Simplified method to survey amphibians will aid conservation

Researchers developed a method to determine which amphibians inhabit a specific area. The new technique will resolve some of the issues with conventional methods, such as capture and observational surveys.

Ryukyu Sword Tailed Newt, or Firebellied Newt. Photo by Neil Dalphin via Creative Commons CC0.

An international collaborative research group of members from seven institutions has developed a method to determine which amphibians (frogs, newts and salamanders) inhabit a specific area. Their work was published in the open-access, peer-reviewed journal Metabarcoding and Metagenomics (MBMG).

To do so, the scientists amplified and analysed extra-organismal DNA (also known as environmental DNA or eDNA) found in the water. This DNA ends up in the water after being 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 newly 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 due to environmental factors, such as climate and season.

The researchers hope that the new method will revolutionise species monitoring, as it will enable anyone to easily monitor the amphibians that inhabit an area by collecting water samples.  

While monitoring in general is crucial to conserve the natural ecosystems, the importance of surveying amphibians is even more pressing, given the pace of their populations’ decline.

Amongst major obstacles to amphibian monitoring, however, are the facts that they are nocturnal; their young (e.g. tadpoles) and adults live in different habitats; and that specialist knowledge is required to capture individuals and identify their species. These issues make it particularly difficult to accurately survey amphibians in a standardised way, and results of individual efforts often contradict each other.

On the other hand, eDNA analysis techniques have already been established in programmes targeted at monitoring fish species, where they are already commonplace. So, the researchers behind the present study joined forces to contribute towards the development of a similar standardised analysis method for amphibians.

First of all, the researchers designed multiple methods for analysing 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 along with the conventional methods (i.e. capture surveys using a net and observation surveys). 

As a result, the newly developed method was able to detect all three orders of amphibians: Caudata (the newts 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. Standardised methods of analysis have already been established for other species, especially fishes, and diversity monitoring using eDNA is becoming commonplace. 

However, eDNA monitoring of amphibians is still at the development 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 specialised 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. 

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Research article: 

Sakata MK, Kawata MU, Kurabayashi A, Kurita T, Nakamura M, Shirako T, Kakehashi R, Nishikawa K, Hossman MY, Nishijima T, Kabamoto J, Miya M, Minamoto T (2022) Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia. Metabarcoding and Metagenomics 6: e76534. https://doi.org/10.3897/mbmg.6.76534

One water bucket to find them all: Detecting fish, mammals, and birds from a single sample

Revolutionary environmental DNA analysis holds great potential for the future of biodiversity monitoring, concludes a new study.

Revolutionary environmental DNA analysis holds great potential for the future of biodiversity monitoring, concludes a new study

Collection of water samples for eDNA metabarcoding bioassessment.
Photo by Till-Hendrik Macher.

In times of exacerbating biodiversity loss, reliable data on species occurrence are essential, in order for prompt and adequate conservation actions to be initiated. This is especially true for freshwater ecosystems, which are particularly vulnerable and threatened by anthropogenic impacts. Their ecological status has already been highlighted as a top priority by multiple national and international directives, such as the European Water Framework Directive.

However, traditional monitoring methods, such as electrofishing, trapping methods, or observation-based assessments, which are the current status-quo in fish monitoring, are often time- and cost-consuming. As a result, over the last decade, scientists progressively agree that we need a more comprehensive and holistic method to assess freshwater biodiversity.

Meanwhile, recent studies have continuously been demonstrating that eDNA metabarcoding analyses, where DNA traces found in the water are used to identify what organisms live there, is an efficient method to capture aquatic biodiversity in a fast, reliable, non-invasive and relatively low-cost manner. In such metabarcoding studies, scientists sample, collect and sequence DNA, so that they can compare it with existing databases and identify the source organisms.

Furthermore, as eDNA metabarcoding assessments use samples from water, often streams, located at the lowest point, one such sample usually contains not only traces of specimens that come into direct contact with water, for example, by swimming or drinking, but also collects traces of terrestrial species indirectly via rainfalls, snowmelt, groundwaters etc. 

In standard fish eDNA metabarcoding assessments, these ‘bycatch data’ are typically left aside. Yet, from a viewpoint of a more holistic biodiversity monitoring, they hold immense potential to also detect the presence of terrestrial and semi-terrestrial species in the catchment.

In their new study, reported in the open-access scholarly journal Metabarcoding and MetagenomicsGerman researchers from the University of Duisburg-Essen and the German Environment Agency successfully detected an astonishing quantity of the local mammals and birds native to the Saxony-Anhalt state by collecting as much as 18 litres of water from across a two-kilometre stretch along the river Mulde.

After water filtration the eDNA filter is preserved in ethanol until further processing in the lab.
Photo by Till-Hendrik Macher.

In fact, it took only one day for the team, led by Till-Hendrik Macher, PhD student in the German Federal Environmental Agency-funded GeDNA project, to collect the samples. Using metabarcoding to analyse the DNA from the samples, the researchers identified as much as 50% of the fishes, 22% of the mammal species, and 7.4% of the breeding bird species in the region. 

However, the team also concluded that while it would normally take only 10 litres of water to assess the aquatic and semi-terrestrial fauna, terrestrial species required significantly more sampling.

Unlocking data from the increasingly available fish eDNA metabarcoding information enables synergies among terrestrial and aquatic biodiversity monitoring programs, adding further important information on species diversity in space and time. 

“We thus encourage to exploit fish eDNA metabarcoding biodiversity monitoring data to inform other conservation programs,”

says lead author Till-Hendrik Macher. 

“For that purpose, however, it is essential that eDNA data is jointly stored and accessible for different biodiversity monitoring and biodiversity assessment campaigns, either at state, federal, or international level,”

concludes Florian Leese, who coordinates the project.

Original source:

Macher T-H, Schütz R, Arle J, Beermann AJ, Koschorreck J, Leese F (2021) Beyond fish eDNA metabarcoding: Field replicates disproportionately improve the detection of stream associated vertebrate species. Metabarcoding and Metagenomics 5: e66557. https://doi.org/10.3897/mbmg.5.66557