Evaluation of eDNA for groundwater invertebrate detection and monitoring: a case study with endangered Stygobromus (Amphipoda: Crangonyctidae)

Abstract

Effective conservation and management of biodiversity is limited by a lack of critical knowledge on species’ distributions and abundances. This problem is particularly exacerbated for species living in habitats that are exceptionally difficult to access or survey, such as groundwater habitats. Environmental DNA (eDNA) represents a rapid, noninvasive, and potentially cost-effective new tool for detection and monitoring of biodiversity that occur in such habitats. In this study, we investigated the utility of eDNA in detecting the federally endangered Hay’s Spring Amphipod Stygobromus hayi and a co-occurring common congener S. tenuis potomacus from unique groundwater-associated habitats—hypotelminorheic seepage springs—in the Washington, DC metro area. We developed taxon-specific primers and probes for each species to amplify Stygobromus DNA using qPCR. In silico and in vitro validation demonstrated specificity of each designed assay. Assays were then used to screen water samples collected from ten seepage springs. Stygobromus hayi was detected at four seepage springs, including one potential new locality, while S. t. potomacus was detected at four springs, two of which were new localities. This study is the first to our knowledge to successfully employ an eDNA approach to detect rare or threatened invertebrates from subterranean ecosystems. Our study highlights challenges of employing an eDNA approach for the detection and monitoring of invertebrates in groundwater habitats that are difficult to study, including accounting for PCR inhibition and the potential for cryptic genetic diversity.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2

References

  1. Barnosky AD, Matzke N, Tomiya S, Wogan GOU, Swartz B, Quental TB, Marshall C, McGuire JL, Lindsey EL, Maguire KC, Mersey B, Ferrer EA (2011) Has the Earth’s sixth mass extinction already arrived? Nature 471:51–57

    Article  PubMed  CAS  Google Scholar 

  2. Barr TC, Holsinger JR (1985) Speciation in cave faunas. Ann Rev Ecol Syst 16:313–337

    Article  Google Scholar 

  3. Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P, Kauserud H (2010) ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases. BMC Microbiol 10:189

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Bohmann K, Evans A, Gilbert MTP, Carvalho GR, Creer S, Knapp M, Yu DW, de Bruyn M (2014) Environmental DNA for wildlife biology and biodiversity monitoring. Trends Ecol Evol 29:358–367

    Article  PubMed  Google Scholar 

  5. Boulton AJ, Fenwick GD, Hancock PJ, Harvey MS (2008) Biodiversity, functional roles and ecosystem services of groundwater invertebrates. Invertebr Syst 22:103–116

    Article  Google Scholar 

  6. Cardoso P, Erwin TL, Borges PAV, New TR (2011) The seven impediments in invertebrate conservation and how to overcome them. Biol Conserv 144:2647–2655

    Article  Google Scholar 

  7. Ceballos G, Ehrlich PR, Barnosky AD, Garcia A, Pringle RM, Palmer TM (2015) Accelerated modern human-induced species losses: entering the sixth mass extinction. Sci Adv 1:e1400253

    Article  PubMed  PubMed Central  Google Scholar 

  8. Chazal AC, Hobson CS (2003) Surveys for the Northern Virginia Well Amphipod (Stygobromus phreaticus) at Fort Belvoir, Virginia. Technical report 03–11. Division of Natural Heritage, Department of Conservation and Recreation, Commonwealth of Virginia, Richmond, p 12

    Google Scholar 

  9. Culver DC, Pipan T (2011) Redefining the extent of the aquatic subterranean biotope—shallow subterranean habitats. Ecohydrology 4:721–730

    Article  Google Scholar 

  10. Culver DC, Pipan T (2014) Shallow subterranean habitats: ecology, evolution and conservation. Oxford University Press, Oxford

    Google Scholar 

  11. Culver DC, Sereg I (2004) Kenk’s spring amphipod (Stygobromus kenki) and other amphipods in Rock Creek Park, Washington. Unpublished report to Rock Creek Park, National Park Service, p 147

  12. Culver DC, Pipan T, Gottstein S (2006) Hypotelminorheic—a unique freshwater habitat. Subterr Biol 4:1–7

    Google Scholar 

  13. Culver DC, Holsinger JR, Feller DJ (2012) The fauna of seepage springs and other shallow subterranean habitats in the mid-Atlantic Piedmont and Coastal Plain, USA. Northeastern Nat 19:1–42

    Article  Google Scholar 

  14. Davis MA, Douglas MR, Webb CT, Collyer ML, Holycross AT, Painter CW, Kamees LK, Douglas MR (2015) Nowhere to go but up: impacts of climate change on demographics of a short-range endemic (Crotalus willardi obscurus) in the sky-islands of southwestern North America. PLoS ONE 10:e0131067

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Edgar R (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acid Res 32:1792–1797

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Espinasas L, McCahill A, Kavanagh A, Espinasa J, Scott A, Cahill A (2015) A troglobitic amphipod in the ice caves of the Shawangunk Ridge: behavior and resistance to freezing. Subterr Biol 15:95–104

    Google Scholar 

  17. Ethridge JZ, Gibson JR, Nice CC (2013) Cryptic diversity within and amongst spring-associated Stygobromus amphipods (Amphipoda: Crangonyctidae). Zool J Linn Soc 167:227–242

    Article  Google Scholar 

  18. Feller D (1997) Aquatic subterranean macroinvertebrate survey of Rock Creek and associated national parks, Washington, DC. Heritage and Biodiversity Conservation Programs Technical Report. Maryland Department of Natural Resources, Annapolis, p 38

    Google Scholar 

  19. Ficetola GF, Miaud C, Pompanon F, Taberlet P (2008) Species detection using environmental DNA from water samples. Biol Lett 23:423–425

    Article  Google Scholar 

  20. Ficetola GF, Coissac E, Zundeyl S, Riaz T, Shehzad W, Bessiere J, Taberlet P, Pompanon F (2010) An in silico approach for the evaluation of DNA barcodes. BMC Genom 11:434

    Article  CAS  Google Scholar 

  21. Fišer C, Pipan T, Culver DC (2014) The vertical extent of groundwater metazoans: an ecological and evolutionary perspective. Bioscience 64:971–979

    Article  Google Scholar 

  22. Geller J, Meyer C, Parker M, Hawk H (2013) Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all-taxa biotic surveys. Mol Ecol Resour 13:851–861

    Article  PubMed  CAS  Google Scholar 

  23. Gibert J, Culver DC, Dole-Olivier MJ, Malard F, Christman MC, Deharveng L (2009) Assessing and conserving groundwater biodiversity: synthesis and perspectives. Freshw Biol 54:930–941

    Article  Google Scholar 

  24. Giordano M (2009) Global groundwater? Issues and solutions. Ann Rev Environ Resour 34:153–178

    Article  Google Scholar 

  25. Gleeson TJ, Alley WM, Allen DM, Sophocleous MA, Zhou Y, Taniguchi M, VanderSteen J (2012) Towards sustainable groundwater use: setting long-term goals, backcasting, and managing adaptively. Groundwater 50:19–26

    Article  CAS  Google Scholar 

  26. Goldberg CS, Sepulveda A, Ray A, Baumgardt J, Waits LP (2013) Environmental DNA as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum). Freshw Sci 32:792–800

    Article  Google Scholar 

  27. Goldberg CS, Strickler KM, Pilliod DS (2015) Moving environmental DNA methods from concept to practice for monitoring aquatic macroorganisms. Biol Conserv 183:1–3

    Article  Google Scholar 

  28. Goldberg CS, Turner CR, Deiner K, Klymus KE, Thomsen PF, Murphy MA, Spear SF, McKee A, Oyler-McCance SJ, Cornman RS, Laramie MB, Mahon AR, Lance RF, Pilliod DS, Strickler KM, Waits LP, Fremier AK, Takahara T, Herder JE, Taberlet P (2016) Critical considerations for the application of environmental DNA methods to detect aquatic species. Meth Ecol Evol 7:1299–1307

    Article  Google Scholar 

  29. Gorički Š, Stanković D, Aljančič M, Snoj A, Kuntner M, Gredar T, Vodnik L, Aljančič G (2016) Searching for black Proteus with the help of eDNA. Nat Sloveniae 18:57–58

    Google Scholar 

  30. Gorički Š, Stanković D, Snoj A, Kuntner M, Jeffery WR, Trontelj P, Pavićević M, Grizelj Z, Năpăruş-Aljančič M, Aljančič G (2017) Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus. Sci Rep 7:45054

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Griebler C, Lueders T (2009) Microbial biodiversity in groundwater ecosystems. Freshw Biol 54:649–677

    Article  Google Scholar 

  32. Griebler C, Malard F, Lefebure T (2014) Current developments in groundwater ecology: from biodiversity to ecosystem function and services. Curr Opin Biotechnol 27:159–167

    Article  PubMed  CAS  Google Scholar 

  33. Harvey MS, Rix MG, Framenau VW, Hamilton ZR, Johnson MS, Teale RJ, Humphreys G, Humphreys WF (2011) Protecting the innocent: studying short-range endemic taxa enhances conservation outcomes. Invertebr Syst 25:1–10

    Article  Google Scholar 

  34. Holsinger JR (1966) Subterranean amphipods of the genus Stygonectes (Gammaridae) from Texas. Am Midl Nat 76:100–124

    Article  Google Scholar 

  35. Holsinger JR (1967) Systematics, speciation, and distribution of the subterranean amphipod genus Stygonectes (Gammaridae). Bull United States Natl Mus 259:1–176

    Google Scholar 

  36. Holsinger JR (1978) Systematics of the North American amphipod genus Stygobromus, part II: species of the eastern United States. Smithsonian Contrib Zool 266:1–144

    Article  Google Scholar 

  37. Hortal J, de Bello F, Diniz-Filho JAF, Lewinsohn TM, Lobo JM, Ladle RJ (2015) Seven shortfalls that beset large-scale knowledge of biodiversity. Annu Rev Ecol Evol Syst 46:523–549

    Article  Google Scholar 

  38. Hubricht L, Mackin G (1940) Description of nine new species of fresh-water crustaceans with notes and new localities for other species. Am Midl Nat 23:187–218

    Article  Google Scholar 

  39. Hutchins B, Culver DC (2008) Investigating rare and endemic pollution-sensitive subterranean fauna of vulnerable habitats in the NCR. Report to National Capital Region. National Park Service, Washington, DC, p 101

    Google Scholar 

  40. Inland Water Crustacean Specialist Group (1996) Stygobromus hayi. IUCN Red List Threat Species 1996:e.T20990A9241906. doi:10.2305/IUCN.UK.1996.RLTS.T20990A9241906.en

    Article  Google Scholar 

  41. Jerde CL, Mahon AR, Chadderton WL, Lodge DM (2011) “Sight-unseen” detection of rare aquatic species using environmental DNA. Conserv Lett 4:150–157

    Article  Google Scholar 

  42. Keany J (2016) Investigating the physico-chemical niche of obligate subterranean amphipods and isopods in shallow subterranean waters of the DC metro area. Unpublished Master’s Thesis. American University, Washington, DC

    Google Scholar 

  43. Kolbert E (2014) The sixth extinction: an unnatural history. Henry Holt and Co., New York

    Google Scholar 

  44. Laramie MB, Pilliod DS, Goldberg CS (2015) Characterizing the distribution of an endangered salmonid using environmental DNA analysis. Biol Conserv 183:29–37

    Article  Google Scholar 

  45. Maurice L, Bloomfield J (2012) Stygobitic invertebrates in groundwater: a review from a hydrogeological perspective. Freshw Rev 5:51–71

    Article  Google Scholar 

  46. McKee AM, Spear SF, Pierson TW (2015) The effect of dilution and the use of a post-extraction nucleic acid purification column on the accuracy, precision, and inhibition of environmental DNA samples. Biol Conserv 183:70–76

    Article  Google Scholar 

  47. Meleg IN, Zakšek V, Fišer C, Kelemen BS, Moldovan OT (2013) Can environment predict cryptic diversity? The case of Niphargus inhabiting Western Carpathian groundwater. PLoS ONE 8:e76770

    Article  CAS  Google Scholar 

  48. Morris BL, Lawrence ARL, Chilton PJC, Adams B, Calow RC, Liknck BA (2003) Groundwater and its susceptibility to degradation: a global assessment of the problem and options for management. Early Warning and Assessment Report Series, RS. 03–3. United Nations Environment Programme, Nairobi

    Google Scholar 

  49. NatureServe (2016) NatureServe Explorer: an online encyclopedia of life [web application]. Version 7.1. http://explorer.natureserve.org

  50. Niemiller ML, Near TJ, Fitzpatrick BM (2012) Delimiting species using multilocus data: diagnosing cryptic diversity in the southern cavefish, Typhlichthys subterraneus (Teleostei: Amblyopsidae). Evol Int J Org Evol 66:846–866

    Article  Google Scholar 

  51. Niemiller ML, McCandless JR, Reynolds RG, Caddle J, Near TJ, Tillquist CR, Pearson WD, Fitzpatrick BM (2013) Effects of climatic and geological processes during the Pleistocene on the evolutionary history of the northern cavefish, Amblyopsis spelaea (Teleostei: Amblyopsidae). Evol Int J Org Evol 67:1011–1025

    Article  Google Scholar 

  52. Pimm SL, Brooks TM (2000) The sixth extinction: how large, where, and when. Nature and human society: the quest for a sustainable world. National Academy Press, Washington, DC

    Google Scholar 

  53. Pipan T, Fišer C, Novak T, Culver DC (2012) Fifty years of the hypotelminorheic: what have we learned? Acta Carsol 42:275–285

    Google Scholar 

  54. Rambaut A, Suchard MA, Xie D, Drummond AJ (2014) Tracer v1.6. http://beast.bio.ed.ac.uk/Tracer

  55. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574

    Article  PubMed  CAS  Google Scholar 

  56. Schliep K, Paradis E, Potts A, Kendall M (2016) Phangorn: phylogenetic analysis in R. https://cran.r-project.org/web/packages/phangorn/

  57. Sigsgaard EE, Carl H, Moller PR, Thomsen PF (2015) Monitoring the near-extinct European weather loach in Denmark based on environmental DNA from water samples. Biol Conserv 183:46–52

    Article  Google Scholar 

  58. Sophocleous M (2002) Interactions between groundwater and surface water: the state of the science. Hydrogeol J 10:52–67

    Article  CAS  Google Scholar 

  59. Stanković D, Gorički Š, Aljančič M, Snoj A, Kuntner M, Aljančič G (2016) Application of environmental DNA for detection of Proteus. Nat Sloveniae 18:55–56

    Google Scholar 

  60. Thomsen PF, Willerslev E (2015) Environmental DNA: an emerging tool in conservation for monitoring past and present biodiversity. Biol Conserv 183:4–18

    Article  Google Scholar 

  61. Thomsen PF, Kielgast J, Iversen LL, Wiuf C, Rasmussen M, Gilbert MTP, Orlando L, Willerslev E (2012) Monitoring endangered freshwater biodiversity using environmental DNA. Mol Ecol 21:2565–2573

    Article  PubMed  CAS  Google Scholar 

  62. Trontelj P, Douady CJ, Fišer C, Gibert J, Gorički S, Lefebure T, Sket B, Zakšek V (2009) A molecular test for cryptic diversity in ground water: how large are the ranges of macro-stygobionts? Freshw Biol 54:727–744

    Article  CAS  Google Scholar 

  63. USFWS (1982) Endangered and threatened wildlife and plants; listing of Hay’s spring amphipod as an endangered species. Fed Regist 47:5425–5427

    Google Scholar 

  64. USFWS (2007) Hay’s spring amphipod (Stygobromus hayi). 5-year review: summary and evaluation. Chesapeake Bay Field Office, US Fish and Wildlife Service, Annapolis

    Google Scholar 

  65. USFWS (2013) Hay’s spring amphipod (Stygobromus hayi). 5-year review: summary and evaluation. Chesapeake Bay Field Office, US Fish and Wildlife Service, Annapolis

    Google Scholar 

  66. Vörös J, Marton O, Schmidt BR, Gal JT, Jelic D (2017) Surveying Europe’s only cave-dwelling chordate species (Proteus anguinus) using environmental DNA. PLoS ONE 12:e0170945

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Wake DB, Vredenburg VT (2008) Are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proc Natl Acad Sci USA 105:11466–11473

    Article  PubMed  PubMed Central  Google Scholar 

  68. Wilcox TM, Carim KJ, McKelvey KS, Young MK, Schwartz MK (2015) The dual challenges of generality and specificity when developing environmental DNA markers for species and subspecies of Oncorhynchus. PLoS ONE 10:e0142008

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by a grant from the Friends of the Capital Crescent Trail and the town of Chevy Chase, Maryland, and the Illinois Natural History Survey. Sampling of S. hayi was authorized by a subpermit under U.S. Fish and Wildlife Service Regional Endangered Species Recovery Permit TE-697823. This research was also authorized under National Park Service permit no. ROCR-2016-SCI-0014. We thank Jonathan Cybulski and Emily Petersen for assistance with field collections of water samples. We thank Michael Slay for providing specimens of non-target species for DNA analyses.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Matthew L. Niemiller.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 20 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Niemiller, M.L., Porter, M.L., Keany, J. et al. Evaluation of eDNA for groundwater invertebrate detection and monitoring: a case study with endangered Stygobromus (Amphipoda: Crangonyctidae). Conservation Genet Resour 10, 247–257 (2018). https://doi.org/10.1007/s12686-017-0785-2

Download citation

Keywords

  • District of Columbia
  • Hypotelminorheic seep, real-time PCR
  • Short-range endemic
  • Stygobromus hayi
  • Stygobromus tenuis potomacus
  • Subterranean