采水量对寡营养海域浮游真核微生物分子多样性评价的影响

Evaluation of the effects of sample size on estimating the molecular diversity of planktonic microeukaryotes in an oligotrophic oceanic region

真核微生物是海洋生态系统中生物多样性高且功能重要的组成部分,但因个体微小且形态特征不明显,传统的分类学方法很难全面评估其多样性。环境DNA结合高通量测序技术为真核微生物多样性研究提供了可靠技术支撑,然而采水量对于评估寡营养海域浮游真核微生物多样性的影响仍知之甚少。本研究在吕宋海峡及菲律宾海盆中设置5个采样点,每个站点各取4个10 L表层海水样品用于真核微生物高通量测序分析。对各站每个重复所获得的真核微生物多样性进行研究,并分析了采水量与真核微生物OTU数量、群落结构及优势种和稀有种之间的关系。研究发现,采水量和真核微生物OTU数量之间呈正相关关系,10 L水所获得的平均OTU数量为40 L水的64%,20 L水可获得82%,30 L水为92%。10 L样品所获得的群落多样性指数显著低于30 L和40 L样品检获的群落,而20 L样品与30 L和40 L样品在多样性指数上无显著差异。随着采水量增加,更多的稀有OTUs被检获,而丰富OTUs数量变化不明显。但统计分析显示,10 L与40 L组样品检获的真核微生物群落结构无显著差异。因此,综合考虑样品的可得性和现场处理时间,最低20 L的采水量可用于评估这一寡营养海域的真核微生物多样性和群落结构特点。

With a high morphological and functional diversity,marine microeukaryotes are considered a vital component of marine ecosystems.Because of their small size and indistinct morphological features,it is difficult to evaluate the community diversity of microeukaryotes using conventional taxonomic methods.The combination of high-throughput sequencing with environmental DNA is a reliable method for assessing the molecular diversity of microeukaryotes;however,the effects of sample size on estimating their diversity remain unclear.Here we established five sampling sites in the Luzon Strait and collected four replicates of 10 L of surface water at each site for the 18S rDNA high-throughput sequencing of microeukaryotes.We compared the microeukaryotic OTU number,community structure,abundant OTUs,and rare OTUs among the samples of different sample sizes.The results showed a positive relationship between the OTU number of microeukaryotes and the sample size.The average OTU number of microeukaryotes obtained with 10 L,20 L,and 30 L seawater samples contributed to approximately 64%,82%,and 92%,respectively,of that obtained with the 40 L sample.The community diversity index of the 10 L sample was significantly lower than that of the 30 L and 40 L samples,but there was no significant difference among the 20 L,30 L,and 40 L samples.The number of rare OTUs increased with increasing sample size,whereas no distinct change was observed for abundant OTUs.Statistical analysis showed that there was no significant difference in the composition of microeukaryotic community between the 10 L and 40 L samples.Considering the sample availability and treatment time,a seawater sample of at least 20 L is recommended for estimating the diversity and community structure of microeukaryotes in the oligotrophic sea region.