Michael J. Sanderson, PhD - US grants

The characteristics of ciliary activity and the periciliary fluid layer of the lung are vital to the lung defense mechanism of mucociliary clearance. Because impaired mucus transport results in chronic obstructive lung disease, it is important to understand the underlying cellular regulatory mechanisms of mucociliary transport. In this proposal the basic electrophysiological characteristics of respiratory tract ciliated and non-ciliated cells from human (normal and cystic fibrosis) and rabbit trachea will be investigated to determine the involvement of membrane properties in the regulation to ciliary activity and the depth and composition of the periciliary fluid layer in the lung. We will measure membrane potential (Vm) and whole-cell currents from human and rabbit ciliated cells using whole-cell patch clamp techniques. Cells will be grown in culture conditions designed to maintain a differentiated cell type. The effects of B-adrenergic agents, cAMP analogs, prostaglandins, Ca++ ionophores and mechanical stimulation on the cell Vm and ion currents, especially C1- and K+, will be examined. With single-channel patch clamp techniques, we intend to identify and characterize the ion channels of isolated human and rabbit ciliated cells. Because C1-, K+ and amiloride-sensitive Na+ channels have been found in other RT cell preparations, we will determine whether these channel types are present in our identified cell types. Preliminary evidence implicates a role for Ca++ and stretch-activated channels in RT ciliated cell physiology, but their presence has not, as yet, been demonstrated. Consequently, we will determine if Ca++ and stretch-activated channels are also present in ciliated cells. The basic properties of the ion channels present will be determined. Regulation of ion channels by Ca++ and cAMP will be investigated. By understanding the cellular mechanisms that regulate late mucociliary activity, the conditions that cause a failure of mucus transport and predispose the lung to disease can be identified and improvements made in preventive and therapeutic pulmonary care.

Airway epithelial cells are vital to normal lung function. By forming and maintaining a cellular barrier and being responsible for mucociliary clearance and transepithelial ion transport, epithelial cells protect he airways from infection, dehydration and accumulation of inhaled material. A failure of one or more of these mechanisms commonly contributes to chronic obstructive lung disease. All of these epithelial functions utilize Ca2+ as a regulatory signal and require cooperative cell activity. However, the mechanisms of Ca2+ signaling and intercellular communication and the consequences that Ca2+ signals have for epithelial physiology is poorly understood. We have discovered that two types of Ca2+ signaling occur in epithelial cells non-propagating Ca2+ oscillations and propagating intercellular Ca2+ waves. We hypothesize that Ca2+ oscillations regulate individual cell function whereas propagating Ca2+ waves coordinate multicellular activity and that the principles underlying these Ca2+ signals are different. We intend to test these hypotheses by characterizing the mechanisms of Ca2+ signaling in airway epithelial cells and by determining the consequences that Ca2+ signaling has for ciliary activity and the control of mucociliary clearance, ion channel activity and the regulation of the periciliary layer, and wound healing to maintain the cellular barrier of the airways. Specifically, we will (1) determine, with digital video microscopy and Ca2+ specific dyes, if intercellular Ca2+ waves via gap junctions by a Ca2+ independent regeneration of inositol triphosphate, (2) characterized the properties of epithelial intracellular Ca2+ oscillations to determine why Ca2+ oscillations do not propagate to adjacent cells, (3)determine if intercellular Ca2+ signaling initiates and orients the early healing responses of airways epithelium to trauma induced by physical or chemical agents, (4) determine the consequences that Ca2+ oscillations and intercellular C2+ waves have for ion channel activity associated with periciliary fluid regulation and (5) determine how Ca2+ signaling regulates ciliary activity and mucociliary transport by epithelia cells. By understanding the role of Ca2+ signaling in these research will not only contribute to our knowledge of the pathophysiology of airway epithelial cells, but will also significantly contribute to our understanding of the principles of signal transduction and intercellular communication in non-excitable cells.

Air flow in the lungs depends on the maintenance of unobstructed airways and this is a major responsibility of the airway epithelial cells. These cells perform muco-ciliary clearance and ion transport to expel inhaled contaminants and prevent surface dehydration. Abnormalities in these airway defense mechanisms lead to decreased airway caliber. For example, compromised mucociliary clearance leads to chronic obstructive lung disease and defective ion transport is characteristic of cystic fibrosis. Another factor regulating air flow is bronchiole caliber and this is influenced by the contractile state and mass of the airway smooth muscle cells (SMCs). A decrease in airway caliber occurs in asthmatic patients due to airway hyper-reactivity and increased SMC mass. These epithelial functions require multicellular activity and are strongly influenced by changes in intracellular calcium concentration ([Ca2+]i). The location and close apposition of epithelial cells to small bronchiole SMCs also suggests that the epithelial cells may detect lumenal stimuli and pass information to the SMCs. Contraction of SMCs is initiated by an increase in (Ca2+]i but, at the tissue level, force production requires the cooperative effort of multiple cells. This cooperation is not mediated by neuronal activity as all airway SMCs are not innervated and the mechanisms coordinating multicellular increases in [Ca2+]i in SMCs are not understood. In airway epithelial cells, slow propagating increases in [Ca2+]i or intercellular Ca2+ waves are mediated by the diffusion of IP3 through gap junctions. We have recently observed that intercellular Ca2+ waves also occur in airway SMCs and between airway epithelial cells and SMCs. As a result, we hypothesize that heterotypic intercellular Ca2+ signaling provides a direct mechanism by which airway epithelial cells can communicate with airway smooth muscle cells. However, the spatial organization of intercellular Ca2+ waves and how these relate to a physiological action still remain poorly understood. Consequently, our aims are: 1) to characterize the properties, messengers and function of intercellular Ca2+ waves between epithelial and SMCs with digital fluorescence microscopy, in tissue cultures and intact tissue, 2) to determine the relationship between the Ca2+ signaling elements of the cell and the three-dimensional organization of Ca2 + waves in airway epithelial and SMCs and 3,) to identify the physiological role of Ca2+ waves in ciliary activity, cell migration and cell contraction of airway epithelial and SMCs. In summary, our overall objective is to understand the mechanisms and function of intercellular Ca2+ signaling between airway cells. With this understanding our ability to design therapies to counter obstructive lung disease and prevent asthmatic attacks will be enhanced.

[unreadable] DESCRIPTION (provided by applicant): Mucociliary clearance is the primary lung defense mechanism that prevents the accumulation of foreign material in the airways and relies on cooperative ciliary activity. In airway epithelial cells, Ca2+ commonly serves as a signal for cell regulation and strongly influences ciliary beat frequency (CBF). However, our understanding of the regulation and coordination of airway ciliary activity by Ca2+ is extremely poor even though this is vital to our comprehension of the physiology and dysfunction of the airway epithelium in obstructive lung disease. Changes in intracellular Ca2+ concentration occur in the form of propagating intercellular Ca2+ waves or asynchronous Ca2+ oscillations. These signals can encode spatial and temporal regulatory information but the mechanisms underlying Ca2+ oscillations and waves in airway cells, their interactions with each other and the consequences each have for airway cell physiology is virtually unknown. Extracellular ATP stimulates both Ca2+ oscillations and increases in CBF in airway epithelial cells. This is extremely important because ATP (or UTP) is released by the airway epitheliurn and may serve as a local regulator of mucociliary transport. However, the mechanism of ATP release is unknown. Consequently, our hypothesis is that Ca2+ oscillations provide temporal-spatial Ca2+ gradients within individual cells to regulate and coordinate ciliary activity and thereby control mucociliary clearance. To test this hypothesis, our specific aims are 1) to determine the molecular mechanisms linking changes in Ca2+ with changes in CBF in airway epithelial cells and the route of ATP release, 2) to characterize the spatial organization of intracellular Ca2+ oscillations and their relevance to ciliary activity of airway epithelial cells and 3) to quantify the relationship between Ca2+, CBF, metachrony and the control of mucociliary transport. By understanding how Ca2+ signals control and coordinate ciliary activity and how intracellular Ca2+ oscillations occur within cells, this research will significantly contribute to our knowledge and understanding of the pathophysiology of airway defense mechanisms, the principles of Ca2+ signal transduction in non-excitable cells and the potential use of ATP or UTP as therapeutic agents for airway disease.

The University of California, Davis, has been awarded a grant to develop new methods and software tools to help construct the genealogical "tree of life" of all biological species. The focus of these efforts will be on extracting information from the vast molecular sequence databases to build collections of smaller trees that can be assembled into larger, more comprehensive pictures of the tree of life. The scale of the data input is large: GenBank, for example, contains tens of millions of sequences sampled from over 100,000 species. Whereas extensive research has focused on the problem of building a tree from a single data set; relatively little is known about extracting these data sets en masse from sequence databases and then assembling a synthesis. The proposal is to study a set of novel computational problems that are as challenging as the basic tree building problem itself. These occur in three broad areas: (1) assessment of the potential information in sequence databases of various kinds; (2) optimal extraction of data from databases to bring the best information to bear on individual tree reconstruction; and (3) integration of these smaller trees into "supertrees" (larger trees assembled from smaller ones that share species in common), especially by identifying target sets of new sequences needed to construct optimal supertrees. Theoretical results will be evaluated by analysis of three diverse databases that pose a range of computational challenges (a subset of GenBank, SWISS-PROT, and the TIGR EGO database). This work will characterize the phylogenetic information content of these sequence sets, identify maximal sets of combinable sequence information, construct nonredundant partitions of the database to permit estimation of collections of trees, and assemble supertrees from these collections. The interdisciplinary team includes phylogenetic biologists and computer scientists with experience in phylogenetic theory, data analysis, and algorithm development and implementation. The project is also collaborating with three existing Tree-of-Life projects (each aimed at reconstructing particular portions of the tree) to provide tests of the sequence targeting algorithms.

[unreadable] DESCRIPTION (provided by applicant): A characteristic of asthma is airway hyper-reactivity, a response acutely mediated by smooth muscle cell (SMC) contraction. SMC contractility is initiated by increases in intracellular calcium concentration ([Ca2+]i) but the mechanisms of Ca2+ signaling in airway SMCs and how these relate to changes in airway caliber are poorly understood. To address this problem, we have developed a unique lung slice preparation that retains many properties of the lung structure, and in which the Ca2+ signaling of the SMCs and the associated airway contraction or relaxation can be measured simultaneously with confocal microscopy. Our data shows that airway SMCs display a graded range of IP3-based Ca2+ signaling that consists of elemental Ca2+ signals, intracellular Ca2+ oscillations and Ca2+ waves and that these signals correlate with the establishment of resting airway tone as well as the initiation and maintenance of SMC and airway contraction. [unreadable] [unreadable] Consequently, we hypothesize that the frequency of the Ca2+ signals in SMCs serves to regulate Ca2+ airway caliber. To test this idea, we plan to determine: 1) the Ca signaling mechanisms responsible for the Ca2+ establishment of resting airway tone by investigating the elemental Ca signaling and mechanism of spontaneous Ca2+ oscillations and contractions of airway SMCs, 2) if airway caliber is regulated by frequency-modulation (FM) mediated by Ca2+ oscillations, and 3) how Ca2+ signaling and contraction is altered in airway SMCs of mouse models for hyper-reactivity and asthma. By understanding the graded mechanisms of elemental Ca2+ signaling and Ca2+ oscillations in lung SMCs and how these events regulate SMC contractility and relate to airway caliber, we can gain the necessary insight needed to approach a therapeutic strategy for modulating airway hyper-reactivity.

[unreadable] DESCRIPTION (provided by applicant): Summary: Asthmatic lungs typically respond to inhaled allergens with exaggerated reductions in airway function. This phenomenon is termed airway hyper-responsiveness (AHR) and can be life threatening. AHR is not a simple reaction but is the culmination of multiple processes that manifest over a huge range of length and time scales. At one extreme, molecular signaling and interactions determine the force generated by airway smooth muscle cells (ASMCs). At the other extreme, contraction of the ASMCs is converted into a dynamic and complex constriction of branched airways that patients perceive by increased difficulty in breathing. Furthermore, asthma therapies are predominately pharmacological and operate at the molecular level, yet clinical outcomes are measured at the level of the whole lung. These two extremes are linked by numerous events operating at intermediate ranges of scale. These complex characteristics of AHR limit our understanding and ability to control asthma and will continue to confound research studies that only address responses at a single scale. Complex multi-scale systems cannot, by their very nature, be understood by studies limited to a few parameters. Consequently, this proposal will follow the innovative and alternative systems approach of developing a multi-scale experimental and computational model of AHR. We will initially determine how Ca2+ oscillations and the kinetics of cross-bridge cycling between actin and myosin molecules determine force production by ASMCs. Subsequently, we will determine how this force production distorts the airway wall and brings about airway narrowing throughout the lung. This will be achieved by the collaboration of a multidisciplinary group of investigators with experimental and mathematical expertise who will integrate our current knowledge and understanding of AHR at different cellular and tissue levels into a mathematical and computational model of AHR. The model will initially include phenomena that meet the criteria of being essential for airway contraction, of clear importance to AHR and experimentally accessible for iterative validation. In future studies, this model will be refined by the addition of relevant details. The model will be used to make specific predictions of molecular, cellular and tissue behavior and suggest critical experiments. In combination with extensive iteration between theory and experimentation, the sub-sections of the model will be refined and validated to identify the fundamental parameters that link the successive processes or scales. The results of this investigation will lead to an improved understanding of the link between the basic cellular pathophysiology and the whole lung response in asthma and other obstructive lung diseases This will improve the diagnosis of cause and effectiveness of treatment of these diseases. In addition, because AHR is clearly a complicated symptom, this investigation will evaluate the effectiveness of addressing disease with a systems biology approach. Many individuals in the USA suffer from asthma, a condition that is characterized by an exaggerated airway contraction or airway hyper-responsiveness (AHR). This response is extremely complicated being initiated at the molecular level by airborne allergens or stimuli and culminating at the organ level with difficulty in breathing. The objective of this research is to develop an understanding of this sequence of events by using a mathematical framework to guide and integrate experimental studies that elucidate the details of each process involved. With this approach, the key events in AHR can be identified and targeted for therapeutic intervention. [unreadable] [unreadable] [unreadable]

Research in the life sciences is undergoing a radical transformation. With the complete genome sequences of many organisms now available, scientists can begin to tackle fundamental questions that were previously intractable. Where do new genes come from? Which genes make us uniquely human? What functions are associated with different regions of the genome? Genomics will reshape biological research in the next twenty years and have a profound impact on medicine and human health, agriculture, engineering, our understanding of the origin of life and of the relationships among living organisms. Research in this new area requires the coordinated interaction of scientists with diverse backgrounds. To address this need, an Integrative Graduate Education and Research Traineeship (IGERT) program in Genomics was established at the University of Arizona five years ago. In recognition of the changing directions of the field, four new activities will take place in this renewal: (1) a lecture and computer laboratory course on comparative genomics, in which all students will learn the foundations of genomics including the skills to participate in (2) a hands-on class on reserach in genomics, (3) teaching and outreach at a local science magnet high school with 70% minority enrollment, and (4) annual symposia with invited speakers from other institutions. The program will also build on existing strengths, including interdisciplinary advising, research rotations, seminars, training in ethics, discussion groups, and internships at other academic institutions or in industry. The impact of this training program will be to equip the next generation of biologists with the tools to tackle the challenges of genome-scale research. This program to date has been highly successful, having trained Ph.D. students in 37 labs from 14 departments at the University of Arizona. A key new component of the renewal is the plan to involve IGERT students in high school science education. This will bring science in general, and genomics in particular, to a wider community, and will encourage minority students to enter careers in science at an early stage of their education. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

Phylogenetic trees, which depict the genealogical relationships of organisms to each other, are a key tool for organizing and analyzing information about biological diversity. Trees are used by many researchers in comparative biology and the demand for them is high. The proposed research will take advantage of the phenomenal breadth of data in the GenBank molecular sequence database (currently including sequences from 185,000 species, or some 10% of all species known to science) to build an electronic repository of one billion phylogenetic trees. The goal of this research is to build a very large number of phylogenetic trees and then construct simple search and retrieval tools to match these trees to any query list of species in which a user may be interested.

The primary impact of this research will be outside of the phylogenetics research community. Users of phylogenetic trees span most areas of modern biology: epidemiologists, genomicists, functional morphologists, conservation biologists, and community ecologists, to name a few. A repository of molecular phylogenies built using a consistent methodology, and satisfying prescribed minimal levels of statistical confidence, will provide users a level of consistency needed for strong inferences within and between comparative biological studies.

PI: Rod A. Wing (University of Arizona)

CoPIs: Scott A. Jackson (Purdue University), Manyuan Long (University of Chicago), Carlos A. Machado (University of Maryland), and Michael J. Sanderson (University of Arizona)

Collaborators: O. Panaud (University of Perpignan, France) and D. Weigel (Max Planck Institute for Developmental Biology, Germany), Doreen Ware (Cold Spring Harbor Laboratory), Qifa Zhang and Sibin Yu (Huazhong Agricultural University, China), Bin Han (National Center for Gene Research, China), Marco Wopereis (Africa Rice Center (WARDA), Benin), Mathias Lorieux (International Center for Tropical Agriculture (CIAT), Columbia, and Georgia Eizenga (DB NRRC, Germany.

Intellectual merit. Asian cultivated rice (Oryza sativa) feeds more people than any other crop. As the rice-dependent population is expected to double in about 25 years, breeders are faced with the enormous task of doubling rice yields with less land, water, and fertilizers, and on poorer soils. It is therefore critical that the scientific community unites to provide the tools and biological/evolutionary insights required to meet future needs. The project's long-term goal is to exploit 15 million years of evolutionary diversification and adaptation that have been locked in the genomes of wild and domesticated Oryza species, to both improve cultivated rice and address fundamental questions in evolutionary and comparative genomics. To accomplish this goal the project will first generate a set of publicly available genomics resources for the genus Oryza including: i) physical maps for three wild relatives of cultivated rice and the closely related outgoup species Leersia perrieri; ii) complete sequences for the short arms of chromosome 3 of these species; iii) complete reference genome sequences for O. barthii (the wild progenitor of West African cultivated rice), and O. punctata (a wild species that will serve as the evolutionary outgroup the eight AA genome species--the species group containing O. sativa); iv) gene and small RNA expression data sets for 11 Oryza species and L. perrieri; and v) DNA polymorphism data sets across the AA genome phylogeny. The project will integrate these new resources with 5 to 8 forthcoming new Oryza reference genome sequences to create the most comprehensive within-genus comparative genomics platform for any plant system. The project will interrogate this comparative genomics platform to discover key evolutionary events and characterize the forces that led to the formation of 24 Oryza species with 10 different genome types. Specific research areas that will be addressed include: analyses of structural variation, phylogenomics, population genomics, genome evolution and the origin of new genes, and the role of transposable elements in genome evolution. Libraries, genome sequences and their annotations will be accessible via public databases and repositories, such as the AGI BAC/TEST Resource Center (http://www.genome.arizona.edu/orders/), NCBI (http://www.ncbi.nlm.nih.gov/), and Gramene (www.gramene.org).

Broader impacts. This project will have broad impacts at several levels, including basic and applied research communities, postdoctoral/graduate/undergraduate and high school student training and mentoring, community outreach, and K-5 children and their families. For basic/applied research communities, the project will enhance and develop a genus-level resource as a platform for accessing new traits and alleles that is unparalleled in the plant world. This transformative platform can be used to address fundamental hypothesis-driven questions as well as provide essential tools to improve the world's most important food crop. The project will provide training and mentoring to postdoctoral scientists, graduate/undergraduate students and high school students interested in genome evolution, plant breeding, and careers in academic and corporate science. Finally, the project will develop a biannual "Plant Science Family Night" program, targeting K-5 students and families, to get children and their families excited about plants and the role plant science plays in ensuring a safe, sustainable, and secure food supply for our planet.

DESCRIPTION (provided by applicant): Asthmatic patients respond to inhaled stimuli with an excessive reduction in airway caliber, a phenomenon known as airway hyperresponsiveness (AHR). AHR is highly complex and reflects multiple processes that manifest over a large range of length and time scales. At one extreme, molecular interactions determine the force generated by airway smooth muscle (ASM). At the other extreme, the spatially distributed constriction of the many branches of the airway tree lead to persistent difficulties in breathing. Similarly, conventional asthma therapies are pharmacological and operate at the molecular level, while clinical outcomes are evaluated in terms of global lung function. These extremes are linked by numerous events operating over intermediate scales of length and time. Thus, AHR is an emergent phenomenon that is extremely challenging to understand in its entirety. This in turn limits our understanding of asthma and confounds the interpretation of experimental studies that each can address physiological mechanisms over only a very limited range of scales. Our solution to this conundrum has been to construct a modular multi-scale mathematical model that links and integrates experimental data from multiple scales. The current manifestation of this model, which is the result of a multi-disciplinary collaboration between 5 investigators with complementary experimental and mathematical expertise, incorporates force production by actin-myosin dynamics, force regulation by Ca2+ dynamics, force-dependent tissue deformation, and airway constriction. While this model demonstrates feasibility for our project and, most importantly, establishes computational algorithms for modeling over a wide range of scales, it currently represents only an initial frame-work. Consequently, in this proposal, we intend to develop our unique multi-scale computational model of the lung to the point where it can be used to make realistic predictions of bronchoconstriction, thereby allowing us to identify those pathophysiologic mechanisms having the greatest impact on AHR. We will include in this extended model: 1) at the molecular level, the kinetics of the contractile proteins during regular cross-bridge cycling and during the latch-state and their contributions to force production, 2) at the cellular level, the Ca2+ signaling mechanisms that regulate ASM force production, 3) at the tissue level, the detailed balance of forces between contracting ASM and the opposing viscoelastic tissue that determine airway narrowing, and 4) at the organ level, the topographic distribution of ASM contraction dynamics that determine changes in mechanical impedance in the normal and hyperresponsive lung. By extensive iteration between theory and experimentation, the modules of the model will be individually validated to identify the key parameters that link between successive scales. The model will then be used to make testable predictions of molecular, cellular and tissue behavior. This will improve our understanding of the link between cellular pathophysiology and the clinical phenotype in asthma. PUBLIC HEALTH RELEVANCE: Many individuals worldwide suffer from asthma, an inflammatory lung disease characterized by excessive constriction of the airways. This excessive constriction, termed airway hyperresponsiveness (AHR) can be life- threatening and is the result of an extremely complicated sequence of events initiated at the molecular level by airborne allergens or other stimuli, and culminating at the organ level with difficulty in breathing. The objective of this research is to understand the sequence of events leading to AHR by using a mathematical model to integrate data from experimental studies that by themselves can only elucidate the details of each step. With this multi-scale approach, we will be able to identify key events in AHR that may serve as targets for therapeutic intervention.

Scientists are using massive data sets of DNA sequences and new computing technologies to place all 2 million biological species into a grand synthesis, the evolutionary tree of life. Evolutionary trees permit predictions about the traits that organisms possess based on traits present in closely related species. This has important practical benefits in areas like medicine, conservation biology, and crop improvement. Although building small trees from DNA from just a few genes is straightforward, many obstacles remain to scaling up this effort to all life. One of the most serious is missing data. Under some conditions, even a few gene sequences missing for a handful of species can produce evolutionary trees that lack detailed resolution. This grant is a collaboration among biologists, mathematicians and computer scientists to understand the circumstances in which missing data cause problems, and to develop methods to overcome them. The research will generate mathematically provable results and new software to help biologists build more reliable large evolutionary trees. These products will be tested with real world data sampled from flowering plants, one of the most diverse branches of the tree of life, with over 250,000 species. The high quality evolutionary trees generated because of this project will provide a societal need by providing clear evolutionary knowledge to inform conservation planning and prioritization. The research team will organize a workshop for graduate students around the country to train them in the use of these new tools, and will develop educational materials for high school students involving computer visualization of the tree of life.


The construction of very large phylogenetic trees from sequence data mined from databases can be challenging because of the recently discovered problem of terraces--potentially vast regions in "tree space" in which all trees have precisely the same optimality score due to missing data. This research focuses first on developing a better conceptual understanding of four problematic impacts of terraces on large tree construction: (i) increased ambiguity, (ii) biased confidence assessments obtained from bootstrapping or Bayesian posterior probabilities, (iii) impediments to tree search algorithms, and (iv) downstream effects on comparative inferences that rely on trees. Next, the research will develop analytical methods and software implementations to overcome these problems. Finally, it will test these new methods by applying them to 27 large-scale phylogenies newly constructed within flowering plants, each with 1000+ species, examining trait evolution, as an exemplar of downstream comparative inference, in a subset of these.

Milkvetch (Neo-Astragalus) species from the legume plant family known as selenium hyperaccumulators are restricted to soils rich in the element selenium, where they accumulate the element in their tissues at levels that are toxic to other organisms. This research will determine the number of origins of selenium hyperaccumulation in milkvetch species by reconstructing the group's evolutionary history using DNA sequence data. The project will also sequence genes thought to be involved in selenium hyperaccumulation to determine if they have been subject to natural selection. This research will provide one graduate student and undergraduates with training in molecular, chemical, and bioinformatics techniques and will also supply molecular resources for population genetic studies of poorly known North American Astragalus species.

Twenty-five Neo-Astragalus species from two traditional taxonomic groups have been identified as selenium hyperaccumulators; however, it is not known if these groups are monophyletic. The researchers will sample these selenium hyperaccumulators and other species from across the clade and reconstruct a phylogeny using a target-enrichment approach for sequencing low-copy nuclear loci in order to infer the number of times selenium hyperaccumulation has evolved. Analytical chemistry methods will be used to survey selenium accumulation in relevant species, and phylogenetic ancestral state reconstruction will be used to characterize the number of origins of selenium hyperaccumulation from the resulting phylogeny. This approach can quantify the relative contribution of ecologically driven speciation and allopatric speciation in light of the number of origins of the cryptic trait, their phylogenetic position, and geographic ranges of the different species.