Diversity Of Planktonic Ciliates On Chongming Island And Its Relationship With Environmental Factors

As a group of the highest and most complex single–celled eukaryotic organisms,ciliates are not only diverse and widely distributed,but also respond quickly to the surrounding environment due to their small size and thin cell membranes,which are highly favored by taxonomists and ecologist researchers.With the continuous innovation of research methods,the current ecological research on ciliates mainly includes traditional ecology methods and environmental DNA sequencing methods.The traditional ecology methods include settling chamber counts and quantitative protargol stain.The differences between the two traditional ecology methods and the environmental DNA method in recent years for studying the relationship between ciliate diversity and community structure and environmental factors and which method is more applicable to the study of the relationship between ciliates and environmental factors is still inconclusive.This study combined morphological observation,traditional ecology methods and environmental DNA methods for the first time to study the diversity of planktonic ciliate protozoa in the river channel of Chongming Island and its relationship with environmental factors.The differences among the three methods for studying the relationship between ciliates,environmental factors and geographic distances were compared and a list of planktonic ciliate protozoa species in the river channel of Chongming Island was provided.According to the research results,it is proposed that the combination of quantitative protein silver method and environmental DNA method supplemented by detailed taxonomic studies will be the main method to study the ecology of ciliates in the future.This study takes the intricate river network in Chongming Island as the research object.A total of 23 sampling sites were set up in Chongming Island,including city–level,district–level,town–level and village–level river channels,and we sampled in July 2020,October 2020,January 2021 and May 2021 separately.The diversity of planktonic ciliate protozoa in the channel of Chongming Island and its relationship with environmental factors were studied by traditional ecology and environmental DNA sequencing methods.The results showed that 24 species of ciliates（including undetermined species）were detected by settling chamber counts method,belonging to7 orders,8 families,and 8 genera,of which Oligotrichida accounted for more than 70%;A total of 131 species（including classified species）of ciliates were identified by quantitative protargol stain method,which belonged to 24 orders,43 families,and 61genera,of which 28%were Prostomatida,25%were Choreotrichida,and 18%were Oligotrichida.The environmental DNA sequencing method detected a total of 258species belonging to 37 orders,107 families and 182 genera,among which Choreotrichida accounted for 41%,Cyclotrichida accounted for 12%,Haptorida accounted for 9%and Oligotrichida accounted for 8%.Species with relatively high relative abundance include Tintinnidium fluviatile,Zosterodasys sp.1,Strombidiidae gen.sp.1,Cyclotrichida fam.sp.1,Strobilidiidae gen.sp.1,Rimostrombidium lacustris,etc.Among them,Tintinnidium fluviatile is significantly positive with Temperature,TP and NH₄–N.There was a significant negative correlation with DO;Rimostrombidium lacustris of the Choreotrichida was significantly positively correlated with Cond,and significantly negatively correlated with Temperature,DO,NH₄–N,NO₂–N and TP.There were significant differences in community structure between seasons.DO,Temperature,NH₄–N,NO₂–N and TP were the main environmental factors affecting the community structure.The main taxa seized by the three methods are basically the same,mainly including Oligotrichida,Choreotrichida,Prostomatida,Haptoria etc.Except some unspecified species,the quantitative protargol stain method can detect most of the species seized by settling chamber counts methods.The environmental DNA sequencing method seizes a large number of ciliates species that have not been detected by traditional ecology methods.On the one hand,the large number of species may be mostly rare species,and the sampling volume of morphological methods may be difficult to find them.On the other hand,it may also be that some species are omitted due to poor fixation and cell lysis during the experimental process of the morphological method.Theα–diversity of ciliate community by traditional ecology method has significant differences with seasonal changes;the ACE index,Chao1 index,goods coverage index and Richness index of ciliate community by environmental DNA sequencing method have no significant difference between seasons.With Pielou evenness index,there is a significant difference between each other except that there is no significant difference between May and July;the Shannon index is only significant between May and October,July and October;the Simpson index only has significant difference in October and other months.The national standard method found that the environmental factors that were significantly correlated with the ciliate community were Transparency,Temperature,DO,p H,NO₂–N,NH₄–N,TP,TN,and Chl–a;the QPS method found that there was a significant correlation with the ciliate community and the environmental factors,including Transparency,Temperature,DO,p H,NO₂–N,NH₄–N,TP,TN;the environmental DNA sequencing method found that the environmental factors significantly correlated with ciliate community were Transparency,Temperature,DO,Cond,p H,NO₂–N,NH₄–N,TP;through the comparison of the three methods,it can be seen that the main environmental factors that can affect the community structure of planktonic ciliates in the river channel of Chongming Island are Transparency,Temperature,DO,p H,NO₂–N,NH₄–N,TP and the three methods were nearly equally effective.However,considering the cost of experiment and time,settling chamber counts method is the most convenient method to reflect water environmental conditions,while QPS method and environmental DNA method have more advantages in reflecting the diversity of ciliate community in water.In addition,in order to better use traditional morphological methods to monitor the protozoa diversity of ciliates in the river channel of Chongming Island,this work provides primary color photos of 34 common ciliates in vivo,and also found two rare ciliates belonging to the same genus.The SSU r RNA 18s DNA sequence differences of the rare species Hastatella radians and Hastatella aesculacantha are concentrated in the V4 region,but there is no difference in the V9 region.If the 18s V9 region is used for diversity survey,the protozoa species diversity of Chongming Island will be underestimated.