eDNA technology for monitoring terrestrial biodiversity: Technical highlights, challenges and progress

doi:10.13287/j.1001-9332.202503.031

Abstract

Abstract: Biodiversity is a fundamental prerequisite for human survival, constituting an indispensable component of global ecosystems. The United Nations Sustainable Development Goal 15 (SDG 15) underscores the significance of conserving terrestrial biodiversity and strives to advance the sustainable utilization of terrestrial ecosystems. To achieve this goal, it is of the utmost importance to establish a comprehensive biodiversity monitoring system. As an emerging monitoring tool, environmental DNA (eDNA) metabarcoding technology offers a number of advantages, including species morphology-independent, rapidity, economical, and high accuracy. Consequently, it provides an effective method for monitoring terrestrial biodiversity. We outlined the key technical aspects of using eDNA metabarcoding as a tool for biodiversity monitoring in terrestrial ecosystems, discussed the challenges of using eDNA metabarcoding technology in biodiversity research, along with strategies for addressing these challenges, surveyed recent research advances regarding eDNA from different sources for terrestrial biodiversity monitoring, and proposed future research directions.

Key words: terrestrial ecosystem, eDNA source, cross-domain biodiversity, specific taxa, metabarcoding primer

LIU Mingqian, ZHANG Zheng, WANG Shang, FENG Kai, GU Songsong, LI Chunge, DENG Ye. eDNA technology for monitoring terrestrial biodiversity: Technical highlights, challenges and progress[J]. Chinese Journal of Applied Ecology, 2025, 36(3): 927-942.

References

[1] Rondon MR, August PR, Bettermann AD, et al. Cloning the soil metagenome: A strategy for accessing the genetic and functional diversity of uncultured microorganisms. Applied and Environmental Microbiology, 2000, 66: 2541-2547
[2] Hebert PD, Cywinska A, Ball SL, et al. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London Series B: Biological Sciences, 2003, 270: 313-321
[3] Deiner K, Bik HM, Mächler E, et al. Environmental DNA metabarcoding: Transforming how we survey animal and plant communities. Molecular Ecology, 2017, 26: 5872-5895
[4] Antil S, Abraham JS, Sripoorna S, et al. DNA barcoding, an effective tool for species identification: A review. Molecular Biology Reports, 2023, 50: 761-775
[5] Miya M. Environmental DNA metabarcoding: A novel method for biodiversity monitoring of marine fish communities. Annual Review of Marine Science, 2022, 14: 161-185
[6] Cristescu ME, Hebert PD. Uses and misuses of environmental DNA in biodiversity science and conservation. Annual Review of Ecology, Evolution and Systematics, 2018, 49: 209-230
[7] Perry WB, Seymour M, Orsini L, et al. An integrated spatio-temporal view of riverine biodiversity using environmental DNA metabarcoding. Nature Communications, 2024, 15: 4372
[8] Kelly RP, Lodge DM, Lee KN, et al. Toward a national eDNA strategy for the United States. Environmental DNA, 2024, 6: e432
[9] Drummond AJ, Newcomb RD, Buckley TR, et al. Evaluating a multigene environmental DNA approach for biodiversity assessment. Gigascience, 2015, 4: s13742-015
[10] Bohmann K, Lynggaard C. Transforming terrestrial biodiversity surveys using airborne eDNA. Trends in Ecology & Evolution, 2023, 38: 119-121
[11] Dickie IA, Boyer S, Buckley HL, et al. Towards robust and repeatable sampling methods in eDNA-based studies. Molecular Ecology Resources, 2018, 18: 940-952
[12] Sirois SH, Buckley DH. Factors governing extracellular DNA degradation dynamics in soil. Environmental Microbiology Reports, 2019, 11: 173-184
[13] 赵枫, 史亚, 王莹, 等. PCR反应条件对16S rRNA基因扩增子测序准确性的影响. 微生物学通报, 2022, 49(7): 2527-2537
[14] Edwards ME, Alsos IG, Yoccoz N, et al. Metabarcoding of modern soil DNA gives a highly local vegetation signal in Svalbard tundra. Holocene, 2018, 28: 2006-2016
[15] Li SZ, Deng Y, Du XF, et al. Sampling cores and sequencing depths affected the measurement of microbial diversity in soil quadrats. Science of the Total Environment, 2021, 767: 144966
[16] Dopheide A, Xie D, Buckley TR, et al. Impacts of DNA extraction and PCR on DNA metabarcoding estimates of soil biodiversity. Methods in Ecology and Evolution, 2019, 10: 120-133
[17] 姜维, 王启军, 邓捷, 等. 以川陕哲罗鲑为目标物种的水样环境DNA分析流程的优化. 应用生态学报, 2016, 27(7): 2372
[18] Frøslev TG, Ejrnæs R, Hansen AJ, et al. Treated like dirt: Robust forensic and ecological inferences from soil eDNA after challenging sample storage. Environmental DNA, 2023, 5: 158-174
[19] 侯卫国, 董海良, 蒋宏忱, 等. 沉积物中古DNA在古生态、古环境和古气候研究中的应用. 地学前缘, 2017, 24(2): 286-291
[20] Coissac E, Riaz T, Puillandre N. Bioinformatic challenges for DNA metabarcoding of plants and animals. Molecular Ecology, 2012, 21: 1834-1847
[21] 周天成, 胡思敏, 林先智, 等. 基于18S rDNA条形码技术的珊瑚礁区塔形马蹄螺(Tectus pyramis)食性分析. 海洋科学, 44(2): 99-107
[22] Li ZP, Shangguan HY, Yao H, et al. Colonization ability and uniformity of resources and environmental factors determine biological homogenization of soil protists in human land-use systems. Global Change Biology, 2024, 3: e17411
[23] Kartzinel TR, Chen PA, Coverdale TC, et al. DNA metabarcoding illuminates dietary niche partitioning by African large herbivores. Proceedings of the National Aca-demy of Sciences of the United States of America, 2015, 112: 8019-8024
[24] Ariza M, Fouks B, Mauvisseau Q, et al. Plant biodiversity assessment through soil eDNA reflects temporal and local diversity. Methods in Ecology and Evolution, 2023, 14: 415-430
[25] Johnson MD, Cox RD, Grisham BA, et al. Airborne eDNA reflects human activity and seasonal changes on a landscape scale. Frontiers in Environmental Science, 2021, 8: 563431
[26] Johnson MD, Cox RD, Barnes MA. Analyzing airborne environmental DNA: A comparison of extraction methods, primer type, and trap type on the ability to detect airborne eDNA from terrestrial plant communities. Environmental DNA, 2019, 1: 176-185
[27] Giguet-Covex C, Pansu J, Arnaud F, et al. Long livestock farming history and human landscape shaping revealed by lake sediment DNA. Nature Communications, 2014, 5: 3211
[28] Brun L, Schneider J, Carrió EM, et al. Focal vs. fecal: Seasonal variation in the diet of wild vervet monkeys from observational and DNA metabarcoding data. Ecology and Evolution, 2022, 12: e9358
[29] Chac LD, Thinh BB. Species identification through DNA barcoding and its applications: A review. Biology Bulletin, 2023, 50: 1143-1156
[30] Parisy B, Schmidt NM, Wirta H, et al. Ecological signals of arctic plant-microbe associations are consistent across eDNA and vegetation surveys. Metabarcoding and Metagenomics, 2023, 7: 155-193
[31] Johnson MD, Fokar M, Cox RD, et al. Airborne environmental DNA metabarcoding detects more diversity, with less sampling effort, than a traditional plant community survey. BMC Ecology and Evolution, 2021, 21: 218
[32] Epp LS, Boessenkool S, Bellemain EP, et al. New environmental metabarcodes for analysing soil DNA: Potential for studying past and present ecosystem. Molecular Ecology, 2012, 21: 1821-1833
[33] Zhang GK, Chain FJJ, Abbott CL, et al. Metabarcoding using multiplexed markers increases species detection in complex zooplankton communities. Evolutionary Applications, 2018, 11: 1901-1914
[34] Baldwin DS, Colloff MJ, Rees GN, et al. Impacts of inundation and drought on eukaryote biodiversity in semi-arid floodplain soils. Molecular Ecology, 2013, 22: 1746-1758
[35] Willerslev E, Davison J, Moora M, et al. Fifty thousand years of Arctic vegetation and megafaunal diet. Nature, 2014, 506: 47-51
[36] Andersen K, Bird KL, Rasmussen M, et al. Meta-barcoding of ‘dirt' DNA from soil reflects vertebrate biodiversity. Molecular Ecology, 2012, 21: 1966-1979
[37] Shehzad W, Riaz T, Nawaz MA, et al. Carnivore diet analysis based on next-generation sequencing: Application to the leopard cat (Prionailurus bengalensis) in Pakistan. Molecular Ecology, 2012, 21: 1951-1965
[38] Shao XN, Lu Q, Xiong MY, et al. Prey partitioning and livestock consumption in the world's richest large carnivore assemblage. Current Biology, 2021, 31: 4887-4897
[39] Lu Q, Xiao LY, Cheng C, et al. Snow leopard dietary preferences and livestock predation revealed by fecal DNA metabarcoding: No evidence for apparent competition between wild and domestic prey. Frontiers in Ecology and Evolution, 2021, 9: 783546
[40] Shao XN, Song DZ, Huang QW, et al. Fast surveys and molecular diet analysis of carnivores based on fecal DNA and metabarcoding. Biodiversity Science, 2019, 27: 543
[41] Kelly RP, Port JA, Yamahara KM, et al. Using environmental DNA to census marine fishes in a large mesocosm. PLoS One, 2014, 9: e86175
[42] Lynggaard C, Frøslev TG, Johnson MS, et al. Airborne environmental DNA captures terrestrial vertebrate diversity in nature. Molecular Ecology Resources, 2024, 24: 3840.
[43] Lynggaard C, Bertelsen MF, Jensen CV, et al. Airborne environmental DNA for terrestrial vertebrate community monitoring. Current Biology, 2022, 32: 701-707
[44] Deiner K, Renshaw MA, Li Y, et al. Long-range PCR allows sequencing of mitochondrial genomes from environmental DNA. Methods in Ecology and Evolution, 2017, 8: 1888-1898
[45] Wilcox TM, McKelvey KS, Young MK, et al. Robust detection of rare species using environmental DNA: The importance of primer specificity. PLoS One, 2013, 8: e59520
[46] Lyman JA, Sanchez DE, Hershauer SN, et al. Mammalian eDNA on herbaceous vegetation? validating a qPCR assay for detection of an endangered rodent. Environmental DNA, 2022, 4: 1187-1197
[47] Newton JP, Bateman PW, Heydenrych MJ, et al. Monitoring the birds and the bees: Environmental DNA metabarcoding of flowers detects plant-animal interactions. Environmental DNA, 2023, 5: 488-502
[48] Kirse A, Bourlat SJ, Langen K, et al. Unearthing the potential of soil eDNA metabarcoding: Towards best practice advice for invertebrate biodiversity assessment. Frontiers in Ecology and Evolution, 2021, 9: 630560
[49] Marullo R, Mercati F, Vono G. DNA Barcoding: A reliable method for the identification of Thrips species (Thysanoptera, Thripidae) collected on sticky traps in onion fields. Insects, 2020, 11: 489
[50] Nagoshi RN, Brambila J, Meagher RL. Use of DNA barcodes to identify invasive armyworm Spodoptera species in Florida. Journal of Insect Science, 2011, 11: 1-11
[51] Dopheide A, Tooman LK, Grosser S, et al. Estimating the biodiversity of terrestrial invertebrates on a forested island using DNA barcodes and metabarcoding data. Ecological Applications, 2019, 29: e01877
[52] Kestel JH, Bateman PW, Field DL, et al. eDNA metabarcoding of avocado flowers: ‘Hass' it got potential to survey arthropods in food production systems? Molecular Ecology Resources, 2023, 23: 1540-1555
[53] Kirtane A, Dietschler NJ, Bittner TD, et al. Sensitive environmental DNA (eDNA) methods to detect hemlock woolly adelgid and its biological control predators Leucotaraxis silver flies and a Laricobius beetle. Environmental DNA, 2022, 4: 1136-1149
[54] Arstingstall KA, DeBano SJ, Li X, et al. Capabilities and limitations of using DNA metabarcoding to study plant-pollinator interaction. Molecular Ecology, 2021, 30: 5266-5297
[55] McPherson C, Avanesyan A, Lamp WO. Diverse host plants of the first instars of the invasive Lycorma delicatula: Insights from eDNA metabarcoding. Insects, 2022, 13: 534
[56] Ahmed M, Back MA, Prior T, et al. Metabarcoding of soil nematodes: The importance of taxonomic coverage and availability of reference sequences in choosing suitable marker(s). Metabarcoding and Metagenomics, 2019, 3: e36408
[57] Porazinska DL, Giblin-Davis RM, Faller L, et al. Eva-luating high-throughput sequencing as a method for metagenomic analysis of nematode diversity. Molecular Ecology Resources, 2009, 9: 1439-1450
[58] Ji YQ, Ashton L, Pedley SM, et al. Reliable, verifiable and efficient monitoring of biodiversity via metabarcoding. Ecology Letters, 2013, 16: 1245-1257
[59] Bienert F, De DS, Miquel C, et al. Tracking earthworm communities from soil DNA. Molecular Ecology, 2012, 21: 2017-2030
[60] Shokralla S, Gibson JF, Nikbakht H, et al. Next-generation DNA barcoding: Using next-generation sequencing to enhance and accelerate DNA barcode capture from single specimens. Molecular Ecology Resources, 2014, 14: 892-901
[61] Hajibabaei M, Janzen DH, Burns JM, et al. DNA barcodes distinguish species of tropical Lepidoptera. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103: 968-971
[62] Rougerie R, Decaëns T, Deharveng L, et al. DNA barcodes for soil animal taxonomy. Pesquisa Agropecuária Brasileira, 2009, 44: 789-802
[63] De-astro AP, Quirino BF, Pappas G, et al. Diversity of soil fungal communities of Cerrado and its closely surrounding agriculture fields. Archives of Microbiology, 2008, 190: 129-139
[64] Wang YC, Dang N, Feng K, et al. Grass-microbial inter-domain ecological networks associated with alpine grassland productivity. Frontiers in Microbiology, 2023, 14: 1109128
[65] Krah FS, March-Salas M. eDNA metabarcoding reveals high soil fungal diversity and variation in community composition among Spanish cliffs. Ecology and Evolution, 2022, 12: e9594
[66] Hunter S, Horner I, Hosking J, et al. Phytophthora communities associated with Agathis australis (kauri) in Te Wao Nui o Tiriwa/Waitākere Ranges, New Zealand. Preprints, 2024, 15: 735
[67] Riddell CE, Frederickson-Matika D, Armstrong AC, et al. Metabarcoding reveals a high diversity of woody host-associated Phytophthora spp. in soils at public gardens and amenity woodlands in Britain. PeerJ, 2019, 7: e6931
[68] Tang CQ, Leasi F, Obertegger U, et al. The widely used small subunit 18S rDNA molecule greatly underestimates true diversity in biodiversity surveys of the meiofauna. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109: 16208-16212
[69] Poczai P, Hyvönen J. Nuclear ribosomal spacer regions in plant phylogenetics: Problems and prospects. Molecular Biology Reports, 2010, 37: 1897-1912
[70] Zhang Z, Li J, Li HJ, et al. Environmental DNA metabarcoding reveals the influence of human activities on microeukaryotic plankton along the Chinese coastline. Water Research, 2023, 233: 119730
[71] Li SZ, Deng Y, Wang ZJ, et al. Exploring the accuracy of amplicon-based internal transcribed spacer markers for a fungal community. Molecular Ecology Resources, 2020, 20: 170-184
[72] Wu YN, Feng K, Wei ZY, et al. ARDEP, a rapid degenerate primer design pipeline based on k-mers for amplicon microbiome studies. International Journal of Environmental Research and Public Health, 2020, 17: 5958
[73] 吴悦妮, 冯凯, 厉舒祯, 等. 16S/18S/ITS扩增子高通量测序引物的生物信息学评估和改进. 微生物学通报, 2020, 47(9): 2897-2912
[74] Chorlton SD. Ten common issues with reference sequence databases and how to mitigate them. Frontiers in bioinformatics, 2024, 4: 1278228
[75] 沈菊培, 张丽梅, 郑袁明, 等. 土壤宏基因组学技术及其应用. 应用生态学报, 2017, 18(1): 212-218
[76] Thomsen PF, Willerslev E. Environmental DNA: An emerging tool in conservation for monitoring past and present biodiversity. Biological Conservation, 2015, 183: 4-18
[77] Darling JA, Mahon AR. From molecules to management: Adopting DNA-based methods for monitoring biological invasions in aquatic environments. Environmental Research, 2011, 111: 978-988
[78] Baena-Bejarano N, Reina C, Martinez-Revelo DE, et al. Taxonomic identification accuracy from BOLD and GenBank databases using over a thousand insect DNA barcodes from Colombia. PLoS One, 2023, 18: e0277379
[79] Elbrecht V, Vamos EE, Meissner K, et al. Assessing strengths and weaknesses of DNA metabarcoding-based macroinvertebrate identification for routine stream monitoring. Methods in Ecology and Evolution, 2017, 8: 1265-1275
[80] Porter TM, Hajibabaei M. Scaling up: A guide to high-throughput genomic approaches for biodiversity analysis. Molecular Ecology, 2018, 27: 313-338
[81] 李莎, 刘雪清, 姜伟, 等. 环境DNA技术在宜昌江段四大家鱼自然繁殖中的应用. 应用生态学报, 2021, 32(6): 2241
[82] Goldberg CS, Turner CR, Deiner K, et al. Critical considerations for the application of environmental DNA methods to detect aquatic specie. Methods in Ecology and Evolution, 2016, 7: 1299-1307
[83] Griffin JE, Matechou E, Buxton AS, et al. Modelling environmental DNA data; Bayesian variable selection accounting for false positive and false negative errors. Journal of the Royal Statistical Society Series C: Applied Statistics, 2020, 69: 377-392
[84] Hassan S, Sabreena, Poczai P, et al. Environmental DNA metabarcoding: A novel contrivance for documenting terrestrial biodiversity. Biology, 2022, 11: 1297
[85] Gotelli NJ, Colwell RK. Quantifying biodiversity: Procedures and pitfalls in the measurement and comparison of species richness. Ecology Letters, 2001, 4: 379-391
[86] Lamb PD, Hunter E, Pinnegar JK, et al. How quantitative is metabarcoding: A meta-analytical approach. Molecular Ecology, 2019, 28: 420-430
[87] Johnson MD, Freeland JR, Parducci L, et al. Environmental DNA as an emerging tool in botanical research. American Journal of Botany, 2023, 110: e16120
[88] Little DP. A DNA mini-barcode for land plants. Molecular Ecology Resources, 2014, 14: 437-446
[89] Zhan A, Bailey SA, Heath DD, et al. Performance comparison of genetic markers for high-throughput sequencing-based biodiversity assessment in complex communities. Molecular Ecology Resources, 2014, 14: 1049-1059
[90] Zhang S, Zhao JD, Yao M. A comprehensive and comparative evaluation of primers for metabarcoding eDNA from fish. Methods in Ecology and Evolution, 2020, 11: 1609-1625
[91] Yarza P, Yilmaz P, Pruesse E, et al. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nature Reviews Microbiology, 2014, 12: 635-645
[92] Droege J, Gregor I, McHardy AC. Taxator-tk: Precise taxonomic assignment of metagenomes by fast approximation of evolutionary neighborhoods. Bioinformatics, 2015, 31: 817-824
[93] 牟铭, 李昂, 赵新宁, 等. 人工模拟条件下环境DNA宏条形码技术的定量分析初探. 渔业科学进展, 2021, 42(5): 24-30
[94] Pinol J, Senar MA, Symondson WOC. The choice of universal primers and the characteristics of the species mixture determine when DNA metabarcoding can be quantitative. Molecular Ecology, 2019, 28: 407-419
[95] 康子清, 张银龙, 吴永波, 等. 环境DNA宏条形码在生物多样性研究与监测中的应用. 生物技术通报, 2022, 38(1): 299-310
[96] Hiiesalu I, Öpik M, Metsis M, et al. Plant species richness belowground: Higher richness and new patterns revealed by next-generation sequencing. Molecular Ecology, 2012, 21: 2004-2016
[97] Khaliq I, Hardy GESJ, White D, et al. eDNA from roots: A robust tool for determining Phytophthora communities in natural ecosystems. FEMS Microbiology Ecology, 2018, 94: fiy048
[98] Brunetti M, Magoga G, Cussigh A, et al. Soil invertebrate biodiversity and functionality within the intensively farmed areas of the Po Valley. Applied Soil Ecology, 2024, 197: 105326
[99] Decaens T, Porco D, James SW, et al. DNA barcoding reveals diversity patterns of earthworm communities in remote tropical forests of French Guiana. Soil Biology & Biochemistry, 2016, 92: 171-183
[100] Lilja MA, Buivydaitė Ž, Zervas A, et al. Comparing earthworm biodiversity estimated by DNA metabarcoding and morphology-based approaches. Applied Soil Ecology, 2023, 185: 104798
[101] Llanos J. Assessing Earthworm Diversity and Population Dynamics in Agroecosystems. PhD Thesis. Sheffield, UK: University of Sheffield, 2021
[102] Fan KK, Chu HY, Eldridge DJ, et al. Soil biodiversity supports the delivery of multiple ecosystem functions in urban greenspaces. Nature Ecology & Evolution, 2023, 7: 113-126
[103] 廖晨阳, 杨明乐, 高庆, 等. eDNA宏条形码技术在城市生物多样性监测中的适用性与展望. 中国城市林业, 2023, 21(3): 167-173
[104] Yao HF, Li ZP, Geisen S, et al. Degree of urbanization and vegetation type shape soil biodiversity in city parks. Science of the Total Environment, 2023, 899: 166437
[105] 金宇斌, 周旭东, 谢雨初, 利用雨养泥炭沼泽及湖泊沉积物重建多氯联苯污染时空变化趋势. 应用生态学报, 2021, 32(1): 309-316
[106] Hou WG, Dong HL, Li GY, et al. Identification of photosynthetic plankton communities using sedimentary ancient DNA and their response to late-holocene climate change on the Tibetan Plateau. Scientific Reports, 2014, 4: 6648
[107] Li GY, Dong HL, Hou WG, et al. Temporal succession of ancient phytoplankton community in Qinghai lake and implication for paleo-environmental change. Scientific Reports, 2016, 6: 19769
[108] Murchie TJ, Monteath AJ, Mahony ME, et al. Collapse of the mammoth-steppe in central Yukon as revealed by ancient environmental DNA. Nature Communications, 2021, 12: 7120
[109] Ficetola GF, Marta S, Guerrieri A, et al. The development of terrestrial ecosystems emerging after glacier retreat. Nature, 2024, 632: 336-342
[110] Roger F, Ghanavi HR, Danielsson N, et al. Airborne environmental DNA metabarcoding for the monitoring of terrestrial insects: A proof of concept from the field. Environmental DNA, 2022, 4: 790-807
[111] Banerjee P, Stewart KA, Dey G, et al. Environmental DNA analysis as an emerging non-destructive method for plant biodiversity monitoring: A review. AoB Plants, 2022, 14: plac031
[112] Weber S, Stothut M, Mahla L, et al. Plant-derived environmental DNA complements diversity estimates from traditional arthropod monitoring methods but outperforms them detecting plant-arthropod interactions. Molecular Ecology Resources, 2024, 24: e13900
[113] Thomsen PF, Sigsgaard EE. Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods. Ecology and Evolution, 2019, 9: 1665-1679
[114] Stothut M, Kühne D, Ströbele V, et al. Environmental DNA metabarcoding reliably recovers arthropod interactions which are frequently observed by video recordings of flowers. Environmental DNA, 2024, 6: e550
[115] Schnell IB, Thomsen PF, Wilkinson N, et al. Screening mammal biodiversity using DNA from leeches. Current Biology, 2012, 22: 1980-1980
[116] Fu A, Wang QY, Fan YW, et al. Fecal DNA metabarcoding reveals the winter diet of Eurasian otter (Lutra lutra) in Northeast China. Global Ecology and Conservation, 2024, 53: e03033