Rapid diversication of coevolving marine

Synechococcus and a virus

Marcia F. Marstona, Francis J. Pierciey, Jr.a, Alicia Sheparda, Gary Gearinb, Ji Qic, Chandri Yandavab, Stephan C. Schusterc,
Matthew R. Hennb, and Jennifer B. H. Martinyd,1
a
Department of Biology and Marine Biology, Roger Williams University, Bristol, RI 02809; bBroad Institute of MIT and Harvard, Cambridge, MA 02142; cCenter
for Comparative Genomics and Bioinformatics, Pennsylvania State University, University Park, PA 16802; and dDepartment of Ecology and Evolutionary
Biology, University of California, Irvine, CA 92697

Edited by Nancy A. Moran, Yale University, West Haven, CT, and approved January 28, 2012 (received for review December 8, 2011)

Marine viruses impose a heavy mortality on their host bacteria,

whereas at the same time the degree of viral resistance in marine
bacteria appears to be high. Antagonistic coevolutionthe reciprocal evolutionary change of interacting speciesmight reconcile
these observations, if it leads to rapid and dynamic levels of viral
resistance. Here we demonstrate the potential for extensive antagonistic coevolution between the ecologically important marine cyanobacterium Synechococcus and a lytic virus. In a 6-mo-long replicated chemostat experiment, Synechococcus sp. WH7803 and the
virus (RIM8) underwent multiple coevolutionary cycles, leading to
the rapid diversication of both host and virus. Over the course of
the experiment, we detected between 4 and 13 newly evolved viral
phenotypes (differing in host range) and between 4 and 11 newly
evolved Synechococcus phenotypes (differing in viral resistance) in
each chemostat. Genomic analysis of isolates identied several candidate genes in both the host and virus that might inuence their
interactions. Notably, none of the viral candidates were tail ber
genes, thought to be the primary determinants of host range in
tailed bacteriophages, highlighting the difculty in generalizing
results from bacteriophage infecting -Proteobacteria. Finally, we
show that pairwise virushost coevolution may have broader community consequences; coevolution in the chemostat altered the sensitivity of Synechoccocus to a diverse suite of viruses, as well as the
virus ability to infect additional Synechococcus strains. Our results
indicate that rapid coevolution may contribute to the generation
and maintenance of Synechococcus and virus diversity and thereby
inuence viral-mediated mortality of these key marine bacteria.
cyanophage

| bacterial diversity | arms race

iruses play a central role in marine biogeochemical cycles by

killing as much as 2040% of marine prokaryotic cells each
day (1). At the same time, bacteria isolated from the marine
environment are often resistant to many viral strains (24).
These two sets of observations about marine virushost interactionshigh virus-induced host mortality on the one hand and
a high degree of resistance on the otherhave yet to be entirely
reconciled (1, 5, 6). As a result, the controls on viral-mediated
nutrient turnover in the oceans remain largely unknown.
In addition to their effects on host mortality, marine viruses
affect host diversity in several ways. First, viruses contribute
genetic material to their hosts via horizontal gene transfer (7).
For instance, photosynthesis genes encoded by both cyanobacteria and their infecting viruses appear to have undergone repeated recombination events (8, 9). Second, viruses, like other
pathogens, are thought to maintain host diversity by frequency
dependence such that host strains (genotypes or taxa) that become relatively abundant are more susceptible to attack (the
kill the winner hypothesis) (1012). Finally, viruses might affect marine host diversity by promoting the emergence of viral
resistance, leading to antagonistic coevolution (the reciprocal
evolution of host resistance and viral infectivity) (13).
Experimental microcosms, focusing primarily on heterotrophic
-Proteobacteria, such as Escherichia coli and Pseudomonas uorescens, have demonstrated that viral-bacterial coevolution can
quickly generate genetic and phenotypic diversity (1417). At the

45444549 | PNAS | March 20, 2012 | vol. 109 | no. 12

same time, however, a classic microcosm study revealed constraints on the potential for coevolutionary arms races between
viruses and heterotrophic bacteria (16). Using E. coli and various
T-phages, that study found that after one or two cycles of coevolution (in which the host evolves resistance and the virus then
evolves to overcome that resistance), a bacterial strain evolves
resistance that the virus cannot overcome. The inability of the
phage to continue to evolve host range mutants suggests that antagonistic coevolution is unlikely to be important in natural systems, at least over ecological time scales (16). Although extensive
coevolution has recently been observed in P. uorescens (15),
possibly in E. coli (17), and indirectly in Archaea (18), the potential
for such coevolution has never been tested in a marine system.
In the present study we tested the potential for antagonistic
coevolution to generate diversity in an ecologically important,
nonheterotrophic marine bacterium. Marine Synechococcus is
a widespread unicellular cyanobacterium that may account for
as much as 20% of primary productivity in coastal and upwelling
regions (19, 20). Viruses that infect and lyse cyanobacteria
(cyanophages) are also abundant and ubiquitous in the ocean (4,
21) and recycle as much as one-quarter of photosynthetically
xed carbon back to dissolved organic material pools (22). We
conducted a 6-mo replicated chemostat experiment to address
three main questions. First, what is the potential for coevolution
and diversication between Synechoccocus and a lytic virus? In
particular, we tested whether virushost coevolution is limited to
one or two coevolutionary cycles, with a cycle dened as the host
evolving resistance to the virus and the virus evolving to overcome that resistance (in contrast to a predatorprey cycle dened by abundances). Second, can we identify candidate genes
that underlie the phenotypic diversication observed in the
chemostats? Currently, the genetic mechanisms underlying Synechococcusvirus interactions (or that of any marine viruses and
its host) are largely unknown and based primarily on inferences
from viruses infecting -Proteobacteria. Finally, do mutations
that arise during pairwise coevolution have consequences for
interactions with other Synechococcus and cyanophage strains?
In natural settings, evolution occurs in the context of a diverse
community; thus, we aimed to test whether antagonistic coevolution might alter these broader community interactions.
Results and Discussion
We established four chemostats with Synechococcus spp.
WH7803 and RIM8 (family Myoviridae) and one control che-

Author contributions: M.F.M. and J.B.H.M. designed research; M.F.M., F.J.P., A.S., and
J.B.H.M. performed research; S.C.S. and M.R.H. contributed new reagents/analytic tools;
M.F.M., F.J.P., A.S., G.G., J.Q., C.Y., S.C.S., M.R.H., and J.B.H.M. analyzed data; and M.F.M.
and J.B.H.M. wrote the paper.
The authors declare no conict of interest.
This article is a PNAS Direct Submission.
Database deposition: The sequences reported in this paper have been deposited in the
GenBank database (accession nos. JF974288, JF974289, and HQ317385).
1

To whom correspondence should be addressed. E-mail: jmartiny@uci.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1120310109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1120310109

mostat with Synechococcus only. Both Synechococcus and RIM8

persisted for the duration of the 6-mo experiment (170 generations). Host abundance was dramatically reduced after the
addition of the virus, but recovered after 5080 d. Thereafter,
cell abundance remained similar to (chemostats C and D) or
somewhat lower than (chemostats A and B) the abundance of
Synechococcus in the control (no virus) chemostat (top third of
the panels in Fig. 1).
To test for the potential of antagonistic coevolution to lead to
the diversication of marine Synechococcus and a lytic virus, we
isolated single cells and viruses from each of the chemostats by
colony isolation and plaque purication at numerous time points.
Using these isolates, we conducted infection assays to determine
the ability of the viral isolates to infect various Synechococcus
isolates from the same chemostat. We then dened the phenotypes of the host and viral isolates based on these patterns of
susceptibility and infectivity.

B
9

10 9

10 7

10 7
10

resistance

R 0-13
R 0-6
R 0-3,5,7

R 0-4,7
R 0-2

infectivity

1
0

R 0-13 R 0-13
R 0-8,11
R 0-10
R 0-9

R 0-6,11,12

8
6
5
4
2

R 0-2
R 0,1
R 0,2
13
10
9
7

11

R 0-4

R0
S

R 0,1
R0

12

2
1

4
3

R 0-4
R 0-3
R 0-2

R 0-3

R 0,1

D
9

10 9

10 7

10 7

10

infectivity

10
R 0-3

0
0

30

R 0-4
R 0-2

R 0-4
R 0-3

R 0,1

R0

60

90

Day

R 0-3

2
120

150

4
3

resistance

10

resistance

cells/ml or pfu/ml

R0,1,2,3R 0-3 R0,1,2,3RR0-3

0,1,2,3
RR0-2
0,1,3
R0,1,2 R0,1,2
R0
R

R 0-3
R 0-2
R 0,1,3

R 0-2
R0

4
1

0
0

30

R0

R0

60

90

120

R0

4
3

150

Day

Fig. 1. Synechococcus sp. WH7803 and virus (RIM8) dynamics in the four replicate chemostats AD. The top third of each panel plots the abundance of Synechococcus cells (red solid line) and infectious viral particles (blue dashed line) over time. For reference, the gray line is cell abundance in the control (no virus)
chemostat. The middle of the panel indicates the host phenotypes detected at six time points, and the bottom of the panel, the viral phenotypes detected at the same
six time points. Each chemostat was inoculated with ancestral virus (0) on day 0. Host range mutants are numbered in their order of infectivity (e.g., 112), with
higher numbers indicating the ability to infect a greater number of host phenotypes. Host phenotypes are labeled by their ability to resist infection by each host range
mutant from the same chemostat. For example, S (sensitive to RIM8) is the ancestral host, and R02 is resistant to 0, 1, and 2. We cannot determine whether
a particular phenotype evolved directly from another phenotype, because some of the same phenotypes might have evolved more than once. Therefore, the dashed
lines connecting the phenotypes are for ease of reading and make only the most parsimonious assumptions. The fully sequenced host and viral isolates are circled in A.

Marston et al.

PNAS | March 20, 2012 | vol. 109 | no. 12 | 4545

EVOLUTION

resistance

10

infectivity

10

infectivity

cells/ml or pfu/ml

In the virus chemostats, Synechococcus spp. WH7803 and

RIM8 underwent at least four cycles of coevolution, and we found
no evidence indicating an imminent end to coevolution (bottom
two-thirds of the panels in Fig. 1). We detected Synechococcus
isolates that evolved resistance to the ancestral virus, viral isolates
that infected this resistant strain, Synechococcus isolates that were
resistant to the evolved virus, and so forth with continuing cycles
of resistance and host range evolution. In each virus chemostat,
we detected at least 4 newly evolved viral phenotypes over the
course of the experiment, and in one chemostat, we found 13 viral
phenotypes (Fig. 1A). At the same time, between 4 and 11 distinct
Synechococcus phenotypes evolved as well. In contrast, in the
control chemostat with no virus, we detected only the ancestral
Synechococcus phenotype (sensitive to the ancestral virus and
host range mutants from the other chemostats). Thus, Synechococcus and RIM8 underwent extensive antagonistic coevolution,
resulting in the rapid (within fewer than 170 generations) appearance of considerable phenotypic diversity in both the host

and virus. These results resemble the dynamics observed for

P. uorescens and the virus Phi 2 over 400 generations (15).
They also lend support to recent evolutionary ecology models that
predict the diversication and coexistence of multiple phenotypes
(quasispecies) of bacteria and virus in chemostats (23).
Both the evolution of viral host range and the evolution of
viral resistance in Synechococcus were directional. Viral infectivity (i.e., the ability to infect various Synechococcus isolates)
of both isolates and whole populations increased over time, such
that on average, viruses from later time points had a broader
host range than those from earlier time points (Fig. 1 and Fig.
S1). Similarly, Synechococcus cells isolated from later time points
were on average more resistant than cells isolated from earlier
time points in terms of both resistance to viral isolates and viral
populations (Fig. 1 and Fig. S2). This directional arms race is
typical of that observed in other laboratory studies of antagonistic coevolution (e.g., refs. 13, 17, but see ref. 24).
Although Synechococcus and virus evolution was generally
directional, the phenotypic pattern was more variable in Synechococcus than in the virus. Viral phenotypes appeared to replace one another over time, such that we usually detected only
one or two host range phenotypes in any one sample. At the same
time, infectivity increased steadily (Fig. 1). In contrast, many
Synechococcus phenotypes were observed at multiple time points,
and up to three host phenotypes co-occurred in a sample. This
greater phenotypic diversity among the hosts versus the viruses is
particularly notable because at each time point we examined only
two to four host isolates versus 10 viral isolates; thus, we likely
greatly underestimated actual host diversity. Further, co-occurring
host phenotypes often differed greatly in their degree of resistance
(Fig. 1); for instance, in chemostat C, a sensitive (S) host phenotype and a R03 phenotype (resistant to four viral phenotypes)
were present on the same day. The high degree of coexistence
among host phenotypes suggests that resistance to RIM8 and its
host range mutants is associated with a tness cost (25, 26). Indeed, previous experiments have reported a tness cost for viral
resistance in Synechococcus (27) and Prochlorococcus (28).
To identify genes that might be involved in the interactions
between Synechococcus and the virus, we sequenced the complete genomes of three viral isolates [the ancestral (0) phenotype from day 56, the 9 phenotype from day 112, and the 12
phenotype from day 167] and two Synechococcus isolates (an S
phenotype from day 0 and an R013 phenotype from day 167)
from chemostat A (circled in Fig. 1A). Among the viral genomes
(171 kb each), we identied eight nonsynonymous nucleotide
substitutions in genic regions, three indels in intergenic regions,
and one large deletion of several genes; one of the nucleotide
substitutions was in the 9 isolate, and all of the other mutations
were in the 12 isolate (Table S1). Notably, none of these
changes occurred in homologs to known tail ber genes, which
are thought to be involved in determining the specicity of
myoviruses (T4-like phages) (29) and have been observed to
evolve in other coevolution experiments (30). Half of the nucleotide substitutions were in a 130-bp region of a 972-bp gene of
unknown function (RIM8.A.HR1_096), suggesting that the region is involved in viralhost interactions. To further explore this
possibility, we sequenced the region in viral isolates obtained at
each sampling time in the four chemostats.
Extensive parallel evolution in RIM8.A.HR1_096 was observed among the four chemostats, similar to that found in other
viral evolution studies (31, 32). Of the 87 viral isolates genoytped,
71 had one or more substitutions in RIM8.A.HR1_096 compared
with the ancestral RIM8 isolate. Nucleotide substitutions were
observed at 11 positions in the gene (Table S2). All substitutions
were nonsynonymous and led to changes in 10 amino acid sites,
although not all changes were to the same amino acid (Table 1).
Most (>60%) of the amino acid changes were observed in more
than one chemostat.
This large number of parallel, nonsynonymous substitutions
suggests that the region was under strong positive selection. Such
a pattern could be caused by host range adaptation or,
4546 | www.pnas.org/cgi/doi/10.1073/pnas.1120310109

alternatively, general adaptation to the chemostat environment.

However, the number of amino acid changes was also highly correlated with the isolates infectivity (R2 = 0.500.96, P < 0.0001 for
all chemostats) (Fig. S3), strongly suggesting that RIM8.A.
HR1_096 is involved in determining viral host range. At the same
time, eight viruses isolated from chemostat A on day 84 had
the same RIM8.A.HR1_096 amino acid sequence, but represented
ve phenotypes (Table 1), indicating the role of additional mutations in other parts of the genome. Thus, host range determination
in this virus appears to involve multiple genes (in addition to this
candidate gene), similar to that observed in heterotrophic bacteriophage (30).
Viral resistance in Synechococcus also appears to be a complex
trait encoded by multiple loci. Sequencing of the two host
genomes revealed four changes (in four different coding regions)
between the ancestral Synechococcus and the derived isolate that
was resistant to 13 viral phenotypes: three nucleotide substitutions
and a single nucleotide deletion, leading to an early stop codon
(Table S3). As in the viral genomes, the mutations observed were
nonsynonymous, suggesting that they are adaptive. Only one mutationa point mutation in the glucose-1-phosphate thymidylyltransferase gene (SynWH7803_0102)was found in isolates from
additional chemostats (using PCR amplication and sequencing),
further supporting the idea that it is an adaptive mutation (Table
S4). This core gene (all sequenced Synechoccocus genomes contain homologs) is located in a highly variable region, identied as
genomic island 1 (ISL1) in Synechococcus sp. WH7803 (33). The
gene encodes the rst enzyme in the dTDP-L-rhamnose biosynthesis pathway. L-rhamnose is one of the important residues of
the O antigen of LPS in Gram-negative organisms (34) and has
been detected in the LPS of marine Synechococcus (35). LPS is
also involved in bacteriophage attachment in WH7803 (36). The
two additional mutations were found in genes encoding a glycosyltransferase, which could be involved in LPS biosynthesis (37),
and a histidine kinase, which could regulate proteins exposed
outside the cell wall to which viruses attach (38) (Table S3).
Two of the mutations were not observed in other chemostat A
Synechococcus isolates (SynWH7803_0140 and SynWH7803_1555).
The lack of shared mutations among the chemostat A Synechococcus isolates is notable, because the isolates are also resistant to
subsets of the same viruses (Fig. 1A). Thus, Synechococcus appears
to evolve resistance to some viruses in more than one way, such that
mutations in entirely different genes can confer resistance to the
same virus (39). This nding, in contrast to that seen in the virus,
also supports the hypothesis that parallel evolution within a gene
occurs more often in bacteriophages than in their hosts (40).
In seawater, viralhost coevolution occurs in the context of a
diverse community of Synechococcus and virus strains. To test
whether coevolution in this experimental setting has potential
consequences in this broader community, we challenged the
evolved Synechococcus populations with 31 genetically distinct
myovirus strains isolated from Rhode Island (RI) waters. Over the
course of the experiment, the number of RIM strains to which the
Synechococcus populations were resistant increased (F1,18 = 90.83,
P < 0.0001, ANCOVA) (Table S5). Similar trends were observed
with Synechococcus isolates, suggesting that this increased resistance is related to the increased resistance of individual cells
(Table S5). Furthermore, resistance to similar RI strains evolved
in parallel among the four chemostats; the phenotypic prole of
Synechococcus populations differed signicantly by sampling day
(R2 = 0.742, P = 0.006, ANOSIM), but not by chemostat (R2 =
0.33, P = 0.186) (Fig. 2). Such pleiotropic effectswith resistance
to one virus also conferring resistance to other viruseshave recently been reported in other marine bacteria, including Prochlorococcus (28) and Flavobacterium (41). Notably, in both
Synechococcus and Prochlorococcus, there are examples where
gaining resistance to one virus simultaneously increases sensitivity
to other viruses (28, 39); for instance, in the present study, some of
the Synechococcus cells coevolving with RIM8 lost their initial
resistance to RIM26 (Table S5).
Marston et al.

Table 1. Comparison of viral phenotypes and RIM8.A.HR1_096 genotypes in the four chemostats

Day Isolate Phenotype

0
28

56

84

112

142

167

RIM8
3
5
8
2
4
5
7
9
10
1
2
3
4
6
7
8
10
1
3
5
6
7
8
9
10
2
4
6
8
2
5
7
9

0 / 0.06

1 / 0.13
0 / 0.06
0 / 0.06
0 / 0.06
1 / 0.13
1 / 0.13
4 / 0.38
2 / 0.13
8 / 0.56
5 / 0.38
8 / 0.56
6 / 0.44
6 / 0.44
6 / 0.44
7 / 0.44
10 / 0.69
9 / 0.63
9 / 0.63
9 / 0.63
9 / 0.63
13 / 0.81
3 / 0.31
11 / 0.69
11 / 0.69
11 / 0.69
11 / 0.69
12 / 0.75
12 / 0.75
12 / 0.75
12 / 0.75

Genotype
GEQFNSSDEG
..........
..........
..........
....K.R...
......R...
......R...
......R...
....K.R...
....K.R...
......RH..
......RH..
......RH..
......RH..
......RH..
......RH..
......RH..
......RH..
......R...
....K.R...
......R...
......R...
....K.R...
....K.R...
RKH..RR...
....K.R...
RQH..RR...
RQH..RR...
RKH..RR...
RKH..RR...
RKH..RR...
RQH..RR...
RQH..RR...
RKH..RR...

Chemostat B
Isolate Phenotype

Genotype

Chemostat C
Isolate Phenotype

Genotype

Chemostat D
Isolate Phenotype

Genotype

0 / 0.11

GEQFNSSDEG
..........

RIM8

0 / 0.07

GEQFNSSDEG

RIM8
2
5

0 / 0.00

GEQFNSSDEG
..........
..........

1
3
5
8

0
0
0
0

0.11
0.11
0.11
0.11

..........
..........
..........
..........

1
4
8

0 / 0.07
0 / 0.07
0 / 0.07

..........
..........
..........

2
5

1 / 0.40
1 / 0.40

.......N..
.......N..

5
6
9

2 / 0.44
2 / 0.44
2 / 0.44

.KH...R...
.KH...R...
.KH...R...

3
5
9

1 / 0.25
1 / 0.25
1 / 0.25

..........
..........
..........

1
7

2 / 0.47
2 / 0.47

....K..N..
....K..N..

1
4
6
8

3
4
3
4

/
/
/
/

0.56
0.78
0.56
0.78

.KH..RR...
RKH..RR...
.KH...R...
RKH..RR...

1
5
8

2 / 0.38
2 / 0.38
2 / 0.38

......R...
......R...
......R...

4
6
8

4 / 1.00
4 / 1.00
4 / 1.00

....K..N..
....K..N..
....K..N..

3
4
7
9
1
3
5
8

4
4
4
4
4
4
4
4

/
/
/
/
/
/
/
/

0.78
0.78
0.78
0.78
0.78
0.78
0.78
0.78

RKH..RR...
RKH..RR...
RKH..RR...
RKH..RR...
R.H..RR...
RKHS.RR...
RKHS.RR...
R.H..RR...

4
5
8

3 / 0.44
3 / 0.44
3 / 0.44

..H..RR...
.....RR...
......R...

4
4
4
3

R.H..RR...
R.H..RR...
R.H..RR...
.....RR...

1
3
5
9
3
5
7
9
18

4
4
4
4
4
4
4
4

....R..N.A
....R..N.A
....R..N.A
....R..N.A
....R..N.A
....R..N.A
....R..N.A
....R..N.A
....R..NKA

RIM8
4

/
/
/
/

4
6
8
10

/
/
/
/

0.69
0.69
0.69
0.44

/ 1.00
/ 1.00
/ 1.00
/ 1.00
/ 1.00
/ 1.00
/ 1.00
/ 1.00

For each isolate, the phenotype column lists the label used in Figure 1 followed by its infectivity score (the fraction of host isolates from the same
chemostat that the virus can infect). The genotype column is the alignment of the genes variable region, where the bolded amino acids in RIM8 indicate
those that vary among all the isolates (see Table S2 for aa positions and nt changes). Shading denotes fully sequenced isolates. A dash indicates that the
phenotype was not determined.

Coevolution with Synechococcus sp. WH7803 also altered the

interactions of the virus with other Synechococcus strains. We
challenged cyanophage isolates from each chemostat with Synechococccus sp. WH8018 and ve strains derived either from
WH7803 or WH8108. These derived strains were previously
selected for resistance to other RIM viruses (39), but were resistant to the ancestral RIM8 virus as well. Coevolution altered
the ability of RIM8 to infect three of the ve previously resistant
strains, including those derived from the genetically distinct
Synechococcus sp. WH8018 (Table 2).
Conclusions
The potential for rapid and extensive antagonistic coevolution
between marine Synechococcus and their viruses has various
implications. In particular, we have shown that persistent, coevolutionary dynamics quickly generate and maintain diversity in
this ecologically important aquatic bacterium. Furthermore, although Synechococcus readily evolved viral resistance, the cells are
often susceptible to co-occurring viral genotypes. Such dynamics
offer a possible explanation for the paradoxical observations of
simultaneously high host mortality and high viral resistance, given
that resistance may be highly temporally dynamic.
Marston et al.

Coevolution within a broader community context might further

maintain Synechococcus diversity. Within the chemostat, Synechococcusvirus coevolution led to a generally directional arms race.
However, this pairwise coevolution also altered interactions with
host and virus strains not in the chemostat. Coevolution conferred
sensitivity in Synechococcus sp. WH7803 to at least one other RIM
virus, whereas RIM8 developed the ability to infect resistant
strains of a different Synechococcus species. This suggests that in
a diverse natural community, pairwise coevolution may be interrupted when changes in resistance and/or host range of the
coevolving pair alter their interactions with other members of the
community. These new interactions might then aid in the coexistence of Synechococcus and infecting viruses by preventing
a particular host strain from winning a directional arms race.
New theoretical models that consider virushost coevolution in
a community context, as well as the complex genetic mechanisms
observed here, are needed to provide insight into marine virus
host dynamics.
The phenotypic diversication in Synechococcus and RIM8
was driven by small genetic changes across a variety of genes.
Although genetic manipulations are needed to pinpoint the
molecular mechanisms involved in virushost interactions, we
identied several candidate genes that may provide alternative
PNAS | March 20, 2012 | vol. 109 | no. 12 | 4547

EVOLUTION

Chemostat A

D-0
C-0
B-0
A-0
X-56
X-0
X-167
D-56
D-28
C-84
C-56
D-28
B-56
A-56
A-28
B-28
D-167
D-84
C-167
A-167
B-70
B-167
20

15

10
5
Distance

genotypes, differing in their sensitivity (for the host) or infectivity

(for the virus) (3, 28, 43, 44). Such ne-scale dynamics may explain
why in natural communities, particular bacterial taxa remain
dominant for extended periods despite high viral abundances (45).
In conclusion, viral coevolution led to rapid diversication and
a highly dynamic degree of viral resistance in Synechococcus.
Selection for viral resistance is known to alter nutrient acquisition rates in marine bacteria (27, 41), and in laboratory
experiments, the fraction of Synechococcus cells that are resistant
to co-occurring cyanophages inuences nutrient turnover rates
(46). Taken together, this evidence suggests that rapid coevolutionary processes may inuence how viruses mediate nutrient cycling in the ocean.

resistant to
2-3 strains

8 strains

13-14 strains

Materials and Methods

26 strains

Fig. 2. Dendrogram of the phenotype of the chemostat Synechococcus

populations, where phenotype is dened as the sensitivity or resistance to 31
virus strains from Rhode Island waters (Table S5). The populations are indicated by chemostat (A, B, C, D, and X for the no-virus control) and sampling day. The number of virus strains to which the populations are resistant
is noted. Of note, the Synechococcus population from the control chemostat
at the end of the experiment (X-167) was similar to populations from day
0 of the experiment, indicating that resistance did not increase in the absence of virus.

markers for hostphage interactions. This study identies host

range genes in cyanophage, and notably none of these candidates
are homologous to tail ber genes in known bacteriophages.
Indeed, the gene with the most nucleotide substitutions in the
present study (RIM8.A.HR1_096) is not even conserved among
cyanophages and apparently exists in only two of the other fully
sequenced cyanophage genomes (Syn1 and P-HM1). Thus, the
genetic mechanisms underlying virushost interactions may be
quite different from those previously characterized for terrestrial
-Proteobacteria systems.
Our ndings also conrm previous studies indicating that neither
Synechococcus susceptibility nor cyanophage infectivity is correlated with the genotype of highly conserved genes (39, 42). Thus,
coevolutionary and/or frequency-dependent (i.e., kill the winner)
dynamics are unlikely to be observed using conserved genetic
markers such as bacterial rpoC and viral g20. Instead, taxa dened
by these markers may be composed of multiple co-occurring

Strains and Chemostats. Synechococcus sp. WH7803 was obtained from the
Woods Hole Collection of Cyanobacteria (Woods Hole Oceanographic Institution). RIM8 was isolated from Narragansett Bay, Rhode Island in 2000
(47). Cells derived from a single colony of WH7803 were used to inoculate
ve chemostats; this Synechococcus culture is nonaxenic, containing heterotrophic bacteria (46). An articial seawater medium (48) was supplied to
each 35-mL chemostat at a dilution of 1.02/d. The contents were stirred
with magnetic stir bars and maintained at 23.0 C on a 14:10 light:dark cycle
at 10 E m2 s1. The cells were allowed to equilibrate for 2 wk before RIM8
(derived from a single plaque) was added to four of the chemostats. The
fth chemostat with no virus was maintained as a control.
Sampling. After the addition of viruses (day 0), cell and virus abundance was
estimated every day for the rst week, then approximately once a week
thereafter until day 167. Cells were counted by epiuorescence microscopy
and viruses were counted by counting plaques on dilution plates (47). Single
cells and viral particles were isolated from each of the chemostats by colony
isolation and plaque purication (39) at eight time points. At each time
point, 610 Synechococcus colonies and 10 viral plaques were chosen and
grown in liquid media (with ancestral host for the virus). To examine
whether the isolate results were biased by selecting for plaques on the ancestral host or colony formation on a solid media, host and virus populations
were collected as well. Synechococcus populations (containing virus) were
collected by transferring an aliquot directly from the chemostats into the AN
medium. Viral populations were obtained by ltering 12 mL of the chemostat sample through a 0.22-m lter. For long-term storage, lysates were
stored in cryogenic tubes at 4 C, and Synechococcus cultures were stored in
7.5% (vol/vol) DMSO at 80 C.
Phenotype Assays. Resistance and infectivity were assayed by challenging the
Synechococcus isolates and populations with the viral isolates and/or populations. All assays were carried out in duplicate in 24-well microtiter plates
as described previously (39). Two control wells containing cells but no virus

Table 2. Susceptibility of Synechococcus spp. WH7803 and WH8018 and ve derived RIM8-resistant strains to viral
isolates from the chemostats
Chemostat Day Isolate Phenotype WH7803 WH7803R17 WH8018 WH8018R16 WH8018R19 WH8018R20 WH8018R34
A

0
28
28
56
112
167
56
112
167
56
112
167
28
28
56
167

RIM8
5
8
5
5
7
1
4
3
4
8
10
2
5
5
9

0
9
12
0
4
4
0
2
3

1
4

S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S

R
R
R
R
S
S
R
S
S
R
R
R
R
R
R
S

S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S

R
R
R
R
R
S
R
S
S
R
R
R
R
R
R
R

R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R

R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R

R
R
R
S
S
S
R
S
S
R
S
S
R
R
S
S

A dash indicates that the phenotype was not determined.

4548 | www.pnas.org/cgi/doi/10.1073/pnas.1120310109

Marston et al.

Two Synechococcus isolates from chemostat A also were fully sequenced,

including the ancestral Synechococcus inoculated into the chemostats (S in
Fig. 1A) and a derived isolate (R013 in Fig. 1A). The regions surrounding four
identied mutations were PCR-amplied (Table S6) and sequenced in 13
isolates from chemostat A (and conrmed in the fully sequenced isolates). If
the mutation was found to be variable in chemostat A, then 19 additional
isolates were sequenced from the other chemostats. Further details of the
bacterial genome sequencing and analysis are provided in the SI Materials
and Methods.

Genetic Analysis. The genomes of three viral isolates from chemostat A were
fully sequenced. Genomic DNA was puried, sequenced by 454 FLX technology,
assembled, and annotated as described previously (49, 50). Coverage ranged
from 44% to 60% for the three genomes. The sequencesJF974288, the ancestral (0) phenotype from day 56; JF974289, the 9 phenotype from day 112;
and HQ317385, the 12 phenotype from day 167are available from GenBank. To further determine genetic variability among the viral populations in
the chemostats, the variable portion of RIM8.A.HR1_096 was PCR-amplied
and sequenced from the ancestral RIM8 isolate and 87 viral isolates with representatives from each time point in each chemostat (Table 1 and Table S6).

ACKNOWLEDGMENTS. We thank the Broad Institutes Genome Sequencing,

Assembly, Annotation, and Finishing teams for their efforts in generating
the genomic data reported here. We also thank Lisa Crummett, Brandon
Gaut, China Hanson, Adam Martiny, and Olivier Tenaillon for constructive
suggestions on the manuscript. This research was supported by National
Science Foundation Grants OCE-0314523 and OCE-1029684 (to M.F.M.)
and OCE-0315645 and OCE-1031783 (to J.B.H.M.), the Gordon and Betty
Moore Foundation (awards to J.B.H.M., M.R.H., and S.C.S.), Rhode Island
Experimental Program to Stimulate Competitive Research Grant 0554548
(to M.F.M.), and the Roger Williams University Foundation to Promote Scholarship (to M.F.M.).

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PNAS | March 20, 2012 | vol. 109 | no. 12 | 4549

EVOLUTION

were also included on each plate. Plates were incubated under constant illumination at room temperature and scored once a week for 4 wk. Synechococcus cultures were scored as susceptible (and the virus as infective) if
growth was visibly inhibited compared with the control wells. Five sets of
these resistance/infectivity assays were performed: (i) host isolates vs. viral
isolates, (ii) host isolates vs. viral populations, (iii) hosts from the no-virus
chemostat vs. ancestral virus, (iv) host populations and isolates vs. other
RI viruses, and (v) viral isolates vs. other Synechococcus species. See the
SI Materials and Methods for details of these assays.