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

The Yellow Sea Cold Water Mass(YSCWM) is a distinct hydrographic phenomenon of the Yellow Sea,and the distribution pattern of meio-and macrobentho s diffe rs 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 macrobentho s,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 adj acent shallow water by using morphology and DNA and complementary DNA(cDNA) high-throughput sequencing of the V4 region of 18 S rRNA gene.Results showed that the diversity of benthic ciliate s detected by DNA(303 OTU s),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.