Nucleomorph ribosomal DNA and telomere dynamics in chlorarachniophyte algae

Chlorarachniophytes are enigmatic marine unicellular algae that acquired photosynthesis by secondary endosymbiosis. Chlorarachniophytes are unusual in that the nucleus of the engulfed algal cell (a green alga) persists in a miniaturized form, termed a nucleomorph. The nucleomorph genome of the model chlorarachniophyte, Bigelowiella natans CCMP621, is 373 kilobase pairs (kbp) in size, the smallest nuclear genome characterized to date. The B. natans nucleomorph genome is composed of three chromosomes, each with canonical eukaryotic telomeres and sub-telomeric ribosomal DNA (rDNA) operons transcribed away from the chromosome end. Here we present the complete rDNA operon and telomeric region from the nucleomorph genome of Lotharella oceanica CCMP622, a newly characterized chlorarachniophyte strain with a genome ～610 kbp in size, significantly larger than all other known chlorarachniophytes. We show that the L. oceanica rDNA operon is in the opposite chromosomal orientation to that of B. natans. Furthermore, we determined the rDNA operon orientation of five additional chlorarachniophyte strains, the majority of which possess the same arrangement as L. oceanica, with the exception of Chlorarachnion reptans and those very closely related to B. natans. It is thus possible that the ancestral rDNA operon orientation of the chlorarachniophyte nucleomorph genome might have been the same as in the independently evolved, red algal-derived, nucleomorph genomes of cryptophytes. A U2 small nuclear RNA gene was found adjacent to the telomere in Gymnochlora stellata CCMP2057 and Chlorarachnion sp. CCMP2014. This feature may represent a useful evolutionary character for inferring the relationships among extant lineages.     

Phylogeny and nucleomorph karyotype diversity of chlorarachniophyte algae

Chlorarachniophytes are flagellated and/or reticulopod-forming marine algae with chlorophyll a- and b-containing plastids of secondary endosymbiotic origin. They are one of only two algal groups known to possess a "nucleomorph" (i.e. the remnant nucleus of the eukaryotic endosymbiont that donated the plastid). Apart from the recently sequenced nucleomorph genome of Bigelowiella natans, little is known about the size, structure, and composition of chlorarachniophyte nucleomorph genomes. Toward the goal of better understanding nucleomorph genome diversity, as well as establishing a phylogenetic framework with which to interpret variation in chlorarachniophyte morphology, ultrastructure, and life cycle, we are studying a wide range of chlorarachniophyte strains from public culture collections and natural habitats. We have obtained 22 new chlorarachniophyte nuclear and nucleomorph 18S rRNA gene (18S rDNA) sequences and nucleomorph genome size estimates for 14 different strains. Consistent with previous studies, all of the chlorarachniophytes examined appear to possess three nucleomorph chromosomes. However, our results suggest considerable variation in nucleomorph genome size and structure, with individual chromosome sizes ranging from approximately 90 to approximately 210 kbp, and total genome sizes between approximately 330 kbp in Lotharella amoebiformis and approximately 610 kbp in unidentified chlorarachniophyte strain CCMP622. The significance of these phylogenetic and nucleomorph karyotype data is discussed.     

Nucleomorph and plastid genome sequences of the chlorarachniophyte Lotharella oceanica: convergent reductive evolution and frequent recombination in nucleomorph-bearing algae

Background

Nucleomorphs are residual nuclei derived from eukaryotic endosymbionts in chlorarachniophyte and cryptophyte algae. The endosymbionts that gave rise to nucleomorphs and plastids in these two algal groups were green and red algae, respectively. Despite their independent origin, the chlorarachniophyte and cryptophyte nucleomorph genomes share similar genomic features such as extreme size reduction and a three-chromosome architecture. This suggests that similar reductive evolutionary forces have acted to shape the nucleomorph genomes in the two groups. Thus far, however, only a single chlorarachniophyte nucleomorph and plastid genome has been sequenced, making broad evolutionary inferences within the chlorarachniophytes and between chlorarachniophytes and cryptophytes difficult. We have sequenced the nucleomorph and plastid genomes of the chlorarachniophyte Lotharella oceanica in order to gain insight into nucleomorph and plastid genome diversity and evolution.

Results

The L. oceanica nucleomorph genome was found to consist of three linear chromosomes totaling ~610 kilobase pairs (kbp), much larger than the 373 kbp nucleomorph genome of the model chlorarachniophyte Bigelowiella natans. The L. oceanica plastid genome is 71 kbp in size, similar to that of B. natans. Unexpectedly long (~35 kbp) sub-telomeric repeat regions were identified in the L. oceanica nucleomorph genome; internal multi-copy regions were also detected. Gene content analyses revealed that nucleomorph house-keeping genes and spliceosomal intron positions are well conserved between the L. oceanica and B. natans nucleomorph genomes. More broadly, gene retention patterns were found to be similar between nucleomorph genomes in chlorarachniophytes and cryptophytes. Chlorarachniophyte plastid genomes showed near identical protein coding gene complements as well as a high level of synteny.

Conclusions

We have provided insight into the process of nucleomorph genome evolution by elucidating the fine-scale dynamics of sub-telomeric repeat regions. Homologous recombination at the chromosome ends appears to be frequent, serving to expand and contract nucleomorph genome size. The main factor influencing nucleomorph genome size variation between different chlorarachniophyte species appears to be expansion-contraction of these telomere-associated repeats rather than changes in the number of unique protein coding genes. The dynamic nature of chlorarachniophyte nucleomorph genomes lies in stark contrast to their plastid genomes, which appear to be highly stable in terms of gene content and synteny.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-374) contains supplementary material, which is available to authorized users.     

Molecular, morphological and phylogenetic characterization of six chlorarachniophyte strains

We compare and contrast the morphological and molecular features of six chlorarachniophyte strains, and examine their evolutionary origins. Electron microscopical studies of nucleomorphs and chloroplasts, characterization of nucleomorph karyotypes, and phylogenetic analyses of small subunit ribosomal RNA (srRNA) genes derived from the nucleomorph and host cell genomes have been used to separate the six strains into three distinct groups. One group, dubbed the‘beast group’, contains the strains Chlorarachnion sp. 242, Chlor-arachnion sp. 621, Chlorarachnion sp. 1408 and Chlorarachnion sp. 1481. Members of the beast group have a novel flagellate form and are apparently picoplank-tonic. The other two groups currently contain only one species each: Chlorarachnion reptans and Lotharella sp. 240. All chlorarachniophyte nucleomorphs examined house three small linear chromosomes each furnished with telomeres and srRNA genes.     

Lotharella reticulosa sp. nov.: a highly reticulated network forming chlorarachniophyte from the Mediterranean Sea

A new chlorarachniophyte Lotharella reticulosa sp. nov. is described from a culture isolated from the Mediterranean Sea. This strain is maintained as strain RCC375 at the Roscoff Culture Collection, France. This species presents a multiphasic life cycle: vegetative cells of this species were observed to be coccoid, but amoeboid cells with filopodia and globular suspended cells were also present in the life cycle, both of which were not dominant phases. Flagellate cells were also observed but remained very rare in culture. The vegetative cells were 9-16 μm in diameter and highly vacuolated, containing several green chloroplasts with a projecting pyrenoid, mitochondria, and a nucleus. The chloroplast was surrounded by four membranes possessing a nucleomorph in the periplastidial compartment near the pyrenoid base. According to ultrastructural observations of the pyrenoid and nucleomorph, the present species belongs to the genus Lotharella in the phylum Chlorarachniophyta. This taxonomic placement is consistent with the molecular phylogenetic trees of the 18S rRNA gene and ITS sequences. This species showed a unique colonization pattern. Clusters of cells extended cytoplasmic strands radially. Then, amoeboid cells being born proximately moved distally along the cytoplasmic strand like on a "railway track". Subsequently the amoeboid cell became coccoid near the strand. In this way, daughter cells were dispersed evenly on the substratum. We also observed that the present species regularly formed a structure of filopodial nodes in mid-stage and later-stage cultures, which is a novel phenotype in chlorarachniophytes. The unique colonization pattern and other unique features demonstrate that RCC375 is a new chlorarachniophyte belonging to genus Lotharella, which we describe as Lotharella reticulosa sp. nov.     

Partenskyella glossopodia (Chlorarachniophyceae) possesses a nucleomorph genome of approximately 1 Mbp

The nucleomorph genome size of the recently described chlorarachniophyte Partenskyella glossopodia, which forms an independent lineage in the phylogeny of chlorarachniophytes, was analyzed by pulse‐field gel electrophoresis and Southern hybridization. These analyses showed that the nucleomorph genome of P. glossopodia is composed of three linear chromosomes that are about 445 kbp, 313 kbp, and 275 kbp in size. Thus, the total genome size is approximately 1033 kbp, which is significantly larger than the known size of chlorarachniophyte nucleomorph genomes, i.e. 330–610 kbp. This is the first study to report a nucleomorph genome that reaches approximately 1 Mbp in size.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Insight into the diversity and evolution of the cryptomonad nucleomorph genome

The cryptomonads are an enigmatic group of marine and freshwater unicellular algae that acquired their plastids through the engulfment and retention of a eukaryotic ("secondary") endosymbiont. Together with the chlorarachniophyte algae, the cryptomonads are unusual in that they have retained the nucleus of their endosymbiont in a miniaturized form called a nucleomorph. The nucleomorph genome of the cryptomonad Guillardia theta has been completely sequenced and with only three chromosomes and a total size of 551 kb, is a model of nuclear genome compaction. Using this genome as a reference, we have investigated the structure and content of nucleomorph genomes in a wide range of cryptomonad algae. In this study, we have sequenced nine new cryptomonad nucleomorph 18S ribosomal DNA (rDNA) genes and four heat shock protein 90 (hsp90) gene fragments, and using pulsed-field gel electrophoresis and Southern hybridizations, have obtained nucleomorph genome size estimates for nine different species. We also used long-range polymerase chain reaction to obtain nucleomorph genomic fragments from Hanusia phi CCMP325 and Proteomonas sulcata CCMP704 that are syntenic with the subtelomeric region of nucleomorph chromosome I in G. theta. Our results indicate that (1) the presence of three chromosomes is a common feature of the nucleomorph genomes of these organisms, (2) nucleomorph genome size varies dramatically in the cryptomonads examined, (3) unidentified cryptomonad species CCMP1178 has the largest nucleomorph genome identified to date at approximately 845 kb, (4) nucleomorph genome size reductions appear to have occurred multiple times independently during cryptomonad evolution, (5) the relative positions of the 18S rDNA, ubc4, and hsp90 genes are conserved in three different cryptomonad genera, and (6) interchromosomal recombination appears to be rapidly changing the size and sequence of a repetitive subtelomeric region of the nucleomorph genome between the 18S rDNA and ubc4 loci. These results provide a glimpse into the genetic diversity of nucleomorph genomes in cryptomonads and set the stage for more comprehensive sequence-based studies in closely and distantly related taxa.     

Nucleus- and nucleomorph-targeted histone proteins in a chlorarachniophyte alga

The plastid of chlorarachniophytes is distinguished by the retention of a relict nucleus (nucleomorph) derived from a green algal endosymbiont, which is located in the periplastidal compartment (PPC). The nucleomorph genome of a chlorarachniophyte, Bigelowiella natans, encodes several plastid-targeted proteins and hundreds of housekeeping proteins, but it lacks many fundamental genes to maintain itself. Here we report the first two host nucleus-encoded genes for proteins targeted to the nucleomorph, histone H2A and H2B. We identified 20 histone genes from the host nuclear genome, and based on phylogenetic analyses predicted that most of these are derived from the host, but that two histone genes are symbiont-derived. The genes both encode N-terminal extensions resembling PPC targeting signals, further suggesting they function in the nucleomorph. Using green fluorescent protein (GFP) fusion proteins expressed in transformed cells, we confirmed that the putative symbiont H2A and H2B were targeted into the nucleomorph, whereas putative host proteins were localized to the host nucleus. Furthermore, we have developed a method to temporarily synchronize B. natans cells, and confirmed that both host and symbiont histone expression is controlled during the cell cycle. Our findings provide the first evidence of how the nucleomorph may be regulated by host-encoded gene products.     

<Emphasis Type="Italic">Norrisiella sphaerica</Emphasis> gen. et sp. nov., a new coccoid chlorarachniophyte from Baja California,Mexico

A new chlorarachniophyte, Norrisiella sphaerica S. Ota et K. Ishida gen. et sp. nov., from the coast of Baja California, Mexico is described. We examined its morphology, ultrastructure, and life cycle in detail, using light microscopy, transmission electron microscopy, and time-lapse videomicroscopy. We found that this chlorarachniophyte possessed the following characteristics: (1) vegetative cells were coccoid and possessed a cell wall, (2) a pyrenoid was slightly invaded by plate-like periplastidial compartment from the tip of the pyrenoid, (3) a nucleomorph was located near the pyrenoid base in the periplastidial compartment, (4) cells reproduced vegetatively via autospores, and (5) a flagellate stage was present in the life cycle. This combination of characteristics differs from any of the described chlorarachniophyte genera, and therefore a new genus is established. Fluorescent microscopic observations suggested that the alga formed multinucleate cells prior to forming autospores. Time-lapse observations during autospore formation showed that cytokinesis occurred simultaneously in the multinucleate cells. Zoospores were also produced, and video sequences captured the release of zoospores from coccoid cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Jam packed genomes--a preliminary,comparative analysis of nucleomorphs

There are two ways eukaryotic cells can permanently acquire chloroplasts. They can take up a cyanobacterium and turn it into a chloroplast or they can engulf an alga that already has a chloroplast. The second method is far more common and there are at least seven major groups of protists that have obtained their chloroplasts, this way. In most cases little remains of the engulfed alga apart from its chloroplast, but in two groups, the cryptomonads and chlorarachniophytes, a small remnant nucleus of the engulfed alga is still present. These tiny nuclei, called nucleomorphs, are the smallest and most compact eukaryotic genomes known and recently the nucleomorph of the cryptomonad alga Guillardia theta, was completely sequenced (551 kilobases). The nucleomorph of the chlorarachniophyte Bigellowiella natans (380 kilobases), is also being sequenced and is about half complete. We discuss some of the similarities and differences that are emerging between these two nucleomorph genomes. Both genomes contain just three chromosomes that encode mainly housekeeping genes and a few proteins for chloroplast functions. The bulk of nucleomorph gene coding capacity, therefore, appears to be devoted to self perpetuation and creating gene and protein expression machineries to make a small number of essential chloroplast proteins. We discuss reasons why both nucleomorphs are extraordinarily compact and why their gene sequences are evolving rapidly.     

Genome-based reconstruction of the protein import machinery in the secondary plastid of a chlorarachniophyte alga

Most plastid proteins are encoded by their nuclear genomes and need to be targeted across multiple envelope membranes. In vascular plants, the translocons at the outer and inner envelope membranes of chloroplasts (TOC and TIC, respectively) facilitate transport across the two plastid membranes. In contrast, several algal groups harbor more complex plastids, the so-called secondary plastids, which are surrounded by three or four membranes, but the plastid protein import machinery (in particular, how proteins cross the membrane corresponding to the secondary endosymbiont plasma membrane) remains unexplored in many of these algae. To reconstruct the putative protein import machinery of a secondary plastid, we used the chlorarachniophyte alga Bigelowiella natans, whose plastid is bounded by four membranes and still possesses a relict nucleus of a green algal endosymbiont (the nucleomorph) in the intermembrane space. We identified nine homologs of plant-like TOC/TIC components in the recently sequenced B. natans nuclear genome, adding to the two that remain in the nucleomorph genome (B. natans TOC75 [BnTOC75] and BnTIC20). All of these proteins were predicted to be localized to the plastid and might function in the inner two membranes. We also show that the homologs of a protein, Der1, that is known to mediate transport across the second membrane in the several lineages with secondary plastids of red algal origin is not associated with plastid protein targeting in B. natans. How plastid proteins cross this membrane remains a mystery, but it is clear that the protein transport machinery of chlorarachniophyte plastids differs from that of red algal secondary plastids.     

Culturing Heterotrophic Protists from the Baltic Sea: Mostly the “Usual Suspects” but a Few Novelties as Well

The study of cultured strains has a long tradition in protistological research and has greatly contributed to establishing the morphology, taxonomy, and ecology of many protist species. However, cultivation‐independent techniques, based on 18S rRNA gene sequences, have demonstrated that natural protistan assemblages mainly consist of hitherto uncultured protist lineages. This mismatch impedes the linkage of environmental diversity data with the biological features of cultured strains. Thus, novel taxa need to be obtained in culture to close this knowledge gap. In this study, traditional cultivation techniques were applied to samples from coastal surface waters and from deep oxygen‐depleted waters of the Baltic Sea. Based on 18S rRNA gene sequencing, 126 monoclonal cultures of heterotrophic protists were identified. The majority of the isolated strains were affiliated with already cultured and described taxa, mainly chrysophytes and bodonids. This was likely due to “culturing bias” but also to the eutrophic nature of the Baltic Sea. Nonetheless, ~ 12% of the isolates in our culture collection showed highly divergent 18S rRNA gene sequences compared to those of known organisms and thus may represent novel taxa, either at the species level or at the genus level. Moreover, we also obtained evidence that some of the isolated taxa are ecologically relevant, under certain conditions, in the Baltic Sea.     

Preliminary characterization of carbohydrate stores from chlorarachniophytes (Division: Chlorarachniophyta)

Chlorarachniophytes are amoeboid/flagellate eukaryotes that harbor reduced green algal endosymbionts. The carbohydrate stores of chlorarachniophyte algae have been investigated using methylation analysis to determine monosaccharide composition. An appreciable quantity of long chain β-1,3 glucan occurs in these algae. Immunogold electron microscopy using an antibody specific for (β-1,3 glucans localized (β-1,3 glucans within a vacuole in the host cell cytoplasm. The results suggest that photosynthate produced by the endosymbiont is stored by the host. Implications of the data for endosymbiosis are discussed.     

Evaluating the Ribosomal Internal Transcribed Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker

Background

DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.

Methodology/Principal Findings

The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name.

Conclusions/Significance

The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.     

Nucleus-encoded periplastid-targeted EFL in chlorarachniophytes

Chlorarachniophytes are cercozoan amoeboflagellates that acquired photosynthesis by enslaving a green alga, which has retained a highly reduced nucleus called a nucleomorph. The nucleomorph lacks many genes necessary for its own maintenance and expression, suggesting that some genes have been moved to the host nucleus and their products are now targeted back to the periplastid compartment (PPC), the reduced eukaryotic cytoplasm of the endosymbiont. Protein trafficking in chlorarachniophytes is therefore complex, including nucleus-encoded plastid-targeted proteins, nucleomorph-encoded plastid-targeted proteins, and nucleus-encoded periplastid-targeted proteins. A major gap in our understanding of this system is the PPC-targeted proteins because none have been described in any chlorarachniophytes. Here we describe the first such protein, the GTPase EFL. EFL was characterized from 7 chlorarachniophytes, and 2 distinct types were found. One is related to foraminiferan EFL and lacks an amino-terminal extension. The second, distantly related, type encodes an amino-terminal extension consisting of a signal peptide followed by sequence sharing many characteristics with transit peptides from nucleus-encoded plastid-targeted proteins and which we conclude is most likely PPC targeted. Western blotting with antibodies specific to putative host and PPC-targeted EFL from the chlorarachniophytes Bigelowiella natans and Gymnochlora stellata is consistent with posttranslational cleavage of the leaders from PPC-targeted proteins. Immunolocalization of both proteins in B. natans confirmed the cytosolic location of the leaderless EFL and a distinct localization pattern for the PPC-targeted protein but could not rule out a plastid location (albeit very unlikely). We sought other proteins with a similar leader and identified a eukaryotic translation initiation factor 1 encoding a bipartite extension with the same properties. Transit peptide sequences were characterized from all 3 classes of targeted protein by comparing all examples of each class from expressed sequence tag surveys of B. natans and G. stellata. No recognizable difference between plastid- and PPC-targeted proteins was observed, but nucleomorph-encoded transit peptides differ, likely reflecting high AT content of nucleomorph genomes. Taken together, the data suggest that the system that directs proteins to the PPC in chlorarachniophytes uses a bipartite targeting sequence, as does the PPC-targeting system that evolved independently in cryptomonads.     

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.     

Nucleomorph genome diversity and its phylogenetic implications in cryptomonad algae

The relationship between phylogeny and nucleomorph genome size was examined in 16 strains of cryptomonad algae using pulsed‐field gel electrophoresis, Southern hybridization and phylogenetic analyses. Our results suggest that all cryptomonads examined in this study contain three nucleomorph chromosomes and their total genome size ranges from 495 to 750 kb. In addition, we estimated the plastid genome size of the respective organisms. The plastid genomes of photosynthetic strains were approximately 120–160 kb in size, whereas the non‐photosynthetic Cryptomonas paramecium NIES715 possesses a genome of approximately 70 kb. Phylogenetic analysis of the nuclear small subunit ribosomal DNA (SSU rDNA) gene showed that nucleomorph genome size varies considerably within closely related strains. This result indicates that the reduction of nucleomorph genomes is a rapid phenomenon that occurred multiple times independently during cryptomonad evolution. The nucleomorph genome sizes of Cryptomonas rostratiformis NIES277 appeared to be approximately 495 kb. This is smaller than that of Guillardia theta CCMP327, which until now was thought to have the smallest known nucleomorph genome size among photosynthetic cryptomonads.     

Integrative taxonomy of the Pavlovophyceae (Haptophyta): a reassessment

The Pavlovophyceae (Haptophyta) contains four genera (Pavlova, Diacronema, Exanthemachrysis and Rebecca) and only thirteen characterised species, several of which are important in ecological and economic contexts. We have constructed molecular phylogenies inferred from sequencing of ribosomal gene markers with comprehensive coverage of the described diversity, using type strains when available, together with additional cultured strains. The morphology and ultrastructure of 12 of the described species was also re-examined and the pigment signatures of many culture strains were determined. The molecular analysis revealed that sequences of all described species differed, although those of Pavlova gyrans and P. pinguis were nearly identical, these potentially forming a single cryptic species complex. Four well-delineated genetic clades were identified, one of which included species of both Pavlova and Diacronema. Unique combinations of morphological/ultrastructural characters were identified for each of these clades. The ancestral pigment signature of the Pavlovophyceae consisted of a basic set of pigments plus MV chl cPAV, the latter being entirely absent in the Pavlova + Diacronema clade and supplemented by DV chl cPAV in part of the Exanthemachrysis clade. Based on this combination of characters, we propose a taxonomic revision of the class, with transfer of several Pavlova species to an emended Diacronema genus. The evolution of the class is discussed in the context of the phylogenetic reconstruction presented.