Algae containing chlorophylls a + c are paraphyletic: molecular evolutionaryanalysis of the Chromophyta.
Eukaryotic algae are nonvascular plants that have been traditionally classified according to plastic characteristics such as accessory pigments and thylakoid structure (Dodge, 1974; Taylor, 1976). The major taxonomic groups include red algae (Rhodophyta), green algae (Chlorophyta), and brown plus golden-brown algae (Chromophyta). Ultrastructure and molecular sequence similiarities demonstrate that chlorophylls a + b containing green algae (except for photosynthetic euglenoids) share a unique evolutionary history with multicellular green plants. In contrast the Rhodophyta and Chromophyta are members of the Protista which is represented by paraphyletic lines of descent that cannot be definitively placed in a phylogenetic context using traditional methods of comparative morphology, physiology, or biochemistry. This is especially true for chromophytes since they can no longer be considered as a monophyletic group on the basis of plastid features (Gunderson et al., 1987; Bhattacharya and Druehl, 1988; Williams, 1991; Andersen, 1991). For example, dinoflagellates have plastids containing chlorophylls a + c and were traditionally placed within the Chromophyta (Christensen, 1980, 1989; South and Whittick, 1987; Dodge, 1989) but molecular studies show their relationship with apicomplexans and ciliated protozoa (Gajadhar et al., 1991).
The Chromophyta are traditionally defined by 10 major classes which can be differentiated on the basis of ultrastructure and plastid characteristics (Green et al., 1989). The classes include the Bacillariophyceae, Chrysophyceae, Dictyochophyceae, Eustigmatophyceae, Pedinellophyceae, Phaeophyceae, Prymnesiophyceae, Raphidophyceae, Synurophyceae and Xanthophyceae. Characters shared by this diverse group of organisms are the presence of tubular mitochondrial cristae (Taylor, 1976), tripartite mastigonemes (flagellar hairs) on the immature flagellum (prymnesiophytes excepted, Bouck, 1972), use of [Beta] -1,3 (or -1,6)-linked glucans as a storage product (Hellebust, 1988) and, in photosynthetic members, chlorophyll a, one or more of the chlorophyll c molecules, and carotenoids (Bjornland and Liaaen-Jensen, 1989; Jeffrey, 1989). With the exception of plastids and their accessory pigments, these ultrastructural features are also characteristic of the oomycete fungi (Barr, 1983; Beakes, 1989). The presence of tripartite mastigonemes and tubular, mitochondrial cristae may be sufficient to describe the chromophyte/oomycete lineage (Patterson, 1989) which may include other protist groups such as opalinids, bicosoecids, labyrinthulids, and thraustochytrids.
Because of the small number of informative characters that can be used to describe membership at the highest taxonomic groupings, the true diversity of chromophyte/oomycete classes has been difficult to describe and phylogenetic relationships within this assemblage have remained unresolved. Molecular data can provide an independent method to infer phylogenetic relationships between these classes. Recent analyses of small subunit ribosomal RNA (16S-like rRNA) and actin coding regions confirm the inclusion of oomycetes in the chromophyte lineage (Gunderson et al., 1987; Bhattacharya and Druehl, 1988; Bhattacharya et al., 1991) which Patterson (1989) collectively describes as stramenopiles.
In this paper, we present 16S-like rRNA sequences of 12 taxa from six classes of chromophyte algae. The Bacillariophyceae, the Phaeophyceae/Xanthophyceae and the Chrysophyceae/Eustigmatophyceae/Synurophyceae define three chromophyte lineages that share a common evolutionary history with the oomycetes. However, the prymnesiophyte, E. huxleyi, represents a line of descent that is unrelated to other chromophyte algae. Phylogenetic inferences, from 16S-like rRNA sequences were compared to evolutionary relationships based upon comparisons of ultrastructural characters.
MATERIALS AND METHODS
Table 1 identifies the source of algal cultures included in this analysis. Complete 16S-like rRNA sequences were determined for: Emiliania huxleyi (Prymnesiophyceae), Bacillaria paxillifer, Cylindrotheca closterium, Nitzschia apiculata, Rhizosolenia setigera, Stephanopyxis cf. broschii (Bacillariophyceae), Nannochloropsis salina (Eustigmatophyceae), Mallomonas striata, Synura spinosa (Synurophyceae), Chromulina chromophila, Hibberdia magna (Chrysophyceae), and Fucus distichus (Phaeophyceae). Cell lysates were prepared from exponentially growing cultures. Cells were mechanically disrupted with a mortar and pestle in the presence of liquid nitrogen, in a glass tissue homogenizer, or by passage through a French pressure cell (Fain et al., 1988; Medlin et al., 1988; Bhattacharya et al., 1991). Total nucleic acids were extracted with phenol and DNA was purified by CsCI equilibrium gradient centrifugation (Maniatis et al., 1982) or by using an anion exchange resin (Tube 20, Qiagen).
Small subunit rRNA coding regions were amplified with the polymerase chain reaction (PCR) protocol (Mullis and Faloona, 1987; Saiki et al., 1988) as modified by Medlin et al. (1988). Oligonucleotide primers complementary to the 5' and 3' termini of 16S-like rRNA coding regions were used to initiate DNA synthesis in the polymerase chain reactions. These primers contain multiple restriction endonuclease sites and do not anneal to organellar or prokaryotic 16S-like rRNA genes. The polylinker sites permitted directional cloning into single-stranded M13 bacteriophages (Medlin et al., 1988) for the dideoxynucleotide-mediated chain-termination sequencing protocol (Sanger et al., 1977). The population of PCR products was sampled by preparing single-stranded sequencing templates from a pool of no fewer than 4, and as many as 15 independent recombinant M13 clones. Sequence heterogeneity, representing microheterogeneity in the different 16S-like rRNA gene copies from a given organism or from potential PCR artifacts, appears as multiple bands at corresponding positions in sequencing gel lanes (Illingsworth et al., 1991); however, such heterogeneities were not observed in any of the 16S-like rRNA coding regions included in this analysis. Both strands (coding and noncoding) of 16S-like rRNA genes were sequenced for the studied taxa with primers complementary to evolutionarily conserved regions of eukaryotic 16S-like rRNA (Elwood et al., 1985). The 5' (21 nucleotides) and 3' (24 nucleotides) terminal sequences were not determined since they corresponded to the amplification primers.
The chromophyte 16S-like rRNA sequences were aligned with those of 175 eu.
karyotes including members of the major eukaryotic groups (i.e., animals, green plants, ciliates, fungi), and diverse protist lineages. Initially the highly conserved sequences were aligned by using a computer assisted procedure. Alignment gaps were introduced to juxtapose similar regions in the sequence collection. We take advantage of our innate color perception and parallel processing capacity to interpret multiple sequence alignments in which bases are color coded. This so called "eye ball" procedure was repeated in order to detect and align regions that displayed lower similarity. If several sequences are considered simultaneously, it is possible to identify very short sequences of weaker similarity that represent homologous positions in the 16S-like rRNA coding regions. The alignment within regions that display length variations were refined by a consideration of phylogenetically conserved higher order structures. Sequence elements that define evolutionarily proven helices were juxtaposed by the appropriate placement of alignment gaps. Only those positions which are in obvious alignment (using the criteria of primary and/or secondary structure conservation) were used for phylogenetic analyses.
Phylogenetic trees were inferred from the aligned 16S-like rRNA sequences with distance matrix and maximum parsimony methods. For the distance technique (Fitch and Margoliash, 1967), pairwise comparisons of sequences were used to calculate similarity values. Similarity is defined as: s = m/(m + u + g/2) where m is the number of sequence positions with matching nucleotides, u is the number of positions with nonmatching nucleotides, and g is the number of sequence gaps. (Only the first five positions in a gap are considered in making the calculations. Large insertions or deletions probably reflect single rare events.) The similarity values were converted to "distance" values (the number of evolutionary changes per 100 positions) using the formula of Jukes and Cantor (1969). These similarity values were converted to phylogenetic trees as described by Olsen (1988). The evaluation of alternative phylogenetic trees was based upon the agreement of the distance data separating pairs of organisms and the sum of tree segment lengths joining the organisms in the tree. Bootstrap methods (Felsentstein, 1985) were used to assess the fraction of positions (using 100 resamplings of the data set) that support a given topological element in the distance matrix trees. The distance boot-strap analysis was limited to 20 taxa which required 15 hours of computer time on a VAX 4000 for 100 resamplings. Maximum parsimony analysis of the 16S-like rRNA sequences was implemented with the PAUP computer package (PAUP 3.0L, Swofford, 1989). Alignment gaps were treated as "missing data" and heuristic procedures using a branch-swapping algorithm (tree bisection-reconnection) and the MULPARS option were utilized. As with the distance methods, bootstrap methods were used to assess support for branching patterns in the parsimony analyses.
Phylogenetic frameworks inferred from the 16S-like rRNA data set were compared to a cladistic analysis of 12 ultrastructural characters identified as being taxonomically significant by Andersen (1991). We also examined the distribution of ultrastructure features in a parsimony analysis of rRNA sequences for a collection of organisms similar to that represented in Andersen's original data set. Of the 16 taxa in Andersen's original data set, 11 identical or very closely related species are represented in our 16S-like rRNA data base. The identical taxa included: Hibberdia, Ochromonas (Chrysophyceae), Mallomonas (M. papillosa), Synura (Synurophyceae), and Phytophthora (Oomycota). In the 16S-like rRNA analysis we replaced the following genera used in Andersen's analysis with related organisms: Costaria for Laminaria (Phaeophyceae), Cylindrotheca for Biddulphia (Bacillariophyceae), Emiliania for Prymnesium (Prymnesiophyceae), Nannochloropsis for Vischeria (Eustigmatophyceae), Prorocentrum for Amphidinium (Dinophyceae) and Tribonema for Heterococcus (Xanthophyceae). The 16S-Like rRNA sequences from species in the reduced taxon list were analyzed by maximum parsimony methods using Branch and Bound search options. Maximum parsimony analysis of the ultrastructure characters for the reduced data set generated a tree topology that is congruent with parsimony trees for Andersen's complete data set (not shown).
Complete 16S-like rRNA sequence alignments for 12 new species plus several closely related taxa are presented in Appendix 1. Since electronic data bases rarely provide alignments and never archive information about sites used in phylogenetic reconstructions, we have displayed nucleotides that we consider to be unambiguously aligned as upper case letters (1,735 positions). Relationships between chromophyte algae and major groups of eukaryotes, as well as branching patterns within the chromophytes were explored using the 16S-like rRNA data set. Similarity and evolutionary distances (Table 2) were calculated for 16S-like rRNAS from eukaryotic assemblages that separated after the earlier divergence of Dictyostelium discoideum (Sogin et al., 1986). A phylogenetic framework inferred from these evolutionary distances is shown in Figure 1A together with a maximum parsimony analysis (based upon 430 phylogenetically informative sites) in Figure 1B. Both trees are in general agreement and portray the chlorophylls a + c containing prymnesiophytes (as represented by E. huxleyi) and dinoflagellates (as represented by Prorocentrum micans) to be independent eukaryotic lines of evolution unrelated to each other or the Chromophyta. In contrast, chromophyte algae plus oomycetes are seen as a cohesive evolutionary assemblage as are the animals, fungi, plants, and ciliates. Membership within these groups is supported by relatively long common branches in distance analyses or high bootstrap values in the parsimony trees. Differences between the topologies, e.g., placement of the red algal representative G. lemaneiformis and the green plants relative to other major groups reflect uncertainties in both the distance and parsimony trees. The parsimony analysis has a length of 1,608 steps but trees with 1,609, 1,610 and three topologies with 1,611 steps were also found. Differences among these parsimony trees reflect uncertainty in the placement of G. lemaneiformis as well as minor rearrangements for H. magna.
Branching patterns within the chromo-phyte/oomycete assemblage were further resolved by restricting the analysis to members of the group (Fig. 2). The larger number of unambiguously aligned positions (1,735 sites) defined in Appendix 1 and additional chromophyte taxa were included in the phylogenetic reconstructions. As determined above, the 16S-like rRNA sequence of E. huxleyi represents a separate lineage and was used to establish the order of branching in both the distance and parsimony trees shown in Figures 2A and 2B. These analyses identify three distinct photosynthetic lineages (the Bacillariophyceae, the Phaeophyceae/ Xanthophyceae and the Chrysophyceae/ Eustigmatophyceae/Synurophyceae) which share a common evolutionary history with nonpigmented oomycetes. Maximum parsimony analysis of chromophyte/oomycete 16S-like rRNA sequences identified two equally parsimonious trees. The strict consensus tree shown in Figure 2B has a total length of 1,368 steps and it is based upon 363 phylogenetically informative sites. The placement of H. magna relative to either the O. danica/C. chromophila group or the Synurophyceae in the parsimony tree is unresolved. The maximum parsimony analysis agrees with the distance matrix phylogenies except for the evolutionary position of O. danica. Bootstrap values support the placement of O. danica at the base of the Synurophyceae radiation in the distance trees but parsimony methods do not provide similar resolutions.
Molecular comparisons provide quantitative phylogenetic frameworks that can be used to evaluate other evolutionary markers. For example, there is remarkable agreement between the rRNA based phylogenies and evolutionary relationships inferred from shared ultrastructure features in ciliated protozoa (Small and Lynn, 1981; Greenwood et al., 1991). Andersen (1991) has recently described a cladistic analysis of 12 ultrastructure, features in 16 chromophyte taxa. Parsimony analyses of ultrastructure features of the reduced data set (11 taxa) were compared to maximum parsimony cladograms of corresponding rRNA sequences. The ultrastructure data yielded three equally most parsimonious phylogenies (identified using a Branch and Bound search.) Figure 3A is a strict consensus of that analysis. A single most parsimonious tree was identified in a Branch and Bound search of the corresponding 16S-like rRNA sequence data sets (Fig. 3B). The phylogenies based on ultrastructure and 16S-like rRNA sequence comparisons are similar with respect to the exclusion of dinoflagellates and prymnesiophytes from the chromophyte/oomycete assemblage as well as the clustering of chrysophytes and synurophytes. When the topology of the 16S-like rRNA phylogeny is used to constrain parsimony analysis of the morphological data, the morphological analysis requires 4 additional steps (35 versus 39 steps). Similarly, when the topology of the ultrastructure-based tree is used to constrain parsimony analysis of the 16S-like rRNA data, 44 additional steps are required to resolve the tree. The major difference between the molecular and morphological based phylogenies is the clustering of eustigmatophytes and xanthophytes with the separation of phaeophytes and xanthophytes in trees inferred from ultrastructure similarities. In the chromophyte/oomycete rRNA phylogenies shown in Figures 2A and 2B, the xanthophyte and phaeophytes appear as sister taxa while the eustigmatophyte is related to chrysophytes/synurophytes.
Comparative analyses of ultrastructural features and plastid characteristics have traditionally delineated three eukaryotic algal groups (red, green, and brown plus golden-brown algae). Our phylogenetic reconstructions based upon 16S-like rRNA sequence similarities provide evidence for three independent chromophyte groups that are not closely related to each other or other photosynthetic eukaryotes. Dinoflagellates are members of an assemblage that includes nonphotosynthetic apicomplexans and ciliated protozoa. The prymnesiophytes (as illustrated by E. huxleyi in this study) represent a discrete lineage. Diatoms, brown algae, golden-brown algae and nonphotosynthetic aquatic fungi are members of a complex evolutionary assemblage recently described as "stramenopiles" (Patterson, 1989). This general perspective of algal evolution is supported by both parsimony and distance analyses of the 16S-like rRNA data set.
Two of these assemblages include one or more nonphotosynthetic taxa. Collectively this pattern can be interpreted as evidence for multiple origins of plastids, presumably a result of independent endosymbiotic events. However, a single plastid origin followed by multiple losses in different lineages cannot be rigorously excluded until fully resolved phylogenies of plastid and host genomes are compared; incongruent topologies would support the multiple origin hypothesis. Unfortunately, the order of branching for the independent cholorophylls a + c assemblages relative to each other or other protist and multicellular groups remains unresolved in the rRNA based phylogenies. Separations of the groups in rRNA based distance matrix analyses correspond to fewer than one nucleotide change per 250 positions and bootstrap values in parsimony trees do not support a preferred branching order.
In terms of morphology and lifestyle, the diversity exhibited by stramenopiles approaches that observed in other "higher" eukaryotic kingdoms. In our rRNA trees, the group is defined by oomycetes plus three major photosynthetic groups; the Bacillariophyceae, the Phaeophyceae/Xanthophyceae and the Chrysophyceae/Eustigmatophyceae/Synurophyceae. The close relationship between synurophytes and chrysophytes is confirmed by similarities between incomplete large subunit rRNA sequences of three chromophyte taxa (Perasso et al., 1989). The Eustigmatophyceae have been considered to be outside the Chromophyta by some authors because of the possession of a distinctive eyespot, the lack of a girdle lamella in chloroplasts and the absence of dictyosomes from zoospores (Hoek, 1978; Hibberd, 1979). These features may not be useful for phylogenetic reconstructions since the 16S-like rRNA analyses as well as comparisons of other ultrastructure features (see below) clearly position the eustigmatophytes as an early divergence from the chrysophyte/synurophyte lineage.
The inclusion of the Phaeophyceae within the stramenopiles is supported by molecular (Bhattacharya and Druehl, 1988) and ultrastructural studies (Loiseaux and West, 1970; O'Kelly, 1987). Due to their complex morphologies and tissue differentiation, phaeophytes have often been considered evolutionarily advanced (Ragan and Chapman, 1978). The present data strengthen this hypothesis with the divergence of the Phaeophyceae occurring relatively late in chromophyte evolution. Given the wealth of ultrastructure data supporting a bacillariophyte or chrysophyte ancestor of the Phaeophyceae (Clayton, 1989; O'Kelly, 1989), it is surprising that the xanthophyte (T. aequale) appears here as their closest evolutionary relation. Phaeophytes and xanthophytes are thought to be distantly related on the basis of differences in cell wall composition, pigmentation, and pyrenoid ultrastructure (Bold and Wynne, 1985).
Prymnesiophytes have been grouped with other chromophytes because they have tubular mitochondrial cristae, chlorophyll-c, and similar storage products. However they lack other ultrastructural features common to stramenopiles (see below) and bear a haptonema complex not found in other groups (Scherffel, 1900). Hibberd (1976) removed prymnesiophytes from the Chrysophyceae because of differences in ultrastructure and swimming behavior. Our molecular framework identifies the prymnesiophyte E. huxleyi as a lineage distinct from stramenopiles. Other molecular studies based upon partial large subunit rRNA sequences (450 nucleotides) position the prymnesiophytes Prymnesium parvum and Crocosphaera roscoffensis) at the base of the stramenopile assemblage (Perasso et al., 1989). Disagreement between these phylogenetic placements can be attributed to the short, statistically unreliable branch lengths uniting prymnesiophytes and other chromophytes in distance matrix analyses of the partial large subunit rRNA sequences.
The rNA trees allow for a clearer understanding of morphological variation and the identification of homologous elements in the chromophyte flagellar apparatus. The simplified rRNA phylogeny in Figure 3B was used to trace the distribution of ultrastructure features in Andersen's original data set. The presence of tripartite mastigonemes are sufficient to differentiate stramenopiles from other chlorophylls a + c containing lineages. Filaments on the tripartite hairs distinguish synurophytes and chrysophytes from other stramenopiles. Flagellar roots are microtubular or fibrous structures associated with basal bodies that terminate in the cytoplasm. They are classified by composition as well as their origin relative to the basal bodies. R1 roots with or without cytoplasmic microtubules are found in prymnesiophytes and most stramenopiles, but were probably lost in diatoms because of their reduced dependence of flagellar motility. Transitional helices are fibrous structures lying between the base of the central pair of microtubules and the nine peripheral doublets of microtubules. They are not present in dinoflagellates or prymnesiophytes; they must have been introduced by the common ancestor to eustigmatophytes, synurophytes, and chrysophytes. Oomycetes and xanthophytes have a similar but not necessarily homologous double helical structure but their closest relatives lack transitional helices of the type found in other stramenopiles. The eukaryotic flagellar apparatus contains more than 200 different polypetides (Luck, 1984) that define diverse ultrastructure elements (Andersen et al., 1991). The distribution of these ultrastructural features on the rRNA based phylogenetic framework provides evidence that certain components in complex biological structures must evolve in a coordinated manner. Those elements which are homologous in different lineages can be used to reconstruct phylogenetic history. The relative order of branching for major lineages within the stramenopile subtree are not well resolved but several morphological clues suggest that oomycetes and diatoms diverged before other members of this complex assemblage. Dinoflagellates, prymnesiophytes, diatoms, and oomycetes lack fibrous roots which except for brown algae, are present in other chromophyte algae. Photoreceptors are found in chrysophytes, synurophytes, brown algae, and xanthophytes, but are absent in oomycetes, diatoms, some dinoflagellates, and other chlorophylls a + c containing algae. Perhaps these morphological features were introduced after the divergence of oomycetes and diatoms from other stramenopiles.
Several characteristics in Andersen's ultrastructure data set including the girdle lamella, fucoxanthin, and carotenoids appear to be randomized in the rRNA trees. However these are plastid characteristics which may have been introduced via independent evolutionary events. The distribution of R3 roots and character states of silica deposition vesicles are also inconsistent with the molecular trees. Metabolism of silica is a shared character widespread among many chromophytes. The diatoms have been the most successful in utilizing silica with its incorporation in the cell walls to form many patterns and ornamentations which are species-specific and precisely reproduced with each cell division (Round et al., 1990). Silica is involved in diatom cell metabolism with an absolute requirement before DNA will replicate (Darley and Volcani, 1969). The chrysophytes have siliceous cysts, synurophytes have siliceous scales, and the xanthophytes have silica in cell walls of some vegetative cells and cysts (Pascher, 1939). Silica has also been reported in the cell walls of some phaeophytes (Parker, 1969). Silica metabolism was most likely present in the ancestor of chromophytes.
The fossil record is of little help in resolving relationships among major stramenopile lineages although it contributes to an understanding of evolution within the Bacillariophyceae. Diatom fossils appear suddenly as morphologically advanced organisms resembling modem genera (Harwood and Gersonde, 1990). However, a progression from centric (radially symmetrical) to pennate (bilaterally symmetrical) forms is evident (Round and Crawford, 1984). For example, Rhizosolenia, with its many scale-like girdle bands and Stephanopyxis, with its domed valves and scale-like girdle bands are abundant and widespread in early diatom fossils (Strelnikova, 1974). In support of these observations, the 16S-like rRNA analyses suggest an early divergence of R. setigera and S. cf. broschii from the diatom lineage prior to the divergence of the other centric species, S. costatum, from the pennate forms (B. paxilifer, C. closterium, N. apiculata). Both S. costatum and the pennate species belong to geologically young families and possess morphological features which are considered to be advanced (Simonsen, 1979).
In conclusion, the 16S-like rRNA phylogenetic frameworks support phylogenetic relationships proposed on the basis of several ultrastructure features associated with the flagellar apparatus. It is clear that the presence of tripartite hairs defines a monophyletic assemblage of diverse organisms. Further resolution within different stramenopile lineages are supported by other elements of the flagellar apparatus including filaments on the tripartite hairs, flagellar R1 roots, and transitional helices. Cavalier-Smith (1986) suggested an unique origin and homology of all tripartite hairs due to their common structure, mode of assembly and function (reversal of thrust). The generality of this kingdom-level character will be tested as 16S-like rRNA sequences are determined for other organisms with tripartite hairs. Groups in this category include the Pedinellophyceae, Dictyochophyceae, Raphidophyceae and nonalgal forms such as opalinids, labyrinthulids, and thraustochytrids. The addition of sequences from these groups to the rRNA data base may consolidate the branching order between major stramenopile lines of descent.
This research was supported by a National Institutes of Health grant (GM32964) to M. L. Sogin, a Sloan Foundation Fellowship for Molecular Studies of Evolution (89-1-6 ME) to D. Bhattacharya, and National Engineering Research Council Grants GR3/ 7533, GR3/7095, GST/02/223, GR3/3095 and Interdisciplinary Fund Grant from the Natural History Museum, London to L. Medlin. The authors also thank R. A. Andersen of the Bigelow Laboratory for Ocean Sciences for provision of algal cultures.
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Aligned 16S-like rRNA sequences of algae containing chlorophylls a + c and ommycetes. Small subunit rRNA sequences were determined from Emiliania huxleyi, Bacillaria paxillifer, Chromulina chromophila, Cylindrotheca closterium, Fucus distichus, Hibberdia magna, Mallomonas striata, Nanochloropsis salina, Nitzschia apiculata, Rhizosolenia setigera, Stephanopyxis cf. broschii, and Synura spinosa. Other sequences included in the alignment are from: Achlya bisexualis, Costaria costata, Ochromonas danica, Skeletonema costatum (Neefs et al., 1990), Lagenidium giganteum, Phytophthora megasperma (Forster et al., 1990), Mallomonas papillosa, Tribonema aequale (Ariztia et al., 1991). Positions used in phylogenetic analyses are shown in upper case. Alignment gaps (-) were used to compensate for sequence length variations. Position numbers, which include alignment gaps, are given on the left side of the sequences while the nucleotide numbering system on the right side is for individual sequences and does not include alignment gaps. Uppercase letters indicate sites that we consider to be unambiguously aligned.