High-throughput sequencing of Diatoms using V4 region of 18S rRNA gene in Bayug Island, Iligan City, Philippines

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DARSHEL ESTER P. ESTOR
SHARON ROSE TABUGO
https://orcid.org/0000-0001-6813-840X

Abstract

Abstract. Estor DEP, Tabugo SR. 2023. High-throughput sequencing of Diatoms using V4 region of 18S rRNA gene in Bayug Island, Iligan City, Philippines. Biodiversitas 24: 6343-6350. Diatoms are the main contributors to aquatic primary production and are the most dominant phytoplankton. Their adaptability and growth in diverse natural resources enable them to thrive in various climates and geographical areas, making them suitable for monitoring and biotechnology applications. This study aimed to analyze the diatom composition in two study sites: a mangrove area and the estuary in Bayug Island. The analysis was conducted using metabarcoding of the V4 region of the 18S rRNA gene, amplified and sequenced on the Illumina MiSeq platform. Four amplicon libraries were generated, representing the diatom composition at the two study sites. Data processing was performed using the Parallel-Meta-Suite software. Following quality control and processing, 20,433 amplicon sequence variants (ASVs) were obtained. Among these, the genera Navicula, Thalassiosira, Chaetoceros, Guinardia, Amphora, and Minidiscus were the most abundant ASVs. The assessment of diatom alpha diversity using the Shannon index indicated a higher diversity in the estuary site. Furthermore, the analysis done within the Parallel Meta Suite suggested that the presence of these specific diatom species can be linked to salinity levels, and may indicate environmental disturbances such as water pollution. The presence of these diatoms has the potential to be utilized in ecological treatments, disease prevention, biotechnology, and as nutrient indicators for other marine organisms. Physico-chemical parameters were recorded in the estuary area (S1), with the average water pH measuring around 7.6, salinity at approximately 36.6 ppt, and water temperature at 26.7?. In contrast, the measurements in the mangrove area (S2) revealed an average water temperature of 30?, salinity of around 35 ppt, and a water pH of 7.7. This research will serve as baseline data for diatom community in the area and a pioneering work on using NGS and eDNA for diatom study.

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References
Almarez DN, Almarez FJS, Baulete EM. 2014. Bayug Island Aquasilvi Program: An Eco-Governance Strategy for Climate Change Adaptation and Mitigation. J Govern Dev 10(2): 35-54. DOI: 10.32890/jgd.
Alongi DM. 2014. Carbon cycling and storage in mangrove forests. Ann Rev Mar Sci 6: 195-219. DOI: 10.1146/annurev-marine-010213-135020.
Balint M, Pfenninger M, Grossart HP, Taberlet P, Vellend M, Leibold MA, Englund G, Bowler D. 2018. Environmental DNA time series in ecology. Eco Evo 33(12):945-957. DOI: 10.1016/j.tree.2018.09.003.
Balqis SA, Yusoff FM, Nishikawa J, Lindsay D, Nishida S. 2019. Influence of environmental parameters on habitat preference of gelatinous zooplankton in various coastal ecosystems, the Straits of Malacca. Reg Stud in Mar Sci 30:100712. DOI: 10.1016/j.rsma.2019.100712.
Beng KC, Corlett RT. 2020. Applications of environmental DNA (eDNA) in ecology and conservation: opportunities, challenges and prospects. Biodiver Con 29: 2089-2121. DOI:10.1007/s10531-020-01980-0.
Bergström, A.-K.; Jonsson, A.; Isles, P.D.F.; Creed, I.F.; Lau, D.C.P. Changes in nutritional quality and nutrient limitation regimes of phytoplankton in response to declining N deposition in mountain lakes. Aqua Sci 82(31):1-16. DOI:101007/s00027-020-0697-1.
Bylemans J, Gleeson DM, Duncan RP, Hardy CM, Furlan EM. 2019. A performance evaluation of targeted eDNA and eDNA metabarcoding analyses for freshwater fishes. Environmental DNA 1(4):402-414. DOI:10.1002/edn3.41.
Carey JC, Fulweiler RW. 2014. Salt marsh tidal exchange increases residence time of silica in estuaries. Lim Ocean J 59(4): 1203-1212. DOI:10.4319/lo.2014.59.4.1203.
Carraro L, Bertuzzo E, Fronhofer EA, Furrer R, Gounand I, Rinaldo A, Altermatt F. 2020. Generation and application of river network analogues for use in ecology and evolution. J Eco Evo 10(14): 7537-7550. DOI: 10.1002/ece3.6479.
Carter JL, Resh VH, Hannaford MJ. 2017. Macroinvertebrates as biotic indicators of environmental quality. In Methods in stream ecology, Acad Press 293-318. DOI:10.1016/B978-0-12-813047-6.00016-4.
Chain FJ, Brown EA, MacIsaac HJ, Cristescu ME. 2016. Metabarcoding reveals strong spatial structure and temporal turnover of zooplankton communities among marine and freshwater ports. J Diver and Dis 22(5): 493-504. DOI: 10.1111/ddi.12427.
Chariton AA, Stephenson S, Morgan MJ, Steven AD, Colloff MJ, Court LN, Hardy CM. 2015. Metabarcoding of benthic eukaryote communities predicts the ecological condition of estuaries. Environ Pol 203:165-174. DOI: 10.1111/ddi.12427.
Chen Y, Li J, Zhang Y, Zhang M, Sun Z, Jing G, Su X. 2022. Parallel?Meta Suite: Interactive and rapid microbiome data analysis on multiple platforms. iMeta 1(1):1-11. DOI:10.1002/imt2.1.
Cloern JE, Foster SQ, Kleckner AE. 2014. Phytoplankton primary production in the world's estuarine-coastal ecosystems. Biogeosciences 11(9):2477-2501. DOI:10.5194/bg-11-2477-2014.
Cui Z, Liu S, Xu Q, Zhao Y, Chen N. 2023. Differential ecological adaptation of diverse Chaetoceros species revealed by metabarcoding analysis. Environment 1-19. DOI:101002/edn3.455.
Dafforn KA, Johnston EL, Ferguson A, Humphrey CL, Monk W, Nichols SJ, Baird DJ. 2015. Big data opportunities and challenges for assessing multiple stressors across scales in aquatic ecosystems. Mar Fresh Res 67(4):393-413. DOI:10.1071/MF15108.
Daniels CJ, Poulton AJ, Esposito M, Paulsen ML, Bellerby R, St John M, martin AP. 2015. Phytoplankton dynamics in contrasting early stage North Atlantic spring blooms: composition, succession, and potential drivers. Biogeosciences 12:2395–2409. DOI:10.5194/bg-12-2395-2015.
Deiner K, Renshaw MA, Li Y, Olds BP, Lodge DM, Pfrender ME. 2017. Long?range PCR allows sequencing of mitochondrial genomes from environmental DNA. Met Eco Evo 8(12):1888-1898. DOI:10.1111/2041-210X.12836.
Depledge MH, Lovell R, Wheeler BW, Morrissey KM, White M, Fleming LE. 2017. Future of the sea: health and wellbeing of coastal communities. http://hdl.handle.net/10871/31606
Desrosiers C, Leflaive J, Eulin A, Ten-Hage L. 2013. Bioindicators in marine waters: benthic diatoms as a tool to assess water quality from eutrophic to oligotrophic coastal ecosystems. Eco Ind 32:25-34. DOI:10.1016/j.ecolind.2013.02.021.
DOI: 10.1371/journal.pone.0177643.
Edgar GJ, Stuart-Smith RD, Willis TJ, Kininmonth S, Baker SC, Banks S, Barrette NS, Becerro, MA, Bernard, ATF, Bekhout J, Buston CD, Campbell SJ, Cooper AT, Davey M, Edgar SC, Forsterra G, Galvan DE, Irigoyen AJ, Kushner DJ, Moura R, Parnel PE, Shears NT, Soler G, Strain EMA, Thomson RJ. 2014. Global conservation outcomes depend on marine protected areas with five key features. Nature 506(7487):216-220. DOI:10.1038/nature13022.
El-Sheek M, Abu-Faddan M, Abo-Shady A, Nassar MZA, Labib W. 2023. Seasonal dynamics of phytoplankton in the northern part of Suez Gulf, Egypt. Environ Monit Assess 195:1060. DOI:10.1007/s10661-023-11688-7.
Evans NT, Shirey PD, Wieringa JG, Mahon AR, Lamberti GA. 2017. Comparative cost and effort of fish distribution detection via environmental DNA analysis and electrofishing. Fisheries 42(2):90-99. DOI: 10.1080/03632415.2017.1276329.
Flanjak L, Vrana I, Kušan AC, Godrijan J, Novak T, Penezi? A, Gašparovi? B. 2022. Effects of high temperature and nitrogen availability on the growth and composition of the marine diatom Chaetoceros pseudocurvisetus, J Exp Bot 73(12):4250–4265. DOI:10.1093/jxb/erac145.
Fonseca VG, Davison PI, Creach V, Stone D, Bass D, Tidbury HJ. 2023. The Application of eDNA for Monitoring Aquatic Non-Indigenous Species: Practical and Policy Considerations. Diversity 15(5):631. DOI: 10.3390/d15050631.
Goldberg CS, Strickler KM, Pilliod DS. 2015. Moving environmental DNA methods from concept to practice for monitoring aquatic microorganisms. Bio Conserv 183:1-3. DOI:10.1016/j.biocon.2014.11.040.
Hardikar R, Haridevi CK, Chowdhury M, Shinde, N, Ram A., Rokade MA, Rakesh PS. 2017. Seasonal distribution of phytoplankton and its association with physico-chemical parameters in coastal waters of Malvan, west coast of India. Environ Mon Assess 189(151):1-16. DOI 10.1007/s10661-017-5835-4.
Hassan HA, Muhamad MH, Ji B, Nazari NA, Jiat KW, Sim SISWA, Poh AFMS. 2023. Revolutionizing wastewater treatment with microalgae: Unveiling resource recovery, mechanisms, challenges, and future possibilities. Eco Eng 197:107117. DOI: 10.1016/j.ecoleng.2023.107117.
Hilaluddin F, Yusoff FM, Natrah FMI, Lim PT. 2020. Disturbance of mangrove forests causes alterations in estuarine phytoplankton community structure in Malaysian Matang mangrove forests. Mar Environ Res 158:104935. DOI: 10.1016/j.marenvres.2020.104935.
Holman LE, de Bruyn M, Creer S, Carvalho G, Robidart J, Rius M. 2019. Detection of introduced and resident marine species using environmental DNA metabarcoding of sediment and water. Sci Rep 9(1), 11559. DOI:10.1038/s41598-019-47899-7.
Ishika T, Bahri PA, Laird DW, Moheimani NR. 2018. The effect of gradual increase in salinityon the biomass productivity and biochemical composition of several marine, halotolerant, and halophilic microalgae. App Phycol J 30, 1453-1464. DOI:10.1007/s10811-017-1377-y.
Jin P, Agustí S. 2018. Fast adaptation of tropical diatoms to increased warming with trade-offs. Sci Rep, 8(1):17771. DOI: 10.1038/s41598-018-36091-y.
Kanniah P, Chelliah P, Thangapandi JR, Thangapandi EJJSB, Kasi M, Sivasubramaniam S. 2022. Benign fabrication of metallic/metal oxide nanoparticles from algae. Nanobiotech for Plant Pro 465-493. DOI:10.1016/B978-0-12-823575-1.00007-X.
Keck F, Vasselon V, Rimet F, Bouchez A, Kahlert M. 2018. Boosting DNA metabarcoding for biomonitoring with phylogenetic estimation of operational taxonomic units’ ecological profiles. Mol Eco Res 18(6): 1299-1309. DOI: 10.1111/1755-0998.12919.
Keeley N, Wood SA, Pochon X. 2018.Development and preliminary validation of a multi-trophic metabarcoding biotic index for monitoring benthic organic enrichment. Eco Indi 85:1044-1057. DOI:10.1016/j.ecolind.2017.11.014.
Klymus KE, Marshall NT, Stepien CA. 2017. Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes. PloS one 12(5):1-24.
Lava Arreola ML. 2020. Philippine Statutory Compliance with Biodiversity Treaty Obligations on Land Reclamation Projects. Phil. LJ 93:1218.
Lobo J, Shokralla S, Costa MH, Hajibabaei M, Costa FO. 2017. DNA metabarcoding for high-throughput monitoring of estuarine microbenthic communities. Sci Rep 7(1):15618. DOI:10.1038/s41598-017-15823-6.
Mago? T, Salzberg SL. 201. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27(21):2957-2963. DOI:10.1093/bioinformatics/btr507.
Matas RF, Popovic M, Cagalj M, Simat V. 2023. The marine diatom Thalassiosira rotula: chemical profile and antioxidant activity of hydroalcoholic extracts. Front Mar Sci 10:1221417. DOI: 10.3389/fmars.2023.1221417.
Matos CR, Berrêdo JF, Machado W, Metzger E, Sanders CJ, Faial KC, Cohen MC. 2022. Seasonal changes in metal and nutrient fluxes across the sediment-water interface in tropical mangrove creeks in the Amazon region. App Geo 138:105217. DOI: 10.1016/j.apgeochem.2022.105217.
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. Sci Rep 9(1):580. DOI:10.1038/s41598-018-37001-y.
McColl?Gausden EF, Weeks AR, Coleman RA, Robinson KL, Song S, Raadik TA, Tingley R. 2021. Multispecies models reveal that eDNA metabarcoding is more sensitive than backpack electrofishing for conducting fish surveys in freshwater streams. Mol Eco 30(13):3111-3126. DOI: 10.1111/mec.15644.
Meon G, Pätsch M, Van Phuoc N, Quan NH. 2014. EWATEC?COAST: Technologies for Environmental and Water Protection of Coastal Regions in Vietnam: Contributions to 4th International Conference for Environment and Natural Resources—ICENR 2014 (Vol. 1). Cuvillier Verlag.
Miya M, Gotoh RO, Sado T. 2020. MiFish metabarcoding: a high-throughput approach for simultaneous detection of multiple fish species from environmental DNA and other samples. Fish Sci 86(6):939-970. DOI: 10.1007/s12562-020-01461-x.
Nakagawa H, Yamamoto S, Sato Y, Sado T, Minamoto T, Miya M. 2018. Comparing local?and regional?scale estimations of the diversity of stream fish using eDNA metabarcoding and conventional observation methods. Fresh Bio 63(6):569-580. DOI: 10.1111/fwb.13094.
Nester GM, De Brauwer M, Koziol A, West KM, DiBattista JD, White NE, Bunce M. 2020. Development and evaluation of fish eDNA metabarcoding assays facilitate the detection of cryptic seahorse taxa (family: Syngnathidae). Environmental DNA 2(4):614-626. DOI: 10.1002/edn3.93.
Nodine ER, Gaiser EE. 2014. Distribution of diatoms along environmental gradients in the Charlotte Harbor, Florida (USA), estuary and its watershed: Implications for bioassessment of salinity and nutrient concentrations. Estuaries and Coast 37:864-879. DOI:10.1007/s12237-013-9729-6.
Nunes M, Lemley DA, Adams JB. 2022. Benthic Diatom Diversity and Eutrophication in Temporarily Closed Estuaries. Estuaries and Coasts 1-20. DOI:10.1007/s12237-022-01126-1.
Nursuhayati AS, Yusoff FM, Shariff M. 2013. Spatial and temporal distribution of phytoplankton in Perak estuary, Malaysia, during monsoon season. Fish Aqua Sci J 8(4):480.
Oliveira MC, Bhattacharya D. 2000. Phylogeny of the Bangiophycidae (Rhodophyta) and the secondary endosymbiotic origin of algal plastids. J Phycol 36:52-52. DOI: 10.1046/j.1529-8817.1999.00001-155.x
Parfait NG, Didier R, Fosah MR, Teke AN, Delphin A. 2023. Influence of some abiotic factors on the diatom densities in the mezam river (Bamenda, North-West Cameroon). African J Bio Res 6(3):40-55. DOI:10.52589/AJBMR-QTSJWH91.
Pawlowski J, Bonin A, Boyer F, Cordier T, Taberlet P. 2021. Environmental DNA for biomonitoring. Mol Eco 30(13):2931. DOI: 10.1111/mec.16023.
Pawlowski J, Bruce K, Panksep K, Aguirre FI, Amalfitano S, Apothéloz-Perret-Gentil L, Baussant T, Bouchez A, Carugati L, Cermakova K, Cordier T, Corinldes C, Costa FO, Danovaro R, Dell’Anno A, Duarte S, Eisendle U, Ferrari BJD, Frontalini F, Fruhe L, Fazi S. 2022. Environmental DNA metabarcoding for benthic monitoring: A review of sediment sampling and DNA extraction methods. Science of the Total Environment, 818:151783. DOI:10.1016/j.scitotenv.2021.151783.
Pérez-Burillo J, Valoti G, Witkowski A, Prado P, Mann DG, Trobajo R. 2022. Assessment of marine benthic diatom communities: insights from a combined morphological–metabarcoding approach in Mediterranean shallow coastal waters. Mar Pol Bul 174:113183. DOI:10.1016/j.marpolbul.2021.113183.
Rahaman SMB, Golder J, Rahaman MS, Hasanuzzaman AFM, Huq KA, Begum S, Bir J. 2013. Spatial and temporal variations in phytoplankton abundance and species diversity in the Sundarbans mangrove forest of Bangladesh. Mar Sci Res Dev J 3(2), 1-9.DOI:10.4172/2155-9910.1000126.
Rashid H, Prakash MM. 2022. Zooplanktons as Bioindicators Of Water Pollution from Vikram Tearth Sarovar Ujjain (MP). Phar Neg Res J 13(5):888-895. DOI:10.47750/pnr.2022.13. S05.140.
Rincón B, Rivas M. 2022. Wastewater, reclaimed water, and seawater utilization in the production of microalgae-based fuels. 3rd Gen Bio 153-173. DOI:10.1016/B978-0-323-90971-6.00017-6.
Ruppert KM, Kline RJ, & Rahman MS. 2019. Past, present, and future perspectives of environmental DNA (eDNA) metabarcoding: A systematic review in methods, monitoring, and applications of global eDNA. Glo Eco Con 17: e00547. DOI:10.1016/j.gecco.2019.e00547.
Sabia A, Clavero E, Pancaldi S, Salvado?-Rovira J. 2018. Effect of different CO2 concentrations on biomass, pigment content, and lipid production of the marine diatom Thalassiosira pseudonana. Appl Microbiol Biotechnol 102(4):1945–1954. DOI: 10.1007/s00253-017-8728-0.
Sahu G, Satpathy KK, Mohanty AK, Sarkar SK. 2012. Variations in community structure of phytoplankton in relation to physicochemical properties of coastal waters, southeast coast of India. Indian J Mar Sci 41(3):223-241. DOI:nopr.niscpr.res.in/handle/123456789/1416.
Saifullah ASM, Abu Hena MK, Idris MH, Halimah AR, Johan I. 2014. Composition and diversity of phytoplankton from mangrove estuaries in Sarawak, Malaysia. J Biol Sci, 14(5), 361-369. DOI:10.3923/jbs.2014.361.369.
Sañe? E, Del Mondo A, Ambrosino L, Smerilli A, Sansone C, Brunet C. 2021. The recent advanced in microalgal phytosterols: bioactive ingredients along with human-health driven potential applications. Food Rev Int 39(4):1859–1878. DOI: 10.1080/87559129.2021.1938115.
Santos M, Amorim A, Brotas, V, Cruz JPC, Borges C, Favareto LR, Veloso V, Damaso-Rodrigues ML, Chainho P, Felix PM, Brito AC. 2022. Spatio-temporal dynamics of phytoplankton community in a well-mixed temperate estuary (Sado Estuary, Portugal). Sci Rep 12:16423. DOI:10.1038/s41598-022-20792-6.
Sharma N, Simon DP, Diaz-Garza AM, Fantino E, Messaabi A, Meddeb-Mouelhi F, Germain H, Desgagne-Penix I. 2021. Diatoms Biotechnology: Various industrial applications for a greener tomorrow. Front Mar Sci 8:1-18. DOI:10.3389/fmars.2021.636613.
Shen R, Ren H, Yu P, You Q, Pang W, Wang Q. 2018. Benthic Diatoms of the Ying River (Huaihe River Basin, China) and Their Application in Water Trophic Status Assessment. Water 10(8):1013. DOI:10.3390/w10081013.
Strickler KM, Fremier AK, Goldberg CS. 2015. Quantifying effects of UV-B, temperature, and pH on eDNA degradation in aquatic microcosms. Bio Con 183:85-92. DOI:10.1016/j.biocon.2014.11.038.
Thomsen PF, Willerslev E. 2015. Environmental DNA–An emerging tool in conservation for monitoring past and present biodiversity. Bio Con 183:4-18. DOI:doi.org/10.1016/j.biocon.2014.11.019.
Trábert Z, Duleba M, Bíró T, Dobosy P, Földi A, Hidas A, Kiss KT, Óvári M, Takács A, Várbíró G, Zaray g, Acs E. 2020. Effect of Land Use on the Benthic Diatom Community of the Danube River in the Region of Budapest. Water 12(2):479. DOI:10.3390/w12020479.
Udalov A, Chikina M, Azovsky A, Basin A, Galkin S, Garlitska L, Mokievsky V. 2021. Integrity of benthic assemblages along the arctic estuarine-coastal system. Ecological Indicators, 121:107-115.
Valentini A, Taberlet P, Miaud C, Civade R, Herder J, Thomsen PF, Bellemain E, Besnard A, Coissac E, Boyer F, Gaboriaud C, Jean P, Poulet N, Roset N, Copp GH, Geniez P, Pont D, Argillier C, Baudoin JM, Peroux T, Crivelli AJ, Olivier A, Acqueberge M, Le Brun M, Moller PR, Willerslev E, Dejean T. 2016. Next?generation monitoring of aquatic biodiversity using environmental DNA metabarcoding. Mol Eco 25(4):929-942. DOI:10.1111/mec.13428.
Valenzuela-Sanchez CG, Pasten-Miranda NM, Enriquez-Ocaña LF, Barraza-Guardado RH, Holguin JV, Martinez-Cordova LR. 2021. Phytoplankton composition and abundance as indicators of aquaculture effluents impact in coastal environments of mid Gulf of California. Heliyon 242:106830. DOI: 10.1016/j.heliyon. 2021.e06203.
Vasselon V, Rimet F, Domaizon I, Monnier O, Reyjol Y, Bouchez A. 2019. Assessing pollution of aquatic environments with diatoms' DNA metabarcoding: experience and developments from France Water Framework Directive networks. Met Met 3(1). DOI: 10.3897/mbmg.3.39646.
Vasselon V, Rimet F, Tapolczai K, Bouchez A. 2017. Assessing ecological status with diatoms DNA metabarcoding: Scaling-up on a WFD monitoring network (Mayotte island, France). Eco Ind 82:1-12. DOI:10.1016/j.ecolind.2017.06.024.
Venturina, REL, Del Prado YLC, Kamir RAC, Balmores MN, Diesmos AC. 2020. A revised checklist of amphibians and reptiles in Camiguin Sur, Misamis Oriental, Mindanao, Philippines. Asain Herpt Res 11(1):28-43. DOI: 10.16373/j.cnki.ahr.190036.
Wang Y Xu H, Li M. 2021. Long-term changes in phytoplankton communities in China's Yangtze Estuary driven by altered riverine fluxes and rising sea surface temperature. Geomorphology 376:107566. DOI:10.1016/j.geomorph.2020.107566.
Ward RD, Friess DA, Day RH, Mackenzie RA. 2016. Impacts of climate change on mangrove ecosystems: a region by region overview. Eco Health Sus 2(4): e01211. DOI: 10.1002/ehs2.1211.
Westfall KM, Therriault TW, Abbott CL. 2020. A new approach to molecular bio surveillance of invasive species using DNA metabarcoding. Global Change Bio26(2):1012-1022.
Wu Y, Campbell DA, Irwin AJ, Suggett DJ, Finkel ZV. 2014. Ocean acidification enhances the growth rate of larger diatoms. Limnology and Oceanography, 59(3), 1027-1034.
Xiong F, Shu L, Zeng H, Gan X, He S, Peng Z. 2022. Methodology for fish biodiversity monitoring with environmental DNA metabarcoding: the primers, databases and bioinformatic pipelines. Water Bio Sec 1(1):100007. DOI:10.1016/j.watbs.2022.100007.
Yamamoto S, Masuda R, Sato Y, Sado T, Araki H, Kondoh M, Miya M. 2017. Environmental DNA metabarcoding reveals local fish communities in a species-rich coastal sea. Sci Rep 7(1):40368.
Zaiko A, Samuiloviene A, Ardura A, Garcia-Vazquez E. 2015. Metabarcoding approach for nonindigenous species surveillance in marine coastal waters. Mar Pol Bul 100(1):53-59. DOI:10.1016/j.marpolbul.2015.09.030.
Zimmermann J, Jahn R, Gemeinholzer B. 2011. Barcoding diatoms: evaluation of the V4 subregion on the 18S rRNA gene, including new primers and protocols. Organisms Diver Evo 11, 173-192. DOI: 10.1007/s13127-011-0050-6.
Zou K, Chen J, Ruan H, Li Z, Guo W, Li M, Liu L. 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. Sci Total Environ 702:134704. DOI:10.1016/j.scitotenv.2019.134704.