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Author for correspondence: Karl J. Niklas Tel: 001 607 255 8727 Email: firstname.lastname@example.org
Developmental constraint or a phyletic legacy?
Green plant phylogeny
The ancestral green plant life cycle
Haplobiontic or diplobiontic life cycles?
Pseudo-archegonia, plasmodesmata, and parenchyma
Genomic re-deployment and embryophyte reproduction
Isomorphic or dimorphic?
The extant land plants are unique among the monophyletic clade of photosynthetic eukaryotes, which consists of the green algae (chlorophytes), the charophycean algae (charophytes), numerous groups of unicellular algae (prasinophytes) and the embryophytes, by possessing, firstly, a sexual life cycle characterized by an alternation between a haploid, gametophytic and a diploid, sporophytic multicellular generation; secondly, the formation of egg cells within multicellular structures called archegonia; and, thirdly, the retention of the zygote and diploid sporophyte embryo within the archegonium. We review the developmental, paleobotanical and molecular evidence indicating that: the embryophytes descended from a charophyte-like ancestor; this common ancestor had a life cycle with only a haploid multicellular generation; and the most ancient (c. 410 Myr old) land plants (e.g. Cooksonia, Rhynia and Zosterophyllum) had a dimorphic life cycle (i.e. their haploid and diploid generations were morphologically different). On the basis of these findings, we suggest that the multicellular reproductive structures of extant charophytes and embryophytes are developmentally homologous, and that those of the embryophytes evolved by virtue of the co-option and re-deployment of ancient algal homeodomain gene networks.
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One of the most important biological events in the history of life was the successful colonization of the terrestrial landscape by green, multicellular plants and their subsequent rapid diversification during the early Paleozoic (Chaloner, 1970; Graham, 1993, 1996; Niklas, 1997; Raven & Edwards, 2001; Taylor et al., 2009). This key event, which was unknown to Darwin (1859), paved the way for terrestrial animal evolution, altered geomorphology by accelerating soil formation and modifying hydrology patterns, and thus irrevocably changed the Earth’s climate (Chaloner & Lawson, 1985; Willis & McElwain, 2002). Authorities differ regarding when the land plants first appeared (Fig. 1a). Some lines of evidence indicate that microfossil assemblages of spores from the Lower Middle Ordovician are the oldest remains of terrestrial plant life, whereas others point to a Silurian–Early Devonian invasion (Gray et al., 1974; Gray, 1985; Strother et al., 1996; Beck & Strother, 2001; Wellman et al., 2003), although reports of Neoproterozoic terrestrial soil crusts containing photosynthetic organisms (of a cyanobacterial nature?) must be considered (see Knauth & Kennedy, 2009). What can be said with more certainty is that the modern-day descendants of the first successful land plants comprise a monophyletic group, the Embryophyta (Kingdom Plantae). The living representatives of this taxon include the paraphyletic nonvascular plant lineages (colloquially referred to as the ‘bryophytes’) and the ‘tracheophytes’ (i.e. lycophytes, ferns, horsetails and seed plants).
Numerous lines of evidence support the contention that the embryophytes are monophyletic and closely related to the green algae (Kingdoms Protista and Protoctista). However, the embryophytes are unique among all extant lineages in possessing three important and interrelated reproductive attributes. First, they possess a sexual life cycle that requires an alternation between a multicellular haploid generation, which produces sperm and egg cells (the gametophyte), and a multicellular diploid generation, which produces meiospores with sporopollenin-rich walls (the sporophyte). Second, they develop multicellular, parenchymatous structures that produce eggs and sperm (called archegonia and antheridia, respectively). Third, they retain the fertilized egg (i.e. the zygote) within the archegonium, wherein the sporophyte embryo is nurtured and protected (Walbot & Evans, 2003). The retention of the diploid embryo within the archegonium is the reason why the land plants are called ‘embryophytes’ and why the older literature referred to them as the Archegoniatae (Campbell, 1905; Bower, 1908).
II. Developmental constraint or a phyletic legacy?
Whether the archegoniate diplobiontic life cycle was essential for (or merely coincidental to) the evolutionary and ecological success of the first multicellular land plants remains problematic. The retention of this life cycle may reflect a developmental constraint or a phyletic legacy, that is, a feature that either could not be or was not lost once acquired by the last common ancestor to all embryophytes. Alternatively, this life cycle may have been retained because it conferred functional advantages that prefigured (or were requisite for) survival and reproductive success in an aerial and potentially desiccating habitat (Fig. 1a). Ad hoc adaptive scenarios can be easily constructed to argue in favor of the latter, whereas recent insights from plant developmental genomics suggest that very ancient algal gene networks were co-opted during the evolution of the embryophyte life cycle and multicellular body plan (Niklas & Kutschera, 2009).
In the light of this uncertainty, this article has two goals. The first is to review the available phycological, paleobotanical, developmental and molecular data that shed light on how the archegoniate diplobiontic life cycle may have evolved. The second is to explore how these data influence the interpretations of developmental homologies among embryophyte reproductive structures (i.e. antheridia, archegonia and sporangia). These goals dictate the structure of this article, which conforms to a concept map dominated by three ‘axes’ (Fig. 2). The first of these axes focuses on the environmental context in which early land plants evolved, grew and reproduced. The environmental context is pivotal to tracing the evolution of any life cycle, because the most ancient embryophytes required access to liquid water for the successful fertilization of their eggs. The second axis focuses on the plant body plan and the transition from the unicellular to the multicellular condition. All the available information indicates that the ancestral condition for each of the major green plant lineages involved a unicellular body plan, and that multicellularity is a derived evolutionary condition. The third axis deals directly with the life cycle concept. For the purposes of our review, only two life cycles are relevant, one in which two multicellular generations occur and another in which only one multicellular generation exists, i.e. the diplobiontic and haplobiontic life cycles, respectively (see Fig. 1b). Because terminology, such as diplobiontic and haplobiontic, may be unfamiliar to some (and used in different ways by others), we provide definitions for these and other technical words and phrases as used in the context of this article (Table 1).
Table 1. Definitions of key words and phrases used in the context of this article
The multicellular, sperm-producing structure of the embryophytes, consisting of a sterile jacket of cells surrounding spermatogenous cells
The multicellular, egg-producing structure of the embryophytes, consisting of a neck, neck canal cells and a venter surrounding the egg
The phenotypic architecture that distinguishes one group of organisms from another; the processes that obtain an organism’s organized growth and development
The presence of substantive phenotypic differences between the haploid and diploid phases (generations) in the life cycle of an organism; in phycology, heteromorphic (i.e. morphologically different haploid and diploid generations)
A life cycle that involves the alternation of two multicellular phases (one haploid and another diploid) to complete sexual reproduction; also known as the ‘alternations of generations’ and as the diplohaplontic life cycle, e.g. mosses and ferns
Multicellular gamete-producing structures, e.g. antheridia, archegonia, globules and nucules
The multicellular haploid phase in a plant life cycle that produces gametes (sperm or eggs, or both)
Sperm-producing multicellular organ of charalean algae
A life cycle involving only one multicellular generation
A haplobiontic life cycle in which the only multicellular generation is diploid, e.g. Homo sapiens and other vertebrates
A haplobiontic life cycle in which the only multicellular generation is haploid; one in which the only diploid phase is the zygote; in phycology, equivalent to haplontic, e.g. charophycean algae
One or more traits characterizing two or more phyletically related taxa that emerge as a result of shared highly conserved ancestral structures, genetic networks or mechanism(s)
The absence of substantive phenotypic differences between the haploid and diploid phases (generations) in the life cycle of an organism
Egg-producing multicellular structure of charalean algae
In phycology, a cell specialized to function as an egg
A tissue composed of not distinctly specialized and generally uniformly appearing living cells with thin primary cell walls
Parenchymatous tissue construction
A tissue in which cells have the capacity to divide in any plain with respect to the principal body axis and in which primary or secondary plasmodesmata develop at the majority of cell walls shared among neighboring cells
Microscopic channels traversing the cell walls of embryophytes and some algae that enable intercellular symplastic transport and communication
The multicellular diploid phase in a plant life cycle that produces spores
An informal taxonomic term referring to the charophyte–embryophyte lineage
Meiosis occurring after the fertilization of the egg without any intervening mitotic cell divisions. In multicellular algae, zygotic meiosis obtains a haplobiontic-haploid life cycle
III. Green plant phylogeny
Concept maps help to establish logical transformational alternatives among critical character states (e.g. terrestrial vs aquatic, unicellular vs multicellular; haplobiontic vs diplobiontic life cycles). However, taken in isolation, they cannot identify the polarity of evolutionary transformations (e.g. aquatic to terrestrial vs terrestrial to aquatic). For our purpose, a stringent cladistic hypothesis is required, because the phylogenetic relationships among the various green plant lineages are very complex and because a well-supported cladogram provides a framework with which to deduce the evolutionary transitions leading to the embryophyte life cycle. In this section, we review the phylogenetic relationships among the green plant lineages as a prelude to mapping life cycle evolutionary transformations. All current palaeobotanical, cytological, physiological and molecular data indicate that the green algae (i.e. the Chlorophyta sensuSmith, 1950) and the Embryophyta share a last common ancestor (Mattox & Stewart, 1984; Mishler & Churchill, 1985; McCourt, 1995; Karol et al., 2001; Scherp et al., 2001; Lewis & McCourt, 2004; Archibald, 2009), which was a unicellular flagellate that evolved as a result of ancient endosymbiotic events involving a prokaryotic host cell and a cyanobacterial-like photoautotroph (Bhattacharya & Medlin, 1995; Kutschera & Niklas, 2004, 2005, 2008) (Fig. 3). Numerous lines of evidence further show that the embryophytes descended from a last common ancestor shared with the Coleocheatophyceae, Charophyceae and possibly other lineages (such as the Zygnemophyceae and Klebsormidiophyceae), which collectively comprise the green algae colloquially called the ‘charophytes’ (Karol et al., 2001; McCourt et al., 2004). Taken in isolation, the Coleocheatophyceae–Charophyceae lineage is relatively small in terms of species’ numbers and includes species with unicellular and multicellular body plans, some of which are adapted to, or at least capable of tolerating, some desiccation (Fig. 3).
The evidence for the monophyly of the charophyte–embryophyte lineage (collectively referred to as the ‘streptophytes’) is extensive. In addition to producing cell walls containing cellulose, chloroplasts with stacked grana and chlorophylls a and b, bi- or multiflagellated cells (when motile cells are present) and starch as their primary photosynthate, the charophytes and embryophytes also share features not found in any other green algae, such as, for example, several enzyme systems (e.g. glycolate oxidase), motor organelles with asymmetrically inserted flagella, dissimilar flagella roots with a multilayered structure, persistent mitotic spindles, open mitosis and phragmoplasts (Mattox & Stewart, 1984; Graham, 1993; McCourt, 1995; Graham & Wilcox, 2000; Karol et al., 2001; Scherp et al., 2001; McCourt et al., 2004).
Nevertheless, although all green plants are monophyletic, the most recent phylogenies consistently identify a deep genomic dichotomy between the streptophytes and three cladistically well-supported lineages (i.e. the Chlorophyceae, Trebouxiophyceae and Ulvophyceae), which are collectively referred to as the ‘chlorophytes’. Like the charophytes, the chlorophytes are ecologically diverse and include species with unicellular and multicellular body plans, some of which can survive in subaerial or emergent habitats (Fig. 3).
The deep streptophyte–chlorophyte ‘divide’ is occupied by an assortment of lineages represented by unicellular species, collectively called ‘prasinophytes’ (Fig. 3), whose phylogenetic relationships remain problematic (Sym & Pienaar, 1993; Lewis & McCourt, 2004). Although the existence of six or seven prasinophyte lineages is supported by molecular data (e.g. Zignone et al., 2002), these algae are best viewed as a grade of cellular organization emerging from the base of the green plant clade. As such, they have the potential to shed light on the features characterizing the last common flagellate ancestor to the entire green plant ‘tree of life’.
IV. The ancestral green plant life cycle
The precise phylogenetic relationships among the prasinophytes, chlorophytes and streptophytes will undoubtedly be modified as more taxa are examined and more data are incorporated into cladistic analyses. However, given current information, three conclusions can be drawn: the green plants are monophyletic; the phyletic dichotomy separating the chlorophytes and the streptophytes is occupied by prasinophytes; and the streptophytes descended from a unicellular freshwater alga. Here, we review data that support two additional conclusions: the ancestral life cycle in all of the major green algal lineages involved zygotic meiosis and was thus haplobiontic (Fig. 1b); and the derived green plant diplobiontic life cycle evolved at least twice, once among the chlorophytes (Ulvophyceae) and again among the streptophytes (charophytes and embryophytes).
These assertions are based on two lines of evidence. First, among extant unicellular and multicellular green algae (i.e. prasinophytes, chlorophytes and charophytes) for which sexual reproduction has been documented, most have a life cycle in which the only diploid cell is the zygote (Smith, 1950; Bold & Wynne, 1978; Graham & Wilcox, 2000; Lee, 2008). Second, although sexual reproduction has been documented for very few species in the basal prasinophyte lineages, those that have been corroborated involve zygotic meiosis (e.g. Nephroselmis olivacea; Suda et al., 1989) (Fig. 4a). Thus, for the majority of green algae, the ‘adult’ or ‘mature’ organism in the sexual life cycle is haploid and functions reproductively as the gametophyte generation in the embryophyte life cycle (Graham & Wilcox, 2000; Niklas & Kutschera, 2009).
As noted, plant life cycles involving only one multicellular individual are called haplobiontic (in contrast with diplobiontic life cycles with two multicellular individuals; Fig. 1b). Two variants of the haplobiontic life cycle are possible. One in which the multicellular generation is diploid (the haplobiontic-diploid life cycle; H–d) and one in which the haploid generation is exclusively multicellular (haplobiontic-haploid; H–d) (see Fig. 2, node 4). Therefore, life cycles involving zygotic meiosis are classified as haplobiontic-haploid (Table 1). Clearly, no multicellular generation exists in the case of unicellular algae. The sexually ‘mature’ individual functions either indirectly or directly (i.e. with or without intervening mitotic cell divisions) as the ‘adult’ organism and as a ‘gamete’. Therefore, the haplobiontic vs diplobiontic terminology is largely irrelevant. Nevertheless, the life cycles of unicellular algae, such as Nephroselmis olivacea, and multicellular algae, such as Monostroma grevillei, are fundamentally the same (Fig. 4a,b, respectively). Both are defined by zygotic meiosis. The only fundamental distinction that conceptually separates the two life cycles is whether (and where) multicellularity is developmentally expressed.
In contrast with the broad phyletic distribution of haplobiontic-haploid life cycles, diplobiontic life cycles occur in only one green algal lineage – the Ulvophyceae (Fig. 3). Three of the six orders within this class are reported to contain species with diplobiontic life cycles (i.e. the Cladopherales, Trentepohliales and Ulvales; see Graham & Wilcox, 2000; Lewis & McCourt, 2004; Lee, 2008 and references therein). Among these species, some diplobiontic life cycles are isomorphic (e.g. Ulva), whereas others are dimorphic (e.g. Derbesia). However, even among the various Ulvophyceae, the diplobiontic life cycle appears to be an evolutionarily derived condition, because molecular data suggest that the Ulotrichales are basal in the Ulvophyceae (O’Kelly et al., 2004), and because ulotrichalean algae have haplobiontic-haploid life cycles, e.g. Ulothrix and Monostroma (Fig. 4b). The haplobiontic-haploid life cycle is also well represented among the acellular (siphonous) ulvophycean marine algae. These lines of evidence indicate that the diplobiontic life cycles of the Embryophyta and Ulvophyceae are the result of convergent evolution (see Fig. 3).
V. Haplobiontic or diplobiontic life cycles?
Was the last common ancestor to the Charophyceae and Embryophyta unicellular or multicellular? This question is important because its answer provides an insight into the evolution of the embryophyte life cycle. Consider that, if the last common ancestor were unicellular, the capacity for multicellularity could have evolved in either the haploid or diploid generation, or both simultaneously (Fig. 5a,b) – a possibility that opens the door to many conceivable life cycle variants as the ancestral condition. Alternatively, if the last common ancestor were multicellular and had a life cycle involving delayed zygotic meiosis, the diploid generation in the embryophyte life cycle (i.e. the sporophyte) would be an evolutionary innovation.
The phyletic distribution of unicellular species at the base of the streptophyte lineage highlights the problematic nature of the answer to this question, as illustrated by the cladistic position of the monotypic Mesostigmatophyceae (Fig. 3). Mesostigma vivida is an asymmetrical cell that was originally classified as a charophyte on the basis of its flagellar ultrastructure (Melkonian, 1989). Subsequent molecular analyses of 18S rRNA sequences indicated that the genus was closely related to Chaetosphaeridum, which led to the establishment of a new class, the Mesostigmatophyceae (Marin & Melkonian, 1999). However, Delwiche et al. (2002) demonstrated that Chaetosphaeridum is a charophyte and, on the basis of rbcl sequences, concluded that Mesostigma is a sister taxon to the streptophytes. Subsequently, Yoshii et al. (2003) argued that Mesostigma represents an early evolutionary lineage and placed the Mesostigmatophyceae among the prasinophytes (Fig. 3).
The phylogenetic position of Mesostigma remains contentious (see Lewis & McCourt, 2004). However, its phyletic position at the base of the chlorophyte–streptophyte ‘divide’, in tandem with the antiquity of lineages containing numerous semi-aquatic and terrestrial unicellular species (i.e. Chlorokybophyceae, Klebsormidiophyceae and Zynemophyceae), suggests that a variety of life cycles may have evolved (and disappeared) over the early course of charophycean evolution (see Fig. 3). Indeed, one intriguing line of speculation is the prospect that important life cycle evolutionary innovations occurred among unicellular or filamentous charophytes serving as phycobionts in ancient lichen-like organisms. Currently, no lichen is known to contain a charophycean phycobiont (Friedl & Bhattacharya, 2006). However, it is possible that the ancient co-evolutionary history of the land plants and mycorrhiza was prefigured by a mutually beneficial relationship between ancient charophytes and fungi that can be broadly thought of as lichen-like symbiotic organisms (McCourt et al., 2004).
Although the nature of the most ancient land plant life cycle cannot be asserted currently, the available evidence indicates that the ancestor to the streptophytes was multicellular and possessed a haplobiontic-haploid life cycle similar to that of Coleochaete and Chara. If this supposition is true, the land plant sporophyte generation was an evolutionary innovation resulting from delayed zygotic meiosis and the intercalation of one or more mitotic cellular divisions. Put differently, the first embryophyte sporophyte was a multicellular zygote. Whether this life cycle first appeared in an aquatic, semi-aerial or aerial environment is conjectural. Although the transition from a haplobiontic-haploid to a diplobiontic life cycle may have come at a cost with regard to the growth of the haploid generation (as indicated by studies on mosses; see Ehrlen et al., 2000; Rydgren & Økland, 2002), the diplobiontic life cycle confers adaptive benefits across a broad range of habitats and environmental conditions (e.g. the numerical amplification of zoospores or meiospores resulting from possibly rare fertilization events, and the possibility to occupy two different niches in the same general environment), as attested by the reproductive and ecological success of ulvophycean algae and embryophytes with free-living gametophytes.
Nevertheless, if the evolutionary transformation from a haplobiontic-haploid to a diplobiontic life cycle occurred in a fresh water or terrestrial habitat, which is almost a certainty, the first sporophytes would hardly qualify as ‘land plants’, as they would have grown on maternal gametophytes attached, in turn, to a hydrated substrate, and thus are more properly thought of as ‘air plants’ (see Fig. 1a).
VI. Pseudo-archegonia, plasmodesmata and parenchyma
Whether the first charophycean algae to evolve a diplobiontic life cycle were archegoniates is another challenging and unresolved question. The fossil record of the earliest land plants is especially sparse and problematic, and there is nothing in the diplobiontic life cycle concept that stipulates the manner in which sperm or eggs are produced (Wellman et al., 2003). In addition, as noted earlier, the diplobiontic life cycle is not unique to the embryophytes (see Fig. 3). However, it is very likely that the last common ancestor to the streptophytes had reproductive structures that functioned in some, if not many, ways like the antheridia and archegonia of embryophytes, such as Equisetum (Lycopodiacae), a seedless land plant (Fig. 6a,b).
This assertion rests on three observations. The zygotes of many, albeit not all, species in the Coleochaetophyceae and all species in the Charophyceae are retained by the gametophyte, during which many species nourish and protect them for short, albeit developmentally substantive, periods of time; and the most evolutionarily derived charophycean species, such as stoneworts of the genus Chara, have multicellular and morphologically complex gametangia (Fig. 6c,d). In the case of Coleochaete (see Fig. 1b), sterile filaments develop around the oogonium after fertilization. In the case of charalean species, sterile cells envelop the oogonium before fertilization. Indeed, the sperm-producing organ of Chara (the globule) is functionally (and developmentally) similar in many ways to the antheridium, whereas the egg-producing structure (the nucule) can be called a ‘pseudo-archegonium’ on the basis of its capacity to protect and provide the egg and zygote with nourishment for a not inconsiderable time (Fig. 6c,d).
Another physiologically important feature shared by the charophytes and embryophytes is the capacity to form plasmodesmata (Graham, 1982), which has evolved independently many times in different algal lineages and independently within the Chlorophyceae and again in the Charophyceae (Raven, 1997). Among the streptophytes, the ability to form these symplastic connections among adjoining cells is restricted to the Coleochaetophyceae (specifically Coleochaete, see Fig. 1b), Charophyceae (Fig. 6c,d) and the Embryophyta (Brown et al., 1994; Cook et al., 1998). The ability to form plasmodesmata permits active polar transport of large molecular weight solutes across adjoining cell walls, which facilitates the targeting and nutrition of specialized cells (see Jansen, 2001; Lucas et al., 2001). It can also play a pivotal role in the physiological control of embryogenesis. More detailed studies are required to determine whether charophycean plasmodesmata are typically primary or secondary in nature. The former develop during cytokinesis and cell wall deposition; the latter develop after cytokinesis and may appear after secondary wall deposition. Whether primary or secondary plasmodesmata form during or after histogenesis is important, because it helps to resolve whether the tissues in which plasmodesmata develop are truly parenchymatous, and because primary and (complex) secondary plasmodesmata have different protein-trafficking functions, which can influence organogenesis (e.g. Itaya et al., 1998). Regardless of these subtleties, primary plasmodesmata have been demonstrated for at least one species of Chara (Brown et al., 1994; Cook et al., 1998), and ultrastructural studies suggest that the nodal regions of Chara have a parenchymatous tissue structure (Pickett-Heaps, 1975; Cook et al., 1998).
VII. Genomic re-deployment and embryophyte reproduction
The ability to form plasmodesmata and parenchyma is not a requisite for the formation of morphologically complex multicellular structures, such as the gametangia of Chara (Fig. 6c,d). The nucule and globule of Chara and other charalean algae are composed of branched filaments, which only give the appearance of having a parenchymatous tissue construction. However, the ability to form parenchyma and plasmodesmata is an important attribute of the embryophytes, because it establishes complex and physiologically integrated symplastic interconnections via primary and secondary plasmodesmata formation that are required for sporophyte embryogenesis and development.
Indeed, recent studies of homeodomain-containing transcription factor genes suggest that an intriguing ‘genomic redeployment strategy’ has attended the evolution of the archegoniate diplobiontic life cycle. For example, among the best known of these genes is the MADS-box gene family, which has been extensively studied in the flowering model organism Arabidopsis thaliana. This gene family is divided into two subfamilies, referred to as type I and type II. There are 45 type II genes, which are also referred to as MIKC factors (for MADS DNA-binding domain, intervening domain, keratin-like domain and C-terminal domain); the type II group can be further subdivided into MIKCC and MIKC* genes on the basis of the inferred evolutionary history of the family. MIKC* proteins tend to have longer I domains and less-conserved K domains than do the MIKCC proteins. Sequences encoding MIKCC and MIKC* factors have been identified in bryophytes and lycopods, as well as in gymnosperms and angiosperms, which suggests that the MIKC* and MIKCC genes have evolved independently for at least 450 Myr. The expression of MIKC-type genes in angiosperms occurs only after the specification of the vegetative to inflorescence meristem transition, which is mediated by the transcription factor encoded by FLORICAULA/LEAFY (FLO/LFY). In ferns, FLO/LFY homologs are expressed predominantly in sporogenous meristematic tissues, but MADS-box gene expression is not closely correlated, suggesting that these genes have not yet been subordinated to FLO/LFY regulation. In the moss Physcomitrella, two FLO/LFY paralogs (PpLFY-1 and PpLFY-2) are required for the first division of the zygote and early sporophyte embryogenesis (Henschel et al., 2002; Tanahashi et al., 2005), whereas MADS-box gene expression occurs during Chara globularis gametangium differentiation and declines after fertilization (Tanabe et al., 2005).
It is therefore reasonable to suggest that MADS-box genes originally functioned in the differentiation of haploid reproductive structures (e.g. the nucule and moss archegonium) and were subsequently redeployed to function in the formation of sporophyte reproductive structures (e.g. the fern sporangium). Such combinatorial homeodomain-based transcriptional control of reproduction may have extremely deep phylogenetic roots. Ectopic expression of the homeoproteins Gsp1 and Gsm1 in the plus and minus strains of the unicellular chlorophyte Chlamydomonas activates vegetative haploid cells to form zygote-like structures (Lee et al., 2008). Likewise, Gsp1 and Gsp2 are members of the TALE (three amino acid loop extension) homeodomain-containing transcription factors, which include the class 1 KNOX and class 2 KNOX proteins. Homeodomain gene networks, similar to those in land plants, have been reported for prasinophytes (e.g. Micromonas), which are postulated to reveal the attributes of the last common ancestor of all green plants (Worden et al., 2009) (see Fig. 3).
VIII. Developmental homologies?
The recruitment and redeployment of homeodomain gene networks underlying much of the evolution of streptophyte reproduction may help to explain why charalean gametangia and embryophyte antheridia, archegonia and sporangia share the same fundamental developmental choreography (Fig. 7).
Perhaps the most obvious shared attribute of these multicellular structures is that each develops from a single superficial meristematic initial. In the case of charalean globule and nucule, this initial is a nodal cell (Fig. 7a); in the case of embryophytes, it is typically an epidermal cell (Fig. 7b). Charalean antheridium induction involves an unequal division of the nodal initial cell. The smaller of the two derivatives develops into a stalk; the larger, apical cell undergoes a series of cellular divisions that eventually produce external shield cells that surround stalks and branched structures (manubria) from which sperm filaments radiate (Pickett-Heaps, 1975). The development of the charalean nucule also begins from a single nodal cell that undergoes unequal cell divisions to form a basal stalk and the tube cells that gyrate around a centrally located oogonium (Pickett-Heaps, 1975). In much the same way, sporangial induction involves periclinal division of one or more epidermal cells. Among eusporangiate species, the innermost derivatives give rise to sporogenous cells, whereas the outermost develop into the sporangium wall (Fig. 7b). Embryophyte gametangia (antheridia and archegonia) development also begins when a single epidermal cell undergoes a periclinal division. The innermost cells resulting from this division develop into spermatogenous cells, or the neck canal and egg cells (for details, see Campbell, 1905; Bower, 1908; Gifford & Foster, 1989).
Numerous differences exist in the development of eu- and leptosporangia and in the development of antheridia and archegonia. For example, the sporogenous cell initials in the eusporangium develop from the innermost periclinal derivatives of the superficial sporangial initials, whereas the sporogenous cells of the leptosporangium trace their developmental origins to outer periclinal derivative cells. Likewise, charalean and embryophyte development differ in many ways. For example, the charalean oogonium occupies an apical (albeit enveloped) position, whereas the embryophyte egg cell develops from a hypodermal derivative. Likewise, the nucule and globule have a ‘pseudo-parenchymatous’ (filamentous) tissue construction, whereas sporangia, antheridia and archegonia are parenchymatous (Gifford & Foster, 1989; Graham & Wilcox, 2000).
Nevertheless, we suggest that there is sufficient developmental and molecular evidence to conclude that the embryophyte sporangium is homologous to the antheridium/archegonium as a result of homeodomain gene network recruitment from the gametophyte generation and redeployment in the sporophyte generation. In this context, we use the concept of homology sensuShubin et al. (1997), namely a correspondence in growth and differentiation resulting from highly conserved and deeply ancestral genetic mechanisms. Likewise, we believe that archegonia and antheridia are developmentally homologous to charalean gametangia, i.e. embryophyte gametangia are homologous to the nucule and globule (Fig. 7). This perspective is consistent with current knowledge of the molecular developmental biology of streptophyte reproductive structures. It is also consistent with the evolution of the embryophyte diplobiontic life cycle from the haplobiontic-haploid life cycle of a charalean common ancestor. The alternative assertion that embryophyte sporangia are homologous to leaves is far less tenable (Kenrick & Crane, 1997a,b).
IX. Isomorphic or dimorphic?
We have argued that the ancient aquatic ancestor to all green eukaryotes (Kingdoms Protoctista and Plantae) was a single-celled flagellate (Fig. 3) with a life cycle that alternated between a haploid generation that functioned as the adult + gamete and a more or less ephemeral single-celled zygote (Fig. 1b). The traditional botanical view precludes assigning such an organism a diplobiontic or a haplobiontic life cycle, because multicellularity is not expressed in this life cycle. Nevertheless, this kind of organism has an ‘alternation of generations’, albeit unicellular ones. Accordingly, any consideration of the life cycle of the earliest ‘Urform’ (prototype) of all land plants necessarily begins by asking whether the immediate ancestor to the streptophytes was unicellular in both phases of its life cycle (as was its aquatic precursor), or whether one or both of its life cycle phases was multicellular. This consideration is cast in botanical tradition by asking whether the life cycle was isomorphic (both life forms are the same) or dimorphic (two different life forms) (see Fig. 2, node 5; Table 1).
There are obvious advantages to multicellularity for an organism living in a subaerial environment, e.g. protection against UV exposure and dehydration (Niklas, 1997; Raven, 1999, 2002). However, the most ancient green land plants may have inhabited the soil or evolved from a lichen-like organism, and thus may have been protected by moisture-laden boundary layers (Stebbins & Hill, 1980; Read et al., 2000). What is clear is that, over many generations, the land plants became multicellular, and we necessarily need to know whether this innovation occurred simultaneously in both the gametophyte and sporophyte generation, or whether one generation acquired multicellularity whilst the other generation subsequently acquired this body plan.
On the basis of the distribution of life cycles among extant green plants (Fig. 3), it is logical to argue that the series of life phase transformations achieving multicellularity began with the gametophyte generation, and that the zygotes of the ancestors of the first green land plants were preprimed for meiosis. If true, the vegetative phase of the gametophyte generation is the primal (ancestral) and archetypical home for land plant gene expression and gene network interactions, as well as the effects of heritable mutations on morphogenesis. Nevertheless, the alternative possibility – that multicellularity arose first in the diploid, sporophytic phase – cannot be excluded, especially from a genetic perspective, as a single mitotic division before meiotic cell division generates eight (not four) potentially recombinant genotypes from each zygote (with consequent selective advantages), in contrast with a mitotic division after meiosis, which generates two (not one) spore recombinant genotypes (with consequent effects on the stochastic loss of a desirable combination of genes).
Another conceptually important, but as yet unresolved, question is whether the most ancient embryophytes possessed an isomorphic or a dimorphic life cycle (Fig. 2, nodes 5 and 6). Kenrick & Crane (1997a,b) and Steemans et al. (2009) have argued that the isomorphic alternation of generations is the most ancient, in part based on three-dimensionally preserved gametophytes in the c. 410 Myr old Rhynie Chert (see Kerp et al., 2004; Taylor et al., 2005; Niklas & Kutschera, 2009). As discussed in the previous section, the gametophytes and sporophytes of the most ancient embryophytes undoubtedly shared similar genomic and developmental repertories, just as they shared genomic similarities with their charalean common ancestor. Thus, in the absence of phenomenologies, such as gene silencing, sex chromosomes or epigenetic effects, differences in ploidy may not have equated to significant gametophyte–sporophyte morphological differences. This perspective is strengthened in the light of some modern-day mosses and ferns, which can generate sporophyte morphologies directly from their gametophytic cells (apogamy) and gametophyte morphologies directly from their sporophyte cells (apospory). Apogamy and apospory show that the haploid and diploid genomes contain much of the information required to construct both the gametophyte and the sporophyte body plans (Niklas & Kutschera, 2009).
A focus on apogamy rather than apospory is justified in the context of our review, because the thesis developed thus far is that the sporophyte generation evolved during the transition from a charalean-like haplobiontic-haploid life cycle to an embryophyte diplobiontic life cycle. Among extant species, apogamy can be induced by cell trauma, low light intensities, suitable concentrations of sugar or auxin. It can also be induced by the deletion of the CURLY LEAF ortholog in the moss Physcomitrella (PpCLF). Okano et al. (2009) have reported that gametophytic cells that usually form protonema or gametophore apical cells generate meristematic apical cells that form branched morphologies, which can be induced to form sporangium-like structures with the exogenous application of PpCLF. The resulting morphologies have been reported to be similar to very ancient tracheophytes, such as Zosterophyllum or Cooksonia (Figs 8 and 9).
These findings suggest that PpCLF regulatory gene networks may have participated in the early evolution of the embryophyte sporophyte (Okano et al., 2009). Spontaneous mutation of the CURLY LEAF ortholog attending fertilization and zygote formation among ancient streptophytes may have participated in delayed zygotic meiosis, and thus the formation of a multicellular diploid phase. It is nevertheless doubtful that the mutation of any single gene was sufficient for this important evolutionary transition. Likewise, it is uncertain whether the sporophyte generation evolved as a consequence of delayed zygotic meiosis or precocious zygotic mitosis. Molecular analyses of angiosperm mega- and microsporogenesis and mega- and microgametogenesis indicate that numerous complex gene networks are involved in the initiation or suppression of meiosis. For example, the male sterile multiple archesporial cell (mac1) mutant in maize (Zea mays), which leads to the production of extra diploid sporocytes in ovules and anthers, appears to contribute to the restriction of the identity of cells competent for meiotic division (see Sheridan et al., 1996; Walbot & Evans, 2003), whereas the Meiosis Arrested at Leptotene1 (MEL1) gene in rice (Oryza sativa) is required for sporocycte meiosis (Nonomura et al., 2007). More data from taxa deeper within the streptophyte lineage are required to shed meaningful light on the gene networks that participate in early sporophyte embryogenesis.
Returning to the antiquity of the isomorphic vs diplobiontic life cycle, a number of lines of evidence indicate that the most ancient embryophyte life cycles were dimorphic. First, extant monoploid embryophytes do not develop apogamous sporophytes, suggesting that gene duplication and subsequent functional divergence presaged the evolution of multicellular sporophytes, which is consistent with the extensive analysis of the Physcomitrella patens genome (Rensing et al., 2008). Second, embryophyte sporophytes and gametophytes normally develop in very different physiological and mechanical environments. Young sporophytes develop within archegonia; ‘free-living’ embryophyte gametophytes develop from dispersed meiospores. Third, numerous environmental factors influence early morphogenesis that fosters dimorphism (Sinnott, 1960). Fourth, if the most ancient sporophytes were an ‘intercalated’ multicellular generation, it is difficult to imagine that they were morphologically elaborate – indeed, they may have been nothing more than the functional equivalent of a sporangium (Niklas, 1997). Fifth, the different functional obligations of the gametophyte and sporophyte generations would have sustained and even amplified their ancestral dimorphism. Sixth, the purported life cycles of Rhynie Chert plants (dated to 410 Myr), such as Rhynia, are not isomorphic (Kerp et al., 2004; Taylor et al., 2005; Niklas & Kutschera, 2009). Seventh, there is some evidence (albeit, at this point, very problematic) that more ancient tracheophytes, such as Zosterophyllum and Cooksonia (Figs. 8,9), may have had dimorphic life cycles (Probst, 1986; Niklas & Banks, 1990; Remy et al., 1993; Gerrienne et al., 2006).
Many details regarding the phylogeny of the green plant lineages (chlorophytes and streptophytes) remain unresolved and will require more data from more taxa, particularly from those residing at the base of this ecologically rich clade. The broad phylogeny of the green plants is nevertheless sufficiently well resolved to permit a reasonably stringent phyletic scaffold upon which to trace the character transformations attending the evolution of the embryophyte diplobiontic life cycle. Using this approach, two conclusions emerge that will probably stand the test of future scrutiny. First, the ancestral organism for the entire green plant clade (and for the lineage leading to the charophytes) was a flagellated eukaryotic photoautotroph (Fig. 3), and, second, the ancestral organism to the streptophytes (Coleochaetophyceae, Charophyceae, and Embryophyta) was a multicellular alga that had a haplobiontic-haploid life cycle and morphologically complex gametangia reminiscent of those of Chara (Fig. 6c,d). With far less certainty, we surmise that the earliest land plants (Figs 1a,8,9) had a dimorphic diplobiontic life cycle in which the haploid (gametophytic) phase dominated. Subsequent evolutionary divergence led to land plant forms that retained this ancestral condition (represented today by the extant bryophytes) and lineages in which the diploid generation became dominant (represented by all extant tracheophytes, notably the seed plants, Fig. 1b). Although speculative, we further suggest that there is sufficient observational and molecular data to indicate that the reproductive organs of embryophytes (i.e. archegonia, antheridia, and eusporangia) are homologous structures (sensu Shubin et al., 1997) that, in turn are homologous with the multicellular gametangia of the charalean algae (the nucule and globule) (Fig. 7). These homologies appear to be the result of the co-option and re-deployment of ancient algal gene networks.
Much speculation surrounds the properties of genes, gene networks, and even entire organisms that favor pivotal evolutionary transformations that nevertheless conserve structural and genomic homologies. Although the evidence is sparse, speculation favors the effects of regulatory genes, in general, and transcription factors, in particular, as the most probable ‘drivers’ of evolutionary innovation (see Doebley & Lukens, 1998; Cronk, 2001). However, the few detailed studies of plant developmental gene interactions are insufficient to justify this view to the exclusion of other potentially equally important mechanisms, as witnessed by the recent finding that the developmental patterning of the highly reduced angiosperm mega-gametophyte depends on an asymmetric, location-specific gradient of auxin synthesis (Pagnussat et al., 2009). For this reason, we conclude that future enquiries into the developmental mechanism(s) by which the embryophyte life cycle evolved would profit from detailed diagnoses of the molecular ‘drivers’ of the plant cell cycle, the induction of meiotic versus mitotic cell division, and a host of other fundamental phenomena. Scrutiny of meiotic gene candidates and factors that contribute to homologous chromosome pairing will be a particularly important and fertile field of inquiry (see Able et al., 2009).
We thank Dr. P. Gerienne for providing Fig. 9a and Dr. J. Raven (the University of Dundee, UK) for many useful suggestions. We also thank the College of Agriculture and Life Sciences (Cornell University, Ithaca, USA) and the Alexander-von Humboldt-Foundation (AvH, Bonn, Germany) for financial support (AvH-fellowship 2009, Stanford/California, USA to U. Kutschera).