Amphibian biodiversity is in decline worldwide and collecting information about their habitats and populations through 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 new technique to identify amphibian species that live in an area through environmental DNA analysis. It is hoped that the new method will revolutionize species tracking, as it will allow anyone to easily study a habitat by taking water samples.
An international collaborative research group with members from 7 institutions has developed a method to determine which amphibian species (types of frog, newt, and salamander) inhabit an area. They achieved this by amplifying extra-organic DNA (environmental DNA) found in water so that it could be analyzed. This DNA ends up in the water as it is expelled from the amphibian’s body along with mucus and feces. 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 the associate professor NISHIKAWA Kanto (Kyoto University).
The technique developed will solve some of the problems encountered with conventional methods such as capture and sighting surveys, which require a specialized 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 allow anyone to easily monitor the amphibians that inhabit an area by taking water samples.
These research results will be published in the online academic journal Metabarcoding and metagenomics.
- Biodiversity monitoring is vital for the conservation of natural ecosystems as a whole. In particular, the importance of amphibian monitoring is increasing, as the number of individuals is drastically decreasing.
- However, amphibians are nocturnal, young (eg tadpoles) and adults live in different habitats, and specialist knowledge is required to capture individuals and identify their species. These issues make it difficult to accurately track amphibians in a standardized way, so results from individual surveys often contradict each other.
- Environmental DNA (eDNA) is extra-organic 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 put in place and are now commonplace. However, a standardized analysis method for amphibians has not yet been established. In order to contribute to its development, this research group has collected and analyzed the DNA of amphibians to propose an analysis method to find out which species of amphibians inhabit a studied area.
- First, the researchers designed several methods for analyzing amphibian eDNA and evaluated their performance to identify the most effective method. Then, they conducted parallel monitoring of 122 sites in 10 farmlands across Japan using the developed eDNA analysis method and conventional methods (catch net surveys and observation surveys) .
- The newly developed method was able to detect all three orders of amphibians: Caudata (the newt and salamanders), Anura (the frogs) and Gymnophiona (the caecilians). Moreover, this new eDNA analysis method was able to detect more species at all field study sites than conventional surveys based on the method, indicating its effectiveness.
The biodiversity of amphibians continues to decline worldwide and collecting baseline information on their habitats and other aspects through monitoring is vital for conservation efforts. Traditional amphibian monitoring methods include visual and auditory sightings and capture records. However, amphibians tend to be small in size and many are nocturnal. The success of surveys varies greatly depending on climate and season, and specialist knowledge is required to identify species. Therefore, it is difficult to monitor a large area and assess habitats. The last decade has seen the significant development of environmental DNA analysis techniques, which can be used to study the distribution of a species by analyzing external DNA (environmental DNA) that is released into the environment along with feces, mucus and other bodily fluids of an organism. .
The fundamentals of this technique involve collecting water from the study site and analyzing the eDNA it contains to determine what species inhabit the area. In recent years, the technique has gained attention as a supplement to conventional monitoring methods. Standardized analysis methods have already been established for other species, in particular fish, and diversity monitoring using eDNA is becoming commonplace.
However, amphibian eDNA monitoring is still in the development stage. One of the reasons for this is that the proposed eDNA analysis method must be tailored to the target species or taxonomic group, and there are still problems with the development and implementation of a method. comprehensive amphibian detection. If such a method could be developed, it would allow monitoring to be carried out even by people who do not have the specialist knowledge to identify species or the experience of surveying. Hopefully, this would become a unified standard for large-scale surveillance investigations, such as those nationwide. This research group’s efforts to develop and evaluate analytical methods will hopefully lay the foundation for eDNA analysis to become a common tool for monitoring amphibians, as well as fish.
Design and evaluation of a universal primer for amphibians
The researchers obtained a total of 1,034 amphibian DNA sequences from an open online database. They located regions of DNA where the base sequence is the same in different species and designed five analytical systems in the mitochondrial region of 16S rRNA that could fully amplify amphibian DNA. After that, they used a computer to study the various characteristics of each set, including how easily amphibian DNA was amplified, the likelihood that non-amphibian DNA would also be amplified (i.e. specificity) and whether they could reliably determine the species (i.e., species-level resolution). Additionally, they conducted experiments using DNA obtained from individual amphibians to confirm that DNA from various species in the class could be amplified. The results revealed that all tests were able to amplify DNA well. From the results of all these pre-studies, the researchers selected the test that had the best specificity and resolution at the species level, and then conducted a field study to assess its effectiveness as a tool. monitoring.
Validation of efficacy through in-depth comparisons based on field investigations with conventional methods
From June to September 2019, field studies were conducted at 122 sites in 10 agricultural ecosystems (rice paddy ecosystems) in Japan to verify the suitability and effectiveness of the designed test for field monitoring. In the field surveys, eDNA analyzes and surveys based on conventional methods (capture/observations) were carried out in agricultural ponds and streams at each site, and the number and composition of amphibians detected were compared. The number of species detected by eDNA analysis was greater than the number detected by conventional methods at all field study sites. This therefore demonstrates that the eDNA analysis method using amphibian assays developed by this research group is highly effective for field monitoring.
- Researchers have developed an analytical method capable of comprehensively detecting amphibian DNA. The eDNA monitoring system using this method could also contribute to a rapid survey of threatened and important species.
- Unlike conventional survey methods such as capture and observation, eDNA analysis is not easily affected by environmental factors, such as season and climate. Therefore, it is a stable means of detecting amphibians and it is hoped that it will become the standard method for large-scale surveys, such as nationwide surveys.
- Collection of baseline data to determine the species from which the amplified DNA originates would allow this technique to be more effectively applied. This would contribute significantly to amphibian monitoring, as the more baseline data is collected, the greater the range of countries and regions where this technique can be used.
Environmental DNA (eDNA): DNA from organisms found in the environment, such as in 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 allows them to estimate and understand the distribution of the target species. Environmental DNA studies are used in a wide range of research fields including species conservation, ecology, taxonomy, microbiology and paleontology. Previous research using eDNA analysis has been conducted on fish species, and eDNA analysis is becoming a common tool for monitoring organisms. However, such amphibian monitoring methods are still being developed, and a detection system that can be used to conduct in-depth biodiversity surveys has not yet been established.
Metabarcoding (from eDNA): Method for the simultaneous detection of several species. A universal primer consists of regions of nucleotide sequence that are the same for the target taxa. Using this universal primer, assembled DNA from multiple species including those of the target taxa can be amplified by PCR. Then, the sequences of the amplified sections are exhaustively determined using massively parallel sequencing. Species can be identified by matching sequences to those recorded in a database.
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Material provided by Kobe University. Note: Content may be edited for style and length.