Phytoplankton Community Structure And Molecular Diversity In Beibu Gulf

Beibu Gulf is located in the northwest of the South China Sea,and is the largest natural semi-enclosed bay in south China.It has excellent port construction conditions,and is an important area for urban development,aquaculture and tourism.With the implementation of economic development policies such as the"China-ASEAN Free Trade Area"and"One Belt,One Road",economy of the Beibu Gulf coastal area has entered a period of rapid growth.The economic development has led to the increase of nutrient levels in the coastal waters,and the enrichment environment would result in the changes of the phytoplankton community structure reflected by the increase of harmful algal blooms.In this study,a total of 70 surface water samples were collected in the Beibu Gulf through two surveys by"2020 Beibu Gulf Autumn Voyage by Marine Environment Monitoring Center Station of Guangxi Zhuang Autonomous Region"and"2021 Beibu Gulf Summer Voyage by the National Natural Sicence Foundation of China".Phytoplankton community were analyzed by both microscopic observation and r DNA metabarcoding,and the phytoplankton community structures were compared with both methods.The feasibility of using metabarcoding in phytoplankton studies was discussed.The research results provided background information for the phytoplankton community structure in Beibu Gulf,revealed the distribution of harmful algal bloom(HAB)species,and thus provide a scientific basis for the prevention and control of algal blooms in Beibu Gulf.Our results play an important information for the sustainable development of Beibu Gulf.The main results of this study are as follows:(1)A total of 118 species in 59 genera,7 classes of 4 phyla were observed in the 70 surface water samples from Beibu Gulf based on microscopic observation.Diatoms were the most abundant phylum,in which 78 species in 40 genera were recorded,followed by dinoflagellates(38species in 17 genera).The phytoplankton cell density ranged from 1.28×10~3-8.98×10~4 cells/L,with an average of 2.46×10~4 cells/L.The average cell density was 3.06×10~4cells/L in the southern sea area of Beibu Gulf in the summer survey of 2021(referred to BS),and were 2.59×10~4 cells/L,2.08×10~4 cells/L and 1.21×10~4 cells/L in the northern sea area of Beibu Gulf in the autumn survey of 2020(referred to BA),in the northern sea area(referred to BN)and Qiongzhou Strait section(referred to QZ)in the summer survey of 2021,respectively.Diatoms were the predominant group in the phytoplankton community,with an average cell density of 2.34×10~4 cells/L,accounting for95.06%of the overall phytoplankton cell density.The average cell density of dinoflagellates was1.15×10~3 cells/L,accounting for 4.68%of the overall phytoplankton.The cell densities of phytoplankton and diatoms distributed in the BS,and the low densities were in the QZ.Higher dinoflagellates occurred in the BS as well,while dinoflagellates were lower in the other sea areas.(2)Chaetoceros spp.was the most abundant phytoplankton species in the Beibu Gulf based on microscope analysis,whose cell density varied from 2.75×10~2 cells/L to 4.25×10~4cells/L with the average of 8.04×10~3 cells/L.The distribution of Chaetoceros spp.was similar to diatoms and overall phytoplankton,with the higher in the BS and BN,and the lower in the QZ.Navicula spp.,Rhizosolenia spp.,Leptocylindrus danicus,Thalassionema frauenfeldii and Thalassionema nitzschioides were also the common dominant diatom species.Scrippsiella acuminata,Gymnodinium spp.,Prorocentrum spp.,and Alexandrium spp.were dominant dinofalgellate species.Through microscope analysis,a total of 24 harmful algal bloom(HAB)species were identified,including 11 diatoms and 13 dinoflagellates.The HAB species,including Leptocylindrus danicus,Thalassionema frauenfeldii,Thalassionema nitzschioides,Prorocentrum micans,Prorocentrum sigmoides,Noctiluca scintillans widely distributed in Beibu Gulf,while other HAB species occurred occasionally.The core species in the four sea areas were mainly those of diatoms and dinoflagellates,including a large number of HAB species.Phytoplankton community structure was similar in the BN and BS,while was quite different in the QZ.Furthermore,phytoplankton community differed in the BA and BN,which is the same sea area however in different sampling seasons.(3)Based on metabarcoding analysis,a total of 1516222 reads and 3647 OTUs of eukaryotic phytoplankton DNA sequences were obtained,including 251 species of eukaryotic phytoplankton in 185 genera,31 classes of 11 phyla.Dinoflagellates and Haptophyta were the dominant phyla,followed by Cryptophyta,Diatoms,Chlorophyta,Chrysophyta and Ochrophyta.Dinoflagellates dominated in DNA sequence,accounting for 79.69%of the total phytoplankton reads,followed by Haptophyta(9.52%),Cryptophyta(4.44%),diatoms(2.99%),Chlorophyta(2.58%),Chrysophyta(0.11%)and Ochrophyta(0.09%).DNA reads of overall phytoplankton,dinoflagellate and Haptophyta were higher in the BS.The Dinoflagellates also had the highest OTU richness,including 3319 OTUs,accounting for 85.52%of phytoplankton OTU richness.There were 146,96,92 and 86 OTUs in diatoms,Haptophyta,Chlorophyta and Cryptophyta,respectively,accounting for 4.00%,2.63%,2.52%and 2.36%,respectively.OTU richness in each sample ranged between 274 and 886 OTUs,with the average of 562 OTUs.The OTU richness of eukaryotic phytoplankton in each sea area from high to low followed as BS>BA>BN>QZ.The alpha diversity indexes,including Chao1,Ace,Shannon,Simpson diversity index and Pielou evenness,were higher in the BS indicating more healthy and stable phytoplankton community in the BS than other sea areas in Beibu Gulf.(4)Karlodinium veneficum was the most abundant species in the metabarcoding analysis,while the Phaeocystis globosa was the more abundant Haptophyta.A total of 53 HAB species were identified in the metabarcoding analysis,including 31 dinoflagellates,17 diatoms,3 Ochrophytes and 2 Haptophytes.47,50,45 and 52 HAB species were identified in the BA、BN、QZ,and BS,respectively.HAB species included the causative species that produce human toxins NSP,Pb Tx-2,AZP,YTX,PSP,DSP,and ASP,such as Karlodinium veneficum,Karenia papilionacea,Azadinium dexteroporum,Gonyaulax spinifera,Alexandrium hiranoi,Prorocentrum minimum,Pseudo-nitzschia caciantha and Nitzschia navis-varingica etc.,and included those that form blooms and cause ecological hazards and fish toxins,such as Prorocentrum micans,Neoceratium furca,Scrippsiella acuminata,Thalassiosira mala,Chattonella subsalsa,Fibrocapsa japonica,Heterosigma akashiwo etc.,and also those which caused algal blooms in Beibu Gulf before,such as Guinardia flaccida,Skeletonema costatum,Leptocylindrus danicus,Akashiwo sanguinea and Phaeocystis globosa etc.(5)Phytoplankton species richness analyzed by metabarcoding was much higher than that obtained by microscope analysis.Species richness was 118 taxa by microscopy,of which 88 staxa were identified at the species level.While a total of 3647 OTUs of phytoplankton were identified by metabarcoding,and 251 species were identified.There were 24 and 53 HAB species recorded by microscopy and metabarcoding,respectively.The results suggest that metabarcoding has the power to detect the hidden and rare species,which may be ignored under microscopic observations.And thus,metabarcoding is feasible in the identification of phytoplankton species,early monitoring of algal blooms,and changes in biodiversity.However,due to the preference of primers,the difference in the DNA copy number of different phytoplankton and the limitation of the database,metabarcoding cannot completely represent the phytoplankton community structure.In order to more comprehensively reveal the structure and distribution of phytoplankton community,it is necessary to combine metabarcoding and microscope analyses to make the research results more credible.