淡水鱼类环境DNA宏条形码引物的筛选及其在千岛湖的应用

基于环境DNA(eDNA)技术的鱼类多样性监测方法因具有灵敏度高、对目标生物无伤害以及成本低等特点,近年来在国内外得到了广泛应用。eDNA方法的有效性往往取决于宏条形码引物的选择,尽管目前已经有一些鱼类环境DNA宏条形码引物,但较少有研究综合评估这些引物的检出效果。本研究评估了来自COI、Cytb、12S rRNA和16S rRNA基因的29对鱼类eDNA宏条形码引物(包括本研究设计的2对和从国内外文献中引用的27对引物),首先基于计算机模拟PCR(in silico PCR)进行了初步分析,随后通过高通量测序对其中效果较好的17对引物开展了更进一步的验证,结果显示:计算机模拟PCR结果良好的引物在进行高通量测序时并非全部表现良好,表明引物的筛选不能仅仅依靠计算机模拟PCR;具有更长扩增片段长度的宏条形码引物并没有获得理想中更好的扩增效果,表现效果好的引物大多是扩增片段长度处于200~300 bp之间的引物;相对于COI和Cytb而言,12S rRNA引物与16S rRNA引物均具有良好的扩增效果,适宜作为鱼类环境DNA宏条形码而用于鱼类多样性研究;同时,鉴于当前的鱼类DNA条形码数据库尚不完备以及不同引物的特性不同,使用多对引物将大大增加物种的检出概率和eDNA研究的可信性。本研究展示了eDNA在评估生物多样性方面的潜力,有助于将来的鱼类eDNA研究,从而为鱼类多样性的保护提供参考。     

基于环境DNA宏条形码技术的水体无脊椎动物多样性研究：以广州海珠湖为例

于2021年1月,利用环境DNA宏条形码（eDNA）技术,对广州海珠国家湿地公园海珠湖的水体和沉积物进行无脊椎动物多样性和群落结构调查,并比较了eDNA和传统形态学鉴定对浮游动物的检出能力。结果表明：eDNA检出海珠湖无脊椎动物9门16纲34目71科93属137个可操作分类单元（OTUs）;其中,水采样品中共检出9门50属68个OTUs,网采样品中共检出6门27属35个OTUs,沉积物休眠卵样品中共检出9门70属103个OTUs。水采和网采样品中,主要的无脊椎动物均为轮虫动物门和节肢动物门;沉积物中主要的无脊椎动物为环节动物门和节肢动物门。沉积物休眠卵OTU丰富度最高,水采样品的OTU丰富度高于网采样品。比较浮游动物的形态鉴定和eDNA鉴定,发现后者在属水平上能鉴定出更多的种类;形态学鉴定的桡足类（100%）和多数轮虫（58.82%）可被eDNA注释出,而eDNA注释出的多数桡足类（71.43%）和轮虫（58.82%）在形态学鉴定中未发现;eDNA未注释出枝角类。结果表明,eDNA在无脊椎动物调查中具有较高的应用潜力,与传统形态学鉴定组合应用能更全面地了解无脊椎动物的种类和水生生物多样...     

生物共现网络原理及其在淡水生态系统评估中的应用

快速有效的生物监测指标对于评估、保护、管理和恢复淡水生态系统至关重要.传统评估方法主要利用指示生物或类群的出现率和多度信息,但是忽略了水体环境中“生物”与“生物”,以及“生物”与“环境”间相互作用的复杂关系,而这些相互作用对淡水生态系统的生物多样性、生态系统服务功能以及生态系统对环境变化的响应有着深刻影响.生物共现网络是群落水平物种互作的结构模型,通过物种在群落出现及丰度数据,描述了物种间潜在的相互作用、群落的基本结构,反映群落在生态系统的功能和结构特性.生物共现网络展示了淡水生态系统中所有生物体之间潜在的相互作用关系,其拓扑结构特性可与特定的生态系统状态相关联,能够揭示生态系统的组织规律及其功能,可作为早期的、灵敏的生物指标,是一种很有应用前景的评估淡水生态系统状态和稳定性的工具.     

基于环境DNA宏条形码的丹江口水库浮游生物多样性及群落特征

为了解丹江口库区浮游生物群落的多样性,本研究于2020年7月在丹江口库区丹库、汉库和入库支流等区域共计9个样点采集水体样品,抽滤并提取总DNA样本后,基于18S和16S分子标记进行单分子实时测序,分别探究真核和原核浮游生物群落的多样性及其群落特征.结果表明：（1）真核浮游生物群落的主要优势类群包括节肢动物、链型植物、绿藻门、硅藻门等;本研究在种水平上鉴定出库区分布广泛且相对丰度较大的物种,包括弯曲隐藻、对蛋白核隐藻和空球藻等,它们与库区化学需氧量密切相关;库区化学需氧量是影响真核浮游生物群落格局的重要环境因子.（2）原核浮游生物群落的主要优势类群为变形菌门;原核生物中相对丰度较大的不动杆菌Acinetobacter是污水污染的指示菌群;群落中起重要作用的Limnohabitans与水体的富营养化密切相关;尽管库区蓝藻相对丰度较低,但与蓝藻关系密切的CL500-29_marine_group和hgcI_clade两类细菌在浮游生物群落中也起着重要作用;原核生物群落中的各类物种均指示丹江口水库的水生态健康存在一定风险,需要加强监测以预防生态环境的恶化.（3）丹江口水库不同区域的浮游生物群落异质性较大,tb-RDA分析显示丹江口的浮游生物群落可以分为丹库型、汉库型和入库支流型,其组间差异要大于组内差异.综上所述,丹江口水库的浮游生物群落具有明显的空间异质性,整个库区的群落结构与水体富营养化、水体有机污染和污水污染等方面相关,需要加强对丹江口库区的水生态监测.     

鄱阳湖流域蚌类环境DNA宏条形码引物的筛选验证

环境DNA技术是一种非侵入、高灵敏、高效率且对环境无破坏性,对生物体无损伤的调查工具.为了筛选适合于蚌类环境DNA生物多样性研究的宏条形码引物,本研究通过对鄱阳湖流域24种常见蚌的基因组DNA进行普通扩增和高通量测序筛选了11对引物(设计了9对通用引物及从相关文献中引用了2对引物),结果显示引物cytb和16S rRN...     

中国南亚热带水库敞水区春季轮虫群落结构:基于分类特征和功能性状的分析

轮虫是淡水后生浮游动物中种类最丰富的类群,是水体生态系统食物链的关键环节.很多种类的轮虫对环境变化敏感,环境条件的改变能够导致其群落结构的快速变化,因此,轮虫群落的组成与变化可从生态位角度得以解释.物种的功能性状可提供更强的生态适应性信号,轮虫的个体大小是反映其代谢与行为的综合性状,而咀嚼器、防御结构及防御行为则反映了它们对食物利用和应对捕食等方面的功能性状,轮虫群落中物种的这些功能性状组成反映了食物资源和捕食压力等对群落组成的选择作用.为了解我国南亚热带水库轮虫群落的结构特征,于2018年春季(3—5月)采集了我国华南地区43座水库敞水区的浮游动物定性样品和定量样品,同时测定了水温、水深、水体透明度及叶绿素a浓度等指标.共采集到轮虫19属45种,主要为营浮游生活的种类,异尾轮属(Trichocerca)和臂尾轮属(Brachionus)的种类最多,分别有10和7种.在轮虫个体大小上,群落中体长≤150 μm个体的丰度和生物量均占绝对优势(分别为83.11%和53.10%),体现出热带、亚热带地区轮虫群落以小个体种类占优势的一般特征;具有槌型咀嚼器、杖型咀嚼器的轮虫为优势咀嚼器功能群,分别占57.78%和33.14%;具背甲与棘刺的轮虫为主要优势防御结构功能群(66.59%).南亚热带水库春季轮虫群落在食物利用及应对捕食压力上的特点明显,冗余分析表明轮虫群落的种类组成及性状功能群的丰度组成显著地受到食物、温度及竞争的影响.     

汉江流域上游支流大型底栖动物群落结构特征与生物多样性

2003年6月至2004年6月,对汉江流域上游支流--黑竹冲河的大型底栖动物群落结构与生物多样性进行了深入研究,结果表明:河流生物群落结构和生物多样性比较复杂,河流功能状况良好.在采集到的85种大型底栖动物中,昆虫占71种;群落表现出明显的时空特点,具边缘效应的第4采样点物种最丰富,2月份物种最多,达50种;生物多样性指数周年动态显示,第1采样点的物种多样性变动最为平稳,第5采样点变动最大,其它各点居中;各微生境中共有种占据优势,物种相似性均大于60%;群落春季的多样性高于夏季,夏季高于秋季;功能摄食群则以收集者占优,共计达68种.群落密度在1月份达到最大,为1843 ind/m2;而生物量则在10月份达到最大,为228.7393 g/m2.     

1988-2016年洞庭湖大型底栖动物群落变化及驱动因素分析

洞庭湖是我国第二大淡水湖泊,其水文条件对湖泊湿地生态系统健康的维系发挥着不可替代的作用.近年来,水环境恶化日益威胁湖区水生态系统健康.然而,有关底栖动物水生态健康评价的研究仍然停留在物种群落结构方面,缺乏底栖动物群落功能对水污染响应的研究,尤其在较长时间尺度上.因而,本研究分析了19882016年近30 a来洞庭湖的水质和底栖动物群落数据,探寻底栖动物群落功能对水环境恶化的响应规律.结果表明,洞庭湖水体总氮浓度是威胁底栖动物物种和功能群落变动的主要因素.此外,不断恶化的水环境驱动底栖动物物种和功能群落结构改变,表现为敏感水生昆虫的比例下降,寡毛类、小型软体动物比例的上升,并伴随着体长为1.00~1.99 cm、背扁型、侧扁型、不移动等功能性状类别比例的下降.同时,水环境恶化降低物种丰富度、功能丰富度和劳氏二次熵多样性.基于距离的冗余分析结果显示,水体氮营养盐、重金属离子和有机污染物共同驱动底栖动物物种群落结构的变异,而营养盐类与无/有机污染物决定着其功能群落结构的变异.鉴于洞庭湖水质不断恶化的状况,本研究建议采取一系列措施,包括合理管控湖区周边废水直排入湖、取缔湖区内的非法采砂以及调控枯水季洞庭湖水位等.生物监测和评价方面,建议将底栖动物物种和功能群落一并纳入评价体系,且优先选用物种丰富度、功能丰富度和劳氏二次熵指数评估换水周期较短的大型浅水湖泊水质变化对底栖动物物种和功能多样性的影响.     

水生生物监测质量保证、质量控制与标准化:动态与展望

水生生物监测是流域水生态状况评价的重要基础,监测数据的准确性和可靠性直接影响水生态环境质量评价与流域水生态管理成效。目前我国水生生物监测质量保证和质量控制体系尚处于持续完善的阶段,关键环节的控制要求和质控目标等亟待明确和细化。本文总结了欧美发达国家水生生物监测质量保证与质量控制体系建设中的经验,通过梳理我国水生生物监测工作基础与进展,较为系统地提出了适合我国现状的水生生物监测质量保证与质量控制体系标准化建设对策建议,包括实验室内部和外部质量保证与质量控制、水生生物监测质控数据库、人工智能识别和环境DNA新技术质控要点等标准化建议,为实现水生生物监测业务化运行,准确评估我国流域水生态质量状况的变化提供技术支撑。     

采用倒置显微镜法定量浮游植物的数据稳定性

浮游植物种类组成细胞密度或生物量的现存量反映其在水生生态系统的结构与功能,准确地对水体中浮游植物进行定量是水质评价和生态功能分析的基础.针对目前国际上推荐使用的倒置显微镜法(即Utermhl计数法),通过采集处于不同营养状态和水体条件(水库和实验围隔)中的浮游植物,分析样品的显微计数量、水体营养状态对浮游植物密度和多样性等指标稳定性的影响,同时比较了多个水体中同一采样点的重复(或平行)样品之间浮游植物定量数据的差别,从而对倒置显微镜法进行较为系统的评估.结果表明,基于浮游植物的显微镜计数效率与定量数据稳定性的综合考虑,选择计数400个个体即可基本保证定量数据的稳定性;在依赖生物量或稀有种进行水质评价时,处于不同营养水平的水体均需要增加样品的平行数来提高定量数据的可靠性,贫营养型水体中单个采样的重复或平行样品更为必要;两种定量方法所得群落数据计算的辛普森指数无显著差异,说明两种方法所获得结果均能反映浮游植多样性;通过样品浓缩法和倒置显微镜法所获得的浮游植物生物量和细胞密度均具有显著差异,因样品浓缩法在样品处理过程中造成浮游植物损失,使通过样品浓缩法所得的浮游植物群落生物量及细胞密度偏小;相比浓缩法,倒置显微镜法沉淀浓缩的水样体积小,样品处理和计数耗时短,更适宜用于应急监测.     

Comparing efficacy of different taxonomic resolutions and surrogates in detecting changes in soft bottom assemblages due to coastal defence structures

Sandy shores on the West coast of the North Adriatic Sea are extensively protected by different types of defence structures to prevent coastal erosion. Coastal defence schemes modify the hydrodynamic regime, the sediment structure and composition thus affecting the benthic assemblages. This study examines the effectiveness in detecting changes in soft bottom assemblages caused by coastal defence structures by using different levels of taxonomic resolution, polychaetes and/or bivalves as surrogates and different data transformations. A synoptic analyses of three datasets of subtidal benthic macrofauna used in studies aimed at assessing the impact of breakwaters along the North Adriatic coast has been done. Analyses of similarities and correlations between distance matrices were done using matrices with different levels of taxonomic resolution, and with polychaetes or bivalves data alone. Lentidium mediterraneum was the most abundant species in all datasets. Its abundance was not consistently related to the presence of defence structures. Moreover, distribution patterns of L. mediterraneum were masking the structure of the whole macrofaunal assemblages. Removal of L. mediterraneum from the datasets allowed the detection of changes in benthic assemblages due to coastal defences. Analyses on different levels of taxonomic resolution showed that the level of family maintained sufficient information to detect the impacts of coastal defence structures on benthic assemblages. Moreover, the outcomes depended on the transformation used. Patterns of distribution of bivalves, used as surrogates, showed low correlations with the patterns of the total macrofaunal species assemblages. Patterns of polychaetes, if identified to the species or genus level showed higher correlations with the whole dataset. However, the identification of polychaetes to species and genus level is as costly as the identification of all macrobenthic taxa at family level.This study provided additional evidences that taxonomic sufficiency is a useful tool in environmental monitoring, also in investigations on the impacts of coastal defence structures on subtidal macrofauna. The use of coarser taxonomic level, being time-efficient, would allow improving sampling designs of monitoring programs by increasing replication in space and time and by allowing long term monitoring studies.     

基于综合指标法的鄱阳湖生态系统健康评价

由于湖泊生态问题日益突出,湖泊生态系统安全状态已经成为人们关注的热点问题,了解湖泊水生态系统的状况并根据湖泊生态系统健康状况开展精准治理和生态修复与保护尤为重要。本文基于对鄱阳湖及其流域生态环境的长期监测数据和资料收集,采用综合指标体系法,从物理形态、水文、水环境、水域生态、湿地生态和社会服务6个方面构建了鄱阳湖生态系统健康评估的指标体系,主要涵盖了湖泊口门状况、“五河”入湖径流变异程度、入湖河流水质达标率等26个指标。依据设置的阈值等级得到鄱阳湖生态系统健康评价各层次健康状况等级,通过对各湖泊生态系统各指标得分进行加权计算,得出生态系统健康评估准则层和目标层的得分,最终对鄱阳湖生态系统健康进行了客观的评价。结果表明,构建的湖泊生态系统健康评价体系针对性强、科学全面、具有可操作性,可为鄱阳湖及类似通江湖泊的生态系统健康评价提供案例和方法借鉴。评价结果表明鄱阳湖健康体征状况目标层得分为73.45分,评价结果为亚健康,鄱阳湖水生态系统健康主要受泄流能力、水文节律变化、富营养化程度和物种多样性的影响。最后根据鄱阳湖的水生态系统健康评分等级探讨了鄱阳湖水生态系统中亟需解决的问题,针对性地提出了...     

Cost/benefit analysis of a benthic monitoring programme of organic benthic enrichment using different sampling and analysis methods

To investigate the combined effects of decreasing taxonomic resolution (i.e. species, family, phylum), the use of different mesh-size (1.0 mm and 0.5 mm) and the type of samplers used (van Veen vs. corers taken by divers) on the quality of data obtained, a comparative study was undertaken with the overall aim of identifying cost efficient methods for routinely monitoring the ecological change caused by Mediterranean fish farming. The results clearly showed that information loss was relatively low as data were aggregated at higher taxonomic levels, particularly up to the level of family or even order. It was also found that the extra information gained by sieving samples through a 0.5 mm sieve did not improve the ability to distinguish the potentially impacted sites from the control stations. Finally, it was found that a relatively large proportion of the available information concerning the community structure such as abundance, biomass or diversity is lost when sampling is carried out with corers. A cost/benefit ratio analysis for the two sampling and the two sieving methods showed minimal values for the van Veen samples (for both sieve fractions) at the family level, indicating that analysis at this level gives the best balance between precision of the results and decrease in taxonomic effort. However, if the time needed to sort the samples is included in the analysis, then samples taken with corers using a 0.5 mm sieve and identified to families seems like a good compromise between precision and cost.     

Estimating the Sampling Uncertainty for the Analysis of Solid Refused Fuel

Uncertainty assessment of the parameters used for the reporting in the emissions trading system (ETS) to characterize fuels includes not only the processes in a laboratory but has to be expanded for inclusion of sampling and sample preparation. Analysis of variance was used to allocate the contributing uncertainties to the steps described. Apart from some specific results for sampling methods, it was observed that the uncertainty of sampling was in the same order of magnitude than the analytical and the sample preparation error. Several measures to enhance quality assurance in sampling and sample preparation could be derived from specific results. The results were used in the European Monitoring and Reporting Regulation, which set out the requirements for ETS. Sampling and sample preparation have to be included in uncertainty assessment as well as sampling procedures and sampling plans (including quality assurance) have to be agreed upon by the laboratories, carrying out the analytics of those samples.     

Efficiency of sampling invertebrates in groundwater habitats

Sampling efficiency is directly linked to (faunistic) stability issues of any sampled site. It is more probable to sample a high proportion of the species occurring at a certain site, if communities remain constant in species composition with time. For this study 64 groundwater monitoring wells in Southwestern Germany were sampled six times to determine sampling efficiency. False-Negative (FN) rates and SIMPER values, two independent models expressing Faunistic values of the sampled bores, were calculated and three groups of faunistic stability could be identified based on calculated SIMPER-values. FN-rates as well as SIMPER values proved to be a valuable approach for the estimation of faunistic stability/instability, as these methods showed a highly negative correlation with each other. To collect 95% of species a number of 1.7–16.4 samples was calculated to be necessary depending on faunistic stability of the sampled habitat. On the six sampling occasions the sites harbouring a stable groundwater meiofauna resulted in 98.2% of occurring species, whereas the percentage decreased over intermediate sites (94.9%) and stressed sites (79.5%).A consequence of the data presented may be to view surveys using only two samples as campaign with orienting character, with further studies of stability issues of groundwater communities which would provide efficient and effective sampling for various kinds of sites.     

Small Purge Method to Sample Vapor from Groundwater Monitoring Wells Screened Across the Water Table

Groundwater monitoring wells are present at most hydrocarbon release sites that are being assessed for cleanup. If screened across the vadose zone, these wells provide an opportunity to collect vapor samples that can be used in the evaluation of vapor movement and biodegradation processes occurring at such sites. This paper presents a low purge volume method (modified after that developed by the U.S. EPA) for sampling vapor from monitoring wells that is easy to implement and can provide an assessment of the soil gas total petroleum hydrocarbon (TPH) and O₂ concentrations at the base of the vadose zone. As a result, the small purge method allows for sampling of vapor from monitoring wells to support petroleum vapor intrusion (PVI) risk assessment. The small purge volume method was field tested at the Hal's service station site in Green River, Utah. This site is well‐known for numerous soil gas measurements containing high O₂ and high TPH vapor concentrations in the same samples which is inconsistent with well‐accepted biodegradation models for the vapor pathway. Using the low purge volume method, monitoring wells were sampled over, upgradient, and downgradient of the light nonaqueous phase liquid (LNAPL) footprint. Results from our testing at Hal's show that vapor from monitoring wells over LNAPL contained very low O₂ and high TPH concentrations. In contrast, vapor from monitoring wells not over LNAPL contained high O₂ and low TPH concentrations. The results of this study show that a low purge volume method is consistent with biodegradation models especially for sampling at sites where low permeability soils exist in and around a LNAPL source zone.