Biodiversity And Distribution Of Ciliates In Sediments From The Yellow Sea And Bohai Sea As Estimated By High Throughput Sequencing And Morphology

The diversity and distribution pattern of microbial eukaryotes in particular ciliates have long been ongoing debates.Ciliates,with the high morphological diversity and important functions,have been utilized as a model organism group to evaluate the diversity and distribution pattern of microbial eukaryotes.The application of recent molecular methods has greatly improved our understanding of the ciliate diversity and distribution.However,the knowledge on the variations of ciliate diversity and community structure detected by the traditional morphological method and molecular techniques and the driving factors of the differences are still far from known.Therefore,we,for the first time,compared and evaluated the ciliate diversity and community structure in marine sediments by utilizing both the morphological method and the 18 S rDNA and cDNA high-throughput sequencing.The methodological comparison indicates that both the morphological examination and cDNA high-throughput sequencing are reliable methods to assess the diversity of benthic ciliates in marine sediments.Thus,we combined the two methods to investigate the diversity and distribution pattern of ciliates in sediments as well as their relationship with environmental factors at 17 stations in the Yellow Sea and Bohai Sea.Our first attempt is to compare the differences of the ciliate diversity and community composition revealed by the morphological method and the 18 S rDNA and cDNA high-throughput sequencing on the basis of sediment samples collected from one station in the South Yellow Sea.The results showed that the morphological method(i.e.the Ludox-QPS method)obtained a total of 97 ciliate morphospecies belonging to 8 classes,20 orders,30 families,and 36 genera.The DNA high-throughput sequencing detected a higher diversity of 174 OTUs belonging to 10 classes,28 orders,55 families,and 76 genera.The highest diversity was obtained by the cDNA high-throughput sequencing,which yielded a total of 284 OTUs belonging to 10 classes,31 orders,68 families,and 99 genera.Compared to the morphological method,the two molecular methods detected much higher diversity and more similar community composition of ciliates.Further analyses showed that the morphological method detected only benthic ciliates,while the DNA high-throughput sequencing detected also a high proportion of sequences related to planktonic ciliates accounting for about 90% of the total DNA sequences.These sequences likely originated from the resting cysts of planktonic ciliates in sediment or dead planktonic forms which sunk into sediment.The cDNA high-throughput sequencing detected also sequences of planktonic ciliates,which accounted for about 7% of the total cDNA sequences.In comparison to the DNA high-throughput sequencing,the cDNA sequencing detected much more similar community composition of benthic ciliates to that detected by the morphological method.Our study indicates that the molecular methods are efficient in estimating the overall ciliate diversity in marine sediments.The DNA high-throughput sequencing can obtain also the ciliate diversity of resting cysts as well as extracellular and historic DNA,while the cDNA high-throughput sequencing has advantages in evaluating active ciliates.Subsequently,we used the three methods to compare the diversity and community composition of ciliates in marine sediments from five stations along an obvious gradient of environmental factors and to assess the relationship between the ciliate diversity and environmental factors.The results showed that the morphological method obtained the lowest diversity of 79 morphospecies,all of which belonged to benthic ciliates.The DNA high-throughput sequencing obtained a total of 308 OTUs,among which the planktonic ciliate sequences accounting for 66% of the total sequences,and the number of Choreotrichia and Oligotrichia OTUs contributed to about 27% of the total OTUs.The cDNA high-throughput sequencing obtained a total of 628 OTUs,among which the Choreotrichia and Oligotrichia OTUs contributed to about 13% of the total OTUs and 12% of the total sequences.Both the morphological and cDNA high-throughput sequencing methods revealed that the ciliate communities at the two stations located in the Yellow Sea Cold Water Mass(YSCWM)were similar,and those at the other three stations were more accordant.By contrast,the DNA high-throughput sequencing uncovered a different community.Several classes/subclasses detected by the morphological and cDNA sequencing methods at certain stations were not covered by the DNA sequencing method.The three methods detected only 5%-8% of common morphospecies/ OTUs at the five stations,where,however,the sequences of these common taxa contributed 20%-90% to the total sequences.The analyses further demonstrated that the morphological and cDNA sequencing methods could detect a similar ciliate community structure from the same site.We suggested that a combination of the morphological and cDNA sequencing methods was practicable to detect the diversity and the distribution pattern of the active ciliates in sediments,and to assess their relationship with the environmental factors.Based on the results of methodological evaluation,we utilized the morphological and cDNA high-throughput sequencing methods to investigate the diversity and distribution pattern of benthic ciliates as well as their relationships with environmental factors at 17 stations in the Yellow Sea and the Bohai Sea.The results showed that the morphological methods identified 216 species from the 17 stations and the cDNA high-throughput sequencing detected 966 ciliates OTUs,among which 861 OTUs belonged to benthic ciliates.The dominant benthic ciliate taxa included Prostomatea,Oligohymenophorea,Litostomatea and Hypotrichia,with Prostomatea as the most abundant ciliates detected by both methods.The cluster analysis based on the OTUs table detected by cDNA sequencing showed that the YSCWM stations shared a similar community of benthic ciliates and were thus grouped together and the non-YSCWM stations were grouped also.This distribution pattern is generally accordant with the usual pattern of benthos in this area.By contrast,no clear pattern was observed by the cluster analysis based on the morphospecies table.Statistical analysis showed that the number of morphospecies and OTUs and the Shannon-Wiener and Margalef index values were all positively correlated with the median grain size of sediments and negatively correlated with the contents of sediment nitrogen and total organic matters.BIOENV analysis showed that the combination of bottom water temperature,salinity and water depth best explained the difference of the community structure of ciliates.In sum,compared to the DNA high-throughput sequencing,the morphological method and the cDNA high-throughput sequencing could detect a more similar community structure and distribution pattern of marine benthic ciliates.The cDNA high-throughput sequencing would detect also a certain proportion of planktonic ciliate sequences,the existence of which might influence the evaluation of the benthic ciliate diversity.In the future,further steps are needed to improve the molecular methods for better evaluating the diversity and distribution pattern of ciliates as well as other microbial eukaryotes,such as the removal of the extracellular DNA/RNA,increasing the length of the marker gene,and improving the pipeline for analyzing sequences.