New and Refined Tools and Guidelines to Expand the Scope and Improve the Reproducibility of Palaeomicrobiological Research

Abstract

Microorganisms vastly outnumber animals and play key roles in our planet’s biosphere. Recent advances in technology and computational tools have made it possible to study the great diversity of microorganisms on Earth rapidly and efficiently. A large fraction of this research has focused on the microbial communities that inhabit the human body—the human microbiota—which account for more than half of the cells we carry and collectively possess >100-fold more genes than the human genome. This research has discovered key coevolutionary relationships between the host and microbiota, many of which have been shaped through human history to the benefit of both partners. Evidence is mounting that disruptions to these microbial communities and to the relationship between the host and microbiota (dysbioses) can have a drastic effect on human health. There is also evidence that recent changes in human societies, such as antibiotic use and exposure to bioactive chemicals, have promoted dysbiosis to the detriment of human wellbeing. Thus, there is great interest in studying human microbiota that existed prior to these recent changes, with the hope of providing insight into the evolution of the human microbiota and informing modern medical strategies and the development of new therapies. The recent finding that ancient microbial DNA is preserved in human dental calculus (calcified dental plaque) offers us the ability to investigate how oral microbiota have changed through human history. Additionally, further advances in DNA sequencing technology and laboratory methods have made it possible to rapidly process ancient specimens and to obtain large quantities of ancient microbial data. However, our ability to analyse this data has not caught up with the speed at which we generate it, and there are many analytical challenges and pitfalls that stand in the way of realising the full potential of ancient microbial DNA studies. This thesis aims to develop and improve methods for analysing ancient microbial DNA and to identify and highlight challenges and pitfalls present in the field in order to increase the quality of future research. Initially, I propose a novel approach of using ancient microbial DNA in dental calculus as a proxy for determining past human migrations. Next, I develop and propose criteria to improve research standards in low-biomass microbiota research. I then assess how characteristics of ancient DNA impact our ability to determine the composition of past microbiota and develop new analytical strategies and methods to improve current taxonomic identification approaches. I also generate and authenticate high-quality data for 132 new ancient dental calculus samples from the Asia-Pacific region, and develop and test two new methods to analyse this data and determine if oral microbiota can be used to infer past human migration and demographic history. Finally, I critically review and respond to three questionable palaeomicrobiological studies with the hope that future researchers, reviewers, and editors will learn from the issues highlighted. Ultimately, this thesis highlights and constructively addresses key pitfalls of palaeomicrobiological research and pushes the field closer to realising its potential.