Volume 43 Issue 2
Mar.  2022
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Hui-Ting Ruan, Rui-Li Wang, Hong-Ting Li, Li Liu, Tian-Xu Kuang, Min Li, Ke-Shu Zou. Effects of sampling strategies and DNA extraction methods on eDNA metabarcoding: A case study of estuarine fish diversity monitoring. Zoological Research, 2022, 43(2): 192-204. doi: 10.24272/j.issn.2095-8137.2021.331
Citation: Hui-Ting Ruan, Rui-Li Wang, Hong-Ting Li, Li Liu, Tian-Xu Kuang, Min Li, Ke-Shu Zou. Effects of sampling strategies and DNA extraction methods on eDNA metabarcoding: A case study of estuarine fish diversity monitoring. Zoological Research, 2022, 43(2): 192-204. doi: 10.24272/j.issn.2095-8137.2021.331

Effects of sampling strategies and DNA extraction methods on eDNA metabarcoding: A case study of estuarine fish diversity monitoring

doi: 10.24272/j.issn.2095-8137.2021.331
Funds:  This work was supported by the National Natural Science Foundation of China (32102793), National Key R&D Program of China (2018YFD0900802), Central Public-Interest Scientific Institution Basal Research Fund, South China Sea Fisheries Research Institute, CAFS (2019TS13, 2021SD18), Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (GML2019ZD0605), Open Fund Project of Key Laboratory of Offshore Fishery Development of Ministry of Agriculture and Rural Affairs (LOF 2020-02), and China-ASEAN Maritime Cooperation Fund (CAMC-2018F)
More Information
  • Environmental DNA (eDNA) integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity. To date, however, no standardized eDNA-based protocol has been established to monitor fish diversity. In this study, we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary (PRE), a highly anthropogenically disturbed estuarine ecosystem. Compared to filtration-based precipitation, direct filtration was a more suitable method for eDNA metabarcoding in the PRE. The combined use of DNeasy Blood and Tissue Kit (BT) and traditional phenol/chloroform (PC) extraction produced higher DNA yields, amplicon sequence variants (ASVs), and Shannon diversity indices, and generated more homogeneous and consistent community composition among replicates. Compared to the other combined protocols, the PC and BT methods obtained better species detection, higher fish diversity, and greater consistency for the filtration water volumes of 1 000 and 2 000 mL, respectively. All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity. Furthermore, combining traditional methods with eDNA analysis enhanced accuracy. These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.
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  • [1]
    Ahn H, Kume M, Terashima Y, Ye F, Kameyama S, Miya M, et al. 2020. Evaluation of fish biodiversity in estuaries using environmental DNA metabarcoding. PLoS One, 15(10): e0231127. doi: 10.1371/journal.pone.0231127
    Barnes MA, Turner CR. 2016. The ecology of environmental DNA and implications for conservation genetics. Conservation Genetics, 17(1): 1−17. doi: 10.1007/s10592-015-0775-4
    Barnes MA, Turner CR, Jerde CL, Renshaw MA, Chadderton WL, Lodge DM. 2014. Environmental conditions influence eDNA persistence in aquatic systems. Environmental Science & Technology, 48(3): 1819−1827.
    Beng KC, Corlett RT. 2020. Applications of environmental DNA (eDNA) in ecology and conservation: opportunities, challenges and prospects. Biodiversity and Conservation, 29(7): 2089−2121. doi: 10.1007/s10531-020-01980-0
    Bessey C, Jarman SN, Berry O, Olsen YS, Bunce M, Simpson T, et al. 2020. Maximizing fish detection with eDNA metabarcoding. Environmental DNA, 2(4): 493−504. doi: 10.1002/edn3.74
    Bhakta D, Meetei WA, Vaisakh G, Kamble S, Das SK, Das BK. 2019. Finfish diversity of narmada estuary in Gujarat of India. Proceedings of the Zoological Society, 72(3): 257−262. doi: 10.1007/s12595-018-0263-1
    Blowes SA, Supp SR, Antão LH, Bates A, Bruelheide H, Chase JM, et al. 2019. The geography of biodiversity change in marine and terrestrial assemblages. Science, 366(6463): 339−345. doi: 10.1126/science.aaw1620
    Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, Knight R, et al. 2013. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nature Methods, 10(1): 57−59. doi: 10.1038/nmeth.2276
    Boulanger E, Loiseau N, Valentini A, Arnal V, Boissery P, Dejean T, et al. 2021. Environmental DNA metabarcoding reveals and unpacks a biodiversity conservation paradox in Mediterranean marine reserves. Proceedings of the Royal Society B:Biological Sciences, 288(1949): 20210112. doi: 10.1098/rspb.2021.0112
    Buxton A, Matechou E, Griffin J, Diana A, Griffiths RA. 2021. Optimising sampling and analysis protocols in environmental DNA studies. Scientific Reports, 11(1): 11637. doi: 10.1038/s41598-021-91166-7
    Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. 2016. DADA2: high-resolution sample inference from Illumina amplicon data. Nature Methods, 13(7): 581−583. doi: 10.1038/nmeth.3869
    Callahan H. 2009. Inhibitor-free DNA purification from water samples. BioTechniques, 46(6): 473. doi: 10.2144/000113159
    Cantera I, Cilleros K, Valentini A, Cerdan A, Dejean T, Iribar A, et al. 2019. Optimizing environmental DNA sampling effort for fish inventories in tropical streams and rivers. Scientific Reports, 9(1): 3085. doi: 10.1038/s41598-019-39399-5
    Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. 2010. QIIME allows analysis of high-throughput community sequencing data. Nature Methods, 7(5): 335−336. doi: 10.1038/nmeth.f.303
    Costello MJ, Basher Z, McLeod L, Asaad I, Claus S, Vandepitte L, et al. 2017. Methods for the study of marine biodiversity. In: Walters M, Scholes RJ. The GEO Handbook on Biodiversity Observation Networks. Cham: Springer, 129–163.
    Coutant O, Cantera I, Cilleros K, Dejean T, Valentini A, Murienne J, et al. 2021. Detecting fish assemblages with environmental DNA: does protocol matter? Testing eDNA metabarcoding method robustness. Environmental DNA, 3(3): 619−630. doi: 10.1002/edn3.158
    Deiner K, Bik HM, Mächler E, Seymour M, Lacoursière-Roussel A, Altermatt F, et al. 2017. Environmental DNA metabarcoding: transforming how we survey animal and plant communities. Molecular Ecology, 26(21): 5872−5895. doi: 10.1111/mec.14350
    Deiner K, Lopez J, Bourne S, Holman L, Seymour M, Grey EK, et al. 2018. Optimising the detection of marine taxonomic richness using environmental DNA metabarcoding: the effects of filter material, pore size and extraction method. Metabarcoding and Metagenomics, 2: e28963. doi: 10.3897/mbmg.2.28963
    Deiner K, Walser JC, Mächler E, Altermatt F. 2015. Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA. Biological Conservation, 183: 53−63. doi: 10.1016/j.biocon.2014.11.018
    Dixon KM, Cary GJ, Worboys GL, Banks SC, Gibbons P. 2019. Features associated with effective biodiversity monitoring and evaluation. Biological Conservation, 238: 108221. doi: 10.1016/j.biocon.2019.108221
    Djurhuus A, Port J, Closek CJ, Yamahara KM, Romero-Maraccini O, Walz KR, et al. 2017. Evaluation of filtration and DNA extraction methods for environmental DNA biodiversity assessments across multiple trophic levels. Frontiers in Marine Science, 4: 314. doi: 10.3389/fmars.2017.00314
    Dubart M, Pantel JH, Pointier JP, Jarne P, David P. 2019. Modeling competition, niche, and coexistence between an invasive and a native species in a two-species metapopulation. Ecology, 100(6): e02700.
    Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics, 27(16): 2194−2200. doi: 10.1093/bioinformatics/btr381
    Foote AD, Thomsen PF, Sveegaard S, Wahlberg M, Kielgast J, Kyhn LA, et al. 2012. Investigating the potential use of environmental DNA (eDNA) for genetic monitoring of marine mammals. PLoS One, 7(8): e41781. doi: 10.1371/journal.pone.0041781
    Gold Z, Sprague J, Kushner DJ, Marin EZ, Barber PH. 2021. eDNA metabarcoding as a biomonitoring tool for marine protected areas. PLoS One, 16(2): e0238557. doi: 10.1371/journal.pone.0238557
    Goldberg CS, Turner CR, Deiner K, Klymus KE, Thomsen PF, Murphy MA, et al. 2016. Critical considerations for the application of environmental DNA methods to detect aquatic species. Methods in Ecology and Evolution, 7(11): 1299−1307. doi: 10.1111/2041-210X.12595
    Heberle H, Vaz Meirelles G, da Silva FR, Telles GP, Minghim R. 2015. InteractiVenn: a web-based tool for the analysis of sets through Venn diagrams. BMC Bioinformatics, 16(1): 169. doi: 10.1186/s12859-015-0611-3
    Hermans SM, Buckley HL, Lear G. 2018. Optimal extraction methods for the simultaneous analysis of DNA from diverse organisms and sample types. Molecular Ecology Resources, 18(3): 557−569. doi: 10.1111/1755-0998.12762
    Hiddink JG, Jennings S, Sciberras M, Bolam SG, Cambiè G, McConnaughey RA, et al. 2019. Assessing bottom trawling impacts based on the longevity of benthic invertebrates. Journal of Applied Ecology, 56(5): 1075−1084. doi: 10.1111/1365-2664.13278
    Irfan S, Alatawi AMM. 2019. Aquatic ecosystem and biodiversity: a review. Open Journal of Ecology, 9(1): 1−13. doi: 10.4236/oje.2019.91001
    Jerde CL, Chadderton WL, Mahon AR, Renshaw MA, Corush J, Budny ML, et al. 2013. Detection of Asian carp DNA as part of a Great Lakes basin-wide surveillance program. Canadian Journal of Fisheries and Aquatic Sciences, 70(4): 522−526. doi: 10.1139/cjfas-2012-0478
    Jeunen GJ, Knapp M, Spencer HG, Taylor HR, Lamare MD, Stat M, et al. 2019. Species-level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization. Ecology and Evolution, 9(3): 1323−1335. doi: 10.1002/ece3.4843
    Kimura M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16(2): 111−120. doi: 10.1007/BF01731581
    Koziol A, Stat M, Simpson T, Jarman S, Dibattista JD, Harvey ES, et al. 2019. Environmental DNA metabarcoding studies are critically affected by substrate selection. Molecular Ecology Resources, 19(2): 366−376. doi: 10.1111/1755-0998.12971
    Kumar G, Eble JE, Gaither MR. 2020. A practical guide to sample preservation and pre-PCR processing of aquatic environmental DNA. Molecular Ecology Resources, 20(1): 29−39. doi: 10.1111/1755-0998.13107
    Lear G, Dickie I, Banks J, Boyer S, Buckley HL, Buckley TR, et al. 2018. Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples. New Zealand Journal of Ecology, 42(1): 10−50A.
    Li GF, Zhou L, Ye SH, Zeng L, Weng SP, Liu L, et al. 2018. Conservations of Fish Diversity and Resource in the Pearl River Estuary. Beijing: China Agriculture Press. (in Chinese)
    Liang ZB, Keeley A. 2013. Filtration recovery of extracellular DNA from environmental water samples. Environmental Science & Technology, 47(16): 9324−9331.
    Liu D, Cai L, Yang YO, Hu JW. 2018. Length‐weight relationships of two fish species caught from the estuary of the Pearl River. Journal of Applied Ichthyology, 34(4): 1071−1072. doi: 10.1111/jai.13713
    Lugg WH, Griffiths J, van Rooyen AR, Weeks AR, Tingley R. 2018. Optimal survey designs for environmental DNA sampling. Methods in Ecology and Evolution, 9(4): 1049−1059. doi: 10.1111/2041-210X.12951
    Magoč T, Salzberg SL. 2011. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics, 27(21): 2957−2963. doi: 10.1093/bioinformatics/btr507
    Majaneva M, Diserud OH, Eagle SHC, Boström E, Hajibabaei M, Ekrem T. 2018. Environmental DNA filtration techniques affect recovered biodiversity. Scientific Reports, 8(1): 4682. doi: 10.1038/s41598-018-23052-8
    Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet Journal, 17(1): 10−12. doi: 10.14806/ej.17.1.200
    Mauvisseau Q, Burian A, Gibson C, Brys R, Ramsey A, Sweet M. 2019. Influence of accuracy, repeatability and detection probability in the reliability of species-specific eDNA based approaches. Scientific Reports, 9(1): 580. doi: 10.1038/s41598-018-37001-y
    Minamoto T, Naka T, Moji K, Maruyama A. 2016. Techniques for the practical collection of environmental DNA: filter selection, preservation, and extraction. Limnology, 17(1): 23−32. doi: 10.1007/s10201-015-0457-4
    Mirimin L, Desmet S, Romero DL, Fernandez SF, Miller DL, Mynott S, et al. 2021. Don't catch me if you can – Using cabled observatories as multidisciplinary platforms for marine fish community monitoring: an in situ case study combining Underwater Video and environmental DNA data. Science of the Total Environment, 773: 145351. doi: 10.1016/j.scitotenv.2021.145351
    Murphy HM, Jenkins GP. 2010. Observational methods used in marine spatial monitoring of fishes and associated habitats: a review. Marine and Freshwater Research, 61(2): 236−252. doi: 10.1071/MF09068
    Nukazawa K, Hamasuna Y, Suzuki Y. 2018. Simulating the advection and degradation of the environmental DNA of common carp along a river. Environmental Science & Technology, 52(18): 10562−10570.
    Piggott MP. 2016. Evaluating the effects of laboratory protocols on eDNA detection probability for an endangered freshwater fish. Ecology and Evolution, 6(9): 2739−2750. doi: 10.1002/ece3.2083
    Polanco FA, Martinezguerra MM, Marques V, Villa‐Navarro F, Pérez GHB, Cheutin MC, et al. 2021. Detecting aquatic and terrestrial biodiversity in a tropical estuary using environmental DNA. Biotropica, 53(6): 1606−1619. doi: 10.1111/btp.13009
    Pont D, Rocle M, Valentini A, Civade R, Jean P, Maire A, et al. 2018. Environmental DNA reveals quantitative patterns of fish biodiversity in large rivers despite its downstream transportation. Scientific Reports, 8(1): 10361. doi: 10.1038/s41598-018-28424-8
    Prié V, Valentini A, Lopes-Lima M, Froufe E, Rocle M, Poulet N, et al. 2021. Environmental DNA metabarcoding for freshwater bivalves biodiversity assessment: methods and results for the Western Palearctic (European sub-region). Hydrobiologia, 848(12): 2931−2950.
    Rajendhran J, Gunasekaran P. 2008. Strategies for accessing soil metagenome for desired applications. Biotechnology Advances, 26(6): 576−590. doi: 10.1016/j.biotechadv.2008.08.002
    Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology Evolution, 4(4): 406−425.
    Sakata MK, Watanabe T, Maki N, Ikeda K, Kosuge T, Okada H, et al. 2021. Determining an effective sampling method for eDNA metabarcoding: a case study for fish biodiversity monitoring in a small, natural river. Limnology, 22(2): 221−235. doi: 10.1007/s10201-020-00645-9
    Sales NG, Wangensteen OS, Carvalho DC, Deiner K, Præbel K, Coscia I, et al. 2021. Space-time dynamics in monitoring neotropical fish communities using eDNA metabarcoding. Science of the Total Environment, 754: 142096. doi: 10.1016/j.scitotenv.2020.142096
    Santos RVS, Ramos S, Bonecker ACT. 2017. Can we assess the ecological status of estuaries based on larval fish assemblages?. Marine Pollution Bulletin, 124(1): 367−375. doi: 10.1016/j.marpolbul.2017.07.043
    Shaw JLA, Clarke LJ, Wedderburn SD, Barnes TC, Weyrich LS, Cooper A. 2016. Comparison of environmental DNA metabarcoding and conventional fish survey methods in a river system. Biological Conservation, 197: 131−138.
    Shu L, Ludwig A, Peng ZG. 2020. Standards for methods utilizing environmental DNA for detection of fish species. Genes, 11(3): 296. doi: 10.3390/genes11030296
    Stat M, John J, Dibattista JD, Newman SJ, Bunce M, Harvey ES. 2019. Combined use of eDNA metabarcoding and video surveillance for the assessment of fish biodiversity. Conservation Biology, 33(1): 196−205. doi: 10.1111/cobi.13183
    Stauffer S, Jucker M, Keggin T, Marques V, Andrello M, Bessudo S, et al. 2021. How many replicates to accurately estimate fish biodiversity using environmental DNA on coral reefs?. Ecology and Evolution, 11(21): 14630−14643. doi: 10.1002/ece3.8150
    Sun DR, Chen Z. 2013. Retrieval of Fish in South China Sea. Beijing: Ocean Press. (in Chinese)
    Taberlet P, Bonin A, Zinger L, Coissac E. 2018. Environmental DNA: for Biodiversity Research and Monitoring. Oxford: Oxford University Press.
    Tringe SG, Rubin EM. 2005. Metagenomics: DNA sequencing of environmental samples. Nature Reviews Genetics, 6(11): 805−814. doi: 10.1038/nrg1709
    Troth CR, Sweet MJ, Nightingale J, Burian A. 2021. Seasonality, DNA degradation and spatial heterogeneity as drivers of eDNA detection dynamics. Science of the Total Environment, 768: 144466. doi: 10.1016/j.scitotenv.2020.144466
    Tsuji S, Takahara T, Doi H, Shibata N, Yamanaka H. 2019. The detection of aquatic macroorganisms using environmental DNA analysis—A review of methods for collection, extraction, and detection. Environmental DNA, 1(2): 99−108. doi: 10.1002/edn3.21
    Turner CR, Barnes MA, Xu CCY, Jones SE, Jerde CL, Lodge DM. 2014. Particle size distribution and optimal capture of aqueous macrobial eDNA. Methods in Ecology and Evolution, 5(7): 676−684. doi: 10.1111/2041-210X.12206
    Valdivia-Carrillo T, Rocha-Olivares A, Reyes-Bonilla H, Domínguez-Contreras JF, Munguia-Vega A. 2021. Integrating eDNA metabarcoding and simultaneous underwater visual surveys to describe complex fish communities in a marine biodiversity hotspot. Molecular Ecology Resources, 21(5): 1558−1574. doi: 10.1111/1755-0998.13375
    Valentini A, Taberlet P, Miaud C, Civade R, Herder J, Thomsen PF, et al. 2016. Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding. Molecular Ecology, 25(4): 929−942. doi: 10.1111/mec.13428
    Wang SP, Yan ZG, Hänfling B, Zheng X, Wang PY, Fan JT, et al. 2021. Methodology of fish eDNA and its applications in ecology and environment. Science of the Total Environment, 755: 142622. doi: 10.1016/j.scitotenv.2020.142622
    Wintzingerode FV, Göbel UB, Stackebrandt E. 1997. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiology Reviews, 21(3): 213−229. doi: 10.1111/j.1574-6976.1997.tb00351.x
    Wittwer C, Nowak C, Strand DA, Vrålstad T, Thines M, Stoll S. 2018. Comparison of two water sampling approaches for eDNA-based crayfish plague detection. Limnologica, 70: 1−9. doi: 10.1016/j.limno.2018.03.001
    Yan HF, Kyne PM, Jabado RW, Leeney RH, Davidson LNK, Derrick DH, et al. 2021. Overfishing and habitat loss drive range contraction of iconic marine fishes to near extinction. Science Advances, 7(7): eabb6026. doi: 10.1126/sciadv.abb6026
    Zhang BJ, Mo JH, Mai JB, Li M, Huang LB, Mai GQ, et al. 2015. The fish population composition and the variations of the fishery resources in Humen Harbor Waters. Freshwater Fisheries, 45(5): 50−58. (in Chinese)
    Zheng CY. 1989. Pearl River Fishes. Beijing: Science Press. (in Chinese)
    Zhou L, Wang GP, Kuang TX, Guo DL, Li GF. 2019. Fish assemblage in the Pearl River Estuary: spatial‐seasonal variation, environmental influence and trends over the past three decades. Journal of Applied Ichthyology, 35(4): 884−895.
    Zou KS, Chen JW, Ruan HT, Li ZH, Guo WJ, Li M, et al. 2020. eDNA metabarcoding as a promising conservation tool for monitoring fish diversity in a coastal wetland of the Pearl River Estuary compared to bottom trawling. Science of the Total Environment, 702: 134704. doi: 10.1016/j.scitotenv.2019.134704
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