Molecular Phylogeny of the Parasitic Dinoflagellate Chytriodinium within the Gymnodinium Clade (Gymnodiniales, Dinophyceae)

The Journal of 

Eukaryotic Microbiology 

Published by 

the International 

Society of 

Protistologists 

Journal of Eukaryotic Microbiology ISSN 1066-5234 

SHORT COMMUNICATION 

Molecular Phylogeny of the Parasitic Dinoflagellate 

Chytriodinium within the Gymnodinium Clade 

(Gymnodiniales, Dinophyceae) 

Fernando Gomez a & Alf Skovgaard b 

a Laboratory of Plankton Systems, Oceanographic Institute, University of S~ao Paulo, S~ao Paulo, Brazil 

b Department of Veterinary Disease Biology, University of Copenhagen, Stigbøjlen 7, DK-1870, Frederiksberg C, Denmark 

Keywords 

Copepod; Dissodinium; Gymnodinium sensu 

stricto; parasite Dinophyta; parasitism. 

Correspondence 

F. Gomez, Laboratory of Plankton Systems, 

sala 100, Oceanographic Institute, University 

of S~ao Paulo, Pracßa do Oceanografico 191, 

Cidade Universitaria, S~ao Paulo, 

SP 05508-900, Brazil 

Telephone number: +55-11-30918963; 

Fax: +55-11-30916607; e-mail: fernando. 

gomez@fitoplancton.com 

ABSTRACT 

The dinoflagellate genus Chytriodinium, an ectoparasite of copepod eggs, is 

reported for the first time in the North and South Atlantic Oceans. We provide 

the first large subunit rDNA (LSU rDNA) and Internal Transcribed Spacer 1 

(ITS1) sequences, which were identical in both hemispheres for the Atlantic 

Chytriodinium sp. The first complete small subunit ribosomal DNA (SSU rDNA) 

of the Atlantic Chytriodinium sp. suggests that the specimens belong to an 

undescribed species. This is the first evidence of the split of the Gymnodinium 

clade: one for the parasitic forms of Chytriodiniaceae (Chytriodinium, Dissodinium), 

and other clade for the free-living species. 

Received: 7 July 2014; revised 31 July 

2014; accepted July 31, 2014. 

doi:10.1111/jeu.12180 

COPEPODS dominate the zooplankton biomass and are 

considered to be the most abundant animals in the ocean. 

The lipid-rich copepod eggs are the target of the specialized 

parasitic dinoflagellates Chytriodinium and Dissodinium 

which dinospores are able to infest crustacean eggs, 

absorb the host content and form successive cysts that 

produce colorless gymnodinioid spores (Cachon and Cachon 

1968; see Video S1 http://youtu.be/nwFZQAAm- 

QaA). 

Our knowledge of the members of the family Chytriodiniaceae 

is limited. Some stages of the life cycle of Dissodinium 

psedolunula, such as the lunate sporangia, are highly 

distinctive and commonly recognized by plankton researchers 

(Gomez and Artigas 2013). In contrast, Chytriodinium 

is absent in identification guides, and it easily goes unnoticed 

especially in fixed samples. The genus comprises 

three species. In Chytriodinium affine, the numerous dinospores 

develops in a coiled chain inside a hyaline spherical 

membrane that was absent in the chain of C. roseum. 

Chytriodinium parasiticum parasitizes larger crustacean 

eggs, and forms a sophisticated stalk apparatus (Cachon 

and Cachon 1968). These species are only known from the 

western Mediterranean Sea, with some recent records of 

C. affine from the Pacific Ocean (Gomez-Gutierrez et al. 

2009; Meave del Castillo et al. 2012). It is clear that abundance 

and ecological role is being underestimated. 

Gomez et al. (2009) provided the first molecular data 

based on the partial SSU rDNA sequence of the species 

Chytriodinium affine and C. roseum from the Mediterranean 

Sea. Chytriodinium branched within the so-called 

Gymnodinium sensu stricto or Gymnodinium clade 

(Daugbjerg et al. 2000). This clade showed a strong diversification 

in the trophic modes with plastids of different 

microalgal origins and development of specialized organelles 

(nematocyst, ocelloid, or piston) that are unknown in 

other dinoflagellate clades. 

The current molecular information on Chytriodinium is 

restricted to partial SSU rDNA sequences of Mediterranean 

specimens. The complete SSU rDNA sequence and 

additional molecular markers (LSU rDNA, ITS1) will allow 

to test whether the heterotrophic Chytriodinium and photosynthetic 

Dissodinium were or maybe are derived from 

a recent common ancestor or the parasitism appeared 

independently in the Gymnodinium clade. 

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists 

Journal of Eukaryotic Microbiology 2014, 0, 1–4 1

Phylogeny of Parasitic Chytriodinium in Gymnodinium Clade 

Gomez & Skovgaard 

MATERIALS AND METHODS 

Specimens of Chytriodinium were isolated from water 

samples collected at two coastal sites, in the North Atlantic, 

in the coasts of the Caribbean Sea at Puerto Rico 

(Bahıa Fosforescente, 17°58 0 19.80″N, 67°0 0 50.73″W), and 

in the South Atlantic Ocean, the coasts of S~ao Paulo 

State, Brazil (S~ao Sebasti~ao Channel, 23°50 0 4.05″S, 

45°24 0 28.82″W). The Caribbean specimens of Chytriodinium 

were collected from the surface using a phytoplankton 

net (20 lm mesh size) during the night of March 8, 2012, 

and they were isolated onboard with a 3030 Accu-scope 

inverted microscope. The Brazilian specimens of Chytriodinium 

were collected in the S~ao Sebasti~ao Channel on 

early morning of April 30, 2013, and they were isolated in 

a coastal laboratory with a Nikon TS-100 inverted microscope. 

After being photographed, each sporangium of 

Chytriodinium containing tens of immature dinospores 

was separated from the egg sac. The sporangium was 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 sterilized seawater. Finally, the sporangium 

of Chytriodinium was placed in a 0.2-ml Eppendorf 

tube filled with several drops of absolute ethanol. The 

methods of PCR amplification and sequencing, and phylogenetic 

analysis are detailed in an appendix as supporting 

information. 

RESULTS AND DISCUSSION 

In the North Atlantic Ocean, detached copepod egg sacs 

infected with Chytriodinium were observed in one survey 

carried out during the night on March 8, 2012 in Bahıa 

Fosforescente, Puerto Rico. The egg sac typically contained 

six copepod eggs of about 50–60 lm in diameter. 

The infected eggs showed one or several sporangia of 

Chytriodinium that reached a diameter slightly larger (~60– 

70 lm) than the infected egg. A chain of dinospores was 

coiled within a fine hyaline membrane of the sporangium. 

The sporangium remained attached to the copepod egg 

and more than 60 dinospores were released when the 

membrane was lysed (see Video S2 as supporting information, 

http://youtu.be/JA_Gu57WkXQ). The epicone and 

hypocone of the recently released dinospores were hemispherical 

with a deep constriction at the cingulum level 

(Fig. S1). 

In the South Atlantic Ocean, the plankton observations 

were carried out during 6 mo in S~ao Sebasti~ao Channel. 

Chytriodinium infecting copepod eggs was observed only 

on April 30, May 20, June 7 and July 5, 2013. Nearly all 

the observations of Chytriodinium corresponded to 

detached sacs of six or more eggs. In a few cases, the 

infected eggs were observed in sacs that still remained 

attached to the copepod such as Oithona cf. robusta. The 

dinospores of about 8–9 lm long were attached to the 

egg, and multi-infections were frequent with different 

degrees of sporangia development. The copepod eggs 

were 40–50 lm in diam, and the sporangium reached a 

diameter of 50–65 lm. The dinospore was attached to the 

host by means of a feeding tube, enlarged at its base. An 

orange ampulla with one large trophic vacuole formed at 

the end of the peduncular disk that gradually absorbed the 

egg cytoplasm. The recently released dinospores showed 

a deep constriction at the cingulum level. The sporangia 

used for PCR analysis were isolated on April 30, and the 

sporangia with successful PCR products are illustrated in 

the Fig. S2. 

We obtained the complete SSU rDNA sequence (1,797 

base pairs) of the isolate #7 of Chytriodinium sp. from Brazil 

(Fig. S2). A BLAST search revealed that the closest 

species based on the entire SSU rDNA sequence was 

Gymnodinium aureolum with an identity of 93%. Only a 

partial sequence Chytriodinium affine from the Mediterranean 

Sea is available (1,206 base pairs, #FJ473380). If the 

first 550 base pairs of our new sequence are removed, 

the closest BLAST match was the Mediterranean C. 

affine. The percentage of identify between the sequences 

of the Mediterranean and Atlantic specimens was 96%. 

In the Bayesian consensus tree, the Chytriodinium 

sequences branched within a sister group to the Gymnodinium 

clade with maximum support (albeit unsupported in 

the ML analysis). The Gymnodinium clade subdivided into 

four weakly supported groups. The first group included 

the sequences of G. impudicum, species with cryptophyte 

endosymbionts, Lepidodinium, Paragymnodinium, G. 

catenatum, warnowiids and Gyrodiniellum; the second 

group included Polykrikos, with Gymnodinium fuscum in a 

basal position; the third group comprised Gymnodinium 

aureolum and Pheopolykrikos, and the fourth group for 

Gymnodinium baicalense. As a sister group of the major 

Gymnodinium clade, the sequence of the Atlantic Chytriodinium 

sp. branched in a distal position of a group with 

Chytriodinium affine and C. roseum, and Dissodinium 

pseudolunula (Fig. 1). 

The first LSU rDNA sequences of the genus Chytriodinium 

were obtained from one sporangium (isolate #2; Fig. 

S1) from Puerto Rico and the other one from Brazil (isolate 

#7, Fig. S2). The LSU rDNA and ITS1 sequences of the 

Caribbean and Brazilian isolates were identical (100%). 

The closest BLAST match of the LSU rDNA sequence 

was Gymnodinium aureolum (85%). Dissodinium pseudolunula 

showed an identity of 88%, although with a lower 

coverage. In the Bayesian consensus tree, the new LSU 

rDNA sequences of Chytriodinium branched in the Gymnodinium 

clade (Fig. S3). This clade subdivided into 13 

subclades, with the Atlantic Chytriodinium branching in a 

separate linage than Dissodinium pseudolunula. The latter 

branched with negligible support with the benthic pseudocolonial 

species Polykrikos lebouriae (Fig. S3). 

This study provides the first observations of Chytriodinium 

in the Atlantic Ocean. The limited records of Chytriodinium 

do not seem to reflect the actual abundance of this 

parasite in the world oceans. The partial SSU rDNA 

sequence of the Mediterranean Chytriodinium affine differs 

from that of the Atlantic Chytriodinium (identity of 

96%). This suggests that the Atlantic specimens belong to 

an undescribed species. The genus Chytriodinium currently 

comprises three species described in 1906. The 

2 


Journal of Eukaryotic Microbiology 2014, 0, 1–4



Figure 1 Phylogeny tree inferred from the SSU rDNA sequences using Bayesian inference method. The species newly sequenced in this study 

are bold. Accession numbers are provided. Posterior probability of 1 is denoted with a black circle; white circles denote posterior probability of 

0.95–0.99. ML bootstrap support (when above 50%) is given near nodes. The scale bar represents the number of substitutions per site. Numbers 

at the end of each taxon name are GenBank accession numbers. 

morphology of the dinospores of the Mediterranean Chytriodinium 

affine has not been examined in detail in order to 

establish differences with other tentative species. No obvious 

differences exist between the Mediterranean Chytriodinium 

affine and Chytriodinium sp. from the tropical 

Atlantic Ocean. The size, shape and general appearance 

of the infective spores of the Mediterranean and Atlantic 

species is similar. The life cycle and appearance of the 


Journal of Eukaryotic Microbiology 2014, 0, 1–4 3



sporangium is also similar. All the reported observations of 

Chytriodinium affine in the Mediterranean Sea concern 

infections of individual eggs of free-spawning copepod 

species (Cachon and Cachon 1968; Gomez et al. 2009). In 

contrast, the Atlantic specimens of Chytriodinium of both 

hemispheres were exclusively observed infecting egg sacs 

of brood-carrying copepod species (Fig. S1–S2). To the 

best of our knowledge this feature is reported for the first 

time in the genus Chytriodinium. At the present, difference 

in host species is the main character distinguishing 

the Atlantic species and Chytriodinium affine. However, it 

is necessary to perform infection experiments to verify 

whether this apparent host specificity is exclusively due to 

the type of egg available. 

Similar to the presence or absence of plastids, parasitism 

is known for numerous groups of dinoflagellates 

(Coats et al. 2010). Gymnodinium aureolum ingests only 

small microalgal preys through a peduncle and divides by 

binary division (Jeong et al. 2010). This mechanism of 

feeding may be essentially the same as in members of 

Chytriodiniaceae that possess a more sophisticated 

peduncle (i.e., C. parasiticum, see Video S1 as supporting 

information, http://youtu.be/nwFZQAAmQaA). Dissodinium, 

Chytriodinium and Myxodinium are able to feed on 

larger preys, and this allows producing a high number of 

dinospores after each infection. This study provides the 

first evidence of the split of the Gymnodinium clade: one 

for the parasitic forms of Chytriodiniaceae (Chytriodinium, 

Dissodinium), and other clade for the free-living species. 

ACKNOWLEDGMENTS 

We thank E. Otero and B. M. Soler for the hospitality 

extended during the sampling in Puerto Rico, and the 

assistance of CEBIMar technical staff (USP, S~ao Sebasti~ao). 

F.G. is currently supported by the Brazilian Conselho 

Nacional de Desenvolvimento Cientıfico e Tecnologico 

(grant number 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. 

LITERATURE CITED 

Cachon, J. & Cachon, M. 1968. Cytologie et cycle evolutif des 

Chytriodinium (Chatton). Protistologica, 4:249–262. 

Coats, D. W., Kim, S., Bachvaroff, T. R., Handy, S. M. & Delwiche, 

C. F. 2010. Tintinnophagus acutus n. g., n. sp. (Phylum Dinoflagellata), 

an ectoparasite of the ciliate Tintinnopsis cylindrica 

Daday 1887, and its relationship to Duboscquodinium collini 

Grasse 1952. J. Eukaryot. Microbiol., 57:468–482. 

Daugbjerg, N., Hansen, G., Larsen, J. & Moestrup, Ø. 2000. 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, 39:302–317. 

Gomez, F. & Artigas, L. F. 2013. The formation of the twin resting 

cysts in the dinoflagellate Dissodinium pseudolunula, a parasite 

of copepod eggs. J. Plankton Res., 35:1167–1171. 

Gomez, F., Moreira, D. & Lopez-Garcıa, P. 2009. Life cycle and 

molecular phylogeny of the dinoflagellates Chytriodinium and 

Dissodinium, ectoparasites of copepod eggs. Eur. J. Protistol., 

45:260–270. 

Gomez-Gutierrez, J., Kawaguchi, S. & Nicol, S. 2009. Epibiotic 

suctorians and enigmatic ecto- and endoparasitoid dinoflagellates 

of euphausiid eggs (Euphausiacea) off Oregon, USA. J. 

Plankton Res., 31:777–785. 

Jeong, H. J., Yoo, Y. D., Kang, N. S., Rho, J. R., Seong, K. A., 

Park, J. W., Nam, G. S. & Yih, W. 2010. Ecology of Gymnodinium 

aureolum. I. Feeding in western Korean waters. Aquat. Microb. 

Ecol., 59:239–255. 

Meave del Castillo, M. E., Zamudio-Resendiz, M. E. & Castillo-Rivera, 

M. 2012. Riqueza fitoplanctonica de la Bahıa de Acapulco 

y zona costera aleda~na, Guerrero, Mexico. Acta Bot. Mex., 

100:405–487. 

SUPPORTING INFORMATION 

Additional Supporting Information may be found in the 

online version of this article: 

Figure S1. Light micrographs of Chytriodinium sp. from 

Bahıa Fosforescente, Puerto Rico. 

Figure S2. Light micrographs of Chytriodinium sp. from 

S~ao Sebasti~ao Channel, Brazil. 

Figure S3. Phylogeny tree inferred from LSU rDNA 

sequences using Bayesian inference method. 

Data S1. Materials and methods. 

Video S1. The parasitic dinoflagellate Chytriodinium from 

Villefranche sur Mer, France, by J. Cachon and M. Cachon, 

http://youtu.be/nwFZQAAmQaA. 

Video S2. The parasitic dinoflagellate Chytriodinium from 

Bahía Fosforescente, Puerto Rico, http://youtu.be/JA_ 

Gu57WkXQ. 

4 


Journal of Eukaryotic Microbiology 2014, 0, 1–4

Molecular Phylogeny of the Parasitic Dinoflagellate Chytriodinium within the Gymnodinium Clade (Gymnodiniales, Dinophyceae)

Create successful ePaper yourself

Delete template?

Save as template?