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Biological Conservation
journal homepage: www.elsevier.com/locate/biocon
a r t i c l e i n f o a b s t r a c t
Article history: The continuous decline in Earths biodiversity represents a major crisis and challenge for the 21st
Received 28 February 2014 century, and there is international political agreement to slow down or halt this decline. The challenge
Received in revised form 4 November 2014 is in large part impeded by the lack of knowledge on the state and distribution of biodiversity especially
Accepted 10 November 2014
since the majority of species on Earth are un-described by science. All conservation efforts to save biodi-
Available online 18 December 2014
versity essentially depend on the monitoring of species and populations to obtain reliable distribution
patterns and population size estimates. Such monitoring has traditionally relied on physical identification
Keywords:
of species by visual surveys and counting of individuals. However, traditional monitoring techniques
Environmental DNA
eDNA
remain problematic due to difficulties associated with correct identification of cryptic species or juvenile
DNA metabarcoding life stages, a continuous decline in taxonomic expertise, non-standardized sampling, and the invasive
Conservation nature of some survey techniques. Hence, there is urgent need for alternative and efficient techniques
Biodiversity for large-scale biodiversity monitoring. Environmental DNA (eDNA) defined here as: genetic material
Monitoring obtained directly from environmental samples (soil, sediment, water, etc.) without any obvious signs of biolog-
Extinction ical source material is an efficient, non-invasive and easy-to-standardize sampling approach. Coupled
with sensitive, cost-efficient and ever-advancing DNA sequencing technology, it may be an appropriate
candidate for the challenge of biodiversity monitoring. Environmental DNA has been obtained from
ancient as well as modern samples and encompasses single species detection to analyses of ecosystems.
The research on eDNA initiated in microbiology, recognizing that culture-based methods grossly
misrepresent the microbial diversity in nature. Subsequently, as a method to assess the diversity of
macro-organismal communities, eDNA was first analyzed in sediments, revealing DNA from extinct
and extant animals and plants, but has since been obtained from various terrestrial and aquatic environ-
mental samples. Results from eDNA approaches have provided valuable insights to the study of ancient
environments and proven useful for monitoring contemporary biodiversity in terrestrial and aquatic
ecosystems. In the future, we expect the eDNA-based approaches to move from single-marker analyses
of species or communities to meta-genomic surveys of entire ecosystems to predict spatial and temporal
biodiversity patterns. Such advances have applications for a range of biological, geological and
environmental sciences. Here we review the achievements gained through analyses of eDNA from
macro-organisms in a conservation context, and discuss its potential advantages and limitations for
biodiversity monitoring.
! 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2. Achievements of eDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1. Ancient environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1.1. Terrestrial sediments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1.2. Aquatic sediments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.1.3. Ice cores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2. Modern environments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
http://dx.doi.org/10.1016/j.biocon.2014.11.019
0006-3207/! 2015 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
P.F. Thomsen, E. Willerslev / Biological Conservation 183 (2015) 418 5
Fig. 1. The overall workflow for environmental DNA (eDNA) studies with examples of organisms that have been identified from environmental samples. Environments and
their respective samplings from left to right: (i) glaciers; (ii) permafrost/tundra; (iii) aquatic sediments; (iv) lakes and streams; (v) terrestrial habitats; (vi) oceans. The first
three are ancient environments while the latter three are modern. Color codes of arrows represent the different steps in the analyses: (blue) environmental sampling (ice
cores, soil/sediment core samples, freshwater/seawater samples); (green) DNA extraction using procedures specific for the individual type of sample; (orange) PCR
amplification of extracted DNA using generic or species-specific primers targeting biodiversity and subsequent sequencing of amplicons (here shown as Illumina MiSeq
technology); (red) bioinformatic data-processing including error trimming, sequence sorting, and identification pipelines leading to various taxonomic level or MOTUs and
the final interpretation and publication of results. Drawing by Lars Holm.
Source References Title Sample habitat Taxons studied Geographical location Aproximate ages
(years BP)
Terrestrial Willerslev et al. (2003) Diverse plant and animal genetic records from Holocene Cave sediments, permafrost Mammals, birds, plants New Zealand; Siberia Present 400,000
sediments and Pleistocene sediments
Hofreiter et al. (2003) Molecular caving Cave sediments Mammals, birds Arizona, USA ca. 10,845
Lydolph et al. (2005) Beringian paleoecology inferred from permafrost- Permafrost Fungi, plants, protists Siberia Present 300,000
preserved fungal DNA 400,000
Haile et al. (2007) Ancient DNA chronology within sediment deposits: Are Cave sediments Birds, plants New Zealand Present 3300
paleobiological reconstructions possible and is DNA
leaching a factor?
Haile et al. (2009) Ancient DNA reveals late survival of mammoth and Perennially frozen sediments Mammals Alaska, USA ca. 700011,000
horse in interior Alaska
Thomsen et al. (2009) Non-destructive sampling of ancient insect DNA Temperate dry sediments Insects New Zealand 3000
Hebsgaard et al. (2009) The farm beneath the sand an archaeological case Sand sediment Mammals South-west Greenland AD 10301530
study on ancient dirt DNA
Snsteb et al. (2010) Using next-generation sequencing for molecular Permafrost Plants Siberia 15,81022,960
7
8
Table 1 (continued)
Source References Title Sample habitat Taxons studied Geographical location Aproximate ages
(years BP)
unexpectedly high fungal diversity
Yoccoz et al. (2012) DNA from soil mirrors plant taxonomic and growth form Boreal, temperate and Plants Norway; France; Present
diversity tropical soil French Guiana
Taberlet et al. (2012) Soil sampling and isolation of extracellular DNA from Grassland soil Plants France Present
large amount of starting material suitable for
metabarcoding studies
Bienert et al. (2012) Tracking earthworm communities from soil DNA Soil Earthworms France Present
Andersen et al. (2012) Meta-barcoding of dirt DNA from soil reflects Soil from animal enclosuers Mammals Denmark Present
vertebrate biodiversity
Freshwater Martellini et al. (2005) Use of eukaryotic mitochondrial DNA to differentiate Wastewater, rivers Human, cow, pig, sheep Quebec, Canada Present
human, bovine, porcine and ovine sources in fecally
contaminated surface water
Ficetola et al. (2008) Species detection using environmental DNA from water Ponds, artificial containers Amphibians (american bull frog) France Present
samples
Jerde et al. (2011) Sight-unseen detection of rare aquatic species using Rivers Fish Illinois, USA Present
9
10 P.F. Thomsen, E. Willerslev / Biological Conservation 183 (2015) 418
revealing DNA from extinct and extant mammals, birds and plants algae and protists, of both local and distant origin, could be
(Willerslev et al., 2003), followed by dry cave sediments, revealing obtained from Holocene ice cores in northern Greenland. Later,
DNA from past cave-dwelling mammals and birds (Hofreiter et al., eDNA from much older, basal ice cores provided insights into a past
2003). Later, it was shown that also DNA from invertebrates such ecosystem of plants and arthropods, indicative of a forested south-
as insects (Epp et al., 2012; Thomsen et al., 2009) as well as fungi ern Greenland 450800 thousand years ago almost 2 million
(Lydolph et al., 2005) is also preserved in ancient sediments. This years younger than previously presumed (Willerslev et al., 2007).
so-called sedimentary ancient DNA (sedaDNA or dirtDNA) has
been shown to be of local origin, and organisms must be physically 2.2. Modern environments
present at the site for their DNA to be deposited (Andersen et al.,
2012; Haile et al., 2009; Lydolph et al., 2005; Yoccoz et al., 2012). 2.2.1. Surface soil
DNA leaching through strata has not been observed in ancient fro- While studies on soil eDNA began on ancient deposits (sedi-
zen sediments (Hansen et al., 2006; Willerslev et al., 2007, 2004) or ments), there have been several recent applications to modern eco-
recently (<1000 years old) frozen sediments (Hebsgaard et al., systems. Total soil DNA includes both intra- and extracellular DNA,
2009), but may occur between layers in non-frozen depositional the latter of which probably being the most significant part (Levy-
settings (Andersen et al., 2012; Haile et al., 2007). Sedimentary Booth et al., 2007; Pietramellara et al., 2008). Metabarcoding of
eDNA has been speculated to derive from faeces, urine, epidermal eDNA from surface soil has thus efficiently been used as a proxy
cells and hair, which is based on the presence of DNA from copro- for plant taxonomic diversity in several different terrestrial ecosys-
philic and keratinophilic fungi found in ancient sediments tems (Taberlet et al., 2012c; Yoccoz et al., 2012). The applicability
(Lydolph et al., 2005). The effectiveness of NGS techniques to of soil eDNA has also been widely demonstrated for fungi
reconstruct paleo-ecosystems from permafrost samples has been (reviewed by Anderson and Cairney, 2004; Chase and Fay, 2009),
demonstrated to reveal rich diversity in past flora (Snsteb for example by using DNA metabarcoding from various forest soils,
et al., 2010) and study recent environmental change in Greenland revealing rich and diverse fungal communities (Buee et al., 2009;
(Jrgensen et al., 2012b). However, some results indicate that OBrien et al., 2005). As for animal eDNA in modern soil samples,
physical remains such as pollen and macrofossils are complemen- Bienert et al. (2012) demonstrated that earthworm communities
tary rather than overlapping with sedaDNA, and in combination, an important indicator group of ecosystem functioning and
reveal more detailed information on ancient plant communities health can be identified using next-generation sequencing,
than can be achieved by each individual approach (Jrgensen whereas Andersen et al. (2012) found that DNA from the soil
et al., 2012a; Parducci et al., 2013). surface reflects overall taxonomic richness and relative biomass
of individual vertebrate species.
2.1.2. Aquatic sediments
Aquatic sediments (freshwater and marine) have also proven to 2.2.2. Freshwater
be rich in eDNA. In fact, the extracellular DNA in marine sediments One of the first studies retrieving macro-organismal eDNA from
is considered by far the largest reservoir of DNA in the oceans freshwater, focused on DNA from human, cow, pig and sheep, as a
(DellAnno and Danovaro, 2005), and especially anoxic conditions method to detect the sources in faecally contaminated surface
reduce nuclease degradation and favours long-term preservation water (Martellini et al., 2005). Later, eDNA from American bull-
of eDNA (Corinaldesi et al., 2011). For example, Bhadury et al. frogs, an invasive amphibian species native to North America,
(2006) studied eDNA from nematodes, as an indicator of the mei- was successfully retrieved from natural pond water samples in
ofauna, in marine and estuarine sediments. Also, eDNA from estu- France (Ficetola et al., 2008). This study initiated a rapidly growing
arine sediments have been used to compare eukaryotic species interest in aquatic ecosystems, which have generating numerous
assemblages to assess human impacts on these ecosystems studies with direct applications to conservation (e.g. see this jour-
(Chariton et al., 2010). Later and more comprehensive analyses of nal special issue). Many subsequent studies continued to focus on
eDNA from marine benthic (Fonseca et al., 2010) and deep-sea sed- detection of invasive species. Dejean et al. (2012) demonstrated
iments (Pawlowski et al., 2011) have deployed NGS, revealing dif- that eDNA can deliver improved detection of invasive species over
ferences in metazoan diversity at microgeographical scales and traditional methods, using the original model species American
DNA from all major groups of eukaryotes and most marine meta- bullfrog. Jerde et al. (2011) studied two species of Asian carps in
zoan groups, respectively. river systems, which demonstrated that the forefronts of the spe-
Matisoo-Smith et al. (2008) was, as far as we know, the first cies were closer to invasion of upstream lake systems than initially
study to show that freshwater lake sediment, traditionally used seen by traditional surveillance methods. This approach were later
for pollen records, also contain macro-organismal eDNA in this expanded significantly (Jerde et al., 2013) and extended to other
case from fish. Later Anderson-Carpenter et al. (2011) successfully related fish species in a study that confirmed presence of eDNA
amplified plant DNA from up to 4600 years old freshwater lake also for low-abundance species in river systems (Mahon et al.,
sediment samples, and found that the results matched the taxo- 2013). Takahara et al. (2013) used eDNA to estimate presence of
nomic identity of the macrofossil in the respective samples. Since the invasive bluegill sunfish on Japanese mainlands and surround-
the DNA preservation is good under anoxic conditions, studies on ing islands. Here eDNA also proved superior to visual detections.
eDNA from aquatic sediments, such as the above, holds potential Recently, the eDNA approach has been expanded to invasive
for a better understanding of the ecological and evolutionary con- reptiles (Piaggio et al., 2014), snails (Goldberg et al., 2013) and
sequences of environmental change (Giguet-Covex et al., 2014; crustaceans (Trguier et al., 2014).
Parducci et al., 2012). Similar to results from terrestrial sediments, Another line of freshwater eDNA reseach concerns endangered
eDNA from lake sediments also complements analyses of pollen species. Here, most studies have focused on detection of amphibi-
and macrofossils, validating the combination of traditional and ans (Goldberg et al., 2011; Olson et al., 2013; Pilliod et al., 2013;
molecular techniques to obtain a more comprehensive picture of Santas et al., 2013; Thomsen et al., 2012b) and even quantification
biodiversity (Parducci et al., 2013; Pedersen et al., 2013). (Pilliod et al., 2013; Thomsen et al., 2012b), which provides a
potential new range of opportunities for estimating relative abun-
2.1.3. Ice cores dance. Quantification of fish biomass from fresh water samples has
Willerslev et al. (1999) showed for the first time that eukaryotic also been attempted (Takahara et al., 2012), as well as species com-
eDNA, representing a diverse assemblage of both plants, fungi, position and communities (Minamoto et al., 2011; Thomsen et al.,
P.F. Thomsen, E. Willerslev / Biological Conservation 183 (2015) 418 11
2012b). Furthermore, it has been demonstrated that also endan- predict which species would go extinct or survive over evolution-
gered freshwater insects, crustaceans, fish and mammals can be ary time scales. Although the current study uses mtDNA from
monitored using eDNA, and that such an approach can account ancient remains and does not employ eDNA as such, it demon-
for entire lake faunas of amphibians and fish using high-through- strates that DNA can extend the time frame of population demog-
put sequencing (Thomsen et al., 2012b). raphy and help reveal the uncertainties associated with extinction
Although Turner et al. (2014) recently found that aqueous predictions, providing relevant considerations for current species
macrobial eDNA likely exists predominantly inside mitochondria conservation attempts.
or small cells, the decay of eDNA in freshwater beyond the thresh- Many extinction models are based on predictions as to when
old of detectability has been demonstrated to happen at a scale of the extinction happened (Nogus-Bravo et al., 2008). Again, such
days or weeks (Dejean et al., 2011; Pilliod et al., 2014; Thomsen estimates rely on dating of macrofossils like teeth and bones. How-
et al., 2012b). This contrasts situations in soil, where eDNA is pro- ever, the chance of finding some of the youngest individuals in a
ven to persist for decades (and perhaps centuries) after deposition dwindling population may be hard or impossible and scientists
(Andersen et al., 2012; Yoccoz et al., 2012). The rapid degradation are very unlikely to come across the last macrofossil left behind.
time in freshwater ecosystems makes eDNA very useful in conser- In this case eDNA from shed skin cells, urine or faces, which is
vation, since a positive detection is likely to be associated with likely to be present and distributed over a larger area, is a valid
contemporary presence of species and populations while poten- supplement to establish last appearance dates of species. This
tially misleading signals from past populations are not picked up. was attempted by Haile et al. (2009) in Stevens Village, Alaska,
Nevertheless, long distance transport of eDNA from hundreds of which is among the places where most bones of woolly mammoth
meters to several kilometres have been reported in river systems, have been dated. Dating of these bones have previously suggested
and should be taken into account for flowing waters (Deiner and an extinction date for woolly mammoth on mainland Alaska some
Altermatt, 2014; Jane et al., 2014). 13,100 years BP. However, the eDNA results suggest survival of
mammoth until some 10,500 years BP ca. 3500 years after first
human arrival into mainland Alaska and thus that man and beast
2.2.3. Seawater
co-existed several thousand years prior to extinction of the latter.
It has been widely demonstrated that microbial (prokaryotic
In a recent study by Willerslev et al. (2014) more than 200 per-
and eukaryotic) biodiversity can be studied by sequencing DNA
mafrost samples from 21 sites across the Arctic covering the past
from filtered seawater samples (Rusch et al., 2007; Sogin et al.,
50,000 years were analyzed for plant chloroplast eDNA. Contrary
2006; Venter et al., 2004; Zinger et al., 2011). Recently however,
to many pollen studies, the results reveal that the coldest and dri-
Thomsen et al. (2012a) showed that it is possible to detect a rich
est stages during the last ice age (the Last Glacial Maximum, LGM)
marine fish fauna by metabarcoding of eDNA from seawater sam-
around 20 thousand years ago was a major bottleneck in plant
ples, and that such an approach can cover the fish diversity better
diversity and that the warmer periods before this bottleneck was
than or equal to any of 9 methods conventionally used in marine
richer and more diverse in plant composition than the Holocene
fish surveys. The approach has been validated in a large sea-tank
period following the LGM, where many new taxa became domi-
mesocosm, where also rank abundance of recovered eDNA corre-
nant. As such the study suggests that not only climatic changes
lated with abundance of corresponding species biomass (Kelly
but also the recent vegetation history determines how ecosystems
et al., 2014a). Furthermore, Foote et al. (2012) showed that eDNA
develop over time.
from whales can also be detected from seawater samples, and an
experimental setup in sea-enclosures indicated the eDNA signal
is evident only within short distances (metres) of animals. These 4. Pitfalls and challenges for eDNA
are the first evidence that marine water samples contain detect-
able eDNA from macro-organisms. An experimental setup has Despite obvious perspectives and useful applications using
shown that even small (100-bp) eDNA fragments in seawater eDNA, several problematic issues associated with the approach
degrades beyond detectability within days (Thomsen et al., needs to be considered. For a more thorough treatment of these
2012a). DNA degradation in seawater has previously been sug- issues see Coissac et al. (2012) and Pedersen et al. (in press).
gested to be substantially faster with an empirical turnover rate
as low as 10 h (DellAnno and Corinaldesi, 2004). These studies
4.1. Contamination
indicate low probability of long-distance dispersal of eDNA in mar-
ine ecosystems. Although further studies are needed to validate the
The most serious pitfall of eDNA is probably the risk of contam-
eDNA approach in contemporary marine environments, initial
ination and hence the possibility of false positive results. Contam-
studies show promising perspectives for future monitoring and
ination of samples can occur from taking the samples in the field to
management of marine biodiversity and resources (Kelly et al.,
every step of analyses in the laboratory. If several localities are
2014b).
sampled after one another in the field, there is a risk of cross-
contamination: target DNA carried unintentionally from one
3. Ancient environments and extinctions locality to another. Lab contamination is especially serious because
of the frequent use of PCR in eDNA studies, generating billions of
Ancient eDNA represents a challenge to obtain and verify given DNA copies, which can readily spread throughout the lab.
that it often has undergone significant degradation (Hansen et al., The use of NGS technologies has further complicated the
2006; Willerslev and Cooper, 2005). Nevertheless, analyzing DNA contamination issue, as they produce a very high throughput of
in an evolutionary timeframe holds an important potential to DNA sequences likely to reveal tiny amounts of lab-source PCR
understand changes in ecosystems and extinctions. This was products. Cross-contamination in the lab seems almost unavoidable,
recently evident by a study of Lorenzen et al. (2011), which ana- and it is essential to apply conservative cut-offs for minimum
lyzed the population history over the past 50,000 years using percentages of sequences obtained in a sample, and/or the
mtDNA from six big-bodied mammals (megafauna). Coupling this amplification success in independent PCR reactions, before includ-
information with climate niche modelling and the archaeological ing a recovered taxon as authentic. A strict clean-lab protocol using
records it became evident that each species reacted individualisti- decontamination procedures and physical separation of labs for
cally to climate changes and that it is basically impossible to pre- and post-PCR work will significantly limit the contamination
12 P.F. Thomsen, E. Willerslev / Biological Conservation 183 (2015) 418
risks (Champlot et al., 2010; Willerslev and Cooper, 2005). Inclu- ity and quantification ability, but is hampered by the limit to detect
sion of DNA extraction blanks and PCR blanks, as well as field only one target organism at a time. For more diverse systems, this
blanks, to monitor contamination is essential. approach quickly becomes cost-inefficient and even impossible
due to lack of DNA extract for multiple reactions. Additionally,
4.2. Inhibition many issues remains for reliable quantification of biomass/individ-
uals by eDNA using qPCR. These include Cycle threshold (Ct) cut-
Humic acids or humic substances, co-extracted with DNA in off values and percentage of positive replicates for defining true
environmental samples, strongly inhibit enzymes such as the Taq positives from background, as well as proper treatment of negative
Polymerase used in PCR reactions to amplify DNA (Matheson qPCR replicates (Ellison et al., 2006; Bustin et al., 2009). A stan-
et al., 2010). This obviously represents a bias in eDNA studies, dardized number of qPCR replicates is also important to be used
which is probably most severe in soil samples, but also occurs in consistently throughout a study. Finally, development of more
water samples contaminated with sediment particles (McKee sophisticated modelling for describing DNA production and degra-
et al., 2015; Sigsgaard et al., 2015). If not addressed sufficiently dation should be undertaken. The above issues remains to be prop-
in each study, inhibition can lead to generation of false negative erly addressed in qPCR based eDNA studies including those from
results. Both false positive and false negative results can have con- our own group (Thomsen et al., 2012a, 2012b). Here, we did not
sequences for downstream conservation effort leading to over- or define strict Ct cut-off values or include non-detect (no Ct) repli-
under-estimation of a species occurrence, respectively. cates as zero in the calculations of average DNA concentrations,
which has been shown to increase accuracy and should be the
4.3. Errors practice (Ellison et al., 2006). Nevertheless, negative replicates
are frequent in eDNA studies where DNA concentration is often
Erroneous DNA sequences will, just as contamination, give rise low and the sample is a complex mixture of non-target DNA and
to biased results. Errors can occur either before sampling in long- environmental particles probably influencing oligo efficiency.
term preserved DNA (Hansen et al., 2006; Willerslev and Cooper, Thus, template amount is a trade-off between avoiding inhibition
2005), during PCR or during sequencing. PCR-generated errors and retaining a detectable concentration of target molecules. Fx.
include point mutations and formation of chimeric molecules Biggs et al. (2015) finds eDNA detection in 1/12 replicates for some
(Acinas et al., 2005). However, most errors are probably generated samples. Accordingly, some of the standard detection and quantifi-
during sequencing, and this has shown to overestimate actual cation thresholds defined in the literature might have to be relaxed
microbial diversity by two orders of magnitude (Kunin et al., for eDNA studies. If high Ct values are included, sequencing of the
2010). For animals, biodiversity signals are also obscured by PCR product for verification is crucial (Thomsen et al., 2012a, 2012b).
and/or sequencing errors (Sefc et al., 2007). Therefore, raw On the other hand, the DNA metabarcoding approach is power-
sequence data must be carefully filtered to limit false positives ful and cost-efficient (Thomsen et al., 2012a, 2012b; Yoccoz et al.,
and to generate a reliable taxon list (Fig. 1). There is obviously a 2012) and the technology for mass DNA sequencing continues to
trade-off between conservative error trimming, and retaining as improve (Schadt et al., 2010; Shokralla et al., 2012). The main
much information on diversity in the samples as possible (Coissac drawback, when using generic primers for metabarcoding, is pri-
et al., 2012). mer affinity bias leading to certain sequences (species) amplifying
less efficiently than others (Deagle et al., 2014). This will in turn
4.4. Reference DNA databases limit the results to species with best primer affinity or to species,
which is already known to be locally present so specific-primers
Identification of DNA sequences derived from environmental can be designed (Riaz et al., 2011; Wilcox et al., 2013). However,
samples depends crucially on reliable reference DNA-sequence such limitations will continuously become less crucial due to opti-
databases. These are skewed in geographical and taxonomic mization and publication of generic primers for metabarcoding
overage (Kvist, 2013), but global initiatives addressing this need studies.
have been established, and databases are rapidly growing High-throughput sequencing has also added another level of
(www.ncbi.nlm.nih.gov/genbank, www.boldsystems.org). Accord- complexity to the analyses of results, and the massive amount of
ingly, there are specific initiatives to provide DNA barcodes of sequences needs to be filtered in a conservative way to remove
all the worlds fish (www.fishbol.org), mammal (www. low abundant sequence reads that might originate from errors or
mammaliabol.org) and bird species, (www.barcodingbirds.org) chimeric sequences. Such denoising is crucial to discriminate
etc. It is clear, though, that the remaining gap in knowledge will authentic molecular diversity from errors and hence to give
for some time impair the usefulness of eDNA monitoring at lower accurate estimation of the biodiversity represented in the
taxonomic levels, where all species have not yet been sequenced. samples and a number of programs are available for sequence
However, the highly frequent COI sequences in these databases data e.g. Amplicon Noise (Quince et al., 2011). A range of tools
are sub-optimal for eDNA metabarcoding (Deagle et al., 2014). has been developed to aid processing of NGS data, such as OBITools
We thus suggest, given the massive increase in DNA sequencing (www.grenoble.prabi.fr/trac/OBITools) and QIIME (Caporaso et al.,
cost-efficiency, that future DNA reference databases focus on com- 2010). The translation of DNA sequence diversity obtained from
plete mitochondrial or even nuclear genomes for much wider metabarcoding of e.g. an environmental sample into actual
applications than traditional DNA barcoding. species richness and diversity is not straightforward. First, retrieved
sequences are clustered in MOTUs representing supposedly discrete
4.5. Single species detection vs. DNA metabarcoding taxons, however this delimitation is sometimes based on arbitrary
criteria (Coissac et al., 2012), and is unlikely to be standardized
Broadly, two different approaches of species detection from across taxa. When taxonomic coverage of the DNA database is
eDNA have been deployed: single species detection by PCR or low or non-existent, sequences will remain clustered into MOTUs.
quantitative PCR (qPCR) and multi-species detection (eDNA meta- The task of retrieving actual species names (important units of con-
barcoding) by NGS. The former is advantageous where the target is servation) from eDNA metabarcoding results are more challenging
one or a few known species, for which species-specific primers and due to: (i) limited knowledge of inter- and intra-specific sequence
probes can be developed (Dejean et al., 2012; Takahara et al., 2013; diversity of the targeted DNA-fragment and considerable gaps in
Thomsen et al., 2012b). The approach has high specificity, sensitiv- DNA sequence databases (Kvist, 2013), (ii) limited genetic variation
P.F. Thomsen, E. Willerslev / Biological Conservation 183 (2015) 418 13
of target genes of some taxa (Waugh, 2007) and (iii) the limitation et al., 2013). Transport of eDNA within an ecosystems remains an
of eDNA to short DNA sequences with poor taxonomic resolution. issue especially in flowing waters (Deiner and Altermatt, 2014;
Nevertheless, similar constraints are also associated with species Jane et al., 2014; Pilliod et al., 2013) and marine environments
identification using morphological characters. Progress are being (Thomsen et al., 2012a), where long-distance transport is possible.
made to standardize and improve the clustering and taxonomic
identification of sequences from metabarcoding studies (Jones 4.8. Skepticism
et al., 2011; Puillandre et al., 2012; Zhang et al., 2013). However,
for obtaining sheer estimates of diversity and richness based on The fate of eDNA-based monitoring of biodiversity relies heavily
sequence diversity, DNA metabarcoding can essentially be inde- on the validity of results from the scientists and other organiza-
pendent of DNA databases and clustering sequences into MOTUs tions applying the approach. Skepticism about the general
can provide estimates comparable between different localities or approach from conservation planners will appear, if false results
at different conditions within a locality. from scientists or companies are unintentionally published. Here,
See further discussion on bioinformatic methods and challenges there is a lesson to be learned from ancient DNA research, where
in Coissac et al. (2012). enthusiasm in the initial research phase generated spectacular
results that were later disproved. These include claims of DNA
4.6. Interpretation of results sequences surviving for millions of years in dinosaur bones
(Woodward et al., 1994) or amber inclusions (Cano et al., 1992).
Another important issue in eDNA studies concerns the critical However, it is the nature of any new research field to make mis-
interpretation of final results. Here, important caveats associated takes, which for ancient DNA meant the adoption of rigorous lab
with eDNA detection compared to traditional surveys includes procedures to avoid contamination and high standards for authen-
the failure to distinguish living vs. dead organisms, particular life tication of results. From this history a plea for strict general proto-
stages (eggs, juveniles, adults) and hybrid species. The latter is cols and authentication of results in conservation-related eDNA
linked to the dominant use of mitochondrial markers. Despite studies emerges. Similarly, the implementation of eDNA in conser-
advantages of being useful as DNA barcodes and plentiful in vation management remains a challenge and is subject of debate
excreted cells, mtDNA can only detect the maternal lineage of a (Darling and Mahon, 2011).
hybrid species. Finally, eDNA, like any other monitoring approach,
will only detect a proportion of the total sites occupied by a given 4.9. Future directions?
species. Using site occupancy models, Schmidt et al. (2013) dem-
onstrated the importance of using rigorous analyses of presence/ Moving from fundamental research to an applied tool in conser-
absence data to obtain more reliable estimates on species occu- vation, several aspects need to be more thoroughly investigated
pancy based on eDNA. Occupancy models can also be used to cal- before eDNA approaches can be fully matured and integrated into
culate the number of eDNA samples needed to reach a cumulative conservation. In this regard we suggest that future research on
detection chance of fx 95% (Schmidt et al., 2013). both terrestrial and aquatic ecosystems should focus on:
4.7. What is the temporal and spatial scale? (i) The temporal and spatial distribution of eDNA in different
habitats, which gives information as to what part of the bio-
For eDNA to be applicable in monitoring and conservation of diversity is monitored in space and time.
contemporary biodiversity, it is crucial that results reflect the pres- (ii) More precise links between eDNA concentration and species
ent state of an ecosystem. While studies of eDNA from water sam- abundance whether this is measured as total biomass or
ples have demonstrated rapid degradation times suggesting that density of individuals.
results are fairly consistent in space and time (Dejean et al., (iii) The exact sources of eDNA, whether this comes from
2011; Thomsen et al., 2012a, 2012b), eDNA in soil seems to be able sloughed epithelial cells, intestinal cells, faeces and urine,
to persist for decades or centuries (Andersen et al., 2012; Yoccoz etc., which might vary with life stages of the target organism
et al., 2012). This obviously questions the use of eDNA in soil from and could therefore greatly influence abundance estimates.
terrestrial ecosystems in a conservation context and necessitate Klymus et al. (2015) shows that an important source of
further studies. However, the potential release of ancient eDNA aquatic eDNA might be faeces as eDNA shedding rates
from bottom sediments to the water column (Turner et al. 2015) increase with feeding.
might also complicate the use of aquatic eDNA as strict contempo- (iv) Physio-chemical factors influencing eDNA availability and
rary biodiversity surveys. An extreme and highly intriguing exam- degradation such as temperature, pH and salinity. Barnes
ple of the borderline between ancient and contemporary eDNA et al. (2014) showed that a high degree of biological activity,
was recently demonstrated (Overballe-Petersen et al., 2013). Here measured as chlorophyll concentration, biological oxygen
the authors show experimentally that bacteria can take up ancient demand (BOD) and total eDNA, decreased the degradation
and damaged mammoth DNA down to just 20 bp by natural trans- rate of eDNA, perhaps due to shielding of eDNA from UV
formation. As a consequence, the diverse pool of DNA in the envi- radiation by algae. Furthermore, Strickler et al. (2015)
ronment could potentially have shaped the evolution of bacteria by explored the effect of various abiotic factors (temperature,
incorporation of this DNA into their genomes through the history UV-radiation and pH) on eDNA degradation, which indicated
of life on Earth. Furthermore, such transformed eDNA fragments that high DNA degradation rates were associated with con-
could even be re-deposited in the environment following cell- ditions favourable for microbial growth.
death, which could make eDNA fragments continuously available
thousands or millions of years after they were first shed from its Finally, we find it intriguing to explore the possibility of obtain-
original source. ing much longer fragments than the usually targeted 100200 bp,
Likewise, the spatial scale of eDNA monitoring is essential to target nuclear genes and test the eDNA metabarcoding approach
consider for any inferences on the proximity of target organisms more thoroughly in truly high-diverse ecosystems such as the tro-
compared to their DNA traces left behind. Transport of target eDNA pics. Such advances would lead the way for important conservation
from another locality by e.g. predators or man-made structures measures like species-level identification and population genetics
represents a potential, yet often insignificant possibility (Jerde using environmental samples.
14 P.F. Thomsen, E. Willerslev / Biological Conservation 183 (2015) 418
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