The world's oldest man-made biological experiment

Biological experiments are often short-lived due to logistical or resource-related challenges, and short-term observations are extrapolated to make long-term predictions. However, the effects of experimental treatments on biological communities and processes take time to develop. Consequently, the robustness of conclusions drawn from observations increases with the duration of the experiment. As a striking real-world example and scattered throughout central Laos, thousands of large stone jars have been left behind from ancient burial rituals. The most famous sites in the Xiengkhouang province are collectively referred to as the Plain of Jars. These jars form a massive biological experiment: for ~2000 years, rainwater has interacted with the geological origin of each jar to create unique yet replicated aquatic ecosystems influenced by different tree coverage. The layout of these jars, with clusters of up to several hundred jars separated by several kilometers, allows for controlled testing of multiple questions within ecology and evolution. Here, we report for the first time how these ancient mesocosms can be used to test ecosystem responses to local abiotic variation and disturbance. We show that tree cover dominates every jar ecosystem’s state, and that variations in tree cover density create gradients in O2 and nutrient concentrations among jar ecosystems. These initial findings show that litter contribution to aquatic ecosystems leads to higher nutrient content and reduces O2 concentration, even in systems under different long-term selection in the oldest man-made ecosystems ever analyzed. This first biological analysis provides a fundamental understanding of a unique environment and offers trajectories for future exploration.
Methods
The data was collected during an initial assessment of environmental conditions in jar-based ecosystems on the Plain of Jars:

Five sites, that have been cleared of unexploded ordnance and are therefore now safely accessible, were selected to study the environmental conditions inside the jars. Each site has a different number of available jars, depending on the size of the site and the condition of the jars, as many are broken and therefore unable to hold water. Site 1 is exposed on the plateau, with only a few trees in the vicinity. Ten jars from three different groups (1, 2, and 4) were studied at this site. Sites 2, 3, 21, and 52 are located in the forest and surrounded by trees. We investigated 6 jars at site 2, 6 jars at site 3, and 15 jars at site 52, respectively. At Site 21, two jars were sampled. We present here data from the samplings in November 2022 and September 2023. At each site, jars were randomly selected during the first visit, but based on their ability to hold water, located via GPS, and photographed. The parameters measured on site depend on the sampling period. Documentation included measurement of water depth inside the jars, water temperature, and oxygen. Water samples were taken and stored in the dark in a fridge before being transported to Copenhagen, where they were stored frozen (-20°C; 2022 samples) / in a fridge (4°C, 2023 samples) until analysis. Laboratory measurements included alkalinity, total nitrogen and total phosphorus. Alkalinity was measured using Gran titration (Gran 1952). Total phosphorus and nitrogen were analyzed following Danish Standards (DS221 and DS292) using an AA3 autosampler (Seal Analytic). In November 2022, we also took pictures of the canopy right above the jars. We then used those pictures to create a binary image (black and white) and the percentage of black canopy was used to estimate canopy cover.