DELWICHE AND COOPER 2015 THE EVOLUTIONARY ORIGIN OF A TERRESTRIAL FLORA

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Current Biology
Review
The Evolutionary Origin of a Terrestrial Flora
Charles Francis Delwiche1,* and Endymion Dante Cooper2
1Cell Biology andMolecular Genetics and theMarylandAgricultural Experiment Station, University ofMaryland, College Park,MD20742, USA
2School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, UK
*Correspondence: Delwiche@umd.edu
http://dx.doi.org/10.1016/j.cub.2015.08.029
Life on Earth as we know it would not be possible without the evolution of plants, and without the transition of
plants to live on land. Land plants (also known as embryophytes) are a monophyletic lineage embedded
within the green algae. Green algae as a whole are among the oldest eukaryotic lineages documented in
the fossil record, and are well over a billion years old, while land plants are about 450–500 million years
old. Much of green algal diversification took place before the origin of land plants, and the land plants are
unambiguously members of a strictly freshwater lineage, the charophyte green algae. Contrary to single-
gene and morphological analyses, genome-scale phylogenetic analyses indicate the sister taxon of land
plants to be the Zygnematophyceae, a group of mostly unbranched filamentous or single-celled organisms.
Indeed, several charophyte green algae have historically been used as model systems for certain problems,
but often without a recognition of the specific phylogenetic relationships among land plants and (other) char-
ophyte green algae. Insight into the phylogenetic and genomic properties of charophyte green algae opens
up new opportunities to study key properties of land plants in closely related model. This review will outline
the transition from single-celled algae to modern-day land plants, and will highlight the bright promise study-
ing the charophyte green algae holds for better understanding plant evolution.
Introduction
Plants dominate the terrestrial environment. Remarkably, a sin-
gle lineage, referred to here as land plants, but more formally
called embryophytes, and sometimes treated as the Kingdom
Plantae [1], accounts for the vast majority of land cover,
biomass, and named biological diversity (among oxygenic pho-
totrophs) [2]. Marine environments are a different story, with a
diversity of oxygenic phototrophs largely unrelated to the terres-
trial flora; we refer here to eukaryotic phototrophs other than land
plants as ‘algae’, although land plants are phylogentically placed
deeply within the green algae. Algae (including land plants) su-
perficially appear to have diverse phylogenetic origins, but the
plastid lineage appears to bemonophyletic [3–5] (but see discus-
sion below), and the possession of a plastid (see Box 1 for a glos-
sary of terms) unites them in a natural grouping.
Two lineages of algae — the red and the green algae — have
ancient fossil records. Red algae account for some of the earliest
unambiguous eukaryotic fossils known, from �1200 Ma sedi-
ments [6]. The fossil record for green algae is more difficult to
interpret, although there are ancient acritarchs that could well
be attributed to green algae (but to several other clades as
well) in sediments�1500Ma [7], andmolecular phylogenetic an-
alyses suggest that red and green algae are of comparable age
[8,9]. It has been suggested that preservation bias favoring ma-
rine over freshwater sediments may account for the relatively
greater age of red algae in the fossil record [8,10], which would
resolve a paradox if red and green algae are indeed sister taxa.
Despite some popular images to the contrary, the link between
the marine and terrestrial floras is found in freshwater. Fresh-
water environments harbor a broad diversity of algae. Among
these are the charophyte green algae (also called basal strepto-
phytes), which are exclusively freshwater. The charophytes
constitute one of two great lineages of green algae, the other
Current Biology 25, R899–R
being the chlorophytes. Chlorophytes account for the bulk of
green algal diversity and are found in both freshwater andmarine
environments [11], but are relatively distantly related to land
plants. As noted above, green algae are extremely ancient eu-
karyotes, and the divergence between the charophyte and chlor-
ophyte lineages may be well over a billion years old [9]. The fact
that the ancestral habitat for charophyte algae was clearly in
freshwater provides strong evidence that the common ancestor
of land plants lived in freshwater as well. This idea is bolstered
by the observation that marine shoreline splash zones in high-
energy environments (i.e., where there are strong waves) are
generally barren, while less energetic shorelines have a very
specialized flora (e.g., mangroves and salt marshes).
Over 470 million years ago, during the Silurian or late Ordovi-
cian Period [12,13], a lineage of charophyte green algae under-
went an evolutionary transition that allowed it to remain hydrated
and reproduce while in full contact with the atmosphere, and
(eventually) to access subsurface water. In so doing, these
organisms gained access to atmospheric CO2 and sunlight unfil-
tered by water. They were probably not the first photosynthetic
organisms to occupy the terrestrial environment (Figure 1) [14],
but they diversified into the land plants that now occupy all but
the harshest terrestrial environments. They constitute the basis
for agriculture, as well as lumber, paper, plant fibers (cotton,
linen, flax, etc.), and other key industrial products. Their diverse
biochemistries give rise to secondarymetabolites that are crucial
pharmaceuticals, recreational drugs, and pesticides. Dead land
plants are the primary source not only of coal, but also of the
organic components of soil, which is the single largest reservoir
of stored carbon on Earth [15]. Thus, the origin of a terrestrial
flora was one of the most profound geobiological transitions in
the history of the planet [16–19], and established the basis for
the environment in which we live. Because land plants are a
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Box 1. Glossary of Terms.
Alternation of Generations: see Diplohaplontic life cycle.
Archaea: phylogenetically, one of the three great Domains of life, and long confused with Bacteria, they are prokaryotic in cellular
organization, but are a separate group with distinct molecular biology and biochemistries.
Bacteria: phylogenetically, one of the three great Domains of life. Bacteria are structurally prokaryotic, and account for themajority
of well characterized, cultured microorganisms.
Bryophyte: a form of classification referring to those land plants that have haploid-dominant alternation of generations. Bryo-
phytes may or may not be a monophyletic group; the major lineages are liverworts, mosses, and hornworts.
Charophyte or charophyte green alga: one of two great clades of green algae, the charophytes also include the land plants. We
follow the nomenclature of Hall and Delwiche [45], but see also Mattox and Stewart [47]. The group is named for the genus Chara,
which is also the basionym for the order Charales and the class Charophyceae S.Str. See ‘Streptophyte’.
Chlorophyte: used here to refer to one of the two great clades of green algae, the chlorophytes are found in bothmarine and fresh-
water environments, and have a diversity of life cycles. They are thought to have diverged from the charophytes over a billion years
ago.
Chloroplast: the plastid of green algae and land plants.
Corticating filaments: in Coleochaete, branches that grow from filaments adjoining a zygote and at maturity completely surround
the zygote with a layer of sterile jacket cells.
Cryptogam: a non-seed plant. The term refers to the ‘hidden reproduction’ in these plants.
Cyanobacteria:a lineage of bacteria typically capable of oxygenic photosynthesis. Green algae and plants carry out oxygenic
photosynthesis thanks to the chloroplast, an organelle that is an endosymbiotic cyanobacterium.
Diazotrophy: an organism that is capable of nitrogen fixation, or canmake use of atmospheric nitrogen to construct more complex
N-containing compounds.
Dinotom: a dinoflagellate with a diatom endosymbiont.
Diplohaplontic life cycle: also referred to as an ‘alternation of generations’, a life cycle that involves both multicellular haploid and
multicellular diploid phases; characteristic of land plants.
Embryophyte: used here synonymously with ‘land plant’, a monophyletic group of photosynthetic eukaryotes characterized by a
life cycle consisting of an alternation of multicellular haploid and multicellular diploid generations; the name refers to the multicel-
lular diploid embryo.
Endosymbiotic organelle: a cellular organelle that is derived from the permanent, intracellular symbiosis of a previously free-living
organism.
Eukaryote/Eukarya: one of the three great Domains of life, eukaryotes are often relatively large, and are characterized by a com-
plex endomembrane system including a nucleus that separates the genome from the cytoplasm of the cell. See Bacteria and
Archaea.
Gametophyte: the haploid, multicellular phase in the land plant life cycle.
Glaucocystophyte: one of three lineages of algae with primary plastids, glaucocystophytes have plastids that retain bacterium-
like peptidoglycan cell walls. Described species are freshwater organisms and are not rare, but they typically do not occur at high
densities.
Green algae: one of three lineages of algae with primary plastids, green algae are found in both marine and freshwater environ-
ments. They are divided into two great lineages (plus a few outgroups), the chlorophytes and the charophytes. Land plants are
placed phylogenetically within the charophyte green algae.
Gymnosperm: a form classification referring to vascular, seed plants that do not produce flowers.
Haplontic life cycle: a life cycle where the majority of the life cycle is spent in a haploid condition, and the only diploid phase is a
zygote.
Higher charophytes: the clade consisting of land plants, Zygnematophyceae, Charophyceae S.Str., and Coleochaetophyceae
(sensu Mattox and Stewart, 1984).
Kleptoplasty: photosynthesis that relies on the acquisition and retention of plastids from prey. Plastids are not passed on to prog-
eny, or at least not consistently so. Kleptoplastic organisms must periodically capture prey to refresh their plastids.
Land plant: used here synonymously with ‘embryophyte’.
Meristem: a localized region of cell division that gives rise to new tissue.
Mesenchyme: the central region of a complex structure, such as the interior of a leaf, typically occupied by loosely connected
tissue.
Mycorrhyzae: a fungus–root symbiosis. These occur in almost all land plants, and are thought to play an important role in plant
nutrient acquisition, while the fungus benefits from access to fixed carbon (photosynthate).
Nucleoklepty: acquisition from prey and retention of functional nuclei which are used to maintain chloroplasts also acquired from
prey. To date only documented in Mesodinium rubrum (= Myrionecta rubra). See Kleptoplasty.
(Continued on next page)
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Current Biology
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Plant: used here to refer to the Archaeplastida of Adl et al. [1], the term is also used colloquially to refer to the land plants or to any
photosynthetic organism.
Plastid: an endosymbiotic organelle derived from a cyanobacterium. Many, but not all, are photosynthetic. The most familiar
plastid is the chloroplast of plants.
Primary plastid: an endosymbiotic organelle derived directly from a cyanobacterium, normally surrounded by two membranes.
Red algae: one of three lineages of algae with primary plastids, red algae occur in both marine and freshwater environments, but
are more common and display greater diversity in marine environments. Red algae entirely lack flagella, and many species have
very complex life cycles.
Secondary plastid: an endosymbiotic organelle derived via symbiosis of two eukaryotes, one already possessing plastids.
Sporophyte: the diploid, multicellular phase in the land plant life cycle.
Streptophyte: used here as synonymously with ‘charophyte’.
Stroma: the interior compartment of a plastid, equivalent to the cytoplasm of a cyanobacterium, and the location of plastid gene
expression.
Streptophyte: a grouping consisting of land plants, the Charophyceae S.Str., and some other taxa, and sometimes used synon-
ymously with charophyte green algae. The circumscription of streptophytes remains uncertain. Originally proposed by Jeffrey in
1967 [60] to consist of Charophyceae S.Str. and embryophytes, Bremer [111] noted that this clade probably includes some
additional taxa, but did not specify what those would be. The term has often been used to refer to the entire Charophyceae sensu
Mattox and Stewart [47] plus embryophytes. Karol et al. [59] proposed that Charophyceae S.Str. were the sister taxon to embryo-
phytes, in which case the Streptophyta sensu stricto would be that clade (Charophyceae S.Str. + embryophytes), but several
recent analyses, including from our own lab [54,64,69], have found a different topology, with zygnematophytes as the sister taxon
to embryophytes, and Coleochaetophyceae and Charophyceae S.Str. somewhat more distant. If this topology stands the test of
time, then Jeffery’s streptophytes would include embryophytes and Mattox and Stewart’s Zygnematales, Coleochaetales, and
Charales (a group sometimes called the ‘higher charophytes’).
Subaerial: living under conditions of continual exposure to the air.
Zooxanthellae: symbiotic algae — typically the dinoflagellate Symbiodinium — that live in association with corals, giant clams,
and other marine animals.
Box 1. Continued
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monophyletic group, the peculiarities of this lineage are respon-
sible for many of the properties of the terrestrial flora (the several
independent colonizations of land by cyanobacteria and algae
not withstanding), and understanding the early history and
biology of land plants and their close relatives, the charophytes,
can provide valuable insights into why land plants are they way
they are.
Oxygenic Photosynthesis and the Origin of Eukaryotic
Phototrophs
The essence of plant biology is oxygenic photosynthesis.
Oxygenic photosynthesis makes use of light energy to remove
electrons (and in the process the hydrogen atoms) from water,
releasing oxygen gas (O2), and using those energetic electrons
to produce ATP and NADPH2, which are in turn used for the con-
version of CO2 into more complex organic molecules. Several
forms of photosynthesis are found amongBacteria, but oxygenic
photosynthesis has substantial energetic advantages over other
forms of photosynthesis, and it is unique to one lineage of Bac-
teria, the cyanobacteria [20,21]. Some eukaryotes (which we
refer to as algae or land plants) are photosynthetic because of
the endosymbiotic incorporation of a cyanobacterium (known
as a plastid, or chloroplast in the green lineage). The cyanobac-
terium, although formerly a free-living organism, is now a fully
incorporated part of the cell, even for the purposes of gene
expression (reviewed in [5,22,23]).
The three lineages of algae with plastids directly incorporated
in the eukaryotic cell are glaucocystophytes, red algae, and
green algae. Glaucocystophytes are obscure freshwater
Current Biology 25, R899–R
organisms, and are not otherwise important, but they are note-
worthy because their plastids retain a peptidoglycan wall similar
to that of cyanobacteria [24]. Prior to the understanding that
plastids are endosymbiotic organelles, these organisms caused
great confusion because it wasunclear whether the structures
were plastids or endosymbiotic cyanobacteria. Now it is under-
stood that these are one and the same, and the glaucocysto-
phytes no longer seem paradoxical. Unlike glaucocystophytes,
red algae and green algae are both species-rich, diverse groups
inhabiting a wide range of environments.
In these three lineages, the plastids are surrounded by two
membranes, and retain small genomes that are transcribed
and translated separately from the nuclear genome. These ge-
nomes are quite small, ranging from 154.5 kb in Arabidopsis,
to 191 kb in the red alga Porphyra, to 420 kb in the chlorophyte
green alga Volvox, and compared to genome sizes in free-living
cyanobacteria that range from 1643 to 12,073 kb [25,26]. This
means that a typical plastid genome is on the order of 5% the
size of a free-living cyanobacterial genome, and consequently
that the organelles are completely dependent upon the host
cell to encode, transcribe, and translate most proteins, as well
as to carry out many other metabolic processes [27]. Thus, two
formerly independent cells, one eukaryotic and the other pro-
karyotic in organization, have become integrated into a single
organism. The host cell is also highly modified from its ancestor,
and is nearly as dependent upon the plastid as the organelle is on
its host. The merging of the two organisms was accompanied by
massive reduction in the genome size and content of one partner
(the organelle), and the large scale integration of genetic material
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Figure 1. Modern examples of non-
charophyte terrestrial vegetation[14].
(A) Lichens living on an exposed rock surface.
Although lichens are important early colonists in
modern terrestrial ecosystems, they do not have
an extensive fossil record, and appear not to
have been present in the Devonian Period [2]
(near Juneau, Alaska). (B) Endolithic green algae
(Cascade Mountains, Washington). (C) Cyano-
bacteria-dominated microbial community leading
to weakly consolidated sediments (Death Valley,
California). (D) A more fully developed microbial
mat, including cyanobacteria, diverse Treboux-
iophycean green algae, and diatoms (Mojave
Desert, California). (E) Trentepohlia mat (Domin-
ican Republic). (F) Nitella growing in shallow water
with emergent shoots (Zion National Park, Utah).
Image F by E.D.C., all others by C.F.D.
Current Biology
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into the other (the host), resulting in a dramatic alteration in the
biology of the host cell [28–30]. The evolutionary consequences
of this integration appear to have been profound and wide-
ranging.
Because land plants are derived from within the charophytes,
they have inherited the same general cellular organization,
including primary plastids surrounded by twomembranes. There
are, however, a number of other lineages of algae that acquired
their plastids by ingesting a second eukaryote, either a red or a
green alga, and retaining the plastid [23,29,31]. One obvious
clue to such ‘secondary’ plastids is that they are surrounded
by more than two membranes. Organisms with secondary plas-
tids include (to name a few) kelps and other brown algae, which
make up enormous offshore ‘forests’ in temperate and polar
marine waters; diatoms, which have beautiful silica cell walls
and are among the most important carbon-fixing organisms on
the planet; dinoflagellates, which include the coral symbionts
known as ‘zooxanthellae’ (Symbiodinium spp.) and whose loss,
referred to as ‘coral bleaching’, is a major environmental
concern; and euglenoids, which are relatives of trypanosomes
(best known as parasites, such as the causative agent of
sleeping sickness) and are important in highly nutrient rich fresh-
water environments.
In contrast to the primary plastids of red, green, and glauco-
cystophyte algae, all of these have plastids that are surrounded
by more than two membranes, and a few (notably cryptomo-
nads, with red plastids, and chlorarachnophytes, with green
plastids) even retain tiny, highly reduced eukaryotic nuclei in
the same compartment as the primary plastid [30]. Although
the situation is most obvious when there are two eukaryotic
nuclei present, a number of lines of evidence ranging from cell
structure and biochemistry to DNA sequence analyses have
demonstrated unambiguously that all lineages of algae other
than the red, green, and glaucocystophyte lineages acquired
their plastids through secondary endosymbiosis [22].
There is evidence of relatively recent endosymbiotic organelle
acquisition, most notably in Paulinella, an amoeba classified
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in the Rhizaria, which appears to repre-
sent an independent acquisition of an
endosymbiotic cyanobacterium [32,33].
Because this organelle is clearly distinct
from the plastids of red, green, and glau-
cocystophyte algae, we do not refer to it here as a ‘plastid’, but
the distinction is phylogenetic, not functional. There are also ex-
amples of ‘tertiary’ plastids, where an organism with secondary
plastids has been acquired as an endosymbiont by another
eukaryote. One clear example is found in the ‘dinotoms’
[34,35], which are dinoflagellates with endosymbiotic diatoms;
they have two easily recognizable eukaryotic nuclei, and six
membranes between the cytoplasm of the host and the plastid
stroma. Tertiary plastids are also found in the brevetoxin-pro-
ducing dinoflagellateKarenia brevis and its close relatives, which
have pigmentation that resembles another group of algae, the
haptophytes, rather than more typical dinoflagellates. Their
plastid ultrastructure also resembles that of haptophytes, and
molecular phylogenetic investigations have demonstrated that
the plastid was acquired from haptophytes [36].
Some interactions are not symbiotic, but provide insights into
how plastids may be acquired. For example, the ciliate Mesodi-
nium rubrum (also known as Myronecta rubra) is functionally
photosynthetic, but has plastids that resemble those of crypto-
monads, and to be cultured must be given a small but steady
supply of cryptomonad prey. It has been shown to ingest the
cryptomonad and selectively retain both the nucleus and plastid
from the prey; the nucleus is maintained in a transcriptionally
active state, and appears to play a key role in maintaining plastid
function [37]. Remarkably, the dinoflagellate Dinophysis also re-
lies on cryptomonad plastids, but seems to be unable to make
direct use of the cryptomonad as prey. Rather, Dinophysis in-
gests the cellular contents ofM. rubrum, and retains the plastids,
which M. rubrum had itself acquired from its cryptomonad prey
[38–41]. This is kleptoplasty, not symbiosis, but it is an example
of a quaternary interaction; a cyanobacterium, symbiotic in a red
alga, became a secondary endosymbiont in the cryptomonad, a
tertiary kleptoplastid (accompanied by a cryptomonad nucleus)
in M. rubrum, and a quaternary kleptoplastid in Dinophysis.
Clearly it can be helpful to have a plastid.
Plastids can also be lost. A particularly dynamic example
has been documented in the freshwater/soil dinoflagellate
Figure 2. Key organisms on the charophyte
lineage.
(A) Klebsormidium nitens, from cultured material.
(B) Nitella hyalina, from cultured material, showing
antheridia (orange) and developing zygotes. (C)
Coleochaete pulvinata from Lake Tomohawk,
Oneida Co., Wisconsin, showing its branched
filamentous structure and zygotes with corticating
filaments growing toward them. (D) Two filamen-
tous Zygnematophyceae, Spirogyra (spiral chlo-
roplast) and Mougeotia (flat chloroplast), from
nature. Note basal branch in Mougeotia (white
arrow) and holdfasts (black arrows). Mougeotia
often has a basal branch, but the filaments do not
otherwise branch, although the formation of
conjugation papillae involves branch-like selective
expansion of the cell wall. (E)The hornworts
Phaeoceros and Anthoceros along with a single
liverwort, Fossombronia (arrow), showing sporo-
phytes. Found growing on a roadside embank-
ment near Big Sur, Monterey Co., California. (F) A
bog in Oneida County, Wisconsin, showing the
dominance of embryophytes in the terrestrial
environment. Although not visible in the photo-
graph, the bog is a floating mass of Sphagnum, a
moss. Prominent are Eriophorum, ‘Cotton Grass’,
a monocot angiosperm, and Larix, ‘Tamarack’, a
gymnosperm in the Pinaceae. All images by C.F.D.
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Esoptrodinium, which is predatory but does retain plastids in
some, but not all, strains [42]. It appears to be an example of
plastid loss occurring in amodern population of cells. Somewhat
more difficult to document is the functional conversion of plas-
tids to other roles. A number of organisms have membrane-
bound pigment- or lipid-filled bodies called eyespots that are
thought to help with phototaxis by shading the light-sensitive re-
ceptors in the flagellar base, or perhaps serve other functions. It
has long been thought that in at least some cases these are
derived from plastids, but because they have not been shown
to contain an organellar genome, it is difficult to demonstrate
the origin of the organelle. Recent work has shown that dinofla-
gellates with diatom endosymbionts have genes that can be
attributed to the native dinoflagellate plastid, and whose gene
product appears to be targeted to an organelle [34]. Although
the target for these proteins has not been definitively shown to
be the eyespot, this is a substantial step toward testing the
long-standing hypothesis that at least some eyespots are
remnant plastids.
Genome-scale analyses of gene contents in a variety of sec-
ondary endosymbiotic systems are painting a complex picture
of genomic integration and gene transfer [29,30,43]. Under-
standing the genomic turbulence that accompanies establish-
ment of endosymbioses helps us understand the molecular
genetic background for the massive radiation of photosynthetic
eukaryotes, first in aquatic systems, and subsequently on land.
An intriguing hypothesis places the origin of plastids in fresh
water, and posits that marine algae are, in fact, only secondarily
marine organisms [44]. In support of this hypothesis is the fact
that all known charophyte green algae are freshwater organisms.
Nevertheless, they constitute just one of the two great, and
certainly ancient, lineages of green algae [9,45]. The second
great lineage, the chlorophytes, is abundant in fresh water and
inhabits a range of ecosystems, but has its greatest diversity in
marine waters. Glaucocystophytes and many of the basal
branches of both green and red algae are freshwater organisms.
Current Biology 25, R899–R
A freshwater origin for the primary-plastid lineages might
partially explain the abundance of secondary-plastid lineages
in marine environments [46], if the host cell were better adapted
to the marine environment than the symbiont. It might also
explain the complete lack of motile cells in red algae, which is
peculiar for amarine organism, and largely keeps them restricted
to rocky shorelines.
The Charophyte Lineage
Although they are not on the whole particularly species-rich,
charophyte green algae do display considerable morphological
diversity (Figure 2) [1,47]. The charophytes are almost exclu-
sively freshwater, and all have a haplontic (haploid-dominated)
life cycle. The term Charophyceae was used by Mattox and
Stewart in a 1984 taxonomic review [47], based largely on ultra-
structural data and lacking any formal phylogenetic analysis, to
refer to a lineage of green algae consisting of what we call here
the Chlorokybophyceae, Klebsormidiophyceae, Zygnemato-
phyceae, Coleochaetophyceae, and Charophyceae S.Str., but
silent on the placement of embryophytes (Mattox and Stewart
referred to these groups as orders; we refer to them here as clas-
ses to better accommodate their known relationship to land
plants). The relationship of Mesostigma to the group was noted
by Melkonian [48–50]. We refer here to Mattox and Stewart’s
orders as Zygnematophyceae, Coleochaetophyceae, and Char-
ophyceae S.Str. (as distinct from Charophyceae sensu Mattox
and Stewart, which are referred to here as the ‘‘Charophyte
Green Algae’’).
Mesostigma viride is an asymmetrical, scaly green flagellate,
which Melkonian insightfully allied with the charophytes on the
basis of its flagellar base ultrastructure [51]. Despite some con-
tradictory evidence, M. viride does appear to be a member of
the charophyte lineage [48,52–54], and to date has no known
close relatives. We treat it here as belonging to its own Class,
the Mesostigmatophyceae. Chlorokybus atmophyticus, like
M. viride, is a monotypic lineage and, like Mesostigma, we treat
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A B
C
D F G H I
E
Land plants Spirogyra Nitella ChlorkybusColeochaete Klebsormidium Mesostigma
Zygnemato-
phyceae
Charophyceae 
S.Str.
Chlorokybo-
phyceae
Coleochaeto-
phyceae
Klebsormidio-
phyceae
Mesostigmato-
phyceae
Land plants
Desmidiales
Zygnematales
Charales ChlorokybalesColeochaetales Klebsormidiales Mesostigmatales
Chlorophytes
Current Biology
Figure 3. Relationships among charophytes
showing the habitats in which they typically
occur.
All are freshwater organisms, and several are
semi-terrestrial organisms. The land plants have
distinctive adaptations to the terrestrial environ-
ment, but many charophytes occur in shallow or
transient water, and some are highly tolerant of
desiccation. Below the photographs showing
members of key lineages of charophytes (A–I) is a
schematic diagram showing their organization
superimposed on a hypothetical landscape with a
phylogenetic tree following the topology that
currently has strongest support [63,64,69].
Despite the diversity of morphologies, several
ancestral nodes are likely reconstructed as a
branched filament as shown. Coleochaete pulvi-
nata is one good modern analog, but a better one
might be the enigmatic alga Awadhiella, which has
been reported only once, from a pond in India
[112]; it is a branched filament with Coleochaete-
like hairs, but Spirogyra-like naked zygotes. (A)
Galanthis nivalis, ‘snow drops’, a land plant in the
flowering plants. (B)Micrasterias sp., a ‘desmid’ in
the Zygnematophyceae. (C) Spirogyra sp., a fila-
mentous Zygnematophyceae. (D) Coleochaete
orbicularis, a discoidal Coleochaetophyceae. (E)
Coleochaete pulvinata, a filamentous Coleochae-
tophyceae. (F) Nitella hyalina, a member of the
Charophyceae S.Str. (G) Klebsormidium nitens, a
member of the Klebsormidiophyceae. (H) Chlor-
okybus atmophyticus, the sole known member of
the Chlorokybophyceae; it grows as small packets
on damp soil. (I)Mesostigma viride, the sole known
member of the Mesostigmatophyceae; it occurs in
ponds and lakes but has also been reported iso-
lated from soil. All images by C.F.D.
Current Biology
Review
it here as belonging to its own Class, the Chlorokybophyceae,
and consists of relatively undifferentiated packets of cells in a
commonmucilage and cell-wall remnants. Some analyses place
the Mesostigmatophyceae and Chlorokybophyceae as sister
taxa, but the large sequence divergence and lack of morpholog-
ical synapomorphies argues for retaining both classes as
distinct. The Klebsormidiophyceae [45] includes the genera
Klebsormidium and Entransia, both of which are simple filaments
found in shallow water or (particularly for some species of Kleb-
sormidium) on damp walls and surfaces such as the splash zone
of drinking fountains and bird baths [55]. Klebsormidium is com-
mon, and it is the first charophyte for which a draft genome
sequence has been published [56]. Sexual reproduction is either
unknown or poorly characterized for any of the three basal line-
ages of charophyte green algae, although Klebsormidium can be
fairly easily induced to producezoospores [57].
The three remaining lineages of charophyte green algae (Char-
ophyceae S.Str., equivalent to the Charales of Mattox and Stew-
art 1984; Coleochaetophyceae; and Zygnematophyceae), along
with land plants, constitute a monophyletic group (the ‘higher
charophytes’), but the branching order among them remains
somewhat controversial [58]. One of us (CFD) participated in
an analysis [59] that placed the Charophyceae S.Str. (i.e., the
stoneworts Chara andNitella and their close relatives) as the sis-
ter taxon to land plants, and seemed to validate the original
concept of the Streptophyta as a clade consisting of Charophy-
ceae S.Str. and land plants [60,61]. However, even at the time,
analyses of plastid-genome data supported an alternative topol-
ogy, with Zygnematophyceae sister to land plants, and with the
R904 Current Biology 25, R899–R910, October 5, 2015 ª2015 Elsevi
Coleochaetophyceae and Charophyceae S.Str. more distantly
related [62,63]. Our own, more recent analyses of large datasets
derived from high-throughput transcriptome sequencing sup-
port this topology [64] (an alternative erroneous topology [65]
was marred by contaminant sequences [66,67]). The Karol
et al. (2001) topology seems largely to have disappeared from
genome-scale analyses [68,69], although it has considerable
appeal because of the striking similarity between Charophyceae
S.Str. and land plants, both on the basis of gross morphology
and of cellular structure.
The topology placing the Zygnematophyceae sister to land
plants is now seen in most genome-scale analyses [64,70],
but is difficult to reconcile with the simple morphology of
Zygnematophyceae, and the more plant-like morphology and
development of other contenders (i.e., Charophyceae S.Str.
and Coleochaetophyceae). It is, however, important to bear in
mind that genome-scale analyses have only been feasible for a
short time, and even now the taxon sampling for multi-gene
datasets is rather poor. With denser taxon sampling and careful
analyses controlling for systematic error another topology may
emerge from the data. Nonetheless, if we assume that the topol-
ogy with Zygnematophyceae sister to land plants is correct and
interpret organismal morphology on that basis, some interesting
observations emerge (Figure 3). Whether Zygnematophyceae or
Charophyceae S.Str. is the sister taxon to land plants, the ances-
tral state for the higher charophyte lineages would be a branched
filament, likely with oogamous reproduction. It would not have
been large, and among living organisms, filamentous species
such as Coleochaete pulvinata would probably be the closest
er Ltd All rights reserved
Current Biology
Review
analog. Each of the lineages then shows specialization from that
form. The Charophyceae S.Str., which are generally found in
relatively permanent and deep water, have evolved a larger
size, elaborate thallus organization, and sterile jacket cells on
both oogonia and antheridia. They are not organized into plant-
like parenchyma, but instead are composed of branching fila-
ments. The apical meristem is a single cell that always divides
in the same plane. In the Coleochaetophyceae, the thalli remain
generally small, but have evolved compact thalli closely
appressed to the substrate, sheathed hairs of uncertain function,
and a life cycle involving post-meiotic cell division [71,72]. Vege-
tative cell division in these organisms is limited to the terminal cell
in each filament, effectively creating a marginal meristem in the
discoidal species. Most interesting are the Zygnematophyceae.
If they are indeed closely allied with the land plants, how can their
seemingly simple structure be reconciled with the complex tis-
sues of land plants? The answer perhaps lies in adaptation to
the hydrological gradient that separates land plants from their
relatives.
To understand the evolution of terrestrial organisms from
aquatic organisms, it is important to remember that life remains
a fundamentally aquatic process. The ancestral habitat for all of
the charophyte lineages is almost certainly freshwater, although
a few of these groups have members that have become second-
arily adapted to brackish or alkaline waters, and many of them
have semi-terrestrial or ‘subaerial’ members. Because rainfall
is essentially distilled water, terrestrial environments are intermit-
tently available to freshwater organisms. However, occupancy of
intermittently wet habitats requires dormancy, desiccation-toler-
ance, or desiccation-resistance mechanisms that allow them to
survive between wetting events. Temporal factors are also
important — if an organism can only survive desiccation in a
specialized dormant state, the hydrated environment must
persist long enough for the organism to emerge, grow, and return
to its dormant state. Conceptualized in this way, all plants inhabit
a hydrological gradient, their location on which is determined by
their ability to maintain hydrated conditions under varying de-
grees of desiccation pressure. Indeed, given that many fresh-
water environments such as pools and streams are subject to
occasional or periodic drying, it is unsurprising that many charo-
phytes (and other lineages of freshwater algae) have terrestrial
members.
It is plausible that the reduced filamentous and unicellular
structures of the Zygnematophyceae reflect adaptation to life
shifted to the drier end of the hydrological gradient. The Zygne-
matophyceae are quite diverse, with around 10,000 named
species, and a correspondingly complex phylogeny, but the
deepest branches are multicellular filaments, with the unicellular
desmids being more derived lineages (some of which have in
turn secondarily reverted to a filamentous form) [73]. Although
the filamentous Zygnematophyceae, such as Zygnema, Spiro-
gyra, and Mougeotia, are generally thought of as unbranched,
they do have some branch-like features, most notably a holdfast
and conjugation papillae (Figure 1). The deposition of callose at
the tip of developing rhizoids in Spirogyra suggests develop-
mental similarity to pollen tubes and root hairs in flowering plants
[74]. Callose is broadly distributed among the charophytes
and other algae, and seems often to be associated with tip
growth [75–77].
Current Biology 25, R899–R
This morphologically reductive evolution evident in the Zygne-
matophyceae is consistent with the hypothesis that Zygnemato-
phyceae are structurally reduced from amore complex ancestor.
Structural simplicity may have been advantageous in shallow
and transient aquatic habitats because of the short time required
for a small organism to grow, and because even a thin film of
water can provide enough room for coverage.Many species pro-
duce copious quantities of mucilage, which can help retain water
close to the cell. Another of the most striking properties of the
Zygnematophyceae is their complete lack of flagella, and use
of gliding motility during sexual reproduction. This has long
been considered an adaptive response to life on land. Working
from this premise, Stebbins and Hill [78] postulated a wholly
extinct lineage of algae, not represented in the fossil record,
that gave rise to both extant charophyte algae and land plants.
This concept seems unjustified, but the underlying premise,
that the characteristic features of the Zygnematophyceae reflect
adaptations to terrestrial and semi-terrestrial environments, is
sound.
The emergence of progressively less expensive, high
throughput DNA sequencing techniques, which has accelerated
greatly within the last decade, has opened up the possibility of
genome-scale analyses in non-model organisms. Such studies
have revealed that the genomes of diverse charophyte algae
are very plant-like [56,79], consistent with and expanding on
the earlier ultrastructural, biochemical, and single-gene phyloge-
netic evidence for a common ancestry [80]. Genomic studies
haverevealed properties that were difficult to study with other
methods. For example, several plant hormone systems previ-
ously thought to be characteristic of land plants have been found
in charophyte algae, largely as a result of high-throughput
sequencing [56,81,82]. Recent work has demonstrated that the
system for the plant hormone ethylene (which is often associated
with stress responses) is present in the Spirogyra genome
[68,70], that it is homologous and functionally similar to the
land plant system, and that exposure to ethylene causes cell
elongation in Spirogyra [83]. In the model moss Physcomitrella
patens, ethylene signaling plays a major role in regulating growth
of the protonemawhen it is submerged (the protonema being the
early, filamentous developmental stage of the gametophyte).
When submerged, Physcomitrella exhibits an ‘escape’ response
in which the protonemal filaments become less densely packed,
peripheral elongation is enhanced, and gametophores develop
preferentially at the periphery [84,85].
This response may help the developing gametophyte regulate
its response to the depth of the water in which it is growing, and it
is plausible that in Spirogyra the hormone plays a similar role. In
fact, because it is a hydrophobic gas, ethylene is well suited to
signaling submersion; because it is five orders of magnitude
less soluble in water than in air [86], submerged filaments pro-
ducing ethylene will rapidly build up a high concentration, which
would rapidly dissipate once the filaments come into contact
with the air. This system then appears to have been coopted
for more diverse stress and developmental responses in the
land plant lineage [87].
Thus, many of the adaptations displayed by land plants seem
to have roots among the charophyte algae, probably because
the freshwater environment is so closely intertwined with the
terrestrial environment. If the Zygnematophyceae are indeed
910, October 5, 2015 ª2015 Elsevier Ltd All rights reserved R905
Current Biology
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the closest living relatives of land plants, then they present a
spectacular example of two sister lineages adapting to much
the same environmental pressures in dramatically different
ways. The Zygnematophyceae remained small (or became
smaller), and evolved to exploit transient hydration, shallow
waters, and interstitial moisture, while the land plants evolved
complex plant bodies, mechanisms such as cuticles and (even-
tually) stomata that permitted regulation of water loss, and
specialized structures for locating and extracting moisture from
well below the surface of the soil. The fascinating, and enigmatic,
question remains: what were the drivers that propelled one line-
age far enough along the hydrological gradient that it became so
spectacularly dominant in most terrestrial ecosystems?
Wet-to-dry Transitions
Key adaptations in the origin of a terrestrial flora have been
examined in detail elsewhere [11,12,80,88]. Some of the most
fundamental adaptations are biochemical, and involve photo-
synthesis at increased CO2 concentrations. Because the
concentration of CO2 in water is typically less than 2% its con-
centration in the overlying atmosphere [89], there is a substantial
benefit to performing photosynthesis in direct contact with the
atmosphere, if this is possible without lethal dehydration. The
higher concentration of CO2 permits relatively rapid photosyn-
thesis, but with the penalty of increased intracellular concentra-
tion of oxygen, which means increased photorespiration and
formation of reactive oxygen species (ROS). Another mixed
blessing of the terrestrial environment is the increased photon
flux, which facilitates rapid photosynthesis, but at the cost of
increased photodamage. This is exacerbated by substantially
higher exposure to ultraviolet light (UV) on land [90–92]. It has
been proposed that one of the key geobiological prerequisites
to the colonization of the land was the formation of an ozone
layer because it led to a reduction in surface UV; an ozone layer
could develop only after oxygenic photosynthesis had converted
the atmosphere to oxidizing [93], although the importance of
ozone for the colonization of the land has been questioned [94].
Another key characteristic of land plants is their symbiotic as-
sociation with fungi, which play a vital role in nutrient acquisition,
and may also facilitate water uptake. Although best character-
ized in vascular plants, mycorrhizae are found in association
with almost all land plants [95], and can be identified deep in
the fossil record [96]. It seems likely that symbiosis with mem-
bers of the Glomales (arbuscular mycorrhizae) either coincided
with or arose soon after the transition to the land [97]. To date
there is no evidence of fungal symbioses in charophyte green
algae [98,99], which is consistent with the hypothesis that such
symbioses are characteristic of land plants, and may be one of
the traits leading to the dominance of the group.
The interaction between root and mycorrhiza is largely medi-
ated by strigolactones — plant hormones that are associated
with root growth and development [11]. Interestingly, despite
the lack of obvious mycorrhizal symbionts, there is evidence
that the charophytes Chara and Nitella (Charophyceae S.Str.),
but not Spirogyra (Zygnematophyceae) or Coleochaete (Coleo-
chaetophyceae) produce and respond to strigolactones, plant
hormones associated with plant–fungus signaling inmycorrhizae
[82]. The most obvious explanation for this would be that strigo-
lactone signaling played a developmental role in the ancestral
R906 Current Biology 25, R899–R910, October 5, 2015 ª2015 Elsevi
charophytes that was unrelated to mycorrhizae, and was later
coopted for mycorrhizal signaling early in the evolution of land
plants. Obviously, if this is the case, either the strigolactone hor-
mone system was lost from both Zygnematophyceae and
Coleochaetophyceae, or else the Karol et al. (2001) [59] phylog-
eny, in which the Charophyceae S.Str. are sister to land plants, is
correct. Whereas reductive evolution characterizes the Zygne-
matophyceae, no such process has been proposed for the
Coleochaetophyceae, and indeed neither the genome of Coleo-
chaete nor that of Spirogyra appears to have reduced coding
potential compared with Nitella [83].
It is striking that in mycorrhizae, the symbiotic partners have
retained distinct identities over hundreds of millions of years.
This observation contrasts with plastids and mitochondria,
where the symbionts have become inextricably intertwined
with the host cell, to the extent that most genetic functions of
the symbiont are carried out by the host. This may stem in part
from the nature of the symbiosis — in order to provide their ser-
vices to the plant, mycorrhizae must be in direct contact with the
environment. Plastids, by contrast, perform a variety of functions
including photosynthesis, isoprenoid, fatty acid, and amino acid
biosynthesis, but none of these requires direct contact with the
environment. In mycorrhizae, gene transfer to the host would
remove the transferred gene from the regulatory context of the
mycorrhiza, which would necessarily make any environmental
responses indirect, and presumably adversely affect fitness.
Raven has looked at this question in detail [100], and postulated
that nitrogen fixation, symbiont biodiversity, and ectosymbiosis
create a situation where ‘cheaters’ are a constant risk, andwhere
strict inheritance of the symbiont would be disadvantageous,
thus prohibiting genetic integration into the host. Mycorrhizae
appear to be characteristic of the land plant lineage, but it is un-
certain whether the mycorrhizal association with land plants
coincided with, or occurred subsequent to, the origin of land
plants.
It is often implicitly assumed that multicellularity is necessary
for structural complexity, but the Charophyceae S.Str., alongwith structurally complex chlorophyte green algae such as
Acetabularia,Halimeda, andCaulerpa clearly show that multicel-
lularity is not a prerequisite for complexity per se. Indeed, in
Caulerpa differentiation of leaf-like ‘assimilators’ and a root-like
‘stolon’ and ‘rhizoids’ seem to be achieved by transcript parti-
tioning in the absence of cell division [101]. However, at least
two structural properties of plants seem difficult to achieve
without three-dimensional, multicellular tissue. The first of these
is the complex hydraulic system that enables plants to maintain
rapid gas exchange in a desiccating environment. Acquisition
of CO2 is essential, and the efficient hydraulic system found in
land plants permits them to maintain a gas-filled mesenchyme
that is fully saturated with water, but able to exchange CO2
and O2 with the atmosphere even when the partial pressure
of water in the atmosphere is quite low. The cells in the interior
of a leaf exist in an environment that is water-saturated, but
has a far higher concentration of CO2 than is typically available
in water. A few land plants, such as mosses, leafy liverworts,
and filmy ferns have their photosynthetic tissue directly exposed
to the atmosphere, but they are able to be physiologically active
only when the surrounding environment is near saturation with
water.
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Current Biology
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This hydraulic system is poorly developed in the earliest
diverging lineages of land plants, but spectacularly successful
in, for example, the towering coast redwood (Sequoia sempervi-
rens). For the most part bryophytes (liverworts, mosses, and
hornworts) cope with life in a desiccating environment by inhab-
iting sheltered sites in humid environments or with moist sub-
strates. Those that survive in drier environments do so through
their capacity to survive near complete loss of cellular water
and rapidly resume normal metabolism when rehydrated, thus
allowing them to make use of transiently available water. The
earliest land plants, like extant bryophytes, presumably lived
close to the ground in moist habitats, but cells specialized for
water transport soon arose, and vascular tissues were well
developed by the time the Rhynie Chert formed in the Early
Devonian, about 410 million years ago [102,103]. The hydraulic
system of extant vascular plants consists of regulated pores
for gas exchange (stomata), an impermeable coating (cuticle)
to control water loss to the atmosphere, a network of pipes
(xylem) to distribute water, and an extensive root system to
extract water from, often deep, underground.
This water management and distribution system enables
plants to develop the second key structural property, large
body size. A great advantage of multicellularity is that it permits
strength. Unlike animals, plants are largely free from the need to
move. That means that they are free from the need to compro-
mise between exo- and endo-skeletons, and in plants nearly all
tissues can play a structural role. Multicellularity permits plants
to grow tall, and indeed, competition for height (presumably to
acquire light) is seen almost immediately following the appear-
ance of land plants in the fossil record [12,104].
In addition to biochemical and structural adaptations, land
plants underwent a dramatic shift in their life history as an adap-
tation to the terrestrial environment. As already mentioned, land
plants are also known as ‘embryophytes’; this term refers to their
peculiar life history, which involves an alternation of multicellular
haploid (gametophyte) and multicellular diploid (sporophyte)
generations. The anatomical and morphological features of the
gametophyte and sporophyte are usually considered non-
homologous. Intriguingly, it has been shown that simply knock-
ing out a transcription factor is sufficient to transfer the entire
gametophyte developmental program to the diploid generation
in the moss Physcomitrella patens. The remarkable ease with
which sporophytes can be induced to develop gametophyte
structure and function illustrates the fact that both are morpho-
logical expressions of the same genome, and calls into question
the assumption that completely independent developmental
programs must play out in each [105].
The embryo is the first multicellular developmental stage of the
diploid phase, and it is unique within the greater charophyte line-
age to land plants. Notwithstanding complex shifts in ploidy in
some charophyte algae (e.g. Coleochaete scutata, Spirogyra
and Sirogonium species, andClosterium ehrenbergii), a true em-
bryo is unknown in charophyte algae [106]. So far as is known, all
are haplontic, meaning that all stages of development consist of
haploid cells with the sole exception of the zygote. Following
fertilization the zygote often enters into a resting stage, surviving
winter or drought conditions, but when it germinates the first cell
division is always meiosis, restoring the haploid condition.
Despite generations of debate concerning the origin of the land
Current Biology 25, R899–R
plant life cycle [72,107], phylogenetic analyses have made it
abundantly clear that land plants are deeply embedded within
a haplontic lineage [61,64,69,108]). It is therefore nearly certain
that it arose through intercalation of a multicellular diploid within
an existing haplontic life cycle, probably to amplify the results of
any successful fertilization, which must have been a rare accom-
plishment for early land plants [72]. This also explains a peculiar-
ity of the plant life cycle — although the sporophyte is dominant
(i.e., the largest and longest-lived phase) in most plant groups,
the sporophyte always initially develops dependent upon the
gametophyte of the previous generation. Only the gametophyte
(in the ‘cryptogams’, i.e., ferns and their allies, lycophytes,
mosses, liverworts, and hornworts) can grow independently
from its germination. This is peculiar, but makes sense once
one understands that the sporophyte is a relatively recent
innovation.
The Promise of Charophyte Green Algae
Despite their close relationship to land plants, at the time of this
writing, the first charophyte draft genome, from Klebsormidium,
has only recently been published [56]. Transcriptomes have
been described from several more taxa [64,70,83], but genomics
remains largely in its infancy for charophyte green algae. The
tools of genetic engineering are also verymuch amatter of devel-
opment. Successful transformation has been described in the
zygnematophytes Closterium [109] and Penium [110], but none
has yet become routine. Other fundamental capabilities are lack-
ing as well — only a few species are readily available in axenic
culture and passed through their complete life cycle under con-
trol. These are not insurmountable obstacles, but rather reflect
the relatively small community working on these organisms.
Genomic studies have helped illustrate the profound similarities
these organisms have to land plants, and this in turn suggests
the valuable potential that they have for study of plant processes.
Crop plants can often take years to pass through a genetic
screen, so the rock cress Arabidopsis has proven to be invalu-
able because of its small size and rapid life cycle. The chloro-
phyte green alga Chlamydomonas has permitted profound
insights into the biology of photosynthetic eukaryotes despite
its relatively distant relationship to land plants. Phylogenetically
placed between these organisms, the charophyte green algae
present great opportunities for model system development,
and offer a wide range of structural features potentially of use,
from simple filamentous and unicellular forms in the Zygnemato-
phyceae; to disk-like branched filaments in the Coleochaetophy-
ceae that show developmental complexity and yet are amenable
to microscopy because in most regions they are a single layer of
cellsthick; to the giant cells and complex thalli in the Charophy-
ceae S.Str. that have already been widely used in electrophysi-
ology. The charophyte green algae present a largely uncharted
resource with great potential to advance plant research.
ACKNOWLEDGEMENTS
Supported in part by US NSF Award DEB-1036506 to C.F.D. This project has
also received funding from the European Union’s Horizon 2020 research
and innovation programme under the Marie Sklodowska-Curie grant agree-
ment No 660003. This article reflects only the authors’ views and that the
Agency is not responsible for any use that may be made of the information it
contains.
910, October 5, 2015 ª2015 Elsevier Ltd All rights reserved R907
Current Biology
Review
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	The Evolutionary Origin of a Terrestrial Flora
	Introduction
	Oxygenic Photosynthesis and the Origin of Eukaryotic Phototrophs
	The Charophyte Lineage
	Wet-to-dry Transitions
	The Promise of Charophyte Green Algae
	Acknowledgments
	References