Archaeologists from the Ca’ Foscari University of Venice and elsewhere have found traces of indigotin — a blue secondary compound, also known as indigo — on unknapped ground stone tools recovered from Dzudzuana Cave, located in the foothills of the Caucasus in Georgia. Indigotin forms through a reaction between atmospheric oxygen and the natural glycoside precursors in the leaves of Isatis tinctoria. This proves that the plant, despite not being edible, was intentionally processed as early as 34,000 years ago.
Five pebbles from Dzudzuana Cave, Georgia. Image credit: Longo et al., doi: 10.1371/journal.pone.0321262.
Modern humans first appear in the archaeological record around 300,000 years ago, in Africa.
Most of the evidence for their cognitive and technological abilities is based on recovered assemblages of chipped stone artifacts and animal bones since these endure far longer in the archaeological record than plants.
Accordingly, the Paleolithic narrative centers primarily on animal hunting and stone tool manufacture.
Perishable materials, the so-called ‘missing majority,’ notably plants for which there is growing evidence for their use as food, string and cordage, weaving and medicine, are largely missing, creating a partial narrative.
There is therefore, a need to identify and demonstrate the use of plants and the roles they played in a wide range of activities, many of which may still be unknown.
“Rather than viewing plants solely as food resources, as is often the case, we highlight their role in complex operations, likely involving the transformation of perishable materials for use in different phases of daily life among Homo sapiens 34,000 years ago,” said Dr. Laura Longo, an archaeologist at the Ca’ Foscari University of Venice.
“While research continues to improve the identification of elusive plant-derived residues, typically absent from conventional studies, our multi-analytical approach opens new perspectives on the technological and cultural sophistication of Upper Paleolithic populations, who skilfully exploited the inexhaustible resource of plants, fully aware of the power of plants.”
In their study, the researchers examined 34,000-year-old stone tools recovered from Dzudzuana Cave in Georgia.
They found traces of mechanical processing of soft and moist materials, compatible with plant materials such as leaves.
Using various microscopy techniques (optical and confocal), they unexpectedly revealed blue residues — sometimes fibrous — alongside starch grains.
These residues were mainly concentrated in the areas of the tools showing visible wear.
To determine the nature of the blue-colored residues, the scientists employed advanced microspectroscopic techniques, notably Raman and FTIR spectroscopy.
These analyses confirmed the presence of the indigotin chromophore in several samples.
“Once the molecule responsible for the blue colour was identified, a new challenge emerged: how and why did these residues become associated with the working surfaces of the tools?” the authors said.
They then investigated the porosity of the stones — a key factor in their ability to trap and preserve biogenic residues.
Both microscopic fragments of the archaeological tools and larger samples from experimental replicas were analyzed using micro-CT tomography.
The analysis confirmed the presence of pores with volumes suitable for retaining micrometric remains.
As a result, the team designed a series of replicative experiments.
First, raw lithic materials similar to those used by the prehistoric inhabitants of Dzudzuana were sourced.
Pebbles were collected by Nino Jakeli from the Nikrisi River, which runs just below the cave.
Controlled experiments followed, mechanically processing various plants, including those used for fiber production (e.g. bast fibers) and those potentially capable of generating indigotin.
“We used a stringent approach to contamination control and biomolecular analysis to provide evidence for a new perspective on human behavior, and the applied technical and ecological knowledge that is likely to have prevailed in the Upper Paleolithic,” the reserchers said.
“Whether this plant was used as a colourant, as medicine, or indeed for both remains unknown, but offers a new perspective on the fascinating possibilities of non-edible plant use.”
The findings were published online in the journal PLoS ONE.
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L. Longo et al. 2025. Direct evidence for processing Isatis tinctoria L., a non-nutritional plant, 32-34,000 years ago. PLoS One 20 (5): e0321262; doi: 10.1371/journal.pone.0321262