Molecular Phylogeny of Symbiotic Dinoflagellates from Planktonic Foraminifera and Radiolaria R. J. Gast and D. A. Caron Biology Department, Woods Hole Oceanographic Institution Recent analyses of the small subunit ribosomal DNA (srDNA) from dinoflagellate symbionts of cnidaria have confirmed historical descriptions of a diverse but well-defined clade, S~mhiodinium, as well as several other inde- pendent symbiont lineages (Rowan 199 1; Rowan and Powers 1992; Sadler et al. 1992; McNally et al. 1994). Dinoflagellates also occur as intracellular symbionts in a number of pelagic protistan taxa, but the srDNA of these symbionts has not been examined. We analyzed the srDNA sequences of the symbiotic dinoflagellates from four planktonic foraminiferal species and six radiolarian species. The symbionts from these sarcodines formed two distinct lineages within the dinoflagellates. Within each lineage, symbionts obtained from different host species showed few, if any, srDNA sequence differences. The planktonic foraminiferal symbionts were most closely related to Gymnodinium simplex and the Symbiodinium clade, whereas the radiolarian symbionts were most closely related to the dinoflagellate symbiont from the oceanic chondrophore, Velella velella. Therefore, although the dinoflagellate symbionts of foraminifera appear to be a sister taxon of the symbionts from benthic foraminifera and invertebrates, the symbionts of radiolaria are distinct and arose from an independent lineage of dinoflagellate symbionts that shares common ancestry with the symbiont of at least one pelagic metazoan. The lack of srDNA variability within the sarcodine symbiont lineages suggests that coevolution of host and symbiont has not occurred. Introduction Planktonic foraminifera and radiolaria are relatively large (> 1 mm), cosmopolitan, marine protists that have significant biological and geological importance in oli- gotrophic oceans. Living specimens form conspicuous biological assemblages that contribute to primary pro- duction, herbivory, and carnivory within epipelagic oce- anic communities (Swanberg and Caron 1991; Caron et al. 1995). In addition, foraminifera form calcium car- bonate skeletons that are used extensively in the analysis of marine sediments and paleoclimatological reconstruc- tion (Bolli, Saunders, and Perch-Nielsen 1985). Many of the extant species of planktonic forami- nifera and radiolaria in the surface waters of tropical and subtropical oceans harbor algal symbionts. Four species of planktonic foraminifera (Globigerinoides congloba- tus, G. ruber, G. sacculifer, and Orbulina universa) and numerous species of radiolaria contain symbiotic dino- flagellates (fig. I). Despite knowledge of the existence of these symbioses among planktonic sarcodines for well over a century, identification of the symbionts has been hindered by the loss of diagnostic morphological features, such as thecae or flagella, when the alga is in the symbiotic state. The dinoflagellate symbionts of the radiolarian Cal- lozoum inerme were first described as Zooxanthella nu- tricula (Brandt 188 I), but emendations to the identifi- cation of radiolarian dinoflagellate symbionts have in- cluded reassignment to the genera Amphidinium and En- dodinium (Blank and Trench 1986). Recently, Banaszak, Iglesias-Prieto, and Trench (1993) proposed identifica- Abbreviations: x-DNA, small subunit ribosomal RNA gene; RFLR restriction fragment length polymorphism. Key words: srDNA, dinoflagellate, symbiont, phylogeny, fora- minifera, radiolaria. Address for correspondence and reprints: R. J. Cast, Biology De- partment, Woods Hole Oceanographic Institution, Woods Hole, Mas- sachusetts 02.543. E-mail: [email protected]. Mol. B/01. EV0l. 139): 1 192-l 197. I996 0 1996 by the Society for Molecular Bdogy and Evolution. ISSN: 0737-4038 1192 tion of the dinoflagellate symbionts from the colonial radiolarian Collozoum inerme as Scrippsiella nutricula based on morphologic similarities with the dinoflagellate symbionts of VeZeZZa velella. Initial investigations of the dinoflagellates of plank- tonic foraminifera indicated that these species were sim- ilar to species of Aureodinium (Spindler and Hemleben 1980, pp. 133-140). More recent studies of the mor- phologic features of symbionts cultured from 0. univ- ersa led Spero to describe these symbionts as Gymno- dinium beii (Spero 1987). The identities of the symbi- onts from the other three dinoflagellate-bearing forami- nifera have not been confirmed, although they have been presumed to be similar to G. beii. Using srDNA se- quences, we investigated the phylogenetic relationship of the symbionts from the planktonic sarcodines to each other, and to Symbiodinium, the symbiotic dinoflagellate commonly found in many benthic cnidaria and the ben- thic foraminifera. Materials and Methods Sample Collection In order to examine the molecular phylogeny of the symbiotic dinoflagellates, foraminifera and radiolaria were collected from several areas in the Sargasso Sea 3-5 miles southeast of Bermuda during September-Oc- tober 1994 and May 1995. Sarcodines were collected individually by divers to ensure that these delicate or- ganisms were in good condition (Be et al. 1977). We collected and analyzed the dinoflagellate symbiont of VeZeZZa velella from the Sargasso Sea in May, 1995. Symbiont Microdissection and DNA Isolation Sarcodines were transferred through three sterile seawater washes and the symbionts were dissected out in filter-sterilized seawater. Symbionts were collected from individual hosts, and each microdissection was considered a single sample. Samples were not pooled. Several hundred symbionts were obtained from each
Transcript
Molecular Phylogeny of Symbiotic Dinoflagellates from Planktonic Foraminifera and Radiolaria
R. J. Gast and D. A. Caron Biology Department, Woods Hole Oceanographic Institution
Recent analyses of the small subunit ribosomal DNA (srDNA) from dinoflagellate symbionts of cnidaria have confirmed historical descriptions of a diverse but well-defined clade, S~mhiodinium, as well as several other inde- pendent symbiont lineages (Rowan 199 1; Rowan and Powers 1992; Sadler et al. 1992; McNally et al. 1994). Dinoflagellates also occur as intracellular symbionts in a number of pelagic protistan taxa, but the srDNA of these symbionts has not been examined. We analyzed the srDNA sequences of the symbiotic dinoflagellates from four planktonic foraminiferal species and six radiolarian species. The symbionts from these sarcodines formed two distinct lineages within the dinoflagellates. Within each lineage, symbionts obtained from different host species showed few, if any, srDNA sequence differences. The planktonic foraminiferal symbionts were most closely related to Gymnodinium simplex and the Symbiodinium clade, whereas the radiolarian symbionts were most closely related to the dinoflagellate symbiont from the oceanic chondrophore, Velella velella. Therefore, although the dinoflagellate symbionts of foraminifera appear to be a sister taxon of the symbionts from benthic foraminifera and invertebrates, the symbionts of radiolaria are distinct and arose from an independent lineage of dinoflagellate symbionts that shares common ancestry with the symbiont of at least one pelagic metazoan. The lack of srDNA variability within the sarcodine symbiont lineages suggests that coevolution of host and symbiont has not occurred.
Introduction
Planktonic foraminifera and radiolaria are relatively large (> 1 mm), cosmopolitan, marine protists that have significant biological and geological importance in oli- gotrophic oceans. Living specimens form conspicuous biological assemblages that contribute to primary pro- duction, herbivory, and carnivory within epipelagic oce- anic communities (Swanberg and Caron 1991; Caron et al. 1995). In addition, foraminifera form calcium car- bonate skeletons that are used extensively in the analysis of marine sediments and paleoclimatological reconstruc- tion (Bolli, Saunders, and Perch-Nielsen 1985).
Many of the extant species of planktonic forami- nifera and radiolaria in the surface waters of tropical and subtropical oceans harbor algal symbionts. Four species of planktonic foraminifera (Globigerinoides congloba- tus, G. ruber, G. sacculifer, and Orbulina universa) and numerous species of radiolaria contain symbiotic dino- flagellates (fig. I). Despite knowledge of the existence of these symbioses among planktonic sarcodines for well over a century, identification of the symbionts has been hindered by the loss of diagnostic morphological features, such as thecae or flagella, when the alga is in the symbiotic state.
The dinoflagellate symbionts of the radiolarian Cal- lozoum inerme were first described as Zooxanthella nu- tricula (Brandt 188 I), but emendations to the identifi- cation of radiolarian dinoflagellate symbionts have in- cluded reassignment to the genera Amphidinium and En- dodinium (Blank and Trench 1986). Recently, Banaszak, Iglesias-Prieto, and Trench (1993) proposed identifica-
Abbreviations: x-DNA, small subunit ribosomal RNA gene; RFLR restriction fragment length polymorphism.
Address for correspondence and reprints: R. J. Cast, Biology De- partment, Woods Hole Oceanographic Institution, Woods Hole, Mas- sachusetts 02.543. E-mail: [email protected].
Mol. B/01. EV0l. 139): 1 192-l 197. I996 0 1996 by the Society for Molecular Bdogy and Evolution. ISSN: 0737-4038
1192
tion of the dinoflagellate symbionts from the colonial radiolarian Collozoum inerme as Scrippsiella nutricula based on morphologic similarities with the dinoflagellate symbionts of VeZeZZa velella.
Initial investigations of the dinoflagellates of plank- tonic foraminifera indicated that these species were sim- ilar to species of Aureodinium (Spindler and Hemleben 1980, pp. 133-140). More recent studies of the mor- phologic features of symbionts cultured from 0. univ- ersa led Spero to describe these symbionts as Gymno- dinium beii (Spero 1987). The identities of the symbi- onts from the other three dinoflagellate-bearing forami- nifera have not been confirmed, although they have been presumed to be similar to G. beii. Using srDNA se- quences, we investigated the phylogenetic relationship of the symbionts from the planktonic sarcodines to each other, and to Symbiodinium, the symbiotic dinoflagellate commonly found in many benthic cnidaria and the ben- thic foraminifera.
Materials and Methods Sample Collection
In order to examine the molecular phylogeny of the symbiotic dinoflagellates, foraminifera and radiolaria were collected from several areas in the Sargasso Sea 3-5 miles southeast of Bermuda during September-Oc- tober 1994 and May 1995. Sarcodines were collected individually by divers to ensure that these delicate or- ganisms were in good condition (Be et al. 1977). We collected and analyzed the dinoflagellate symbiont of VeZeZZa velella from the Sargasso Sea in May, 1995.
Symbiont Microdissection and DNA Isolation
Sarcodines were transferred through three sterile seawater washes and the symbionts were dissected out in filter-sterilized seawater. Symbionts were collected from individual hosts, and each microdissection was considered a single sample. Samples were not pooled. Several hundred symbionts were obtained from each
G. beii (Globigerinoides conglobatus) Foraminiferal symbionts
* Gymnodinium sanguineum
0.10
FIG. 4.-Maximum-likelihood phylogenetic reconstruction. The scale bar represents a length equivalent to 10 changes per 100 nucle- otides. Both sarcodine symbiont lineages are highlighted. Benthic fo- raminiferal symbiont species are indicated by asterisks.
We also found that the dinoflagellate symbionts from the radiolaria examined were identical to each oth- er, despite the rather wide taxonomic positions of the hosts. All of the identified hosts were spumellarian ra- diolaria. However, Thalassicolla nucleata and the co- lonial species were from different families within the suborder Sphaerocollina, and Spongostaurus is a newly constructed genus within the suborder Sphaerellarina (Lee, Hutner, and Bovee 1985; Swanberg, Anderson, and Bennett 1985). This was in contrast to the situation with the foraminifera where all of the extant host species that harbor dinoflagellate symbionts were derived from one (or potentially two) lineages (Kennett and Sriniva- san 1983).
Sequences from the radiolarian symbionts were very similar to the symbiont of Velella velella (four dif- ferences out of 1,802 bp), and quite distinct from G. beii (85 bases out of an aligned 1,802 bp). Based on mor- phologic criteria, Banaszak, Iglesias-Prieto, and Trench (1993) described the dinoflagellate symbionts from V. velella in the Pacific as Scrippsiella velellae. They found it very similar to, but morphologically distinct from, the dinoflagellate symbiont of V. velella from the Mediter- ranean and the dinoflagellate symbiont of the colonial radiolarian C. inerme. For these two symbionts, they proposed the names Scrippsiella chattonii and Scripps- iella nutricula, respectively, retaining the original spe- cies distinctions (Brandt 1881; Taylor 1971) but emend- ing the genus.
Our srDNA data for the symbionts from the Sar- gasso Velella and the Sargasso radiolaria support the conclusion that the symbionts of Velella and the radi-
Molecular Phylogeny of Symbiotic Dinoflagellates 1195
olaria are closely related (Banaszak, Iglesias-Prieto, and Trench 1993). However, the srDNA data (four base dif- ferences) do not support a distinction between the ra- diolarian and Velella symbionts isolated from the Sar- gasso. Based on precedence (Brandt 1881), we propose the name Scrippsiella nutricula for both of these sym- bionts. It is unclear if the slight morphologic differences noted by Banaszak, Iglesias-Prieto, and Trench (1993) indicate intraspecific morphologic variation or closely related, but different, symbionts in organisms from dif- ferent oceans. A comparison of srDNA sequences from Pacific and Mediterranean symbionts could be helpful in establishing the validity of historical descriptions of these symbionts and their relationship to the Sargasso Sea specimens in this study.
We believe that the sequences we have obtained are representative of the symbiont populations in the plank- tonic sarcodines that we have collected. We have cul- tured the symbionts from the sarcodines and have found their srDNA sequences to be the same as those that we have amplified directly from microdissected tissues. The cellular morphology of the free-living symbionts is sim- ilar to what has been previously described (Spero 1987; Banaszak, Iglesias-Prieto, and Trench 1993). It would be very unlikely to obtain the same sequences from dif- ferent types of samples (microdissected vs. cultured), as well as from different organisms, if we were not ampli- fying the symbiont. It is possible that our PCR-based analyses obscured some of the diversity that exists in the natural symbiont population. However, our experi- ments were designed to determine the broad phyloge- netic relationships of the symbionts, and microhetero- geneity that might exist within (or between) populations would not change our conclusions.
Phylogenetic reconstructions based on srDNA se- quences were accomplished to examine the relationship between symbiotic and nonsymbiotic dinoflagellates. Maximum-parsimony analysis indicated that G. beii (fo- raminiferal symbiont) is related to but still distinct from the Symbiodinium clade (fig. 3). Although G. simplex branched prior to the divergence of Symbiodinium and G. beii, this node was not well supported (bootstrap value of 59%). It does appear that the Symbiodinium species and G. beii shared a recent common ancestor, but the identification of “ancestral” lineages within the dinoflag- ellates has been problematic (McNally et al. 1994).
The Symbiodinium clade included two benthic fo- raminiferal dinoflagellate symbionts (Lee, Wray, and Lawrence 1995). Interestingly, the benthic foraminiferal symbionts were more closely related to the symbionts from benthic invertebrates than to the symbionts of the planktonic foraminifera. This result could be explained if one considers that the two groups of foraminifera oc- cupy distinctly different environments. The benthic fo- raminifera co-occur with many symbiont-bearing ben- thic invertebrates and perhaps the establishment of sym- bioses within the benthic foraminifera is based upon availability or environmental conditions rather than in- heritance (Lee, Wray, and Lawrence 1995).
More surprising than the relationship of the benthic foraminiferal symbionts relative to the planktonic fora-
1196 Cast and Caron
miniferal symbionts was the dissimilarity of the sym- bionts of planktonic foraminifera and radiolaria. The di- vergence of the radiolarian dinoflagellate symbionts from the foraminiferal symbionts was evident from their position within both trees (fig. 3 and 4). Note that there is no significance to the branch order within the sarco- dine symbiont groups in either reconstruction. The geo- graphical, temporal, and vertical distributions of the two planktonic sarcodine groups overlap significantly, as do their life histories and feeding behaviors (Swanberg and Caron 199 1). Given the close relationship observed for the symbionts of benthic foraminifera and invertebrates, one might have expected the symbionts of the plankton- ic sarcodines to be more closely related than they were.
When maximum-likelihood was used for phyloge- netic reconstruction, the branch lengths emphasized the similarity of the sequences within either the planktonic foraminiferal or the radiolarian symbiont groups (fig. 4). The close relationship between G. beii and G. simplex also becomes more evident. G. simplex was the only nonsymbiotic dinoflagellate that grouped closely with either the radiolarian or foraminiferal symbionts. Am- phidinium belaunese, the symbiont of a flatworm, and Gloeodinium viscum, the symbiont of a hydrocoral, were both unrelated to G. beii, the Symbiodinium species, and the Scrippsiella species.
Conclusions
Our molecular characterization of the sarcodine di- noflagellate symbionts was striking for two reasons. First, foraminifera and radiolaria, organisms that have been considered to be related, have similar lifestyles, and occupy the same environment, have distinctly dif- ferent symbionts. Second, the degree of symbiont spec- ificity is impressive because these symbioses must be reestablished in each individual host every generation. Reproduction among planktonic sarcodines is thought to be a sexual process involving the division of an adult into several hundred thousand gametes (Be and Ander- son 1976; Spindler et al. 1978). There is no evidence that symbionts are passed directly to the next generation via the gametes, and in at least one of the foraminiferal species the symbionts are digested immediately preced- ing gametogenesis (Be et al. 1983). In nature, reinfection occurs sometime during early ontogeny as relatively small juvenile foraminifera can be collected which al- ready possess symbiotic dinoflagellates.
Very little is known about the highly selective, yet frequently occurring, process of symbiont reacquisition in the pelagic protozoa. It is presumed that symbionts are ingested with the wide variety of phytoplankton and zoo- plankton prey consumed by these species (Caron and Swanberg 1990; Swanberg and Caron 1991) and that ap- propriate dinoflagellates are retained as symbionts rather than digested. No information is available at present on the distribution of free-living symbiotic dinoflagellates because it has not been feasible to identify free-living cells in water samples. The srDNA sequence information described here, however, will allow the development of rRNA-targeted oligonucleotide probes that can be used
to detect and identify free-living symbiotic dinoflagellates and study their distribution and acquisition.
The information we have obtained from an srDNA- based analysis of the symbionts suggests that coevolu- tion of symbiont and planktonic sarcodine host has not occurred. We do not have the corresponding molecular phylogenetic information for the planktonic foraminifera and radiolaria, but the lack of molecular variation within the two sarcodine symbiont lineages, compared to the taxonomic diversity of the hosts, suggests that while specificity for a single species of symbiont may exist, speciation of the host has occurred independently. Lang- er and Lipps (1994) also found that the phylogeny of the dinoflagellate symbionts indicated that they evolved independently of their benthic foraminiferal hosts.
The instances where similar sarcodines have com- pletely different algal symbionts (Anderson 1983, p. 12 1; Faber et al. 1988) and the similarity of the sym- bionts from VeZeZZa and the radiolaria are further evi- dence that although the symbiotic relationships are spe- cific within the hosts, they are not unique to a particular host. The presence of very similar symbionts in very different hosts is extremely interesting because it may indicate the existence of genetic characters in these di- noflagellates that are universally involved in establish- ment and maintenance of the symbiotic association.
Recent work by Rowan and Knowlton (1995) in- dicated that different species of zooxanthellae could in- habit the same species of coral host, perhaps in response to light zonation at different depths. Our work has shown a very specific association of symbiont and host over time and space. At the present time, we have found only one type of symbiont present in a host. There was no evidence for different species of dinoflagellate sym- bionts within either the foraminifera or the radiolaria over three different collecting trips (preliminary data from third trip not shown), each at least 6 months apart and in different locations in the Sargasso Sea. There appears to be some strain variation (detectable only by sequence analysis) for Gymnodinium beii isolates from 0. universa, but this did not correlate with the different sampling times or locations. Because we have only ob- tained samples from the Sargasso, there is the potential that different dinoflagellate symbionts exist in sarco- dines from other oceans. srDNA sequence information from those organisms would help determine whether the symbiont specificity is universal or regional.
Acknowledgments
We thank M. Sogin, R. Norris, L. Amaral-Zettler, and E. L. Lim for discussions and comments on the manuscript. We also thank L. Maranda for our Sym- biodinium culture and D. Anderson for the culture of Gymnodinium sanguineum. We are grateful to A. Mi- chaels, H. Trapido-Rosenthal, members of their labs, and the staff and crew at the Bermuda Biological Station for Research for making the fieldwork possible. This work was supported by NSF Grant OCE-93 14533 and the WHO1 Education Program (WHO1 contribution #9243).
Molecular Phylogeny of Symbiotic Dinoflagellates 1197
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