Enigmatic Diphyllatea eukaryotes: culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution.

BACKGROUND: The class Diphyllatea belongs to a group of enigmatic unicellular eukaryotes that play a key role in reconstructing the morphological innovation and diversification of early eukaryotic evolution. Despite its evolutionary significance, very little is known about the phylogeny and species diversity of Diphyllatea. Only three species have described morphology, being taxonomically divided by flagella number, two or four, and cell size. Currently, one 18S rRNA Diphyllatea sequence is available, with environmental sequencing surveys reporting only a single partial sequence from a Diphyllatea-like organism. Accordingly, geographical distribution of Diphyllatea based on molecular data is limited, despite morphological data suggesting the class has a global distribution. We here present a first attempt to understand species distribution, diversity and higher order structure of Diphyllatea. RESULTS: We cultured 11 new strains, characterised these morphologically and amplified their rRNA for a combined 18S-28S rRNA phylogeny. We sampled environmental DNA from multiple sites and designed new Diphyllatea-specific PCR primers for long-read PacBio RSII technology. Near full-length 18S rRNA sequences from environmental DNA, in addition to supplementary Diphyllatea sequence data mined from public databases, resolved the phylogeny into three deeply branching and distinct clades (Diphy I - III). Of these, the Diphy III clade is entirely novel, and in congruence with Diphy II, composed of species morphologically consistent with the earlier described Collodictyon triciliatum. The phylogenetic split between the Diphy I and Diphy II + III clades corresponds with a morphological division of Diphyllatea into bi- and quadriflagellate cell forms. CONCLUSIONS: This altered flagella composition must have occurred early in the diversification of Diphyllatea and may represent one of the earliest known morphological transitions among eukaryotes. Further, the substantial increase in molecular data presented here confirms Diphyllatea has a global distribution, seemingly restricted to freshwater habitats. Altogether, the results reveal the advantage of combining a group-specific PCR approach and long-read high-throughput amplicon sequencing in surveying enigmatic eukaryote lineages. Lastly, our study shows the capacity of PacBio RS when targeting a protist class for increasing phylogenetic resolution.

Correction to: Enigmatic Diphyllatea eukaryotes: culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution.

In the original publication of this article [1] there was an error in an author name. In this correction article the correct and incorrect name are indicated.

Collodictyon--an ancient lineage in the tree of eukaryotes.

The current consensus for the eukaryote tree of life consists of several large assemblages (supergroups) that are hypothesized to describe the existing diversity. Phylogenomic analyses have shed light on the evolutionary relationships within and between supergroups as well as placed newly sequenced enigmatic species close to known lineages. Yet, a few eukaryote species remain of unknown origin and could represent key evolutionary forms for inferring ancient genomic and cellular characteristics of eukaryotes. Here, we investigate the evolutionary origin of the poorly studied protist Collodictyon (subphylum Diphyllatia) by sequencing a cDNA library as well as the 18S and 28S ribosomal DNA (rDNA) genes. Phylogenomic trees inferred from 124 genes placed Collodictyon close to the bifurcation of the "unikont" and "bikont" groups, either alone or as sister to the potentially contentious excavate Malawimonas. Phylogenies based on rDNA genes confirmed that Collodictyon is closely related to another genus, Diphylleia, and revealed a very low diversity in environmental DNA samples. The early and distinct origin of Collodictyon suggests that it constitutes a new lineage in the global eukaryote phylogeny. Collodictyon shares cellular characteristics with Excavata and Amoebozoa, such as ventral feeding groove supported by microtubular structures and the ability to form thin and broad pseudopods. These may therefore be ancient morphological features among eukaryotes. Overall, this shows that Collodictyon is a key lineage to understand early eukaryote evolution.

Sulcozoa revealed as a paraphyletic group in mitochondrial phylogenomics.

Recently, phylogenomic analyses have been used to assign the vast majority of eukaryotes into only a handful of supergroups. However, a few enigmatic lineages still do not fit into this simple picture. Such lineages may have originated early in the history of eukaryotes and are therefore of key importance in deduction of cellular evolution. In this study, we focus on two deeply diverging lineages, Diphyllatea and Thecamonadea. They are classified in the same phylum, Sulcozoa, but previous multigene phylogenetic analyses have included only one of these two lineages. It is therefore unclear whether they constitute one group or two distinct lineages. The study of rare genomic changes reveals that both have the fused dihydrofolate reductase (DHFR) and thymidylate synthase (TS) genes (i.e. DHFR-TS), which are separated in all other unikonts that have been investigated, indicating a possible close relationship. Their phylogenetic positions have implications for the classification of Sulcozoa and the early eukaryote evolution. Here we present a phylogenomic analysis of these species that include Illumina and 454 transcriptome data from two Collodictyon strains. A total of 42 mitochondrial proteins, which correspond to orthologs published from Thecamonas trahens (Thecamonadea), were used to reconstruct their phylogenies. In the resulting trees, Collodictyon appears as sister to Amoebozoa, whereas Thecamonas branches as the closest relative of Opisthokonta (i.e. the animal, fungi and unicellular Choanozoa). In contrast, the position of another early diverging eukaryote, Malawimonas, is unresolved. The separation of Collodictyon and Thecamonas in our studies suggests that the recently proposed Sulcozoa group is most likely paraphyletic. Furthermore, the data support the hypothesis that the two supergroups Opisthokonta and Amoebozoa, which comprise a great diversity of eukaryotes, have originated from a sulcozoan ancestor.

Marine-freshwater colonizations of haptophytes inferred from phylogeny of environmental 18S rDNA sequences.

The Haptophyta is a common algal group in many marine environments, but only a few species have been observed in freshwaters, with DNA sequences available from just a single species, Crysochromulina parva Lackey, 1939. Here we investigate the haptophyte diversity in a high mountain lake, Lake Finsevatn, Norway, targeting the variable V4 region of the 18S rDNA gene with PCR and 454 pyrosequencing. In addition, the freshwater diversity of Pavlovophyceae was investigated by lineage-specific PCR-primers and clone library sequencing from another Norwegian lake, Lake Svaersvann. We present new freshwater phylotypes belonging to the classes Prymnesiophyceae and Pavlovophyceae, as well as a distinct group here named HAP-1. This is the first molecular evidence of a freshwater species belonging to the class Pavlovophyceae. The HAP-1 and another recently detected marine group (i.e. HAP-2) are separated from both Pavlovophyceae and Prymnesiophyceae and may constitute new higher order taxonomic lineages. As all obtained freshwater sequences of haptophytes are distantly related to the freshwater species C. parva, the phylogeny demonstrates that haptophytes colonized freshwater on multiple independent occasions. One of these colonizations, which gave rise to HAP-1, took place very early in the history of haptophytes, before the radiation of the Prymnesiophyceae.

Detection and quantification of the crayfish plague agent in natural waters: direct monitoring approach for aquatic environments.

Aphanomyces astaci, a specialised parasite of North American freshwater crayfish, is the disease agent of crayfish plague that is lethal to European freshwater crayfish. The life cycle of A. astaci has been inferred from experimental laboratory studies, but less is known about its natural sustainability and ecology. To address such questions, tools for monitoring of A. astaci directly in aquatic environments are needed. Here, we present an approach for detecting and quantifying A. astaci directly from water samples using species-specific TaqMan minor groove binder real-time PCR. Samples of a 10-fold dilution series from approximately 10(4) to approximately 1 spore of A. astaci were repeatedly tested, and reliable detection down to 1 spore was demonstrated. Further, to simulate real-life samples from natural water bodies, water samples from lakes of various water qualities were spiked with spores. The results demonstrated that co-extracted humic acids inhibit detection significantly. However, use of bovine serum albumin or the TaqMan Environmental Master Mix largely removes this problem. The practical application of the approach was successfully demonstrated on real-life water samples from crayfish farms in Finland hosting infected North American signal crayfish Pacifastacus leniusculus. Direct monitoring of A. astaci from aquatic environments may find application in the management of wild noble crayfish Astacus astacus stocks, improved aquaculture practices and more targeted conservation actions. The approach will further facilitate studies of A. astaci spore dynamics during plague outbreaks and in carrier crayfish populations, which will broaden our knowledge of the biology of this devastating crayfish pathogen.

A phylogenetic mosaic plastid proteome and unusual plastid-targeting signals in the green-colored dinoflagellate Lepidodinium chlorophorum.

BACKGROUND: Plastid replacements through secondary endosymbioses include massive transfer of genes from the endosymbiont to the host nucleus and require a new targeting system to enable transport of the plastid-targeted proteins across 3-4 plastid membranes. The dinoflagellates are the only eukaryotic lineage that has been shown to have undergone several plastid replacement events, and this group is thus highly relevant for studying the processes involved in plastid evolution. In this study, we analyzed the phylogenetic origin and N-terminal extensions of plastid-targeted proteins from Lepidodinium chlorophorum, a member of the only dinoflagellate genus that harbors a green secondary plastid rather than the red algal-derived, peridinin-containing plastid usually found in photosynthetic dinoflagellates. RESULTS: We sequenced 4,746 randomly picked clones from a L. chlorophorum cDNA library. 22 of the assembled genes were identified as genes encoding proteins functioning in plastids. Some of these were of green algal origin. This confirms that genes have been transferred from the plastid to the host nucleus of L. chlorophorum and indicates that the plastid is fully integrated as an organelle in the host. Other nuclear-encoded plastid-targeted protein genes, however, are clearly not of green algal origin, but have been derived from a number of different algal groups, including dinoflagellates, streptophytes, heterokonts, and red algae. The characteristics of N-terminal plastid-targeting peptides of all of these genes are substantially different from those found in peridinin-containing dinoflagellates and green algae. CONCLUSIONS: L. chlorophorum expresses plastid-targeted proteins with a range of different origins, which probably arose through endosymbiotic gene transfer (EGT) and horizontal gene transfer (HGT). The N-terminal extension of the genes is different from the extensions found in green alga and other dinoflagellates (peridinin- and haptophyte plastids). These modifications have likely enabled the mosaic proteome of L. chlorophorum.

Telonemia-specific environmental 18S rDNA PCR reveals unknown diversity and multiple marine-freshwater colonizations.

BACKGROUND: Recent surveys of eukaryote 18S rDNA diversity in marine habitats have uncovered worldwide distribution of the heterotrophic eukaryote phylum Telonemia. Here we investigate the diversity and geographic distribution of Telonemia sequences by in-depth sequencing of several new 18S rDNA clone libraries from both marine and freshwater sites by using a Telonemia-specific PCR strategy. RESULTS: In contrast to earlier studies that have employed eukaryote-wide PCR design, we identified a large and unknown diversity of phylotypes and the first rigorous evidence for several freshwater species, altogether comprising 91 unique sequences. Phylogenies of these and publicly available sequences showed 20 statistically supported sub-clades as well as several solitary phylotypes with no clear phylogenetic affiliation. Most of these sub-clades were composed of phylotypes from different geographic regions. CONCLUSIONS: By using specific PCR primers we reveal a much larger diversity of Telonemia from environmental samples than previously uncovered by eukaryote-wide primers. The new data substantially diminish the geographic structuring of clades identified in earlier studies. Nevertheless, since these clades comprise several distinct phylotypes we cannot exclude endemicity at species level. We identified two freshwater clades and a few solitary phylotypes, implying that Telonemia have colonized freshwater habitats and adapted to the different environmental and ecological conditions at independent occasions.

Draft genome assembly and transcriptome sequencing of the golden algae Hydrurus foetidus (Chrysophyceae).

Hydrurusfoetidus is a freshwater chrysophyte alga. It thrives in cold rivers in polar and high alpine regions. It has several morphological traits reminiscent of single-celled eukaryotes, but can also form macroscopic thalli. Despite its ability to produce polyunsaturated fatty acids, its life under cold conditions and its variable morphology, very little is known about its genome and transcriptome. Here, we present an extensive set of next-generation sequencing data, including genomic short reads from Illumina sequencing and long reads from Nanopore sequencing, as well as full length cDNAs from PacBio IsoSeq sequencing and a small RNA dataset (smaller than 200 bp) sequenced with Illumina. The genome sequences were combined  to produce an assembly consisting of 5069 contigs, with a total assembly size of 171 Mb and a 77% BUSCO completeness. The new data generated here may contribute to a better understanding of the evolution and ecological roles of chrysophyte algae, as well as to resolve the branching patterns at a larger phylogenetic scale.

Diversification of unicellular eukaryotes: cryptomonad colonizations of marine and fresh waters inferred from revised 18S rRNA phylogeny.

The cryptomonads is a well-defined lineage of unicellular eukaryotes, composed of several marine and freshwater groups. However, the evolutionary relationships among these groups are unclear due to conflicting inferences between morphological and molecular phylogenies. Here, we have inferred the evolutionary relationships among marine and freshwater species in order to better understand the importance of the marine-freshwater boundary on the historical diversification patterns of cryptomonads. We have constructed improved molecular phylogenies by taking into account rate variation both across sites and across sequences (covarion substitutions), and by analysing the vast majority of publicly available cryptomonad 18S rRNA sequences and related environmental phylotypes. The resulting phylogenies included 55 sequences, and revealed two novel freshwater cryptomonad clades (CRY1 and CRY2) and a large hidden diversity of cryptomonads. CRY1 was placed deeply within the cryptomonad phylogeny together with all the major freshwater lineages (i.e. Goniomonas and Cryptomonas), while CRY2 was placed within a lineage of marine species identified as Plagioselmis-like with the aid of a new sequence generated from a cultured species. The inferred phylogenies suggest only few successful marine-freshwater transitions over the history of cryptomonads. Most of the transitions seem to have occurred from marine to fresh waters, but re-colonizations of marine habitats have also taken place. This implies that the differences in the biogeophysical conditions between marine and fresh waters constitute a substantial barrier for the cross-colonization of these environments by cryptomonads.

Analysis of environmental 18S ribosomal RNA sequences reveals unknown diversity of the cosmopolitan phylum Telonemia.

Telonemia has recently been described as a new eukaryotic phylum with uncertain evolutionary origin. So far, only two Telonemia species, Telonema subtilis and Telonema antarcticum, have been described, but there are substantial variations in size and morphology among Telonema isolates and field observations, indicating a hidden diversity of Telonemia-like species and populations. In this study, we investigated the diversity and the global distribution of this group by analyzing 18S rDNA sequences from marine environmental clone libraries published in GenBank as well as several unpublished sequences from the Indian Ocean. Phylogenetic analyses of the identified sequences suggest that the Telonemia phylum includes several undescribed 18S rDNA phylotypes, probably corresponding to a number of different species and/or populations. The Telonemia phylotypes form two main groups, here referred to as Telonemia Groups 1 and 2. Some of the closely related sequences originate from separate oceans, indicating worldwide distributions of various Telonemia phylotypes, while other phylotypes seem to have limited geographical distribution. Further investigations of the evolutionary relationships within Telonemia should be conducted on isolated cultures of Telonema-like strains using multi-locus sequencing and morphological data.

Discovery of the toxic dinoflagellate Pfiesteria in northern European waters.

Several dinoflagellate strains of the genus Pfiesteria were isolated by culturing techniques from sediment samples taken in the Oslofjord region of Norway. Pfiesteria piscicida, well known as a fish killer from the Atlantic coast of America, was identified by genetic methods and light microscopy. The related species Pfiesteria shumwayae was attracted from the sediment by the presence of fish, and has proved toxic. This present survey demonstrates the wide distribution of these potentially harmful species, but so far they have not been connected with fish kills in Europe.

Botulism outbreaks in natural environments - an update.

Clostridium botulinum comprises a diverse group of botulinum toxin-producing anaerobic rod-shaped spore-forming bacteria that are ubiquitously distributed in soils and aquatic sediments. Decomposition of plants, algae, and animals creates anaerobic environments that facilitate growth of C. botulinum, which may then enter into food webs leading to intoxication of animals. Via saprophytic utilization of nutrients, the bacteria rapidly sporulate, creating a reservoir of highly robust spores. In the present review, we focus on the occurrence of C. botulinum in non-clinical environments, and examine factors influencing growth and environmental factors associated with botulism outbreaks. We also outline cases involving specific environments and their biota. In wetlands, it has been found that some C. botulinum strains can associate with toxin-unaffected organisms--including algae, plants, and invertebrates--in which the bacteria appear to germinate and stay in the vegetative form for longer periods of time. We suggest the need for future investigations to resolve issues related to the environments in which C. botulinum spores may accumulate and germinate, and where the vegetative forms may multiply.

Host-specificity and dynamics in bacterial communities associated with Bloom-forming freshwater phytoplankton.

Many freshwater phytoplankton species have the potential to form transient nuisance blooms that affect water quality and other aquatic biota. Heterotrophic bacteria can influence such blooms via nutrient regeneration but also via antagonism and other biotic interactions. We studied the composition of bacterial communities associated with three bloom-forming freshwater phytoplankton species, the diatom Aulacoseira granulata and the cyanobacteria Microcystis aeruginosa and Cylindrospermopsis raciborskii. Experimental cultures incubated with and without lake bacteria were sampled in three different growth phases and bacterial community composition was assessed by 454-Pyrosequencing of 16S rRNA gene amplicons. Betaproteobacteria were dominant in all cultures inoculated with lake bacteria, but decreased during the experiment. In contrast, Alphaproteobacteria, which made up the second most abundant class of bacteria, increased overall during the course of the experiment. Other bacterial classes responded in contrasting ways to the experimental incubations causing significantly different bacterial communities to develop in response to host phytoplankton species, growth phase and between attached and free-living fractions. Differences in bacterial community composition between cyanobacteria and diatom cultures were greater than between the two cyanobacteria. Despite the significance, major differences between phytoplankton cultures were in the proportion of the OTUs rather than in the absence or presence of specific taxa. Different phytoplankton species favoring different bacterial communities may have important consequences for the fate of organic matter in systems where these bloom forming species occur. The dynamics and development of transient blooms may also be affected as bacterial communities seem to influence phytoplankton species growth in contrasting ways.

Radiolaria divided into Polycystina and Spasmaria in combined 18S and 28S rDNA phylogeny.

Radiolarians are marine planktonic protists that belong to the eukaryote supergroup Rhizaria together with Foraminifera and Cercozoa. Radiolaria has traditionally been divided into four main groups based on morphological characters; i.e. Polycystina, Acantharia, Nassellaria and Phaeodaria. But recent 18S rDNA phylogenies have shown that Phaeodaria belongs within Cerocozoa, and that the previously heliozoan group Taxopodida should be included in Radiolaria. 18S rDNA phylogenies have not yet resolved the sister relationship between the main Radiolaria groups, but nevertheless suggests that Spumellaria, and thereby also Polycystina, are polyphyletic. Very few sequences other than 18S rDNA have so far been generated from radiolarian cells, mostly due to the fact that Radiolaria has been impossible to cultivate and single cell PCR has been hampered by low success rate. Here we have therefore investigated the mutual evolutionary relationship of the main radiolarian groups by using the novel approach of combining single cell whole genome amplification with targeted PCR amplification of the 18S and 28S rDNA genes. Combined 18S and 28S phylogeny of sequences obtained from single cells shows that Radiolaria is divided into two main lineages: Polycystina (Spumellaria+Nassellaria) and Spasmaria (Acantharia+Taxopodida). Further we show with high support that Foraminifera groups within Radiolaria supporting the Retaria hypothesis.

Freshwater Perkinsea and marine-freshwater colonizations revealed by pyrosequencing and phylogeny of environmental rDNA.

Protist parasites are ecologically important, as they can have great impact on host population dynamics and functioning of entire ecosystems. Nevertheless, little is known about their prevalence in aquatic habitats. Here, we investigate the diversity and distributional patterns of the protist parasites Perkinsus and Parvilucifera (Perkinsea). Our approach included 454 pyrosequencing of the 18S rDNA gene obtained from a high-altitude lake (Lake Finsevatn, Norway) and phylogenetic analyses of all publicly available sequences related to Perkinsea. The applied PCR primers target a 450 bp region that encompass the variable V4 region of the 18S rDNA gene and have been optimized for the Titanium upgrade of the 454 technology. Nearly 5000 sequences longer than 150 bp were recovered from nearly all eukaryotic supergroups, and of those, 13 unique sequences were affiliated to Perkinsea. Thus, our new strategy for 454 amplicon sequencing was able to recover a large diversity of distantly related eukaryotes and previously unknown species of Perkinsea. In addition, we identified 40 Perkinsea sequences in GenBank generated by other recent diversity surveys. Importantly, phylogenetic analyses of these sequences identified 17 habitat-specific marine and freshwater clades (PERK 1-17). Hence, only a few successful transitions between these habitats have taken place over the entire history of Perkinsea, suggesting that the boundary between marine and fresh waters may constitute a barrier to cross-colonizations for intracellular parasites.

Large-scale phylogenomic analyses reveal that two enigmatic protist lineages, telonemia and centroheliozoa, are related to photosynthetic chromalveolates.

Understanding the early evolution and diversification of eukaryotes relies on a fully resolved phylogenetic tree. In recent years, most eukaryotic diversity has been assigned to six putative supergroups, but the evolutionary origin of a few major "orphan" lineages remains elusive. Two ecologically important orphan groups are the heterotrophic Telonemia and Centroheliozoa. Telonemids have been proposed to be related to the photosynthetic cryptomonads or stramenopiles and centrohelids to haptophytes, but molecular phylogenies have failed to provide strong support for any phylogenetic hypothesis. Here, we investigate the origins of Telonema subtilis (a telonemid) and Raphidiophrys contractilis (a centrohelid) by large-scale 454 pyrosequencing of cDNA libraries and including new genomic data from two cryptomonads (Guillardia theta and Plagioselmis nannoplanctica) and a haptophyte (Imantonia rotunda). We demonstrate that 454 sequencing of cDNA libraries is a powerful and fast method of sampling a high proportion of protist genes, which can yield ample information for phylogenomic studies. Our phylogenetic analyses of 127 genes from 72 species indicate that telonemids and centrohelids are members of an emerging major group of eukaryotes also comprising cryptomonads and haptophytes. Furthermore, this group is possibly closely related to the SAR clade comprising stramenopiles (heterokonts), alveolates, and Rhizaria. Our results link two additional heterotrophic lineages to the predominantly photosynthetic chromalveolate supergroup, providing a new framework for interpreting the evolution of eukaryotic cell structures and the diversification of plastids.

Phenotypically different microalgal morphospecies with identical ribosomal DNA: a case of rapid adaptive evolution?

The agents driving the divergence and speciation of free-living microbial populations are still largely unknown. We investigated the dinoflagellate morphospecies Scrippsiella hangoei and Peridinium aciculiferum, which abound in the Baltic Sea and in northern temperate lakes, respectively. Electron microscopy analyses showed significant interspecific differences in the external cellular morphology, but a similar plate pattern in the characteristic dinoflagellate armor. Experimentally, S. hangoei grew in a wide range of salinities (0-30), whereas P. aciculiferum only grew in low salinities (0-3). Despite these phenotypic differences and the habitat segregation, molecular analyses showed identical ribosomal DNA sequences (ITS1, ITS2, 5.8S, SSU, and partial LSU) for both morphospecies. Yet, a strong interspecific genetic isolation was indicated by amplified fragment length polymorphism (F (ST) = 0.76) and cytochrome b (cob) sequence divergence ( approximately 1.90%). Phylogenetic reconstructions based on ribosomal (SSU, LSU) and mitochondrial (cob) DNA indicated a recent marine ancestor for P. aciculiferum. In conclusion, we suggest that the lacustrine P. aciculiferum and the marine-brackish S. hangoei diverged very recently, after a marine-freshwater transition that exposed the ancestral populations to different selective pressures. This hypothetical scenario agrees with mounting data indicating a significant role of natural selection in the divergence of free-living microbes, despite their virtually unrestricted dispersal capabilities. Finally, our results indicate that identical ITS rDNA sequences do not necessarily imply the same microbial species, as commonly assumed.

Combined heat shock protein 90 and ribosomal RNA sequence phylogeny supports multiple replacements of dinoflagellate plastids.

Dinoflagellates harbour diverse plastids obtained from several algal groups, including haptophytes, diatoms, cryptophytes, and prasinophytes. Their major plastid type with the accessory pigment peridinin is found in the vast majority of photosynthetic species. Some species of dinoflagellates have other aberrantly pigmented plastids. We sequenced the nuclear small subunit (SSU) ribosomal RNA (rRNA) gene of the "green" dinoflagellate Gymnodinium chlorophorum and show that it is sister to Lepidodinium viride, indicating that their common ancestor obtained the prasinophyte (or other green alga) plastid in one event. As the placement of dinoflagellate species that acquired green algal or haptophyte plastids is unclear from small and large subunit (LSU) rRNA trees, we tested the usefulness of the heat shock protein (Hsp) 90 gene for dinoflagellate phylogeny by sequencing it from four species with aberrant plastids (G. chlorophorum, Karlodinium micrum, Karenia brevis, and Karenia mikimotoi) plus Alexandrium tamarense, and constructing phylogenetic trees for Hsp90 and rRNAs, separately and together. Analyses of the Hsp90 and concatenated data suggest an ancestral origin of the peridinin-containing plastid, and two independent replacements of the peridinin plastid soon after the early radiation of the dinoflagellates. Thus, the Hsp90 gene seems to be a promising phylogenetic marker for dinoflagellate phylogeny.

Heterotachy processes in rhodophyte-derived secondhand plastid genes: Implications for addressing the origin and evolution of dinoflagellate plastids.

Serial transfer of plastids from one eukaryotic host to another is the key process involved in evolution of secondhand plastids. Such transfers drastically change the environment of the plastids and hence the selection regimes, presumably leading to changes over time in the characteristics of plastid gene evolution and to misleading phylogenetic inferences. About half of the dinoflagellate protists species are photosynthetic and unique in harboring a diversity of plastids acquired from a wide range of eukaryotic algae. They are therefore ideal for studying evolutionary processes of plastids gained through secondary and tertiary endosymbioses. In the light of these processes, we have evaluated the origin of 2 types of dinoflagellate plastids, containing the peridinin or 19'-hexanoyloxyfucoxanthin (19'-HNOF) pigments, by inferring the phylogeny using "covarion" evolutionary models allowing the pattern of among-site rate variation to change over time. Our investigations of genes from secondary and tertiary plastids derived from the rhodophyte plastid lineage clearly reveal "heterotachy" processes characterized as stationary covarion substitution patterns and changes in proportion of variable sites across sequences. Failure to accommodate covarion-like substitution patterns can have strong effects on the plastid tree topology. Importantly, multigene analyses performed with probabilistic methods using among-site rate and covarion models of evolution conflict with proposed single origin of the peridinin- and 19'-HNOF-containing plastids, suggesting that analysis of secondhand plastids can be hampered by convergence in the evolutionary signature of the plastid DNA sequences. Another type of sequence convergence was detected at protein level involving the psaA gene. Excluding the psaA sequence from a concatenated protein alignment grouped the peridinin plastid with haptophytes, congruent with all DNA trees. Altogether, taking account of complex processes involved in the evolution of dinoflagellate plastid sequences (both at the DNA and amino acid level), we demonstrate the difficulty of excluding independent, tertiary origin for both the peridinin and 19'-HNOF plastids involving engulfment of haptophyte-like algae. In addition, the refined topologies suggest the red algal order, Porphyridales, as the endosymbiont ancestor of the secondary plastids in cryptophytes, haptophytes, and heterokonts.