With over 300,000 plant species on the planet and more than 90% of them relying on pollen for reproduction, palynology, the study of pollen grains plays a vital role in many research fields. One of them is forensic palynology, which uses pollen as a proxy to link individuals or objects to a location or instance. It relies on the fact that (1) pollen is an ever-present feature of the environment; (2) different locations have different pollen signatures, allowing for inference related to spatial tracking; (3) plants bloom at different times, allowing for temporal inference; and (4) pollen is exceptionally durable and can be used for forensic studies decades after sample collection. In addition, forensic palynology has played a role in many criminal investigations, mainly investigations dealing with homicide, violent assault, rape, genocide, terrorism, and suspected terrorism. Identifying plant species from their pollen relies on two traditional methods: microscopy or genetic analysis. On the one hand, microscopy relies on identifying pollen grains using their morphology (i.e., size, shape, and wall structure) and comparing it to an image library for accurate identification. On the other hand, genetic analysis characterizes pollen species using a short DNA sequence from a universal standard in the genome. Both methods have so far been mutually exclusive. The standard procedure for microscopy is to clean the grain through acetolysis, which destroys any genetic material in the sample. Studies involving genomic characterization of the plant material require the release of the genomic material by mechanically crushing the grain, which can no longer be analyzed for morphology through microscopic methods. While the number of forensic palynological studies increases, they usually rely on only one of the two techniques above and rarely show the potential for an efficient analysis of individual grains within an assemblage to statistically evaluate the species distribution in the mixture of grains that can be the evidence. During this Ph.D. research, a new method for pollen DNA extraction was developed that does not destroy the pollen grain, getting around both crushing and acetolysis approaches. After evaluating the non-destructive nature of the new protocol by microscopy, single pollen grains from a variety of common species were examined using universally accepted genetic markers (rbcL, matK, and ITS2) for DNA analysis. The sequencing of the different species was also performed and discussed to evaluate the potential for single species identification from databases. Finally, the developed approach for non-destructive DNA extraction was evaluated on a complex object, a car cabin air filter, showing how microscopic plant evidence (pollen and debris) analysis can easily provide information in an investigation.

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