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Green algae are the eukaryotic base of green plant evolution. Based on the presence of these traits in both Chlorophytes and Charophytes, the ancestors to these clades probably possessed basics such as mating types, plant hormones, cell polarity, the ability to osmoregulate and retain turgor of the central vacuole, tropisms (e.g., phototropism and gravitropism) and circadian rhythms. Taxa in the Chlorophytes and Charophytes that live in different habitats (marine and freshwater), varies in thallus size (micro- to macroalgae), morphology (simple flagellate to complex sessile), and organization (unicellular, colonial and true multicellular) were chosen. Having BAC resources to compare Chlorophytes to Charophytes is important because it will lead us to understanding which basic traits were essential to survival and evolution on land and which were not.

Chlorophytes: In the Chlorophytes are the only two genetic models among the green algae, Volvox and Acetabularia. Volvox acts as an important counterpart to the 2 siphonous species, Caulerpa and Acetabularia both in body habitus (colonial versus siphonous unicells), coupling of cellular and nuclear division (coupled in Volvox versus uncoupled in Caulerpa and Acetabularia) and in reproductive strategy. Whereas Volvox makes specialized reproductive cells, Caulerpa and Acetabularia donate all of the cytoplasm and all of the parental nuclei to their progeny. The siphonous body plans of Caulerpa and Acetabularia beg the question: was differentiation of cytoplasmic regions necessary for cellularization of the multicellular vegetative body plans of green land plants? We chose the siphonous Chlorophytes, Acetabularia and Caulerpa, to compare with the siphonous Charophyte, Chara.
Volvox carteri, a multicellular freshwater green alga with fully differentiated germ and somatic cells, and Chlamydomonas reinhardtii, the well-known unicellular green flagellate, shared a common ancestor as recently as 35 MYA. Together they provide a unique opportunity to explore the genetic basis for the evolution of multicellularity and cellular differentiation. Studies in progress (D. Kirk, pers. comm to D.F. Mandoli) are exploring changes in the structure and functions of orthologous gene products that have accompanied this important transition. Although a BAC library exists for C. reinhardtii, these comparative studies are inhibited by the absence of a comparable library for V. carteri.
Caulerpa species are multinucleate unicells that fascinate plant biologists, because they bear a striking superficial resemblance to land plants: a tube or "stolon" grows along the sea floor differentiating root-like structures from its lower surface, and leaves, supported on a leaf-stem, from its upper surface. Caulerpa species respond to gravity much as flowering plants do and, consistent with this, they make and respond to land plant hormones. They excel at rapid wound healing. Their secondary biochemistry is renown, producing unusual sequiterpenoids that act as cytoskeletal, neural and developmental toxins. Caulerpa taxifolia is a marine weed with potential for commercial impact. First found in the NW Mediterranean and Italy, it quickly spread to the SW Mediterranean and then to California. DNA fingerprinting suggests that this weedy strain of C. taxifolia came from an aquarium. Current studies focus on how to eradicate this ecological pest.
Classic developmental studies in Acetabularia acetabulum revealed the role of the nucleus and predicted the existence of mRNA for the first time in any eukaryote. Like green land plants, this giant, marine unicell grows by both tip and diffuse growth even though it lacks meristems and undergoes juvenile and adult development. It makes a morphologically elaborate thallus (-body plan) including a specialized reproductive structure even though it has just one nucleus for most of its life cycle. Important for cell biological studies, it is amenable to grafting within and between species and graft chimeras express intermediate morphologies. Evidence suggests that mRNAs involved in reproductive differentiation of the apex have a half-life of 20 days. This species shares an alternate codon usage with the ciliates. It makes a cell wall typical of a group of algae, "the mannan weeds" that differs fundamentally from the cellulosic wall of land plants. Evidence indicates that all but one of the DNA synthesis enzymes are on the chloroplast genome. This begs the question how the nuclear and organellar genomes co-regulate DNA synthesis required for reproduction (2-6 million progeny/cell/generation) and may provide fundamental insights into different resolutions of endosymbiotic problems. Mutants in morphology and development are now available. Well preserved in a 570 MY fossil record, the morphological innovations in thallus, laterals and specialized reproductive structures in the Dasycladales are remarkable. Interestingly, these regions may also be physiologicallydistinct which may represent an early drive to organogenesis. Genomic and cDNA libraries as well as EST arrays for both juvenile and adult phases (Mandoli et al, unpub) are available.

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The fossil record indicates that relatives of the modern charaleans were the dominant macrophytic vegetation in freshwaters for -300 MY ( The freshwater Charophytes are considered ancestral to land plants based on ultrastructural, biochemical and molecular evidence of extant species. Sequence analyses confirm the close relationship of the Charophyte algae to land plants. Charophyte innovations, such as the division apparatus, the "phragmoplast', were clearly built upon by green land plants.
Based on morphological and molecular analyses, Mesostigma viride, a scaly, freshwater flagellate in the Prasinophyceae, is thought to be an early divergent species most closely related to charophyceans. It is essential to understanding the evolution of green plants. Of all the taxa discussed, this is the most basal, retaining the most primitive features of green plants. One key feature, a "multilayered structure" is common to Mesostigma viride, the reproductive cells of charophyceans and land plants. Data from mitochondrial, chloroplast and nuclear genes support Mesostigma viride as basal to evolution of the Charophyta. BAC library would be invaluable because there are virtually no molecular resources available to study this evolutionarily important species.
Chara, which are sister to land plants, are branched, filamentous species reaching lengths of A m. The size of its internode cells (95 cm long) makes it ideal for electrophysiology and many of these ion channels resemble those in green land plants. A specialized apical cell remains meristematic, cutting off daughters only from its lower surface. Charalean cells use a land plant-like phragmoplast during cell division, but differ from green land plant division in regulation of cytokinesis. Like Coleochaete, Chara sp. control the plane of cell division and make plasmodesmata. Chara cells also undergo asymmetric cell division, an ability correlated with formation of special differentiated cells in green land plants, e.g. stomatal complexes. They make a thick, layered cell wall that contains cellullose. As in Coleochaete, vegetative cells form elaborate structures which grow in coordinated fashion to enclose sexual cells of Chara, presaging the evolution of the embryophytes. In addition, nodal complexes are actually tissue-like or parenchymatous. Each internode cell contains thousands of nuclei. C. corallina internodes contains nuclei ranging from 5.4 to 10.9 pg/nucleus (A. Arumuganathan, M. Bisson & DF Mandoli, unpub.) which may well reflect different stages of cell division. If the genome of C. corallina is smaller than that of C. aspera , it will be used instead of C. aspera. Because Chara internode nuclei may be endoreduplicated, we will also determine the IC value of the nucleus in the meristematic cell of Chara sp, with confocal microscopy in order to avoid making too large a BAC library.
Coleochaete orbicu/aris is an epiphyte that grows as a monolayer of cells resembling tissues (the protonemata) of the early divergent moss Sphagnum. An important morphological innovation, Coleochaete differentiates cell types making terminal or marginal meristems and is capable of controlling the plane of cell division. Like charaleans and green land plants, Coleochaete cells are interconnected by plasmodesmata. Like Mesostigma, Coleochaete possesses a multi-layer structure. Species with tissue-like organization tend to grow in shallow waters and may presage evolution of green land plant tissues. Given that this species is frequently exposed to desiccation in its native habitat and can survive in moist air, the evolution of physiological and physical features for water conservation essential to survival on land is plausible. Consistent with this, the mucilage of this species forms ridges that have some ultrastructural similarities to cuticles of green land plants. Grana in Coleochaete thylakoids are organized more like those in green land plants than in other algae. Two features of reproduction in Coleochaete presage evolution of the embryophyta. Unlike other algae, egg cells of Coleochaete orbicularis are retained on the thallus. In addition, vegetative cells surrounding the egg transfer nutrients to the embryo. Like the pollen of land plants, Coleocheate makes sporopollenin and phenolics using them to protect reproduction.
Alternative green algae. Construction of BAC libraries for Chlorelia sp. (Trebouxiophyceae), K/ebsormidium flaccidum (Klebsormidiales), and Mougeotia transeaui (Zygonematales) would round out sampling of major lineages in the algal phylogeny. Note that several polyphyletic groups, such as the Prasinophytes that includes Mesostigma, are not encompassed by our taxomonic sampling. However, the phylogenetic data to make rational decisions are simply not yet available in these clades.
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The extant non-seed embryophytes include three major groups: the bryophytes (liverworts, hornworts and mosses), lycophytes (Lycopodaceae, Selaginellaceae and Isoetaceae) and the moniliforms (psilotophytes, equisetophytes and the eusporangiate and leptosporangiate ferns). The embryophytes, including seed plants, are a monophyletic group originating from an ancestor most closely related to the charophycean algae. The phylogenetic relationships of the extant embryophytes and the innovations that preceded their appearance during land plant evolution are well-documented. We will make BAC libraries from species representing each major lineage in the non-seed embryophytes.
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Bryophytes: These are simple, small, rootless plants that lack lignified vascular tissue. Their life cycle consists of a dominant gametophytic phase and sporophytes that are dependent, unbranched and monosporangiate. Bryophytes are ancestral to vascular plants and are most representative of the earliest land plants. They hold clues to understanding the innovations necessary for the transition from life in water to dry land. These innovations include a cuticle to prevent water loss, stomata to enable gas exchange, enclosure of the egg in a protective archegonium; protection against UV and pathogens; and the beginnings of a diploid sporophyte generation and apical meristem. Questions relating to the evolution of the genes regulating these traits can be addressed with BAC libraries from bryophytes. Marchantia polymorpha (a liverwort) and Anthoceros sp, (a hornwort) are included in this proposal because they are morphologically very distinct and may represent independently derived lineages. Marchantia polymorpha, a common greenhouse weed, is particularly attractive because it can be transformed.
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Lycophytes: Vascular plants are thought to have followed two lines of evolution: the Lycophytina (lycophytes) and the Euphyllophytina (all other vascular plants). Extant lycophytes include members of the Lycopodiaceae, Selaginellaceae and Isoetaceae; species within each family have been included in this proposal. Lycophytes were the first Glade to evolve stem/leaf/root organography, reflecting an increase in the complexity of the sporophyte generation. The lycophytes form a monophyletic group that diverged prior to the evolution of stem/leaf/root organography, heterosporous reproduction, secondary growth, and complex modes of branching in the vascular plant lineage. The lycophytes are therefore an important group for determining if independently evolved characters in divergent groups of plants involved the same (homologous) or different genes and control mechanisms. Lycopodium clavatum is homosporous, while Selaginella kraussiana and Isoetes engelmanii are heterosporous. Although Lycopodium clavatum, has a large genome size, it is the smallest known in this genus. Selaginella kraussiana could become a new model plant species because it has a small genome size [-30% that of Arabidopsis], is easy to culture and can be crossed. Isoetes englemannii was chosen because it has a relatively small genome and is easy to culture.
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Pteridophytes: Pteridophytes are a Glade including Equisetum, Psilotum, and the ferns. Recent palaeontological and molecular analysis indicate that pteridophytes form a monophyletic group sister to the seed plants. Psilotum nudum was chosen for its unique morphology, which has caused much debate about its phylogenetic placement. Angiopteris, one of six extant genera of the Marattiales (eusporangiate, homosporous ferns), is related to the tree ferns that thrived in the Carboniferous swamps; the morphology of Angiopteris erecta has been well-characterized. Of the leptosporangiate ferns (in which the sporangium develops from a single cell), Ceratopteris richardii (homosporous) and Marsilea quadrifolia (heterosporous) are choice because they are genetically and experimentally tractable. Since the common ancestor of pteridophytes and seed plants lacked defined leaves and roots, the pteridophytes are also an important group in understanding the evolution of traits in land plants. The common ancestor of pteridophytes and seed plants lacked defined leaves and roots, secondary growth, bipolar growth, axillary branching, the heterosporous life cycle, seeds and pollen. Therefore, a thorough understanding of the comparative genomics for different pteridophytes and seed plant groups will provide the crucial data needed to decipher the phylogenetic and developmental origins of these vital characters.
Alternative non-seed plants. The choice of species for this group was simple, since there are few species to select from, and even fewer for which all criteria (see above) hold. Although the moss Physcomitrella patens is amenable to reverse genetic techniques, a P. patens BAC library is publicly available (courtesy R. Quatrano, Washington University). Mosses were therefore excluded from this proposal. Equisetum (the only representative of the equisetophytes) was also excluded because the genome sizes of all species examined to date are very large [1C>110,000 Mbp].

The genetic systems that generate flowers, fruits and body form in angiosperms echo an evolutionary heritage to the origins of seed plants: to non-seed plants, green algae, and beyond. To understand the genetic complexity of modern plants, including crops and model organisms, one must appreciate how these systems arose evolutionarily. We will sample taxa that occupy pivotal phylogenetic positions, make linkages to model plants and provide sampling of key basal lineages in the basal angiosperms, eudicots, and outgroups. Inclusion of basal angiosperms is important because the origins of many of the innovations elaborated on in seed plants can be traced back to the basal angiosperms. Hence, we include exemplars from the earliest-branching lineages of angiosperms (i.e., Amborellaceae, Nymphaeales). We also chose basal exemplars of the eumagnoliids (Liriodendron), the monocots (Acorus) and the basal eudicots (Eschscholzia) which have vast structural diversity. For linkage of these basal seed groups to more derived and `typical' eudicot lineages, we selected two important microevolutionary model plants, Silene and Mimulus.
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Gymnosperms: Gymnosperms include the earliest living lineages with innovations including a greatly reduced male gametophyte (pollen), pollination, seeds, and true secondary growth of the vascular tissues to produce "wood.". Recent molecular phylogenetic studies indicate that the five major lineages of extant gymnosperms (cycads, Ginkgo, Gnetales, Pinaceae, and all other conifers) form a monophyletic group that is sister to angiosperms, although an alternate placement of Gnetales as sister to the angiosperms cannot be unambiguously rejected. Because the genomes of gymnosperms are extremely large, our priority is to build one new library from the basal most lineage of gymnosperms, the cycads (Zamia). Cycads represent the likely sister lineage to all other extant gymnosperms and are consistently included in comparative developmental and molecular systematic studies. As a basal lineage, cycads provide exemplars to help ascertain the generalized gymnosperm reproductive features from which flowering plant morphology and genetic controls were likely modified. Zamia will also be the target of extensive sequencing and expression profiling in the NSF Floral Genome Project.
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Basal Angiosperm lineages: Angiosperm innovations include the flower; double fertilization, endosperm, "true" vessels, fibers, and great physiological and biochemical diversity. Although most species of angiosperms are either monocots (especially grasses and orchids) or eudicots, the greatest diversity of structure occurs in the basal angiosperms. Recent molecular phylogenies have clearly identified 8-10 of the major basal flowering plant lineages. We chose four of these major lineages to represent this diversity so that a comprehensive picture of the ancestral angiosperm can be developed from the BAC library resource, and capture the genetic and genomic changes that led to key angiosperm innovations. The FGP will simultaneously conduct intensive EST and gene expression studies, including in situ hybridization on app. 300 genes and microarrays of 2,000 to 5,000 genes. The four proposed basal angiosperms are: Amborella, Nuphar, Liriodendron and Acorus.
Amborella trichopoda is widely recognized as the sister to all other extant angiosperms. This species is primitively vesselless, has unsealed carpels and has tepals rather than distinct petal-sepal differentiation. Because of its unique phylogenetic position, Amborella is crucial to understanding angiosperm ancestry and development. Amborella is endemic to New Caledonia but has recently been brought into cultivation and is now easily maintained.
Nuphar adventa (waterlily, spatterdoc) is a second critical basal angiosperm. Distinct petals and sepals first appeared with the waterlilies, and they possess unusual "primitive" vessel types. This second basal branch of angiosperms, or perhaps the shared basal branch with Amborella [see Amborella refs], differs morphologically and anatomically from Amborella and thus equally important for understanding the origin of key angiosperm innovations. Nuphar is becoming the focus of intensive developmental, reproductive, and evolutionary genomic study, including floral biology and gene expression [N. Friedman, pers. comm. to C. dePamphilis].
The Eumagnoliid Glade is species rich and contains much floral, structural, and biochemical diversity, including oil cells, cyanogenic glycocides, terpenoids, and benzyquinaline alkaloids. Liriodendron tulipifera (yellow or tulip poplar; Magnoliaceae) is a valuable timber tree that is unusual among harvested species for being relatively free from serious pest problems, including resistance to attack by the exotic gypsy moth. It is transformable and the focus of intensive genetic studies. It has the lowest chromosome number in Magnoliaceae (2n = 38) and a relatively small genome size (784 Mbp).
Acorus gramineus (sweet flag; Acoraceae) The monocot proposed for BAC library construction is Acorus gramineus (2n = 18) which has a very small genome (392 Mbp). A large EST set and detailed expression studies will soon be available. Acorus is a widely studied medicinal botanical species producing pharmacologically active antibiotic resistance inhibitors, essential oils, neurotransmitter sedatives, antioxidants, and inhibitors of excitotoxic neuronal death . Because of its unique phylogenetic position as sister to all other monocots. Acorus gramineus will be very important for understanding the evolution of monocot genomes as well as morphological and developmental features. An Acorus BAC library will enable comparisons to genomes of several cereal grains for which BAC libraries already exist.
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Eudicot developmental & microevolutionary model systems: Most flowering plant diversity (outside of the monocots) is found in two large eudicot groups, the rosids and the asterids. Genomic resources for this group have been driven by economic importance, not by evolutionary significance or (except for Arabidopsis) depth of interest in the research community. We have selected one basal eudicot lineage and two microevolutionary model organisms that hold key taxonomic positions in the evolution of angiosperm diversity. BAC libraries for these plants will be rapidly put to use by large and active research communities.
Inclusion of an early-diverging eudicot, Eschscholzia californica (California poppy; Papaveraceae), is critical for the broad evolutionary comparisons proposed, because it will provide a root for all genomic-scale analysis for more derived eudicot systems. Poppies are intensely studied as a model for floral molecular genetics and evolution, and for studies of the molecular basis of alkaloid (opiate) chemistry, and latex chemistry. The California poppy has a smaller genome
than the more widely studied opium poppy (Papaversomniferum) and does not require research permits. We will thus also use E. californica for FGP EST and gene expression studies. E. californica is the most widespread poppy species in North America and is highly variable in structure and life history. It is transformable and a variety of floral mutants are available.
The genus Mimulus (monkeyflower, Scrophulariaceae) is one of the premier model systems for the study of ecological and genetic mechanisms of evolutionary change. Several species are the subject of genomewide studies of inbreeding depression, mating system evolution, adaptation to toxic soils, and different elevational habitats, divergence of floral development and pollination hybridization and introgression, and reproductive isolation mechanisms. Mimulus guftatus, is easily cultivated and crossed, has a small genome size and complete genetic map with evolutionarily important quantitative trait loci. In addition, large permanent mapping populations and an EST database are being developed (Willis letter).
Silene latifolia (catch fly, Caryophyllaceae) is one of the most intensely used wild plant models for a wide variety of ecological and evolutionary processes including parasite-host interactions, sex determination and development, sex ratio evolution, and sex chromosome evolution, and metapopulation genetics (Kesseli letter). S. latifolia (2n=24) has a chromosomal based sex determination system in which males (XY) and females (XX) are easily distinguished by flower structure or by genetic markers on the sex chromosomes. A BAC library will speed progress in all aspects of Silene functional genomics, but will be particularly useful for cloning of critical genes on non recombining Y-chromosome regions.
Alternative seed plant. The following taxa in the remaining major lineages were also carefully considered, but were relegated to alternates for budgetary reasons. These taxa include innovations of interest, are economically important (e.g., floral fragrance, wood), are a model for microevolution or a specific feature of interest (e.g., wood formation, parasitism, ring-like anthers) and many have other resources available.
GvmnosDerms: Welwitschia mirabilis (Genetales) (6,720 Mbp). Pinus taeda (n=12; 21,700 Mbp).
Basal Angiosperms: Yucca angustissima (n = 30; 2,500Mbp).
Eudicots: Aquilegia formosa (columbine; Ranunculales) (n=14; 539 Mbp) (Hodges letter). Arceuthobium tsugense (Viscaceae; Santalales). (7,154 Mbp) (Nickrent letter), Vaccinium corymbosum (blueberries: Ericaceae) (2n = 24, 1078 Mbp) (Rowland letter).
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