Tag: metabarcoding

Shifting sands: how coastal protection affects unseen beach life

Sand nourishments are routinely applied coastal protection measures that have a significant impact on meiofauna communities.

Guest blog post by Iryna Kapshyna, Gritta Veit-Köhler, Leon Hoffman and Sahar Khodami.

During a relaxing beach vacation, most people probably give little thought to whether the beach would still be there if it was not regularly replenished.

In fact, sand nourishment is a common and frequently used coastal protection measure whereby sand is sucked up from the seabed by a flushing ship, transported to the coast, washed up and spread with bulldozers.

An infographic showing different levels of sand on a beach during the sand nourishment process.
Beach extent before and its changes after sand nourishment: Immediately before the sand nourishment (T0, beige); After the sand nourishment the beach reached its maximum extent (T1, burgundy); Two more sampling dates (T2 and T3, dark and light pink).

Due to continuous erosion – the removal of sand by storms, waves and currents – sand nourishment has to be repeated regularly. Otherwise, uncontrolled erosion would mean the loss of beaches, shore areas, coastal cliffs and dunes.

But, while they are important, coastal protection measures such as sand nourishments incur high costs and often lead to the disturbance of ecosystems.

In a study published in the open-access journal Metabarcoding & Metagenomics, A research team from Senckenberg am Meer in Wilhelmshaven show how such disturbances and the subsequent recovery can be monitored efficiently and reliably.

At Ahrenshoop on the Baltic Sea, researchers investigated the effects of sand nourishment on the meiofauna – organisms less than one millimeter in size – and found significant results.

Two women collecting sand samples on a beach.,
We took a total of 246 sediment samples for the investigation of meiofauna from the beach at Ahrenshoop and analysed them in the laboratory in Wilhelmshaven. Photo: Leon Hoffman

“State-of-the-art genetic methods and the traditional method of identifying and counting the animals under the microscope show the same result. The communities of meiofauna changed drastically after the sand was washed up and slowly recovered over the course of a year.”

Project coordinator, Dr Gritta Veit-Köhler.

Immediately after the impact, mites (Acari) and annelid worms (Annelida) had almost completely disappeared from the swash zone, copepods (Copepoda) declined significantly, while the number of flatworms (Platyhelminthes) increased.

Meiofauna organisms are the most numerous animals on the seabed and play an important role in the food webs there. They are well suited as ‘bioindicators’ to detect and study environmental impacts and various forms of ecosystem disturbance, including those caused by humans. Due to their small body size, ubiquity and large numbers, their communities can be studied with small sample sizes.

Over a period of one and a half years, the researchers took and analysed a total of 246 sand samples from the beach-water interface.

“Using the classic taxonomic method, we identified 27,445 individuals under the microscope, which we assigned to ten higher taxonomic groups such as nematodes and copepods. But it was only the genetic analysis that brought the full diversity of species to light.”

Iryna Kapshyna, doctoral student and first author of the study.
Meiofauna organisms collected on the beach at Ahrenshoop. Six purple organisms on a black background.
Meiofauna organisms collected on the beach at Ahrenshoop. Top left: Tardigrade, flatworm, top right: copepod, nematode, bottom left to right: marine mite, annelid. Scales 0.1 mm each. Images: Iryna Kapshyna, Olena Uzun, Tobias Fischer

Using the ‘metabarcoding’ method, in which all animals in a sample are analysed together and differences in specific gene segments (here V1&V2) are searched for, a large number of samples can be analysed quickly and reliably.

The researchers were able to identify a total of 843 so-called ‘operational taxonomic units’ (OTUs) – in simple terms, different species.

“843 species sounds like a lot – but in fact, the beach studied had a lower diversity of meiofauna compared to the deep sea or other marine areas.”

Dr Sahar Khodami, Senckenberg am Meer.

The size of the organisms studied means they have previoulsy been difficult to study and have not received as much attention as larger species.

“When considering the effects of coastal protection measures on ecosystems, the smallest marine animals should not be overlooked! Metabarcoding can replace the traditional morphological method, after initial studies using both methods.”

The research team.

Original study:

Kapshyna I, Veit-Köhler G, Hoffman L, Khodami S (2024) Impact of a coastal protection measure on sandy-beach meiofauna at Ahrenshoop (Baltic Sea, Germany): results from metabarcoding and morphological approaches are similar. Metabarcoding and Metagenomics 8: e127688. https://doi.org/10.3897/mbmg.8.127688

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Celebrating scientific excellence: Dr. Paul D. N. Hebert awarded the Benjamin Franklin Medal

Dr. Paul D. N. Hebert, known as “the father of DNA barcoding,” has been honoured with the prestigious Benjamin Franklin Medal, a testament to his trailblazing contributions to biodiversity science.

Dr. Paul D. N. Hebert. Photo credit Åge Hojem, NTNU Vitenskapsmuseet/NTNU University Museum, used under a CC BY 2.0 licence

Dr. Hebert’s innovative work has advanced our understanding of global biodiversity, making the identification of species easier, which in turn helps support global conservation efforts. By devising a method that allows the quick and efficient discerning of species, he has transformed biodiversity science.

DNA barcoding has many applications in the classification and monitoring of biodiversity. It can help protect endangered species, control agriculture pests, and identify disease vectors.

Founder and Director of the Centre for Biodiversity Genomics and Chief Executive Officer of the International Barcode of Life consortium (iBOL), Dr. Hebert is one of the leading voices of today’s biodiversity innovation and research.

Dr. Hebert is also chair of the advisory board of Pensoft’s journal Metabarcoding and Metagenomics. He has authored 13 papers in ZooKeys, substantially contributing to untangling the taxonomy of braconid wasps, butterflies, and other insects.

Acylomus ergoti, one of the many insect species Dr. Hebert has worked on.

His work has also appeared in other Pensoft-published journals, such as Biodiversity Data Journal, Nota Lepidopterologica, and Deutsche Entomologische Zeitschrift.

His innovative approach has sparked discussions and debates around the role of novel methodologies in taxonomy.

Dr. Hebert’s recognition with the Benjamin Franklin Medal demonstrates the critical role of biodiversity studies in dealing with global challenges such as the biodiversity crisis. He has inspired a generation of scientists to push the boundaries of knowledge and drive innovation in research technology.

We at Pensoft extend our heartfelt congratulations to Dr. Paul D. N. Hebert on this well-deserved recognition. He continues to lead the way in unravelling the complexities of global biodiversity.

All good things come from above! DNA-based food analysis in the Leisler’s bat

Through the analysis of DNA traces in the droppings of a Leisler’s bat colony, researchers at LIB have now identified over 350 different insect species that were consumed by the bats.

Adequate food supply is a fundamental need and requirement for survival. To protect a species, it is often very helpful to know what that species prefers and frequently consumes. Through the analysis of DNA traces in the droppings of a Leisler’s bat colony, researchers at LIB (Leibniz Institute for the Analysis of Biodiversity Change) have now identified an astonishingly high number —over 350— different insect species that were consumed by the bats.

Portrait of the studied species Nyctalus leisleri, Leisler’s bat. © M. Koch

Especially for small animal species and those that are nocturnal, it can be extremely difficult to determine what they feed on. Identifying small prey insects or their remains is also rarely possible down to the exact species or family. In the case of the studied bat species, there is the additional challenge that it is a forest bat species that needs to be located first. “Following bats equipped with radio transmitters in the forest at night is quite special,” says Martin Koch, co-initiator of the study.

Design and installation of the guano trap (3 m) and roost entrance (9 m).

Fortunately — but also complicating matters — there are about 13 different bat species living in the investigated area near Bonn, in the forests of the Natura 2000 area ‘Waldreservat Kottenforst.’ Initially, as part of an EU Life+ project, roosts — the trees where the bats live — of the Leisler’s bats were identified, from which the study’s starting material was then obtained. This was done using a specially developed “guano trap.” The trap consists of approximately 2.2 square meters of mosquito netting stretched rectangularly.

It was installed about 3 meters high on the tree trunk, below the entrance to the roosting cavity at about 9 meters high. During the so-called “twilight swarming” after the nightly insect hunt, the bats return to the roosting cavity and initially circle the tree. They frequently perch briefly next to the cavity entrance and stick a small guano pellet to the trunk. Regularly, pellets fall and land in the mosquito netting under the cavity entrance. This “bat guano” was collected, fixed, and further processed in the laboratory.

“It’s fascinating how much DNA you can extract from a small amount of droppings and how much information we can draw from the DNA: from which bat species does the droppings come, and what has the bat eaten?” explains Dr. Kathrin Langen. Using the DNA contained in the droppings, our researchers were able to determine nine samples from nine different nights when only the target species swarmed around the roosting tree. On six other nights, other bats and a species of mouse were also active around the roosting tree. From the nine samples containing only the guano of the evening bat, an astonishingly rich menu was then reconstructed: the group consumed at least 126 different species of moths, 86 different species of flies and mosquitoes, 48 species of beetles, and a few dozen other various species of bugs, mayflies, caddisflies, and lacewings. Occasionally, spiders, harvestmen, lice, and other small animals were also consumed.

Timeline showing arthropod community composition at order level in the guano of N. leisleri, all three markers combined (COImldg, COIArt, 16S). With the exception of plots showing RRA assigned to major groups depending on sampling date (4C and 4F), read counts were not taken into account. A, D Number of species of each arthropod order detected at each time point; B, E Relative number of species per arthropod order as a percentage of the diet; C, F Species detected in each arthropod order, based on relative read abundances.

From the results, the team was able to deduce which of the three molecular genetic markers used worked best and provided the most species detections, a total of 358. “It’s incredibly satisfying to see what species lists come out at the end of all the lab work and bioinformatics,” says Dr. Sarah Bourlat, Head of the Metabarcoding Section at LIB, Bonn. However, the temporal course of the composition of the consumed insects was also interesting to observe: from late March to late June, the number of species in the guano steadily increases, only to decrease again by mid-August. This aligns very well with the activity patterns of certain insect groups.

The beech moth was the most frequently consumed butterfly, and a mayfly known as the transient virgin or ‘Uferaas’, was the most frequently consumed mayfly. The author team has listed the most important ecological parameters for the 18 key prey species in the study to contribute to better protecting the Leisler’s bat and the habitats needed by its prey insects.

Research article:
Bourlat SJ, Koch M, Kirse A, Langen K, Espeland M, Giebner H, Decher J, Ssymank A, Fonseca VG (2023) Metabarcoding dietary analysis in the insectivorous bat Nyctalus leisleri and implications for conservation. Biodiversity Data Journal 11: e111146. https://doi.org/10.3897/BDJ.11.e111146

News announcement originally published by the Leibniz Institute for the Analysis of Biodiversity Change. Republished with permission.

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Biodiversity in a bird’s nest: DNA as a tool for bird conservation

Researchers employ eDNA to investigate ‘fascinating microcosms’ inside birds’ nests.

Researchers have found that advanced DNA technologies can get a detailed snapshot of insect diversity within a bird’s nest, showing everything from the bird’s last meal to disease-causing parasites.

A bird’s nest. Photo by Farrini

“Birds’ nests are fascinating microcosms, but until now, studies have only examined the living insects that can be seen crawling and flying around the nests,” says Valerie Levesque-Beaudin, lead author on the study and a leading expert in Diptera taxonomy at the Centre for Biodiversity Genomics (CBG) at the University of Guelph (U of G).

With newer DNA-based methods, researchers can pick up traces of environmental DNA to get a snapshot of all the species in these tiny ecosystems. “The analysis of nest contents and environmental DNA, or ‘eDNA’ as it’s called, via metabarcoding helps us to gain more insight into a bird’s diet, parasites, and other factors that could impact a bird’s health and breeding success,” says Levesque-Beaudin.

For the study, published in Metabarcoding and Metagenomics, researchers collected 20 birds’ nests from the 162-hectare Arboretum at U of G. They examined the nests using DNA barcoding to identify insects to species and DNA metabarcoding to look at the entire nest ecosystem.

Organisms leave traces of DNA behind as they move through the environment, and researchers can use metabarcoding to build a comprehensive picture of life in the nest. Metabarcoding pulls all DNA traces in a bulk sample – in this case, parts of dead insects, debris, and dust from birds’ nests. This method differs from DNA barcoding, where a single specimen – an insect in this case – is DNA sequenced to identify it to species level.

The CBG team used emergence traps for a first sweep of the nest’s contents followed by a second, deeper probe using DNA metabarcoding to identify all the species encountered in the nest. Researchers passed the nests through a sieve, collecting insect remains and the dust for DNA extraction. “We not only found insects making a living in the nest, but traces of prey, parasites, and many other things,” says Levesque-Beaudin. “The most unexpected was the amount of information gained on other birds’ species whose feathers were either used for nest building or whose nests were essentially overbuilt by the nesting species.”

A tent-like emergence trap is used to contain and collect insects living inside the nest debris. PHOTO: VALERIE LEVESQUE-BEAUDIN

“This approach has the potential to revolutionize how we study bird nests as a micro-ecosystem. It unravels connections between different ecological guilds within the nest and connections of the birds with their environment, which would otherwise remain hidden,” says Dr. Bettina Thalinger, senior author of the study.

The CBG’s Associate Director of Analytics, Dr. Dirk Steinke, says the study has positive implications for bird conservation efforts. He says his students have already begun looking at American Kestrels, a threatened bird of prey, to find out if there are clues in the nest communities via metabarcoding and if DNA can help scientists determine if lack of prey or increased parasitism could be among the causes of nestling mortalities.

Darwin’s finch, also called Galápagos finch. PHOTO BY CHRIS HO, CENTRE FOR BIODIVERSITY GENOMICS.

Galapagos finches are another species threatened by the avian vampire fly – a parasite that attacks nestlings – and treatments include pesticides. Steinke notes that one of his graduate students has begun using DNA metabarcoding in the finches’ nests to understand better the potential impact of pesticide treatment on the entire arthropod nest community.

Research article:

Levesque-Beaudin V, Steinke D, Böcker M, Thalinger B (2023) Unravelling bird nest arthropod community structure using metabarcoding. Metabarcoding and Metagenomics 7: e103279. https://doi.org/10.3897/mbmg.7.103279

News piece originally published by the Centre of Biodiversity Genomics. Republished with permission.

Plants quick to let their flowers fade for protection, show new field experiments and microbiome

Microbes growing on flowers have adverse effects on their fruit yields. This is why plants are quick to shed their flowers, reveals a new study involving both field experiments and plant microbiome analyses.

The present study looked into the wild ginger in Japan (Alpinia japonica, Zingiberaceae). Its flowers open in the morning and wither around sunset, as many one-day flower plants do.
Photo by Shoko Sakai.

Microbes growing on flowers have adverse effects on their yields. This is why plants are quick to shed their flowers, reveals a new study involving both field experiments and plant microbiome analysis.

The findings – made by a joint team of researchers from the Centre for Research on Ecology and Forestry Applications (CREAF, Spain) and Kyoto University (Japan) – are published in the open-access scholarly journal Metabarcoding and Metagenomics.

Scientifically speaking, flowers are a reproductive structure of a plant. Unlike mammals, though, perennial plants develop those de novo every season and only retain them for as long as needed.

While a few earlier studies have already looked into the variation in flower lifespan among species, they were mainly concerned with the tradeoff between plants spending energy on producing and maintaining their flowers, and the benefit they would achieve from retaining their reproductive organs.

Most flowers complete their role and wither or drop within only several days or even less.
Photo by Shoko Sakai.

Prior to the present study, however, the team found another perspective to look at the phenomenon: why did plants invest their energy –  even if the ‘cost’ was minimal – to produce fragile flowers that would wither in a matter of days, rather than investing a bit more of it to produce a lot more durable ones, thereby increasing their reproductive success?

“Interestingly, flower lifespan is negatively correlated with temperature; the hotter the environment where they bloom, the shorter the period a plant retains them. The phenomenon has been known for a long time.

Then, at some point, I came up with the hypothesis that antagonistic microbes, such as bacteria and fungi growing on flowers after the flower bud opens, must be the driver that shortens the lifespan of a flower. I doubted that it was a coincidence that microbes grow faster in higher temperatures,”

comments Shoko Sakai, author of the present study.

Flowers provide various habitats for microbes. They attract pollinators by secreting nectar, which is rich in sugars, and often contains other nutrients, such as amino acids and lipids. The stigma is a germination bed for pollen grains connected to a growth chamber for pollen tubes. It maintains humidity and nutrients necessary for pollen tube growth. Not surprisingly, abundance of the microbes increases over time on individual flowers after it opens.

Before jumping to their conclusions, the scientists set out to conduct field experiments to see what microbial communities would appear on flowers if their longevity was prolonged.

To do this, they took microbes from old flowers of wild ginger (Alpinia japonica) – a species found in Japan and blooming in the early summer when the hot and humid weather in the country is ideal for microbial growth. Then, they transferred the microbes to other wild ginger plants, whose flowers had just opened.

In line with their initial hypothesis, the research team noted that the plant produced significantly fewer fruits, yet there were no visible symptoms on the flowers or fruits to suggest a disease. However, an analysis of the plants’ microbiomes revealed the presence of several groups of bacteria that were increasing with time. As these bacteria can also be found on the flower buds of flowers that have not been treated, the bacteria is categorised as “resident” for the plant.

“So far, flower characteristics have mostly been studied in the context of their interactions with pollinators. Recent studies have raised the question whether we have overlooked the roles of microbes in the studies of floral characteristics.

For example, flower volatiles – which are often regarded as a primary pollinator attractant – can also function to suppress antagonistic microbes. The impacts of microbes on plant reproductive ecology may be more deeply embedded in the evolution of angiosperms than we have considered,”

Sakai concludes.
Flowers have various organs rich in nutrients, and each organ harbours a distinct microbiome. Flower visitors transfer microbes between and within flowers.
Photo by Shoko Sakai.

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Research article:

Jiménez Elvira N, Ushio M, Sakai S (2022) Are microbes growing on flowers evil? Effects of old flower microbes on fruit set in a wild ginger with one-day flowers, Alpinia japonica (Zingiberaceae). Metabarcoding and Metagenomics 6: e84331. https://doi.org/10.3897/mbmg.6.84331

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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

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.” 

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

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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

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 Metagenomics, German 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

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

DNA metabarcoding detects ecological stress within freshwater species

Metabarcoding allows scientists to extract DNA from the environment, in order to rapidly detect species inhabiting a particular habitat. While the method is a great tool that facilitates conservation activities, few studies have looked into its applicability in monitoring species’ populations and their genetic diversity, which could actually be critical to assess negative trends early on. The potential of the method is confirmed in a new study, published in the peer-reviewed scholarly journal Metabarcoding & Metagenomics.

In a new study, German scientists confirm that responses below species level can be inferred with DNA metabarcoding

Metabarcoding allows scientists to extract DNA from the environment (known as environmental DNA or eDNA), for example, river water or, as in the case of the study by the team from the University of Duisburg-Essen (Essen, Germany) within the German Barcode of Life project (GBOL II): Vera Zizka, Dr Martina Weiss and Prof Florian Leese, from individuals in bulk samples. Thus, they are able to detect what species inhabit a particular habitat.

However, while the method has already been known to be of great use in getting an approximate picture of local fauna, hence facilitating conservation prioritisation, few studies have looked into its applicability to infer responses below species level. That is, how the populations of a particular species fare in the environment of interest, also referred to as intraspecific diversity. Meanwhile, the latter could actually be a lot more efficient in ecosystem monitoring and, consequently, biodiversity loss mitigation.

The potential of the method is confirmed in a new study, published in the peer-reviewed scholarly journal Metabarcoding & Metagenomics. To do so, the researchers surveyed the populations of macroinvertebrate species (macrozoobenthos) in three German rivers: Emscher, Ennepe and Sieg, where each is subject to a different level of ecological disturbance. They were looking specifically at species reported at all of the survey sites by studying the number of different haplotypes (a set of DNA variations usually inherited together from the maternal parent) in each sample. The researchers point out that macrozoobenthos play a key role in freshwater ecosystem functionality and include a wide range of taxonomic groups with often narrow and specific demands with respect to habitat conditions.

“As the most basal level of biodiversity, genetic diversity within species is typically the first to decrease, and the last to regenerate, after stressor’s impact. It consequently provides a proxy for environmental impacts on communities long before, or even if never visible on species diversity level,”

explain the scientists.

Emscher is an urban stream in the Ruhr Metropolitan Area that has been used as an open sewage channel for the past hundred years, and is considered to be a very disturbed environment. Ennepe – regarded as moderately stressed – runs through both rural and urban sites, including ones with sewage treatment plant inflow. Meanwhile, Sieg is considered as a stable, near-natural river system with a good ecological and chemical status.

As a result, despite their original assumption that Sieg would support the most prominent diversity within populations of species sensitive to organic pollution, such as mayflies, stoneflies and caddisflies, the scientists reported no significant difference to the medium stressed river Ennepe. This was also true for overall biodiversity. On the other hand, the team discovered higher intraspecific diversity for species resilient to ecological disturbance like small worms and specialised crustaceans in the heavily disturbed Emscher. The latter phenomenon may be explained with low competition pressure for these species, their ability to use organic compounds as resources and, consequently, increased population growth.

“[T]his pioneer study shows that the extraction of intraspecific genetic variation, so-called ‘haplotypes’ from DNA metabarcoding datasets is a promising source of information to assess intraspecific diversity changes in response to environmental impacts for a whole metacommunity simultaneously,”

conclude the scientists.

However, the researchers also note limitations of their study, including the exclusion of specialist species that only occured at single sites. They prompt future studies to also carefully control for the individual number of specimens per species to quantify genetic diversity change specifically.

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Original source:

Zizka VMA, Weiss M, Leese F (2020) Can metabarcoding resolve intraspecific genetic diversity changes to environmental stressors? A test case using river macrozoobenthos. Metabarcoding and Metagenomics 4: e51925. https://doi.org/10.3897/mbmg.4.51925