Syst Parasitol (2015) 90:125–135 DOI 10.1007/s11230-014-9538-8

The molecular phylogeny of the type-species of Oodinium Chatton, 1912 (Dinoflagellata: Oodiniaceae), a highly divergent parasitic dinoflagellate with non-dinokaryotic characters Fernando Go´mez • Alf Skovgaard

Received: 10 October 2014 / Accepted: 20 November 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Oodinium pouchetii (Lemmermann, 1899) Chatton, 1912, the first described parasitic dinoflagellate, is the type of the Oodiniaceae Chatton, 1920. In the taxonomical schemes, this family of metazoan parasites includes Amyloodinium Brown & Hovasse, 1946 and Piscinoodinium Lom, 1981 that are responsible of important damages in fish aquaculture. Species of Oodinium Chatton, 1912 have unique characteristics such as the possession of both nondinokaryotic and dinokaryotic nuclei within the lifecycle, and the absence of the transversal (cingulum) and longitudinal (sulcus) surface grooves in the parasitic stage. We provide the first molecular data

Electronic supplementary material The online version of this article (doi:10.1007/s11230-014-9538-8) contains supplementary material, which is available to authorized users. F. Go´mez (&) Laboratory of Plankton Systems, Oceanographic Institute, University of Sa˜o Paulo, Prac¸a do Oceanogra´fico 191, Cidade Universita´ria, Butanta˜, Sa˜o Paulo 05508-900, Brazil e-mail: [email protected] A. Skovgaard Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbøjlen 7, 1870 Frederiksberg C, Denmark Present Address: A. Skovgaard Department of Environmental, Social and Spatial Change, University of Roskilde, 4000 Roskilde, Denmark

for the genus Oodinium from specimens of O. pouchetii infecting the chordate Oikopleura sp. (Tunicata: Appendicularia) off the coasts of Brazil. Although O. pouchetii lacks dinokaryotic characters in the parasitic stage, the SSU rDNA phylogeny revealed that it forms a distinct fast-evolved clade that branches among the dinokaryotic dinoflagellates. However, there is no clear relationship with other dinoflagellates. Hence, the taxonomic affinity of the family Oodiniaceae is unclear at the moment.

Introduction A large number of dinoflagellates are known to parasitise marine vertebrates and invertebrates (e.g. Chatton, 1920; Cachon & Cachon, 1987; Shields, 1994). The first parasitic dinoflagellate to be described was Oodinium pouchetii (Lemmermann, 1899) Chatton, 1912 (= Gymnodinium pulvisculus (Ehrenberg, 1832) Stein, 1878 sensu Pouchet, 1885), an ectoparasite on the tail of the tunicate appendicularian Oikopleura dioica Fol (see Pouchet, 1884, 1885). The genus Oodinium Chatton, 1912 also includes another parasite of appendicularians, O. fritillariae Chatton, 1912, the parasite of annelids O. dogielii J. Cachon & M. Cachon, 1971, the parasites of chaetognaths O. jordanii McLean & Nielsen, 1989 and O. inlandicum Horiguchi & Ohtsuka, 2001, and an undescribed species that parasitises ctenophores and cnidarians (Chatton, 1912; Cachon & Cachon, 1971;

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McLean & Nielsen, 1989; Mills & McLean, 1991; Horiguchi & Ohtsuka, 2001). The oodinids that parasitise fishes have been removed from the genus Oodinium. Oodinium pillularis Scha¨perclaus, 1954 and O. limneticum Jacobs, 1946 have been transferred to the genus Piscinoodinium Lom, 1981 and O. ocellatum Brown, 1931 has been transferred to Amyloodinium Brown & Hovasse, 1946, together with the parasite of salps Amyloodinium amylaceum (Bargoni, 1894) Brown & Hovasse, 1946 (see Brown & Hovasse, 1946; Lom, 1981). Oodinium cyprinodontum Lawler, 1967 has been transferred to Crepidoodinium Lom & Lawler, 1981, a genus that includes other two species (Lom et al., 1993). Another parasite of fishes, the type-species of the monotypic genus Oodinioides Reichenbach-Klinke, 1970, is also classified within the family Oodiniaceae Chatton, 1920 (see Cachon & Cachon, 1987). Species of the genera that infect fishes cause great damage in aquaculture (Lauckner, 1984). Historically, most species of parasitic dinoflagellates have been classified as members of the orders Blastodiniales or Syndiniales (Fensome et al., 1993). Molecular phylogeny has demonstrated that Blastodiniales is an artificial assemblage with several of its members now distributed among the dinokaryotic dinoflagellates (Litaker et al., 1999; Saldarriaga et al., 2001; Ku¨hn & Medlin, 2005; Levy et al., 2007; Skovgaard et al., 2007; Go´mez et al., 2009a; Coats et al., 2010; Go´mez & Skovgaard, 2014). Even genera such as Amyloodinium and Piscinoodinium, classified within the family Oodiniaceae, belong to different phylogenetic clades. Amyloodinium ocellatum branches with thecate dinoflagellates, the fish-killer pfiesterids and cryptoperidiniopsoids, the parasites of diatoms of the genus Paulsenella Chatton, 1920 and the parasites of tintinnid ciliates of the genus Tintinnophagus Coats, 2010 (see Litaker et al., 1999; Ku¨hn & Medlin, 2005; Coats et al., 2010). Piscinoodinium spp. branch with the so-called ‘thin-walled’ dinoflagellates with mutualistic symbionts of reef-forming invertebrates and planktonic rhizarians (Pelagodinium Siano, Montresor, Probert & de Vargas, 2010; Symbiodinium Freudenthal, 1962), and free-living photosynthetic species (Polarella Montresor, Procaccini & Stoecker, 1999 and Biecheleria Moestrup, Lindberg & Daugbjerg, 2009) (see Levy et al., 2007; Siano et al., 2010). Whether one or both of the clades Amyloodiniumpfiesterids or Piscinoodinium-‘thin-walled’ dinoflagellates

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belongs to the family Oodiniaceae depends on the phylogenetic position of the type Oodinium pouchetii. However, up to date no sequence from the type-genus of Oodiniaceae is available. The heterotrophic dinoflagellates of the genus Oodinium have been the subject of numerous cytological investigations that revealed the possession of both non-dinokaryotic and dinokaryotic nuclei within the life-cycle (Chatton, 1920; Hovasse, 1935; Cachon & Cachon, 1971, 1977; McLean & Nielsen, 1989; Horiguchi & Ohtsuka, 2001). During the early vegetative stage, the cell is ovoid and attached to its host by a peduncle through which adsorption of metabolites takes place (Cachon & Cachon, 1971). It lacks typical dinoflagellate characters such as flagella and transverse or longitudinal grooves. On the other hand, it has organelles such as trichocysts and a pusule. At this stage, the parasite and its nucleus grow tremendously without any cellular division. The cell covering of the trophont comprises thecal plates. However, assignment of these plates into conventional Kofoid’s tabulation system is difficult, due to the lack of conventional reference points for the recognition of plate series (Hovasse, 1935; Horiguchi & Ohtsuka, 2001). All these unique characters make Oodinium a key genus for inferring dinoflagellate evolution. During plankton surveys along the coasts of Brazil, specimens of the appendicularian Oikopleura sp. infected with Oodinium pouchetii were collected. This study describes the life-cycle, with the first micrographs of the dinospores, and provides the first molecular data for O. pouchetii, the type-species of the family Oodiniaceae and the first described parasitic dinoflagellate.

Materials and methods Sampling and isolation of materials Specimens of O. pouchetii were isolated in the South Atlantic Ocean at two locations off the coast of Sa˜o Paulo State, Brazil, at Sa˜o Sebastia˜o Channel (23°500 4.0500 S, 45°240 28.820 W) in July 2013 and off Ubatuba (23°320 20.1500 S, 45°50 58.9400 W) in June 2014. The specimens were collected from the surface using a phytoplankton net (20 lm mesh size). The live, concentrated samples were examined in Utermo¨hl chambers at magnification of 920 with an inverted microscope (Eclipse TS-100, Nikon, Tokyo) and photographed with digital camera (Cyber-shot DSC-

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W300, Sony, Tokyo) mounted on the microscope eyepiece. Trophonts that had detached before the first division were micropipetted individually with a fine capillary into a clean chamber and washed several times in a series of drops of 0.2 lm-filtered and sterilised seawater. Finally, trophonts of O. pouchetii were placed in 0.2 ml Eppendorf tubes filled with several drops of absolute ethanol. We used naturally detached trophonts instead of cutting the attachment peduncle of trophonts still attached to the host. In the case of other ectoparasites that do not naturally detached from the host (i.e. species of Ellobiopsis Caullery, 1910), this manipulation increases the probability of contamination with DNA from the damaged host (Go´mez et al., 2009a). Samples were kept at room temperature and in darkness until molecular analysis could be performed. Life-cycle observations After obtaining samples for DNA analysis, specimens of O. pouchetii were used to investigate cell division, and the morphology and behaviour of the dinospores. Recently detached trophonts prior the first division were individually placed in Utermo¨hl chambers with 0.2 lm-filtered seawater, and periodically observed under the microscope. Other recently detached trophonts were individually placed in 12-well tissue culture plates with 0.2 lm-filtered seawater. In order to have controlled environmental conditions, the plates were placed in an incubator used for microalgae culturing, at 23°C, 100 lmol photons m-2s-1 from cool-white tubes and 12:12 h Light:Dark photoperiod. The cell division and the swarmers were periodically photographed under light microscope. PCR amplification and sequencing The sample tubes containing specimens of O. pouchetii in ethanol were centrifuged and dried by placing them overnight in a desiccator at room temperature. Then 30 ll of sterile DNase-free water was added to each sample tube and the samples were sonicated through three 10-second pulses at an output setting of 1.0 (Coats et al., 2010) using a Virsonic 600 sonicator (SP Scientific, Gardiner, NY) equipped with a microtip. Ten microlitres of the crude cell lysate was used for each polymerase chain reaction (PCR) amplification. SSU rDNA was amplified using the primers EukA and EukB (Medlin et al., 1988). A single PCR was in one case (isolate #58) sufficient for obtaining a

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PCR product suitable for sequencing. In two cases (isolates #23 and #54), a nested PCR was performed using 0.5 ll of the initial PCR product as template for a second PCR with the newly-designed primers Ask1F (5’-GAT TAA GCC ATG CAT GTC TCA G-3’) and Ask2R (5’-GAA ACC TTG TTA CGA CTT CTC C-3’). All PCR amplifications were performed in 25 ll reaction volumes containing 1.25 unit of Biotaq polymerase (Bioline Reagents Limited, London, UK), buffer supplied with the polymerase, MgCl2 at 3.0 mM, dNTPs at 1.6 mM, and the forward and reverse primers at 1.0 mM. The PCR was run in a T100TM Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) under the following conditions: initial denaturation (94°C/2 min); 35 cycles of denaturation (94°C/15 s), annealing (57°C/30 s), and extension (72°C/2 min); final extension (72°C/7 min). Nested PCR was run as touchdown PCR for 30 cycles: initial denaturation (94°C/2 min); then 10 cycles of ‘touchdown’ PCR with denaturation (94°C/15 s), 10 annealing steps (30 s) decreasing from 65 down to 56°C (1°C decrease with each cycle), and extension (72°C/2 min); followed by 20 cycles of denaturation (94°C/15 sec), annealing (55°C/30 sec), and extension (72°C/2 min); and final extension (72°C/7 min). PCR products were purified using Illustra GFX PCR DNA and Gel Purification Kit (GE Healthcare, Little Chalfont, UK) and sequenced bi-directionally with an ABI3730xl sequencer (Macrogen Europe, Amsterdam, The Netherlands) using the same primers as used for PCR and additional internal primers [ND2F, ND7R, ND9R (Ekelund et al., 2004); 528f (Elwood et al., 1985); 1209f (Giovannoni et al., 1988)]. Sequence reads were aligned and assembled using the software ChromasPro 1.75 (Technelysium, Brisbane, Australia). Phylogenetic analyses Three phylogenetic analyses were performed based on nearly complete SSU rDNA sequences. One analysis (Alveolata tree) was based on an alignment of 73 sequences for species of the major alveolate groups plus four cercozoan sequences serving as the outgroup. The second analysis (Dinokaryota tree) comprised sequences for dinokaryotes most similar to O. pouchetii as identified through BLAST search (http:// blast.ncbi.nlm.nih.gov/Blast.cgi; Altschul et al., 1997). Furthermore, sequences of a wide selection of dinokaryotes were included, aiming at including species of all mutualist symbiotic and parasitic

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dinokaryote genera for which sequences were available. Two perkinsid and two syndinean sequences were used as outgroups. The final matrix contained 64 sequences. The Dinokaryota tree analysis was repeated with the addition of the two shorter O. pouchetii sequences and three short sequences with highest similarity to O. pouchetii according to a BLAST search. Sequences were aligned using Clustal X v2.1 (Larkin et al., 2007) and non-informative sites were removed using Gblocks (Castresana, 2000) with parameters set for less stringent conditions (minimum number of sequences for a flanking position: 28; minimum length of a block: 5; allow gaps in half positions). Final alignments of the SSU rDNA sequences spanned over 1,630 and 1,737 positions (Alveolata and Dinokaryota trees, respectively). Bayesian phylogenetic trees were constructed with MrBayes v3.2 (Huelsenbeck & Ronquist, 2001). MrBayes settings for the best-fit model (GTR?I?G) were selected by AIC in MrModeltest 2.3 (Nylander, 2004). Four simultaneous Monte Carlo Markov chains were run from random trees for a total of 2,000,000 generations in two parallel runs. A tree was sampled every 100 generations, and the first 2,000 trees (burnin) were discarded before calculating posterior probabilities and constructing Bayesian consensus trees. The newly-generated sequences were deposited in DDBJ/EMBL/GenBank under accession numbers KM879217–KM879219.

Results Life-cycle Specimens of the appendicularian Oikopleura sp. were observed with ectoparasitic trophonts of O. pouchetii. The tails of the appendicularians contained up to sixteen trophonts of different sizes (Fig. 1A–B). Larger trophonts (up to 200 lm) showed dark brown pigmentation and an oval shape (Fig. 1C–D); smaller specimens were pyriform with yellowish pigmentation (Fig. 1A–B; see video at http://youtu.be/DlCPkpK7 oSM). In younger specimens, the nucleus was distally placed (opposite side of the attachment peduncle), while the nucleus was centrally located in the larger specimens. These large trophonts with dark brown pigmentation were usually found detached from the

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host at the bottom of the settling chambers; these were isolated for PCR analysis. There was a high proportion of detached mature trophonts on the bottom of the settling chambers compared with those still attached to the hosts. This may suggest that the manipulation due to plankton net sampling and/or the stress or damage of the captured hosts induce the final of the trophic life when the parasite leaves its host by breaking its peduncle attachment. The rupture of the stalk always preceded sporogenesis. When the plankton sample was allowed to settle for several hours, aggregations of c.200 sporocysts were observed (Fig. 1Q–R). Detached trophonts were individually placed in filtered seawater in order to observe the different steps of the sporogenesis process avoiding the influence of other organisms and under controlled environmental conditions. The divisions proceeded without interruption to form progressively smaller sporocysts that eventually became liberated as flagellated dinospores after 8–12 hours (Fig. 1F–W). The palintomic sporogenesis began with the retraction of the cell cytoplasm (Fig. 1F, H). The cleavage of the trophont was longitudinal (slightly oblique to the axis of the attachment peduncle) (Fig. 1G, I). In the first generation, the two daughter cells sometimes remained inside the theca (Fig. 1K) and later they left behind an empty theca (Fig. 1J). Further divisions always occurred outside the theca. The nucleus divided before the cytoplasmic furrow reached the nuclear level. The cell cytokinesis began before the complete division of the nuclei (Fig. 1L, M). Different steps of the development of the first generation of daughter cells are shown in Figs. 1G, I, K–O and the second generation of daughter cells are illustrated in Fig. 1P–O. The dark brown pigmentation of the adult trophont was progressively diluted along the successive divisions. This suggested that this pigmented substance is a reserve compound consumed during the sporogenesis. The sporocyst generations followed one after the other without any compensating growth until swarmers with dinoflagellate characteristics appeared. The sporogenic divisions proceeded without pause and were synchronic in the first generations. However, this synchrony was lost in the last steps of the sporogenesis (Fig. 1Q, S). This made it difficult to account the final number of infective dinospores produced by each trophont because the dinospores, swarmers, began to swim when sporocysts were yet devoid of flagella. An aggregation of c.200 immotile sporocysts was

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Fig. 1 Life-cycle stages of the ectoparasite Oodinium pouchetii from the coast of Brazil. Ectoparasitic phase of young trophonts infecting Oikopleura sp. (A–E) and phase of multiplication (palintomic sporogenesis) (D–W). A, B, Young trophonts; C–E, Mature trophont; F–H, Recently detached trophonts (note the retraction of the cytoplasm inside the theca and the large round nucleus); G, I, K, L–O. First generation of daughter cells; G–I, The cleavage is longitudinal, slightly oblique to the axis of the attachment peduncle. The daughter cells and the nuclei are oval; J, Empty theca; K–O, Division of the first generation; P, Note that cell division begins with segmentation of the cell; P, Q, Second generation of daughter cells; R, Sporocysts of the penultimate generation; S, Sporocysts of the penultimate generation (large) and sporocysts of the last generation that formed each a pair of swarmers. The inset shows the last division; T–W, Recently formed dinospores, swarmers, forming couples; U, V, Note the longitudinal flagellum (lf), and the transversal flagellum (tf) that vibrates outside the cingulum. Scale-bars: E–R, 20 lm; S–W, 10 lm

observed after eight hours at a temperature of 23°C (Fig. 1R). In the last step, these immotile sporocysts divided again and formed a couple of dinospores each

that developed two flagella. The couple of dinospores remained together, and began to move the flagella (Fig. 1S, T). The transversal flagellum first vibrated

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outside the cingulum, but eventually beat inside the cingulum when the cells began to swim (Fig. 1U–W; see video at http://youtu.be/DlCPkpK7oSM). The swarmers began to disperse while other dinospores were yet devoid of flagella. The small dinospores swam with the typical dinoflagellate rotation, following a straight trajectory at different levels in the settling chamber, and with sudden accelerations. This feature made it difficult to record the trajectories at high magnification. We counted c.200 immotile sporocysts from each detached trophont. Subsequently each immotile sporocyst divided into two swarmers. With this last division the total number of swarmers from each detached trophont would be 512, implying nine successive binary divisions (29 = 512). The swarmers (c.9 lm long and c.7 lm wide) showed a hemispherical contour of the hyposome and an episome more conical with a round apex, and a wellmarked cingulum. The swarmers contained small yellow-greenish body inclusions, and a round nucleus located in the centre of the cell (Fig. 1T–W). Molecular phylogeny Three partial SSU rDNA sequences (up to 1,713 bp) for O. pouchetii were obtained: one sequence from detached trophonts isolated at Sa˜o Sebastia˜o Channel on 10 July 2013 (isolate #23; KM879217), and two sequences from detached trophonts isolated off Ubatuba on 6 June 2014 (isolate #54; KM879218) and on 13 June 2014 (isolate #58; KM878219). The three sequences were identical. A BLAST search was conducted on the new sequences to find related sequences in the GenBank database. Initial BLAST comparisons showed that, with the exception of the partial environmental sequence (643 bp; EF539018, retrieved from the western Pacific coast, which shared 99% identity with O. pouchetii), the closest identified relatives in the database were dinokaryotes such as the thecate parasites Blastodinium spp. (JX473665 and DQ317538) and Duboscquodinium collinii Grasse´, 1952 (HM483399), and free-living thecate species [e.g. Peridinium umbonatum Stein, 1883 (GU001637) and Scrippsiella trochoidea (Stein, 1883) Balech ex Loeblich III, 1965 (HM483396)]. However, similarities were low in all cases (84%). We studied the phylogenetic position of O. pouchetii in three SSU rDNA phylogenetic trees. The first tree contained diverse representatives of the alveolate lineages, with ciliates, apicomplexans, perkinsids,

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euduboscquellids (Marine Alveolate Group I), syndineans (Marine Alveolate Group II) and dinokaryotes. This tree (Supplementary file 1, Fig. S1) placed unequivocally O. pouchetii within the dinokaryotic lineage. Then, we studied the phylogenetic position of O. pouchetii using a dataset including a variety of dinoflagellate sequences, especially with representatives of the parasitic dinokaryotes, including the oodinioid dinoflagellates (species of Amyloodinium and Piscinoodinium), other parasitic dinoflagellates, and a diverse representation of the dinokaryotic lineages, and rooted using perkinsid and syndinean sequences as an outgroup (Fig. 2). We carried out an additional analysis that also included shorter sequences, especially partial SSU rDNA sequences of environmental clones and other known dinoflagellates (Supplementary file 1, Fig. S2). The clade of O. pouchetii in the resulting tree is included as an inset in Fig. 2. In the Bayesian consensus tree (Fig. 2), the SSU rDNA phylogeny revealed that sequences for Oodinium spp. formed a distinct clade among the dinokaryotic dinoflagellates. The three sequences for O. pouchetii formed a highly supported lineage (maximum posterior probability value) with an environmental sequence (EF539018) from the port of Hong Kong, Western Pacific. In the tree containing short sequences (Supplementary file 1, Fig. S2), this lineage branched with strong support with an environmental sequence from the sub-surface waters of the equatorial Pacific (AJ402340), and two environmental sequences (AY046653; AY046659) from the surface waters of the South Pacific Ocean. The Oodinium clade branched within the dinokaryotic dinoflagellates. However, it was not possible to find any close genetically characterised relatives of the family Oodiniaceae.

Discussion The life-cycle of the trophont and the morphology of the dinospores of O. pouchetii studied here coincide with the original description of O. pouchetii. Pouchet (1885, figure 27) illustrated a pair of recently formed dinospores with long flagella and a cell contour similar to those observed in the present specimens (Fig. 1T– U). Chatton (1920) demonstrated that along the growth of the Oodinium trophont, it progressively losses the typical nuclear characteristic of the

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Fig. 2 Phylogenetic tree of the Dinokaryota based on phylogenetic analysis of 64 SSU rDNA sequences using Bayesian inference. Perkinsozoa is used as outgroup. Parasitic taxa are highlighted. The species newly-sequenced in this study are in bold. Inset: The Oodinium-clade from a corresponding tree (Supplementary file 1, Fig. S2) in which short sequences were included. Posterior probabilities (when above 0.5) are given at nodes. The scale-bar represents the number of substitutions per site

dinokaryotic dinoflagellates (condensed rod-like chromosomes). Hovasse (1935) found that the nucleus of swarmer cells possess a typical dinokaryon, and only

gradually acquire eukaryote-like organisation during the feeding phase as ectoparasite. The characteristic morphological features of a dinoflagellate do indeed

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disappear during the vegetative stage and can only be seen again in the last stages of sporogenesis. Consequently, O. pouchetii alternates between ultrastructure features of basal dinoflagellates and dinokaryotes. Also the trophonts of Noctiluca scintillans (Macartney, 1810) Kofoid, 1920 do not retain the shared characters of typical dinoflagellates such as a transverse flagellum and permanently condensed chromosomes. The swarmers of species of Noctiluca Suriray, 1836 maintain the dinokaryotic characteristics including two grooves, slightly differentiated flagella and condensed chromosomes (Fukuda & Endoh, 2006). Species of Noctiluca and other noctilucoids (species of Spatulodinium J. Cachon & M. Cachon, 1968; Kofoidinium Pavillard, 1928) do not branch within the dinokaryotic lineage (Go´mez et al., 2010). Despite the absence of dinokaryotic characters in the parasitic phase, the molecular phylogeny reveals that O. pouchetii is truly a member of the dinokaryotic lineage. The type of life-cycle has traditionally been used for classification of the parasitic dinoflagellates into families (Fensome et al., 1993). However, molecular phylogeny has revealed that species of closely related genera (i.e. Dissodinium Klebs, 1916 and Chytriodinium Chatton, 1912) have different life-cycles (Go´mez et al., 2009a). The morphology of dinospores seems to be more informative for the classification of these parasites. However, it is not always easy to observe the detailed morphology of the small swarmers when compared with the trophonts. For example, before Skovgaard et al. (2007), the thecate swarmers of Blastodinium Chatton, 1906 were usually referred to as naked, gymnodinioid dinoflagellates (Chatton, 1920; Taylor, 2004). The swarmers of Amyloodinium ocellatum were first described as naked gymnodinioid dinoflagellate (Brown, 1934; Lom, 1981), but further analysis with scanning electron microscopy revealed that the swarmers were thecate (Landsberg et al., 1994). Paulsenella vonstoschii Drebes & Schnepf branches within a clade of thecate dinoflagellate. However, the thecal plates have not yet been reported (Ku¨hn & Medlin, 2005). Due to instrumental constraints, we were not able to discern the tabulation of the dinospores of O. pouchetii. The thecal plates of the trophonts of this species consist of four equatorial series and are thin, with no visible ornamentation (Hovasse, 1935). The assignment of these plates into conventional Kofoid’s tabulation system is difficult,

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due to the lack of conventional reference points (apical pore plate, cingulum or sulcus) for the recognition of plate series. To date, Amyloodinium is still classified within the family Oodiniaceae, but we have here shown that it is distantly related to the type-species of the family. Hence, O. pouchetii remains the only morphologically identified member of Oodiniaceae with a sequence in the GenBank database. Several environmental sequences are related to O. pouchetii and thus may represent potential unidentified members of the Oodiniaceae (Fig. 2; Supplementary file 1, Fig. S2). Assuming that these environmental clones are not artefacts and correspond to true organisms, unidentified relatives of Oodinium spp. are present in different marine habitats. An environmental sequence from coastal eutrophic waters unequivocally corresponded to O. pouchetii (see Cheung et al., 2008). Sequences for relatives of O. pouchetii were retrieved from surface and subsurface waters of the oligotrophic waters of the Pacific Ocean (Moon-van der Staay et al., 2001; Lie et al., 2014). We do not know whether these environmental clones branching with O. pouchetti are parasitic or free-living organisms. Our results suggest that there are dinoflagellates belonging to the family Oodiniaceae that have not yet been morphologically and ecologically characterised. We must conclude that the parasite of fishes, Amyloodinium and Piscinoodinium, should not be placed in the family Oodiniaceae. In the classical taxonomical schemes, Fensome et al. (1993) grouped under the Oodiniaceae ectoparasites with suboval to fusiform in outline trophonts, with a well-developed peduncle or invasive organ that consists of a complex rhizoid-like absorptive structure and with reproduction by palintomic sporogenesis. Another character of this family is the possession of both non-dinokaryotic and dinokaryotic nuclei within the life-cycle (Fensome et al., 1993). There is no evidence for the presence of non-dinokaryotic nuclei in species of Amyloodinium and Piscinoodinium. The thecate Amyloodinium ocellatum branched with other parasites such as Paulsenella vonstoschii and Tintinnophagus acutus Coats (as in Coats et al., 2010). The trophont of O. pouchetii is also covered by thecal plates (Hovasse, 1935), and we can expect a relationship between these taxa. Amyloodinium ocellatum can be distinguished from O. pouchetii by the possession of rhizoid- and root-like processes for attachment and by the

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production of starch grains. Species of the genus Oodinium, on the other hand, are characterised by the possession of a disk, rather than rhizoids, for attachment and by the lack of starch grains (Brown & Hovasse, 1946). Several of the congeneric species that parasitise other metazoan groups show marked morphological differences in comparison with the typespecies O. pouchetii (see McLean & Nielsen, 1989; Mills & McLean, 1991; Horiguchi & Ohtsuka, 2001). This raises questions of the correct generic affiliation of these species. Other parasitic genera with some morphological resemblance such as Cachonella Rose & Cachon, 1952, Crepidoodinium, Protoodinium Hovasse, 1935 or Oodinioides also lack molecular information. Unfortunately, the strong bias in the availability of sequences towards photosynthetic and cultivable dinoflagellates hinders the advances in dinoflagellate phylogeny. The percentage of parasitic dinoflagellates for which at least one DNA sequence is available is very low (7%; Go´mez, 2014). Proper taxon sampling is one of the greatest challenges to our understanding of the phylogenetic relationships of the parasitic dinoflagellates. Analyses of environmental marine rDNA sequences have revealed an extensive diversity of ribotypes related to dinoflagellates, especially Syndiniales, widely distributed throughout the oceans (Lo´pez-Garcı´a et al., 2001; Guillou et al., 2008). The closest relatives to the dinoflagellates, apicomplexans and perkinsids, and nearly all the basal dinoflagellates (Syndiniales, euduboscquellids and ellobiopsids), are parasites. These basal dinoflagellates lack the characteristics of dinokaryotic dinoflagellates such as the condensed chromosomes in interphase. The proportion of parasites is low (3%) among the core dinoflagellates (Go´mez, 2012). In our phylogenetic analyses, we have included the available sequences for other parasitic dinokaryotes. The parasite species of the genera Amyloodinium, Tintinnophagus, Piscinoodinium, Duboscquodinium Grasse´, 1952, Chytriodinium and Dissodinium branched with strong support in different clades dominated by free-living dinoflagellates (Litaker et al., 1999; Ku¨hn & Medlin, 2005; Levy et al., 2007; Go´mez et al., 2009a, b; Coats et al., 2010). The phylogenetic position of Blastodinium remains unclear with a weak relationship with peridinioid clades (Skovgaard et al., 2007, 2012), and Blastodinium is not always a monophyletic group in SSU rDNA phylogenies (Coats et al., 2008; Skovgaard &

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Salomonsen, 2009). With exception of Amyloodinium/ Paulsenella and Chytriodinium/Dissodinium, the clades of parasitic dinokaryotes are not related to each other. Haplozoon Dogiel, 1906 and Oodinium have in common that they comprise both fast-evolved dinokaryotes without any close known relatives (Fig. 2; see also Saldarriaga et al., 2001; Rueckert & Leander, 2008). We observed in the SSU rDNA phylogeny that the sequences for species of Amyloodinium, Tintinnophagus, Piscinoodinium, Chytriodinium, Haplozoon and Oodinium have long branches when compared with the typical short-branched species of the main clades of the core dinoflagellates. This has been considered with caution because the branches of these parasites are shorter in ingroup trees with a rich taxonomic sampling of their relatives (Go´mez & Skovgaard, 2014). A longer branch usually is interpreted as either a longer time period since that taxon split from the rest of the organisms in the tree or faster evolutionary change in a lineage. We can only hypothesise accelerated rates of evolutionary change in parasitic dinoflagellates when compared with their free-living counterparts. Oodinium pouchetii is obviously highly derived not only in relation to its morphology, but also to SSU rDNA sequence. In this, as well as in many other parasitic dinoflagellates, the detached trophont, its sporogenesis stages and the small dinospores are only recognised during routine phytoplankton analysis by well-trained observers. Researchers focused on plankton metazoans usually preserve the samples in formalin and the parasites are unrecognisable due to fixationinduced distortion (Skovgaard & Saiz, 2006). Consequently, the abundance and role of Oodinium spp. in the world oceans remains understudied. Acknowledgements F.G. was supported by the Brazilian Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (grant no. BJT 370646/2013-14). A.S. was supported through the project IMPAQ - IMProvement of AQuaculture high quality fish fry production, funded by the Danish Council for Strategic Research (Grant No. 10-093522).

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