SCIENCE CHINA Life Sciences, Volume 62 , Issue 2 : 203-214(2019) https://doi.org/10.1007/s11427-018-9422-5

Further analyses of variation of ribosome DNA copy number and polymorphism in ciliates provide insights relevant to studies of both molecular ecology and phylogeny

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  • ReceivedSep 8, 2018
  • AcceptedNov 6, 2018
  • PublishedJan 21, 2019


Sequence-based approaches, such as analyses of ribosome DNA (rDNA) clone libraries and high-throughput amplicon sequencing, have been used extensively to infer evolutionary relationships and elucidate the biodiversity in microbial communities. However, recent studies demonstrate both rDNA copy number variation and intra-individual (intra-genomic) sequence variation in many organisms, which challenges the application of the rDNA-based surveys. In ciliates, an ecologically important clade of microbial eukaryotes, rDNA copy number and sequence variation are rarely studied. In the present study, we estimate the intra-individual small subunit rDNA (SSU rDNA) copy number and sequence variation in a wide range of taxa covering nine classes and 18 orders of the phylum Ciliophora. Our studies reveal that: (i) intra-individual sequence variation of SSU rDNA is ubiquitous in all groups of ciliates detected and the polymorphic level varies among taxa; (ii) there is a most common version of SSU rDNA sequence in each cell that is highly predominant and may represent the germline micronuclear template; (iii) compared with the most common version, other variant sequences differ in only 1–3 nucleotides, likely generated during macronuclear (somatic) amplification; (iv) the intra-cell sequence variation is unlikely to impact phylogenetic analyses; (v) the rDNA copy number in ciliates is highly variable, ranging from 103 to 106, with the highest record in Stentor roeselii. Overall, these analyses indicate the need for careful consideration of SSU rDNA variation in analyses of the role of ciliates in ecosystems.

Funded by

the National Natural Science Foundation of China(31772428)

the National Science Foundation of the USA(1541511)

Young Elite Scientists Sponsorship Program by CAST

and Fundamental Research Funds for the Central Universities(201841013,201762017)


This work was supported by the National Natural Science Foundation of China (31772428), the National Science Foundation of the USA (1541511), Young Elite Scientists Sponsorship Program by CAST, and Fundamental Research Funds for the Central Universities (201841013 and 201762017). We thank Prof. Weibo Song, Ocean University of China (OUC), for the helpful suggestions in drafting this manuscript. Many thanks are also due to Wen Song, Chunyu Lian, Mingjian Liu, Borong Lu, Song Li, Rui Wang, Lun Wang and Yang Bai, graduate students in OUC, for their help in species identification.

Interest statement

The author(s) declare that they have no conflict of interest.



Figure S1 The liner relationships between the cycle threshold (Ct) and the logarithms of rDNA copy number of standards in all 11 times of qPCR reactions in this study. Correspondence between reactions and species: A, Deviata sp., Paramecium caudatum, Caenomorpha medusula and Stentor roeselii; B, Strombidium stylifer, Diophrys scutum and Uronychia binucleate; C, Vorticella sp.-1,2, Apodileptus visscheri and Favella ehrenbergii; D, Oxytricha trifallax and Chilodontopsis depressa; E, Coleps sp.; F, Loxodes sp.; G, Vorticella sp.-3, Plagiopyla sp. and Euplotes vannus; H, Pseudocohnilembus persalinus and Tetrahymena thermophila-1; I, Tetrahymena thermophila-2; J, Trithigmostoma sp.; K, Tetrahymena thermophila-3 and Litonotus sp.

Table S1 Portion of common sequence and rDNA copy numbers of the three individuals of the 20 species

Table S2 Genetic distances and polymorphic sites of SSU rDNA in the 20 species

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    (Color online) Relationships among the 20 species analyzed in the present study. Topology based on Gao et al. (2016).

  • Figure 2

    (Color online) The proportion of clones with single nucleotide sites and the distribution of single nucleotide polymorphisms (SNPs) in small subunit rDNA in the 20 species. A, The 20 species cover nine classes in Ciliphora, which are shown in A–I in figure respectively. B, Polymorphic sites are indicated by red solid circles. The dark blue, yellow and light blue segments indicate the variable regions 2, 4 and 9 respectively. Lengths of sequences are drawn to scale.

  • Figure 3

    (Color online) The position of polymorphic sites compared with common sequence (com). For the numbers x-y behind the species name, x means the individual number while y means the clone number of this individual. A, Diophrys scutum; B, Deviata sp.; C, Caenomorpha medusula; D, Oxytricha trifallx; E, Euplotes vannus; F, Trithigmostoma sp.; G, Chilodontopsis depressa; H, Coleps sp.; I, Apodileptus visscheri; J, Favella ehrenbergii; K, Strombidium stylifer; L, Tetrahymena thermophila; M, Stentor roeselii; N, Paramecium caudatum; O, Uronychia binucleata; P, Pseudocohnilembus persalinus; Q, Loxodes sp.; R, Plagiopyla sp.; S, Litonotus sp.

  • Figure 4

    (Color online) The ML tree based on the small subunit ribosomal DNA (SSU rDNA) sequences showing phylogenetic positions of all haplotypes of the newly sequenced 20 species (in red and bold). Numbers near the branches denote bootstrap values for ML and posterior probability value for BI. Asterisk indicates the disagreement in topology between ML and BI trees. Fully supported (100%/1.00) nodes are represented by solid circles. Red triangles represent all haplotypes of the species can form a monophyly while red rectangle shows that all haplotypes of this species group with other species. All branches are drawn to scale. Scale bar corresponds to 5 substitutions per 100 nucleotide positions.

  • Figure 5

    (Color online) Estimated rDNA copy numbers in different ciliates according to present and previous studies (Gong et al., 2013; Huang and Katz, 2014; Wang C et al., 2017).

  • Table 1   SSU rDNA polymorphisms and rDNA copy numbers of the 20 species


    SNP sites of each individual


    of each individual

    Common sequence of each


    Copy numbers of each


    Oxytricha trifallax





    Deviata sp.





    Favella ehrenbergii





    Strombidium stylifer





    Uronychia binucleata





    Diophrys scutum





    Euplotes vannus





    Litonotus sp.





    Apodileptus visscheri





    Chilodontopsis depressa





    Trithigmostoma sp.





    Pseudocohnilembus persalinus





    Vorticella sp.





    Paramecium caudatum





    Tetrahymena thermophila





    Coleps sp.





    Plagiopyla sp.





    Caenomorpha medusula





    Stentor roeselii





    Loxodes sp.





  • Table 2   The number of defined OTUs for V2, V4 and V9 regions of the 20 species when 97% to 99% cutoffs are used

















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