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.