基于形态学与核糖体DNA及其cDNA高通量测序的海洋沉积物中纤毛虫多样性比较

摘要

高通量测序技术广泛应用于环境真核微生物多样性的研究,可检获较之传统形态学方法更高的多样性.然而,检获的高分子多样性与基于形态的物种多样性在构成上的差异仍然不明.本研究首次对比分析了基于形态学的南黄海沉积物中的纤毛虫物种多样性与基于核糖体18S DNA和cDNA高通量测序的多样性异同.结果表明:形态鉴定获得8纲、20目、30科、36属共97种纤毛虫;DNA测序检获10纲、28目、55科、76属共174个OTUs;而通过cDNA测序获取的纤毛虫多样性最高,获10纲、31目、68科、99属共284个OTUs.研究发现,形态学方法检获的纤毛虫均为底栖生纤毛虫;两种分子手段检获的多样性更高,群落结构更为近似,覆盖了形态鉴定所获的绝大部分类群.但DNA测序还检获了序列比例高达90％的浮游类群,可能源于包囊或死亡沉降的物种;雨cDNA测序检获了约7％的浮游纤毛虫序列.较之DNA测序,cDNA法检获的底栖纤毛虫在群落结构上与形态学结果更为接近.本研究表明,分子手段有助于更全面地揭示沉积物中的纤毛虫多样性,DNA测序可同时揭示休眠包囊、胞外DNA及过去群落的信息,而cDNA测序在研究活动纤毛虫多样性上更有优势.%The high-throughput sequencing is widely used to estimate the diversity of eukaryotic microbes including ciliates,and can detect much higher diversity than traditional morphological methods.However,the differences between the two types of methods in ciliate diversity and composition remain unclear.We compared the ciliate diversity and community composition in marine sediment for the first time using morphological method and the DNA and cDNA high-throughput sequencing of 18S rRNA gene.The morphological method detected 97 ciliate morphospecies belonging to 8 classes,20 orders,30 families,and 36 genera.By contrast,the DNA sequencing detected a higher level of diversity of 174 OTUs belonging to 10 classes,28 orders,55 families,and 76 genera.The highest diversity was obtained by the cDNA sequencing,which yielded 284 OTUs belonging to 10 classes,31 orders,68 families,and 99 genera.Our analyses indicate that morphological method detected only benthic ciliates.The two molecular methods detected higher biodiversity and uncovered most of the ciliate groups detected by the morphological method,with a more similar community composition.The DNA high-throughput sequencing detected also sequences of planktonic ciliates occupying about 90％ of the total DNA sequences,in which the planktonic sequences likely originated from the resting cysts of planktonic ciliates in sediment or dead planktonic forms sunk into sediment.The cDNA high-throughput sequencing detected also sequences of planktonic ciliates occupying about 7％ of the total cDNA sequences.In comparison to the DNA high-throughput sequencing,the cDNA high-throughput sequencing detected a more similar community composition of benthic ciliates to the morphological method.Our study indicates that the molecular methods are more efficient in estimating the overall ciliate diversity in marine sediment.The DNA high-throughput sequencing can obtain also the ciliate diversity of resting cysts and extracellular and historic DNA,while the cDNA high-throughput sequencing has advantages in evaluating active ciliates.