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2 Department of Botany, Institute of Biosciences, University of São Paulo, R. Matão, Travessa 14, N. 321, São Paulo, SP, Brazil CEP 05508–900; and 3 Department of Biological Sciences, University of Iowa, 239 Biology Building, Iowa City, Iowa 52242-3110 USA
Received for publication March 30, 1999. Accepted for publication July 29, 1999.
ABSTRACT
The Rhodophyta (red algae) are composed of the subclasses Bangiophycidae and Florideophycidae. Two evolutionarily interesting features of the Bangiophycidae are: (1) they are the ancestral pool from which the more morphologically complex taxa in the Florideophycidae have arisen and (2) they are the sources of the plastids, through secondary endosymbioses, for the Cryptophyta, Haptophyta, and the Heterokonta. To understand Bangiophycidae phylogeny and to gain further insights into red algal secondary endosymbioses, we sequenced the plastid-encoded small subunit ribosomal DNA (rDNA) coding region from nine members of this subclass and from two members of the Florideophycidae. These sequences were included in phylogenetic analyses with all available red algal plus chlorophyll a + c algal plastid rDNA coding regions. Our results are consistent with a monophyletic origin of the Florideophycidae with these taxa forming a sister group of the Bangiales. The Bangiophycidae is of a paraphyletic origin with orders such as the Porphyridiales polyphyletic and distributed over three independent red algal lineages. The plastids of the heterokonts are most closely related to members of the Cyanidium–Galdieria group of Porphyridiales and are not directly related to cryptophyte and haptophyte plastids. The phylogenies provide strong evidence for the independent origins of these "complex" algal plastids from different members of the Bangiophycidae.
Key Words: Bangiophycidae • phylogeny • plastid • Rhodophyta • secondary endosymbiosis • small subunit rRNA
The Rhodophyta (red algae) comprise a distinct lineage that arises from the "crown" of the eukaryotic radiation (Bhattacharya et al., 1990
; Van de Peer et al., 1996
; Stiller and Hall, 1997
). Red algae are united by a suite of characters that do not occur together in any other eukaryote. These include a complete lack of flagellated stages and basal bodies, a two-membraned "simple" plastid (Bhattacharya and Medlin, 1995
) that lacks chlorophyll b or c, contains unstacked thylakoids, and food reserves stored as floridean starch (Garbary and Gabrielson, 1990
). Traditionally, systematists have divided the red algae into two subclasses, the Bangiophycidae and the Florideophycidae (Gabrielson, Garbary, and Scagel, 1985
). The Florideophycidae include morphologically complex red algae in orders such as the Gigartinales and the Ceramiales, and is widely believed to be a derived, monophyletic group (Garbary and Gabrielson, 1990
; Ragan et al., 1994
; Freshwater et al., 1994
; Saunders and Kraft, 1997
). The Bangiophycidae, on the other hand, are thought to form the ancestral pool from which the Florideophycidae has evolved. The Bangiophycidae may have a paraphyletic origin because of a lack of shared synapomorphic characters (Gabrielson et al., 1990
; Garbary and Gabrielson, 1990
). Molecular sequence comparisons generally confirm these views of red algal evolution (Ragan et al., 1994
; Freshwater et al., 1994
), although a paucity of data has left unclear the phylogeny of the Bangiophycidae (Dixon, 1973
; Garbary, Hansen, and Scagel, 1980
; Saunders and Kraft, 1997
).
A second, equally interesting and as yet unexplored issue regarding the Bangiophycidae concerns its central position in the endosymbiotic origin of algal plastids (Gray, 1992
; Gibbs, 1993
; Whatley, 1993
; Bhattacharya and Medlin, 1995, 1998
). One part of this theory holds that the plastids of many algal groups have originated, not through a primary endosymbiosis involving a cyanobacterium, but rather through the uptake of an existing photosynthetic eukaryote (secondary endosymbiosis—Ludwig and Gibbs, 1987
; Häuber et al., 1994
; Gilson and McFadden, 1996
; Palmer and Delwiche, 1996
). Molecular analyses have substantiated this view and have indicated that a number of algal lineages have gained their so-called "complex" morphology (Sitte, 1993
) plastids (three or more bounding membranes) through a secondary endosymbiosis involving, in some cases, a eukaryotic red alga (viz., Cryptophyta, Haptophyta, Heterokonta) and in other cases, a eukaryotic green alga (viz., Chlorarachniophyta, Euglenophyta). An important question that remains regarding red algal secondary endosymbioses is the number of events involved. In other words, have all of these plastids arisen from multiple, independent endosymbioses of red algae or do some of them (and the "host" cells) trace their origins to a single endosymbiotic event followed by separation of the nuclear lineages over evolutionary time? This is an important question because the genome sequence and phylogenetic data that are presently available, although clearly supportive of a red algal origin of the complex plastids of the Cryptophyta, Haptophyta, and the Heterokonta, are more ambiguous about the number of events that gave rise to them (Kowallik, 1997
; Douglas and Penny, 1999
). This is because the plastid genomes of these algae are sufficiently different from each other to preclude a clear understanding of their interrelationships. Phylogenies of the host cells, based on nuclear-encoded rRNA sequences, are similarly ambiguous in establishing phylogenetic relationships among these algal lineages relative to other crown group eukaryotes (Bhattacharya et al., 1995
). To address these issues and to gain insights into the evolutionary relationships of the Bangiophycidae, we sequenced the plastid-encoded small subunit ribosomal DNA (SSU rDNA) coding region from nine members of this subclass and from two members of the Florideophycidae. These sequences were included in an alignment with all available plastid rDNA sequences from the red algae and taxa containing plastids of a red algal origin and were analyzed with different phylogenetic methods to understand plastid phylogeny.
MATERIALS AND METHODS
The red algal cultures were obtained from the Sammlung von Algenkulturen (SAG) at the University of Göttingen (Schlösser, 1994
). The species and strain numbers of these taxa are listed in Table 1. Algal material (100–400 mg fresh mass) was ground in liquid nitrogen and total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Santa Clarita, California, USA). Polymerase chain reaction (PCR) amplifications were performed using synthetic oligonucleotide primers that recognize conserved sequences at the termini of the plastid-encoded SSU rDNA genes (Huss and Giovannoni, 1989
; see Table 2). PCR products were purified using the QIAquick PCR Purification kit (Qiagen) and cloned using the TA cloning kit and the plasmid vector pCR2.1 (Invitrogen, Carlsbad, California, USA). Plasmid DNA was isolated and purified using the Plasmid Midi Kit (Qiagen). The rDNA sequences were determined over both strands using an ABI-310 Genetic Analyser (Perkin-Elmer), and the primers listed in Table 2, with the Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Norwalk, Connecticut, USA). These plastid coding regions were included in a secondary-structure-based alignment of SSU rDNAs that included sequences from eubacteria and members of all the major algal groups except the dinoflagellates.
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) and a total of 1370 sequence positions were used in the phylogenetic analyses. Two data sets (available from D. B.) were created from the large alignment. The first included cyanobacteria and representatives of the different plastid lineages (567 parsimony informative sites), and the second data set was restricted to the red algae and plastids derived from these taxa with the green algae as the outgroup (489 parsimony informative sites). A matrix was calculated from the first data set using the LogDet (Lockhart, Steel, and Penny, 1994
) transformation, and a tree was built using the neighbor-joining method (Saitou and Nei, 1987
). This data set was also analyzed with a distance method in which the matrix was calculated with the HKY-85 model (Hasegawa, Kishino, and Yano, 1985
) and the tree built with the neighbor-joining method. Bootstrap analyses (Felsenstein, 1985
; 2000 replications) were done using both the LogDet transformation and distance method. Missing data and gaps were excluded from the LogDet and distance analyses. All phylogenetic analyses were implemented with the PAUP* version 4.0b1 computer program (Swofford, 1999
). We did not implement the parsimony method with the first data set because previous analyses (e.g., Helmchen, Bhattacharya, and Melkonian, 1995
; Nelissen et al., 1995
) show that this (and often other) method artifactually groups the secondary plastids of the Euglenophyta within the red plastid lineage. This results from divergence rate differences (Van de Peer et al., 1996
) and a biased nucleotide content of the euglenophyte rDNA sequences (Helmchen, Bhattacharya, and Melkonian, 1995
). The LogDet transformation is applicable under such circumstances (Lockhart, Steel, and Penny, 1994
). In our analyses, the distance method also provided the predicted result of a sister-group relationship between the chlorophyll a + b containing plastids of the chlorarachniophytes/euglenophytes and the green algae and, therefore, was also used to calculate bootstrap support for nodes in the LogDet tree.
The second data set was analyzed with the maximum likelihood method (in PAUP*). Starting trees were obtained with stepwise additions (randomly drawn, five rounds) and re-arranged with tree bisection-reconnection. The HKY-85 distance model was used in this analysis with the transition/transversion ratio set to two, empirical determination of nucleotide frequencies, equal divergence rates over all sites, and the starting branch-lengths calculated with the Rogers-Swofford method (Swofford, 1999
). The maximum parsimony, LogDet transformation, and distance methods were also used to infer trees with the second data set. In the maximum parsimony analysis, starting trees were obtained with stepwise sequence addition (randomly drawn, ten rounds) and rearranged with tree bisection-reconnection. The sequence positions were weighted in the parsimony analysis (rescaled consistency index over an interval of 1–1000) to reduce the influence of highly divergent sites in the phylogeny reconstruction (Bhattacharya, 1996
). The LogDet transformation and distance method were done as described above. Bootstrap analyses (2000 replications) were done with all phylogenetic methods except maximum likelihood. In the bootstrap analyses, consensus trees were calculated, and the resulting bootstrap values were included (for shared monophyletic groups) at the branches of the maximum likelihood tree.
We also tested for congruence of the trees inferred with the maximum likelihood, maximum parsimony, LogDet, and distance trees using the Kishino-Hasegawa test (Kishino and Hasegawa, 1989
[within PAUP*]). In this analysis, all trees were compared to the topology of the maximum likelihood phylogeny to test for significant (P < 0.05) differences among them. In addition, we changed the topology of the maximum likelihood tree using the MacClade computer program (version 3.07; Maddison and Maddison, 1997
) and compared these rearranged trees to the "best" tree to test different hypotheses about plastid evolution. The taxa used in our study and GenBank accession numbers (the prefix GBAN- has been added to link the online version of American Journal of Botany to GenBank but is not part of the actual accession number) are shown in Table 3. The OM (off Cape Hatteras, North Carolina, USA) and OCS (off the mouth of Yaquina Bay, Oregon, USA) clones derive from open-ocean material collected and analyzed by Rappé et al. (1998)
. These are environmental samples in which the diverse array of algae were identified through cloning and sequence analysis of rDNA isolated from plankton collections (for details, see Rappé et al., 1998
).
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The SSU rDNAs isolated and sequenced in this study did not contain any significant insertions or deletions. These coding regions were easily aligned with homologous rDNAs from the other plastids. The LogDet transformation resulted in a phylogeny that is consistent with previous analyses of plastid SSU rDNAs (e.g., Bhattacharya and Medlin, 1998
; Martin et al., 1998
). This tree (Fig. 1) shows that the plastids form a monophyletic group with respect to the cyanobacteria included in the analyses, an observation that is consistent with a single primary endosymbiotic origin of these organelles (Cavalier-Smith, 1982
; Bhattacharya and Medlin, 1995
). Within the plastids, there is a separation into three lineages: (1) defined by the cyanelles of the Glaucocystophyta (strong support); (2) the chloroplasts and secondary endosymbionts derived from these algae (moderate suport); and (3) the rhodoplasts and secondary endosymbionts derived from these algae (weak support). Within the rhodoplasts, the complex plastids of the Cryptophyta, Haptophyta, and Heterokonta form three strongly supported lineages (see also Fig. 2) with weaker bootstrap support for cryptophyte/haptophyte monophyly (69% LogDet, 60% distance). The LogDet and distance trees were largely congruent, with the only difference restricted to the divergence point of the cyanelles within the three plastid groups. The LogDet tree (Fig. 1) showed the cyanelles to be positioned as a sister group of the rhodoplasts (51% bootstrap support, not shown), whereas the distance tree showed the cyanelles to be the earliest divergence within plastids (42% bootstrap support, not shown). Previous rDNA analyses using parsimony and distance have also failed to resolve clearly the evolutionary relationships between the three plastid lineages (Helmchen, Bhattacharya, and Melkonian, 1995
; Rappé et al., 1998
). A recent analysis of concatenated protein sequences (11 039 amino acids) from nine different completely sequenced plastid genomes provides, however, strong support for the early divergence of cyanelles within the plastid radiation (Martin et al., 1998
).
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) or as members of a separate order, Cyanidiales (Ott and Seckbach, 1994
).
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The Kishino-Hasegawa test was used to compare the trees shown in Fig. 3. The maximum likelihood tree was used as the standard for these analyses and the different rearrangements tested were: tree 1 forced monophyly of the heterokont and haptophyte plastids, tree 2 forced monophyly of the cryptophyte and haptophyte plastids, tree 3 forced monophyly of the heterokont and cryptophyte plastids, tree 4 forced monophyly of the heterokont, haptophyte, and cryptophyte plastids, tree 5 forced monophyly of plastids of the Cyanidium–G. sulphuraria group and the Porphyridiales group that includes C. richteriana and G. alsidii, and tree 6 forced monophyly of plastids of the Cyanidium–G. sulphuraria group and the Porphyridiales group of F. sanguinaria and P. aerugineum. The results of these tree comparisons are shown in Table 4.
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DISCUSSION
Phylogeny of the Bangiophycidae
Our results indicate that the Bangiales is a monophyletic order. Porphyra appears, however, to be paraphyletic, with P. purpurea being more closely related to B. fuscopurpurea than to P. leucosticta. Both of these observations are supported by comparisons of nuclear-encoded SSU rDNA coding regions (Ragan et al., 1994
; Oliveira et al., 1995
; Muller et al., 1998
). The Bangiales is positioned as a sister group of the Florideophycidae (Figs. 1, 2), being more closely related to the Florideophycidae than to the other lineages of Bangiophycidae. This relationship is supported by the association of the Golgi apparatus with the mitochondrion in these groups (see Table 5; Garbary and Gabrielson, 1990
) and the results of rbcL sequence comparisons (Freshwater et al., 1994
). Ragan et al. (1994) have shown, based on nuclear SSU rDNA analyses, that the Florideophycidae is monophyletic, with the Hildenbrandiales as the first divergence and the NAP complex (Nemaliales, Acrochaetiales, and Palmariales) as the second diverging lineage. Our data also suggest that the Florideophycidae is a monophyletic group.
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) are not a monophyletic order, nor is the family Porphyridiaceae. Garbary and Gabrielson (1990)
have shown that the Porphyridiales remain the most problematic order of red algae in terms of classification and evolutionary relationships. This order has been considered a heterogeneous assemblage by many authors (Fritsch, 1945
; Lee, 1974
; Ott, 1976
; Scott, 1986
). Others regard the Porphyridiales as an assemblage of organisms united solely by their unicellular to palmelloid habit and suggested that it may be polyphyletic (Garbary, Hansen, and Scagel, 1980
; Gabrielson, Garbary, and Scagel, 1985
; Scott and Gabrielson, 1987
). The well-supported grouping composed of four species of the Porphyridiales, C. richteriana, C. ramosum, S. alsidii, and R. marinus, includes both unicells and filaments, with only one chloroplast and one pyrenoid per cell (Table 5). The close relationship of Chroodactylon and Chroothece has been previously pointed out by different authors (Lewin and Robertson, 1971
; Garbary, Hansen, and Scagel, 1980
). A second Porphyridiales grouping, which is more closely related to the Compsopogonales (however, with no bootstrap support), includes F. sanguinaria and P. purpureum. Both of these species are unicells with no cell walls and contain a single chloroplast that lacks peripheral thylakoids (Table 5). Scott (1986)
pointed out that cell division in Flintiella was similar to that in Batrachospermum and, on this basis, Garbary and Gabrielson (1990)
suggested that Flintiella may have evolved through reduction from a Batrachospermum-like ancestor. This scenario is not supported by our phylogenetic analyses.
Compsopogon coeruleus (Compsopogonales) is a complex, multiseriate alga with pit connections, cellulose in the cell walls, and one or more chloroplasts per cell with no pyrenoid (Table 5; Scott and Broadwater, 1989
). Glaucosphera vacuolata, which groups weakly with C. coeruleus, is classified within the Glaucocystophyta (Kies and Kremer, 1986
). Previous analyses of both nuclear- and plastid-encoded SSU rDNA sequences have demonstrated that G. vacuolata is not directly related to other glaucocystophytes and is a member of the Rhodophyta (Bhattacharya et al., 1995
; Helmchen, Bhattacharya, and Melkonian, 1995
). Ultrastructural analyses suggest that G. vacuolata is a member of the Porphyridiales (Broadwater et al., 1995
). Regarding Cyanidium spp. and G. sulphuraria, Seckbach and Ott (1994) have proposed that these genera should be united in a separate order, the Cyanidiales, within the Bangiophycidae. Our analyses support this idea. There is, however, presently no justification to unite both genera, as has been proposed by Seckbach and Ott (1994).
Due to a lack of synapomorphic characters to unite bangiophyte orders into a monophyletic group, Gabrielson, Garbary, and Scagel (1985)
have recommended that a single class, Rhodophyceae, be recognized that includes all red algal lineages. Ragan et al. (1994)
, using comparisons of nuclear SSU rDNA sequences, showed that the Bangiophycidae are the basal rhodophyte line, comprising at least three distinct lineages. Our results add to the bulk of data that indicates a paraphyletic origin of the Bangiophycidae within the Rhodophyta. The generic use of the term bangiophytes is, however, undeniable and it continues to be useful to differentiate the earlier diverging, less complex, red algal lineages from the monophyletic, and more recently diverged florideophytes. Usage of the terms bangiophyte and florideophyte may therefore be used primarily for convenience, and these designations need not necesarily imply acceptance of previous taxonomic definitions of these taxa (Gabrielson and Garbary, 1987
).
Secondary endosymbiotic origin of red algal plastids
The origin of the plastids in the Cryptophyta, Haptophyta, and the Heterokonta from a red algal secondary endosymbiont has been previously documented on the basis of sequence comparisons and plastid gene order (Douglas et al., 1991
; Bhattacharya and Medlin, 1995
; Palmer and Delwiche, 1996
; Daugbjerg and Andersen, 1997a
; Medlin et al., 1997
; Douglas and Penny, 1999
; Durnford et al., 1999
). The recently sequenced plastid genome of the cryptophyte Guillardia theta, for example, shows remarkable conservation of synteny groups with the genome of the rhodophyte, Porphyra purpurea (Douglas and Penny, 1999
). Such data "establish" a red algal origin of the G. theta, and possibly all other cryptophyte plastids. Douglas and Penny (1999)
suggest that the cryptophyte endosymbiont may have been a taxon closely related to P. purpurea, not only on the basis of conserved synteny groups, but also based on the presence of a protein intein in the dnaB genes of these taxa and the putative origin, through reciprocal recombination, of the inverted repeat in G. theta from the nonidentical, directly repeated rDNA cistrons in P. purpurea. Gene order data also show conservation of the plastid genomes of the heterokont (diatom) Odontella sinensis and P. purpurea (Kowallik et al., 1995
; Kowallik, 1997
). A haptophyte plastid genome has not yet been sequenced. Given this information, then what can we say about the possible interrelationships of the heterokont and cryptophyte plastids?
Douglas and Penny (1999)
noted that the conservation of synteny groups between the G. theta and P. purpurea genomes was much greater than that between O. sinensis and P. purpurea. The diatom genome has undergone much greater rearrangement. This was interpreted as indicating either a longer divergence time between the cryptophyte and heterokont endosymbioses (presumably from a closely related red algal cell) or that the host cells had "captured" evolutionarily distantly related red algae (Douglas and Penny, 1999
). Kowallik (1997)
noted the large number of rearrangements between the plastids of the heterokonts, Dictyota dichotoma and O. sinensis, and suggested that these plastids may have had independent origins. Plastid gene order is, therefore, largely consistent with independent origins of the plastids of the cryptophytes and heterokonts from red algal sources. The alternative hypothesis of a relatively higher rate of gene rearrangement in heterokonts relative to other red algae or red algal-derived plastids cannot, however, be excluded with these data. Our results support the hypothesis of independent plastid origins in the cryptophytes and heterokonts. First, we find a consistent grouping, with weak to moderate bootstrap support, of members of the Cyanidiales with the heterokonts to the exclusion of all other complex plastids (Fig. 2). This suggests that a species related to C. caldarium str. RK-1 was the direct ancestor of heterokont plastids, a result that is consistent with previous analyses of smaller data sets (e.g., Bhattacharya and Medlin, 1995
; Leblanc, Boyen, and Loiseaux-de Goër, 1995
; Medlin et al., 1995, 1997
). That the rearranged trees 5 and 6 (Fig. 3) were significantly different from the best tree shown in Fig. 2 is also consistent with the idea that the Cyanidiales are not directly related to other Porphyridiales. This suggests that its divergence at the base of the Heterokonta is not an artifact of our tree-reconstruction methods.
Second, we find no evidence for independent endosymbiotic origins of the different heterokont plastids. This is consistent with extensive analyses of plastid-encoded rbcL sequences from different heterokont and red algae (Daugbjerg and Andersen, 1997a, b
). In addition, we have included a partial (665 nt) SSU rDNA sequence of D. dichotoma in the second data set to determine its position within the red algal phylogeny. Both the parsimony method and LogDet transformation show D. dichotoma to be closely related to the phaeophyte Pylaiella littoralis. This result shows that the great differences in gene order of the D. dichotoma and O. sinensis plastids are not due to the polyphyletic origins of these organelles from different red algal secondary endosymbionts. Third, our data do not support a monophyletic origin of the heterokont and cryptophyte plastids. The rearranged tree 3 (Fig. 3), which forced the monophyly of these complex plastids, was significantly different from the best tree (P < 0.0001). Regarding a possible close evolutionary relationship between the plastids of Porphyra spp. and the cryptophytes (i.e., G. theta), we presently find no support for this theory. The plastids of the cryptophytes and Porphyra spp. (Bangiales) do not group together in any of the phylogenies. The plastids of the Bangiales do, however, form a sister group of the Florideophycidae in all the trees, with bootstrap support in the LogDet transformation and distance analyses (see Fig. 2). What about evidence regarding plastid origin based on comparisons of host cell sequences?
Previous phylogenetic analyses of the nuclear-encoded SSU rDNA trees show conclusively that the photosynthetic members of the Heterokonta are rooted within nonphotosynthetic taxa such as bicosoecids and labyrinthulomycetes (Leipe et al., 1994
; Sogin et al., 1996
). In addition, rDNA analyses support a sister-group relationship between cryptophytes and glaucocystophytes (Bhattacharya et al., 1995
; Van de Peer et al., 1996
) with the nonphotosynthetic Goniomonas truncata as the earliest divergence within the Cryptophyta (McFadden, Gilson, and Hill, 1994
). Actin and rDNA trees also do not provide any evidence for the monophyly of the Cryptophyta and the Heterokonta (e.g., Bhattacharya and Weber, 1997
; Van de Peer et al., 1996
). Taken together, these data provide strong support for separate evolutionary histories of the heterokont and cryptophyte hosts and are consistent with the plastid data that show separate secondary endosymbiotic origins of their plastids from different red algae. A similar story is emerging regarding the origin of the plastid in haptophytes. Analyses of host rDNA and actin coding regions fail to show a sister group relationship between haptophytes and heterokonts (Van de Peer et al., 1996
; Bhattacharya, Stickel, and Sogin, 1993
), the group with which they have most often been allied (for details, see Daugbjerg and Andersen, 1997a
; Medlin et al., 1997
).
Consistent with this view, we do not find any evidence for the monophyly of heterokont and haptophyte plastids in our analyses. These rDNAs form a weakly supported cluster in Fig. 1 and in trees in which the cyanelle sequences are used to outgroup-root the taxa in the second data set. We interpret these results as consistent with the view that closely related red algae gave rise independently to the heterokont and haptophyte plastids and not that the host lineages share a monophyletic origin. The positioning of the Cyanidiales rDNA sequences between these complex plastids suggests that these organelles result from independent endosymbiotic events. Detailed phylogenetic analyses of rbcL and rDNA sequences support this hypothesis (Daugbjerg and Andersen, 1997a, b
; Medlin et al., 1997
). And finally, we find no support for the scenario that all rhodoplast-derived complex plastids (and, thereby, their host cells) form a monophyletic group, an idea that was put forth largely on the basis of plastid characters, such as the existence of a plastid endoplasmic reticulum in these taxa (Cavalier-Smith, 1982
). The rearranged tree 4 that forced the monophyly of the cryptophyte, haptophyte, and heterokont plastids was significantly different from the best maximum likelihood tree shown in Fig. 2 (P = 0.0231).
In summary, our expanded phylogenetic analyses help to clarify the evolutionary histories of the Bangiophycidae and the Florideophycidae. Most importantly, the subclass Bangiophycidae is found to be a paraphyletic assemblage that should be included in a single red algal class, the Rhodophyceae, which also includes the Florideophycidae (Gabrielson, Garbary, and Scagel, 1985
). In addition, we suggest that the cryptophyte, haptophyte, and heterokont plastids were derived from independent secondary endosymbiotic events involving members of the Bangiophycidae. This is supported by the rDNA phylogeny of the plastids and of the host nuclei and is consistent with the differing pigmentations in these organelles. We see our data as a start toward understanding the complex forces that have shaped algal evolution. In this respect, single-gene phylogenies are valuable for providing a broad perspective on cellular evolution and we hope that such data are superceded and improved upon by future plastid genome and multiple-sequence comparisons using the taxa included in this study.
FOOTNOTES
1 DB thanks the College of Liberal Arts (University of Iowa) and a grant from the Carver Foundation and K. Weber (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany) for supporting this research. MCO thanks FAPESP and CNPq for research grants and the DAAD for a visiting grant at the Max Planck Institute for Biophysical Chemistry, E. C. Oliveira for useful comments, and C. Bird for help with nomenclature. 
4 Author for correspondence (Ph: 319-335-1977, FAX: 319-335-1069, e-mail: dbhattac{at}blue.weeg.uiowa.edu
).
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60% are shown. Arrows are used to show the position of bootstrap values when they do not fit on the branches. The complex plastid lineages are shown with thick branches. Chlorara. = chlorarachniophytes. Sequences determined in this study have been underlined.
