MBE Advance Access published August 25, 2012

Extreme Conservation and Non-Neutral Evolution of the cpmA

Circadian Locus in a Globally Distributed Chroococcidiopsis
sp. from Naturally Stressful Habitats
Volodymyr Dvornyk* and Akhee Sabiha Jahan
School of Biological Sciences, University of Hong Kong, Pokfulam, Hong Kong S.A.R., Peoples Republic of China
*Corresponding author: E-mail: dvornyk@hku.hk.
Associate editor: Jennifer Wernegreen
DNA sequences reported in this article have been deposited in the Genbank accessions
HQ113413HQ113425, HQ113427HQ113442, HQ113448HQ113458, JQ669824JQ669833.

Cyanobacteria are among the most ancient organisms known to have circadian rhythms. The cpmA gene is involved in
controlling the circadian output signal. We studied polymorphism and divergence of this gene in six populations of a
stress-tolerant cyanobacterium, Chroococcidiopsis sp., sampled in extreme habitats across the globe. Despite high haplotype
diversity (0.774), nucleotide diversity of cpmA is very low ( = 0.0034): the gene appears to be even more conserved than
housekeeping genes. Even though the populations were sampled thousands kilometers apart, they manifested virtually no
genetic differentiation at this locus (FST = 0.0228). Using various tests for neutrality, we determined that evolution of cpmA
significantly departures from the neutral model and is governed by episodic positive selection.
Key words: circadian phase modifier A, Chroococcidiopsis, selection, diversity, populations.

Introduction
The circadian system controls the expression of many genes
in a genome and thus is essential for maintaining cellular
homeostasis. Circadian rhythms have been observed in cyanobacteria since the 1990s, making them one of the simplest
known organisms to possess circadian rhythmicity (Kondo
et al. 1993). This circadian programming can enhance the
ability of an organism to anticipate important cyclic changes
in the environment and generate appropriate responses
(Woelfle et al. 2004). Cyanobacteria are a major model
system in prokaryotes for analyzing and testing predictions
about circadian rhythms and possible effects of evolutionary
forces on them (Mackey et al. 2011).
Having an internal clock that can match the external light/
dark cycle is thought to be advantageous for cyanobacteria as
photosynthetic organisms (Johnson et al. 1998; Ouyang et al.
1998). Cyanobacteria are quite versatile and have a high adaptive potential that allows them to occupy the most extreme
habitats on our planet (Whitton 1987). Given its significance
for controlling essential physiological processes, such as cell
division, regulation of nitrogen fixation, and photosynthesis
(Johnson and Golden 1999), the circadian system is important
for adaptability of cyanobacteria.
Macroevolutionary studies of various circadian genes
showed that their evolution was shaped by many factors,
including duplications, lateral transfers, and various types of
selection (Dvornyk et al. 2003; Dvornyk 2006, 2009; Baca et al.
2010). However, very little is known about the factors, which
govern evolution of circadian genes at the population level.
Previously, we reported multiple duplications and an elevated

neutral mutation rate in the core circadian genes, kaiB and

kaiC, from a filamentous cyanobacterium Nostoc linckia
under acute environmental stress (Dvornyk et al. 2002).
Apart from this, no other reports are available.
The cpmA (circadian phase modifier) gene is an element
of the output pathway of the circadian clock (Katayama
et al. 1999). Mutations in the cpmA gene alter the phasing
of the circadian rhythm of a restricted subset of genes,
which results in severe growth defects (Katayama et al.
1999). The gene was reported to consist of two domains
of similar length (N- and C-terminal domains), which
harbor several highly conserved regions of potential circadian function (Dvornyk 2006). The C-terminal domain
shares partial homology with the N-terminus of the AIR
carboxylase (AIRC) (pfam00731) and the NCAIR mutase
(COG0041) (Dvornyk 2006).
Chroococcidiopsis is a coccoid unicellular cyanobacterium
first reported from hot deserts (Fewer et al. 2002).
Chroococcidiopsis is not only capable of surviving extreme
desiccation but also shows a remarkable resistance to high
doses of ultraviolet radiation (Cordoba-Jabonero et al. 2005).
Resemblance of morphological characteristics of Chroococcidiopsis to proterozoic microfossils suggests it as one of the
evolutionary oldest cyanobacteria (Friedmann 1980;
Friedmann and Ocampo-Friedmann 1995).
This study was designed to examine polymorphism and
diversity of the cpmA gene in the Chroococcidiopsis strains
sampled from various extremely stressful habitats around the
Earth and to determine factors that govern microevolution of
this gene.

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Mol. Biol. Evol. doi:10.1093/molbev/mss191 Advance Access publication July 25, 2012

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Research
article

Abstract

MBE

Dvornyk and Jahan . doi:10.1093/molbev/mss191

Table 1. Characteristics of the Four Sampling Sites Used in This Study.
Population Name
AC (chasmolith)
AE (endolith)
AH (hypolith)

Coordinates
S77 24.5950 E161 11.7470

Canadian Arctic Circle, Devon Island

CH (hypolith)

N75 23.2240 W89 40.3350

Taklimakan Desert, China

TH (hypolith)

N38 24.2330 E88 53.8060

Kalahari Desert, Botswana

SH (hypolith)

S 22 11.00 E29 7.00

Materials and Methods

Environmental Samples and Cyanobacterial Strains
Environmental samples were collected from four locations representative of extreme arid climate regimes
(Bahl et al. 2011; Caruso et al. 2011). Major environmental
parameters and geographical coordinates are given in table 1.
To standardize the nature of samples collected, hypoliths
were targeted. These comprise biofilms that colonize the ventral surface of quartz, a relatively inert, and ubiquitous substrate in deserts worldwide. Hypoliths develop independently
of the surrounding soil and are dominated by cyanobacteria
(Pointing et al. 2009). The sampling sites were Antarctic Dry
Valleys (McKelvey Valley), Canadian Arctic (Devon Island),
cold desert (Taklimakan, China), and hot desert (Kalahari,
Southern Africa). Climate was delineated according to the
standards based on the long-term mean annual precipitation
and temperature (Peel et al. 2007).
The collected environmental samples were subcultured
to establish cyanobacterial cultures containing predominantly
Chroococcidiopsis sp. The cultures were maintained on BG-11
medium in conical flasks at 25 C in sterilized incubators with
constant light. In addition to the environmental cultures, a
strain of Chroococcidiopsis ATCC 27 900 was used in the analyses for identification purposes and comparisons.

Environmental Conditions at the Site

Strong winds
Ice cap deposits
Mean annual rainfall <100 mm
Average yearly temperature 20 C to 35 C
High levels of solar radiation
Variation in salinity
Mean annual rainfall <500 mm
Average yearly temperature less than 5 C
Large fluctuation in temperature
Shifting sand
Salt accumulation
Mean annual rainfall <25 mm
Average temperature less than 20 C
(winter) and <40 C (summer)
Dry air
Shifting sands
Large fluctuation in temperature
Mean annual rainfall <200 mm
Average yearly temperature more than 11 C
(winter) and >40 C (summer)

tag: AM1_4350), Trichodesmium erythraeum IMS101

(NC_008312, locus tag: Tery_4722), Microcystis aeruginosa
NIES-843
(NC_010296,
locus
tag:
MAE_62660),
Thermosynechococcus elongatus BP-1 (NC_004113, locus
tag: tll1189), and Nostoc sp. PCC 7120 (BA000019, locus tag:
alr3885).
The PCR was conducted using forward primer 5 F (50 -ACC
GGATTTCCCGAAGTGATTTGG-30 ) and reverse primer 4 R
(50 -GCCGCACCAAATCCATTATC-30 ). The master mix of
50 l contained 38.5 l of water, 5 l of 10 RED Taq
buffer (Sigma-Aldrich), 2 l of dNTPs (Sigma-Aldrich), 1 l
of each primer, 0.3 l of Red Taq polymerase (SigmaAldrich), 2 l of MgCl2, and 1.2 l of Tween-20 were used.
The PCR was conducted using the following profile: denaturation at 92 C for 1 min, annealing at 50 C, and extension at
72 C both for 2 min with 40 cycles. The PCR products were
then purified using a Qiagen purification kit and cloned into a
pDrive cloning vector (Qiagen, Inc.) following the manufacturers protocol.
The cloned gene fragments were isolated and sequenced
using an automated sequencer (3730xl DNA Analyzer,
Applied Biosystems) in Genome Research Center
(University of Hong Kong) using the dye primer method
(Applied Biosystems).

Data Analyses
DNA Extraction, Amplification, Cloning,
and Sequencing
Genomic DNA was isolated by the cetyltrimethylammonium
bromide DNA extraction protocol with minor modifications
(Stewart and Via 1993).
The polymerase chain reaction (PCR) primers were
designed using Net Primer (http://www.premierbiosoft.com/
netprimer) and the cpmA nucleotide sequences of the
following cyanobacterial species: Acaryochloris marina
MBIC11017 (GenBank accession no. NC_009925, locus
2

The sequence chromatograms were proofread and trimmed

for unresolved bases. The obtained nucleotide sequences
were aligned using ClustalW (Thompson et al. 1994) as
implemented in the BioEdit software v. 7.0.9 (Hall 1999).

Phylogenetic Analysis and Taxonomical Identification

of Sequences
The likelihood ratio test as implemented in the ProtTest 3.0
software (Darriba et al. 2011) was used to determine the most
appropriate substitution model for the data set. According to

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Sampling Sites
McKelvey Valley, Antarctica

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cpmA Locus in Chroococcidiopsis . doi:10.1093/molbev/mss191

Analysis of Sequence Polymorphism and Population

Genetic Diversity
The sequences were grouped into six populations according
to their location and type of microbial community (table 1).
Intra- and interpopulation nucleotide diversity was computed assuming the Kimura two-parameter model (Kimura
1980). The analyses were carried out using MEGA5 (Tamura
et al. 2011).
For each population, the following parameters were calculated: average number of nucleotide differences (K), number
of haplotypes (H), haplotype diversity (Hd), and  per site
(Watterson 1975). These analyses were performed using
DnaSP v5.10 (Librado and Rozas 2009). Interpopulation differentiation was estimated by FST computed with the analysis
of molecular variance (AMOVA) (Excoffier et al. 1992) as
implemented in the Arlequin 3.5 software (Excoffier et al.
2005) and with Josts D (Jost 2008) as implemented in
SPADE (Chao and Shen 2010).
The recombination parameter R and the minimum
number of recombination events in the sample, Rm, were
estimated according to Hudson (Hudson 1987). The genetic
association between polymorphic sites in the whole sample
was measured by the ZnS statistic (Kelly 1997), and the effect
of intragenic recombination on the observed DNA variation
was estimated by the ZZ statistics (Rozas et al. 2001). The
confidence intervals for the above estimates were obtained by
coalescent simulations with 10,000 replicates under an assumption of no recombination. DnaSP v5.10 (Librado and
Rozas 2009) was used for these computations.

Tests for Neutrality and Positive Selection

Correspondence of the obtained data to the neutral expectations was examined using several estimates: Tajimas D
(Tajima 1989), Fay and Wus H (Fay and Wu 2000), Fus Fs

test of selective neutrality (Fu 1997), Fu and Lis D* and

F* (Fu and Li 1993), and Achaz Y/Y* (Achaz 2008) as implemented online at http://wwwabi.snv.jussieu.fr/achaz/
neutralitytest.html. To detect positive selection, we applied
a compound DHEW test (Zeng et al. 2007), which combines
Tajimas D, Fay and Wus normalized H, and Ewens
Watterson estimates of neutrality. The synonymous (dS)
and nonsynonymous diversity (dN), and the dN/dS ratio
were calculated using the modified NeiGojobori method
(Nei and Gojobori 1986). The analyses were conducted
using DnaSP v5.10 (Librado and Rozas 2009).

Analysis of Population History

We analyzed the data for signs of historical population size
changes using two estimates: Fus Fs (Fu 1997) and R2 statistics
(Ramos-Onsins and Rozas 2002). Extensive computer simulations suggested that these tests are most robust for detecting
population growth/decline (Ramos-Onsins and Rozas 2002).
We also used a Bayesian analysis as implemented in LAMARC
2.1.6 to compute the exponential population growth rate, g
(Kuhner 2006). The growth rate relates to the scaled timedependent mutation parameter  as follows: t = present time
exp(gt), where t is time before present (Kuhner 2006).

Results
Nucleotide Polymorphism and Intrapopulation
Diversity of the cpmA Locus in Chroococcidiopsis sp.
The phylogenetic analysis of the obtained cpmA sequences of
Chroococcidiopsis sp. yielded a completely unresolved tree
(supplementary fig. S1, Supplementary Material online). For
the determined 26 unique haplotypes (alleles), there were 35
segregating sites out of 513 sequenced. Among the polymorphisms, 11 were synonymous and 23 nonsynonymous.
One polymorphism, a singleton at position 283, was either
one depending on the evolutionary path. Figure 1 shows distribution of the polymorphism along the sequenced region of
the gene.

FIG. 1. The distribution of nucleotide polymorphism along the 50 partial sequences of the Chroococcidiopsis sp. cpmA gene. Sliding window of
100 bp with increments of 10 bp. The putative functional domains are
shaded with tints of gray. The two hydrophobic motifs (positions
376408 and 433513) are depicted by black boxes .

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the Bayesian information criterion, the LG model (Le and

Gascuel 2008) with gamma distribution ( = 0.55) turned
out to fit our data best.
Because the cpmA gene was amplified from enriched but
nonaxenic cultures, it was necessary to verify which of the
obtained cpmA gene sequences belong to Chroococcidiopsis
sp. This was done by constructing a maximum likelihood
phylogenetic tree using the CpmA sequences of various cyanobacteria (supplementary table S1, Supplementary Material
online) and that of Chroococcidiopsis thermalis PCC 7203
(GenBank accession no. HQ113459.1) as a control for comparison. The statistical significance of the tree was evaluated
by a bootstrap resampling with 100 replications. The
sequences, which appeared on the tree outside the 100%
bootstrap support clade with the control sequence, were
assumed not to belong to Chroococcidiopsis sp. According
to the sequencing of 4 kbp of the SSU/ITS/LSU region,
the used strains are all Chroococcidiopsis, which currently is
considered as a monospecific genus (Bahl et al. 2011). Finally,
a total of 50 sequences were retained for the analyses. From
these sequences, 26 were unique haplotypes. This analysis was
conducted using PhyML 3.0 (Guindon and Gascuel 2003).

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Dvornyk and Jahan . doi:10.1093/molbev/mss191

Table 2. Genetic Diversity Parameters of the Studied Populations.

Population
AC
AE
AH
CH
SH
TH
Average
for the species

H
5
6
6
6
3
5
4.3

Hd
0.933
0.893
0.889
0.641
0.833
0.647
0.774

K
1.67
1.93
2.56
1.08
2.50
1.40
1.74

dS
0.0033
0.0070
0.0034
0.0023
0.0038
0.0030
0.0036

dN
0.0035
0.0026
0.0056
0.0020
0.0053
0.0026
0.0033

hg
0.0043
0.0053
0.0072
0.0044
0.0053
0.0048
0.0152

p
0.0033
0.0038
0.0049
0.0021
0.0049
0.0028
0.0034

NOTE.H, number of haplotypes; Hd, haplotype (gene) diversity; K, average no. of

differences; , theta (per site) from the total number of mutations; , intrapopulation diversity; dS, synonymous substitutions; dN, non-synonymous substitutions.

On the other hand, all populations manifested high haplotype diversity, which ranged from 0.641 to 0.993, averaging at
0.774 for the whole species.

Recombination and Linkage Disequilibrium

The analysis yielded the overall R value of 61.2 and Rm value of
2. The values of ZnS and ZZ were 0.0247 and 0.0092, respectively. The coalescent simulations showed that Rm, ZnS, and ZZ
are not significant. These results suggest that intragenic
recombination is not a significant factor for the observed
nucleotide variation at the cpmA gene.

Between-Population Diversity
The between-population differentiation at the cpmA locus
was very low (table 3). The genetic distances between the
populations ranged from 0.0024 to 0.0049 with the mean
interpopulation distance value of 0.0034  0.0006, that is,
the same as the mean intrapopulation nucleotide diversity.
The matrix of pairwise FST and Josts D values (table 3) provides further support for the low between-population differentiation. The average interpopulation FST was only 0.0228
and Josts D was only 0.011. Overall, the results of AMOVA
indicated that 97.72% of the variability resided within
populations.

Tests for Neutrality

The results of all tests for neutrality suggested that the cpmA
locus did not follow neutral expectations at the species level
(table 4). The compound DHEW test, which analyzes data for
presence of positive selection (Zeng et al. 2007), yielded a
highly significant P value of 0.0034. There were signs of
non-neutral evolution and positive selection in particular
populations too. The dN/dS ratio was above 1 in three populations: AC, AH, and SH (table 4).

Population History
Both results of the Fus Fs (Fu 1997) and R2 (Ramos-Onsins
and Rozas 2002) tests showed significant departure from the
constant population size. The Fs and R2 values (30.0980 and
0.0244, respectively) were far outside their 95% confidence
intervals as determined by coalescent simulations: (4.2967
to 4.7621) and (0.0501 to 0.1966), respectively. Along with the
highly significant Tajimas D (table 4), these results point out
to the population expansion of Chroococcidiopsis sp. Further

Table 3. Genetic Differentiation between the Studied Populations.

Population
AC
AE
AH
CH
SH
TH

AC
0.0035
0.0042
0.0026
0.0041
0.0030

AE
0.0064/0
0.0046
0.0030
0.0044
0.0033

AH
0.0127/0.103
0.0370/0.068
0.0037
0.0049
0.0040

CH
0.0018/0.153
0.0266/0.086
0.0466*/0.185
0.0035
0.0024

SH
0.0201/0.125
0.0355/0.066
0.0237/0.167
0.0889/0.033
0.0038

NOTE.Below diagonal, pairwise between-population genetic distance; above diagonal, pairwise FST values/Josts D.
*P < 0.01

TH
0.0065/0.141
0.0109/0.075
0.0254/0.175
0.0034/0
0.0515/0.029

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The average total nucleotide diversity of the gene for the

whole species (  SE) was 0.0034  0.0005; the synonymous
diversity was 0.0036, almost the same as the nonsynonymous
diversity (0.0033). The total DNA diversity of the two gene
domains was slightly different: 0.0030 and 0.0036 for N- and
C-terminal domains, respectively. The dN values were a bit
higher in the N-terminal domain (0.0037  0.0013 and
0.0032  0.0008, respectively), whereas the rate of synonymous substitutions was significantly higher at the C-terminal
domain (0.0049  0.0014) than at the N-terminal domain
(0.0010  0.0010). Interestingly, six replacement substitutions
versus only one synonymous substitution occurred in one of
the two hydrophobic motifs at the C-terminal domain (positions 126146 and 155171 in the translated CpmA protein
sequence of Chroococcidiopsis sp.). Some of these
replacements are radical. For example, highly hydrophobic
cysteine at position 138 is replaced by hydrophilic positively
charged arginine, and hydrophobic nonpolar leucine at position 159 is replaced by polar hydrophilic serine.
All studied populations and the whole species manifested
very low genetic diversity at the cpmA locus (table 2). The
highest value of total nucleotide diversity ( = 0.0049) was
determined in populations AH and SH, and the lowest in
population Canadian Arctic Circle (CH;  = 0.0021).
Populations AH and SH also showed the highest rate of nonsynonymous substitutions (0.0056 and 0.0053, respectively),
whereas the highest rate at synonymous sites (0.0070) was
recorded for population AE. Overall, the populations from
Canadian Arctic Circle (CH) and Taklimakan Desert (TH)
were the least polymorphic.

MBE

NOTE.D, Tajimas D (Tajima 1989); H, Fay and Wus H (Fay and Wu 2000); Fs, Fus Fs test of selective neutrality (Fu 1997); D* and F*, Fu and Lis D* and F* test statistics (Fu and Li 1993); Y and Y*, Achaz Y and Y* test statistics (Achaz 2008); E, test
for directional selection (P values) (Zeng et al. 2006); DHEW, compound test for positive selection (P values) (Zeng et al. 2007); dN/dS, ratio of nonsynonymous to synonymous substitutions; NA, not available.

P = 0.001

P = 0.1922

P = 0.0419
P = 0.2173

Y
NA
2.4510,
0.7952,
NA
1.2248,
NA
2.4699,
P > 0.10
P > 0.10
P > 0.10
P < 0.02
P > 0.10
P < 0.05
P < 0.02
F*
1.4501,
1.5029,
1.5486,
2.6914,
 0.7529,
2.3170,
4.7860,
P > 0.10
P > 0.10
P > 0.10
P < 0.02
P > 0.10
P < 0.05
P < 0.02
D*
1.3683,
1.3604,
1.3748,
2.4988,
 0.7968,
2.1369,
4.7781,
P = 0.012
P = 0.012
P = 0.115
P = 0.005
P = 0.501
P = 0.083
P < 0.001
Fs
2.5180,
2.7262,
1.5490,
2.9045,
0.4611,
1.4301,
30.0980,
P = 0.783
P = 0.2306
P = 0.8899
P = 0.6698
P = 0.3782
P = 0.7324
P = 0.049
0.9361,
0.2882,
0.9685,
0.6275,
0.2531,
0.7643,
1.7418,

P = 0.06
P = 0.100
P = 0.076
P = 0.006
P = 0.179
P = 0.015
P < 0.001
D
1.3369,
1.3593,
1.4219,
1.9819,
0.7968,
1.8391,
2.6443,
Population
AC
AE
AH
CH
SH
TH
Average for
the species

Table 4. Results for Tests of Neutrality.

support for this conclusion comes from the Bayesian estimates of the exponential population growth parameter,
g, which ranged from 851 to 933 for all six studied
populations.

Discussion
Low Level of Intra- and Interpopulation Nucleotide
Diversity at the cpmA Locus of Chroococcidiopsis sp.
Our results are in further support of very low nucleotide diversity at circadian genes of cyanobacteria. Despite the
extreme environments and the very large geographical distances between the sampling locations, both the
intrapopulation (table 2) and interpopulation diversity
(table 3) of Chroococcidiopsis sp. at the cpmA locus were
quite low. Previously, we studied microevolution of the two
core circadian genes, kaiB and kaiC, in a filamentous cyanobacterium Nostoc linckia from the environmentally contrasting slopes of the ecological model microsites, Evolution
Canyons I and II (Israel) (Dvornyk et al. 2002). These genes
manifested approximately 1,000-fold higher mutation rate in
the cyanobacterial strains from the environmentally stressful
south-facing slopes when compared with the strains from the
temperate north-facing slopes.
Data on intra- and interpopulation DNA diversity of protein-coding bacterial genes are very limited and fragmentary;
some available information of several housekeeping genes of
some bacterial species in comparison with the respective
values for cpmA is provided in table 5. The cpmA gene appears to be even less polymorphic than housekeeping genes,
which are thought to be extremely conserved due to their
significance for basic functions of an organism (Jordan et al.
2002). For example, the total nucleotide diversity of the cpmA
gene is at least 2-fold lower than that of the most conserved
housekeeping genes among those presented in table 5, rplL
and gyrA of Clostridium perfringens.
More comparable evidence comes from the study of another cyanobacterium, toxic Microcystis aeruginosa (Tanabe
et al. 2007). The average nucleotide diversity reported for
seven housekeeping genes of this prokaryote was 0.023, ranging from 0.013 (recA) to 0.043 (pgi), which is up to 10-fold
higher than that for the cpmA gene (table 5).
Recent multilocus study using the 16S rRNA-5.8S ITS-23S
rRNA gene region sequences of globally distributed
Chroococcidiopsis sp. reported differentiation between the
ecotypes of the cyanobacterium from cold and hot deserts
(Bahl et al. 2011). It was suggested that this differentiation
was driven by adaptation to specific environmental conditions rather than by geographic isolation. We did not find any
such differentiation at the cpmA locus. This inconsistency
may be due to the much higher conservation of cpmA
even when compared with the rRNA genes.
The observed extreme conservation of the circadian gene,
cpmA, may result from two main factors. First, cpmA belongs
to the AIRC superfamily. Members of this superfamily are of
critical importance, as they are involved into de novo biosynthesis of purines, an essential component of DNA (Luckens
and Buchanan 1959). Taking account of this, cpmA may
5

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Y*
NA
1.0615, P = 0.1746
1.3463, P = 0.0910
NA
NA
NA
2.3260, P = 0.002

E
0.9044
0.7167
0.0628
0.4386
0.1525
0.5489
0.0143

DHEW
0.7331
0.5792
0.3723
0.3416
1
0.4369
0.0034

dN/dS
1.06
0.37
1.64
0.87
1.39
0.87
0.92

cpmA Locus in Chroococcidiopsis . doi:10.1093/molbev/mss191

MBE

Dvornyk and Jahan . doi:10.1093/molbev/mss191

Table 5. Polymorphism of Some Housekeeping and Symbiotic Genes of Bacteria in Comparison with cpmA of Chroococcidiopsis sp.
Species
Clostridium perfringens

Bradyrhizobium elkanii

Bradyrhizobium canariense

Rhizobium gallicum

Chroococcidiopsis sp.

Hd

0.263
0.238
0.238
0.928
0.902
0.863
0.939
0.923
0.903
0.855
0.939
0.948
0.969
0.938
0.956
0.957
0.951
0.774

dS
0.042
0.019
0.029

0.1003
0.0473
0.0455

0.0036

dN
0.004
0.004
0.001

0.0016
0.0012
0.0014

0.0033

h/hg
9.349/
4.415/
9.868/

/0.0246
/0.0103
/0.0108
/0.0248
/0.0256
/0.0442
/0.0876

7.814/0.0152

p
0.014
0.007
0.007
0.0350
0.0257
0.0386
0.0271
0.0127
0.0117
0.0230
0.0209
0.0458
0.1243
0.026
0.025
0.023
0.017
0.043
0.013
0.019
0.0034

References
Rooney et al. (2006)

Perrineau et al. (2011)

Vinuesa et al. (2005)

Silva et al. (2005)

Tanabe et al. (2007)

NOTE.atpD, beta subunit of ATPase; ftsZ, cell division protein FtsZ; glnA, glutamine synthetase; gltX, glutamyl-tRNA synthetase; gyrB, DNA gyrase subunit B; pgi,
glucose-6-phosphate isomerase; recA, recombination protein RecA; tpi, triosephosphate isomerase; gyrA, DNA gyrase subunit A; pfoS, regulatory protein PfoS; rplL, 50 S ribosomal
protein; dnaK, heat shock chaperone protein; glnII, glutamine synthetase II; nifH, dinitrogenase reductase; nodB, N-acetylglucosmine deacetylase; , theta (per gene) from the total
number of mutations; , theta (per site) from the total number of mutations; , not provided.

actually be considered as a housekeeping gene. Furthermore,

the circadian system as a whole is critically important for
maintenance of intracellular homeostasis and adaptation to
environment (Johnson 2005). According to the estimates by
different methods, a proportion of genes that are expressed
rhythmically in the cyanobacterial genome varies from 2% in
Synechocystis sp. PCC 6803 (Kucho et al. 2005) to up to 30%
in Synechococcus elongatus PCC 7942 (Liu et al. 1995). The
importance of the circadian system assumes the importance
of all its elements. Indeed, mutations in cpmA significantly
reduce a growth rate of the cyanobacterium (Katayama
et al. 1999)
The high haplotype diversity of the cpmA gene is not quite
surprising. It has been frequently reported for many genes in
bacteria (table 5 and references therein). However, high haplotype diversity usually results in significant interpopulation
differentiation of bacteria (Silva et al. 2005; Vinuesa et al.
2005), which is not a case for Chroococcidiopsis sp. Even the
Josts D test (Jost 2008), which is particularly sensitive to private alleles, failed to detect significant differentiation between the populations of Chroococcidiopsis sp. (table 3).
One of the possible reasons for the high gene diversity of
bacteria and, particularly, Chroococcidiopsis sp. may be the
existence of multiple ecotypes in the same habitat, which
are adapted to ecological microniches (Tanabe et al. 2007).
This assumption is plausible, given the extreme environmental conditions and quite pronounced fluctuations in environmental factors (temperature, light, humidity, etc.) at the
sampling sites.
6

Non-Neutral Evolution of the cpmA Locus in

Chroococcidiopsis sp.
CpmA is one of the genes, which control the circadian output
in cyanobacteria (Katayama et al. 1999). There is limited information about its physiological function. It was reported
that inactivation of cpmA results in a significantly earlier
phase and lower amplitude of rhythmic expression of a
core circadian gene, kaiA, and severe decrease in growth of
a cyanobacterium (Katayama et al. 1999). Based on these
data, it was suggested that cpmA might contribute to basic
cell metabolism (Katayama et al. 1999). Although there is no
direct evidence of whether cpmA itself has adaptive significance or does not, but signals for positive selection in the
whole gene and, specifically, the character of some replacement substitutions in its hydrophobic motifs suggest that this
possibility should not be excluded. Moreover, this seems likely
considering the adaptive significance of the circadian system
as a whole (Johnson et al. 1998; Ouyang et al. 1998).
Although data about primarily non-neutral evolution and
positive selection in various genes of bacteria are abundant,
they are quite limited with respect to the circadian genes. As
to prokaryotes, positive selection at the population level was
documented for two core circadian genes, kaiB and kaiC, in a
filamentous cyanobacterium, Nostoc linckia, from two model
ecological microsites known as Evolution Canyons I and II
(Dvornyk et al. 2002). Results of other studies (Dvornyk
et al. 2004; Dvornyk 2005) suggested positive selection at
the above-species level for two other circadian genes, ldpA

Downloaded from http://mbe.oxfordjournals.org/ at Indian Institute Of Chemical Biology (Iicb) on November 5, 2012

Microcystis aeruginosa

Genes
pfoS
rplL
gyrA
recA
dnaK
glnII
recA
atpD
glnII
glnII
atpD
nifH
nodB
ftsZ
glnA
gltX
gyrB
pgi
recA
tpi
cpmA

MBE

cpmA Locus in Chroococcidiopsis . doi:10.1093/molbev/mss191

Small Effective Population Size and Mutation-Drift

Equilibrium
The obtained results are in favor of a relatively small effective
population size of Chroococcidiopsis sp. We found high linkage disequilibrium and very low recombination rate in our
data set. Given that linkage disequilibrium at neutral sites of a
haploid organism depends on effective population size Ne and
a recombination rate c and is determined by 1/(2Nec). Since c
is very small, Ne should also be small to explain the high
disequilibrium. Using the obtained values for ZnS (0.0247)
and c (0.0004), the Ne was estimated to be only 50,000
individuals. Populations with small Ne should experience significant genetic drift, which reduces their diversity. Indeed, the
observed nucleotide diversity of the studied populations was
very low. However, despite the very large distances between
the sampled populations, they show almost no genetic differentiation. This suggests virtually unlimited gene flow
between them.
We also examined population history of the data, that is,
whether there is any departure from demographic equilibrium. One of the signs for a recent expansion is an excess of

singleton mutations, which can be measured by Tajimas D or

Fus Fs. Both these statistics yielded statistically significant
negative values (table 4). Two other tests, R2 (RamosOnsins and Rozas 2002) and g (Kuhner 2006), also supported
recent expansion of the Chroococcidiopsis sp. populations. In
addition, the obtained results suggest that Chroococcidiopsis
sp. probably consists of many relatively small populations
occupying various environmental microniches within a particular habitat.
Such population structure of Chroococcidiopsis sp. is not
something unusual. Many bacteria, particularly endosymbiotic and parasitic ones, may have extremely large population
size at the global scale and small effective size at the population level due to bottlenecks experienced by bacterial populations during transmission between hosts (Sharp et al. 2005).
Given the harsh environmental conditions of their habitats,
micropopulations of Chroococcidiopsis sp. may experience a
bottleneck during their recovery after various climatic
extremes, such as low temperature or high UV.

Concluding Remarks
This study provides compelling evidence for extremely high
conservation and non-neutral evolution of a circadian locus,
cpmA, in a stress-tolerant cyanobacterium Chroococcidiopsis
sp. from extreme environments around the globe. The signs
for positive selection detected in some of the studied populations and the whole species suggest that cpmA evolution is
likely of adaptive nature.

Supplementary Material
Supplementary figure S1 and table S1 are available at
Molecular Biology and Evolution online http://www.mbe
.oxfordjournals.org.

Acknowledgments
The authors express their deepest gratitude to Dr Stephen
Pointing (University of Hong Kong) for providing the
Chroococcidiopsis sp. strains and his expert opinion on the
article, and Dr Claus Vogl (University of Veterinary Medicine
Vienna) for his comments on the early draft of the manuscript. This work was supported by grant 10208127 from the
University of Hong Kong.

References
Achaz G. 2008. Testing for neutrality in samples with sequencing errors.
Genetics 179:14091424.
Baca I, Sprockett D, Dvornyk V. 2010. Circadian input kinases and their
homologs in cyanobacteria: Evolutionary constraints vs architectural
diversification. J Mol Evol. 70:453465.
Bahl J, Lau MC, Smith GJ, et al. (12 co-authors). 2011. Ancient origins
determine global biogeography of hot and cold desert cyanobacteria. Nat Commun. 2:163.
Caruso T, Chan Y, Lacap DC, Lau MC, McKay CP, Pointing SB. 2011.
Stochastic and deterministic processes interact in the assembly of
desert microbial communities on a global scale. ISME J. 5:14061413.
Chao A, Shen T-J. 2010. Program SPADE (Species Prediction And
Diversity Estimation). Program and Users Guide. Distributed by
the author. Available from: http://chao.stat.nthu.edu.tw.

Downloaded from http://mbe.oxfordjournals.org/ at Indian Institute Of Chemical Biology (Iicb) on November 5, 2012

and sasA, which are elements of the circadian input and

output, respectively. On the other hand, the previous macroevolutionary study of cpmA in prokaryotes did not determine
any positive selection on the gene (Dvornyk 2006). The discrepancies between the results of the cpmA studies at microand macrolevels may be due to fairly low intensity and an
episodic character of selection pressure on the gene. As such,
the existing methods may not be powerful enough to detect
positive selection at the above-species level, unless this selection is sufficiently strong and operates during reasonably long
evolutionary period. On the other hand, selection may be
population specific, as was suggested for the human period
2 gene (Cruciani et al. 2008).
Data about selection on circadian genes in eukaryotes are
more abundant. In this respect, gene period of Drosophila has
received much attention due to the report about a latitudinal
cline in its threonineglycine repeat polymorphism in
European Drosophila melanogaster, which was implicated in
selection for clock temperature compensation (Costa et al.
1992). A majority of the studies, which subsequently examined this and other polymorphisms of period in different
populations and species of Drosophila, reported various
types of selection, which might shape the observed polymorphism (Kliman and Hey 1993; Rosato et al. 1994;
Sawyer et al. 1997, 2006). Natural selection was also reported
to operate on another Drosophila circadian gene, timeless
(Tauber et al. 2007). Recently, six clock-associated genes of
a plant, Populus tremula, were found to experience positive
selection (Hall et al. 2011).
Altogether, the above data suggest that selection is among
the most important factors operating on circadian genes of
prokaryotes and eukaryotes. Evidence for positive selection
further supports the previous reports about the adaptive significance of the circadian clock system (Johnson et al. 1998;
Ouyang et al. 1998).

Dvornyk and Jahan . doi:10.1093/molbev/mss191

Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment

editor and analysis program for Windows 95/98/NT. Nucl Acids
Symp Ser. 41:9598.
Hudson RR. 1987. Estimating the recombination parameter of a finite
population model without selection. Genet Res. 50:245250.
Johnson CH. 2005. Testing the adaptive value of circadian systems.
Methods Enzymol. 393:818837.
Johnson CH, Golden SS. 1999. Circadian programs in cyanobacteria:
adaptiveness and mechanism. Annu Rev Microbiol. 53:389409.
Johnson CH, Golden SS, Kondo T. 1998. Adaptive significance of circadian programs in cyanobacteria. Trends Microbiol. 6:407410.
Jordan IK, Rogozin IB, Wolf YI, Koonin EV. 2002. Essential genes are more
evolutionarily conserved than are nonessential genes in bacteria.
Genome Res. 12:962968.
Jost L. 2008. GST and its relatives do not measure differentiation. Mol
Ecol. 17:40154026.
Katayama M, Tsinoremas NF, Kondo T, Golden SS. 1999. cpmA, a gene
involved in an output pathway of the cyanobacterial circadian
system. J Bacteriol. 181:35163524.
Kelly JK. 1997. A test of neutrality based on interlocus associations.
Genetics 146:11971206.
Kimura M. 1980. A simple method for estimating evolutionary rates of
base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 16:111120.
Kliman RM, Hey J. 1993. DNA sequence variation at the period locus
within and among species of the Drosophila melanogaster complex.
Genetics 133:375387.
Kondo T, Strayer CA, Kulkarni RD, Taylor W, Ishiura M, Golden SS,
Johnson CH. 1993. Circadian rhythms in prokaryotes: luciferase as
a reporter of circadian gene expression in cyanobacteria. Proc Natl
Acad Sci U S A. 90:56725676.
Kucho K, Okamoto K, Tsuchiya Y, Nomura S, Nango M, Kanehisa M,
Ishiura M. 2005. Global analysis of circadian expression in the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol. 187:
21902199.
Kuhner MK. 2006. LAMARC 2.0: maximum likelihood and Bayesian
estimation of population parameters. Bioinformatics 22:768770.
Le SQ, Gascuel O. 2008. An improved general amino acid replacement
matrix. Mol Biol Evol. 25:13071320.
Librado P, Rozas J. 2009. DnaSP v5: a software for comprehensive analysis
of DNA polymorphism data. Bioinformatics 25:14511452.
Liu Y, Tsinoremas NF, Johnson CH, Lebedeva NV, Golden SS, Ishiura M,
Kondo T. 1995. Circadian orchestration of gene expression in cyanobacteria. Genes Dev. 9:14691478.
Luckens LN, Buchanan JM. 1959. Biosynthesis of the purines. XXIV. The
enzymatic synthesis of 5-amino-1-ribosyl-4-imidazolecarboxylic acid
5-phosphate from 5-amino-1-ribosylimidazole 5-phosphate and
carbon dioxide. J Biol Chem. 234:17991805.
Mackey SR, Golden SS, Ditty JL. 2011. The itty-bitty time machine genetics of the cyanobacterial circadian clock. Adv Genet. 74:1353.
Nei M, Gojobori T. 1986. Simple methods for estimating the numbers of
synonymous and nonsynonymous nucleotide substitutions. Mol
Biol Evol. 3:418426.
Ouyang Y, Andersson CR, Kondo T, Golden SS, Johnson CH. 1998.
Resonating circadian clocks enhance fitness in cyanobacteria. Proc
Natl Acad Sci U S A. 95:86608664.
Peel MC, Finlayson BL, McMahon TA. 2007. Updated world map of the
Koppen-Geiger climate classification. Hydrol Earth Syst Sci. 11:
16331644.

Downloaded from http://mbe.oxfordjournals.org/ at Indian Institute Of Chemical Biology (Iicb) on November 5, 2012

Cordoba-Jabonero C, Zorzano MP, Selsis F, Patel MR, Cockell CS. 2005.

Radiative habitable zones in martian polar environments. Icarus 175:
360371.
Costa R, Peixoto AA, Barbujani G, Kyriacou CP. 1992. A latitudinal cline
in a Drosophila clock gene. Proc R Soc Lond B. 250:4349.
Cruciani F, Trombetta B, Labuda D, Modiano D, Torroni A, Costa R,
Scozzari R. 2008. Genetic diversity patterns at the human clock gene
period 2 are suggestive of population-specific positive selection. Eur J
Hum Genet. 16:15261534.
Darriba D, Taboada GL, Doallo R, Posada D. 2011. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27:
11641165.
Dvornyk V. 2005. Molecular evolution of ldpA, a gene mediating circadian input signal in cyanobacteria. J Mol Evol. 60:105112.
Dvornyk V. 2006. Subfamilies of cpmA, a gene involved in circadian
output, have different evolutionary histories in cyanobacteria.
Microbiology 152:7584.
Dvornyk V. 2009. The circadian clock gear in cyanobacteria: assembled
by evolution. In: Ditty JL, Mackey S, Johnson CH, editors. Bacterial
circadian programs. Berlin-Heidelberg: Springer. p. 241258.
Dvornyk V, Deng HW, Nevo E. 2004. Structure and molecular
phylogeny of sasA genes in cyanobacteria: insights into
evolution of the prokaryotic circadian system. Mol Biol Evol. 21:
14681476.
Dvornyk V, Vinogradova ON, Nevo E. 2002. Long-term
microclimatic stress causes rapid adaptive radiation of kaiABC
clock gene family in a cyanobacterium, Nostoc linckia, from the
"Evolution Canyons" I and II, Israel. Proc Natl Acad Sci U S A. 99:
20822087.
Dvornyk V, Vinogradova ON, Nevo E. 2003. Origin and evolution of
circadian clock genes in prokaryotes. Proc Natl Acad Sci U S A. 100:
24952500.
Excoffier L, Laval G, Schneider S. 2005. Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol
Bioinform Online. 1:4750.
Excoffier L, Smouse PE, Quattro JM. 1992. Analysis of molecular variance
inferred from metric distances among DNA haplotypes: application
to human mitochondrial DNA restriction data. Genetics 131:
479491.
Fay JC, Wu CI. 2000. Hitchhiking under positive Darwinian selection.
Genetics 155:14051413.
Fewer D, Friedl T, Budel B. 2002. Chroococcidiopsis and heterocystdifferentiating cyanobacteria are each others closest living relatives.
Mol Phylogenet Evol. 23:8290.
Friedmann EI. 1980. Endolithic microbial life in hot and cold deserts. Orig
Life. 10:223235.
Friedmann EI, Ocampo-Friedmann R. 1995. A primitive cyanobacterium
as pioneer microorganism for terraforming Mars. Adv Space Res. 15:
243246.
Fu YX. 1997. Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics 147:
915925.
Fu Y-X, Li W-H. 1993. Statistical tests of neutrality of mutations. Genetics
133:693709.
Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm to
estimate large phylogenies by maximum likelihood. Syst Biol. 52:
696704.
Hall D, Ma XF, Ingvarsson PK. 2011. Adaptive evolution of the Populus
tremula photoperiod pathway. Mol Ecol. 20:14631474.

MBE

cpmA Locus in Chroococcidiopsis . doi:10.1093/molbev/mss191

Tajima F. 1989. Statistical method for testing the neutral mutation

hypothesis by DNA polymorphism. Genetics 123:585596.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011.
MEGA5: Molecular Evolutionary Genetics Analysis using maximum
likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 28:27312739.
Tanabe Y, Kasai F, Watanabe MM. 2007. Multilocus sequence typing
(MLST) reveals high genetic diversity and clonal population structure of the toxic cyanobacterium Microcystis aeruginosa.
Microbiology 153:36953703.
Tauber E, Zordan M, Sandrelli F, et al. (13 co-authors). 2007. Natural
selection favors a newly derived timeless allele in Drosophila melanogaster. Science 316:18951898.
Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix
choice. Nucleic Acids Res. 22:46734680.
Vinuesa P, Silva C, Werner D, Martinez-Romero E. 2005. Population
genetics and phylogenetic inference in bacterial molecular
systematics: the roles of migration and recombination in
Bradyrhizobium species cohesion and delineation. Mol Phylogenet
Evol. 34:2954.
Watterson GA. 1975. On the number of segregating sites
in genetical models without recombination. Theor Pop Biol. 7:
256276.
Whitton BA. 1987. Survival and dormancy of algae. In: Hennis Y, editor.
Survival and dormancy of microorganisms. New York: John Wiley.
p. 109167.
Woelfle MA, Ouyang Y, Phanvijhitsiri K, Johnson CH. 2004. The adaptive
value of circadian clocks: an experimental assessment in cyanobacteria. Curr Biol. 14:14811486.
Zeng K, Fu YX, Shi S, Wu CI. 2006. Statistical tests for detecting positive
selection by utilizing high-frequency variants. Genetics 174:
14311439.
Zeng K, Shi S, Wu CI. 2007. Compound tests for the detection of
hitchhiking under positive selection. Mol Biol Evol. 24:18981908.

Downloaded from http://mbe.oxfordjournals.org/ at Indian Institute Of Chemical Biology (Iicb) on November 5, 2012

Perrineau MM, Le Roux C, de Faria SM, de Carvalho Balieiro F, Galiana A,

Prin Y, Bena G. 2011. Genetic diversity of symbiotic Bradyrhizobium
elkanii populations recovered from inoculated and non-inoculated
Acacia mangium field trials in Brazil. Syst Appl Microbiol. 34:
376384.
Pointing SB, Chan Y, Lacap DC, Lau MC, Jurgens JA, Farrell RL. 2009.
Highly specialized microbial diversity in hyper-arid polar desert. Proc
Natl Acad Sci U S A. 106:1996419969.
Ramos-Onsins SE, Rozas J. 2002. Statistical properties of new neutrality
tests against population growth. Mol Biol Evol. 19:20922100.
Rooney AP, Swezey JL, Friedman R, Hecht DW, Maddox CW. 2006.
Analysis of core housekeeping and virulence genes reveals cryptic
lineages of Clostridium perfringens that are associated with distinct
disease presentations. Genetics 172:20812092.
Rosato E, Peixoto AA, Barbujani G, Costa R, Kyriacou CP. 1994.
Molecular polymorphism in the period gene of Drosophila simulans.
Genetics 138:693707.
Rozas J, Gullaud M, Blandin G, Aguade M. 2001. DNA variation at the
rp49 gene region of Drosophila simulans: evolutionary inferences
from an unusual haplotype structure. Genetics 158:11471155.
Sawyer LA, Hennessy JM, Peixoto AA, Rosato E, Parkinson H, Costa R,
Kyriacou CP. 1997. Natural variation in a Drosophila clock gene and
temperature compensation. Science 278:21172120.
Sawyer LA, Sandrelli F, Pasetto C, Peixoto AA, Rosato E, Costa R,
Kyriacou CP. 2006. The period gene Thr-Gly polymorphism in
Australian and African Drosophila melanogaster populations: implications for selection. Genetics 174:465480.
Sharp PM, Bailes E, Grocock RJ, Peden JF, Sockett RE. 2005. Variation in
the strength of selected codon usage bias among bacteria. Nucleic
Acids Res. 33:11411153.
Silva C, Vinuesa P, Eguiarte LE, Souza V, Martinez-Romero E. 2005.
Evolutionary genetics and biogeographic structure of Rhizobium
gallicum sensu lato, a widely distributed bacterial symbiont of diverse
legumes. Mol Ecol. 14:40334050.
Stewart CN Jr, Via LE. 1993. A rapid CTAB DNA isolation technique
useful for RAPD fingerprinting and other PCR applications.
Biotechniques 14:748750.

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