DNA Barcoding Of Macrobenthic Invertebrates And Nektons In The Yangtze Estuary

Species identification is the cornerstone of biological research.Most known species were identified by using morphological characteristics.Traditional taxonomists generally use morphological characteristics for species identification,which depends on long-term professional training and experience accumulation.Although the traditional method plays a role in the identification of species,it also has the problems of high professional requirements,long time-consuming,and difficulty in accurately identify species.DNA barcoding technology is a new molecular identification technology developed in recent years.This technology uses the universal primers to amplify homologous DNA sequences to set up a database of many species.Unknown species can be identified by comparing its sequence with those in database.It provides a standard taxonomy information and becomes the most rapidly progress in research fields.It's a powerful complement to the traditional way.In addition,on the basis of the accurate identification of species,the formation mechanism of species can be inferred through the study of kinship and divergence time among species or within species.The Yangtze estuary has a large variety of macrobenthic invertebrates and nektons and is rich in biodiversity.However,there are still some problems on species identification,such as rapid and accurate identification of species,identification of insect larvae,and identification of residues.The development of DNA barcoding technology provides a new method to solve the above problems.Meanwhile,the mechanism of species formation in the estuary of the Yangtze River is not yet known.Based on accurate species identification and phylogenetic approaches,the species divergence time can be inferred,which is conducive to the study of the estuarine species formation mechanism.This paper intends to conduct the research of the following three aspects.1)Adding DNA sequences to barcoding database of macrobenthic invertebrates in Yangtze estuary for rapid and accurate species identification;2)Using the DNA barcode to identify the species in the Yangtze estuary which are difficult to identify using traditional methods;3)Trying to construct the evolutionary tree using the molecular clock theory and multi-gene combination to calculate the divergence time of the estuarine species and marine species of the Yangtze estuary,and then correcting it based on geological events and inferring its formation mechanism.The specimen in this study were from the museum of Yangtze estuary Wetland Station and some of supplementary specimen were collected from September 2015 to December 2017.The COI gene,Cyt b gene and the control area of mitochondrial DNA sequences;18s,and H3 of nuclear DNA sequence were selected as molecular markers.The main results were as follows:(1)The 47 sequences of COI were successfully amplified by using the universal barcoding primers of 87 specimens of the macrobenthic invertebrates and fishes in Yangtze estuary.There were 45 species of COI in macrobenthic invertebrates,belong to 45 species(3 annelid,5 molluscs and 37 arthropods).Two fish sequences,belong to 2 species.Comparing with the homologous sequences,33 sequences' similarity degree were greater than 95%,which could be determined as the same species;14 sequences' were less than 90%,which could not be determined.The above findings laid the foundation for the rapid identification of species in the estuary of the Yangtze river and is a powerful supplement to traditional species identification methods.(2)Using the animal universal primers LCO1490 and HCO2198 to identify the benthic larvae of the Yangtze estuary and amplified 9 sequences.Through phylogenetic trees which were constructed by using the sequences and known homologous sequences to identify a larva as Macratriinae,a larva as Ischnura senegalensis(Rambur,1842),a larva Ceratopogonidae,a larva as Dolichopodidae,a larva as Donacia,two larvae as Polypedilum nubifer(Skuse,1889)and Glyptotendipes barbipes(Staeger,1839).In addition,genomic DNA was extracted from the whale wrack in the area of the Yangtze estuary and Hangzhou bay.The sequences of control region,Cyt b gene and COI gene were successfully amplified.Compared with the homologous sequences,only one base pair in the control region is different from the sequence of Balaenoptera physalus,KC572816 and KC572776.The body was unequivocally identified as Balaenoptera physalus(Linnaeus,1758).Moreover,there were multiple termination codons in COI and Cyt b and they should be pseudogenes.The Onchidium sample of chongming dongtan was identified as the Onchidium reevesii(Gray,1850)by using DNA barcoding technology which was mistakenly thought as Onchidium struma.(3)Time tree was constructed by using 18 S,and H3 genes to analyze the divergence time of the Palaemonidae species in Yangtze estuary,including fresh water species Exopalaemon modestus(Heller,1862),estuarine species Exopalaemon carinicauda(Holthuis,1950),Exopalaemon annandalei(Kemp,1917)and Palaemon gravieri(Yu,1930),and marine species Palaemon macrodactylus and Palaemon tenuidactylus Liu,Liang & Yan,1990.Among them,the divergence time of the estuarine species Exopalaemon annandalei and Exopalaemon carinicauda was 0.5186Mya(95%HPD : 0.3683—0.6783),the divergence time of Exopalaemon annandalei and Exopalaemon carinicauda was 0.4866 Mya(95%HPD : 0.3445—0.6408),the divergence time of Palaemon gravieri and Palaemon tenuidactylus was 0.0883Mya(95%HPD:0.0300—0.1660).We hypothesize that the ancestors of the Palaemonidae entered the Yangtze estuary with the melting of the Quaternary glaciers.After seawater receded,they were geographically isolated from the closely related marine species and eventually formed new species.In summary,DNA barcoding technology can be applied to identify the insect larvae,animal residues and doubtful Onchidium species in the Yangtze estuary.Moreover,we can infer the formation mechanism of estuarine species on the basis of accurate identification of species,combined with phylogenetic analysis methods.