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EVOLUTION & DEVELOPMENT 15:6, 466–474 (2013) DOI: 10.1111/ede.12055 The evo‐devo of multinucleate cells, tissues, and organisms, and an alternative route to multicellularity Karl J. Niklas,a,* Edward D. Cobb,a and David R. Crawfordb a b Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA Department of Philosophy, University of Bristol, Bristol, BS 8 1TB, UK *Author for correspondence (e‐mail: [email protected]) SUMMARY Multinucleate cells, tissues, or organisms occur in 60 families of land plants and in five otherwise diverse algal lineages (Rhodophyceae, Xanthophyceae, Chlorophyceae, Ulvophyceae, and Charophyceae). Inspection of a morphospace constructed out of eight developmental processes reveals a large number of possible variants of multinucleate cells and organisms that, with two exceptions, are represented by one or more plant species in one or more clades. Thus, most of these permutations of developmental processes exist in nature. Inspection of the morphospace also shows how the siphonous body plan (a multinucleate cell with the capacity for indeterminate growth in size) can theoretically serve as the direct progenitor of a multicellular organism by a process similar to segregative cell division observed in siphonocladean algae. Using molecular phylogenies of algal clades, different evolutionary scenarios are compared to see how the multicellular condition may have evolved from a multinucleate unicellular progenitor. We also show that the siphonous progenitor of a multicellular organism has previously passed through the alignment‐of‐fitness phase (in which genetic similarity among cells/nuclei minimizes internal genomic conflict) and the export‐of‐fitness phase (in which genetically similar cells/nuclei collaborate to achieve a reproductively integrated multicellular organism). All that is theoretically required is the evolutionary acquisition of the capacity to compartmentalize its cytoplasm. INTRODUCTION coenocytes (Biao et al. 1988; Ding et al. 1999; Niklas and Kutschera 2009), i.e., embryophytes are multinucleate supra‐ cellular organisms whose continuous cytoplasm is incompletely partitioned by a cell wall infrastructure (Niklas and Kaplan 1991). The appearance of multinucleate cells in 177 land plant species representing 60 families (Beer and Arber 1919, see however Burkholder and McVeigh 1941) and in algal clades as different as the Rhodophyceae (e.g., Griffithsia), Xanthophyceae (e.g., Vaucheria), Chlorophyceae (e.g., Pediastrum), Ulvophyceae (e.g., Cladophora), and Charophyceae (e.g., Nitella) raises a number of important but as yet unresolved questions. For example, are the mechanisms giving rise to multinucleate cells homologous across different clades? Does this condition provide an analogue to autopolyploidy without the early negative consequences of polyvalent chromosomes? Indeed, does it provide any metabolic or developmental advantage, or does it simply appear under conditions of neutral or relaxed selection? Did the siphonous body plan evolve in some cases into a multicellular body plan, or is it evolutionarily derived from the latter? These and other questions have been addressed in different ways depending on the frame of reference of different authors (e.g., Strasburger 1913; McNaughton and Goff 1990; Mine et al. 2008). However, all perspectives share a comparative approach because of the multiple evolutionary origins of multinucleate cells, tissues, and organisms. The division of the nucleus is synchronized with the division of the cytoplasm in the majority of plant and animal cells. However, many fungi, algae, plants, and animals are composed entirely or in part of multinucleate cells, e.g., the giant amoeba Chaos chaos (aka C. carolinensis), the multicellular green alga Cladophora, and the basal cell of the Pinus embryo. Likewise, there are tissues and entire organisms that are transiently composed of multinucleate cells, e.g., the free‐nuclear division phase in endosperm and gymnosperm megagametophyte development. These occurrences demonstrate that karyokinesis is not inextricably coupled to cytokinesis (as noted by Strasburger 1913), in much the same way that the duplication of chloroplasts and mitochondria is not intrinsically coupled to the division of either the nucleus or the cytoplasm (Imoto et al. 2011). Likewise, there are phylogenetically unrelated organisms that consist of a single multinucleate cell that can grow indefinitely in size, e.g., the siphonous (¼ coenocytic) ulvophycean alga Derbesia and the xanthophycean alga Asterosiphon. Some of these organisms are externally and internally intricate, which shows that multicellularity is not required to achieve morphological or anatomical complexity (Fig. 1). Finally, the distributions of plasmodesmata within the land plants (embryophytes) establish different but interconnecting physiological domains that are functionally analogous to 466 © 2013 Wiley Periodicals, Inc. Niklas et al. The evo‐devo of multinucleate cells, tissues, and organisms, and an alternative 467 Fig. 1. A portion of the thallus of the siphonous, holocarpic green alga Caulerpa cactoides. The objective of this paper is to explore the developmental and evolutionary implications of the multinucleate condition and the siphonous body plan in plants, which are here broadly defined as photosynthetic eukaryotes to include the algae as well as the embryophytes. We specifically focus on the possible origin of a multicellular organism from a siphonous ancestor. In order to achieve this objective, (1) a previously published morphospace for the body plans of plants elaborated to identify all possible variants of the multinucleate condition, (2) the existence of these variants in the different plant clades is assessed, (3) the phylogenies of different plant clades are examined to determine whether the siphonous body plan could give rise to a multicellular organism, and (4) the evolutionary advantages and disadvantages of multinucleate cells are reviewed and discussed to determine whether such a transition provides an evolutionarily expeditious route to multicellularity. A BODY PLAN MORPHOSPACE A morphospace is a representation of all theoretically possible forms of a specific organism or group of organisms (McGhee 1999). Each axis in a morphospace represents a variable that describes a phenotypic character (with one or more character states). Each point in a morphospace represents an individual with the characters states specified by the variables identified by intersecting axes. A morphospace for the body plans of plants was previously constructed using four developmental features (Niklas 2000; see also Niklas and Newman 2013): (1) whether or not cytokinesis and karyokinesis are synchronous, (2) whether or not cells remain aggregated after they divide, (3) whether or not symplastic continuity is maintained among the cross walls of neighboring cells, (4) whether or not individual cells continue to grow indefinitely in size (Fig. 2). The juxtaposition of these features identifies four Fig. 2. A morphospace for the four major plant body plans shown in bold (unicellular, siphonous/coenocytic, colonial, and multicellular) resulting from the intersection of five developmental processes (e.g., karyokinesis and cytokinesis). Note that the siphonous/coenocytic body plan may evolve from a unicellular or a multicellular progenitor. The lower panels dealing with the plane of cell division, localization of cellular division, and symmetry pertain to the evolution of complex multicellular organisms. Adapted from Niklas (2000). major body plans, each of which can be in theory either uninucleate or multinucleate: (1) the unicellular body plan, which is characterized by the complete separation of cells after division coupled with determinate growth in cell size, (2) the siphonous body plan, which is characterized by the indeterminate growth of a unicellular body plan, (3) the colonial body plan, which is characterized by the lack of symplastic continuity among normally aggregated cells, and (4) the multicellular body plan in which all adjoining cells have the potential to establish and maintain symplastic continuity. The addition of a fifth developmental feature –– the orientation of cell division –– distinguishes among the different tissue constructions of the multicellular body plan: (1) the unbranched filament, which results when each cell has the capacity to divide in only one plane of reference with respect to the body axis, (2) the branched filament (with or without a pseudoparenchymatous tissue construction), which requires that each cell has the ability to divide in two planes of reference, and (3) the parenchymatous tissue construction, which requires that cells have the ability to divide in all three planes of reference (Fig. 2). 468 EVOLUTION & DEVELOPMENT Vol. 15, No. 6, November–December 2013 A review of algal cytology and morphology summarized by Smith (1950), Fritsch (1965), Lee (2008), and Graham et al. (2009) shows that all but two of the 14 theoretically possible variants are represented by one or more algal species. For example, the unicellular multinucleate variant with determinate growth is represented by the chlorophycean alga Bracteacoccus and the ulvophycean alga Chlorochytridium; the colonial multinucleate body plan is represented by the chlorophycean algae Pediastrum and Hydrodictyon; the siphonous body plan is represented by the ulvophycean alga Caulerpa and the xanthophycean alga Vaucheria; and the multicellular multinucleate (siphonocladous) condition is represented by the rhodophycean alga Griffithsia and the ulvophycean alga Cladophora (see Graham et al. 2009). Among the siphonocladous variants differing in tissue construction, the unbranched and branched filamentous condition are represented by the ulvophycean algae Urospora and Acrosiphonia, respectively; the multicellular body plan with a pseudoparenchymatous tissue construction is represented by species assigned to the ulvophycean alga Codium. The two variants that are not represented by any extant or extinct species are the uninucleate siphonous body plan and the parenchymatous siphonocladous variants. The absence of the former may be the result of physiological constraints imposed by the volume of cytoplasm that a single nucleus can sustain, a hypothesis proposed by Julius Sachs (1892; see also Baluška et al. 2012). An explanation for the absence of a parenchymatous siphonocladous body plan is more problematic. Hommersand and Fredericq (1990) suggest that morphogenetic constraints imposed by the necessity of forming cross walls to sequester reproductive cells is responsible for the lack of parenchymatous tissues in the rhodophytes. A more detailed rendering of the multinucleate domain within the plant body plan morphospace is achieved with the addition of three developmental features, each of which has two character states: (1) whether or not the adult plant dies after reproducing (i.e., holocarpic or non‐holocarpic life cycles), (2) whether or not the plant produces uninucleate or multinucleate asexual propagules, and (3) whether or not all cells in a body plan are multinucleate. The addition of these three developmental features provides a more diverse landscape for inspection (Fig. 3). Nevertheless, a survey of the literature once again summarized by Smith (1950), Fritsch (1965), Lee (2008), and Graham et al. (2009) shows that, with the aforementioned two exceptions, all of the remaining variants exist in nature. For example, the siphonous holocarpic variant with uninucleate propagules is represented by xanthophycean alga Botrydium and the chlorophycean alga Characiosiphon, whereas its counterpart with multinucleate propagules is represented by the xanthophycean alga Vaucheria and the chlorophycean alga Protosiphon. The presence of uninucleate and multinucleate cells in the same organism is represented by the external and rhizoidal cells of the ulvophycean alga Ulva and the apical and sub‐apical cells of the florideophycean alga Gigartina. INFERRED CHARACTER POLARITIES The preceding review permits two conclusions. First, as noted, almost all of the theoretically possible multinucleate variants are Fig. 3. An elaboration of the multinucleate condition among the four body plans (see Fig. 2) resulting from the intersection of three additional developmental processes (e.g., holocarpic life cycle) that result in 28 possible variants. A survey of the plant literature shows that all but two of all possible variants exist in one or more clades. Niklas et al. The evo‐devo of multinucleate cells, tissues, and organisms, and an alternative represented by one or more plant species (i.e., the multinucleate condition has been extensively evolutionarily explored), although it is evident that some plant lineages occupy a greater volume of this morphospace than others. Second, extensive convergence (or homoplasy) among these variants has occurred, particularly between the Xanthophyceae and the Ulvophyceae. This raises a number of questions. For example, the conventional view concerning the evolutionary origins of multicellularity is that a unicellular progenitor evolved into a colonial organism that subsequently evolved multicellularity as a consequence of passing through an alignment‐of‐fitness phase and an export‐of‐ fitness phase (Grosberg and Strathman 2007; Folse and Roughgarden 2010), i.e., “unicellular ) colonial ) multicellular” transformation series of body plans. Evidence for this scenario is found among a number of current phylogenies for the rhodophytes, stramenopiles, and chlorobionta (Niklas and Newman 2013). In addition, an “unbranched ) branched ) pesudoparenchymatous ) parenchymatous” tissue transformation series occurs within lineages that have achieved complex multicellularity (Niklas and Newman 2013). The evolution and development of the volvocine green algae provide several cases of the unicellular (uninucleate) ¼> colonial (uninucleate) transition series (Huxley 1912; Kirk 2005; Herron and Michod 2008). The ancestral state of volvocines is unicellular (e.g., Chlamydomonas reinhardtii). Transformation of the unicellular cell wall into and extracellular matrix (seen in the Tetrabaenaceae ¼> Goniaceae ¼> Volvocaceae transformation series), incomplete cytokinesis (seen in the Goniaceae ¼> Volvocaceae transformation series), and the appearance of additional derived traits produce forms ranging from simple cellular aggregates (e.g., Tetrabaena socialis) to colonies with complex, asymmetric cell division, to organisms with full germ‐ soma division of labor (e.g., Volvox carteri) (Kirk 2005; Herron and Michod 2008). Models for these transitions in the volvocines focus on “cooperation‐conflict‐conflict mediation cycles” that allowed these algae to achieve both the alignment‐of‐fitness phase at the cellular level and the export‐of‐fitness phase at the colony level (Herron and Michod 2008). Evidence for the transition from colonial to multicellular plant life‐forms is reviewed by Niklas and Newman (2013). In contrast, little is known about the transformation series involving the siphonous/coenocytic body plan, particularly the possibility that it may serve as the progenitor of a multicellular body plan. Inspection of the more elaborate multinucleate morphospace shows that the siphonous body plan can be derived from a unicellular uni‐ or multinucleate progenitor with the acquisition of indeterminate growth in cell size (Figs. 2–3). However, it can also serve as the ancestral condition for a multicellular organism (by the developmental elaboration of cross wall formation) just as it may be evolutionarily derived from a multicellular plant (by the developmental suppression of cross wall formation). Therefore, the “siphonous ) multicellular” and the “multicellular ) siphonous” body plan transforma- 469 tion series are both theoretically possible. Indeed, the “siphonous ) multicellular” transformation series actually occurs during the ontogeny of siphonocladean algae by means of a process called segregative cell division (Fig. 4), which is posited to have evolved by the co‐option of a wound healing response mechanism (Børgesen 1905; Le Claire 1982; Graham et al. 2009) as for example the wound response of the siphonous green alga Boergesenia forbesii. Analogs of segregative cell division are seen in endosperm free‐nuclear development (Schnarf 1931), the early embryology of gymnosperms such as Welwitschia (Bower 1881), Drosophila embryogenesis preceding the formation of the blastoderm (Zalokar and Erk 1976; Foe and Alberts 1983), and zoospore differentiation of Blastocladiella and other chytrids (Lessie and Lovett 1968). These and other speculations about the evolution of multinucleate cells, tissues, or organisms can be evaluated with parsimony‐based character state reconstructions of the phylogeny of selected plant lineages. Barring extensive character state reversals, it should be possible to infer whether the siphonous body plan is ancestral or derived in one or more algal lineages containing uni‐ and multinucleate unicellular, colonial, and multicellular species. Unfortunately, despite great advances made in algal phylogenetics (e.g., Lewis and McCourt 2004; Maistro et al. 2007; Cocquyt et al. 2010), sufficiently detailed parsimony‐based cytological and morphological character state reconstructions of most algal clades are unavailable. Indeed, detailed yet broad‐scale phylogenies based on molecular data are rare and difficult to produce, particularly for the algae, in part because the species used to construct algal phylogenies are often limited to those that can be cultivated under laboratory conditions (and may not be representative of the larger taxa they purportedly represent). Another limitation arises when one or more lineages within a phylogeny contain species with diverse body plans, since this can obscure inferences concerning which among alternative body plans may have been the progenitor of more evolutionarily derived variants (Fig. 5). Nevertheless, it is possible to examine hypothetical character polarities in light of the distribution of body plans on current Fig. 4. A schematic for segregative cell division in which the protoplasm of a multinucleate organism (left) simultaneously separates into spherical portions (middle) that develop new cell walls to form a multicellular (siphonocladous) body plan (right). Among siphonocladean algae, the resulting cells remain multinucleate. 470 EVOLUTION & DEVELOPMENT Vol. 15, No. 6, November–December 2013 Fig. 5. Possible difficulties in interpreting the character polarities among variants of different plant body plans (see insert to right) in a hypothetical plant phylogeny with three lineages (A – D) when more than one body plan resides on one or more basal lineages (lineages A – B). Arrows indicate theoretically possible and competing body plan transformation series the resolution of which requires knowing the transformation series within lineages A and B. reconstructions of algal phylogeny. For example, among the red algae, multinucleate cells occur in the multicellular Florideophyceae (e.g., Chondrus), but are unknown among species assigned to the Bangiophyceae, which most authorities consider to be ancient within this clade (Cole and Sheath 1990; Goff and Coleman 1990; Graham et al. 2009). It is reasonable therefore to suggest that the multinucleate condition is a derived condition within the red algae. Likewise, using a phylogeny for the Ulvophyceae based on seven nuclear genes, small subunit nuclear ribosomal DNA, and two plastid markers, Cocquyt et al. (2010) considered three hypotheses about the evolution of multicellularity, among which one posits a “siphonous ) multicellular” transformation series (Cocquyt et al. 2010, their Fig. 3). All three hypotheses share the assumption that the ancestral ulvophyte was a unicellular, uninucleate organism (represented by the prasinophytes), since “early‐branching lineages are of this type” [of organism] (Cocquyt et al. 2010). Another feature common to all three hypotheses is that multicellularity evolved multiple times. Inspection of their phylogeny shows that the unicellular, colonial, and multicellular uninucleate body plans are all represented by species in the Trebouxiophyceae, that the colonial multinucleate and the siphonocladous variants are represented by species in the Chlorophyceae, and that the siphonous body plan is represented in the Trentepohliales and in other phyletically affiliated orders (Fig. 6). This distribution of body plans is consistent with the traditional “unicellular (uninucleate) ) colonial (uninucleate) ) multicellular (uninucleate)” evolutionary scenario for the origin of multicellularity (Fig. 6). Indeed, Cocquyt et al. (2010) concluded that the siphonous body plan is an evolutionarily dead‐end body plan that was most likely derived from a unicellular ancestor. However, other algal phylogenies provide support for the evolution of a “siphonous ) multicellular” transformation series. For example, inspection of a phylogeny for the Tribonematales (Xanthophyceae) constructed by Maistro et al. (2007) based on the plastid genes rbcL and psaA shows that the unicellular uninucleate and the colonial uninucleate body plans (represented by Chlorellidium tetrabotrys and two species of Heterococcus) occur in what authorities consider to be ancient lineages, while the siphonous body plan (represented by Asterosiphon dichotomus and Vaucheria terrestris) occurs in species on shorter presumably more recent branches within this phylogeny. The multicellular uninucleate body plan (represented by species of Tribonema and Xanthonema) appears on even shorter branches (Fig. 7). It is therefore not unreasonable to Fig. 6. Multinucleate body plans (see insert) mapped onto a redacted phylogeny of the Ulvophyceae (based on the molecular phylogeny of Cocquyt et al. 2010) and the charophycean‐embryophyte (streptophyte) lineages within the chlorobionta (green algae). Note that all of the body plans known for each branch of this clade are mapped regardless of the particular species used to construct this phylogeny because there is no a priori assumption that the particular species used to construct the phylogeny are representative of their higher taxon. Theuninucleate unicellular body plan (represented by the prasinophytes for the entire clade and particularly by the prasinophycean alga Mesostigma for the streptophytes) is represented by species assigned to the earliest divergent lineages in both portions of this cladogram. Niklas et al. The evo‐devo of multinucleate cells, tissues, and organisms, and an alternative 471 Fig. 7. Multinucleate body plans (see insert) mapped onto a redacted phylogeny of the Tribonematales (based on the molecular phylogeny of Maistro et al. 2007) in the Xanthophyceae. Note that all of the body plans known for each branch of this clade are mapped regardless of the particular species used to construct the phylogeny because there is no a priori assumption that the particular species used are representative of their higher taxon. The uninucleate unicellular body plan and the uninucleate colonial body plan (represented by Chlorellidium and species of Heterococcus, respectively) occur at the bottom of the cladogram. The siphonous body plan maps on a branch represented by Asterosiphon and Vaucheria that precedes the earliest appearance of the multicellular uninucleate body plan represented by species of Xanthonema. suggest that, within the Tribonematales, the “unicellular or colonial (uninucleate) ) siphonous” transformation series evolved at least once. This inference is strengthened by the occurrence of siphonocladous species that achieve their multicellular condition ontogenetically by means of segregrative cell division. THE MULTINUCLEATE CONDITION: HOW AND WHY The evolutionary appearance and diversity of the multinucleate condition in unrelated plant clades raises a number of important questions, not the least of which is how and why it evolved multiple times, and whether it is an ancient or derived condition. There are two basic ways a multinucleate plant cell can be formed under normal physiological conditions: (1) as a result of repeated mitotic nuclear division unattended by cytokinesis (e.g., during the formation of the Plasmodium schizont stage, non‐articulated laticifers, endosperm, and Chara internodal cells), or (2) by the fusion or dissolution of plasma membranes (e.g., during the formation of syncytia as in the tapetum, the megagametophytic storage cells of the conifer Pseudotsuga menziesii, and the development of mammalian musculoskeletal system). The mechanism responsible for the first of these two routes to the multinucleate condition appears to differ among clades (Mine et al. 2008), although some basic commonalities exist (Baluška et al. 2012). Molecular studies of cytokinesis in the model embryophyte Arabidopsis reveal a number of genetic mutations that may account for this form of defective cytokinesis. For example, the gene products KEULE and KNOLLE mediate membrane fusion at the cell plate and keule and knolle mutants are characteristically multinucleate (reviewed by Assaad 2001). In addition, some of the cells in keule mutants have incomplete cell wall extensions (Assaad et al. 2001; Lukowitz et al. 2001) that are similar in appearance to the trabeculae that extend into the cytoplasm of the siphonous green algae Caulerpa (Fagerberg et al. 2010). Among these and other mutants, the ontogeny of multinucleate phenotypes indicates that nuclear division can be completed even when cytokinesis is incomplete, whereas cytokinesis can only be initiated once the nuclear cycle is complete (for a review, see Nacry et al. 2000). However, the mechanisms of cytokinesis are not likely to be homologous among the different plant clades (see for example McDonald and Pickett‐Heaps 1976; McNaughton and Goff 1990; Pickett‐Heaps et al. 1999). For example, embryophyte cytokinesis involves a phragmoplast composed of microtubules and the formation of a new cell plate that starts at the center of the cell and proceeds centrifugally outward toward the parental cell wall (phragmoplastic cytokinesis) (Pickett‐Heaps et al. 1999), a process that involves a complex sequence of cytoskeletal and membrane dynamics with a vast array of molecular players (Dhonukske et al. 2006; McMichael and Bednarek 2013). With the exception of the charophycean algae and the Trentepohliales, cytokinesis among the various algal lineages is accomplished by a diaphragm‐like furrowing of the plasma membrane and associated cell wall layers that develops as a centripetally growing septum (phycoplastic cytokinesis) (Graham 1996). A hybrid of these two forms of cytokinesis is reported for the charophycean (Zygnematales) alga Spirogyra in which a diaphragm‐like furrow organizes the centripetal in‐ growth of a furrow and a phragmoplastic‐like array of microtubules is associated with the centrifugal development of cell plate vesicles (Sawitzky and Grolig 2001). Animal cytokinesis is based on an actomyosin‐containing contractile ring and begins at the cell perimeter as a cleavage furrow, which is drawn toward the derivative nuclei into the dividing cytoplasm (centripetal cytokinesis). Despite these cytological differences, cytokinesis appears to involve the participation of the endosomal protein ESCRT III in the final act of cytokinetic membrane constriction across the majority of eukaryotes, since endocytosis 472 EVOLUTION & DEVELOPMENT Vol. 15, No. 6, November–December 2013 fails in ESCRT III mutants (Carlton and Martin‐Serrano 2007, 2009). As to why the multinucleate condition evolved, there are obvious disadvantages as well as advantages to being coenocytic. On the one hand, the absence of cross walls allows for the systemic spread of a pathogen once the cell wall is breached. It is not surprising therefore that at least six different rapid‐wound response mechanisms have evolved among the siphonous algae (Menzel 1988). Yet another disadvantage of the siphonous body emerges for holocarpic organisms (i.e., those that completely evacuate their cytoplasm in the act of reproduction), since the adult must sacrifice itself to reproduce. On the other hand, there are potential advantages of having multinucleate cells or a siphonous body plan, e.g., (1) rapid intracellular mass‐energy exchange when coupled with cytoplasm streaming, (2) a buffer against deleterious mutations as a consequence of multiple genome copies (or, conversely, a platform to segregate defective nuclei during the formation of uninucleate gametes or multinucleate asexual propagules), (3) higher metabolic and growth rates due to amplified chromosomal copies of ribosomal RNA cistrons (as an analog of one of the possible effects of endoreduplication or polyploidy), and (4) a multinucleate (siphonous) cell has the potential to occupy diverse microenvironments when coupled with indeterminate growth (which, for a tubular organism with a large vacuole, can confer the additional advantage of increasing the effective surface area with respect to volume for mass and energy exchange). Naturally, the null hypothesis is that multinucleate cells, tissues, and organisms are subject to neutral selection and reflect homoplasy rather than convergent adaptive evolution. Major evolutionary innovations are not likely to be retained within a lineage if they are incompatible with successful reproduction. However, it is not always the case that every transition requires a large or even measurable advantage (Grosberg and Strathmann 2007; Anderson et al. 2011), nor is it the case that phenotypic responses to selection are invariably in the direction of an adaptive advantage (Bonduriansky and Day 2009; Gray et al. 2010; Lynch 2012). A DIFFERENT EVOLUTIONARY SCENARIO FOR MULTICELLULARITY? We have shown that it is theoretically possible for a multicellular organism to evolve directly from a siphonous/coenocytic progenitor via a developmental process analogous to segregative cell division (see Fig. 4). Here, we argue that such a progenitor is an integrated phenotype that has already passed through both the alignment‐of‐fitness phase and the export‐of‐fitness phase (processes reviewed by Folse and Roughgarden 2010; see also Niklas and Newman 2013), while bypassing the colonial body plan, which is traditionally postulated in the unicellular to multicellular transformation series (see also Niklas and Newman 2013). All that is required to achieve multicellularity in this scenario is the acquisition of the capacity to partition cytoplasm into individual cells. All plant life cycles, including those of all known siphonous algae, have at least one unicellular “bottleneck” that establishes genomic uniformity among subsequently produced nuclei. The bottleneck can take the form of a zygote, an asexual uninucleate propagule, or an asexual propagule containing genetically identical nuclei (Niklas and Newman 2013). Further, no siphonous plant is known to propagate as a result of the somatic fusion of genetically dissimilar individuals, and most are haploid. Thus, baring extensive somatic mutations, the nuclei in a siphonous alga are as genetically similar as the nuclei in multicellular organism. Consequently, every siphonous organism has passed though an alignment‐of‐fitness phase as a consequence of its sexual or asexual reproductive cycle. Traditionally, the export‐of‐fitness phase is said to occur when cells evolve fitness‐specific tasks to give rise to a multicellular entity that reproduces similar entities with a heritable fitness (Folse and Roughgarden 2010). The appearance of a division of labor between germ‐ and soma‐functionalities provides evidence that this evolutionary phase has been attained (Buss 1987). However, obligate sexual reproduction is not required to override the conflict between the individual organism and its constituent cells (Michod 1997; Folse and Roughgarden 2010), or to be more precise in the context of this discussion, the individual organism and its constituent nuclei. All that is theoretically required is the evolution of one or more multistable gene regulatory networks (MGRP) capable of producing different cellular/nuclear functionalities (Laurent and Kellershohn 1999; Libby and Rainey 2013), e.g., an organism containing nuclei capable of existing in multiple states of gene expression. MGRP occur in unicellular bacteria as well as in uninucleate algae, yeast, and amoebae exhibiting alternative stable states of gene activity during their life cycles. Further, mathematical models indicate that cellular/nuclear differentiation can emerge even among genetically identical adjoining cells as a response to poor compatibility among competing physiological processes (Ispolatov et al. 2012). Arguably, a minimum number of different cellular/nuclear functionalities is required before it becomes necessary to establish methods to control functional differentiation (Arnellos et al. 2013). However, once again, this condition is achieved by a reproductively viable siphonous organism. We therefore argue that the multinucleate unicellular coenocyte possesses all of the preconditions required for the evolution of multicellularity in that it has achieved a level of cooperation among nuclei capable of differential genomic expression that acheieved delegated functionalities, albeit within a shared cytoplasm. All that is required is the evolution of the capacity for cellularization of a siphonous progenitor. In this context, three features evident among multinucleate plants are Niklas et al. The evo‐devo of multinucleate cells, tissues, and organisms, and an alternative relevant: (1) many siphonous algae achieve elaborate vegetative morphological features as a result of regional differential genomic expression patterns (e.g., the ulvophycean algae Caulerpa; see Fig. 1), (2) many also form temporary cell walls to sequester “germ” from “somatic” nuclei during their asexual or sexual life cycles (e.g., the ulvophycean algae Acetabularia), and (3), as previously noted, siphonocladous algae begin their life cycles as coenocytes, but subsequently become multicellular by means of segregative cell division. The transition to multicellularity via a unicellular multinucleate progenitor has been proposed for account for aspects of animal evolution (see Hadzi 1953; Hanson 1977), but it is incompatible with our current understanding of metazoan phylogenetics. Nevertheless, we believe that this scenario is consistent with certain aspects of plant evolution. This proposition can be tested experimentally by detailed analyses of the phyletic relationships within algal lineages containing uni‐ and multinucleate cells and siphonous and siphonocladous organisms. Such analyses require resolution of character state polarities at the ordinal, familial, and perhaps at the generic level. However, regardless of its frequency of occurrence or the exact mechanisms by which it is achieved, the scenario of a unicellular multinucleate cell evolving into a multicellular organism represents a theoretically viable hypothesis that requires consideration when discussing the origins of multicellularity. Acknowledgements The authors thank Dr. Linda E. Graham (University of Wisconsin, Madison), Drs. Dominick J. Paolillo Jr. and Thomas Owens (Cornell University), and two anonymous reviewers for insightful suggestions and comments. 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