The oldest traces of blue indigo on 34,000-year-old grinding tools: New Scenarios for the use of plants in the Palaeolithic

An international research team coordinated by Ca’ Foscari University of Venice has identified the presence of indigotin - a blue dye compound - on stone pebbles dating back to the Upper Palaeolithic. This molecule, derived from the leaves of Isatis tinctoria L., a biennial plant in the Brassicaceae family native to the Caucasus and commonly known as woad, was found on ground stone tools.

This is the first time indigotin, a blue secondary compound, also known as indigo, has been identified on such ancient artefacts. The molecule forms through a reaction between atmospheric oxygen and the natural glycoside precursors in Isatis tinctoria L. leaves, released from the cellular vacuoles. This proves that the plant, despite not being edible, was intentionally processed as early as 34,000 years ago.

This discovery, published in the scientific journal PLOS One, offers new insights into the complexity of early Homo sapiens' interaction with plant resources—not only as food but also for more sophisticated uses such as dyeing and medicine. Indeed, Isatis tinctoria L., has a long history of use in both dyeing and healing.

“Rather than viewing plants solely as food resources, as is often the case,” explains Laura Longo, archaeologist at Ca’ Foscari University of Venice, “this study highlights 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. 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 Palaeolithic populations, who skilfully exploited the inexhaustible resource of plants, fully aware of the power of plants.”

Stone Tools from the Caucasus

The discovery was made through microscopic analyses of ancient unknapped ground stone tools recovered from Dzudzuana Cave, located in the foothills of the Caucasus in Georgia. The tools were excavated from a layer dating to around 34,000 years ago, during excavations conducted in the 2000s under the direction of Ofer Bar-Yosef (Harvard University, USA), Tengiz Meshveliani, Nino Jakeli (Georgian National Museum), and Anna Belfer-Cohen (Hebrew University of Jerusalem, Israel).

The stone collection was studied and sampled at the Georgian National Museum in Tbilisi by archaeologist Laura Longo (Ca’ Foscari University of Venice), who, together with scientist Elena Badetti, conducted the research thanks to funding from The Leakey Foundation (USA) and the Italian Archaeological Unit, as part of the Ministry of Foreign Affairs and International Cooperation (MAECI), through Office VI of the Directorate General for Public and Cultural Diplomacy (DGDP – Contributions for Italian Archaeological, Anthropological and Ethnological Missions Abroad).

The sampling in Tbilisi was conducted by Ana Tetruashvili (European University, Tbilisi) following a strict protocol to enable the later use of advanced analytical methods.

Discovery of Plant Residues

The research initially focused on understanding the function of the stone tools. It later revealed traces of mechanical processing of soft and moist materials, compatible with plant materials such as leaves. Further study using various microscopy techniques (optical and confocal) 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-coloured residues, the team employed advanced microspectroscopic techniques, notably Raman and FTIR spectroscopy. These analyses confirmed the presence of the indigotin chromophore in several archaeological residues. The equipment used for these investigations was available at the University of Padua through the SYCURI research infrastructure, with the involvement of the Interdepartmental Research Centre for Cultural Heritage (CIBA).

Open Hypotheses: Medicinal or Dye Use?

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 research team thus investigated the porosity of the stones—a key factor in their ability to trap and preserve biogenic residues. Analyses were carried out using synchrotron radiation at the SYRMEP beamline of Elettra Sincrotrone Trieste. Both microscopic fragments of the archaeological tools and larger samples from experimental replicas were analysed using micro-CT tomography. The analysis confirmed the presence of pores with volumes suitable for retaining micrometric remains.

As a result, the Ca’ Foscari team designed a series of replicative experiments (documented in a video). 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 fibre production (e.g. bast fibres) and those potentially capable of generating indigotin.

These experiments took place over three summers (during the harvest season of woad) at Corte Badin (Marano di Valpolicella, Verona), where Isatis tinctoria was cultivated with the full support of Giorgio Bonazzi and processed with selected pebbles. The resulting materials were used to build a reference collection, enabling the identification of use-wear traces and, in particular, plant residues.

This careful and innovative approach has been fundamental in shedding light on the early use of plant-based compounds in the Palaeolithic, opening up new perspectives on the complex behaviour of prehistoric human groups.

The research team made fruitful use of a long and profitable collaboration between the groups in Venice and Padua. From Ca’ Foscari University of Venice: Laura Longo, Clarissa Cagnato, Giusi Sorrentino, Antonio Marcomini, and Elena Badetti; and from the University of Padua: Mauro Veronese, Moreno Meneghetti, Alfonso Zoleo, and Gilberto Artioli. Thanks to the ERC project “Powerful Plants”, Ca’ Foscari’s collaboration network has expanded to include Karen Hardy from the University of Glasgow.