The importance of DNA barcode reference libraries and selection primer pair in monitoring fish diversity using environmental DNA metabarcoding




Abstract. Roesma DI, Tjong DH, Syaifullah, Nofrita, Janra MN, Prawira FDL,Salis VM, Aidil DR. 2023.The importance of DNA barcode reference libraries and selection primer pair in monitoring fish diversity using environmental DNA metabarcoding. Biodiversitas 24: 2251-2260. Environmental DNA (eDNA) metabarcoding has become an alternative method used for biodiversity monitoring of an ecosystem. The eDNA metabarcoding has advantages compared to the conventional method because it is non-invasive, quick, and requires less cost. However, the effectiveness of the eDNA method is highly dependent on the coverage of the DNA barcode reference and primerpair. A study using the eDNA method was conducted for fish biodiversity monitoring in Singkarak Lake. Two-liter water samples were collected using sterile bottle samples at each sampling site (five sites). The universal primers (Fish FI and Fish R1) used for Next-generation sequencing (GRIDION, Nanopore, Oxford Technologies). The study detected 152 fish species using eDNA metabarcoding. Ten species out of the 30 originally reported in Singkarak Lake were detected using eDNA metabarcoding. The low percentage of fish detected is thought to be due to several factors; incomplete/unavailability of freshwater fish DNA barcodes in Indonesia registered in the database repository, inappropriate primer pair selection, low DNA quality, and the absence of target species DNA in collected water samples. The results demonstrated the significance of correctly registering DNA barcodes to the database and appropriate primer pair selection to identify eDNA metabarcoding. This study provides recommendations using eDNA metabarcoding for monitoring in future work.


Andriyono S, Alam MDJ, Kim H. 2019. Environmental DNA (eDNA) metabarcoding: Diversity study around the Pondok Dadap fish landing station, Malang, Indonesia. Biodiversitas 20 (12): 3772-3781. DOI: 10.13057/biodiv/d201241.
Andruszkiewicz EA, Starks HA, Chavez FP, Sassoubre LM, Block BA, Boehm AB. 2017. Biomonitoring of marine vertebrates in Monterey Bay using eDNA metabarcoding. Plos One 12 (4): e0176343. DOI: 10.1371/journal.pone.0176343.
Aylagas E, Borja A, Rodriguez-Ezpeleta N. 2014. Environmental status assessment using DNA metabarcoding: towards a genetics based marine biotic index (gAMBI). PLoS One 9:e90529–e90511.
Bean TP, Greenwood N, Beckett R, Biermann L, Bignell JP, Brant JL, Copp GH, DevlinMJ, Dye S, Feist SW, Fernand L et al. 2017. A review of the tools used for marine monitoring in the UK: combining historic and contemporary methods with modeling and socioeconomics to fulfill legislative needs and scientific ambitions. Front Mar Sci 4: 263. 2017.00263
Bohmann K, Evans A, Gilbert MTP, Carvalho R, Creer S, Knapp M, Yu DW, Bruyn M. 2014. Environmental DNA for wildlife biology and biodiversity monitoring. Trends Ecol Evol 29: 358–367
Carew ME, Pettigrove, VJ, Metzeling L, Hoffmann AA. 2013. Environmental monitoring using Next-generation sequencing: rapid identification of macroinvertebrate bioindicator species. Front Zool 10: 45.
Carim KJ, McKelvey KS, Young MK, Wilcox TM, Schwartz MK. 2016. A Protocol for Collecting Environmental DNA Samples from Streams. Gen Tech Rep RMRS-GTR-355. Department of Agriculture, Forest Service, Rocky Mountain Research Station. Fort Collins, Colorado, United states of America.
Darling JA, Mahon AR. 2011. From molecules to management: Adopting DNA based methods for monitoring biological invasions in aquatic environments. Environ Res 111: 978-988. DOI: 10.1016/j.envres.2011.02.001.
de Coster W, D'Hert S, Schultz DT, Cruts M, van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34: 2666-2669. 10. 1093/ bioinformatics/bty149.
Ellingsen KE, Yoccoz NG, Tveraa T, Hewitt JE, Thrush SF (2017) Longterm environmental monitoring for assessment of change: measurement inconsistencies over time and potential solutions. Environ Monit Assess 189:595.
Ficetola GF, Miaud C, Pompanon F, Taberlet P. 2008. Species detection using environmental DNA from water samples. Biol Lett 4 (4): 423-425. DOI :10.1098/Rsbl. 2008.0118.
Goldberg CS, Strickler KM, Pilliod DS. 2015. Moving environmental DNA methods from concept to practice for monitoring aquatic macroorganisms. Biol Conserv 183:1-3. doi:10.1016/j.biocon.2014.11.040
Goldberg C, Strickler K. 2017. Environmental DNA Protocol for Freshwater Aquatic Ecosystems version 2.2.Washington State University, Washington.
Hebert PDN, Cywinska A, Ball SL, deWaard JR. 2003. Biological identifications through DNA barcodes. Proc R Soc B: Biol sci 270: 313–321.
Hebert PDN, Ratnasingham S, Zakharov EV, Telfer AC, Levesque-Beaudin V, Milton MA, Pedersen S, Jannetta P, deWaard JR. 2016. Counting animal species with DNA barcodes: Canadian insects. Philos Trans R Soc B: Biol Sci. 371(1702) 20150333. doi:10.1098/rstb.2015.0333
Hestetun JT, Bye-Ingebrigtsen E, Nilsson RH, Glover AG, Johansen P, Dahlgren TG. 2020. Significant taxon sampling gaps in DNA databases limit the operational use of marine macrofauna metabarcoding. Marine Biodiversity 50: 70
Hogg ID, Banks SM, Wood SM. 2018. Biodiversity Genomics: Monitoring Restoration Efforts Using DNA Barcoding and Environmental DNA
In: D. P. Hamilton et al. (eds.) Lake Restoration Handbook. University of Waikato, Hamilton, New Zealand
Kelly RP, Port JA, Yamahara KM, Crowder LB. 2014. Using environmental DNA to census marine fishes in a large mesocosm. Plos One 9 (1): e86175. DOI: 10.1371/journal. pone.0086175.
Kim D, Song L, Breitwieser FP and Salzberg SL. 2016 Centrifuge: Rapid and sensitive classification of metagenomic sequences. Genome Res 26(12): 1721-1729. doi: 10.1101/gr. 210641.116.
Knowlton N. 1993. Sibling species in the sea. Annu Rev Ecol Syst 24: 189-216.
Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, errorprone reads using repeat graphs. Nat Biotechnol 37:540–546.
Laramie MB, Pilliod DS, Goldberg CS, Strickler KM. 2015. Environmental DNA sampling protocol-Filtering water to capture DNA from aquatic organisms: U.S. Geological Survey Techniques and Methods. U.S. Geological Survey, Reston, Virginia.
Menning D, Simmons T, Talbot S. 2018. Using redundant primer sets to detect multiple native Alaskan fish species from environmental DNA. Conserv Gen Resour 12: 109-123
Oxford Nanopore Technologies. 2008. Nanopore Sequencing.
Pawlowski J, Esling P, Lejzerowicz F, Cedhagen T, Wilding TA. 2014. Environmental monitoring through protist next-generation sequencing metabarcoding: assessing the impact of fish farming on benthic foraminifera communities. Mol Ecol Resour 14(6): 1129–1140.
Roesma DI. 2011. Species Diversity and Genetic Relationship of Cyprinidae in Lakes and the Adjoining Rivers in West Sumatra. Dissertation. Andalas University, Padang. [Indonesian]
Roesma DI, Djong HT, Munir W, Aidil DR. 2018. New record species of Puntius (Pisces: Cyprinidae) from West Sumatra Based on Cytochrome Oxidase I Gene. Int J Adv Sci Eng Inf Technol 8 (1): 250-256. DOI: 10.18517/ijaseit.8.1.4170
Roesma DI, Tjong DH, Karlina W, Aidil DR. 2019. Taxonomy confirmation of puntius cf. binotatus from Gunung Tujuh lake, Jambi, Indonesia based on cytochrome oxidase I (COI) gene. Biodiversitas 20 (1): 54-60. DOI: 10.13057/biodiv/d200107.
Roesma DI, Tjong DH, Aidil DR. 2020. Phylogenetic analysis of transparent gobies in three Sumatran lakes, inferred from mitochondrial Cytochrome Oxidase I (COI) gene. Biodiversitas 21 (1): 43-48. DOI: 10.13057/biodiv/d210107.
Roesma DI, Tjong DH, Janra MN, Aidil RA. 2021a. Fish diversity monitoring in Maninjau Lake, West Sumatra using the eDNA with the next generation sequencing (NGS) technique. IOP Conf Ser : Earth Environ Sci 819 012045 p 12
Roesma DI, Tjong DH, Janra MN, Aidil RA. 2021b. Freshwater vertebrates monitoring in Maninjau Lake, West Sumatra, Indonesia using environmental DNA. Biodiversitas 22: 2794-2802. doi: 10.13057/biodiv/d220543
Roesma DI, Tjong DH, Janra MN, Aidil RA. 2022. DNA barcoding of freshwater fish in Siberut Island, Mentawai Archipelago, Indonesia. Biodiversitas 23: 1795-1806. doi: 10.13057/bio div/d230411
Schmitter-Soto JJ. 1999. Distribution of continental fishes in northern Quintana Roo, Mexico. Southwest Nat 44: 166–172.
Schwentner M, Zahiri R, Yamamoto S, Husemann M, Kullmann B, Thiel R. 2021. eDNA as a tool for non-invasive monitoring of the fauna of a turbid, well-mixed system, the Elbe estuary in Germany. Plos One 16(4): e0250452. pone.0250452
Shelton AO, O’Donnell JL, Samhouri JF, Lowell N, Williams GD, Kelly RP. 2016. A framework for inferring biological communities from environmental DNA. Ecol Appl 26 (6): 1-47. DOI: 10.1890/15-1733.1.
Shokralla S, Spall JL, Gibson JF, Hajibabaei M. 2012. Next generation sequencing technologies for environmental DNA Research. Mol Ecol 21: 1794-1805. DOI: 10.1111/J.1365-294x.2012.05538.X.
Specchia V, Tzafesta E, Marini G, Scarcella S, D’Attis S, Pinna M. 2020. Gap Analysis for DNA Barcode Reference Libraries for Aquatic Macroinvertebrate Species in the Apulia Region (Southeast of Italy). J Mar Sci Eng 8: 538. doi:10.3390/jmse8070538
Syandri H. 2008. Threats to the germplasm of Bilih fish (Mystacoleucus padangensis Blkr) and conservation efforts in Singkarak Lake. Scientific Oration. University of Bung Hatta, Padang, 08 Juli 2008.[Indonesian]
Taberlet P, Coissac E, Hajibabaei M, Rieseberg, LH. 2012. Environmental DNA. Mol Ecol 21: 1789-1793. DOI: 10.1111/j.1365-294X. 2012. 05542.x.
Thomsen PF, Willerslev E. 2015. Environmental DNA an emerging tool in conservation for monitoring past and present biodiversity. Biol Conserv 183: 4-18. DOI: 10.1016/j.biocon.2014.11.019.
Valdez-Moreno M, Ivanova NV, Elias-Gutie rrez M, Pedersen SL, Bessonov K, Hebert PDN. 2019. Using eDNA to biomonitor the fish community in a tropical oligotrophic lake. Plos one 14: e0215505.
Valentini A, Pompanon F, Taberlet P. 2009. DNA barcoding for ecologists. Trends Ecol Evol 24(2): 110–117. doi:10.1016/j.tree.2008.09.011
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. Mol Ecol 25: 929-942. PMID: 26479867
von Rintelen K, Arida E, Hauser C. 2017. A review of biodiversity-related issues and challenges in megadiverse Indonesia and other Southeast Asian countries. RIO 3: e20860.
Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN. 2005. DNA barcoding Australia's fish species. Phil Trans R Soc B 360: 1847–1857. doi:10.1098/rstb.2005.1716
Weber M and de Beaufort L F 1913 The Fishes of the Indo-Australian Archipelago Vol. I. EJ Brill, Leiden, Netherland
Weber M and de Beaufort L F 1916 The Fishes of the Indo-Australian Archipelago Vol. II. EJ Brill, Leiden, Netherland
Wick RR, Judd LM, Holt KE. 2019 Performance of neural network basecalling tools for Oxford Nanopore . Genome Biol 20: 129.
Widjaja EA, Rahayuningsih Y, Rahajoe JS, Ubaidillah R, Maryanto I, Walujo EB, Semiadi G. 2014. Kekinian Keanekaragaman Hayati Indonesia 2014. LIPI Press, Jakarta
Yamamoto S, Masuda R, Sato Y, Sado T, Araki H, Kondoh M, et al. 2017. Environmental DNA metabarcoding reveals local fish communities in a species-rich coastal sea. Scientific Reports. 7: 40368.