Invasive species monitoring based on eDNA multiplex PCR sequencing

501 Invasive species monitoring based on eDNA multiplex PCR sequencing Zheyuan Liu1*, Xiaoru Du1*, Zehua Zhang2, Yawen Mu3, Jianghua Yang1, Jiaxin Yang1, Xiaowei Zhang2,4 1 School of Marine Science and Engineering, Nanjing Normal University, Nanjing, 210046, China 2 State Key Laboratory of Pollution Control & Resource, School of the Environment, Nanjing University, Nanjing, 210023, China 3 Jiangsu Provincial Environmental Monitoring Center, Nanjing 210019, China 4 School of Ecology and Environmental Science, Yunnan University, Kunming 650500, China Corresponding author: Jianghua Yang ([email protected]) Copyright: © Zheyuan Liu et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Invasive species pose a significant threat to global biodiversity and the stability of ecosystems. Although environmental DNA (eDNA)-based quantitative PCR is considered effective, its limited multiplexing capacity makes it impractical for large-scale monitoring of invasive species. To address this limitation, we developed a novel and efficient approach for invasive species monitoring by combining multiplex PCR amplification with high-throughput sequencing. In this study, we screened 46 aquatic invasive species of major concern in China. We have integrated and designed 91 pairs of primers that can simultaneously amplify these species in a single PCR system. The validated method was applied to field monitoring in the Pearl River Basin to evaluate its practical performance. Multiplex PCR sequencing successfully detected 28 invasive species, with over 90% of environmental samples testing positive for invasive species DNA, demonstrating the method’s high sensitivity and broad applicability. Furthermore, all 11 invasive species identified through metabarcoding were also consistently detected by multiplex PCR sequencing, showing a strong positive correlation and high concordance across all monitoring sites. In conclusion, multiplex PCR sequencing represents a powerful and cost-effective tool for simultaneously detecting multiple aquatic invasive species in the early stages of invasion. It significantly improves detection efficiency, reduces monitoring costs and provides a solid foundation for developing a scientific and scalable monitoring system for aquatic invasive species. Key words: Amplicon sequencing, biodiversity monitoring, estuary ecosystem, metabarcoding, tilapia Introduction Invasive Alien Species (IAS) are defined as non-native organisms capable of forming self-sustaining populations in new ecosystems, often leading to substantial ecological disruption, economic losses or societal challenges (Early et al. 2016; Pysek et al. 2020). As globalisation accelerates and human activities intensify, the frequency of species transport and introduction has Academic editor: Gert-Jan Jeunen Received: 25 May 2025 Accepted: 28 August 2025 Published: 14 October 2025 Citation: Liu Z, Du X, Zhang Z, Mu Y, Yang J, Yang J, Zhang X (2025) Invasive species monitoring based on eDNA multiplex PCR sequencing. Metabarcoding and Metagenomics 9: e159944. https://doi.org/10.3897/ mbmg.9.159944 Metabarcoding and Metagenomics 9: 501–518 (2025) DOI: 10.3897/mbmg.9.159944 * These two authors contributed equally to this paper. 502 Metabarcoding and Metagenomics 9: 501–518 (2025), DOI: 10.3897/mbmg.9.159944 Zheyuan Liu et al.: eDNA multiplex PCR for invasive species monitoring increased, leading to a rise in the occurrence of IAS (Havel et al. 2015). IAS are especially notable for their high adaptability and reproductive capacity, which are facilitated by the open and connected nature of aquatic ecosystems. These characteristics enable them to rapidly establish, spread and alter local ecological dynamics within a short timeframe (Jerde et al. 2011; Abdo et al. 2013; Ray and Umapathy 2022). For example, Eichhornia crassipes (water hyacinth), a floating plant native to South America, often forms a dense floating layer that blocks sunlight and reduces oxygen levels in the water, inhibiting the growth of submerged vegetation and ultimately disrupting the ecological balance of freshwater ecosystems (Wilson et al. 2005). Currently, species monitoring primarily relies on physical identification methods, including visual surveys and the use of fishing nets. While these methods are simple, intuitive and reliable (Thomsen and Willerslev 2015), they have several limitations, including a cumbersome monitoring process, time consumption, low species resolution, poor accuracy and high costs (Shen et al. 2016). Traditional techniques, such as electrofishing and netting, can also have environmental impacts, especially in freshwater ecosystems (Lundberg et al. 2000; Relyea 2005). Consequently, there is a pressing need for more efficient and environmentally friendly monitoring technologies. In recent years, environmental DNA (eDNA) technology has emerged as a highly sensitive method for detecting aquatic species by collecting DNA fragments from water or soil. Amongst these, metabarcoding has become a focal point in ecological research, utilising high-throughput DNA sequencing for comprehensive monitoring of species. Olds et al. (2016) compared the effectiveness of electrofishing and eDNA metabarcoding in American streams and demonstrated that eDNA can detect more fish species than traditional methods, highlighting its unique advantages in species monitoring. Additionally, metabarcoding can be used as a monitoring tool to screen for invasive species; however, it has limitations, such as reduced sensitivity for rare or low-abundance taxa and the potential for non-target amplification. These drawbacks have prompted the development of species-specific assays, such as qPCR and ddPCR, which offer higher sensitivity and specificity (Thomas et al. 2019; Feng et al. 2023; Nynatten et al. 2023; Zaiko et al. 2023). However, this method has several limitations: (1) It can only detect a limited number of species at once. When screening for multiple invasive species, repeated testing is necessary, resulting in high costs. Repeated testing with qPCR requires additional sample material and increases costs, which can be a limiting factor for large-scale or long-term monitoring programmes; (2) it is challenging to detect invasive species in the early stages of invasion; (3) it is challenging to detect invasive species at trace DNA levels in the early stages of invasion. In such cases, nested PCR or digital PCR is typically needed, increased number of experimental steps and the reliance on more expensive equipment to the process; (4) its demand for comprehensive databases in the aquatic field is often incomplete, which leads to identification problems (Hindson et al. 2011; Whale et al. 2012; Wilcox et al. 2013; Wu et al. 2022). Multiplex PCR (mPCR) technology enables the simultaneous amplification of multiple targets within a single PCR system (Chen et al. 2005; Zhong et al. 2020; Li et al. 2024). This not only reduces the amount of DNA required, but also enhances detection efficiency. mPCR has been widely applied to detect pathogenic bacteria (Altinok et al. 2008). In aquatic ecology, mPCR is commonly used 503 Metabarcoding and Metagenomics 9: 501–518 (2025), DOI: 10.3897/mbmg.9.159944 Zheyuan Liu et al.: eDNA multiplex PCR for invasive species monitoring for identifying economically important species, distinguishing closely-related species and monitoring fish pathogen outbreaks (Zhong et al. 2020). The ability of mPCR to simultaneously amplify multiple target genes makes it a valuable tool for monitoring invasive species. For example, Wozney and Wilson (2017) utilised multiplex qPCR to investigate the presence of Asian Carp in the Great Lakes (Wozney and Wilson 2017). Amplicon sequencing not only offers high throughput, but also accurately distinguishes closely-related species, based on DNA sequence differences, enabling semi-quantitative monitoring through sequence abundance (Lundberg et al. 2013; Spilsberg et al. 2024). Due to its reliability and cost-effectiveness, amplicon sequencing has been widely utilised in bacterial community analysis and the development of whole-genome markers for animals and plants. The combination of amplicon sequencing and mPCR holds promise for advancing the screening of alien invasive species. This study aims to develop an mPCR-based monitoring method for aquatic invasive species and apply it in the Pearl River Basin to achieve precise monitoring and minimise DNA requirements. This method allows for the simultaneous detection of multiple targets, significantly enhancing detection efficiency while reducing costs. It supports the efficient and accurate monitoring of aquatic invasive species, providing crucial technical support for future management and ecological conservation of these species. Materials and methods China common invasive species list and mPCR primers design By reviewing the literature, official documents and open-source databases (Xu et al. 2004; Xu et al. 2012; Xiong et al. 2015; Wang et al. 2016; Xian et al. 2022), 46 key invasive species were selected, based on their harmfulness (Suppl. material 1: table S1). A total of 91 pairs of primers for mPCR amplification, including 31 pairs for 20 species, based on former studies (Suppl. material 1: table S2) and 60 pairs for 31 species newly designed from the mitochondrial genome (Suppl. material 1: table S3). Both D-loop and COI are considered to have high sequence variability and are suitable for designing species-specific primers (Ravago et al. 2002; Hebert et al. 2003; Aminisarteshnizi et al. 2024; Sumana et al. 2024). We verified all primers individually using the corresponding species’ tissue DNA and found that all primers could be successfully amplified. Invasive species standard plasmid design Tissues of alien species were obtained through historical surveys and purchases. DNA was extracted from each invasive species (n = 46) using the Tissue DNA Kit (Egenomics, Nanjing, China), following the manufacturer’s instructions. DNA concentration was measured using a Nanodrop spectrophotometer. The 91 pairs of invasive species-specific primers were used to amplify the tissue DNA and PCR products were obtained for each primer pair. Amplicons of the expected sizes were purified using an Agarose Gel DNA Purification Kit and then subcloned into the pMD-19T cloning vector (TaKaRa, Japan). Positive clones containing inserts of the expected size were sequenced using M13 primers. The PCR was conducted with an initial denaturation step at 94 °C for 504 Metabarcoding and Metagenomics 9: 501–518 (2025), DOI: 10.3897/mbmg.9.159944 Zheyuan Liu et al.: eDNA multiplex PCR for invasive species monitoring 5 minutes, followed by 35 cycles of denaturation at 94 °C for 10 seconds, annealing at 50 °C for 30 seconds and extension at 72 °C for 30 seconds. A final extension step was performed at 72 °C for 10 minutes. Each 20 μl reaction mixture contained 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 mM of each primer, 1 U of Taq DNA polymerase and 5 ng of DNA. Mix plasmid amplification All plasmids were diluted to 100 ng/μl and 5 μl of each was mixed together. The upstream and downstream primers of the 91 species-specific primer pairs (0.02 μM each) were combined to create a primer mix. PCR amplification was then performed using the mixed plasmids as templates. The amplification system consisted of 10 μl of 2×Hieff NGS® HG Multiplex PCR Master Mix, 4 μl of multi-primer mix, 4 μl of ddH2O and 2 μl of DNA template (100 ng/μl). PCR was carried out for 5 minutes at 94 °C, followed by 35 cycles of 94 °C for 10 seconds, 63 °C for 30 seconds and 72 °C for 30 seconds. A final extension step was performed at 72 °C for 10 minutes. PCR products were purified for sequencing. Pearl river environmental DNA collection and extraction The Pearl River Estuary, a critical aquatic ecosystem in southern China, is vulnerable to the introduction of invasive species that pose significant threats to biodiversity and ecosystem stability. In July 2021, thirty-two sampling sites (initially planned for 40 sites, but eight sites did not collect samples) were established in the Pearl River Estuary (Suppl. material 1: fig. S1). Three water samples were collected from each site using a 1 litre sampling bottle and then filtered through three mixed cellulose filter membranes (47 mm, 0.45 μm, Millipore, USA), respectively. After filtration, the membranes were transferred into 5 ml sterilised tubes and transported to the laboratory with dry ice. All samples were stored at -20 °C until DNA extraction. DNA was extracted from the filter membranes using the Water DNA Kit (Egenomics, Nanjing, China). DNA concentration and purity were measured by Qubit 2.0 and Nanodrop. The extracted DNA was stored at -20 °C. Filter 500 ml of ddH2O as a negative control during the sampling, DNA extraction and PCR process. The plasmids for each species are for positive PCR controls. mPCR and metabarcoding with environmental samples Environmental sample mPCR amplification was performed in a total volume of 30 μl, consisting of 15 μl of 2×Hieff NGS® HG Multiplex PCR Master Mix, 6 μl of Primer Mix (0.01 μM), 8 μl of ddH2O and 1 μl of DNA template. The reaction conditions of mPCR when amplifying plasmids and eDNA are consistent, except for the annealing temperature. The reaction conditions included pre-denaturation at 95 °C for 3 minutes, followed by 38 cycles of denaturation at 95 °C for 20 seconds, annealing at 55 °C for 30 s and extension at 72 °C for 30 seconds. A final extension was performed at 72 °C for 5 minutes, followed by storage at 4 °C. Fish metabarcoding amplification of environmental sample using the 12S universal primers (Yang et al. 2023). The amplification reaction volume was 25 μl, consisting of 12 μl of 2×Taq Plus Master Mix II (Dye Plus) DNA polymerase (Novozymes, China), 2 μl of primer, 9 μl of ddH2O and 2 μl of DNA template. 505 Metabarcoding and Metagenomics 9: 501–518 (2025), DOI: 10.3897/mbmg.9.159944 Zheyuan Liu et al.: eDNA multiplex PCR for invasive species monitoring The reaction conditions included pre-denaturation at 95 °C for 3 minutes, followed by 30 cycles of denaturation at 95 °C for 15 seconds, annealing at 62.4 °C for 20 seconds and extension at 72 °C for 20 seconds. A final extension was performed at 72 °C for 5 minutes, followed by storage at 4 °C. High-throughput sequencing and bioinformatics analysis The sequencing libraries of mPCR, metabarcoding and mixed plasmid were constructed using the same method. PCR amplification products of three repeated samples at each location were combined in equal volumes and purified using VAHTS DNA Clean Beads (Vazyme, Nanjing, China). The concentration of the purified products was measured using Qubit™ dsDNA HS Assay Kits (Invitrogen, USA). Sequencing adaptors were connected to purified DNA fragments using the Ion Xpress Plus Fragment Library Kit (Life Technologies, USA). Different libraries are assigned different indices when ligating the sequencing adapters. After assessing the quality and concentration of the sequencing libraries with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), the libraries were diluted to 100 pM and subjected to high-throughput sequencing on an Ion Torrent S5 sequencer (Life Technologies, USA). Low-quality (< Q20) and short reads (< 100 bp) were removed from the raw FASTQ files using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline (Yang et al. 2017). The FASTQ files were then converted to FASTA format using Mothur (Schloss et al. 2009). Sequences with no mismatches in tags and a maximum of two mismatches in primers were identified using the “split_libraries. py” script with the parameters “-w 50 -s 20 -M 2” and retained for further analysis (Zhang et al. 2020). Unique sequences were identified using the “derep_fulllength” script in Vsearch. Sequences were clustered into Operational Taxonomic Units (OTUs) using USEARCH. Species annotation was performed using a 97% similarity threshold with the previously constructed database and the Basic Local Alignment Search Tool (BLAST) (Zhang et al. 2023). All data analyses were conducted in R (version 4.1.2) using the Vegan package (Oksanen et al. 2022). All plots were generated using the ggplot2 package (Wickham et al. 2016). Pearson’s correlation analysis was conducted to examine the relationships between invasive species detection by mPCR and metabarcoding. Results Validation of mPCR amplification using mixed standard plasmids Of the 91 primer pairs tested, 70 successfully amplified target sequences, including 15 pairs adapted from published literature and 55 pairs newly designed in this study (Fig. 1a). The literature-derived primers primarily targeted the mitochondrial COI and Cytb gene regions, whereas the newly-designed primers primarily targeted the COI and D-loop regions (Fig. 1b). A total of 41 out of 46 aquatic invasive species were successfully detected, resulting in a species detection rate of 90%. Five species —Pomacea canaliculata, Macrochelys temminckii, Oncorhynchus mykiss, Oreochromis mossambicus and Chelydra serpentina – failed to amplify. Most amplicons were approximately 150 bp and the annealing temperatures of the primers were primarily around 65 °C (Fig. 1c, d). 506 Metabarcoding and Metagenomics 9: 501–518 (2025), DOI: 10.3897/mbmg.9.159944 Zheyuan Liu et al.: eDNA multiplex PCR for invasive species monitoring Invasive species monitoring in the Pearl River Estuary using mPCR In the mPCR assay, 33 primer pairs successfully amplified target seque-nces, detecting a total of 28 aquatic invasive species (Suppl. material 1: table S4). Amongst these, five fish species, including Coptodon zillii, Cirrhinus mrigala, Oreochromis aureus, Sarotherodon galilaeus and Labeo rohita were detected by two primer pairs (Fig. 2a). The invasive invertebrate species Amphibalanus amphitrite had the highest relative abundance in the Pearl River Basin, followed by Tinca tinca, C. zillii and O. niloticus (Fig. 2b). Of the 33 successfully amplified primer pairs, 23 targeted the COI region, accounting for 69.7%, while seven primer pairs targeted the D-loop region, accountin-g for 21.2%, those all being fishes (Fig. 2c). Several invasive species, including Pygocentrus nattereri, Amphibalanus Amphitrite Anguilla anguilla Anguilla rostrata Atractosteus spatula Channa striata Chelydra serpentina Cirrhinus mrigala Clarias batrachus Clarias gariepinus Coptodon zillii Crassostrea gigas Eleotris fusea Gambusia affinis Ictalurus punctatus Ictiobus cyprinellus Labeo rohita Lates calcarifer Lepisosteus oculatus Lepomis auratus Lepomis macrochirus Limnoperna fortunei Macroclemys temminckii Micropterus salmoides Morone saxatilis Mytilus galloprovincialis Notopterus notopterus Oncorhynchus kisutch Oncorhynchus mykiss Oreochromis aureus Oreochromis mossambicus Oreochromis niloticus Parachromis managuensis Perca fluviatilis Polyodon spathula Pomacea canaliculata Procambarus clarkii Prochilodus lineatus Pseudorasbora parva Pterygoplichthys disjunctivus Pterygoplichthys pardalis Pygocentrus nattereri Rana catesbeiana Sarotherodon galilaeus Sciaenops ocellatus Tinca tinca Trachemys scriptaelegans reference new.design Number of successful primers 1 2 a 12S 16S Cytb Dloop COI reference new.design b 0.0 0.2 0.4 0.6 55 60 65 70 PCR Tm Density c 0.000 0.002 0.004 0 200 400 Amplicon length (bp) Density d b Figure 1. Multiplex PCR primers for common aquatic invasive species of China. a PCR primers for each species. b Genetic classification of PCR primer targets. c Distribution of primer annealing temperatures. d Distribution of PCR amplicon lengths. 507 Metabarcoding and Metagenomics 9: 501–518 (2025), DOI: 10.3897/mbmg.9.159944 Zheyuan Liu et al.: eDNA multiplex PCR for invasive species monitoring Oncorhynchus kisutch, Oreochromis niloticus, Labeo rohita and Sciaenops ocellatus were detected at all sampling sites (Fig. 2d). The number of invasive species detected at sites L2, L3, L10, L31, L38 and L40 was higher than that at other sites (species number > 22) (Fig. 2e). At each site, species such as A. Amphitrite, Tinca tinca and C. zillii, all occupied a relatively high sequence abundance (Suppl. material 1: fig. S2). At estuarine sites (e.g. L26, L27, L37, L40), A. amphitrite exhibits a significantly dominant relative abundance; in inland river basins (e.g. L3, L13, L34), O. niloticus shows a significantly dominant relative abundance (Fig. 3). Comparison of metabarcoding and mPCR A total of 91 fish species were detected using 12S rRNA metabarcoding, including 11 invasive fish. All invasive fish detected by 12S rRNA metabarcoding could also be monitored by mPCR (Suppl. material 1: fig. S3). Compared with metabarcoding, mPCR can detect more species with fewer sequences (Fig. 4a, b). When only considering the 11 alien species jointly detected by the two Figure 2. Invasive species monitoring in the Pearl River Estuary using mPCR. a Number of successful mPCR primers. b The sequence composition. c Classification of successful mPCR primers, based on target regions. d Detection rates of invasive species. e Number of invasive species detected at each site. Amphibalanus Amphitrite Anguilla anguilla Anguilla rostrata Atractosteus spatula Channa striata Chelydra serpentina Cirrhinus mrigala Clarias batrachus Clarias gariepinus Coptodon zillii Crassostrea gigas Eleotris fusea Gambusia affinis Ictalurus punctatus Ictiobus cyprinellus Labeo rohita Lates calcarifer Lepisosteus oculatus Lepomis auratus Lepomis macrochirus Limnoperna fortunei Macroclemys temminckii Micropterus salmoides Morone saxatilis Mytilus galloprovincialis Notopterus notopterus Oncorhynchus kisutch Oncorhynchus mykiss Oreochromis aureus Oreochromis mossambicus Oreochromis niloticus Parachromis managuensis Perca fluviatilis Polyodon spathula Pomacea canaliculata Procambarus clarkii Prochilodus lineatus Pseudorasbora parva Pterygoplichthys disjunctivus Pterygoplichthys pardalis Pygocentrus nattereri Rana catesbeiana Sarotherodon galilaeus Sciaenops ocellatus Tinca tinca Trachemys scriptaelegans reference new.design Number of successful primers 1 2 a species Amphibalanus Amphitrite Tinca tinca Coptodon zillii Oreochromis niloticus Limnoperna fortunei Polyodon spathula Oncorhynchus kisutch Sciaenops ocellatus Other b 12S Cytb Dloop COI reference ne w design cb 3.125 9.375 15.625 15.625 18.75 25 25 28.125 50 50 68.75 68.75 75 78.125 87.5 90.625 96.875 100 100 100 100 100 100 100 100 100 100 100 Anguilla rostrata Parachromis managuensis Pterygoplichthys pardalis Sarotherodon galilaeus Gambusia affinis Morone saxatilis Perca fluviatilis Pterygoplichthys disjunctivus Oreochromis aureus Notopterus notopterus Limnoperna fortunei Prochilodus lineatus Micropterus salmoides Channa striata Clarias gariepinus Polyodon spathula Ictiobus cyprinellus Amphibalanus Amphitrite Lepisosteus oculatus Cirrhinus mrigala Tinca tinca Crassostrea gigas Coptodon zillii Sciaenops ocellatus Labeo rohita Oreochromis niloticus Oncorhynchus kisutch Pygocentrus nattereri 0 25 50 75 100 Frequency d 23 19 22 17 16 15 15 14 16 23 19 15 18 17 20 20 20 15 19 20 20 21 20 21 20 24 18 19 18 24 25 17 L40 L39 L38 L37 L36 L35 L34 L33 L32 L31 L30 L29 L28 L27 L26 L25 L24 L23 L18 L17 L16 L15 L14 L13 L11 L10 L6 L5 L4 L3 L2 L1 0 5 10 15 20 25 Species Number Site ed 508 Metabarcoding and Metagenomics 9: 501–518 (2025), DOI: 10.3897/mbmg.9.159944 Zheyuan Liu et al.: eDNA multiplex PCR for invasive species monitoring methods, the 12S rRNA metabarcoding revealed that O. niloticus was the most abundant species across all sampling sites, followed by C. zillii and O. aureus. The mPCR method, on the other hand, showed the highest relative abundance of C. zillii and O. niloticus, followed by O.aureus and C. mrigala (Fig. 4c). The two methods jointly detected more sequences of the species at the upstream sites, such as L2, L3, L13 and L14 (Fig. 4d, f). For low-abundance species, such as Pterygoplichthys pardalis, which was clearly detected at sites L3 and L10 using 12S metabarcoding, it was rarely observed through mPCR (Fig. 4e, g). A correlation analysis was conducted on 11 co-detected invasive species, revealing significant correlations in seven species (Fig. 5a) and no correlations in four species (Fig. 5b). Five species - C. zillii, O. aureus, O. niloticus, P. pardalis and G. affinis - exhibited “highly significant” correlations (p < 0.01). Two species, C. striata and C. gariepinus, showed “significant” correlations (p < 0.05). C. zillii and O. niloticus demonstrated particularly strong correlations with R2-values exceeding 0.75, indicating high goodness-of-fit. A comparative analysis between metabarcoding and mPCR methodologies for detecting invasive species revealed a significant positive correlation (p < 0.05), as supported by the regression curve equation and R2-value (Fig. 5c). This demonstrates strong methodological consistency in species detection capability between these two molecular approaches. Figure 3. Top 10 invasive species in the Pearl River Estuary detected by mPCR. 509 Metabarcoding and Metagenomics 9: 501–518 (2025), DOI: 10.3897/mbmg.9.159944 Zheyuan Liu et al.: eDNA multiplex PCR for invasive species monitoring Discussion Accuracy of mPCR sequencing All 91 pairs of species-specific primers successfully amplified in individual tests, but only 70 primer pairs yielded effective amplification in the mPCR system, with five species not detected. This outcome can be attributed to the inherent complexity of mPCR technology. First, primer interactions may reduce the amplification efficiency of some primers at high annealing temperatures. Unlike single Figure 4. Comparison of invasive species that were detected by both metabarcoding and mPCR. a Read counts of all sequences. b All invasive species detected by metabarcoding and mPCR. c Relative abundances of invasive species that were detected by both methods. d Metabarcoding read counts of both detected species at each site. e Site-specific relative abundances of invasive species, based on metabarcoding. f mPCR read counts of both detected species at each site. g Site-specific relative abundances of detected species, based on mPCR. 0e+00 2e+05 4e+05 metabarcoding mPCR Reads number a 0 10 20 metabarcoding mPCR Species number b 0.00 0.25 0.50 0.75 1.00 metabarcoding Proportion mPCR species c Anguilla rostrata Channa striata Cirrhinus mrigala Clarias gariepinus Coptodon zillii Gambusia affinis a 0e+00 2e+04 5e+04 8e+04 1e+05 1e+05 Reads d 0.00 0.25 0.50 0.75 1.00 L1 L2 L3 L4 L5 L6 L10 L11 L13 L14 L15 L16 L17 L18 L23 L24 L25 L26 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L37 L38 L39 L40 Reads number e d 0 1000 2000 3000 4000 Reads f 0.00 0.25 0.50 0.75 1.00 L1 L2 L3 L4 L5 L6 L10 L11 L13 L14 L15 L16 L17 L18 L23 L24 L25 L26 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L37 L38 L39 L40 Relative abundance g f Micropterus salmoides Prochilodus lineatus Oreochromis aureus Oreochromis niloticus Pterygoplichthys pardalis 516 Metabarcoding and Metagenomics 9: 501–518 (2025), DOI: 10.3897/mbmg.9.159944 Zheyuan Liu et al.: eDNA multiplex PCR for invasive species monitoring Olds BP, Jerde CL, Renshaw MA, Li Y, Evans NT, Turner CR, Deiner K, Mahon AR, Brueseke MA, Shirey PD, Pfrender ME, Lodge DM, Lamberti GA (2016) Estimating species richness using environmental DNA. 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Journal of Bioengineering 36: 171–179. https://doi.org/10.13345/j.cjb.190206 Supplementary material 1 Invasive Species Monitoring Based on eDNA Multiplex PCR Sequencing Authors: Zheyuan Liu, Xiaoru Du, Zehua Zhang, Yawen Mu, Jianghua Yang, Jiaxin Yang, Xiaowei Zhang Data type: docx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mbmg.9.159944.suppl1