Browsing by Subject "Polyploidy"

The Polypodium vulgare complex (Polypodiaceae) comprises a well-studied group of fern taxa whose members are cryptically differentiated morphologically and have generated a confusing and highly reticulate species cluster. Once considered a single species spanning much of northern Eurasia and North America, P. vulgare has been segregated into approximately 17 diploid and polyploid taxa as a result of cytotaxonomic work, hybridization experiments, and isozyme studies conducted during the 20th century. Despite considerable effort, however, the evolutionary relationships among the diploid members of the P. vulgare complex remain poorly resolved, and several taxa, particularly allopolyploids and their diploid progenitors, remain challenging to delineate morphologically due to a dearth of stable diagnostic characters. Furthermore, compared to many well-studied angiosperm reticulate complexes, relatively little is known about the number of independently-derived lineages, distribution, and evolutionary significance of the allopolyploid species that have formed recurrently. This dissertation is an attempt to advance systematic knowledge of the Polypodium vulgare complex and establish it as a "model" system for investigating the evolutionary consequences of allopolyploidy in ferns.

Chapter I presents a diploids-only phylogeny of the P. vulgare complex and related species to test previous hypotheses concerning relationships within Polypodium sensu stricto. Analyses of sequence data from four plastid loci (atpA, rbcL, matK, and trnG-trnR) recovered a monophyletic P. vulgare complex comprising four well-supported clades. The P. vulgare complex is resolved as sister to the Neotropical P. plesiosorum group and these, in turn, are sister to the Asian endemic Pleurosoriopsis makinoi. Divergence time analyses incorporating previously derived age constraints and fossil data provide support for an early Miocene origin for the P. vulgare complex and a late Miocene-Pliocene origin for the four major diploid lineages of the complex, with the majority of extant diploid species diversifying from the late Miocene through the Pleistocene. Finally, node age estimates are used to reassess previous hypotheses, and to propose new hypotheses, about the historical events that shaped the diversity and current geographic distribution of the diploid species of the P. vulgare complex.

Chapter II addresses reported discrepancies regarding the occurrence of Polypodium calirhiza in Mexico. The original paper describing this taxon cited collections from Mexico, but the species was omitted from the recent Pteridophytes of Mexico. Originally treated as a tetraploid cytotype of P. californicum, P. calirhiza now is hypothesized to have arisen through hybridization between P. glycyrrhiza and P. californicum. The allotetraploid can be difficult to distinguish from either of its putative parents, but especially so from P. californicum. These analyses show that a combination of spore length and abaxial rachis scale morphology consistently distinguishes P. calirhiza from P. californicum and confirm that both species occur in Mexico. Although occasionally found growing together in the United States, the two species are strongly allopatric in Mexico, where P. californicum is restricted to coastal regions of the Baja California peninsula and neighboring Pacific islands and P. calirhiza grows at high elevations in central and southern Mexico. The occurrence of P. calirhiza in Oaxaca, Mexico, marks the southernmost extent of the P. vulgare complex in the Western Hemisphere.

Chapter III examines a case of reciprocal allopolyploid origins in the fern Polypodium hesperium and presents it as a natural model system for investigating the evolutionary potential of duplicated genomes. In allopolyploids, reciprocal crosses between the same progenitor species can yield lineages with different uniparentally inherited plastid genomes. While likely common, there are few well-documented examples of such reciprocal origins. Using a combination of uniparentally inherited plastid and biparentally inherited nuclear sequence data, we investigated the distributions and relative ages of reciprocally formed lineages in Polypodium hesperium, an allotetraploid fern that is broadly distributed in western North America. The reciprocally-derived plastid haplotypes of Polypodium hesperium are allopatric, with populations north and south of 42˚ N latitude having different plastid genomes. Biogeographic information and previously estimated ages for the diversification of its diploid progenitors, lends support for middle to late Pleistocene origins of P. hesperium. Several features of Polypodium hesperium make it a particularly promising system for investigating the evolutionary consequences of allopolyploidy. These include reciprocally derived lineages with disjunct geographic distributions, recent time of origin, and extant diploid progenitor lineages.

This dissertation concludes by demonstrating the utility of the allotetraploid Polypodium hesperium for understanding how ferns utilize the genetic diversity imparted by allopolyploidy and recurrent origins. Chapter IV details the use of high-throughput sequencing technologies to generate a reference transcriptome for Polypodium, a genus without preexisting genomic resources, and compare patterns of total and homoeolog-specific gene expression in leaf tissue of reciprocally formed lineages of P. hesperium. Genome-wide expression patterns of total gene expression and homoeolog expression ratios are strikingly similar between the lineages--total gene expression levels mirror those of the diploid progenitor P. amorphum and homoeologs derived from P. amorphum are preferentially expressed. The unprecedented levels of unbalanced expression level dominance and unbalanced homoeolog expression bias found in P. hesperium supports the hypothesis that these phenomena are pervasive consequences of allopolyploidy in plants.

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.

Whole-genome duplication (WGD) generates polyploid cells possessing more than two copies of the genome. These events commonly occur during the evolution of human tumors across tissue types and mutational drivers, affecting an estimated 30-37% of all tumors. The frequency of WGD increases in advanced and metastatic tumors, and WGD is associated with poor prognosis in diverse tumor types, suggesting a functional role for polyploidy in tumor progression. Experimental evidence suggests that polyploidy has both tumor-promoting and suppressing effects. The polyploidization of a normally diploid cells often compromises genomic stability. In this way, WGD may be capable of promoting tumor formation, growth and progression, by facilitating the evolution of genetic heterogeneity on which selection can act. However, while some features of polyploidy can promote tumor growth, these features can also be countered by associated tumor suppressive qualities of polyploidization and associated cellular stresses. Chromosomal instability and resulting aneuploidy often have negative effects on cellular fitness; this can occur through the induction of proteotoxic stress, replication stress and delayed proliferation. Polyploidization can also be opposed by cell intrinsic and extrinsic pathways, including p53, the Hippo pathway and immunosurveillance. How these diverse and multifaceted features of polyploid cells work together to regulate tumor progression remains unclear.

Using a genetically engineered mouse model of HER2-driven breast cancer, we explored the prevalence and consequences of whole-genome duplication during tumor growth and recurrence. While primary tumors in this model are invariably diploid, nearly 40% of recurrent tumors undergo WGD. WGD in recurrent tumors was associated with increased chromosomal instability, decreased rates of proliferation and increased survival in stress conditions. The effects of WGD on tumor growth were dependent on tumor stage. Surprisingly, in recurrent tumor cells, WGD slowed tumor formation, tumor growth rate and opposed the process of recurrence, while WGD promoted the growth of primary tumors. Our findings highlight the importance of identifying conditions that promote the growth of polyploid tumors, including the cooperating genetic mutations that allow cells to overcome the barriers to WGD tumor cell growth and proliferation.

While our results revealed fitness disadvantages for recurrent polyploid tumor cells, the paradox remains that WGD is common in cancer cells despite this, suggesting that cells must evolve ways to overcome barriers to tumorigenesis. These findings suggest that a polyploid cancer cell may be delicately balanced, relying on certain pathways or processes to compensate for its cellular deficiencies more than their diploid counterparts. Ploidy-specific lethality describes the phenomenon in which inhibiting the activity or expression of a specific protein results in death of polyploid cells but not their diploid counterparts. To interrogate this idea, we next employed our models of recurrent polyploid cells to explore the impact of polyploidization on gene expression and signaling dependencies. Using RNA sequencing we uncovered that tetraploid cells exhibited decreased expression of genes of the cGAS-STING pathway. We performed two loss-of-function CRISPR screens against the kinome, one in vitro and one in vivo, to identify ploidy-specific lethal genes. The in vivo screen revealed candidates for ploidy-specific lethal genes including Srpk1, Mark4 and Ryk. Together these results demonstrated that polyploid recurrent tumor cells exhibit unique gene expression patterns that may reflect selection pressure of the immune system and may rely on unique survival mechanisms in vivo.

Mitosis involves the faithful segregation of two identical copies of chromosomes into two daughter cells. This process is highly regulated to maintain genome integrity, as mis-segregation of partial or whole chromosomes can lead to genomic instability. Cells are constantly exposed to both endogenous and exogenous forms of DNA damage, which if left unattended to, can contribute to mitotic errors. Cells therefore possess DNA damage responses (DDRs) which involves enacting cell cycle checkpoints, DNA damage repair, and in cases of extreme damage – cell death or senescence.While several lines of investigation have identified key mechanisms of the DDR during interphase of the cell cycle, there are several key questions that remain with regards to how cells deal with damage that persists into mitosis. Further, there is currently a gap in knowledge on the mechanisms, timing, and conditions in which different aspects of the DDR are active and coordinated. In this dissertation, I will demonstrate how I implemented genetic and imaging tools using our laboratory’s previously established model system, Drosophila rectal papillar cells [hereafter papillar cells]. Using this model, I studied (1) mechanisms of the DDR during mitosis, (2) mechanisms that act in the absence of key DDR components, and (3) novel regulators and protein-protein interactions of the mitotic DDR. This body of work contributes to the growing knowledge of how cells tolerate DNA damage that persists into mitosis.

A fundamental question of biology is how tissues are organized. Tissues can be composed of many small cells or comparatively fewer large cells that add nuclear content to facilitate tissue growth. The cells can be separate, discrete units or interconnected collectives. The nuclear composition of a tissue has functional consequences from the tissue physiology to likelihood of cancers and hyperproliferation to the response to stress and tissue damage. These two decisions, to be small or large and to be distinct (mononucleated) or joined (multinucleated), and, especially, the interaction between these choices are poorly understood. In this dissertation, I identify the Drosophila rectal papillae as a new model to study tissue interconnectivity, multinuclearity, and the interaction between nuclear content and cytoplasm-sharing. I played a major role in the discovery that the adult Drosophila rectal papillae share cytoplasm and proteins up to at least 62 kDa. This sharing is developmentally regulated and requires membrane trafficking and gap junction genes instead of canonical cell-cell fusion or incomplete cytokinesis factors. This mechanism of sharing does not appear to involve plasma membrane breaches, a novel way for tissues to share contents. Additionally, I advance the Drosophila larval heart as a model to study nuclear content (ploidy) in heart development and physiology. Together, my work explores how tissues use mononucleate and multinucleate ploidy in development and physiology.

Given the emerging epidemic of renal disease in HIV+ patients and the fact that HIV DNA and RNA persist in the kidneys of HIV+ patients despite therapy, it is necessary to understand the role of direct HIV-1 infection of the kidney. HIV-associated kidney disease pathogenesis is attributed in large part to viral proteins. Expression of Vpr in renal tubule epithelial cells (RTECs) induces G2 arrest, apoptosis and polyploidy. The ability of a subset of cells to overcome the G2/M block and progress to polyploidy is not well understood. Polyploidy frequently associates with a bypass of cell death and disease pathogenesis. Given the ability of the kidney to serve as a unique compartment for HIV-1 infection, and the observed occurrence of polyploid cells in HIV+ renal cells, it is critical to understand the mechanisms and consequences of Vpr-induced polyploidy.

Here I determined effects of HIV-1 Vpr expression in renal cells using highly efficient transduction with VSV.G pseudotyped lentiviral vectors expressing Vpr in the HK2 human tubule epithelial cell line. Using FACS, fluorescence microscopy, and live cell imaging I show that G2 escape immediately precedes a critical junction between two distinct outcomes in Vpr+ RTECs: mitotic cell death and polyploidy. Vpr+ cells that evade aberrant mitosis and become polyploid have a substantially higher survival rate than those that undergo complete mitosis, and this survival correlates with enrichment for polyploidy in cell culture over time. Further, I identify a novel role for ATM kinase in promoting G2 arrest escape and polyploidy in this context. In summary, my work identifies ATM-dependent override of Vpr-mediated G2/M arrest as a critical determinant of cell fate Vpr+ RTECs. Further, our work highlights how a poorly understood HIV mechanism, ploidy increase, may offer insight into key processes of reservoir establishment and disease pathogenesis in HIV+ kidneys.

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte proliferation have been identified as potential candidates for functional heart repair following myocardial infarction (MI). Mature adult CMs are highly resistant to mitogenic stimuli, with many mitogens only slightly raising adult CM proliferation rates in vivo. Conversely, immature neonatal CMs are more proliferative, permitting early neonatal rodent hearts to partially regenerate following injury. It is therefore of importance to improve our understanding of the mechanisms behind CM maturation and cell cycle regulation, which could be aided by the development and utilization of more relevant in vitro systems to study CM proliferation. Of particular interest, CMs in vivo undergo polyploidization, an increase in cellular DNA content without division, during postnatal maturation. This has been shown to correlate with a decline in their proliferative capacity and an increase in cell size and maturation. The goals of this dissertation have been to utilize in vitro cardiac tissue models to: 1) identify potent mitogenic stimuli capable of inducing CM proliferation, 2) describe the underlying mechanisms behind how this stimulus promotes CM proliferation, and 3) investigate the link between CM proliferation, maturation, and polyploidy.

First, we examined how CM-specific lentiviral expression of various candidate mitogens affects human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) in vitro. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues (ECTs) generated from NRVMs, a constitutively active mutant form of the membrane receptor of mitogen activated protein kinase (MAPK) signaling pathway, human Erbb2 (cahErbb2), was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM cell cycle entry and mitosis, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of MEK or ERK signaling. These results suggested transient activation of Erbb2/ERK axis in cardiomyocytes as a potential strategy for regenerative heart repair.

We then explored whether activating ERK via a constitutively active mutant of a more downstream effector of MAPK pathway, BRAF (BRAF-V600E, caBRAF), can induce pro-proliferative effects in NRVM ECTs. Sustained CM-specific caBRAF expression induced chronic ERK activation, significant tissue growth, deficit in sarcomeres and contractile function, and tissue stiffening, all of which persisted for at least 4 weeks of culture. CaBRAF-expressing CMs in ECTs exhibited broad transcriptomic changes, shift to glycolytic metabolism, loss of connexin-43, and a pro-migratory phenotype. Transient, doxycycline-controlled caBRAF expression revealed that the induction of CM cycling is rapid, precedes functional decline, and the effects are reversible only with short-lived ERK activation. Together, direct activation of the BRAF kinase was sufficient to modulate CM cycling and functional phenotype, offering mechanistic insights into roles of ERK signaling in the context of cardiac development and regeneration.

In the aforementioned in vitro studies, we relied on use of the more mature and less-proliferative NRVM vs. hiPSC ECT model system for identifying cardiac mitogens. Unlike predominantly polyploid adult CMs, immature hiPSC-CMs are primarily mononuclear and diploid, which makes them permissive to proliferation and therefore potentially less suitable for mitogen screens. We therefore studied whether induction of polyploidy alone in hiPSC-CMs would be sufficient to promote their maturation in vitro. Lentiviral overexpression of dominant negative ECT2 (dnECT2), an important cytokinesis gene, was sufficient to induce polyploidy in hiPSC-CMs and NRVMs. Upon screening of small molecules for polyploidy induction, we identified transient small molecule inhibition of AURKB as an even more effective approach than dnECT2 expression in producing polyploid hiPSC-CMs. Compared to diploid hiPSC-CMs, small molecule-induced polyploid hiPSC-CMs exhibited increased cell size, mitochondrial density, and rates of transcription and translation. Polyploid hiPSC-CMs were also less proliferative than diploid hiPSC-CMs, suggesting an improved potential for use in screening for cardiac mitogens in vitro.

In summary, this dissertation describes the effects of MAPK activation on CMs and shows a causative relationship between polyploidy and some aspects of CM maturation. We produced multiple RNAseq datasets of NRVM ECTs overexpressing different constitutively active MAPK genes, which could be of interest to research beyond cardiac biology. Further investigation of MAPK pathway activation in CMs may show that some downstream ERK effectors mediate the pro-proliferative effects in CMs, while others coordinate CM functional changes or changes in cell hypertrophy and maturation. Dissecting these mechanisms could enable the design of more targeted MAPK pathway modifiers with utility in cardiac regenerative biology and anticancer therapeutics.

In an ever-changing world, evolution is an essential process that may allow organisms to adapt to their environment through natural selection. All evolutionary processes act through a single fundamental medium: genetic variation. Polyploidy, or whole genome duplication, is a major mechanism for evolutionary change because it is both widespread across taxa and results in a proliferation of genetic material that evolution can act upon. The key questions addressed here are: (1) How does chromosome pairing during meiosis in allopolyploids affect the magnitude of genetic variation?, (2) How does the genome of polyploids evolve following formation, and what genetic mechanisms govern this evolution?, and (3) How does genetic and genomic evolution in polyploids affect phenotypic evolution? I use the shy monkeyflower, Mimulus sookensis, a tetraploid of hybrid origin between Mimulus guttatus and Mimulus nasutus, to address these focal questions. In order to develop a foundation to aid in interpretation of my findings, I first investigate the evolutionary history of M. sookensis. Chromosome counts establish that M. sookensis is indeed an allotetraploid, and a review of taxonomic literature reveals that this species is heretofore undescribed. By analysing the patterns of genetic variation at chloroplast and nuclear loci in M. guttatus, M. nasutus, and M. sookensis, I show that M. sookensis has recurrently formed from M. guttatus and M. nasutus. Crossing experiments within M. sookensis indicate that recurrent origins can contribute to genetic diversity without contributing to reproductive isolation among independently arisen polyploid lineages.

To address my focal questions, I take advantage of an intriguing and striking difference in flower size among M. sookensis, M. guttatus, and M. nasutus. The flowers of M. sookensis and M. nasutus are small and remarkably similar to one another, while the flowers of M. guttatus and diploid and tetraploid F1 hybrids between M. guttatus and M. nasutus are large and showy. This phenotypic divergence in flower size between M. sookensis and M. guttatus-like hybrids indicates that small flower size has evolved in M. sookensis. Using genetic marker data and phenotypic measurements from synthetic neoallotetraploid Mimulus, I demonstrate that there are low levels of fragment loss and phenotypic variation in neoallotetraploids; this suggests that homeologous pairing and recombination following polyploidization is not a major source of genetic variation or phenotypic evolution in M. sookensis. Analysis of the whole genome sequence of two M. sookensis lines reveals that M. sookensis is a fixed heterozygote throughout its entire genome, in that it has retained both a M. guttatus-like and M. nasutus-like subgenome, neither of which have been removed through homeologous recombination. These subgenomes have been homogenized by widespread gene conversion, and do not appear to have been differentially affected by deletions or deleterious mutations. Finally, to directly characterize the genetic architecture of flower size in M. sookensis, I cross a large-flowered synthetic neoallotetraploid Mimulus to small-flowered M. sookensis. I then employ a novel genotyping-by-sequencing approach to identify quantitative trait loci (QTL) associated with flower size. I find that there is one locus that accounts for a large proportion of phenotypic variation, and four other loci also contribute to flower size variation between the parental lines. Some of these loci co-localize with previously identified loci for flower size in diploid Mimulus, while others do not. Altogether, genetic marker data, phenotypic analysis of neoallotetraploids, whole genome sequence data, and QTL mapping data suggest that the genetic variation necessary for flower size evolution was likely caused by both gene conversion and new mutations, but not homeologous recombination. These results suggest that trait evolution in polyploids may be affected by the unique attributes of polyploids, but that new mutations are always an important source of genetic variation, regardless of ploidy level.