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Ancient dental calculus research currently relies on destructive techniques whereby archaeological specimens are broken down to determine their contents. Two strategies that could partly remediate a permanent loss of the original sample and enhance future analysis and reproducibility include: 1) structural surface characterization through spectroscopy along with crystallographic and spectroscopic analysis of its molecular structure, and 2) surface decontamination protocols in which the efficacy of cleaning dental calculus prior to extraction is demonstrated. Dental calculus provides ancient starch research a niche where granules may be adsorbed to minerals, coated, overgrown, entrapped, and/or protected from chemical degradation. While encapsulation offers protection from degradation, it does not shield the sample’s surface from contamination. The most common approach to retrieving microbotanical particles from archaeological calculus has been the direct decalcification of the sample, after a cleaning stage variously consisting of immersion in water, acids, and mechanical dislodgment via gas, sonication, and/or toothbrushes. Little is known about the efficiency of these methods for a complete removal of sediment/soil and unrelated microbotanical matter. In this paper, controlled laboratory experimentation leads to chemical structural characterization and a decontamination protocol to eradicate starch granules. Several concentrations of acids, bases, and enzymes were tested at intervals to understand their potential to gelatinize and fully destroy starch granules; arriving at a procedure that effectively eradicates modern starch prior to dissolution without damaging the matrix or entrapped starch microremains. This is the first attempt at creating synthetic calculus to understand and systematically test effective decontamination protocols for ancient starch research.

The assumption that taxonomy can be ascertained by starch granule shape and size has persisted unchallenged since the late nineteenth and early twentieth century biochemistry. More recent work has established that granule morphological affinity is scattered throughout phylogenetic branches, morphotype proportions vary within the genus, granules from closely related genera can differ dramatically in shape, and size variations do not reflect phylogenetic relationships. This situation is confounded by polymorphism at the species and tissue level, resulting in redundancy and multiplicity. This paper classifies morphological features of starch granules from 77 species, 31 families, and 22 orders across three African ecoregions. This is the largest starch reference collection published to date, rendering the dataset uniquely well suited to explore i) the diagnostic power of unique morphometric classifiers and their frequency, ii) morphotypes that cut across taxonomic boundaries, and iii) issues surrounding the minimum counts needed to accurately reflect granule polymorphism, variability, and identification. In a collection of 23,100 granules, taxonomic identification occurred very rarely. In the instances it did, it was at the species level, with no occurrences of a single morphotype or complement identifying all species within a family or genus. Some families cannot be uniquely identified, and morphometric types are shared despite taxonomic distance for three quarters of the taxa. However, this reference collection boasts 98 unique identifiers located in the Arecaceae, Convolvulaceae, Cyperaceae, Dioscoreaceae, Fabaceae, Musaceae, Pedaliaceae, Poaceae, and Zamiaceae. Therefore, the identification of starch granules is possible in some cases only.

Ancient starch research illuminates aspects of human ecology and economic botany that drove human evolution and cultural complexity over time, with a special emphasis on past technology, diet, health, and adaptation to changing environments and socio-economic systems. However, lapses in prevailing starch research demonstrate the exaggerated expectations for the field that have been generated over the last few decades, including an absence of explanation for the millennial survivability of a biochemically degradable polymer, and difficulties in proving authenticity and taxonomic identification. These flaws perpetuate skepticism and place credibility at risk. By applying new criteria and using a model that puts greater emphasis on detailed authentication procedures, including bio-geochemical characterization, starch granule preservation pathways will be clarified. Future work must consider growing demands from readers, editors, and reviewers that look for objective compositional identification of putatively ancient starch granules.