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Applied and Environmental Microbiology, August 2001, p. 3501-3513, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3501-3513.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Rapid Evolution of a Sexual Reproduction Gene in
Centric Diatoms of the Genus Thalassiosira
E. Virginia
Armbrust* and
H. M.
Galindo
Marine Molecular Biotechnology Laboratory,
School of Oceanography, University of Washington, Seattle,
Washington 98195
Received 2 February 2001/Accepted 8 May 2001
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ABSTRACT |
Sexual reproduction is commonly assumed to occur in the vast
majority of diatoms due to the intimate association of this process with cell size control. Surprisingly, however, little is known about
the impact of sexual events on diatom population dynamics. The
Sig1 gene is strongly upregulated during sexual
reproduction in the centric diatom Thalassiosira
weissflogii and has been hypothesized to encode a protein
involved in gamete recognition. In the present study, degenerate PCR
primers were designed and used to amplify a portion of
Sig1 from three closely related species in the
cosmopolitan genus Thalassiosira, Thalassiosira
oceanica, Thalassiosira guillardii, and
Thalassiosira pseudonana. Identification of
Sig1 in these three additional species facilitated
development of this gene as a molecular marker for diatom sexual
events. Examination of the new sequences indicated that multiple copies
of Sig1 are probably present in the genome. Moreover,
compared to the housekeeping gene
-tubulin, the Sig1
genes of isolates of T. weissflogii collected from
different regions of the Atlantic and Pacific oceans displayed high
levels of divergence. The Sig1 genes of the four closely related Thalassiosira species also displayed high levels
of sequence divergence compared to the levels observed with a second
gene, Fcp, probably explaining why Sig1
could not be amplified from more distantly related species. The high
levels of sequence divergence both within and between species suggest
that Sig1 is rapidly evolving in a manner reminiscent of
the manner observed in other genes that encode gamete recognition
proteins. A simple model is presented for Sig1 evolution
and the implications of such a rapidly evolving sexual reproduction
gene for diatom speciation and population dynamics.
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INTRODUCTION |
Diatoms are the most species-rich
group of phytoplankton known. Conservative estimates suggest that tens
of thousands of different species of diatoms are distributed throughout
marine and freshwater ecosystems (27). Explosive
diversification of diatom species has therefore occurred over the last
200 million years (27, 30). Typically, tens to perhaps
hundreds of species of diatoms comprise the phytoplankton community of
any given body of water. Under most circumstances, diatoms are likely
to be essentially indifferent to whether neighboring cells are
the same or different species. During sexual events, however, the
ability of a given species to distinguish between itself and all other
species becomes critical.
The onset of sexual reproduction in diatoms is commonly coupled to
control of cell size. Due to physical and developmental constraints
associated with generation of the silica frustule, each mitotic
division results in the formation of two daughter cells of different
sizes, one that is the same size as the parent and one that is slightly
smaller. Thus, over successive generations the mean cell size of a
diatom population decreases (26, 37). Interestingly, only
relatively small cells within a population respond to environmental
signals and undergo sexual reproduction, an event that ultimately
restores cell size (10). Consequently, multiple species of
diatoms may undergo sexual reproduction simultaneously in a single body
of water (reviewed in reference 11). In centric diatoms,
flagellated sperm formed during sexual events must distinguish not only
between vegetative cells and egg cells still encased within their
frustule but also between vegetative and egg cells of different species.
The molecular basis of species-specific gamete recognition during
external fertilization has been examined in marine
invertebrates such as abalone (42, 45), sea
urchin (31), and teguline gastropods (17). A
common feature of sexual recognition proteins appears to be rapid
diversification of amino acid sequences in closely related species
(33, 43). In many instances, strong selection for sequence
variation, known as positive Darwinian selection, appears to occur. The
unicellular freshwater algal genus Chlamydomonas is the only
phytoplankton genus in which evolution of a sex-related protein has
been examined (12). The C. reinhardtii protein,
MID, is required for gamete differentiation, and the sequences
from two closely related species display dramatic differences, although
positive selection does not appear to underlie the evolution of this
protein. Ultimately, rapid diversification of sexual recognition proteins is expected to lead to speciation, although it remains unclear
what forces underlie the evolution of new species (12, 45).
We recently identified in the centric diatom Thalassiosira
weissflogii a gene family, composed of Sig1,
-2, and -3, whose transcription is highly
upregulated during the onset of sexual reproduction. The proteins
encoded by these genes appear to be part of the extracellular matrix
and have been hypothesized to play a role in mediating sperm-egg
recognition (1). Our initial goal in the present study was
to determine whether Sig homologues could be identified in
other species of centric diatoms and whether upregulation of these
genes could serve as a molecular marker for the occurrence of sexual
reproduction in field populations. In this work, we found that the gene
on which we focused, Sig1, is a multicopy gene that appears
to be undergoing rapid divergence both within and between species, a
feature that has come to be expected for genes encoding proteins
involved in sexual recognition.
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MATERIALS AND METHODS |
Culture conditions.
Seven T. weissflogii
isolates (CCMP1336 clone Actin from Long Island Sound, New York;
CCMP1049 clone 4C from Long Island Sound, New York; CCMP1050 clone
WTFLU from Del Mar Slough, California; CCMP1051 clone THALA7 from King
Kalakaua's Fishpond, Hawaii; CCMP1052 clone TTW1 from Segerrak
Sea, Norway; CCMP1053 clone SA from Portugal; CCMP1587 clone JA92I from
Jakarta Harbor, Indonesia) and isolates of four additional
Thalassiosira species, Thalassiosira guillardii (CCMP988 clone 7-15 from the North Atlantic Ocean), Thalassiosira oceanica (CCMP1005 clone 13-1 from the Sargasso Sea),
Thalassiosira rotula (CCMP1647 from the Bay of Naples,
Italy), and Thalassiosira pseudonana (CCMP1335 clone3H from
Moriches Bay, New York) were purchased from the Provasoli-Guillard
National Center for Culture of Marine Phytoplankton (CCMP), Bigelow
Laboratory for Ocean Sciences. A Thalassiosira antarctica
isolate was obtained from R. J. Olson of Woods Hole Oceanographic
Institution (Table 1). All cultures were
maintained in f/2-enriched seawater (14). All species
except T. guillardii and T. antarctica were
maintained at 20°C with continuous illumination at 120 µmol of
photons · m
2 · s1. The T. guillardii culture was
maintained at 14°C with a cycle consisting of 16 h of light (66 µmol of photons · m2 · s1) and 8 h of darkness. The T. antarctica culture was maintained at 2°C with constant
illumination at 20 µmol of photons · m2 · s1.
Clonal isolates of
T. weissflogii clone Actin were obtained
by plating cells on f/2-enriched seawater solidified with 1.5%
agar
(Difco). Individual colonies were then transferred to and
maintained in
liquid f/2 media. The
T. weissflogii clone Actin
isolates
were induced to undergo sexual reproduction by interrupting
exponential
growth in continuous light with 12 h of darkness (
1,
3).
Nucleic acid isolation and generation of cDNAs.
Genomic DNA
was isolated with a DNeasy Plant Mini Kit (Qiagen). Total RNA
was isolated with an RNeasy Plant Mini Kit (Qiagen). First-strand cDNAs were generated from 500 ng of total RNA with a 1st
Strand cDNA synthesis kit (Clontech).
Detection of DNA polymorphisms.
DNA polymorphisms were
examined in two gene fragments, -tubulin
(2) and Sig1 (1). An internal
fragment of the -tubulin gene spanning a
single intron was amplified by using two gene-specific PCR primers,
5'-TTCGACCGGATAACTTTG-3' (forward) and
5'-CGACTAGTCAAAGGAGC-3' (reverse). PCR amplifications in
reaction mixtures (final volume, 20 µl) containing each
deoxynucleoside triphosphate (dNTP) at a concentration of 0.1 mM, 2.5 mM MgCl2, 10 pmol of each primer, and 0.75 U of
Taq DNA polymerase (Display) began with a 2-min denaturation step at 94°C, which was followed by 35 cycles of 94°C
for 10 s, 50°C for 30 s, and 72°C for 90 s and then
by a final extension at 72°C for 10 min. The genomic fragment was
eluted from a low-melting-point agarose gel (40), cloned
into pCR2.1-TOPO, and transformed into TOP10 Escherichia
coli cells with a TOPO TA cloning kit (Invitrogen). Positive
transformants containing inserts of the correct size were identified by
PCR using the vector-specific M13F and M13R primers. Plasmid DNAs were
isolated from the transformants with a Mini Prep kit (Qiagen), were
sequenced with a DYEnamic ET dye terminator kit (Amersham Pharmacia
Biotech Inc.), and were analyzed with a MegaBACE 1000 (Molecular Dynamics).
Sig1 DNA fragments were obtained in the following manner.
Blockmaker (
http://blocks.fhcrc.org/blockmkr/make_blocks.html)
was
used to identify conserved amino acid domains within the SIG1,
SIG2, and SIG3 proteins (
1). Based on the amino acid
sequences
of the conserved domains, degenerate PCR primers were
designed
by using the CODEHOP algorithm (
36) to amplify a
fragment from
the
Sig1 and
Sig3 genes. The
forward
Sig primer was
5'-AACGCTGCTCTGGCCACGGNWCTTGYGG-3',
and the reverse primer
was 5'-GGGCCGGTATATCCAGGATCRCAYTTRCAWCC-3'.
PCR
amplifications in reaction mixtures (final volume, 20 µl)
containing
each dNTP at a concentration of 0.1 mM, 2.5 mM
MgCl
2,
10 pmol of each primer, and 0.75 U of
Taq DNA polymerase (Display)
began with a 2-min denaturation
step at 94°C, which was followed
by 30 cycles of 94°C for 10 s, 62°C for 30 s, and 72°C for 90
s and then by a final
extension at 72°C for 10 min. Genomic or
cDNA fragments corresponding
to
Sig1 were cloned, and the resulting
transformants were
screened as described above for inserts of
the correct
size.
Positive
Sig1 transformants were PCR amplified a second time
with the degenerate
Sig-specific primers as described above,
but this time the forward primer was labeled with FAM (Operon).
The resulting fluorescent PCR products were analyzed by using
single-strand conformational polymorphism (SSCP) (
23). PCR
products
were diluted 1:1 with deionized formamide, denatured for 5 min
at 95°C, and immediately placed on ice before they were loaded
onto a
10% acrylamide (ratio of acrylamide to bisacrylamide, 99:1)
gel (20 by
20 cm). The gel was electrophoresed in a water-cooled
apparatus (Owl
Scientific Inc.) at 6 V for 17 h. Fluorescent products
were
detected with a FluorImager 595 (Molecular
Dynamics).
Clones whose PCR products displayed unique SSCP patterns were chosen
for DNA sequencing. Plasmid DNAs were isolated from the
original
transformants as described above. Either the DNAs were
sequenced with a
Thermosequenase II dye terminator cycle sequencing
kit (Amersham
Pharmacia Biotech Inc.) and analyzed with a 373A
DNA sequencer (Applied
Biosystems), or they were sequenced with
a DYEnamic ET dye
terminator kit (Amersham Pharmacia Biotech Inc.)
and analyzed with a
MegaBACE 1000 (Molecular
Dynamics).
All sequence data were compiled and analyzed by using a combination of
the Wisconsin Package (version 10.0) of the Genetics
Computer Group,
Madison, Wis.; Sequencher 4.0.5 (Gene Codes);
and SeqApp
(
http://ftp.bio.indiana.edu/soft/molbio/seqapp).
Phylogenetic analysis.
The 18S rRNA genes from each species
were isolated by using the universal 18sA and 18sB primers lacking the
5' restriction sites (28). PCR amplifications in reaction
mixtures (final volume, 10 µl) containing each dNTP at a
concentration of 0.1 mM, 3.125 mM MgCl2, 10 pmol
of each primer, and 0.75 U of Taq DNA polymerase (Promega)
began with a 2-min denaturation step at 94°C, which was followed by
35 cycles of 94°C for 10 s, 50°C for 30 s, and 72°C for
60 s and then by a final extension at 72°C for 10 min. PCR
products of the correct size were cloned into pCR2.1-TOPO as described
above and were sequenced by using a combination of vector-specific and
gene-specific primers. The 18S rRNA gene-specific forward primers were
5'-CTGCCCTATCAGCTTTGG-3' (primer C) and
5'-TTGACTCAACACGGGAAAAC-3' (primer E); the 18S rRNA
gene-specific reverse primers were 5'-CGGCCATGCACCACC-3' (primer D) and 5'-ATCCAAAGCTGATAGGGCAG-3' (primer F).
Phylogenetic analyses were performed by using the default settings of
the PAUP program (Smithsonian Institution, 1997) accessed through the
Genetics Computer Group. Consensus (50% majority rule) trees
were constructed by using neighbor-joining distances with 1,000 bootstrap replicates and were viewed by using TREEVIEW
(32). Complete coding and intron sequences of
Sig1 and -tubulin gene fragments
were used for phylogenetic analyses. For phylogenetic analysis of the
18S rRNA gene, 1,635 nucleotides of an informative sequence were used.
Nucleotide sequence accession numbers.
Nucleotide sequences
have been deposited in the GenBank database under the following
accession numbers: 18S rRNA genes, AF374477 to AF374482;
-tubulin genes, AF374483 to AF374489;
Sig1 genomic DNAs, AF374490 to AF374539; and Sig1
cDNAs, AF374540 to AF374552.
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RESULTS |
Multiple copies of Sig1 are transcribed shortly
after T. weissflogii cells undergo sexual
reproduction.
Identification of five highly conserved amino acid
domains in SIG1, SIG2, and SIG3 proteins of T. weissflogii
(1) suggested that these regions could represent
functional domains that might also be conserved in SIG homologues
in different species of Thalassiosira. The CODEHOP algorithm
(36) was used to design degenerate PCR primers to
amplify DNA encoding the region spanning two of the domains, domains I
and IV (1), which display the greatest amino acid identity
for SIG1 and SIG3. Although the SIG2 protein also displayed significant
amino acid identity with the other SIG proteins in these two domains,
the level of degeneracy needed to recognize Sig2 as well was
quite high, and no attempts were made to amplify this gene.
The utility of the newly designed degenerate
Sig primers was
tested by using genomic DNA isolated from a culture that had
originated
from a single cell of
T. weissflogii clone Actin, the
clone
in which the
Sig genes were originally identified
(
1).
As expected, two fragments, which were 706 and 483 bp
long, were
amplified; the sizes corresponded to the sizes predicted for
genomic
fragments (each with a single intron) of
Sig1 and
Sig3, respectively.
To ensure that the degenerate
Sig primers were
Sig specific, the
706-bp
fragment was cloned, and DNA inserts from three transformants
were
sequenced. Pairwise comparisons of the new sequences and
the previously
published
Sig1 sequence indicated that the four
sequences
differed from one another by anywhere from 1 to 5 bp
(data not shown).
Assuming that the diploid clone from which the
DNA was isolated was a
heterozygote, the presence of four distinct
Sig1 sequences
implied that at least two
Sig1 loci might be present
in an
individual.
SSCP was used to provide an estimate of the number of unique copies of
Sig1 in an individual. Analysis of SSCP patterns can
be used
to detect, relatively quickly, sequence differences between
DNA
fragments of the same length due to sequence-dependent rates
of
migration of denatured, single-stranded DNA fragments in a
nondenaturing gel (
23). When SSCP was used with the
Sig1 clones,
at least 19 variants that migrated differently
were detected (Fig.
1A). Assuming that
the clone from which the genomic DNA was isolated
was heterozygous at
every
Sig1 locus, the SSCP results suggested
that
Sig1 was composed of at least 10 different loci.
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FIG. 1.
Representative SSCP patterns of genomic (A) and cDNA (B)
versions of Sig1, each amplified from 20 E.
coli transformants. The asterisks indicate a subset of the
original clones that were sequenced to completion.
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This potentially high number of
Sig1 loci in a single
individual was unexpected. To confirm the predicted sequence diversity,
the original plasmids corresponding to 15 of the 19
Sig1
inserts
that migrated differently were sequenced. SSCP analysis proved
to be an extremely sensitive predictor of DNA sequence variation
as
each clone with a different DNA sequence migrated differently
in the
SSCP gel (although two clones differed only in the primer
sequence),
indicating that the high level of SSCP variation was
not simply an
artifact of a second round of PCR amplification.
One
Sig1
genomic fragment displayed a 1-bp insertion, and two
fragments
displayed 1-bp deletions in the coding sequence (data
not shown). These
copies were not analyzed further since they
were assumed to represent
nonfunctional
pseudogenes.
The DNA sequences of the remaining 11 unique copies differed at 30 positions scattered throughout the 645-bp fragment (not
including the
two primer sites). Surprisingly, only four variable
sites were located
within the 87-bp intron (Table
2). At six
positions an identical sequence change was found in two copies,
and at
one position an identical change was present in three copies.
Pairwise
comparisons of all sequences indicated that the greatest
number of
substitutions for any two copies was seven, corresponding
to a
maximum sequence difference of about 1.1%. Relative to the
reference
sequence, two fragments displayed seven substitutions,
five displayed
four substitutions, two displayed two substitutions,
and only one
displayed one substitution (Table
2). Assuming that
the potential
Taq replication error rate is 0.07% per base
(
24),
then about one substitution in 1,400 bp is expected
to occur simply
due to a PCR artifact (background). The substitution
rate observed
with
Sig1 sequences was about sixfold greater
than this, which
indicates that multiple copies of
Sig1 are
present in an individual.
Interestingly, there was no obvious grouping
of the different
sequences on the basis of shared similarities, as has
been seen
with other multicopy genes (
4).
When the intron sequence was excluded from the 11 unique
Sig1 genomic sequences, 558-bp open reading frames predicted
to encode
186 amino acids were identified, suggesting that the
different
gene copies might be transcribed. To determine whether
multiple
copies of the
Sig1 gene were in fact transcribed,
the
T. weissflogii clone used for genomic DNA analysis was
induced to undergo sexual
reproduction. RNA was isolated 5 h into
sexual reproduction and
reverse transcribed into cDNAs. When
first-strand cDNAs were used
as templates for PCR with the degenerate
Sig primers, two bands,
at 558 and 400 bp corresponding to
Sig1 and
Sig3 mRNAs, respectively,
were
obtained.
The cDNA fragment that was the size of
Sig1 was cloned, and
34 different transformants were analyzed with SSCP. The differences
in
the migration rates of cDNA fragments were not as great as
the
differences in the migration rates of genomic copies, which
made it
more difficult to unambiguously identify cDNA variants
using SSCP alone
(Fig.
1B). Based on apparent differences in SSCP
migration patterns,
plasmids corresponding to 15 cDNA fragments
were chosen for sequencing.
Thirteen of the 15 cDNA sequences
were unique, and the same sequence
change was observed at three
positions in both genomic and cDNA clones
(Table
2). The same
sequence change was found at a single nucleotide
position in two
copies of the cDNA fragment and at a single position in
three
copies of the fragment. One cDNA sequence was identical to the
genomic coding sequence. There were fewer substitutions per cDNA
copy
than per genomic copy: four cDNA sequences displayed a single
substitution, seven displayed two substitutions, and only one
displayed
five substitutions. Regardless, the substitution rate
was about four
times greater than the background rate, suggesting
that multiple
copies of
Sig1 were also transcribed. As with the
genomic
copies, there was no obvious grouping of the different
cDNA
sequences.
A total of 56 nucleotide substitutions were present in the coding
sequence of the cDNA and genomic clones. Nineteen of these
were
first-position substitutions, 19 were second-position substitutions,
and 18 were third-position substitutions. Only 15 third-position
substitutions were silent and resulted in no change in the predicted
amino acid sequence. All other nucleotide substitutions resulted
in
amino acid changes. Thus, there appeared to be no bias towards
silent
substitutions, as would be expected under conditions of
stabilizing
selection in which amino acid divergence is selected
against
(
19).
The 15 cDNAs were predicted to encode 11 unique proteins with different
amino acid sequences. When genomic sequences with
the intron excluded
were included in this analysis, an additional
nine unique amino acid
sequences were observed; thus, a total
of 20 potentially unique
proteins were encoded by
Sig1 loci (Table
3). Thirty-seven amino acid changes were
scattered throughout
the predicted protein sequences. Amino acid
substitutions can
be categorized as conservative, moderate, radical, or
very radical
depending on the predicted change in composition,
polarity, and
molecular volume (
13). Eighteen of the amino
acid changes were
conservative, 12 were moderate, and 7 were radical
(Table
3).
Different protein variants were expected to display slightly
different
characteristics, suggesting that individuals might express
multiple
SIG1 proteins. Furthermore, this potential variation in SIG1
within
an individual suggested that different individuals likely
possessed
different combinations of
Sig1 copies; this was
particularly true
of individuals isolated from different locations.
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TABLE 3.
Variable amino acids in predicted open reading frames of
either genomic or cDNA copies of Sig1 amplified from
T. weissflogii clone Actin
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Sig1 displays relatively high levels of sequence
divergence in T. weissflogii isolates collected from
different ocean regions.
Intraspecific DNA sequence divergence is
expected to mirror the extent to which populations are geographically
isolated from one another (33). Intraspecific sequence
divergence was compared for two gene fragments,
-tubulin, which is required for cellular housekeeping (8), and Sig1. Genomic fragments
corresponding to the two genes were amplified from a number of T. weissflogii isolates that had been collected from different
oceanic regions over the course of 34 years (Table 1).
When
-tubulin-specific primers
(
2) were used with genomic DNAs isolated from the seven
T. weissflogii isolates, a single
671-bp fragment was
obtained, and it was subsequently cloned.
The gene encoding
-tubulin
appears to be a single-copy gene in
T. weissflogii
(
2), and only one clone was sequenced for each
isolate.
The DNA sequences of the four Atlantic isolates were
identical except
for a single transition in the intron of the
Norwegian isolate. The DNA
sequences of the three Pacific isolates
showed slightly more variation;
two nucleotide positions in the
intron and one position in the coding
sequence were variable (Table
4). In
contrast, the DNA sequences of the
-tubulin
fragment
from the four Atlantic isolates differed from the DNA
sequences
of the
-tubulin fragment from the
three Pacific isolates at 32
positions; 17 of the variable positions
were localized to the
intron, and 15 were localized to the coding
sequence (Table
4).
The
-tubulin fragment from
T. weissflogii clone Actin, the isolate
for which the
primers were originally developed (
2), has an
87-bp intron
and 582 bp of coding sequence. This means that 19.5%
of the sites in
the intron and 2.6% of the sites in the coding
sequence varied for
-tubulin sequences from different isolates.
Evolutionary distances between
-tubulin
sequences were calculated by using the Jukes-Cantor model of DNA
sequence divergence,
in which substitutions among nucleotides are
assumed to occur
at the same rate at the different nucleotide
positions and each
nucleotide can change with equal probability
to the other three
nucleotides (for a discussion, see reference
15). Based on this
model, the
-tubulin DNA coding sequences from the
Atlantic and
Pacific isolates displayed a maximum divergence of
2.8%. A distance-based
analysis grouped the three Pacific
strains together and the four
Atlantic strains together (Fig.
2A), which suggested that the
two groups
of isolates had been physically separated for a long
time. All
nucleotide changes in the coding sequences of different
isolates were
silent, which resulted in identical amino acid sequences
for all
-tubulin molecules regardless of the ocean from which
the organisms
originated. Thus, although DNA sequences distinguished
the Atlantic and
Pacific isolates, stabilizing selection appears
to have maintained the
same amino acid sequence for each predicted
protein.
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FIG. 2.
Unrooted, neighbor-joining trees for
-tubulin gene fragments (A) and
Sig1 gene fragments isolated from seven T.
weissflogii isolates from Long Island Sound, New York
(CCMP1336, CCMP1049); Norway (CCMP1052); Portugal (CCMP1053);
California (CCMP1050); Hawaii (CCMP1051); and the Java Sea
(CCMP1587). The greatest estimated distances are the distances for the
Atlantic and Pacific isolates (A) and the distances for the Long Island
and Hawaii-South Pacific isolates (B).
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The
Sig1 genomic fragment was amplified, cloned, and
sequenced from the same seven
T. weissflogii isolates. DNA
sequence polymorphisms
were observed for sequences from each
T. weissflogii isolate,
confirming the multicopy nature of this gene.
Five different
Sig1 genomic DNA sequences of each isolate
were chosen randomly for
comparison with clone Actin genomic sequences
(Table
5). Even
greater overall sequence
variation was observed in
T. weissflogii Sig1 sequences when
the comparison included genomic clones from
all the isolates; 86 nucleotide positions displayed variation.
Due to this high number of
polymorphisms, only data for substitutions
present in two or more
clones are summarized in Table
5. More
variable positions were observed
if cDNA clones were included
in the comparison (Tables
2 and
5). Only
16 of the 86 variable
positions in
Sig1 were localized to
the intron. For example, the
intron from the South Pacific isolate had
a 2-bp insertion relative
to other isolates, and the 3' splice site was
TAG rather than
CAG. In contrast to what was observed with
-tubulin, more than
80% of sequence variation
between isolates occurred within the
coding sequence of
Sig1. Similar to the
-tubulin
intron sequence
variation, about 19% of the sites in the 84-bp
Sig1 intron were
variable. In contrast, about 12.5% of the
sites in the
Sig1 coding
region were variable; this value
was nearly five times higher
than the value observed for the coding
sequence of
-tubulin.
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TABLE 5.
Variable nucleotides present in two or more copies of the
Sig1 gene fragment amplified from seven isolates of T. weissflogii
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Despite the frequent occurrence of within-individual nucleotide
polymorphisms (Table
2), the same DNA substitution was observed
at 30 different positions in all
Sig1 copies examined from
Hawaiian
and South Pacific isolates. Five positions had a substitution
found only in South Pacific copies; three positions had a substitution
found only in Hawaiian copies; one position had a substitution
found
only in Portuguese copies; and one position had a substitution
found in
all South Pacific copies and four of the five Hawaiian
copies (the
fifth Hawaiian copy had a C rather than a T at this
position) (Table
5). The greatest divergence between isolates
was the divergence between
the Long Island clone Actin isolate
and the Hawaiian and South Pacific
isolates; the estimated distance
was about 6.5% (Fig.
2B), almost
2.5-fold greater than the distance
found with
-tubulin (Fig.
2A).
Remarkably, the isolate from California did not display variation at
the same positions as other Pacific isolates (Table
5).
In fact, the
divergence between the
Sig1 sequences of the California
isolate and the Long Island clone Actin isolate was only 1.4%,
which
was comparable to the divergence observed within individual
isolates.
The California isolate clustered with and was essentially
indistinguishable from other Atlantic isolates (Fig.
2B). This
result
directly contrasts with what was observed with the
-tubulin phylogeny (Fig.
2A) and suggests that
perhaps with
Sig1 the groups
are not determined by ocean
basin but instead are determined by
a division between tropical or
subtropical regions and temperate
regions, with the South Pacific and
Hawaiian isolates belonging
to the tropical-subtropical
group.
In contrast to the uniformity of
-tubulin amino acid sequences,
pairwise comparisons of different SIG1 amino acid sequences
identified
five changes that were isolate specific (Table
6).
The Portuguese isolate was
characterized by a moderate amino acid
change of alanine to serine at
position 37 (the position is the
amino acid position in the fragment).
The South Pacific isolate
was characterized by a conservative amino
acid change of histidine
to arginine at position 173. Both the South
Pacific and Hawaiian
isolates were distinguished from other isolates by
three amino
acid changes, a conservative change of leucine to valine at
position
48, a conservative change of phenylalanine to tyrosine at
position
169, and a moderate change of valine to alanine at position
184.
Stabilizing selection appears to have maintained the same amino
acid sequence in
-tubulin, whereas SIG1 appears to be less
constrained.
The high level of within-species polymorphism suggested
that the
levels of divergence of
Sig1 between species might
also be high.
View this table:
[in this window]
[in a new window]
|
TABLE 6.
Variable amino acids in predicted open reading frames of
Sig1 genomic copies amplified from seven isolates of
T. weissflogii
|
|
Sig1 homologues display high levels of divergence in
Thalassiosira species.
Five species of
Thalassiosira were chosen for comparison with T. weissflogii. Three non-chain-forming species, T. pseudonana, T. oceanica, and T. guillardii,
were chosen based on their presumed close relationships to one another
and to T. weissflogii (6, 16). The
chain-forming species T. rotula was chosen because it is a
common member of diatom blooms, and T. antarctica
was chosen because of its presumed distant relationship to the
temperate species T. weissflogii.
The nucleus-encoded 18S rRNA gene of each species was sequenced to
confirm these predicted relationships. A well-supported
distance-based
phylogeny revealed that
T. oceanica,
T. guillardii, and
T. pseudonana formed a tight cluster
closely related to
T. weissflogii (Fig.
3). In fact, the
T. oceanica
sequence differed
by only 0.4% from the
T. weissflogii
sequence. As expected,
T. antarctica was only distantly
related to
T. weissflogii.
View larger version (7K):
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|
FIG. 3.
Unrooted, neighbor-joining tree for the 18S ribosomal
gene from T. weissflogii CCMP1336, T.
oceanica CCMP1005, T. guillardii CCMP988,
T. pseudonana CCMP1335, T. rotula
CCMP1647, T. antarctica, and S. costatum
(GenBank accession no. X85395). Bootstrap values are indicated at the
nodes.
|
|
When total genomic DNAs of the five
Thalassiosira species
other than
T. weissflogii were used as templates for PCR
with the
degenerate
Sig primers, two fragments, at 706 and
483 bp, were
readily amplified from
T. guillardii and
T. oceanica; only the
706-bp fragment was amplified from
T. pseudonana. Neither fragment
was amplified from
T. antarctica or
T. rotula despite numerous
modifications
to the amplification protocol and redesign of the
PCR primers (data not
shown).
The
Sig1-sized fragment from
T. guillardii,
T. pseudonana, and
T. oceanica was cloned, and
SSCP was used to determine that
multiple copies of the
Sig1
homologue were likely to be present
in each species (data not shown).
Three copies of
Sig1 were sequenced
for each species. The
Sig1 DNA sequences fell into three distinct
groups, one
composed of sequences from
T. weissflogii and
T. oceanica,
one composed of sequences from
T. pseudonana,
and one composed
of sequences from
T. guillardii. The
Sig1 DNA sequences from
T. oceanica and the
Atlantic and California isolates of
T. weissflogii were very similar to one another, displaying no more
variation
than that observed within single isolates. In contrast,
the
Sig1 DNA sequences from members of different groups
differed from one
another at more than 225 positions in the 538-bp
coding
sequence.
The intron occurred at the same site in each
Sig1 fragment
and had very similar 5' and 3' splice sites. The
T. oceanica
intron
sequences were readily recognized as they were essentially
identical
to those of
T. weissflogii clone Actin. In
contrast, the intron
sequences from the other species had diverged so
dramatically
from one another that it was not possible to align them
(data
not shown). The
T. guillardii intron was 91 bp long,
and the
T. pseudonana intron was 95 bp long; both of these
introns were larger
than the
T. weissflogii and
T. oceanica introns, which were 87
bp long. The 5' splice site
for the
T. guillardii and
T. pseudonana introns was GTGAG rather than GTAAG, as found in
T. weissflogii.
The 3' splice site in
T. pseudonana,
T. oceanica, and each
T. weissflogii isolate except the
South Pacific isolate was CAG.
The South Pacific isolate and
T. guillardii both had a TAG 3'
splice
site.
Due to the high level of DNA divergence among
Sig1 genes
from members of the different groups, the best DNA alignment was
achieved by using two steps. The intron was excluded to determine
the
predicted amino sequence of the resulting open reading frames.
The DNA
sequences were then aligned based on amino acid alignment,
although
there were two regions consisting of four to five amino
acids at
positions 126 to 129 and 146 to 150 where alignment was
ambiguous. In
this manner, one 3-bp gap was introduced at the
same position into the
T. guillardii, T. oceanica, and
T. weissflogii DNA sequences, and one 3-bp gap was introduced into the
T. pseudonana DNA sequences at a different position. As expected, the
smallest
evolutionary distance was the distance between
T. weissflogii and
T. oceanica; this distance was only
1.3%, which was comparable
to the intraspecific variation in
T. weissflogii. The greatest
evolutionary distance was the distance
between the
Sig1 sequences
from
T. guillardii and
T. weissflogii clone Actin and the distance
between the
T. guillardii and
T. pseudonana Sig1
sequences. Both
of these comparisons resulted in a
maximum evolutionary distance
of 37% (the evolutionary distance
between
T. weissflogii and
T. pseudonana was only slightly less, around 35%), a remarkably high
value given the extremely close relationship of these three species
(Fig.
3). Even this value probably underestimates the true divergence
since the Jukes-Cantor model assumes that the probabilities of
nucleotide change are equal and does not take into account the
possibility of
saturation.
The only other nuclear gene fragment that has been isolated from
different
Thalassiosira species is the multicopy gene
Fcp,
which encodes fucoxanthin chlorophyll
a/c binding protein. Sequences
of
Fcp cDNA
fragments have been reported previously for
T. weissflogii clone Actin (
22) and
T. pseudonana (GenBank
accession number
U66184). An
Fcp cDNA (
41) has
also been isolated from the
relatively closely related centric diatom
Skeletonema costatum (
29). The estimated
distance for the
Fcp genes of
T. weissflogii and
T. pseudonana was 15%, about 2.5 times less than the
distance
observed for the
Sig1 fragments from the same two
species. Even
more striking is the fact that the evolutionary distance
between
Fcp copies is only 20% in
T. weissflogii
and
S. costatum, and
only 20% in
T. pseudonana and
S. costatum.
SIG1 and FCP amino acid sequences from the different species were
compared to determine if low levels of DNA sequence identity
between
Sig1 groups reflected a high number of synonymous
substitutions
at third positions of codons. The percentages of
identical codons
in SIG1 in members of the three groups of
Thalassiosira species
ranged from 38 to 43%. In contrast,
64 to 71% of the codons were
identical in FCP regardless of
whether
T. weissflogii and
T. pseudonana or
T. weissflogii and
S. costatum were
compared. Furthermore,
24% (45 of 186) of the amino acids in SIG1
differed when
T. weissflogii and
T. pseudonana
were compared (Table
7). In contrast,
only
7% (8 of 110) of the amino acids in FCP differed in the two
species.
Moreover, pairwise comparisons of the three
Thalassiosira SIG1
protein groups indicated that a
relatively high percentage of
the amino acid differences were radical
or very radical substitutions
(Table
7). Finally, the SIG1 amino acid
sequences reflected higher
numbers of synonymous changes between
T. weissflogii and
T. pseudonana (36% for SIG1
and 24% for FCP).
View this table:
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|
TABLE 7.
Amino acids that are fixed in all SIG1 copies in a given
species or isolate but differ between T. guillardii and
T. pseudonana and T. weissflogii isolates
from Long Island Sound, Portugal, the South Pacific, and Hawaii.
|
|
Similar to the within-species comparison of
-
tubulin and
Sig1, a between-species comparison of
Fcp and
Sig1 also suggested
that different selective forces shaped
Sig1 diversity. A commonly
used means of determining
divergence between genes from different
species is to compare the
number of synonymous changes per synonymous
site
(
Ks) and the number of nonsynonymous
changes per nonsynonymous
site (
Ka)
(
19). When
T. weissflogii and
T. pseudonana were compared,
Ks was 1.46 ± 0.40 for
Sig1 and 0.43 ± 0.11 for
Fcp. The number
of
synonymous changes per site was almost 3.5-fold higher in
Sig1 than in
Fcp. The
Ka values for the same comparison were
0.17 ±
0.04 for
Sig1 and 0.06 ± 0.03 for
Fcp. The number of nonsynonymous
changes per nonsynonymous
site was about threefold higher for
Sig1 than for
Fcp. Both results suggested that
Sig1 genes are
diverging more rapidly than
Fcp genes. Positive selection, a
potential
driving force for divergence, can be invoked when the
Ka/
Ks ratio
is greater than 1 (
19). The
Ka/
Ks ratio
for
Sig1 was 0.12, and
the ratio for
Fcp was
0.13, indicating that in neither case is
positive selection at
work.
| |
DISCUSSION |
Sexual reproduction in diatoms is intimately connected to the
control of cell size (38). Consequently, the vast majority of species are assumed to undergo sexual reproduction, even if they do
so only infrequently. In reality, however, the sexual cycles of only
about 200 of the more than 10,000 species of diatoms have been
described. Furthermore, sexual events in natural populations of marine
species have only rarely been documented (9, 46), due in
large part to inherent difficulties in identifying sexual stages in
mixed diatom communities. Thus, our understanding of the frequency of
sexual events and the potential generation of genetic diversity within
diatom populations remains limited (however, see reference
39).
The genes in the gene family composed of Sig1, -2, and
-3 in T. weissflogii were recently identified as
potential molecular markers for sexual reproduction in centric diatoms
since transcription of these genes is strongly upregulated during the
sexual cycle (1). Detection of transcription of genes
encoding key enzymes, such as ribulose bisphosphate carboxylase
(34) or nitrogenase (47), has proven to
be an extremely sensitive means of determining when and where key
processes occur in natural populations. In the present study,
degenerate PCR primers were designed that amplified Sig1
homologues from four closely related species of
Thalassiosira, T. weissflogii, T. oceanica,
T. guillardii, and T. pseudonana. Identification of
Sig1 in different species of this cosmopolitan genus should
facilitate determination of when and where sexual reproduction occurs
in natural populations of these target species.
Examination of Sig1 DNA sequences suggests that the likely
reason that this gene could be isolated only from closely related Thalassiosira species is that the gene is undergoing rapid
sequence divergence. For example, 24% of the amino acids in the
predicted SIG1 proteins from T. weissflogii and T. pseudonana differed; in contrast, a comparison of FCP proteins
from the same two species indicated that only 7% of the amino acids
differed. High levels of sequence divergence between closely related
species appear to characterize proteins involved in sexual recognition
(43), the hypothesized role of SIG1 (1). In
animals, positive Darwinian selection is believed to underlie extreme
sequence divergence and is commonly assumed to be a molecular signature
for recognition proteins (7). No evidence of positive
selection was observed with Sig1. However, a recent study
with abalone indicated that an egg receptor protein also does not
display positive selection (42). Interestingly, no
evidence of positive selection was found with a full-length gamete
differentiation gene that nonetheless displayed high levels of
divergence in two species of the unicellular algal genus
Chlamydomonas (12). Thus, evidence of
positive selection does not appear to be required for all recognition
and/or differentiation genes.
The most striking feature of Sig1 is the presence of high
levels of intraspecific variation and intraindividual variation in
addition to high levels of interspecific divergence. The marine invertebrate recognition genes described thus far are all present as single-copy genes, and only the sea urchin gene bindin
displays evidence of intraspecific polymorphisms (31).
Sig1, on the other hand, is repeated multiple times in the
genomes of individual T. weissflogii cells and multiple
times in the genomes of all Sig1-positive species.
Significantly, multiple copies of different Sig1 genes
are transcribed in T. weissflogii, and presumably the same
is true for other Sig1-positive species. The
multicopy nature of Sig1 suggests that different
variants of the protein are expressed during sexual reproduction.
Assuming that only a subset of SIG1 proteins are required for proper
function, selective pressure on any individual gene copy would be
reduced, presumably permitting the observed high levels of
intraindividual polymorphisms to persist (25).
The comparison of between-individual polymorphisms in Sig1
and the housekeeping gene -tubulin is
particularly intriguing. Divergence in
-tubulin is relatively easy to explain.
Isolates from the Atlantic and Pacific oceans have apparently
been physically separated from one another long enough (about 2 million
years since North America and South America joined) to display slight DNA sequence divergence (about 2.8%) in their
-tubulin genes, but amino acid sequence
divergence is not evident due to purifying selection (19).
In contrast, the patterns of divergence in Sig1 are more
complicated. As expected, Atlantic isolates have similar Sig1 sequences. What is surprising, however, is the fact
that the Sig1 sequence from the California isolate is
indistinguishable from Atlantic sequences but is very different (6.5%
divergence) from Hawaiian and South Pacific sequences.
How could this difference in Sig1 sequences between
California and other Pacific isolates be maintained? Based on average surface circulation patterns, for example, one would expect Hawaiian and California populations to mix, with genetic recombination during
sexual reproduction homogenizing any molecular divergence. The apparent
differences between these two populations could simply reflect a
sampling error; if Sig1 from a second isolate from
California, for example, was analyzed, perhaps less divergence would be
observed. Unfortunately, no other California isolates of T. weissflogii are currently available to test this hypothesis.
However, Sig1 sequences from two Long Island Sound isolates
collected 10 years apart were analyzed. These two sets of
Sig1 sequences were very similar to one another (Table 5).
The maintenance of sequence similarity for 10 years suggests that the
largest source of Sig1 diversity in a given population may
be diversity within individuals rather than diversity between
individuals. This suggests that the differences in the Sig1
DNA sequences from California and Hawaiian isolates may reflect true
divergence rather than a sampling artifact. Regardless of the
explanation, some form of regional selection pressure associated with
the Hawaiian and South Pacific environment, rather than drift, appears
to drive the high level of intraspecific Sig1 divergence.
Potential selective pressures that underlie rapid protein evolution
remain unclear. The most intuitive hypotheses have been developed for
proteins displayed on cell surfaces. For example, the surface proteins
of pathogens appear to evolve rapidly, presumably to avoid recognition
by the immune system (18). Similarly, surface proteins of
externally fertilized gametes have been hypothesized to evolve rapidly
to avoid recognition by pathogens (44, 45). If
Sig1 does encode a protein displayed in the extracellular
matrix, similar pathogen-induced evolution could occur. Two dominant
types of diatom pathogens are viruses (35) and
parasitoids (21), both of which appear to possess
species-specific recognition mechanisms. Sexually reproducing diatoms
could be particularly vulnerable to infection since sperm entirely, and
auxospores temporarily, lack the silica frustule. SIG1 variants with
amino acid substitutions that prevent infection by a pathogen could
presumably sweep through a population to fixation. Region-specific
differences in pathogen communities could then generate region-specific
differences in SIG1 sequences, such as the apparent temperate and
tropical divergence. Thus, there is the intriguing possibility that
avoidance of infection by pathogens in the ocean might actually lead to
speciation and further division of niche space (20).
An alternate, more recent hypothesis is that no external forces drive
divergence of surface molecules; the process simply results from the
repetitive structure common to these kinds of proteins. In abalone, for
example, the egg surface receptor protein is composed of multiple,
tandemly repeated amino acid domains, each of which binds the cognate
sperm protein to some degree (42). Selective pressures on
individual repeat units are relaxed, and concerted evolution is
predicted to propagate identical nucleotide changes throughout the
molecule. A rapidly changing amino acid sequence of the egg receptor is
believed to drive rapid evolution of the cognate sperm protein
(42). An analogous mechanism could drive Sig1
evolution. Instead of multiple tandemly repeated sequences in a single
gene, the entire Sig1 sequence appears to be repeated multiple times as distinct genes. It is interesting that multicellular organisms commonly combine into a single protein domains that are
present as individual proteins in unicellular organisms
(5). If Sig1 is tandemly repeated, then
concerted evolution could propagate particular nucleotide changes to
the multiple gene copies.
The simplest explanation for the observed intraspecific and
interspecific divergence is that different forces, acting on different times scales, combine to shape Sig1 evolution. First,
selective pressure on individual copies of Sig1 has
apparently been relaxed, allowing high levels of intraindividual
polymorphisms to arise, either because Sig1 duplicated
relatively recently (25) or because Sig1 has
somehow escaped selection pressures. Superimposed on this high rate of
divergence appears to be a lower rate of convergent evolution, in which
specific nucleotide changes are spread throughout all gene copies. The
effect of these two processes in combination with stochastic factors
could lead to large-scale region-specific differences in
Sig1 sequences without any need for external driving forces.
Small-scale regional differences would be expected to persist if
particular SIG1 variants conveyed a region-specific selective
advantage, such as resistance to infection by pathogens. If, for
example, the selective forces in the tropics differ from those in more
temperate environments, collection of T. weissflogii isolates from tropical regions in the Atlantic Ocean could provide insight into these processes. A true understanding of the evolutionary pressures acting on SIG1, however, awaits determination of the exact
role played by this protein.
The potential scenarios described here, although speculative,
should not be considered specific to diatoms. Similar mechanisms could
explain speciation in any group of phytoplankton in which multiple,
closely related species simultaneously undergo sexual reproduction in a
body of water.
| |
ACKNOWLEDGMENTS |
We thank Paul Bentzen, Mike Canino, and Tatiana Rynearson for
many helpful discussions.
This work was supported in part by National Science Foundation grant
OCE 9702158 (to E.V.A.), by Office of Naval Research DURIP award
N000140010597 (to E.V.A.), and by a Mary Gates Endowment for Students
undergraduate research training grant (to H.M.G.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: University
of Washington School of Oceanography, Box 357940, Seattle, WA 98195. Phone: (206) 616-1783. Fax: (206) 685-6651. E-mail:
armbrust{at}ocean.washington.edu.
| |
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Applied and Environmental Microbiology, August 2001, p. 3501-3513, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3501-3513.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
