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Advances in the aquatic sciences
RESEARCH ARTICLE

Improving reliability in environmental DNA detection surveys through enhanced quality control

Elise M. Furlan A B and Dianne Gleeson A
+ Author Affiliations
- Author Affiliations

A Institute for Applied Ecology, University of Canberra, University Drive, Bruce, ACT 2617, Australia.

B Corresponding author. Email: elise.furlan@canberra.edu.au

Marine and Freshwater Research 68(2) 388-395 https://doi.org/10.1071/MF15349
Submitted: 14 September 2015  Accepted: 13 February 2016   Published: 27 April 2016

Abstract

Species-specific environmental DNA (eDNA) surveys are increasingly being used to infer species presence in an environment. Current inadequacies in quality control increase concern for false negatives, which can have serious ramifications for both the management of invasive species and the conservation of native species. eDNA surveys involve a multi-step process to sample, capture, extract and amplify target DNA from the environment. We outline various positive control options and show that many of the commonly used controls are capable of detecting false negatives arising during the amplification stage only. We suggest a secondary, generic primer, designed to co-amplify endogenous DNA sampled during species-specific eDNA surveys, constitutes a superior positive control to monitor method success throughout all stages of eDNA analysis. We develop a species-specific European carp (Cyprinus carpio) assay and a generic fish assay for use as an endogenous control for eDNA surveys in Australian freshwater systems where fish are known to be abundant. We use these assays in a multiplex on eDNA samples that are simultaneously sampled, captured, extracted and amplified. This positive control allows us to distinguish method error from informative non-amplification results, improving reliability in eDNA surveys, which will ultimately lead to better informed conservation management decisions.

Additional keywords: detection, false negative, positive control, sensitivity, type II error.


References

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology 215, 403–410.
Basic local alignment search tool.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3MXitVGmsA%3D%3D&md5=7b370601ae69b72b6c1f2992335d019eCAS | 2231712PubMed |

Amberg, J. J., Grace Mccalla, S., Monroe, E., Lance, R., Baerwaldt, K., and Gaikowski, M. P. (2015). Improving efficiency and reliability of environmental DNA analysis for silver carp. Journal of Great Lakes Research 41, 367–373.
Improving efficiency and reliability of environmental DNA analysis for silver carp.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXlvVemt7w%3D&md5=db98a440b896f4e10b151e949b7a7fb4CAS |

Ardura, A., Zaiko, A., Martinez, J. L., Samulioviene, A., Semenova, A., and Garcia-Vazquez, E. (2015). eDNA and specific primers for early detection of invasive species – a case study on the bivalve Rangia cuneata, currently spreading in Europe. Marine Environmental Research 112, 48–55.
eDNA and specific primers for early detection of invasive species – a case study on the bivalve Rangia cuneata, currently spreading in Europe.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXhs1aktb7I&md5=55a69d9aa644ec257a5ef4c7309c4bf9CAS | 26453004PubMed |

Baay, M. F., Quint, W. G., Koudstaal, J., Hollema, H., Duk, J. M., Burger, M. P., Stolz, E., and Herbrink, P. (1996). Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas. Journal of Clinical Microbiology 34, 745–747.
| 1:CAS:528:DyaK28XhvVantL0%3D&md5=5dd8b02f2654162060c9bec1ff34471bCAS | 8904451PubMed |

Baker-Austin, C., Morris, J., Lowther, J. A., Rangdale, R., and Lees, D. N. (2009). Rapid identification and differentiation of agricultural faecal contamination sources using multiplex PCR. Letters in Applied Microbiology 49, 529–532.
Rapid identification and differentiation of agricultural faecal contamination sources using multiplex PCR.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1MnisFShuw%3D%3D&md5=89e504fc948f28c75f8d9ca27e1142d8CAS | 19708886PubMed |

Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., and Sayers, E. W. (2009). GenBank. Nucleic Acids Research 37, D26–D31.
GenBank.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhsFejtLzF&md5=77c382402ed728f25c52e5158e04e587CAS | 18940867PubMed |

Berry, O., Sarre, S. D., Farrington, L., and Aitken, N. (2007). Faecal DNA detection of invasive species: the case of feral foxes in Tasmania. Wildlife Research 34, 1–7.
Faecal DNA detection of invasive species: the case of feral foxes in Tasmania.Crossref | GoogleScholarGoogle Scholar |

Biggs, J., Ewald, N., Valentini, A., Gaboriaud, C., Dejean, T., Griffiths, R. A., Foster, J., Wilkinson, J. W., Arnell, A., Brotherton, P., Williams, P., and Dunn, F. (2015). Using eDNA to develop a national citizen science-based monitoring programme for the great crested newt (Triturus cristatus). Biological Conservation 183, 19–28.
Using eDNA to develop a national citizen science-based monitoring programme for the great crested newt (Triturus cristatus).Crossref | GoogleScholarGoogle Scholar |

Bunce, M., Oskam, C. L., and Allentoft, M. E. (2012). Quantitative real-time PCR in aDNA research. In ‘Ancient DNA: Methods and Protocols’. (Eds B. Shapiro and M. Hofreiter.) pp. 121–132. (Humana Press: USA.)

Cooper, A., and Poinar, H. N. (2000). Ancient DNA: do it right or not at all. Science 289, 1139.
Ancient DNA: do it right or not at all.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXmt1Sqtbo%3D&md5=2cda28b1fd4fd34b424bbfb1f6c2b625CAS | 10970224PubMed |

Darling, J. A., and Mahon, A. R. (2011). From molecules to management: adopting DNA-based methods for monitoring biological invasions in aquatic environments. Environmental Research 111, 978–988.
From molecules to management: adopting DNA-based methods for monitoring biological invasions in aquatic environments.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXht1Kmt77E&md5=d7ede06ac60a932c79441f3f48b75b5aCAS | 21353670PubMed |

Deagle, B. E., Bax, N., and Patil, J. G. (2003). Development and evaluation of a PCR-based test for detection of Asterias (Echinodermata: Asteroidea) larvae in Australian plankton samples from ballast water. Marine and Freshwater Research 54, 709–719.
Development and evaluation of a PCR-based test for detection of Asterias (Echinodermata: Asteroidea) larvae in Australian plankton samples from ballast water.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXovVGmur8%3D&md5=34b2419839b4a2646bf55c00bb7e568fCAS |

Dejean, T., Valentini, A., Miquel, C., Taberlet, P., Bellemain, E., and Miaud, C. (2012). Improved detection of an alien invasive species through environmental DNA barcoding: the example of the American bullfrog Lithobates catesbeianus. Journal of Applied Ecology 49, 953–959.
Improved detection of an alien invasive species through environmental DNA barcoding: the example of the American bullfrog Lithobates catesbeianus.Crossref | GoogleScholarGoogle Scholar |

Dell’Anno, A., and Corinaldesi, C. (2004). Degradation and turnover of extracellular DNA in marine sediments: ecological and methodological considerations. Applied and Environmental Microbiology 70, 4384–4386.
Degradation and turnover of extracellular DNA in marine sediments: ecological and methodological considerations.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXmtFWmsr4%3D&md5=b92c64980baa64728dfbea7bee57af5cCAS | 15240325PubMed |

Eichmiller, J. J., Bajer, P. G., and Sorensen, P. W. (2014). The relationship between the distribution of common carp and their environmental DNA in a small lake. PLoS One 9, e112611.
The relationship between the distribution of common carp and their environmental DNA in a small lake.Crossref | GoogleScholarGoogle Scholar | 25383965PubMed |

Eichmiller, J. J., Miller, L. M., and Sorensen, P. W. (2016). Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish. Molecular Ecology Resources 16, 56–68.
Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXitV2jsbbP&md5=f3be4494dd345042a57b7997c525e2e8CAS | 25919417PubMed |

Ficetola, G. F., Miaud, C., Pompanon, F., and Taberlet, P. (2008). Species detection using environmental DNA from water samples. Biology Letters 4, 423–425.
Species detection using environmental DNA from water samples.Crossref | GoogleScholarGoogle Scholar | 18400683PubMed |

Foote, A. D., Thomsen, P. F., Sveegaard, S., Wahlberg, M., Kielgast, J., Kyhn, L. A., Salling, A. B., Galatius, A., Orlando, L., and Gilbert, M. T. P. (2012). Investigating the potential use of environmental DNA (eDNA) for genetic monitoring of marine mammals. PLoS One 7, e41781.
Investigating the potential use of environmental DNA (eDNA) for genetic monitoring of marine mammals.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xht12htLzI&md5=eecf5b63edfcd0d797b5fa0e5ba05daeCAS | 22952587PubMed |

Furlan, E. M., Gleeson, D. M., Hardy, C. M., and Duncan, R. P. (2016). A framework for estimating the sensitivity of eDNA surveys. Molecular Ecology Resources 16, 641–654.
A framework for estimating the sensitivity of eDNA surveys.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC28XlslWjs7s%3D&md5=590f0a766c6c9406ac44084c544cd8e4CAS | 26536842PubMed |

Gill, P., Whitaker, J., Flaxman, C., Brown, N., and Buckleton, J. (2000). An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA. Forensic Science International 112, 17–40.
An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXksFels7w%3D&md5=e82f5801575fa150b6f66f275695f0beCAS | 10882828PubMed |

Goldberg, C. S., Sepulveda, A., Ray, A., Baumgardt, J., and Waits, L. P. (2013). Environmental DNA as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum). Freshwater Science 32, 792–800.
Environmental DNA as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum).Crossref | GoogleScholarGoogle Scholar |

Green, H. C., and Field, K. G. (2012). Sensitive detection of sample interference in environmental qPCR. Water Research 46, 3251–3260.
Sensitive detection of sample interference in environmental qPCR.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XmsF2ltL8%3D&md5=17f0a90a5e57ba3269ef61ac67b79859CAS | 22560896PubMed |

Gu, W., and Swihart, R. K. (2004). Absent or undetected? Effects of non-detection of species occurrence on wildlife–habitat models. Biological Conservation 116, 195–203.
Absent or undetected? Effects of non-detection of species occurrence on wildlife–habitat models.Crossref | GoogleScholarGoogle Scholar |

Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis. Nucleic Acids Symposium Series 41, 95–98.
| 1:CAS:528:DC%2BD3cXhtVyjs7Y%3D&md5=bc87ebd59d3449e8e2cb27069fecf75cCAS |

Hardy, C. M., Adams, M., Jerry, D. R., Court, L. N., Morgan, M. J., and Hartley, D. M. (2011). DNA barcoding to support conservation: species identification, genetic structure and biogeography of fishes in the Murray–Darling River Basin, Australia. Marine and Freshwater Research 62, 887–901.
DNA barcoding to support conservation: species identification, genetic structure and biogeography of fishes in the Murray–Darling River Basin, Australia.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhtVOgu7jF&md5=ce3c03b726ffb532d67dcc3b3b2f41b2CAS |

Haugland, R. A., Siefring, S. C., Wymer, L. J., Brenner, K. P., and Dufour, A. P. (2005). Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis. Water Research 39, 559–568.
Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtFejs7o%3D&md5=0231a25acdd3c46712d7054d27dac88eCAS | 15707628PubMed |

Hughes, J. P., and Totten, P. (2003). Estimating the accuracy of polymerase chain reaction-based tests using endpoint dilution. Biometrics 59, 505–511.
Estimating the accuracy of polymerase chain reaction-based tests using endpoint dilution.Crossref | GoogleScholarGoogle Scholar | 14601751PubMed |

Jerde, C. L., Mahon, A. R., Chadderton, W. L., and Lodge, D. M. (2011). “Sight-unseen” detection of rare aquatic species using environmental DNA. Conservation Letters 4, 150–157.
“Sight-unseen” detection of rare aquatic species using environmental DNA.Crossref | GoogleScholarGoogle Scholar |

King, C. E., Debruyne, R., Kuch, M., Schwarz, C., and Poinar, H. N. (2009). A quantitative approach to detect and overcome PCR inhibition in ancient DNA extracts. BioTechniques 47, 941–949.
A quantitative approach to detect and overcome PCR inhibition in ancient DNA extracts.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsFSntrnI&md5=aee6ffddba870baa25245bf4ef759a61CAS |

Leonard, J. A., Shanks, O., Hofreiter, M., Kreuz, E., Hodges, L., Ream, W., Wayne, R. K., and Fleischer, R. C. (2007). Animal DNA in PCR reagents plagues ancient DNA research. Journal of Archaeological Science 34, 1361–1366.
Animal DNA in PCR reagents plagues ancient DNA research.Crossref | GoogleScholarGoogle Scholar |

Lintermans, M. (2001). ‘Fish in the Upper Murrumbidgee Catchment. A Review of Current Knowledge.’ (Wildlife Research & Monitoring: Canberra, ACT.)

Moyer, G. R., Díaz-Ferguson, E., Hill, J. E., and Shea, C. (2014). Assessing environmental DNA detection in controlled lentic systems. PLoS One 9, e103767.
Assessing environmental DNA detection in controlled lentic systems.Crossref | GoogleScholarGoogle Scholar | 25079969PubMed |

Ogram, A., Sayler, G. S., and Barkay, T. (1987). The extraction and purification of microbial DNA from sediments. Journal of Microbiological Methods 7, 57–66.
The extraction and purification of microbial DNA from sediments.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1cXlt1ekuw%3D%3D&md5=5d632a1c8621ad43e57e1cce461514bbCAS |

Olson, Z. H., Briggler, J. T., and Williams, R. N. (2012). An eDNA approach to detect eastern hellbenders (Cryptobranchus a. alleganiensis) using samples of water. Wildlife Research 39, 629–636.
An eDNA approach to detect eastern hellbenders (Cryptobranchus a. alleganiensis) using samples of water.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhsVCgt7zP&md5=d758fdc59d8f76d9693cd30c7bab3787CAS |

Pääbo, S., Poinar, H., Serre, D., Jaenicke-Després, V., Hebler, J., Rohland, N., Kuch, M., Krause, J., Vigilant, L., and Hofreiter, M. (2004). Genetic analyses from ancient DNA. Annual Review of Genetics 38, 645–679.
Genetic analyses from ancient DNA.Crossref | GoogleScholarGoogle Scholar | 15568989PubMed |

Pilliod, D. S., Goldberg, C. S., Arkle, R. S., and Waits, L. P. (2013). Estimating occupancy and abundance of stream amphibians using environmental DNA from filtered water samples. Canadian Journal of Fisheries and Aquatic Sciences 70, 1123–1130.
Estimating occupancy and abundance of stream amphibians using environmental DNA from filtered water samples.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhtVehtL3P&md5=959be7aa732843f4ad874fd25e32d6c8CAS |

Richardson, B. A., Hughes, J. P., and Benki, S. (2007). Statistical methods for determining the accuracy of quantitative polymerase chain reaction-based tests. Statistics in Medicine 26, 895–902.
Statistical methods for determining the accuracy of quantitative polymerase chain reaction-based tests.Crossref | GoogleScholarGoogle Scholar | 16685704PubMed |

Schmidt, T., Hummel, S., and Herrmann, B. (1995). Evidence of contamination in PCR laboratory disposables. Naturwissenschaften 82, 423–431.
Evidence of contamination in PCR laboratory disposables.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXosVKisrY%3D&md5=28225da3f85e094d40e5c979e85ad2ebCAS | 7477415PubMed |

Schmidt, B. R., Kéry, M., Ursenbacher, S., Hyman, O. J., and Collins, J. P. (2013). Site occupancy models in the analysis of environmental DNA presence/absence surveys: a case study of an emerging amphibian pathogen. Methods in Ecology and Evolution 4, 646–653.
Site occupancy models in the analysis of environmental DNA presence/absence surveys: a case study of an emerging amphibian pathogen.Crossref | GoogleScholarGoogle Scholar |

Shen, Z., Qu, W., Wang, W., Lu, Y., Wu, Y., Li, Z., Hang, Z., Wang, X., Zhao, D., and Zhang, C. (2010). MPprimer: a program for reliable multiplex PCR primer design. BMC Bioinformatics 11, 143.
MPprimer: a program for reliable multiplex PCR primer design.Crossref | GoogleScholarGoogle Scholar | 20298595PubMed |

Sint, D., Raso, L., and Traugott, M. (2012). Advances in multiplex PCR: balancing primer efficiencies and improving detection success. Methods in Ecology and Evolution 3, 898–905.
Advances in multiplex PCR: balancing primer efficiencies and improving detection success.Crossref | GoogleScholarGoogle Scholar | 23549328PubMed |

Taberlet, P., Coissac, E., Hajibabaei, M., and Rieseberg, L. H. (2012). Environmental DNA. Molecular Ecology 21, 1789–1793.
Environmental DNA.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XptVGksLw%3D&md5=010b460ffc6e0e6bb91f35ff263e0a39CAS | 22486819PubMed |

Takahara, T., Minamoto, T., and Doi, H. (2013). Using environmental DNA to estimate the distribution of an invasive fish species in ponds. PLoS One 8, e56584.
Using environmental DNA to estimate the distribution of an invasive fish species in ponds.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXjsF2isLw%3D&md5=8f706e3a23106d5f645d5c089842ba63CAS | 23437177PubMed |

Thomsen, P. F., and Willerslev, E. (2015). Environmental DNA – an emerging tool in conservation for monitoring past and present biodiversity. Biological Conservation 183, 4–18.
Environmental DNA – an emerging tool in conservation for monitoring past and present biodiversity.Crossref | GoogleScholarGoogle Scholar |

Thomsen, P. F., Kielgast, J., Iversen, L. L., Wiuf, C., Rasmussen, M., Gilbert, M. T. P., Orlando, L., and Willerslev, E. (2012). Monitoring endangered freshwater biodiversity using environmental DNA. Molecular Ecology 21, 2565–2573.
Monitoring endangered freshwater biodiversity using environmental DNA.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xht1Wlu7vO&md5=eae79c85d30a5c1c2c774c65132d1e14CAS | 22151771PubMed |

Tréguier, A., Paillisson, J. M., Dejean, T., Valentini, A., Schlaepfer, M. A., and Roussel, J. M. (2014). Environmental DNA surveillance for invertebrate species: advantages and technical limitations to detect invasive crayfish Procambarus clarkii in freshwater ponds. Journal of Applied Ecology 51, 871–879.
Environmental DNA surveillance for invertebrate species: advantages and technical limitations to detect invasive crayfish Procambarus clarkii in freshwater ponds.Crossref | GoogleScholarGoogle Scholar |

Turner, C. R., Miller, D. J., Coyne, K. J., and Corush, J. (2014). Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.). PLoS One 9, e114329.
Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.).Crossref | GoogleScholarGoogle Scholar | 25474207PubMed |

Turner, C. R., Uy, K. L., and Everhart, R. C. (2015). Fish environmental DNA is more concentrated in aquatic sediments than surface water. Biological Conservation 183, 93–102.
Fish environmental DNA is more concentrated in aquatic sediments than surface water.Crossref | GoogleScholarGoogle Scholar |

Vuong, N.-M., Villemur, R., Payment, P., Brousseau, R., Topp, E., and Masson, L. (2013). Fecal source tracking in water using a mitochondrial DNA microarray. Water Research 47, 16–30.
Fecal source tracking in water using a mitochondrial DNA microarray.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhsFeqsrvF&md5=925e3a45602387268c7288d12564e9eeCAS | 23084117PubMed |

Whiley, D. M., and Sloots, T. P. (2005). Sequence variation in primer targets affects the accuracy of viral quantitative PCR. Journal of Clinical Virology 34, 104–107.
Sequence variation in primer targets affects the accuracy of viral quantitative PCR.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtVWit7zI&md5=968fe22a57f3c5a10e459ef5b98d5ea3CAS | 16157260PubMed |

Wilcox, T. M., Schwartz, M. K., Mckelvey, K. S., Young, M. K., and Lowe, W. H. (2014). A blocking primer increases specificity in environmental DNA detection of bull trout (Salvelinus confluentus). Conservation Genetics Resources 6, 283–284.
A blocking primer increases specificity in environmental DNA detection of bull trout (Salvelinus confluentus).Crossref | GoogleScholarGoogle Scholar |

Wilcox, T. M., Mckelvey, K. S., Young, M. K., Lowe, W. H., and Schwartz, M. K. (2015). Environmental DNA particle size distribution from Brook Trout (Salvelinus fontinalis). Conservation Genetics Resources 7, 639–641.
Environmental DNA particle size distribution from Brook Trout (Salvelinus fontinalis).Crossref | GoogleScholarGoogle Scholar |

Wilson, I. G. (1997). Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology 63, 3741–3751.
| 1:CAS:528:DyaK2sXms1Sgsrg%3D&md5=a2c5f69311824fd72218b36be9157849CAS | 9327537PubMed |

Wilson, C., Wright, E., Bronnenhuber, J., Macdonald, F., Belore, M., and Locke, B. (2014). Tracking ghosts: combined electrofishing and environmental DNA surveillance efforts for Asian carps in Ontario waters of Lake Erie. Management of Biological Invasions 5, 225–231.
Tracking ghosts: combined electrofishing and environmental DNA surveillance efforts for Asian carps in Ontario waters of Lake Erie.Crossref | GoogleScholarGoogle Scholar |

Yoccoz, N. G., Nichols, J. D., and Boulinier, T. (2001). Monitoring of biological diversity in space and time. Trends in Ecology & Evolution 16, 446–453.
Monitoring of biological diversity in space and time.Crossref | GoogleScholarGoogle Scholar |