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基于eDNA技术的造礁珊瑚属级分类多样性监测:多引物联用有效性评估
段晓伟, 洪淼, 王建佳, 黄丁勇, 周曦杰, 郑新庆
海洋学研究 ›› 2026, Vol. 44 ›› Issue (3) : 76-82.
PDF(2454 KB)
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基于eDNA技术的造礁珊瑚属级分类多样性监测:多引物联用有效性评估
Monitoring genus-level diversity of scleractinian corals based on eDNA technology: effectiveness evaluation of multi-primer combination
环境DNA(eDNA)宏条形码技术作为一种新兴工具,已被广泛应用于海洋生物多样性调查与研究。但受引物特异性不足、珊瑚分子参考数据库准确性有限等因素制约,该技术在珊瑚多样性监测中的应用效果仍有待系统评估。本研究采用CoralITS2、CoralITS2_acro与12S三对引物,对珊瑚属组成明确的养殖珊瑚体系开展eDNA宏条形码检测,探究不同引物单独使用或组合联用对造礁珊瑚属级分类多样性的检出差异与应用效果。结果显示,CoralITS2与12S引物均可检出19个隶属于石珊瑚目的珊瑚属,而CoralITS2_acro引物仅检出11个属,不同引物的物种检测结果存在明显互补性。多引物联用扩增共计检出27个石珊瑚目下的珊瑚属,覆盖体系内实际珊瑚属总数的87.5%。相较于单一引物检测,多引物联用能够有效缩小生物检测盲区、显著提升造礁石珊瑚属级类群的检出率。多引物联用策略可有效优化造礁石珊瑚群落的eDNA监测方案,是提升珊瑚群落调查完整性与准确性的有效手段。本研究同时发现,eDNA测序注释结果中出现6个研究体系内未实际分布的珊瑚属,表明该技术存在一定的假阳性风险。后续可通过完善本地造礁珊瑚参考序列数据库、构建标准化的多引物联合扩增分析流程,进一步提升eDNA宏条形码技术在珊瑚多样性监测中的精准性和可靠性。
Environmental DNA (eDNA) metabarcoding has been widely applied in marine biodiversity monitoring and research. However, its effectiveness in coral diversity monitoring requires further evaluation due to limitations in primer specificity and the accuracy of coral molecular databases. In this study, three primer pairs—CoralITS2, CoralITS2_acro, and 12S—were used to conduct eDNA metabarcoding detection in a coral aquaculture system with a well-defined genus composition, aiming to investigate the differences in detection and application effectiveness of individual primers and their combinations on the genus-level taxonomic diversity of scleractinian corals. The results showed that both CoralITS2 and 12S primers detected 19 coral genera belonging to the order Scleractinia, whereas CoralITS2-acro detected only 11 genera, indicating clear complementarity among the primers. The combined amplification using multiple primers detected a total of 27 scleractinian coral genera, representing 87.5% of the total coral genera known to be present in the system. Compared with single-primer detection, the multi-primer combination effectively narrowed detection blind spots and significantly improved the detection rate of scleractinian coral genus-level taxa. The multi-primer strategy can effectively optimize the eDNA monitoring protocol for scleractinian coral communities and serves as an effective means to enhance the completeness and accuracy of coral community surveys. Meanwhile, we also found that the eDNA sequencing annotation results identified six coral genera that were not actually distributed in the study system, indicating a certain risk of false positives in this technology. Future improvements in the precision and reliability of eDNA metabarcoding in coral diversity monitoring could be pursued by refining local reference sequence databases for scleractinian corals and establishing a standardized multi-primer combined amplification and analysis pipeline.
石珊瑚 / eDNA / 12S / ITS2 / 分子标记 / 物种多样性 / 生物多样性监测
scleractinian corals / eDNA / 12S / ITS2 / molecular marker / species diversity / biodiversity monitoring
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Atmospheric carbon dioxide concentration is expected to exceed 500 parts per million and global temperatures to rise by at least 2 degrees C by 2050 to 2100, values that significantly exceed those of at least the past 420,000 years during which most extant marine organisms evolved. Under conditions expected in the 21st century, global warming and ocean acidification will compromise carbonate accretion, with corals becoming increasingly rare on reef systems. The result will be less diverse reef communities and carbonate reef structures that fail to be maintained. Climate change also exacerbates local stresses from declining water quality and overexploitation of key species, driving reefs increasingly toward the tipping point for functional collapse. This review presents future scenarios for coral reefs that predict increasingly serious consequences for reef-associated fisheries, tourism, coastal protection, and people. As the International Year of the Reef 2008 begins, scaled-up management intervention and decisive action on global emissions are required if the loss of coral-dominated ecosystems is to be avoided.
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The assessment of species distribution is a first critical phase of biodiversity studies and is necessary to many disciplines such as biogeography, conservation biology and ecology. However, several species are difficult to detect, especially during particular time periods or developmental stages, potentially biasing study outcomes. Here we present a novel approach, based on the limited persistence of DNA in the environment, to detect the presence of a species in fresh water. We used specific primers that amplify short mitochondrial DNA sequences to track the presence of a frog (Rana catesbeiana) in controlled environments and natural wetlands. A multi-sampling approach allowed for species detection in all environments where it was present, even at low densities. The reliability of the results was demonstrated by the identification of amplified DNA fragments, using traditional sequencing and parallel pyrosequencing techniques. As the environment can retain the molecular imprint of inhabiting species, our approach allows the reliable detection of secretive organisms in wetlands without direct observation. Combined with massive sequencing and the development of DNA barcodes that enable species identification, this approach opens new perspectives for the assessment of current biodiversity from environmental samples.
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Cumulative anthropogenic pressures have triggered a global decline in the health of marine ecosystems, and coral reefs, in particular, are in crisis. With climate and population-related pressures predicted to intensify in the coming decades, it is increasingly crucial to develop cost-effective and accurate monitoring tools to document changes to these important ecosystems. Environmental DNA (eDNA) coupled with metabarcoding is a powerful tool for surveying a wide variety of biota. Here, we develop a baseline eDNA toolkit targeting scleractinian corals and validate its performance in conjunction with data derived on traditional diver-based visual surveys at the Cocos (Keeling) Islands. Three assays targeting the ITS2 and 16S barcoding regions were designed, which broadly detected diversity within Scleractinia and Porifera. Our eDNA assays recovered 78 ITS2 operational taxonomic units (OTUs) from 25 scleractinian genera which is comparable to the level of diversity recorded on visual surveys (68 species from 26 genera). There were some notable differences in the species detected using eDNA versus visual records that may relate to either misidentifications, intragenic variation, differential assay performance or cryptic species. Our data demonstrate that a multi-assay eDNA analytical approach, applied on surface water collections, represents a powerful and complementary way to survey diversity that can also reveal fine scale spatial differentiation in community composition. With further refinement and improved reference databases, we envisage eDNA to become a powerful complement to visual surveys and to play a key role in monitoring the health and diversity of complex coral reefs ecosystems.
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黄林韬, 黄晖, 江雷. 中国造礁石珊瑚分类厘定[J]. 生物多样性, 2020, 28(4): 515-523.
造礁石珊瑚是珊瑚礁框架建造者, 具有维持珊瑚礁生态系统功能和稳定性的重要作用, 其分类对于造礁石珊瑚和珊瑚礁的研究与保护至关重要。目前, 随着分子系统学的不断发展, 造礁石珊瑚的分类体系发生改变, 伴随着出现大量同物异名。近年来也出现许多无中文学名的中国造礁石珊瑚新记录种, 这些都给物种认定和命名带来困难, 阻碍了中国造礁石珊瑚的研究与保护工作。为此, 本文收集了中国造礁石珊瑚物种记录文献资料, 采用最新的造礁石珊瑚分类体系, 确认同物异名, 形成中国造礁石珊瑚物种名录, 并对中国造礁石珊瑚物种的中文名进行统一的规范和命名。结果表明, 中国共有造礁石珊瑚2个类群16科77属445种。与《中国动物志·腔肠动物门·珊瑚虫纲·石珊瑚目·造礁石珊瑚》相比, 科级分类阶元新增7个科, 变更5个科; 属级分类阶元新增26属, 变更1属, 合并3属; 种级分类阶元新增291种, 变更13种, 合并20种, 新命名305个物种的中文名。并且筛选出187个同物异名。此外, 由于造礁石珊瑚分类体系现仍有部分争议, 文章也进行了讨论说明。
As the major reef framework builder, hermatypic corals play a key role in maintaining the stability and function of coral reef ecosystems. Understanding the taxonomy of hermatypic corals is critical to furthering research and conservation of reef corals and the ecosystem they construct. The application of molecular and small-scale morphological analyses has identified synonymous species and have challenged the traditional taxonomy of hermatypic corals, which was based merely on skeletal morphology. Moreover, many newly recorded species appeared without Chinese scientific names. These aforementioned issues hinder the progress of research and conservation of hermatypic corals in China. Here, we present a comprehensive species list of all hermatypic corals reported in China by collecting historical records, incorporating revised systematics, confirming synonymous species, and updating Chinese scientific names. Our results show that there are 445 known species of hermatypic corals in China, belonging to 16 families and 77 genera. Compared to Fauna Sinica · Coelenterata · Anthozoa · Scleractinia · Hermatypic Coral, we added 291 species, revised 13 species, synonymized 20 species, and designated Chinese name to 305 species. We added 26, revised 1 and synonymized 3 genera, and we added 7 and revised 5 families. Though we identified 187 synonymous species from historical records, there are still several discrepancies in the current taxonomy. We discuss these discrepancies and explain our classification rationale herein. |
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Biological sequence data is accumulating rapidly, motivating the development of improved high-throughput methods for sequence classification.UBLAST and USEARCH are new algorithms enabling sensitive local and global search of large sequence databases at exceptionally high speeds. They are often orders of magnitude faster than BLAST in practical applications, though sensitivity to distant protein relationships is lower. UCLUST is a new clustering method that exploits USEARCH to assign sequences to clusters. UCLUST offers several advantages over the widely used program CD-HIT, including higher speed, lower memory use, improved sensitivity, clustering at lower identities and classification of much larger datasets.Binaries are available at no charge for non-commercial use at http://www.drive5.com/usearch.
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Mitochondrial genes have been used extensively in population genetic and phylogeographical analyses, in part due to a high rate of nucleotide substitution in animal mitochondrial DNA (mtDNA). Nucleotide sequences of anthozoan mitochondrial genes, however, are virtually invariant among conspecifics, even at third codon positions of protein-coding sequences. Hence, mtDNA markers are of limited use for population-level studies in these organisms. Mitochondrial gene sequence divergence among anthozoan species is also low relative to that exhibited in other animals, although higher level relationships can be resolved with these markers. Substitution rates in anthozoan nuclear genes are much higher than in mitochondrial genes, whereas nuclear genes in other metazoans usually evolve more slowly than, or similar to, mitochondrial genes. Although several mechanisms accounting for a slow rate of sequence evolution have been proposed, there is not yet a definitive explanation for this observation. Slow evolution and unique characteristics may be common in primitive metazoans, suggesting that patterns of mtDNA evolution in these organisms differ from that in other animal systems.
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The genomic revolution has fundamentally changed how we survey biodiversity on earth. High-throughput sequencing ("HTS") platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed "environmental DNA" or "eDNA"). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called "eDNA metabarcoding" and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.© 2017 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.
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