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Application of molecular methods to the study of diversity and biogeography of marine ciliates
George
1Dept.
1
McManus ([email protected]),
Oona
Marine Sciences, University of Connecticut, Groton CT;
Introduction
Ciliates are an important component of zooplankton communities. They are often the dominant herbivores,
and they serve as food for copepods and other mesozooplankton, including larvae of commercially-important
fish species. They can be challenging to study because they are difficult to cultivate and may be destroyed
beyond recognition by commonly used preservatives (e.g. formaldehyde). For these and other reasons,
traditional morphologically-based taxonomic descriptions of species are problemmatic. This has resulted in
difficulties in both species level and higher taxonomy, and in developing an understanding of ciliate
phylogeny. On the other hand, ciliates are ideal for use in molecular studies for several reasons:
2
Snoeyenbos-West ,
2Dept.
Laura
2
Katz ,
Huan
1
Zhang ,
Senjie
1
Lin
Biological Sciences, Smith College, Northampton MA
I. Molecular phylogeny vs traditional taxonomy
II. Endemism, morphological variation, and cryptic
species within the tintinnids
tintinnids
Tintinnids
1. Ciliates contain two different kinds of nuclei. The transcriptionally-active macronucleus contains a highlyamplified genome, with thousands of copies of some genes. With such high copy numbers, PCR products can
be obtained from very few ciliates, in some cases from a single individual.
2. Most ciliates are large enough and abundant enough to be amenable to hand-picking under a stereomicroscope,
obviating the need for preservatives or concentration by filtration.
oligotrichs
Ecological questions
1. Does endemism exist among ciliate species? A long-held view in microbial ecology is summed up by the
phrase “Everything is everywhere”. In this view, there is no true endemism among microbes, including
ciliates, because small organisms have such large absolute population sizes and high dispersal rates that
extinction and geographical isolation of populations are virtually impossible.
choreotrichs
?
2. How well do morphologically-based descriptions of species match up with DNA sequences? And how well
do higher-level morphological groupings of species compare with DNA-based phylogenies?
3. Can we use highly-variable DNA marker sequences to discriminate among populations of individual ciliate
species as they vary over space and time in the sea?
4. Can we use DNA sequences in the chloroplast genome to identify the source of enslaved chloroplasts in
natural ciliate populations? Many herbivorous ciliates have the interesting habit of retaining and “enslaving”
chloroplasts from their food. These chloroplasts remain photosynthetically active, to the ciliate’s benefit.
Methods
We have been using sequences from various genes to address the questions outlined above. In particular,
1. 18S ribosomal RNA and alpha-tubulin genes for higher-level phylogeny.
Figure 1. Molecular phylogenies based on the 18S rDNA and ITS genes produced the
same topologies (shown here), and were consistent with traditional, morphologicallybased higher-level taxonomy. The three major groups of planktonic ciliates, tintinnids,
oligotrichs, and choreotrichs, clustered as distinct groups. An exception was the
freshwater species Halteria grandinella, which had already been proposed for removal
to a different subclass (Stichotricha) by morphological taxonomists (grey box in
figure). An unknown ciliate that grew up in one of our tintinnid cultures (indicated by
the “?”) did not cluster with any of the three groups. The phylogenetic tree produced
with the alpha-tubulin sequences was not concordant with either traditional taxonomy
or the 18S and ITS trees (not shown).
2. 5.8S ribosomal RNA genes, with adjacent internally-transcribed spacer regions (ITS1 and ITS2; see figure
below) for species and population-level questions.
III. Source of enslaved chloroplasts
rbcL-1 tree
3. Form I rubisco large subunit gene (rbclI) from chloroplast DNA for questions about enslaved chloroplast
source.
Ciliates hand-picked with a drawn-out capillary from natural populations or cultures (usually < 2 weeks old)
were extracted and the resulting DNA subjected to PCR. PCR products were cloned and sequenced, and
phylogenetic trees were created using CLUSTAL W, in some cases with additional sequences obtained from
GenBank.
Four tintinnid ciliates. This protist group is characterized by the lorica or outer covering. Traditionally,
species descriptions are based on size and shape of the lorica, which can be variable.
99
100
90
S. oculatum DUB(1)
ThalassiosiraAB018007
KarlodiniumAF463410
Emiliania D45845
Porphyra AF319460
Aureococcus AF117905
Heterosigma X61918M
Cryptomonas sp P14957
Fucus AF195515
Nostoc P00879
Pyramimonas AJ404887
Euglena X70810
DunaliellaAJ001877
Chlamydomonas AB084334
Platydorina D86827
94
Volvox AB076084
Gonium AB006821
Caulerpa AB038486
Derbesia AF212142
Codium AB038481
100
Tetraselmis U30284
Prasiola AF189064
Chlorella AF499684
Ulotrichales
Ulothris AF499683
Strombidium oculatumDUB(1)
93 100
S. oculatum DUB(1)
S. oculatum DUB(1)
S.
oculatum GAL(1)
88
Enteromorpha intestinalisAF189070
81
S. stylifer LIS(8)
E. clathrata AF525939
95 Ulvaria AF499673
Ulvales
Ulva AF499669
Enteromorpha-like DUB-A(3)
S. oculatum IOM(4),DUB(1),GAL(3)
0.1
90 Enteromorpha-like DUB-B(1)
Enteromorpha-like GAL(2)
Red
-like
Green
-like
Ulvophyceae
Figure 3. Strombidium oculatum (top photo) and S. stylifer (bottom) are oligotrichs that
live in tide pools. Both contain grass-green chloroplasts retained from ingested algae.
Although the color of the chloroplasts would suggest a chlorophyte or prasinophyte
origin, light microscopy cannot reveal more than that. We sequenced the rbclI gene from
chloroplasts in both ciliates and analyzed the results by constructing an rbclI tree based
on GenBank sequences and those from macroalgae collected with the ciliates. The
surprising result is that these ciliates apparently eat macroalgae (seaweeds). Probably,
the ciliates ingest gametes or zoospores released by green algae in the tide pools.
Figure 2. The tintinnids live in a kind of shell called a lorica. This may be clear
(hyaline), as in the top two pictures in the figure above, or agglutinated (with attached
organic or inorganic particles), as in the lower seven pictures above. The
agglutinated forms, typified by the genus Tintinnopsis here, present particularly
difficult taxonomic problems as there are few morphological traits with which loricas
can be distinguished. This detailed tree of ITS sequences illustrates several points.
First, the agglutinated Tintsp00C, which we identified as a Tintinnopsis sp., is not
monophyletic with the other Tintinnopsis representatives, suggesting that the
morphological definition of the genus is not adequate. Second, the sequences for
Ttub99 and Tintsp00B indicated them to be the same species, even though the loricas
are quite different, again pointing up problems with using the lorica as a
morphological entity in species descriptions. The same can be said of Tvas01 and
Tintsp02F, which are identical by molecular methods, but not by morphology. On the
other hand, species that appear quite similar by lorica morphology may be only
distantly related, as illustrated by the isolates Ttub99 and Tintsp00A. Finally, two
pairs of ciliates (Tvas01/Tintsp02F and Tintsp00A/Tintsp02E) showed virtually
identical sequences even though they were collected far apart in space (Long Island
Sound vs The Irish Sea) and time (more than 1 year apart). This supports the idea
that there is little or no endemism among ciliates. Similarly, we have obtained
identical DNA sequences for Strombidium oculatum, an oligotrich ciliate that lives
only in tide pools, in samples from The Isle of Man, Ireland, and Maine, suggesting
that even species from restricted habitats can disperse over long distances.
Future work
 Find more variable genetic markers to examine
intraspecific variation at the population level
 Develop probes from our library of sequences to
identify species in a sample using F.I.S.H.
 Develop arrays of probes for examination of DNA
extracted from bulk seawater samples
 New methods for in situ examination of ciliates DNA?