The Plant Tree of Life: an Overview and Some Points of View1

American Journal of Botany 91(10): 1437±1445. 2004.

THE PLANT TREE OF LIFE: AN OVERVIEW AND SOME POINTS OF VIEW1

JEFFREY D. PALMER,2,5 DOUGLAS E. SOLTIS,3 AND MARK W. C HASE4

2Department of Biology, Indiana University, Bloomington, Indiana 47405-3700 USA; 3Department of Botany and the Genetics Institute, University of Florida, Gainesville, Florida 32611 USA; and 4Molecular Systematics Section, Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, TW9 3DS, United Kingdom

We provide a brief overview of this special issue on the plant tree of life, describing its history and the general nature of its articles. We then present our estimate for the overall topology and, for land plants, divergence times of the plant tree of life. We discuss several major controversies and unsolved problems in resolving portions of this tree. We conclude with a few thoughts about the prospects for obtaining a comprehensive, robustly resolved, and accurately dated plant tree of life and the importance of such a grand endeavor.

Key words: algae; endosymbiosis; Gnepines hypothesis; land plants; phylogeny; plastids.

To mark the 90th anniversary of this journal, Scott Russell, eukaryotic phylogeny and plastid primary and secondary sym- the then-Immediate Past President of the Botanical Society of biosis. Such an overview is essential to have proper perspec- America, and Karl Niklas, the long-term Editor-in-Chief of the tive on the origin and phylogenetic relationships of land plants American Journal of Botany, jointly asked the three of us to and the many diverse groups of ``algae,'' as well as those serve as guest editors for a special issue devoted to the re- otherwise unrelated groups (especially fungi) that nonetheless markable recent progress in reconstructing the evolutionary have traditionally been allied with plants and which are still history of plant life, also known as the plant tree of life. Our part of the purview of this journal and its governing society. invitations to prospective authors went out in June of 2003. The group-speci®c authors were given free rein as to how to For this issue to appear in print only 16 months later, with approach and cover their groups. Thus, you will see a wide 100% delivery of solicited articles and each of them outstand- range of treatments. The central, common thread, however, to ing, is nothing short of remarkable. This testi®es to the com- all 11 of these articles is a description, usually encompassing mitment and enthusiasm of the authors; to the editorial exper- a critical assessment, of our current state of understanding of tise and dedication of Karl Niklas, the journal's of®ce manager the phylogeny of each group. Although this phylogenetic un- Caroline Spellman, copy editor Beth Hazen, and production derstanding increasingly relies on gene sequence data, many editors Beth Hazen and Elizabeth Lawson; and to the timely of the articles also provide a more or less thorough integration cooperation of the two to four expert scientists chosen to pro- of phylogeny with the overall evolution (principally from a vide rigorous, anonymous, and speedy review of each article. morphological standpoint) and classi®cation of the group in Although the rapid gestation of this issue ensures that it is up- question. to-date and timely, these articles should also, we believe, prove The coverage represented by the 11 taxon-speci®c papers to be of relatively long-lasting and enduring value. We trust re¯ects the amount of effort and progress made in elucidating that readers of this ®rst-ever special issue of this journal will the phylogeny of each group, as well as their size and eco- be as delighted with the outcome of this effort as are we. nomic and ecological importance. Thus, angiosperms, the larg- est, most important, and best studied group of land plants, are NATURE OF THE ARTICLES IN THIS ISSUE covered by three articles (Soltis and Soltis, 2004; Chase, 2004; Judd and Olmstead, 2004), whereas three much smaller but The other 18 articles in this issue fall into two general cat- considerably older groups of land plantsÐseed plants (Bur- egories. Twelve are taxon-oriented, while the other six are top- leigh and Mathews, 2004), ferns (Pryer et al., 2004), and bryo- ical in nature. Of the ®rst set, 11 are taxon-speci®c, providing phytes (Shaw and Renzaglia, 2004)Ðare each given but a sin- broad coverage of current understanding of the phylogeny and gle article. Likewise, green algae, the parent group of land overall evolution of the major groups of ``plants,'' as de®ned plants, are treated in a single paper (Lewis and McCourt, either phylogenetically or historically. The twelfth, by Keeling 2004), as are the sister group to green algae/land plants, the (2004), was commissioned to provide a broad overview of red algae (Saunders and Hommersand 2004). Two papers cov- er three of the largest and best studied groups of secondary 1 Manuscript received 29 June 2004; revision accepted 2 July 2004. plastid-containing algae, the dino¯agellates (Hackett et al., We wish to thank all those responsible for helping put together this special 2004) and the putatively related heterokonts (brown algae, di- issue of this journal, including the journal's editor-in-chief Karl Niklas, its of®ce manager Caroline Spellman, its copy editor Beth Hazen, its production atoms, chrysophytes, etc.) and haptophytes (Andersen, 2004). editor Elizabeth Lawson, and, of course, the many authors of the various Finally, Lutzoni et al. (2004) take on all the fungi. Page limits papers that comprise the issue. We also thank Gordon Burleigh, Chuck Del- to this issue made for hard choices, and we regret that four wiche, Les Goertzen, Patrick Keeling, Sarah Mathews, Kathleen Pryer, and fascinating but mostly poorly studied groups with secondary SasÏa Stefanovic for comments on this manuscript, Walt Judd and Patrick Keel- plastids (apicomplexans, chlorarachniophytes, cryptomonads, ing for communicating unpublished data, and the US National Institutes of and euglenids), as well the primary plastid-containing glau- Health (JDP), US National Science Foundation (DES and MWC), and the Royal Botanic Gardens, Kew (MWC), for funding to study plant phylogeny cophytes, could be given only brief mention in the overview and genome evolution. by Keeling (2004). 5 E-mail: [email protected]. With three exceptions, all 12 taxon-oriented articles are es- 1437 1438 AMERICAN JOURNAL OF BOTANY [Vol. 91 sentially review articles, providing a critical overview and syn- taining plastids). As described in the next paragraph, this is thesis of the recent literature pertinent to their respective sub- necessarily set in the broader context of overall eukaryotic jects. The article by Lutzoni et al. (2004) on fungal phylogeny phylogeny. Figure 2 shows our estimate of both phylogenetic is the ®rst paper from a large team of investigators whose relationships and divergence times of extant land plants. These currently funded goal (from the U.S. National Science Foun- trees are based entirely on DNA sequence data, generated and dation Tree-of-Life Program) is to reconstruct phylogenetic re- analyzed in far too many studies to cite in this brief overview lationships of 1500 fungi using eight genes (ca. 10 kb of DNA (for speci®c citations, see the 12 taxon-oriented articles in this sequence) and represents a major achievement in fungal phy- issue and references therein). We emphasize that these trees logeny akin to the earlier large-scale collaborative molecular are an estimate of phylogenyÐof evolutionary historyÐand, studies on angiosperms (e.g., Chase et al., 1993; Soltis et al., in the case of the global tree (Fig. 1), of only one dimension 2000). The article by Pryer et al. (2004) on fern phylogeny at that, of branching order (but not divergence time). As with stands out among all of the papers in this issue by virtue of all science, we can never be certain we have obtained ``the presenting the only fully integrated study of both the branch- truth.'' At the same time, however, the power of the DNA ing pattern and divergence times of a particular group of revolution makes it likely that we will soon approach relatively plants, both derived from the same set of gene sequence data. great certainty in estimating most or all of the branching orders The article by Burleigh and Mathews (2004) on seed plant of extant plant phylogeny and will gradually achieve greater phylogeny establishes a seemingly robust framework tree for and greater understanding of the time dimension as well. Im- gymnosperms and provides important evidence for the single portantly, as Crane et al. (2004) and Crepet et al. (2004) cau- most controversial and revolutionary hypothesis generated tion, a more complete understanding of the tree of life can from molecular analyses of any group of plants, namely, that only be accomplished with the integration of fossils. conifers are paraphyletic, with Gnetales sister to Pinaceae to As reviewed by Keeling (2004), at its deepest level, the the exclusion of all other conifers. Although these three arti- history of plants is a history of endosymbiosis, of their birth cles largely focus on new molecular data sets and phylogenetic by primary, eukaryotic/cyanobacterial endosymbiosis and of analyses, in keeping with the nature of this issue, the authors multiple secondary, tertiary, etc., eukaryotic/eukaryotic endo- have also broadened their remit beyond that of the usual orig- symbioses that have spread plastids into several other regions inal article, to provide a somewhat longer than normal over- of the eukaryotic world. Endosymbiosis dominates our global view of the group in question. perspective of plant evolution, as portrayed in Fig. 1. Of the The six topical articles focus on selected issues that are ®ve supergroups of eukaryotes tentatively identi®ed by Keel- highly relevant to reconstructing and understanding the plant ing (2004) and mapped onto Fig. 1, two, Primoplantae and tree of life. Crane et al. (2004) emphasize the importance of Chromoalveolates, are largely if not entirely de®ned by a his- integrating fossil evidence with data, both morphological and tory of endosymbiosis. The Primoplantae were by de®nition molecular, from extant plants, and also stress that a compre- born of primary, cyanobacterial endosymbiosis, and the hensive understanding of some groups (e.g., seed plants) will Chromalveolates were possibly born of red algal secondary require a thorough reassessment of available fossils. Kellogg symbiosis (and if not, were then repeatedly subjected to red and Bennetzen (2004) review the structure, evolution, and, to algal symbiosis; discussed later and Fig. 1). Two of the other a limited extent, phylogenetic utility of plant nuclear genomes. three supergroups of eukaryotes have also acquired plastids Linder and Rieseberg (2004) discuss the increasingly powerful and evolved ``plantlike'' attributes through secondary symbi- molecular approaches used to dissect the role of hybridization in plant evolution. Whereas almost all articles in this issue osis, both times via green algae endosymbionts (Fig. 1). focus on reconstructing the branching pattern (or, sometimes, Although whole-organism symbiosis represents horizontal the network) of plant evolutionary history, Sanderson et al. gene transfer (HGT) on the grandest scale possible, there is (2004) and Crepet et al. (2004) explore a relatively recent de- little reason to suspect that HGT occurs commonly enough velopment in molecular phylogenetics, albeit one that we ex- within eukaryotic evolution to compromise signi®cantly our pect to achieve great importance, namely, the application of prospects for recovering the metaphorical plant tree of life. In gene sequence data to estimate divergence times. Crepet et al. the prokaryotic world, in contrast, HGT has shaken the very (2004) do so as part of a broader, original effort to integrate concept of whether an underlying tree structure exists to re- the fossil record more thoroughly with the molecular record. cover, the metaphor of a network of gene trees being preferred Sanderson et al. (2004) note that despite the limitations of by some authorities (Gogarten et al., 2002). As noted in the methods for estimating divergence times, recent analyses are next section, the prevalence of HGT in the bacterial, including converging on similar and reasonable age estimates of angio- cyanobacterial, world may provide important limits on our sperms. Finally, Friedman et al. (2004) provide a timely re- ability to answer some of the deepest, most fundamental ques- view of a topic of rapidly growing interest in plant biology, tions about the origin and earliest evolution of plants/plastids. namely, the evolution of plant development. The view of land plant phylogeny expressed in Fig. 2 has Although these articles do not cover every possible topic several messages. It tells us that most of the major groups of and group that are relevant to efforts to reconstruct and inter- extant land plants are monophyletic. Extant angiosperms are pret the plant tree of life, we believe that in sum they provide monophyletic, as are gymnosperms, seed plants, ferns (albeit suf®ciently broad and deep coverage to give a vivid sense of slightly broadened to include horsetails), euphyllophytes, ly- the excitement, progress, questions, and prospects in this im- cophytes, and vascular plants. Bryophytes, shown unresolved, portant and booming area of botanical research. are the conspicuous exception here, most likely being a grade that is paraphyletic to vascular plants (Shaw and Renzaglia, THE PLANT TREE OF LIFE: A 2004 ESTIMATE 2004). Figure 2 emphasizes how asymmetric the tree of extant Figure 1 shows our current estimate of the overall relation- plant life is with respect to species richness. At the extreme, ships of extant ``plants'' (de®ned here as all organisms con- Amborella trichopoda, a single species endemic to New Ca- October 2004] PALMER ET AL.ÐOVERVIEW OF PLANT TOL 1439

Fig. 1. Cladogram showing phylogenetic relationships among the major groups of extant eukaryotes, with emphasis on plastid-containing groups and their evolutionary connections via primary and secondary endosymbiosis. This estimate of relationships is modi®ed from Keeling (2004) and Baldauf et al. (2004), which should be consulted for more inclusive trees showing additional groups of nonphotosynthetic eukaryotes. The names and circumscriptions of the ®ve eukaryotic supergroups (Excavates, etc.) are from Keeling (2004), except that we have used ``Primoplantae'' (J. D. Palmer, unpublished manuscript) in place of ``Plants.'' Colors distinguish the three lineages of primary plastid-containing eukaryotes (Primoplantae) and also mark those eukaryotes with secondary plastids of red or green algal origin. The exact placement of the ``symbiosis'' arrows is arbitrary; essentially nothing is currently known about the timing of these events or the speci®c nature of the donor lineages. Two, probably independent, green algal secondary symbioses are shown, whereas the number of red algal symbioses could be as few as one (as shown) or, less likely, as many as ®ve (see text). The three slashes indicate loss of plastids under the hypothesis of a single early red algal secondary symbiosis and Chromalveolate monophyly. ``Other charophytes'' denotes what is most likely a grade of four orders from which the Charales/land plant clade has arisen (Karol et al., 2001; Lewis and McCourt, 2004). Groups covered by a particular article in this special issue are circled and connected to the names of the article's authors. Branch lengths in this cladogram are entirely arbitrary; no implications with respect to time are intended. ledonia, is probably the sister group to all other living angio- at the base of several major groupsÐangiosperms, gymno- sperms, numbering over 250 000 species. Similar asymmetries sperms, and fernsÐimplies relatively rapid emergence of the are evident at various places on the tree (Fig. 2), making it major lineages of each group from a common ancestor. Most clear that rates of speciation and extinction vary widely over of the diversity of these clades is not, however, represented on time and from group to group. The series of short internodes this tree. For species-rich groups such as the eudicots and 1440 AMERICAN JOURNAL OF BOTANY [Vol. 91

Fig. 2. Chronogram showing estimates of phylogenetic relationships and divergence times among the major groups of extant land plants. The estimate of relationships is synthesized from the following papers in this issue: Burleigh and Mathews (2004), Pryer et al. (2004), Shaw and Renzaglia (2004), and Soltis and Soltis (2004). Divergence time estimates are mostly based on analyses of molecular data with fossil constraints (WikstroÈm et al., 2001; Pryer et al., 2004) and are augmented by fossil evidence (Kenrick and Crane, 1997; Wellman et al., 2003). Estimates of the number of species in each group are from Judd et al. (2002) and W. S. Judd (personal communication). Groups covered by a particular article in this special issue are circled and connected to the names of the article's authors. ``Other conifers'' refers to the clade consisting of all conifers except for Pinaceae (see Burleigh and Mathews, 2004). ``Lepto. ferns'' refers to leptosporangiate ferns. monocots, we can barely imagine how many more ®nely 2004) speaks volumes to the power of thousands of (nucleo- spaced internodes must exist to account for the vast number tide) characters, and of judicious taxon sampling, to resolve of extant species in these groups. That so much progress has close divergences and heralds success in ultimately resolving already been made in resolving many of these internodes (e.g., all but the most rapid and anciently compressed of radiations see Chase, 2004, on resolving the backbone of the monocot as data sets head towards the tens and even hundreds of thou- tree; also see Judd and Olmstead, 2004, and Soltis and Soltis, sands of characters. October 2004] PALMER ET AL.ÐOVERVIEW OF PLANT TOL 1441

TOPOLOGICAL CONTROVERSIES IN THE PLANT bacteria (Zhaxbayeva et al., 2004) to bedevil attempts to ever TREE OF LIFE completely settle the issue of how many primary plastid ori- gins and, especially, to identify the cyanobacterial progeni- Although the papers in this issue amply document and re- tor(s) of plastids. view the great progress made in recent years in elucidating the plant tree of life, there is of course much work still to be done. What happened after plastids arose?ÐIf plastids did in- Many plant groups have barely been touched by phylogenetic deed arise only once, then a major ensuing issue is, what is inquiry, and for others little resolution has been obtained. Even the branching order among the three lineages of Primoplantae? in such a well-studied group as angiosperms, much important Keeling (2004) summarizes evidence that leads him to con- work remains, and not just at the tips of the trees where most clude that it ``seems likely'' that the glaucophytes are sister to species-level problems await resolution in this immense clade green algae plus red algae (as shown in Fig. 1), whereas we of over 250 000 species. For example, Soltis and Soltis (2004) view the issue as largely unsettled. The strongest support for point out a number of major groups within angiosperms for this relationship among plastid genes has always come from which signi®cant resolution is currently lacking. These include plastid small subunit rDNA (e.g., Turner et al., 1999). How- not only the deep level pentachotomy shown in Fig. 2 (of ever, a recent analysis of plastid large subunit rDNA, either eudicots, monocots, Chloranthaceae, Ceratophyllaceae, and alone or, more importantly, in combination with small subunit magnoliids), but also the major groups that make up the im- rDNA, ®nds instead strong (99% bootstrap) support for green mense (175 000 species) clade known as eudicots. algae as sister to red algae plus glaucophytes (S. Turner, K. We will not discuss any further these many areas of non- M. Pryer, and J. D. Palmer, unpublished data). Although most controversial poor resolution. Likewise, we will not explore analyses of very large character sets (of ca. 40 plastid protein here any of the major methodological controversies that sur- genes) ®nd strong support for glaucophytes being ``deepest'' round the business of inferring the plant tree of life. Some of (e.g., Martin et al., 2002), at least one is essentially unresolved these are, however, touched on in the following sections on on this issue (Turmel et al., 1999). Critically, all such analyses case studies of topological controversy, and include such top- are plagued by woefully inadequate taxon sampling (only a ics as the appropriate generation and use of DNA vs. other single glaucophyte, a single cyanobacterial outgroup, and just evidence in inferring phylogeny (e.g., Scotland et al., 2003), a few each of red and green algae), which, especially at this trade-offs between sampling more taxa vs. more characters level of divergence, raises the specter of obtaining strong sup- (Chase et al., 1993; Soltis et al., 2004), the limits and pitfalls port for an incorrect topology (Soltis et al., 2004). Although of using molecular data to date plant divergence times (Crepet the only relevant nuclear multigene analysis appears to lack et al., 2004; Sanderson et al., 2004), and how best to construct resolution on this issue (Moreira et al., 2000), red and green and assess phylogenetic trees from DNA sequence data (Fel- algae do share a nuclear gene duplication/plastid targeting senstein, 2004; Albert, 2005). Instead we focus on six current event to the exclusion of glaucophytes (Rogers and Keeling, controversies in plant phylogeny, chosen because they all con- 2003). cern deep and important issues in plant evolutionary history, Many more sequence data (especially from glaucophytes) relate to taxa shown in Figs. 1 and 2, and illustrate a variety are clearly needed to resolve this key issue, which greatly af- of biological and methodological issues in phylogeny recon- fects interpretation of various important aspects of plastid evo- struction. lution, such as their history of gene loss (Martin et al., 2002) and cell wall loss. For example, glaucophytes uniquely retain How many origins of plastids/plants?ÐThe two deepest a bacterial-like, peptidoglycan cell wall around their plastids: questions in plant phylogeny concern the birth of plastids/ Did green and red plastids lose their cell wall independently plants via cyanobacterial endosymbiosis: Did plastids arise or in a common ancestor? Unfortunately, the apparent predi- once or more than once, i.e., was there but a single or as many lection of cyanobacteria (the only good outgroups for this in- as three cyanobacterial endosymbioses to establish the three quiry) to engage in HGT (Zhaxbayeva et al., 2004) compro- lineages of clearly monophyletic, primary plastid-containing mises, perhaps severely or even irrevocably, our ability to an- eukaryotes (green algae, red algae, and glaucophytes)? And swer this question. relatedly, which lineage(s) of cyanobacteria gave rise to plas- tids? Although we have no clue of an answer to the second How many secondary symbioses of red algae?ÐPerhaps question, longstanding controversy over the former has sub- the most important question relating to the symbiotic history sided over the past decade. This is because DNA sequence- of plastids concerns the number of red algal secondary sym- based trees and a number of features of plastid genomes, the bioses. The diversity of eukaryotes with secondary red plastids plastid protein import apparatus, and photosynthetic pigment is immense, and these are phylogenetically interspersed with proteins have converged to provide clear (to some observers, groups that lack plastids (Fig. 1). This has led many observers overwhelming) evidence for a single origin of plastids (Fig. to postulate multiple secondary symbioses involving a red al- 1; Palmer, 2003; Keeling, 2004; McFadden and van Dooren, gal endosymbiont. As reviewed by Keeling (2004), however, 2004), although there are still reasons (Palmer, 2003; Stiller, recent DNA evidence, both sequence-based and rearrange- 2003; Stiller et al., 2003) to be cautious in assuming that this ment-based, now provides reason to think that all secondary issue is completely settled. It should also be emphasized that red algal plastids trace back to a common secondary symbiosis the evidence for a common origin of plastids is much stronger (Fig. 1, red arrow), in the common ancestor of a putative eu- for green and red algae than for the comparatively poorly stud- karyotic supergroupÐChromalveolatesЮrst postulated by ied glaucophytes (only a single glaucophyte plastid genome Cavalier-Smith (1998). If Chromalveolates are indeed mono- has been sequenced and scant information is available on glau- phyletic, arising by only a single red algae symbosis, then this cophyte nuclear and mitochondrial genomes). Although very means that the many diverse lineages within the group that rare in plastids, HGT may be suf®ciently common in cyano- apparently lack plastids (see Keeling, 2004, for many such 1442 AMERICAN JOURNAL OF BOTANY [Vol. 91 examples beyond the three shown in Fig. 1) must have once Figure 2 shows a number of surprising placements and re- had them. In the case of ciliates and oomycetes (Fig. 1), initial lationships that have emerged solely from and/or found strong analysis of complete nuclear genomes has failed to ®nd any support only from DNA sequences. Within ferns, these include signi®cant vestiges of putative plastid-derived genes (P. J. the placement of horsetails within the ferns and the sister- Keeling, University of British Columbia, personal communi- group relationship of the enigmatic, vegetatively reduced Psi- cation). Either ciliates and oomycetes have been cleansed of lotales and the ophioglossoid ferns (Pryer et al., 2004). Within their plastid heritage to an extent unthinkable for land plants angiosperms, we note the deep positions of the monotypic, (Martin et al., 2002) or else the evidence for Chromalveolate New Calendonian-endemic, Amborellaceae, and of Nymphae- monophyly and a single red algal secondary symbiosis is mis- aceae and Austrobaileyales (Soltis and Soltis, 2004). Also not leading. Clearly, much more phylogenetic and genomic evi- shown are the many other surprising placements that have dence is needed. emerged within groups of land plants (and also nonland plants) not covered by the obviously scanty representation afforded Where does Mesostigma belong?ÐAs reviewed by Lewis by these two global, framework trees. In almost all these cases, and McCourt (2004), a major puzzle in green algal phylogeny however, the surprise has been one of delight (``Ah ha, we've concerns the placement of Mesostigma, an unusual asymmet- ®nally found a place for that troublesome species X.'') rather rical unicell. With good taxon sampling, a four-gene, three- than one of dismay or disbelief (``There's no way species Y genome data set strongly supports Mesostigma within charo- belongs in that group; something has got to be wrong with phytes, probably as sister to the rest of this clade (Karol et al., these DNA data.''). Frequently, careful assessment of mor- 2001), whereas a two-gene plastid data set places it with equal- phology reveals characters that agree with the ``DNA sur- ly strong support as sister to all green algae, prior to what is prise,'' although in many instances these characters may be otherwise regarded as the fundamental split in green algal evo- cryptic (Judd and Olmstead, 2004). lution (between charophytes and chlorophytes; Turmel et al., 2002a; Fig. 1). With poor taxon sampling, a similar con¯ict Are Gnetales really sister to Pinaceae?ÐThe most radical, arises for much larger character sets. Some analyses of com- shocking, and controversial placement of any group of plants bined matrices of ca. 40 plastid genes place Mesostigma within concerns Gnetales, a small, relatively obscure group of gym- the charophytes (e.g., Martin et al., 2002), whereas other such nosperms. Whereas a notable hypothesis from morphological analyses and those of 19 mitochondrial genes place it as sister cladistic studies had been that Gnetales were sister to angio- to all other green algae (Lemieux et al., 2000; Turmel et al., sperms, many molecular studies instead found gymnosperms 2002b). As with many critical phylogenetic issues in plants to be monophyletic and placed Gnetales within conifers, as and other organisms, robust resolution of the position of Me- sister to Pinaceae (e.g., Bowe et al., 2000; Chaw et al., 2000; sostigma may require both large character sets and better taxon reviewed in Burleigh and Mathews, 2004). Although other sampling. Resolving the placement of Mesostigma is important molecular studies found support for monophyly of conifers to understanding the early evolution of green algae, as well as and instead placed Gnetales either as sister to conifers or as evolutionary trends in organellar gene content (Lemieux et al., sister to all other seed plants, the original analyses in the paper 2000; Turmel et al., 2002b) and photosynthetic pigments by Burleigh and Mathews (2004) in this issue provide strong (Yoshii et al., 2003). evidence for the ``gnepines'' hypothesis (Bowe et al., 2000; Chaw et al., 2000), i.e., that Gnetales and Pinaceae are sister Who's at the base of land plants?ÐA major controversy taxa. Burleigh and Mathews (2004) sampled moderately ex- in land plant phylogeny concerns the base of the tree (Fig. 2). tensively within gymnosperms and examined more genes (13 Traditionally, land plants have been divided into two groups, total; ®ve plastid, four nuclear, and four mitochondrial) and vascular plants and bryophytes. Although vascular plants are characters (almost 19 000 nucleotides) than in any other pre- strongly supported as monophyletic based on both DNA evi- vious study on this issue. Contrary to previous claims (e.g., dence (e.g., Nickrent et al., 2000) and morphology (Kenrick Rydin et al., 2002) that earlier, three-genome analyses favoring and Crane, 1997), bryophytes are now generally thought to the gnepines topology rested ``almost exclusively'' on mito- comprise a grade of three monophyletic lineages (mosses, liv- chondrial genes and that there is a ``severe con¯ict between erworts, and hornworts) of uncertain relationship to each other the mitochondrial and other genomes [that] should not be sup- and to vascular plants. Many studies (listed in Shaw and Ren- pressed or ignored,'' Burleigh and Mathews (2004) found that zaglia, 2004) have addressed these relationships. In our view all three genomic data partitions support the gnepines result. there is as yet no clear answer, and therefore we show the base This was true in all maximum likelihood analyses and in those of land plants as a tetrachotomy (Fig. 2). Most commonly, parsimony analyses in which the most rapidly evolving sites either liverworts or hornworts emerge as sister to all land were excluded. Indeed, contra Rydin et al. (2002), the mito- plants, but we see little reason to favor one result over the chondrial partition actually provided only slightly lower boot- other based on current data. Those who do, e.g., favoring liv- strap support for gnepines in these analyses than did the nu- erworts-sister on the basis of ``compelling evidence'' (Fried- clear or plastid partitions. Moreover, when all 13 genes were man et al., 2004) from so-called ``rare genomic markers'' (mi- combined (and with the fastest sites excluded for parsimony), tochondrial intron presence/absence; Qiu et al., 1998; Dom- both parsimony and maximum likelihood gave 100% bootstrap brovska and Qiu, 2004) are in our view privileging a select support for gnepines. few (not so rarely changing) characters over thousands of other Only in parsimony analyses that included all sites did Bur- characters for which we have a long history of robust use. leigh and Mathews (2004) fail to recover the gnepines topol- Such purportedly rare events can be subject to high levels of ogy. Tellingly, however, these analyses (with all 13 genes) homoplasy when examined with intensive sampling (Adams placed Gnetales in a radically different position, as sister to et al., 2002; McPherson et al., 2004), and thus we do not really all other seed plants, prompting Burleigh and Mathews (2004) know how reliable any of these sorts of characters are. to conclude that the ``Gnetales-sister signal is restricted to sites October 2004] PALMER ET AL.ÐOVERVIEW OF PLANT TOL 1443

[largely in plastid genes] in the fastest two of nine evolution- (Chase, 2004; Judd and Olmstead, 2004; Soltis and Soltis, ary rate categories'' and re¯ects bias ``in the most rapidly 2004). evolving sites to which parsimony is particularly sensitive.'' The next 20 years will undoubtedly see an ever more rapid In short, the Gnetales-sister placement is a likely example of accumulation of DNA sequence data and the generation of long-branch-attraction (between the long branches leading to comprehensive, robustly resolved, and increasingly well-dated Gnetales and to the outgroups), and the gnepines topology is phylogenetic trees. Phylogenetics is now hitched to a powerful likely to be correct. Nonetheless, we also caution that this issue engineÐthe economic, largely biomedical engine that has should not be considered settled; even though receiving 100% been driving the development of faster and cheaper technol- bootstrap support in most 13-gene analyses, the gnepines to- ogies for sequencing DNA and for high-throughput robotic pology is, as Burleigh and Mathews (2004) point out, ``very handling of DNA in general. Even at the current rate of data similar'' to the gnetifer topology (Gnetales sister to a mono- accumulation, nearly all of the remaining problems in plants phyletic conifers) ``and potentially even a small amount of are tractable, and it is likely that there will continue to be error or bias could in¯uence [these] phylogenetic results.'' improvements that will increase output and hasten progress. We emphasize this particular case because it nicely illus- The next 20 years may even see radical breakthroughs in se- trates a variety of issues in phylogenetics. It illustrates the quencing and other DNA technologies, breakthroughs that potential to get artifactual results (e.g., the Gnetales-sister could allow the virtually limitless collection of gene and even placement) under the following evolutionary conditions: (1) whole genome data from most plants (especially from the high levels of extinction (on, among others, the outgroup small but gene-rich genome of plastids). If so, then we will branch and the branches leading to Gnetales, angiosperms, and certainly look back on this era as a small waystation on the Ginkgo), (2) high rates of sequence evolution (in Gnetales), path toward reconstructing the complex and fascinating evo- and (3) biased evolution at a subset of sites (rapidly evolving lutionary history of the botanical world. sites in plastid and nuclear genes). The ®rst two of these fac- The prospects for achieving a robustly resolved and well- torsÐextensive extinction and rapid sequence evolutionÐare dated tree of plant life are exhilarating. To rephrase Dobzhan- probably the greatest intrinsic problems in molecular phylo- sky (1973), ``Nothing in biology makes sense except in light genetics, especially when operating in concert, as with the of phylogeny.'' Only with an accurate tree in hand can we Gnetales placement. This case vividly illustrates how different properly make sense of the profound richness and diversity of partitions of a sequence data set can generate highly supported the botanical world. Only then can we fully appreciate evo- trees with radically different topologies and also points to an lution at all levels of organization, from the ecological and apparently useful methodological approach to deal with this morphological to the biochemical and genomic. Speci®c ap- intragenic con¯ict. It also illustrates the superior performance plications of phylogenetic studies, of ``tree thinking,'' are too of maximum likelihood over parsimony under these challeng- numerous to list here, ranging from the applied (Yates et al., ing conditions (whereas parsimony generally performs well in 2004) to fundamental studies of evolutionary and ecological situations where extinction has not eliminated the potential for processes (36 such applications are listed in Table 3.1 of Fu- extensive taxon sampling to break up the long branches that tuyma, 2004). Hillis (2004) aptly argues that ``as the Tree of are particularly problematic for it). Finally, it shows the po- Life becomes more complete, its applications are also ex- tential for morphology to be dif®cult to interpret and even panding exponentially. A complete Tree of Life would allow unhelpful in phylogenetic inference. At the same time, it is analyses that we would never contemplate today.'' He also important to note that the gnepines topology con¯icts with contends that before long the phylogenetic revolution will relatively few morphological characters, especially ones that have permeated the way we study all areas of biology. The provide a clear pattern (Burleigh and Mathews, 2004; Soltis study of the plant tree of life has, through the collaborative et al., in press). This points to the dangerously seductive in- spirit of the ®eld and its remarkable progress, set the standard ¯uence that ``charismatic singular characters,'' be they mor- for tree of life efforts, and we look forward to accelerating phological (Scotland et al., 2003) or ``rare genomic structural'' rates of progress in the years to come. (McPherson et al., 2004), can exert compared to the numeri- cally overwhelming but unlovable mass of nucleotide se- LITERATURE CITED quence characters that are the foundation of virtually all well- supported phylogenetic trees. ADAMS, K. L., Y.-L. 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