Complementary DNA sequencing (cDNA): an effective approach for assessing the diversity and distribution of marine benthic ciliates along hydrographic gradients -- Journal of Oceanology and Limnology,2021,39(1):208-222

	Cite this paper:
	Pingping HUANG, Feng ZHAO, Kuidong XU. Complementary DNA sequencing (cDNA): an effective approach for assessing the diversity and distribution of marine benthic ciliates along hydrographic gradients[J]. Journal of Oceanology and Limnology, 2021, 39(1): 208-222

Complementary DNA sequencing (cDNA): an effective approach for assessing the diversity and distribution of marine benthic ciliates along hydrographic gradients

Pingping HUANG^1,3, Feng ZHAO^1,2,4, Kuidong XU^1,2,3,4

1 Laboratory of Marine Organism Taxonomy and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
2 Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China;
3 University of Chinese Academy of Sciences, Beijing 100049, China;
4 Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China

Abstract:

The Yellow Sea Cold Water Mass (YSCWM) is a distinct hydrographic phenomenon of the Yellow Sea, and the distribution pattern of meio- and macrobenthos differs inside and outside of the YSCWM. However, such a pattern has never been observed in the microbenthic ciliate communities. Therefore, we hypothesized that benthic ciliates followed a similar distribution pattern as meio- and macrobenthos, but this pattern has not been uncovered by morphological methods. We evaluated the diversity and distribution of benthic ciliates at five stations along hydrographic gradients across the YSCWM and adjacent shallow water by using morphology and DNA and complementary DNA (cDNA) high-throughput sequencing of the V4 region of 18S rRNA gene. Results showed that the diversity of benthic ciliates detected by DNA (303 OTUs), and the cDNA (611 OTUs) sequencing was much higher than that detected by the morphological method (79 species). Morphological method detected roughly different ciliate communities inside and outside of the YSCWM, but without statistical significance. No clear pattern was obtained by DNA sequencing. In contrast, cDNA sequencing revealed a distinct distribution pattern of benthic ciliate communities like meioand macrobenthos, which coincided well with the results of the environmental parameter analysis. More than half of the total sequences detected by DNA sequencing belonged to planktonic ciliates, most (if not all) of which were recovered from historic DNA originating through the sedimentation of pelagic forms because none of them were observed morphologically. The irrelevant historic DNA greatly influenced the recovery of rare species and thus limited the understanding of the benthic ciliate diversity and distribution. Our research indicates that the methods used have significant effects on the investigation of benthic ciliate communities and highlights that cDNA sequencing has great advantages in estimating the diversity and distribution of benthic ciliates, as well as the potential for benthic environmental assessments.

Key words: benthic ciliates|cDNA high-throughput sequencing|community comparison|DNA highthroughput sequencing|morphology

Received: 2019-09-19 Revised: 2019-11-18

Tools

PDF (1339 KB) Free

Print this page

Add to favorites

Email this article to others

Authors

Articles by Pingping HUANG

Articles by Feng ZHAO

Articles by Kuidong XU

References:
Abad D, Albaina A, Aguirre M, Laza-Martínez A, Uriarte I, Iriarte A, Villate F, Estonba A. 2016. Is metabarcoding suitable for estuarine plankton monitoring? A comparative study with microscopy. Marine Biology, 163(7):149,https://doi.org/10.1007/s00227-016-2920-0.
Appeltans W, Ahyong S T, Anderson G, Angel M V, Artois T, Bailly N, Bamber R, Barber A, Bartsch I, Berta A, Błazewicz-Paszkowycz M, Bock P, Boxshall G, Boyko C B, Brandão S N, Bray R A, Bruce N L, Cairns S D, Chan T Y, Cheng L, Collins A G, Cribb T, Curini-Galletti M, Dahdouh-Guebas F, Davie P J F, Dawson M N, de Clerck O, Decock W, de Grave S, de Voogd N J, Domning D P, Emig C C, Erséus C, Eschmeyer W, Fauchald K, Fautin D G, Feist S W, Fransen C H J M, Furuya H, Garcia-Alvarez O, Gerken S, Gibson D, Gittenberger A, Gofas S, GómezDaglio L, Gordon D P, Guiry M D, Hernandez F, Hoeksema B W, Hopcroft R R, Jaume D, Kirk P, Koedam N, Koenemann S, Kolb J B, Kristensen R M, Kroh A, Lambert G, Lazarus D B, Lemaitre R, Longshaw M, Lowry J, Macpherson E, Madin L P, Mah C, Mapstone G, McLaughlin P A, Mees J, Meland K, Messing C G, Mills C E, Molodtsova T N, Mooi R, Neuhaus B, Ng P K L, Nielsen C, Norenburg J, Opresko D M, Osawa M, Paulay G, Perrin W, Pilger J F, Poore G C B, Pugh P, Read G B, Reimer J D, Rius M, Rocha R M, Saiz-Salinas J I, Scarabino V, Schierwater B, Schmidt-Rhaesa A, Schnabel K E, Schotte M, Schuchert P, Schwabe E, Segers H, SelfSullivan C, Shenkar N, Siegel V, Sterrer W, Stöhr S, Swalla B, Tasker M L, Thuesen E V, Timm T, Todaro M A, Turon X, Tyler S, Uetz P, van der Land J, Vanhoorne B, van Ofwegen L P, van Soest R W M, Vanaverbeke J, Walker-Smith G, Walter T C, Warren A, Williams G C, Wilson S P, Costello M J. 2012. The magnitude of global marine species diversity. Current Biology, 22(23):2 189-2 202, https://doi.org/10.1016/j.cub.2012.09.036.
Azovsky A I, Mazei Y A. 2018. Diversity and distribution of free-living ciliates from high-arctic Kara sea sediments.Protist, 169(2):141-157, https://doi.org/10.1016/j.protis. 2018.01.001.
Bachy C, Dolan J R, López-García P, Deschamps P, Moreira D. 2013. Accuracy of protist diversity assessments:morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study.The ISME Journal, 7(2):244-255, https://doi.org/10.1038/ismej.2012.106.
Botnen S S, Davey M L, Halvorsen R, Kauserud H. 2018.Sequence clustering threshold has little effect on the recovery of microbial community structure. Molecular Ecology Resources, 18(5):1 064-1 076, https://doi.org/10.1111/1755-0998.12894.
Caporaso J G, Kuczynski J, Stombaugh J, Bittinger K, Bushman F D, Costello E K, Fierer N, Peña A G, Goodrich J K, Gordon J I, Huttley G A, Kelley S T, Knights D, Koenig J E, Ley R E, Lozupone C A, McDonald D, Muegge B D, Pirrung M, Reeder J, Sevinsky J R, Turnbaugh P J, Walters W A, Widmann J, Yatsunenko T, Zaneveld J, Knight R. 2010. QⅡME allows analysis of high-throughput community sequencing data. Nature Methods, 7(5):335-336, https://doi.org/10.1038/nmeth.f.303.
Corinaldesi C, Danovaro R, Dell'Anno A. 2005. Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments. Applied and Environmental Microbiology, 71(1):46-50, https://doi.org/10.1128/AEM.71.1.46-50.2005.
Dolan J R, Stoeck T. 2011. Repeated sampling reveals differential variability in measures of species richness and community composition in planktonic protists.Environmental Microbiology Reports, 3(6):661-666, https://doi.org/10.1111/j.1758-2229.2011.00250.x.
Edgar R C, Haas B J, Clemente J C, Quince C, Knight R. 2011.UCHIME improves sensitivity and speed of chimera detection. Bioinformatics, 27(16):2 194-2 200, https://doi.org/10.1093/bioinformatics/btr381.
Edgar R C. 2013. UPARSE:highly accurate OTU sequences from microbial amplicon reads. Nature Methods, 10(10):996-998, https://doi.org/10.1038/nmeth.2604.
Esteban G F, Finlay B J. 2007. Exceptional species richness of ciliated protozoa in pristine intertidal rock pools. Marine Ecology Progress Series, 335:133-141, https://doi.org/10.3354/meps335133.
Fenchel T. 2008. The microbial loop-25 years later. Journal of Experimental Marine Biology and Ecology, 366(1-2):99-103, https://doi.org/10.1016/j.jembe.2008.07.013.
Filker S, Kühner S, Heckwolf M, Dierking J, Stoeck T. 2019.A fundamental difference between macrobiota and microbial eukaryotes:protistan plankton has a species maximum in the freshwater-marine transition zone of the Baltic Sea. Environmental Microbiology, 21(2):603-617, https://doi.org/10.1111/1462-2920.14502.
Foissner W. 2008. Protist diversity and distribution:some basic considerations. Biodiversity and Conservation, 17(2):235-242, https://doi.org/10.1007/s10531-007-9248-5.
Foissner W. 2014. An update of ‘basic light and scanning electron microscopic methods for taxonomic studies of ciliated protozoa’. International Journal of Systematic and Evolutionary Microbiology, 64(1):271-292, https://doi.org/10.1099/ijs.0.057893-0.
Giner C R, Forn I, Romac S, Logares R, de Vargas C, Massana R. 2016. Environmental sequencing provides reasonable estimates of the relative abundance of specific picoeukaryotes. Applied and Environmental Microbiology, 82(15):4 757-4 766, https://doi.org/10.1128/AEM.00560-16.
Gong J, Dong J, Liu X H, 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(3):369-379, https://doi.org/10.1016/j.protis.2012.11.006.
Gong J, Shi F, Ma B, Dong J, Pachiadaki M, Zhang X L, Edgcomb V P. 2015. Depth shapes α- and β-diversities of microbial eukaryotes in surficial sediments of coastal ecosystems. Environmental Microbiology, 17(10):3 722-3 737, https://doi.org/10.1111/1462-2920.12763.
Gonzalez J M, Portillo M C, Belda-Ferre P, Mira A. 2012.Amplification by PCR artificially reduces the proportion of the rare biosphere in microbial communities. PLoS One, 7(1):e29973, https://doi.org/10.1371/journal.pone.0029973.
Groendahl S, Kahlert M, Fink P. 2017. The best of both worlds:a combined approach for analyzing microalgal diversity via metabarcoding and morphology-based methods. PLoS One, 12(2):e0172808, https://doi.org/10.1371/journal.pone.0172808.
Haas B J, Gevers D, Earl A M, Feldgarden M, Ward D V, Giannoukos G, Ciulla D, Tabbaa D, Highlander S K, Sodergren E, Methé B, DeSantis T Z, The Human Microbiome Consortium, Petrosino J F, Knight R, Birren B W. 2011. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Research, 21(3):494-504, https://doi.org/10.1101/gr.112730.110.
Hausmann K, Hülsmann N, Polianski I, Schade S, Weitere M. 2002. Composition of benthic protozoan communities along a depth transect in the eastern Mediterranean Sea.Deep Sea Research Part I:Oceanographic Research Papers, 49(11):1 959-1 970, https://doi.org/10.1016/S0967-0637(02)00095-X.
Hu S K, Campbell V, Connell P, Gellene A G, Liu Z F, Terrado R, Caron D A. 2016. Protistan diversity and activity inferred from RNA and DNA at a coastal ocean site in the eastern North Pacific. FEMS Microbiology Ecology, 92(4):fiw050, https://doi.org/10.1093/femsec/fiw050.
Jing H M, Zhang Y, Li Y D, Zhu W D, Liu H B. 2018. Spatial variability of picoeukaryotic communities in the Mariana Trench. Scientific Reports, 8(1):15 357, https://doi.org/10.1038/s41598-018-33790-4.
Jousset A, Bienhold C, Chatzinotas A, Gallien L, Gobet A, Kurm V, Küsel K, Rillig M C, Rivett D W, Salles J F, van der Heijden M G A, Youssef N H, Zhang X W, Wei Z, Hol W H G. 2017. Where less may be more:how the rare biosphere pulls ecosystems strings. The ISME Journal, 11(4):853-862, https://doi.org/10.1038/ismej.2016.174.
Karl D M, Bailiff M D. 1989. The measurement and distribution of dissolved nucleic acids in aquatic environments.Limnology and Oceanography, 34(3):543-558, https://doi.org/10.4319/lo.1989.34.3.0543.
Lara E, Berney C, Harms H, Chatzinotas A. 2007. Cultivationindependent analysis reveals a shift in ciliate 18S rRNA gene diversity in a polycyclic aromatic hydrocarbonpolluted soil. FEMS Microbiology Ecology, 62(3):365-373, https://doi.org/10.1111/j.1574-6941.2007.00387.x.
Lejzerowicz F, Voltsky I, Pawlowski J. 2013. Identifying active foraminifera in the Sea of Japan using metatranscriptomic approach. Deep Sea Research Part Ⅱ:Topical Studies in Oceanography, 86-87:214-220, https://doi.org/10.1016/j.dsr2.2012.08.008.
Li G H, Su L, Zhang Q Q, Zhang X L, Gong J. 2019. Molecular diversity and biogeography of benthic ciliates in the Bohai Sea and Yellow Sea. Acta Oceanologica Sinica, 38(2):78-86, https://doi.org/10.1007/s13131-018-1236-y.
Liu X S, Zhang Z N, Huang Y. 2007. Sublittoral meiofauna with particular reference to nematodes in the southern Yellow Sea, China. Estuarine, Coastal and Shelf Science, 71(3-4):616-628, https://doi.org/10.1016/j.ecss.2006.09.013.
Lynn D H, Small E B. 2002. Phylum ciliophora doflein, 1901.In:Lee J J, Bradbury P C, Leedale G F eds. An Illustrated Guide to the Protozoa. Allen Press, Lawrence, Kansas.p.371-656.
Lynn D H. 2008. The Ciliated Protozoa:Characterization, Classification, and Guide to the Literature, 3^rd edn.Springer, Dordrecht. 605p.
Magoč T, Salzberg S L. 2011. FLASH:fast length adjustment of short reads to improve genome assemblies.Bioinformatics, 27(21):2 957-2 963, https://doi.org/10.1093/bioinformatics/btr507.
Malviya S, Scalco E, Audic S, Vincent F, Veluchamy A, Poulain J, Wincker P, Iudicone D, de Vargas C, Bittner L, Zingone A, Bowler C. 2016. Insights into global diatom distribution and diversity in the world's ocean.Proceedings of the National Academy of Sciences of the United States of America, 113(11):E1516-E1525, https://doi.org/10.1073/pnas.1509523113.
Massana R, Gobet A, Audic S, Bass D, Bittner L, Boutte C, Chambouvet A, Christen R, Claverie J M, Decelle J, Dolan J R, Dunthorn M, Edvardsen B, Forn I, Forster D, Guillou L, Jaillon O, Kooistra W H C F, Logares R, Mahé F, Not F, Ogata H, Pawlowski J, Pernice M C, Probert I, Romac S, Richards T, Santini S, Shalchian-Tabrizi K, Siano R, Simon N, Stoeck T, Vaulot D, Zingone A, de Vargas C. 2015. Marine protist diversity in European coastal waters and sediments as revealed by highthroughput sequencing. Environmental Microbiology, 17(10):4 035-4 049, https://doi.org/10.1111/1462-2920.12955.
Meng Z C, Xu K D, Dai R H, Lei Y L. 2012. Ciliate community structure, diversity and trophic role in offshore sediments from the Yellow Sea. European Journal of Protistology, 48(1):73-84, https://doi.org/10.1016/j.ejop.2011.08.001.
Mieczan T, Adamczuk M, Pogorzelec M. 2018. Ciliates as restoration indicators in peatbogs-10 years of study.European Journal of Protistology, 62:11-23, https://doi.org/10.1016/j.ejop.2017.10.002.
Moss J A, McCurry C, Tominack S, Romero I C, Hollander D, Jeffrey W H, Snyder R A. 2015. Ciliated protists from the nepheloid layer and water column of sites affected by the Deepwater Horizon oil spill in the Northeastern Gulf of Mexico. Deep Sea Research Part I:Oceanographic Research Papers, 106:85-96, https://doi.org/10.1016/j.dsr.2015.10.001.
Nascimento F J A, Lallias D, Bik H M, Creer S. 2018. Sample size effects on the assessment of eukaryotic diversity and community structure in aquatic sediments using highthroughput sequencing. Scientific Reports, 8(1):11 737, https://doi.org/10.1038/s41598-018-30179-1.
Ngando T S, Groliere C A. 1991. Quantitative effects of fixatives on the storage of freshwater planktonic ciliates.Archiv fur Protistenkunde, 140(2-3):109-120, https://doi.org/10.1016/S0003-9365(11)80179-X.
Nielsen K M, Johnsen P J, Bensasson D, Daffonchio D. 2007.Release and persistence of extracellular DNA in the environment. Environmental Biosafety Research, 6(1-2):37-53, https://doi.org/10.1051/ebr:2007031.
Novitsky J A. 1986. Degradation of dead microbial biomass in a marine sediment. Applied and Environmental Microbiology, 52(3):504-509.
Orsi W, Biddle J F, Edgcomb V. 2013. Deep sequencing of subseafloor eukaryotic rRNA reveals active fungi across marine subsurface provinces. PLoS One, 8(2):e56335, https://doi.org/10.1371/journal.pone.0056335.
Penton C R, Gupta V V S R, Yu J L, Tiedje J M. 2016. Size matters:assessing optimum soil sample size for fungal and bacterial community structure analyses using high throughput sequencing of rRNA gene amplicons.Frontiers in Microbiology, 7:824, https://doi.org/10.3389/fmicb.2016.00824.
Prokopowich C D, Gregory T R, Crease T J. 2003. The correlation between rDNA copy number and genome size in eukaryotes. Genome, 46(1):48-50, https://doi.org/10.1139/g02-103.
Santoferrara L F, Grattepanche J D, Katz L A, McManus G B. 2014. Pyrosequencing for assessing diversity of eukaryotic microbes:analysis of data on marine planktonic ciliates and comparison with traditional methods. Environmental Microbiology, 16(9):2 752-2 763, https://doi.org/10.1111/1462-2920.12380.
Scheckenbach F, Hausmann K, Wylezich C, Weitere M, Arndt H. 2010. Large-scale patterns in biodiversity of microbial eukaryotes from the abyssal sea floor. Proceedings of the National Academy of Sciences of the United States of America, 107(1):115-120, https://doi.org/10.1073/pnas. 0908816106.
Seimon T A, Olson S H, Lee K J, Rosen G, Ondzie A, Cameron K, Reed P, Anthony S J, Joly D O, McAloose D, Lipkin W L. 2015. Correction:adenovirus and herpesvirus diversity in free-ranging great apes in the sangha region of the Republic of Congo. PLoS One, 10(11):e0142766, https://doi.org/10.1371/journal.pone.0142766.
Sherr E B, Sherr B F. 2002. Significance of predation by protists in aquatic microbial food webs. Antonie van Leeuwenhoek, 81(1-4):293-308, https://doi.org/10.1023/A:1020591307260.
Sogin M L, Morrison H G, Huber J A, Welch D M, Huse S M, Neal P R, Arrieta J M, Herndl G J. 2006. Microbial diversity in the deep sea and the underexplored "rare biosphere". Proceedings of the National Academy of Sciences of the United States of America, 103(32):12 115-12 120, https://doi.org/10.1073/pnas.0605127103.
Song W B, Warren A, Hu X Z. 2009. Free-living ciliates in the Bohai and Yellow Seas. Science Press, Beijing. p.1-518.(in Chinese)
Sonntag B, Posch T, Psenner R. 2000. Comparison of three methods for determining flagellate abundance, cell size, and biovolume in cultures and natural freshwater samples.Archiv fur Hydrobiologie, 149(2):337-351. https://doi.org/10.1127/archiv-hydrobiol/149/2000/337
Stock A, Edgcomb V, Orsi W, Filker S, Breiner H W, Yakimov M M, Stoeck T. 2013. Evidence for isolated evolution of deep-sea ciliate communities through geological separation and environmental selection. BMC Microbiology, 13:150, https://doi.org/10.1186/1471-2180-13-150.
Stoeck T, Bass D, Nebel M, Christen R, Jones M D M, Breiner H W, Richards T A. 2010. Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Molecular Ecology, 19(S1):21-31, https://doi.org/10.1111/j.1365-294X.2009.04480.x.
Stoeck T, Breiner H W, Filker S, Ostermaier V, Kammerlander B, Sonntag B. 2014. A morphogenetic survey on ciliate plankton from a mountain lake pinpoints the necessity of lineage-specific barcode markers in microbial ecology.Environmental Microbiology, 16(2):430-444, https://doi.org/10.1111/1462-2920.12194.
Stoeck T, Kochems R, Forster D, Lejzerowicz F, Pawlowski J. 2018. Metabarcoding of benthic ciliate communities shows high potential for environmental monitoring in salmon aquaculture. Ecological Indicators, 85:153-164, https://doi.org/10.1016/j.ecolind.2017.10.041.
Stoeck T, Zuendorf A, Breiner H W, Behnke A. 2007. A molecular approach to identify active microbes in environmental eukaryote clone libraries. Microbial Ecology, 53(2):328-339, https://doi.org/10.1007/s00248-006-9166-1.
Stokell J R, Hamp T J, Steck T R. 2016. Examining changes in bacterial abundance in complex communities using nextgeneration sequencing is enhanced with quantitative PCR.Antonie van Leeuwenhoek, 109(8):1 161-1 166, https://doi.org/10.1007/s10482-016-0707-4.
Tang Y Z, Ma Z P, Hu Z X, Deng Y Y, Yang A A, Lin S H, Yi L, Chai Z Y, Gobler C J. 2019.3, 000 km and 1, 500-year presence of Aureococcus anophagefferens reveals indigenous origin of brown tides in China. Molecular Ecology, 28(17):4 065-4 076, https://doi.org/10.1111/mec.15196.
Torti A, Lever M A, Jørgensen B B. 2015. Origin, dynamics, and implications of extracellular DNA pools in marine sediments. Marine Genomics, 24:185-196, https://doi.org/10.1016/j.margen.2015.08.007.
Wang F P, Xie Y Y, Wu W X, Sun P, Wang L, Huang B Q. 2019.Picoeukaryotic diversity and activity in the Northwestern Pacific Ocean based on rDNA and rRNA high-throughput sequencing. Frontiers in Microbiology, 9:3 259, https://doi.org/10.3389/fmicb.2018.03259.
Wang Y, Liu R Y, Su J L. 2013. Chinese Marine Geography.Science Press, Beijing. 331p. (in Chinese)
Wickham S, Gieseke A, Berninger U G. 2000. Benthic ciliate identification and enumeration:an improved methodology and its application. Aquatic Microbial Ecology, 22(1):79-91, https://doi.org/10.3354/ame022079.
Xin M, Ma D Y, Wang B D. 2015. Chemicohydrographic characteristics of the Yellow Sea Cold Water Mass. Acta Oceanologica Sinica, 34(6):5-11, https://doi.org/10.1007/s13131-015-0681-0.
Xu D P, Li R, Hu C, Sun P, Jiao N Z, Warren A. 2017. Microbial eukaryote diversity and activity in the water column of the South China Sea based on DNA and RNA high throughput sequencing. Frontiers in Microbiology, 8:1 121, https://doi.org/10.3389/fmicb.2017.01121.
Xu K D, Du Y F, Lei Y L, Dai R H. 2010. A practical method of Ludox density gradient centrifugation combined with protargol staining for extracting and estimating ciliates in marine sediments. European Journal of Protistology, 46(4):263-270, https://doi.org/10.1016/j.ejop.2010.04.005.
Yang J, Ma L A, Jiang H C, Wu G, Dong H L. 2016. Salinity shapes microbial diversity and community structure in surface sediments of the Qinghai-Tibetan Lakes. Scientific Reports, 6:25078, https://doi.org/10.1038/srep25078.
Zhang J L, Xu F S, Liu R Y. 2012. Community structure changes of macrobenthos in the South Yellow Sea.Chinese Journal of Oceanology and Limnology, 30(2):248-255, https://doi.org/10.1007/s00343-012-1076-0.
Zhang S W, Wang Q Y, Lü Y, Cui H, Yuan Y L. 2008.Observation of the seasonal evolution of the Yellow Sea Cold Water Mass in 1996-1998. Continental Shelf Research, 28(3):442-457, https://doi.org/10.1016/j.csr.2007.10.002.
Zhao F, Xu K D. 2016. Molecular diversity and distribution pattern of ciliates in sediments from deep-sea hydrothermal vents in the Okinawa Trough and adjacent sea areas. Deep Sea Research Part I:Oceanographic Research Papers, 116:22-32, https://doi.org/10.1016/j.dsr.2016.07.007.
Zhao F, Xu K D. 2017. Distribution of ciliates in intertidal sediments across geographic distances:a molecular view.Protist, 168(2):171-182, https://doi.org/10.1016/j.protis. 2017.01.001.
Zhao Y, Yi Z Z, Gentekaki E, Zhan A B, Al-Farraj S A, Song W B. 2016. Utility of combining morphological characters, nuclear and mitochondrial genes:an attempt to resolve the conflicts of species identification for ciliated protists.Molecular Phylogenetics and Evolution, 94:718-729, https://doi.org/10.1016/j.ympev.2015.10.017.
Zhou B L, Xu K D. 2016. Spatiotemporal variation in community structure of marine benthic ciliates in the Yellow Sea during and after macroalgal and giant jellyfish blooms. Chinese Journal of Oceanology and Limnology, 34(4):629-641, https://doi.org/10.1007/s00343-016-5115-0.