Biodiversity monitoring method fusing eDNA macro bar code and morphology

Abstract

The invention provides a biodiversity monitoring method fusing an eDNA macro bar code and morphology. The method comprises the following steps: collecting an eDNA water sample and a morphological sample in parallel at the same sampling point and the same moment; the eDNA water sample is subjected to DNA extraction, PCR amplification, high-throughput sequencing and bioinformatics analysis, and a species screening result is obtained; preparing a morphological sample, performing targeted screening on key species according to a species screening result, and performing morphological identification on the key species; and according to a pre-constructed comprehensive evaluation model, fusing the species screening result and the morphological identification result, carrying out weight distribution and abundance correction, and outputting a monitoring result. By fusing the high efficiency of the eDNA macro bar code technology and the reliability of morphological verification, the accurate monitoring method with high sensitivity, high accuracy and comprehensiveness is provided, and a scientific basis is provided for aquatic ecological environment evaluation, pollution abatement and biodiversity protection.

Description

Technical Field

[0001] This application relates to the field of aquatic ecological environment monitoring technology, and in particular to a biodiversity monitoring method that integrates eDNA macrobarcoding and morphology. Background Technology

[0002] Aquatic plankton, as a key component of aquatic ecosystems, directly reflects the quality of the aquatic environment through changes in its diversity, and is one of the core indicators for ecological health assessment. Currently, monitoring of aquatic plankton biodiversity mainly relies on two types of methods: traditional morphological identification and single molecular biology techniques.

[0003] Traditional morphological identification methods rely on microscopic observation, with taxonomists identifying and counting species based on morphological characteristics of plankton (such as size, structure, and pigmentation). The advantage of this method is that it directly obtains morphological evidence of species and is relatively mature in identifying some common dominant species. However, it has significant limitations: First, it requires highly skilled personnel with extensive taxonomic experience, resulting in a long training cycle and difficulty in meeting the needs of large-scale monitoring. Second, for small-sized (e.g., phytoplankton less than 20 μm), morphologically similar closely related species, or juvenile species, the accuracy rate is extremely low, easily leading to missed or incorrect identifications. Third, the sampling and identification process is time-consuming and labor-intensive, inefficient, and cannot achieve rapid, high-frequency dynamic monitoring. Fourth, some plankton are prone to morphological changes or death after sampling due to environmental changes, further affecting the reliability of the identification results.

[0004] With the development of molecular biology techniques, eDNA macrobarcoding technology has been increasingly applied to plankton monitoring. This technology collects environmental DNA from water samples, performs PCR amplification using universal primers, and then performs high-throughput sequencing on the amplified products, combined with bioinformatics analysis to identify species. Its advantages include convenient sampling, high detection efficiency, and the ability to simultaneously detect multiple species, especially those difficult to morphologically identify. However, this technology also has inherent limitations: First, eDNA may have a residual effect in water, meaning that DNA from dead species can remain in the water for a period of time, leading to inaccurate monitoring results reflecting the current status of species. Second, PCR amplification involves primer bias; some species cannot be effectively amplified due to low primer binding efficiency, resulting in false negatives. Third, the large amount of data generated by high-throughput sequencing may contain exogenous contaminant DNA (such as DNA from terrestrial species introduced by sampling tools), leading to false positives. Fourth, it cannot obtain key information such as species morphology, size, and developmental stage, making it difficult to comprehensively assess community structure.

[0005] Existing single monitoring methods cannot simultaneously meet the monitoring requirements of accuracy, efficiency, and comprehensiveness: traditional morphological identification is inefficient and lacks accuracy; eDNA macrobarcoding technology suffers from false positives, false negatives, and cannot reflect the current status of species.

[0006] Therefore, there is an urgent need for an integrated monitoring method that can combine the advantages of two technologies and complement each other's shortcomings, in order to solve the problems of insufficient accuracy, low efficiency, and incomplete information in existing aquatic planktonic biodiversity monitoring methods. Summary of the Invention

[0007] The purpose of this application is to provide a biodiversity monitoring method that integrates eDNA macrobarcoding and morphology. By combining the high efficiency of eDNA macrobarcoding technology with the reliability of morphological verification, this method provides a precise monitoring method that is highly sensitive, accurate, and comprehensive, providing a scientific basis for aquatic ecological environment assessment, pollution control, and biodiversity conservation.

[0008] To achieve the above objectives, this application provides a biodiversity monitoring method integrating eDNA macrobarcoding and morphology. The method includes: collecting eDNA water samples and morphological samples in parallel at the same sampling point and time; performing DNA extraction, PCR amplification, high-throughput sequencing, and bioinformatics analysis on the eDNA water samples to obtain species screening results; preparing morphological samples, targeting and screening key species based on the species screening results, and performing morphological identification on the key species; and integrating the species screening results and morphological identification results according to a pre-constructed comprehensive evaluation model, performing weight allocation and abundance correction, and outputting monitoring results.

[0009] The biodiversity monitoring method integrating eDNA macrobarcoding and morphology, as described above, includes the parallel collection of eDNA water samples and morphological samples at the same sampling point and at the same time, comprising: Based on the water body type, hydrological characteristics, and pollution status of the monitoring area, sampling points were set up using a grid-based method. At the sampling point, eDNA water samples were collected underwater using sterile sampling bottles; Morphological samples were collected at the sampling points.

[0010] The biodiversity monitoring method integrating eDNA macrobarcoding and morphology, as described above, involves DNA extraction, PCR amplification, high-throughput sequencing, and bioinformatics analysis of eDNA water samples to obtain species screening results, including: DNA was extracted from the eDNA water sample. Select primers; Using the selected primers, PCR amplification was performed using the extracted DNA as a template; For qualified PCR products, high-throughput sequencing and bioinformatics analysis are performed to obtain species screening results.

[0011] The biodiversity monitoring method integrating eDNA macrobarcoding and morphology, as described above, includes the preparation of morphological samples, targeted screening of key species based on species screening results, and morphological identification of key species, comprising: Preparation of morphological samples; Key species were targeted for selection based on species screening results; Morphological identification of key species; Record the results of morphological identification.

[0012] The biodiversity monitoring method integrating eDNA macrobarcoding and morphology, as described above, integrates species screening results and morphological identification results based on a pre-constructed comprehensive evaluation model, performs weight allocation and abundance correction, and outputs monitoring results including: Based on the pre-constructed comprehensive evaluation model, the results of species screening and morphological identification are compared to conduct a consistency test. Weights are assigned to the species identified through the consistency test; The relative abundance of eDNA was corrected for abundance, and the corrected eDNA abundance was calculated. Integrate the corrected species list and abundance data, calculate the diversity index, and output the final result.

[0013] The biodiversity monitoring method that integrates eDNA macrobarcoding and morphology as described above has the following formula for calculating the corrected eDNA abundance: eDNA abundance = eDNA relative abundance × (morphological count / predicted eDNA count).

[0014] The biodiversity monitoring method that integrates eDNA macrobarcoding and morphology as described above includes: comparing species screening results and morphological identification results, classifying them into congruent species, divergent species, and conflicting species; Among them, consistent species are those that are detected in both species screening and morphological identification results and have the same species name; divergent species are those that are detected only in either species screening or morphological identification results; conflicting species are those that are detected in both species screening and morphological identification results and have different names.

[0015] The biodiversity monitoring method that integrates eDNA macrobarcoding and morphology, as described above, involves collecting 1-2 L of eDNA water samples and adding EDTA preservative to the eDNA water samples; morphological samples are collected using a planktonic net trawling method and fixed with 4% formaldehyde.

[0016] The biodiversity monitoring method that integrates eDNA macrobarcoding and morphology, as described above, includes at least three parallel sampling points in each monitoring area.

[0017] The biodiversity monitoring method that integrates eDNA macrobarcoding and morphology as described above includes the following steps for extracting DNA from eDNA water samples: taking 1L of eDNA water sample, enriching microorganisms by vacuum filtration through a filter membrane, and extracting total DNA from the filter membrane using a silica gel membrane column purification method.

[0018] The beneficial effects achieved by this application are as follows: (1) This application effectively reduces false positives and false negatives through the dual guarantee of high-throughput screening of eDNA and morphological targeted verification, and the species identification accuracy rate is ≥95%, which solves the problem of insufficient accuracy of single methods.

[0019] (2) The eDNA technology of this application enables high-throughput species screening, and morphological verification is only performed on key species. Compared with traditional full-sample microscopic examination, the time consumption is reduced by more than 50%, which can meet the needs of large-scale and high-frequency monitoring.

[0020] (3) This application simultaneously obtains the molecular information and morphological characteristics of species, and combined with abundance correction data, it can calculate a variety of diversity indices, providing a more comprehensive scientific basis for ecological assessment.

[0021] (4) This application is applicable to different water body types such as lakes, rivers, and reservoirs. It can monitor various planktonic organisms such as phytoplankton and zooplankton, and is not limited by species size or developmental stage. It can be widely used in aquatic ecological environment monitoring, pollution source tracing, biodiversity protection, fishery resource assessment and other fields, providing strong support for environmental management decision-making. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings.

[0023] Figure 1 This is a flowchart illustrating a biodiversity monitoring method that integrates eDNA macrobarcoding and morphology, according to an embodiment of this application.

[0024] Figure 2 This is a flowchart illustrating a method for parallel collection of eDNA water samples and morphological samples at the same sampling point and at the same time, according to an embodiment of this application.

[0025] Figure 3This is a flowchart illustrating a method for weighting and abundance correction based on a pre-constructed comprehensive evaluation model, integrating species screening results and morphological identification results, according to an embodiment of this application. Detailed Implementation

[0026] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0027] like Figure 1 As shown, this application provides a method for biodiversity monitoring that integrates eDNA macrobarcoding and morphology, the method comprising: Step S1: Collect eDNA water samples and morphological samples in parallel at the same sampling point and at the same time, and set up blank control and positive control simultaneously.

[0028] like Figure 2 As shown, step S1 includes the following steps: Step S110: Based on the water body type, hydrological characteristics and pollution status of the monitoring area, sampling points are set up using a grid-based sampling method.

[0029] Specifically, based on the water body type (such as lakes, rivers, and reservoirs), hydrological characteristics (such as flow velocity and water depth), and pollution status of the monitoring area, sampling points are set up using a grid-based method, with at least three parallel sampling points set up in each monitoring area to ensure representativeness.

[0030] Step S120: At the sampling point, collect eDNA water samples underwater using a sterile sampling bottle.

[0031] Specifically, 1-2L of water samples were collected at a depth of 0.5m below the water surface using sterile sampling bottles. EDTA (ethylenediaminetetraacetic acid) preservative (final concentration 0.1mol / L) was added to obtain eDNA water samples, which were then transported under refrigeration at 4℃. At the same time, blank control samples (sterile ultrapure water) were collected for subsequent contamination investigation.

[0032] Step S130: Collect morphological samples at the sampling point.

[0033] Specifically, morphological samples were obtained by horizontal trawl sampling at the same sampling point using a No. 25 planktonic net (0.064 mm aperture). The trawl distance was 50 m and the speed was 0.5 m / s. The net-collected samples were collected into sterile centrifuge tubes, fixed with 4% formaldehyde solution, and brought back to the laboratory for later use.

[0034] Step S140: Set up blank control and positive control.

[0035] Specifically, a blank control was set up using sterile water as a template. A positive control was set up using a known mixture of planktonic DNA.

[0036] Step S2 involves extracting DNA from the eDNA water sample, performing PCR amplification, high-throughput sequencing, and bioinformatics analysis to obtain species screening results.

[0037] Specifically, DNA extraction, PCR amplification, high-throughput sequencing, and bioinformatics analysis are performed on eDNA water samples to obtain species screening results, enabling efficient and accurate screening of phytoplankton species information in water bodies.

[0038] Step S2 includes the following steps: Step S210: Extract DNA from the eDNA water sample.

[0039] Specifically, 1L of eDNA water sample was taken and enriched with microorganisms by vacuum filtration through a 0.22μm filter membrane. Total DNA was extracted from the filter membrane using a silica gel membrane column purification method. The DNA concentration (≥20ng / μL) and purity (A260 / A280=1.8-2.0) were detected using Nanodrop to ensure DNA quality.

[0040] Step S220: Select primers.

[0041] Specifically, for phytoplankton, the V4-V5 region of the 18S rRNA gene was selected as the target fragment, and the primer sequences were upstream primer 5'-CCAGCASCYGCGGTAATTCC-3' and downstream primer 5'-ACTTTCGTTCTTGATYRA-3'. Specifically, for zooplankton, the conserved region of the COI gene was selected as the target fragment, and the primer sequences were: upstream primer 5'-GGTCAACAAATCATAAAGATATTGG-3'; downstream primer 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'; and amplification conditions were optimized by gradient PCR (annealing temperature 55-60℃, cycle number 30-35) to reduce primer bias.

[0042] Step S230: Using the selected primers, perform PCR amplification with the extracted DNA as a template.

[0043] Specifically, PCR amplification (polymerase chain reaction) was performed using the extracted DNA as a template. The reaction system (25 μL) included: 2.5 μL of 10×PCR Buffer, 2 μL of dNTPs (2.5 mmol / L), 1 μL each of forward and reverse primers (10 μmol / L), 0.2 μL of Taq enzyme (5 U / μL), 1 μL of DNA template, and 17.3 μL of sterile water. A blank control (using sterile water as a template) and a positive control (a mixture of known planktonic DNA) were also set up. The amplification products were detected by 1.5% agarose gel electrophoresis to ensure amplification specificity.

[0044] Specifically, the PCR amplification included: phytoplankton primers for the 18S rRNA V4-V5 region, annealing temperature of 58℃, and 32 cycles; zooplankton primers for the COI gene, annealing temperature of 56℃, and 35 cycles; the blank control showed no amplification product, and the positive control showed normal amplification.

[0045] Step S240: For qualified PCR products, perform high-throughput sequencing and bioinformatics analysis to obtain species screening results.

[0046] Specifically, qualified PCR products were subjected to high-throughput sequencing (Illumina MiSeq platform, 250bp paired ends); after quality control (filtering low-quality reads and removing adapter sequences), the sequencing data were clustered into OTUs using USEARCH software (97% similarity), and aligned to a planktonic reference database (integrating databases such as NCBI, Silva, and BOLD) using BLAST to obtain species annotation information, while also calculating the relative abundance of each species.

[0047] Step S3: Prepare morphological samples, target and screen key species based on species screening results, and perform morphological identification on key species.

[0048] This invention provides targeted validation of eDNA analysis results, thereby improving monitoring accuracy.

[0049] Among them, the key species for targeted screening include: dominant species with a relative abundance of ≥0.1%, suspected species with a database matching degree of 70%-90%, closely related species with a sequence similarity of 95%-99%, and ecological indicator species.

[0050] Step S3 includes: Step S310: Prepare the morphological sample.

[0051] Specifically, the fixed morphological sample was centrifuged and concentrated (3000 r / min, 5 min), the supernatant was removed, and 1 mL of precipitate was retained; 0.1 mL of precipitate was added to a glass slide, a coverslip was placed on top, and a temporary slide was prepared.

[0052] Step S320: Targeted screening of key species is conducted based on the species screening results.

[0053] Specifically, based on the eDNA sequencing results, a list of species to be verified is determined, including: ① dominant species with a relative abundance of ≥0.1%; ② suspected positive species (database matching degree of 70%-90%); ③ closely related species (sequence similarity of 95%-99%); ④ ecological indicator species (such as sensitive species and pollution-tolerant species).

[0054] Step S330: Morphological identification of key species.

[0055] Specifically, optical microscopes (10×40x) are used to observe slides, and morphological identification and counting of target species are performed based on authoritative data such as the "Atlas of Freshwater Plankton in China". For species that are difficult to identify, scanning electron microscopes are used to observe fine structures to improve the accuracy of identification.

[0056] Step S340: Record the morphological identification results.

[0057] Specifically, the names, numbers, morphological characteristics, and confidence levels (high / medium / low) of the identified species are recorded to form a morphological identification report.

[0058] Step S4: Based on the pre-constructed comprehensive evaluation model, the species screening results and morphological identification results are integrated, and weight allocation and abundance correction are performed to output the monitoring results.

[0059] Specifically, the process involves integrating eDNA macrobarcode and morphological verification results based on a pre-constructed comprehensive evaluation model. The comprehensive evaluation model includes algorithms for result consistency testing, weight allocation, abundance correction, and diversity index calculation, which are either written as scripts or embedded into the LIMS system.

[0060] like Figure 3 As shown, step S4 includes the following steps: Step S410: Based on the pre-constructed comprehensive evaluation model, compare the species screening results and morphological identification results to conduct a consistency test.

[0061] Specifically, by comparing species screening results and morphological identification results, species are categorized into congruent species (both detected and with the same species name), divergent species (detected only by eDNA or only by morphology), and conflicting species (detected with different species names). Congruent species are those detected in both species screening and morphological identification results and have the same species name; divergent species are those detected only by either species screening or morphological identification results; and conflicting species are those detected with different names in both species screening and morphological identification results.

[0062] Of these, the same species accounted for 83.3% (phytoplankton) and 87.8% (zooplankton).

[0063] Step S420: Assign weights to the species identified by the consistency test.

[0064] Specifically, weights are set according to species type. Among them, the weight of consistent species is 0.8, the weight of species with only morphological detection (due to unsuccessful eDNA amplification) is 0.7, the weight of species with only eDNA detection and database matching degree ≥90% is 0.6, and conflicting species need to be re-verified and identified.

[0065] Step S430: Perform abundance correction on the relative abundance of eDNA.

[0066] Specifically, based on the morphological counting results, the relative abundance of eDNA is corrected.

[0067] The corrected formula for calculating eDNA abundance is as follows: eDNA abundance = eDNA relative abundance × (morphological count / predicted eDNA count).

[0068] The eDNA abundance is corrected by morphological count. For example, if the eDNA predicts that the relative abundance of cyanobacteria is 35% and the morphological count accounts for 32%, the corrected value is 35% × (32% / 35%) = 32%.

[0069] Step S440: Integrate the corrected species list and abundance data, calculate the diversity index, and output the final result.

[0070] Specifically, the corrected species list and abundance data are integrated to calculate diversity indices (such as the Shannon-Wiener index, Simpson index, and Pielou evenness index) and generate accurate monitoring reports.

[0071] For example, the calculated diversity index is 3.82 in late spring and 3.25 in midsummer, indicating that planktonic biodiversity is higher in late spring.

[0072] As a specific embodiment of the present invention, the monitoring of planktonic biodiversity in a freshwater lake is used as an implementation case. The monitoring area is about 5 km², and five sampling points are set up, located at the lake entrance, center, outlet and near shore area respectively. The monitoring time is May (late spring) and August (midsummer) in 2024 to compare the changes in biodiversity in different seasons.

[0073] Sample collection: Collect 1L of water sample (0.5m below the surface) from each sampling point using a sterile sampling bottle, add 0.1mol / L EDTA, and use as the eDNA water sample. Keep the eDNA water sample refrigerated at 4℃; simultaneously collect two blank control samples (sterile ultrapure water). Use a 25-gauge planktonic net to drag the sample 50m from the same sampling point (the same sampling point as the eDNA water sample) to obtain morphological samples. Collect the samples into centrifuge tubes, fix with 4% formaldehyde, and add 3 drops of glycerol to each sample to prevent dehydration.

[0074] The collected samples underwent eDNA extraction and macrobarcode analysis, including: eDNA extraction: A 1L water sample was filtered through a 0.22μm filter membrane, and DNA was extracted using the DNeasy PowerWater Kit. The DNA concentration was found to be 25-35 ng / μL, and A260 / A280 = 1.85-1.95.

[0075] PCR amplification: Phytoplankton primers were 18S rRNA V4-V5 region, annealing temperature 58℃, cycle number 32; zooplankton primers were COI gene, annealing temperature 56℃, cycle number 35; blank control showed no amplification product, positive control showed normal amplification.

[0076] Sequencing and Analysis: Illumina MiSeq sequencing yielded 400,000-600,000 valid reads per sample. After OTU clustering and comparison with the reference database, a total of 126 phytoplankton species and 89 zooplankton species were detected.

[0077] Morphological verification includes: Sample preparation: Centrifuge the fixed morphological sample to concentrate it to 1 mL, and take 0.1 mL to prepare a slide.

[0078] Targeted screening: 32 phytoplankton species and 25 zooplankton species with a relative abundance of ≥0.1% detected by eDNA, as well as 15 suspected species with a matching degree of 75%-90%, were selected for morphological identification.

[0079] Morphological identification results were obtained: a total of 118 phytoplankton species and 82 zooplankton species were identified. Among them, 105 phytoplankton species and 73 zooplankton species were consistent with the eDNA results; 8 phytoplankton species and 9 zooplankton species were detected only morphologically (eDNA amplification was unsuccessful); 11 phytoplankton species and 6 zooplankton species were detected only eDNA (not observed morphologically, presumably due to their small size or low abundance).

[0080] Specifically, compared with traditional morphological methods (sampling at the same time and location, full sample microscopic examination), the species detection rate of the method of this invention is increased by 42%, the identification time is shortened by 60%, and the accuracy rate is increased from 75% of the traditional method to 96%. Compared with the single eDNA method, the false positive rate is reduced from 12% to 3%, and the false negative rate is reduced from 18% to 5%, which fully demonstrates the accuracy and efficiency of this method.

[0081] This application also provides a computer storage medium storing computer instructions, which, when invoked, execute the address mapping method of the large-capacity solid-state drive. The computer storage medium includes one or more program instructions, which are executed by a processor as a biodiversity monitoring method integrating eDNA macrobarcoding and morphology.

[0082] The embodiments disclosed in this invention provide a computer-readable storage medium storing computer program instructions that, when executed on a computer, cause the computer to perform the aforementioned biodiversity monitoring method integrating eDNA macrobarcoding and morphology.

[0083] This invention provides a processor for processing the above-described biodiversity monitoring method that integrates eDNA macrobarcoding and morphology.

[0084] In this embodiment of the invention, the processor can be an integrated circuit chip with signal processing capabilities. The processor can be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, or discrete hardware components.

[0085] The various methods, steps, and logic diagrams disclosed in the embodiments of this invention can be implemented or executed. The general-purpose processor can be a microprocessor or any conventional processor. The steps of the methods disclosed in the embodiments of this invention can be directly implemented by a hardware decoding processor, or implemented by a combination of hardware and software modules in the decoding processor. The software modules can reside in random access memory, flash memory, read-only memory, programmable read-only memory, electrically erasable programmable memory, registers, or other mature storage media in the art. The processor reads information from the storage medium and, in conjunction with its hardware, completes the steps of the above methods.

[0086] The storage medium can be memory, such as volatile memory or non-volatile memory, or may include both volatile and non-volatile memory.

[0087] The non-volatile memory can be read-only memory (ROM), programmable read-only memory (PROM), erasable programmable read-only memory (EEPROM), electrically erasable programmable read-only memory (EEPROM), or flash memory. The volatile memory can be random access memory (RAM), which is used as an external cache. By way of example, but not limitation, many forms of RAM are available, such as Static Random Access Memory (SRAM), Dynamic Random Access Memory (DRAM), Synchronous DRAM (SDRAM), Double Data Rate SDRAM (DDRSDRAM), Enhanced Synchronous DRAM (ESDRAM), Synchlink DRAM (SLDRAM), and Direct Rambus RAM (DRRAM).

[0088] The beneficial effects achieved by this application are as follows: (1) This application effectively reduces false positives and false negatives through the dual guarantee of high-throughput screening of eDNA and morphological targeted verification, and the species identification accuracy rate is ≥95%, which solves the problem of insufficient accuracy of single methods.

[0089] (2) The eDNA technology of this application enables high-throughput species screening, and morphological verification is only performed on key species. Compared with traditional full-sample microscopic examination, the time consumption is reduced by more than 50%, which can meet the needs of large-scale and high-frequency monitoring.

[0090] (3) This application simultaneously obtains the molecular information and morphological characteristics of species, and combined with abundance correction data, it can calculate a variety of diversity indices, providing a more comprehensive scientific basis for ecological assessment.

[0091] (4) This application is applicable to different water body types such as lakes, rivers, and reservoirs. It can monitor various planktonic organisms such as phytoplankton and zooplankton, and is not limited by species size or developmental stage. It can be widely used in aquatic ecological environment monitoring, pollution source tracing, biodiversity protection, fishery resource assessment and other fields, providing strong support for environmental management decision-making.

[0092] In the description of this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the stated features. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0093] In the description of this application, the word "for example" is used to mean "used as an example, illustration, or description." Any embodiment described as "for example" in this application is not necessarily to be construed as being more preferred or advantageous than other embodiments. The following description is provided to enable any person skilled in the art to make and use the invention. Details are set forth in the following description for purposes of explanation. It should be understood that those skilled in the art will recognize that the invention can be made without using these specific details. In other instances, well-known structures and processes will not be described in detail to avoid obscuring the description of the invention with unnecessary detail. Therefore, the invention is not intended to be limited to the embodiments shown, but is consistent with the broadest scope of the principles and features disclosed in this application.

[0094] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for monitoring biodiversity by integrating eDNA macrobarcoding and morphology, characterized in that, The method includes: eDNA water samples and morphological samples were collected in parallel at the same sampling point and at the same time. DNA extraction, PCR amplification, high-throughput sequencing, and bioinformatics analysis were performed on the eDNA water samples to obtain species screening results. Morphological samples were prepared, key species were targeted for screening based on species screening results, and morphological identification of key species was performed. Based on a pre-constructed comprehensive evaluation model, the results of species screening and morphological identification are integrated, and weights are assigned and abundance is corrected to output the monitoring results.

2. The biodiversity monitoring method integrating eDNA macrobarcoding and morphology according to claim 1, characterized in that, Parallel collection of eDNA water samples and morphological samples at the same sampling point and at the same time included: Based on the water body type, hydrological characteristics, and pollution status of the monitoring area, sampling points were set up using a grid-based method. At the sampling point, eDNA water samples were collected underwater using sterile sampling bottles; Morphological samples were collected at the sampling points.

3. The biodiversity monitoring method integrating eDNA macrobarcoding and morphology according to claim 1, characterized in that, DNA extraction, PCR amplification, high-throughput sequencing, and bioinformatics analysis were performed on the eDNA water samples to obtain species screening results, including: DNA was extracted from the eDNA water sample. Select primers; Using the selected primers, PCR amplification was performed using the extracted DNA as a template; For qualified PCR products, high-throughput sequencing and bioinformatics analysis are performed to obtain species screening results.

4. The biodiversity monitoring method integrating eDNA macrobarcoding and morphology according to claim 1, characterized in that, Morphological samples were prepared, key species were targeted for screening based on species screening results, and morphological identification of key species was performed, including: Preparation of morphological samples; Key species were targeted for selection based on species screening results; Morphological identification of key species; Record the results of morphological identification.

5. The biodiversity monitoring method integrating eDNA macrobarcoding and morphology according to claim 1, characterized in that, Based on a pre-constructed comprehensive evaluation model, the results of species screening and morphological identification are integrated, and weights are assigned and abundance is corrected. The output monitoring results include: Based on the pre-constructed comprehensive evaluation model, the results of species screening and morphological identification are compared to conduct a consistency test. Weights are assigned to the species identified through the consistency test; The relative abundance of eDNA was corrected for abundance, and the corrected eDNA abundance was calculated. Integrate the corrected species list and abundance data, calculate the diversity index, and output the final result.

6. The biodiversity monitoring method integrating eDNA macrobarcoding and morphology according to claim 5, characterized in that, The corrected formula for calculating eDNA abundance is: eDNA abundance = relative eDNA abundance × (morphological count / predicted eDNA count).

7. The biodiversity monitoring method integrating eDNA macrobarcoding and morphology according to claim 5, characterized in that, include: Based on the comparison of species screening results and morphological identification results, they were divided into congruent species, divergent species, and conflicting species. Among them, consistent species are those that are detected in both species screening and morphological identification results and have the same species name; divergent species are those that are detected only in either species screening or morphological identification results; conflicting species are those that are detected in both species screening and morphological identification results and have different names.

8. The biodiversity monitoring method integrating eDNA macrobarcoding and morphology according to claim 2, characterized in that, The volume of eDNA water samples collected was 1-2L, and EDTA preservative was added to the eDNA water samples; morphological samples were collected by trawling with a planktonic net, and 4% formaldehyde was added to the collected morphological samples for fixation.

9. The biodiversity monitoring method integrating eDNA macrobarcoding and morphology according to claim 2, characterized in that, At least three parallel sampling points should be set up in each monitoring area.

10. The biodiversity monitoring method integrating eDNA macrobarcoding and morphology according to claim 3, characterized in that, DNA extraction from eDNA water samples includes: taking 1L of eDNA water sample, enriching microorganisms by vacuum filtration through a filter membrane, and extracting total DNA from the filter membrane using a silica gel membrane column purification method.

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