Diversity,Spatio-temporal Dynamics,and Biogeographical Modeling Of Diazotrophic Community Structures In The Northern South China Sea

Nitrogen(N)is an important element that makes up living organisms.The marine nitrogen cycle is a major control on the primary productivity in the global ocean.As the largest marginal sea in the Western Pacific Ocean,the South China Sea(SCS)is a huge oligotrophic water body with very limited influx of nitrogenous nutrients due to its lower N:P ratio.This suggests that microbial dinitrogen(N2)fixation plays an important role in the production of bioavailable nitrogen.In the South China Sea,N2 fixation not only provides available ammonia for marine plankton,but also couples a series of important biogeochemical cycles associated with carbon(C),phosphorus(P),and silicon(Si)in the ocean,so nitrogen cycle plays a vital role in the ocean.In order to assess the functional diversity of N2-fixing microbes(diazotrophs)in the northern South China Sea(nSCS),we sequenced the PCR amplicons of the nitrogenase iron protein-coding nifH gene,from DNA and cDNA,respectively,in 52 samples with the High Throughput Sequencing(HTS)technique.Using TaqMan qPCR assay,we also quantified the representative diazotrophs including the filamentous Trichodesmium,the unicellular nitrogen-fixing cyanobacteria(UCYN)in groups A1,A2,B,and C(UCYN-A1,UCYN-A2,UCYN-B,and UCYN-C),the heterocystous cyanobacteria symbiotic with diatoms(Het-1,Het-2,and Het-3),a gammaproteobacterium(?-24774A11)and an alpha-proteobacterium(?-MH144511).The main results are as follows:(1)The dominant N2-fixing microbes in the nSCS are Trichodesmium,UCYN-B and ?-24774A11.In 2018,the composition of diazotrophs at different stations were significantly different,while in 2017,the composition of representative diazotrophs were more uniform among different samples;(2)TaqMan qPCR assay identifies three N2-fixing phylotypes with obvious circadian rhythms,namely Trichodesmium,UCYN-B,and Het-1.Among them,Trichodesmium fixes N2 during the day,UCYN-B and Het-1 fix N2 at night,and the nifH gene copies in Trichodesmium does not change significantly with the sampling depth,but those in UCYN-B,UCYN-C,and Het-1 decrease with the increasing sampling depths.Network analysis of ten N2-fixing phylotypes suggests a strong negative correlation between daytime N2-fixing Trichodesmium and UCYN-B and Het1 that fix N2 at night.The abundant Trichodesmium and UCYN-B are negatively correlated with other N2-fixing phylotypes,while some of the less abundant N2-fixing microbes are positively correlated with other N2-fixing microbes.This phenomenon suggests that the interrelationship between nitrogen fixers may be related to their abundance and circadian rhythm.(3)HTS shows that the N2-fixing phylotypes can be better distinguished by sampling depths than the sampling time.Some environmental factors affecting the distribution of N2-fixing microbial communities also vary with sampling years and water depths.Further OTU annotation demonstrates sample-specific pattern of different N2-fixing microbes:those with high abundance are prevalent in most samples,but those with low abundance tend to distribute in individual samples.This result may be related to the specific hydrological characteristics and environmental factors in the northern South China Sea.(4)The HTS data were fitted to the neutral model,and the effects of environmental and spatial variables on the N2-fixing microbial communities were analyzed through Mental test.The degrees of fit between the samples and the neutral model are 0.324 and 0.527 for the cruises in 2017 and 2018,respectively.Compared with 2017,the samples in 2018 were greatly affected by the stochastic process,which may be related to typhoon,Kuroshio,and sampling stations.In building the N2-fixing microbial community,the dominant species had a great influence.(5)The N2 fixation rate assay reveals that the highest nitrogenase activity is associated with the samples collected in station SCS3 where Trichodesmium dominates.The rates of N2 fixation vary with stations,but they are relatively stable at specific locations.Although the N2 fixation rates of samples collected at the same station in different water layers during the day or at night are relatively close,the overall N2 fixation rate differs greatly from that at day and night,and the N2 fixation rate also varies greatly between different stations.These indicate that the N2 fixation capacity may vary with the specific sampling condition.