基因组学技术在生物多样性保护研究中的应用

在分子生物学、细胞生物学、微生物学、遗传学等学科的推动下, 生物多样性研究从仅关注宏观表型的博物学, 迅速演化为涵盖生态系统、物种和遗传多样性等多个维度的综合性生命科学。组学技术, 尤其是DNA测序技术的更新和发展, 使获取DNA序列所需的成本大幅下降, 促进了近年来其在生物多样性研究中取得的一系列令人瞩目成就。本文将从物种水平的遗传多样性和群落水平的物种多样性两个层面总结和介绍与DNA相关的组学技术在生物多样性研究中的一些创新和应用。其中, 物种水平主要是总结单一个体的基因组和单物种多个体在时空多个维度上的群体遗传研究; 而群落水平的物种多样性层面主要总结现有的分子鉴定技术(metabarcoding, eDNA, iDNA等), 以及上述新技术在群落多样性评估、旗舰保护物种监测以及物种间相互作用关系等研究中的应用。     

环境DNA metabarcoding及其在生态学研究中的应用

环境DNA metabarcoding(eDNA metabarcoding)是指利用环境样本(如土壤、水、粪便等)中分离的DNA进行高通量的多个物种(或高级分类单元)鉴定的方法。近年来,该方法引起了学者的广泛关注,逐渐应用于生物多样性研究、水生生物监测、珍稀濒危物种和外来入侵物种检测等生态学领域。介绍环境DNA metabarcoding的含义和研究方法;重点介绍环境DNA metabarcoding在物种监测、生物多样性研究和食性分析等生态学领域中的应用;总结环境DNA metabarcoding应用于生态学研究领域面临的挑战并对该方法的发展进行展望。     

从物种识别到生物多样性评估——DNA条形码与DNA metabarcoding技术

在生命科学研究中,物种识别和生物多样性评估是重要的基础环节。从基于形态学特征分类的经典分类学,到近来被分子分类学家广泛接受的DNA条形码,以及在高通量测序技术基础上衍生出的DNA metabarcoding研究方法,人类正以前所未有的速度与深度重新认识生命世界。DNA metabarcoding可快速、简便、有效地从多物种混合DNA样本中还原其中的生物类群及其居群结构,实现从物种的有效识别到生物多样性的评估。概述了DNA条形码与DNA metabarcoding的概念、技术方法、应用及最新进展。     

基于DNA metabarcoding技术的如意金黄散处方成分鉴定研究

中药的有效性和安全性是国内外关注的热点,本研究以如意金黄散为例,基于DNA metabarcoding技术,尝试从投料药材真伪角度评价中药的有效性和安全性.首先,收集如意金黄散处方成分药材及其混伪品样品161份,从已发表文献中下载密切相关物种序列154条,构建"如意金黄散DNA条形码鉴定数据库",该数据库包含10属119个物种.收集不同批次的如意金黄散样品3份,以ITS2序列为条形码,使用Illumina HiSeq平台进行PE250深度测序,利用本研究构建的"如意金黄散DNA条形码鉴定数据库"进行投料药材基原物种识别,结果表明,基于DNA metabarcoding技术可检测到除厚朴外的全部处方成分,不同药材的测序量存在显著差异;检测到苍术药材的混伪品朝鲜苍术和天南星药材的混伪品虎掌南星,提示如意金黄散临床使用的有效性和安全性存在潜在风险;不同批次鉴定结果具有稳定性和可重复性.本研究表明,基于DNA metabarcoding技术的如意金黄散处方成分鉴定方法符合中药复杂体系特点,有望成为中成药鉴定的新方法,并为中成药的有效性和安全性评价提供参考.     

丛枝菌根真菌最新分类系统与物种多样性研究概况

丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)是自然界分布最广泛的一类植物共生真菌,能够与大部分高等植物的根系形成共生关系.由于它们在农林、环境等领域的巨大应用潜力,国内外关于AMF物种多样性的研究一直受到较高的关注.然而,AMF专性共生的特征以及研究方法不够理想等因素长期阻碍了AMF物种多样性的研究进展.近年来,研究方法的改进与新技术的应用为AMF物种多样性的研究提供了极好的机遇.简述了AMF的最新分类系统及全球物种数量、AMF物种多样性影响因素以及AMF物种多样性研究方法三个方面的研究进展,并分析了今后在AMF物种多样性相关领域值得关注的研究方向.     

丛枝菌根真菌物种多样性研究进展

丛枝菌根真菌(Arbuscular mycorrhizal fungi,AMF)在不同生态系统均发挥至关重要的作用,研究其多样性能够为AMF物种资源的保护和利用提供科学依据。AMF不能被离体纯培养以及自身的高变异性等因素严重阻碍了对其进行深入研究,随着研究方法的不断改进,尤其是新一代测序技术的运用,极大加速了人们对AMF物种多样性的认识。本文主要从AMF分类系统、不同宿主植物和不同生境中的AMF物种多样性及AMF物种多样性研究方法(包括形态鉴定、Sanger测序和高通量测序)方面介绍AMF物种多样性研究进展,并且探讨AMF物种多样性研究中存在的主要问题,认为在今后AMF物种多样性研究中不仅要注重运用新的研究手段,还应该着重解决AMF不能离体纯培养的问题。     

环境DNA宏条形码在生物多样性研究与监测中的应用

环境DNA宏条形码(eDNA metabarcoding)技术通过提取水体、土壤、空气中的环境DNA,使用引物PCR扩增与高通量测序,进行物种鉴定与生物多样性评估.作为一种新的监测技术,相比于传统监测技术更加快捷、准确以及对自然环境的破坏小,因此在一定程度上改变了我们调查地球生物多样性的方式.本文综述了环境DNA宏条形...     

戴云山国家级自然保护区访花昆虫DNA条形码数据集

访花和传粉昆虫对于维持生态系统功能具有重要作用,但我国相关昆虫类群的本底数据非常缺乏。作为基于特定基因序列的物种划分方法, DNA条形码在标本鉴定、新物种发现、生物多样性保护、种群遗传和进化等研究领域具有重要的应用价值。本文报道了福建戴云山国家级自然保护区双翅目、膜翅目和鞘翅目3个类群访花昆虫的815条线粒体COI条形码数据,并详细提供了所获样品的海拔分布信息。该数据集可为地区性昆虫多样性的DNA条形码数据库构建、隐存种发现、海拔梯度物种遗传多样性和生物多样性保护等方面研究提供帮助。     

生物多样性保护廊道构建方法研究进展

生物多样性保护廊道对遏制生态系统退化及生物多样性丧失,改善生态系统服务功能,消除生境破碎化对生物多样性的影响,恢复珍稀濒危物种的种群数量,维护自然生态系统平衡稳定具有极为重要的作用。在近20年(1997—2017)国内外生物多样性保护廊道的相关研究分析的基础上,对廊道的概念、构建理论及方法应用进行了系统总结与探讨,分析了廊道构建理论的发展过程及适用性,分类总结了现有的廊道构建方法和17种廊道构建模型工具。研究分析表明,廊道作为一种新的生物多样性保护模式,已成为目前国际生态领域研究的热点之一,随着对物种景观运动过程认识的加深,廊道构建理论逐渐趋于成熟,与之匹配的廊道构建方法及模型工具进展迅速。借助遥感与地理信息技术,大范围,高精度的获取廊道模拟数据,并集成综合模型实现目标物种廊道的构建、保护和管理是今后生物多样性保护廊道构建研究的发展方向。最后,对当前该领域的研究现状和不足展开讨论并展望了未来发展,为我国生物多样性保护廊道的应用与实践及国家生态廊道体系的建设完善提供借鉴与参考。     

生物多样性保护廊道构建方法研究进展

生物多样性保护廊道对遏制生态系统退化及生物多样性丧失,改善生态系统服务功能,消除生境破碎化对生物多样性的影响,恢复珍稀濒危物种的种群数量,维护自然生态系统平衡稳定具有极为重要的作用。在近20年(1997—2017)国内外生物多样性保护廊道的相关研究分析的基础上,对廊道的概念、构建理论及方法应用进行了系统总结与探讨,分析了廊道构建理论的发展过程及适用性,分类总结了现有的廊道构建方法和17种廊道构建模型工具。研究分析表明,廊道作为一种新的生物多样性保护模式,已成为目前国际生态领域研究的热点之一,随着对物种景观运动过程认识的加深,廊道构建理论逐渐趋于成熟,与之匹配的廊道构建方法及模型工具进展迅速。借助遥感与地理信息技术,大范围,高精度的获取廊道模拟数据,并集成综合模型实现目标物种廊道的构建、保护和管理是今后生物多样性保护廊道构建研究的发展方向。最后,对当前该领域的研究现状和不足展开讨论并展望了未来发展,为我国生物多样性保护廊道的应用与实践及国家生态廊道体系的建设完善提供借鉴与参考。     

Fungal diversity living in the root and sporophore of the endemic Korean fern Mankyua chejuense

Ferns represent the basal group of vascular plants and are known to have fungal interactions with arbuscular mycorrhizal fungi, but diversity of endophytic fungi from ferns is rarely studied. Moreover, fungal diversity associated with ferns is likely underestimated as most studies have been performed based on a microscopic or culture-dependent approach. In this study, we investigated the endophytic fungal diversity within roots and sporophore of an endangered Korean fern (Mankyua chejuense), and compared it to fungi in surrounding soil using a metabarcoding approach. A high diversity of endophytic fungi (236 OTUs), mostly belonging to Ascomycota, was detected and fungal richness and composition were significantly different between habitats. Indicator species analysis showed that endophytic fungi have similar ecological characteristics to fungal species found from other land plants. Our results suggest that various fungal species are associated with ferns, thus understanding fern-associated fungal diversity can have a great implication for fern biology and conservation.     

Vector soup: high‐throughput identification of Neotropical phlebotomine sand flies using metabarcoding

Phlebotomine sand flies are haematophagous dipterans of primary medical importance. They represent the only proven vectors of leishmaniasis worldwide and are involved in the transmission of various other pathogens. Studying the ecology of sand flies is crucial to understand the epidemiology of leishmaniasis and further control this disease. A major limitation in this regard is that traditional morphological‐based methods for sand fly species identifications are time‐consuming and require taxonomic expertise. DNA metabarcoding holds great promise in overcoming this issue by allowing the identification of multiple species from a single bulk sample. Here, we assessed the reliability of a short insect metabarcode located in the mitochondrial 16S rRNA for the identification of Neotropical sand flies, and constructed a reference database for 40 species found in French Guiana. Then, we conducted a metabarcoding experiment on sand flies mixtures of known content and showed that the method allows an accurate identification of specimens in pools. Finally, we applied metabarcoding to field samples caught in a 1‐ha forest plot in French Guiana. Besides providing reliable molecular data for species‐level assignations of phlebotomine sand flies, our study proves the efficiency of metabarcoding based on the mitochondrial 16S rRNA for studying sand fly diversity from bulk samples. The application of this high‐throughput identification procedure to field samples can provide great opportunities for vector monitoring and eco‐epidemiological studies.     

Unlocking biodiversity and conservation studies in high‐diversity environments using environmental DNA (eDNA): A test with Guianese freshwater fishes

Determining the species compositions of local assemblages is a prerequisite to understanding how anthropogenic disturbances affect biodiversity. However, biodiversity measurements often remain incomplete due to the limited efficiency of sampling methods. This is particularly true in freshwater tropical environments that host rich fish assemblages, for which assessments are uncertain and often rely on destructive methods. Developing an efficient and nondestructive method to assess biodiversity in tropical freshwaters is highly important. In this study, we tested the efficiency of environmental DNA (eDNA) metabarcoding to assess the fish diversity of 39 Guianese sites. We compared the diversity and composition of assemblages obtained using traditional and metabarcoding methods. More than 7,000 individual fish belonging to 203 Guianese fish species were collected by traditional sampling methods, and ~17 million reads were produced by metabarcoding, among which ~8 million reads were assigned to 148 fish taxonomic units, including 132 fish species. The two methods detected a similar number of species at each site, but the species identities partially matched. The assemblage compositions from the different drainage basins were better discriminated using metabarcoding, revealing that while traditional methods provide a more complete but spatially limited inventory of fish assemblages, metabarcoding provides a more partial but spatially extensive inventory. eDNA metabarcoding can therefore be used for rapid and large‐scale biodiversity assessments, while at a local scale, the two approaches are complementary and enable an understanding of realistic fish biodiversity.     

Enhancing DNA metabarcoding performance and applicability with bait capture enrichment and DNA from conservative ethanol

Metabarcoding is often presented as an alternative identification tool to compensate for coarse taxonomic resolution and misidentification encountered with traditional morphological approaches. However, metabarcoding comes with two major impediments which slow down its adoption. First, the picking and destruction of organisms for DNA extraction are time and cost consuming and do not allow organism conservation for further evaluations. Second, current metabarcoding protocols include a PCR enrichment step which induces errors in the estimation of species diversity and relative biomasses. In this study, we first evaluated the capacity of capture enrichment to replace PCR enrichment using controlled freshwater macrozoobenthos mock communities. Then, we tested if DNA extracted from the fixative ethanol (etDNA) of the same mock communities can be used as an alternative to DNA extracted from pools of whole organisms (bulk DNA). We show that capture enrichment provides more reliable and accurate representation of species occurrences and relative biomasses in comparison with PCR enrichment for bulk DNA. While etDNA does not permit to estimate relative biomasses, etDNA and bulk DNA provide equivalent species detection rates. Thanks to its robustness to mismatches, capture enrichment is already an efficient alternative to PCR enrichment for metabarcoding and, if coupled to etDNA, is a time‐saver option in studies where presence information only is sufficient.     

Spineless and overlooked: DNA metabarcoding of autonomous reef monitoring structures reveals intra- and interspecific genetic diversity in Mediterranean invertebrates

The ability to gather genetic information using DNA metabarcoding of bulk samples obtained directly from the environment is crucial to determine biodiversity baselines and understand population dynamics in the marine realm. While DNA metabarcoding is effective in evaluating biodiversity at community level, genetic patterns within species are often concealed in metabarcoding studies and overlooked for marine invertebrates. In the present study, we implement recently developed bioinformatics tools to investigate intraspecific genetic variability for invertebrate taxa in the Mediterranean Sea. Using metabarcoding samples from Autonomous Reef Monitoring Structures (ARMS) deployed in three locations, we present haplotypes and diversity estimates for 145 unique species. While overall genetic diversity was low, we identified several species with high diversity records and potential cryptic lineages. Further, we emphasize the spatial scale of genetic variability, which was observed from locations to individual sampling units (ARMS). We carried out a population genetic analysis of several important yet understudied species, which highlights the current knowledge gap concerning intraspecific genetic patterns for the target taxa in the Mediterranean basin. Our approach considerably enhances biodiversity monitoring of charismatic and understudied Mediterranean species, which can be incorporated into ARMS surveys.     

Using spatial analysis to monitor tree diversity at a large scale: a case study in Northeast China Transect

Aims Monitoring and assessing diversity change at a large scale is important for any meaningful biodiversity conservation and management. Spatial analysis techniques can provide information about different aspects of diversity distribution including change. We applied some common spatial analysis methods and additive partitioning of species diversity in the Northeast China Transect as a case study to show how to characterize the distribution and change of tree diversity in this area from different perspectives.Methods The field data were collected from the permanent plots conducted every 4 km. The additive partitioning of species diversity was used to characterize the diversity of tree species at different scales. Moran's I was used for identifying the spatial scale of autocorrelation, lacunarity was studied for diversity patch contagion and dispersion and spectral entropy was used for assessing the overall spatial distribution.Important findings Data collected from 1986 to 1994 indicate that the change of α diversity was not significant in the study area, but the change of β diversity was significant. The percentage of α diversity in total diversity (γ) increased from 14.2 to 17.2%, and the percentage of β diversity decreased from 85.8 to 82.8%. For both α and β diversities, the scale of spatial autocorrelation decreased at the scale of 25–40 km and increased around 15–20 and 200 km. The lacunarity of α diversity decreased significantly and there was a sudden change at the scale of 56–68 km, but the lacunarity of β diversity increased across scales. The spectral entropy decreased slightly in α diversity and remained similar for β diversity. By using spatial analysis, we can monitor the diversity change over a large area and also assess the effectiveness of the current conservation strategies.     

Performance of amplicon and shotgun sequencing for accurate biomass estimation in invertebrate community samples

New applications of DNA and RNA sequencing are expanding the field of biodiversity discovery and ecological monitoring, yet questions remain regarding precision and efficiency. Due to primer bias, the ability of metabarcoding to accurately depict biomass of different taxa from bulk communities remains unclear, while PCR‐free whole mitochondrial genome (mitogenome) sequencing may provide a more reliable alternative. Here, we used a set of documented mock communities comprising 13 species of freshwater macroinvertebrates of estimated individual biomass, to compare the detection efficiency of COI metabarcoding (three different amplicons) and shotgun mitogenome sequencing. Additionally, we used individual COI barcoding and de novo mitochondrial genome sequencing, to provide reference sequences for OTU assignment and metagenome mapping (mitogenome skimming), respectively. We found that, even though both methods occasionally failed to recover very low abundance species, metabarcoding was less consistent, by failing to recover some species with higher abundances, probably due to primer bias. Shotgun sequencing results provided highly significant correlations between read number and biomass in all but one species. Conversely, the read–biomass relationships obtained from metabarcoding varied across amplicons. Specifically, we found significant relationships for eight of 13 (amplicons B1FR‐450 bp, FF130R‐130 bp) or four of 13 (amplicon FFFR, 658 bp) species. Combining the results of all three COI amplicons (multiamplicon approach) improved the read–biomass correlations for some of the species. Overall, mitogenomic sequencing yielded more informative predictions of biomass content from bulk macroinvertebrate communities than metabarcoding. However, for large‐scale ecological studies, metabarcoding currently remains the most commonly used approach for diversity assessment.     

Efficacy of metabarcoding for identification of fish eggs evaluated with mock communities

There is urgent need for effective and efficient monitoring of marine fish populations. Monitoring eggs and larval fish may be more informative than that traditional fish surveys since ichthyoplankton surveys reveal the reproductive activities of fish populations, which directly impact their population trajectories. Ichthyoplankton surveys have turned to molecular methods (DNA barcoding & metabarcoding) for identification of eggs and larval fish due to challenges of morphological identification. In this study, we examine the effectiveness of using metabarcoding methods on mock communities of known fish egg DNA. We constructed six mock communities with known ratios of species. In addition, we analyzed two samples from a large field collection of fish eggs and compared metabarcoding results with traditional DNA barcoding results. We examine the ability of our metabarcoding methods to detect species and relative proportion of species identified in each mock community. We found that our metabarcoding methods were able to detect species at very low input proportions; however, levels of successful detection depended on the markers used in amplification, suggesting that the use of multiple markers is desirable. Variability in our quantitative results may result from amplification bias as well as interspecific variation in mitochondrial DNA copy number. Our results demonstrate that there remain significant challenges to using metabarcoding for estimating proportional species composition; however, the results provide important insights into understanding how to interpret metabarcoding data. This study will aid in the continuing development of efficient molecular methods of biological monitoring for fisheries management.     

基于环境DNA-宏条形码技术的底栖动物监测及水质评价研究进展

底栖动物是淡水生态系统中物种多样性最高的类群,也是应用最广泛的水质监测指示生物之一。传统的底栖动物监测以形态学为基础,耗时费力,无法满足流域尺度大规模监测的需求。环境DNA-宏条形码技术是一种新兴的生物监测方法,其与传统方法相比优势在于采样方法简单、低成本、高灵敏度,不受生物样本和环境状况的影响,不依赖分类专家和鉴定资料,能够快速准确地对多个类群进行大规模、高通量的物种鉴定。然而,在实际应用中该方法的效果受诸多因素的影响,不同的方法、流程往往会产生差异较大的结果。鉴于此,着重分析总结了应用环境DNA-宏条形码技术监测底栖动物的关键影响因素,包括样品采集与处理流程、分子标记选择、引物设计、PCR偏好性、参考数据库的完整性及相应的优化。并基于此探讨了提高环境DNA-宏条形码技术在底栖动物监测效率和准确率的途径,以期为底栖动物环境DNA-宏条形码监测方案的制定提供可靠的参考。最后对该技术在底栖动物监测和水质评价中的最新发展方向进行了展望。     

Environmental monitoring through protist next‐generation sequencing metabarcoding: assessing the impact of fish farming on benthic foraminifera communities

The measurement of species diversity represents a powerful tool for assessing the impacts of human activities on marine ecosystems. Traditionally, the impact of fish farming on the coastal environment is evaluated by monitoring the dynamics of macrobenthic infaunal populations. However, taxonomic sorting and morphology‐based identification of the macrobenthos demand highly trained specialists and are extremely time‐consuming and costly, making it unsuitable for large‐scale biomonitoring efforts involving numerous samples. Here, we propose to alleviate this laborious task by developing protist metabarcoding tools based on next‐generation sequencing (NGS) of environmental DNA and RNA extracted from sediment samples. In this study, we analysed the response of benthic foraminiferal communities to the variation of environmental gradients associated with salmon farms in Scotland. We investigated the foraminiferal diversity based on ribosomal minibarcode sequences generated by the Illumina NGS technology. We compared the molecular data with morphospecies counts and with environmental gradients, including distance to cages and redox used as a proxy for sediment oxygenation. Our study revealed high variations between foraminiferal communities collected in the vicinity of fish farms and at distant locations. We found evidence for species richness decrease in impacted sites, especially visible in the RNA data. We also detected some candidate bioindicator foraminiferal species. Based on this proof‐of‐concept study, we conclude that NGS metabarcoding using foraminifera and other protists has potential to become a new tool for surveying the impact of aquaculture and other industrial activities in the marine environment.