Molecular phylogeny of ocelloid-bearing dinoflagellates (Warnowiaceae) as inferred from SSU and LSU rDNA sequences
© Hoppenrath et al; licensee BioMed Central Ltd. 2009
Received: 24 February 2009
Accepted: 25 May 2009
Published: 25 May 2009
Dinoflagellates represent a major lineage of unicellular eukaryotes with unparalleled diversity and complexity in morphological features. The monophyly of dinoflagellates has been convincingly demonstrated, but the interrelationships among dinoflagellate lineages still remain largely unresolved. Warnowiid dinoflagellates are among the most remarkable eukaryotes known because of their possession of highly elaborate ultrastructural systems: pistons, nematocysts, and ocelloids. Complex organelles like these are evolutionary innovations found only in a few athecate dinoflagellates. Moreover, the taxonomy of warnowiids is extremely confusing and inferences about the evolutionary history of this lineage are mired by the absence of molecular phylogenetic data from any member of the group. In this study, we provide the first molecular phylogenetic data for warnowiids and couple them with a review of warnowiid morphological features in order to formulate a hypothetical framework for understanding character evolution within the group. These data also enabled us to evaluate the evolutionary relationship(s) between warnowiids and the other group of dinoflagellates with complex organelles: polykrikoids.
Molecular phylogenetic analyses of SSU and LSU rDNA sequences demonstrated that warnowiids form a well-supported clade that falls within the more inclusive Gymnodinium sensu stricto clade. These data also confirmed that polykrikoids are members of the Gymnodinium sensu stricto clade as well; however, a specific sister relationship between the warnowiid clade and the polykrikoid clade was unresolved in all of our analyses. Nonetheless, the new DNA sequences from different isolates of warnowiids provided organismal anchors for several previously unidentified sequences derived from environmental DNA surveys of marine biodiversity.
Comparative morphological data and molecular phylogenetic data demonstrate that the polykrikoid and the warnowiid clade are closely related to each other, but the precise branching order within the Gymnodinium sensu stricto clade remains unresolved. We regard the ocelloid as the best synapomorphy for warnowiids and infer that the most recent common ancestor of polykrikoids and warnowiids possessed both nematocysts and photosynthetic plastids that were subsequently lost during the early evolution of warnowiids. Our summary of species and genus concepts in warnowiids demonstrate that the systematics of this poorly understood group is highly problematic and a comprehensive revision is needed.
Dinoflagellates represent a major lineage of unicellular eukaryotes with unparalleled diversity and complexity in morphological features, molecular processes, nutritional modes and symbioses with distantly related organisms [1–3]. The ecological importance of dinoflagellates is also extraordinary; members of the group play key roles as marine primary producers, coral reef zooxanthellae, and (micro)consumers in aquatic communities around the globe. The monophyly of dinoflagellates and their relationship to other alveolate taxa – particularly apicomplexans and ciliates – have been convincingly demonstrated with congruent molecular phylogenetic data [e.g., [1, 4–9]]. However, the interrelationships among dinoflagellate lineages still remain largely unresolved, especially near the phylogenetic backbone of the group [e.g., [10–12]]. Although significant events in the evolutionary radiation of dinoflagellate diversity have been inferred from comparative analyses of morphological characters alone [e.g., ], the coupling of these data with molecular phylogenetic data, including environmental DNA surveys of biodiversity, has more robustly demonstrated delimitations between 'species' and the monophyly of several dinoflagellate subgroups [e.g., [10, 11, 14]].
Brief history of athecate dinoflagellate systematics
Understanding the phylogenetic relationships of athecate (syn. unarmored or naked) dinoflagellates has been problematic for more than a century, because of difficulties in identifying reliable morphological characters with light microscopy. For instance, overlapping and ambiguous criteria, such as episome dimensions and the displacement of the cingulum, have been used in the past to distinguish genera from one another. Improved methods incorporating both molecular and morphological data have been used to re-investigate the type species of different athecate genera that have long been recognized to be polyphyletic, such as Gymnodinium Stein, Gyrodinium Kofoid et Swezy, and Amphidinium Claparède et Lachmann [10, 15–18]. More precise re-definitions of these genera have caused many of the species that were formerly assigned to them to be considered "sensu lato taxa". Accordingly, several new genera have been established over the past decade to accommodate these newly recognized lineages, such as Akashiwo Hansen and Moestrup,Karenia Hansen and Moestrup, Karlodinium Larsen, Takayama de Salas, Bolch, Botes et Hallegraeff, Togula Flø Jørgensen, Murray et Daugbjerg, Prosoaulax Calado et Moestrup, and Apicoporus Sparmann, Leander et Hoppenrath [10, 19–22].
Apical surface structures found in athecate dinoflagellates using electron microscopy, such as 'acrobases' (apical grooves) and apical pores with hook-like protrusions, have turned out to be phylogenetically meaningful features that are consistent with more natural classification systems [10, 19, 22–24]. Moreover, variable features of chloroplasts (or more generally 'plastids') can be diagnostic at both the generic level, such as Karenia and Lepidodinium, and the species level, such as within Gymnodinium and Polykrikos Bütschli [e.g., [25–27]]. Characters like the formation of pseudocolonies and the possession of complex organelles are evolutionary innovations found only in a few athecate dinoflagellates, namely polykrikoids and warnowiids [27–29]. Although the value of these characteristics for establishing robust phylogenetic hypotheses is expected to be high, molecular phylogenetic data are still unavailable for most of these lineages. This is mainly due to the fact that polykrikoids and warnowiids are uncultivated and difficult to both find and isolate from natural marine samples [27, 28, 30].
Complex organelles in athecate dinoflagellates
Perhaps the most complex organelle found in any dinoflagellate so far happens to be synapomorphic for warnowiids, namely a distinctive multilayered photoreceptor called an 'ocelloid'  (see also Figure 1e). Ocelloids are composed entirely of subcellular components and are highly reminiscent in general organization to the multicellular camera eyes that evolved independently in several different lineages of metazoans (e.g. cubozoans, scallops, cephalopods and vertebrates) . The complexity of these subcellular systems is so distinctive that ocelloids have been described as "the most extraordinarily sophisticated differentiations of grouped structures for a single function in protists" . Ocelloids are comprised of two main components: a hyalosome and a melanosome . The translucent haylosome consists of a layered cornea-like structure and a lens-like inclusion that are bounded at the base by iris-like constriction rings; the melanosome is a highly ordered and pigmented retina-like body that is separated from the hyalosome by a seawater chamber. In fact, the melanosome (syn. pigment cup or retinal body) appears to be a highly derived plastid with thylacoids that can be recognized when the melanosome becomes relatively unordered during cell division and daughter cell formation; by contrast, the hyalosome appears to be synthesized by the cell and is disassembled during cell division before being reassembled in each daughter cell [; this study]. The structural details of ocelloids are specific to different warnowiid lineages (e.g. the number of constriction rings and the position of the ocelloid in the dinoflagellate cell) , and this variation provides important insights into the evolutionary history of the clade.
Other general features of warnowiids
Systematics of warnowiid genera (Warnowiaceae Lindemann 1928).
Fensome et al. 1993 
Sournia 1986 
Steidinger & Tangen 1997 
Gómez 2005 
Warnowia Lindemann 1928 (type genus)
Syn.: Pouchetia Schütt 1895
Syn.: ? Proterythropsis partim
Erythropsidinium Silva 1960
Syn.: Erythropsis Hertwig 1884
Syn.: Pouchetia partim
Syn.: Pouchetia partim
Greuetodinium Loebl. III 1980
taxon of doubtful validity
Syn.: Leucopsis Greuet 1968
taxon of doubtful validity
Nematodinium Kof. et Sw. 1921
Nematopsides Greuet 1973
taxon of doubtful validity
Proterythropsis Kof. et Sw. in Kofoid 1920
Proterythropsis Kof. et Sw. 1921
Protopsis Kof. et Sw. 1921
taxon of doubtful validity
Morphological features of warnowiid dinoflagellate genera.
position in the cell
~cell middle or posterior
ventral to anteriorly
# constriction rings
band, continuity with upper retinal body
ring, isolated from retinal body
ring, totally independent from retinal body
# per cell
1 (or 2*)
yes (and no, see Hulburt 1957
yes (and no, see Kofoid & Swezy 1921)
no (but see Hertwig 1884)
yes and no
no (but see Hulburt 1957)
upper cell half
upper cell half
middle or upper cell half
right upper cell half
median upper cell half
outward left spiral, 1 turn (or little more) = loop in Gymn. s.s.
outward left spiral, 1 turn (or little more) = loop in Gymn. s.s.
inward left spiral, 1.5–2.0 turns, some species plus outward left spiral
angled left 'spiral', 2 turns
? (ventral below the cingulum)
1 turn, steep descending dorsally
< 1–2.0 turns
< 1 turn
nearly one turn
cell 'extension' (tentacle)
1 (seldom 2*)
In this study, we provide the first molecular phylogenetic data for warnowiids: small subunit (SSU) rDNA sequences were obtained from two species of 'Warnowia' (one from British Columbia and one from Florida), two isolates of 'Nematodinium sp.' (both from British Columbia), and two isolates of 'Proterythropsis sp.' (both from British Columbia); partial large subunit (LSU) rDNA sequences were obtained from one species of 'Nematodinium' (from British Columbia) and one species of Warnowia (from British Columbia). Moreover, we obtained partial LSU rDNA sequences from three polykrikoid species: Polykrikos kofoidii (from British Columbia), Polykrikos lebourae (from British Columbia), and 'Pheopolykrikos' hartmannii (from Maryland). These molecular phylogenetic data were coupled with a review of warnowiid morphological data (Table 2) in order to formulate a hypothetical framework for understanding character evolution within the group.
Collection of organisms and light microscopy
Near surface plankton samples were collected in the morning hours with a small net (mesh-size 20 μm) at the docks of the Bamfield Marine Sciences Center, Vancouver Island, BC (48°50.0' N, 125°8.0' W) in June 2005, April 2006, April 2007 and May 2007. Immediately after sampling, single cells of the species were identified at 40× to 250× magnifications and isolated from the mixed plankton sample by micropipetting for the preparations described below. Cells were observed directly and micromanipulated with a Leica DMIL inverted microscope connected to a PixeLink Megapixel color digital camera. For DIC light microscopy, micropipetted cells were placed on a glass specimen slide and covered with a cover slip. Images were produced directly with either the PixeLink Megapixel color digital camera or a Zeiss Axioplan 2 imaging microscope connected to a Leica DC500 color digital camera. Sand samples containing Polykrikos lebourae were collected with a spoon during low tide at Centennial Beach, Boundary Bay, BC (49°0.0' N, 123°8.0' W) in May 2007. The sand samples were transported directly to the laboratory, and the flagellates were separated from the sand by extraction through a fine filter (mesh size 45 μm) using melting seawater-ice . The flagellates accumulated in a Petri dish beneath the filter and were then identified at 40× to 250× magnifications. Cells were isolated by micropipetting for the preparations described below.
Samples containing Erythropsidinium and an unidentified "Warnowia sp." (Florida) were collected from the Gulf Stream off of Ft. Pierce, Florida (N27° 28.25' and W79° 53.62') on August 28, 2007. Pheopolykrikos hartmannii was collected from the Rhode River, MD at the Smithsonian Environmental Research Center (SERC) dock (N38° 53.1' W 76° 32.5') on July 31, 2007. A horizontal plankton tow was taken from the surface layer using a net with a mesh-size of 35 μm. Samples were held at ambient temperature and transported to the lab. The sample was screened using a 250 μm-mesh Nitex sieve to remove large zooplankton, and diluted with seawater to enhance viability during transport. Cells were visualized through a dissecting microscope and individually picked using drawn glass tubing and mouth aspiration. Each cell was washed six times with 0.2 μm filtered station water, photographed, placed into a sterile 1.5-mL microfuge tube containing 40 μL of lysis buffer, amended with Igepal instead of nonidet P40, and frozen at -80°C .
Scanning electron microscopy
A mixed-extraction sample containing 'Proterythropsis' sp. was fixed with OsO4 for 30 min at room temperature. Cells were transferred onto a 5-μm polycarbonate membrane filter (Corning Separations Div., Acton, MA), washed with distilled water, dehydrated with a graded series of ethanol and critical point dried with CO2. The filter was mounted on a stub, sputter-coated with gold and viewed under a Hitachi S4700 Scanning Electron Microscope.
DNA extraction, PCR amplification and sequencing
Primers used for PCR.
Date of collection, number of isolated cells, method of DNA extraction and primer combination for each DNA sequence reported in this study.
# of cells
'Proterythropsis' sp. 1
24. June 2005
'Proterythropsis' sp. 2
25. June 2005
'Nematodinium' sp. 1
27. April 2006
'Nematodinium' sp. 2
28. April 2006
Warnowia sp. (BC)
02. May 2007
"Warnowia sp." (Florida)
28. Aug. 2007
28. April 2007
28. April 2007
Warnowia sp. (BC)
02. May 2007
18. May 2007
31. July 2007
Warnowiid cells collected from the east coast of North America were frozen in microfuge tubes before being thawed and sonicated using a probe tipped sonicator (Heat Systems Ultrasonic, Inc. Model W-225R, Plain View, NY) set to a power level of 3 and a 30% duty cycle. The sonicator probe was immersed in the sample, and three to five pulses of sonication were used over ~5 seconds. The probe was washed between each sample with 10% bleach solution, rinsed with distilled water, and wiped dry with a kimwipe. Dummy samples without cells were sonicated and used as negative controls. Ribosomal DNA regions were amplified with the following primer combinations EukA-EukB (SSU), Dino1662F-25R1 (ITS and part of LSU) and 25F1-LSUR2 (last part of LSU) for 'Pheopolykrikos' hartmannii (Table 3) [58–62]. The resulting products were sequenced with the same primers they were amplified with and the primers D3A and DLSUR2 (Table 3)  and assembled into a contig of 3,637 bases covering the SSU-ITS-LSU region. For the "Warnowia sp." (Florida), the EukA-EukB PCR product was used as a template for the following nested PCR amplifications: EukA-DinoR, SR4–SR9 and SR8–SR12 (Table 3) [58, 59, 61, 63]; single PCR bands derived from each primer pair were sequenced with internal primers shown in Table 3. PCR experiments were run in 20-μL volumes with the following final concentrations: 500 mg/mL BSA (Sigma A2053), 50 mM Tris HCl (pH 8.3), 3 mM Mg, 10 μM dNTPs, 0.12 units of Promega Go-Taq, and 4 μL of sample (1 μL in the case of nested PCR). Cycling conditions were 95°C for 2 min, followed by 40 cycles of the following: 95°C for 30 s, 55°C for 30 s, 72°C for 1.5 min. This was followed with a 72°C step for 5 min after which the reactions were held at 4°C. The products from these reactions were visualized on ethidium bromide stained 1.5% agarose gels, precipitated using PEG (20% w/v polyethylene glycol, mw 8000, 2.5 M NaCl solution), washed with 70% ethanol, briefly air dried, resuspended in 10 μL of distilled water, and sequenced using Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystem, Foster City, California) and an ABI 3730 sequencer. Sequence identity was evaluated initially by BLAST using the NCBI nonredundant database  and then by phylogenetic analyses.
Alignments and molecular phylogenetic analyses
The new SSU and LSU rDNA sequences were aligned with other alveolate sequences using MacClade 4 , forming a 45-taxon and 36-taxon alignment respectively. However, we also analyzed our new LSU sequences within the context of several shorter-length sequences retrieved from GenBank, forming a 47-taxon alignment, and concatenated our SSU-LSU rDNA sequences where possible, forming a 17-taxon alignment. These four alignments are available on request. Maximum likelihood (ML) and Bayesian methods using the General Time Reversible (GTR) model of nucleotide substitutions were performed on all four alignments, this model was selected with MODELTEST version 3.06 . All gaps were excluded from the alignments prior to phylogenetic analysis. The alpha shape parameters were estimated from the data using GTR, a gamma distribution with invariable sites and four rate categories (45-taxon SSU alignment with 1693 sites: ∝ = 0.378, i = 0.282; 36-taxon LSU alignment with 855 sites: ∝ = 0.509, i = 0.115; 47-taxon LSU alignment with 358 sites: ∝ = 0.742, i = 0.132; 17-taxon SSU-LSU alignment with 2549 sites: ∝ = 0.469, i = 0.339). ML trees analyzed using the parameters listed above were constructed with PhyML [67, 68]. ML bootstrap analyses were also performed with PhyML (GTR+I+G model) on five hundred re-sampled datasets (one heuristic search per dataset) from each of the four alignments.
The four alignments were analyzed with Bayesian methods using the MrBayes program 3.1.2 [69, 70]. The program was set to operate with a gamma distribution and four Monte-Carlo-Markov chains (MCMC) starting from a random tree. A total of 2,000,000 generations were calculated with trees sampled every 50 generations and with a prior burn-in of 100,000 generations (2000 sampled trees were discarded; burn-in was checked manually). A majority rule consensus tree was constructed from 38,001 post-burn-in trees. Posterior probabilities correspond to the frequency at which a given node was found in the post-burn-in trees. Independent Bayesian runs on each alignment yielded similar results.
Results & Discussion
Warnowiids evolved from within the Gymnodinium sensu stricto clade
The phylogenetic relationships within the warnowiid clade were poorly resolved near the backbone (Figure 3). However, the molecular phylogenetic analyses did demonstrate that the two isolates of 'Nematodinium' grouped strongly together, and this clade formed the nearest sister group to the Warnowia sp. collected from British Columbia (Figures 3 and 4, Additional files 1 and 2). The two isolates of 'Proterythropsis' also grouped strongly together with an environmental sequence, namely EF527120, collected from Framvaren Fjord, Norway, (Figure 3) [A. Behnke, pers. comm.]. The "Warnowia sp." collected from Florida was much more divergent and clustered strongly with several environmental sequences collected from Sargasso Sea (Figure 3) [Armbrust et al., unpublished]; two of the environmental sequences, namely AY664983 and AY665026, formed the nearest sister lineages to this "Warnowia sp." (Florida) (Figure 3). The molecular phylogenetic analyses of SSU rDNA also indicated that the following environmental sequences probably represent a single, potentially undescribed, warnowiid species: AY664914, AY664912, AY664911 and AY664896 (Figure 3). The generated SSU rDNA sequence for Erythropsidinium was too divergent to be included into the analysis.
Daugbjerg et al.  hypothesized that the warnowiid Nematodinium armatum is related to the Gymnodinium s.s., because of the presence of a nuclear fibrous connector and the loop-shaped acrobase. This is the only published hypothesis about the possible relationship of a warnowiid taxon to other dinoflagellate taxa, and our molecular phylogenetic analyses reinforced this hypothesis. The characteristic feature for the Gymnodinium s.s. clade is the loop-shaped acrobase which has been demonstrated in the Gymnodinium species within this clade, Lepidodinium, 'Pheopolykrikos' hartmannii, Polykrikos spp., and Nematodinium armatum [e.g., [24, 26–28]]. We have shown for the first time that a loop-shaped acrobase is also present in 'Proterythropsis' sp. (Figure 2) and hypothesize, based on our molecular phylogenetic data, that a similar acrobase morphology is present in the 'Nematodinium' and Warnowia species isolated from British Columbia (Figure 3).
We present the first LSU rDNA sequence for P. lebourae. Although a strongly supported Polykrikos clade, within the Gymnodinium s.s. clade, was shown previously in SSU phylogenies [27, 28] (Figure 3), this clade did not receive strong statistical support in our phylogenetic analyses of LSU rDNA sequences (Figure 4 and Additional file 1) .
Character evolution in warnowiids
The ocelloid is perhaps the most striking feature of all dinoflagellates and is the best synapomorphy for the warnowiid clade as inferred from SSU- and LSU-rDNA sequences (Figures 3, 4 and 5). Greuet  performed the pioneering ultrastructural research on warnowiids, and the structural diversity found in different ocelloids led him to formulate a hypothesis about character evolution within the group; variations of ocelloids have been described in Nematodinium, 'Warnowia morphotype II', and Erythropsidinium [35, 42–48]. Greuet  hypothesized that during the evolution of warnowiids, the ocelloid increased in complexity:
(1) the number of (iris-like) constriction rings increased from 1 → 2 → 3, (2) the ocelloid increased in size, and (3) the position of the ocelloid gradually shifted toward the anterior end of the cell (Table 2, Figure 5). We have extended this hypothesis by incorporating the following events: (4) the presence of nematocysts is an ancestral state that was subsequently lost in more derived lineages, (5) the piston was gained in the most recent ancestor of Erythropsidinium and Greuetodinium, and (6) the lense region or 'hyalosome' of the ocelloid was multiplied in the most recent ancestor of Greuetodinium (Figure 5). Moreover, because Nematodinium armatum contains nematocysts and is the only known photosynthetic species, we hypothesize that photosynthesis involving typical peridinin-containing dinoflagellate plastids is an ancestral state for warnowiids that was subsequently lost early in the history of the clade (Figure 5). Mornin and Francis  wrote that the plastids of N. armatum lack thylakoids, which would preclude photosynthesis; however, one of the TEM images published in this study (namely, Figure "a" on plate III) shows a plastid with many thylakoids. Because the relatively low magnification of this image does not allow us to determine whether the plastids have the usual dinoflagellate morphology (i.e., thylakoids in stacks of three and three outer membranes), an ultrastructural reinvestigation of this species is needed.
The species from which we were able to acquire SSU- and LSU-rDNA sequences represent only a part of the morphological diversity found in warnowiid dinoflagellates. All of the species we examined possessed an ocelloid located near the posterior end of the cell, which is inferred to be an ancestral state for the group. However, one species, namely 'Proterythropsis' sp., possessed nematocysts, and one species, namely 'Nematodinium' sp., was photosynthetic. Interestingly, the taxa without nematocysts, namely 'Nematodinium' sp. and Warnowia sp. (British Columbia), clustered together in the phylogenies inferred from SSU rDNA sequences, albeit with weak support (Figure 3); these taxa also clustered together in the phylogeneis inferred from LSU rDNA sequences, but they were the only two warnowiid species in the analyses (Figure 4 and Additional files 1 and 2). The "Warnowia sp." (Florida) showed the most divergent position in the phylogenies inferred from SSU rDNA sequences and clustered strongly with environmental sequences (Figure 3). Although all of these results are consistent with the hypothetical framework(s) shown in Figure 5, SSU- and LSU-rDNA sequences from relatively scarce planktonic warnowiids – like Warnowia morphotype II, Erythropsidinium, and Greuetodinium – will be required to more comprehensively evaluate character evolution within the group.
A survey of the literature on warnowiids reveals several taxonomic problems associated with the genera Warnowia, Nematodinium, and Proterythropsis (Table 2). For instance, Warnowia currently contains two very different morphotypes with respect to the cingulum, sulcus, acrobase(s), and the position of the ocelloid [24, 29, 36, 37] (Table 2). Species in the genus are characterized by the absence of features, such as plastids, nematocysts, pistons, and posterior cell extensions (Table 2). Warnowia morphotype I is similar to Nematodinium armatum, and the cells of Warnowia morphotype II are distinctively elongated (Figure 5). Nematodinium species also show a wide range of cingulum and sulcus morphologies, but are defined by the presence of nematocysts and a posterior ocelloid and the absence of a posterior cell extension (Table 2). However, Hulburt  reported specimens of Nematodinium armatum without nematocysts. These cells also fit the circumscription of Warnowia type I, but they are photosynthetic [34, 35, 49]. The genus Proterythropsis is recognized by the presence of a posterior cell extension and is circumscribed as having a posterior ocelloid and nematocysts (Table 2, Figure 5) [36, 38]; however, Kofoid and Swezy  reported Proterythropsis cells without nematocysts. Therefore, it is unclear whether nematocysts are a stable taxonomic character at any level in the phylogenetic hierarchy. In fact, it is not even clear whether all specimens of a species possess nematocysts during all stages of their life cycle or whether there are situations when nematocysts are simply unrecognizable with light microscopy during some developmental stages [e.g., ].
Species concepts within warnowiids are also highly problematic. It has been shown that the structure, color and position of the ocelloid can change during the course of cell development and division  (also see our Figures 1a and 1d of the same species). Because of this variability, many previously described species probably represent conspecifics [e.g., [36–38]].
These taxonomic ambiguities have made the identification of warnowiid genera and species extremely challenging for us when isolating cells from natural samples. Therefore, in this study, we have decided not to name the taxa to the species level and to demarcate ambiguous genus assignments with quotation marks. Our 'Proterythropsis' sp. has a relatively short posterior cell 'extension' and nematocysts (Figures 1a, b, c, d, e and 1f); it essentially possesses morphological features that are intermediate between Proterythropsis sensu stricto and heterotrophic Nematodinium cells. Although our 'Nematodinium' sp. looks most like Nematodinium armatum, we were unable to detect nematocysts (Figures 1m, n, o and 1p). It is possible that this species best represents an undescribed photosynthetic version of Warnowia morphotype I; however, because these cells could also represent Nematodinium armatum without detectible nematocysts, as observed by Hulburt , we decided to tentatively assign this species to Nematodinium. The "Warnowia sp." (Florida) (Figures 1j and 1k) possessed morphological features that were intermediate between morphotypes I and II, but we are not able to confirm the presence or absence of nematocysts; only one specimen was available for our investigations. The Warnowia sp. (British Columbia) possessed the morphological features of morphotype I (Figures 1g, h and 1i) and divided within hyaline cysts. The genus Erythropsidinium was the easiest to identify due to the conspicuous piston and the anterior position of the ocelloid (Figures 1q and 1r).
Warnowiid environmental sequences
The DNA sequences we report here demonstrate that warnowiid dinoflagellates have been unknowingly recorded in previous environmental PCR surveys of biodiversity (Figure 3, accession numbers represent environmental sequences of uncultured eukaryotes with unknown morphology/identity). 'Proterythropsis' sp. or a very close relative (EF527120, Figure 2) was sequenced/detected in a sample from Framvaren Fjord, Norway, in September 2005 (A. Behnke, pers. comm.). The sampled water was anoxic with measurable H2S (unpublished data, A. Behnke, pers. comm.). To the best of our knowledge, this is the first report of a warnowiid from anoxic habitats. Relatives of "Warnowia sp." (Florida) were sequenced from nanoplankton samples taken from Sargasso Sea eddies (Armbrust et al. unpublished, from GenBank, acc. no. AY664983 and AY665026). Four additional sequences of that survey (Armbrust et al. unpublished, from GenBank, acc. no. AY664911, AY664912, AY664914, and AY664896) clustered together as sister lineage(s) to the "Warnowia sp." (Florida) clade (Figure 3). How warnowiids could be part of the nanoplankton fraction of a water sample is difficult to explain with present data and might indicate a more complex lifecycle involving nanoflagellated stages; all described species of warnowiids belong to the microplankton (> 20 μm). Contamination of the nanoplankton sample with free-floating DNA from ruptured microplanktonic organisms could also explain these findings. Nonetheless, the warnowiid environmental sequences demonstrate previously undetected diversity in the group and the sequences that we report here help provide cellular identities to these clades.
This study reports the first molecular phylogenetic data from uncultivated warnowiid dinoflagellates collected from both the Pacific and Atlantic Oceans, namely SSU- and LSU-rDNA sequences from probably three different genera of warnowiids ('Nematodinium', Warnowia and 'Proterythropsis'), and partial LSU-rDNA sequences from three different species of polykrikoids (P. kofoidii, P. lebourae, and 'Pheopolykrikos' hartmannii). All of the investigated species clustered within the Gymnodinium sensu stricto (s.s.) clade with very strong support, which was concordant with comparative morphological data. The warnowiid sequences clustered together in one well-supported clade, which reinforced the ocelloid as the best synapomorphy for this group. Comparative morphological data and molecular phylogenetic data demonstrate that the polykrikoid clade and the warnowiid clade are closely related to each other, but the precise branching order within the Gymnodinium s.s. clade remains unresolved. Nonetheless, the most parsimonious scenario of character evolution suggests that the most recent common ancestor of polykrikoids and warnowiids possessed nematocysts, and probably photosynthetic plastids, that were subsequently lost during the early evolution of the warnowiid clade. Species and genus concepts in warnowiids are highly problematic and a comprehensive taxonomic revision is needed in order to better understand the evolutionary history of the group. However, additional molecular and morphological data is severely hindered by the extraordinary rarity that these planktonic dinoflagellates are encountered in natural samples. Accordingly, this study represents a first step toward meeting these aims and provides a set of preliminary DNA barcodes for warnowiids that not only helps advance the systematics of the group, but also improves inferences about the evolutionary history that gave rise to some of the most sophisticated organelles ever discovered in eukaryotic cells: ocelloids.
List of abbreviations
- GTR model:
general time-reversable model
- HKY model:
- LSU rDNA:
large subunit ribosomal DNA
polymerase chain reaction
- SSU rDNA:
small subunit ribosomal DNA.
TRB and SH would like to thank Dr. D. Wayne Coats for help with sample isolation and photography. This article is contribution number 780 from the Smithsonian Marine Station in Ft. Pierce, FL, U.S.A. This work was supported by a postdoctoral research salary to MH from the Deutsche Forschungsgemeinschaft (grant Ho3267/1-1) and operating funds from the National Science Foundation – Assembling the Tree of Life (NSF #EF-0629624) and the National Science and Engineering Research Council of Canada (NSERC 283091-04). BSL is a fellow of the Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity.
- Taylor FJR: The biology of dinoflagellates. 1987, Oxford: Blackwell Scientific Publications
- Hackett JD, Anderson DM, Erdner DL, Bhattacharya D: Dinoflagellates: a remarkable evolutionary experiment. Am J Bot. 2004, 91 (10): 1523-1534.View ArticlePubMed
- Taylor FJR, Hoppenrath M, Saldarriaga JF: Dinoflagellate diversity and distribution. Biodivers Conserv. 2008, 17: 407-418.View Article
- Maroteaux L, Herzog M, Soyer-Gobillard MO: Molecular organization of dinoflagellate ribosomal DNA: molecular implications of the deduced 5.8S rRNA secondary structure. Bioystems. 1985, 18: 307-319.View Article
- Cavalier-Smith T: Kingdom Protozoa and its 18 phyla. Microbiol Rev. 1993, 57: 953-994.PubMed CentralPubMed
- Fensome RA, Taylor FJR, Norris G, Sarjeant WAS, Wharton DI, Williams GL: A classification of living and fossil dinoflagellates. Am Mus Nat Hist. 1993, 7: 1-351.
- Patterson D: The diversity of eukaryotes. Am Nat. 1999, 154: 96-124.View Article
- Leander BS, Keeling PJ: Morphostasis in alveolate evolution. Trends Ecol Evol. 2003, 18: 395-402.View Article
- Leander BS, Keeling PJ: Early evolutionary history of dinoflagellates and apicomplexans (Alveolata) as inferred from hsp90 and actin phylogenies. J Phycol. 2004, 40: 341-350.View Article
- Daugbjerg N, Hansen G, Larsen J, Moestrup Ø: Phylogeny of some of the major genera of dinoflagellates based on ultrastructure and partial LSU rDNA sequence data, including the erection of three new genera of unarmoured dinoflagellates. Phycologia. 2000, 39: 302-317.View Article
- Saldarriaga JF, Taylor FJR, Cavalier-Smith T, Menden-Deuer S, Keeling PJ: Molecular data and the evolutionary history of dinoflagellates. Europ J Protistol. 2004, 40: 85-111.View Article
- Murray S, Flø Jørgensen M, Ho SYW, Patterson DJ, Jermiin LS: Improving the analysis of dinoflagellate phylogeny based on rDNA. Protist. 2005, 156: 269-286.View ArticlePubMed
- Taylor FJR: On dinoflagellate evolution. BioSystems. 1980, 13: 65-108.View ArticlePubMed
- Taylor FJR: Illumination or confusion? Dinoflagellate molecular phylogenetic data viewed from a primarily morphological standpoint. Phycol Res. 2004, 52: 308-324.View Article
- Hansen G, Moestrup O, Roberts KR: Light and electron microscopical observations on the type species of Gymnodinium, G. fuscum (Dinophyceae). Phycologia. 2000, 39: 365-376.View Article
- Hansen G, Daugbjerg N: Ultrastructure of Gyrodinium spirale, the type species of Gyrodinium (Dinophyceae), including a phylogeny fo G. dominans, G. rubrum and G. spirale deduced from partial LSU rDNA sequences. Protist. 2004, 155: 271-294.View ArticlePubMed
- Takano Y, Horiguchi T: Surface ultrastructure and molecular phylogenetics of four unarmored heterotrophic dinoflagellates, including the type species of the genus Gyrodinium (Dinophyceae). Phycol Res. 2004, 52: 107-116.View Article
- Flø Jørgensen M, Murray S, Daugbjerg N: Amphidinium revisited. I. Redefinition of Amphidinium (Dinophyceae) based on cladistic and molecular phylogenetic analyses. J Phycol. 2004, 40: 351-365.View Article
- De Salas MF, Bolch CJS, Botes L, Nash G, Wright SW, Hallegraeff GM: Takayama gen. nov. (Gymnodiniales, Dinophyceae), a new genus of unarmored dinoflagellates with sigmoid apical grooves, including the description of two new species. J Phycol. 2003, 39: 1233-1246.View Article
- Flø Jørgensen M, Murray S, Daugbjerg N: A new genus of athecate interstitial dinoflagellates, Togula gen. nov., previously encompassed within Amphidinium sensu lato: Inferred from light and electron microscopy and phylogenetic analyses of partial large subunit ribosomal DNA sequences. Phycol Res. 2004, 52: 284-299.View Article
- Calado AJ, Moestrup Ø: On the freshwater dinoflagellates presently includeed in the genus Amphidinium, with a description of Prosoaulax gen. nov. Phycologia. 2005, 44: 112-119.View Article
- Sparmann SF, Leander BS, Hoppenrath M: Comparative morphology and molecular phylogeny of taxa of the new marine benthic dinoflagellate genus Apicoporus, classified formerly within Amphidinium sensu lato. Protist. 2008, 159: 383-399.View ArticlePubMed
- Biecheler B: Recherches sur les péridiniens. Bull Biol France Belgique, Suppl. 1952, 36: 1-149.
- Takayama H: Apical grooves of unarmored dinoflagellates. Bull Plankton Soc Japan. 1985, 32 (2): 129-140.
- De Salas MF, Bolch CJS, Hallegraeff GM: Karenia umbella sp. nov. (Gymnodiniales, Dinophyceae), a new potentially ichthyotoxic dinoflagellate species from Tasmania, Australia. Phycologia. 2004, 43 (2): 166-175.View Article
- Hansen G, Botes L, de Salas M: Ultrastructure and large subunit rDNA sequences of Lepidodinium viride reveal a close relationship to Lepidodinium chlorophorum comb. nov. (= Gymnodinium chlorophorum). Phycol Res. 2007, 55: 25-41.View Article
- Hoppenrath M, Leander BS: Morphology and phylogeny of the pseudocolonial dinoflagellates Polykrikos lebourae and Polykrikos herdmanae n. sp. Protist. 2007, 158: 209-227.View ArticlePubMed
- Hoppenrath M, Leander BS: Character evolution in polykrikoid dinoflagellates. J Phycol. 2007, 43: 366-377.View Article
- Greuet C: Complex organelles. The Biology of Dinoflagellates. Edited by: Taylor FJR. 1987, Oxford: Blackwell Science Publishers, Botanical Monographs, 21: 119-142.
- Gómez F: Erythropsidinium (Gymnodiniales, Dinophyceae) in the Pacific Ocean, a unique dinoflagellate with an ocelloid and a piston. Eur J Prostistol. 2008, 44 (4): 291-298.View Article
- Westfall JA, Bradbury PC, Townsend JW: Ultrastructure of the dinoflagellate Polykrikos. J Cell Sci. 1983, 63: 245-261.PubMed
- Shostak S, Kolluri V: Symbiogenetic origins of cnidarian cnidocysts. Symbiosis. 1995, 19 (1): 1-29.
- Leander BS: Different modes of convergent evolution reflect phylogenetic distances. Trends Ecol Evol. 2008, 23: 481-482.View ArticlePubMed
- Dogiel V: Beiträge zur Kenntnis der Peridineen. Mitt Zool Staatsinst Neapel. 1906, 18: 1-45.
- Mornin L, Francis D: The fine structure of Nematodinium armatum, a naked dinoflagellate. J Microscopie. 1967, 6: 759-772.
- Kofoid CA, Swezy O: The free-living unarmored Dinoflagellata. 1921, Berkeley, University of California Press; Memoirs of the University of California, 5: 1-559.
- Elbrächter M: On the taxonomy of unarmoured dinophytes (Dinophyta) from the Northwest African upwelling region. "Meteor" Forsch-Ergebnisse. 1979, D (30): 1-22.
- Greuet C: Les critères de determination chez les Péridiniens Warnowiidae Lindemann. Protistologica. 1972, 8 (4): 461-469.
- Sournia A: Atlas du Phytoplankton Marin. Vol. I: Introduction, Cyanophycées, Dictyochophycées, Dinophycées et Raphidophycées. 1986, Edition du CNRS, Paris, 26-98.
- Steidinger KA, Tangen K: Dinoflagellates. Identifying Marine Phytoplankton. Edited by: Tomas CR. 1997, San Diego: Academic Press, 387-584.View Article
- Gómez F: A list of free-living dinoflagellate species in the world's oceans. Acta Bot Croat. 2005, 64 (1): 129-212.
- Greuet C: Organisation ultrastructurale de l'ocelloide de Nematodinium. Aspect phylogénétique de l'évolution du photorécepteur des Péridiniens Warnowiidae Lindemann. Cytobiologie. 1978, 17: 114-136.PubMed
- Francis D: On the eyespot of the dinoflagellate, Nematodinium. J Exp Biol. 1967, 47: 495-501.PubMed
- Greuet C: Structure fine de l'ocelle d'Erythropsis pavillardi Hertwig, Péridinien Warnowiidae Lindemann. C R Acad Sc Paris. 1965, 261: 1904-1907.
- Greuet C: Organisation ultrastructurale de l'ocelle de deux Péridiniens Warnowiidae, Erythropsis pavillardi Kofoid et Swezy et Warnowia pulchra Schiller. Protistologica. 1968, 4 (2): 209-230.
- Greuet C: Étude morphologique et ultrastructurale du trophonte d'Erythropsis pavillardi Kofoid et Swezy. Protistologica. 1969, 5 (4): 481-503.
- Greuet C: Étude ultrastructurale et évolution des cnidocystes de Nematodinium, Péridinien Warnowiidae Lindemann. Protistologica. 1971, 7 (3): 345-355.
- Greuet C: Évolution structurale et ultrastructurale de l'ocelloide d'Erythropsis pavillardi Kofoid et Swezy (Péridinien Warnowiidae Lindemann) au cours des divisions binaire et palintomiques. Protistologica. 1977, 13 (1): 127-143.
- Hulburt EM: The taxonomy of unarmored Dinophyceae of shallow embayments on Cape Cod, Massachusetts. Biol Bull. 1957, 112 (2): 196-219.View Article
- Hertwig R: Erythropsis agilis. Eine neue Protozoe. Gegenb Morphol Jahrb. 1884, 10: 204-212.
- Kofoid CA, Swezy O: On the orientation of Erythropsis. Univ Calif Publ Zool. 1917, 18 (6): 89-102.
- Greuet C: Organisation ultrastructurale du tentacule d'Erythropsis pavillardi Kofoid et Swezy Péridinien Warnowiidae Lindemann. Protistologica. 1967, 3 (3): 335-345.
- Greuet C: Leucopsis cylindrica, nov. gen., nov. sp., Péridinien Warnowiidae Lindemann considerations phylogénétiques sur les Warnowiidae. Protistologica. 1968, 4 (2): 419-422.
- Uhlig G: Eine einfach Methode zur Extraktion der vagilen, mesopsammalen Mikrofauna. Helgol Wiss Meeresunters. 1964, 11: 178-185.View Article
- Galluzzi L, Penna A, Bertozzini E, Vila M, Garces E, Magnani M: Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a dinoflagellate). Appl Environ Microb. 2004, 1199-1206.
- Leander BS, Clopton RE, Keeling PJ: Phylogeny of gregarines (Apicomplexa) as inferred from small subunit rDNA and beta-tubulin. Int J Syst Evol Microbiol. 2003, 53: 345-354.View ArticlePubMed
- Scholin CA, Herzog M, Sogin M, Anderson DM: Identification of group- and strain-specific genetic markers for globally distributed Alexandrium (Dinophyceae). II. Sequence analysis of a fragment of the LSU rRNA gene. J Phycol. 1994, 30: 999-1011.View Article
- Yamaguchi A, Kawamura H, Horiguchi T: A further phylogenetic study of the heterotrophic dinoflagellate genus, Protoperidinium (Dinophyceae) based on small and large subunit ribosomal RNA gene sequences. Phycol Res. 2006, 54: 317-329.View Article
- Medlin L, Elwood HJ, Stickel S, Sogen ML: The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene. 1988, 71: 491-499.View ArticlePubMed
- Handy SM, Demir E, Hutchins DA, Portune KJ, Whereat EB, Hare CE, Rose JM, Warner M, Farestad M, Cary SC, Coyne KJ: Using quantitative real-time PCR to study competition and community dynamics among Delaware Inland Bays harmful algae in field and laboratory studies. Harmful Algae. 2008, 7: 599-613.View Article
- Yamaguchi A, Horiguchi T: Molecular phylogenetic study of the heterotrophic dinoflagellate genus Protoperidinium (Dinophyceae) inferred from small subunit rRNA gene sequences. Phycol Res. 2005, 53: 30-42.View Article
- Kogame K, Horiguchi T, Masuda M: Phylogeny of the order Scytosiphonales (Phaeophyceae) based on DNA sequences of rbcL, partial rbcS, and partial LSU rDNA. Phycologia. 1999, 38: 496-502.View Article
- Takano Y, Horiguchi T: Acquiring scanning electron microscopical, light microscopical and multiple gene sequence data from a single dinoflagellate cell. J Phycol. 2006, 42: 251-256.View Article
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Bio. 1990, 215: 403-410.View Article
- Maddison DR, Maddison WP: 2000, MacClade Sunderland, MA. Sinauer Associates, Inc.
- Posada D, Crandall KA: MODELTEST: testing the model of DNA substitution. Bioinformatics. 1998, 14: 817-818.View ArticlePubMed
- Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003, 52: 696-704.View ArticlePubMed
- Guindon S, Lethiec F, Duroux P, Gascuel O: PHYML Online – a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res. 2005, W: 557-559.View Article
- Huelsenbeck JP, Ronquist F: MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics. 2001, 17: 754-755.View ArticlePubMed
- Ronquist F, Huelsenbeck JP: MrBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003, 19: 1572-1574.View ArticlePubMed
- Kim K-Y, Iwataki M, Kim C-H: Molecular phylogenetic affiliations of Dissodinium pseudolunula, Pheopolykrikos hartmannii, Polykrikos cf. schwartzii and Polykrikos kofoidii to Gymnodinium sensu stricto species (Dinophyceae). Phycol Res. 2008, 56: 89-92.View Article
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