By collecting and analyzing DNA floating in the water, researchers working in the Mediterranean found that more species of fish live outside government-protected areas than within them, highlighting a "biodiversity conservation paradox" in marine nature reserves.

The study, published April 28 in Proceedings of the Royal Society B: Biological Sciences, contributes to a growing debate about the efficacy of marine protected areas. 

With less than 6% of the world's oceans under protection, skeptics question whether marine protected areas are enough to ensure species survival. Proponents of marine protected areas maintain that they provide a safe space for marine species. Research has also demonstrated that if protected areas are strategically selected, they can help combat climate change and global loss of biodiversity and other environmental problems.

"[Our results] definitely don't mean that marine protected areas are not working, because when you look at the abundance of certain species and the biomass inside [these areas], many studies have shown that we have higher abundances and higher densities inside protected areas," said first author Emilie Boulanger, a Ph.D. candidate studying marine conservation at the University of Montpellier.

However, the findings do indicate that the effect of marine protection on biodiversity is more complicated than previously thought. To Boulanger, the findings suggest that species richness, the overall number of species in an area without consideration of their populations, "maybe is not the best indicator" of the success of these areas.

The study also fills a methodological gap, as marine protected areas are usually studied with visual observation techniques such as live divers. Submarine cameras can also be used to sample areas where it's unsafe to send humans. But both these methods come with biases.

"They often miss the pelagic species that will pass ahead, or the cryptobenthic species that will be hiding in crevices or under rocks," Boulanger said. "So we wanted to see, OK, what can we learn, if we apply this new method of environmental DNA, which promises to actually detect all the species present in the ecosystem, going from the smallest to the biggest, the resident and the transient."

Environmental DNA refers to genetic material that is sloughed off from cells but persists in the surrounding environment. It has been detected in the air, in soil and even in substances produced by animals, such as honey. However, one of the best places to harvest environmental DNA is the ocean.

"All living things and all species leave traces in their environments," Boulanger said. "In the case of fishes, that can be excrements or gametes or even skin cells or slime. It's kind of like those forensic methods where you go to a crime scene and you look for traces of your criminal you're looking for."

However, instead of dusting for fingerprints or looking at blood spatters, the researchers gathered many liters of seawater from six marine protected areas in the Mediterranean and the surrounding fishable areas. A filtering system onboard the boat allowed the team to parse out the organic material from the water away from the lab, letting the team gather a much more comprehensive set of samples than would otherwise have been possible. 

"We can extract the DNA, we amplify it, and then we can actually target a specific gene that we know is present in all the fishes in the area but sufficiently different, so we can differentiate them," Boulanger said.

This approach, of identifying large groups of organisms from a mixture of their DNA, is called metabarcoding, and it is a common technique when working with environmental DNA because the samples usually contain DNA of many species.

The results of this metabarcoding were not what the researchers expected: They found more species with increasing distance from the protected areas.

"That was very surprising," Boulanger said. "So then we came and looked at, which species are they and why are they outside or inside? And we do find that marine protected areas had more of a tendency to host the more vulnerable, higher-trophic level species, whereas outside we'd find more of those lower-trophic level species that are not targeted by fishing."

In other words, areas protected from fishing seem to best protect larger, predaceous fish. When fishing is allowed and those fish are removed, the smaller species have a greater chance of survival.

Boulanger maintains that the counterintuitive results do not spell doom for marine protected areas. However, she does believe that the findings provide insight that could inform more targeted protection strategies for marine areas, particularly because "protected status" tends to mean different things in different areas.

"Even most protection areas are often not fully protected," she said.

For now, the team is excited about the potential of using environmental DNA and metabarcoding to answer more nuanced questions about what pressures, particularly human pressures, are driving trends in biodiversity around the world.

"There are still a lot of a lot of advances to be made," Boulanger said. "But it's cool that even though everything is not perfectly figured out, we can already start applying it and start looking for new processes and new answers."

The study, "Environmental DNA metabarcoding reveals and unpacks a biodiversity conservation paradox in Mediterranean marine reserves," published April 28 in Proceedings of the Royal Society B: Biological Sciences, was authored by Emilie Boulanger, Nicolas Loiseau, Véronique Arnal, Nacim Guellati, Jean-Baptiste Juhel, Stéphanie Manel and David Mouillot, University of Montpellier; Alice Valentini and Tony Dejean, SPYGEN; Pierre Boissery, Agence de l'Eau Rhône-Méditerranée-Corse; Julie Deter and Florian Holon, Andromède Océanologie; and Philippe Lenfant, University of Perpignan.