Biological invasion is a major threat to multiple ecosystems across the globe, causing severe damages to ecological integrity, loss of biodiversity, economic and social development and even human health. With the rapid development in aquaculture, shipping and aquarium and ornamental trades in the past several decades, China has become one of the countries most influenced by invasive species. Studies have clearly shown that the development and application of robust early monitoring and warning is one of the most effective ways to prevent and possibly control invasive species in aquatic ecosystems. Compared to terrestrial ecosystems, there remain several technical difficulties for developing early monitoring and warning in aquatic habitats. The technical challenges are mainly due to several features of aquatic biological communities such as high biodiversity and complex structure, a large number of microscopic species, extremely low population density and lack of available taxonomic keys for species identification. With the rapid development of high-throughput sequencing techniques, environmental DNA (eDNA)-metabarcoding has become the top priority method for developing the early monitoring and warning programs in aquatic ecosystems. In this review, we aim to synthesize the research progress on eDNA-metabarcoding and its application to early monitoring and warning of invasive species in aquatic ecosystems. In addition, we briefly discuss the technological advantages of eDNA-metabarcoding for the early monitoring and warning programs. Finally, we propose research perspectives for solutions to technical issues for false positive and false negative errors in the eDNA-metabarcoding process.

Background: Research techniques in molecular biology, cell biology, microbiology and genetics have been accelerated by rapid development of modern genomic technologies. These advances have rapidly evolved the field of biodiversity research, once a branch of natural history focusing on morphology, into an integrated life science. Modern biodiversity studies can now investigate and link element of ecological systems, the species within them, and their genetic diversity. DNA related technologies, among other omics techniques, have continued to develop and launch new sequencing platforms, leading to a reduction of DNA sequencing costs that has already outstripped Moore’s Law, which also facilitates a series of breakthroughs in the research fields of biodiversity.
Prospects: Here, we introduce emerging trends in DNA-based omics techniques applied in biodiversity research, including species-level genomics as well as genetic diversity and community-levels species diversity. The former includes genomes obtained based on single individuals and genetic diversity of focal populations in both spatial and temporal dimensions, while the latter includes molecular identification approaches, such as metabarcoding, eDNA, iDNA etc. These new methods can be applied in biodiversity estimation for various communities, as well as in monitoring and conservation of flagship species and interspecific interactions.

Environmental DNA (eDNA) refers to DNA fragments that organisms leave behind in their surrounding environment (such as soil, sediment and water). eDNA technology sequences these DNA fragments and can provide information on taxonomic composition of benthic macroinvertebrate communities. Compared with traditional biological survey methods, eDNA technology is more sensitive, efficient and noninvasive. As a novel method for surveying aquatic organisms, eDNA techniques have been widely used in biodiversity assessments of aquatic organisms, including monitoring of endangered, rare and invasive species. In this review, we summarize recent developments in eDNA technology and focus primarily on the operational procedure and its application for freshwater benthic macroinvertebrate analyses. Finally, we discuss the advantages and potential caveats of current eDNA practices.

Aims: We used eDNA metabarcoding to detect fish diversity in three river systems of Beijing during summer and autumn. The aim of this study is to explore new methods applicable to monitor and protect fish diversity and study the spatio-temporal patterns of fish community in Beijing.
Methods: A total of 34 sites were surveyed simultaneously using the eDNA metabarcoding and ground cage methods. The main processes of the eDNA method are water sample collection and enrichment, DNA extraction with DNeasy Tissue and Blood DNA, PCR amplification with prism of MiFish-U, high-throughput sequencing by Illumina Miseq and bioinformatics analysis. The data of eDNA metabarcoding, environmental factors and human activity were used for statistical analysis between fish communities and influencing factors. The main processes of the ground cage with three replications were placed at each site waiting 24 h to be collected for species identification, and the species with number were recorded for subsequent analysis.
Results: The result showed that 55 fish species were detected by eDNA metabarcoding was higher than that captured by the ground cage (35 species). The fish community composition was dominated by Cypriniformes and Perciformes. The diversity of clearwater fish in mountain rivers is significantly higher than urban rivers. Urban rivers (the Beiyun River System) were dominated by pollution-tolerant species such as Carassius auratus, Pseudorasbora parva and Misgurnus anguillicaudatus, with a relatively homogeneous community structure. Mountain rivers (Chaobai River System and Daqing River System) are dominated by Zacco platypus, Rhynchocypris lagowskii and Opsariichthys uncirostris. The community structure of clearwater fish was affected by electrical conductivity and total dissolved solids in summer but by altitude and temperature in autumn. The correlation between the abundance of clearwater fish with environmental factors and human activities showed that the abundance of clearwater fish decreased significantly with the increase by turbidity of water and human activities, and was significant correlation with altitude and temperature.
Conclusion: The study used eDNA metabarcoding to show the diversity and spatio-temporal patterns of major rivers in Beijing, with a focus on the distribution of clearwater fish. The results demonstrate the feasibility of eDNA metabarcoding for monitoring fish diversity and spatial-temporal distribution in Beijing.

Aims: Hydrological connectivity is essential for maintaining biodiversity in coastal wetlands, and it is important to understand the response of fish diversity, as a major component of biodiversity, to different hydrological connectivity intensities. This study aimed to investigate the response of fish diversity to hydrological connectivity in the typical tidal creek system and verify the applicability of eDNA metabarcoding for monitoring and conserving fish diversity in the Yellow River Delta.

Methods: Water samples were collected from six sites in a typical tidal creek system in the Yellow River Delta in September 2022. Fish diversity was analyzed by using eDNA metabarcoding, in which, eDNA extracted from the water samples source were amplified by PCR using the 12S rRNA classical fish primers MiFish-E and high-throughput sequencing was performed by Illumina Miseq. The OTU representative sequences were aligned to the NCBI nucleotide sequence database to obtain species taxonomic annotation information. Thirteen environmental physicochemical indicators were measured in the water column. The relationship between fish communities and environmental factors was explored based on redundancy analysis.

Results: A total of 55 fish species were detected, including 27 native and 28 non-native fish species, and Perciformes dominated the fish composition. The fishes with high sequence abundance at each sample site included Acanthogobius hasta, Planiliza haematocheilu, Acanthogobius elongatus, etc. Fish diversity of the tidal creeks with different hydrological connectivity was different. Significantly higher community diversity and richness index, the highest number of species and a more even distribution of the number of individual species were found in the secondary tidal creek than in the other two tidal creeks. SiO₃^2--Si, NO₃^--N, pH, salinity, NH₄⁺-N, and dissolved oxygen were significantly correlated (P < 0.05) with fish community structure shown by RDA analysis. Acanthogobius hasta with higher sequence abundance was positively correlated with silicate, and the sequence abundance of Planiliza haematocheilus was negatively correlated with salinity.

Conclusion: This study confirmed the feasibility of using environmental DNA metabarcoding to monitor fish diversity of typical tidal water bodies in the Yellow River estuary by comparing it with previous traditional sampling data and indicated that hydrological connectivity of tidal creek systems has a significant impact on fish community structure and diversity. The results of this study are helpful to further understand the influence mechanism of coastal wetland hydrological connectivity on biological communities.

Background & Aims: Amphibians and reptiles are important indicators of ecosystem health, and they are vulnerable to changes in the environment. Many of their populations are undergoing rapid decline and species extinction in the worldwide. A recent assessment of Red List of China’s Vertebrates found that 37.05% of amphibians and 30.5% of reptiles are threatened. Therefore, building a national biodiversity monitoring network, to research and analyze the trend and threats of amphibians and reptiles is a prerequisite for conservation. As part of the Chinese Biodiversity Monitoring and Research Network (Sino BON), the Sino BON-Amphibian & Reptile has covered 11 key areas with rich biodiversity and high habitat heterogeneity across China. This program aims to combine intensive field surveys and ecological modeling techniques to evaluate population dynamics and community structures of amphibian and reptile species in the study areas.

Review Results: Currently, the Sino BON-Amphibian & Reptile adheres to focusing on the research of amphibian and reptile diversity. A total of 16 species were newly discovered in China, including 13 new species and 3 national new record species. Significant progress has been made in the national need for the protection of Critically Endangered amphibian (CR) Chinese giant salamander (Andrias davidianus) and Endangered (EN) amphibian Taliang crocodile newt (Liangshantriton taliangensis), the environmental DNA (eDNA) technology and radio tracking technology applied in wild, the ecological impact of desertification habitats on reptiles, and invasive alien species, which have provided strong scientific support for protecting biodiversity of amphibians and reptiles in China.

Prospects: For further progresses, it is still necessary to continuously enhance monitoring and research on amphibians and reptiles, strengthen monitoring network construction and application of advanced technologies, emphasize the protection of amphibians and reptiles from the level of laws and regulations, establish and optimize the protected areas, and implement the necessary ex situ and in situ conservation, so as to achieve more effective protection of rare amphibian and reptile resources.

Aim: Global climate change and anthropogenic impacts are placing enormous pressures on coastal ecosystems, and networks of marine protected areas have been recognized as an effective means of dealing with these dual pressures. However, there have been few investigations of biodiversity and connectivity of marine protected areas, in particular a lack of studies at the multispecies level. As one of the most biologically rich regions in China and an important contributor to China’s rapid socio-economic development, a number of protected areas have been established in the East China Sea at the end of the last century, but not much research has been conducted on biodiversity and connectivity to support the conservation and sustainable development.
Methods: In this study, six national marine protected areas were selected in the East China Sea, including Chengzhou Island National Marine Park (CZD), Shenhu Bay National Nature Reserve (SHW), Meizhou Island National Marine Park (MZD), Haitan Bay National Marine Park in Pingtan (PTD), Dongtou National Marine Park (DTD) and Yuhuan National Marine Park (YH). Water samples were collected from sandy coast. Environmental DNA was amplified with MiFish-U primers and high-throughput sequencing was performed by Illumina NovaSeq. Permutational multivariate analysis of variance was performed to determine the differences in community composition among protected areas. Network analysis was performed to assess robustness within each marine protected area.
Results: A total of 71 samples were obtained, including 5,069,193 reads and 46,309 MOTUs, from six protected areas in different seasons. Further analysis revealed that the abundance of MOTUs shared between protected areas accounted for more than 40% of the total MOTUs abundance. Although the environments differed significantly among protected areas, there were no spatial differences in biodiversity and community structure among protected areas (P > 0.05), and only seasonal differences observed. DTD, SHW and YH had a more complex network structure, while MZD and PTD were relatively simpler.
Conclusion: The results of the study suggest that there is a strong connectivity among these protected areas, which has the potential to establish a network of marine protected areas. The community network structures of the protected areas reveal that the protection effects vary from different protected areas, which may be related to the size and management of the protected areas. This study not only provides a reference for the study of nearshore community connectivity in the East China Sea, but also offers direct support for the management and construction of protected areas network in East China Sea.

Aims: Environmental DNA (eDNA) technology provides a non-invasive approach for biodiversity monitoring. Recent studies have demonstrated that airborne eDNA collected from the air can be utilized to monitor wildlife in forest ecosystems. Compared with other eDNA survey methods, airborne eDNA offers greater flexibility in sampling site selection, especially in survey areas lacking environmental media such as water bodies. Therefore, airborne eDNA holds significant potential for biodiversity monitoring in forest ecosystems. This study aims to assess the effectiveness of airborne eDNA in monitoring terrestrial vertebrate diversity in tropical rainforests.

Methods: The research was conducted in a 20-ha forest dynamics plot in Xishuangbanna, China. Airborne eDNA technology was employed to survey terrestrial vertebrate diversity, and results were compared with those obtained from infrared camera monitoring data. A total of 20 airborne eDNA samplers were deployed, and three 24-hour sampling sessions were conducted over six days in November 2023. Collected samples were amplified using 12SV05 primers targeting the 12S rRNA gene fragment and sequenced on the Illumina NovaSeq 6000 platform. Sequences data were taxonomically annotated, and species detection efficiency was compared between airborne eDNA and infrared cameras.

Results: Across the three airborne eDNA sampling sessions, 66 operational taxonomic units (OTUs) were detected, representing birds, mammals, reptiles, and amphibians. In comparison, 20 infrared cameras placed at the same sites recorded 15 mammal species and 15 bird species over 5,682 camera days. Comparative analyses revealed that airborne eDNA was more effective in detecting species diversity. Additionally, evaluation of the alpha diversity accumulation curve indicated that the diversity plateaued when 10 airborne eDNA samples were collected. Suggesting that within the current experimental conditions, 10 samples collected over three days were sufficient for maximizing species detection.

Conclusion: This study demonstrates that airborne eDNA serves as an effective tool for monitoring terrestrial vertebrate diversity in tropical rainforests, enabling rapid and comprehensive biodiversity assessments. Compared to infrared cameras, airborne eDNA shows distinct advantages in rapid species detection. Although the technology is still under development and its stability and accuracy under specific environmental conditions require further improvement, advancing technology is likely to establish airborne eDNA as a critical tool for cross-trophic and multi-species biodiversity monitoring. It holds strong potential to support large-scale, standardized biodiversity monitoring networks in China.

Aims: Scientific protection and effective management of wildlife highly rely on baseline data regarding biological resources, and the precision of survey methods directly affects the objectivity and accuracy of resource assessments. Environmental DNA (eDNA) technology has been widely used in monitoring of fish community, but its application in amphibian surveys still remains relatively limited and is mostly focused on detecting specific species. This study takes Hunan Mangshan National Nature Reserve as a case study to evaluate the detection efficiency and accuracy of eDNA technology in amphibian surveys.

Methods: eDNA test samples were collected from 19 water sampling sites within the reserve between July to August 2023. eDNA data were then compared with data collected during the same period using traditional transect survey methods. By calculating α and β diversity, a comprehensive evaluation of the two methods results was conducted.

Results: Both methods detected 34 amphibian species, with 24 species identified by both approaches. Significant differences (P < 0.05) were observed between the two methods in terms of the Shannon-Wiener diversity index, Simpson dominance index, and Pielou evenness index. PCoA and ANOSIM analysis showed significant differences in species composition between the two methods (P < 0.05), potentially influenced by species behavior, sampling location and timing, and environmental disturbances.

Conclusion: This study demonstrates that while eDNA technology offers clear advantages in amphibian resource survey, it cannot yet completely replace traditional transect method. It is suggested that eDNA techniques be integrated with traditional approaches to obtain more comprehensive and accurate baseline data on amphibian resources, thereby supporting more informed conservation and management efforts.

Aims: The Yarlung Zangbo River is a crucial river ecosystem on the Tibetan Plateau, and protozoa, as an essential component of eukaryotic microorganisms, play a key role in aquatic ecosystems. This study aimed to investigate the diversity patterns and geographical distribution of protozoan communities in the middle reaches of the Yarlung Zangbo River during the dry season, focusing on their community structure, diversity, and environmental response mechanisms in both the mainstream and tributaries.

Methods: In the dry season of 2021, we conducted a comprehensive study of protozoan communities in the middle reaches of the Yarlung Zangbo River. We analyzed community structure, diversity, and environmental drivers using high-throughput sequencing, generating amplicon sequence variants (ASVs). Statistical analyses included distance- decay, environmental-decay, neutral and null models, and co-occurrence network analysis to assess spatial and environmental influences on protozoan assemblages.

Results: A total of 6,066 ASVs were detected, encompassing 23 phyla, 55 classes, 86 orders, 114 families, 281 genera, and 323 species. Significant differences in community structure were observed between the mainstream and tributaries, with species turnover identified as the primary driver of β-diversity variations. Distance-decay and environmental-decay analyses revealed that protozoan communities in tributaries were more sensitive to geographical and environmental changes. Neutral and null model analyses indicated that community assembly in both habitats was primarily driven by homogenous selection, though stochastic processes played a greater role in tributaries. Co-occurrence networks showed distinct environmental influences on protozoan communities in the mainstream versus tributaries.

Conclusion: These findings highlight the spatial heterogeneity and environmental response mechanisms of protozoan communities in river ecosystems. These insights contribute to a deeper understanding of microbial ecology in high-altitude river systems.