The BNIP project: studying the distribution of protected amphibians in 1,000 ponds

Context of the study

The Belgian Nature Integrated Project (BNIP) is a strategic project that aims to contribute to the fulfilment of the goals of Natura 2000 as well as European nature conservation targets thanks to around 50 different initiatives. The project brings together many partners who are actively working together to consolidate the protection of Europe’s most valuable and threatened species and habitats.

Some of the flagship initiatives being pursued within the framework of the project include using techniques exploiting environmental DNA to explore some 1,000 ponds throughout Wallonia. This ambitious inventory programme was designed to improve our understanding of the distribution area of two threatened species: the great crested newt (Triturus cristatus) and the common midwife toad (Alytes obstetricans).

Under the supervision of the Service public de Wallonie, E-BIOM’s scientists worked alongside the non-profit organisation Natagora to complete this major inventory programme.

Mare naturelle
Formation des experts de Natagora

Sampling and laboratory analysis

The E-BIOM team has trained naturalists and trainees from the Natagora association, several Nature Parks (Pays des collines, Plaines de l’Escaut and Burdinale-Mehaigne) and various River Contracts (Dender and Escaut Lys) in taking samples.

More than 30 people took part in this extensive inventory campaign: more than 330 days in the field, nearly 3,000 ponds visited and nearly 1,000 ponds sampled!

Each sampling process involved taking 40ml of water from about 20 locations evenly distributed within the pond, maximising the sub-habitats suitable for the two target species. Once the samples had been taken, the 800ml of water collected was filtered through a filtration capsule to concentrate the DNA.

In parallel, herpetological inventories were carried, identifying the species of amphibians present in certain ponds on the basis of morphological characteristics in order to calibrate the method.

Back in the laboratory, DNA was extracted from the filtration capsules and then amplified through qPCR using specific primers targeting great crested newts and midwife toads. This method, known as environmental DNA barcoding, is particularly well suited to the study of species that are rare or hard to observe.

In order to guarantee the quality of our analyses and the reliability of our data, positive and negative controls were carried out to validate each step of the experimental process.

Filtration de l'eau et concentration de IADN
Alyte accoucheur

Results and conclusion

Calibration of the method has shown that the results obtained by the environmental DNA analysis agree with direct observations in nearly 90% of cases.

The presence of the great crested newt was highlighted in more than 150 pools and that of the midwife toad in more than 200!  Genetic analyses have thus made it possible to review the distribution of the great crested newt and the midwife toad in Wallonia.

The data collected in the form of the presence or absence of the species is used by the LIFE teams to create or restore nearly 500 ponds and the surrounding terrestrial habitats that are suitable for maintaining and developing these two species.