Browsing by Subject "Botany"

The 2010 Deepwater Horizon disaster remains one of the largest oil spills in history. This event caused significant damage to coastal ecosystems, the full extent of which has yet to be fully determined. Crude oil contains both toxic substances that are detrimental to microbes and compounds that may be used as food and energy resources by some microbial species. As a result, oil spills have the potential to cause significant shifts in microbial communities. In this study, we assessed the impact of oil contamination on the function of endophytic microbial communities associated with saltmarsh cordgrass (Spartina alterniflora). Soil samples were collected from two locations in coastal Louisiana, USA: one severely affected by contamination from the Deepwater Horizon oil spill and one relatively unaffected location. Spartina alterniflora seedlings were grown in both soil samples under greenhouse conditions, and GeoChip 5.0 was used to evaluate the endophytic microbial metatranscriptome shifts in response to host oil exposure. Microbial functional shifts were detected in functional categories related to metal homeostasis, organic remediation, and phosphorus utilization. These findings show that host oil exposure elicits multiple changes in metabolic response from their endophytic microbial communities, producing effects that may have the potential to impact host plant fitness.

Fungal endophytes are ubiquitous inhabitants of plants and can have a wide range of effects on their hosts, from pathogenic to mutualistic. These fungal associates are important drivers of plant success and therefore contribute to plant community structure. The majority of endophyte studies have focused on seed plants, but in order to understand the dynamics of endophytes at the ecosystem scale, as well as the evolution of these fungal associations, investigations are also necessary in earlier-diverging clades of plants, such as the non-vascular bryophytes (mosses, liverworts, and hornworts). This dissertation presents a survey of the diversity of fungal endophytes found in the liverwort Marchantia polymorpha L. and develops a gnotobiotic experimental system for testing the effects of these fungi on their liverwort host. The survey reveals a diverse community of fungi in M. polymorpha, with some fungi that are associated with this host across geographically distant sites. The laboratory experiments demonstrate that culturable endophytes of M. polymorpha can, in isolation, cause positive, negative, or neutral effects on host success and that these effects change in response to nutrient levels and the presence of multiple endophytes. The experimental system developed in this dissertation has great potential in the growing field of plant microbiota research to answer questions that range in scale from molecular mechanisms to ecosystem function.

Environmentally cued development is widespread across the natural world. Many organisms rely on abiotic and biotic cues to undergo developmental transitions like budburst, flowering, and mating at the appropriate times of year. The study of the timing of these transitions is known as phenology. Because the timing of development determines the environment experienced by the next life history stage, it has the potential to affect evolutionary processes that occur after development. Further, because the timing of development can filter the environment experienced by the next life history stage, it can be considered a form of “habitat tracking.” In this dissertation, I use manipulative laboratory and field experiments to quantify how germination phenology can alter the postgermination environment, show that the postgermination environment can itself be genetically determined, show that germination phenology is a form of habitat tracking, and test how germination phenology can affect trait expression, natural selection, and fitness.

In my first chapter, using the ecological and genetic model species Arabidopsis thaliana, I showed that when the timing of development is genetically controlled, and the timing of development affects the environment experienced by the next developmental stage, then the environment experienced after development can itself be inherited and can evolve. Further, I demonstrated that germination phenology is a form of “habitat tracking”, by enabling seeds to establish seedlings in a subset of the full environmental conditions available. In my second chapter, using ecologically diverse A. thaliana genotypes, I show that the timing of germination can affect natural selection on postgermination traits, and that postgermination traits can affect selection on germination phenology. In my third chapter, using two plants native to North Carolina, Phacelia purshii and P. fimbriata, I show that populations can vary naturally in their propensity to germinate in response to different environmental cues, that populations preferentially germinate in habitats that are beneficial for seedlings, and when placed in new geographic locations, populations may use phenology to track novel but beneficial environmental conditions.

My dissertation placed the common process of cued development into the well- established theoretical framework of habitat tracking and habitat selection. By doing so, I was able to generate and test novel predictions about potential consequences of phenological cueing that have not yet been explored—namely, that the post- development environment itself can be inherited, that the magnitude and frequency of natural selection can vary with changes in habitat tracking, that habitat tracking itself may evolve in response to traits expressed and environments experienced after development, and that habitat tracking through phenology may be an important mechanism that organisms can use to cope with climate change.

Not all ferns grow in moist and shaded habitats. One notable example is the ecologically unusual clade of notholaenids. With approximately 40 species, the notholaenids have adapted to and diversified within the deserts of Mexico and the southwestern United States. In my dissertation, I studied the evolution and diversification of notholaenid ferns, using an approach that integrates data from multiple sources: biochemistry, biogeography, cytology, ecological niche modeling, molecular phylogeny, morphology, and physiology.

In Chapter 1, I infer a species phylogeny for notholaenid ferns using both nuclear and plastid DNA sequences, and reconstruct the evolutionary history of “farina” (powdery exudates of lipophilic flavonoid aglycones), a characteristic drought-adapted trait, that occurs on both the gametophytic and sporophytic phases of members of the the clade. Forty-nine notholaenid and twelve outgroup samples were selected for these analyses. Long (ca. 1 kb) low-copy nuclear sequences for four loci were retrieved using a recently developed amplicon sequencing protocol on the PacBio Sequel platform and a bioinformatics pipeline PURC; plastid sequences from three loci were retrieved using Sanger sequencing. Each nuclear/plastid dataset was first analyzed individually using maximum likelihood and Bayesian inference, and the species phylogeny was inferred using *BEAST. Ancestral states were reconstructed using likelihood (re-rooting method) and MCMC (stochastic mapping method) approaches. Ploidy levels were inferred using chromosome counts corroborated by spore diameter measurements. My phylogenetic analyses results are roughly congruent with previous phylogenies inferred using only plastid data; however, several incongruences were observed between them. Hybridization events among recognized species of the notholaenid clade appear to be relatively rare, compared to what is observed in other well-studied fern genera. All characters associated with farina production in the group appear to be homoplastic and have complex evolutionary histories.

In Chapter 2, I focus on the infraspecific diversification of Notholaena standleyi, a species that thrives in the deserts of the southwestern United States and Mexico and has several “chemotypes” that express differences in farina color and chemistry. Forty-eight samples were selected from across the geographic distribution of N. standleyi. Phylogenetic relationships were inferred using four plastid makers and five single/low-copy nuclear markers. Sequences were retrieved using PacBio and the PURC pipeline. Ploidy levels were inferred from relative spore size measurements calibrated with chromosome counts, and farina chemistry was compared using thin-layer chromatography (TLC). My studies of Notholaena standleyi reveal a complex history of infraspecific diversification traceable to a variety of evolutionary drivers including classic allopatry, parapatry with or without changes in geologic substrate, and sympatric divergence through polyploidization. Four divergent clades were recognized within the species. Three roughly correspond to previously recognized chemotypes: gold (G), yellow (Y), and pallid/yellow-green (P/YG). The fourth clade, cryptic (C), is newly reported here. The diploid clades G and Y are found in the Sonoran and Chihuahuan Deserts, respectively; they co-occur (and hybridize) in the Pinaleño Mts. of eastern Arizona. Clades G and Y are estimated to have diverged in the Pleistocene, congruent with the postulated timing of climatological events that divide these two deserts. Clade P/YG is tetraploid and partially overlaps the distribution of clade Y in the eastern parts of the Chihuahuan Desert. However, PY/G is apparently confined to limestone, a geologic substrate rarely occupied by members of the other clades. The newly discovered diploid clade, cryptic (C), is distributed in the southern Mexican states of Oaxaca and Puebla and is highly disjunct from the other three clades.

In Chapter 3, I study the ecological niche differentiation among the three major chemotypes––G, Y, and P/YG. Using both ordination and species distribution modelling techniques, the ecological niches for each chemotype were characterized and compared. The main environmental drivers for their distributions were identified, their suitable habitats in both geographic and environmental spaces were predicted, and their niche equivalencies and similarities were tested. My ecological niche analyses results suggest that all three chemotypes are ecologically diverged. The ecological niches of the two parapatric, sister diploid chemotypes, G and Y, are significantly different from one another. Chemotype G occupies a very extreme niche with higher solar radiation, and lower rainfall and higher temperatures in the wettest quarter. The niche space of tetraploid chemotype P/YG is similar but not equivalent to the other two chemotypes. Its distribution model is highly influenced by the high percentage of Calcids and warmer temperatures in the wet season, reflecting the fact that it is confined to limestone in areas of lower elevation/latitude.

In Chapter 4, I gather together all my other studies related to Notholaena standleyi, including: 1) morphological and anatomical observations of its desiccation-tolerant leaf, with special focus on the farina; 2) two cases of hybridization between the chemotypes, one between the diploid chemotype Y and tetraploid chemotype YG, and another between diploid chemotypes Y and G; and 3) morphological and physiological comparisons between the two diploid chemotypes Y and G. My plan is to finalize these studies and submit them for publication in the near future.

In Chapter 5, I summarize collaborative contributions that I made to other fern studies during my Ph.D.

The kingdom Fungi is one of the major groups of the plant microbiome(Hardoim et al., 2015; Vandenkoornhuyse et al., 2015; Peay et al., 2016). Of the various plant-fungus interactions, mycorrhizal fungi that form mutualistic associations with host plants are the best studied symbiotic system(Bonfante & Genre, 2010; van der Heijden et al., 2015). Fungal endophytes represent another major type of plant-fungus symbioses(Rodriguez et al., 2009; Porras-Alfaro & Bayman, 2011). Defined as endosymbionts inhabiting a wide range of plant and lichen hosts without causing obvious symptoms, endophytes are now considered both ubiquitous and hyperdiverse (Stone, 2004; Rodriguez et al., 2009; U'Ren et al., 2012). Yet most of these fungi have to be identified using a phylogenetic approach (Arnold et al., 2009; Gazis et al., 2012; Chen et al., 2015) and remain unknown at lower taxonomic ranks (e.g., genus and species) and undefined in terms of their function in their symptomless hosts(Arnold et al., 2003; Busby et al., 2016). It is now understood that some endophytes are capable of switching to pathogenic(Wipornpan Photita et al.; Ávarez-Loayza et al., 2011) or saprotrophic(U'Ren et al., 2010; Zuccaro et al., 2011; Kuo et al., 2014) modes, but the genetic mechanisms of these switches remain unexplored. Bryophytes are a major component of the vegetation in boreal and arctic regions, where ecosystems are most vulnerable to global climate change(Turetsky et al., 2012; Jassey et al., 2013). It has been proposed that early land plants adopted a terrestrial lifestyle with the help of fungi(Heckman et al., 2001; Field et al., 2015). Mosses do not have mutualistic fungal symbionts such as mycorrhizal fungi(Davey & Currah, 2006; Field et al., 2015), but they are known to harbor diverse fungal endophytes of uncertain functions(U'Ren et al., 2010; Davey et al., 2012; Davey et al., 2013). The growth form of the moss Dicranum scoparium provided an ideal system for studying functional transitions between endophytism and saprotrophism across a senescent gradient. My PhD thesis focuses on the evolutionary history (Chapter 1) and functionality (Chapter 2, 3) of endophytic fungi.

In Chapter 1, I investigated the phylogenetic placements of fungal endophytes within the pharmaceutically and agriculturally important class Eurotiomycetes. The class Eurotiomycetes (Pezizomycotina, Ascomycota) includes various fungi with different ecological traits, including animal pathogens, saprotrophs, ectomycorrhizae, plant pathogens, rock-inhabiting fungi, lichens and endophytes(Geiser et al., 2006; Schoch et al., 2009; Gueidan et al., 2015). Phylogenetic affiliations of eurotiomycetous fungal endophytes with their ecologically diverse relatives had not been evaluated, leaving a gap in our understanding of the major evolutionary trends and ecological breadth of Eurotiomycetes as a whole. To fill this gap, we recently inferred the phylogenetic and taxonomic affinities of representatives of class 3 endophytes within Eurotiomycetes (Chen et al., 2015). Our results based on seven loci and 157 taxa revealed an undescribed new order (Phaeomoniellales) composed mainly of fungal endophytes and plant pathogens, and to a lesser extent, endolichenic and lichen-forming fungi. However, most of the deep nodes within this order were poorly supported. Interestingly, while described species of the order Phaeomoniellales are mostly plant pathogens on angiosperms (e.g., Genera Vitis, Nephelium and Prunus(Groenewald et al., 2001; Damm U. et al., 2010; Rossman et al., 2010; Thambugala et al., 2014)), endophytes within this order were mostly isolated from leaves of gymnosperms (Fig.1). These results, first-authored by the Co-PI, have been published in the journal Molecular Phylogenetic and Evolution(Chen et al., 2015).

In Chapter 2, I used metatranscriptomes of fungal ribosomal RNA to detect active fungal communities across a gradual gradient of senescence in wild-collected gametophytes of Dicranum scoparium (Bryophyta) to understand the distribution and the active component of fungal communities at a given time in adjacent living, senescing, and dead tissues. My results suggested that Ascomycota generally were more prevalent and active in living tissues, whereas Basidiomycota were more prevalent and active in senescing and dead tissues. Differences in community assembly detected by metatranscriptomics were echoed by amplicon sequencing of cDNA and compared to culture-based inferences and observation of fungal fruit bodies in the field. The combination of metatranscriptomics and amplicon sequencing of cDNA is promising for studying symbiotic systems with complex microbial diversity, allowing simultaneous detection of microbial presence, abundance and metabolic activity in symbiotic systems.

In Chpater3, I investigated the functions of D. scoparium across its naturally occurring senescence gradient and the associated fungal nutrient transporter (carbon, amino acid, phosphorus and nitrogen) activities. Higher fungal nutrient-related transporter activities were detected toward the bottom layer of the moss gametophytes. Among the four fungal nutrient types (Amino acid, carbon, nitrogen, phosphorus), the activities of nitrogen-related transporters had a drastic increase proportionally toward the bottom layer. In parallel, nitrogen breakdown was detected as the most enriched Gene Ontology term of D. scoparium for those transcripts having higher expression in the bottom layers. I analyzed the most abundant fungal nitrogen-related transporters in my dataset, the ammonium transporters, using a phylogenetic approach. I revealed that all ammonium transporters actively expressed in association with D. scoparium belong to the MEPg clade. Different sets of potential plant-microbe communication/defense/symbiosis-related genes are highly expressed in top vs. bottom layers, which suggest different mechanisms are involved in plant-fungus associations in photosynthetic vs. decomposing tissues.

Plants in the genus Sphagnum (peat moss) are the dominant biotic features of boreal peatlands that store nearly one-third of Earth’s terrestrial carbon. Peat mosses are ecosystem engineers and create the peatlands that they inhabit through the accumulation of peat, or partially decayed biomass, and the functional traits underlying this extended phenotype. Interspecific functional trait variation is hypothesized to promote niche differentiation through the creation and maintenance of ecological gradients along which species sort within communities. One prominent gradient relates to height-above-water-table wherein some species produce hummocks raised up to a meter above the water table, while others live in hollows at or near the water table. However, it is unclear how these traits evolved during Sphagnum diversification, to what extent natural selection produced functional trait variation, and which genes might contribute to such phenotypes.

In Chapter 2, a meta-analysis of data from recent studies is used to relate patterns of functional trait variation to the phylogeny of Sphagnum. The results suggest that interspecific variation in various measures of growth, decomposability, and litter biochemistry is phylogenetically conserved in Sphagnum, meaning that closely related species tend to be more similar in trait values than species selected at random from the phylogeny. Furthermore, these results suggest that patterns of trait covariation might represent adaptive syndromes related to niche. This is the first study to formally relate functional trait variation in Sphagnum to its evolutionary history.

In Chapter 3, a field experiment and phylogenetic comparative methods are used to show that natural selection is responsible for shaping interspecific variation in Sphagnum decomposability and its coevolution with niche. In the largest experiment of its kind to date, litter decomposability was measured for over 50 species of Sphagnum under natural conditions. Models of trait evolution were competed against one another to determine which best explained the evolution of this important functional trait. The best model was a multiple-peak Ornstein-Uhlenbeck process wherein the predominantly hummock and hollow clades of Sphagnum possess separate adaptive optima towards which trait values are pulled. Furthermore, the results suggest that shifts in trait optima occurred concomitantly with shifts in realized niche along the hummock-hollow gradient.

In Chapter 4, comparative genomics is used to identify genes involved in the biosynthesis of an ecologically-important class of secondary metabolites, the anthocyanins, and determine when this biosynthetic pathway first evolved in embryophyte land plants. In Sphagnum, complex phenolic molecules embedded in the cell walls influence litter decomposability and, in turn, the rate of peat accumulation. One group of such phenolics, the sphagnorubins, confer red-violet pigmentation to some species of Sphagnum. Sphagnorubins are thought to be homologous to anthocyanin pigments, known best from flowering plants, that have numerous roles including mitigation of abiotic stress. Phylogenetic analyses using full genome sequences representing nearly all major green plant lineages show that the entire anthocyanin biosynthetic pathway was not intact until the most recent common ancestor of seed plants. Furthermore, orthologs of many downstream enzymes in the pathway are absent from seedless plants including mosses, liverworts, and ferns. These results suggest that the production of red-violet flavonoid pigments in seedless plants, including sphagnorubins, requires the activity of novel enzymes and represents convergent evolution of red-violet coloration across land plants.

In Chapter 5, comparative genomics is used to test for molecular adaptation in Sphagnum genomes. Two reference-quality peat moss genomes were compared to those from other plants to identify genes bearing signatures of positive selection and gene families marked by significant rates of expansion in the Sphagnum lineage. Gene Ontology enrichment analyses were then used to identify over-represented classes of genes that might have been particularly important during the evolution of peat mosses. The results suggest adaptive evolution largely occurred in genes and gene families related to epigenetic regulation, secondary metabolism, stress response, and transmembrane transport. Together, these data suggest that selection favored changes to genes involved in response to environmental stress and provide candidate loci that might underlie adaptation to the harsh conditions of boreal peatlands.

How geographical distance and historical events affect patterns of population divergence and gene flow is an important question in evolution and biogeography. Sphagnum subgenus Acutifolia is one of the four major subgenera of Sphagnum peatmoss comprising numerous species with broad geographic ranges and diverse ecological niches within wetland habitats. One group of particular interest within the subgenus is the S. capillifolium complex which contains at least seven closely related species. Five species within the complex are circumboreal and all have overlapping geographic ranges, one species is endemic to subtropical region of eastern North America, and one species is in the tropical regions of Central and South America. The presence of both species with overlapping and disjunct distributions makes the Sphagnum capillifolium complex a natural experiment to investigate gene flow and population divergence in multiple phylogenetic scales (within and between species). Chapter 1 describes patterns of phylogenetic discordance across the genome in the Sphagnum capillifolium complex using whole genome resequencing data. The species tree phylogeny was generally well supported but phylogenetic discordance among genomic regions was prevalent, especially at nodes in the backbone. Alternative topologies for each of the backbone nodes were not random, suggesting the presence of introgression, in addition to incomplete lineage sorting (ILS). Analyses of introgression signals using ABBA/BABA tests and branch length distributions (QuIBL) showed that there were several possible introgression events within the S. capillifolium complex involving both extant and ancestral species. Most of the introgression events occurred between species that currently have overlapping geographic ranges. Further investigation of one introgression event using comparisons of terminal branch lengths showed that the biased pattern of shared derived alleles likely derives from introgression, not ancient polymorphism. These findings show that introgression played a significant role in generating phylogenetic incongruence within the S. capillifolium complex. We also show that the use of multiple phylogenomic methods and investigating localized genomic regions are essential to infer complex introgression scenarios. Chapter 2 describes phylogenetic structure of Sphagnum subgenus Acutifolia and population structure of circumboreal species within the S. capillifolium complex. Genome scale data (RAD-seq) was generated for the subgenus, with an intensive population sampling of circumboreal species within the S. capillifolium complex. Most of the species are resolved as monophyletic, although relationships among species are weakly supported in some parts of the phylogeny. Some currently recognized species are phylogenetically discernable while others are not distinguishable from the well-supported species. Within the S. capillifolium complex, five circumboreal species show similar patterns of population structure. One population system comprises plants in eastern North America and Europe, and sometimes includes plants from eastern Eurasia and the Pacific Northwest of North America. Another group comprises plants in the Pacific Northwest, or around the Beringian and Arctic regions. Our results suggest that populations of circumboreal species survived in multiple refugia during the last glacial maximum (LGM). Long-distance dispersal out of refugia, population bottlenecks, and possible adaptations to conditions unique to each refugium contribute to current geographic patterns. There are patterns of genetic admixture among distinct genotype groups within species in some restricted areas. Alaska is a hotspot for both intraspecific genetic diversity and admixture. These genetic results indicate the important role of historical events, especially Pleistocene glaciation, in shaping the complex population structure of plants with broad distribution ranges. Chapter 3 assesses the pattern of gene flow between a pair of sister Sphagnum species within the S. capillifolium complex: S. warnstorfii and S. talbotianum. The two species have different distribution ranges: S. warnstorfii is circumboreal while S. talbotianum is circumarctic, but they overlap in Alaska. Genetic data from chapter 2 were used in this chapter. Analyses of interspecific gene flow and population sizes were accomplished using coalescent simulations of site frequency spectra (SFSs), and the signature of gene flow was further corroborated by ABBA/BABA statistics. Our results indicate that S. warnstorfii and S. talbotianum were isolated after divergence. S. warnstorfii was relatively recently established in Alaska and Alaska is the only region that shows evidence of gene flow between S. talbotianum and S. warnstorfii. Gene flow occurred in only one direction from S. talbotianum into S. warnstorfii, which can possibly help S. warnstorfii survive in subarctic conditions. Molecular evidence further suggests gene flow from Alaska S. warnstorfii to other regional populations of that species. S. warnstorfii suffered a stronger population bottleneck than S. talbotianum, suggesting that Beringia could have harbored larger populations during the last glacial maximum than other, likely more southern, refugia. Although the two species are very closely related, S. talbotianum has larger pores on the convex surfaces of branch leaf apices than S. warnstorfii. Our results represent a case study of a recent gene flow between geographically sympatric peatmoss species using genomic data. Our results also support S. talbotianum as a distinct species from S. warnstorfii.

Why do species exist where they do? Understanding the forces and processes that shape species’ ranges—and that affect their dispersal and range expansion—have long fascinated biologists. In this work, I focus on understanding diversity and dispersal in Cheilanthes ferns. I first describe a species new to science, Cheilanthes ecuadorensis, from among the understudied South American members of this genus. I then turn to studying the widely distributed, asexual, Australasian species C. distans. Careful review of samples from this species allowed me to find sexual specimens previously unknown to science that exist in a narrow range, as well as to catalogue extensive spore diversity that has gone unrecorded. I find strong evidence for trade-offs related to spore size, with larger spores having higher germination while smaller spores have greater dispersal. Excitingly, I find that spores previously catalogued as abortive are in fact viable, and contribute to the spore size diversity I observe. I then place these findings into phylogenetic context by building a phylogeny for all Australasian Cheilanthes, and use it to explore the relationships of sexual and asexual lineages, of different ploidy levels, and of geographic distributions. These analyses reveal that most dispersal in C. distans occurs over shorter rather than longer distances, in contrast to previous hypotheses posited by fern biologists. I observe that lineages are not limited to particular geographic regions, as well finding that dispersal is asymmetrical and seems to be tracking trade winds. For all my work I rely heavily on herbarium specimens and use them to catalogue morphological variation as well as to obtain DNA sequences that are used for phylogenetic analysis. I implement a variety of statistical and systematic analyses to explore correlations between spore size, reproductive mode, ploidy, germination, and dispersal. While this work expands our knowledge of fern diversity and biogeography, much still remains to be understood, including cataloguing possible novel species, understanding the biology behind spore size determination, and exploring the role of niche in the dispersal and range expansion of C. distans.

Science has an inspiring capacity to change the world around us as it informs of the details governing life. From feats of engineering to medical breakthroughs, it has rapidly changed the way we live. Though it is woven into the fabric of all we do, there are still gaps between science and society—such as how science is shared from the academy and connects to communities. This has always fascinated me and the resulting body of this dissertation is three parts biology, exploring the symbiosis between the small aquatic fern, Azolla, and its cyanobacterial symbiont, Nostoc azollae; and one-part sociology, examining ways to reconnect science to the society it impacts so deeply and to which it owes so much. My hope is that within these pages you will find a new enthusiasm for plants—especially the tiny wonder, Azolla—and that you will see the value I do in strengthening the connection between science and the communities around it. With everything we do as scientists we have the power to affect the world around us, and it is our responsibility to think deeply about how we engage and join science and society together for the betterment of us all.

Chapters 2 and 3 focus on the biological aspects of the symbiosis between the aquatic fern, Azolla filiculoides, and its symbiont, N. azollae. It is not so coincidental that this symbiosis ended up being the subject of my research and has sweeping connections to societies around the globe—including its use as a green fertilizer in China for over 1500 years. Azolla is a small genus of aquatic fern with immense green potential to positively impact the globe. It owes this distinction to the nitrogen-fixing cyanobacterial symbiont, N. azollae, it harbors within specialized cavities within its photosynthetic leaves. These two partners have been living together for over 90 million years. There are many plant microbial symbioses, however, what makes the Azolla-Nostoc symbiosis unique among the others is that the cyanobiont is intimately associated with the fern perpetually throughout both organisms’ life cycles. The two are rarely—if ever—seen apart. This symbiosis has long captured the curiosity of scientists, who have explored various aspects in detail, such as what compounds are exchanged between the two partners, how the leaf cavities develop, and what structures are present within the cavities. Presently, we are at a stage to delve deeply into understanding the way this symbiosis thrives by exploring the genome, transcriptome, and connecting these to features of the leaf cavity.

Chapter 2 details a visual examination of the symbiosis using confocal microscopy. I used a clearing protocol coupled with confocal microscopy to image the leaves of Azolla filiculoides as the symbiosis develops to visualize the symbiotic cavity and labeled different cellular components with the fluorescent dyes calcofluor white and 4’,6-diamidino-phenylindole (DAPI). I imaged the cavity trichomes within the leaf pocket in whole leaves, as well as the trichomes at the apex that facilitate movement of the cyanobiont into the megaspores. These trichomes are the main plant structures that interact with the cyanobiont. The ultimate goal is to use this technique alongside the genomics and transcriptomics data (chapters 3 and 4) to identify the functions of the trichomes, eventually outlining the strategy A. filiculoides uses to engage in its symbiosis.

Chapter 3 takes a closer look at the putative symbiosis genes. I examined the expression of ammonium assimilation genes and their potential post-translational modifications; as well as how the larger pool of putative symbiosis genes may be transcriptionally regulated and what their functional categories are using gene ontology analysis. The RNA-sequencing analysis revealed 160 putative symbiosis genes. These genes included nutrient transporters for compounds like ammonium and molybdate, but did not include glutamine synthetase, glutamate synthase genes, or sucrose transporters. We also found that the nitrogen assimilation genes in A. filiculoides lack the post-translational modifications used in other plants to regulate their activity, leading to questions about how Azolla does this differently. This work provides the groundwork for establishing new ideas for how the Azolla-Nostoc symbiosis works, which factors are used to communicate between the two partners and what is used to regulate the exchange of nutrients, all of which allows their life cycles to be linked together.

This dissertation concludes with a departure from the biology of the Azolla-Nostoc symbiosis, and transitions into a survey of how science and society can be reconnected. In chapter 4, I detail three case studies addressing problems that keep science and society separated— (1) the inaccessibility of science to certain groups, (2) the inability of scientists to build trusting relationships with non-scientific audiences, and (3) the lack of innovative ways to engage the public about science. The first case study specifies programs I have been involved in to engage underrepresented minority students in the sciences. I also detail work to improve science to make it an inclusive environment for these students to succeed and thrive. The second case study focuses on training and preparing scientists to interact with the public. This is crucial to share science in ways that are accessible and resonate with people. I remark on my use of improvisational comedy as a way of making scientists more attentive to their audiences as they are presenting as well as improving their own body awareness. I also discuss work to introduce them to the basics of good science communication, and outreach opportunities to put this training into practice. In the third case study, I make a case for joining the arts and sciences as a means to powerfully connect science and people. This comes from a science and art exhibit that I launched with fellow graduate students, that has sparked a wave of other science-art minded endeavors. The combination of art, science, and community-engagement hit upon a way to captivate the public and pull them into the stories behind science. Together, these seek to be examples of how we can rejoin science and society in meaningful ways, allowing all people to share in science and see how science weaves into our lives.

Theoretical and empirical studies have consistently shown that the optimal timing of seed germination reduces exposure to physical stress and minimizes competitive interactions with neighbors. However, this research has not accounted for facilitative (positive) interactions among plants, which become more pronounced as environmental stress increases. Facilitation is more likely to occur early in a plant's life when it is more susceptible to stress. In seasonal environments, the stress a given individual experiences can change throughout the year, and some years are more stressful than others. These sources of temporal variation in stress will dictate the facilitation-competition balance that individuals experience. However, it remains unclear how this balance affects the optimal timing of germination. My dissertation research asks how the timing of germination responds to neighbors, how those responses affect the facilitation-competition balance individuals experience, and how that balance in turn affects fitness and demography. More generally, it asks how the timing of germination and other types of emergence affect the facilitation and competition that individuals experience throughout their lives.

I used laboratory, greenhouse, and field experiments to examine how the timing of germination in the winter annual Arabidopsis thaliana (Brassicaceae) responds to cues of neighbors and how those responses affect interactions with neighbors. I then developed a mathematical model of population growth in an annual plant to examine how intraspecific facilitation and competition over ontogeny affect the optimal degree of investment in dormancy (i.e., delayed germination) in variable environments.

My experiments revealed that seeds of A. thaliana typically delay germination in response to neighbors and that these responses can promote facilitative interactions and reduce competitive ones with neighbors. Selection against delayed germination, which occurs because of stress later in the season, can be mitigated by facilitation. Further, delaying germination can be beneficial by increasing the difference in sizes between seedlings and their neighbors, which may promote resource partitioning. In the theoretical study, I found that increasing the degree of investment in the fraction of dormant seeds (i.e., delaying germination) can promote the persistence of populations that experience both facilitation and competition in variable environments. This occurs because increased dormancy prevents high juvenile densities that promote facilitation and consequently limit reproduction in large populations. The findings of this research indicate that plant-plant interactions depend strongly on temporal context, and they reveal that the facilitation-competition balance determined by temporal variation in stress plays a key role in how germination and dormancy traits will evolve in variable environments.