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Fishing for mammals: Landscape-level monitoring of terrestrial and semi-aquatic communities using eDNA from riverine systems

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Abstract

1. Environmental DNA (eDNA) metabarcoding has revolutionized biomonitoring in both marine and freshwater ecosystems. However, for semi-aquatic and terrestrial animals, the application of this technique remains relatively untested. 2. We first assess the efficiency of eDNA metabarcoding in detecting semi-aquatic and terrestrial mammals in natural lotic ecosystems in the UK by comparing sequence data recovered from water and sediment samples to the mammalian communities expected from historical data. Secondly, using occupancy modelling we compared the detection efficiency of eDNA metabarcoding to multiple conventional non-invasive survey methods (latrine surveys and camera trapping). 3. eDNA metabarcoding detected a large proportion of the expected mammalian community within each area. Common species in the areas were detected at the majority of sites. Several key species of conservation concern in the UK were detected by eDNA sampling in areas where authenticated records do not currently exist, but potential false positives were also identified. 4. Water-based eDNA metabarcoding provided comparable results to conventional survey methods in per unit of survey effort for three species (water vole, field vole and red deer) using occupancy models. The comparison between survey 'effort' to reach a detection probability of ≥.95 revealed that 3-6 water replicates would be equivalent to 3-5 latrine surveys and 5-30 weeks of single camera deployment, depending on the species.
J Appl Ecol. 2020;00:1–10. wileyonlinelibrary.com/journal/jpe
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  1© 2020 British Ecological Society
Received: 3 Octob er 2019 
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  Accepted: 21 January 2020
DOI : 10.1111/136 5-2664.13592
RESEARCH ARTICLE
Fishing for mammals: Landscape-level monitoring of terrestrial
and semi-aquatic communities using eDNA from riverine
systems
Naiara Guimarães Sales1| Maisie B. McKenzie1| Joseph Drake2|
Lynsey R. Harper3| Samuel S. Browett1| Ilaria Coscia1| Owen S. Wangensteen4|
Charles Baillie1| Emma Bryce5| Deborah A. Dawson6| Erinma Ochu1|
Bernd Hänfling3| Lori Lawson Handley3| Stefano Mariani1,7 | Xavier Lambin5|
Christopher Sutherland2,8 | Allan D. McDevitt1
1Environm ent and Ecosyste m Research Centre, Scho ol of Science, Engi neeri ng and Environment, Universit y of Salfo rd, Salford, UK ; 2Department of
Environmental C onser vatio n, Unive rsity of Massachuset ts-Am herst, Amherst, USA; 3Department of Bio logical and Marine Sciences, U niversity of Hu ll,
Kings ton upon H ull, UK ; 4Norwegian College of Fis hery Science , Univer sity of Troms ø, Tromsø, Nor way; 5School of Biologic al Sciences, University of Aberdeen,
Aberdeen, UK; 6Department of Animal an d Plant Sciences , Univer sity of S heff ield, Sheffield, UK; 7School of Natur al Sciences and Psychology, Liverpool John
Moores University, Liver pool, UK and 8Centre for Research into Ecological an d Environ mental Modelling, University of St Andrews, St Andrews, UK
Naiara G uimarães Sa les, Ma isie B. McKenzie an d Joseph Drake co ntributed eq ually to t his work.
Correspondence
Allan D. McDevitt
Email: a.mcdevitt@salford.ac.uk
Funding information
British Ecological Society, Grant/Award
Number : SR17/1214; University of Salfor d;
University of Massachusetts
Handling Editor: Brittany Mosher
Abstract
1. Environmental DNA (eDNA) metabarcoding has revolutionized biomonitoring in
both marine and freshwater ecosystems. However, for semi-aquatic and terres-
trial animals, the application of this technique remains relatively untested.
2. We first assess the efficiency of eDNA metabarcoding in detecting semi-aquatic
and terrestrial mammals in natural lotic ecosystems in the UK by comparing
sequence data recovered from water and sediment samples to the mammalian
communities expected from historical data. Secondly, using occupancy mod-
elling we compared the detection efficiency of eDNA metabarcoding to mul-
tiple conventional non-invasive survey methods (latrine surveys and camera
trapping).
3. eDNA metabarcoding detected a large proportion of the expected mammalian
community within each area. Common species in the areas were detected at the
majority of sites. Several key species of conservation concern in the UK were de-
tected by eDNA sampling in areas where authenticated records do not currently
exist, but potential false positives were also identified.
4. Water-based eDNA metabarcoding provided comparable results to conventional
survey methods in per unit of survey effort for three species (water vole, field vole
and red deer) using occupancy models. The comparison between survey ‘effort’ to
reach a detection probability of ≥.95 revealed that 3–6 water replicates would be
equivalent to 3–5 latrine surveys and 5–30 weeks of single camera deployment,
depending on the species.
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1 | INTRODUCTION
Environmental DNA (eDNA) metabarcoding (the simultaneous iden-
tification of multiple taxa using DNA extracted from an environmen-
tal sample, e.g. water, soil, based on short amplicon sequences) has
revolutionized the way we approach biodiversity monitoring in both
marine and freshwater ecosystems (Deiner et al., 2017; Valentini
et al., 2016). Successful applications include tracking biological in-
vasions, detecting rare and endangered species and describing en-
tire communities (Holman et al., 2019). Most eDNA metabarcoding
applications on vertebrates to date have focused on monitoring
fishes and amphibians (Hänfling et al., 2016; Valentini et al., 2016).
What has become apparent from studies in lentic systems (ponds
and lakes) is that semi-aquatic and terrestrial mammals can also be
detected (Hänfling et al., 2016; Harper et al., 2019). As a result, there
has been an increasing focus on the use of both vertebrate (Harper
et al., 2019) and mammal-specific primer sets (Leempoel, Herbert, &
Hadly, 2020; Sal es, Kaizer, et al., 2020; Ushio et al., 2017) for detect-
ing mammalian communities using eDNA metabarcoding.
Mammals include some of the most imperiled taxa, with over
one-fifth of species considered to be threatened or declining
(Visconti et al., 2011). Monitoring of mammalian biodiversit y is
therefore essential. Given that any optimal survey approach is likely
to be species-specific, ver y few species can be detected at all times
when they are present. This imperfect detection (even greater for
elusive and rare species) can lead to biased estimates of occurrence
and hinder species conservation (Mackenzie et al., 2002). For mam-
mals, repeated sur veys using several monitoring methods are usually
applied. These include indirect observations such as latrines, faeces,
hair or tracks, or direct observations such as live-trapping or cam-
era trapping sur veys over short time intervals such that closure/in-
variance can be assumed and detectability estimated (Nichols et al.,
2008). Each of these methods has associated efficiency, cost and
required expertise trade-offs, which become more challenging as
the spatial and temporal scales increase.
Environmental DNA sampling yields species-specific presence/
absence data that are likely to be most valuable for inferring spe-
cies distributions using well-established analytical tools such as
occupancy models (MacKenzie et al., 2002). These models resolve
concerns around imperfect detection of difficult to obser ve species.
When coupled with location-specific detec tion histories, these can
be used to infer true occurrence states, factors that influence oc-
cupancy rates, colonization-extinction probabilities and estimates
of detection probability (MacKenzie et al., 2017). The use of eDNA
sampling to generate species-specific detection data has unsurpris-
ingly increased in recent years, and in many cases has outperformed
or at least matched conventional survey methods (Lugg, Griffiths,
van Rooyen, Weeks, & Tingley, 2018; Tingley, Greenlees, Oertel,
van Rooyen, & Weeks, 2019). Although comparisons between eDNA
analysis and conventional surveys for multi-species detection are
numerous (see table S1 in Lugg et al., 2018), studies focusing on de-
tection probability estimates for multiple species identified by me-
tabarcoding are rare (Abrams et al., 2019; Valentini et al., 2016).
The aim of this study was to assess the efficiency of eDNA me-
tabarcoding for detecting semi-aquatic and terrestrial mammals in
natural lotic systems in the UK. We conducted eDNA sampling in
rivers and streams in two areas (Assynt, Scotland and Peak District
National Park, England). Together these locations have the major-
ity of UK semi-aquatic and terrestrial mammalian species present
(Table S1). Our objectives were twofold: first, we sought to estab-
lish whether eDNA metabarcoding is a viable technique for moni-
toring semi-aquatic and terrestrial mammals by comparing it to the
mammalian communities expected from historical data, a group for
which eDNA sampling has rarely been evaluated in a natural set-
ting. Secondly, we evaluate the detection ef ficiency of water- and
sediment-based eDNA sampling in one of these areas (Assynt) for
multiple species compared to multiple conventional non-invasive
survey methods (latrine surveys and camera trapping).
2 | MATERIALS AND METHODS
2.1 | Latrine surveys
Assynt, a heather-dominated upland landscape in the far north-
west of the Scottish Highlands, UK (Figure 1a), is the location of
5. Synthesis and applications. eDNA metabarcoding can be used to generate an initial
‘distribution map’ of mammalian diversity at the landscape level. If conducted dur-
ing times of peak abundance, carefully chosen sampling points along multiple river
courses provide a reliable snapshot of the species that are present in a catchment
area. In order to fully capture solitary, rare and invasive species, we would cur-
rently recommend the use of eDNA metabarcoding alongside other non-invasive
surveying methods (i.e. camera traps) to maximize monitoring efforts.
KEY WORDS
biomonitoring, camera trapping, eDNA metabarcoding, latrine surveys, mammals, occupancy
modelling, rivers
  
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an ongoing 20 -year met apopulation study of water voles Arvicola
amphibius led by the University of Aberdeen (Figure S1). Here, we
mainly focus only on data collected in 2017. The metapopulation is
characterized by 116 discrete linear riparian habitat patches (ranging
from 90 m to nearly 2.5 km) distributed sparsely (4% of waterway
network) throughout the 140 km2 study area (Sutherland, Elston, &
Lambin, 2014). Water voles use promin ently placed latrines for terri-
tory marking (Figure S2a). Using latrine surveys, a reliable method of
detection (Sutherland et al., 2014), water vole occupancy status was
determined by the detection of latrines that are used for territory
marking (Sutherland, Elston, & Lambin, 2013). During the breed-
ing season (July and August), latrine surveys were conducted twice
at each site. In addition to water vole latrines, field vole Microtus
agrestis pellets are also easily identifiable, and so field vole detec-
tions were also recorded along waterways as a formal part of the la-
trine survey protocol. Live-trapping was then carried out at patches
deemed to be occupied by water voles according to latrine surveys
to determine their abundances (this was used to determine which
sites were sampled for eDNA; Figure 1a).
2.2 | Camera trap data
Camera traps were deployed at the beginning of July and thus over-
lapped temporally with the latrine survey in Assynt. Data were col-
lected from cameras deployed at seven of these patches. Within
each of these patches, cameras were deployed at the midpoint of
the areas where active signs (latrines, grass clipping, burrows) were
detected, and if no signs were detected, at the midpoint of historical
water vole activity (J. Drake, C. Sutherland and X. Lambin, pers.
comm.). These will also capture images of any species present in the
area that come within close proximity of the camera (Figure S3a–f).
Camer as were deployed app roximately 1 m above-gro und on iron
‘u-posts’ to avoid flooding, prevent knock-down by wind/wildlife and
optimize both depth of field and image clarity. Cameras (Bushnell
HD Trophy Cam) were set at normal detection sensitivity (to reduce
false-triggers from grass/shadows), low night time LED intensity (to
prevent image white out in near depth of field), three shot burst
(to increase chance of capturing small, fast moving bodies) and 15-
min intervals between bursts (to increase temporal independence
of captures and decrease memory burden). The area each camera
photographed was approximately 12 m2. Animals were identified
on images and information was stored as metadata tags using the
r (R Core Team, 2018) package cam trapr following the procedures
described in Niedballa, Courtiol, and Sollmann (2018). Independence
between detections was based on 60 -min intervals between species-
specific detections.
2.3 | eDNA sampling
A total of 18 potential water vole patches were selected for eDNA
sampling in Assynt from 25 to 27 October 2017. The time lag be-
tween the latrine/live-trapping and eDNA surveys was because of
two main reasons: (a) legitimate concerns around cross-site DNA
contamination during latrine/live-trapping where researchers moved
on a daily basis between sites as well as regularly handled and pro-
cessed live animals (for decontamination procedures see Supporting
FIGURE 1 (a) Environmental DNA (eDNA) sampling sites in A ssynt, Scotland; the size of sites corresponds to abundance categories based
on summer live-trapping. (b) A bubble graph representing presence/absence and categorical values of the number of reads retained (after
bioinformatic filtering) for eDNA (water in blue and sediment in orange) from each wild mammal identified in each site in Assynt (A1–A18)
(a) (b)
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Information) and (b) the selec tion of eDNA sampling sites was based
on the latrine surveys and abundance data provided by live-trapping
so could only occur af ter this was completed by August 6th. Water
and sediment samples were collected from patches where water
voles were determined to be absent (five sites; A1–A5); with 1–2 in-
dividuals present (three sites; A9, A16 and 18); 3–5 individuals (five
sites; A6, A8, A11, A14 and A17); and 711 individuals (five sites;
A7, A10, A12, A13 and A15; Figure 1a). Each of these streams/rivers
differed in their characteristics (in terms of width, depth and flow)
and a representation of the sites is depicted in Figure S4a–d. Three
water (two litres each) and three sediment (~25 ml) replicates were
taken at each patch (further details of sample collection are provided
in Appendix S1).
In addition to Assynt, eDNA sampling was also conducted on a
smaller scale in the Peak District National Park, England (Figure S5)
to incorporate additional mammals that are not known to be present
in Assynt ( Table S1). Here, the occurrence of water vole was identi-
fied by the presence of latrines in two sites (P1 and P2) at the time of
eDNA sampling (Figure S2a), whilst no latrines were identified at one
site (P3). At site P1, an otter Lutra lutra spraint was identified at the
time of eDNA sampling (Figure S2b). These three sites were sampled
in March 2018 using the same methodology as in Assynt but were
taken in close proximity (<50 cm) to water vole latrines where pres-
ent (Figure S2a).
2.4 | eDNA laboratory methods
DNA was extracted from the sediment samples using the DNeasy
PowerMax Soil kit and from the water samples using the DNeasy
PowerWater Kit (both QIAGEN Ltd.) following the manufacturer's
instructions in a dedicated eDNA laboratory in the University of
Salford. In order to avoid the risk of contamination during this step,
DNA extraction was conducted in increasing order of expected
abundance of water voles in the eDNA samples (all field blanks
were extracted first, followed by the sites with supposedly zero
water vole abundance, up to the highest densities last). Along with
field blanks (Assynt = 8, Peak District = 2), six lab extraction blanks
were included (one at the end of each daily block of extractions).
A decontamination stage using a Phileas 25 Airborne Disinfection
Unit (Devea SAS) was undertaken before processing samples from
different locations. Additional information regarding decontamina-
tion measures and negative controls can be found in the Supporting
Information.
A complete description of PCR conditions, library preparation
and bioinformatic analyses is provided in Appendix S1. Briefly, eDNA
was amplified using the MiMammal 12S primer set (MiMammal-U-F,
5′-GGGTTGGTAAATTTCGTGCCAGC-3′; MiMammal-U-R, 5-CATA
GTGGGGTATCTAATCCCAGTTTG-3′; Ushio et al., 2017) targeting
a ~170 bp amplicon from a variable region of the 12S rRNA mito-
chondrial gene. A total of 147 samples, including field collection
blanks (10) and laboratory negative controls (12, including six DNA
extraction blanks and six PCR negative controls), were sequenced
in two multiplexed Illumina MiSeq runs. To minimize bias in individ-
ual reactions, PCRs were replicated three times for each sample and
subsequently pooled. Illumina libraries were built using a NextFlex
PCR-free library preparation kit according to the manufacturer's
protocols (Bioo Scientific) and pooled in equimolar concentrations
along with 1% PhiX (v3, Illumina). The libraries were run at a final
molarit y of 9 pM on an Illumina MiSeq platform using the 2 × 150 bp
v2 chemistry.
Bioinformatic analysis was conducted using OBItOOls metabar-
coding package (Boyer et al., 2016) and the taxonomic assignment
was conduc ted using ecotag against a custom reference database
(see Appendix S1). To exclude MOTUs/reads putatively belonging to
sequencing errors or contamination, the final dataset included only
MOTUs that could be identified to species level (>98%), and MOTUs
cont aining <10 reads and with a similarity to a sequence in the refer-
ence database lower than 98% were discarded (Cilleros et al., 2019).
The maximum number of reads detected in the controls for each
MOTU in each sequencing run was removed from all samples (Table
S7). For water voles, field voles and red deer (the most abundant wild
mammals in terms of sequence reads in our dataset), this equated
to a sequence frequency threshold of ≤0.17%, within the bounds
of previous studies on removing sequences to account for contam-
ination and tag jumping (Cilleros et al., 2019; Hänfling et al., 2016;
Schnell, Bohmann, & Gilbert, 2015).
2.5 | Occupancy/detection analysis in Assynt
The data collection from the different sur vey types described above
(water-based eDNA, sediment-based eDNA, latrine and camera
traps) produced the following site-specific detection/non-detection
data:
1. Latrine: two latrine surveys at 116 patches.
2. w-eDNA: three water-based eDNA samples at 18 of the 116
patches surveyed.
3. s-eDNA: three sediment-based eDNA samples at 18 of the 116
patches surveyed.
4. Camera: six 1-week occasions of camera trapping data at seven of
the 18 patches sur veyed by both Latrine and eDNA (w-eDNA +
s-eDNA) surveys.
We chose to focus on three species that were detected by at
least three of the four methods: water voles, field voles and red
deer Cervus elaphus. Water voles and field voles were recorded
using all four survey methods and had detection histories for 14
surveying events ((Latrine × 2) + (w-eDNA × 3) + (s-eDNA × 3) +
( C am e ra × 6) ). Re d d e er w e r e no t r e co rd ed d u r in g l a tr i n e su r ve ys a n d
had detection histories for 12 surveying events ((w-eDNA × 3) +
(s-eDNA × 3) + (Camera × 6)). To demonstrate the relative effi-
cacy of the four sur veying methods, we restricted the analyses
to the 18 sites where both latrine surveys were conducted and
eDNA samples were taken, seven of which had associated camera
  
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trapping data. Although each surveying method differs in terms
of effort and effective area sur veyed, each is a viable surveying
method that is readily applied in practice. A unit of survey effort
here is defined as one latrine survey, one w-eDNA replicate, one
s-eDNA replicate or 1 week of c amera tra pping. So, while the spe-
cific units of effort are not directly comparable, the relative de-
tection efficacy per surveying method-specific unit of effort is of
interest and will provide important context for designing future
monitoring studies and understanding the relative merits of each
surveying method. Analysing the data using occupancy models al-
lowing for method-specific detectability enables such a compari-
son in per unit effort efficacy between eDNA metabarcoding and
multiple conventional survey methods.
A single season occupancy model (MacKenzie et al., 2002) was
applied to the ensemble data where detection histories were con-
structed using each of the surveying events as sampling occasions
(MacKenzie et al., 2017). The core assumption here is that the un-
derlying occupancy state (i.e. occupied or empty) is constant over
the sampling period, and therefore, every sampling occasion is a
potentially imper fect observation of the true occupancy status.
Because occasions represent method-specific surveying events,
we used ‘surveying method’ as an occasion-specific covariate on
detection (Latrine, w-eDNA, s-eDNA and Camera). Our primary
objective was to quantify and compare method-specific detect-
ability, so we did not consider any other competing models. For
comparing the methods, we compute accumulation curves as
(MacKenzie & Royle, 20 05):
where
p
smk
is the cumulative probability of detecting species s, when
species s is present, using method m after k sur veying events based
on the estimated surveying method-specific detection probability for
each species (
̂
psm
). We vary k from 1 to a large number and find the
value of k that results
p
smk
≥ .95. We con ducted the same analysis sepa-
rately for water voles, field vole s and red deer. An alysis was conducted
in r (R Core Team, 2018) usi ng the package unmarked (Fis ke & Chandler,
2011).
3 | RESULTS
3.1 | Mammal detection via eDNA metabarcoding
The two sequencing runs generated 23,276,596 raw sequence
reads and a total of 15,463,404 sequences remained following
trimming, merging and length filtering. After bioinformatic analy-
sis, the final ‘filtered dataset contained 23 mammals (Tables S2
and S3). For mammals, ~12 million reads were retained after ap-
plying all quality filtering steps (see Appendix S1). Reads from hu-
mans, cat tle Bos taurus, pig Sus scrofa, horse Equus ferus, sheep
Ovis aries and dog Canis lupus familiaris, were not considered fur-
ther as the focus of this study was on wild mammals (Table S4).
Felis was included because of the potential of it being wildcat Felis
silvestris or domestic cat F. catus/wildcat hybrids. A final dataset
comprising ~5.9 million reads was used for the downstream analy-
ses (Table S4).
In Assynt, the wild species identified were the red deer (18/18
sites); water vole (15/18); field vole (13/18); wood mouse Apodemus
sylvaticus—9/18; pygmy shrew Sorex minutus—4/18; wild/domes-
tic cat Felis spp.—4/18; mountain hare Lepus timidus—4/18; rabbit
Oryctolagus cuniculus—3/18; water shrew Neomys fodiens—3/18;
common shrew Sorex araneus2/18; edible dormouse Glis glis
2/18; grey squirrel Sciurus carolinensis1/18; pine marten Martes
martes—1/18; brown rat Rattus norvegicus1/18; red fox Vulpes
vulpes1/18 and badger Meles meles—1/18 (Figure 1b). All of these
species are distributed around/within Assynt (Table S1), with the
exception of the edible dormouse and the grey squirrel. These are
unequivocally absent from the region. The edible dormouse is only
present in southern England and the grey squirrel is not distributed
that far north in Scotland (Mathews et al., 2018).
Of the wild mammals in the Peak District, the water vole, field
vole, wood mouse and otter were found in two sites (P1 and P2).
The red deer, pygmy shrew, common shrew, water shrew, red squir-
rel Sciurus vulgaris, grey squirrel, pine marten and badger were each
found at a single site (Figure S5). Only rabbit was found in site P3. All
species identified are currently distributed within the Park (Table S1),
except the red squirrel and pine marten. The pine marten, which is
critically endangered in England, has only two reliable records that
have been confirmed in the Park since 2000 and the red squirrel
has not been present for over 18 years (Alston, Mallon, & Whiteley,
2012).
Overall, water samples yielded better results than sediment
samples regarding species detection and read count for both areas
sampled (Figure 1b; Figure S5). In Assynt, only the wild/domestic cat
was exclusively detected in sediment samples (four sites), whereas
water samples recovered eDNA for ten additional species not found
in the sediment samples. The red deer, water vole, field vole, moun-
tain hare and pygmy shrew were also found in sediment samples
in Assynt (Figure 1b), and water vole and wood mouse in the Peak
District sediment samples (Figure S5).
3.2 | Occupancy analysis
Of the 18 sites where both latrine and eDNA surveys were con-
ducted, water voles were detected at 13 and field voles were de-
tected at 11. A total of seven wild mammals were recorded at the
seven sites with a camera trap from 10 July to 25 October 2017
(Figure S3; Table S5). There were several incidences where a shrew
could not be identified to species level using camera traps. For cam-
era traps, water voles were recorded at all sites, red deer at five out
of seven, field voles and weasels at three sites, water shrews and
otters at two and a red fox at a single site.
For the 18 sites in Assynt, estimated site occupancy (with 95%
confidence intervals) from the combined surveying methods was
p
smk
=1(1
̂
p
sm
)
k,
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SALES E t AL.
0.91 (0.63–0.98) for water voles and 0.88 (0.57–0.98) for field
voles. Red deer were observed at every patch by at least one of
the methods, and therefore occupancy was 1 (Table 1). For all three
species, per sample detection probability was higher for eDNA
taken from water than for eDNA taken from sediment (Table 1;
Figure 2). The surveying method-specific efficacy pattern was sim-
ilar for water voles and field voles (Table 1; Figure 2): latrine sur-
veys had the highest probability of detecting the species (.77 and
.52 respectively), followed by eDNA from water (.57 and .40 re-
spectively), then camera trapping (.50 and .20 respectively) and
finally eDNA from sediment (.27 and .02 respectively). Detection
probability was higher for water voles than field voles using all four
methods (Table 1; Figure 2). No effort was made to record red deer
pr ese nce duri ng la tri n e sur veys . Lik e the wate r vol es an d fiel d vol es,
red deer detection was higher using eDNA from water (0.67, CI:
0.53–0.78) compared to eDNA from sediment (0.10, CI: 0.04–0.21).
Un li ke th e vol es, wh ich were more dete c tab l e by ca mer a s tha n sed i-
ment eDNA, red deer detection on cameras was similar to sediment
eDNA (0.10, CI: 0.04–0.24).
The patterns described above detail surveying event-specific de-
tectability. We also computed the cumulative detection probabilit y
for each method and each species (
̂
psm
). The cumulative detection
curves over 15 surveying events are shown in Figure 2. The num-
ber of surveying event s, k, required to achieve
p
psm
≥ .95 for water
voles was three surveys, four samples, 10 samples and 5 weeks, for
latrines, water eDNA, sediment eDNA and cameras respectively.
The number of surveying events, k, required to achieve
p
psm
≥ .95 for
field voles was five sur veys, six samples, 141 samples and 14 weeks,
for latrines, water eDNA, sediment eDNA and cameras respectively.
The number of surveying events, k, required to achieve
p
psm
≥ .95 for
red deer was three samples, 30 samples and 29 weeks, for water
eDNA, sediment eDNA and cameras respectively (see also Figure 2).
TABLE 1 Estimated site occupancies and detection probabilities, with associated 95% confidence inter vals in brackets, obtained for
water-based eDNA (w-eDNA), sediment-based eDNA (s-eDNA) and conventional survey methods (Latrine and Camera) in Assynt, Scotland
Species Occupancy
Detection probability
Latrine w-eDNA s-eDNA Camera
Water vole 0.91 (0.63–0.98) 0.77 (0.59–0.89) 0.57 (0.43–0.71) 0.27 (0.16–0.41) 0.50 (0.35–0.65)
Field vole 0.89 (0.57–0.98) 0.52 (0.34–0.69) 0.40 (0.26–0.55) 0.02 (0.00–0.14) 0.20 (0.10–0.37)
Red deer 1.00 (1.00–1.00) 0.67 (0.53–0.78) 0.10 (0.04–0.21) 0.10 (0.09–0.24)
FIGURE 2 Figures on the left show
estimated detection probabilities of
each sur vey method for each of three
focal species; the vertical lines are 95%
confidence intervals. Figures on the right
show the method- and species-specific
cumulative detection probability with
increasing number of sampling events; the
horizontal dashed line shows a probability
of .95 for reference
Red deer
Field vole
SedimentWater Latrine Camera
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
Red deer
Field vole
4812
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
Method
Sediment
Water
Latrine
Camera
  
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 7
Journal of Applied Ecolog
y
SALES E t AL.
4 | DISCUSSION
Despite the increasing potential of eDNA metabarcoding as a bio-
monitoring tool (Deiner et al., 2017), its application has largely been
focused on strictly aquatic or semi-aquatic animals, thus restrict-
ing management and conservation efforts of the wider ecosystem
(Williams, Huyvaert, Vercauteren, Davis, & Piaggio, 2018). Here, we
demonstrate the ability of eDNA metabarcoding to provide a valu-
able ‘terre strial divide nd’ for mammals from freshwater lotic ecosys-
tems, with a large proportion of the expected species from the wider
landscape being detected in each of the two study locations. In par-
ticular, we have demonstrated that water-based eDNA sampling
offers a promising and complementary tool to conventional survey
methods for the detection of whole mammalian communities.
4.1 | Detection of mammalian communities using
eDNA metabarcoding
Of the species known to be common in both Assynt and the Peak
District, eDNA metabarcoding readily detected the water vole,
field vole and red deer at the majority of sites surveyed (Figure 1b;
Figure S5). Pygmy, common and water shrews, wood mice and moun-
tain hares were also detected by eDNA metabarcoding at multiple
sites in Assynt (Figure 1b). A higher eDNA detection rate is expected
for aquatic and semi-aquatic mammals compared to terrestrial mam-
mals in aquatic environments due to the spatial and temporal stochas-
ticity of opportunities for terrestrial mammals to be in contact with
the water (Ushio et al., 2017). The semi-aquatic water vole was gen-
erally detected by eDNA metabarcoding where we expected to find
it and at relatively high read numbers (Figure 1b; Figures S1 and S5).
This is in line with previous studies in lentic systems (Harper et al.,
2019). However, the red deer was the only terrestrial species de-
tected by eDNA sampling at all sites in Assynt, and the terrestrial
field vole at over 70% of surveyed sites.
In addition to lifestyle (semi-aquatic or terrestrial), the number
of individuals of each species (i.e. group-living) may be important
for eDNA detection (Williams et al., 2018). As a counter example
to this, otters and weasels were notably absent in the eDNA sam-
ples in Assynt despite being captured by camera traps (Figure S3;
Table S5). Otters were present in the water eDNA samples at two
sites in the Peak District, albeit at a lower number of reads in com-
parison to most of the other species detected (Figure S5; Table S2).
This mirrors previous studies where eDNA analysis has performed
relatively poorly for otter detection in captivity and the wild (Harper
et al., 2019; Thomsen et al., 2012). Carnivores were generally de-
tected on fewer occasions (e.g. red foxes, badgers and pine mar tens;
Figure 1b; Figure S5) or not at all (e.g. stoats and American mink in
addition to those discussed above) in comparison to smaller mam-
mals and red deer, and a similar pattern has been shown with North
American carnivores in a recent study using eDNA from soil samples
(Leempoel et al., 2020). For some of these species, species ecology/
behaviour such as a relatively large home range and more solitary
nature (e.g. red foxes) may go some way towards explaining a lack of,
or few, eDNA records. Furthermore, as demonstrated by Ushio et al.
(2017) poor efficiency for amplifying some mammal species might be
associated to suboptimal experimental conditions (e.g. inadequate
primer design, primer bias, DNA concentration, species masking
and/or annealing temperatures).
Regarding the sampling medium for eDNA, we demonstrated
that water is a more effective method for detection of mammal
eDNA than sediment (Table 1; Figure 1b; Figure S5). For one of our
foc al species, the water vole, 75% of site s which were deemed un oc-
cupied by latrine surveys and those with ≤2 individuals (eight sites) in
Assynt, returned a non-detection for sediment eDNA as opposed to
37.5% of sites for wate r (F igu re 1a,b ; Fi gur e S1) . Disti nct temp ora l in -
ferences are provided by eDNA recovered from water and sediment
samples. DNA bound to sediments can remain detectable for a lon-
ger period (i.e. up to hundreds of years) and provide historical data,
whereas, eDNA retrieved from water samples provide more contem-
porary data due to a faster degradation in the water column (Turner,
Uy, & Everhart, 2015). It is worth investigating further if sediment
eDNA could indicate the presence of a more ‘established’ popula-
tion, where a cert ain threshold of individuals and long-term occupa-
tion (i.e. historical) is required for detection in sediment (Figure S1;
Leempoel et al., 2020; Turner et al., 2015).
Importantly, sparse or single eDNA records should be carefully
verified. The edible dormouse and grey squirrel sequences identi-
fied within the Assynt samples (Figure 1b) and red squirrel within the
Peak District (Figure S5) highlight the caveats associated with this
technique. If management decisions had relied on eDNA evidence
alone, false positives for these species could lead to unnecessary
resources being allocated for management/eradication programmes
as the edible dormouse and grey squirrel are classified as invasive
species within Great Britain. These potentially arose due to sample
carryover from a previous sequencing run on the same instrument
(a known issue with Illumina sequencing platforms; Nelson, Morrison,
Benjamino, Grim, & Graf, 2014) which included those species for the
reference database construction. Controlling for false positives is
certainly a huge challenge in eDNA metabarcoding and the need
to standardize and optimize thresholds for doing so is an ongoing
debate (Ficetola et al., 2015; Harper et al., 2019).
Even with these concerns around false positives highlighted, two
records are potentially notewor thy in a conservation contex t for UK
mammals because of the relatively high read number associated with
these records (Tables S2 and S3). The first of these is the Felis records
in sediment samples in multiple sites in Assynt (Figure 1b). Even with
a ‘pure’ F. silvestris as a reference sequence, it was not possible to
distinguish between the wild and domesticated species for this 12S
fragment (data not shown). Despite ongoing conservation efforts,
there may now be no ‘pure’ Scottish wildcats left in the wild in the UK
but isol at ed popu la tions (pe rhaps of hybr id origin) may exis t in this re-
gion (Sainsbury et al., 2019). Given that these eDNA detections were
all from sediment samples, it is possible that they may be historical
rather than contemporary (see above). The other significant eDNA
record was the pine mar ten in the Peak District. The pine mar ten
8 
|
  
Journal of Applied Ecology
SALES E t AL.
Mar tes martes is know n to occur in the Scot tis h Hi ghlan ds bu t ha d dis-
appeared from most of the UK and recently has been recovering from
historical persecution, including a potential expansion of its range.
Still, authentic records from northern England are scarce or lacking
altogether (Alston et al., 2012; Sainsbury et al., 2019). However, a
record of a recent roadkill exists from just outside the Park's bound-
ary (BBC News, 2018). The high number of reads recovered for the
Peak District sample (4,293 reads vs. 25 in the Assynt sample) adds
credence to this positive eDNA detection but further investigations
are warranted into the potential presence of this species in the area.
4.2 | Comparisons between surveying methods
Comparisons of species detection by traditional survey approaches and
eDNA analysis are now numerous in the literature, and mainly focus on
what is and what is not detected within and across different methods
(Hänfling et al., 2016; Leempoel et al., 2020). Yet, there has been grow-
ing incorporation of occupancy modelling to estimate the probability of
detecting the focal species, in comparison to one other survey method,
either for a single species (Lugg et al., 2018; Tingley et al., 2019) or mul-
tiple species (Abrams et al., 2019; Valentini et al., 2016). Simultaneous
multi-method comparisons for multiple species have been lacking and
this study directly addresses this for the first time.
The probability of detecting the water vole and field vole was
higher for the latrine surveys than eDNA sampling (both water and
sediment) and camera traps (Table 1; Figure 2). However, when consid-
ering confidence intervals, there was considerable overlap between
latrine, water-based eDNA metabarcoding and camera traps for both
species, with only sediment-based eDNA metabarcoding yielding
a low probability of detection (Table 1). Detection probabilities for
water-based eDNA metabarcoding and camera traps were similar for
water voles, with camera tr aps less likely to detec t the field vole than
water-based eDNA . For the red deer (for which no latrine survey was
undertaken), water-based eDNA metabarcoding had a much higher
probability of detection than either sediment-based eDNA metabar-
coding or camera traps (which performed similarly; Table 1). Despite
the increasing adoption of camera traps in providing non-invasive de-
tections for mammals (Hofmeester et al., 2019), camera traps were
outperformed by water-based eDNA metabarcoding for the three
focal species in this component of the study. Here, camera traps were
deployed so as to sample the habitat of the water vole (see Figure
S3), which may explain lower detection for other terrestrial species
in comparison to eDNA metabarcoding (see above). Studies focusing
on a single species often repor t that eDNA analysis outperforms the
conventional survey method in terms of detec tion probabilities (e.g.
Lugg et al., 2018). For metabarcoding, there is clearly a need to care-
fully consider the potential for cross contamination between samples
an d ho w fal se posi tives (an d neg at ive s) cou ld im pac t det ect ion pro ba-
bilities using occupancy modelling with eDNA data (Brost, Mosher, &
Davenport, 2018; Lahoz-Monfort, Guillera-Arroita, & Tingley, 2016).
Amon g th e re co mm endations mad e by Lahoz-Monfort et al. (2 016) to
account for these uncertainties, one was the simultaneous collection
of data from more conventional sur veying methods. Here, we have
demonstrated general congruence between surveying methods for
the water vole (Table S5; Figure S1) and using certain sp ecies to app ly
a multiple detection methods model would be appropriate in further
studies (Lahoz-Monfort et al., 2016). Alternatively, using repeated
samplin g and known neg at ive controls in oc cupanc y models that ful ly
incorporate false-positive errors could be applied in the absence of
other surveying data (Brost et al., 2018). Overall, multi-species me-
tabarcoding studies may trade-off a slightly lower (but comparable)
detection probability than other survey methods for individual spe-
cies (Figure 2) in favour of a better overall ‘snapshot’ of occupancy of
the whole mammalian community (Ushio et al., 2017).
The comparison between survey ‘effort’ for the four methods
to reach a probability of detection of ≥.95 is highly informative
and provides a blueprint for future studies on mammal monitoring.
Focusing on the water vole for example, three latrine surveys would
be required. A total of four water-based and 10 sediment-based
eDNA replicates or 5 weeks of camera trapping would be required
to achieve the same result (Figure 2). This increases for the field vole
in the same habitat, with five latrine surveys and six water-based
eDNA replicates. Sediment-based eDNA metabarcoding would
be impractical for this species and camera trapping would take
14 weeks. What is important here is the spatial component and the
amount of effort involved in the field. Taking 4–6 water-based eDNA
replicates from around one location within a patch could provide
the same probability of detecting these small mammals with three
latrine surveys. In many river catchments, there may be 100 s to
1,000s of kilometres to survey that would represent suitable habi-
tat, and only a fraction of that may be occupied by any given species.
This is par ticularly relevant in the context of recovery of water vole
populations post-translocation or in situations where remnant pop-
ulations are bouncing back after invasive American mink Neovison
vison control has been instigated. On a local scale, finding signs of
water voles through latrine surveys is not necessarily dif ficult, but
monitoring the amount of potential habitat (especially lowland) for a
species which has undergone such a massive decline nationally is a
huge undertaking (Morgan, Cornulier, & Lambin, 2019).
The use of eDNA metabarcoding from freshwater systems to gen-
erate an initial, coarse and rapid ‘distribution map’ for vertebrate bio-
diversity (and at a relatively low cost) could transform biomonitoring at
the landscape level. For group-living (i.e. deer) and small mammal spe-
cies, carefully chosen sampling points (with at least five water-based
replicates) along multiple river courses could provide a reliable indica-
tion of what species are present in the catchment area if conducted
during times of peak abundance (i.e. Summer and Autumn). Then, on
the basis of this, practitioners could choose to further investigate spe-
cific areas for confirmation of solitary, rare or invasive species (e.g. car-
nivores) with increased effort in terms of both the number of sampling
sites and replicates taken. At present, we would recommend the use of
eDNA metabarcoding alongside other non-invasive surveying meth-
ods (e.g. camera traps) when monitoring invasive species or species of
conservation concern to maximize monitoring efforts (Abrams et al.,
2019; Sales, Kaizer, et al., 2020).
  
|
 9
Journal of Applied Ecolog
y
SALES E t AL.
It is clear that eDNA metabarcoding is a promising tool for moni-
toring semi-aquatic and terrestrial mammals in both lotic (this study)
and lentic systems (Harper et al., 2019; Ushio et al., 2017). We de-
tected a large proportion of the expected mammalian community
(Table S1). Water-based eDNA metabarcoding is comparable or
outperforms other non-invasive survey methods for several species
(Figure 2). However, there remain challenges for the application of this
technique over larger spatial and temporal scales. Technical issues of
metabarcoding in laboratory and bioinformatic contexts have been
dealt with elsewhere (Harper et al., 2019) but understanding the dis-
tribution of eDNA transport in the landscape and its entry into natural
lotic systems is at an early stage (and incorporating such variables in
occupancy modelling approaches). This clearly requires more detailed
and systematic eDNA sampling than undertaken here, particularly in
an interconnected river/stream network with organisms moving be-
tween aquatic and terrestrial environments. Leempoel et al. (2020)
recently demonstrated the feasibility for detecting terrestrial mammal
eDNA in soil samples but this study has shown that sampling a few
key areas in freshwater ecosystems (e.g. larger rivers and lakes) within
a catchment area could potentially provide data on a large propor-
tion (if not all) of the mammalian species within it, even when some
species are present at low densities (Deiner et al., 2017). In this re-
gard, future studies might also investigate the value of citizen science,
where trained volunteers can contribute to data collection at key sites,
thus scaling up the reach of research whilst raising public awareness
and the significance of mammalian conservation concerns (Parsons,
Goforth, Costello, & Kays, 2018).
ACKNOWLEDGEMENTS
The eDNA component of this project was funded by the British
Ecological Society (grant no. SR17/1214) and a University of Salford
Internal Research Award awarded to A.D.M. J.D. was supported by
the University of Massachusetts Organismal and Evolutionary Biology
Research Grant and Spring 2018 Graduate School Fieldwork Grant.
We thank Kristy Deiner for enlightening conversations about these
results. We are grateful to Jerry Herman and Andrew Kitchener for the
tissue samples from National Museums Scotland. Christine Gregory,
Douglas Ross and Sarah Proctor provided water vole and otter infor-
mation for sampling in the Peak District and Sara Peixoto provided
sequence assemblies. We thank the various landowners for permis-
sion to sample on their property. We thank Brittany Mosher and the
anonymous reviewers for significantly improving the manuscript. The
authors declare that no conflict of interest exists.
AUTHORS' CONTRIBUTIONS
A.D.M., X.L., C .S., O.S.W., I.C., S.M., N.G.S., S.S.B., E.O., B.H. and
L.L.H. conceived the study; Monitoring and live-trapping of water
voles was pa rt of X.L ., C. S., E. B. and J.D.'s ongoing work in As synt;
J.D. analysed the camera trap data; D.A.D. advised on primer set/
data validation and provided information and data on mammals in
the Peak District; A.D.M., N.G.S., S.S.B. and M.B.M. carried out
the eDNA sampling; M.B.M., N.G.S., S.S.B., C.B. and A.D.M. per-
formed the laboratory work; N.G.S., O.S.W., L.R.H., M.B.M., C .B.
and A. D.M. c arried out the bioinfo rmatic analyses; N.G .S., A. D.M.,
I.C. and M.B.M. analysed the eDNA data; C.S. and J.D. conducted
the occupancy modelling; A.D.M., N.G.S., C.S., J.D., M.B.M. and
L. R .H. wr ot e the pape r, wit h all auth ors con tri butin g to ed iting and
discussions.
DATA AVA ILAB ILITY STATE MEN T
Data are available via the Dryad Digit al Repository ht tps://doi.
org/10.5061/dryad.d51c5 9zzf (Sales, McKenzie, et al., 2020).
ORCID
Naiara Guimarães Sales https://orcid.org/0000-0002-2922-3561
Joseph Drake https://orcid.org/0000-0003-0458-3533
Lynsey R. Harper https://orcid.org/0000-0003-0923-1801
Stefano Mariani https://orcid.org/0000-0002-5329-0553
Christopher Sutherland https://orcid.org/0000-0003-2073-1751
Allan D. McDevitt https://orcid.org/0000-0002-2677-7833
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Sales NG , McKenzie MB, Drake J,
et al. Fishing for mammals: Landscape-level monitoring of
terrestrial and semi-aquatic communities using eDNA
from riverine systems. J Appl Ecol. 2020;00:1–10.
https://doi .org /10.1111/1365-26 64.13592
... eDNA surveys that are based on metabarcoding can actually acquire information across the taxonomic tree of life 5,6,11,32,33 . However, eDNA that originates from different taxonomic groups has a different probability of being left in the environment and input into water 6,8,9,34 . van Bochove et al. inferred that the eDNA contained inside of cells and mitochondria is especially resilient against degradation (i.e., intracellular vs. extracellular effects) 28 . ...
... In previous studies, the effectiveness of using water eDNA to monitor terrestrial organisms was indicated by the detection probability 8,9,34 , and the effectiveness of using downstream water eDNA to monitor upstream organisms was indicated by the detectable distance 7,12,17,19,20,35 . In this study, we approximated the biodiversity information monitoring effectiveness by the WBIF transportation effectiveness and proposed its assessment framework, in which we described the riparian-to-river monitoring effectiveness with the proportion of biodiversity information in riparian soil that was detected by using riverine water eDNA samples. ...
... eDNA metabarcoding surveys are relatively cheaper, more efficient, and more accurate than traditional surveys in aquatic systems 10,13 , although this is certainly not true in all circumstances 36 . Sales et al. show that the detection probability of using riverine water eDNA to monitor the semi-aquatic and terrestrial mammals in natural lotic ecosystems in the UK was 40-67%, which provided comparable results to conventional survey methods per unit of survey effort for three species (water vole, field vole and red deer); in other words, the results from 3 to 6 water replicates would be equivalent to the results from 3 to 5 latrine surveys and 5-30 weeks of single camera deployment 9 . In the current case, the riverine water eDNA samples detected 53.52% of eukaryotic species from riparian soil samples. ...
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Both aquatic and terrestrial biodiversity information can be detected in riverine water environmental DNA (eDNA). However, the effectiveness of using riverine water eDNA to simultaneously monitor the riverine and terrestrial biodiversity information remains unidentified. Here, we proposed that the monitoring effectiveness could be approximated by the transportation effectiveness of land-to-river and upstream-to-downstream biodiversity information flows and described by three new indicators. Subsequently, we conducted a case study in a watershed on the Qinghai–Tibet Plateau. The results demonstrated that there was higher monitoring effectiveness on summer or autumn rainy days than in other seasons and weather conditions. The monitoring of the bacterial biodiversity information was more efficient than the monitoring of the eukaryotic biodiversity information. On summer rainy days, 43–76% of species information in riparian sites could be detected in adjacent riverine water eDNA samples, 92–99% of species information in riverine sites could be detected in a 1-km downstream eDNA sample, and half of dead bioinformation (the bioinformation labeling the biological material that lacked life activity and fertility) could be monitored 4–6 km downstream for eukaryotes and 13–19 km downstream for bacteria. The current study provided reference method and data for future monitoring projects design and for future monitoring results evaluation.
... Then, for sections where chance of detecting signs is low, more efficient monitoring methods can be applied to reduce survey effort (e.g. Sales et al., 2020). ...
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Assessing protected areas (PA) effectiveness for aquatic species is essential, as they are frequently recognised ineffective for freshwater ecosystems. By using spatially correlated replicates, the occupancy model with Markovian spatial dependence is well-suited to network-constrained environments. We applied this model to a semi-aquatic mammal, Galemys pyrenaicus, across the river network of the French Pyrenees. We found that occupancy is mainly influenced by climatic and hydrographic factors. Rainfall, forest cover and flow variability influence the overall high faeces detection. We then assessed the efficiency of the PA network to protect suitable streams for G. pyrenaicus by combining conservation gap analyses with two types of model outputs (i.e. occupancy probabilities and binary predictions). Using complementary indices and permutation tests, we found that about 25% of stream sections protected by PA are highly suitable for G. pyrenaicus and less than 5.5% of the most suitable sections benefit from a moderate to strong level of protection. Some highly suitable unprotected areas for G. pyrenaicus were identified where conservation measures should urgently be implemented. This study presents an innovative and integrative approach that opens future perspectives for development and additional applications to other taxa, difficult-to-survey or network-constrained environments.
... This approach represents a clear advantage with respect to DNA barcoding, as it allows the simultaneous processing of many specimens at once, greatly reducing the workload and processing time. In addition, it is more cost efficient for large numbers of specimens, as the number of sequences obtained from a single metabarcoding run is in the order of millions (Sales et al., 2020;Watts et al., 2019). The downside of using this approach is that individual specimens cannot be traced back or are sometimes even lost in the process of preparing the bulk sample, making it impossible to revise the voucher specimens if interesting sequences were found. ...
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Although arthropods are the largest component of animal diversity, they are traditionally underrepresented in biological inventories and monitoring programs. However, no biodiversity assessment can be considered informative without including them. Arthropod immature stages are often discarded during sorting, despite frequently representing more than half of the collected individuals. To date, little effort has been devoted to characterising the impact of discarding non‐adult specimens on our diversity estimates. Here, we used a metabarcoding approach to analyse spiders from oak forests in the Iberian Peninsula, to assess (1) the contribution of juvenile stages to local diversity estimates, and (2) their effect on the diversity patterns (compositional differences) across assemblages. We further investigated the ability of metabarcoding to inform on abundance. We obtained 363 and 331 species as adults and juveniles, respectively. Including the species represented only by juveniles increased the species richness of the whole sampling in 35% with respect to those identified from adults. Differences in composition between assemblages were greatly reduced when immature stages were considered, especially across latitudes, possibly due to phenological differences. Moreover, our results revealed that metabarcoding data are to a certain extent quantitative, but some sort of taxonomic conversion factor may be necessary to provide accurate informative estimates. Although our findings do not question the relevance of the information provided by adult‐based inventories, they also reveal that juveniles provide a novel and relevant layer of knowledge that, especially in areas with marked seasonality, may influence our interpretations, providing more accurate information from standardised biological inventories.
... If abiotic factors are accurately accounted for, this methodology could be optimized to estimate ranges and the population abundance of specific sea turtle species in coastal waters. Environmental DNA monitoring data are increasingly being coupled with mathematical models for more robust population dynamics, range and abundance estimates, with varying success (Schmelzle & Kinziger, 2015;Keck et al., 2022;Martel et al., 2020;Sales et al., 2020;Burian et al., 2021). For example, occupancy models that estimate occurrence and detection probability indicated that eDNA detection was positively related to an index of target species density (Strickland & Roberts, 2018). ...
Article
Elusive aquatic wildlife, such as endangered sea turtles, are difficult to monitor and conserve. As novel molecular and genetic technologies develop, it is possible to adapt and optimize them for wildlife conservation. One such technology is environmental (e)DNA – the detection of DNA shed from organisms into their surrounding environments. We developed species‐specific green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtle probe‐based qPCR assays, which can detect and quantify sea turtle eDNA in controlled (captive tank water and sand samples) and free ranging (oceanic water samples and nesting beach sand) settings. eDNA detection complemented traditional in‐water sea turtle monitoring by enabling detection even when turtles were not visually observed. Furthermore, we report that high throughput shotgun sequencing of eDNA sand samples enabled sea turtle population genetic studies and pathogen monitoring, demonstrating that non‐invasive eDNA techniques are viable and efficient alternatives to biological sampling (e.g. biopsies and blood draws). Genetic information was obtained from sand many hours after nesting events, without having to observe or interact with the target individual. This greatly reduces the sampling stress experienced by nesting mothers and emerging hatchlings, and avoids sacrificing viable eggs for genetic analysis. The detection of pathogens from sand indicates significant potential for increased wildlife disease monitoring capacity and viral variant surveillance. Together, these results demonstrate the potential of eDNA approaches to ultimately help understand and conserve threatened species such as sea turtles.
... We discuss its use primarily from two sources proven to be useful in wildlife studies, either hair roots or scat. Potential sources also exist in environmental DNA (eDNA) from trace amounts of DNA from shed epithelial cells, saliva, or urine that can be sampled from water, soil, snow, and air (Bohmann et al., 2014;Ushio et al., 2017; Rupert et al., 2019;Sales et al., 2020;Clare et al., 2021;Mena et al., 2021). Invertebrate-derived DNA (iDNA, Schnell et al., 2015) using blood-feeding leeches (Haemadipsa picta), blow-flies (Diptera: Calliphoridae) have been used to detect sun bears and Asiatic black bears (Drinkwater et al., 2020;Tilker et al., 2020) as well as dung beetle-derived DNA to sample forest vertebrates (Lee et al., 2016;Ji et al., 2020;Drinkwater et al., 2021). ...
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Efficient and effective monitoring methods are required to assess population status and gauge efficacy of conservation actions for threatened species. Here we review the spectrum of field methods useful for monitoring distribution, occupancy, abundance, and population trend for the five species of Asian terrestrial bears. Methods reviewed include expert opinion, local knowledge, bear sign, visual observations, camera traps, DNA-based methods (hair and scat derived), and radio telemetry. We examine the application of each method in terms of realizing specific monitoring objectives, their assumptions, challenges, and advantages. Our goal is to assist researchers in matching appropriate field methods with sought-after project objectives and to highlight shortfalls and trade-offs. Methods vary greatly in terms of cost, logistics, required number and expertize of staff, and the reliability of the data they provide. Many Asian bear population assessments have relied on expert opinion, local interviews, and sign surveys to provide estimates of distribution, abundance, and trend, in part because these are inexpensive and relatively easy to employ. However, increasing use of camera traps and DNA-based methods now allow for better monitoring via occupancy or rigorous capture–recapture population estimation, with the caveat that these methods may be restricted by inadequate budgets or logistical constraints. For distribution monitoring, camera traps and DNA yield the most definitive records of presence, but in low density bear populations, sign and local knowledge may be more effective. For occupancy, camera traps and DNA are advantageous in providing definitive detections in known time periods. For abundance/density or population trend monitoring in relatively small areas (
... In this context, traditional field monitoring of otters can be highly demanding in terms of time and costs. eDNA analysis could be a valuable alternative, especially to assess the species occurrence in peripheral areas of the species range (Sales et al., 2020). In the present study, we tested for the very first time in Italy an eDNA-based experimental workflow to detect L. lutra DNA from water samples by a target qPCR assay. ...
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The assessment of the occurrence of rare and endangered species in freshwater environments is crucial for ecological studies and conservation issues, but it can be time-consuming and challenging in harsh environments. Detecting DNA traces from the environment (environmental DNA, eDNA) can provide innovative and reliable solutions for the monitoring and conservation of rare and elusive species, such as the Eurasian otter Lutra lutra. We tested an experimental workflow based on target qPCR assay to detect L. lutra eDNA from water samples as a speditive monitoring tool at large scale to be coupled with fine-scale traditional field surveys. This is the first application of an eDNA-based approach to monitor the presence of L. lutra in Italy. We compared the eDNA-based results with traditional survey observations and confirmed the reliability of this innovative approach for the large-scale monitoring of such aquatic elusive species.
... In contrast, sampling for eDNA is more standardized for the type of environmental sample, be it water, soil or air, and can be consistently sampled regardless of the ecosystem type, season, or taxa of interest. The reliability of eDNA to detect communities has repeatedly been shown to be on par or better than traditional sampling methods (Valentini et al., 2016;Sales et al., 2020;Seymour et al., 2020). In addition, the standardized molecular and bioinformatics approaches used to process and analyze eDNA data are often faster and cheaper to routinely conduct compared to traditional taxonomic identification methods (Davy et al., 2015) and avoid the potential bias induced by using multiple or inexperienced taxonomic identifiers . ...
Chapter
Aim: The aim of this chapter is to introduce the concepts and applications of environmental DNA (eDNA) for species detection and biomonitoring of freshwater ecosystems. Environmental assessment of inland waters is currently undergoing a revolution due to the increased utilization of eDNA and major advancements in molecular techniques. Several aspects of ecology and conservation biology across the academic, private and government sectors are already utilizing eDNA-based approaches with more applications being rapidly developed. Therefore, this chapter disseminates current fundamental understanding of the dynamics and applications of eDNA in freshwater environments. Main concepts covered: Environmental DNA (eDNA) is DNA extracted from environmental samples without targeting a particular organism or group of organisms. Within the realm of inland waters, eDNA samples typically include water, sediment or biofilm samples, though there is potential for other environmental sources. The ecology of eDNA (e.g., transport, degradation rate, molecular state) greatly affects the detectability of eDNA, as well as the interpretation of the results drawn from eDNA-based assessment. In particular, understanding the difference between eDNA detection dynamics in lotic (e.g., rivers and streams) versus lentic (e.g., ponds and lakes) environments is key to understanding and applying eDNA-derived information. Main methods covered: A major characteristic of eDNA-based research is the non-targeted aspect of species and community detection. It is therefore paramount that an understanding of the sampling and DNA extraction methods are outlined, and that concerns regarding potential inhibition (false negatives) and contamination sources (false positives) are addressed. The use of eDNA also requires additional experimental design considerations, particularly regarding replication and spatial resolution, due to the need to cross validate findings and the increased complexity of the data created compared to traditional taxonomic-based approaches. Currently, eDNA-based research can be divided into two main groups, population- and community-based analyzes. Population-based analyzes rely primarily on single-targeted (i.e., species) methods such as qPCR, which are lower cost and easier for smaller institutes or individuals to independently implement. Community-based approaches rely largely on high throughput sequencing (HTS) and require additional molecular and bioinformatics specialization and support, but result in greater potential for data generation and analytical power, given the proper study design. Future eDNA work will include applying PCR free-based methods to population analyzes and combining multi-dimensional environmental data for environmental community analyzes. Other advances in eDNA research may look to assess the transcriptional profiles of eDNA samples to assess functional community diversity. Conclusion/Outlook: This chapter provides an overview of current molecular and eDNA-based approaches for inland water assessment. There are many aspects of eDNA that are still largely unknown, but the ability to apply standardized non-invasive sampling with high throughput data is hard to ignore in the modern age.
Technical Report
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The installation of marine energy systems may affect marine environments, and by extension, marine fish communities. Therefore, biomonitoring is an integral part of assessing impacts on species. Environmental DNA (eDNA) provides a noninvasive alternative to conventional monitoring surveys and the possibility of a more accurate assessment of species richness. Yet, its cost efficiency compared to traditional methods of monitoring is relatively unknown, especially when applied to monitoring around tidal, wave, and offshore wind energy installations. For this study, 202 peer-reviewed journal articles were dissected to inventory the diversity of supplies used for collecting and processing eDNA samples and to compile the average cost of eDNA surveys. Information collected included the type, volume, and brand of containers used in sampling; material, size, and brand of filters; and extraction methods. Cost information was gathered for the most common supplies, and a total cost was estimated for a hypothetical eDNA survey in Sequim Bay, WA, to compare with traditional methods of surveying such as beach seining and scuba surveys. The results showed a higher-than-expected diversity of supplies to collect and process eDNA samples. The most common supplies were 1 L Nalgene bottles at an average cost of 7.96 USD for collecting samples, 0.45 μm glass fiber Merck Millipore filters at an average cost of 1.51 USD for filtering samples, and the Qiagen DNeasy Blood and Tissue kit at 3.54 USD per sample for extracting DNA. When compared to beach seine and scuba surveys, eDNA surveys undertaken by senior researchers are less expensive for both initial surveys with all new materials as well as for follow-up surveys reusing some of the supplies. However, when surveys are done solely by students, eDNA surveys are more expensive than scuba surveys when no prior supplies are available and more than both beach seine and scuba surveys for follow-up surveys reusing supplies. In a professional sphere, where surveys are less often conducted by teams of students only, eDNA surveys are an effective and less-costly alternative to conventional methods. We anticipate that the development and refinement of eDNA methodology will continue to decrease surveying costs.
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The crisis of declining biodiversity¹ exceeds our current ability to monitor changes in ecosystems. Rapid terrestrial biomonitoring approaches are essential to quantify the causes and consequences of global change. Environmental DNA² has revolutionized aquatic ecology,³ permitting population monitoring⁴ and remote diversity assessments matching or outperforming conventional methods of community sampling.3, 4, 5 Despite this model, similar methods have not been widely adopted in terrestrial ecosystems. Here, we demonstrate that DNA from terrestrial animals can be filtered, amplified, and then sequenced from air samples collected in natural settings representing a powerful tool for terrestrial ecology. We collected air samples at a zoological park, where spatially confined non-native species allowed us to track DNA sources. We show that DNA can be collected from air and used to identify species and their ecological interactions. Air samples contained DNA from 25 species of mammals and birds, including 17 known terrestrial resident zoo species. We also identified food items from air sampled in enclosures and detected taxa native to the local area, including the Eurasian hedgehog, endangered in the United Kingdom. Our data demonstrate that airborne eDNA concentrates around recently inhabited areas but disperses away from sources, suggesting an ecology to airborne eDNA and the potential for sampling at a distance. Our findings demonstrate the profound potential of air as a source of DNA for global terrestrial biomonitoring.
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Biodiversity assessments are indispensable tools for planning and monitoring conservation strategies. Camera traps (CT) are widely used to monitor wildlife and have proven their usefulness. Environmental DNA (eDNA)-based approaches are increasingly implemented for biomonitoring, combining sensitivity, high taxonomic coverage and resolution, non-invasiveness and easiness of sampling, but remain challenging for terrestrial fauna. However, in remote desert areas where scattered water bodies attract terrestrial species, which release their DNA into the water, this method presents a unique opportunity for their detection. In order to identify the most efficient method for a given study system, comparative studies are needed. Here, we compare CT and DNA metabarcoding of water samples collected from two desert ecosystems, the Trans-Altai Gobi in Mongolia and the Kalahari in Botswana. We recorded with CT the visiting patterns of wildlife and studied the correlation with the biodiversity captured with the eDNA approach. The aim of the present study was threefold: (a) to investigate how well waterborne eDNA captures signals of terrestrial fauna in remote desert environments, which have been so far neglected in terms of biomonitoring efforts; (b) to compare two distinct approaches for biomonitoring in such environments; and (c) to draw recommendations for future eDNA-based biomonitoring. We found significant correlations between the two methodologies and describe a detectability score based on variables extracted from CT data and the visiting patterns of wildlife. This supports the use of eDNA-based biomonitoring in these ecosystems and encourages further research to integrate the methodology in the planning and monitoring of conservation strategies.
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Before environmental DNA (eDNA) can establish itself as a robust tool for biodiversity monitoring, comparison with existing approaches is necessary, yet is lacking for terrestrial mammals. Moreover, much is unknown regarding the nature, spread and persistence of DNA shed by animals into terrestrial environments, or the optimal experimental design for understanding these potential biases. To address some of these challenges, we compared the detection of terrestrial mammals using eDNA analysis of soil samples against confirmed species observations from a long-term (approx. 9-year) camera-trapping study. At the same time, we considered multiple experimental parameters, including two sampling designs, two DNA extraction kits and two metabarcodes of different sizes. All mammals regularly recorded with cameras were detected in eDNA. In addition, eDNA reported many unrecorded small mammals whose presence in the study area is otherwise documented. A long metabarcode (≈220 bp) offering a high taxonomic resolution, achieved a similar efficiency as a shorter one (≈70 bp) and a phosphate buffer-based extraction gave similar results as a total DNA extraction method, for a fraction of the price. Our results support that eDNA-based monitoring should become a valuable part of ecosystem surveys, yet mitochondrial reference databases need to be enriched first.
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The application of environmental DNA (eDNA) metabarcoding as a biomonitoring tool has greatly increased, but studies have focused on temperate aquatic macro‐organisms. We apply eDNA metabarcoding to detecting the mammalian community in two high‐biodiversity regions of Brazil: the Amazon and Atlantic Forests. We identified Critically Endangered and Endangered mammalian species and found overlap with species identified via camera trapping. We highlight the potential for using eDNA monitoring for mammals in biodiverse regions and identify challenges: we need a better understanding of the ecology of eDNA within variable environments and more appropriate reference sequences for species identification in these anthropogenically impacted biomes. Mammalian families (shown with stylised drawings of representative species within hexagons) identified from environmental DNA samples in the Amazon and Atlantic Forests of Brazil. Water and sediment samples were taken from rivers (‘Meeting of the Waters’; top left) and streams (bottom left and right) and subjected to DNA metabarcoding analyses using mammal‐specific primers. A total of 28 molecular operational taxonomic units (MOTUs) were identified, and 13 of these could be identified to species level. This case study has highlighted the potential of this non‐invasive genetic technique to detect (and potentially monitor) terrestrial and aquatic mammals in these biodiverse Neotropical regions.
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Environmental DNA (eDNA) surveys are increasingly being used for biodiversity monitoring, principally because they are sensitive and can provide high resolution community composition data. Despite considerable progress in recent years, eDNA studies examining how different environmental sample types can affect species detectability remain rare. Comparisons of environmental samples are especially important for providing best practice guidance on early detection and subsequent mitigation of non-indigenous species. Here we used eDNA metabarcoding of COI (cytochrome c oxidase subunit I) and 18S (nuclear small subunit ribosomal DNA) genes to compare community composition between sediment and water samples in artificial coastal sites across the United Kingdom. We first detected markedly different communities and a consistently greater number of distinct operational taxonomic units in sediment compared to water. We then compared our eDNA datasets with previously published rapid assessment biodiversity surveys and found excellent concordance among the different survey techniques. Finally, our eDNA surveys detected many non-indigenous species, including several newly introduced species, highlighting the utility of eDNA metabarcoding for both early detection and temporal / spatial monitoring of non-indigenous species. We conclude that careful consideration on environmental sample type is needed when conducting eDNA surveys, especially for studies assessing community change.
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Obtaining reliable species observations is of great importance in animal ecology and wildlife conservation. An increasing number of studies use camera traps (CTs) to study wildlife communities, and an increasing effort is made to make better use and reuse of the large amounts of data that are produced. It is in these circumstances that it becomes paramount to correct for the species-and study-specific variation in imperfect detection within CTs. We reviewed the literature and used our own experience to compile a list of factors that affect CT detection of animals. We did this within a conceptual framework of six distinct scales separating out the influences of (a) animal characteristics, (b) CT specifications, (c) CT setup protocols, and (d) environmental variables. We identified 40 factors that can potentially influence the detection of animals by CTs at these six scales. Many of these factors were related to only a few overarching parameters. Most of the animal characteristics scale with body mass and diet type, and most environmental characteristics differ with season or latitude such that remote sensing products like NDVI could be used as a proxy index to capture this variation. Factors that influence detection at the microsite and camera scales are probably the most important in determining CT detection of animals. The type of study and specific research question will determine which factors should be corrected. Corrections can be done by directly adjusting the CT metric of interest or by using covariates in a statistical framework. Our conceptual framework can be used to design better CT studies and help when analyzing CT data. Furthermore, it provides an overview of which factors should be reported in CT studies to make them repeatable, comparable, and their data reusable. This should greatly improve the possibilities for global scale analyses of (reused) CT data.
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Containing the spread of established invasive species is critical for minimizing their ecological impact. Effective containment requires sensitive sampling methods capable of detecting new introductions when invaders are at low density. Here we explore whether environmental DNA (eDNA) sampling could be used as a surveillance tool to detect new incursions of aquatic invasive species on offshore islands. We develop an eDNA molecular assay for invasive cane toads (Rhinella marina) in Australia, validate our assay on the mainland, and apply it to an offshore island (Moreton Island) that is a target of ongoing cane toad surveillance. Our eDNA assay correctly identified four mainland sites at which cane toads were observed, as well as a fifth site within 1 km of known populations. Five additional sites outside the cane toad’s current distribution tested negative for cane toad eDNA. Site occupancy detection models indicated that two water samples and three qPCR replicates were sufficient to achieve a cumulate detection probability > 0.95. Applying our eDNA assay to samples from 19 sites on an offshore island over a 2-year period revealed the absence of cane toad eDNA, in line with our current understanding of cane toad distribution. Our results suggest that eDNA sampling could be strategically applied to meet the Australian Commonwealth’s objective of maintaining cane toad-free offshore islands.
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Environmental DNA (eDNA) metabarcoding can identify terrestrial taxa utilising aquatic habitats alongside aquatic communities, but terrestrial species' eDNA dynamics are understudied. We evaluated eDNA metabarcoding for monitoring semi-aquatic and terrestrial mammals, specifically nine species of conservation or management concern, and examined spatiotemporal variation in mammal eDNA signals. We hypothesised eDNA signals would be stronger for semi-aquatic than terrestrial mammals, and at sites where individuals exhibited behaviours. In captivity, we sampled waterbodies at points where behaviours were observed (‘directed’ sampling) and at equidistant intervals along the shoreline (‘stratified’ sampling). We surveyed natural ponds (N = 6) where focal species were present using stratified water sampling, camera traps, and field signs. eDNA samples were metabarcoded using vertebrate-specific primers. All focal species were detected in captivity. eDNA signal strength did not differ between directed and stratified samples across or within species, between semi-aquatic or terrestrial species, or according to behaviours. eDNA was evenly distributed in artificial waterbodies, but unevenly distributed in natural ponds. Survey methods deployed at natural ponds shared three species detections. Metabarcoding missed badger and red fox recorded by cameras and field signs, but detected small mammals these tools overlooked, e.g. water vole. Terrestrial mammal eDNA signals were weaker and detected less frequently than semi-aquatic mammal eDNA signals. eDNA metabarcoding could enhance mammal monitoring through large-scale, multi-species distribution assessment for priority and difficult to survey species, and provide early indication of range expansions or contractions. However, eDNA surveys need high spatiotemporal resolution and metabarcoding biases require further investigation before routine implementation.
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1.Invertebrate‐derived DNA (iDNA), in combination with high throughput sequencing, has been proposed as a cost‐efficient and powerful tool to survey vertebrate species. Previous studies, however, have only provided evidence that vertebrates can be detected using iDNA, but have not taken the next step of placing these detection events within a statistical framework that allows for robust biodiversity assessments. 2.Here, we compare concurrent iDNA and camera‐trap surveys. Leeches were repeatedly collected in close vicinity to 64 camera‐trap stations in Sabah, Malaysian Borneo. We analyse iDNA‐derived mammalian detection events in a modern occupancy model that accounts for imperfect detection and compare the results with those from occupancy models parameterized with camera‐trap‐derived detection events. We also combine leech‐iDNA and camera‐trap data in a single occupancy model. 3.We found consistent estimates of occupancy probabilities produced by our camera‐trap and leech datasets. This indicates that the metabarcoding of leech‐iDNA method provides reasonable estimates of occupancy and may be a suitable method for studying and monitoring mammal species in tropical rainforests. However, we also show that a more extensive collection of leeches would be needed to assess mammal biodiversity with a robustness similar to that of camera traps. As certain taxa were only detected in leeches, we see great potential in complementing camera‐trap studies with the iDNA approach, as long as the collection of leeches follows a robust and standardized sampling scheme. 4.Synthesis and applications. Here, we describe an approach to analyse detection records of mammals derived from leech samples using an occupancy framework that accounts for leech‐specific factors influencing the detection probability. We further combined camera‐trap and leech data, which lead to increased confidence in occupancy estimates. Our approach is not restricted to the processing of leech samples, but can be used for the analysis of other invertebrate DNA (iDNA) and environmental DNA (eDNA) data. Our study is the first step to shift the application of iDNA studies from opportunistic ad‐hoc collections to the systematic surveys required for long‐term management of wildlife populations. This article is protected by copyright. All rights reserved.
Article
The potential ranges of many species are shifting due to changing ecological conditions. Where populations become patchy towards the range edge, the realised distribution emerges from colonisation‐persistence dynamics. Therefore, a greater understanding of the drivers of these processes, and the spatial scales over which they operate, presents an opportunity to improve predictions of species range expansion under environmental change. Species reintroductions offer an ideal opportunity to investigate the drivers and spatial scale of colonisation dynamics at the range edge. To this effect, we performed and monitored experimental translocations of water voles to quantify how colonisation and local persistence were influenced by habitat quality and occupancy. We used a novel statistical method to simultaneously consider effects across a range of spatial scales. Densely occupied neighbourhoods were highly persistent and frequently colonised. Persistence was more likely in high quality habitat, whereas influence of habitat quality on colonisation was less clear. Colonisation of suitable habitat in distant, sparsely occupied areas was much less frequent than expected from the well documented high dispersal ability of the species. Persistence of these distant populations was also low, which we attribute to the absence of a rescue effect in sparsely populated neighbourhoods. Our results illustrate a mismatch between the spatial scales of colonisation dynamics in the core and edge of a species range, suggesting that recolonisation dynamics in established populations may be a poor predictor of colonisation dynamics at the range edge. Such a mismatch leads to predictions of long lags between the emergence and colonisation of new habitat, with detrimental consequences for a species realised distribution, conservation status and contribution to ecosystem function. Conservation translocations that also reinforce existing populations at the range edge might stimulate the rescue effect and mitigate lags in expansion. This article is protected by copyright. All rights reserved.
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Occupancy Estimation and Modeling: Inferring Patterns and Dynamics of Species Occurrence, Second Edition, provides a synthesis of model-based approaches for analyzing presence-absence data, allowing for imperfect detection. Beginning from the relatively simple case of estimating the proportion of area or sampling units occupied at the time of surveying, the authors describe a wide variety of extensions that have been developed since the early 2000s. This provides an improved insight about species and community ecology, including, detection heterogeneity; correlated detections; spatial autocorrelation; multiple states or classes of occupancy; changes in occupancy over time; species co-occurrence; community-level modeling, and more. Occupancy Estimation and Modeling: Inferring Patterns and Dynamics of Species Occurrence, Second Edition has been greatly expanded and detail is provided regarding the estimation methods and examples of their application are given. Important study design recommendations are also covered to give a well rounded view of modeling.