New discoveries at the base of the green plant phylogeny

Cite as: Leliaert F., Verbruggen H. & Zechman F.W. (2011) Into the deep: New discoveries at the base of the green plant phylogeny. BioEssays 33: 683-692


Abstract. Recent data have provided evidence for an unrecognized ancient lineage of green plants that persists in marine deep-water environments. The green plants are a major group of photosynthetic eukaryotes that have played a prominent role in the global ecosystem for millions of years. A schism early in their evolution gave rise to two major lineages, one of which diversified in the world’s oceans and gave rise to a large diversity of marine and freshwater green algae (Chlorophyta) while the other gave rise to a diverse array of freshwater green algae and the land plants (Streptophyta). It is generally believed that the earliest-diverging Chlorophyta were motile planktonic unicellular organisms, but the discovery of an ancient group of deep-water seaweeds has challenged our understanding of the basal branches of the green plant phylogeny. In this review, we discuss current insights into the origin and diversification of the green plant lineage.

A brief history of green plant evolution

Green plants are one of the most dominant groups of primary producers on earth. They include the green algae and the embryophytes, which are generally known as the land plants. While green algae are ubiquitous in the world’s oceans and freshwater ecosystems, land plants are major structural components of terrestrial ecosystems (Lewis & McCourt, 2004; O’Kelly, 2007). The green plant lineage is ancient, probably over a billion years old (Yoon et al., 2004; Herron et al., 2009), and intricate evolutionary trajectories underlie its present taxonomic and ecological diversity.

The green plants originated following an endosymbiotic event, where a heterotrophic eukaryotic cell engulfed a photosynthetic cyanobacterium-like prokaryote that became stably integrated and eventually evolved into a membrane-bound organelle, the plastid (Gould et al., 2008; Keeling, 2010). This single event marked the origin of oxygenic photosynthesis in eukaryotes and gave rise to three autotrophic lineages with primary plastids: the green plants, the red algae and the glaucophytes. From this starting point, photosynthesis spread widely among the eukaryotes via secondary endosymbiotic events that involved the capture of either green or red algae by diverse non-photosynthetic eukaryotes, thus transferring the captured cyanobacterial endosymbionts (i.e., the plastids) laterally among eukaryotes (Gould et al., 2008). Some of these secondary endosymbiotic partnerships have, in turn, been captured by other eukaryotes, known as tertiary endosymbiosis, resulting in an intricate history of plastid acquisition (reviewed in Gould et al., 2008; Archibald, 2009; Keeling, 2010). Three groups of photosynthetic eukaryotes now have plastids derived from a green algal endosymbiont: the chlorarachniophytes, a small group of mixotrophic algae from tropical seas; the euglenophytes, which are especially common in freshwater systems, and some green dinoflagellates. A much wider diversity of photosynthetic eukaryotes, including the dinoflagellates, haptophytes, cryptophytes, chrysophytes, diatoms and brown seaweeds have obtained plastids from a red algal ancestor, either by a single or by repeated endosymbiotic events (Bodyl et al., 2009; Keeling, 2010).

An early split in the evolution of green plants gave rise to its two principal lineages, which have subsequently followed radically different evolutionary trajectories (Fig. 1) (Lewis & McCourt, 2004; Lemieux et al., 2007; Rodríguez-Ezpeleta et al., 2007). One lineage, the Chlorophyta, diversified as plankton in the oceans and gave rise to the modern prasinophytes and the core chlorophytes that radiated in marine coastal and freshwater environments. The Chlorophyta now encompass a large diversity of green algae with a bewildering variety of body forms, eco-physiological traits and life cycle strategies (Lewis & McCourt, 2004). The second lineage, the Streptophyta, evolved in freshwater and damp terrestrial habitats and colonised dry land approximately 476-432 million years ago, giving rise to the land plants (McCourt et al., 2004). Contemporary streptophytes comprise a diverse array of mainly freshwater algae (collectively termed the charophytes) and the vastly species-rich land plants (McCourt et al., 2004).

Figure 1. Phylogenetic relationships among the main lineages of green plants. The tree topology is a composite on accepted relationships based on molecular phylogenetic evidence (Lewis & McCourt, 2004; McCourt et al., 2004; Lemieux et al., 2007; Rodríguez-Ezpeleta et al., 2007; Turmel et al., 2007b; Becker & Marin, 2009; Turmel et al., 2009; Cocquyt et al., 2010; Finet et al., 2010; Marin & Melkonian, 2010; Zechman et al., 2010). Uncertain phylogenetic relationships are indicated by polytomies. The divergence times are rough approximations based on the fossil record and molecular clock estimates (Colbath, 1983; Knoll, 1992; Yoon et al., 2004; O'Kelly, 2007; Herron et al., 2009). These age estimates should be interpreted with care as different molecular clock studies have shown variation in divergence times between major green plant lineages. Drawings illustrate representatives of each lineage: (1) Acetabularia, (2) Pediastrum, (3) Chlorella, (4) Tetraselmis, (5) Picocystis, (6) Ostreococcus, (7) Micromonas, (8) Crustomastix, (9) Monomastix, (10) Pyramimonas, (11) Pycnococcus, (12) Pseudoscourfieldia, (13) Nephroselmis, (14) Prasinococcus, (15) Verdigellas (a: general habit, b: individual cells in a gelatinous matrix), (16) Mesostigma, (17) Chlorokybus, (18) Klebsormidium, (19) Chara, (20) Xanthidium, (21) Coleochaete, (22) Chaetosphaeridium, (23) Ranunculus.
Figure 1. Phylogenetic relationships among the main lineages of green plants. The tree topology is a composite on accepted relationships based on molecular phylogenetic evidence (Lewis & McCourt, 2004; McCourt et al., 2004; Lemieux et al., 2007; Rodríguez-Ezpeleta et al., 2007; Turmel et al., 2007b; Becker & Marin, 2009; Turmel et al., 2009; Cocquyt et al., 2010; Finet et al., 2010; Marin & Melkonian, 2010; Zechman et al., 2010). Uncertain phylogenetic relationships are indicated by polytomies. The divergence times are rough approximations based on the fossil record and molecular clock estimates (Colbath, 1983; Knoll, 1992; Yoon et al., 2004; O’Kelly, 2007; Herron et al., 2009). These age estimates should be interpreted with care as different molecular clock studies have shown variation in divergence times between major green plant lineages. Drawings illustrate representatives of each lineage: (1) Acetabularia, (2) Pediastrum, (3) Chlorella, (4) Tetraselmis, (5) Picocystis, (6) Ostreococcus, (7) Micromonas, (8) Crustomastix, (9) Monomastix, (10) Pyramimonas, (11) Pycnococcus, (12) Pseudoscourfieldia, (13) Nephroselmis, (14) Prasinococcus, (15) Verdigellas (a: general habit, b: individual cells in a gelatinous matrix), (16) Mesostigma, (17) Chlorokybus, (18) Klebsormidium, (19) Chara, (20) Xanthidium, (21) Coleochaete, (22) Chaetosphaeridium, (23) Ranunculus.

The early evolutionary history of the Chlorophyta in the oceans of the Meso- and Neoproterozoic (between 700 and 1500 million year ago) is marked by a radiation of planktonic unicellular organisms (O’Kelly, 2007). These ancestral green algae were of fundamental importance to the eukaryotic greening that shaped the geochemistry of our planet (Worden et al., 2009). Although the fossil record is clearly incomplete, analysis of microfossils suggests that green algae were prevalent in the eukaryotic oceanic phytoplankton of the Paleozoic Era (Colbath, 1983; Knoll, 1992; O’Kelly, 2007). Subsequently, the red plastid-containing dinoflagellates, coccolithophores and diatoms increased in abundance to largely displace the green algae in the phytoplankton from the end-Permian extinction to the present. This evolutionary transition has been related to a long-term change in the chemistry of the ocean during the Mesozoic combined with specific eco-physiological traits of the red plastid-containing lineages (Katz et al., 2004). Trace element usage in algae with a red-type plastid differs from that of the green algae, which may have been advantageous following a shift in the redox conditions of the oceans (Falkowski et al., 2004). The pigment sets of red plastids provide for higher underwater photosynthetic efficiency compared to green plastids, and may be another explanation for the red dominance in the seas (O’Kelly, 2007; Simon et al., 2009). In addition, the success of lineages with red-type plastids has been explained by better portability of red-type plastids via secondary endosymbiosis to diverse eukaryotic hosts (Falkowski et al., 2004), although this hypothesis has been questioned (Keeling et al., 2004).

Despite this red dominance in the phytoplankton, green algae continue to play prominent roles in contemporary marine environments. Prasinophytic picoplanktonic species (i.e., with cells smaller than 3 µm) can dominate both photosynthetic biomass and production in open oceans and coastal systems (Vaulot et al., 2008). In addition, the green seaweeds of the class Ulvophyceae, which radiated in marine benthic habitats in the Neoproterozoic (Butterfield, 2009; Verbruggen et al., 2009; Cocquyt et al., 2010) (Fig. 1), form key components in many contemporary coastal environments.

The first eukaryotic algae in freshwater environments were probably unicellular streptophytes, which prevailed in these ecosystems in the Proterozoic (Becker & Marin, 2009). During the Paleozoic, the two principal multicellular groups of charophytes, the conjugating green algae (Zygnematophyceae) and stoneworts (Charophyceae) radiated, and the latter dominated freshwater macrophytic communities between the Permian and Early Cretaceous (Martín-Closas, 2003). In the Late Cretaceous and Tertiary, they were largely replaced by freshwater angiosperms. Two classes of the Chlorophyta, the Chlorophyceae and Trebouxiophyceae, adapted to freshwater environments during the Neoproterozioc (Herron et al., 2009) (Fig. 1) and dominated freshwater planktonic assemblages during the Paleozoic and Mesozoic eras while the diversity and abundance of charophytes gradually decreased (Martín-Closas, 2003; Becker & Marin, 2009). The demise of green dominance of freshwater phytoplankton began with the appearance of freshwater dinoflagellates in the Early Cretaceous, and the radiation of diatoms and chrysophytes during the Cenozoic.

The dominance of algae with red-type plastids in the seas (and to a lesser extent in freshwater environments) is in sharp contrast to the situation on land where photosynthesis has been dominated by the green land plants ever since they colonised the terrestrial environment in the Ordovician (Kenrick & Crane, 1997).

Deep branches of the Chlorophyta

Molecular phylogenetic, ultrastructural and biochemical studies have identified the prasinophytes as a paraphyletic assemblage of free-living unicells with a wide variety of cell shapes (Fig. 1), flagellar numbers and behaviour, body scale shapes, mitotic processes, biochemical features and photosynthetic pigment signatures (Nakayama et al., 1998; Fawley et al., 2000; Zingone et al., 2002; Guillou et al., 2004; Latasa et al., 2004).

The critical phylogenetic position of the prasinophytes, diverging early from the remaining Chlorophyta (Fig. 1), reinforced the notion that the ancestral chlorophytes were marine planktonic unicellular flagellates with characters typical of extant prasinophytes such as the presence of organic body scales (Melkonian, 1990; Sym & Pienaar, 1993). The nature of this hypothetical ancestral green flagellate (AGF), however, still remains uncertain. Moestrup (1991) proposed that small, simple flagellate cells most closely resemble the AGF. Other researchers have interpreted the food-uptake mechanism of some complex flagellates as a character inherited from a phagotrophic ancestor of the green plants (Moestrup et al., 2003; O’Kelly, 2007; Turmel et al., 2009).

A better understanding of prasinophytic diversity and relationships is crucial to elucidate the nature of the common ancestor of the green plants. Originally, only flagellate unicells covered with organic body scales were classified in the prasinophytes (Melkonian, 1990). The discovery of several new species and the application of environmental sequencing have revealed a greater morphological and ecological diversity (Zingone et al., 2002; Marin & Melkonian, 2010; Jouenne et al., 2011). Non-motile (coccoid) forms have been identified in several of the major prasinophytic lineages and many members lack scales or have other types of cell coverings (Table 1). Prasinophytes are primarily marine but several representatives have adapted to freshwater environments.

Although there is little doubt that sex pre-dates diversification of extant eukaryotes (Schurko et al., 2009; Wilkins & Holliday, 2009), it has rarely been observed in prasinophytes. A notable exception is Nephroselmis, where sexual reproduction has been detected in cultures (Suda et al., 1989; Suda et al., 2004). However, circumstantial evidence points towards a much wider occurrence of sex among prasinophytes. For example, members of the Pyramimonadales produce walled cysts that contain two chloroplasts, suggestive of gamete fusion (Moestrup et al., 2003). In addition, sexual reproduction has been implied in Ostreococcus and Micromonas based on the occurrence of sex-related and meiosis-specific genes in their genomes (Derelle et al., 2006; Worden et al., 2009).

Several studies have aimed at resolving the relationships among the prasinophytic lineages, which has proven to be a difficult task due to the antiquity of these divergences. Small subunit nuclear ribosomal DNA (18S rDNA) sequences have long been the main source of data for phylogenetic inference within the green plant lineage (Pröschold & Leliaert, 2007). Although 18S data have been useful in delineating the main prasinophytic lineages (Fawley et al., 2000; Guillou et al., 2004; Marin & Melkonian, 2010), analyses of these single gene datasets have not resolved the relationships among them. A robust phylogeny for an ancient lineage, such as that of green plants, requires analysis of a large number of genes.

Multi-gene data derived from chloroplast genomes, which are presently available for five prasinophytes, are just beginning to shed light on the ancient divergences of the Chlorophyta. A recent chloroplast phylogenomic analysis identified Nephroselmis (Nephroselmidophyceae) as the earliest-branching chlorophytic lineage (Turmel et al., 2009) (Fig. 1). This flagellate with a complex covering of scales and two unequal flagella (Fig. 2A,B, Table 1) might thus represents our best guess of what the AGF might have looked like. Interestingly, Nephroselmis is one of the few prasinophytes in which sexual reproduction has been well documented (Suda et al., 2004).

The close relationship between the Pyramimonadales and the Mamiellophyceae was an unexpected result from chloroplast phylogenomic studies (2009) (Fig. 1). The Pyramimonadales are relatively large flagellates with complex body scale coverings (Fig. 2C-D), and, as mentioned above, some of its members are unique among green plants in possessing a food uptake apparatus (Moestrup et al., 2003). The Mamiellophyceae is a large group comprising the morphologically and ecologically diverse Mamiellales and two smaller clades, the Monomastigales and Dolichomastigales (Marin & Melkonian, 2010). The phylogenetic affinity of the latter two has long been uncertain because several of their members lack scales and have atypical surface structures (Table 1). The Mamiellales are probably the largest and most diverse group of prasinophytes (Table 1). Several members (e.g., Ostreococcus and Micromonas) may form major components of marine picoeukaryotic communities (O’Kelly et al., 2003; Not et al., 2004; Vaulot et al., 2008). These algae have cell sizes smaller than those of many bacteria and show highly reduced cellular complexity and unusually compact genomes (Derelle et al., 2006; Palenik et al., 2007; Worden et al., 2009). These minute unicells have been regarded as “the bare limits of life as a free-living photosynthetic eukaryote” (Derelle et al., 2006) and likely evolved through secondary reduction from larger and more complex flagellates (Turmel et al., 2009).

There are several other groups of early-branching prasinophytes that we cannot place in the phylogenetic tree with any great precision, either because only single-gene data are available or because genome-scale phylogenetic analyses generate equivocal results.

1. The Pycnococcaceae is a small clade of marine flagellates and coccoids (Fig. 1, Table 1). Some studies based on 18S rDNA sequences have related this clade with the Nephroselmidophyceae (Fawley et al., 2000; Guillou et al., 2004) but this relationship has not been supported by chloroplast multi-gene analyses (Turmel et al., 2009).

2. The Prasinococcales includes a few marine coccoid prasinophytes (Hasegawa et al., 1996; Sieburth et al., 1999) (Fig. 2E, Table 1) and has been suggested to form an early-diverging clade based on 18S data (Guillou et al., 2004) (Fig. 1). Multi-gene data has not yet been generated for this group.

3. The Picocystis clade has been identified by environmental and culture-based sequencing. It includes a number of undescribed coccoid prasinophytes along with the saline lake-dwelling coccoid Picocystis (Table 1). 18S and multi-gene phylogenies have allied this clade with the core chlorophytes (Fig. 1), but support for this relationship is not strong (Guillou et al., 2004; Marin & Melkonian, 2010; Matsumoto et al., 2011).

4. Environmental sequencing of photosynthetic picoeukaryotic communities has identified two additional prasinophytic clades with uncertain affinities (termed clades VIII and IX) (Viprey et al., 2008; Lepère et al., 2009; Shi et al., 2009). As these organisms are only known from DNA sequence data, nothing is known about their morphology.

Figure 2. Representatives of some early-diverging lineages of the Chlorophyta. A: Light micrograph of Nephroselmis (Nephroselmidophyceae) showing two laterally inserted, unequal flagella, and a cup-shaped chloroplast (image courtesy of William Bourland). B: Scanning electron micrograph of Nephroselmis showing a complex covering of organic body scales (image courtesy of Shoichiro Suda, University of the Ryukyus). C: Light micrograph of Pyramimonas (Pyramimonadales) showing four similar flagella emerging from an anterior depression, a large cup-shaped chloroplast, and eyespot located lower left (image courtesy of David Patterson and Bob Andersen, Provasoli-Guillard National Center for Culture of Marine Phytoplankton). D: Scanning electron micrograph of Pyramimonas showing body and flagella covered with different types of scales (image courtesy of Rick van den Enden, Australian Antarctic Division © Commonwealth of Australia). E: Light micrograph of Prasinococcus (Prasinococcales) showing non-motile unicells, each embedded in gelatinous capsules (image courtesy of Daniel Vaulot, Station Biologique de Roscoff). F: Cross-section of a Palmophyllum plant (Palmophyllales), composed of coccoid cells embedded in a gelatinous matrix. G: Verdigellas, a member of the Palmophyllales growing in deep-water habitats (100-200 m) of the western Atlantic Ocean, is attached to the substrate by a central holdfast structure (not visible) above which the rest of the body expands (image courtesy of Mark and Diane Littler, National Museum of Natural History, Smithsonian Institution). H: Palmophyllum, forming irregularly lobed crusts that are tightly fixed to the substrate, is shown growing in the Mediterranean Sea down to depths of 130 m (image courtesy of Véronique Lamare, Données d'Observations pour la Reconnaissance et l’Identification de la faune et de la flore Subaquatiques). I: Palmoclathrus, a genus from deep-water habitats (down to 60 m deep) of Southern Australia, characterized by perennial stalks from which seasonal, netlike blades grow (image courtesy of Kevin Branden © Board of the Adelaide Botanic Gardens).
Figure 2. Representatives of some early-diverging lineages of the Chlorophyta. A: Light micrograph of Nephroselmis (Nephroselmidophyceae) showing two laterally inserted, unequal flagella, and a cup-shaped chloroplast (image courtesy of William Bourland). B: Scanning electron micrograph of Nephroselmis showing a complex covering of organic body scales (image courtesy of Shoichiro Suda, University of the Ryukyus). C: Light micrograph of Pyramimonas (Pyramimonadales) showing four similar flagella emerging from an anterior depression, a large cup-shaped chloroplast, and eyespot located lower left (image courtesy of David Patterson and Bob Andersen, Provasoli-Guillard National Center for Culture of Marine Phytoplankton). D: Scanning electron micrograph of Pyramimonas showing body and flagella covered with different types of scales (image courtesy of Rick van den Enden, Australian Antarctic Division © Commonwealth of Australia). E: Light micrograph of Prasinococcus (Prasinococcales) showing non-motile unicells, each embedded in gelatinous capsules (image courtesy of Daniel Vaulot, Station Biologique de Roscoff). F: Cross-section of a Palmophyllum plant (Palmophyllales), composed of coccoid cells embedded in a gelatinous matrix. G: Verdigellas, a member of the Palmophyllales growing in deep-water habitats (100-200 m) of the western Atlantic Ocean, is attached to the substrate by a central holdfast structure (not visible) above which the rest of the body expands (image courtesy of Mark and Diane Littler, National Museum of Natural History, Smithsonian Institution). H: Palmophyllum, forming irregularly lobed crusts that are tightly fixed to the substrate, is shown growing in the Mediterranean Sea down to depths of 130 m (image courtesy of Véronique Lamare, Données d’Observations pour la Reconnaissance et l’Identification de la faune et de la flore Subaquatiques). I: Palmoclathrus, a genus from deep-water habitats (down to 60 m deep) of Southern Australia, characterized by perennial stalks from which seasonal, netlike blades grow (image courtesy of Kevin Branden © Board of the Adelaide Botanic Gardens).

Table 1. Characteristics of the major prasinophytic lineages. Numbers between round brackets refer to the drawings of the organisms in Fig. 1.

Lineage and members Morphology and life cycle Ecology
Picocystis cladePicocystis (5) and several undescribed taxa Scale-less coccoids surrounded by a thin cell wall (Lewin et al., 2000).Sexual reproduction unknown. Picoplanktonic communities in saline lakes (Picocystis) or oceans (Lewin et al., 2000; Shi et al., 2009).
Mamiellophyceae: MamiellalesOstreococcus (6), Bathycoccus, Micromonas (7), Mantoniella, Mamiella Structurally simple, wall-less unicells, including scaly coccoids (Bathycoccus), naked coccoids (Ostreococcus), naked uniflagellates (Micromonas) and scaly biflagellates (Mantoniella, Mamiella) (Marin & Melkonian, 2010). Scales (when present) with a typical spider-web pattern (Sym & Pienaar, 1993).Micromonas and Mantoniella with palmelloid phase in the life cycle (Sym & Pienaar, 1993). Indirect evidence for sexual reproduction from genomic data (Derelle et al., 2006; Worden et al., 2009). Marine planktonic. Ostreococcus and Micromonas can form major components of picoeukaryotic communities (O’Kelly et al., 2003; Not et al., 2004; Vaulot et al., 2008).
Mamiellophyceae: DolichomastigalesCrustomastix (8), Dolichomastix Biflagellates with cells covered with spider-web or circular patterned scales (Dolichomastix) (Sym & Pienaar, 1993; Zingone et al., 2002) or cells scale-less and covered with a thin, double-layered membrane (Crustomastix) (Nakayama et al., 2000; Zingone et al., 2002).Sexual reproduction unknown. Mainly marine planktonic; a few species from freshwater environments (Marin & Melkonian, 2010).
Mamiellophyceae: MonomastigalesMonomastix (9) Flagellates with a single mature flagellum (second flagellum present as a basal body only), cells covered with very thin imbricate scales, resembling those of chrysophytes and prymnesiophytes (Moestrup, 1991).Only known to reproduce asexually, involving cyst formation. Freshwater habitats (Marin & Melkonian, 2010).
PyramimonadalesPyramimonas (10), Cymbomonas, Halosphaera, Pterosperma, Prasinopapilla Large flagellates, generally with four (sometimes eight or sixteen) flagella, covered with diverse and complex body scales in multiple layers (Melkonian, 1990; Sym & Pienaar, 1993). Some mixotrophic species of Cymbomonas and Pyramimonas possess a food uptake apparatus (Bell & Laybourn-Parry, 2003; Moestrup et al., 2003).Indirect evidence for sexual reproduction from resistant cysts containing two chloroplasts (Moestrup et al., 2003). Some Pyramimonas species with a palmelloid phase in the life cycle (Sym & Pienaar, 1993). Marine and freshwater habitats (Sym & Pienaar, 1993).
PycnococcaceaePycnococcus (11), Pseudoscourfieldia (12) Scale-less coccoids surrounded by a thin cell wall (Pycnococcus) (Sym & Pienaar, 1993) or wall-less flagellates with two unequal flagella, surrounded with simple scales (Pseudoscourfieldia) (Sym & Pienaar, 1993; Nakayama et al., 2007).Culture observations and sequence data indicate that both morphologies may represent different phases of the life cycle (Sym & Pienaar, 1993; Fawley et al., 1999; Zingone et al., 2002; Guillou et al., 2004). Marine picoplanktonic communities (Sym & Pienaar, 1993; Nakayama et al., 2007).
NephroselmidophyceaeNephroselmis (13) Relatively large, asymmetrical cells with a complex covering of diverse scales in multiple layers (Sym & Pienaar, 1993), and two laterally inserted, unequal and heterodynamic flagella (Nakayama et al., 2007).Sexual reproduction detected in culture (Suda et al., 1989; Suda et al., 2004). Marine and freshwater environments (Yamaguchi et al., 2011).
PrasinococcalesPrasinococcus (14), Prasinoderma Small, scaleless coccoids with thick cell walls (Jouenne et al., 2011). Cells of Prasinococcus are embedded in gelatinous capsules, secreted by complex pores (“Golgi-decapore complex”) (Sieburth et al., 1999).Only known to reproduce asexually. Marine habitats (Sieburth et al., 1999; Jouenne et al., 2011).
clade VIII Known from environmental sequencing only. Marine picoplanktonic communities (Viprey et al., 2008; Lepère et al., 2009).
clade IX Known from environmental sequencing only. Marine picoplanktonic communities (Viprey et al., 2008; Shi et al., 2009).

One of the ancestral prasinophytic lineages has given rise to the ecologically and morphologically diverse core chlorophytes (Fig. 1). This group includes the early-diverging Chlorodendrophyceae, a small clade of marine and freshwater quadriflagellates (Guillou et al., 2004). The three other clades are more diverse and comprise unicellular as well as multicellular organisms. The core chlorophytes are characterized by a new mode of cell division that is mediated by a phycoplast, which was subsequently lost in the Ulvophyceae (Lewis & McCourt, 2004). Several eco-physiological adaptations have allowed successful radiation of the Trebouxiophyceae and Chlorophyceae in freshwater and terrestrial habitats. The Ulvophyceae, which are best known as the green seaweeds, have mainly diversified along marine shorelines where they frequently dominate rocky shores and tropical lagoons. This clade has evolved an unequalled diversity of body forms, ranging from microscopic unicells to multicellular or giant-celled algae with unique cytological and physiological features (Cocquyt et al., 2010). Several members of the core chlorophytes live in symbiosis with various eukaryotic organisms, including fungi to form lichens, ciliates, cnidarians, foraminifera and vertebrates (Friedl & Bhattacharya, 2002; Lewis & Muller-Parker, 2004; Kerney et al., 2011).

An ancient lineage of deep-water green seaweeds

A recently published study has provided evidence for another early-diverging chlorophytic lineage, the Palmophyllales (Zechman et al., 2010). This group includes the little-known benthic seaweeds Palmophyllum, Verdigellas, and possibly Palmoclathrus, three genera from marine deep-water and other dimly lit environments. Although gene sequence-based phylogenies support a deeply-branching Palmophyllales, its exact phylogenetic placement remains uncertain. Analyses of the plastid genes rbcL and atpB placed the Palmophyllales sister to the remaining Chlorophyta. However, analysis of nuclear 18S rDNA sequences allied the Palmophyllales with the early-diverging Prasinococcales (Fig. 1). The latter relationship is supported by some shared cytological characteristics, such as a mucus-secreting system (Pueschel et al., 1997; Sieburth et al., 1999) and similarities in cell division (O’Kelly, 1988; Hasegawa et al., 1996; Jouenne et al., 2011).

Members of the Palmophyllales are characterized by a unique type of multicellularity. They form well-defined macroscopic bodies composed of small spherical cells embedded in a firm gelatinous matrix (palmelloid organization) (Womersley, 1984; Nelson & Ryan, 1986; Ballantine & Norris, 1994; Pueschel et al., 1997). Although the cells are separated and undifferentiated (Fig. 2F), several Palmophyllales have evolved large, complex erect bodies. For example, species of Verdigellas (Figs 1, 2G) attach to the substrate by means of a holdfast structure above which the rest of the body expands, resulting in umbrella-like plants that are well-adapted to capture the dim light in deep-water habitats. Palmoclathrus, a genus from temperate waters, are characterized by perennial stalks from which seasonal, netlike blades grow (Womersley, 1984) (Fig. 2I). Palmophyllum is morphologically simpler, forming irregular lobed crusts that are tightly attached to the substrate (Fig. 2H). Despite careful investigation, motile stages or ultrastructural traces from flagella have never been observed (Nelson & Ryan, 1986; O’Kelly, 1988; Pueschel et al., 1997). Interestingly, a number of prasinophytes have been described to have palmelloid stages in their life cycle, although they never form large and complex bodies like the Palmophyllales (Table 1). The early-diverging nature of the non-flagellate Palmophyllales and Prasinococcales, along with the wide phylogenetic distribution of non-motile prasinophytes, raises questions about the nature of the green ancestor. Although there is little doubt that flagella must have been present in a life cycle stage of the green plant ancestor, it may be possible that this ancestor was a non-motile unicell with transient motile stages.

It is remarkable that an ancient lineage like the Palmophyllales is restricted to deep-water or other dimly lit habitats. Low-light ecosystems present a challenging environment for photosynthetic organisms and relatively few algae live in such habitats (Littler et al., 1985). Verdigellas has been recorded from depths down to 200 m (Ballantine & Norris, 1994; Zechman et al., 2010), where only about 0.05% of the irradiance at the water surface remains (Littler et al., 1985). This results in extremely low primary productivity in Verdigellas compared to shallow-water green seaweeds (Littler et al., 1986). Palmophyllum and Palmoclathrus species generally grow between 40 and 100 m deep (Womersley, 1984; Nelson & Ryan, 1986). Palmophyllum is also found in shallower, shady areas like crevices and under rock overhangs (Nelson & Ryan, 1986).

Member of the Palmophyllales lack the green light-harvesting photosynthetic pigments siphonoxanthin and siphonein, which are found in several low-light adapted green algae (Nelson & Ryan, 1986; O’Kelly, 1988). Instead, they maintain high concentrations of chlorophyll b, which absorbs the blue-green light of deeper water more efficiently than does chlorophyll a (Sartoni et al., 1993).

The ability to grow in deep, low-light habitats may be of key importance to the Palmophyllales’ persistence. Compared to shallow habitats, deep-water environments are characterized by diminished abiotic stressors (e.g., wave action and temperature variation) and reduced grazing and competition for substrate. Whereas the more recently evolved green seaweeds (Ulvophyceae) of the core chlorophytes possess morphological and biochemical adaptations that allow them to withstand such stresses (Duffy & Hay, 1990), the Palmophyllales lack protective attributes such as calcification or cortication, and they may have found refuge from competition and herbivory in deep-water habitats (Zechman et al., 2010).

Marine deep-water environments are home to phylogenetic relicts of other lineages of organisms such as the hagfishes (Jorgensen et al., 1997), chimaeras and cow sharks (Weitzman, 1997), stalked crinoids and other invertebrates (Briggs, 1974). The onshore-offshore hypothesis posits the shallow-water origination and deep-water retreat of marine lineages in the fossil record (Jablonski et al., 1983). The early-branching position of the species-poor, deep-water Palmophyllales as compared to the diverse and predominantly shallow-water prasinophytes and core chlorophytes may be interpreted as an example of this phenomenon in photosynthetic organisms (Zechman et al., 2010).

Ancient streptophytes and the progenitors of land plants

The origin of land plants was a key event in the history of life and has led to important changes in the earth’s environment, including the development of the entire terrestrial ecosystem (Kenrick & Crane, 1997). Many studies have focused on the relationship among charophytes and have sought to determine the origins of land plants (Karol et al., 2001; Lemieux et al., 2007; Rodríguez-Ezpeleta et al., 2007; Finet et al., 2010; Wodniok et al., 2011).

The charophytes are mostly freshwater green algae with diverse morphologies ranging from simple unicells and filaments to complex and highly specialized macrophytes. Morphological and molecular data have revealed six distinct groups of charophytes: Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Zygnematophyceae, Charophyceae and Coleochaetophyceae (McCourt et al., 2004) (Fig. 1). Phylogenetic analyses of multi-gene datasets have clarified the relationships among these lineages, although some important questions remain (Karol et al., 2001; Turmel et al., 2003; Turmel et al., 2006; Lemieux et al., 2007; Rodríguez-Ezpeleta et al., 2007; Finet et al., 2010; Wodniok et al., 2011).

Molecular phylogenies have provided evidence that the morphologically simple charophytes Mesostigma (Mesostigmatophyceae) and Chlorokybus (Chlorokybophyceae) form the earliest-diverging streptophytic lineages (Fig. 1) (Turmel et al., 2006; Lemieux et al., 2007; Rodríguez-Ezpeleta et al., 2007; Finet et al., 2010). This result is consistent with ultrastructural features of their cells (Sym & Pienaar, 1993; Lewis & McCourt, 2004) and discrete molecular characteristics such as shared multi-gene families or gene duplications (Nedelcu et al., 2006; Petersen et al., 2006). Some phylogenies inferred from nuclear multi-gene data have placed Mesostigma a sister group to the remaining Streptophyta (Cocquyt et al., 2010; Finet et al., 2010), a position that is supported by the fact that Mesostigma is the only streptophyte with a motile vegetative stage, a presumed ancestral feature of the green plants. Conversely, phylogenies based on complete chloroplast genomes have suggested a sister relationship between Mesostigma and Chlorokybus (Lemieux et al., 2007; Rodríguez-Ezpeleta et al., 2007). Mesostigma is a freshwater biflagellate unicell with a unique suite of photosynthetic pigments. Like many prasinophytes, the cell and flagella are covered with diverse organic scales. Chlorokybus is found in moist terrestrial environments where it forms groups of a few non-motile cells (McCourt et al., 2004).

Gene sequence-based phylogenies unambiguously show that the freshwater or terrestrial filamentous Klebsormidiophyceae diverged after the Mesostigmatophyceae and Chlorokybophyceae (Karol et al., 2001; Turmel et al., 2002; Finet et al., 2010) (Fig. 1), a phylogenetic position that is further supported by several chloroplast genomic features (Turmel et al., 2007b).

Interestingly, sexual reproduction has not been observed in any of these early-diverging lineages and is only known in the later-diverging streptophytes (McCourt et al., 2004). However, determining whether these lineages are truly asexual will require genomic screening, as numerous allegedly asexual chlorophytic members have been shown to have cryptic potential for sex by the presence of meiosis and sex-related genes in their genomes (Derelle et al., 2006; Worden et al., 2009; Blanc et al., 2010).

In contrast to the three early-diverging streptophytic lineages (Mesostigmatophyceae, Chlorokybophyceae and Klebsormidiophyceae) that undergo cell division by furrowing, the cluster consisting of the Charophyceae, Zygnematophyceae, Coleochaetophyceae and the land plants evolved a new mechanism of cell-wall formation during cell division, which involved the production of a phragmoplast. In addition, most of the later-diverging streptophytes have cell-walls with plasmodesmata, facilitating cytoplasmic communication between cells and development of complex tissues (Graham et al., 2000).

Numerous studies have focussed on identifying the closest living relative of land plants, and different charophytes have been suggested based on morphological, biochemical and molecular data (McCourt et al., 2004; Becker & Marin, 2009). Gene sequence-based phylogenies have been sensitive to taxon and gene sampling and have revealed the morphologically complex Charophyceae (Karol et al., 2001; Turmel et al., 2007a; Cocquyt et al., 2010) or Coleochaetophyceae (Turmel et al., 2009; Finet et al., 2010), or the structurally simpler Zygnematophyceae (Turmel et al., 2006; Lemieux et al., 2007; Rodríguez-Ezpeleta et al., 2007; Wodniok et al., 2011) as the sister lineage of the land plants.

The colonization of dry land involved many challenges such as desiccation, increased temperature fluctuations, exposure to UV radiation and gravity (Waters, 2003; Floyd & Bowman, 2007; Lang et al., 2008). Several physiological and morphological innovations have led to a successful adaptation to terrestrial life (Graham et al., 2000; Waters, 2003; Becker & Marin, 2009). Some of these are also found in one or more charophytes and thus likely evolved before the origin of land plants, including cellulosic cell walls, multicellularity, differentiated cells and tissues, intercellular communication networks (plasmodesmata and plant hormones), zygote retention and placenta (Graham et al., 2000; Becker & Marin, 2009). Other innovations, such as a sexual life history involving an alternation of two multicellular bodies, and protected embryos appear to be unique to land plants (Graham et al., 2000). Additional adaptations to life on dry land included enhanced osmoregulation, desiccation and freezing tolerance, and heat resistance (Waters, 2003; Rensing et al., 2008).

Comparative genomic studies have indicated that the molecular bases of many land plant innovations evolved before the transition to land (Becker & Marin, 2009; Timme & Delwiche, 2010; Wodniok et al., 2011). For example, several genes that were thought to be important in the evolution of land plants (Graham et al., 2000) may have true orthologs with similar function in the Coleochaetophyceae and/or Zygnematophyceae (Timme & Delwiche, 2010; Wodniok et al., 2011). The diversification of embryophytes and evolution of complex plants was associated with expansion of numerous gene families, including MADS box genes (Tanabe et al., 2005), homeobox genes (Mukherjee et al., 2009), OPR genes (Li et al., 2009) and genes involved in signalling pathways, such as auxin, ABA and cytokinin (Rensing et al., 2008; Timme & Delwiche, 2010; De Smet et al., 2011). Expansion of the glutaredoxins gene family likely resulted in proteins with novel functions such as development and pathogenesis response (Ziemann et al., 2009). The typical life history of land plants possible evolved through expansion of homeodomain gene networks (Tanabe et al., 2005).

Conclusions and prospects

Molecular phylogenetic studies have drastically reshaped our views of green plant evolution (Lewis & McCourt, 2004; O’Kelly, 2007; Pröschold & Leliaert, 2007). It is now generally accepted that the green plants evolved into two discrete lineages (Fig. 1). One lineage, the Chlorophyta, includes several early-diverging clades of free-living unicells (the prasinophytes) and the morphologically diverse core chlorophytes. The other lineage, the Streptophyta, comprises the early-branching charophytic green algae and the land plants.

Resolving the relationships between these early-branching clades is crucial to addressing questions about the origin of the green plant lineage and to learn about the evolutionary trajectories responsible for the remarkable diversity of green algae and the emergence of the land plants. It has become clear that to achieve a reliable phylogenetic resolution for ancient groups like the green plants, many genes from many species must be analysed by applying state of the art phylogenetic techniques (Philippe & Telford, 2006; Verbruggen et al., 2010). Multi-gene phylogenetic investigations are just starting to shed light on the basal branches of the green plant phylogeny (Lemieux et al., 2007; Rodríguez-Ezpeleta et al., 2007; Turmel et al., 2009). High-throughput DNA sequencing techniques can facilitate broader gene and taxon sampling and will undoubtedly lead to more robust phylogenies (Finet et al., 2010; Wodniok et al., 2011).

The identification of deep-branching lineages is crucial to make inferences about the nature of the common ancestor of the green plant lineage. Sequencing of culture collections and environmental picoplankton samples has led to the discovery of several ancient green algal lineages (Fawley et al., 2000; Guillou et al., 2004; Viprey et al., 2008; Lepère et al., 2009; Shi et al., 2009; Marin & Melkonian, 2010). In addition, sampling from challenging habitats such as marine deep-water ecosystems has recently revealed a previously unrecognized deep-branching lineage of green plants (Zechman et al., 2010). Further exploration of diversity in understudied ecosystems such as deep marine waters, tropical coral reefs and sand habitats may lead to the discovery of other ancient groups and further alter our understanding of the early evolution of green plants.

GlossaryBiflagellate: Having two flagellaBody scales: organic (non-mineralized) structures, produced within the Golgi apparatus, and covering the cell surface of many prasinophytic species. Prasinophytic body scales are remarkably diverse, including plate-like, hair-like, and complex, three dimensional structures (Melkonian, 1990; Sym & Pienaar, 1993).

Coccoid: Spherical, non-motile unicellular microorganism.

Flagella: long whip-like organelles that propels cells through a liquid medium. Flagella contain a highly conserved (9 + 2) arrangement of microtubules. They are homologous with cilia, but generally longer and less numerous.

Flagellate: Noun: Motile unicellular eukaryotic microorganism, which swim by means of flagella. Flagellates include photosynthetic and non-photosynthetic heterotrophic species, which do not form a natural group of organisms but are distributed in several distantly related eukaryotic groups. Adjective: bearing one or more flagella.

Mixotrophic: Having partly autotrophic and partly heterotrophic nutrition.

Palmelloid: A kind of organization of the algal body with cells that are separate but remain enclosed within a mucilage envelope.

Paraphyletic group: A group of organisms that has evolved from a common ancestor but which does not contain all descendants of that ancestor. Green algae and charophytes are paraphyletic groups because they do not include the land plants. Similarly, prasinophytes are paraphyletic with the exclusion of the core chlorophytes. Paraphyletic groups are characterized by shared primitive (plesiomorphic) characters. For the green algae these include the presence of double membrane-bound plastids containing chlorophyll a and b, and several ultrastructural features of the chloroplast and flagella, all of which are also shared with the land plants.

Phagotroph: Heterotrophic or mixotrophic organism that ingests nutrients by engulfing solid particles.

Phragmoplast: Array of microtubules oriented perpendicularly to the plane of cell division, determining the formation of the cell plate and new cell wall. Phragmoplasts occur in land plants and their closest charophytic relatives, Charophyceae, Zygnematophyceae and Coleochaetophyceae.

Phycoplast: Array of microtubules oriented parallel to the plane of cell division, determining the formation of a new cell wall. Phycoplasts occur in the core chlorophytic classes Chlorodendrophyceae, Trebouxiophyceae and Chlorophyceae.

Picoplankton: The fraction of plankton comprising cells of between 0.2 and 3 µm.

Plasmodesmata: Cytoplasmic threads running transversely through cell walls that connect the cytoplasm of adjacent cells.

Quadriflagellate: Having four flagella.

Red-type plastid: plastids derived from a red alga via secondary or tertiary endosymbiosis.

Siphonein and siphonoxanthin: Xanthophyll accessory pigments found in Ulvophyceae and some prasinophytes. The possession of these pigments is believed to be an adaptation to life in deep-water, because they are well suited to harvesting of the green light that penetrates to these depths (Sartoni et al., 1993).

Uniflagellate: Having a single flagellum.

Acknowledgements. We thank Ive De Smet, Frederik Hendrickx and an anonymous reviewer for useful comments, and Shoichiro Suda, Nathalie Simon, Daniel Vaulot, Véronique Lamare, Rick van den Enden, William Bourland, David Patterson, Mark and Diane Littler for kindly providing photographs. This work was supported by the Research Foundation (FWO) Flanders.

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Diversity and Evolution of Algae

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