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

  • Yurui Wang
  • Chundi Wang
  • Yaohan Jiang
  • Laura A. Katz
  • Feng GaoEmail author
  • Ying YanEmail author
Research Paper


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 intraindividual 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.


ciliates SSU rDNA sequence variation phylogenetic analyses rDNA copy number ecological significance 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



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.

Supplementary material

11427_2018_9422_MOESM1_ESM.doc (426 kb)
Further analyses of variation of ribosome DNA copy number and polymorphism in ciliates provide insights relevant to studies of both molecular ecology and phylogeny


  1. Alverson, A.J., and Kolnick, L. (2005). Intragenomic nucleotide polymorphism among small subunit (18s) rDNA paralogs in the diatom genus Skeletonema (bacillariophyta). J Phycol 41, 1248–1257.CrossRefGoogle Scholar
  2. Anson, E.L., and Myers, E.W. (1997). ReAligner: a program for refining DNA sequence multi-alignments. J Comput Biol 4, 369–383.CrossRefGoogle Scholar
  3. Bachvaroff, T.R., Kim, S., Guillou, L., Delwiche, C.F., and Coats, D.W. (2012). Molecular diversity of the syndinean Genus Euduboscquella based on single-cell PCR analysis. Appl Environ Microbiol 78, 334–345.CrossRefGoogle Scholar
  4. Bass, D., and Cavalier-Smith, T. (2004). Phylum-specific environmental DNA analysis reveals remarkably high global biodiversity of Cercozoa (Protozoa). Int J Syst Evol Microbiol 54, 2393–2404.CrossRefGoogle Scholar
  5. Bobola, M.S., Eckert, R.T., and Klein, A.S. (1992). Restriction fragment variation in the nuclear ribosomal DNA repeat unit within and between Picearubens and Picea mariana. Can J For Res 22, 255–263.CrossRefGoogle Scholar
  6. Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., et al. (2010). QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7, 335–336.CrossRefGoogle Scholar
  7. Caron, D.A., Countway, P.D., Savai, P., Gast, R.J., Schnetzer, A., Moorthi, S.D., Dennett, M.R., Moran, D.M., and Jones, A.C. (2009). Defining DNA-based operational taxonomic units for microbial-eukaryote ecology. Appl Environ Microbiol 75, 5797–5808.CrossRefGoogle Scholar
  8. Chen, X., Gao, S., Liu, Y., Wang, Y., Wang, Y., and Song, W. (2016). Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macronuclei of the ciliated model organism, Tetrahymena thermophila. Sci China Life Sci 59, 909–919.CrossRefGoogle Scholar
  9. Cline, J., Braman, J.C., and Hogrefe, H.H. (1996). PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucl Acids Res 24, 3546–3551.CrossRefGoogle Scholar
  10. Dunthorn, M., Klier, J., Bunge, J., and Stoeck, T. (2012). Comparing the hyper-variable V4 and V9 regions of the small subunit rDNA for assessment of ciliate environmental diversity. J Eukaryot Microbiol 59, 185–187.CrossRefGoogle Scholar
  11. Engberg, J., and Pearlman, R.E. (1972). The amount of ribosomal RNA genes in Tetrahymena pyriformis in different physiological states. Eur J Biochem 26, 393–400.CrossRefGoogle Scholar
  12. Fu, R., and Gong, J. (2017). Single cell analysis linking ribosomal (r)DNA and rRNA copy numbers to cell size and growth rate provides insights into molecular protistan ecology. J Eukaryot Microbiol 64, 885–896.CrossRefGoogle Scholar
  13. Galluzzi, L., Penna, A., Bertozzini, E., Vila, M., Garces, E., and Magnani, M. (2004). Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a dinoflagellate). Appl Environ Microbiol 70, 1199–1206.CrossRefGoogle Scholar
  14. Ganley, A.R.D., and Kobayashi, T. (2007). Highly efficient concerted evolution in the ribosomal DNA repeats: total rDNA repeat variation revealed by whole-genome shotgun sequence data. Genome Res 17, 184–191.CrossRefGoogle Scholar
  15. Gao, F., Warren, A., Zhang, Q., Gong, J., Miao, M., Sun, P., Xu, D., Huang, J., Yi, Z., and Song, W. (2016). The all-data-based evolutionary hypothesis of ciliated protists with a revised classification of the phylum Ciliophora (Eukaryota, Alveolata). Sci Rep 6, 24874.CrossRefGoogle Scholar
  16. Gao, F., Huang, J., Zhao, Y., Li, L., Liu, W., Miao, M., Zhang, Q., Li, J., Yi, Z., El-Serehy, H.A., et al. (2017). Systematic studies on ciliates (Alveolata, Ciliophora) in China: progress and achievements based on molecular information. Eur J Protistol 61, 409–423.CrossRefGoogle Scholar
  17. Gao, S., Xiong, J., Zhang, C., Berquist, B.R., Yang, R., Zhao, M., Molascon, A.J., Kwiatkowski, S.Y., Yuan, D., Qin, Z., et al. (2013). Impaired replication elongation in Tetrahymena mutants deficient in histone H3 Lys 27 monomethylation. Genes Dev 27, 1662–1679.CrossRefGoogle Scholar
  18. Gibbons, J.G., Branco, A.T., Yu, S., and Lemos, B. (2014). Ribosomal DNA copy number is coupled with gene expression variation and mitochondrial abundance in humans. Nat Commun 5, 4850.CrossRefGoogle Scholar
  19. Godhe, A., Asplund, M.E., Härnström, K., Saravanan, V., Tyagi, A., and Karunasagar, I. (2008). Quantification of diatom and dinoflagellate biomasses in coastal marine seawater samples by real-time PCR. Appl Environ Microbiol 74, 7174–7182.CrossRefGoogle Scholar
  20. Gong, J., Dong, J., Liu, X., and Massana, R. (2013). Extremely high copy numbers and polymorphisms of the rDNA operon estimated from single cell analysis of oligotrich and peritrich ciliates. Protist 164, 369–379.CrossRefGoogle Scholar
  21. Govindaraju, D.R., and Cullis, C.A. (1992). Ribosomal DNA variation among populations of a Pinus rigida Mill. (pitch pine) ecosystem: I. Distribution of copy numbers. Heredity 69, 133–140.Google Scholar
  22. Grattepanche, J.D., McManus, G.B., and Katz, L.A. (2016a). Patchiness of ciliate communities sampled at varying spatial scales along the New England shelf. PLoS ONE 11, e0167659.CrossRefGoogle Scholar
  23. Grattepanche, J.D., Santoferrara, L.F., McManus, G.B., and Katz, L.A. (2016b). Unexpected biodiversity of ciliates in marine samples from below the photic zone. Mol Ecol 25, 3987–4000.CrossRefGoogle Scholar
  24. Gribble, K.E., and Anderson, D.M. (2005). High intraindividual, intraspecific, and interspecific variability in large-subunit ribosomal DNA in the heterotrophic dinoflagellates Protoperidinium, Diplopsalis, and Preperidinium (Dinophyceae). Phycologia 46, 315–324.CrossRefGoogle Scholar
  25. Hall, T.A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41, 95–98.Google Scholar
  26. Heyse, G., Jönsson, F., Chang, W.J., and Lipps, H.J. (2010). RNAdependent control of gene amplification. Proc Natl Acad Sci USA 107, 22134–22139.CrossRefGoogle Scholar
  27. Huang, J., and Katz, L.A. (2014). Nanochromosome copy number does not correlate with RNA levels though patterns are conserved between strains of the ciliate morphospecies Chilodonella uncinata. Protist 165, 445–451.CrossRefGoogle Scholar
  28. Huang, J., Luo, X., Bourland, W.A., Gao, F., and Gao, S. (2016). Multigene-based phylogeny of the ciliate families Amphisiellidae and Trachelostylidae (Protozoa: Ciliophora: Hypotrichia). Mol Phylogenets Evol 101, 101–110.CrossRefGoogle Scholar
  29. Huang, L., Lu, X., Zhu, C., Lin, X., and Yi, Z. (2017). Macronuclear Actin copy number variations in single cells of different Pseudokeronopsis (Alveolata, Ciliophora) populations. Eur J Protistol 59, 75–81.CrossRefGoogle Scholar
  30. Ide, S., Miyazaki, T., Maki, H., and Kobayashi, T. (2010). Abundance of ribosomal RNA gene copies maintains genome integrity. Science 327, 693–696.CrossRefGoogle Scholar
  31. Izhaki, I., Fridman, S., Gerchman, Y., and Halpern, M. (2013). Variability of bacterial community composition on leaves between and within plant species. Curr Microbiol 66, 227–235.CrossRefGoogle Scholar
  32. Jerome, C.A., Lynn, D.H., and Simon, E.M. (1996). Description of Tetrahymena empidokyrea n. sp., a new species in the Tetrahymena pyriformis sibling species complex (Ciliophora, Oligohymenophorea), and an assessment of its phylogenetic position using small-subunit rRNA sequences. Can J Zool 74, 1898–1906.CrossRefGoogle Scholar
  33. Jones, A.C., Hambright, K.D., and Caron, D.A. (2017). Ecological patterns among bacteria and microbial eukaryotes derived from network analyses in a low-salinity lake. Microb Ecol 75, 917–929.CrossRefGoogle Scholar
  34. Kapler, G.M. (1993). Developmentally regulated processing and replication of the Tetrahymena rDNA minichromosome. Curr Opin Genets Dev 3, 730–735.CrossRefGoogle Scholar
  35. Kathol, M., Norf, H., Arndt, H., and Weitere, M. (2009). Effects of temperature increase on the grazing of planktonic bacteria by biofilmdwelling consumers. Aquat Microb Ecol 55, 65–79.CrossRefGoogle Scholar
  36. Kim, S.Y., Yang, E.J., Gong, J., and Choi, J.K. (2010). Redescription of Favella ehrenbergii (Claparède and Lachmann, 1858) Jörgensen, 1924 (Ciliophora: Choreotrichia), with phylogenetic analyses based on small subunit rRNA gene sequences. J Eukaryotic Microbiol 57, 460–467.CrossRefGoogle Scholar
  37. Librado, P., and Rozas, J. (2009). DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452.CrossRefGoogle Scholar
  38. Liu, X., and Gong, J. (2012). Revealing the diversity and quantity of peritrich ciliates in environmental samples using specific primer-based PCR and quantitative PCR. Microb Environ 27, 497–503.CrossRefGoogle Scholar
  39. Luo, X., Gao, F., Yi, Z., Pan, Y., Al-Farraj, S.A., and Warren, A. (2017). Taxonomy and molecular phylogeny of two new brackish hypotrichous ciliates, with the establishment of a new genus (Ciliophora, Spirotrichea). Zool J Linn Soc 179, 475–491.Google Scholar
  40. Mahé, F., de Vargas, C., Bass, D., Czech, L., Stamatakis, A., Lara, E., Singer, D., Mayor, J., Bunge, J., Sernaker, S., et al. (2017). Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests. Nat Ecol Evol 1, 0091.CrossRefGoogle Scholar
  41. Medinger, R., Nolte, V., Pandey, R.V., Jost, S., Ottenwälder, B., Schlötterer, C., and Boenigk, J. (2010). Diversity in a hidden world: potential and limitation of next-generation sequencing for surveys of molecular diversity of eukaryotic microorganisms. Mol Ecol 19, 32–40.CrossRefGoogle Scholar
  42. Medlin, L., Elwood, H.J., Stickel, S., and Sogin, M.L. (1988). The characterization of enzymatically amplified eukaryotic 16S-like rRNAcoding regions. Gene 71, 491–499.CrossRefGoogle Scholar
  43. Nylander, J. (2004). MrModeltest, a program to evaluate the fit of several models of evolution to a given data and unrooted tree (version 2.2). Program distributed by the author (Evolutionary Biology Centre, Uppsala University, Sweden).Google Scholar
  44. Orias, E., and Flacks, M. (1975). Macronuclear genetics of Tetrahymena. I. Random distribution of macronuclear genecopies in T. pyriformis, syngen 1. Genetics 79, 187–206.Google Scholar
  45. Paredes, S., Branco, A.T., Hartl, D.L., Maggert, K.A., and Lemos, B. (2011). Ribosomal DNA deletions modulate genome-wide gene expression: “rDNA-sensitive” genes and natural variation. PLoS Genet 7, e1001376.CrossRefGoogle Scholar
  46. Petz, W., Valbonesi, A., Schiftner, U., Quesada, A., and Cynan Ellis-Evans, J. (2007). Ciliate biogeography in Antarctic and Arctic freshwater ecosystems: endemism or global distribution of species? FEMS MicroBiol Ecol 59, 396–408.CrossRefGoogle Scholar
  47. Pillet, L., Fontaine, D., and Pawlowski, J. (2012). Intra-genomic ribosomal RNA polymorphism and morphological variation in Elphidium macellum suggests inter-specific hybridization in Foraminifera. PLoS ONE 7, e32373–264.CrossRefGoogle Scholar
  48. Prescott, D.M. (1994). The DNA of ciliated protozoa. Microbiol Rev 58, 233–267.Google Scholar
  49. Prokopowich, C.D., Gregory, T.R., and Crease, T.J. (2003). The correlation between rDNA copy number and genome size in eukaryotes. Genome 46, 48–50.CrossRefGoogle Scholar
  50. Rehnstam-Holm, A.S., Godhe, A., and Anderson, D.M. (2002). Molecular studies of Dinophysis (Dinophyceae) species from Sweden and North America. Phycologia 41, 348–357.CrossRefGoogle Scholar
  51. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A., and Huelsenbeck, J.P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systatic Biol 61, 539–542.CrossRefGoogle Scholar
  52. Santoferrara, L.F., Grattepanche, J.D., Katz, L.A., and McManus, G.B. (2016). Patterns and processes in microbial biogeography: do molecules and morphologies give the same answers? ISME J 10, 1779–1790.CrossRefGoogle Scholar
  53. Santoferrara, L.F., McManus, G.B., and Alder, V.A. (2013). Utility of genetic markers and morphology for species discrimination within the order Tintinnida (Ciliophora, Spirotrichea). Protist 164, 24–36.CrossRefGoogle Scholar
  54. Sela, I., Ashkenazy, H., Katoh, K., and Pupko, T. (2015). GUIDANCE2: accurate detection of unreliable alignment regions accounting for the uncertainty of multiple parameters. Nucl Acids Res 43, W7–W14.CrossRefGoogle Scholar
  55. Simon, D., Moline, J., Helms, G., Friedl, T., and Bhattacharya, D. (2005). Divergent histories of rDNA group I introns in the lichen family Physciaceae. J Mol Evol 60, 434–446.CrossRefGoogle Scholar
  56. Simon, U.K., and Weiss, M. (2008). Intragenomic variation of fungal ribosomal genes is higher than previously thought. Mol Biol Evol 25, 2251–2254.CrossRefGoogle Scholar
  57. Stackebrandt, E., and Ebers, J. (2006). Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 6, 152–155.Google Scholar
  58. Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313.CrossRefGoogle Scholar
  59. Stock, A., Edgcomb, V., Orsi, W., Filker, S., Breiner, H.W., Yakimov, M. M., and Stoeck, T. (2013). Evidence for isolated evolution of deep-sea ciliate communities through geological separation and environmental selection. BMC Microbiol 13, 150.CrossRefGoogle Scholar
  60. Stoeck, T., Bass, D., Nebel, M., Christen, R., Jones, M.D.M., Breiner, H. W., and Richards, T.A. (2010). Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Mol Ecol 19, 21–31.CrossRefGoogle Scholar
  61. Stoeck, T., Przybos, E., and Dunthorn, M. (2014). The D1-D2 region of the large subunit ribosomal DNA as barcode for ciliates. Mol Ecol Resour 14, 458–468.CrossRefGoogle Scholar
  62. Strauss, S.H., and Tsai, C.H. (1988). Ribosomal gene number variability in Douglas-Fir. J Heredity 79, 453–458.CrossRefGoogle Scholar
  63. Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596–1599.CrossRefGoogle Scholar
  64. Taniguchi, A., Onishi, H., and Eguchi, M. (2011). Quantitative PCR assay for the detection of the parasitic ciliate Cryptocaryon irritans. Fish Sci 77, 607–613.CrossRefGoogle Scholar
  65. Terrado, R., Medrinal, E., Dasilva, C., Thaler, M., Vincent, W.F., and Lovejoy, C. (2011). Protist community composition during spring in an Arctic flaw lead polynya. Polar Biol 34, 1901–1914.CrossRefGoogle Scholar
  66. Torres-Machorro, A.L., Hernández, R., Cevallos, A.M., and López-Villaseñor, I. (2010). Ribosomal RNA genes in eukaryotic microorganisms: witnesses of phylogeny? FEMS Microbiol Rev 34, 59–86.CrossRefGoogle Scholar
  67. Tucker, S.J., McManus, G.B., Katz, L.A., and Grattepanche, J.D. (2017). Distribution of abundant and active planktonic ciliates in coastal and slope waters off New England. Front Microbiol 8, 2178.CrossRefGoogle Scholar
  68. Wancura, M.M., Yan, Y., Katz, L.A., and Maurer-Alcalá, X.X. (2018). Nuclear features of the heterotrich ciliate Blepharisma americanum: genomic amplification, life cycle, and nuclear inclusion. J Eukaryot Microbiol 65, 4–11.CrossRefGoogle Scholar
  69. Wang, C., Zhang, T., Wang, Y., Katz, L.A., Gao, F., and Song, W. (2017). Disentangling sources of variation in SSU rDNA sequences from single cell analyses of ciliates: impact of copy number variation and experimental error. Proc R Soc B 284, 20170425.CrossRefGoogle Scholar
  70. Wang, P., Wang, Y., Wang, C., Zhang, T., Al-Farraj, S.A., and Gao, F. (2017). Further consideration on the phylogeny of the Ciliophora: analyses using both mitochondrial and nuclear data with focus on the extremely confused class Phyllopharyngea. Mol Phylogenets Evol 112, 96–106.CrossRefGoogle Scholar
  71. Wang, Y., Wang, Y., Sheng, Y., Huang, J., Chen, X., Al-Rasheid, K.A.S., and Gao, S. (2017a). A comparative study of genome organization and epigenetic mechanisms in model ciliates, with an emphasis on Tetrahymena, Paramecium and Oxytricha. Eur J Protistol 61, 376–387.CrossRefGoogle Scholar
  72. Wang, Y., Chen, X., Sheng, Y., Liu, Y., and Gao, S. (2017b). N6-adenine DNA methylation is associated with the linker DNA of H2A.Zcontaining well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena. Nucl Acids Res 45, 11594–11606.CrossRefGoogle Scholar
  73. Wang, Y., Sheng, Y., Liu, Y., Pan, B., Huang, J., Warren, A., and Gao, S. (2017c). N 6-methyladenine DNA modification in the unicellular eukaryotic organism Tetrahymena thermophila. Eur J Protistol 58, 94–102.CrossRefGoogle Scholar
  74. Wilbert, N. (1975). Eine verbesserte technik der protargolimprägnation für ciliaten. Mikrokosmos 64, 171–179.Google Scholar
  75. Wylezich, C., Nies, G., Mylnikov, A.P., Tautz, D., and Arndt, H. (2010). An evaluation of the use of the LSU rRNA D1-D5 domain for DNA-based taxonomy of eukaryotic protists. Protist 161, 342–352.CrossRefGoogle Scholar
  76. Xu, H., Zhang, W., and Jiang, Y. (2014). Do early colonization patterns of periphytic ciliate fauna reveal environmental quality status in coastal waters? Environ Sci Pollut Res 21, 7097–7112.CrossRefGoogle Scholar
  77. Xu, K., Doak, T.G., Lipps, H.J., Wang, J., Swart, E.C., and Chang, W.J. (2012). Copy number variations of 11 macronuclear chromosomes and their gene expression in Oxytricha trifallax. Gene 505, 75–80.CrossRefGoogle Scholar
  78. Yan, Y., Rogers, A.J., Gao, F., and Katz, L.A. (2017). Unusual features of non-dividing somatic macronuclei in the ciliate class Karyorelictea. Eur J Protistol 61, 399–408.CrossRefGoogle Scholar
  79. Yao, M.C., Kimmel, A.R., and Gorovsky, M.A. (1974). A small number of cistrons for ribosomal RNA in the germinal nucleus of a eukaryote, Tetrahymena pyriformis. Proc Natl Acad Sci USA 71, 3082–3086.CrossRefGoogle Scholar
  80. Zhao, F., Filker, S., Stoeck, T., and Xu, K. (2017). Ciliate diversity and distribution patterns in the sediments of a seamount and adjacent abyssal plains in the tropical Western Pacific Ocean. BMC Microbiol 17, 192.CrossRefGoogle Scholar
  81. Zhao, X., Wang, Y., Wang, Y., Liu, Y., and Gao, S. (2017). Histone methyltransferase TXR1 is required for both H3 and H3.3 lysine 27 methylation in the well-known ciliated protist Tetrahymena thermophila. Sci China Life Sci 60, 264–270.CrossRefGoogle Scholar
  82. Zhao, Y., Yi, Z., Gentekaki, E., Zhan, A., Al-Farraj, S.A., and Song, W. (2016). Utility of combining morphological characters, nuclear and mitochondrial genes: an attempt to resolve the conflicts of species identification for ciliated protists. Mol Phylogenets Evol 94, 718–729.CrossRefGoogle Scholar
  83. Zhao, Y., Yi, Z., Warren, A., and Song, W.B. (2018). Species delimitation for the molecular taxonomy and ecology of the widely distributed microbial eukaryote genus Euplotes (Alveolata, Ciliophora). Proc R Soc B 285, 20172159.CrossRefGoogle Scholar
  84. Zhu, F., Massana, R., Not, F., Marie, D., and Vaulot, D. (2005). Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene. FEMS Microbiol Ecol 52, 79–92.CrossRefGoogle Scholar

Copyright information

© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Evolution & Marine BiodiversityOcean University of ChinaQingdaoChina
  2. 2.Key Laboratory of MaricultureOcean University of China, Ministry of EducationQingdaoChina
  3. 3.Department of Biological SciencesSmith CollegeNorthamptonUSA

Personalised recommendations