SUMMARY

Multicellularity has evolved in several eukaryotic lineages leading to
plants, fungi, and animals. Theoretically, in each case, this involved
(1) cell-to-cell adhesion with an alignment-of-fitness among cells,
(2) cell-to-cell communication, cooperation, and specialization
with an export-of-fitness to a multicellular organism, and (3)
in some cases, a transition from “simple” to “complex”
multicellularity. When mapped onto a matrix of morphologies based
on developmental and physical rules for plants, these three phases
help to identify a “unicellular ⇒ colonial ⇒ filamentous
(unbranched ⇒ branched) ⇒ pseudoparenchymatous ⇒ parenchymatous”
morphological transformation series that is consistent with
trends observed within each of the three major plant clades. In
contrast, a more direct “unicellular ⇒ colonial or
siphonous ⇒ parenchymatous” series is observed in fungal and
animal lineages. In these contexts, we discuss the roles played by the
cooptation, expansion, and subsequent diversification of ancestral
genomic toolkits and patterning modules during the evolution of
multicellularity. We conclude that the extent to which multicellularity
is achieved using the same toolkits and modules (and thus the extent to
which multicellularity is homologous among different organisms) differs
among clades and even among some closely related lineages.

INTRODUCTION

One of the most remarkable events in evolutionary history was the
emergence and radiation of eukaryotic multicellular organisms (Valentine
1978; Bonner 2012; Maynard Smith and Szathmáry 1995; Knoll 2011). Perhaps
even more remarkable is that this “event” occurred independently in
different clades. Estimates of the exact number vary depending on how
multicellular is defined. When defined simply as cellular aggregation,
a conservative estimate is that multicellularity evolved over 25 times
(Grosberg and Strathmann 2007). More stringent definitions requiring
sustained cell-to-cell interconnection and communication obtain an
estimate of ten eukaryotic events, that is, once in the Animalia, three
in the Fungi (chytrids, ascomycetes, and basidiomycetes), and six in the
three major plant clades (twice each in the rhodophytes, stramenopiles,
and chlorobionta).

Regardless of the number, the multiple origins of multicellularity and
their subsequent consequences evoke a number of biologically important,
but largely unanswered, questions. For example, do multicellular lineages
share a common morphological transformational series? What if any are the
selection barriers to (and the drivers toward) multicellularity? Were
the ancestors of some lineages predisposed to engender multicellular
organisms, or is multicellularity the result of random events leading
toward larger organisms. Put differently, are the morphological motifs
that emerge in multicellular lineages the result of adaptive evolution,
or the inevitable consequences of physical laws and processes? Indeed,
are the multiple origins of multicellularity truly independent given
that all life ultimately shared a last common ancestor?

These and other questions about multicellularity have been addressed
in different ways (e.g., Bonner 2012; Niklas 2000; Kirk 2005; Newman
and Bhat 2008, 2009; Newman 2011; Knoll 2011). However, all perspectives
share three features: (1) a comparative approach (because of the multiple
origins of multicellularity, sometimes even within the same clade); (2)
a treatment of how “information” is exchanged among cells and between
cells and their external environment (because coordinated signaling among
cells is one of the defining characteristics of multicellular biology; see
Mian and Rose 2011); and (3) a consideration of functional morphological
features (because these govern energy–mass exchange rates between an
organism and its environment; see Gates 1980).

We address these features as well. However, our primary objective is
to determine, as best as currently possible, whether the evolutionary
trajectory toward multicellularity manifests a common trend across as well
as within clades and, if so, whether this trend is the result of genomic
or physical commonalities among otherwise diverse organisms. Although we
discuss trends in the fungal and animal clades, our focus is primarily
on plants, which we define broadly as eukaryotic photoautotrophs
(Niklas 1997, 2000) to encompass the algae as well as the monophyletic
land plants (embryophytes). This phyto-centrism is adopted because (1)
multicellularity evolved independently at least six times in the three
major plant clades, which permits extensive interphyletic comparisons,
and (2) the origins of plant multicellularity have been largely neglected
in a primarily zoo-centric literature. A third reason for focusing on
plants is that all plant clades evolved cell walls that, in contrast
to animals, can restrict intercellular aggregation and communication
(and requires somatic embryogenesis in multicellular plants).

In the following, we (1) characterize multicellular organisms in terms
of intercellular adherence and cell-to-cell and cell-to-environment
communication, (2) explore the requisite transition from fitness defined
at the level of individual cells to fitness defined at the level of
a truly multicellular entity (Wolpert and Szathmáry 2002; Michod et
al. 2003; Grosberg and Strathmann 2007; Folse and Roughgarden 2012),
(3) assess the transition from simple to complex multicellularity (sensu
Knoll 2011), (4) compare character polarities among the different plant,
fungal, and animal clades, and (5) discuss whether the evolution of
multicellular organisms was instigated by physically based patterning
modules mobilized by shared or unique molecular toolkits (Newman and
Bhat 2009; Hernández-Hernández et al. 2012).

We will affirm that the evolution of multicellular organisms typically
involved intermediate body plans that were achieved by similar
developmental mechanisms in different lineages, but not necessarily by
mechanisms sharing the same physical or biochemical components. Much
like the plant organs collectively called “leaves,” which evolved
independently in different lineages, multicellularity is a recurrent
feature of morphological evolution that was reached in many different
ways. Consequently, the extent to which multicellular organisms are
developmentally homologous at the most basic levels requires careful
analyses, particularly since selection acts on functional traits and not
on their underlying generative mechanisms, enabling different mechanisms
to achieve the same functional traits (Marks and Lechowicz 2006).
EVOLUTIONARY PHASES

Multicellularity has been defined in different ways because of different
taxonomic, developmental, morphological, physiological, or genomic frames
of reference. Here, instead of attempting to define this condition,
we focus on two features that enter into virtually all discussions
of multicellular organisms—cell-to-cell adherence, the condicio
sine qua non of multicellularity across all clades, and cell-to-cell
communication, the basis of multicellular development. However, despite
these commonalities, the cellular and molecular bases of the modes
of adherence and communication differ markedly among clades. Thus,
the Ca2+-rhamnogalacturonanic-dominated composition of the middle
lamella that binds embryophyte cell walls together differs chemically
from the Type-1 transmembrane cadherin proteins responsible for animal
cell adhesion (Hulpiau and van Roy 2009), or the glycoprotein-based
glues produced by many fungi (Epstein and Nicholson 2006). Likewise,
the intercellular interconnections in the green alga Volvox differ
significantly from embryophyte plasmodesmata, mammalian gap-junctions, or
fungal intercellular septal pores that all nevertheless provide avenues
for cell-to-cell communication. Just as in cladistics, “cell-to-cell
adhesion” and “intercellular communication” can be thought of as
“characters” that assume different “character states” depending
on a lineage's phyletic legacy.

If cell-to-cell adhesion and communication are requisite for the evolution
of multicellular organisms, their basic elements must have ancestral
unicellular character states but not necessarily manifesting the same
functionalities. For example, molecular analyses identify a diversity
of cadherins in the unicellular choanoflagellate Monosiga brevicollis
(a descendent of the unicellular metazoan progenitor) that likely
function in environment-responsive intracellular signal transduction,
for example, tyrosine kinase and hedgehog signaling (Abedin and King
2008). Likewise, adherens junctions tethering metazoan cells occur in
sponge epithelia, albeit in a rudimentary form (Abedin and King 2010;
Suga et al. 2012). One pathway to fungal multicellularity illustrated by
dictyostelid slime molds (Bonner 2012) shares elements with the evolution
of animal multicellularity. Upon starvation, a developmental cascade
is instigated involving diverse cell-to-cell-to-substrate adhesion,
for example, membrane bound DdCAD-proteins facilitating cell contact
play a role similar to that of animal cadherins (Nelson 2008; Bonner
2012). Indeed, the evolution of multicellularity in the fungi may
have been rapid. Using differences in settling velocities to separate
unicellular and clustered Saccharomyces cerevisiae cells, Ratcliff et
al. (2012) isolated isogenic “snowflake” genotypes with a simple
cellular division of labor and multicellular propagules.

Multicellular organisms can evolve along different pathways even within
the same clade. Consider the chlorobionta. In the volvocine algae,
multicellularity likely evolved by differential modifications of cell
wall layers in a Chlamydomonas-like progenitor (Kirk 2005). Specifically,
the walls of unicellular volvocines (e.g., Chlamydomonas) are composed
primarily of hydroxyproline-rich glycoproteins and are separated into a
structured outer layer and a more amorphous inner layer (Fig. 1). Among
colonial volvocines (e.g., Gonium), adhering cells are interconnected
by struts that preserve the features of the outer cell wall layer
(Fig. 1). However, among multicellular volvocine algae (e.g., Pandorina
and Volvox), the entire plant is surrounded by a layer that preserves
features of the ancestral outer wall layer by virtue of an unusual pattern
of cell division (palintomy) in which cells undergo multiple divisions
without increasing in total cytoplasmic volume (Herron et al. 2010). High
protein sequence similarities among unicellular and multicellular
volvocines support this scenario; for example, homologs of the
Chlamydomonas outer cell wall protein GP2 occur in Pandorina and Volvox
(Adair and Appel 1989). In contrast, cell adhesion in the evolutionarily
related embryophytes involves a middle lamella enriched with pectins,
a functionally and structurally diverse class of galacturonic acid-rich
polysaccharides (Caffall and Mohnen 2009). Chemical modification of
pectins contributes to changing the mechanical properties of cell walls
(Peaucelle et al. 2011; Wolf and Greiner 2012), which includes cell wall
relaxation in concert with indole-3-acetic-acid (Niklas and Kutschera
2012). Because similar systems are reported for unicellular green algae
(Musatenko 2005), it is possible that embryophyte cell adhesion evolved
by modifications of the fundamental mechanism of cell wall expansion
operating in unicellular progenitors.  image

Figure 1. Putative evolution of multicellularity in the volvocine algae
(adapted from Kirk 2005). The inner cell wall layer of a unicellular
Chlamydomonas-like progenitor is modified into an expanded extracellular
matrix (ECM) wherein multicellularity is achieved by intercellular
cytoplasmic strands (e.g., Volvox). The outer cell wall layer adheres
cells in colonial organisms (e.g., Gonium).

Under any circumstances, cell-to-cell adhesion in one form or another
participates in the life cycles of nearly every unicellular organism
(e.g., Chlamydomonas gamete-aggregation) and there is ample evidence
that cell adhesion (and even limited cellular cooperation) is ancient;
for example, the expression of the flocculation FLO1 gene resulting
in S. cerevisiae cell clustering (Smukalla et al. ) and Chlorella (a
unicellular green alga) genome homologues of Arabidopsis auxin receptors
(Blanc et al. 2010). However, although it is a necessary condition for
a multicellular body plan, cell adhesion is insufficient. What prevents
palmelloid Chlamydomonas, colonial choanoflagellates, and other similar
“cellular aggregate” life-forms from being multicellular organisms
is the failure to constitute biological entities possessing sustained
cell-to-cell communication and cooperative interactions among formerly
individualized cells (Buss 1987), a feature that permits opportunities
for the evolution of cellular specialization (Wolpert and Szathmáry
2002; Michod et al. 2003). In multicellular plants and fungi, this
capacity may have evolved by the co-option of prokaryotic two-component
signaling pathways involving histidine kinases, response regulators,
and in some cases histidine-containing phosphotransfer proteins (see
Schaller et al. 2011), which have been identified in a broad spectrum of
eukaryotes including the centric diatom Thalassiosira, Chlamydomonas,
Dictyostelium, a variety of fungi, and Arabidopsis (Anatharaman et
al. 2007). Indeed, the molecular basis for cell-to-cell adhesion and
communication may have evolved simultaneously in some cases; for example,
metazoan tetraspanin-enriched protein/membrane microdomains participate
in cell–cell adhesion and communication as well as membrane fusion
and cell migration (Bailey et al. 2011; Wang et al. 2012).

But how does a cellular aggregate achieve individuality? One suggestion is
that two evolutionary stages are required—an alignment-of-fitness phase
in which genetic similarity among cells prevents cell–cell conflict
and an export-of-fitness phase in which cells become interdependent
and collaborate in a sustained effort (reviewed by Folse and Roughgarden
2012). The first phase can be achieved by any “unicellular bottleneck”
in an organism's life cycle, for example, spores or zygotes. The second
phase requires intercellular cooperation sufficient to shift selection
from the level of individual cells to the level of a multicellular
entity that reproduces similar entities with a heritable fitness,
typically followed by some degree of cellular specialization. Although
the separation of soma and a germ-line sometimes occurs, obligate
sexual reproduction is not required to override the conflict between the
individual and its constituent cells (Buss 1987; Michod 1997; Nanjundiah
and Sathe 2011). Certainly, in the absence of somatic mutations, the
presence of a zygote in any life cycle assures genetic homogeneity by
providing a unicellular bottleneck regardless of the type of life cycle
(Fig. 2). Likewise, it can be difficult for asexual organisms to escape
the consequences of Muller's ratchet (the inevitable accumulation of
deleterious mutations). However, asexual multicellular organisms also
experience an alignment-of-fitness by means of unicellular or cloned
propagules (Fig. 2).  image

Figure 2. Schematics of four contrasting life cycles each with one or more
“unicellular bottlenecks” (indicated by an asterisk). Diploid cells
and organisms are shaded; haploid cells and organisms are unshaded. For
simplicity, each organism is depicted as a hermaphrodite. (A) The
haplobiontic-haploid life cycle in which the multicellular phase
develops after zygotic meiosis. (B) The haplobiontic-diploid life cycle
in which the multicellular phase develops from zygotic mitosis. (C)
The diplobiontic life cycle containing two multicellular individuals,
one developing after zygotic mitosis and another developing from
meiosis. (D) An asexual life cycle in which the multicellular organism
and its unicellular (or cloned) propagules resulting from mitotic cellular
division (or fragmentation).

Likewise, multicellularity is not required for the evolution of cellular
specialization. Unicellular bacteria, algae, yeast, and amoeba exhibit
alternative stable states of gene activity and even morphology during
their life cycles, often as a result of competing processes, for example,
motility versus mitosis. The origin of cellular differentiation therefore
may reside in the inherent multistability of complex gene regulatory
networks (Laurent and Kellershohn 1999) with somatic or reproductive
functional roles for different cell-types possibly established ad hoc
by natural selection. Thus, mathematical models indicate that cellular
differentiation can emerge among genetically identical cells as a
response to poor compatibility among competing physiological processes
(Ispolatov et al. 2012). In more derived lineages, an alignment of fitness
can compensate for conflicts of interest among cellular components such
that a division of cellular labor becomes possible and even necessary.
SIMPLE VERSUS COMPLEX MULTICELLULARITY

Some authors distinguish between “simple” and “complex”
multicellularity (Butterfield 2000; Schlichting 2003). This distinction
has focused recently on whether all cells make contact with their
external environment, or whether some are internalized (Knoll 2011),
for example, unbranched filamentous morphologies and organisms with
tissue systems, respectively. This difference is important because
(1) it is correlated with differences in cell specialization, energy
consumption per gene expressed, and increases in non-protein-coding
DNA (Bonner 2004; Lane and Martin 2010; see, also, Lozada-Chavez et
al. 2011), (2) it helps to assess the likelihood that a multicellular
organism can evolutionarily revert to a unicellular state (e.g., a complex
multicellular to unicellular transition is far less likely than a colonial
to unicellular transition), and (3) it helps to identify when a simple
multicellular organism evolutionarily reaches a size or morphology that
necessitates tissue specialization for the bulk transport of nutrients.

Consider the consequence of size on passive diffusion as shown by
a variant of Fick's law of diffusivity, which estimates the time
required for the concentration of a neutral molecule i initially absent
within a cell to reach 50% of the external concentration, that is,
t0.5 = [V/(APi)] ln (ct=0/ct=0.5), where V is cell volume, A is cell
surface area, Pi is the permeability coefficient of i, and ct=0 and ct=0.5
are the concentrations of i at time zero and at time 50% concentration
(Niklas and Spatz 2012). This formula shows that the time it takes i
to diffuse into a spherical cell from all directions and reach ct=0.5
is linearly proportional to cell radius, increases with increasing
volume, and decreases with increasing surface area. Consequently,
passive diffusion can be sufficient for the metabolic demands of small
or attenuated cells, filaments, or thin sheets of cells, but it becomes
increasingly insufficient as cells or cell aggregates increase in size.

Indeed, diffusion is a mode of communication that can drive
cell specialization because increasingly steep gradients in
nutrient-availability resulting from increasing size or distance can
give rise to a reaction–diffusion (R–D) morphogenetic system. For
example, an Anabena or Nostoc filament is a one-dimensional R–D
system. Heterocysts manufacture a diffusible inhibitor that prevents
heterocyst formation unless its concentration falls below a specific
threshold (Wilcox et al. 1973; Risser et al. 2012). As the distance
between two heterocysts increases (due to intervening vegetative cellular
divisions), the first undifferentiated cell to be triggered midway
develops into a heterocyst, releases the inhibitor, and reiterates the
process. Analogous R–D systems operating in two- or three-dimensions are
posited for the development of stomata and root hairs (Torii 2012). Even
more complex examples can be drawn from the evolution of land plants or
extant macroscopic algae. Thus, as early embryophytes evolved greater
height, passive diffusion eventually failed to provide water to aerial
tissues at sufficient rates, thereby requiring bulk water flow via xylem
(Niklas 1997). Likewise, the inner cortex of the stipes of the great
kelp Macrocystis contain specialized “trumpet” cells that transport
photosynthates much like the phloem of vascular plants (Bruggeln et
al. 1985).

Nevertheless, the organization of differentiated cells into two-
and three-dimensional patterns, shapes, and forms need not reflect the
immediate consequences of selection (Gilbert 2010). Although the adaptive
advantage of a morphological novelty is clear (such as in the development
of plant and animal vascular systems), morphological or behavioral
novelties can also arise via phenotypic plasticity (West-Eberhard 2003;
Niklas 2009), or as a consequence of the inherent physical properties of
embryonic tissues and condition-dependent developmental systems (Newman
1994; Newman and Müller 2000; Manning et al. 2010), only to become
assimilated subsequently into the organism's developmental program (see,
e.g., Palmer 2004). The former may help to explain why colonial life-forms
often presage the appearance of multicellularity in some lineages since
phenotypic plasticity among genetically identical cells contributes to
fitness (Yokota and Sterner 2010). Likewise, the colonial body plan
has distinct advantages over the unicellular life form; for example,
non-defective cells can compensate for the defects of neighboring cells,
or provide resources to other cells that are then free to specialize.
CHARACTER POLARITIES WITHIN MULTICELLULAR CLADES

The preceding provides some insight into how unicellular organisms
evolved into simple (and later complex) multicellular forms because
it establishes a hypothetical morphological transformation series,
viz., unicellular ⇒ colonial ⇒ simple multicellular
life forms. For plants, this series can be refined further based on
simple developmental and physical principles that identify four major
body plans, that is, unicellular, siphonous/coenocytic, colonial, and
multicellular (Fig. 3). Among variants of the plant multicellular body
plan, an unbranched filament of cells is the simplest biophysically
because it requires a single plane of cell division while reducing
drag forces, maximizing light interception, and maintaining a
constant surface area to volume relationship (Niklas 2000). Further
elaboration requires cell division in two and then three planes of
reference, which respectively gives rise to branched filaments (and
the potential for a pseudoparenchymatous tissue construction) and a
parenchymatous tissue construction (Fig. 3). (We define parenchymatous
tissues as those in which cells can divide in all three planes of
reference.) Accordingly, for plants, the evolution of multicellularity is
predicted to conform to a unicellular ⇒ colonial ⇒ filamentous
(unbranched ⇒ branched) ⇒ pseudoparenchymatous ⇒ parenchymatous
transformation series. This hypothesis can be evaluated empirically by
mapping the different body plans and tissue constructions onto current
phylogenies for plants, fungi, and animals. In theory, unicellular,
colonial, and simple multicellular morphologies should map onto ancestral
(basal) branches within each clade, whereas more complex tissue
constructions should map onto more derived branches.  image

Figure 3. The four major plant body plans shown in bold (unicellular,
siphonous/coenocytic, colonial, and multicellular) configured in a matrix
of the developmental phenomena that give rise to them, two of which are
critical for the evolution of multicellularity: cell-to-cell adherence
and cytoplasmic (symplastic) continuity among adjoining cells. Note
that the siphonous/coenocytic body plan may evolve from a unicellular
or a multicellular progenitor. This body plan can become multicellular
directly. 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).

Although recent plant phylogenies inform this hypothesis, the cladistic
resolution within some clades currently lacks sufficient precision
to identify the ancestral body plans even among some closely related
lineages. Perhaps the most difficult clade to resolve is the red
algae (rhodophytes). Molecular data identify two divergent lineages
(one extending from the Porphyridiophyceae and another from the
Bangiophyceae) that share the unicellular Cyanidiophyceae as their
sister taxon (Saunders and Hommersand 2004; Yoon et al. 2009). The
porphyridian lineage contains unicellular, colonial, and what are
generally believed to be more derived multicellular algae, whereas the
Compsopogonophyceae contains filamentous algae (Fig. 4). The taxonomic
relationships of the Rhodochaetales and Erythropeltidales with the
Compsopogonophyceae and other related rhodophytes remain unresolved (Yoon
et al. 2009). However, in the filamentous Bangiophyceae, the immediate
ancestral condition is unicellular and the derived condition in the
“floridean” red algae is pseudoparenchymatous. Molecular analyses
of the monophyletic stramenopiles (Anderson 2004; Maistro et al. 2009)
identify three major lineages—the eustigmatophytes, dictyochophytes,
and the pelagophytes (Fig. 5). Within the eustigmatophyte and the
dictyochophyte lineages, the colonial body plan is derived and the
filamentous body plan is observed only among the most derived genera. The
various branches emerging from the pelagophyte lineage have a complex
pattern. Nonetheless, the unicellular ⇒ colonial ⇒ filamentous
(unbranched ⇒ branched) ⇒ pseudoparenchymatous ⇒ parenchymatous
transformation series is consistent with current views regarding the
phylogenetic relationships within the brown algae (phaeophytes) and
especially within the yellow-green algae (xanthophytes; see Cocquyt et
al. 2010) (Fig. 5). Perhaps the strongest evidence among the three major
plant clades for the unicellular ⇒ colonial ⇒ filamentous
(unbranched ⇒ branched) ⇒ pseudoparenchymatous ⇒ parenchymatous
transformational series comes from the molecular phylogenetics of
the monophyletic green plants (chlorobionta) (Lewis and McCourt 2004;
Leliaert et al. 2012). This series occurs within the “green algae”
and within the streptophytes (the charophycean algae and the embryophytes)
(Fig. 6).  image

Figure 4. Tentative phylogeny of the monophyletic red algae (rhodophytes)
based on the phylogenetic analyses of Saunders and Hommersand (2004; see
also Graham et al. 2009, pp. 310–311). The body plans characteristic
of terminal branches in the phylogeny are indicated by schematics
(see insert); all groups with multicellular body plans are indicated
in bold. The grey arrow indicates that the uppermost branch in the red
algal clade is traditionally placed in the Bangiophyceae.  image

Figure 5. A phylogeny of the monophyletic stramenopiles (brown and
golden-brown algae) based on the analyses of Anderson (2004). The body
plans characteristic of terminal branches in the cladogram are indicated
by schematics (see insert); all groups with multicellular body plans
are indicated in bold.  image

Figure 6. A phylogeny of the monophyletic chlorobionta based on the
analyses of Lewis and McCourt (2004) and Leliaert et al. (2012). Two major
lineages are recognized: the green algae (chlorophytes) and a lineage
containing the charophycean algae and the land plants (streptophytes). The
body plans characteristic of terminal branches in the cladogram are
indicated by schematics (see insert); all groups with multicellular body
plans are indicated in bold.

In contrast to the plants, the transformational series observed for
the fungi and animals are more truncated. Molecular analyses indicate
that unicellular or siphonaceous (coenocytic) fungi gave rise to the
multicellular condition in the asco- and basidomycetes. Although the
monophyletic status of the chytrids and the lack of resolution at the
base of fungal phylogeny make it difficult to resolve the details of the
last common ancestor (Lutzoni et al. 2004; Schoch et al. 2009) (Fig. 7),
the colonial body plan is absent, whereas multicellular fungi consist
of unbranched filaments that achieve a pseudoparenchymatous tissue
construction in asco- and basidomycete crown-taxa, that is, fungal
evolution conforms to a unicellular or siphonous ⇒ unbranched
filament ⇒ pseudoparenchymatous transformation series. Among
animals, analyses identify the choanoflagellates as basal (Fig. 7). The
phyletic relationships among the Placozoa, Ctenophora, and Cnidaria
remain uncertain (e.g., Halanych 2004; Dunn et al. 2008). Within
the monophyletic choanoflagellates, the unicellular body plan is
traditionally interpreted as ancestral and the colonial body plan
as more derived. Although the phylogenetic relationship between
the choanoflagellates and the Holozoa requires extensive revision,
analyses indicate that the choanoflagellates and Metazoa shared a last
common unicellular ancestor (Carr et al. 2008). Within the Metazoa,
the colonial body plan evolved independently (corals and bryozoans),
although it may be a derived condition. The phyletic relationships among
the Placozoa, Ctenophora, and Cnidaria remain uncertain (e.g., Halanych
2004; Dunn et al. 2008). Thus, within the Holozoa, we see a comparatively
simple and direct unicellular ⇒ colonial ⇒ parenchymatous
transformational series (Fig. 7). Subsequent, evolution gave rise
to organisms with successively inclusive morphological motifs, that
is, functionally differentiated cell types (sponges), multilayering
(placozoans), interior cavities (ctenophorans, cnidarians), and eventually
Bilateria with additional layers and body cavities (ecdysozoans, mollusks,
etc.) and segments (annelids, etc.) (Newman 2012) (Fig. 8).  image

Figure 7. Redacted phylogeny for the Fungi and Animalia and their
respective putative ancestors. The body plans characteristic of terminal
branches in this phylogeny are indicated by schematics (see insert);
lineages with multicellular body plans are indicated in bold.  image

Figure 8. The elaboration of the animal body plans (shown in bold)
involved additions of successive morphological motifs based on the
mobilization of newly relevant physical effects once the multicellular
state evolved. These effects were harnessed by molecules that had
first evolved to serve unicellular functions but that later became
developmental “toolkit” molecules. While aggregates of cells with the
relevant genes would have been capable of generating these motifs in a
heritable fashion, blastulae resulting from cleavage of an enlarged cell
(a “proto-egg”) would have been genetically uniform thus exhibiting a
greater alignment-of-fitness (Newman 2011). Adapted from Newman and Bhat
(2009).

The foregoing polarities clearly need to be examined using finer levels
of taxonomic resolution (e.g., Cocquyt et al. 2010) at the ordinal
and family levels. However, they indicate that the colonial body
plan is a consequence of cell adherence mediated by typically lineage
specific molecules with functionalities that predate the multicellular
condition. In the following section, we argue that other motifs are
similarly the result of the automatic mobilization of physical processes
and effects by appropriate cell-interaction-mediating molecules within
the multicellular state. The self-organizational capabilities of these
“dynamical patterning modules” (Newman and Bhat 2008, 2009) are
realized in cell clusters arising by aggregation, by failure to separate
after division, or by subdivision of an enlarged cell (palintomy in
plants, cleavage in animals). In evolutionary terms, genetic uniformity
of entities that exhibit developmental activity can arise therefore
before or after the appearance of the relevant morphogenetic mechanisms
(Newman et al. 2006). Since adaptive selection has little to do with
such physically based morphogenetic effects, framing the origin of
multicellular body plans exclusively in terms of an alignment-of-fitness
phase and an export-of-fitness phase is perhaps unduly restrictive, an
issue that we return to in our concluding remarks.  SHARED AND UNIQUE
TOOLKITS AND PATTERNING MODULES

We have emphasized five evolutionary features of multicellular organisms:
cell adhesion, cellular communication, cellular specialization, tissue
pattern formation and morphogenesis, and “individuality” at the level
of a multicellular entity. Molecular analyses indicate that each of the
major multicellular clades contains a characteristic set of developmental
“toolkit” genes, some of which are shared among disparate lineages
(e.g., tetraspanin genes in plants, protozoa, insects, fungi, and mammals;
Wang et al. 2012), either by virtue of sharing a last common ancestor,
or as a result of lateral gene flow (e.g., Sun et al. 2010). However,
many are unique to their respective clades (e.g., cell polarity in
plants and animals involves PIN- and PAR-polarity modules, respectively;
Gelder 2009) and some even differ among closely related lineages (e.g.,
cell division mechanisms in unicellular and filamentous ascomycetes;
Seiler and Justa-Schuch 2010).

A substantial number of toolkit genes in each clade are developmental
transcription factors (DTFs), such as MADS box gene products in plants and
Hox gene products in animals, which, along with their cis-acting target
sequences on other such genes, form multistable regulatory networks
whose alternative stationary states determine cell type identity
(Furusawa and Kaneko 2002; Kaneko 2011). Others mediate cell–cell
interaction and communication and constitute an “interaction toolkit”
(Newman et al. 2009). Although DTFs cross cell borders only rarely
during animal embryogenesis (Prochiantz and Joliot 2003), they do so
more typically in plants, making them part of the interaction toolkit
in that clade. Other interaction plant toolkit molecules include the
adhesive components described previously and phytohormones such as auxin
(Niklas and Kutschera 2009; Boot et al. 2012). In animals, these include
cadherins, Notch and Wnt and their respective ligands, BMP, Hedgehog,
and extracellular matrices. Regardless of the roles they may have played
in unicellular ancestors, these molecules assume entirely new roles in
pattern formation and morphogenesis in the multicellular state, typically
by virtue of the newly relevant physical processes they mobilize at the
mesoscale (Forgacs and Newman 2005; Hamant et al. 2008; Newman and Bhat
2008, 2009; Manning et al. 2010; Müller 2012). This has led to animal
body plans that are (as noted) variously, multilayered, hollow or with
nested cavities, elongated, segmented and appendage-bearing, and to
organs with similar morphological motifs (Fig. 8). Among embryophytes,
interaction toolkits are implicated in branching, phyllotaxy, apical
dominance, and the development of vascular tissues.

Considering the shared and specific interaction toolkits of the various
clades in relation to the physical forces and effects they mobilize helps
explain how phyletically different organisms use genetically homologous
components to construct phenotypically dissimilar but functionally similar
(analogous) structures often without common ancestors exhibiting the
character (Newman 2006). Subtle variations in the physical environment
under which cell aggregation occurs, or minor genetic changes in
adhesion molecules, can result in alternative forms such as solid or
hollow spheroids, filaments, or multilayered tissues. With the advent
of complex multicellularity, the physical and physiological context and
microenvironments of distinct subpopulations of cells (e.g., interior
vs. exterior) can yield different developmental fates. Eventually the
conditional effects that contribute to such morphological variability may
become developmentally canalized under selection when their host organisms
find suitable ecological niches. Random gene-based modifications in body
mass or dimension likewise would alter a priori the rates or magnitudes
of size-dependent physical phenomena, such as passive diffusion and
mechanical stresses, in ways that would allow natural selection to filter
from a variety of possible forms those better adapted to available or
novel ecological settings.  FUTURE DIRECTIONS

The origination of multicellular organisms required the evolution of
mechanisms capable of achieving and maintaining cellular differentiation
temporally as well as spatially. The unicellular progenitor of
each multicellular lineage therefore must have had the capacity to
express alternative states of gene expression temporally in response
to changing environmental conditions and the pre-existing regulatory
mechanisms responsible for this developmental variability were co-opted
to provide the capacity to differentiate spatially in the multicellular
context. This new phenotypic context, mediated by preexisting attachment
and matrix molecules, led to the mobilization of newly relevant physical
effects and self-organizing dynamics that provided the early basis for
spatiotemporal regulation in multicellular organisms (Newman and Bhat
2008, 2009; Hernández-Hernández et al. 2012).

The evolutionary expansion of pre-existing gene families encoding
regulatory proteins in combination with novel physical and regulatory
interactions resulting from such expansions also played critical roles
and may even have driven the evolution of multicellular complexity (e.g.,
Feller et al. 2011; Pires and Dolan 2012), as illustrated by the basic
helix-loop-helix (bHLH) protein family involved in diverse cellular
developmental processes in both plants and animals (reviewed by Feller
et al. 2011). Indeed, many intracellular functions involve bHLH–bHLH
interactions as well as synergistic interactions with other regulatory
proteins, such as MYB, to form complexes that either repress or activate
the expression of sets of target genes (Ramsay and Glover 2005; Feller
et al. 2011). Future research is required to identify these targets and
to determine their participation in developmental processes. However,
as noted, it is critical to determine the extent to which the details of
transcription factor regulation and gene network architecture carry over
from one organism to another, since sequence homologies do not necessarily
imply the conservation of function. Likewise, functional homologies are
not invariably the result of genomic or developmental homology, as is
evident from the broad spectrum of molecules providing cell adhesion
and intercellular communication. Nevertheless, we believe that future
research will show that that three vastly different plant clades achieved
multicellularity along a similar morphological transformation series,
e.g., unicellular ⇒ colonial or siphonous ⇒ filamentous
(unbranched ⇒ branched) ⇒ pseudoparenchymatous ⇒ parenchymatous.

Finally, we have side stepped the question of whether multicellularity
confers any selective advantage. Certainly, the current abundance of
multicellular organisms gives the impression that it does. Likewise,
it allows an organism to exceed the size limits imposed by passive
diffusion. Although we are sympathetic to the notion that major
evolutionary innovations will not be retained within a lineage if they
are incompatible with survival, it is not always the case that every
transition requires a large or even measurable advantage (Grosberg
and Strathmann 2007), nor is it the case that phenotypic responses to
selection invariably are in the direction of an adaptive advantage
(Bonduriansky and Day 2009). Thus, a recent theoretical model for
filamentous bacteria shows that strains with the same fitness can
produce genotypes differing in cell-number as a result of differences
in cell division and death rates, or as a result of changes in the
environmental carrying capacity (Rossetti et al. 2011). This model,
which has empirical support, also shows that differences in fitness
attributable to morphology are not required a priori for the evolution
of life cycles with multicellular entities (Rossetti et al. 2011),
although advantages may arise subsequently (Koschwanez et al. 2011). The
retention of multicellularity in some lineages therefore may reflect
“diffusion from the left (unicellular) wall of life” (sensu Gould
1989; Marcot and McShea 2007). Indeed, although examples of a unicellular
organism descending from a multicellular organism are known (Velicer et
al. 1998; Schirrmeister et al. 2011), once an organism passes through
the export-of-fitness phase and achieves complex multicellularity,
its capacity for contingent evolutionary reversion to a simpler state
is reduced for reasons that have little or nothing to do with selection
on fitness.  Acknowledgments

The authors thank Drs. Randy Wayne and Dominick J. Paollilo, Jr. (Cornell
University), Dr. Michael Christianson (University of California, Berkeley)
and two anonymous reviewers for helpful suggestions and acknowledge
support from the College of Agriculture and Life Sciences, Cornell
University, and from the New York Medical College, Valhalla. This article
is dedicated to Prof. John T. Bonner (Princeton University) for his
seminal contributions to understanding the origins of multicellularity.
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