Getting to the Roots of Plant Evolution: Genomics and the Reconstruction of the Tree of Life

1. Getting to the Roots of Plant Evolution: Genomics and the Reconstruction of the Tree of Life Sponsored by the National Science Foundation, the Deep Gene Research Coordination Group, CIPRES, and the Jepson Herbarium, UC Berkeley.

2. Introduction to the Green Plants Land plants first appeared in the Ordovician (~460 million years ago) but did not begin to resemble modern plants until the Late Silurian. By the close of the Devonian, about 360 million years ago, there were a wide variety of shapes and sizes of plants, including tiny creeping plants and tall forest trees. Today, with more than 250,000 species, they are second in size only to the insects. We now know that plants, like all living organisms, had aquatic ancestors. A specific group of freshwater green algae are the closest relatives to the land plants. The story of plant evolution is therefore inseparably linked with their progressive occupation of the land and their increasing independence from water for reproduction. 1. A comparison of conditions faced by algae and plants

3. The origin of terrestrial plants from their aquatic ancestorsHow did plants colonize land? What obstacles did they have to overcome? How did they cope, and eventually thrive in their new terrestrial environment? What were some of the key innovations that led to the diversification of the land plants? The land plants evolved slowly, and in stages. The fossil record chronicles four major periods of plant evolution.The first major period of plant evolution:Plants move onto landThe figure on the previous page provides a comparison of aquatic and terrestrial environments. By understanding the differences, we can begin to think about some of the challenges faced by plants as they transitioned to life on land.

4. Introduction to the Green Algae The Charophyceae, a group of fresh water green algae, are the closest relatives to the land plants. Like the land plants, green algae contain two forms of chlorophyll (a and b), which they use to capture light energy to make sugars that are stored as starch inside plastids (specialized organelles). Green algae differ from plants in many ways. Because they live in the water, they don't have a specialized transport or support systems. Their bodies are supported by the water, and almost all of the cells photosynthesize and have access to the nutrients present in the water. Therefore, transport of nutrients is not necessary. Even though they photosynthesize, green algae do not have true leaves (which are characterized by the presence of vascular tissue). Additionally, green algae lack cuticles (a waxy layer on the outer wall of epidermal cells) and stomata (specialized cells for gas exchange). 2. Chara, a green alga. The gametophyte is the dominant life stage.

5. Early land plants The early land plants, represented today by the bryophytes (mosses, liverworts, and hornworts), possessed two important features that allowed them to live on land: 1) a waxy cuticle to protect against desiccation (drying out) and 2) protection of gametes and embryos in a protective jacket made of cells. Even with these adaptations, early land plants were still closely tied to water, even if only in small amounts such as morning dew. They had swimming sperm and required water to complete their life cycle. Early land plants also lacked true vascular tissue to carry water from the soil to the aerial parts of the plant. Water was imbibed as it moved over the plant body (like a sponge) and distributed by relatively slow processes like diffusion. This mode of hydration helps explain why damp, shady places are the most common habitats for the modern descendants of early land plants, the bryophytes.

6. Introduction to the Bryophyta (The mosses, liverworts and hornworts) After flowering plants and ferns, mosses are the most diverse group of plants, with more than 10,000 species in 700 genera. This makes mosses almost twice as diverse as mammals. Despite their diversity, bryophytes don't receive as much attention as flowering plants, ferns, or conifers because most bryophytes are small and inconspicuous. They have no vascular tissue (no true xylem or phloem) to lend them structural support, nor do they have true leaves or showy flowers. This does not mean that bryophytes are not important; mosses in particular, play important roles in reducing erosion along streams, water and nutrient cycling in tropical forests, and insulating the arctic permafrost. 3. A moss. 4. A liverwort.

7. The earliest land plants were probably very similar to modern-day bryophytes. In bryophytes, the gametophytes are nutritionally independent of the sporophytes and the sporophytes are either completely or partially dependent on the gametophytes. Sperm are free swimming and require water to reach the egg. One key difference between the mosses, hornworts, and liverworts is that liverworts have pores on their surface but lack stomata. Mosses and hornworts (and all other land plants) have stomata. 5. (left) pore on surface of a liverwort (right) stomate on land plant leaf Interesting note: Some mosses have water conducting tubes, but it is not yet resolved what the origin of those structures are. So, for now, we refer to mosses as nonvascular plants. Introduction to the Bryophyta, continued

8. The second major period of plant evolution:The diversification of vascular plants Relatively early in the history of plants, the evolution of efficient fluid-conducting systems, consisting of xylem and phloem, solved the problem of water and food transport throughout the plant body. The ability to synthesize lignin (a plant polymer), which is incorporated into the cell wall of supporting and water conducting cells, was also a pivotal step in the evolution of plants. Lignin adds rigidity to cell walls, making it possible for vascularized plants to reach great heights. The shoot system of plants (stems and leaves) was well suited to the demands of life on land - namely, the acquisition of energy from the sun and carbon dioxide from the atmosphere. The reproductive systems of plants were also changing. The gametophytic stage remained free-living, requiring water for fertilization, but over time, the gametophytic generation underwent a progressive reduction in size - the sporophyte phase became the dominant phase of the life cycle. The earliest vascular plants lacked seeds, a condition still represented by ferns (and a few other groups not discussed here). Therefore, the second major lineage of land plants to evolve is referred to as the seedless, vascular plants. Seedless vascular plants dominated the landscape in shallow swamp like forests of the Carboniferous period about 300-350 mya. Dead plants did not completely decay in the stagnant water, and organic rubble (called peat) accumulated. The swamps were later covered by the sea, and marine sediments piled on top of the peat. Heat and pressure gradually converted the peat to coal, thus the name of the geologic period (Carboniferous). Four divisions of seedless vascular plants are represented in the modern flora; Psilophyta (Psilotum), Lycophyta (lycopods), Sphenophyta (horsetails), and Pterophyta (ferns). Interesting note: Seedless vascular plants grew along side primitive seed plants but the seed plants were not dominant at that time. The seed plant rose to prominence only after the swamps began to dry up at the end of the Carboniferous. 6. Reconstruction of a Carbiniferous swamp (the coal age)

9. Introduction to the Lycophyta (Club mosses and scale trees) 7. Lycopodium, a lycophyte with microphylls The most significant feature of lycophytes is the microphyll, a kind of leaf that has arisen and evolved independently from the leaves of other vascular plants (megaphylls). The microphyll has only a single unbranched strand of vascular tissue (xylem and phloem), whereas megaphylls have multiple veins, usually branching one or more times within the leaf. According to one widely accepted theory (diagrammed below), microphylls evolved as outgrowths, called enations, of the main axis of the plant. Megaphylls evolved by fusion of branch systems. Microphylls cover the sporophyte, the dominant life phase in Lycophytes. The lycophytes are a small and inconspicuous group of plants today, but in the Carboniferous some lycophytes were forest-forming trees more than 35 meters tall. Lycophytes are the oldest extant group of vascular plants, and they dominated major habitats for 40 million years. The club mosses (Lycopodiales) are usually evergreen, and have been used as Christmas decorations, though their flammable spores and increasing rarity has made this illegal in some states. Other lycophytes, such as Selaginella, may form extensive carpets in the understory of wet tropical forests. 8. Evolution of microphylls (showing enations) and megaphylls.

10. Introduction to the Pterophyta (The ferns) Even though ferns have free living gametophytes, the sporophyte is the dominant phase of the fern life cycle. Ferns produce spores (not seeds) that are borne on megaphylls (often called fronds). The pattern of spore distribution is often an important taxonomic character. The ferns are an ancient lineage of plants, dating back to at least the Devonian. Today, there are approximately 11,000 species of ferns; they are the second largest group of plants and are the most diverse in both form and habit. Only about 380 species of ferns occur in the United States, most of the diversity is found in tropical areas. Approximately 1/3 of all species of tropical ferns grow on the trunks or branches of trees as epiphytes. 9. Fiddle head of new fern frond 10. Spores on the back of a fern frond

11. The seed, a specialized unit of reproduction, advanced the colonization of land by further providing a food source for the plant embryo and protecting it from harsh conditions. Seeds are also an important unit of dispersal (replacing the spore as the stage of the life cycle that disperses offspring). The seeds of gymnosperms are not enclosed in any special chambers (or ovary, as they are in the flowering plants); the name gymnosperm means, literally, “naked seed”. The gametophytes of seed plants became even more reduced than the gametophytes of ferns and other vascular plants. The reduction of the gametophyte set the stage for another major innovation - pollination. Pollination replaced swimming as the mechanism for delivering sperm (in the male gametophyte) to the egg (on the female gametophyte). Water was no longer needed to disseminate sperm cells. Wind, insects, or other animals could do the job. This became increasingly important because as the gymnosperms diversified, the climate was becoming drier. The third major period of plant evolution:The origin of the seed

12. Introduction to the Gymnosperms The gymnosperms descended from a group of Devonian plants, the progymnosperms. They coexisted with the bryophytes, ferns, and other seedless vascular plants, but their adaptive radiation occurred during the Carboniferous and early Permian when the climate became warmer and drier. The gymnosperms formed vast forests that dominated the landscape for more than 200 million years. The pine tree, a representative gymnosperm, is a sporophyte. The gametophyte generation develops from spores that are produced in male and female cones. The pollen (male gametophyte) is transferred to the ovule (female gametophyte) via wind. After fertilization, the seed begins to develop. The entire process, from cone production to seed production, can take up to three years. All gymnosperm leaves, including the needle like leaves of conifers, are megaphylls. Most gymnosperms are evergreen but some, like Ginkgo, are deciduous. We get most of lumber and paper pulp from the wood of conifers. What we call wood is actually an accumulation of lignified xylem tissue. Tracheids, special conducting cells, are the primary components of xylem in gymnosperms. There are four divisions of gymnosperms; Cycadophyta (the cycads), Ginkgophyta (Ginkgo), Gnetophyta, and Coniferophyta (the conifers). The largest division, the conifers, are almost all large trees, and include pines, firs, spruce, junipers, and redwoods. Although there are only about 550 species, conifers dominate vast forested regions of the Northern Hemisphere. 11. A pine with female cones Interesting note: The bluish-white structures of the juniper are often referred to as "juniper berries." Berries are a type of fruit and because fruit develops from the ovary of a flower, angiosperms are the only plants that have fruit.

13. The fourth major episode in the evolutionary history of plants:The emergence of the flower The angiosperms arose during the early Cretaceous period about 130 mya. The main feature that led to their success was the evolution of flowers and fruits. The flower is a complex reproductive structure that bears seeds within protective chambers called ovaries. The presence of the ovary is one of the major differences between angiosperms (the flowering plants) and the gymnosperms (the naked seed plants). The ovary develops into the fruit, which is an important structure for seed dispersal. Flowers also allow for specialized pollination by attracting and rewarding pollinators. Vascular tissue also became more refined during angiosperm evolution. Vessel elements, present in almost all angiosperms, are shorter and wider than trachieds (the xylem tissue in ferns and gymnosperms), and allow for more efficient water transport. Leaves (megaphylls) also became more diverse and specialized. While these changes in the plant body were important, it was really the evolution of the flower that contributed most significantly to the success of the angiosperms. 12. The parts of a flower Today, the flowering plants are by far the most diverse and geographically widespread of all plants. They are important in many ways above and beyond their aesthetic appeal. Not a day goes by in which our lives are not affected by a flowering plant. Nearly all of our food comes from flowering plants; grains, beans, nuts, fruits, vegetables, herbs, and spices almost entirely come from plants with flowers, as do tea, coffee, chocolate, wine, beer, tequila, and cola. Much of our clothing comes from them as well; cotton and linen are made from "fibers" of flowering plants, as are rope and burlap, and many commercial dyes are extracted from other flowering plants. We owe flowering plants credit for a large number of our drugs, including over-the-counter medicines such as aspirin and prescribed drugs such as digitalis and atropine. 13. A bee pollinating a flower

14. 14. The geological time scale

15. Getting to the Roots of Plant Evolution - Exercises Now that you know all about the characteristics of plants and how they made their move from fresh water to land, you can use this knowledge to reconstruct the evolutionary relationships of some plant groups that are alive today. In addition to the morphological characteristics, such as the cuticle and seeds that we discussed in the previous section, there are other types of characters, present in the genomes of plants, that can also help us understand their evolutionary relationships. While molecular characters such as these used to be very difficult to obtain, recent advances in fast, high volume DNA sequencing has made it possible to get large amounts of genetic sequence data for plants. One nice source of this sort of sequence data is the circular genome of the plant chloroplast, because it is smaller and more easily sequenced than the entire nuclear plant genome. And, because the chloroplast is a plant organelle, (having been derived from a bacterial endosymbiont), its genome does not undergo recombination; this makes reconstructing evolutionary relationships much less complicated, because each genetic trait in the chloroplast can be traced directly back (in time) through a lineage of mothers and daughters. On the next page, you’ll find a selection of schematics representing various chloroplast genomes and their arrangements for several groups of plants. These representations of the chloroplast genome show the positions of several genes (A-E) as well as the position of a distinctive region known as the inverted repeat. Genome-level characters such as gene position and structural rearrangements are very useful for reconstructing deep evolutionary relationships, because they are believed to occur fairly infrequently; it is unlikely that two groups of plants would have the same unique gene rearrangement due to chance alone. With this genomic information and all you learned about land plants in the previous section, you will be able to complete the land plant data matrix and use it to construct a cladogram.

18. Phylogenetic Analysis of the Green Plants Data Matrix

19. Phylogenetic Analysis of the Green Plants Data Matrix-Solution

22. Questions for discussion 1) Contrast a seed plant to an alga in terms of adaptation for life on land versus water. 2) What evidence is there to support a charophyte ancestry for plants? 3) Bryophytes and vascular plants share a number of characteristics that distinguish them from charophytes and that adapt them for existence on land. What are those characteristics? 4) What is coal? How was it formed? What plants were involved in its formation? 5) What is a seed, and why was the evolution of the seed such an important innovation for plants? 6) How do the mechanisms by which sperm reach the egg differ between gymnosperms and seedless vascular plants? 7) Why was the flower such an important innovation? 8) What role do insects, animals and wind play in plant reproduction?

23. AcknowledgmentsWe gratefully acknowledge the following for use of text and/or information: Biology of Plants. P. H. Raven, R. F. Evert, and S. E. Eichorn. W. H. Freeman and Company, New York, New York. Biology. N. A. Campbell. The Benjamin/Cummings Publishing Company, Inc. Menlo Park, California. Introductory Plant Biology. K. R. Stern. Wm. C. Brown Communications, Inc. Dubuque, Iowa. The Museum of Paleontology, UC Berkeley. http://www.ucmp.berkeley.edu/ We thank the following sources for use of illustrations: Figure 1. Redrwawn from Biology Figure 6. UCMP website: http://www.ucmp.berkeley.edu Figure 8. Redrawn from Biology of Plants Figure 14. Redrawn from Biology • Alternation of Generations Handout: • 1. Redrawn from Biology of Plants • 2. Redrawn from Biology • 3. Redrawn from Biology • 4. Redrawn from Biology of Plants • 5. Redrawn from Biology • 6. Redrawn from Biology