SedaDNA metabarcoding for assessing Upper Palaeolithic plant use in Armenia

Aghitu-3 Cave contains a 15,000-year long record (from ~39,000–24,000 cal BP) of Upper Paleolithic human settlement and environmental variability in the Armenian Highlands. Finds from this cave include stone artifacts, faunal remains, bone tools, shell beads, charcoal, and pollen, among others. We applied sedimentary ancient DNA (sedaDNA) metabarcoding to the Aghitu-3 sedimentary sequence and combined this with pollen data to obtain a temporal reconstruction of plant assemblages. Our results reveal a stratification of plant abundance and diversity especially where sedaDNA reflects periods of human occupation, showing higher diversity in layers with increased human activity. Low pollen concentrations combined with high sedaDNA abundance indicates plant remains may have been brought into the cave by animals or humans during the deposition of the lower two archaeological horizons. The majority of the recovered plants were found to be possibly useful as food, flavor, medicine, and/or technical purposes, demonstrating the potential of the environment around Aghitu-3 Cave to support humans during the Upper Paleolithic. Moreover, we identified several specific plant taxa that strengthen previous findings about Upper Paleolithic plant use in this region (i.e., for medicine and the manufacturing and dyeing of textiles). This study represents the first application of plant sedaDNA analysis of cave sediments for the investigation of potential plant use by prehistoric humans.