Robert A. Andersen

The New Higher Level Classification of Eukaryotes with Emphasis on the Taxonomy of Protists

Abstract. This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional “kingdoms.” The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.

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.1

Pelagomonas calceolata gen. et sp. nov., an ultra‐planktonic marine alga, is described using electron microscopy and the cytoplasmic small subunit (18S) ribosomal RNA (rRNA) gene sequence. Cells are uniflagellate, about 1.5 × 3 μm in size. The flagellium has two rows of bipartite hairs, the paraxonemal rod has a dentate appearance, and a two‐gyred transitional helix is present between two transitional plates. Microtubular roots, striated roots, and a second basal body are absent. A thin organic theca surrounds most of the cell. There is a single chloroplast with a girdle lamella and a single, dense mitochondrion with tubular cristae. A single Golgi body with swelled cisternae lies beneath the flagellum, and each cell has an ejectile organelle that putatwely releases a cylindrical structure. A vacuole, or cluster of vacuoles, contains the putative carbohydrate storage product. The 18S rRNA gene was sequenced completely in both directions, excluding three primer regions. When compared to the same gene sequence from other organisms, Pelagomonas calceolata gen. et sp. nov. occupies an unresolved position among other chromophyte algae and is distinct from members of any of these classes. Based on morphological, ultrastructural, and molecular data, we describe this alga as a new species, and we place this highly unusual new species in a new genus, family, order, and class.

A comparison of HPLC pigment signatures and electron microscopic observations for oligotrophic waters of the North Atlantic and Pacific Oceans

Abstract The use of HPLC pigment analysis has become a primary tool for investigating the taxonomic composition of natural phytoplankton populations. In this study, we compare, for the first time, the taxonomic composition based upon HPLC pigment signatures with direct electron microscopic taxonomic identifications from two sets of open ocean oligotrophic field samples. Electron microscopic observations at sites in the Atlantic and Pacific Oceans (Hydrostation S and Station ALOHA, respectively) agree with taxonomic partitioning based upon HPLC algorithms in the, upper water-column samples, but there is increasing disagreement between the two methods in deeper water samples. This disparity probably results from depth-dependent changes in cellular pigment content and accessory pigment-to-chlorophyll ratios. At both locations, the eukaryotic ultraplankton was similar in taxonomic composition, at least at the class level, and the Prymnesiophyceae and the newly described Pelagophyceae were the two most abundant groups of eukaryotes.

Phylogenetic reconstructions of the Haptophyta inferred from 18s ribosomal DNA sequences and available morphological data

Abstract Most haptophytes are unicellular, photosynthetic flagellates, although some have coccoid, colonial, amoeboid, or filamentous stages. Nearly all have a characteristic filamentous appendage, the haptonema, arising between the two flagella. The small subunit ribosomal RNA gene (l8S ribosomal DNA) from 18 haptophyte species has been sequenced, and the sequences aligned with those of more than 300 published and unpublished chlorophyll a + c algae. Phylogenies were constructed using maximum likelihood, neighbor-joining, and weighted maximum parsimony analyses. The high divergence (6%) between members of Pavlova and the remaining haptophytes supports the division of Haptophyta into two classes: Prymnesiophyceae and Pavlovophyceae. Three major clades that correspond to known taxa within the Prymnesiophyceae were identified: one clade embraces Phaeocystis spp.; the second includes members of the genera Chrysochromulina, Prymnesium, and Imantonia; and the third includes coccolithophorid genera and the genus Isochrysis. Two other clades contain taxa whose sequences were derived from a gene clone library. These taxa are not strongly related to any of the cultured taxa included in this study. Based on 18S ribosomal DNA sequence data and available information on morphological structure and ultrastructure, we propose that the class Prymnesiophyceae be divided into four orders: Phaeocystales ord. nov., Prymnesiales, Isochrysidales, and Coccolithales. A total of 1–2% divergence at this level in the 18S ribosomal RNA gene analysis warrants a separation above the level of family. Within the Pavlovophyceae, a new genus is established, Rebecca J.C. Green gen. nov., into which Pavlova salina and Pavlova helicata are moved.

Biology and systematics of heterokont and haptophyte algae

In this paper, I review what is currently known of phylogenetic relationships of heterokont and haptophyte algae. Heterokont algae are a monophyletic group that is classified into 17 classes and represents a diverse group of marine, freshwater, and terrestrial algae. Classes are distinguished by morphology, chloroplast pigments, ultrastructural features, and gene sequence data. Electron microscopy and molecular biology have contributed significantly to our understanding of their evolutionary relationships, but even today class relationships are poorly understood. Haptophyte algae are a second monophyletic group that consists of two classes of predominately marine phytoplankton. The closest relatives of the haptophytes are currently unknown, but recent evidence indicates they may be part of a large assemblage (chromalveolates) that includes heterokont algae and other stramenopiles, alveolates, and cryptophytes. Heterokont and haptophyte algae are important primary producers in aquatic habitats, and they are probably the primary carbon source for petroleum products (crude oil, natural gas).

SYMBIODINIUM (PYRRHOPHYTA) GENOME SIZES (DNA CONTENT) ARE SMALLEST AMONG DINOFLAGELLATES

Using flow cytometric analysis of fluorescence, we measured the genome sizes of 18 cultured “free‐living” species and 29 Symbiodinium spp. isolates cultured from stony corals, gorgonians, anemones, jellyfish, and giant clams. Genome size directly correlated with cell size, as documented previously for most eukaryotic cell lines. Among the smallest of dinoflagellates, Symbiodinium spp. (6–15 μm) possessed the lowest DNA content that we measured (1.5–4.8 pg·cell−1). Bloom‐forming or potentially harmful species in the genera Alexandrium, Karenia, Pfiesteria, and Prorocentrum possessed genomes approximately 2 to 50 times larger in size. A phylogenetic analysis indicated that genome/cell size has apparently increased and decreased repeatedly during the evolution of dinoflagellates. In contrast, genome sizes were relatively consistent across distantly and closely related Symbiodinium spp. This may be the product of intracellular host habitats imposing strong selective pressures that have restricted symbiont size.

A molecular phylogeny of the heterokont algae based on analyses of chloroplast-encoded rbcL sequence data

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.

Phylogenetic relationships of the ‘golden algae’ (haptophytes, heterokont chromophytes) and their plastids

The phylogenetic relationships of the “golden algae”, like all algae, were rarely addressed before the advent of electron microscopy because, based upon light microscopy, each group was so distinct that shared characters were not apparent. Electron microscopy has provided many new characters that have initiated phylogenetic discussions about the relationships among the “golden algae”. Consequently, new taxa have been described or old ones revised, many of which now include non-algal protists and fungi. The haptophytes were first placed in the class Chrysophyceae but ultrastructural data have provided evidence to classify them separately. Molecular studies have greatly enhanced phylogenetic analyses based on morphology and have led to the description of additional new taxa. We took available nucleotide sequence data for the nuclear-encoded SSU rRNA, fucoxanthin/ chlorophyll photosystem I/II, and actin genes and the plastid-encoded SSU rRNA, tufA, and rbcL genes and analysed these to evaluate phylogenetic relationships among the “golden algae”, viz., the Haptophyceae (= Prymnesiophyceae) and the heterokont chromophytes (also known as chromophytes, heterokont algae, autotrophic stramenopiles). Using molecular clock calculations, we estimated the average and earliest probable time of origin of these two groups and their plastids. The origin of the haptophyte host-cell lineages appears to be more ancient than the origin of its plastid, suggesting that an endosymbiotic origin of plastids occurred late in the evolutionary history of this group. The pigmented heterokonts (heterokont chromophytes) also arose later, following an endosymbiotic event that led to the transfer of photosynthetic capacity to their heterotrophic ancestors. Photosynthetic haptophytes and heterokont chromophytes both appear to have arisen at or shortly before the Permian-Triassic boundary. Our data support the hypothesis that the haptophyte and heterokont chromophyte plastids have independent origins (i.e., two separate secondary endosymbioses) even though their plastids are similar in structure and pigmentation. Present evidence is insufficient to evaluate conclusively the possible monophyletic relationship of the haptophyte and heterokont protist host cells, even though haptophytes lack tripartite flagellar hairs. The molecular data, albeit weak, consistently fail to present the heterokont chromophytes and haptophytes as monophyletic. Phylogenetic resolution among all classes of heterokont chromophytes remains elusive even though molecular evidence has established the phylogenetic alliance of some classes (e.g., Phaeophyceae and Xanthophyceae).

EVOLUTION OF MACROCYSTIS SPP. (PHAEOPHYCEAE) AS DETERMINED BY ITS1 AND ITS2 SEQUENCES1

Macrocystis (Lessoniaceae) displays an antitropical distribution, occurring in temperate subtidal regions along western North America in the northern hemisphere and throughout the southern hemisphere. We used the noncoding rDNA internal transcribed spacer regions (ITS1 and ITS2) to examine relatedness among (1) Macrocystis and several genera of Laminariales, (2) four species of Macrocystis (M. integrifolia Bory from the northern hemisphere, M. angustifolia Bory and M. laevis Hay from the southern hemisphere, and M. pyrifera[L.] C. Ag. from both hemispheres), and (3) multiple clones of several individuals. Of the taxa included in our phylogenetic analysis, the elk kelp, Pelagophycus porra (Lem.) Setch., was the sister taxon to Macrocystis spp. Macrocystis individuals from the southern hemisphere (representing three species) formed a strongly to moderately supported clade, respectively, when the ITS1 and ITS2 sequences were analyzed separately. No distinction was detected between the two species in the northern hemisphere. Thus, Macrocystis may be a monospecific genus (M. pyrifera). A northern‐hemisphere‐to‐southern‐hemisphere pattern of dispersal was inferred, because northern‐hemisphere individuals were more diverse and displayed paraphyletic clades, whereas southern‐hemisphere individuals were less diverse and formed a monophyletic clade. High intraindividual variation in ITS1 sequences was observed in one individual from Santa Catalina Island (CA), suggesting very recent and rapid mixing of genotypes from areas to the north and Baja California (Mexico) or introgressive hybridization with Pelagophycus.

Environmental Barcoding Reveals Massive Dinoflagellate Diversity in Marine Environments

Background Dinoflagellates are an ecologically important group of protists with important functions as primary producers, coral symbionts and in toxic red tides. Although widely studied, the natural diversity of dinoflagellates is not well known. DNA barcoding has been utilized successfully for many protist groups. We used this approach to systematically sample known “species”, as a reference to measure the natural diversity in three marine environments. Methodology/Principal Findings In this study, we assembled a large cytochrome c oxidase 1 (COI) barcode database from 8 public algal culture collections plus 3 private collections worldwide resulting in 336 individual barcodes linked to specific cultures. We demonstrate that COI can identify to the species level in 15 dinoflagellate genera, generally in agreement with existing species names. Exceptions were found in species belonging to genera that were generally already known to be taxonomically challenging, such as Alexandrium or Symbiodinium. Using this barcode database as a baseline for cultured dinoflagellate diversity, we investigated the natural diversity in three diverse marine environments (Northeast Pacific, Northwest Atlantic, and Caribbean), including an evaluation of single-cell barcoding to identify uncultivated groups. From all three environments, the great majority of barcodes were not represented by any known cultured dinoflagellate, and we also observed an explosion in the diversity of genera that previously contained a modest number of known species, belonging to Kareniaceae. In total, 91.5% of non-identical environmental barcodes represent distinct species, but only 51 out of 603 unique environmental barcodes could be linked to cultured species using a conservative cut-off based on distances between cultured species. Conclusions/Significance COI barcoding was successful in identifying species from 70% of cultured genera. When applied to environmental samples, it revealed a massive amount of natural diversity in dinoflagellates. This highlights the extent to which we underestimate microbial diversity in the environment.