Diversity Of Dinoflagellate Cysts In The Yellow Sea And Distribution Of Gymnodinium Catenatum Cysts In The Sediment Of The Coastal Seas Of China

Harmful algal blooms(HABs)have occurred frequently in the offshore waters of more than 80% of the world ’s coastal countries in the past few decades and caused serious damages to fisheries,aquaculture,tourism and ecosystems,and thus have become a hot issue in the field of marine ecology.Literature reviews have shown that75% of the HABs are caused by dinoflagellates,and dinoflagellates account for 54% of the known toxic microalgae and 44% of the HABs-causing species.Therefore,dinoflagellates take a central position in the ecology of harmful algal blooms.More than 200 dinoflagellate species are proved to produce resting cysts during their life cycle.The cysts not only help the dinoflagellates survive the adverse environment,but also “inoculate” the HABs like plant seeds,and can be geographically dispersed via natural or anthropogenic vectors.Therefore,it is of great significance to detect the species,distribution and abundance of dinoflagellate resting cysts in marine sediments,particularly from those HABs-prone areas.Harmful algal blooms caused by different species of dinoflagellates have occurred frequently in the Yellow Sea(YS)in the past40 years,some of these species have only been reported in recent years.This indicated that there possibly exist many potential HABs-causing species that have never been identified in the YS.Most previous species identification of dinoflagellate cysts in the YS mainly relied on conventional morphological methods,while studies using molecular methods have been rare.The YS extends a long coastline with many closed or semi-closed bays,while some of them having particular ecological and economic significance(such as Sanggou Bay and Rizhao Bay)have not been surveyed for their cyst diversity yet.Therefore,this research,combining the high-throughput sequencing and single-cyst morpho-molecular identification,aimed to explore the species diversity and distribution pattern of dinoflagellate cysts in the YS.This work fully identified many dinoflagellate cyst species,including many HABs species and unknown species.Based on the finding of the cysts of Gymnodinium catenatum,one of the most important paralytic shellfish toxin-producing harmful dinoflagellates,in the YS,this work extended the investigation for the existence and abundance of this species to the four seas of China.Blooms of G.catenatum have occurred occasionally in different areas of China and caused tremendous economic loss and even threatened human health.However,the distribution and abundance of G.catenatum cysts in the sediment along the coastal seas of China have never been extensively investigated.Therefore,in this research,specific primers and probes were designed and then used to detect the distribution and abundance of G.catenatum cysts in the sediment along the entire coastal water of China by Taq Man quantitative PCR(q PCR)assays.The main findings of the research are summarized as follows:(1)Species diversity of dinoflagellate cysts in the Yellow Sea.A total of 28 surface sediment samples(～ 2cm thick)were collected from the YS offshore stations(water depth >17m)and 20 surface samples were collected from the YS coastal sediment(including 6 bays: Yantai,Sanggou Bay,Aoshanwei,Liuqing River,Jiaozhou Bay,and Rizhao Bay).The dinoflagellate cyst assemblages in these 48 samples were firstly detected via metabarcoding high-throughput sequencing.A total of2080 OTUs(Operational Taxonomic Units)annotated as dinoflagellate were obtained,from which 45 species(136 OTUs)were accurately identified,with 41 annotated to well-described species and 4 annotated to species having not been taxonomically described.Most OTUs(1704)could not be accurately annotated to the species level or genus level.Secondly,four of the abovementioned samples(collected from Yantai Bay,Sanggou Bay,Jiaozhou Bay,and Rizhao Bay)were selected to detect cyst species by picking up individual cyst under an inverted microscope after a concentration process,photographing each for their morphology,and molecularly identified with single-cyst PCR,cloning,and sequencing that targeted at ～1400 bp fragment of the LSU r RNA gene.This single-cyst morpho-molecular approach successfully identified 212 out of468 dinoflagellate cysts(more than one sequence obtained for each cyst),which were annotated to 79 species,with 45 fully identified as well-described species and 7 as species having not been taxonomically described.The two approaches together fully identified 76 cyst species at the species level,including 22 HABs-causing species(e.g.Alexandrium pacificum,Azadinium poporum,Gonyaulax spinifera,Gymnodinium catenatum,Karlodinium veneficum),3 HABs-causing species that have never been reported from the YS(Alexandrium ostenfeldii,Kryptoperidinium triquetrum,Stoeckeria algicida),11 species that have never been unambiguously reported in China(Alexandrium concavum,A.pohangense,Archaeperidinium saanichi,Azadinium obesum,Ensiculifera carinata,Gonyaulax digitale,Impagidinium pallidum,Scrippsiella bicarinata,S.kirschiae,Thecadinium kofoidii,Yihiella yeosuensis),and 10 species that have never been reported as cyst producers(Alexandrium concavum,A.pohangense,Ansanella granifera,Azadinium obesum,Gonyaulax digitale,Gymnodinium smaydae,Pelagodinium beii,Scrippsiella bicarinata,S.kirschiae,Thecadinium kofoidii).It is noteworthy that both methods detected many species that have not been detected or described yet,indicating that there are still many dinoflagellate species completely “unknown” to taxonomists in the YS,and some of them may be potentially toxic or HABs-causing species,as at least one con-generic species of them has been proven to be toxic or HABs-causing.Besides the cyst diversity,two types of morphological diversity were also found.Different cyst morphotypes existed in one species,while cysts of different species are morphologically indistinguishable.These results greatly enriched the understanding of the diversity of dinoflagellate cysts in the YS,identified important open questions for the future research in the taxonomy and cyst diversity of dinoflagellates in the region,and also provided basic database and insights for the development of HABs ecology and strategies of early warning and forecasting of HABs.(2)Distribution pattern of dinoflagellate cysts in the Yellow Sea.The dinoflagellate cysts assemblages in 48 sediment samples collected from the Yellow Sea identified via the bioinformatic analysis exhibited significant difference between the offshore and nearshore areas.Further analyses for the community structures of cyst assemblages from offshore samples revealed that the Yellow Sea Cold Water Mass(YSCWM)significantly affected both the species composition and abundances of cysts in the area.As a result,the cyst community structure within the YSCWM differed significantly from that off the YSCWM(i.e.,in-and off-YSCWM group).In addition,the off-YSCWM samples could be subdivided further into two clusters: the south and the north groups.For the cyst communities in the coastal samples,no distribution regularity(i.e.grouping or gradient)was recognized.The relative influences of environmental factors(longitude,latitude,temperature,salinity,and water depth)on the spatial distribution of dinoflagellate cysts as observed from RDA analysis showed that,for the offshore samples,longitude,latitude,temperature,salinity,and water depth all significantly affected the cyst community respectively,for the coastal samples,none of these factors could significantly explain the variations in cyst communities among the samples.Further analysis of the relative abundance of the most abundant 34 cyst species revealed that the in-and off-YSCWM samples also differed in the distribution of these abundant species.Correlation analysis showed that the relative abundance of the most abundant species was affected by different environmental factors among the longitude,latitude,temperature,salinity,and water depth.These results provided novel insights into the distribution pattern(geographic range)of dinoflagellate cysts in the HABs-prone YS,and exhibited to be of broad importance in understanding the geography of many species of dinoflagellates.(3)Distribution and abundance of Gymnodinium catenatum cysts in the sediment along the coastal seas of China.A total of 199 sediment samples were collected from the coastal seas of China,including 31 samples from the Bohai Sea(BS),71 from the Yellow Sea(YS),59 from the East China Sea(ECS),and 38 from the South China Sea(SCS).The northernmost sample was collected from the Beidaihe in the BS and the southernmost one was from the Nansha Islands of the SCS.Specific primers and probes targeting the D1-D2 region of 28 S r DNA of G.catenatum were designed,and then used in Taq Man fluorescence quantitative PCR(q PCR)to detect the distribution and abundance of G.catenatum cysts in the abovementioned coastal sediment samples of China.The detection unveiled that G.catenatum cysts were widely present in the sediments of the coastal seas of China,ranging from 10.01°N to 39.85°N(ca 3000 km)and from 108.33°E to 124°E,covering the vast sea area from Nansha Islands in the SCS to Beidaihe in the BS.The average percentage of positive detections of all samples was 63.32%,with that of the BS samples being 93.55%,the YS 74.65%,ECS 42.37%,and SCS 50%.The single-cyst morpho-molecular identification in the sediment samples from Beidaihe,BS confirmed the existence of G.catenatum cysts in the BS,which also partly validated the positive detections of G.catenatum cyst using the q PCR methods.While G.catenatum cysts were widely distributed in the sediments of all four seas of China,the average abundance was low(1 cyst per gram of wet sediment),with the highest abundance in the BS being 8 cysts/g,the YS being 12 cysts/g,the ECS being 23 cysts/g,and the SCS being 6 cysts/g.Three samples from the ECS contained cysts of G.catenatum that were relatively higher than other sea areas,suggesting a pertinence to frequent occurrences of G.catenatum blooms in the area during the most recent years.In addition,cysts havd been detected in both Pearl River Estuary and Lianyungang coastal area,where the G.catenatum blooms had been recorded.Although the detected cyst abundance of G.catenatum was generally low in all samples,cysts in the sediments may be still abundant enough to provide “seeds” for the initiation of blooms,given a favorite environmental condition and enough time for the vegetative growth prior to the blooming season.