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Home , Blepharisma, Gymnodinium, Noctiluca scintillans, Peridinium, Perkinsus, Prorocentrales

I. Ed. Micwhiol.. 46(2). 1999 pp. 194-197 6 1999 by the Sociely of Protozoologists The Phylogenetic Position of Amoebophrya sp. Infecting sanguineum

JOHN H. GUNDERSON,* ' SUSAN H. GOSS** and D. WAYNE COATS*** *Centerfor the Management. Ufilrzation and Proreiction of Water Resources, Tennessee Technological Unrversi~y,PO Box 5033, Cookeville, Tennessee 38505, USA, and **Department of Biology, Tennessee Technological University. Cookeville, Tennessee 38505, and ***P.O. Box 28. Smithsonian Environmental Research Center, Edgewater, Maryland 21037, USA

ABSTRACT The small-subunit rRNA sequence of a species of Amoebophrya infecting Gymnodinium sangutneum in Chesapeake Bay was obtamed and compared to the small subunit rRNA sequences of other protlsts. Phylogenetlc trees constructed with the new sequence place Amoebophrya between the rema~ningdinoflagellates and other . Supplementary key words. , rRNA, Syndiniophyceae.

HE motile stages of the parasitic syndiniophyceans have the fractionation using an autoclaved Sterifil holder (Millipore, T general appearance of other , and have a Bedford, MA) fitted with a 12-pm pore Nuclepore filter. The typical dinoflagellate transverse . However, they differ < 12 pm fraction was transferred to sterile 15-ml conical from most other dinoflagellates in having chromosomes and a tubes and centrifuged at 1000 g for 20 min to produce dinos- mitotic pattern more typical of other 131. They also pore pellets. The supernatant overlying each pellet was re- lack , which many other dinoflagellates possess. moved using a sterile micropipette and pellets were immme- This combination of characteristics could be explained if the diately resuspended in a solution of 4 M guanidium isothio- lineage containing the syndiniophyceans diverged from other cyanate and 0.1% beta-mercaptoethanol to promote cell lysis dinoflagellates before the nucleus had developed the aberrant and stabilize parasite DNA. Genomic DNA was purified with characteristics so typical of the group and before chloroplasts a Wizard PCR Preps DNA Purification System (Promega, had been acquired [16]. Madison, WI) according to the manufacturer's instructions, Ribosomal RNA sequences have been used to infer phylog- except that the water used for eluting DNA was at a temper- eny within the dinoflagellates [I, 10, 12, 13, 15, 16, 181, but ature of 80" C. The high temperature permitted large frag- the sequences used in these analyses have not, to date, included ments of DNA to be eluted. any from the syndiniophyceans. We present here the results of Ampli@cation of rRNA sequences. The small-subunit phylogenetic analyses that include the small-subunit rRNA se- rRNA coding region was amplified with the primers described quence from an Amoeboph~yaspecies infecting, Gymnodinium elsewhere [Ill. Thirty five amplification cycles were used, each sanguineum in Chesapeake Bay. containing a 30-s denaturation step at 92" C, a 1-min annealing This strain has been referred to in earlier papers as Amoe- step at 42" C, a 3-min ramp to 72" C, and a 2-min extension at bophrya ceratii [4, 51 as it matches the original description of 72" C. After the final cycle, the reaction mixture was incubated A. ceratii. However, morphological and developmental d~ffer- at 720 c for 9 min. Each 100-~1reaction volume contained 1 ences exist among strains of "A. ceratii" infecting different unit of Taq DNA polymerase (Promega), 10 pl lox buffer dinoflagellate species in Chesapeake Bay [41, something also (Promega), 0.2 mM dNTPs (Promega), and 0.2 pM each primer. noted by Cachon [2] in Arnoebophrya infecting dinoflagellates Amplified DNA was purified with a Wizard pCR Preps DNA in the Mediterranean Sea. It is possible that "A. ceratii" rep- Purification System according to the manufacturer's instruc- resents a species complex rather than a single species, and be- tions. cause we are not certain that the strain studied by US is identical DNA sequencing. A SequiThem Cycle sequencing- it to the organism originally given that name, we are referring to @picentre Technologies, Madison, WI) was used for direct se- it as Arnoebophrya sp. quencing of the PCR product. Primers listed by Elwood et al. [6] were labelled with 33P(ICN, Costa Mesa, CA) according to MATERIALS AND METHODS the kit manufacturer's instructions and used in sequencing re- Source and growth of cells- Stock cultures of a Chesapeake actions with 25 ng of amplified DNA. Twenty cycles were used Bay isolate (DWC) of the photosynthetic dinoflagellate Gym- in each sequencing reaction, each containing a 30-s denatur- nodiniurn sanguineurn Hirasaka were grown in fl2-Si medium ation step at 95" C, a 30-s annealing step at 42" C, a 3-min [9] formulated using 15%0Chesapeake Bay water supplemented ramp to 720 C, and a 2-min extension at 720 C. Sequencing w~th5% (v/v) soilwater [GR+l [171. Parasitic dinoflagellates reactions were electrophoresed on 5% or 8% polyacrylamide conforming in mor~holog~and life cycle to AmoeboPh~acer- gels, and sequencing ladders detected by exposure to Biomax atzz (Koeppen) were established in culture by adding a single MR film (Eastman Kodak, Rochester, Ny). cell of G. sanguineurn in late-stage infection to an exponentially Sequence analysis. The Amoebophva sequence was aligned growing culture of the same host species [51. Stock cultures with the other protistan sequences listed in Table 1. The number the parasite were subsequently maintained by transferring ah- aligned positions used in constructing trees varied between quots of infected host culture to uninfected G. sanguineurn 1669 and 1725, according to which sequences were used in stocks at approximately three-day intervals. All cultures were building trees. Trees were built using the parsimony programs maintained at 20-23" C on a 14:lO 1ight:dark cycle, with cool DNAPARS or DNAPENNY, the neighbor-joining program white fluorescent bulbs providing - 100 pE m-= s-'. NEIGHBOR in which distances were produced using the Ki- Isolation of DNA. Dinospores, the infective dispersal stage mura z~~~~~~~~model in DNADIST, and the maximum-like- of the ~arasite[s], were separated from size lihood program DNAML. Data sets for bootstrapping were pro- duced by SEQBOOT, and majority-rule consensus trees pro- ' To whom the correspondence should be addressed Telephone; (931) duced with CONSENSE- All of these programs are from PHye 372-3250; Emall, [email protected] LIP 3.57~[7]. Bootstrapped DNAML and DNAPENNY trees 194 GUNDERSON ET AL.-AMOEBOPHRYA PHYLOGENETIC POSITION

Table 1. List of species used in constructing phylogenetic trees. from T. gondii and sp. ranged from 60-89%, with GenBank accession numbers are listed to the right of each species. the average for the 32 trees being 76%. Alternative topologies placed Noctiluca, Amoebophrya or Crypthecodiniurn between Species Accession number T. gondii and Perkinsus. The support for a dinoflagellate clade Alexandrium fundyense U09048 that excluded Amoebophrya, that is, for a tree topology in belauense L13719 Amphidinium carrerae (Sogin lab, unpublished) which Amoebophrya was separated from Noctiluca and americanum M97909 emerged by itself from the base of the dinoflagellates, was BBSR 323 USU52356 much lower. Bootstrap value for such a dinoflagellate clade Cerarium tenue Am22 192 ranged from 37-62%, with the average value being 48%. Al- Collozoum caudatum symbiont usu52354 Cryprhecodinium cohnii M34847 though this arrangement occurred less than 50% of the time, Dictyostelium discoideum KO264 1 the alternative topologies occurred even less frequently. The Gloeodinium viscum L13716 usual alternative topology was one in which Noctiluca and Gymnodinium catenatum Am22 193 Gymnodinium fuscum Am22 194 Amoebophrya emerged together on a common branch diverging Gymnodinium mikimoroi Am22195 from the other dinoflagellates. Gymnodinium sanguineurn GSU41085 Figure 1 shows a bootstrapped neighbor-joining tree. The Gymnodinium sp. Am22196 speed of the algorithm allows many more species to be included Gyrodinium impudicum Am22 197 Heterocapsb triquerra Am22 198 in a tree, but possibly at the cost of accuracy. Its topology is Lepidodinium viride Am22199 in general agreement with the relationships revealed by the Am22200 smaller trees (although the bootstrap figures are different), and Pentapharsodinium tyrrhenicum Am2220 1 sp. Am22202 is therefore a useful summary figure. Perkinsus sp. Lo7375 Prorocentrum micans MI4649 DISCUSSION Scrippsiella nurricula SNU52357 Spongostaurus symbiont usu52355 No single placement of Amoebophrya in our trees is so microadriaticum M88521 ~eirah~menarhermophila MI0932 strongly supported by bootstrapping as to exclude the likelihood U03070 of a different arrangement. However, all of the alternatives place Amoebophrya in a lineage at or near the base of the di- noflagellates. The two most strongly supported alternatives were limited to 9 organisms in order to keep the computation (which taken together probably do make other arrangements time reasonable. Sixteen sequence sets were used with each of unlikely) are one in which a branch containing only Amoebo- these two types of algorithms. All contained Toxoplasma gon- phrya emerges at the base of the dinoflagellates (as seen in Fig.. dii, Perkinsus sp., Amoeb~phryasp., Noctiluca scintillans, and 1) and one in which the most deeply diverging lineage among five other randomly chosen dinoflagdlate species. Each con- the dinoflagellates contains both Amoebophrya and Noctiluc@. sensus tree was produced from 200 bootstmp replicates. Trees The syndiniophycean nuclear state is comparable to that of ot&- were drawn with TREEVIEW 1.5.0 [14]. er eukaryotes, and the phylogenetic position of Amoebophrya in relation to other dinoflagellates is one more indication thai RESULTS the ancestral dinoflagellates had a typical eukaryotic nucleus. Amplification of Amoebophrya genomic DNA with primers A possible association with Noctiluca isn't entirely surpris-, complementary to the ends of eukaryotic small-subunit rRNA ing. Noctiluca has a dinokaryotic nucleus only during the hap- coding regions yielded a product 1826 nucleotides long loid stage of the life cycle. The morphological and life cycle (GenBank Accession No. AF069516). 72 of these positions be- characteristics of Noctiluca place it outside of the typical di- ing complementary to the primers. Nucleotide usage was un- nophyceans [8], and it was the most divergent dinoflagellate in ambiguous; there was no evidence for the existence of multiple trees constructed from a set of small-subunit rRNA sequences slightly different rRNA gene copies in the genome. which, however, did not include sequences from or Initial parsimony trees constructed with DNAPARS and con- syndiniophyceans {16]. The Amoebophrya-Noctiluca relation- taining Dictyostelium discoideum, the , apicomplexans, ship might be better resolved once more sequences are obtained and dinoflagellates always placed Amoebophrya with the api- from more deeply diverging dinoflagellate lineages. It would be complexans and dinoflagellates, as would be expected. D. dis- desirable to have sequences from Oxyrrhis, representatives of coideum and the ciliates were then removed from subsequent the Blastodiniophyceae, and a few more syndiniophyceans. analyses so that more nearly complete sequences could be used The remaining sections of our trees are in general agreement for tree construction. with trees produced from both SSU and LSU rRNA sequences In the majority of trees produced with either DNAPENNY or DNAML, a branch leading only to Amoebophrya diverged by other authors 112, 13, 161. All indicate that Crypthecodinium between Perkinsus sp. and the remaining dinoflagellates. How- and Noctiluca are in lineages that diverged early in dinoflagel- ever, many of the most parsimonious trees produced with DNA- late evolution rather than being derived forms, and that the lin- PENNY united Noctiluca and Amoebophrya in the most deeply eage containing Prorocentrum appeared more recently in di- diverging dinoflagellate clade, while fkw f the trees produced noflagellate evolution, so the Prorocentrum morphotype cannot with DNAML displayed this topology. The results of bootstrap- represent the form of primitive dinoflagellates. A lineage that ping show that this apparent difference in trees produced by diverged early in dinoflagellate evolution contains Alexandrium, the two methods is an illusion. Neither method strongly sup- Amphidinium, Crypthecodinium, and some other genera not ap- ports the divergence of a lineage at the base of the dinoflagellate pearing in our tree. A large number of organisms belong to a tree which contains only Amoebophrya, and the bootstrap val- group that appeared more recently. This group includes the ues for branches produced in DNAPENNY and DNAML trees , , and the . Relation- were usually very similar when produced from the same data ships within this group are very poorly resolved by the avail- set. The bootstrap value for a dinoflagellate clade separated able sequence information. J. EUK. MICROBIOL., VOL. 46, NO. 2, MARCH-APRIL 1999

Toxoplasma gondii Perkinsus sp. - Amoebophrya sp. - Noctiluca scintillans 100 Peridinium sp. - I Symbiodinium microadriaticum - 91 Gloeodinium viscum

- --: Gymnodinium mikimotoi 50 - 42 - Promcentrum micans 23 - Heterocapsa triquetra - - 39 - Pentapharsodinium tyrrhenicum 59 Gymnodinium sanguineum

51 Scrippsiella nutricula - BBSR323 - 100 - 98 -- spongostaum symbiont 98 - Collozoum caudatum symbiont I-- Gyrodinium impudicum Gymnodinium catenatum Lepidodinium viride Gymnodinium sp. Gymnodinium~cum 1- 1- Crypthecodinium cohnii Amphidinium belauense Amphidinium carterae Ceratiurn tenue Alexandrium hndyense GUNDERSON ET AL.-AMOEBOPHRYA PHYLOGENETIC POSITION

ACKNOWLEDGMENTS 10. Cast, R. J. & Caron, D. A. 1996. Molecular phylogeny of sym- biotic dinoflagellates from planktonic foraminifera and radiolaria. Mol. Supported in part by NSF grant no. WE-931772. We thank Biol. Evol., 13: 1192-1 197. Mr. Jeremy Adam for his technical assistance. 11. Gunderson, J., Hinkle, G., Leipe, D., Morrison, H. G., Stickel, S. K., Odelson, D. A., Breznak,J. A., Nerad, T. A., Muller, M. & Sogin, LITERATURE CmD M. L. 1995. Phylogeny of trichomonads inferred from small-subunit 1. Adachi, M., Sako, Y. & Ishida, Y. 19%. Analysis of Alexandriwn rRNA sequences. J. Euk. Microbiol., 42:411-415. () species using sequences of the 5.8s ribosomal DNA and 12. Lenaers, G., Maroteaux, L., Michot, B. & Herzog, M. 1989. Di- internal transcribed spacer regions. J. Phycol., 32:424-432. noflagellates in evolution. A molecular phylogenetic analysis of large- 2. Cachon, J. 1964. Contribution a l'btude des Hridiniens parasites. subunit ribosomal RNA. J, Molec. Evol., 29:40-51. Cytologie, cycles tvolutifs. Ann. Sci. Nut. Zool., 6:l-158. 13, McNally, K. L., Govind, N. S., Thom6, I? E. & Trench, R. K. 3. Cachon, J. & Cachon, M. 1987. Parasitic dinoflagellates. In: Tay- 1994. Small-subunit ribosomal DNA sequence analyses and a recon- lor, E J. R. (ed.), The Biology of Dinoflagellates. Botanical Mono- struction of the inferred phylogeny among symbiotic dinoflagellates graphs, Blackwell Scientific, Oxford. 21:571-610. (Pyrrophyta). J. Phycol., 30:316-329. 4. Coats, D. W., Adam, E. J., Galbgos, C. L. & Hedrick, S. 1996. 14. Page, R. D. M. 1996. TREEVIEW An application to display of photosynthetic dinoflagellates in a shallow subestuary of phylogenetic trees on personal computers. CABIOS, 12357-358. Chesapeake Bay, USA. Aquat. Microb. Ecol., 11: 1-9. 15. Rowan, R. 1992. Ribosomal RNA sequences and the diversity 5. Coats, D. W. & Bockstahler, K. R. 1994. Occurrence of the par- of symbiotic dinoflagellates (zooxanthellae). Proc. Natl. Acad. Sci. asitic dinoflagellate Amebophrya ceratii in Chesapeake Bay popula- USA, 89:3639-3643. tions of Gymnodinium sanguinewn. J. Euk Microbiol., 41:586-593. 16. Saunders, G. W., Hill, D. R. A., Sexton, J. F? & Andersen, R. A. 6. Elwood, H. J., Olsen, 6. J. & Sogin, M. L. 1985. The small sub- 1997. Small-subunit ribosomal RNA sequences from selected dinofla- unit RNA gene sequences from the hypotrichous ciliates Oxytricha nova gellates: testing classical evolutionary hypotheses with molecular sys- and pust&a. Mol. Biol. Evol., 2:399-410. tematic methods. P1. Syst. Evol. (Suppl.), 11:237-259. 7. Felsenstein, J. 1993. PHYLIP: phylogeny inference package, ver- 17. Starr, R. C. & Zeikus, J. A. 1993. UTEX-The culture collection sion 3.5~.University of Washington, Seattle, WA. of at the University of Texas at Austin. J. Phycol., 29(Suppl.). 8. Fensome, R. A., Taylor, l? J. R., Norris, G., Sajeant, W. A. S., 18. Zardoya, R., Costas, E., Mpez-Rodas, V., Garrido-Pertierra, A. Wharton, D. I. & Willliams, G. L. 1993. A classification of living and & Bautista, J. M. 1995. Revised dinoflagellate phylogeny inferred from fossil dinoflagellates. Micropaleontol. Spec. Publ. 7. molecular analysis of large-subunit ribosomal RNA gene sequences. J. 9. Guillard, R. R. L. & Ryther, J. H. 1962. Studies on marine plank- Molec. EvoL, 41:637-645. tonic . I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can. J. Microbiol., 8:229-239. Received 6-1 7-98, 1-15-99; accepted 1-20-99

Fig. 1. Consensus neighbor-joining tree illustrating relationships among the dinoflagellates. The topology is in general agreement with the arrangement of species seen in smaller maximum likelihood and branch-and-bound parsimony trees; all trees indicate that the lineage containing Amoebophrya diverges at or near the base of the dinoflagellates. Bootstrap values are given at the nodes, and represent percentages of 200 replicates. The bootstrap value for a dinoflagellate clade (63%) is lower than was normally seen in parsimony and maximum likelihood trees. CONSENSE produces trees in which branch lengths are proportional to the frequency of a clade in the input trees rather than the number of substitutions per site, so the tree is drawn as a rectangular cladogram without branch lengths.