Evolution Beyond DNA: Long-Term Experimental Evolution in Pristionchus pacificus

Abstract

Evolution is a continuous and historical process that can be studied experimentally in organisms with rapid generation times. Because evolution ultimately depends on the generation and inheritance of phenotypic variation, a key unresolved question is the extent to which environmental factors can produce heritable changes. Therefore, environmental effects on trait inheritance remain debated because it is often difficult to distinguish true transgenerational inheritance from short-term parental influences. While mammalian systems face technical limitations, nematodes provide powerful experimental models. In this work, I investigated transgenerational epigenetic inheritance (TEI) of environmentally induced traits using Pristionchus pacificus, a nematode that exhibits mouth-form plasticity, including predation. I established long- term environmental induction experiments with alternative diets by propagating 110 isogenic lines over 101 generations, together with complementary food-reversal assays. I found that a Novosphingobium diet enriched in vitamin B12 induces the predatory mouth morph, which is subsequently transmitted across generations and occasionally becomes canalized at low frequency. To uncover the underlying mechanisms, I performed large-scale genetic screening and identified the target- directed microRNA degradation (TDMD) factor EBAX-1/ZSWIM8. I showed that Ppa- EBAX-1 specifically destabilizes the expanded miR-35/miR-2235a microRNA cluster in P. pacificus, revealing an unexpected repressive role of microRNAs in regulating TEI of the predatory morph. By defining the dietary trigger landscape, I further demonstrated that vitamin B12 is sufficient to induce TEI in a concentration-dependent manner and uncovered a complex regulatory network of potential RNA triggers linked to TDMD-like processes. Overall, my work over the past five years provides conceptual and experimental advances on nematode TEI. This expeirmental framework leverages the natural organismal readout of plasticity in P. pacificus and offers key insights into developmental plasticity, its transgenerational inheritance, and low-frequency canalization of traits. Together, these findings establish a foundation for addressing broader evolutionary questions and provide a mechanistic link between environmental responsiveness and heritable phenotypic change.