Researchers weed out a way to identify plants using environmental DNA

What do you call seaweed that grows in ponds? Pondweed, of course. This diverse group of freshwater plants provides food and shelter for freshwater fish, birds, invertebrates, and plankton. And it’s the focus of high-tech genetic analysis.

The various pondweed species are tuned to certain water conditions, making them excellent indicators of water quality and habitat. The temperature, flow, and chemical composition of the water in a given spot all influence which pondweeds grow there. This variability makes them effectively small green canaries in an aquatic coal mine, and a gauge of the water’s expected biological community.

However, identifying them by sight is difficult for non-experts, and getting to them in the middle of a lake is a challenge even for scientists.

 

Traditional ecological surveys of freshwater communities require many hours of painstaking work, usually by species experts and trained assistants, to identify the species of many specimens collected in the field. With pondweeds, they normally examine the physical features, such as fruiting structures, under a microscope to determine the species.

A research team in Canada has tested the use of environmental DNA (eDNA) to speed the process of identifying and surveying these and other aquatic plants.

The researchers in the current study developed and tested new genetic markers and methods to detect pondweeds using eDNA, which is made up of the fragments of genetic material left behind by plants and animals. It is plentiful in water and soil: cells of tiny organisms, blood, saliva, fish scales, as well as plant spores, pollen, and resin all contain the organism’s unique genetic material that can potentially be obtained from water or soil samples.

A key component of analyzing the DNA of an unknown plant or animals is a reference library of sample DNA sequences of known species to use as a comparison. Unlike animal DNA, no universal plant DNA markers exist that can be applied across a wide number of plant species and still effectively identify samples to the species level.

Lead author Maria Kuzmina and colleagues at the Centre for Biodiversity Genomics at the University of Guelph focused on pondweeds to make their markers as effective as possible in identifying species from the samples.

“The goal was to design a method that can be used to detect rare or endangered species of pondweeds,” Kuzmina said in a statement. “Narrowing the search allowed the method to be more sensitive and interpretation of the results more reliable.”

The scientists tested their method on Ontario’s 30 species of pondweeds (of the roughly 40 found in North America), for which they had to develop the reference library themselves. For each target species, the researchers extracted DNA from three confirmed specimens and designed primers based on regions of the DNA. Finding these sequences in the water samples would indicate the presence of that species. They combined the successful primers into a reference DNA barcode library for the 30 species, which are members of three plant genera: Potamogeton (26 species), Stuckenia (3), and Zannichellia (1).

Armed with the reference library, the research team collected water samples from the Grand River in a local research reserve. eDNA is often comprised of short fragments of genetic material. The researchers amplified these fragments, using their pondweed primers, and recorded species using the local DNA reference library and GenBank, a secure and publicly accessible reference database that houses the Barcode of Wildlife Project’s collection of barcodes.

The research team detected five of the pondweed species in their samples, three of which were new to the reserve, demonstrating that eDNA analysis can detect plant species in water samples, including those missed by earlier studies.

The scientists state in their paper that effectively detecting species using eDNA “strongly” depends on the completeness and accuracy of the reference libraries. With targeted markers and sufficient reference data, though, eDNA analysis has the potential to identify locations of rare and protected species or locations where sensitive species are absent, as well as changes in species presence over time.

“In broader application, the combination of pondweed species may be used as a ‘fingerprint’ of freshwater ecosystems,” Kuzmina said, “indicating quality of water and showing how this system is suitable for other freshwater organisms such as fishes and invertebrates.”

Importantly for researchers is the efficiency that eDNA analysis may be able to provide. Once the primers and reference library exist, scientists can analyze more eDNA samples relatively inexpensively and thus be able to broaden the scale of a given study.

Nevertheless, Kuzmina said, while “eDNA definitely becomes more and more cost-effective for ecological monitoring, it will never replace an expert for accurate identification of an individual specimen. These two approaches exist in two different dimensions, ideally complementing each other.”

Citation

Kuzmina, M. L., Braukmann, T. W., & Zakharov, E. V. (2018). Finding the pond through the weeds: eDNA reveals underestimated diversity of pondweeds. Applications in Plant Sciences, 6(5), e01155.