Download Molecular Phylogeny of Symbiotic

Molecular Phylogeny of Symbiotic Dinoflagellates
Foraminifera and Radiolaria
from Planktonic
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 independent 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 oligotrophic oceans. Living specimens form conspicuous
biological assemblages
that contribute to primary production, herbivory, and carnivory within epipelagic oceanic communities
(Swanberg and Caron 1991; Caron et
al. 1995). In addition, foraminifera
form calcium carbonate skeletons that are used extensively in the analysis
of marine sediments and paleoclimatological
reconstruction (Bolli, Saunders, and Perch-Nielsen
1985).
Many of the extant species of planktonic foraminifera and radiolaria in the surface waters of tropical and
subtropical oceans harbor algal symbionts. Four species
of planktonic
foraminifera
(Globigerinoides
conglobatus, G. ruber, G. sacculifer, and Orbulina universa) and
numerous species of radiolaria contain symbiotic dinoflagellates (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 Callozoum inerme were first described as Zooxanthella nutricula (Brandt 188 I), but emendations
to the identification of radiolarian dinoflagellate
symbionts have included reassignment to the genera Amphidinium and Endodinium (Blank and Trench 1986). Recently, Banaszak,
Iglesias-Prieto,
and Trench (1993) proposed identificaAbbreviations:
x-DNA, small subunit ribosomal
RFLR restriction fragment length polymorphism.
Key words: srDNA,
minifera, radiolaria.
dinoflagellate,
symbiont,
RNA
gene;
phylogeny,
fora-
Address for correspondence
and reprints: R. J. Cast, Biology Department, Woods Hole Oceanographic
Institution, Woods Hole, Massachusetts 02.543. E-mail: [email protected].
1192-l 197. I996
0 1996 by the Society for Molecular Bdogy
Mol. B/01. EV0l. 139):
1192
and Evolution.
ISSN: 0737-4038
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 planktonic foraminifera indicated that these species were similar to species of Aureodinium
(Spindler and Hemleben
1980, pp. 133-140). More recent studies of the morphologic features of symbionts cultured from 0. universa led Spero to describe these symbionts as Gymnodinium beii (Spero 1987). The identities of the symbionts from the other three dinoflagellate-bearing
foraminifera have not been confirmed, although they have been
presumed to be similar to G. beii. Using srDNA sequences, 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 benthic 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-October 1994 and May 1995. Sarcodines were collected
individually
by divers to ensure that these delicate organisms 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
Molecular
Phylogeny
of Symbiotic
Dinoflagellates
1195
I
Symbiont (Collozoum caudatum)
Symbiont (Thalassicolla nucleata)
Symbiont (Spongostaurus spp.)
Radiolarian
symbionts
Symbiont (UCR 153)
Symbiont (UCR 159)
Symbiont (USR 332)
I
Symbiont (Veleila velella)
Symbiodinium spp. (Marginopora
kudakajimensis)
*
Symbiodinium spp.
Symbiodinium corculorum
Gymnodinium beii (Orbulina universa)
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 nucleotides. Both sarcodine symbiont lineages are highlighted. Benthic foraminiferal symbiont species are indicated by asterisks.
We also found that the dinoflagellate
symbionts
from the radiolaria examined were identical to each other, despite the rather wide taxonomic positions of the
hosts. All of the identified hosts were spumellarian
radiolaria. However, Thalassicolla
nucleata and the colonial 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 Srinivasan 1983).
Sequences
from the radiolarian
symbionts
were
very similar to the symbiont of Velella velella (four differences out of 1,802 bp), and quite distinct from G. beii
(85 bases out of an aligned 1,802 bp). Based on morphologic 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 Mediterranean and the dinoflagellate
symbiont of the colonial
radiolarian
C. inerme. For these two symbionts,
they
proposed the names Scrippsiella chattonii and Scrippsiella nutricula, respectively,
retaining the original species distinctions (Brandt 1881; Taylor 1971) but emending the genus.
Our srDNA data for the symbionts from the Sargasso Velella and the Sargasso radiolaria support the
conclusion
that the symbionts of Velella and the radi-
olaria are closely related (Banaszak, Iglesias-Prieto,
and
Trench 1993). However, the srDNA data (four base differences) do not support a distinction
between the radiolarian and Velella symbionts isolated from the Sargasso. Based on precedence (Brandt 1881), we propose
the name Scrippsiella nutricula for both of these symbionts. 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 different 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 planktonic sarcodines that we have collected. We have cultured 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 similar 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 different types of samples (microdissected
vs. cultured), as
well as from different organisms, if we were not amplifying the symbiont. It is possible that our PCR-based
analyses obscured some of the diversity that exists in
the natural symbiont population.
However, our experiments were designed to determine the broad phylogenetic relationships
of the symbionts, and microheterogeneity that might exist within (or between) populations
would not change our conclusions.
Phylogenetic
reconstructions
based on srDNA sequences were accomplished to examine the relationship
between
symbiotic
and nonsymbiotic
dinoflagellates.
Maximum-parsimony
analysis indicated that G. beii (foraminiferal symbiont) is related to but still distinct from
clade (fig. 3). Although G. simplex
the Symbiodinium
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 dinoflagellates has been problematic (McNally et al. 1994).
The Symbiodinium
clade included two benthic foraminiferal
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 occupy distinctly different environments.
The benthic foraminifera co-occur with many symbiont-bearing
benthic invertebrates and perhaps the establishment
of symbioses within the benthic foraminifera
is based upon
availability
or environmental
conditions rather than inheritance (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 symbionts of planktonic foraminifera and radiolaria. The divergence
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 sarcodine symbiont groups in either reconstruction.
The geographical, 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 planktonic sarcodines to be more closely related than they were.
When maximum-likelihood
was used for phylogenetic 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. Amphidinium 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 dinoflagellate
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 different symbionts. Second, the degree of symbiont specificity 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 Anderson 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 preceding gametogenesis
(Be et al. 1983). In nature, reinfection
occurs sometime during early ontogeny
as relatively
small juvenile foraminifera
can be collected which already 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 zooplankton prey consumed by these species (Caron and
Swanberg 1990; Swanberg and Caron 1991) and that appropriate 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 srDNAbased analysis of the symbionts suggests that coevolution 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.
Langer 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 completely different algal symbionts
(Anderson
1983, p.
12 1; Faber et al. 1988) and the similarity of the symbionts from VeZeZZa and the radiolaria are further evidence that although the symbiotic relationships are specific 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 dinoflagellates that are universally
involved in establishment and maintenance
of the symbiotic association.
Recent work by Rowan and Knowlton (1995) indicated that different species of zooxanthellae
could inhabit 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
symbionts 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 obtained samples from the Sargasso, there is the potential
that different dinoflagellate
symbionts
exist in sarcodines 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 Symbiodinium culture and D. Anderson for the culture of
Gymnodinium
sanguineum. We are grateful to A. Michaels, 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
LITERATURE
CITED
ANDERSON, 0. R. 1983. Radiolaria.
Springer-Verlag,
New
York.
BANASZAK, A. T., R. IGLESIAS-PRIETO, and R. K. TRENCH.
1993. Scrippsiella
velellae sp. nov. (Peridiniales)
and
Gloeodinium viscum sp. nov. (Phytodiniales),
dinoflagellate
symbionts of two Hydrozoans
(Cnidaria). J. Phycol. 29:
517-528.
BB, A. W. H., and 0. R. ANDERSON. 1976. Gametogenesis
in
planktonic foraminifera. Science 192:890-892.
BB, A. W. H., 0. R. ANDERSON, W. W. FABER JR., and D. A.
CARON. 1983. Sequence of morphological and cytoplasmic
changes during gametogenesis in the planktonic foraminifer
Globigerinoides sacculifer (Brady). Micropaleontology
29:
310-325.
BB, A. W. H., C. HEMLEBEN, 0. R. ANDERSON, M. SPINDLER,
J. HACUNDA, and S. TUNTIVATE-CHOY. 1977. Laboratory
and field observations
of living planktonic foraminifera.
Micropaleontology
23: 155-179.
BILOFSKY, H. S., and C. BURKS. 1988. The GenBank genetic
sequence data bank. Nucleic Acids Res. 16: 1861-1864.
BLANK, R. J., and R. K. TRENCH. 1986. Nomenclature of endosymbiotic dinoflagellates. Taxon 35:286294.
BOLLI, H. M., J. B. SAUNDERS, and K. PERCH-NIELSEN. 1985.
Plankton stratigraphy.
Cambridge University Press, New
York.
BRANDT, K. 188 1. Ueber das Zusammenleben
von Thieren und
Algen. Verhandlungen
der Physikalische
Gesellschaft
zu
Berlin.
CARON, D. A., A. E MICHAELS, N. R. SWANBERG, and E A.
HOWSE. 1995. Primary productivity
by symbiont-bearing
planktonic sarcodines (Acantharia, Radiolaria, Foraminifera) in surface waters near Bermuda. J. Plankton Res. 17:
103-129.
CARON, D. A., and N. R. SWANBERG. 1990. The ecology of
planktonic sarcodines. Rev. Aquat. Sci. 3: 147-180.
CILIA, V., B. LAFAY, and R. CHRISTEN. 1996. Sequence heterogeneities
among 16s ribosomal RNA sequences, and
their effect on phylogenetic
analyses at the species level.
Mol. Biol. Evol. 13:45 1-461.
FABER, W. W., 0. R. ANDERSON, J. L. LINDSEY, and D. A.
CARON. 1988. Algal-foraminiferal
symbiosis in the plankGlobigerinella aequilateralis: I. Occurtonic foraminifer
rence and stability of two mutually exclusive chrysophyte
endosymbionts
and their ultrastructure.
J. Foraminiferal
Res. 18:334-343.
FELSENSTEIN, J. 1989. PHYLIP-phylogeny
inference package. Version 3.2. Cladistics 5: 164-166.
Fox, G. E., J. D. WISOTZKEY, and I? JURTSHUK. 1992. How
close is close: 16s rRNA sequence identity may not be
sufficient to guarantee species identity. Intl. J. Syst. Bacteriol. 42: 166-170.
KENNETT, J. l?, and M. S. SRINIVASAN. 1983. Neogene planktonic foraminifera:
a phylogenetic
atlas. Hutchinson Ross
Publishing Company, Stroudsburg, Pa.
LANGER, M. R., and J. H. LIPPS. 1994. Phylogenetic incongruence between dinoflagellate endosymbionts
(Symbiodin&m)
and their host foraminifera (Sorites): small-subunit ribosoma1 RNA gene sequence evidence. Mar. Micropaleontol.
26:179-186.
LEE, J. J., S. H. HUTNER, and E. C. BOVEE. 1985. An illustrated
guide to the protozoa. Society of Protozoologists,
Lawrence, Kans.
LEE, J. J., C. G. WRAY, and C. LAWRENCE. 1995. Could foraminiferal zooxanthellae
be derived from environmental
Phylogeny
of Symbiotic
Dinoflagellates
1197
pools contributed by different coelenterate hosts? Acta Protozool. 34:75-85.
LENAERS, G., C. A. SCHOLIN, Y. BHAUD, D. SAINT-HILAIRE,
and M. HERZOG. 1991. A molecular phylogeny of dinoflagellate protists (Pyrrhophyta) inferred from the sequence of
the 24s rRNA divergent domains Dl and D8. J. Mol. Evol.
32:53-63.
MCNALLY, K. L., N. S. GOVIND, I? E. THOMI& and R. K.
TRENCH. 1994. Small-subunit
ribosomal DNA sequence
analyses and a reconstruction
of the inferred phylogeny
among symbiotic dinoflagellates
(Pyrrophyta).
J. Phycol.
30:3 16-329.
MEDLIN, L., H. J. ELWOOD, S. STICKEL, and M. L. SOGIN.
1988. The characterization
of enzymatically
amplified eukaryotic 16S-like rRNA-coding regions. Gene 71:491-499.
ROWAN, R. 1991. Molecular systematics of symbiotic algae. J.
Phycol. 27:661-666.
ROWAN, R., and N. KNOWLTON. 1995. Intraspecific diversity
and ecological zonation in coral-algal symbiosis. Proc. Natl.
Acad. Sci. USA 92:2850-2853.
ROWAN, R., and D. A. POWERS. 1991a. A molecular genetic
classification of zooxanthellae and the evolution of animalalgal symbiosis. Science 251: 1348-135 1.
1991 b. Molecular genetic identification of symbiotic
dinoflagellates (zooxanthellae). Mar. Ecol. Prog. Ser. 71:6573.
-.
1992. Ribosomal RNA sequences and the diversity of
symbiotic dinoflagellates (zooxanthellae). Proc. Natl. Acad.
Sci. USA 89:3639-3643.
SADLER, L. A., K. L. MCNALLY, N. S. GOVIND, C. E BRUNK,
and R. K. TRENCH. 1992. The nucleotide sequence of the
small subunit ribosomal RNA gene from Symbiodinium piZosum, a symbiotic dinoflagellate. Cm-r. Genet. 21:409-416.
SAIKI, R., I? S. WALSH, C. LEVENSON, and H. ERLICH. 1988.
Primer-directed
enzymatic amplification
of DNA with a
thermostable DNA polymerase. Science 239:487-494.
SPERO, H. J. 1987. Symbiosis in the planktonic foraminifer,
Orbulina universa, and the isolation of its symbiotic dinoflagellate, Gymnodinium beii sp. nov. J. Phycol. 23:307317.
SPINDLER, M., 0. R. ANDERSON, C. HEMLEBEN, and A. W. H.
BB. 1978. Light and electron microscopic observations of
gametogenesis
in Hastigerina pelagica (Foraminifera).
J.
Protozool. 25:427-433.
SPINDLER, M., and C. HEMLEBEN. 1980. Endocytobiology
endosymbiosis
and cell biology. Walter de Gruyter & Co.,
Berlin.
SWANBERG, N. R., 0. R. ANDERSON, and I? BENNETT. 1985.
Spongiose spumellarian radiolaria: the functional morphology of the radiolarian skeleton with a description of Spongostaurus, a new genus. Mar. Micropaleontol.
9:455-464.
SWANBERG, N. R., and D. A. CARON. 199 1. Patterns of sarcodine feeding in epipelagic oceanic plankton. J. Plankton
Res. 13:287-3 12.
SWOFFORD, D. L. 1990. PAUP: phylogenetic
analysis using
parsimony. Version 3.0. Illinois Natural History Survey,
Champaign.
TAYLOR,D. L. 197 1. Ultrastructure of the ‘zooxanthella’ Endodinium chattonii in situ. J. Mar. Biol. Assoc. UK 51:227-234.
WALSH, I? S., D. A. METZGER, and R. HIGUCHI. 1991. Chelex
100 as a medium for simple extraction of DNA for PCR-based
typing from forensic material. Biotechniques 10:506-5 13.
MITCHELL SOGIN, reviewing
Accepted
July
18, 1996
editor