A forensic look at biodiversity

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A syringe is used to pass river water through a filter. Any DNA molecules will r
A syringe is used to pass river water through a filter. Any DNA molecules will remain trapped. (Photograph: ETH Zurich / Annick Ramp)
From the giant blue whale to minuscule microbes, all creatures on this earth continuously shed traces of their DNA. These clues help researchers to determine the degree of biological diversity.

ETH doctoral candidate Anish Kirtane stands in the middle of the River Limmat near the Werdinsel island, below the city of Zurich, wearing a large pair of rubber boots. For an afternoon in late September, the weather is exceptionally warm. Sunlight sparkles on the rippled surface of the water; people loll on the banks of the river, enjoying the autumn sunshine; there are even some intrepid bathers, drifting downstream on the current.

Kirtane dips a measuring beaker into the water, holds it up for inspection, pours off a little, and then wades back to the bank. Awaiting him, in the shade of a willow tree, are the postdoc researcher Cátia Lúcio Pereira and Master’s student Zora Doppmann. They relieve him of the sample. Using a large syringe, Pereira extracts the water and fires it through a flat, square-shaped filter.

Doppmann produces a felt tip and notes the temperature, date, time and place at which the sample was taken. She then casts a final, inquisitive eye over the filter. Is there anything there? As yet, she is unable to say. Back in the lab, however, the three scientists will discover whether it contains any DNA traces from living organisms. That’s what they’re hoping to find - just like the forensic experts in the TV crime series CSI: Miami.

Set the ball rolling

Every living being sheds genetic material into the environment, be it in the form of faeces, flakes of skin, mucus or cells. These molecules of DNA end up in soil, water, the sediment of a lake, the branches of a tree and even in the suspended matter carried in the air.

This project aims to extract DNA molecules from samples collected from the environment and then analyse them according to their fundamental building blocks. Using sophisticated computer programs, the researchers will then compare these DNA sequences with those in reference databases, looking for matches with sequences that are known to belong to a particular species or group of organisms. This will give them an idea of which creatures might be present in a particular area.

Although the method is not new, it is only in recent years that it has started to become established. The first attempts to identify bacteria on the basis of DNA traces in water and soil samples date back to the late 1980s. But it wasn’t until 2008 that European researchers first demonstrated the presence of frog DNA in a sample of water. Since then, this method has taken off.

At the same time, the emergence of new techniques for rapid and comprehensive DNA sequencing has provided a further boost for scientists working on environmental DNA. These include Kristy Deiner, Professor of Environmental DNA at ETH Zurich. She heads up the group that includes Anish Kirtane, Cátia Pereira and Zora Doppmann.

As of 2015, high-throughput sequencers have been routinely used for environmental DNA analysis. These devices can rapidly decode unsorted mixtures containing millions of different DNA molecules in just one run. "We used to have to separate each strand of DNA from the others and purify it before we could analyse its sequence," Deiner explains. "This is the technical revolution that really set the ball rolling."

Cheap and fast

In the meantime, the three researchers have delivered their samples to the lab. Pereira and Doppmann are in the clean room. Dressed in white protective suits, they look like astronauts. Through a pane of glass, Kirtane watches his colleagues process the filter pad and treat it with solutions to wash out material containing DNA. They then purify and prepare the samples so that the solution contains only DNA.

"It’s vital to prevent any contamination," Doppmann explains. Even a minuscule trace of DNA, either from themselves or from outside, could render the samples useless. Before entering the clean room, the researchers must therefore pass through an airlock and put on protective suits, all of which takes time. Furthermore, all the air pumped into the clean room is first filtered. At night, UV lamps are turned on to break down any DNA molecules that might have been accidentally introduced. After each test, all the surfaces must be cleaned with bleach.

Working with genetic material gathered from the environment remains a complex and expensive procedure, not least because it requires a sophisticated lab infrastructure, special chemicals and expensive instruments. Nevertheless, this new approach is cheaper and faster than traditional methods that involve collecting and possibly killing organisms in order to determine their species. "Environmental DNA analysis is non-invasive," Deiner emphasises. "No harm comes to any animal or plant when we extract their DNA from water or soil samples." Moreover, researchers require only a very small amount of DNA to determine a species.

And sample collection is easy - a fact that Deiner and her team intend to exploit. In a soon to be launched project, for which she has secured an ERC Starting Grant, Deiner plans to extend this form of research beyond the scientific community and enlist the support of informal helpers worldwide. On the International Day for Biological Diversity (22 May 2024), volunteers will be asked to take water samples from 1,200 lakes around the globe, filter the water on site and then send the filters to ETH Zurich for analysis. There, any DNA will be extracted, decoded and compared with reference data. "It’s a great example of a citizen-science project," says Pereira, who is coordinating the project and will help with analysis.

One goal is to identify as many species as possible and compare species diversity at the different collection sites. In addition, the researchers will investigate whether a monitoring system based on environmental DNA is feasible on a global scale. In return for their help, participants will receive access to the data and information about which species have been detected in their samples.

Alphabet soup on the screen

Once the researchers have peeled off their protective suits, they take the samples down to the Genetic Diversity Centre, two floors below. One of the rooms contains a seemingly unremarkable piece of equipment that is, in fact, one of the expensive DNA sequencers. "Once we’ve been in here, we’re not allowed back into the clean room, even if we’ve forgotten something," Pereira explains. "So, we have to make sure we plan everything properly."

She taps the screen in front of her with her index finger. The computer has generated DNA sequences from a previous water sample. The document onscreen shows endless sequences of the same four letters, A, C, G and T, which stand for the four building blocks of DNA. A comparison with reference data reveals that one of the sequences can be assigned to a tree - the sycamore - and another to a stinging nettle. Other sequences have no name. "There are still a lot of gaps in the reference database, so we can’t always say which species or group the sequences belong to," she says. The research team therefore hopes that one day other researchers will systematically process genome reference data from a wide variety of organisms and then store this in publicly accessible databases.

Despite these shortcomings, Pereira firmly believes that the environmental DNA method is fundamentally changing the way science captures biodiversity. "It won’t ever replace traditional methods," she says. "But the e-DNA approach will certainly complement them. We’re always going to need experts in taxonomy and ecology, because a species list only ever makes sense in terms of the specific habitat."
Peter Rüegg