Among land plants, one of the most cryptic lineages is the genus Isoëtes L., both in terms of public awareness and appearance. Taxonomically, \textit{Isoëtes} are members of the vascular plant Division Lycopodiophyta, the sister lineage to the more well-known and lineage rich Euphyllophyta, which includes both the Monilophytes (ferns) and Spermatophytes (seed plants). Lycophytes are comprised of three extant lineages, the homosporous order Lycopodiales, which have the greatest extant generic diversity within the lineage, and the heterosporous Selaginellales and Isoetales, both of which are contemporarily monogeneric. Lycophytes are united by a few key features, including the microphyll, a leaf type unique to the lineage, and adaxial placement of the sporangia on the sporophylls. Like the Monilophytes, Lycophytes are free-sporing plants. As such, they have a pronounced alternation of generations, with a large, dominant diploid sporophyte producing haploid spores via meiosis, which go on to germinate into haploid gametophytes. These gametophytes produce either or both haploid eggs and sperm, which unite within the gametophyte’s archegonia to produce a new diploid sporophyte embryo.

The Isoëtalean lycopods, both extinct and extant, are particularly unique among their relatives due to both a suite of unusual characters. One of the first and most obvious of them is their growth form. Unlike their fellow lycophytes, which have unipolar growth, meaning they grow and elongate via a terminal apical meristem on the shoot system, and produce adventitious roots from this stem axis, Isoëtaleans have bipolar growth like most Spermatophytes. And, like the non-monocot Spermatophytes, the Isoëtaleans undergo secondary growth, adding both cortical tissue and secondary vascular tissue via a meristematic region known as the prismatic layer. In the extinct Isoëtalians, these two traits allowed them to become arborescent, with some of the largest members, such as Lepidodendron Sternberg growing to over ten meters tall.

Today, Isoëtes is the only remaining member of Isoëtales Prantl. The genus appears to have arisen in the Triassic and can be found throughout the non-polar regions of the world in seasonally to permanently hydric to aquatic habitats. Morphologically, Isoëtes have a highly conserved base bodyplan; almost all of them are small, perennial, semi-herbaceous geophytes whose body is comprised of a highly reduced 1-3 lobed, corm-like trunk, an apical rosette of long, simple, linear leaves, and numerous roots that emerge from their basal furrow. Species are identified through a combination of habitat, spore color, ornamentation and size, leaf morphology, corm lobe numbers, and size of the plants themselves. Because their morphology is so conserved, species identification can be difficult in areas where multiple taxa overlap, especially when the taxa in question are close relatives, as the plants are known to readily hybridize with one another, or form allopolyploids.

Modern Isoëtes can be broken up into five distinct sub-clades, which predominantly correspond to their geographic range. There is the Gondwanan clade, which is found in Southern Africa, South America, India and Australia. The Laurasian clade, which occur in the Mediterranean region of Europe and North Africa, North America, and India. The Italian clade, which occurs in and around the Italian peninsula. The Austro-Asian clade, found in Eastern and Southern Asia, India and Australia. And the American clade, which is found in North and South America, as well as a few circumboreal species, and the only known species in Oceania. Because Isoëtes is so morphologically conserved, prior to molecular phylogenetics it was assumed that spore morphology or habitat types defined the taxonomic groups, which we now know not to be the case. In fact, these traits are quite labile, particularly when polyploids are involved.

In this dissertation, I explore the evolution of one of the traits in extant Isoëtes through ancestral state reconstruction, as well as conduct multi-locus population genetic and phylogenetic analyses to determine if the species composition in one of the sub-clades found on the West Coast of North America.

Polyploidy or whole-genome duplication (WGD) is a ubiquitous phenomenon in the evolutionary history of land-plants. As WGD often induces many new novel, adaptive, or transgressive phenotypes, it can have cascading ecological effects on biotic interactions and potentially the microbiome. There is a growing body of research on the impact that the microbiome plays in plant ecology, but few studies have looked at the potential interactions between ploidy, microbiomes, and pathogens in shaping the ecology of newly established polyploids. This study uses synthetic auto-tetraploid Arabidopsis accessions and a synthetic microbiome representative of natural commensal bacteria in order to assay how these interactions impact host phenotype with respect to pathogen response.

In Chapter 1, I describe how the induction of polyploidy does not change the beta diversity of the phyllosphere but does alter the selection of various taxa of the synthetic community. In Chapter 2, I describe a phenomenon whereby polyploids fare better than diploids when treated with a pathogen regardless of inoculation with a protective microbiome, but where diploids treated with a microbiome better arrest the growth of pathogens than the non-treated diploids. In Chapter 3, I perform an RNA-Seq experiment and find a pattern where defense-associated genes are expressed less in diploid accessions than in polyploids when treated with a microbiome. Together, these chapters for the first time demonstrate that a potential consequence of whole genome duplication may be a loss of control over the composition of the microbiome. Finally in chapter 4, I review and synthesize the literature on somatic polyploidy to assess whether endopolyploidy and whole-genome duplication have shared underlying evolutionary rules.

Traditional botany focuses on the morphology, anatomy, and evolution of above-ground plant parts, but remarkable variation also exists underground. Underground storage organs (USOs), one example of understudied underground botany, include corms, bulbs, rhizomes, and stem- and root-derived tubers. These odd organs characterize geophytes, plants that produce perennating buds below ground and often store nutrients such as starch and water in USOs. Diverse underground morphology is particularly evident in the monocotyledenous order Liliales.

In this dissertation, I examine the evolution and development of USOs across the order Liliales, or the ’liliids’. In Chapter 1, I take a macroevolutionary perspective to ask if plants with different USOs are evolving towards different climatic niche adaptive peaks across the order. I find that the presence of root tubers, especially rotund root tubers, is associated with lower temperature seasonality. Furthermore, I develop and describe a new analysis pipeline in statistical comparative phylogenetics for testing adaptive hypotheses. In Chapter 2, I zoom in on a particular liliid geophyte, Bomarea multiflora, to identify genes underlying root tuber formation by comparing the transcriptomes of root tubers vs. fibrous roots. I compare the genes identified in this study with patterns from USOs produced by other taxa to characterize to what extent processes are shared across non-homologous USOs and across deep evolutionary divergences. I find that many processes are shared despite these differences, indicating that parallel molecular mechanisms may underlie USO development. In Chapter 3, I describe a new R package, RevGadgets, that can process and visualize the output of complex phylogenetic analyses from the RevBayes phylogenetic graphical modelling software. RevGadgets is designed to provide user-friendly modular workflows and thus increase accessibility to more complex phylogenetic models. I illustrate core RevGadgets functionality through six use cases and provide examples of code and resulting figures.

Together, these projects bring light to the outstanding diversity of below ground forms and begin the work of characterizing the evolution and development of this diversity. This work also illustrates the utility of establishing reproducible and user-friendly pipelines to increase the accessibility and versatility of complex statistical methods in comparative biology.

Paullinia L. is a genus of ~220 species of neotropical lianas, with one species extending to tropical Africa. This genus is a part of the large monophyletic Paullinieae tribe of lianas, together comprising 1/3 of the species diversity in Sapindaceae. Paullinia and the five other genera, Cardiospermum, Serjania, Urvillea, Lophostigma, and Thinouia, are united by their climbing habit, paired inflorescence tendrils, and stipulate compound leaves. Vegetatively quite similar, these genera are best distinguished from each other by their fruit, which range from hard or papery capsules to schizocarp samaras with dorsal or ventral wings. Several stem developmental trajectories are also present across the tribe, ranging from the regular stem development typical of trees and shrubs, but also including a collection of vascular cambial variants. Recent phylogenetic analyses based on two loci, have confirmed the monophyly of Paullinieae, however the relationships among genera were largely unresolved.

Paullinia is distinct in the Paullinieae in having hardened septifragal capsular fruits. These fruits open to display three brown-black glossy seeds enveloped by a white fleshy aril. Within Paullinia, variations on the pericarp morphology have been the main focus for infrageneric classification, resulting in the classic 13-section system established in 1895 by Ludwig Radlkofer. Capsule fruits with wings (alate), without wings (exalae), or with spiny projections (echinate) are all present and are thought to have systematic value. Another feature of interest is the presence of vascular camvia variants that in Paullinia include phloem wedges, lobed xylem, compound stems, and successive cambia. The presence and type of cambial variant has been highlighted by previous authors in connection to a larger evolutionary correlation of the liana habit with unusual wood morphologies, which are thought to aid in the climbing habit. All vascular cambial variants in Paullinia are additionally found in other liana lineages, except the compound wood type that is restricted to the Sapindaceae lianas.

In this dissertation, I develop the first molecular phylogeny of Paullinia and explore the evolution of fruit morphologies, and the evolution of development of cambial variants in a phylogenetic context.

In the first chapter, I develop a bioinformatic pipeline that leverages publicly available genomic and transcriptomic data to target informative single-copy intron nuclear markers and demonstrate its efficacy in generating data for species-level phylogenetics across the Paullinieae. First, transcriptome reads from Dimocarpus lognan (Sapindaceae) are aligned to single isoform genes from the Cirtus sinensis (Rutaceae) genome with introns of a desired size (500-1100). Second, single-nucleotide polymorphisms are called, and at these positions, the base pair is changed to the majority rule base pair–this generates a set of consensus sequences (“pseudoreferences”) that are “closer” to Paullinieae. Next, several filters are applied to meet the criteria of single-copy nuclear loci (i.e. reciprocal BLAST to remove paralogs; BLAST to ribosomal, transposons mitochondrial, chloroplast to remove non-nuclear genes; removal of low coverage sequences (<20x average gene coverage; removal of RepeatMasker hits). Finally, I designed primers in the conserved coding sequences of these putative single copy nuclear markers flanking the targeted introns. Using this pipeline, I developed nine novel and variable (53.7–94.3% pairwise identity) molecular markers.

In the second chapter, I generate a robust molecular phylogeny of Paullinia and of the infrageneric relationships across the Paullinieae tribe using nine single-copy nuclear markers developed from the bioinformatic pipeline outlined in Chapter 1, plus two commonly used variable markers (ITS and trnH-psbA). To generate sequence data, I utilized microfluidics PCR to amplify loci using Fluidigm™ technology, then sequenced those amplicons on an Illumina MiSeq. Given this novel phylogenetic hypothesis, I: 1) discuss the taxonomic implications in relation to the traditional infrageneric classification, and 2) conduct an ancestral state estimation of fruit morphologies along the tree. Paullinia is supported as monophyletic and is sister to Cardiospermum L., which together are sister to Serjania Mill + Urvillea Kunth. I discuss seven major clades are discussed that largely correspond to sections defined by morphology. The ancestral condition of fruit morphology in Paullinia is reconstructed as exalate, and seven transitions are inferred: five transition from exalate to alate, one transition from exalate to echinate, and one reversal from alate to exalate. Although the differences in fruit morphologies suggest changes in dispersal mode, because it is the seed (as opposed to the fruit) that is the dispersal diaspore and most species are dehiscent, I conclude that the repeated transitions in fruit morphology represents various strategies to enhance visual display to attract animal dispersers, as opposed to a shift from animal to wind dispersal.

In the third chapter, I describe six stem ontogenies that capture the diversity observed in Paullinia by studying three stages of stem development (primary growth, intermediate (onset of secondary growth), and mature wood) in 18 species. Most Paullinia species are angular in cross-sectional view at the shoot apex, which becomes reinforced by the unequal distribution of vascular bundles around the circumference of the young stem. Although rare among woody plants, this is the basic bauplan of primary growth in Paullinia, from which five of the six mature stem types develop. To explore the evolution of stem ontogenies in Paullinia and across the other Sapindaceae lianas, I employed phylogenetic comparative methods to reconstruct the ancestral primary plant body shape and the ancestral mature stem type across the Paullinieae tribe. Additionally, I tested the hypothesis that the evolution of cambial variants is contingent on first evolving the irregular angular primary growth confirmation. The results identify a critical relationship between primary and secondary growth in both the development of cambial variants of an individual plant, and the emergence of these novel forms through evolutionary time.

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