2025 Theses Doctoral

It’s Not Just a Phase: Development, Evolutionary History, and Consequences of the Free-Living Gametophyte Phase in the Life Cycles of Ferns

Sorojsrisom, Elissa Suphapun

An alteration to the life cycle was a fundamental early step in the evolution of land plants. The ancestors of land plants likely spent most of their lives as a multicellular haploid organism, known as the gametophyte phase, and entered a single-celled diploid zygote phase only rarely. This changed in early land plants, which developed a multicellular diploid embryo that grew into a multicellular diploid body called the sporophyte phase. Since then, land plants have evolved the full spectrum of relative dependence between phases, i.e. they exhibit sporophyte-dominant, gametophyte-dominant, and co-dominant life cycles. Such foundational changes to the plant life cycle were critical for the establishment of early plants on land and have remained a central aspect of land plant evolution, but beyond this, little detail is known about the early steps in land plant evolution.

Ferns are the only extant land plant lineage in which both life phases are independent and photosynthetic. They also occupy a key phylogenetic position as sister to the most diverse group of land plants (seed plants), thus both their life cycle and their evolutionary history is intermediate between those of the other major plant lineages. Ferns have been historically underutilized in genomic studies due to the difficulty of sequencing their genomes.

However, with advances in sequencing technology and reduction in costs, it has recently become feasible to develop genomic resources in ferns. Additionally, the fern gametophyte – once thought to be relatively unimportant, or even a “handicap” in the fern life cycle – has been an overlooked, but consequential, aspect of land plant evolution. The free-living gametophyte is a salient feature of the fern life cycle and more broadly the life cycles of early-diverging groups of land plants, while the fern sporophyte is similar to the sporophyte of seed plants, which predominate in botanical research. Thus, research in ferns bridges a gap in existing data from disparate lineages.

In this dissertation I utilize ferns, and the fern gametophyte in particular, to address open questions about the origin, evolution, and development of land plants. In Chapter 1, I develop a computational tool called shadie for simulating genome evolution under realistic plant life cycle models to explore the evolutionary consequences of co-dominant fern life cycles compared to those with less independent stages. Simulations using this tool demonstrate that fixation probability of new mutations and patterns of linkage change due to life cycle alone, suggesting that the relative dominance of phases in plant life cycles has likely influenced the long-term evolution of the major lineages in different ways and may be a key characteristic for understanding why they are so distinct from one another.

In Chapter 2 I focus on a key trait of the gametophyte, the gametangia (sex organs), to generate testable hypotheses about this aspect of the evolution and development of land plants and larger trends in land plant evolution. I review 150 years of botanical literature on the subject of gametangia morphology and development to generate an eight-stage ontogenic framework that unites the development of the female and male sex organs of land plants and proposes homologies across all land plant lineages. This review synthesizes data from diverse fields and sometimes elusive sources to identify clear future research directions.

In Chapter 3 I collect the first high resolution transcriptomic time series dataset of gametophyte development in the model fern, Ceratopteris richardii. I use this dataset to characterize differential gene expression changes throughout the fern life cycle, identify transcription factors specific to developmental stages, assign gene specificity to one phase or the other, and test a century-old hypothesis about the origin of land plants. In Chapter 4 I extend this pipeline to a comparative study utilizing 11 species of field-collected sporophytes and lab-reared gametophytes and implement the McDonald-Kreitman test to measure selection. Results from these chapters reveal a consistently high degree of gene expression overlap between the gametophyte and sporophyte, adding to growing evidence that ferns may experience a high degree of pleiotropy between life phases. We also discover that phase-specific genes experience higher rates of adaptive evolution than genes that are shared by both phases, which could suggest that gene-sharing between phases causes selection to act with less efficiency.

Finally, in Chapter 5 I reconstruct the evolution of an ancient family of homeodomain proteins called TALE, incorporating sequences from ferns, lycophytes, and gymnosperms, which were not available for use in earlier reconstructions. I then use in situ hybridization to characterize the spatial gene expression of all fourteen copies of TALE genes in C. richardii in both gametophyte and sporophyte tissues. This study contributes to resolving the TALE phylogeny, which has proven difficult to reconstruct, and assigns previously unidentified TALE genes to their respective clades. This study clarifies evolutionary relationships between TALE genes in land plants, characterize their expression patterns in ferns, and results suggest that at least some copies of ferns retain their ancestral functions. This study is also the first to demonstrate clear evidence that TALE proteins are involved in gametophyte body plan patterning in land plants.