Evolutionary relationships among heterokont algae (the autotrophic stramenopiles) based on combined analyses of small and large subunit ribosomal RNA

In order to study the phylogenetic relationships within the stramenopiles, and particularly among the heterokont algae, we have determined complete or nearly complete large-subunit ribosomal RNA sequences for different species of raphidophytes, phaeophytes, xanthophytes, chrysophytes, synurophytes and pinguiophytes. With the small- and large-subunit ribosomal RNA sequences of representatives for nearly all known groups of heterokont algae, phylogenetic trees were constructed from a concatenated alignment of both ribosomal RNAs, including more than 5,000 positions. By using different tree construction methods, inferred phylogenies showed phaeophytes and xanthophytes as sister taxa, as well as the pelagophytes and dictyochophytes, and the chrysophytes/synurophytes and eustigmatophytes. All these relationships are highly supported by bootstrap analysis. However, apart from these sister group relationships, very few other internodes are well resolved and most groups of heterokont algae seem to have diverged within a relatively short time frame.     

THE AUTOFLUORESCENT FLAGELLUM: A NEW PHYLOGENETIC ENIGMA1

Epifluorescence microscopy reveals the presence of fluorescence in the living cells of at least three classes of flagellates. In Ochromonas cells, the fluorescence is blue-green in color and is found only in the short flagellum, both in the flagellar swelling and throughout the length of the flagellum. As recognized by the locale and color of the flagellar fluorescence, the same fluorescence is observed in only certain other heterokont algal groups but is also found in one of the two isokont flagella of the prymnesiophyte Prymnesium parvum.     

Glossomastix chrysoplasta n. gen., n. sp. (Pinguiophyceae), a new coccoidal, colony-forming golden alga from southern Australia

Glossomastix chrysoplasta gen. et sp. nov. is described from cultures isolated from sandstone rubble, Sorrento Back Beach, Mornington Peninsula, Victoria, Australia. The alga forms wall‐less, coccoidal vegetative cells that congregate in mucilaginous colonies and reproduce by successive bipartition. Plastids have girdle lamellae and partially embedded pyrenoids that are traversed by cytoplasmic channels. Zoospores are uniflagellate and swim poorly; a narrow lingulate pseudopod provides their primary form of motion. The single flagellum, which lacks hairs, a flagellar swelling, and autofluorescence, is the equivalent of the posterior flagellum in other golden algae. The anterior flagellum is absent; the basal body with which it would normally be associated is blind. The flagellar apparatus has two basal bodies, three microtubular roots, and a rhizoplast. The posterior (elder) basal body has a transitional helix that is proximal to the basal plate. Glossomastix chrysoplasta, placed in the Pinguiophyceae on the basis of molecular sequence and biochemical data, shares some ultrastructural features with other members of the class, especially Polypodochrysis teissieri, which has similar zoospores, but it also differs from other pinguiophytes in many respects. Glossomastix chrysoplasta is the pinguiophyte with, on average, the largest cells (exclusive of external materials), and it is the only one with a colonial habit.     

A MOLECULAR PHYLOGENY OF THE HETEROKONT ALGAE BASED ON ANALYSES OF CHLOROPLAST-ENCODED rbcL SEQUENCE DATA1

Nearly complete ribulose-1,5-bisphosphate carboxylase/ oxygenase (rbcL)sequences from 27 taxa of heterokont algae were determined and combined with rbcL sequences obtained from GenBank for four other heterokont algae and three red algae. The phylogeny of the morphologically diverse haterokont algae was inferred from an unambiguously aligned data matrix using the red algae as the root, Significantly higher levels of mutational saturation in third codon positions were found when plotting the pair-wise substitutions with and without corrections for multiple substitutions at the same site for first and second codon positions only and for third positions only. In light of this observation, third codon positions were excluded from phylogenetic analyses. Both weighted-parsimony and maximum-likelihood analyses supported with high bootstrap values the monophyly of the nine currently recognized classes of heterokont algae. The Eustigmatophyceae were the most basal group, and the Dictyochophyceae branched off as the second most basal group. The branching pattern for the other classes was well supported in terms of bootstrap values in the weightedparsimony analysis but was weakly supported in the maximum-likelihood analysis (<50%). In the parsimony analysis, the diatoms formed a sister group to the branch containing the Chrysophyceae and Synurophyceae. This clade, charactetized by siliceous structures (frustules, cysts, scales), was the sister group to the Pelagophyceae/Sarcinochrysidales and Phaeo-/Xantho-/ Raphidophyceae clades. In the latter clade, the raphido-phytes were sister to the Phaeophyceae and Xanthophyceae. A relative rate test revealed that the rbcL gene in the Chrysophyceae and Synurophyceae has experienced a significantly different rate of substitutions compared to other classes of heterokont algae. The branch lengths in the maximum-likelihood reconstruction suggest that these two classes have evolved at an accelerated rate. Six major carotenoids were analyzed cladistically to study the usefulness of carotenoid pigmentation as a class-level character in the heterokont algae. In addition, each carotenoid was mapped onto both the rbcL tree and a consensus tree derived from nuclear-encoded small-subunit ribosomal DNA (SSU rDNA) sequences. Carotenoid pigmentation does not provide unambiguous phylogenetic information, whether analyzed cladistically by itself or when mapped onto phylogenetic trees based upon molecular sequence data.     

PIGMENT COMPOSITION AND rbcL SEQUENCE DATA FROM THE SILICOFLAGELLATE DICTYOCHA SPECULUM: A HETEROKONT ALGA WITH PIGMENTS SIMILAR TO SOME HAPTOPH Y TES

Monophyly of plastids in the morphologically diverse heterokont algae has rarely been questioned. However, HPLC analysis revealed that the pigment composition of the silicoflagellate Dictyocha speculum Ehrenberg is similar to that observed in a group of haptophytes (“type 4”sensu Jeffrey and Wright 1994 . Dictyocha speculum and type 4 haptophytes possess acylfucoxanthins (19′-butanoyloxy- and 19′-hexanoyloxyfucoxanthin) in addition to fuco-, diadino-, and diatoxanthin and chl a, c, and c₃. The pigment composition of two pedinellids (Apedinella radians[Lohmann] Campbell and Mesopedinella arctica Daugbjerg), a sister group to D. speculum, deviates from D. speculum by lack of chl c ₃ and acylfucoxanthins. The distinct pigment composition suggested that plastid evolution in D. speculum differs from that of other heterokont algae. This prompted determination of the plastid-encoded rbcL gene from D. speculum to gain further insight into the evolutionary history of plastids in heterokont algae and haptophytes. A phylogenetic inference based on parsimony, maximum likelihood, and LogDet transformation methods included 35 heterokonts, 19 haptophytes, 8 red algae, and 1 cryptomonad. Three proteobacteria possessing type I RUBISCO were used to root the tree. In phylogenetic analyses, D. speculum was closely related to Rhizochromulina sp. and pedinellids, despite the latter possessing a different pigment composition. Surprisingly, the Dictyochophyceae clustered outside the lineage of heterokont algae but not within the haptophytes. Hence, analyses deduced from rbcL sequences indicated that the plastids in heterokont algae might have a more complex evolutionary history and that the shared pigment composition in D. speculum and type 4 haptophytes could be explained by convergent evolution or gene transfer. The pigment composition in D. speculum may have implications for pigment-based characterization of phytoplankton community structure in natural samples.     

Chlorophyll c-containing plastid relationships based on analyses of a multigene data set with all four chromalveolate lineages

The chlorophyll c-containing algae comprise four major lineages: dinoflagellates, haptophytes, heterokonts, and cryptophytes. These four lineages have sometimes been grouped together based on their pigmentation, but cytological and rRNA data had suggested that they were not a monophyletic lineage. Some molecular data support monophyly of the plastids, while other plastid and host data suggest different relationships. It is uncontroversial that these groups have all acquired plastids from another eukaryote, probably from the red algal lineage, in a secondary endosymbiotic event, but the number and sequence of such event(s) remain controversial. Understanding chlorophyll c-containing plastid relationships is a first step towards determining the number of endosymbiotic events within the chromalveolates. We report here phylogenetic analyses using 10 plastid genes with representatives of all four chromalveolate lineages. This is the first organellar genome-scale analysis to include both haptophytes and dinoflagellates. Concatenated analyses support the monophyly of the chlorophyll c-containing plastids and suggest that cryptophyte plastids are the basal member of the chlorophyll c-containing plastid lineage. The gene psbA, which has at times been used for phylogenetic purposes, was found to differ from the other genes in its placement of the dinoflagellates and the haptophytes, and in its lack of support for monophyly of the green and red plastid lineages. Overall, the concatenated data are consistent with a single origin of chlorophyll c-containing plastids from red algae. However, these data cannot test several key hypothesis concerning chromalveolate host monophyly, and do not preclude the possibility of serial transfer of chlorophyll c-containing plastids among distantly related hosts.     

A HYPOTHESIS FOR PLASTID EVOLUTION IN CHROMALVEOLATES1

Four eukaryotic lineages, namely, haptophytes, alveolates, cryptophytes, and heterokonts, contain in most cases photosynthetic and nonphotosynthetic members—the photosynthetic ones with secondary plastids with chl c as the main photosynthetic pigment. These four photosynthetic lineages were grouped together on the basis of their pigmentation and called chromalveolates, which is usually understood to imply loss of plastids in the nonphotosynthetic members. Despite the ecological and economic importance of this group of organisms, the phylogenetic relationships among these algae are only partially understood, and the so‐called chromalveolate hypothesis is very controversial. This review evaluates the evidence for and against this grouping and summarizes the present understanding of chromalveolate evolution. We also describe a testable hypothesis that is intended to accommodate current knowledge based on plastid and nuclear genomic data, discuss the implications of this model, and comment on areas that require further examination.     

Pinguiococcus pyrenoidosus gen. et sp. nov. (Pinguiophyceae), a new marine coccoid alga

A coccoid marine alga, collected from an aquaculture tank and maintained in culture as CCMP1144, was examined using light and electron microscopy. Young, rapidly growing cells were mostly spherical in shape, approximately 4–6 μm in diameter. Older cells often produced protrusions and pseudopodia‐like extensions, giving cells an amoeboid‐like appearance, but no amoeboid movement was observed and the pseudopodia‐like extensions exhibited no active movement. The single chloroplast had a typical photosynthetic stramenopile ultrastructure. A large stalked pyrenoid was easily observed by light microscopy. Ultrastructurally, the granular portion of the pyrenoid was divided into sections by a penetrating chloroplast envelope. A mitochondrion was often, but not always, adjacent to the pyrenoid, and in some cases the mitochondrion formed a ‘cap’ over the protruding pyrenoid. The Golgi cisternae were (when viewed in cross‐section) curved toward the nucleus. A peripheral network of anastomosing tube‐like membranes was located immediately beneath the plasmalemma. Two centrioles were located adjacent to the nuclear envelope. Lipid‐like and electron transparent vacuoles were present. Based on this investigation and data published elsewhere (large percentage of eicosapentaenoic acid, 18S rRNA and rbcL genes), this alga was described as Pinguiococcus pyrenoidosus gen. et sp. nov.     

EFFECT OF TAXON SAMPLING,CHARACTER WEIGHTING,AND COMBINED DATA ON THE INTERPRETATION OF RELATIONSHIPS AMONG THE HETEROKONT ALGAE1

Nuclear ribosomal small subunit and chloroplast rbcL sequence data for heterokont algae and potential outgroup taxa were analyzed separately and together using maximum parsimony. A series of taxon sampling and character weighting experiments was performed. Traditional classes (e.g. diatoms, Phaeophyceae, etc.) were monophyletic in most analyses of either data set and in analyses of combined data. Relationships among classes and of heterokont algae to outgroup taxa were sensitive to taxon sampling. Bootstrap (BS) values were not always predictive of stability of nodes in taxon sampling experiments or between analyses of different data sets. Reweighting sites by the rescaled consistency index artificially inflates BS values in the analysis of rbcL data. Inclusion of the third codon position from rbcL enhanced signal despite the superficial appearance of mutational saturation. Incongruence between data sets was largely due to placement of a few problematic taxa, and so data were combined. BS values for the combined analysis were much higher than for analyses of each data set alone, although combining data did not improve support for heterokont monophyly.