21st Century Directions in Biology Unfurling Fern Biology in the Genomics Age MICHAEL S. BARKER AND PAUL G. WOLF Twenty-first century technology is addressing many of the questions posed by 20th-century biology. Although the new approaches, especially those involving genomic data and bioinformatic tools, were first applied to model organisms, they are now stretching across the tree of life. Here, we review some recent revelations in the ferns. We first examine how DNA sequence data have contributed to our understanding of fern phylogeny. We then address evolution of the fern plastid genome, including reports of high levels of RNA editing. Recent studies are also shedding light on the evolution of fern nuclear genomes. Initial analyses of genomic data suggest that despite their very high chromosome numbers homosporous ferns may have experienced relatively few rounds of genome duplication. Genomic data are enabling researchers to examine speciation rates and the mechanisms underlying the formation of new fern species. We also describe genetic tools that have been used to study gene function and develop- ment in ferns. Recent findings in fern biology are providing insights that are not only pertinent to this major component of the land flora but can also help to provide an improved evolutionary context for research on flowering plants. Keywords: expressed sequence tag, polyploidy, speciation, hybridization, development enomics and related tools and concepts were initially and places research on economically important plants into Gdeveloped using model organisms, yet their applica- better evolutionary context. tions are now shifting to the rest of the tree of life—the Which plants are considered ferns? As our knowledge of unexpected results of which will ultimately influence our evolutionary relationships expands, we must adjust how broader understanding of biology. Here we illustrate such names are applied and introduce new names. Although this developments by focusing on a plant group that has an ex- can be frustrating for those not familiar with the group in tensive fossil history and remains a conspicuous component question, it is essential for conveying information accu- of the land flora: the ferns. Ferns have several characteristics rately. Figure 1 depicts our current understanding of the that distinguish them from the more familiar seed plants, relationships among the major groups of vascular plants. making ferns an ideal system for addressing previously in- This includes all the land plants (embryophytes) except tractable questions. But ferns are also a major clade of land for mosses, liverworts, and hornworts. The tree is based on plants, and knowledge of their basic biology and evolutionary an accumulation of data from many sources and research history is essential if we are to make appropriate inferences groups, more details of which will be presented as we focus about the seed plants, including the economically important on relationships within ferns. Here we need to put the ferns flowering plants. We first describe some chief characteristics in context. Evidence from DNA sequences of several genes, of ferns, centering on their life cycles, and place them in an as well as information on genome structure (Raubeson evolutionary context. We then address a series of general and Jansen 1992), indicates that a major split occurred, themes, many of which are broadly applicable to all organ- probably about 400 million years ago (MYA). This split isms, especially plants. Within each theme we present some gave rise to the extant lycophytes and a clade containing unanswered questions, both old and new; we review some the remaining vascular plants (Pryer et al. 2004). The lyco- genomic tools and explain how they have enabled us to go phytes include the extant club mosses (Lycopodiaceae) and further than ever before in addressing those questions; and spike mosses (Selaginellaceae), as well as several extinct we describe some of the current limitations (of both the lineages. The remaining vascular plant lineage underwent tools and the information hidden in genomes), the need a later split into “monilophytes” and seed plants; the latter for new (and more-balanced) data, and the areas where we comprise the gymnosperms (among them the conifers) believe more research is needed. Although our own research and the angiosperms (the species-rich and economically is on what some might consider a rather esoteric group of important flowering plants). The monilophytes comprise plants, we seek to illustrate that the research implications four extant lineages: leptosporangiate ferns (about 11,000 extend beyond ferns to the evolution of genomes in general, species), marattioid ferns (including the large king fern), BioScience 60: 177–185. ISSN 0006-3568, electronic ISSN 1525-3244. © 2010 by American Institute of Biological Sciences. All rights reserved. Request permission to photocopy or reproduce article content at the University of California Press’s Rights and Permissions Web site at www.ucpressjournals.com/ reprintinfo.asp. doi:10.1525/bio.2010.60.3.4 www.biosciencemag.org March 2010 / Vol. 60 No. 3 s "IO3CIENCE 21st Century Directions in Biology 21st Century Directions in Biology Core How do ferns differ from seed leptosporangiates plants? All plants have an alternation of generations: Gametes (egg and Heterosporous sperm) are produced by mitosis dur- ferns ing the haploid gametophyte stage 11,000 species 0 genomes of the life cycle; fertilization restores Schizaeoid 2 EST diploidy in the zygote; and then the ferns Leptosporangiate zygote divides mitotically to become ferns the sporophyte, in which meiosis re- Gleichenioid sults in haploid spores that germinate ferns and divide to become the next gen- eration of gametophytes. Thus, the Filmy plant life cycle differs fundamentally ferns from that of animals, in which gam- etes are produced by meiosis rather Osmundaceae than mitosis. For seed plants, most Monilophytes people are familiar with the sporo- = "ferns" phyte, the large plant body that we see 100 species Psilophytes with the naked eye. Gametophytes of 0 genomes 0 EST seed plants are nutritionally depen- dent on the sporophytes (the male 200+ species Marattioid gametophytes are the pollen grains). ferns 0 genomes 0 EST However, in ferns, the spores germi- nate to become independently grow- 15 species Horsetails 0 genomes ing gametophytes, often large enough 0 EST to see (if you know where to look) approximately 300,000 species without a microscope. We will discuss Angiosperms >16 genomes the genomic and evolutionary conse- Seed plants 155 EST quences of this type of life cycle later. A 1100 species second feature that differentiates ferns Gymnosperms 1 genome from seed plants is that most ferns are 11 EST homosporous. Seed plants and other approximately 1200 species heterosporous plants produce two 1 genome Lycophytes 1 EST kinds of spores: (1) large megaspores that develop into the larger female Figure 1. Working phylogenetic framework, based primarily on Pryer and gametophytes in which eggs or egg colleagues (2004). The core leptosporangiate ferns represent about 90% of fern cells form, and (2) small microspores species. For each major clade we list the approximate number of species, the that develop into microgametophytes number of complete genomes sequenced and available, and the number of spe- in which sperm cells form. In con- cies for which expressed sequence tag (EST) data are available. Genome and EST trast, homosporous plants produce a information was obtained from the Plant Genome Database (www.plantgdb. single type of spore and gametophyte, org). Note that for the seed plants there are 17 genomes and 166 EST collections, although fern gametophytes in na- whereas for the sister group, the ferns, there are no genomes and only two EST ture are usually unisexual because of a collections. pheromonal sex-determination system (Schneller et al. 1990, Hamilton and psilophytes (adder’s tongue ferns, moonworts, and whisk Lloyd 1991). Heterospory has evolved independently several ferns), and the horsetails (see figure 1). For the purpose times, including in the relatively small clade of aquatic ferns of this review we will use the general term “fern” for all within the (otherwise homosporous) leptosporangiate fern monilophytes. The important concept here is that the ferns lineage (Pryer et al. 2004). are the sister group to all seed plants, thereby providing information needed for comparative studies. For example, Fern phylogeny any differences between gymnosperms and angiosperms The evolutionary position of ferns relative to other vascular could be a result of changes that evolved in either group. plants is described above. Until fairly recently, several con- It is only by comparison with the outgroup, ferns, that the flicting phylogenetic hypotheses (within ferns) appeared direction of evolutionary changes can be inferred and thus equally likely, but these were made on the basis of intuitive studied in an appropriate context. interpretations of morphological change, rather than on "IO3CIENCE s March 2010 / Vol. 60 No. 3 www.biosciencemag.org 21st Century Directions in Biology 21st Century Directions in Biology hard data. Part of the problem was the lack of phylogeneti- 20 kb, resulted in the Adiantum gene order (Wolf and Roper cally useful characters. Ferns lack complex structures such 2008). Previous attempts to infer these inversion events (Stein as seeds and flowers that provide such a wealth of informa- et al. 1992) failed because