New simple method for surveying amphibians: A vital contribution to conservation efforts

Date:
February 21, 2022
Source:
Kobe University
Summary:
Amphibian biodiversity is declining worldwide and collecting
information about their habitats and populations via monitoring
is vital for conservation efforts. However, it is difficult to
accurately monitor amphibians using conventional methods. To
address these issues, scientists have developed a novel technique
to identify the amphibious species that live in an area through
environmental DNA analysis. It is hoped that the new method will
revolutionize species monitoring, as it will enable anyone to
easily survey a habitat by collecting water samples.



FULL STORY ========================================================================== Amphibian biodiversity is declining worldwide and collecting information
about their habitats and populations via monitoring is vital for
conservation efforts. However, it is difficult to accurately monitor
amphibians using conventional methods. To address these issues, a
collaborative research team has developed a novel technique to identify
the amphibious species that live in an area through environmental DNA
analysis. It is hoped that the new method will revolutionize species monitoring, as it will enable anyone to easily survey a habitat by
collecting water samples.


==========================================================================
An international collaborative research group consisting of members from
7 institutions has developed a method of determining which amphibious
species (types of frog, newt and salamander) inhabit an area. They
achieved this by amplifying extra-organismal DNA (environmental DNA)
found in the water so that they could be analyzed. This DNA ends up in
the water because it is expelled from the amphibian's body along with
mucus and excrement. The research group included Postdoctoral Researcher
SAKATA K. Masayuki and Professor MINAMOTO Toshifumi (Kobe University), Associate Professor KURABAYASHI Atsushi (Nagahama Institute of Bio-Science
and Technology), NAKAMURA Masatoshi (IDEA Consultants, Inc.) and Associate Professor NISHIKAWA Kanto (Kyoto University).

The developed technique will resolve some of the issues with conventional methods such as capture and observational surveys, which require a
specialist surveyor who can visually identify species. Conventional
surveys are also prone to discrepancies in results caused by environmental factors such as climate and season. It is hoped that the new method will revolutionize species monitoring, as it will enable anyone to easily
monitor the amphibians that inhabit an area by collecting water samples.

These research results will be published in the online academic journal Metabarcoding and Metagenomics .

Main Points
1. Monitoring biodiversity is vital for the conservation of natural
ecosystems as a whole. In particular, the importance of monitoring
amphibians is increasing, as the number of individuals is declining
significantly.

2. However, amphibians are nocturnal, young (e.g. tadpoles) and
adults live
in different habitats, and specialist knowledge is required to
capture individuals and identify their species. These issues
that make it difficult to accurately monitor amphibians in a
standardized way, thus the results of individual surveys often
contradict each other.

3. Environmental DNA (eDNA) is extra-organismal DNA that has been
released
into the environment. In recent years, eDNA analysis techniques have
been used to monitor species. Methods for monitoring fish species in
particular have been established and are now commonplace. However,
a standardized analysis method for amphibians has yet to be
established. In order to contribute towards its development,
this research group collected and analyzed the DNA of amphibia to
come up with an analysis method for investigating which amphibious
species inhabit a surveyed area.

4. First of all, the researchers designed multiple methods for
analyzing the
eDNA of amphibians and evaluated their performance to identify the
most effective method. Next, they conducted parallel monitoring of
122 sites in 10 farmlands across Japan using the developed eDNA
analysis method and conventional methods (capture surveys using
a net and observation surveys).

5. The newly developed method was able to detect all three orders of
amphibian: Caudata (the newt and salamanders), Anura (the frogs),
and Gymnophiona (the caecilians). Furthermore, this novel eDNA
analysis method was able to detect more species across all field
study sites than the conventional method-based surveys, indicating
its effectiveness.

Research Background Amphibian biodiversity is continuing to decline
worldwide and collecting basic information about their habitats and other aspects via monitoring is vital for conservation efforts. Traditional
methods of monitoring amphibians include visual and auditory observations,
and capture surveys. However, amphibians tend to be small in size and
many are nocturnal. The success of surveys varies greatly depending on
the climate and season, and specialist knowledge is required to identify species. Consequently, it is difficult to monitor a wide area and
assess habitats. The last decade has seen the significant development of environmental DNA analysis techniques, which can be used to investigate
the distribution of a species by analysing external DNA (environmental
DNA) that is released into the environment along with an organism's
excrement, mucus and other bodily fluids.



==========================================================================
The fundamentals of this technique involve collecting water from
the survey site and analysing the eDNA contained in it to find
out which species inhabit the area. In recent years, the technique
has gained attention as a supplement for conventional monitoring
methods. Standardized methods of analysis have already been established
for other species, especially fish, and diversity monitoring using eDNA
is becoming commonplace.

However, eDNA monitoring of amphibians is still in the developmental
stage. One reason for this is that the proposed eDNA analysis method
must be suitable for the target species or taxonomic group, and there are
still issues with developing and implementing a comprehensive method for detecting amphibians. If such a method could be developed, this would
make it possible for monitoring to be conducted even by people who do
not have the specialized knowledge to identify species nor surveying experience. Hopefully, this would be established as a unified standard for large-scale monitoring surveys, such as those on a national scale. This research group's efforts to develop and evaluate analysis methods will hopefully lay the foundations for eDNA analysis to become a common tool
for monitoring amphibians, as well as fish.

Research Methodology Design and Evaluation of a Universal Primer for
Amphibia The researchers obtained a total of 1,034 amphibian DNA sequences
from an online open database. They located the DNA regions where the base sequence is the same across different species and designed five analytical systems in the mitochondrial 16SrRNA region that could comprehensively
amplify amphibian DNA.

After that, they used a computer to investigate the various
characteristics of each set, including the ease of amplifying amphibian
DNA, the likelihood of non-amphibian DNA being amplified as well
(i.e. the specificity), and whether they could reliably determine the
species (i.e. the species-level resolution).

In addition, they conducted experiments using DNA obtained from
individual amphibians to confirm that the DNA of various species in the
class could be amplified. The results revealed that all of the assays
could amplify the DNA well. From the findings of all these prestudies,
the researchers selected the assay that had the best specificity and species-level resolution and then carried out a field study to evaluate
its efficiency as a monitoring tool.



========================================================================== Validating efficiency thorough field survey-based comparisons with
conventional methods From June to September 2019, field studies were
carried out at 122 sites across 10 agricultural ecosystems (paddy field ecosystems) in Japan in order to check the suitability and efficiency of
the designed assay for field monitoring. In the field surveys, both eDNA analysis and conventional method-based surveys (capture/observations)
were conducted in the ponds and agricultural waterways at each site, and
the number and composition of the detected amphibia were compared. The
number of species detected through eDNA analysis was greater than the
number detected through conventional methods across all field study sites.

This consequently demonstrates that the eDNA analysis method using
amphibian assays developed by this research group is highly effective
for field monitoring.

Further Developments
1. The researchers developed an analysis method that can
comprehensively
detect amphibian DNA. The eDNA monitoring system using this method
could also contribute towards rapid surveying of endangered and
important species.

2. Unlike conventional survey methods like capture and observations,
eDNA
analysis is not easily affected by environmental factors, such
as season and climate. Therefore, it is a stable way to detect
amphibians and it is hoped that it will become the standardized
method for large-scale surveys, such as nationwide monitorings.

3. A collection of reference data to determine which species the
amplified
DNA originates from would enable this technique to be applied
more efficiently. This would significantly contribute towards the
monitoring of amphibians as the more reference data is collected,
the greater the range of countries and regions where this technique
can be utilized.

Glossary Environmental DNA (eDNA):DNA originating from organisms that
is found in the environment, such as in the water or on the ground. By analyzing environmental DNA using PCR, researchers can comprehensively
identify species that inhabit an area, even if they no longer live
there. This in turn enables them to estimate and gain an understanding
of the target species' distribution. Environmental DNA surveys are used
in a wide range of research fields, including species conservation,
ecology, taxonomy, microbiology and palaeontology. Prior research using
eDNA analysis has been carried out on fish species, and eDNA analysis is becoming a common tool for monitoring organisms. However, such methods
for monitoring amphibians are still a work in progress, and a detection
system that can be used to conduct thorough biodiversity surveys has
yet to be established.

Metabarcoding (of eDNA):A method of detecting multiple species
simultaneously.A universal primer consists of the nucleotide sequence
regions that are the same for the target taxa. Using this universal
primer, the collated DNA of multiple species that includes those in the
target taxa can be amplified using PCR.

Next, the sequences of the amplified sections are comprehensively
determined using a massively parallel sequencing. Species can be
identified by matching the sequences to those registered in a database.

========================================================================== Story Source: Materials provided by Kobe_University. Note: Content may
be edited for style and length.


========================================================================== Journal Reference:
1. Masayuki K. Sakata, Mone U. Kawata, Atsushi Kurabayashi, Takaki
Kurita,
Masatoshi Nakamura, Tomoyasu Shirako, Ryosuke Kakehashi, Kanto
Nishikawa, Mohamad Yazid Hossman, Takashi Nishijima, Junichi
Kabamoto, Masaki Miya, Toshifumi Minamoto. Development
and evaluation of PCR primers for environmental DNA (eDNA)
metabarcoding of Amphibia. Metabarcoding and Metagenomics, 2022;
6 DOI: 10.3897/mbmg.6.76534 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/02/220221085741.htm

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