3 Progress of domestic and international eDNA-based surveys of
amphibians
3.1 Species diversity
surveys
With the signing of the Convention on Biological Diversity (CBD) and the
15th Conference of the Parties (COP15) to the United Nations Framework
Convention on Climate Change (UNFCCC), global biodiversity has received
wide attention. eDNA technology combined with Next-generation sequencing
offers a more sensitive and efficient method for biodiversity surveys.
Svenningsen et al. used simultaneously traditional survey methods and
eDNA metabarcoding to detect amphibian diversity in Danish lakes and
ponds, and evaluated the detection efficiency of the two methods from
the perspectives of species richness, the average number of species
detected at various points, and frequency of detection. They found that
both eDNA metabarcoding and traditional surveys yielded a large number
of observations unique to the respective method, suggesting that eDNA
metabarcoding should be combined with traditional methods as a
complement to enhance determination of the total composition of species
(Svenningsen et al., 2022).
eDNA detection of amphibians has yielded good results in temperate
regions (Deiner et al., 2017; Thomsen & Willerslev, 2015; Yates et al.,
2019). However, it has been demonstrated that high temperatures and
strong UV light favor the growth of microorganisms and promote the
degradation of DNA. To validate eDNA metabarcoding surveys of amphibian
diversity in the tropics, Sasso et al. analyzed four streams in the
Atlantic Forest of southeastern Brazil during a five-year period of
traditional field surveys (using visual and acoustic methods) as well as
short-term sampling (4 days) of eDNA. A total of 10 species of aquatic
amphibians were detected over the 5-year period of the traditional
survey, while nine species were detected by eDNA metabarcoding,
including one species that had only been detected once during the 5-year
traditional survey. There was a high degree of similarity in stream
community composition between the results of the two methods (Sasso et
al., 2017). Li et al. used eDNA metabarcoding to assess amphibian
diversity at 288 sites in 18 regions of Hainan Island and found 15
species, including a number difficult to detect with traditional
monitoring methods (Li et al., 2021). These studies demonstrate the
effectiveness of the eDNA metabarcoding technique in detecting
amphibians in the tropics.
3.2 Detection of invasive
species
Alien invasive species are one of the main causes of global biodiversity
loss and homogenization (Ehrenfeld, 2010; Ficetola et al., 2007; Pysek
& Richardson, 2010; Vitousek et al., 1997). Invasive species may
disrupt local ecological balance and disturb the ecosystem, with
substantial impacts on the variety and abundance of native species
(Ficetola et al., 2008). The early presence of invasive species is
difficult to detect in a timely manner using traditional methods, while
eDNA technology allows for rapid and sensitive monitoring of invasive
organisms. Invasive amphibians were the first vertebrates for which
species distribution was successfully assessed by extracting DNA from
water samples (Ficetola et al., 2008). Specific primers were used to
expand short mitochondrial DNA sequences to trace the presence of
invasive bullfrogs (Rana catesbeiana ) in controlled environments
and natural wetlands. Similarly, Dejean et al. assessed the distribution
of this species in ponds in western France using traditional monitoring
(visual and acoustic surveys) and eDNA. Bullfrogs were found in only
14% of sites in the traditional survey, while the eDNA survey detected
a much higher presence (78%), demonstrating substantial effectiveness
and sensitivity (Dejean et al., 2012).
3.3 Detection of rare and endangered
species
eDNA has great potential for detecting rare and endangered species.
Pierson et al. investigated the occurrence of the tiny, rare,
multi-toothed salamander Urspelerpes brucei by using both the
litter bag survey and eDNA methods. The results showed that the
detection probability of the eDNA survey was 0.788, much higher than
that of the litter bag survey (0.048). The eDNA method also imposes
weaker disturbances on aquatic habitats and has lower costs than the
litter bag method (Pierson et al., 2016). Goldberg et al. designed
specific primers to detect two rare species in the United States,Ascaphus montanus and Dicamptodon aterrimus . In the
experiment, water samples were collected from five rivers with different
densities of the target species, and PCR amplification was performed to
detect whether the species’ DNA existed in the samples.Dicamptodon was detected in all water samples and Ascaphusin four of the five rivers, proving the effectiveness of eDNA detection
for surveys of these rare species (Caren S Goldberg et al., 2011).
McKee et al. studied the species Ambystoma cingulatum ,Ambystoma bishopi , Notophthalmus perstriatus andLithobates capito in the southeastern longleaf pine region of the
United States using both eDNA and traditional methods (mainly
dip-netting, trapping, and visualization). The eDNA method succeeded in
detecting the target species at six sites where traditional methods did
not show a presence, again indicating that eDNA can be used as a
supplement to traditional methods for investigating endangered species
(A. M. McKee et al., 2015). Voros et al. used eDNA to detect the
presence of the rare cave-dwelling amphibian Proteus anguinus in
all of 15 sampled caves in Croatia, five of which constituted first
records for the species (Voros et al., 2017). Lopes et al. analyzed
water samples from the Atlantic Coast Forest and six mountainous areas
of the adjacent Cerrado grasslands in Brazil for traces of DNA of rare
and endangered amphibian species. They successfully detected four
declining species (Hylodes ornatus , Hylodes regius ,Crossodactylus timbuhy and Vitreorana eurygnatha ), two
species considered locally extirpated (Phasmahyla exilis andPhasmahyla guttata ), and a species not seen since 1968
(Megaelosia bocainensis ) (Lopes et al., 2021). These studies
successfully confirmed the presence of species not detected by
traditional methods, and underline the effectiveness of the eDNA method
for biodiversity monitoring at low population densities.
3.4 Species abundance and biomass
assessment
eDNA allows for the assessment of the relative abundance and biomass of
faunal communities. In 2019, Kelly et al. analyzed eDNA macro-barcodes
of three simulated biomes with different biomass ratios. The effects of
these ratios on biodiversity estimates were analyzed by assuming that
the amount of DNA collected in the environment was proportional to
abundance of individuals in the water, and that the DNA shedding rate
and amplification efficiency of certain primers of isotaxa was constant.
The analysis results showed that in the case of high amplification
efficiency, such as primer adaptation, the ratio index of eDNA
macro-barcode readings was highly indicative of trends in group biomass
(Kelly et al., 2019). This result has been replicated in amphibian
studies. For example, Thomsen et al. investigated six different species
including two amphibians (Pelobates fuscus and Triturus
cristatus ) using eDNA and qPCR methods, finding that the eDNA
concentrations of these two species in water samples correlated with
density and biomass (P. F. Thomsen, J. Kielgast, L. L. Iversen, C. Wiuf,
et al., 2012). Pilliod et al. used eDNA and traditional methods to
assess the diversity and abundance of two amphibian species,Ascaphus montanus and Dicamptodon aterrimus , in 13 streams
in central Idaho, USA. They reported that eDNA reading proportion was
positively correlated with density and biomass as measured by
traditional methods, and showed that the accuracy of eDNA-based
abundance assessments increased with the amount of eDNA in water and the
number of replicate samples collected (Pilliod et al., 2014). Everts et
al. used droplet digital PCR (ddPCR) analysis of eDNA to detect the
abundance of bullfrogs in the United States, and found that eDNA
concentration increased significantly with increasing abundance of
bullfrog eggs and tadpoles (Teun Everts et al., 2021).
3.5 Reconstruction of ancient
ecosystems
eDNA for amphibian surveys is mainly used to detect the presence of
target species in current communities. However, it has been shown that
since DNA molecules can exist in the environment for a long time,
ancient ecosystems can be assessed by extracting remnant DNA molecules
from environmental sources. Ficetola and Taberlet detected eDNA of
several amphibian species (Bufo bufo , Rana temporaria andIchthyosaura alpestris ) in 1,000-year-old sediments from a lake
in the French Alps. This suggests that eDNA stored in lake sediments can
be used to understand changes in species distribution over time. In
addition, it can be combined with other paleoecological data to
understand species’ responses to environmental changes (such as habitat
alterations, climate change, and the introduction of alien species)
(Giguet-Covex et al., 2019).
3.6 Amphibian disease
surveillance
In addition to factors such as habitat degradation and environmental
pollution, viral infections have also emerged as a significant
contributor to the decline of amphibian populations (Rachowicz et al.,
2006). According to Scheele et al., the amphibian chytrid fungus
(Batrachochytrium dendrobatidis ; Bd) has led to the decline of at
least 501 amphibian species worldwide, with 90 species having gone
extinct, including the Darwin’s frog (Rhinoderma darwinii )
(Scheele et al., 2019). In recent years, eDNA methods have demonstrated
remarkable capabilities for detecting Bd due to their convenience,
sensitivity, and non-invasive nature.
Researchers like Stephen et al. have successfully determined the
presence and quantity of chytrid pathogens by collecting skin swabs from
amphibian hosts, extracting DNA from these swabs, and employing qPCR
techniques (Boyle et al., 2007). Beyond skin swab collection, pathogenic
DNA can also be detected from environmental samples. Walker et al., for
instance, successfully detected the presence of the Bd in small-volume
(<1 L) water samples by filtering and utilizing qPCR (Walker
et al., 2007). Similarly, Julie et al. employed filtration to capture
zoospores from water samples and subsequently detected pathogen DNA from
filtered particles at concentrations as low as 0.06 zoospores per water
sample, highlighting the high analytical sensitivity of the eDNA method
(Kirshtein et al., 2007).
The potential of eDNA technology for early detection of Bd in the
environment is noteworthy. Colleen et al. collected eDNA from filtered
water samples and detected Bd at three sites, all of which a month later
turned out to experience Bd-induced mortality, while Bd was not detected
in sites without later mortality (Kamoroff & Goldberg, 2017). This
underscores the potential of eDNA technology for early detection of Bd
presence.