Accelerated Evolutionary Rate in Sulfur-Oxidizing Endosymbiotic Bacteria
Associated with the Mode of Symbiont Transmission
Andrew S. Peek,1 Robert C. Vrijenhoek, and Brandon S. Gaut1
Center for Theoretical and Applied Genetics, Rutgers University
The nearly neutral theory of molecular evolution predicts that the rate of nucleotide substitution should accelerate
in small populations at sites under low selective constraint. We examined these predictions with respect to the
relative population sizes for three bacterial life histories within chemolithoautotrophic sulfur-oxidizing bacteria: (1)
free-living bacteria, (2) environmentally captured symbionts, and (3) maternally transmitted symbionts. Both relative
rates of nucleotide substitution and relative ratios of loop, stem, and domain substitutions from 1,165 nt of the
small-subunit 16S rDNA were consistent with expectations of the nearly neutral theory. Relative to free-living
sulfur-oxidizing autotrophic bacteria, the maternally transmitted symbionts have faster substitution rates overall and
also in low-constraint domains of 16S rDNA. Nucleotide substitition rates also differ between loop and stem
positions. All of these findings are consistent with the predictions that these symbionts have relatively small effective
population sizes. In contrast, the rates of nucleotide substitution in environmentally captured symbionts are slower,
particularly in high-constraint domains, than in free-living bacteria.
Introduction
One prediction of the nearly neutral theory of molecular evolution is that rates of nucleotide substitution
should be negatively correlated with population size
(Ohta 1987). This prediction applies only to nearly neutral substitutions, because highly deleterious mutations
are removed by selection and absolutely neutral mutations are affected only by the rate of mutation (Kimura
1983; Ohta 1992). Stated precisely, the nearly neutral
theory predicts that the rate of very slightly deleterious
(nearly neutral) substitutions is higher in small populations, while substitution rates of deleterious mutations
are influenced more by purifying selection than by population size.
The relationship between nucleotide substitution
rate and hypothesized population size was recently investigated for endosymbiotic bacteria and their free-living relatives (Moran 1996). Effective population sizes
(Ne) are expected to be small in obligately symbiotic
bacteria relative to free-living bacteria, because the symbionts probably experience a population bottleneck during transmission from one host generation to the next.
This host-mediated reduction in population size is expected to result in a faster rate of substitution at nearly
neutral sites in symbionts compared with their free-living relatives. Moran (1996) documented such rate acceleration in cytoplasmically transmitted symbiotic bacteria. A similar rate acceleration was observed for fungi
that have mutualistic relationships with algae relative to
free-living fungi (Lutzoni and Pagel 1997).
Here, we study nucleotide substitution rates of 16S
rDNA in sulfur-oxidizing gamma subdivision Proteo1 Present address: Department of Ecology and Evolutionary Biology, University of California at Irvine.
Key words: nearly neutral substitution rate, bacterial symbiosis,
chemoautotrophic sulfur oxidation, 16S rDNA domains, loop/stem, selective constraint.
Address for correspondence and reprints: Andrew S. Peek, Department of Ecology and Evolutionary Biology, 321 Steinhaus Hall,
University of California at Irvine, Irvine, California 92697-2525. Email: [email protected].
Mol. Biol. Evol. 15(11):1514–1523. 1998
q 1998 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
1514
bacteria. This bacterial group has three distinct life histories: maternally transmitted endosymbionts, environmentally captured endosymbionts, and free-living bacteria. The maternally transmitted symbionts live within
the cells of their metazoan hosts and undergo vertical
transmission through host eggs. Maternally transmitted
symbionts are found in three molluscan host groups: solemyid bivalves (Gustafson and Reid 1988; Cary 1994;
Krueger, Gustafson, and Cavanaugh 1996), vesicomyid
clams (Endow and Ohta 1990; Cary and Giovannoni
1993), and mytilid mussels (Nelson and Fisher 1995).
In contrast, environmentally captured symbionts are acquired de novo each host generation from a freely living
or dormant bacterial source. Two metazoan host groups
contain environmentally captured endosymbionts: lucinid clams (Gros et al. 1996) and vestimentiferan tubeworms (Cary et al. 1993; Laue and Nelson 1997; Feldman et al. 1997). The free-living Proteobacteria considered in this study have no known association with a
metazoan host. Many free-living gamma subdivision
Proteobacteria oxidize reduced sulfur, including the chemoautotrophic bacterial genera Thiomicrospira, Thiothrix, Beggiatoa, Thioploca, and Thiobacillus, and some
use reduced sulfur in photosynthesis (e.g., the genera
Chromatium and Ectothiorhodospira).
The 16S rDNA molecule consists of stems and
loops. Intramolecular hydrogen-bonding between nucleotides in the stem regions of the 16S molecule contributes to higher order structures that are required for its
proper function (Gutell, Larsen, and Woese 1994). Nucleotides in loop regions also contribute to function
(e.g., Lee et al. 1997) but are under less selective constraint on average than sites in stem positions (Dixon
and Hillis 1993; Vawter and Brown 1993; Springer and
Douzery 1996). The 16S rDNA molecule can also be
partitioned into domains of specific functionality that
occupy discrete regions of the mature ribosomal small
subunit (Dahlberg 1989; Moore 1988). Two peripheral
domains (I and III) tend to be under lower selective
constraint than the single central domain (II) (Dixon and
Hillis 1993; Van de Peer et al. 1996; Golding 1994). In
total, nucleotide substitutions at sites in loop regions and
16S rDNA Rate Variation in Symbiotic Bacteria
in domains I and III are likely more nearly neutral than
nucleotide substitutions at sites in stem regions and domain II.
Given the life histories of sulfur-oxidizing gamma
Proteobacteria and the pattern of selective constraints in
16S rDNA molecules, the nearly neutral theory leads to
two predictions. First, the overall rate of nucleotide substitution should be faster in small populations. Second,
the rate of nucleotide substitution should be preferentially faster at sites under low selective constraint. We
examine these predictions in symbiotic and free-living
groups of sulfur-oxidizing gamma Proteobacteria.
Materials and Methods
Data
The bacterial small-subunit (SSU) 16S nucleotide
sequences used in this study were all taken from
GenBank (table 1). The choice of sequences was based
on three criteria. First, known free-living and symbiotic
bacterial sequences were selected from the gamma subdivision, and outgroup sequences were selected from
beta subdivision Proteobacteria. Second, to limit the
analyses to a tractable number of organisms from the
diverse gamma subdivision, we used only sequences
from autotrophic bacteria that utilize reduced sulfur.
Third, only nearly complete 16S rDNA sequences were
used.
Phylogenetic analyses were performed on aligned
areas of the 16S-rDNA sequences from 46 bacteria, and
rate analyses were performed on the autotrophic subset
of 39 bacteria. Alignments were performed manually
with GDE (Smith et al. 1994), and ambiguously aligned
portions were excluded from further analyses. The
aligned portions of the sequences consisted of 1,165 bp
that corresponded to positions 112–187, 219–440, 481–
834, 849–1138, and 1141–1363 relative to Escherichia
coli. We could not align three nucleotide positions with
the E. coli secondary structure and excluded them from
subsequent analyses of secondary structure. Stem and
loop positions were inferred from the E. coli secondary
structure model (Gutell, Larsen, and Woese 1994). Altogether, 655 positions involved in intramolecular bonding were designated stem positions, and 507 positions
were designated loop positions. Nucleotide positions for
domain analysis consisted of 379 positions within domain I (sites 1–560 relative to E. coli), 345 positions
within domain II (sites 561–919), and 441 positions
within domain III (sites 920–1398).
The 16S rDNA sequences from sulfur-oxidizing
bacteria were divided into three categories based on bacterial life histories. The first category contained 15 maternally transmitted symbionts from the bivalve families
Mytilidae (Distel et al. 1988), Solemyidae (Eisen, Smith,
and Cavanaugh 1992; Distel, Felbeck, and Cavanaugh
1994; Krueger and Cavanaugh 1997), and Vesicomyidae
(Peek et al. 1998). The second category included 12
environmentally captured symbionts from the bivalve
family Lucinidae (Distel et al. 1988; Distel and Wood
1992; Distel and Cavanaugh 1994; Durand et al. 1996)
and vestimentiferan tubeworms (Feldman et al. 1997).
1515
Table 1
Bacteria Studied for Phylogeny and Relative Rate, with
GenBank Accession Numbers
Bacteria
Gamma subdivision proteobacteria
Maternally transmitted symbionts
Vesicomyidae
Calyptogena elongata . . . . . . . . . . . . . . . . . .
Vesicomya gigas . . . . . . . . . . . . . . . . . . . . . .
Ectenagena extenta . . . . . . . . . . . . . . . . . . . .
Calyptogena kilmeri . . . . . . . . . . . . . . . . . . .
Calyptogena magnifica . . . . . . . . . . . . . . . . .
Calyptogena phaseoliformis . . . . . . . . . . . . .
Calyptogena n. sp. . . . . . . . . . . . . . . . . . . . .
Vesicomya lepta . . . . . . . . . . . . . . . . . . . . . .
Calyptogena pacifica . . . . . . . . . . . . . . . . . .
Mytilidae
Bathymodiolus thermophilus . . . . . . . . . . . .
Solemyidae
Solemya reidi . . . . . . . . . . . . . . . . . . . . . . . . .
Solemya velum . . . . . . . . . . . . . . . . . . . . . . . .
Solemya pusilla . . . . . . . . . . . . . . . . . . . . . . .
Solemya terraregina . . . . . . . . . . . . . . . . . . .
Solemya occidentalis . . . . . . . . . . . . . . . . . . .
Environmentally captured symbionts
Lucinidae
Andontia phillipiana . . . . . . . . . . . . . . . . . . .
Codakia costata . . . . . . . . . . . . . . . . . . . . . . .
Codakia obicularis . . . . . . . . . . . . . . . . . . . .
Lucina floridana . . . . . . . . . . . . . . . . . . . . . .
Lucina nassula . . . . . . . . . . . . . . . . . . . . . . .
Lucina aequizonata . . . . . . . . . . . . . . . . . . . .
Thyasira flexuosa . . . . . . . . . . . . . . . . . . . . .
Vestimentifera
Escarpia spicata . . . . . . . . . . . . . . . . . . . . . .
Lamellibrachia columna . . . . . . . . . . . . . . . .
Lamellibrachia sp. . . . . . . . . . . . . . . . . . . . .
Ridgia piscesae . . . . . . . . . . . . . . . . . . . . . . .
Riftia pachyptila . . . . . . . . . . . . . . . . . . . . . .
Free-living bacteria
Thiomicrospira
Cycloclasticus pugetii . . . . . . . . . . . . . . . . . .
Thiomicrospira pelophila . . . . . . . . . . . . . . .
Thiomicrospira crunogena . . . . . . . . . . . . . .
Thiothrix
Thiothrix nivea . . . . . . . . . . . . . . . . . . . . . . .
Thiothrix ramosa . . . . . . . . . . . . . . . . . . . . . .
Beggiatoa
Beggiatoa alba . . . . . . . . . . . . . . . . . . . . . . .
Thioploca ingrica . . . . . . . . . . . . . . . . . . . . .
Cardiobacteria
Thiobacillus hydrothermalis . . . . . . . . . . . . .
Chromatium
Chromatium tepidum . . . . . . . . . . . . . . . . . .
Ectothiorhodospira halochloris . . . . . . . . . .
Ectothiorhodospira shaposhnikovi . . . . . . . .
Other gamma subdivision bacteria
Escherichia coli . . . . . . . . . . . . . . . . . . . . . . .
Pseudomonas stutzeri . . . . . . . . . . . . . . . . . .
Vibrio harvyei . . . . . . . . . . . . . . . . . . . . . . . .
Beta subdivision Proteobacteria
Thiobacillus thiooxidans . . . . . . . . . . . . . . . .
Thiobacillus caldus . . . . . . . . . . . . . . . . . . . .
Thiobacillus ferrooxidans . . . . . . . . . . . . . . .
Pseudomonas testosteroni . . . . . . . . . . . . . .
Rhodocyclus purpureus . . . . . . . . . . . . . . . . .
GenBank
Accession No.
AF035719
AF035726
AF035725
AF035720
AF035721
AF035724
AF035722
AF035727
AF035723
M99445
L25709
M90415
U62130
U62131
U41049
L25711
L25712
M99447
L25707
X95229
M99448
L01575
U77482
U77481
U77479
U77480
U77478
L34955
L40809
L40810
L40993
U32940
L40994
L40998
M90662
M59150
M59152
M59151
J01859a
D84024
X56578
X72851
Z29975
Y11595
M11224
M34132
NOTE.—The taxonomic designations of their metazoan hosts are given for
symbiotic bacteria.
a For brevity, only a single reference is given.
1516
Peek et al.
The third category included 11 sequences from free-living bacteria (Lane et al. 1992; Durand et al. 1993; Muyzer et al. 1995; Teske et al. 1995). A fourth category
(the outgroup) contained sequence from a beta subdivision bacterium, Thiobacillus thiooxidans. Phylogenetic placement of the outgroup taxon varies. Some authors
have placed it in the beta subdivision (Lane et al. 1992),
and others have placed it deeply within the gamma subdivision (McDonald et al. 1997). In either case, the phylogenetic placement for this taxon is consistent with its
use as an outgroup in the present study.
Phylogenetic relationships between selected members of the gamma and beta subdivision were reconstructed by maximum likelihood with fastDNAml (Olsen, Matsuda, and Hagstrom 1994). Maximum-likelihood analyses were performed by randomizing the taxa
input order 40 times with global rearrangements and an
empirically estimated transition/transversion (ts/tv) ratio
of 1.9.
Rate Analysis
We used the relative-rate test of Muse and Weir
(1992) to test for deviations from rate equality between
bacterial 16S rDNA sequences. All possible pairwise
comparisons between gamma subdivision bacteria with
different life history strategies were performed, with the
beta subdivision bacterium T. thiooxidans as outgroup.
For example, the relative-rate comparison between 15
maternally transmitted symbionts and 11 free-living bacteria resulted in 165 comparisons. These pairwise tests
have multiple degrees of interdependence and should be
interpreted as descriptive. A total of 297 pairwise comparisons were made between symbiotic and free-living
groups, while 180 comparisons were made between maternal and environmental symbiotic groups. Equal numbers of pairwise comparisons were made for the loop,
stem, and domain data partitions.
To partially account for the lack of independence
between multiple pairwise comparisons, we also performed the two-group relative-rate test of Li and Bousquet (1992), using Kimura (1980) two-parameter distances. Thiobacillus thiooxidans was used as the outgroup taxon for all tests of rate variation between bacterial groups.
Rate Dynamics
In order to compare rate dynamics between regions
of 16S rDNA, we estimated the ratio of substitution
rates at stem positions between two taxa by the statistic
rs:
rs 5 ds1/ds2,
(1)
where ds1 is the estimated substitution rate at stem positions from taxon 1 and the common ancestor of taxa
1 and 2. In this case, ds1 and ds2 were the maximumlikelihood distance estimates generated from pairwise
relative-rate tests (Muse and Weir 1992). When there is
rate equality between taxa 1 and 2, rs 5 1 for stem
positions. The ratio of substitution rates at loop positions
between two taxa had an analogous test statistic given
by rl. The relative ratios of loop and stem substitution
rates were compared for all pairwise combinations of
taxa between groups with the function
d5
5
0
1
(rl /rs # 1)
(rl /rs . 1)
(2)
and
Od,
m
D5
i
(3)
i
where m is the total number of comparisons made between groups. We used D to contrast rate dynamics between loop and stem regions as well as between domains. The relative ratio of loop to stem substitutions
was designated Dl-s, and analogous statistics were used
for relative ratios of substitution between the 16S rDNA
domains, (i.e., DI-II).
Summary statistics for relative-rate comparisons
and relative-ratio comparisons were made for the freeliving and symbiotic bacterial groups. We estimated average pairwise branch lengths and ratios within a group.
These average pairwise branch lengths between groups
were normalized such that relative values greater than
1.0 were above the average and those less than 1.0 were
below the average. Base composition at stem and loop
positions was tested for homogeneity with the chisquare method as implemented in PAUP 3.1 (Swofford
1993).
Results
Phylogenetic Analysis
Sulfur-oxidizing bacteria occur in all subdivisions
of the Proteobacteria except the delta subdivision (Lane
et al. 1992). The beta and gamma subdivisions are each
other’s closest relatives (Woese 1987), and the gamma
subdivision contains free-living (Durand et al. 1993;
Muyzer et al. 1995; Teske et al. 1995; Caumette et al.
1997; Overmann and Tuschak 1997) and symbiotic
(Distel et al. 1988; Eisen, Smith, and Cavanaugh 1992)
sulfur-oxidizing bacteria. Since relative-rate tests rely on
two closely related ingroup taxa, with a third taxon representing the outgroup, we desired to confirm the outgroup status of the sulfur-oxidizing chemoautotrophic
beta subdivision bacteria. Furthermore, the statistical
power for the relative-rate test is maximized when the
outgroup taxon is closely related to the ingroup taxa
(Muse and Weir 1992). To verify the ancestral status of
the beta subdivision bacteria used as outgroup taxa in
this study, we performed a broad phylogenetic analysis
of the gamma and beta Proteobacteria.
Maximum-likelihood analysis confirms the outgroup status for the beta subdivision (fig. 1). Additionally, the phylogeny upheld previous results for two
clades of symbionts (Distel et al. 1988). One clade
(group I) contained the sulfur-oxidizing symbionts from
bivalves in the families Solemyidae and Lucinidae, and
vestimentiferan tubeworms. This group consists of both
maternally transmitted symbionts and environmentally
captured symbionts (table 1). A second symbiont clade
(group II) contained the sulfur-oxidizing gill bacteria
16S rDNA Rate Variation in Symbiotic Bacteria
1517
FIG. 1.—Maximum-likelihood phylogeny (LnLi 5 212469.69) based on 1,165 positions of 16S rDNA from 46 bacteria, rooted with the
beta subdivision Proteobacteria. All internodes with bootstrap support .50% are indicated. All bacteria are from the gamma subdivision of the
Proteobacteria except those labeled as being from the beta subdivision. Two groups containing endosymbiotic bacteria are labeled group I and
group II. All of the presented bacteria are sulfur-oxidizing and autotrophic, except for heterotrophic members of the gamma subdivision (Escherichia coli, Pseudomonas stutzeri, and Vibrio harveyi) and the beta subdivision (Rhodocyclus purpureus and Pseudomonas testosteroni).
Relationships between most bacteria examined within the gamma subdivision are not well resolved by bootstrap measures or topological
rearrangements. The scale bar is proportional to the estimated nucleotide divergence.
from the clam family Vesicomyidae and the mussel family Mytilidae. All of these group II bacteria are maternally transmitted symbionts. Phylogenetic relationships
among most lineages of the sulfur-oxidizing gamma
Proteobacteria were not well resolved. Thiomicrospira
may be the closest free-living relatives to the group I
symbionts, and Thiothrix may be the closest free-living
relatives to the group II symbionts, but neither of these
results are supported by bootstrap resamplings or topology rearrangements. Equivocal phylogenetic placements
of symbiotic bacteria and their closest free-living relatives suggested the use of rate methods that minimize
phylogenetic assumptions.
Pairwise Relative Rates
To test predictions of the nearly neutral theory, we
compared the rates of substitution in 16S rDNA sequences among autotrophic sulfur-oxidizing bacteria
with three different life histories. Overall substitution
rates appear to be faster in maternally transmitted symbionts than in free-living bacteria (table 2). Of the 165
pairwise rate tests between maternally transmitted symbionts and free-living bacteria, 119 (72%) estimated that
maternally transmitted symbionts evolved more rapidly
than free-living bacteria. Of these 165 tests, 99 reject
the null hypothesis of rate homogeneity between maternally transmitted symbionts and free-living bacteria, and
1518
Peek et al.
Table 2
Pairwise Rate Acceleration Tests with Summaries of All Comparisons and Only Those Comparisons with Significant
Differences in Rate Between Maternally Transmitted, Environmentally Captured, and Free-Living Bacterial Life
Histories
GROUP
2
r1 . r2
Total
Frequency
r1 . r2
Total
Frequency
Free-living
Environmental capture
Free-living
119
172
30
165
180
132
0.721
0.956
0.227
81
142
5
99
142
72
0.818
1.0
0.069
1
Maternal transmission
Maternal transmission
Environmental capture
a
SIGNIFICANT COMPARISONSa
TOTAL COMPARISONS
a 5 0.05; H0: equal rates of change between individual pairwise comparisons, r1 5 r2.
81 (81%) of the 99 significant differences favored faster
rates in maternally transmitted symbionts. The clear
trend is that maternally transmitted symbionts have faster nucleotide substitution rates than do free-living bacteria.
Rates of nucleotide substitutions are also faster in
maternally transmitted symbionts than in environmentally captured symbionts. Of the 180 pairwise rate tests
between maternally transmitted symbionts and environmentally captured symbionts, 172 (96%) estimated that
maternally transmitted symbionts evolved more rapidly
than environmentally captured symbionts. Also, all of
the 142 significant accelerations were in the maternally
transmitted symbionts.
In contrast, rates of substitution tend to be slower
in environmentally captured symbionts than in free-living bacteria. Altogether, 102 (78%) of the 132 pairwise
tests revealed slower rates in environmentally captured
symbionts than in free-living bacteria. Also, 67 (93%)
of the 72 significant accelerations were in free-living
bacteria compared with environmentally captured symbionts (table 2). Overall pairwise rate estimates between
bacterial life histories suggest that maternally transmitted symbionts have the fastest nucleotide substitution
rates, that free-living bacteria have intermediate rates,
and that environmentally captured symbionts have relatively slow rates.
expected from previous comparisons, the rates of maternally transmitted symbionts were also significantly (Z
5 5.953; P , 1025) accelerated when compared with
those of environmentally captured symbionts. Overall,
relative rates for grouped comparisons are consistent
with pairwise comparisons, which indicate that maternally transmitted symbionts evolve most rapidly, freeliving bacteria are intermediate, and environmentally
captured symbionts evolve most slowly.
The Relative Ratio of Substitution Rates Between
Loops and Stems
The nearly neutral theory predicts that the rate of
fixation of nearly neutral nucleotide substitutions is dependent on both population size and the level of selective constraint. This relationship implies that changes in
rate mediated by population size effects should be more
pronounced in regions of molecules that are subject to
low selective constraint. We examined this hypothesis
by comparing rate dynamics between loop and stem
regions of 16S rDNA. We compared the relative ratios
of loop to stem substitution rates between maternally
transmitted symbionts and free-living bacteria with the
statistic Dl-s. If rate differences between maternally
transmitted symbionts and free-living bacteria have affected rates and stems similarly, then d should be equal
to 1 in roughly half of the comparisons for Dl-s. However, d was equal to 1 in 76% of these comparisons
(table 3), suggesting that rate differences between maternally transmitted symbionts and free-living bacteria
are more pronounced at nucleotide sites within loops
than at nucleotide sites within stems. Comparison of maternally transmitted symbionts to environmentally captured symbionts yields an even higher frequency
(;84%; table 3). The relative ratio of loop to stem rates
also differed from 50% between environmentally transmitted symbionts and free-living bacteria, but not to the
degree seen in other comparisons. These comparisons
suggest that rate differences between taxa are more pronounced at loops than at stems.
Two-Group Relative Rates
Statistical inferences from multiple nonindependent
comparisons can be difficult to interpret. To partially
account for the lack of independence between multiple
pairwise relative-rate comparisons, we tested for the relative rates of substitution between bacterial life history
groups using the relative-rate test of Li and Bousquet
(1992). As a group, maternally transmitted symbionts
evolved significantly (Z 5 3.601; P 5 0.0002) faster
than did free-living bacteria. In contrast, environmentally captured symbionts had significantly (Z 5 22.081;
P 5 0.0188) slower rates than free-living bacteria. As
Table 3
Relative Ratios Between Loops and Stems and Between Domains I, II, and III of 16S rDNA
GROUP
1
NO.
2
Maternal transmission Free-living
Maternal transmission Environmental capture
Environmental capture Free-living
OF
COMPARISONS
165
180
132
LOOP/STEM
DOMAINS
Dl-s Frequency
DI-II Frequency DI-III Frequency DIII-II Frequency
126
152
78
119
116
109
0.764
0.844
0.591
0.721
0.644
0.826
79
136
57
0.479
0.756
0.432
114
44
98
0.691
0.245
0.743
16S rDNA Rate Variation in Symbiotic Bacteria
1519
Table 4
Li and Bousquet Correction for Multiple Comparisons Between Groups: Maternally Transmitted, Environmentally
Captured, and Free-Living Bacterial Life Histories
GROUP
DOMAIN
I ........
II . . . . . . .
III . . . . . .
1
2
Maternal transmission
Maternal transmission
Environmental capture
Maternal transmission
Maternal transmission
Environmental capture
Maternal transmission
Maternal transmission
Environmental capture
Free-living
Environmental capture
Free-living
Free-living
Environmental capture
Free-living
Free-living
Environmental capture
Free-living
K1 2 K2
V(K1 2 K2)
Z
P
0.023405
0.033281
20.009876
20.001580
0.030253
20.031833
0.030011
0.027363
0.002648
3
3
3
3
3
3
3
3
3
2.516
3.736
21.068
20.147
3.075
22.965
3.736
3.478
0.329
0.0060a
,0.0001a
0.1423
0.4404
0.0011a
0.0015a
,0.0001a
0.0003a
0.3707
8.7
7.9
8.5
11.5
9.7
11.5
6.5
6.2
6.5
25
10
1025
1025
1025
1025
1025
1025
1025
1025
NOTE.—Domains of 16S rDNA are based on the secondary-structure model of Gutell; nucleotide positions are numbered relative to Escherichia coli; domain
I—positions 1–560, domain II—positions 561–919, domain III—positions 920–1398. Domain I—379 nucleotide sites, domain II—345 nucleotide sites, domain
III—441 nucleotide sites.
a a 5 0.05; H : equal rates of change between groupwise comparisons, K 5 K .
0
1
2
16S rDNA Domain Relative Ratios
A second test of the prediction that differing constraints at nucleotide positions influence substitution
rates involved the relative ratio of substitutions between
domains I, II, and III of the 16S rDNA. Comparisons of
the relative ratios for three domains between maternal
transmission symbionts and free-living bacteria suggest
that fast rates for both domains I and III are more pronounced relative to domain II, but there is no substantial
difference in rates between domains I and III (table 3).
This same pattern is evident in comparisons between
environmentally captured symbionts and their free-living relatives. In contrast, the relative ratios between domains for the two symbiont types were not as clear. The
relative ratios of substitution between domains I and II
for maternal versus environmental symbionts are consistent with previous accelerations at domain I. However, relative ratios involving comparisons with domain
III were reversed compared with previous results, in
which domain III was decelerated when compared with
both domain I and domain II (table 3).
16S rDNA Domain Rates
To further investigate the relative rates of nucleotide substitution across 16S rDNA structural domains,
we performed the Li and Bousquet (1992) relative-rate
test on each domain separately. Comparisons between
maternally transmitted symbionts and free-living bacteria revealed significant rate differences at domain I (P
5 0.0060) and domain III (P , 0.0001) but no significant rate difference at domain II (table 4). In contrast,
comparisons between environmentally captured symbi-
onts and free-living bacteria revealed significantly
slower rates in domain II (P 5 0.0011) of environmentally captured symbionts, but no significant rate differences at domains I and III. Comparisons between maternally transmitted symbionts and environmentally captured symbionts reveal significantly faster rates across
all domains in maternally transmitted symbionts (table
4).
Summary Statistics
To provide an overview of the effects of maternal
transmission and environmental capture symbioses on
16S rDNA rate, we calculated summary rank statistics
for both relative-rate and relative-ratio analyses. The relative branch lengths from each bacterial life history
were calculated and standardized to an average branch
length from all pairwise rate tests. A summary value of
1.0 indicates equal rates between groups; values greater
than one indicate faster rates in group 1 than in group
2, and values less than one indicate that group 2 evolves
more rapidly. Maternally transmitted symbionts were,
on average, 1⅓ times as fast as free-living bacteria and
twice as fast as environmentally captured symbionts (table 5). Most of this acceleration took place at loop positions and in domains I and III. Environmentally captured symbionts were three quarters as fast as free-living
bacteria and there was some difference between stem
and loop positions, with loops being slightly faster, and
domains I and III were faster than domain II. Relative
ranks of substitution between maternally transmitted and
environmentally captured symbionts show patterns consistent with previous analyses, including a lower-than-
Table 5
Relative Average Branch Lengths (d1/d2) for All Sites and for Loop, Stem, and Domain Regions Between Bacterial Life
History Groups
GROUP
1
Maternal transmission
Maternal transmission
Environmental capture
DOMAINS
2
ALL SITES
LOOP
STEM
I
II
III
Free-living
Environmental capture
Free-living
1.314
2.217
0.727
1.933
4.058
0.804
1.276
2.118
0.791
1.345
2.874
0.756
0.978
2.345
0.493
1.583
1.768
0.972
1520
Peek et al.
expected rate of acceleration at domain III relative to
other sites (table 5).
Discussion
Phylogenetic relationships among most free-living
and symbiotic members of the sulfur-oxidizing gamma
Proteobacteria are not well resolved by the current study
of 16S rDNA. However, the beta subdivision bacteria
used as outgroups in relative-rate analyses were found
to be ancestral to bacteria within the gamma subdivision
of the Proteobacteria, as per previous studies (Lane et
al. 1992; McDonald et al. 1997). The phylogeny also
suggests that there are two groups of symbiotic bacteria
(Distel et al. 1988). These two groups are interspersed
by free-living bacterial lineages, but the relationships
among these lineages are not well resolved.
Our studies indicate that there is rate heterogeneity
among groups of sulfur-utilizing gamma Proteobacteria
with different life histories. Explanations for differences
in rates of molecular evolution have been attributed to
several factors (reviewed in Mindell and Thacker 1996),
including (1) incident mutation during DNA duplication,
(2) metabolic environment having thermal or oxidative
differences, (3) efficiency or bias in mutation repairs,
(4) recombination, (5) generation times, (6) differing selective constraints, and (7) population size. It is difficult
to examine the effects of these different factors in the
absence of information on metabolic rates, gene repair,
generation time, and other variables. However, many of
our observations are consistent with the predictions of
the nearly neutral theory.
The nearly neutral theory predicts that the rate of
substitution in populations is controlled by two interacting factors: (1) population size (Ne) and (2) selective
constraint (s). If selection coefficients are similar between two populations of different sizes, then nucleotide
substitution rates are expected to be faster in the smaller
population. Population sizes probably differ among bacteria as a function of life history. For example, the relative population size for maternally transmitted symbionts is likely smaller than that for free-living bacteria
due to population bottlenecks associated with symbiont
transmission through the egg. We observe a general rate
increase in maternally transmitted symbionts relative to
both free-living bacteria and environmentally captured
symbionts (tables 1 and 2). Under the assumption that
maternally transmitted symbionts have relatively small
population sizes, rapid evolutionary rates in maternally
transmitted bacteria are consistent with predictions of
the nearly neutral theory.
Further support for nearly neutral rate dynamics
comes from our observations that rate differences are
more pronounced in regions of 16S rDNA that are
thought to be under low selective constraint. For example, the relative ratio of loops to stems suggests that
loops evolve relatively faster than stems between maternally transmitted symbionts and bacteria with other
life histories. Similarly, relative rates and ratios based
on comparisons of maternally transmitted symbionts and
free-living bacteria indicate that domains I and III have
been more affected by rate changes than has domain II
Table 6
Scaled Ranks for the Rates and Loop-to-Stem Ratios for
Groups Within Bacterial Life Histories
Bacterial Group
Maternal transmission
Mytilidae . . . . . . . . . . . . . . . .
Vesicomyidae . . . . . . . . . . . . .
Solemyidae . . . . . . . . . . . . . .
Free-living
Thiomicrospira . . . . . . . . . . . .
Thiothrix . . . . . . . . . . . . . . . .
Beggiatoa . . . . . . . . . . . . . . . .
Cardiobacteria . . . . . . . . . . . .
Chromatium . . . . . . . . . . . . . .
Environmental capture
Lucinidae . . . . . . . . . . . . . . . .
Vestimentifera . . . . . . . . . . . .
Overall Rate
dl/ds
1.43
1.65
1.10
2.68
1.79
1.26
1.64
1.16
1.00
0.88
0.71
0.59
0.69
0.67
1.35
1.30
0.73
0.66
1.03
0.77
(tables 3 and 4). In addition, relative ratios based on
comparisons between maternally transmitted symbionts
and environmentally captured symbionts indicate that
rate variation is more pronounced in domain I than in
domain II (table 3). In short, rate heterogeneity among
lineages is most pronounced in those regions of the molecule that are most likely to experience nearly neutral
mutations.
It is possible, however, that the rate heterogeneity
we have documented is more closely associated with
phylogenetic history than with life history strategy. We
believe that this is not the case for the following reason.
The group I symbionts in figure 1 consist of several
distinct lineages, of which some undergo maternal transmission and others undergo environmental capture. We
ranked these and other phylogenetic groups based on
both overall rate and loop-to-stem ratio to provide insight into rate heterogeneity associated with phylogenetic history (table 6). The maternally transmitted symbiont groups Mytilidae (group II; fig. 1), Vesicomyidae
(group II), and Solemyidae (group I) had above average
rates at all positions and also had above average rates
for loop-to-stem ratios. The maternal symbionts were
the only groups to have above average rates for both
rate and ratio measures, despite the fact that the Solemyidae symbionts are phylogenetically distinct from
other maternal symbionts. In contrast, phylogenetic
groups of environmentally captured symbionts (Lucinidae and Vestimentifera) had below-average rates at all
positions. Some individual groups of free-living bacteria, such as Thiomicrospira and Thiothrix, were relatively fast overall, but this acceleration was associated
with a low ratio of loop-to-stem substitutions. Other
free-living bacterial groups, such as Chromatium, evolve
relatively slowly but had a high loop-to-stem ratio. In
summary, the maternally transmitted symbionts appear
to have similar rate dynamics regardless of phylogenetic
history, and these rate dynamics are largely consistent
with expectations based on population size and selective
constraint. However, the low number of phylogenetically independent origins of maternally transmitted symbionts (i.e., two) precludes a strong conclusion; here, we
can report only consistent trends.
16S rDNA Rate Variation in Symbiotic Bacteria
The different rate dynamics of environmentally
captured and maternally transmitted symbionts indicates
that rapid nucleotide substitution rates are not a characteristic of symbiosis per se. Different patterns of nucleotide substitution may be a function of the mechanism of symbiont inheritance. For example, environmentally captured symbionts have both a free-living
component and a symbiont component and may thus
have a large effective population size. Slow rates of nucleotide substitution in environmentally captured symbionts could reflect efficient purifying selection in a
large population.
Although the observed rate heterogeneity between
symbiotic and free-living bacteria is largely consistent
with nearly neutral rate variation, it may also be associated with other differences. For example, selective
constraint on 16S rDNA could vary among evolutionary
lineages. Slow evolutionary rates in environmentally
captured symbionts could reflect a few possible selective
scenarios. First, constraints involved in symbiont recapture by the host may place these symbionts in a selective
environment that decreases the overall rate of evolution.
Second, fluctuating environmental conditions (i.e., alternation between an intracellular environment and a freeliving environment) may increase the overall stabilizing
selective pressure and subsequently decrease the rate of
evolution. Reduced temperature fluctuation is one mechanism thought to be responsible for the increase in protein evolutionary rate in homeothermic organisms when
compared with poikilothermic organisms (Martin and
Palumbi 1993). Third, differing cellular metabolic environments might exist between bacteria; for example,
form I Rubisco has been found within vestimentiferan
tubeworms, but form II Rubisco occurs in the Solemyidae and Mytilidae symbionts (Robinson and Cavanaugh
1995). Metabolic differences associated with these differing forms of Rubisco (summarized in Haygood 1996)
could conceivably contribute to rate variation in these
bacterial groups. However, the distribution of form I versus form II Rubisco in autotrophic bacteria is not simple
(Delwiche and Palmer 1996), and, to complicate matters, some bacteria possess both forms (English et al.
1992). Discriminating among these and other possibilities for the observed rate heterogeneity between symbionts and free-living bacteria is not possible at present.
A number of other causes could fuel rate heterogeneity among bacterial lineages, including adaptation
to high-temperature environments. It has been suggested
that high G1C content at stem positions should correlate with high temperature, because increased stability
of G1C nucleotide bonding permits a higher thermal
tolerance (Galtier and Lobry 1997). This suggestion is
supported by the observation that G1C content increases for rRNA and tRNA stem positions with an increase
in the bacterial optimal growth temperature (Galtier and
Lobry 1997). However, the free-living and symbiotic
bacteria examined here are mesophilic, and there was no
significant deviation from the average 65% (60%–72%)
G1C content for 16S rDNA stem positions.
Previous arguments for rate acceleration in symbionts involved reduced recombination rates and re-
1521
duced population size, i.e., Muller’s ratchet (Moran
1996). Our results are consistent with the expectation of
Muller’s ratchet, a mutational advance in small asexual
lineages, but we find no reason to invoke reductions in
recombination rates. Another study showed that the rate
of nucleotide substitution in fungi was associated with
mutualistic associations with algae and was most consistent with an increased exposure to UV irradiation
(Lutzoni and Pagel 1997). This conclusion was based
on the high frequency of AA and TT dinucleotide substitutions in mutualistic fungi. In the bacterial lineages
examined here, we find no evidence for biased dinucleotide distributions. Furthermore, some of the fast-evolving lineages have limited potential for UV exposure (i.e.,
the Vesicomyidae inhabit the deep sea), while some
slowly-evolving lineages are sunlight-dependent (i.e.,
Chromatium). Alternative explanations for our observations should account for the overall rate dynamics between sites under different selective constraint as well
as the rate differences among life histories.
Acknowledgments
This is New Jersey Agriculture Experiment Station
Contribution No. D-32104-4-98, supported by state
funds, NSF grant OCE-96–33131, and NIH grant
PHSTW00735–01 to R.C.V., and an award from the Alfred P. Sloan foundation to B.S.G.
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Accepted August 3, 1998