Species Diversity Of Rotifer Communities And Primarily Application Of Mitochondrial Metagenomics To Rotifer Communities In Reservoirs Of Hanjiang River Basin,Guangdong Province

Rotifers are a group of zooplankton dominating in freshwater and have high specific diversity,which play an important role in food webs and ecosystems.In the present study,we investigated species diversity of rotifers with morphological taxonomy in reservoirs of Hanjiang River basin in Guangdong Province,and try to apply mitochondrial metagenomics to investigate the diversity of this species.In this study,we collected samples from 23 reservoirs in the Hanjiang River Basin of Guangdong Province in November of 2019 to January of 2019.And their physical and chemical factors were measured.We used morphologic methods to estimate the species diversity of rotifers and multivariate analysis including PCA and RDA to detect the influencing factors of community structure in the watershed.Meanwhile,we designed new barcoding primers for rotifers using the results of metagenomic assembly,and verified the effectiveness of mitochondrial enrichment techniques in the mitochondrial metagenomic method.The main results are summarized as follows:1.A total of 47 rotifers from 19 genera were identified through morphological classification in all the 23 investigated reservoirs.Among them,Trichocerca,Brachionus and Lecane had the most species.The dominant species have dorsal spines or small bodies.Redundancy analysis showed that conductivity,chlorophyll a and nitrate nitrogen were significant factors explaining the variation in rotifer community structure.The analysis of variance decomposition indicated that physical variables explained the variation in rotifer community structure more than chemical and biological variables.Analysis of indicator species showed that Pompholyx sulcata and B.forficula can be indicator species for high-conductivity waters.K.tropica,T.pusilla and B.angularis can be indicator species for high nitrate concentration waters.And S.stylata can be the indicator species in the water with high chlorophyll a concentration.2.We used the assembly information of rotifer metagenomic data to design specific primers for amplification of the rotifer barcode region.We test the new primers with 22 species of rotifers(including 6 families and 9 genera).The amplification efficiency of COI primers 30 F and 885 R for rotifers reached up to 86%,which was higher than that of dg LCO/dg HCO(32%)and Folmer primers(59%).The COI sequences of 37 species from 17 genera in the Hanjiang River Basin were amplified by the new pair primers.These sequences are the prerequisite for large-scale application of mitochondrial metagenomics in rotifer community ecology.We obtained 93 rotifer mitochondrial contigs with length greater than 5000 bp from 8 metagenomic data sets,among which 55 sequences were identified to genus or species,and some of the contigs were from the same species.Finally,we obtained mitochondrial contigs from a total of 18 rotifer species.The number of mitochondrial contigs obtained from the mitochondria enriched samples was about 9 times that of the unenriched samples.We verified the effectiveness of the mitochondrial enrichment method.The mitochondrial contigs of rotifer species were annotated,and a large number of protein-coding genes were rearranged in mitochondrial genes of many rotifers.The sequence of mitochondrial genes from B.diversicornis obtained in this study were consistent with those published in the previous study,but the sequence of mitochondrial genes from K.cochlearis were not consistent with those published in this species.Since the mitochondrial genome of some species contains two circular genomes,and there exist a lot of gene rearrangements.Thus,it is the focus of the further work that the development of more genes marker to label the rotifer circular mitochondrial genomes.In conclusion,rotifers in reservoirs in Hanjiang River Basin of Guangdong Province are mainly characterized by dorsalis or spines.Physical variables explain the differences in rotifer community structure more than chemical and biological variables.The rotifer COI primers 30 F / 885 R designed in this study had higher amplification efficiency than the primers dg LCO/dg HCO and LCO2198 / HCO1490.In addition,this study verified that the method of mitochondrial enrichment can effectively improve the assembly efficiency of rotifer mitochondrial contigs in the metagenome.A large number of protein-coding genes were rearranged in the mitochondrial genomes of rotifers of different genera and species.