.g., Rothmaler, 1949; Prescott, 1951; Chadefaud, 1950, 1960; Christensen, 1962, 1964; Silva, 1980; Lee, 1989; van den Hoek, Mann, and Jahns, 1995). Ultrastructurally, both freshwater and marine genera have mucocysts, they share several unusual features in the flagellar apparatus, their cells contain several Golgi bodies, and they have relatively large naked cells (Mignot, 1967, 1976; Hara and Chihara, 1985, 1987; Hara, Inouye, and Chihara, 1985; Vesk and Moestrup, 1987; Heywood, 1989, 1990). Within the Xanthophyceae, the siphoneous genus Vaucheria has had a long and varied taxonomic history. Vaucheria was once classified with the siphoneous green algae (e.g., Oltmanns, 1922; Smith, 1933; Fritsch, 1935; Iyenger, 1951). Pigment analyses provided evidence for its classification in the Xanthophyceae rather than the Chlorophyceae (see Smith, 1950), and flagellar features and chloroplast ultrastructure supported the placement of Vaucheria in the Xanthophyceae (Koch, 1951; Descomps, 1963; Moestrup, 1970). For almost 150 yr, the morphological and reproductive similarity between ¨ omycete water mold, Vaucheria and Saprolegnia, an O has been suggested as a basis for a close relationship between those two genera, and more broadly, for a re¨ omycota lationship between the Xanthophyceae and the O (Pringsheim, 1858; Sachs, 1882; Ott and Brown, 1974a,b, 1975, 1978). Previous phylogenetic analysis using 18S ¨ omycetes, and rRNA gene sequences from green algae, O the xanthophyte Tribonema aequale Pascher did not sup¨ omport a close relationship between xanthophytes and O ycetes (Ariztia, Andersen, and Sogin, 1991), however, gene sequences for Vaucheria were not available at the time. In this paper we examine the evolutionary relationships of the Raphidophyceae and Xanthophyceae, including Vaucheria, with molecular and combined data sets; we also address the relationship between the freshwater and marine raphidophytes. We use the molecular data as an independent means for evaluating Xanthophyceae classification schemes based upon gross morphology (e.g., Ettl, 1978; Hibberd, 1990; Christensen, 1994). These classifications generally follow the Pascher (1937– 1939) scheme with ordinal ranks for flagellate, amoeboid, palmelloid, coccoid, filamentous, and siphoneous life forms. Finally, we comment briefly on the phylogenetic relationships of chromophytes based upon the sequence data presented herein.
MATERIALS AND METHODS Cultures were obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP), Boothbay Harbor, Maine, USA [Chattonella subsalsa Biecheler strain CCMP217, Heterosigma carterae (Holbert) Taylor strain CCMP452, Vaucheria bursata (O.F. Mu¨ller) C. Ag. strain CCMP1084], from the Culture Collection of Algae at the University of Texas, Austin, Texas, USA [Botrydium stoloniferum Mitra strain UTEX156, Botrydiopsis intercedens Vischer et Pascher strain UTEX 296, Bumilleriopsis filiformis Vischer strain UTEX309, Heterococcus caespitosus Vischer strain UTEX 385, Mischococcus sphaerocephalus Vischer strain UTEX150, Xanthonema debile (Vischer) Silva strain UTEX155], and from the Sammlung von Algenkulturen der Universita¨t Go¨ttingen, Germany [Vacuolaria virescens Cienk. strain SAG1195-1]. Cells were grown in DYIV medium (Andersen, Jacobson, and Sexton, 1991), soil-water medium (Pringsheim, 1946), or K Medium (Keller and Guillard, 1985) using a 12:12 L:D cycle. The 18S rRNA gene was amplified from either genomic DNA extracts (Doyle and Doyle, 1987) or cell lysates via the polymerase chain reaction (PCR) using the Perkin Elmer Gene Amp II kit and two external primers (Andersen et al., 1993). PCR products were isolated using a 0.8% agarose gel and the DNA was extracted using a Gene Clean II kit (Bio 101). Complete nucleotide sequences of primary and secondary PCR products were determined, in both directions, using the Perkin Elmer AmpliTaq Cycle Sequencing Kit with 13 59-biotinylated sequencing primers. Sequencing reaction products were separated on 6% Long Ranger (AT Biochem) acrylamide gels and transferred to nylon membranes (Millipore). Sequences were visualized by treating the membranes with the New England Biolabs Phototope Detection Kit and exposing them to x-ray film. The sequences were combined with those of representatives from ¨ omycetes, and one haptophyte. The Alaria marginheterokont algae, O ata Postels et Ruprecht sequence was taken from Saunders and Druehl (1992) and the Mallomonas striata Harris et Bradley sequence was taken from Bhattacharya et al. (1992). The following sequences (with GenBank accession numbers) were obtained from GenBank: Achlya bisexualis (M32705), Apedinella radians (Lohmann) Campbell (U14384), Bacillaria paxillifer (O.F. Mu¨ller) Hendy (M87325), Chromulina chionophila Stein (M87332), Cylindrotheca closterium (Ehr.) Reimann et Lewin (M87326), Dictyocha speculum Ehr. (U14385), Emiliania huxleyi (Lohm.) Hay et Mohler (Lo4957), Fucus distichus L.(M97959), Hibberdia magna (Belcher) Andersen (M87331), Lagenidium giganteum (M54939), Nannochloropsis salina Hibberd (M87328), Ochromonas danica Prings.(M32704), Pelagococcus subviridis Norris (U14386), Pelagomonas calceolata Andersen et Saunders (U14389), Phytophthora megasperma Drechler (X54265), Rhizochromulina sp. (U14388), Stephanopyxis cf. broschii (M87330), Synura spinosa Korsh. (M87336), and Tribonema aequale Pascher (M55286). Sequences were aligned using MALIGN (Wheeler and Gladstein, 1993) and ambiguously aligned sites were excluded from phylogenetic analyses. The complete alignments are available on request. Parsimony analysis was conducted using PAUP (Swofford, 1993). Phylogenetic bootstrapping was implemented in PAUP to assess relative support for branches in the most parsimonious trees (Felsenstein, 1985; 100 replicates for each data set). Ultrastructural and biochemical features (traditional data) were coded as cladistic characters (see Saunders et al., 1995). Missing values were assigned when information for a character was not applicable (e.g., chloroplast pigmentation for water molds), and we assumed that some taxa have the same character states as other members of their taxonomic class when data was not available (Tables 1, 2). The traditional and molecular data sets were combined and directly analyzed with PAUP version 3.1.1 (Swofford, 1993). Bootstrapping was implemented in PAUP as described above.
RESULTS The complete 18S rRNA gene sequences determined in this study were deposited in GenBank with the follow-
TABLE 1. Characters and character states of the traditional data set (see Saunders et al., 1995). Character
1. R1 and R3 flagellar roots 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
0 1 R2 and R4 flagellar roots 0 1 System II fiber 0 1 Flagellar hairs 0 1 Flagellar hair structure 0 1 Paraxonemal rod 0 1 Transitional helix 0 1 2 Flagellum number 0 1 Basal body number 0 1 199-butanoyloxyfucoxanthin 0 1 Diatoxanthin 0 1 Basal body on nucleus 0 1 Sinking mitotic spindle 0 1 Golgi body located on poste- 0 rior nuclear surface 1
5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
present absent present absent present absent present absent smooth shafts with lateral filaments present absent absent present, above major plate present, below major plate one two one two present absent present absent present absent present absent present absent
ing accession numbers: Botrydiopsis intercedens, U41647; Botrydium stoloniferum, U41648; Xanthonema debile, U43277, Vaucheria bursata, U41646; Vacuolaria virescens, U41651; Chattonella subsalsa, U41649; and Heterosigma carterae, U41650. In addition, partial sequences were determined and deposited in GenBank for Bumilleriopsis filiformis (U81591), Heterococcus caespitosus (U81592), and Mischococcus sphaerocephalus (U81593). Phylogenetic analyses of the complete sequences combined with the published sequences for Tribonema aequale and representatives of other heterokont lineages, with the haptophyte Emiliania huxleyi included as the outgroup, produced six equally parsimonious trees, and the strict consensus tree was constructed (Fig. 1). The analysis showed the Xanthophyceae were more closely related to the Phaeophyceae (bootstrap 5 61%) than they were to the Raphidophyceae. The freshwater raphidophyte Vacuolaria was always a sister taxon to the two marine raphidophytes, and the Raphidophyceae were only distantly related to any xanthophyte. Furthermore, Chattonella and Heterosigma showed no close relationship to the chrysophytes. Vaucheria occupied a basal position with respect to the other xanthophytes, and its branch length was long relative to the other taxa in the analysis (Fig. 2). The analysis also indicated that for the taxa studied, the Xanthophyceae and Raphidophyceae are both monophyletic assemblages with substantial bootstrap support (87 and 100%, respectively) (Fig. 1). The position of the Raphidophyceae varied in the six equally parsimonious trees: in three trees they were sister to the Xanthophyceae/Phaeophyceae clade and in the oth-
TABLE 2. The ultrastructural and biochemical database used in the cladistic analyses (see Table 1, Saunders et al. 1995). The Emiliania huxleyi is designated as the outgroup. Missing values are represented by ‘‘?’’. Emiliania huxleyi Achlyla bisexualis Lagenidium giganteum Phytophthora megasperma Stephanopyxis cf. broschii Bacillaria paxillifer Pelagomonas calceolata Pelagococcus subviridis Apendinella radians Tribonema aequale Hibberdia magna Synura spinosa Mallomonas striata Alaria marginata Fucus distichus Dictyocha speculum Rhizochromulina sp. Ochromonas danica Nannochloropsis salina Botrydiopsis intercedens Vaucheria bursata Botrydium stoloniferum Xanthonema debile Vacuolaria virescens Chattonella subsalsa Heterosigma carterae
0011?1?1100111 00?001111??110 00?001111??110 00?001111??110 1110000001000? 1110000001000? 11100020000000 11100020000000 111000201?00?0 00000111110111 00001111111111 01001111111111 01001111111111 00100101111111 00100101111111 111000201000?0 11100?201?00?0 00001111111111 000001111111?? 00000111110111 00100101110111 00000111110111 00000111110111 00000101111111 000001011111?1 000001011111?1
Fig. 1. Strict consensus tree for six most parsimonious trees based ¨ omycetes and hetupon 18S rRNA gene sequences of representative O erokont algae, rooted with Emiliana huxleyi (ambiguous sites removed) (length 5 846; consistency index 5 0.64; retention index 5 0.68). Bootstrap values are based upon 100 replicates.
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Fig. 2. One of six most parsimonious trees used to construct Fig. 1, expressed as the number of changes from the node (numbers above the branches). Note the long branch length for Vaucheria.
er three trees they were sister to the Eustigmatophyceae/ Chrysophyceae/Synurophyceae clade (see Fig. 2). The six trees also differed in the position of the clade that includes the Pelagophyceae, Dictyocha, Rhizochromulina, and Apedinella. In two trees, this clade was sister to all heterokont algae except the diatoms; in two others, it was sister to the Raphidophyceae/Eustigmatophyceae/ Synurophyceae/Chrysophyceae; and in the remaining two it was sister to the Xanthophyceae/Phaeophyceae. In three of the trees, the two representatives of Chrysophyceae formed a monophyletic group sister to the Synurophyceae; in the other three, they were paraphyletic with respect to the latter, with Ochromonas in the basal position. All trees agreed in the following respects: (1) The ¨ omycetes appeared as sister to a clade that included all O heterokont algae. (2) The Bacillariophyceae were sister to the remaining heterokont algae. (3) The Pelagophyceae were sister to a clade that included Dictyocha, Rhizochromulina, and Apedinella. (4) Nannochloropsis salina (Eustigmatophyceae) had a sister relationship to a clade that included members of the Chrysophyceae and Synurophyceae. (5) The Xanthophyceae were sister to the Phaeophyceae. In an effort to obtain more phylogenetic resolution and better support for branching patterns, we combined the
Fig. 3. One of four most parsimonious trees based upon both the 18S rRNA gene sequences (ambiguous sites removed) and the traditional characters (Table 2) (length 5 871, consistency index 5 0.63, retention index 5 0.70). See text for description of the other three most parsimonious trees. Bootstrap values are based upon 100 replicates.
gene sequence data with the traditional data set (Tables 1, 2). The analysis produced four most parsimonious trees, and one of the four trees, with bootstrap values, is shown (Fig. 3). In two trees the reduced flagellar clade (diatoms, Pelagophyceae, Dictyochophyceae) formed the deepest branch of the heterokont chromophyte algae whereas in the other two trees the Chrysophyceae/Synurophyceae/Eustigmatophyceae clade formed the deepest branch. Also, the branching order differed for four Xanthophyceae taxa (Tribonema, Botrydium, Xanthonema, and Botrydiopsis). The Raphidophyceae clade was a sister taxon to the Phaeophyceae/Xanthophyceae clade in all four trees, but the bootstrap support was low (40%). In this combined analysis, the diatom genera joined (bootstrap 5 80%) the other reduced flagellar apparatus taxa (Pelagomonas, Pelagococcus, Apedinella, Rhizochromulina, and Dictyocha) (Fig. 3). The analysis that produced Fig. 1 did not resolve the relationships within the Xanthophyceae, and this was probably due to the removal of ambiguously aligned nucleotides prior to analysis. Therefore, we conducted an analysis using the complete sequences of the Xanthophyceae taxa (using the brown alga Fucus distichus as an outgroup). This analysis gave a single most parsimonious
Fig. 4. The single most parsimonious tree of the Xanthophyceae taxa (Fucus 5 outgroup) based upon complete sequences with no sites removed (length 5 431, consistency index 5 0.88, retention index 5 0.61).
tree (Fig. 4). Vaucheria was on the most deeply divergent branch of the xanthophytes. A second siphoneous xanthophyte, Botridium stoloniferum, was more closely related to Tribonema than it was to Vaucheria. To obtain preliminary data regarding the congruence between 18S rRNA data and existing morphology-based classification schemes for the Xanthophyceae, we determined partial 18S rRNA nucleotide sequences for Bumilleriopsis filiformis, Heterococcus caespitosus, and Mischococcus sphaerocephalus and combined these with the same sequence segment from Botrydiopsis intercedens, Botrydium stoloniferum, Tribonema aequale, Vaucheria bursata, and Xanthonema debile. The partial sequences were ; 330 nucleotides in length, corresponding at their 59 end to nucleotide position 601 in Pelagomonas calceolata (Andersen et al., 1993). A single most parsimonious tree was found (Fig. 5). The ordinal classification of three authors was added (Ettl, 1978; Hibberd, 1990; Christensen, 1994), showing conflicts between the molecular data and the classification scheme. DISCUSSION The Raphidophyceae appear to be a monophyletic group that includes both the freshwater and marine representatives, and this is supported with high (100%) bootstrap values. Therefore, we recognize this group as a distinct algal class (e.g., Silva, 1980; Heywood, 1990; van den Hoek, Mann, and Jahns, 1995), and we do not follow the suggestions of others to split the class or to fuse it with other classes (e.g., Hovasse, 1945; Scagel et al., 1966; Leadbeater, 1969; Gibbs, Chu, and Magnussen, 1980; Christensen, 1980, 1994). Despite the similarity in carotenoid pigments between the freshwater raphidophytes and the xanthophytes, the 18S rRNA data presented here do not support a close relationship between the Xanthophyceae and the Raphidophyceae. Assuming no lateral transfer of the 18S rRNA gene during the evolutionary history of these groups, gene sequence comparisons con-
tinue to suggest that, as first reported by Ariztia, Andersen, and Sogin (1991), the Xanthophyceae have a close evolutionary relationship with the Phaeophyceae (see also Bhattacharya et al., 1992; Leipe et al., 1994; Saunders et al., 1995; Potter et al., 1997). Because chromophyte chloroplasts are postulated to be the result of eukaryotic endosymbiotic events (Gibbs, 1981), it is possible that the similarity in carotenoids for Xanthophytes and Raphidophytes results from an unknown endosymbiotic event (Vesk and Moestrup, 1987). Although we cannot test the endosymbiotic hypothesis with our 18S rRNA data, a comparison of nucleotide sequences for chloroplast-encoded genes may be enlightening. Alternatively, the modern Raphidophyceae may be derived from an ancestral stock that gave rise to two differently pigmented groups, but in this case the 18S rRNA gene sequences lack sufficient phylogenetic information to demonstrate the relationships. The lack of a well-resolved and well-supported phylogenetic relationship of the Raphidophyceae and other chromophyte algal classes was disappointing. There was less than 50% bootstrap support for branches having the raphidophytes and any other algal class (Figs. 1, 3). Using 28 S rRNA gene sequence data, Perasso et al. (1989) showed the Raphidophyceae to be a sister taxon to the Chrysophyceae and Synurophyceae, but the study included only heterokont chomophytes from these three classes. Previous studies using nonmolecular data showed marine and freshwater raphidophytes in distinct clades (Williams, 1991) or as a sister taxon to a clade of chrysophytes, synurophytes, eustigmatophytes, and xanthophytes (Andersen, 1991). Heywood (1990) reviewed the literature for the Raphidophyceae and concluded that they showed no clear evolutionary relationships with other chromophyte algae. Although a thorough examination of the Xanthophyceae was beyond the scope of this study, our preliminary results do not corroborate the current classifications for the class (Fig. 5). Ordinal classification is based upon the dominant life form (e.g., coccoid, filamentous, and flagellate) and familial classification is based upon habit (e.g., free-living or attached, solitary or colonial, branching types) (Hibberd, 1990). The morphological data used in the current classifications show little congruence with the 18S rRNA gene comparisons. These preliminary results suggest additional investigation is necessary. Vaucheria diverged most deeply of all the Xanthophyceae we examined, and was only distantly related to the other siphoneous xanthophyte, Botrydium. Botrydium has been placed with Vaucheria in the order Vaucheriales
Fig. 5. The single most parsimonious tree based upon partial 18S rRNA gene sequences for xanthophytes and rooted with the brown alga Fucus distichus (length 5 173, consistency index 5 0.79, retention index 5 0.53). Ordinal and familial classification are added on the right columns. xxx 5 Botrydiales (Ettl, 1978), 5 Vaucheriales (Hibberd, 1990), 5 Mischococcales (Christensen, 1994).
ET AL.—PHYLOGENY FOR THE
(Hibberd, 1990), with members of Mischococcales (Christensen, 1994) or in a separate order, the Botrydiales (Ettl, 1978). Our data do not support the classification of Botrydium in the Vaucheriales and may suggest that the siphoneous habit has arisen twice independently within the Xanthophyceae. The 18S rRNA data do not place Vaucheria with the ¨ omycetes. This result supports a cell wall analysis that O found little evidence for a close relationship between Vaucheria and water molds (Parker, Preston, and Fogg, 1963). Thus, the similarities between Vaucheria and the ¨ omycetes appear to be the result of parallel evolution. O Examination of the 18S rRNA gene from Saprolegnia would be helpful, and perhaps the question can be re¨ omycetes? stated: Is Saprolegnia a member of the O ¨ omycetes to the heterThe sister relationship of the O okont algae found in this analysis is consistent with the results of earlier studies (Gunderson et al., 1987; Ariztia, Andersen, and Sogin, 1991; Bhattacharya et al., 1992; Leipe et al., 1994; Saunders et al., 1995). A grouping of Pelagophyceae with the diatoms and a clade with Rhizochromulina, Dictyocha, and pedinellids was first supported by phylogenetic analyses of combined molecular and traditional data (Saunders et al., 1995), and the same relationship was resolved in this study when the combined analysis was conducted (Fig. 3). In most previous studies of 18S rRNA genes, just as in this study (Fig. 1), the diatoms appear as sister to the remaining heterokont algae, but that relationship is always weakly supported (Bhattacharya et al., 1992; Leipe et al., 1994; Potter et al., 1997). Little has been published on a hierarchical classification of the chromophyte algae, and molecular data to date have shed little light on the subject. The lack of phylogenetically significant information in the 18S rRNA gene sequence data for deep branches may suggest that the heterokont chromophyte classes radiated rapidly (Leipe et al., 1994). Whether or not there was a rapid radiation remains to be proved, but it is becoming more apparent that the deep branching order is difficult to determine using only 18S rRNA gene sequence comparisons. Additional data, e.g., other genes, may be necessary before a consensus higher classification can be achieved. LITERATURE CITED ANDERSEN, R. A. 1987. Synurophyceae classis nov., a new class of algae. American Journal of Botany 74: 337–353. . 1991. The cytoskeleton of chromophyte alga. Protoplasma 164: 143–159. , D. M. JACOBSON, AND J. P. SEXTON. 1991. Provasoli-Guillard Center for Culture of Marine Phytoplankton Catalog of Strains. CCMP, W. Boothbay Harbor, ME. , G. W. SAUNDERS, M. P. PASKIND, AND J. P. SEXTON. 1993. The ultrastructure and 18s rRNA gene sequence for Pelagomonas calceolata gen. et sp. nov., and the description of a new algal class, the Pelagophyceae classis nov. Journal of Phycology 29: 701–15. ARIZTIA, E. V., R. A. ANDERSEN, AND M. L. SOGIN. 1991. A new phylogeny for chromophyte algae using 16S-like rRNA sequences from Mallomonas papillosa (Synurophyceae) and Tribonema aequale (Xanthophyceae). Journal of Phycology 27: 428–436. BHATTACHARYA, D., L. MEDLIN, P. O. WAINWRIGHT, E. V. ARIZTIA, C. BIBEAU, S. K. STICKEL, AND M. L. SOGIN. 1992. Algae containing chlorophylls a and c are paraphyletic: molecular evolutionary analysis of the Chromophyta. Evolution 46: 1801–17. BJØRNLAND, T., AND S. LIAAEN-JENSEN. 1989. Distribution patterns of
carotenoids in relation to chromophyte phylogeny and systematics. In J. C. Green, B. S. C. Leadbeater, and W. L. Diver [eds.], The chromophyte algae: problems and prospectives, 37–60. Systematics Association Special Volume Number 38, Clarendon Press, Oxford. BOLD, H. C., AND M. J. WYNNE. 1978. Introduction to the algae: structure and reproduction. Prentice-Hall, Englewood Cliffs, NJ. CAVALIER-SMITH, T., E. E. CHAO, AND M. T. E. P. ALLSOPP. 1995. Ribosomal RNA evidence for chloroplast loss within Heterokonta: Pedinellid relationships and a revised classification of Ochristan algae. Archiv fu¨r Protistenkunda 145: 209–220. CHAPMAN, D. D., AND F. T. HAXO. 1966. Chloroplast pigments of Chloromonadophyceae. Journal of Phycology 2: 89–91. CHADEFAUD, M. 1950. Les cellules nageuses des algues dans l’embranchment des Chromophyce´es. Compte rendu hebdomadaire des se´ances de l’acade´mie des sciences, Paris 231: 788–790. . 1960. Les ve´ge´taux non vasculaires (Cryptogamie). In M. Chadefaud and L. Emberger [eds.], Traite´ de botanique Systematique. Tome I. Masson, Paris. CHRISTENSEN, T. 1962. Alger. In T. W. Bo¨cher, M. Lange and T. Sørensen [eds.], Botanik, vol. II/2, 1–178. Munksgaard, Copenhagen. . 1964. The gross classification of algae. In D. F. Jackson [ed.], Algae and man, 59–64. Plenum Press, New York, NY. . 1980. Algae, a taxonomic survey. Fasc. 1. AiO Tryk, Odense. . 1994. Algae: a taxonomic survey, 195–205. AiO Print Ltd., Odense. DESCOMPS, S. 1963. Observations sur l’infrastructure de l’enveloppe des chloroplastes de Vaucheria (Xanthophycees). Compte rendu hebdomadaire des se´ances de l’acade´mie des sciences, Paris 257: 727–729. DOYLE, J. J., AND J. L. DOYLE. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin 19: 11–15. ETTL, H. 1978. Xanthophyceae. In H. Ettl, H. J. Gerloff, and H. Heynig [eds.], Su¨sswasserflora von Mitteleuropa, Bd. 3, 1. Teil, Gustav Fischer, Stuttgart. FELSENSTEIN, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–91. FRITSCH, F. E. 1935. The structure and reproduction of the algae, vol. 1. Cambridge University Press, Cambridge. GIBBS, S. P. 1981. The chloroplasts of some algal groups may have evolved from endosymbiotic eukaryotic algae. Annals of the New York Academy of Science 361: 193–208. , L. L. CHU, AND C. MAGNUSSEN. 1980. Evidence that Olisthodiscus luteus is a member of the Chrysophyceae. Phycologia 19: 173–177. GUNDERSON, J. H., H. ELWOOD, A. INGOLD, K. KINDLE, AND M. L. SOGIN. 1987. Phylogenetic relationships between chlorophytes, chryso¨ omycetes. Proceedings of the National Academy of phytes and O Sciences, USA 84: 5823–5827. HARA, Y., AND M. CHIHARA. 1985. Ultrastructure and taxonomy of Fibrocapsa japonica (Class Raphidophyceae). Archiv fu¨r Protistenkunda 130: 133–141. , AND . 1987. Morphology, ultrastructure and taxonomy of the raphidophycean alga Heterosigma akashiwo. Botanical Magazine of Tokyo 100: 151–163. , I. INOUYE, AND M. CHIHARA. 1985. Morphology and ultrastructure of Olisthodiscus luteus (Raphidophyceae) with special reference to the taxonomy. Botanical Magazine of Tokyo 98: 251–262. HEYWOOD, P. 1978. Ultrastructure of mitosis in the chloromonadophycean alga Vacuolaria virescens. Journal of Cell Science 31: 37– 51. . 1989. Some affinities of the Raphidophyceae with other chromophyte algae. In J. C. Green, B. S. C. Leadbeater, and W. L. Diver [eds.], The chromophyte algae. Problems and perspectives, 279–293. Clarendon Press, Oxford. . 1990. Phylum Rapidophyta. In L. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman [eds.], Handbook of Protoctista, 318–325. Jones and Bartlett, Boston, MA. HIBBERD, D. J. 1990. Phylum Xanthophyta. In L. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman [eds.], Handbook of Protoctista, 686–697. Jones and Bartlett, Boston, MA. , AND G. F. LEEDALE. 1971. A new algal class—the Eustigmatophyceae. Taxon 20: 523–525.
HOVASSE, R. 1945. Contribution a l’e´tude des Chloromonadines: Gonyostomum semen Diesing. Archives de Zoologie Experimentale et Generale 84: 239–269. IYENGAR, M. O. P. 1951. Chlorophyta. In G. M. Smith [ed.], Manual of phycology, an introduction to the algae and their biology, 21– 67. Chronica Botanica, Waltham, MA. KELLER, M. D., AND R. R. L. GUILLARD. 1985. Factors significant to marine dinoflagellate culture. In D. M. Anderson, A. W. White, and D. G. Baden [eds.], Toxic dinoflagellates, 113–116. Elsevier. New York, NY. KOCH, W. J. 1951. A study of the motile cells of Vaucheria. Journal of the Elisha Mitchell Scientific Society 67: 123–131. LEADBEATER, B. S. C. 1969. A fine structural study of Olisthodiscus luteus. Carter. British Phycological Journal 4: 3–17. LEE, R. E. 1989. Phycology, 2d. ed. Cambridge University Press, Cambridge. LEIPE, D. D., P. O. WAINRIGHT, J. H. GUNDERSON, D. PORTER, D. J. PATTERSON, F. VALOIS, S. HIMMERICH, AND M. L. SOGIN. 1994. The stramenopiles from a molecular perspective: 16S-like rRNA sequences from Labyrintuloides minuta and Cafeteria roesbergensis. Phycologia 33: 369–377. MIGNOT, J.-P. 1967. Structure et ultrastructure de quelques Chloromonadines. Protistologica 3: 5–23. . 1976. Comple´ments a l’e´tude des Chloromonadines ultrastructure de Chattonella subsalsa Biecheler flagelle´ d’eau saumaˆtre. Protistologica 12: 279–293. MOESTRUP, Ø. 1970. On the fine structure of the spermatozoids of Vaucheria sescuplicaria and on later stages in spermatogenesis. Journal of the Marine Biological Association of the United Kingdom 50: 513–523. NICHOLS, P. D., J. K. VOLKMAN, G. M. HALLEGRAEFF, AND S. I. BLACKBURN. 1987. Sterols and fatty acids of the red tide flagellates Heterosigma akashiwo and Chattonella antiqua (Raphidophyceae). Phytochemistry 26: 2537–2541. , , AND R. B. JOHNS. 1983. Sterols and fatty acids of the marine unicellular alga, FCRG 51. Phytochemistry 22: 1447–1452. OLTMANNS, F. 1922. Morphologie und Biologie der Algaen. Band I. Chrysophyceae–Chlorophyceae. Gustave Fischer, Jena. OTT, D. W., AND R. M. BROWN, JR. 1974a. Developmental cytology of the genus Vaucheria. I. Organization of the vegetative filament. British Phycological Journal 9: 111–126. , AND . 1974b. Developmental cytology of the genus Vaucheria. II. Sporogenesis in V. fontinalis (L.) Christensen. British Phycological Journal 9: 333–351. , AND . 1975. Developmental cytology of the genus Vaucheria. III. Emergence, settlement and generation of the mature zoospore of V. fontinalis (L.) Christensen. British Phycological Journal 10: 49–56. , AND . 1978. Developmental cytology of the genus Vaucheria. IV. Spermatogenesis. British Phycological Journal 13: 69–85. PARKER, B. C., R. D. PRESTON, AND G. E. FOGG. 1963. Studies on the structure and chemical composition of the cell walls of Vaucheri-
aceae and Saprolegniaceae. Proceedings of the Royal Society, London B 158: 435–445. PASCHER, A. 1937–1939. Heterokonten. In L. Rabenhorst [ed.], Kryp¨ sterreich und der Schweiz. Lietogamen-Flora von Deutschland, O ferung 2, Band XI. Akademische Verlagsgellschaft, Leipzig. PE´RASSO, R., A. BAROIN, L. H. QU, J. P. BACHELLERIE, AND A. ADOUTTE. 1989. Origin of the algae. Nature 339:142–144. POTTER, D., T. C. LAJEUNESSE, G. W. SAUNDERS, AND R. A. ANDERSEN. 1997. Convergent evolution masks extensive biodiversity among marine coccoid picoplankton. Biodiversity and Conservation 6: 99– 107. PRESCOTT, G. W. 1951. Algae of the western Great Lakes area. Wm. C. Brown, Dubuque, IA. PRINGSHEIM, E. G. 1946. Pure cultures of algae, their preparation and maintenance. Cambridge University Press, Cambridge. PRINGSHEIM, N. 1858. Beitra¨ge zur Morphologie und Systematik der Algen. II. Die Saprolegnieen. Jahrbuch fu¨r wissenschaftliche Botanik, Berlin 1: 284–306. ROTHMALER, W. 1949. Die Natu¨rliche ordnung der Lebewesen. Urania (Jena) 12: 466–474. SACHS, J. 1882. Text-book of botany morphological and physiological. Clarendon Press, Oxford. SAUNDERS, G. W., AND L. D. DRUEHL. 1992. Nucleotide sequences of the small-subunit ribosomal RNA genes from selected Laminariales (Phaeophyta): implications for kelp evolution. Journal of Phycology 28: 544–9. , D. POTTER, M. P. PASKIND, AND R. A. ANDERSEN. 1995. Cladistic analyses of combined traditional and molecular data sets reveal an algal lineage. Proceedings of the National Academy of Sciences, USA 92: 244–248. SCAGEL, R. F., R. J. BANDONI, G. E. ROUSE, W. B. SCHOFIELD, J. R. STEIN, AND T. M. C. TAYLOR. 1966. An evolutionary survey of the plant kingdom. Wadsworth, Belmont, CA. SILVA, P. C. 1980. Names of classes and families of living algae. Regnum Vegetabile 103: 1–156. SMITH, G. M. 1933. The fresh-water algae of the United States, 1st. ed. McGraw-Hill, New York, NY. . 1950. The fresh-water algae of the United States, 2d. ed. McGraw-Hill, New York, NY. STEWART, W. D. P. 1974. A note on taxonomy. In W. D. P. Stewart [ed.], Algal physiology and biochemistry, 909–917. University of California Press, Berkeley, CA. SWOFFORD, D. L. 1993. PAUP, Phylogenetic Analysis Using Parsimony, version 3.1, program and documentation. Illinois Natural History Survey, University of Illinois, Champaign, IL. VAN DEN HOEK, C., D. G. MANN, AND H. M. JAHNS. 1995. Algae, an introduction to phycology. Cambridge University Press, Cambridge. VESK, M., AND Ø. MOESTRUP. 1987. The flagellar root system in Heterosigma akashiwo (Raphidophyceae). Protoplasma 137: 15–28. WHEELER, W., AND D. GLADSTEIN. 1993. MALIGN, program and documentation, American Museum of Natural History, New York, NY. WILLIAMS, D. M. 1991. Phylogenetic relationships among the chromista: a review and preliminary analysis. Cladistics 7: 141–156.