Taxonomy And Phylogeny Of Alexandrium Tamarense Species Complex And Endosymbiont Of Noctiluca Scintillans By DNA Barcoding And Morphology

DNA barcode is a standardized,sufficiently variable,easily amplified and relatively short DNA fragment that can represent different species.Since 2003,when Hebert proposed the concept of DNA barcoding,this technology in the animal and plant community has made great progress.COI gene,the originally proposed universal DNA barcode,has already attained more than 100,000 sequences.The biological diversity of marine ecosystems is much higher than that of land or fresh water,especially the existence of cryptic species.DNA barcoding technology can effectively detect and evaluate the species diversity of marine life.Compared with marine fish and invertebrate research,DNA barcoding in marine algae is more complex,as no single widely applicable barcode marker like the COI gene in animals has been found.Therefore,the barcoding research in algae is still focused on the evaluation of multiple genes in different groups to identify species accurately,which requires comparison and evaluation 2-3 barcode markers.In this study,compared with the morphological methodology,DNA barcoding technique was used to address taxonomic questions related to two groups of harmful algae:(1)Alexandrium tamarense speciks complex(Atama complex);(2)the endosymbiont Pedinomonas noctilucae Sweeney inside of Noctiluca scintillans.Different DNA markers were compared and evaluated,and species in the Atama complex and the endosymbiont in N.scintillans were finally identified,classified and named according to the phylogenetic analysis results.The dinoflagellate genus Alexandrium is one of the major harmful algal bloom-forming genera.The classification and identification of species in this group is difficult due to limited morphological differences,especially for Atama complex,which composed of three morphological species(A.catenella,A.tamarense and A.fundyense),so far there is no accurate classification conclusion.In this study,under the classification framework based on the LSU and SSU rDNA,we selected 27 strains that represented the three morphospecies of the Atama complex from different locations around the world including the China coast,to valuate the effectiveness of ITS region in Atama complex.We further investigated the presence of intragenomic polymorphism in ITS,SSU and LSU and its interference in species delimitation.The results are as follows:(1)similar to the previous SSU and LSU results,our ITS-based phylogenies divided the Atama complex to five clusters;(2)the length of ITS fragment is suitable(510-520 bp),the efficiency of amplification and sequencing is very high;(3)the presence of genomic polymorphism in this group does not impact the taxon-resolving power of this gene;(4)according to the phylogenetic species concept,these five different clusters should be defined as five different species.In the last decade,field studies in the northern Arabian Sea showed widespread blooms of the green dinoflagellate Noctiluca,which caused serious ecological problems.Previous study proposed that the survival ofN.scintillans may be tied to the extraordinary ability of its endosymbiont P.noctilucae to fix carbon under hypoxic waters.However,the taxomony of this endosymbionts is limited to morphological evidence and previous studies suggested that P.noctilucae was classified incorrectly.In this study,we used the 18 S rDNA,rbcL and chloroplast 16S rDNA as gene markers,in combination with the previously reported morphological features,to re-examine the phylogenetic position of this endosymbiotic algal species.Phylogenetic trees inferred from these genes consistently indicated that P.noctilucae is distantly related to the genotype of Pedinomonas.The sequences formed an independent genus within the family Marsupiomonadaceae.Based on the phylogenetic affiliation and ecological characteristics of this alga as well as the priority rule of nomenclature,we reinstate the genus Protoeuglena and reclassify the endosymbiont as Protoeuglena noctilucae.For the first time in this study,the application of ITS as DNA barcode in Atama conplex has been evaluated systematically and comprehensively,which solved the problem of identification of Atama complexe that has existed for several decades.We redefined the previous three morphospecies into five,and the effects of polymorphisms in species delimitation were studied in depth.The result will be helpful for further research on the formation of blooms and the mechanism of toxin production.In addition,this is the first time the molecular data were collected and analyzed for the endosymbiont P.noctilucae of Noctiluca,and its transfer into another order paves the way for further evolutionary as well as physiological and transcriptomic studies in the future.