【作者】 孟昭翠；

【导师】 徐奎栋；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2011， 博士

【摘要】 微型底栖生物是底栖微食物网的主要组成部分,由于其形态和功能上的复杂性和多样性及研究方法的制约,对该类群的研究一直欠缺。本研究对2007年6月采自黄海48个站位和2008年7月(浒苔暴发末期)采自黄海33个站位的微型底栖生物的群落结构、多样性、分布及其生态特点和潜在作用进行了研究,并结合底层水温度、盐度、水深、沉积物有机质含量、叶绿素a、脱镁叶绿素a及中值粒径等环境因子,对微型底栖生物的群落结构及分布特点进行了综合分析。近海的微型底栖生物因样品采集和保存的局限,对于DAPI荧光染色技术的定量效能尚缺必要的测试。本研究比较了冷冻和冷藏两种保藏方式以及保存时间对荧光计数底栖细菌、蓝细菌、硅藻、不同粒级自养(PNF)和异养微型鞭毛虫(HNF)的影响。对黄海3个站位表层2 cm的底栖样品进行的4°C冷藏与–20°C冷冻避光保存的对比研究表明,两种保藏方式下,其中两个站位的所有研究对象均无显著差异,但另一站位的PNF(2–5μm和5–10μm)的冷藏保存显著优于冷冻保存。对选取的另外两个站位(0–2 cm和2–5 cm分层)的样品经1个月和4个月冷藏保存后的比较分析表明,对于底栖细菌、蓝细菌、PNF(5–10μm)、PNF(>10μm)、HNF(>10μm)和硅藻在保存1个月和4个月后的计数没有显著差异,而对于PNF(2–5μm)、HNF(2–5μm)、HNF(5–10μm)保存4个月的数量明显低于保存1个月的样品,如其中一个站位的0–2 cm分层的PNF(2–5μm)丰度减少了47.4%,2?5 cm分层的丰度减少了59.6%,HNF(2–5μm)和HNF(5–10μm)经4个月后丰度难以检测到。本研究表明,对底栖细菌、蓝细菌以及原生生物的定量计数可因样品、保藏方式及保存时间的不同而产生差异,因此对于微型底栖生物样品短期内宜采用避光、冷藏保存,并在带回实验室后尽快分析。利用DAPI荧光计数法和Ludox-QPS(硅胶提取结合定量蛋白银染色)法,对2007年夏季黄海冷水团及邻近海域共48个站位的微型底栖生物组成、分布以及沉积环境因子进行了研究。所调查站位表层5 cm沉积物中微型底栖生物丰度为(1.15 ? 0.38)? 109 cells/cm3,生物量为120 ? 54μg C/cm3(? 6009 ? 2673μg C/10 cm2),北黄海(17站位)的丰度及生物量较南黄海(31站位)分别高12%和34%,冷水团区域丰度和生物量较非冷水团区域分别高12%和17%。在表层5 cm沉积物中,细菌丰度为(1.14 ? 0.38)?109 cells/cm3,较PNF及HNF高3个数量级(丰度分别为(2.64 ? 1.67)? 106 cells/cm3,(1.52 ? 1.35)? 106 cells/cm3),较蓝细菌/蓝藻高4个数量级((8.52 ? 6.04)? 105 cells/cm3)。在表层8 cm沉积物中,硅藻的丰度为(3.54 ? 15.92)? 103 cells/cm3,远高于异养小鞭毛虫(HMF,31 ? 33 cells/cm3)及纤毛虫的丰度(33 ? 36 cells/cm3)。在生物量上,底栖细菌贡献最多(平均50.26 ?g C/cm3),其次是PNF(40.73 ?g C/cm3),HNF(19.34 ?g C/cm3),以及蓝细菌(8.84 ?g C/cm3)。而硅藻(0.84 ?g C/cm3),纤毛虫(0.15 ?g C/cm3)和HMF(0.03 ?g C/cm3)的生物量较低。在垂直分布上,0–2 cm分层中的数量高于2–5 cm及5–8 cm分层,其中在数量上约有95%的硅藻,77%的纤毛虫及56%的HMF分布在0–2 cm,其它类群垂直分层不明显。相关分析表明,纤毛虫丰度与叶绿素a含量显著正相关;硅藻丰度与水深显著负相关,与叶绿素a含量显著正相关;HMF丰度与底层水盐度、有机质含量、中值粒径显著负相关;其余微型生物丰度与各环境因子无显著相关。与黄海浮游生物各类群相比较,微型底栖生物各类群约高1–4个(平均为3个)数量级。通过叶绿素a估算的底栖微藻的生物量远高于直接计数(底栖硅藻+蓝藻+PNF)所得,可能超微级(pico级)的底栖藻类对初级生产力贡献巨大。对微型和小型底栖动物的新陈代谢率的估算发现,底栖原生动物(纤毛虫及异养鞭毛虫)的新陈代谢率占微型与小型底栖动物总量的90?,其主要贡献来自HNF。此外,底栖细菌的生物量约为小型底栖动物的4倍,生产力更高达17倍。由此可见微型底栖生物在底栖生态系统中具有十分重要的作用。在本航次中共发现纤毛虫198种,隶属于16纲/亚纲,27目,89属,其中前口纲(Prostomatea)为优势类群,其丰度占纤毛虫总丰度的45%,生物量占58%。肉食性纤毛虫为主要的摄食类群,其生物量占总生物量的64%,其次是菌食性(占21%)、藻食性(12%)及杂食性(3%)。相关分析表明,纤毛虫的物种数与底层水温度呈极显著正相关,而与底层水盐度、水深及沉积物有机质含量呈极显著负相关。此外,Margalef、Shannon多样性指数及Pielou均匀度指数与底层水盐度均呈极显著负相关,而Shannon多样性指数及Pielou均匀度指数与水深、有机质含量呈显著负相关,与沉积物中值粒径呈显著正相关。纤毛虫群落结构与环境因子的统计分析表明,温度对纤毛虫群落结构具有重要的影响。通过对纤毛虫食菌率及食藻率的估算发现,在黄海沉积物中纤毛虫摄食对细菌现存量影响很小,但是对硅藻的现存量有重要的影响。在2008年夏季浒苔暴发末期,对黄海共33个站位的微型底栖生物组成、分布以及环境因子的研究表明,所调查站位表层5 cm沉积物中微型底栖生物丰度为(6.43 ? 2.54)? 108 cells cm-3,生物量为64.14 ? 49.78 ?g C/cm3,现存量较2007年低了约45%,并在浒苔暴发区域下降尤为明显。微型底栖生物的丰度和生物量在南北黄海之间、冷水团及其外围之间的分布情况与2007年相似,北黄海(9站位)的丰度及生物量较南黄海(24站位)分别高15%和54%,冷水团区域丰度及生物量较非冷水团区域分别高19%和35%。在表层5 cm沉积物中,细菌丰度为(6.40 ? 2.51)? 108 cells/cm3,较PNF及HNF高2个数量级(丰度分别为(1.75 ? 1.24)? 106 cells/cm3,(0.66 ? 1.16)? 106 cells/cm),而较蓝细菌高三个数量级((2.72 ? 5.12)? 105 cells/cm3)。在表层8 cm沉积物中,硅藻丰度为(2.12 ? 5.45)? 103 cells/cm3,远高于HMF(6 ? 103 cells/cm3)及纤毛虫(25 ? 29 cells/cm3)。在生物量上,细菌贡献最多(平均28.17 ?g C/cm3),其次是PNF(21.27 ?g C/cm3),HNF(11.18 ?g C/cm3),以及蓝细菌(2.83 ?g C/cm3)。而硅藻(0.52 ?g C/cm3),纤毛虫(0.11 ?g C/cm3)和HMF(0.07 ?g C/cm3)的生物量较低。在垂直分布上,0–2 cm分层中的数量高于2–5 cm及5–8 cm分层,其中约87%的HMF,85%的硅藻,65%的纤毛虫分布在0–2 cm表层,其它类群垂直分层不明显。统计分析显示,在南北黄海沉积物中环境因子对微型底栖生物各类群的影响不同,在北黄海除HMF丰度与沉积物Chl a含量、PNF与底层水叶绿素含量呈负相关外,其余类群与环境因子均未表现出相关性;在南黄海,环境因子对纤毛虫及硅藻丰度的影响较大,但纤毛虫和硅藻丰度与各环境因子的正负相关关系完全相反。除HMF外,其余微型底栖生物各类群的丰度和生物量均较2007年降低,可见浒苔的暴发对微型底栖现存量产生了明显的抑制作用。在2008年夏季航次中共发现的纤毛虫种数也较2007年低,共132种,隶属于13纲/亚纲,24目,55属。前口纲在丰度及生物量上仍是最优势的类群(丰度占71%,生物量占64%)。肉食性纤毛虫仍为主要的功能群(其生物量占总生物量的53%),其次为菌食性(33%)、藻食性(11%)及杂食性(1%)。相关分析表明,纤毛虫的丰度与底层水盐度、水深及有机质含量呈显著正相关;纤毛虫生物量及物种数与各环境因子均没有表现出相关性;Margalef多样性指数与底层水温度呈显著正相关;Shannon多样性指数及Pielou均匀度指数均与水深及有机质含量呈显著负相关,与中值粒径呈显著正相关;而Pielou均匀度指数同时与底层水盐度呈极显著负相关。BIOENV分析表明,底层水温度、盐度及沉积物中值粒径的环境因子组合与纤毛虫群落具有最大的相关性。除Margalef多样性指数2008年较2007年航次略有增大外,纤毛虫的丰度、生物量、物种数、Shannon多样性及Pielou均匀度指数均较2007年降低,浒苔的暴发可能对纤毛虫的群落结构及多样性产生了明显的抑制作用。总体来讲,即使在黄海表层5 cm的沉积物中,其单位面积的微型底栖生物现存量也是水体中相应类群的2?4个数量级,其在数量上的显著性必然联系着功能上的重要性,由此在黄海底栖乃至整个生态系统的初级和次级生产和能流中起着重要的作用。对其资源存量和多样性特点及动态的深入研究和观测,将有助于对底栖乃至整个生态系统的结构和功能的理解。更多还原

【Abstract】 Microbenthos are important components in benthic microbial food web. However, there is little attention to the research of microbenthic ecology because of their complexity in biodiversity and ecological functions and the limitation of research methods. In the present study, we investigated the community structure, distribution, and the qualitative and quantitative importance of microbenthos in the Yellow Sea sediment based on samples collected from 82 stations during the cruises in 2007 and 2008. Benthic environmental variables including water temperature, salinity, water depth and sediment organic matter, chlorophyll a, phaeophytin a, and sediment median grain size were analyzed and discussed their possible relationships with the community structure and distribution of microbenthos.Firstly, we tested the effects of different storage manners and preservation duration on enumeration of microbenthos. The method of epifluorescence microscopy with DAPI has been widely used in the quantitative research on planktonic bacteria and protists, but scarcely applied in benthic study. We tested the effects of cold and freezing storage and preservation duration on the enumeration of marine benthic bacteria, cyanobacteria, and protists including diatoms, phototrophic nanoflagellates (PNF) and heterotrophic nanoflagellates (HNF) by DAPI epifluorescence microscopy. The test samples were collected from 0?2 cm and 2?5 cm sediment layers in the Yellow Sea, preserved with 2% glutaraldehyde, and stored at 4°C and ?20°C in the dark, respectively. The results suggested no significant differences in abundance between the 0?2 cm surface samples stored at 4°C and ?20°C in two stations, while in one station the abundances of PNF (2?5μm, 5?10μm) stored at 4°C were higher than that at ?20°C. The preservation duration effect was further tested using sediment samples collected from 0?2 cm and 2?5 cm sediment layers and stored at 4°C in the dark. The abundances of bacteria, cyanobacteria, diatoms, PNF and HNF were counted after one and four months. Statistical analyses show that storage time had no significant effect on the enumeration of benthic bacteria, cyanobacteria, PNF (5?10μm), PNF (>10μm), HNF (>10μm) and diatom. However, the abundances of PNF (2?5μm), HNF (2?5μm) and HNF (5?10 μm) were distinctly decreased after four months. Our experiments suggested that preservation types and duration as well as sediment types could influence the enumeration efficiency of marine benthic microorganisms. Thus, it is recommended that marine benthic samples can be temporarily stored at 4°C in the dark and should be enumerated as soon as possible.Using epifluorescence microscopy and the Ludox density centrifugation combining the quantitative protargol staining techniques, we investigated the spatial distribution and composition of microbenthos in the seafloor sediments from 48 stations of the Yellow Sea during the cruise on June 15-28, 2007. The microbenthic abundance was on average (1.15 ? 0.38) ? 109 cells/cm3 and biomass was 120 ? 54μg C/cm3 (? 6009 ? 2673μg C/10 cm2) in the upper 5 cm of the sediments. The abundance and biomass of microbenthos in the northern Yellow Sea were separately 12% and 34% higher than those in the southern Yellow Sea. Similarly, the abundance and biomass of microbenthos in the Yellow Sea Cold Water Mass (YSCWM) were separately 12% and 17% higher than those in the area outside the YSCWM. The bacterial abundance ranged from 2.36 ? 108 to 1.85 ? 109 cells/cm3 wet sediment, about three orders of magnitude higher than that of phototrophic (PNF, from 6.43 ? 105 to 8.80 ? 106 cells/cm3 ) and heterotrophic nanoflagellates (HNF, from 5.78 ? 104 to 5.91 ? 106 cells/cm3), and about four orders of magnitude higher than that of cyanobacteria (from 2.31 ? 104 to 2.31 ? 106 cells/cm3) in the upper 5 cm of the sediments. The diatom abundance highly varied from 3 to 1.06 ? 105 cells/cm3 in the upper 8 cm of the sediments, whereas that of heterotrophic microflagellates (HMF, 1?182 cells/cm3) and ciliates (1?221 cells/cm3) was less variable and lower. The biomass partitioning indicated the primary importance of benthic bacteria (on average 50.26 ?g C/cm3), followed by PNF (40.73 ?g C/cm3), HNF (19.34 ?g C/cm3), and cyanobacteria (8.84 ?g C/cm3). Benthic diatoms (0.84 ?g C/cm3), ciliates (0.15 ?g C/cm3) and HMF (0.03 ?g C/cm3) contributed relatively small proportions to the total biomass of microbenthos. About 95% of diatoms, 77% of ciliates and 56% of HMF were distributed in the upper 2 cm of the sediments, while no distinct vertical distributions were observed for bacteria, cyanobacteria, PNF and HNF. The statistical analyses show that the abundance of ciliates and diatoms was significantly positively correlated with sediment Chl-a concentrations while the diatom abundance was also negatively correlated with water depth. The HMF abundance was significantly negatively correlated with bottom water salinity, organic matter contents and sediment median grain size. The microbenthos were quantitatively important in the shallow sea floor, where their main components were about one to four, on average three orders of magnitude higher in abundance than corresponding planktonic ones in the same sea area. The comparison of the direct count of microphytobenthos and the estimation based on the ratio of biomass to Chl a indicated that pico-sized phytobenthos might contribute a large proportion to the primary production. Benthic ciliates and heterotrophic flagellates contributed about 90? to the estimated combined metabolic rate of micro- and meiobenthic consumers in the whole sea area, with nanoheterotrophs accounting for the majority. Benthic bacteria were 3.5 times higher in biomass than that of meiobenthos while it was even 17.5 times higher in production than that of meiobenthos. The data suggest the potential for rapid primary and secondary productions of microbenthos and detrital utilization in the shallow sea floor sediments of the Yellow Sea.A total of 198 morphospecies, representing 16 classes/subclasses, 27 orders, and 89 genera, were identified from the samples collected in 2007. Prostomatea was the most predominant group accounting for 45% of total abundance and 58% of total biomass. The carnivores constituted the primary feeding type occupying about 64% of total biomass, followed by the bacterivores (21%), algivores (12%), and omnivores (3%). Statistical analyses show that the species number was significantly positively correlated with bottom water temperature and negatively with bottom water salinity, water depth, and median grain size. Besides, both Margalef and Shannon-Wiener diversity and Pielou evenness indices showed significantly negative correlation with bottom water salinity, while Shannon-Wiener diversity and Pielou evenness indices also had negative correlation with water depth and organic matter and positive with median grain size. The BIOENV analysis show that bottom water temperature affecting the ciliate community structure. Based on the estimated ciliate bacterivory and herbivory, we indicate that ciliate ingestion had only a marginal influence on bacterial abundance but possibly had an important impact on diatoms in the sediments of the Yellow Sea.The abundance of microbenthos was on average (6.43 ? 2.54) ? 108 cells/cm3 and biomass was 64.14 ? 49.78 ?g C/cm3 in the upper 5 cm of the sediments from 33 stations in the Yellow Sea at the late stage of E. prolifera bloom in summer 2008, which was about 45% lower in standing crops than that in 2007. The microbenthos standing crops decreased more obviously in the stations heavily affected by the E. prolifera bloom. The abundance and biomass of microbenthos in the northern Yellow Sea were separately 15% and 54% higher than those in the southern Yellow Sea. While the abundance and biomass were separately 19% and 35% higher inside than outside the YSCWM. The abundance of bacteria was on average 6.40 ? 108 cells/cm3, about two orders of magnitude higher than that of phototrophic (PNF, on average 1.75 ? 106 cells/cm3) and heterotrophic nanoflagellates (HNF, 0.66 ? 106 cells/cm3) and three orders of magnitude higher than that of cyanobacteria (2.72 ? 105 cells/cm3) in the upper 5 cm of the sediments. The abundance of diatoms was about 2.12 ? 103 cells/cm3 in the upper 8 cm of the sediments, but distinctly higher than that of heterotrophic microflagellates (HMF, 6 cells/cm3) and ciliates (25 cells/cm3). The biomass partitioning indicate the primary importance of benthic bacteria (on average 28.17 ?g C/cm3), followed by PNF (21.27 ?g C/cm3), HNF (11.18 ?g C/cm3), and cyanobacteria (2.83 ?g C/cm3). Benthic diatoms (0.52 ?g C/cm3), ciliates (0.11 ?g C/cm3) and HMF (0.07 ?g C/cm3) contributed relatively small fractions to the total biomass of microbial communities. About 87% of HMF, 55% of diatoms and 65% of ciliates were distributed in the surface 0?2 cm sediment layer, while there were no distinct vertical distributions for bacteria, cyanobacteria, PNF and HNF. The statistical analyses show that the correlations between microbenthos abundance and environmental factors in the northern Yellow Sea were different from those in the southern Yellow Sea. The microbenthic abundance in the northern Yellow Sea had no correlation with environmental factors except that the negative correlations between HMF abundance and sediment Chl-a concentrations and betweet PNF abundance and bottom water Chl concentrations, while in the southern Yellow Sea, the correlations between ciliate abundance and environmental factors were distinct from those between diatom abundance and environmental factors. The abundance and biomass of microbenthos assemblages were lower than those in 2007 except of HMF, indicating inhibition of microbenthos growth caused by the green algal bloom of E. prolifera.A total of 132 morphospecies, representing 13 classes/subclasses, 24 orders, and 55 genus were identified from the samples collected in summer 2008. Prostomatea was also the most predominant group accounting for 71% of total abundance and 64% of total biomass. The carnivores constituted about 53% of total biomass, followed by the bacterivores (33%), algivores (11%), and omnivores (1%). Statistical analyses show that ciliate abundance had positively correlated with bottom water salinity, water depth, and sediment organic matter content. Margalef diversity showed only positively correlation with bottom water temperature. Both Shannon-Wiener diversity and Pielou evenness indices showed negative correlations with water depth and organic matter content and positive with sediment median grain size, while the latter also showed significantly negative correlation with bottom water salinity. The BIOENV analysis show that bottom water temperature and salinity and sediment median grain size affecting the ciliate community structure. Except of Margalef diversity index, the standing crops, species number, Shannon diversity, and Pielou evenness indices of ciliates during the cruise in 2008 were lower than those in 2007, indicating inhibition of ciliate community structure and diversity caused by the green algal bloom of E. prolifera.Overall, the standing crops of microbenthos in the upper 5 cm of the sediments of the Yellow Sea were about two to four orders of magnitude higher than those of corresponding planktonic ones in the same sea area. The quantitively importance of microbenthos might relate with their functional importance, which indicate their important role in matter cycle and the transfer to higher trophic levels. An in-depth study of the standing crops and diversity dynamics of microbenthos will contribute to the understanding of the structure and function of benthic ecosystem.更多还原