Protist, Vol. 164, 369–379, May 2013
http://www.elsevier.de/protis
Published online date 24 January 2013
ORIGINAL PAPER
Extremely High Copy Numbers and
Polymorphisms of the rDNA Operon
Estimated from Single Cell Analysis
of Oligotrich and Peritrich Ciliates
Jun Gonga,b,1 , Jun Donga,c , Xihan Liua,c , and Ramon Massanad
aLaboratory
of Environmental Microbiology, Yantai Institute of Coastal Zone Research, Chinese Academy of
Sciences, Yantai 264003, China
bCollege of Life Sciences, South China Normal University, Guangzhou 510631, China
cInstitute of Marine Diversity and Evolution, Ocean University of China, Qingdao 266003, China
dDepartment of Marine Biology and Oceanography, Institut de Ciències del Mar, CSIC, Passeig Marítim de la
Barceloneta 37-49, 08003 Barcelona, Catalonia, Spain
Submitted June 18, 2012; Accepted November 30, 2012
Monitoring Editor: David Moreira
The copy number and sequence variation of the ribosomal DNA (rDNA) operon are of functional significance in evolution and ecology of organisms. However, the relationship between copy number and
sequence variation of rDNA in protists has been rarely studied. Here we quantified rDNA copy numbers of oligotrich and peritrich ciliate species using single-cell quantitative PCR. We also examined
the rDNA sequence variation by using single-cell PCR, cloning, and sequencing of multiple clones. We
found that the rDNA copy numbers per cell were extremely high and different among even congeners,
with the highest record of about 310,000. There was substantial intraindividual haplotype diversity and
nucleotide diversity for the rDNA markers, with sequence differences primarily characterized by single
nucleotide polymorphisms. Haplotype and nucleotide diversity was positively correlated to the rDNA
copy number. Our findings provide evidence that: (1) ciliates generally have much higher rDNA copy
numbers than other protists and fungi, which could lead to overestimation of the relative abundance
of ciliates in environmental samples when rDNA sequence-based methodologies are used; and that (2)
the rDNA might not always evolve in a strictly concerted manner in ciliates, which may raise problems
in rDNA-based inference of species richness and phylogeny.
© 2012 Elsevier GmbH. All rights reserved.
Key words: Ciliophora; copy number; intragenomic variability; rDNA; quantitative PCR; sequence polymorphism.
Introduction
The ribosomal DNA (rDNA) operons, which are
composed of the encoding regions for the small
1
Corresponding author; fax +86 535 2109 000
e-mail [email protected] (J. Gong).
© 2012 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.protis.2012.11.006
subunit (SSU) rRNA, the large subunit (LSU) rRNA
and the 5.8S rRNA, the noncoding regions of the
internal transcribed spacers (ITS1 and ITS2) and
the intergenic spacer (IGS), are typically organized in tandem with multiple repeats in eukaryotes.
These rDNA repeats within an eukaryotic genome
often evolve “in concert”, and consequently they
370 J. Gong et al.
are more similar to each other than they are to
“orthologous” repeats in a related species (Brown
et al. 1972; Ganley and Kobayashi 2007). The conserved and variable regions of rDNA have been
widely used for clarifying phylogenetic relationships
between species and populations as well as for
barcoding eukaryotic microbes (Woese et al. 1990;
Wylezich et al. 2010). In particular, the SSU rRNA
gene has become a universal phylogenetic marker
and represents the main criterion by which environmental microbes including protists and fungi
are identified and classified (Edgcomb et al. 2002;
Marie et al. 2006; Pace 1997). In addition, the copy
number and sequence variation of rDNA are of
functional significance with influences on the evolutionary ecology of organisms (Weider et al. 2005).
Knowing the copy numbers and the variations
of rDNA sequences within individuals of singlecelled eukaryotes is important for interpreting the
rDNA-based diversity surveys (Amaral-Zettler et al.
2011; Crosby and Criddle 2003; Farrelly et al.
1995; Herrera et al. 2009; Not et al. 2009; Thornhill
et al. 2007). This is especially true as rDNAbased barcoding and microbial diversity studies
using high-throughput sequencing are becoming
more and more popular (Amaral-Zettler et al. 2009;
Stoeck et al. 2009). A broad range of rDNA copy
numbers has been estimated in different protistan groups, such as diatoms (Galluzzi et al. 2004;
Godhe et al. 2008), dinoflagellates (Godhe et al.
2008), and a set of microalgal strains (Zhu et al.
2005), among which diatoms hold the highest estimate with around 37,000 copies per cell (Godhe
et al. 2008). It was also suggested that rDNA copy
numbers are correlated significantly with cell length
and biovolume in marine protists (Godhe et al.
2008; Zhu et al. 2005).
Repetitive gene families like rRNA genes are
generally considered undergoing strict concerted
evolution that tends to homogenize all repeats to
be identical (Dover 1982). However this notion
has been questioned by some recent findings. For
example, low levels of intragenomic rDNA polymorphisms were found in several genome-available
fungal species (Ganley and Kobayashi 2007), but
high variations were detected using a cloning
and sequencing approach in some other fungi
(Simon and Weiß 2008), dinoflagellates (Gribble
and Anderson 2007; Miranda et al. 2012), and
Foraminifera (Pillet et al. 2012).
Community profiling methods based on
sequence polymorphisms and relative abundances of SSU rDNA has been commonly used
to describe microbial community diversity (Marie
et al. 2006; Zhu et al. 2005). However, it has
been demonstrated that the presence of multiple
heterogeneous SSU rDNA copies potentially leads
to overestimation of the diversity of a bacterial
community, when DGGE or T-RFLP are applied
(Kang et al. 2010). The rDNA copy number per
genome is also required for SSU rDNA-based
quantification technique such as quantitative realtime PCR (qPCR) (Zhu et al. 2005). Medinger et al.
(2010) compared high-throughput sequencing and
traditional morphological analyses for characterizing environmental eukaryotic communities, and
concluded that the rDNA copy number variation
among taxa could be one of the main reasons for
the incongruent results of the two approaches, as
alveolate (e.g. ciliate and dinoflagellate) sequences
constitute the largest fraction of sequence reads.
Intragenomic variation in the ITS rDNA region
may obscure phylogenetic relationships and inflate
estimates of operational taxonomic units in fungi
(Lindner and Banik 2011).
Ciliophora, in particular those within the class
Spirotrichea, are well known for having multiple
nanochromosomes in their macronuclear genomes
(Prescott 1994). For instance, Tetrahymena thermophila (called T. pyriformis in Yao et al. 1974)
contains a single chromosomally integrated rDNA
copy in the micronucleus and ∼ 200 copies in the
macronucleus (Yao and Gall 1977; Yao et al. 1974);
later, this species has been re-estimated to have ∼
9,000 extrachromosomal rDNA copies, which are
produced by amplification during the formation of
the macronucleus (Kapler 1993). About 100,000
rDNA copies per macronucleus were estimated in
Oxytricha nova (Prescott 1994). Recent studies
using qPCR have also demonstrated that the copy
numbers of rDNAs in the macronucleus are as high
as ∼200,000 in the spirotrichean ciliate Stylonychia lemnae (Heyse et al. 2010), or ∼3,000 in the
parasite prostomatean ciliate Cryptocaryon irritans
(Taniguchi et al. 2011). Despite these records, data
on rDNA copy numbers in ciliates are still limited,
compared with their large known diversity. Intraspecific rDNA variation has been frequently detected
in ciliate species (e.g. Coleman 2005; Gong et al.
2007; Miao et al. 2004; Strüder-Kypke et al. 2001;
Wright 1999), but again the data available is scarce
when compared with the large ciliate diversity.
In this study, we estimated the rDNA copy
number in a range of species or isolates from
two ecologically important and species-rich ciliate
groups, the oligotrichs (class Spirotrichea) and the
peritrichs (class Oligohymenophorea). This study
was conducted by single cell molecular analysis (PCR-cloning and sequencing; qPCR), so it
was independent of culturing each ciliate species.
Ciliate rDNA Copy Number and Variation 371
Moreover, DNA-sequencing from individual cells
has the advantage of avoiding certain PCR and
cloning biases that distort results obtained from
community DNA (e.g. Heywood et al. 2011). We
found extremely high rDNA copy numbers per cell
and different numbers between even congeners.
The highest copy number (more than 310,000)
was estimated from a peritrich species. We then
hypothesized that this high rDNA copy number
was accompanied with a high level of sequence
polymorphism. To test this “high copy number-high
polymorphism” hypothesis, we obtained multiple
sequences from single cells. The results demonstrated that ciliates indeed had a substantial level of
rDNA polymorphisms. Evolutionary and ecological
implications of these findings are discussed.
Results
Linking Morphotypes to SSU rDNA
Sequences
Individual ciliate specimens were observed and
identified under the microscope and then subjected
to a PCR amplification using 18S rDNA primers.
The full or partial 18S rDNA gene was sequenced
per each isolate. As usual, a consensus SSU rDNA
sequence was obtained in order to link the morphotype to the genetic marker, so ciliates investigated
could be well classified (Table 1) and 18S rDNA
sequences could be compared among isolates of
the same or different species (Supplementary Table
S2). The SSU rDNA sequences of the two Epistylis
colonial isolates share a similarity of 99.9%, with
a single polymorphic site; the two Zoothamnium
isolates share a similarity of 97.4% and have 45
polymorphic sites; the Vorticella isolates from four
different species have similarities ranging from 90.2
to 98.8%, with 45 polymorphic sites; and the two
isolates of the same species (V. sp.3 isolates 3 and
4) are 99.8% similar, differing in a single position
(see Supplementary Table S2).
Intraindividual rDNA Copy Numbers of
Oligotrich and Peritrich Ciliates
The DNA from single ciliate cells or single colonies
was extracted and subjected to a qPCR analysis to
determine the rDNA copy number. The qPCR assay
was based on the SYBR Green dye, so it was important to reduce non-specific amplicons. According
to the melting curves, the melting temperatures
(Tm) for PCR products derived from the standards and samples were identical or quite similar,
indicating specific amplifications (Supplementary
Fig. S1). The linear relationship obtained between
the cycle threshold (CT ) and the rDNA copy
number with the oligotrich standard (Tintinnopsis)
was CT = -3.2709 × lg (rDNA copies/␮l) + 36.954,
with an amplification efficiency of 102.2% and
R2 of 0.9969 (Supplementary Fig. S2A). For peritrichs, the linear relationship obtained with the
Epistylis standard was CT = -3.4943 × lg (rDNA
copies/␮l) + 32.04, with an efficiency of 93.3%
and R2 of 0.9999, while the relationship with the
Vorticella standard was CT = -3.5187 × lg (rDNA
copies/␮l) + 32.035, with an efficiency of 92.4% and
R2 of 0.999 (Supplementary Fig. S2B and C).
Based on these standard curves and the CT
values obtained from each single-cell, the rDNA
copies per cell was estimated for each isolate
(Table 1). Generally speaking, the rDNA copies
per cell were extremely high in all 14 specimens
examined (on average 94,834), being higher in peritrichs (on average 109,142) than in oligotrichs (on
average 80,525). The highest value was found in
Vorticella sp.2, with 315,786 ± 7,100 copies per
cell, and the lowest was found in a Zoothamnium
species (3,385 ± 392).
The rDNA copy numbers in two isolates
of the same species Epistylis, whose SSU
sequences were almost identical (99.9%), were
not significantly different (64,865±15,089 vs.
88,161±20,699, see Table 1). The high standard
deviations could be related to the many (30 and
38) cells in one colony of Epistylis (Supplementary
Table S1). All the five Vorticella isolates (SSU
rDNA identity 90.2-99.8%) have rDNA copy numbers ranging from 61,226 to 315,786. The two
isolates of the same species of Vorticella sp.3
seemingly have significantly different copy numbers
(99,376 ± 5,482 vs. 61,226 ± 2,417). An extreme
case is that of the two Zoothamnium species
(SSU identity of 97.4%), which differ about 10fold in the rDNA copy number (40,675 ± 4,145 vs.
3,385 ± 392).
High Sequence Variations within the
rDNA Copies
For cloning and sequencing of SSU rDNA multiple
copies, we chose four single-celled isolates representing different species. Clone libraries from these
individuals contained many unique sequences, with
haplotype diversities ranging from 0.758 to 0.998
(Table 2). For the SSU rDNA genes, Pseudotontonia sp. had 45 unique sequences (n = 47) and
the highest haplotype diversity (0.998), whereas
Strombidium sp. had 16 unique sequences (n=30)
372 J. Gong et al.
Table 1. Estimated rDNA copy numbers in single ciliate cells and in other eukaryotic groups.
Organism
Oligotrichs
Favella sp.
Pseudotontonia sp.
Strombidinopsis sp.
Strombidium sp.
Tintinnopsis sp.
Peritrichs
Epistylis sp. iso.1
Epistylis sp. iso.2
Vorticella sp.1
Vorticella sp.2
Vorticella sp.3 iso.3
Vorticella sp.3 iso.4
Vorticella sp.5
Zoothamnium sp.1
Zoothamnium sp.2
Oligohymenophorea
Tetrahymena thermophila#
Tetrahymena thermophila
Spirotrichea
Oxytricha nova*
Stylonychia lemnae*
Prostomatea
Cryptocaryon irritans
Other groups
Microalgae
Diatoms
Dinoflagellates
Fungi
Animals
Plants
Accession
No. (SSU)
Copies per cell
Standard deviation
Reference
JX178773
JX178769
JX178771
JX178772
JX178770
46,498
172,889
30,247
34,647
126,372
555
9,832
6,576
3,465
1,368
This study
This study
This study
This study
This study
JX178765
JX178766
JX178760
JX178761
JX178762
JX178763
JX178764
JX178767
JX178768
64,865
88,161
161,355
315,786
99,376
61,226
82,194
40,675
3,385
15,089
20,699
11,498
7,100
5,482
2,417
4,927
4,145
392
This study
This study
This study
This study
This study
This study
This study
This study
This study
-
170-200
∼9,000
-
Yao et al. 1974
Kapler 1993
AF164124
200,000
400,000
-
Prescott 1994
Heyse et al. 2010
AB608054
∼3,000
-
Taniguchi et al. 2011
-
1-12,000
61-36,896
200-1,200
1,057-12,812
60-220
39-19,300
150-26,048
-
Zhu et al. 2005
Godhe et al. 2008
Galluzzi et al. 2004
Godhe et al. 2008
Simon et al. 2005
Prokopowich et al. 2003
Prokopowich et al. 2003
# Named Tetrahymena pyriformis in Yao et al. (1974).
* Species with two mature macronuclei in one cell.
- Data not available.
and the lowest haplotype diversity (0.759). Differences among clones were characterized primarily
by single nucleotide polymorphisms (SNP), which
were more or less evenly distributed along the rRNA
genes and the ITS region (Fig. 1). In the 50 clones
sequenced for SSU rDNA of Tintinnopsis sp., a
total of 45 unique sequences and 118 polymorphic
sites were detected, representing the highest level
of polymorphisms (6.7%) among all rDNA regions
or all species examined. The nucleotide diversity
(␲) of SSU rDNA varied between 0.087 × 10-2 (in
Strombidium) to 0.297 × 10-2 (in Pseudotontonia
sp.).
Since a cutoff (e.g. 0.01, 0.03, 0.05) of genetic
distance in the SSU rDNA marker is usually applied
for defining operational taxonomic units (OTUs)
based on environmental sequences (Caron et al.
2009), we calculated the pairwise distances to
assess whether the intraindividual polymorphisms
of rDNA might affect the definition of ciliate OTUs
(Table 2). The minimal similarity among two copies
from the same specimen was 99.1% whereas, on
average, the similarity among copies of the same
cell was 99.7 to 99.9%. Mean distances of intraindividual rDNA sequences of a given species were
less than 0.003, so even the stringent distance cutoff of 0.01 is higher than the average intragenomic
variability.
In single-celled isolate 4 of the species Vorticella
sp.3, we cloned and sequenced part of the SSU and
0.291
0.283
0.203
0.237
0.21
0.20
0.10
0.10
0.89
0.96
0.69
0.58
568
422
1450
2440
0. 29
0. 28
0. 20
0. 24
0
0
0
0
118 (6.7)
118 (6.6)
21(1.2)
0.15
0.12
0.07
0.92
0.68
0.28
SSU (full-length)
Tintinnopsis sp.
Pseudotontonia sp.
Strombidium sp.
Vorticella sp. 3 iso. 4
SSU (partial)
ITS
LSU (D1-D5)
SSU+ITS+LSU
1761
1776
1762
0.29
0.30
0.09
0
0
0
SD
Max
Mean
Min
23 (4.0)
13 (3.1)
49 (3.4)
86 (3.5)
0.811
0.758
0.936
0.998
34
34
34
34
50
47
30
0.288
0.297
0.087
0.991
0.998
0.759
n
␲ (×10-2 )
Hd
No. of
polymorphic
sites (p)
Pairwise genetic distance (× 10-2 )
Length (bp)
Species /marker
Table 2. Genetic distances and polymorphic sites in nuclear ribosomal DNA markers (SSU, ITS1-5.8S-ITS2, D1–D5 region of LSU) obtained
from single cells. ␲, nucleotide diversity; Hd, haplotype diversity; ITS, a combination of ITS1, 5.8S rDNA and ITS2; Max, Min, the maximum and
minimum value of pairwise genetic distances calculated using the K-2 parameter model; n, number of sequences; p, numbers of polymorphic
sites in relation to fragment length in %; SD, standard deviation.
Ciliate rDNA Copy Number and Variation 373
Figure 1. Distribution of single nucleotide polymorphisms in the SSU, ITS (comprising ITS1, the 5.8S
gene and ITS2), and LSU D1-D5 regions of rDNA.
Polymorphic sites were indicated by short vertical
lines. Length of sequences was drawn to scale.
LSU rDNA genes and the complete ITS region. The
D1-D5 region of LSU had a higher haplotype diversity (0.936) than that of ITS (0.758) or partial SSU
(0.811) (Table 2). Surprisingly, this cell contained
less polymorphic sites in the ITS (13 sites, 3.1% of
the region) than in the SSU or LSU regions examined. Nevertheless, the ITS region had a higher ␲
value (0.283 × 10-2 ) than that of the LSU region
(0.203 × 10-2 ) or the combination of SSU, ITS and
LSU regions (0.237 × 10-2 ).
Rarefaction analysis of haplotypes showed that
the rarefaction curves of all markers were far
from reaching a plateau (data not shown), indicating that there were more haplotypes in a given
individual cell of ciliates than we had sampled.
Overall, of all 357 detected polymorphic sites,
281 (78.71%) were caused by transitions, 60
(16.81%) by transversions, and 16 (4.48%) by
indels. Transitions between A and G (42.58%) were
more common than transitions between C and T
(36.13%) (Fig. 2).
Correlations Between rDNA Copy
Number and SSU Polymorphisms
Pearson’s correlation analysis showed that Hd
(r = 0.976, p < 0.05) and ␲ values (r = 0.809,
p > 0.05) of SSU rDNA sequences were positively
correlated with the rDNA copy numbers (Fig. 3).
The relationship between Hd and copy numbers
was significant, but the correlation between copy
numbers and ␲ values did not reach the 95% significant level, which was probably due to the low
number of cases analyzed (n = 4).
374 J. Gong et al.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Tin.-SSU Pse.-SSU Str.-SSU
Vor.SSU
Vor.-ITS Vor.-LSU
Indels
3
4
4
0
2
3
Transversions
22
21
5
1
4
7
Transions C/T
39
49
10
7
4
20
Transions A/G
56
48
7
15
5
21
Haplotype or nucleode diversity
Figure 2. Proportion (percentage of all detected polymorphisms) of indels, transversions, and transitions
for each rDNA marker and species. Transitions are
further split into substitutions between A and G and
between C and T. Tin, Tintinnopsis sp.; Pse, Pseudotontonia sp.; Str, Strombidium sp.; Vor, Vorticella sp.3
isolate 4.
1.2
1
y = 0.3814x - 0.9853
R² = 0.95204
0.8
Hd
π(× E-2)
0.6
y = 0.2641x - 1.0577
R² = 0.65476
0.4
0.2
0
4.4
4.6
4.8
5
5.2
5.4
Log (rDNA copy number per cell)
Figure 3. Correlation between haplotype diversity
(Hd; r = 0.933, p = 0.067) and nucleotide diversity (␲;
r = 0.912, p = 0.088) of small subunit ribosomal RNA
gene and rDNA copy number in single ciliate cells of
Tintinnopsis, Pseudotontonia, Strombidium and Vorticella sp. 3 isolate 4.
Discussion
Extremely High rDNA Copy Number in
Ciliates
The present paper reports for the first time the
intraindividual rDNA copy numbers and sequence
polymorphisms in two ecologically important and
species-rich ciliate groups, the oligotrichs and peritrichs, which together comprise about 30% of all
known ciliate species (Liu and Gong 2012; StrüderKypke and Lynn 2003). Using group-specific
primer-based qPCR assays, we determined the
rDNA copy number per cell for 14 isolates of 5
oligotrich and 7 peritrich species (Table 1). The
table also shows previously reported values for
other species. Since ciliates are unique protists
with the germline diploid micronucleus and the
somatic polyploid macronucleus (Prescott 1994),
the copy numbers determined in this study are in
theory the sum of rDNA copies in both genomes.
However, it is reasonable to assume that the
main determinant is the macronucleus, since the
few micronuclear copies can be virtually negligible when compared with the extremely large copy
number in the macronucleus.
We found extremely high rDNA copy numbers in
ciliates, not only in taxa within the class Spirotrichea
(e.g. Oxytricha, Stylonychia and Tintinnopsis), but
also in the subclass Peritrichia. Interestingly, the
peritrichs (e.g. Vorticella, Epistylis etc.), which hold
on average 101,891 copies (n=9) and the highest
record (315,786) in a Vorticella species, have far
higher copy numbers than the hymenostomatids
(∼200 and ∼9,000 in T. thermophila), though they
are phylogenetically related and classified in the
same class Oligohymenophora (Kapler 1993; Yao
et al. 1974). For species within the Spirotrichea, the
intraindividual rDNA copy numbers were estimated
to be on average 82,130 copies in oligotrichs (n = 5),
which is comparable with previously reported values for the stichotrich ciliates Oxytricha nova
(∼200,000) and Stylonychia lemnae (∼400,000)
(Heyse et al. 2010; Prescott 1994). One Zoothamnium species has about 3,385 rDNA copies, much
like that found in the prostomatean Cryptocaryon
irritans (∼3,000) (Taniguchi et al. 2011).
The new data we obtained, combined with existing records for ciliates, provide evidence for the
notion that ciliates generally host the highest rDNA
copy number in a single cell than any other protists and fungi, though copy numbers per genome
may vary greatly within a given group (Table 1).
In a set of microalgal strains, 1 to more than
12,000 rDNA copies have been estimated (Zhu
et al. 2005); in diatoms, numbers range from 61
to 36,896 (Godhe et al. 2008); in dinoflagellates
range from 200 to 1,200 (Galluzzi et al. 2004) or
1,057 to 12,812 (Godhe et al. 2008); and in fungi
range from 60-220 (Simon et al. 2005). Moreover,
copy numbers in ciliates are generally higher than
that in animals (39-19,300) and plants (150-26,048)
(Prokopowich et al. 2003), except for Xenopus
oocytes in which there are about 2 × 106 rDNA
copies per cell (Hourcade et al. 1973).
Ciliate rDNA Copy Number and Variation 375
The extremely high copy number of rDNA in the
macronuclear genome of ciliates, as previously
found and also corroborated in this study, is
understandable after considering the life history,
large cell size and rapid growth of these organisms
(Cavalier-Smith 2005; Gall 1981). For instance,
peritrich ciliates need to develop a stalk in a short
time for attaching to a substrate during their life
cycle, so having many copies of rDNA in the
macronuclear genome should be an advantage for
faster transcription and synthesis of proteins. This
is also supported by the clear positive correlation
found between rDNA copy number and cell size
in marine protists (Godhe et al. 2008; Zhu et al.
2005). On the other hand, although there are
numerous copies of rDNA in ciliate macronucleus,
it is likely that only a small portion of these genes
are transcriptionally active in ciliates, as previously
shown for other eukaryotes (Reeder 1999). While
epigenetic mechanisms on the regulation of
activation and inactivation of rRNA gene copies
has been proposed recently (McStay and Grummt
2008), ciliates with very high rDNA copy numbers may serve as an ideal model in addressing
rDNA-related epigenetic issues.
The high copy number of rDNA indicates that
a large percentage of the macronuclear genomic
DNA is devoted to rDNA in ciliates. If the genome
size was positively correlated to rDNA copy number
as shown in plants and animals (Prokopowich et al.
2003), one might expect extremely large genomes
in many ciliate species, which is, however, contrasting to the recent findings that ciliates usually
have relatively small macronuclear genomes sizes
(50-104 Mb) (Aury et al. 2006; Coyne et al. 2011;
Eisen et al. 2006). This may be explained by DNA
elimination which occurs during the development
of macronucleus (Ammermann et al. 1974). The
maintenance of very high copy numbers of rDNA
genes in the macronucleus is therefore intriguing,
and this surely deserves further investigations at
the biological level.
rDNA Copy Number Variation among and
within Species of Ciliates
The significant variations of rDNA copy numbers
among and within ciliate species reflect the
dynamic nature of macronuclear genome in ciliates, that is, extensive processing through genome
amplification and chromosome fragmentation during the development of the somatic macronucleus
(Parfrey et al. 2008; Prescott 1994). Our data also
suggest that these modifications are somewhat
independent of the taxonomic position of the
ciliates. For example, rDNA copy numbers
vary 5-fold among the Vorticella spp. 1-3 with
SSU sequence divergence of only 1.2-1.7%
(Supplementary Table S2). On the other hand,
the different rDNA copy numbers (99,376±5,482
vs. 61,226±2,417) observed from two isolates of
the same species Vorticella sp.3 (SSU identity
99.8%) might reflect different steps in the growth
phase of these individuals, as previously reported
in Tetrahymena (Engberg and Pearlman 1972).
This is consistent with the results published by
Rogers and Bendich (1987), who found that the
variability in rDNA copy numbers existed not only
between distantly related species, but also among
individuals of the same population of a given plant.
In fact, the rDNA copy number in the macronucleus
of Tetrahymena pyriformis seems to vary under
different nutritional conditions (Prescott 1994 and
references therein).
Positive Correlation between rDNA Copy
Number and Sequence Polymorphisms
We found that higher rDNA copy number per cell
tends to yield higher sequence polymorphisms in
ciliates (Fig. 3). This seems also true when fungi
and ciliates are compared: the ␲ values of ciliate rDNA (0.087 – 0.297 × 10-2 ) are much higher
than those previously reported for fungi (0.001 –
0.018 × 10-2 , Ganley and Kobayashi 2007; or 0.071
– 0.156 × 10-2 , Simon and Weiß 2008), which is
coincident with the large (at least about 150 –
800 fold) difference in intraindividual rDNA copy
numbers between these two groups. The rDNA
intraindividual polymorphisms in ciliates seems
comparable with that of some dinoflagellates or
Foraminifera (Gribble and Anderson 2007; Miranda
et al. 2012; Pillet et al. 2012).
Ecological Implications
The much higher rDNA copy number of ciliates
relative to other microbial eukaryotes partially
explains why ciliate SSU sequences were
frequently found to be very important in environmental samples (Massana and Pedrós-Alió 2008;
Medinger et al. 2010; Terrado et al. 2011). The
rDNA copy number variation between and within
ciliate species highlights the difficulty of using the
rDNA sequence number-based approach to infer
the relative abundance of microbial eukaryotic cells
in environmental samples (Medinger et al. 2010;
Terrado et al. 2011; von Wintzingerode et al. 1997).
Moreover, each individual genome contains a substantial level of sequence polymorphism, which
376 J. Gong et al.
also could overestimate diversity estimates from
environmental surveys if a distance cut-off level of
0 was considered. Environmental sequences with
a very low variation level could indeed derive from
the same genome. Fortunately, this intragenomic
variability would be certainly excluded when using
a 0.01 distance cut-off level.
In conclusion, we explored, for the first time,
the relationship between rDNA copy number and
sequence variation in eukaryotes by single-cell
analysis of ciliated protozoa. We found extremely
high rDNA copy numbers per cell in oligotrich
and peritrich ciliates, which is consistent with
previous studies on the spirotricheans. We also
observed substantial variation in rDNA copy number among even closely related morphospecies,
reflecting the dynamic nature of ciliate genomes
during life cycles and under resource conditions.
The newly obtained data support our hypothesis
that high copy number of rDNA is accompanied with
a substantial sequence polymorphism. Our findings
stress that data derived from rDNA sequencebased methodologies (e.g. qPCR, clone libraries,
and pyrosequencing) in microbial eukaryotic ecology have to be interpreted with caution, because
ciliates generally have many more rDNA copies
in single cells than other protists and fungi, which
easily leads to overestimation of their relative abundance, and thus distorts estimation of community
structure and diversity. Furthermore, we showed
that the 1% similarity cut-off for clustering SSU
rDNA sequences in OTUs was generally safe
to exclude the intragenomic sequence variations
when estimating ciliate OTU counts. Nevertheless,
intraspecific and interspecific rDNA sequence variations in ciliates deserve further investigations for
improving the rDNA-based taxonomy of ciliates. At
last, but not least, why ciliates contain so many
rDNA copies is an open question in evolutionary
biology, cell biology, and protistan ecology.
Methods
Organisms: Specimens were collected from the Guangdang
River and a coastal beach in Yantai, China (37◦ 28 N, 121◦ 28 E)
in August and September of 2011, respectively. Water samples were maintained for several days at room temperature
(25 ◦ C), and examined for oligotrich and peritrich ciliates under
a stereoscope with a magnification of 45X. Observation of
living morphology followed Song et al. (1999). In brief, recognized specimens were picked up with a micropipette, and
transferred to glass slides with a drop of water, and observed
under a microscope (Olympus BX61) for living characters and
behavior. Five oligotrich species from genera Pseudotontonia,
Tintinnopsis, Strombidinopsis, Strombidium and Favella, and
seven species of Vorticella, Epistylis and Zoothamnium were
morphologically identified according to taxonomic literatures
(Song et al. 1999).
DNA extraction, rDNA amplification and sequencing: Genomic DNA extraction was performed as previously
described (Gong et al. 2007). Briefly, a single cell or a colony
of peritrich cells was washed with filtered water for three times
using a micropipette and then transferred to a PCR microfuge
tube with a minimum volume of water. Genomic DNA was
extracted using REDExtract-N-Amp Tissue PCR Kit (Sigma, St.
Louis, MO) according to the manufacturer’s protocol, modified
such that only 1/10 of the suggested volume for each solution
was used. Isolation of individual cells (zooids) from colonial
Epistylis and Zoothamnium was rather difficult in practice,
because they easily contract as a single unit when disturbed.
Thus, we had to use the whole colony for DNA extraction, and
the copy number per cell was calculated on the basis of the
number of zooids (Supplementary Table S1). Since no wash or
filtration steps were involved in this procedure, it was assumed
that loss of the genomic DNA did not take place. The volume
of the final extraction solution was measured by an Eppendorf pipette (Supplementary Table S1). SSU rRNA genes were
amplified with primers EukA/EukB (Medlin et al. 1988) or EukAPeri1403R (Liu and Gong 2012). Primers Peri979F/LR7 were
used for amplifying a fragment comprising partial SSU, ITS and
LSU D1-D5 regions of Vorticella sp.3 isolate 4 (Bunyard et al.
1994; Liu and Gong 2012). The PCR amplification, cloning and
sequencing were performed as previously described (Liu and
Gong 2012).
Quantitative real-time PCR assays: Linear plasmids were
constructed for qPCR assays as previously described (Liu
and Gong 2012; Zhu et al. 2005). In order to avoid eukaryotic contaminations (e.g. microalgal or fungal symbionts), we
used ciliate clade specific-primers instead of eukaryotic universal ones. Briefly, peritrichia-specific Peri974F/Peri1403R (Liu
and Gong 2012) and oligotrich-specific (1199+/1765-) primers
(Doherty et al. 2007) were used for amplifying 441-bp and 506bp amplicons from Epistylis sp. (accession number JX178765)
and Tintinnopsis sp. (accession number JX178770), respectively. These fragments were gel-purified, ligated to pTZ57-T
vector (Fermentas, Germany), and cloned into E. coli. Longer
PCR products (598 bp for Epistylis and 662 bp for Tintinnopsis)
amplified from positive clones using M13F/M13R primers were
used as standards following gel-purification, and concentration measurement with a NanoDrop 2000C spectrophotometer
(Thermo, Wilmington, DE, USA).
A serial tenfold dilutions (10-1 to 10-8 ) were used to obtain
standard curves. Reactions were performed in a final volume of
20 ␮l using DyNAmo Flash SYBR Green qPCR kit (Finnzymes,
Espoo, Finland) containing 10 ␮l 2 × master mix, 0.4 ␮M of
each primer, 0.08 ␮l of bovine serum albumin (BSA, 100 ␮g/␮l),
2 ␮l of template DNA, and 6.32 ␮l of RNase-free water. The
primer sets Peri974F/Peri1403R and 1199+/1765- were used
for peritrichs and oligotrichs, respectively. All reactions were
performed in triplicate with an ABI 7500 Fast Real-Time PCR
System (Applied Biosystems, Foster City, CA, USA). The PCR
program started with an initial soaking step at 50 ◦ C for 2 min
and 95 ◦ C for 7 min, followed by 40 cycles of denaturation at
95 ◦ C for 15 s, annealing at 61 ◦ C for 30 s, and extension at
72 ◦ C for 30 s, and finally a melting curve stage (preprogrammed
in system as follows: 95 ◦ C for 15 s, 60 ◦ C for 1 min, 95 ◦ C for
30 s, and 60 ◦ C for 15 s). Data were retrieved at the extension
step.
The
number
of
molecules
in
the
standard
was
calculated
using
the
following
formula:
molecules/␮l = a/(n × 660) × 6.022 × 1023 , where a is the
linear DNA concentration (g/␮l), n is the length of linear
Ciliate rDNA Copy Number and Variation 377
fragments (662 bp for oligotrichs and 598 bp for peritrichs), 660
is the average molecular weight of one base pair for double
strand DNA, and 6.022 × 1023 is the molar constant (Zhu et al.
2005). The efficiency of the amplification (E) was calculated
as: E = (10-1/k -1) × 100%, where k is the slope of standard
curve.
Intraindividual DNA polymorphism and nucleotide diversity: For four ciliate cells (Tintinnopsis sp., Pseudotontonia sp.,
Strombidium sp. and Vorticella sp.3 isolate 4), multiple transformed clones were randomly selected for Sanger sequencing
in order to assess the polymorphisms of rDNA markers in a
single cell of each species (Table 2). Chromatograms were
inspected individually to confirm that polymorphisms were
indeed real. In order to clarify that the observed sequence
variations are due to rDNA polymorphisms rather than errors
occurring during PCR or sequencing, we randomly picked one
clone containing the SSU gene of Tintinnopsis sp. By taking that
clone as template, we amplified and cloned the PCR product
again under the conditions described above. Thirty re-cloned
clones were selected from sequencing and these “secondgeneration” clones of Tintinnopsis sp. were 100% identical to
the “first-generation” sequence, suggesting that the observed
rDNA sequence polymorphism was not due to PCR or sequencing errors.
Sequences were aligned using ClustalX (Thompson et al.
1997). Percentage of polymorphisms (p = numbers of polymorphic sites in relation to fragment length) was calculated for
complete SSU rDNA of Pseudotontonia sp., Tintinnopsis sp.,
and Strombidium sp., and for partial SSU, ITS1-5.8S-ITS2,
D1–D5 region of LSU of Vorticella sp.3 isolate 4. Nucleotide
diversity (␲) based on Nei and Li (1979) and haplotype diversity (Hd) were calculated using the program DnaSP version 5
(Librado and Rozas 2009).
Rarefaction analysis of haplotypes: Rarefaction analysis
was conducted to estimate whether the full diversity of SSU
rDNA sequences had been sampled for each species. Rarefaction allows the calculation of sequence diversity for a given
number of sampled clones and it was calculated with Analytic
Rarefaction 1.3 (http://strata.uga.edu/software/index.html).
Accession Numbers: Sequences have been deposited in
GenBank database under the accession numbers JX178760 to
JX178934.
Acknowledgements
This work was supported by the One Hundred
Talent Program of CAS, the Natural Science
Foundation of China (grant Nos. 40976099 and
41176143), the Natural Science Foundation for
Distinguished Young Scholars of Shandong (No.
JQ201210), and a grant from FANEDD (No.
2007B27) to JG.
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