Green Algae and the Origins of Multicellularity in the ... · cell type differentiation occurs in...

Green Algae and the Origins of Multicellularityin the Plant Kingdom

James G. Umen

Donald Danforth Plant Science Center, St. Louis, Missouri 63132

Correspondence: [email protected]

The green lineage of chlorophyte algae and streptophytes form a large and diverse clade withmultiple independent transitions to produce multicellular and/or macroscopically complexorganization. In this review, I focus on two of the best-studied multicellular groups of greenalgae: charophytes and volvocines. Charophyte algae are the closest relatives of land plantsand encompass the transition from unicellularity to simple multicellularity. Many of theinnovations present in land plants have their roots in the cell and developmental biologyof charophyte algae. Volvocine algae evolved an independent route to multicellularity that iscaptured by a graded series of increasing cell-type specialization and developmental com-plexity. The study of volvocine algae has provided unprecedented insights into the innova-tions required to achieve multicellularity.

The transition from unicellular to multicellu-lar organization is considered one of the ma-

jor innovations in eukaryotic evolution (Szath-mary and Maynard-Smith 1995). Multicellularorganization can be advantageous for severalreasons. Foremost among these is the potentialfor cell-type specialization that enables moreefficient use of scarce resources and can openup new adaptive niches (Stanley 1973; Szath-mary and Maynard-Smith 1995; Ispolatov etal. 2012). In addition, multicellularity allowsorganismal size to scale independently of cellsize, thus freeing organisms from the constraintsof individual cells and allowing them to evolvefundamentally new relationships with theirphysical and biological surroundings (Beardallet al. 2009). A third potential advantage of mul-ticellularity and increased organismal size is es-cape from microscopic predators such as ciliates

and rotifers that are limited by prey size (Bell1985; Boraas et al. 1998). Reciprocally, increasedsize might also entail advantages in capturingmore or larger prey.

There is some debate about how easy ordifficult it has been for unicellular organismsto evolve multicellularity (Grosberg and Strath-mann 2007). A conceptual division of multi-cellularity into two classes, simple and complex,has also been proposed (Knoll 2011). How-ever, the argument that simple multicellulari-ty is easier to achieve than complex multicellu-larity may be less compelling than it seems atfirst glance. In the �2-billion year history ofeukaryotic life, “simple” multicellularity hasarisen about two dozen times (Grosberg andStrathmann 2007; Knoll 2011; Adl et al. 2012).In contrast, complex multicellularity (definedby three-dimensional body plans and multiple

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cell types) has only arisen a few times withanimals and plants being clear examples, andfungi, brown algae and red algae achievingsomewhat lesser extents of complexity. In anycase, because complex multicellularity almostcertainly evolved from simple multicellularity,the odds of going from simple to complex mul-ticellularity are quite high (approximately onein 12), whereas the initial transition from uni-cellular to simple multicellular seems far moredifficult (one in thousands).

Achieving the benefits of multicellularity re-quired evolutionary innovations. At minimum,multicellularity requires adhesive interactionsbetween cells to maintain a coherent, physicallyconnected form. Many multicellular organismsmaintain not only adhesive connections be-tween cells, but also physical bridges that allowthe transfer of cytoplasmic materials directlybetween adjacent cells and mediate cell–cellcommunication. Diffusible extracellular mole-cules such as hormones are another means ofsignaling between cells that is common in mul-ticellular organisms. In addition to adhesionand cell–cell communication, the overall sizeand shape of multicellular organisms typicallyfollows a defined architecture or patterning thatinvolves spatially coordinated growth and divi-sion among groups of cells. A final requirementfor differentiated multicellular organization isregulation of reproductive potential. Reproduc-tion in multicellular organisms is often con-fined to a subset of cells (germ cells or stemcells). This division of labor serves at least twopurposes: First, by partitioning or restrictingreproductive capacity to a subset of cells, geneticconflicts associated with a transition of fitnessfrom individual cells to cooperating groups ofcells can be mitigated (Michod and Roze 2001).Second, in multicellular organisms with inde-terminate body plans such as land plants, vege-tative stem cells play a critical role in integratinginternal and external signals to regulate and es-tablish overall organismal architecture (Smolar-kiewicz and Dhonukshe 2013).

An outstanding question in the evolution ofmulticellularity is its early origins. It is increas-ingly clear that the path to multicellular evolu-tion is dependent on the genetic and cellular

“toolkits” available in unicellular ancestors, aswell as on the specific circumstances and envi-ronment in which multicellularity arose (Rokas2008; Knoll 2011; Bowman 2013; Niklas andNewman 2013; Smolarkiewicz and Dhonukshe2013). By examining the origins of multicellu-larity in diverse lineages, it can be determinedwhether commonalities exist that transcendlineage-specific solutions to achieve multicellu-lar organization.

UNIQUE ASPECTS OF MULTICELLULARITYIN PLANTS AND GREEN ALGAE

Plants and animals are interesting focal groupsbecause they arose from unicellular ancestorsthat appear to have had very different propen-sities or success rates in experimenting withmulticellular organization. The closest unicellu-lar ancestors to animals, the choanoflagellates,show a limited degree of colonial or multicellu-lar organization outside of their one big hit inthe metazoan lineage (Dayel et al. 2011; Alegadoand King 2014). The streptophytes (charophytealgae þ embryophytes [land plants]) and theirsister group, the chlorophytes (green algae),have repeatedly generated multicellular taxa aswell as macroscopic unicellular forms that showmany of the traits that are typically consideredhallmarks of multicellularity (Fig. 1) (De Clercket al. 2012; Leliaert et al. 2012). This diversity inthe green lineage represents a potential treasuretrove of information on the evolution of multi-cellular development. A second reason to focuson plants and algae involves the fundamentalconstraint that a cell wall places on the abilityof cells to move and reorganize spatially. Thisconstraint, which is not present in metazoans,has shaped the means by which multicellularorganization arose in the green lineage.

Cellular Diversity in the Green Lineage

Among unicellular green algae are found spec-tacular amounts of cellular diversification(Lewis and McCourt 2004; Leliaert et al. 2012).The ancestral species in the green lineage werelikely to be small unicellular marine biflagellatesthat originated around 1 billion years ago (De

J.G. Umen

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Unicellular Cladophorales Y

NANA

YN

Y

Y

Y

Y

Y

N

N

N

N

N

N

N

N

N

N

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Y

Y

Y

N

Differentiatedcell types?Ulvophyceae

Chlorophyceae

Trebouxiouphyceae

Prasinophytes

Streptophytes

Glaucophytes

Red algae

Charophyte algaeMestostigmatophyceae

Chlorokybophyceae

Kelbsormidiophyceae

Charophyceae

Zygnematophyceae

Coleochaetophyceae

Embryophytes(land plants)

Volvocales

Chlorophytes

Greenlineage

Dasycladales

Bryopsidales

Trentepohliales

Ignatius clade

Ulvales/Ulotrichales

Oltmannsiellopsidales

Oedogoniales

Oocystaceae

Chlorellales

Microthamniales

Trebouxiales

Prasiola

Pedinophyceae

Chlrodendrophyceae

Picocystis clade

Mamiellales

Dolichomastigales

PyramimonadalesMonomastigales

Pycnococcaceae

Clade VIII

Clade IX

Prasinococcales

Neophoselmidophyceae

Palmophylalles

Botryococcus clade

ChaetophoralesChaetopeltidales

Sphaeropleales

Chlamydomonadales

Cellular/multicellularorganization

Small cluster

Sheet/blade

Sarcinoid

Unbranched filament

Branched filament

Large spheroidal

Large multinucleate(siphonocladous)

Giant uninucleate

Complex twodimensional

Branched threedimensional

Multicellular/multinucleate(siphonocladous)

Figure 1. Multicellularity and morphological complexity in the green lineage. Simplified cladogram based ondata in Leliaert et al. (2012) and De Clerck et al. (2012) showing relationships among the green lineage(chlorophytes and streptophytes) and their sister groups, red algae and glaucophytes, which together comprisethe Archaeplastida. The major classes in the green lineage are in bold and, in some cases, further expanded intosubgroups. Note that the prasinophytes are a paraphyletic grouping of the most primitive chlorophyte algae. Thestreptophytes are divided into the charophyte algae (a paraphyletic grade) and the embryophytes (land plants).To the right are depicted the most complex type of cellular/multicellular organization in each clade and whethercell type differentiation occurs in multicellular forms. NA, not applicable for giant unicellular forms.

Multicellularity in Green Algae and Plants

Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016170 3

Clerck et al. 2012), and this form is still prevalentamong modern aquatic algae. A well-studiedexample of a unicellular biflagellate is Chlamy-domonas reinhardtii whose highly stereotypedcell architecture is described later in this review.Figure 1 shows a simplified phylogenetic treedepicting the major lineages of green algae andtheir land plant relatives discussed in this review.

Reductions in cell size and cellular complex-ity have occurred multiple times among greenlineage resulting in small coccoid species suchas Ostreococcus that have lost flagella and prob-ably underwent genomic reduction and stream-lining (Derelle et al. 2006). On the other hand,some of the largest and most complex singlecells on earth are found among green algae,including species such as Acetabularia whosewine-cup-shaped cells are measured in centime-ters (Mine et al. 2008). Additional cellular diver-sity in microalgae includes different cell shapessuch as crescents, rectangles, ovoids, elongatedspines, and flattened discs. Some species have nocell wall, whereas walled species use a variety ofwall polymers including cellulose, pectins, chi-tin-like molecules, or hydroxyproline-rich gly-coproteins. Besides sugar-polymer-based walls,some species cover themselves with hard calci-fied scales (Popper et al. 2011; Domozych et al.2012; Leliaert et al. 2012).

Cell division in green organisms can occurthrough binary fission, but a form of divisioncalled multiple fission (or palintomy) is alsocommon in chlorophyte algae (Pickett-Heaps1975; Setlık and Zachleder 1984; Graham et al.2009). In multiple fission cell cycles, mothercells divide more than once in rapid successionto produce 2n daughters (2,4,8,16, . . .) within asingle mother cell wall. Multiple fission has beenexploited by green algae as a means of formingmulticellular colonies, most notably in the vol-vocine algae that are discussed below. Differentmechanisms of cytokinesis have also evolved inthe green lineage including a specialized micro-tubule structure called the phycoplast, as wellas an analogous structure, the phragmoplast,which is used in streptophytes (Pickett-Heaps1976). The former mechanism involves mem-brane furrowing guided by microtubules thatare parallel to the plane of division, whereas

the latter involves formation of new cross-wallsand plasma membrane at the overlap regionbetween parallel microtubules emanating fromeach daughter nucleus after mitosis.

Chlorophyte and charophyte algae general-ly have haploid dominant life cycles with 1n ga-metophyte cells that can reproduce vegetative-ly (i.e., asexually). In gametophyte or haploiddominant life cycles, growth and prolifera-tion are generally restricted to the haploid (1n)phase, whereas the diploid (2n) phase is a zygoticspore. Nonetheless, the Ulvophytes (a predom-inantly multicellular order of chlorophyte algae)contain genera with a significant diploid sporo-phytic stage. This derived state of sporophyticgrowth and development also evolved indepen-dently in the embryophytes (land plants) inwhich the gametophyte stage was concomitantlyreduced and the sporophyte dominant life cyclepredominates (Niklas and Kutschera 2009).

Many species also have a sexual cycle inwhich at least one of the gametes is a unicellu-lar biflagellate. Anisogamy and oogamy (spermand egg-mating systems) evolved more thanonce in different chlorophyte subgroups fromisogamous mating systems seen in most unicel-luar species. Although aflagellate gametes arefound in some charophyte algae such as Zyg-nematales that mate via a specialized processcalled conjugation (Graham et al. 2009; Seki-moto et al. 2012), the ancestral state for sexualreproduction in early land plants was presum-ably isogamy with oogamous mating evolvingin advanced orders (McCourt et al. 2004). Thesubsequent evolution of aflagellate, desiccation-resistant pollen in advanced land plants freedtheir male gametes from the requirement formoisture and allowed them to disperse throughthe air or via animal pollinators.

Macroscopic Complexity in Green AlgaeEvolved Using Different Strategies

Green algae challenge the notion that equatesmulticellularity with organismal complexity.Described below are three strategies used inthe green lineage to achieve macroscopic com-plexity, two of which do not involve conven-tional multicellularity.

J.G. Umen


Multicellular Algae

Independent transitions to multicellularity ormacroscopic forms occurred multiple times inthe chlorophyte lineage (Fig. 1). True multicel-lular forms are characterized by having separatecells, each with its own nucleus, plasma mem-brane, and cell wall. Streptophytes and manymulticellular chlorophyte algae are organizedin this manner. It should be noted that connec-tions between neighboring cells (e.g., plasmo-desmata) evolved multiple times in the greenlineage and provide direct cytoplasmic continu-ity between cells in many multicellular algaeand land plants, thus allowing them some de-gree of supracellular organization (Raven 1997).Some common multicellular forms are un-connected groups of cells embedded in extra-cellular matrix, short chains, unbranched andbranched filaments with and without differen-tiated cell types, geometric networks, large leaf-like blades, discoidal colonies, and spheroidalcolonies (Fig. 1).

Giant Uninucleate Algae

An alternative form of complex and macroscop-ic unicellular organization is found in giant uni-cellular algae that possess a single nucleus. Themost well-known species of this type are in thegenus Acetabularia (an ulvophyte) that is fa-mous for transplantation experiments demon-strating nuclear control over cell morphology(Mandoli 1998; Mine et al. 2008).

Coenocytic Algae

Coenocytic algae (also referred to as siphonousalgae) are large single cells that contain multi-ple nuclei housed within a common cytoplasm.The sizes of these forms are measured in centi-meters and meters making them some of thelargest single cells on earth. Despite their lackof multicellular organization, coenocytic algaecan form remarkably complex structures withmultiple types of specialized subdomains. Forexample, the multinucleate cells of the marinealga Caulerpa reach meters in size and containthree distinct tissue types that function similarlyto roots, stems, and leaves (Jacobs 1970). More-

over, some green algae are both multicellularand coenocytic (termed siphonocladous) be-cause they are composed of multiple cells, eachof which contains multiple nuclei (e.g., Clado-phora). Cytoplasmic continuity and develop-mental patterning in coenocytic macroscopicalgae are beyond the scope of this review, butare covered in other recent reviews (Baluskaet al. 2004; Cocquyt et al. 2010; Niklas et al.2013).

CHAROPHYTE GREEN ALGAE ANDTHE ORIGINS OF LAND PLANTMULTICELLULARITY

Overview

Charophyte algae have long been recognized asthe closest algal relatives of land plants basedon cellular and morphological features (Stewartand Mattox 1975). This view has been corrob-orated and refined using molecular phyloge-netics that have begun to clarify relationshipswithin charophyte algae and with their sisterclade, the land plants (embryophytes) (Turmelet al. 2002; Finet et al. 2010; Wodniok et al.2011; Laurin-Lemay et al. 2012; Timme et al.2012; Zhong et al. 2013).

A number of land plant multicellular traitsoriginated within the streptophytes (charo-phyte algae þ embryophytes). These includeapical meristems, cells that undergo asymmetricdivision and differentiation, complex branch-ing patterns, matrotrophic support for a multi-cellular sporophyte, and plasmodesmata thatform symplastic connections between cells. Ad-ditional cellular and biochemical traits that arestreptophyte specific include a hexameric cellu-lose synthase complex that synthesizes the pri-mary cell wall, the phragmoplast as a mecha-nism for cytokinesis, synthesis, and transportof phytohormones, and several metabolic spe-cializations (Graham 1996; Graham et al. 2000;Waters 2003; Becker and Marin 2009).

Later changes and innovations that oc-curred within the embryophytes well after theyachieved multicellularity include three-dimen-sional growth and cell division to produce com-plex tissues and organs, cuticular waxes, and



stomatal pores to control evaporative losses, vas-cular transport systems, root and shoot apicalmeristems, transition to a sporophyte dominantlife cycle, specialized and complex hormonalsignaling, nonflagellated and desiccation-re-sistant male gametes (i.e., pollen), flowers, andlignified secondary cell walls (Langdale andHarrison 2008). Interestingly, molecular evi-dence for proteins related to some of these laterinnovations in embryophytes have been foundin charophytes as described below.

It has been argued that the existing charo-phyte algal orders are actually the remnants of aterrestrial invasion that gave rise to land plants,but whose members were outcompeted by theembryophytes. The remaining extant orders ofcharophyte algae ended up secondarily aquaticwhere they were able to compete more effec-tively than on land (Stebbins and Hill 1980).Although this idea is difficult to test directly,the finding that a nominally aquatic charophytespecies, Coleochaete orbicularis, can grow in ter-restrial environments and even show adaptivechanges in body plan under these conditionssupports this idea (Graham et al. 2012). It hasalso been argued that early streptophytes werepreadapted to the colonization of land by be-ing the first algal group to conquer freshwaterhabitats, whereas chlorophyte algae, which pre-dominate in marine environments, moved intofreshwater much later (Becker and Marin 2009).

A closer examination of charophyte evolu-tion may provide an outline for understand-ing some of the early multicellular innovationsin the embryophytes. It is important to notethat the extant charophyte lineages are highlydiverged from each other and have probablyundergone secondary character losses that com-plicate comparisons and inferences about theprogression to multicellularity in the embryo-phytes (McCourt et al. 2004; Timme et al. 2012;Bowman 2013). Moreover, the fossil record ofcharophyte algae is incomplete and does notextend earlier than that of land plants, so isnot a reliable source of information on timingfor emergence of early embryophytes whoseearliest fossils appear �500 million years ago(Leliaert et al. 2012). As noted above, the char-ophyte algal order that is most closely related to

land plants is unresolved, but it is clear thatthe most developmentally complex order, Char-ophyceae (stoneworts), is probably not the clos-est relative of the embryophyte lineage (Wod-niok et al. 2011; Laurin-Lemay et al. 2012;Timme et al. 2012; Zhong et al. 2013). Despitethis ambiguity and the likelihood of both char-acter loss and gain in the extant charophyte algalorders, some of the steps leading to multicellu-larity in the embryophytes can still be inferred.

To date, there are no sequenced charophytegenomes, so molecularevidence regarding genesand pathways that are shared within strepto-phytes is somewhat piecemeal. Current under-standing of molecular evolution of charophytesalgae has been guided mostly by expressed se-quence tag (EST) sequences and directed inves-tigations of specific gene families (Simon et al.2006; Timme and Delwiche 2010; Vannerumet al. 2011; Wodniok et al. 2011).

Key Innovations in the StreptophyteTransition to Multicellularity

Given the uncertainties surrounding the phy-logeny of charophyte algae and the apparentgain and loss of some traits, the order of topicslisted below is meant only to convey a plausiblepicture of how multicellular innovations wereacquired in the embyrophyte lineage as summa-rized in Figure 2.

The Closest Unicellular Relative of Land Plants

The unicellular ancestor of Streptophytes wasa freshwater biflagellate whose most similar ex-tant relative is in the genus Mesostigma (Bhat-tacharya et al. 1998; Marin and Melkonian 1999;Lemieux et al. 2000, 2007; Rodriguez-Ezpeletaet al. 2006). Mesotigma does not produce cellu-lose or any type of cell wall, but instead has cal-cified scales. It divides by furrowing rather thanthrough formation of a phragmoplast (Mantonand Ettl 1965). This ancestral form likely hadan open mitosis as observed in early divergingcharophytes (Stewart and Mattox 1975; Pickett-Heaps 1976). A systematic investigation of itsgenetic toolkit has not been performed, but sev-eral studies have identified streptophyte-spe-

J.G. Umen


cific gene families in Mesostigma (Nedelcu et al.2006; Petersen et al. 2006; Simon et al. 2006;Grauvogel and Petersen 2007). A genome se-quence and development of molecular genetictools would allow deeper investigation into thecell biology of these unicellular plant relativesand how much of the developmental toolkit forEmbyrophytes was present in their most recent-ly diverged unicellular ancestor.

Filamentous Growth and Cytokinesis

The early multicellular forms produced byprimitive embyrophytes were probably undif-ferentiated filaments, a growth habit that is stillcommon in many green algal lineages includingextant charophyte algae such as Klebsormi-diales, which are among the three most primi-tive orders of this grade. In all orders exceptMesostigmaphyceae, charophyte algae lost mo-tility in the vegetative stage, but with the ex-ception of Zygnematales, retained flagellatedzoospores (Stewart and Mattox 1975). The pre-valence of filamentous forms in both aquat-ic and terrestrial environments and diverse algallineages suggests that filamentation has multi-

ple selective advantages. Filaments can be usedto anchor colonies in place efficiently while al-lowing maximal contact with the surroundingenvironment along each cell’s surface, a propertythat is likely to be useful for nutrient exchange inboth aquatic and terrestrial habitats (Niklas2000). Filaments would also serve as protectionagainst grazing predators by being too large toingest, an idea that has been tested experimen-tally with filamentous bacteria (Gude 1979; Shi-kano et al. 1990). A third advantage of filamentsis their utility for nutrient foraging because theydirect growth along a vector that maximizes ex-ploration of surrounding space. Even withoutresource sharing between cells (i.e., symplasticconnections), the clonal nature of filaments en-sures a benefit to growth-directed foraging be-cause all cells in the filament share the samegenotype.

Filament formation requires at least oneaxis of cell polarity to ensure that growth oc-curs longitudinally and the plane of cytokine-sis is perpendicular to the plane of growth. Aninnovation required for filamentation in char-ophytes involved the deposition of common cellwall material between daughters. Descriptive

Cellulose wallChlorokybophyceae

Klebsormidiophyceae

Charophyceae

Zygnematophyceae

Coleochaetophyceae

Embryophytes(land plants)

Mestostigmatophyceae

Filamentous growth

Multicellular embryo, sporophyte dominant life cycle,

stomata, waxy cuticle, vasculature, multicellular organs,

meristems with >2 cutting faces, seeds, flowers

Complex cell wall, phragmoplast, plasmodesmata, polarized

growth, asymmetric division, stem cells, oogamy,

matrotrophy, phytohormones, three-dimensional patterning,

apical growth

Figure 2. Multicellular innovations in charophyte algae and embryophytes. Simplified cladogram based on datain Leliaert et al. (2012) showing relationships among major orders of charophyte algae and embryophytes. To theright are shown multicellular innovations associated with different groups. Note that reductions or losses oftraits occurred in some orders of advanced charophyte algae (Charophyceae, Zygnematophyceae, Colechaeto-phyceae), most notably in the Zygnematophyceae (McCourt et al. 2004), so the set of innovations in this groupmay not apply to all three orders.



and comparative studies have been performedon cytokinesis in primitive filamentous charo-phytes that occurs after an open mitosis, butdoes not appear to involve a phragmoplast orphycoplast (Floyd et al. 1972; Pickett-Heaps1976; Lokhorst and Star 1985; Katsaros et al.2011). The phragmoplast is still considered animportant innovation and originated in theZygnematales, a transitional group wherein dif-ferent types of cytokinetic patterns coexist.Some groups in this order divide centripetally(e.g., filamentous desmids) (Hall et al. 2008)whereas others, such as the genus Spirogyra, ap-pear to use a reduced but recognizable phrag-moplast (Fowke and Pickett-Heaps 1969; Gal-way and Hardham 1991), although with somefunctional distinction from embryophytephragmoplasts (Sawitzky and Grolig 1995). Itis not known whether the shared wall betweenprimitive filamentous charophyte algal cellsare specialized in any way, but it is known thattheir walls lack many of the complex carbohy-drate polymers present in advanced charophytesand embryophytes (Sørensen et al. 2010, 2011;Domozych et al. 2012). The more advancedcharophyte algae (Charophyceae, Coleochaeto-phyceae) do have a recognizable phragmoplastthat resembles those of embryophytes (Pickett-Heaps 1967; Marchant and Pickett Heaps 1973).Coincident with the evolution of the phragmo-plast in advanced charophyte algae is the ap-pearance of what might be a preprophase bandof microtubules that marks the future plane ofcell division (Galway and Hardham 1991). De-tailed molecular information on primitive char-ophyte cell division is still limited, but these ear-ly filamentous divisions in Spirogyra describedabove may have paved the way for evolution ofthe phragmoplast and preprophase band as anefficient mechanism for marking division sitesand subdividing cells with a rigid wall.

Regulated Division Plane, CellularDifferentiation, and Polarized Growth

Contrasting with simple filamentous forms inwhich cell growth occurs similarly in all constit-uent cells, polarized growth involves some formof tissue organization that dictates which cells in

a filament or thallus (body) grow, and the di-rectionality of growth. Species from both theColeochaetophyceae and Charophyceae havespatially regulated division planes. Coleochae-tales show diverse vegetative forms that includeflat, disk-shaped thalli, branched filaments, andpseudoparenchymatous filaments that are heldtogether within an extracellular matrix (Gra-ham et al. 2009). The discoid species Coleo-chaete orbicicularis grows outward by a com-bination of cell expansion and either anti- orpericlinal cell divisions along its marginal cells(Cook 2004). Cell division planes are set accord-ing to a set of simple rules based on positionwithin the thallus, cell size, and cell shape (Du-puy et al. 2010). Moreover, the division planeorientation behavior of C. orbicularis can bemodeled as an energy minimization functionthat can also be applied to embryophytes whereit may be an inherited mechanism that specifiesa default pattern of symmetrical cell division(Besson and Dumais 2011).

Asymmetric and/or branched histogenet-ic cell divisions are critical for development inthe Coleochatelaes and Charales (Graham 1996;Graham et al. 2000), although little is knownhow such divisions are controlled. In angio-sperms, asymmetric divisions are also crucialfor development and regulated by different ge-netic pathways depending on tissue type (DeSmet and Beeckman 2011). Although a ho-molog of a gene required for asymmetric divi-sion in Arabidopsis zygotes, GNOM, has beenidentified in Coleochaetes, the significance ofthis finding is unknown (Timme and Delwiche2010). The relationships between charophytealgal asymmetric cell divisions and those in em-bryophytes remain to be investigated.

Although more distantly related to landplants than Coleochaetphyceae and Zygnema-tophyceae, Charaophyceae (e.g., Chara and Ni-tella) (Fig. 2) show morphologies and growthpatterns that more closely resemble those ofland plants. These similarities include a long,branched body axis and indeterminate api-cal meristem cell. Charophyceans do not havemulticellular tissues, but their giant multinucle-ated cells show differentiation into specializedtypes including long intermodal cells, nodal

J.G. Umen


cells that undergo asymmetric cell divisions toform branches, and rhizoid cells that act asholdfasts and show tip-polarized growth(Cook et al. 1998; Braun and Limbach 2006).Charophyceae also have male (antheridia) andfemale (oogonia) sexual reproductive structures(see below) that are formed by complex divisionpatterns to generate simple three-dimension-al multicellular tissues (Fritsch 1948; Pickett-Heaps 1968; Kwiatkowska 2003; Graham et al.2009).

Polarized gravitropic growth of the apicalmeristem and rhizoid cells in Chara and Nitellahas been investigated, particularly with respectto actin cytoskeletal dynamics (Braun and Lim-bach 2006). However, little is known aboutother developmental patterning cues in charo-phytes such as light signaling (Rethy 1968;Dring 1988). The discovery of plant hormonesin some charophyte algal species (see below)suggests that phytohormones may be an earlyinnovation that enabled the evolution of char-ophyte multicellularity.

Symplastic Connections and IntercellularCommunication

In land plants, plasmodesmata connect adja-cent cells allowing metabolites and some mac-romolecules to pass between them, as well asallow tissue-level interactions among cells (Lu-cas and Lee 2004). Plasmodesmata are foundin two of the advanced orders of charophytealgae, Coleochaetophyceae and Charophyceae,but intercellular connections in algae are wide-spread and not unique to charophytes (Stewartand Mattox 1975; Raven 1997). In primitiveembryophytes, connections between adjacentcells would enable communication, allow me-tabolite/resource sharing, and promote cellspecialization. The multifaceted roles of plas-modesmata in shaping plant development andevolution has been reviewed recently (Lucas andLee 2004; Hernandez-Hernandez et al. 2012).Information on how intercellular communica-tion modulates development in charophyte al-gae is limited, but has been studied in somedepth in male reproductive tissue (antheridia)in Chara, in which there is compelling evidence

that remodeling of plasmodesmata is importantfor maintaining and coordinating cell divisionsynchrony and developmental fate (Kwiatkow-ska 2003). In both embryophytes and charo-phyte algae, plasmodesmata can be formed dur-ing cytokinesis or secondarily in interphase cellsby remodeling of walls and fusion of adjacentplasma membranes (Hepler 1982; Franceschiet al. 1994), but the molecular details of plas-modesmata formation in charophyte algae andtheir degree of similarity to embryophyte plas-modesmata have not been established (Faulkneret al. 2005; Timme and Delwiche 2010).

Hormonal Signaling

Phytohormones are essential regulators of plantdevelopment and environmental responses. Be-sides contributing to developmental plasticity,small diffusible hormones facilitate long-dis-tance communication that may occur on a mac-roscopic scale to coordinate responses acrosstissues and cell types in a multicellular organ-ism. The presence of growth-promoting phy-tohormones in algae has been investigated indiverse taxa (Bradley 1991; Johri 2004; Tara-khovskaya et al. 2007; Stirk et al. 2013a,b), butthe molecular bases of phytohormone action inalgae is not well understood. Physiological stud-ies on the effects of plant hormones have shownthat charophyte algae produce and respond toseveral phytohormones, indicating an early or-igin of these chemical signals. Because genomesequences are not available, inferences aboutthe presence of known signaling and biosyn-thetic machinery for Embyrophyte phytohor-mones in charophyte algae can only be madefrom the presence of gene families, but notfrom their absence.

Indole derivatives (e.g., indole acetic acid)in the auxin family are produced in Chara(Sztein et al. 2000) and are known to affect de-velopment byaltering shoot elongation, branch-ing, and rhizoid elongation, for example (Ima-hori and Iwasa 1965; Klambt et al. 1992; Cookeet al. 2002), and may act through changes incytoskeletal organization (Jin et al. 2008). Di-rectional transport of auxin in long, giant inter-modal cells of Chara has been shown recently,



suggesting that this phenomenon may be an-cient (Boot et al. 2012; Raven 2013). Some ofthe auxin biosynthetic enzymes entered thegreen lineage before the streptophyte–chloro-phyte divergence, but the response and trans-port proteins characteristic of the well-studiedauxin-signaling networks in angiosperms firstappeared in charophytes, and the full comple-ment of core auxin-signaling proteins becameestablished in early embryophytes (Lau et al.2009; Prigge et al. 2010; De Smet et al. 2011;Wodniok et al. 2011).

Abscisic acid (ABA) is an isoprenoid-derived stress and drought hormone whosefunction can be traced back to the earliest em-bryophyte clade, the bryophytes (Cutler andRodriguez 2010; Khandelwal et al. 2010; Hauseret al. 2011). Although its function in land plantsis associated with the transition to terrestrial life,ABA is also produced by a wide variety of aquat-ic green algae, although its function in thesealgae is unknown (Hartung 2010). G protein–coupled receptor G proteins in plants have beenlinked to ABA signaling in Arabidoposis and haverecently been identified in Chara, but not in anychlorophyte algae (Pandey and Assmann 2004;Hackenberg et al. 2013). Studies on spore ger-mination in Chara suggest a possible inhibitoryrole for ABA in this process that is antagonizedby another hormone, gibberrellic acid (GA),which also promotes growth (Imahori and Iwasa1965; Sabbatini et al. 1987; Sederias and Colman2007). The apparently antagonistic roles of GAand ABA in Chara are reminiscent of similarroles in Angiosperm seed germination, suggest-ing an ancient role for this hormonal system inregulating dormancy that may have precededa role for ABA in drought responses. GA hasalso been reported to affect growth and develop-ment of male reproductive cells (antheridia) inChara by up-regulating sperm production andendoreplication of support cells (Kwiatkowskaet al. 1998). Classical GA-signaling and percep-tion proteins have not been found in charophytealgae (Vandenbussche et al. 2007), although ge-nomic information is unavailable so this nega-tive result awaits confirmation.

Strigolactones (SLs) are cyclic derivatives ofcarotenoids that have roles in biotic interactions

of angiosperms and developmental control ofbranching architecture (de Saint Germain etal. 2013). In the Bryophyte Physcomitrella, SLsinfluence branching and sensing of adjacentcolonies (Proust et al. 2011). Remarkably, SLshave been identified in the Charophyceae (butnot other charophyte algae orders) and assays ofSL-treated Chara revealed a developmental re-sponse of rhizoid cell elongation (Ruyter-Spiraand Bouwmeester 2012).

Much less is known about other land planthormones in charophyte algae including brassi-nosteroids, cytokinins, and ethylene. Brassino-steroids and cytokinins have been detected incharophyte algae, and at least one study hasreported an effect of cytokinins on growth ofChara (Imahori and Iwasa 1965). Ethylene isreported to bind to Chara (Wang et al. 2006),whereas some ethylene biosynthesis and per-ception genes were found in charophyte algaeESTs (Timme and Delwiche 2010; Wodnioket al. 2011), but data on the effect of ethylenein charophyte algae growth or physiology is cur-rently unavailable.

Oogamous Sexual Reproduction

In complex multicellular organisms, sexual re-production is typically oogamous with smallmotile sperm and large immotile eggs, whereasunicellular or simple multicellular organismsare typically isogamous (having equal-sizedgametes). Various explanations for the evolu-tion of anisogamy and oogamy have been putforth (Randerson and Hurst 2001), but one ofthe earliest by Parker may be particularly rele-vant to the evolution of multicellularity (Parkeret al. 1972). The crux of this argument is thefitness advantage of making a large zygotic cellthat is capable of forming a functioning multi-cellular progeny. Disruptive selection drives onegamete type to be much larger than a single cellso that it can provide resources to the zygote,and the other to be as small and numerous aspossible to ensure efficient fertilization.

Sexual reproduction in charophyte algae hasbeen described in the higher orders Charophy-ceae, Zygnematophyceae, and Coleochaetophy-ceae, but has not been observed in the lower

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orders in which it is assumed to be isogamous(McCourt et al. 2004). It is also assumed thatcharophyte algae have a haploid dominant lifecycle similar to most chlorophyte algae andprimitive embryophytes, although this assump-tion has been questioned (Haig 2010). Zygne-matales have the best-studied sexual cycles inthe charophyte algae, but have secondarily lostflagella and reproduce through isogamous con-jugation (Graham et al. 2009; Sekimoto et al.2012). Both Charophyceae and Coleochaeto-phyceae have oogamous sexual cycles with spe-cialized egg and sperm cells. Very little is knownabout the genetics of sex determination in thesetwo orders in which sexual development isusually monoecious (male and female gametesformed in the same organism). As mentionedabove, the Charophyceae have particularly strik-ing and elaborate reproductive tissues (male an-theridia and female oogonia) that include bothgametic cells and support cells (Fritsch 1948;Pickett-Heaps 1968; Sawa and Frame 1974;Beilby and Casanova 2014). Understanding thegenetic programs that give rise to reproductivetissues in charophyte algae and how they relateto those of embryophytes may shed light onwhether oogamy is a synapomorphic trait with-in streptophytes.

Matrotrophic Support of the Zygote

A conserved feature in embryophytes is mater-nal support provided to zygotes and sporophyteembryos after fertilization (Ligrone et al. 1993).This innovation may be related to oogamy be-cause it increases resource allocation to a devel-oping multicellular offspring. The attachmentand nourishment of the developing embryo bymaternal tissue may have been crucial for estab-lishing a later change in embrophyte evolution,the sporophyte dominant life cycle (Grahamand Wilcox 2000). The best-documented oc-currence of matrotrophy in charophyte algaecomes from studies of Coleochate in which thedeveloping zygote remains associated with thevegetative thallus (Graham 1984). These studiesidentified regions of the vegetative maternalcells specifically at the zygote interface that ap-peared specialized for maximal cell–cell contact

and nutrient transport (Graham and Wilcox1983; Delwiche et al. 1989). The molecular de-tails of these junctions and how they form isnot known, but their presence in advancedcharophyte algae and similarity to embryophytematernal-zygotic junctions suggests a commonorigin.

Multicellular Innovations in Land Plantsthat May Be Rooted in the Green Lineage

The above innovations in the charophyte algaecan be directly tied to the transition to multi-cellularity in the streptophyte lineage. Otherimportant properties of embryophytes that arenot directly related to acquisition of multicellu-larity may also have roots in charophyte algaeand the green lineage. Cellulosic walls are foundthroughout the green algal lineage and in othertaxa, but a hexameric cellulose synthase com-plex evolved uniquely in streptophytes (Tsekos1999). Other distinctive characteristics of em-bryophyte cell walls, including complex carbo-hydrate polymer linkages, first appeared in ad-vanced charophyte green algae (Van Sandt et al.2007; Popper et al. 2011; Sørensen et al. 2011;Domozych et al. 2012; Proseus and Boyer 2012).The recent discovery of expansin genes in theZygnematophycean alga Micrasterias suggeststhat these critical cell wall remodeling proteinsfrom embryophytes originated in charophytealgae (Cosgrove 2000; Vannerum et al. 2011).Lignified secondary walls were a critical inno-vation in land plants for desiccation tolerance,load-bearing structural integrity, and decay re-sistance (Boyce et al. 2004). Although charo-phyte algae do not have secondary walls, ligninor lignin-like molecules have been identifiedin this group and may contribute to resistanceagainst microbial attack (Gunnison and Alex-ander 1975; Delwiche et al. 1989; Sørensen etal. 2011). Sporopollenin is a complex moleculefound in the walls of angiosperm pollen thatprotects cells from physical and chemical deg-radation (Ariizumi and Toriyama 2011; Wallaceet al. 2011). However, the origins of sporo-pollenin-like molecules goes as far back as thecharophyte algae in which sporopollenin-likesubstances were identified in the zygotes of Co-



leochate (Delwiche et al. 1989). Both lignin- andsporopollenin-like substances are found outsideof the streptophyte lineage, so without molecu-lar data about the origins of biosynthetic genesfor these compounds in charophyte algae, thepossibility of convergence or horizontal transferinto embryophytes cannot be ruled out (Brooksand Shaw 1978; Emiliani et al. 2009; Martoneet al. 2009).

Although not directly associated with thetransition to multicellularity in land plants,the alteration of generations is a hallmark ofthe embryophytes in which simple, multicel-lular sporophytes first appeared (Graham andWilcox 2000). Interestingly, the genetic programthat directs sporophyte development in thebryophyte moss Physcomitrella is directed bya KNOX family homeodomain transcriptionfactor whose homologs in the chlorophytealga C. reinhardtii direct development of thedormant diploid zygospore formed after fertil-ization (Lee et al. 2008; Nishimura et al. 2012;Sakakibara et al. 2013). The role of these home-odomain proteins in charophyte algae has notbeen examined, but they are predicted to par-ticipate in zygote development.

How Much of the Embryophyte MolecularGenetic Toolkit Originated in CharophyteAlgae?

Although data are incomplete, some progresshas been made in identifying charophyte algalhomologs of key developmental regulators usedby embryophytes (Bowman et al. 2007; Piresand Dolan 2012; Bowman 2013). Without com-plete genome sequences this list is relativelyshort and incomplete, but provides a sampleof potential discoveries that might be madewith increased knowledge of charophyte algalgenomes and developmental patterning mech-anisms.

MADS-box transcription factors are foundwithin and outside the green lineage, but a spe-cific MADS protein subtype, MIKC, defined bythe presence of four characteristic domains,is found only in streptophytes and underwenta dramatic expansion in the embryophytes inwhich members control many aspects of spor-

ophyte developmental patterning (Smaczniaket al. 2012). MIKC-type MADS box genes arealso present in advanced charophyte orders inwhich their expression was analyzed (Tanabeet al. 2005). In Chara, MADS-box gene expres-sion was restricted to reproductive tissues (an-theridia and oogonia), whereas expression wasdetected at multiple stages in a Zygnematophy-cean, Closterium. In bryophytes, MIKC MADSbox genes are incompletely characterized, butappear to have roles in both gametophyte andsporophyte development (Quodt et al. 2007;Singer et al. 2007; Zobell et al. 2010).

Homeodomain leucine zipper (HD-ZIP)transcription factors regulate diverse aspects ofangiosperm sporophyte development includingshoot apical meristem identity, abaxial-adaxialleaf patterning, and epidermal development(Elhiti and Stasolla 2009). The origin of twomajor groups of HD-ZIP proteins (class IIIand class IV) has been traced back to charophytealgae (Floyd et al. 2006; Zalewski et al. 2013).Although HD-ZIP proteins are also found inbryophytes, their function in lower plants isnot well characterized (Sakakibara et al. 2001;Prigge and Clark 2006). Interestingly, a micro-RNA binding site that regulates expression ofclass III HD-ZIP expression in angiosperms isconserved in lower plants, but not in Chara,suggesting that gene family expansion and reg-ulatory evolution contributed to functionalnovelty in embryophyte HD-ZIPs (Floyd et al.2006; Floyd and Bowman 2007). Wuschel-likehomeodomain proteins that are important forAngiosperm shoot apical meristem identity arealso present in charophyte algae, but their rolein this group and bryophytes has not been ex-plored (Timme and Delwiche 2010).

VOLVOCINE ALGAE: A LIVING SNAPSHOTOF MULTICELLUAR EVOLUTION

Overview of Volvocine Algae

Volvocine algae provide an interesting contrastto embryophytes whose body plans are con-structed through polarized growth and cell di-vision of indeterminate meristems. Instead,multicellular volvocine algae undergo a singleseries of embryonic cell divisions and morpho-

J.G. Umen


genesis to produce a determinate body plan.How and why did volvocine algae evolve thisform of multicellularity? The answer to thisquestion can, potentially, be found by examin-ing the cell biology of unicellular volvocine al-gae such as C. reinhardtii, which closely approx-imate the ancestral state of this lineage.

Volvocine algae are particularly well suitedfor investigating the origins of multicellularity.They are monophyletic and contain genera thatare unicellular and multicellular. Two of the keyvolvocine species, unicellular C. reinhardtii andmulticellular Volvox carteri, are model organ-isms with sequenced genomes and establishedmolecular-genetic toolkits that make themamenable to molecular and genetic experimen-tation (Merchant et al. 2007; Prochnik et al.2010; Umen and Olson 2012). V. carteri mutantswith developmental phenotypes have been es-pecially illuminating for understanding the mo-lecular genetic origins of multicellularity (Kirk1998, 2003).

The genus Chlamydomonas has more than600 species whose properties are typical of uni-cellular chlorophytes. Although the genus isnow recognized as polyphyletic, the modelspecies C. reinhardtii is closely related to multi-cellular volvocine algae that together form amonophyletic clade with C. reinhardtii as anoutgroup (Fig. 3A) (Proschold et al. 2001,2005; Herron and Michod 2008; Nakada et al.2008). The last common ancestor of this cladewas estimated to have lived �200 million yearsago (Herron et al. 2009), but it is worth notingthat the single fossil chlorophyte alga used forcalibration in the above dating study has pro-duced incongruences in other analyses and maynot be part of the taxonomic group it was orig-inally identified with (Berney and Pawlowski2006). Multicellular or colonial volvocine algalgenera can be arranged in a stepwise progressionof increasing cell number, colony size, and de-gree of cell-type specialization (Fig. 3B). Thissimplistic arrangement that is termed the volvo-cine lineage hypothesis is probably a good ap-proximation of how complexity evolved withinspecific sublineages, but does not reflect the factthat some grades of organization such as Volvoxarose several times independently (Fig. 3A).

The simplest colonial species are in the ge-nus Gonium that form planar colonies of be-tween four and 16 cells that show a slight cur-vature and have their flagella positioned on theconvex side of the colony. The next step in thevolvocine lineage was the formation of spheroi-dal colonies of 16 to 32 cells with a definedinterior and exterior as typified in the genusPandorina. In the genus Eudorina, larger spher-oidal colonies of 32 to 64 cells are formed by theaddition of an extensive secreted extracellularmatrix (ECM) in which cells of the colony areembedded. The addition of ECM in Eudorinaallows the colonies to increase in volume with-out any significant change in cell number orsize. Up to the genus Eudorina, all the cells ina colony are reproductive; they grow as flagellat-ed cells to provide motility and then synchro-nously divide to produce a set of new daughtercolonies. Starting with the genus Pleodrina, col-onies begin to show germ-soma differentiation.In most species of Pleodorina, there is a clearlyvisible anteroposterior axis with anterior cellsceasing growth and remaining permanentlyflagellated to serve a nonreproductive motilityfunction. The posterior cells in a Pleodorina col-ony show a partial division of labor. They startout as flagellated cells, but continue to growand, subsequently, retract their flagella and di-vide into new colonies. The partial germ-somadivision of labor shown by Pleodorina becomesa fixed dichotomy in the genus Volvox whosecolonies can contain up to 50,000 cells insome species and in which there are dedicatedgroups of sterile somatic cells and reproductivegerm cells. The progression from simpler colo-nial forms to Volvox was probably stepwise asoutlined in the volvocine lineage hypothesis,but variations of this progression led to at leastthree independent origins of Volvox-like colonyorganization, each with distinct properties (Fig.3) (Nozaki et al. 2006b; Herron and Michod2008; Coleman 2012).

The Cell Biology of Chlamydomonas

The key to understanding the proximal mecha-nisms underlying multicellularity in Volvocinealgae lies in the cell biology of their unicellular



ancestor that was similar to C. reinhardtii. C.reinhardtii is a walled biflagellate with a pair ofapical flagella and single large cup-shaped chlo-roplast situated basally that occupies around2/3 of the cell volume. C. reinhardtii cells havea highly polarized and stereotyped organizationcentered on their two basal bodies that residejust under the plasma membrane at the cell an-terior. The basal bodies nucleate the flagella andorganize the internal microtubule cytoskeletonwhose principal axes are defined by two- andfour-membered microtubule rootlets (Holmes

and Dutcher 1989). Basal bodies also coordi-nate the placement of the cell division planeand the microtubule-based phycoplast usedfor cytokinesis (Johnson and Porter 1968; Ehleret al. 1995; Ehler and Dutcher 1998). Besidesapical-basal polarity, C. reinhardtii cells alsohave radial polarity that is evident from an eye-spot located equatorially and always positionedalong the four-membered microtubule rootletemanating from the younger of the two basalbodies (Holmes and Dutcher 1989). The eye-spot allows swimming cells to perceive the di-

Chlamydomonas reinhardtii

Gonium sociale

A B

Gonium pectorale

Volvolina steinii

Pandorina morum

Platydorina caudata

Volvox globator

Volvox rousseletti

Volvox barberi

Yamagishiella unicocca

Eudorina elegans

Pleodorina thompsonii

Pleodorini starii

Pleodorini indica

Eudorina illinoisensis

Eudorina cylindrica

Eudorina elegans

Volvox gigas

Volvox powersiiOogamy

Partial germ-somadivision of labor

Anisogamy

ECM expansion

Full inversion

Gonium

Pandorina

Eudorina

Pleodorina

Organismal polarity and basal body rotation

Genetic modulation of cell number

Incomplete cytokinesis

Cell–cell attachmentsPartial inversion

Asymmetric division

Bifurcated embryonic cell cycle

Complete germ-soma division of laborPleodorina japonica

Pleodorina californica

Volvox aureus

Volvox obversus

Volvox dissipatrix

Volvox tertius

Volvox africanus

Volvox carteri

Volvox carteri

Eudorina unicocca

Figure 3. Multicellular innovations in the volvocine algae. (A) Cladogram based on data in Kirk (2005) andUmen and Olson (2012) showing representative volvocine algal lineages with shading used to highlight differentlevels of organization in each genus. Note that the genus Volvox is polyphyletic with at least three independentlyderived clades. (B) Simplified cladogram based on data in Kirk (2005) showing key innovations in the stepsleading from C. reinhardtii to V. carteri. Schematic cartoons of each genus are also shown.

J.G. Umen


rection of illumination and signal to the flagellato enable phototaxis (Hegemann 2008). Thebasic cellular architecture and polarity of C.reinhardtii cells is conserved in colonial specieswith some modifications, and is likely to haveplayed a role in establishing organismal-levelpolarity.

The wall surrounding C. reinhardtii cells iscomposed mostly of hydroxyproline-rich glyco-proteins (HRGPs) that form rod-shaped poly-proline-II helices with globular domains atone or both ends (Adair et al. 1987; Lee et al.2007). As described below, HRGPs are also ma-jor components of the extracellular matrix ofVolvox and other colonial volvocine algae (Hall-mann 2003).

Like many green algae, Chlamydomonascells and nearly all volvocine algae divide by amechanism called multiple fission (or palint-omy) that is described in the Unique Aspectsof Multicellularity in Plants and Green Algaesection and whose features are intimately relat-ed to multicellularity in volvocine algae. Mostimportant, multiple fission produces clonalclusters of 2n daughter cells within a commonmother cell wall at the end of every divisioncycle. Clonality (versus aggregation) aligns thefitness of each genome in the colony and largelyobviates genetic conflicts between cells over re-productive potential (Buss 1987; Queller 2000).Multiple fission also explains why cell numberper colony in volvocine algae is usually a powerof 2 (i.e., 4,8,16,32,64,128, . . .).

A second important feature of multiple fis-sion is the spiral-like pattern of successive cleav-age divisions caused by rotation of the mitoticspindle axis by 90˚ in daughters with respectto mothers after each division cycle. As a result,postmitotic daughter cells end up clustered in aconfiguration with their basal bodies and flagel-la oriented inward (Johnson and Porter 1968;Kirk et al. 1991). Although this inside-out con-figuration presents no obstacle for individualC. reinhardtii cells that soon hatch from theirmother cell wall and swim their separate ways, itis a topological constraint for colonial species inwhich cells remain connected through cytoplas-mic bridges. The morphogenetic process of in-version that is discussed below is an innovation

that overcomes this constraint and is one of themost remarkable aspects of volvocine algal de-velopment and evolution.

A third aspect of multiple fission that hasimpacted the evolution of multicellularity inVolvocine algae is the relationship between cellsize and division. In C. reinhardtii, there is in-herent flexibility in cell division so that num-bers of daughters can range from 2 (1 division)to 16 or even 32 (4 or 5 divisions) dependingon mother cell size. A sizer mechanism oper-ates to ensure that the appropriate number ofS phase and mitosis cycles is executed to pro-duce daughters of uniform size (Donnan andJohn 1983; Umen 2005). In multicellular spe-cies, cell number per colony is much less vari-able, so the flexibility seen in C. reinhardtii hasbeen overridden by a more stereotyped relation-ship between mother cell size and the initiationof cell division to maintain colony uniformity.

As noted above, during division, Chlamydo-monas cells resorb their flagella and cannotswim. Although this temporary lack of motilityprobably does not impact the fitness of Chla-mydomonas, it may do so for the larger colo-nial volvocine genera such as Pleodorina andVolvox that are thought to have evolved germ-soma specialization as a response to their ances-tral flagellation constraint (Koufopanou 1994).As colonies grow larger, embryonic cell divi-sions take longer to complete, meaning thatthey must endure increasing lengths of timespent immotile and sinking in the water columnduring mitosis. The evolution of a permanentlyflagellated caste of somatic cells to provide con-tinuous motility is an innovation that is likelyto have enabled the evolution of ever-larger col-onies without suffering the cost of lost motilityduring cell division.

Multicellular Innovations in the VolvocineLineage Are Modifications of UnicellularVolvocine Algal Cell Biology

In his review of multicellularity and the evolu-tion of Volvox, Kirk (2005) outlines 12 steps thatdefine the minimum changes or innovations re-quired to convert a C. reinhardtii-type cell to amulticellular V. carteri spheroid. These innova-



tions are described below in an updated contextthat is informed by knowledge of the genomesfrom these two species and new findings thathave been published in the interim. Two addi-tional innovations (anisogamy and oogamy)have also been added (Nishii and Miller 2010).

Six of the 12 innovations outlined by Kirk(2005) occur in the initial multicellular transi-tion from Chlamydomonas to Gonium. The factthat half of the innovations in the lineage oc-curred at this step suggests that forming smallintegrated functional colonies might be themost difficult aspect of the transition to multi-cellularity. The idea of a bottleneck at this step isconsistent with the finding that the simplestcolonial forms are all basal to the more complexvolvocine genera. This basal positioning canbe interpreted to mean that Gonium-like colonyorganization evolved successfully just once, andthe more complex volvocine genera arose justonce from among the Goniaceae (Herron andMichod 2008; Hayama et al. 2010). The appar-ent difficulty of the transition between uni-cellular Chlamydomonas and colonial Goniumappears to contradict findings of experimentalevolution in which unicellular yeasts and Chla-mydomonas could be converted to facultativecolonial forms in a short number of generationsby artificial selection (Ratcliff et al. 2012, 2013).However, this discrepancy is not so paradoxicalconsidering the degree of evolved functionalitythat arose in Gonium that distinguishes it fromthe disorganized cell aggregates produced dur-ing experimental evolution. It may be easy toform such aggregates, but they may not trulyrecapitulate the first steps that led to the evolu-tion of Gonium-like colonies.

Some of the innovations below are groupedtogether in cases in which they are related, so thenumbering does not correspond to that de-scribed by Kirk (2005), but the progression isthe same.

1. Genetic modulation of cell number. As de-scribed above, Chlamydomonas cells showinherent flexibility in mother cell size andnumber of mitotic cell divisions, producingtwo daughters under poor growth condi-tions and up to 16 daughters under favorable

growth conditions (Donnan and John 1983;John 1987). Colonial species, on the otherhand, have more stereotyped numbers of di-visions to produce colonies that typicallyvary in cell number by a factor of 2 (Cole-man 2012). Moreover, unlike Chlamydomo-nas cells that are competent to divide as soonas they double in size, cells in colonial spe-cies must grow large enough between repro-ductive cycles so that new colonies can beformed with the appropriate number of cells.In Chlamydomonas, the retinoblastoma tu-mor suppressor pathway controls cell sizeand division number (Umen and Good-enough 2001; Umen 2005; Fang et al. 2006),and may have been modified in colonial spe-cies to increase the minimum size thresholdat which division can occur.

2. Incomplete cytokinesis. Incomplete cytoki-nesis is observed in all colonial volvocinespecies and can result in either temporaryor perduring cytoplasmic connections orbridges between postmitotic cells. Ultra-structural studies show that the bridges arespatially organized within and between cells,and contain specialized substructures thatmay represent mechanical reinforcementsand/or facilitate intercellular communica-tion much like plasmodesmata in plants.Bridges can also be removed or resolved asoccurs postembryonically in some species(Green et al. 1981; Coleman 2012).

3. and 4. Organismal polarity and basal bodyrotation. 16-cell Gonium colonies that have aplanar 4 � 4 arrangement of cells show po-larity that is evident by comparing the ar-rangements of basal bodies and flagella ofinterior versus peripheral cells. The four cellson the interior of the colony have their basalbodies oriented with 180˚ rotational sym-metry and beat in opposite directions in abreast stroke motion just as they do in Chla-mydomonas. The 12 cells on the edge, how-ever, rotate their basal bodies and flagella to aparallel orientation that cause the colony torotate in a pinwheel-like fashion as it swimsforward (Greuel and Floyd 1985). The basalbody reorientation observed in Gonium pe-

J.G. Umen


ripheral cells occurs in all of the motile cellsof spheroidal volvocine genera and is crucialfor coordinated swimming (Hoops 1984;Ueki et al. 2010). Visible signs of organismalpolarity are present in other aspects of colo-ny development in larger volvocine generathat appear during inversion, germ-somadifferentiation, and asymmetric cell divisionas described below.

5. and 6. Partial and complete inversion. As aresult of the spiral division pattern of volvo-cine algal cells described above, postmitoticembryos have their apical ends and basalbodies clustered on the interior of the em-bryo (or the concave surface for Gonium).This configuration of interior-facing basalbodies and flagella must be reversed by in-version for the embryo to swim. Inversioninvolves a coordinated series of cell-shapechanges resulting in a complete reversal ofembryonic curvature. Although inversionhas been best studied in Volvox, it has alsobeen observed in all other colonial volvocinegenera (Hallmann 2006a). The partial in-version process that causes a reversal of cur-vature in Gonium embryos is presumablyrelated to the process of full inversion inspheroidal volvocine embryos in which a hol-low ball of cells turns itself completely insideout.

Full inversion of spheroidal colonies hasbeen examined carefully in V. carteri and V. glo-bator (Viamontes and Kirk 1977; Nishii andOgihara 1999; Nishii 2003; Ueki and Nishii2009; Hohn and Hallmann 2011), which usedifferent but related mechanisms to invert.In both species, inversion is driven by coordi-nated cell-shape changes that move through thespheroid along the anteroposterior axis anddrive cell sheet bending and involution. Inver-sion in many ways parallels gastrulation in an-imals in which similar cell-shape changes areused to remodel the hollow, ball-like blastulaand generate primordial embryonic germ layersof the gastrula (Solnica-Krezel and Sepich2012). The sequence of events and types ofcell-shape changes that drive inversion in V. car-teri (termed type A inversion) and V. globator

(termed type B inversion) differ substantially(Hohn and Hallmann 2011) and underscorethe fact that these two species belong to inde-pendent clades of Volvox (Fig. 3A). In both typesof inversion, cytoplasmic bridges are crucial formaintaining mechanical integrity of the cellsheets and fixed relationships between neigh-boring cells.

V. carteri inversionless mutants have pro-vided insights into the processes governingtype A inversion. One such mutant, invA, affectsa volvocine algal kinesin that localizes to the cy-toplasmic bridge region of cells (Nishii 2003).The function of InvA protein is postulated toinvolve moving the cytoplasmic bridges relativeto the rest of the cell along longitudinal micro-tubules to effect cell-shape changes and tissuebending. It is noteworthy that the highly similarC. reinhardtii ortholog of invA, IAR1, can func-tionally substitute for invA in Volvox despiteC. reinhardtii lacking any known process anal-ogous to inversion (Nishii and Miller 2010).Additional inv loci define a second extraembry-onic function required for inversion associatedwith enlargement of the glycoprotein vesicle thatencapsulates each embryo. When this vesiclefails to enlarge as it does in invB and invCmutants, the inverting embryo is physically con-strained and becomes stuck midway throughinversion (Ueki and Nishii 2008, 2009). invBand invC encode enzymes involved in sugartransport or metabolism and both have C. rein-hardtii orthologs. It is likely that the C. rein-hardtii orthologs of invB and invC proteins areinvolved in cell wall biosynthesis, but this idearemains to be tested.

7. and 8. Transformation of the cell wall intoECM and ECM expansion. Although indi-vidual Gonium cell walls have a tripartitestructure similar to that of Chlamydomonas,theyalso have an added outer layer that main-tains colony cohesion and contains special-ized regions of electron-dense material atcell–cell attachment points (Nozaki 1990).

Further modifications of the ECM arefound in the more complex colonial genera.These include formation of a colony boundarylayer in spheroidal genera (Pandorina, Eudori-



na, Pleodorina, and Volvox) that resembles thetripartite structure of the cell wall surroundingindividual Chlamydomonas and Gonium cells.In Eudorina, Pleodorina, and Volvox, the ECMis expanded to fill large spaces between cells andwithin the colony interior (Coleman 2012). Al-though Volvox colonies have more than onethousand cells, ECM expansion accounts for.99% of their volume and makes them six or-ders of magnitude larger than an individualC. reinhardtii cell. ECM expansion in volvocinealgae has some parallels with vacuolar expan-sion in land plants that also allows massive or-ganismal growth without the addition of newcells or significant protein biomass (Robagliaet al. 2004). Although the ECM in volvocinealgal colonies appears transparent to the eye, ithas an elaborate internal structure that has beeninvestigated in detail for V. carteri in which eachcell has its own ECM domain and the colony as awhole has distinguishable layers of ECM (Kirket al. 1986; Hallmann 2003). Paralleling thisECM expansion has been a notable increase inthe number of ECM genes from two families inV. carteri relative to counterparts in C. reinhard-tii (Prochnik et al. 2010). The expanded familiesare pherophorins that have structural and sig-naling roles (Hallmann 2006b) and the VMPs(Volvox metalloproteinases) that function inECM remodeling (Heitzer and Hallmann 2002).There are dozens of other uncharacterized vol-vocine algal protein families that are differen-tially expanded in V. carteri that may also play arole in multicellularity (Prochnik et al. 2010).

9. and 10. Partial and complete germ-somadifferentiation. Perhaps the most well-stud-ied multicellular innovation in volvocine al-gae is the differentiation of germ and somaticcell types that is observed in the genera Pleo-dorina and Volvox.

The genetic basis for germ-soma differenti-ation has been investigated in V. carteri in whichdevelopmental mutants exist that alter cell fate.In regA (somatic regenerator) mutants, somaticcells form normally after embryogenesis, butthen redifferentiate into gonidia; they withdrawtheir flagella, grow, and subsequently divideinto new embryos (Starr 1970; Huskey and Grif-

fin 1979). regA is a nuclear protein that is relatedto transcriptional repressors and expressed insomatic cells and required to maintain theirfate (Kirk et al. 1999; Stark et al. 2001). regAbelongs to a family of VARL (Volvocine algaeregA-like) proteins with 14 members in Volvoxand 12 in Chlamydomonas (Duncan et al. 2006,2007). Phylogenies of VARL proteins indicatecomplex patterns of gene duplication and lossin both algal lineages, with no ortholog of regApresent in C. reinhardtii. It has been hypothe-sized that VARL proteins evolved in unicellularand simple colonial species to coordinate tem-poral cell differentiation with environmentalcues (Nedelcu and Michod 2006).

A second class of mutants in V. carteri is atlag loci (late gonidia), whose gene productshave not been identified. In lag mutants, cellsthat are normally destined to form gonidia in-stead adopt a somatic or quasi-somatic cell fateand become flagellated and stop growing. Even-tually, however, lag mutant gonidia withdrawtheir flagella and redifferentiate as reproductivecells (Kirk 1988).

The phenotypes of regA and lag mutantssuggest that establishment and maintenance ofcell fate in V. carteri are separable functions con-trolled by multiple loci (Kirk 1988). Continuedwork on these mutants and isolation of newregulatory loci promises to further clarify theorigins of cell-type specification in this lineage.

11. and 12. Asymmetric cell division and abifurcated cell division program. As de-scribed above, the genus Volvox is poly-phyletic with germ-soma differentiationestablished by different mechanisms in sep-arate groups (Desnitski 1995; Coleman2012). In V. carteri and its close relatives,the vegetative germ line is established earlyin embryonic development by means ofasymmetric cell divisions. At embryonic di-vision 6 (32! 64 cell stage), a subset of 16anterior cells in the embryo divides asym-metrically to produce a large and smalldaughter. The large daughters divide asym-metrically one or two more times and thencease dividing, whereas the smaller daugh-ters continue dividing five or six more

J.G. Umen


times along with the remaining embryoniccells that divide symmetrically. Asymmetriccell divisions combined with early exit fromthe cell cycle of the large daughters resultsin an embryo with approximately 2000small cells and 16 large cells. The 16 largecells will mature and differentiate as gonid-ia, whereas the small cells become somatic(Kirk et al. 1991).

Mutants that disrupt asymmetric cell divi-sion have been identified and at least one locus,glsA, has been cloned (Miller and Kirk 1999).glsA is in a conserved family of eukaryotic pro-teins that contain a dnaJ-like chaperone domainthat mediates an essential interaction withHSP70-type heat shock proteins (Cheng et al.2005), as well as SANT domains that mediatechromatin association in V. carteri (Pappas andMiller 2009). Although glsA is distributed uni-formly through the embryo, its HSP70A partneris distributed in a decreasing anterior-to-poste-rior concentration gradient at the time of asym-metric division and may provide positionalcues for localizing asymmetric cell divisions tothe anterior of the embryo (Cheng et al. 2005).During mitosis, glsA localizes to the spindlesuggesting that it might be directly involved inspindle positioning, but this localization wasinsufficient to mediate its function that mayalso involve transcriptional or translational reg-ulation (Cheng et al. 2005; Pappas and Miller2009). Although it is not known to divide asym-metrically, C. reinhardtii has a glsA ortholog,GAR1, that can complement a glsA mutant(Cheng et al. 2003). The function of GAR1 inC. reinhardtii is not known, but the above find-ing suggests that at least some of the machineryfor asymmetric cell division existed in the uni-cellular ancestor of colonial volvocine algae.

Elegant experiments involving manipula-tion of embryonic division and daughter cellsize showed that cell size (rather than positionwithin the embryo) is the primary determinantof cell fate in V. carteri (Kirk et al. 1993). Thenature of this cell-size signal and how it controlssubsequent differentiation is unknown, but itmay be coupled to the retinoblastoma pathwaythat functions in Chlamydomonas to sense and

control cell size (Fang et al. 2006). Interestingly,comparative genomic studies of cell cycle genesrevealed an expansion of the D-type cyclinsin V. carteri compared with C. reinhardtii (Proch-nik et al. 2010). D-type cyclins in plants andanimals are activating subunits for cyclin-de-pendent kinases that regulate retinoblastoma-related proteins through phosphorylation (Gut-zat et al. 2012). Whether the expanded D-cyclinrepertoire in V. carteri enabled modifications ofthe embryonic cell division program or contrib-utes to germ-soma differentiation remains to bedetermined.

13. and 14. Anisogamy and oogamy. Volvo-cine algae recapitulate the transition fromisogamy to anisogamy to oogamy and are amodel system in which to investigate theevolution of these traits (Nozaki et al.2000; Umen 2011). C. reinhardtii is isoga-mous with two mating types, plus and mi-nus, that are determined by a complex, mul-tigenic mating locus (MT), with MTþ andMT2 haplotypes (Umen 2011; De Hoffet al. 2013). Gonium and Pandorina arealso isogamous, whereas Eudorina and re-lated genera show a mixture with someisogamous species and anisogamous spe-cies (Coleman 2012). The larger and morecomplex spheroids in the genus Pleodorinaare anisogamous, whereas all Volvox speciesare oogamous (Nozaki 1996). Other thanC. reinhardtii MT, V. carteri is the only othervolvocine algal mating locus characterizedto date. V. carteri MT is syntenic with Chla-mydomonas MT, but its male and femalehaplotypes are genetically more complexand share several properties with sex chro-mosomes (Ferris et al. 2010). The develop-mental and evolutionary bases for oogamyand anisogamy that are controlled by MTremain to be determined, but may involvethe transcription factor MID (minus dom-inance) that specifies sexual differentiationin Chlamydomonas and is found in eitherthe minus mating type or males of all vol-vocine algae examined to date (Ferris andGoodenough 1997; Nozaki et al. 2006a;Hamaji et al. 2008, 2013).



Future Perspectives for Volvocine Algaeand Multicellularity

Volvocine algae allow detailed inferences to bemade regarding the cell biological and develop-mental events required for the transition tomulticellularity, but have only just begun to beexploited to gain mechanistic insights into howunderlying processes evolved. The finding thatdevelopmental mutants in Volvox can be com-plemented with orthologous genes from Chla-mydomonas should spur further research intohow these genes are used in Chlamydomonasand what has allowed their function to be al-tered for development in Volvox. The genomesof other volvocine algae will also soon be se-quenced and help clarify the origins of genefamilies that contribute to developmental inno-vations in the lineage. The identification andcharacterization of new developmental mutantsand investigation of existing mutants in bothV. carteri and C. reinhardtii promises to furtherdeepen our understanding of how multicellu-larity and evolutionary innovation occur at anunprecedented level of detail.

CONCLUDING REMARKS

The specific mechanisms used to achieve mul-ticellular innovation differ between lineages,but some of the emerging themes and principlesdiscovered in charophyte and volvocine algaemay be more universal. First, it is clear thatunderstanding the transition to multicellularityrequires a detailed understanding of the life cy-cle, cell biology, and ecology of simpler ances-tor(s) that gave rise to multicellular descendantlineages. Indeed, research into the origins ofanimal multicellularity have been transformedby investigating their closest unicellular ances-tors including choanoflagellates and others(King et al. 2008; Fairclough et al. 2013; Sebe-Pedros et al. 2013; Alegado and King 2014).Gene family expansion and novel protein do-main organization are observed in metazoansand embryophytes and associated with somekey innovations (Floyd and Bowman 2007; De-gnan et al. 2009; Srivastava et al. 2010). Howev-er, wholesale changes in numbers or types of

protein families and domain structures neednot underlie the innovations required to achievemulticellularity as evidenced by the similar-ity between the C. reinhardtii and V. carterigenomes (Prochnik et al. 2010), and shared or-igins with C. reinhardtii of key developmen-tal regulators in V. carteri. Similar ideas haveemerged from investigating the origins of devel-opmental pathways from land plants that arosein charophyte algae or the earliest embryophytelineages (Preston et al. 2011; Pires and Dolan2012; Bowman 2013). Third, lineage-specificprotein families such as the Chlamydomonascell wall/Volvox ECM proteins may provide aparticularly rich source of raw material for evo-lutionary innovation. Although very little isknown about such proteins in the volvocine lin-eage, there are many examples of proteins whoseorigins are unique to the animal/choanoflagel-late lineage that play key roles in metazoan de-velopment (King et al. 2008; Fairclough et al.2013). It can be anticipated that full genomesequences of charophyte algae will reveal addi-tional examples of such proteins that contributeto the evolution of land plants.

ACKNOWLEDGMENTS

I thank Gavriel Matt and Patrick Keeling forcomments and editorial suggestions on thismanuscript, and Joe Heitman for helpingto involve me in this collection. Research onvolvocine algal cell biology and evolution inmy laboratory is supported by National Insti-tutes of Health grants R01GM092744 andR01GM078376.

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