Extensive practical guide to DNA-based biodiversity assessment methods published as a ‘living’ document by DNAqua-Net COST Action

Between 2016 and 2021, over 500 researchers collaborated within the DNAqua-Net international network, funded by the European Union’s European Cooperation in Science and Technology programme (COST), with the goal to develop and advance biodiversity assessment methods based on analysis of DNA obtained from the environment (e.g. river water) or from unsorted collections of organisms. 

Such innovative methods are a real game changer when it comes to large-scale assessment of biodiversity and ecological monitoring, as collecting environmental samples that are sent to the lab for analysis is much cheaper, faster and non-invasive, compared with capturing and examining live organisms. However, large-scale adoption has been hindered by a lack of standardisation and official guidance. 

Recognising the urgent need to scale up ecological monitoring as we respond to the biodiversity and climate crises, the DNAqua-Net team published a guidance document for the implementation of DNA-based biomonitoring tools.

The guide considers four different types of samples: water, sediments, invertebrate collections and diatoms, and two primary analysis types: single species detection via qPCR and similar targeted methods; and assessment of biological communities via DNA metabarcoding. At each stage of the field and laboratory process the guide sets out the scientific consensus, as well as the choices that need to be made and the trade-offs they entail. In particular, the guide considers how the choices may be influenced by common practical constraints such as logistics, time and budget. Available in an Advanced Book format, the guidelines will be updated as the technology continues to evolve.

Leaders of DNAqua-Net are Prof. Dr. Florian Leese of the University of Duisburg-Essen (Germany) and Dr. Agnès Bouchez of the French National Institute for Agriculture, Food, and Environment (INRAE). The core writing team for the present guide book involves Dr. Micaela Hellström (MIX Research AB, Sweden), Dr. Kat Bruce (NatureMetrics Ltd., UK), Dr. Rosetta Blackman (University of Zurich and EAWAG, Switzerland), Dr. Sarah Bourlat (LIB/Museum Koenig, Germany), and Prof. Kristy Deiner (ETH Zurich and SimplexDNA AG, Switzerland).

“Back in 2016 we realised that all around the globe researchers are testing new eDNA methods, developing individual solutions and products. While this is excellent, we need to reach a consensus and provide this consensus to stakeholders from the applied sectors”, 

says Florian Leese.
This video was created as part of EU COST Action DNAqua-Net (CA15219) and shows how environmental DNA (eDNA) can be sampled and analysed from aquatic ecosystems. It shows the whole cycle from the start to final results. 
Credit: DNAqua-Net

The guide’s lead author Dr. Kat Bruce adds:

“The urgency of addressing the twin biodiversity and climate crises means that we need to accelerate the adoption of new technologies that can provide data and insights at large scales. In doing so, we walk a tricky line to agree on sufficiently standardised methods that can be usefully applied as soon as they add value, while still continuing to develop them further and innovate within the field. It was a daunting task to seek consensus from several hundred scientists working in a fast-moving field, but we found that our technology is based on a strong foundation of knowledge and there was a high level of agreement on the core principles – even if the details vary and different users make different choices depending on their environmental, financial or logistical constraints.”

Looking back on the last four years that culminated in the publication of a “living” research publication, Prof. Dr. Kristy Deiner says:

“The document took many twists and turns through more than ten versions and passionate discussions across many workshops and late night drinks. All in the days when we could linger at conferences without fear of the pandemic weighing on us. As we worked to find consensus, one thing was clear: we had a lot to say and a standard review paper was not going to cut it. With the knowledge and experience gathered across the DNAqua-Net, it made sense to not limit this flow of information, but rather to try and tackle it head on and use it to address the many questions we’ve all struggled with while developing DNA-based biodiversity survey methods.”

Now that the document – or at least its first version – is publicly available, the researchers are already planning for the next steps and challenges.

“The bottom line is we’ve come a long way in the last ten years. We have a buffet of methods for which many produce accurate, reliable and actionable data to the aid of biodiversity monitoring and conservation. While there is still much work to be done, the many unanswered questions are because the uptake is so broad. With this broad uptake comes novel challenges, but also new insights and a diversity of minds with new ideas to address them. As said this is planned to be a living document and we welcome continued inputs no matter how great or small,” says Deiner.

Dr. Micaela Hellström recalls:

“The book evolved over the four years of COST Action DNAqua-Net which made it possible for the many scientists and stakeholders involved to collaborate and exchange knowledge on an unprecedented scale. Our whole team is well aware of the urgent need to monitor biodiversity loss and to provide accurate species distribution information on large scales, to protect the species that are left. This was a strong driving force for all of us involved in the production of this document. We need consensus on how to coherently collect biodiversity data to fully understand changes in nature.”

“It was a great and intense experience to be a part of the five-person core writing team. In the months prior to submitting the document, we spent countless hours, weekends and late nights researching the field, communicating with researchers and stakeholders, and joining vivid Zoom discussions. As a result, the present book provides solid guidance on multiple eDNA monitoring methods that are – or will soon become – available as the field moves forward.” 

***

The DNAqua-Net team invites fellow researchers and practitioners to provide their feedback and personal contributions using the contacts below.

***

Original source:

Bruce K, Blackman R, Bourlat SJ, Hellström AM, Bakker J, Bista I, Bohmann K, Bouchez A, Brys R, Clark K, Elbrecht V, Fazi S, Fonseca V, Hänfling B, Leese F, Mächler E, Mahon AR, Meissner K, Panksep K, Pawlowski J, Schmidt Yáñez P, Seymour M, Thalinger B, Valentini A, Woodcock P, Traugott M, Vasselon V, Deiner K (2021) A practical guide to DNA-based methods for biodiversity assessment. Advanced Books. https://doi.org/10.3897/ab.e68634

Scientists took a rare chance to prove we can quantify biodiversity by ‘testing the water’

Recent study conducted at a UK fishery farm provides new evidence that DNA from water samples can accurately determine fish abundance and biomass

Organisms excrete DNA in their surroundings through metabolic waste, sloughed skin cells or gametes, and this genetic material is referred to as environmental DNA (eDNA).

As eDNA can be collected directly from water, soil or air, and analysed using molecular tools with no need to capture the organisms themselves, this genetic information can be used to report biodiversity in bulk. For instance, the presence of many fish species can be identified simultaneously by sampling and sequencing eDNA from water, while avoiding harmful capture methods, such as netting, trapping or electrofishing, currently used for fish monitoring.

While the eDNA approach has already been applied in a number of studies concerning fish diversity in different types of aquatic habitats: rivers, lakes and marine systems, its efficiency in quantifying species abundance (number of individuals per species) is yet to be determined. Even though previous studies, conducted in controlled aquatic systems, such as aquaria, experimental tanks and artificial ponds, have reported positive correlation between the DNA quantity found in the water and the species abundance, it remains unclear how the results would fare in natural environments.

However, a research team from the University of Hull together with the Environment Agency (United Kingdom), took the rare opportunity to use an invasive species eradication programme carried out in a UK fishery farm as the ultimate case study to evaluate the success rate of eDNA sampling in identifying species abundance in natural aquatic habitats. Their findings were published in the open-access, peer-reviewed journal Metabarcoding and Metagenomics.

“Investigating the quantitative power of eDNA in natural aquatic habitats is difficult, as there is no way to ascertain the real species abundance and biomass (weight) in aquatic systems, unless catching all target organisms out of water and counting/measuring them all,”

explains Cristina Di Muri, PhD student at the University of Hull.
Drained pond after fish translocation.
Photo by Dr. Watson H.V.

During the eradication, the original fish ponds were drained and all fish, except the problematic invasive species: the topmouth gudgeon, were placed in a new pond, while the original ponds were treated with a piscicide to remove the invasive fish. After the eradication, the fish were returned to their original ponds. In the meantime, all individuals were counted, identified and weighed from experts, allowing for the precise estimation of fish abundance and biomass.

“We then carried out our water sampling and ran genetic analysis to assess the diversity and abundance of fish genetic sequences, and compared the results with the manually collected data. We found strong positive correlations between the amount of fish eDNA and the actual fish species biomass and abundance, demonstrating the existence of a strong association between the amount of fish DNA sequences in water and the actual fish abundance in natural aquatic environments,”

reports Di Muri.
Environmental DNA sampling using water collection bottles
Photo by Dr. Peirson G.

The scientists successfully identified all fish species in the ponds: from the most abundant (i.e. 293 carps of 852 kg total weight) to the least abundant ones (i.e. one chub of 0.7 kg), indicating the high accuracy of the non-invasive approach.

“Furthermore, we used different methods of eDNA capture and eDNA storage, and found that results of the genetic analysis were comparable across different eDNA approaches. This consistency allows for a certain flexibility of eDNA protocols, which is fundamental to maintain results comparable across studies and, at the same time, choose the most suitable strategy, based on location surveyed or resources available,”

elaborates Di Muri.

“The opportunity of using eDNA analysis to accurately assess species diversity and abundance in natural environments will drive a step change in future species monitoring programmes, as this non-invasive, flexible tool is adaptable to all aquatic environments and it allows quantitative biodiversity surveillance without hampering the organisms’ welfare.”

***

Original publication:

Di Muri C, Lawson Handley L, Bean CW, Li J, Peirson G, Sellers GS, Walsh K, Watson HV, Winfield IJ, Hänfling B (2020) Read counts from environmental DNA (eDNA) metabarcoding reflect fish abundance and biomass in drained ponds. Metabarcoding and Metagenomics 4: e56959. https://doi.org/10.3897/mbmg.4.56959

Scientists use forensic technology to genetically document infanticide in brown bears

Modern open-source software helped the researchers identify the male that killed a female and her two cubs

Scientists used a technology designed for the purposes of human forensics, to provide the first genetically documented case of infanticide in brown bears, following the murder of a female and her two cubs in Trentino, the Italian Alps, where a small re-introduced population has been genetically monitored for already 20 years.

The study, conducted and authored by Francesca Davoli, The Italian Institute for Environmental Protection and Research (ISPRA), Bologna, and her team, is published in the open access journal Nature Conservation.

To secure their own reproduction, males of some social mammalian species, such as lions and bears, exhibit infanticidal behaviour where they kill the offspring of their competitors, so that they can mate with the females which become fertile again soon after they lose their cubs. However, sometimes females are also killed while trying to protect their young, resulting in a survival threat to small populations and endangered species.

“In isolated populations with a small number of reproductive adults, sexually selected infanticide can negatively impact the long-term conservation of the species, especially in the case where the female is killed while protecting her cubs,” point out the researchers.

“Taking this into account, the genetic identification of the perpetrators could give concrete indications for the management of small populations, for example, placing radio-collars on infanticidal males to track them,” they add. “Nevertheless, genetic studies for identifying infanticidal males have received little attention.”

Thanks to a database containing the genotypes of all bears known to inhabit the study site and an open-source software used to analyse human forensic genetic profiles, the scientists were able to solve the case much like in a television crime series.

orsa occultata - leggeraUpon finding the three corpses, the researchers were certain that the animals had not been killed by a human. In the beginning, the suspects were all male brown bears reported from the area in 2015.

Hoping to isolate the DNA of the perpetrator, the researchers collected three samples of hairs and swabbed the female’s wounds in search for saliva. Dealing with a relatively small population, the scientists expected that the animals would share a genotype to an extent, meaning they needed plenty of samples.

However, while the DNA retrieved from the saliva swabs did point to an adult male, at first glance it seemed that it belonged to the cubs’ father. Later, the scientists puzzled out that the attacker must have injured the cubs and the mother alternately, thus spreading blood containing the inherited genetic material from the father bear. Previous knowledge also excluded the father, since there are no known cases of male bears killing their offspring. In fact, they seem to distinguish their own younglings, even though they most likely recognise the mother.

To successfully determine the attacker, the scientists had to use the very small amount of genetic material from the saliva swabs they managed to collect and conduct a highly sophisticated analysis, in order to obtain four genetic profiles largely overlapping with each other. Then, they compared them against each of the males reported from the area that year. Eventually, they narrowed down the options to an individual listed as M7.

“The monitoring of litters is a fundamental tool for the management of bear populations: it has allowed the authors to genetically confirm the existence of cases of infanticide and in the future may facilitate the retrieval of information necessary to assess the impact of SSI on demographic trends,” conclude the researchers.

###

Original source:

Davoli F, Cozzo M, Angeli F, Groff C, Randi E (2018) Infanticide in brown bear: a case-study in the Italian Alps – Genetic identification of perpetrator and implications in small populations. Nature Conservation 25: 55-75. https://doi.org/10.3897/natureconservation.25.23776