A phylogenetic analysis of molecular (ITS, trnL-F trnK including the matK coding region) and morphological data is presented for the pantropical monocot family Costaceae (Zingiberales), including 65 Costaceae taxa and two species of the outgroup genus Siphonochilus (Zingiberaceae). Taxon sampling included all four currently described genera in order to test the monophyly of previously proposed taxonomic groups. Sampling was further designed to encompass geographical and morphological diversity of the family to identify trends in biogeographic patterns and morphological character evolution. Phylogenetic analysis of the combined data reveals three major clades with discrete biogeographic distribution: (1) South American, (2) Asian, and (3) African-neotropical. The nominal genus Costus is not monophyletic and its species are found in all three major clades. The Melanesian genus Tapeinochilos is monophyletic and included within the Asian clade. Monocostus and Dimerocostus are sister taxa and form part of the South American clade. The African-neotropical clade is composed entirely of the genus Costus; moreover, there is support for previously recognized subgeneric groupings within the Costus clade. Evolutionary trends in floral morphology show that close associations with pollinators have evolved several times from an ancestral generalist pollinator floral form. Bee pollination has evolved once in the family, arising in Africa from an open-flowered (generalist) ancestor. Bird pollination has evolved multiple times: once from an open-flowered ancestor in Southeast Asia and multiple times from a bee-pollinated ancestor in the neotropics. Additional morphological characters not traditionally used to define taxonomic groups, but having high consistency in the current phylogenetic analysis, are discussed.

Understanding patterns of speciation is of fundamental importance to evolutionary biologists. Species diversity can be maintained and generated through processes originating from interactions with climate, biota, and geography. In order to investigate the role of these factors on diversity in a species-rich group of plants, we developed a phylogeny of the core Bromelioideae including Aechmea and related genera with the specific goals of investigating monophyly of genera, biogeographic history, morphological trait evolution, and environmental niche diversification. The phylogenetic estimation encompassed 20 genera and 4 genomic regions including chloroplast, low-copy, and ribosomal nuclear genes. Subsequently, morphologies were re-explored to understand species relationships that had not been previously recognized and to determine if morphological synapomorphies could define well supported clades. Species were coded as present or absent in 16 geographic regions to reconstruct ancestral areas. Finally, to understand how species within this group divided climatic niche space, research was focused on two clades that diversified independently in Central America. Niche models were generated for each species and for the clades as a whole and were compared for overlap. The distribution of each environmental variable for all occurrence records was compared at each node of divergence within each clade to understand divergence in environmental conditions in a method free from model uncertainty. From these analyses we found: (1) Lack of monophyly in many genera and subgenera (2) Closely related species often were found in geographic sympatry (at the scale of co-occurrence in the same country or state) such that taxonomic groups that had been thought to include geographically disjunct species, actually were representative of distinct evolutionary histories (3) Morphological traits that had been used to assign species to genera were often homoplastic (through convergence or reversals of plesiomorphic states) (4) Although a single unique synapomorphy for each clade was not found, morphological traits were often consistent within clades, providing independent support for the molecular phylogenetic estimation and a basis for future taxonomic work (5) Most well supported clades diversified within one of eight geographic areas, although the ancestral areas for several larger clades remains uncertain (6) Both clades native to Central America are found in a unique environmental niche (i.e. environmental niche models of each clade do not overlap and there is significant divergence of climatic variables between the clades) (7) Species within the clade composed of species with greater morphological variation had more environmental niche overlap than the clade with little morphological variation. These findings add to the evolutionary knowledge of the Bromeliaceae and suggest that geographic range should be taken into account when assigning species to genera. Centers of diversity of the core Bromelioids are found within eastern and Amazonian Brazil, northern south America and Central America and each radiation could have been influenced by climate in a variety of ways. In the two studied examples, species divergence is coincident with divergence in environmental niche occupation. However, environmental niche evolution was limited such that each clade as a whole was not modeled in the niche of the other clade, suggesting either some degree of niche conservatism or competition preventing parapatric niche expansion.

Phylogenetic relationships among carnivorous plants of the angiosperm order Caryophyllales are explored using Bayesian statistics and maximum-likelihood based searches of phylogeny. Nuclear ribosomal (ITS) and chloroplast intergenic spacer (PY-IGS) regions, along with previously- sequenced DNA are utilized for phylogenetic reconstructions. Taxonomic relationships across genera are refined and three strongly supported clades are identified: monophyletic Droseraceae, Nepenthaceae, and a third clade containing Ancistrocladaceae, Dioncophyllaceae, and Drosophyllaceae. In combination with phylogenetic reconstruction, stochastic character mapping is utilized to assess evolutionary changes in the morphology of glands found on the lamina and involved in the digestion of prey. Adaptive changes resulting in the evolution of the carnivorous gland are discussed, which may have occurred either by emargination of the leaf blade or homologous transformation of pinnae.

A variety of enzymes are excreted from the carnivorous gland that aid in prey digestion. Within the carnivorous plants of the Caryophyllales, two subclasses of class I chitinases have been identified to play a role in the digestion of prey. Proteins produced by the large and diverse chitinase gene family are involved in the hydrolyzation of glycosidic bonds in chitin, a polymer of N-acetylglucosamines. Members of these subclasses, depending on the presence or absence of a C-terminal extension, can be secreted from specialized digestive glands found within morphologically diverse traps that develop from plant leaves. Homology among carnivorous plant class I chitinases and the method by which these enzymes have been adapted for the carnivorous habit are investigated. Novel class I chitinase homologs are recovered from Ancistrocladus, Dionaea, Drosera, Nepenthes, and Triphyophyllum, in addition to class I chitinases available from sequenced angiosperm genomes. Substitutions specific to carnivorous plant class I chitinases are revealed by detecting sites under positive selection, which may confer functional differences as indicated by protein structure homology-modeling.

To study gene function in non-model organisms, a virus induced gene silencing (VIGS) method was developed. VIGS has been shown to be effective for transient knockdown of gene expression in plants to analyze the effects of specific genes in development and stress related responses. It is demonstrated that the barley stripe mosaic virus (BSMV) is able to infect two species within the Zingiberaceae, and that BSMV-VIGS can be applied to specifically downregulate phytoene desaturase in the culinary ginger Zingiber officinale. BSMV-VIGS is likely to be effective in other angiosperms susceptible to BSMV infection. This should enable targeted studies for identifying gene function to be carried out in ecologically and evolutionarily important groups.

Recent cladistic analysis Of Multiple molecular data from chloroplast and nuclear genomes as well as morphological data have indicated that a reclassification of the family Costaceae is necessary in order to appropriately reflect phylogenetic relationships. The previously described genera Tapeinochilos, Monocostus, and Dimerocostus are all upheld in the new classification. Monocostus and Dimerocostus are found to be sister taxa. The large pantropical genus Costus is found to be polyphyletic and is thus divided into four genera, three of which are new (Cheilocostus, Chamaecostus, Paracostus). Costus has now a more concise generic concept including morphological synapomorphies previously absent due to the polymorphic nature of the prior nonmonophyletic assemblage. Of the three new genera, one (Parocostus) was previously recognized as a subgenus of Costus. Cheilocostus comprises several Asian taxa and is sister to Tapeinochilos, whereas Chamaecostus comprises entirely neotropical taxa and is sister to a neotropical Monocostus + Dimerocostus clade. A reevaluation of the traditional taxonomy indicates that floral characters and pollination syndromes commonly used to identify groups exhibit homoplasy when analyzed in a cladistic framework and are thus unreliable as taxonomic indicators.

Abstract

The Development and Evolution of Floral Symmetry in the Zingiberales and Interactive Tools for Teaching Evolution (ArborEd)

by

Riva Anne Bruenn

Doctor of Philosophy in Plant Biology

University of California, Berkeley

Professor Chelsea D. Specht, Chair

Floral symmetry is a key innovation in the evolution of flowering plants. Zygomorphy, or single-planed symmetry, is associated with the diversification of many flowering plant lineages. The model system for floral symmetry is the snapdragon (Antirrhinum majus). In A. majus flowers, a set of TCP and MYB-related transcription factors form a core gene regulatory network necessary for zygomorphy. The genes involved in this network have been implicated in several independent transitions to zygomorphy from actinomorphy (many-planed symmetry). Although the TCP components of the symmetry network have been investigated across flowering plants, MYB-related transcription factors remain largely unstudied outside of the Asterid group containing A. majus and close relatives. Here we investigate the evolution of MYB-related genes DIVARICATA-like (DIV-like), RADIALIS-like (RAD-like), and DIVARICATA and RADIALIS INTERACTING FACTOR-like (DRIF-like) across flowering plants, and their expression patterns in the developing flowers of two zygomorphic species of the monocot order Zingiberales.

We found that RAD-like and DIV-like are sister MYB-related genes which diverged before the diversification of flowering plants. Each gene contains one MYB-like domain that has been closely conserved throughout flowering plant evolution. Furthermore, we identified candidate homologs to A. majus RAD and DIV in several monocot taxa, with at least three copies of each in the Zingiberales.

In the Zingiberales, RAD-like and DIV-like genes are expressed in Costus spicatus (Costaceae) and Musa basjoo (Musaceae) in patterns consistent with roles in floral symmetry. Using Reverse Transcription PCR and in situ hybridization we recovered asymmetric expression patterns for some RAD-like genes across the dorsal/ventral plane of developing flowers, and universal expression of DIV-like genes, consistent with the model known from Antirrhinum majus.

We identified DRIF-like genes across flowering plants, recovering a previously undescribed duplication in eudicot DRIF Group 1 genes. Furthermore, we recovered candidate DRIF-like genes in Musa basjoo (Musaceae: Zingiberales) with expression patterns similar to those described in A. majus DRIF1 and DRIF2.

Finally, we developed a tutorial for high school and college students to investigate a coevolutionary hypothesis in sharpshooters and their bacterial endosymbionts. This tool will help students understand how comparative evolutionary research is performed, and give them hands-on experience performing common analyses.

Gene duplication plays an important role in the evolution of novel protein functions. Gene duplications in Polycomb Repressive Complex 2 (PRC2) proteins could have led to novel biochemical functions. However, since PRC2 regulates gene expression by marking its target genes with Histone 3 Lysine 27 trimethylation (H3K27me3), gene duplication might not alter the complex’s biochemical function, but could instead affect its set of target genes. Thus, gene duplication could result in new genomic targets for one of the duplicate PRC2s, subdivision of the genomic targets between the duplicate PRC2s, or some combination of these two scenarios. To determine where gene duplication, and possible functional divergence, occurred we first reconstructed the evolutionary history of the four core PRC2 proteins in eukaryotes. Gene duplications were detected in each of the four PRC2 core proteins: Enhancer of zeste [E(z)]; extra sex combs (esc); Suppressor of zeste 12 [Su(z)12]; and Chromatin assembly factor 1, p55 subunit (Caf1-55). Vertebrates and plants experienced duplications in their E(z), Su(z)12, and Caf1-55 homologs, whereas invertebrates only underwent a small-scale esc duplication in the Schizophora clade of flies. The fungal Caf1-55 genes duplicated at least twice, but these duplications were only found in fungal clades that had lost the other PRC2 core proteins. The consequences of these PRC2 duplications are discussed—these duplications may have resulted in novel biochemical activity, new genes targeted for repression, or partitioning of PRC2 activity throughout development.

Three different PRC2 complexes exist in Arabidopsis. While some of the three Arabidopsis E(z) homologs can be used interchangeably in PRC2s, each of the three Su(z)12 homologs is unique to its PRC2. The model moss, Physcomitrella patens, also possesses three Su(z)12 homologs, although they arose from independent gene duplications. Unlike Arabidopsis, there is only a single copy of the other PRC2 proteins, so we investigated the evolution and function of the only multi-copy P. patens PRC2 core protein. We amplified five conserved exons from the Su(z)12 genes in mosses and detected multiple lineage-specific Su(z)12 duplication events. To determine the functional consequence of the duplications, we made single gene knockouts for each of the three P. patens Su(z)12 genes and then characterized the phenotypes of the mutants. Although there exists some degree of redundancy among the three Su(z)12 copies, each regulates development of the haploid gametophyte generation differently.

While the P. patens Su(z)12 genes affect gametophyte growth, moss E(z) and esc homologs are required to prevent sporophyte development during the gametophyte generation. A handful of other candidate genes important in gametophyte and sporophyte development have been identified. To identify additional genes required for either gametophyte or sporophyte development, we performed RNA-Seq on RNA from both generations. We found that a large proportion of genes and transcripts were differentially expressed between the two tissue types. These differentially expressed genes were enriched for Gene Ontology categories related to translation and photosynthesis in the gametophyte and DNA binding and vesicle-mediated transport in the sporophyte. The most highly differentially expressed genes between gametophyte and sporophyte tissues could be investigated in future studies to determine their roles in moss development.

The rapid rise and diversification of the angiosperms has puzzled biologists for centuries; processes leading to current angiosperm diversity remain a key question in evolutionary biology, with particular focus on the morphological diversity of flowers. The Zingiberales are an order of tropical monocots that represent an ideal group of plants to study the evolution of floral morphology. The order contains approximately 2,500 species, many of which form specialized pollination relationships with bees, birds, bats, dung beetles, moths, butterflies, and primates (lemurs) via alterations in floral form. After developing a technique for visualizing and then studying gene expression in floral apices, I investigated the role of two candidate gene families, the GLOBOSA (GLO)-like genes and the CYCLOIDEA/ TEOSINTE BRANCHED 1 (CYC/TB1)-like genes, in the evolution of floral morphology in the Zingiberales.

Evolutionary developmental biology often combines methods for examining morphology (e.g. Scanning Electron Microscopy) with analyses of gene expression (e.g. RNA in situ hybridization). Due to differences in tissue preparation for SEM and gene expression analyses, the same specimen cannot be used for both sets of techniques. I developed a method that couples extended-depth-of-field (EDF) epi-illumination microscopy to in situ hybridization in a sequential format, enabling both surface microscopy and gene expression analyses to be carried out on the same specimen (Chapter 1). I first created a digital image of inflorescence apices using epi-illumination microscopy and commercially available EDF software. I then performed RNA in situ hybridizations on photographed apices to assess expression of two developmental genes: Knotted1 (Kn1) in Zea mays (Poaceae) and a GLO homolog in Musa basjoo (Musaceae). I demonstrate that expression signal is neither altered nor reduced in the imaged apices as compared with unphotographed controls. The demonstrated method reduces the amount of sample material necessary for developmental research and enables individual floral development to be placed in the context of the entire inflorescence. While the technique presented is particularly relevant to floral developmental biology, it is applicable to any research where observation and description of external features can be fruitfully linked with analyses of gene expression.

The MADS box transcription factor family has long been identified as an important contributor to the control of floral development. It is often hypothesized that the evolution of floral development across angiosperms and within specific lineages may occur as a result of duplication, functional diversification, and changes in regulation of MADS box genes. In Chapter 2 I examine the role of GLO-like genes, members of the B-class MADS box gene lineage, in the evolution of floral development within the monocot order Zingiberales. I assessed changes in perianth and stamen whorl morphology in a phylogenetic framework. I identified GLO homologs from 50 Zingiberales species and investigated the evolution of this gene lineage. Expression of two GLO homologs was assessed in Costus spicatus Swartz (Costaceae) and Musa basjoo Siebold (Musaceae). Based on the phylogenetic data and expression results, I propose several family-specific losses and gains of GLO homologs that appear to be associated with key morphological changes. The GLO-like gene lineage has diversified concomitant with the evolution of the dimorphic perianth and the staminodial labellum. Duplications and expression divergence within the GLO-like gene lineage may have played a role in floral diversification in the Zingiberales.

In the Zingiberales, evolutionary shifts in symmetry occur in all floral whorls, making this an ideal group of plants in which to study the evolution of this important ecological and developmental trait. The CYC/TB1-like genes have been implicated in the development and evolution of floral symmetry in divergent angiosperm lineages, and I thus chose them as a candidate gene family to investigate their role in the evolution of floral symmetry within the Zingiberales (Chapter 3). I identified both Zingiberales-specific gene duplications and a duplication in the TB1-like (TBL) lineage that predates the divergence of the commelinid monocots. I examined the expression of two TBL genes in Costus spicatus (Costaceae) and Heliconia stricta (Heliconiaceae), two Zingiberales taxa with divergent floral symmetries. I found that TBL gene expression shifts concomitant with shifts in floral symmetry.

Through this body of work we have gained some insight into the mechanics of angiosperm evolution. Duplications in the GLO-like gene lineage in the Zingiberales may have allowed for gene sub- or neofunctionalization and the evolution of new morphologies; in particular, the evolution of differentiated sepals and petals and of the staminodial labellum. In addition, this study adds to the growing body of evidence that CYC/TB1-like genes have been repeatedly recruited through the course of evolution to generate bilateral floral symmetry (zygomorphy). Although this work certainly doesn't preclude the involvement of as yet uncharacterized genes and gene families, it adds to the growing body of evidence that angiosperms as a group do indeed have a genetic `toolkit': a core set of genes that have been variously deployed through evolutionary time to generate both convergent and divergent floral morphologies.

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.

Premise of the study:

We developed a bioinformatic pipeline that leverages a publicly available genome and published transcriptomes to design primers in conserved coding sequences flanking targeted introns of single-copy nuclear loci. Paullinieae (Sapindaceae) is used to demonstrate the pipeline.

Methods and Results:

Transcriptome reads phylogenetically closer to the lineage of interest are aligned to the closest genome. Single-nucleotide polymorphisms are called, generating a “pseudoreference” closer to the lineage of interest. Several filters are applied to meet the criteria of single-copy nuclear loci with introns of a desired size. Primers are designed in conserved coding sequences flanking introns. Using this pipeline, we developed nine single-copy nuclear intron markers for Paullinieae.

Conclusions:

This pipeline is highly flexible and can be used for any group with available genomic and transcriptomic resources. This pipeline led to the development of nine variable markers for phylogenetic study without generating sequence data de novo.