Cooperation and conﬂict in the evolution of individuality IV. … · 2008-12-02 · BioSystems 69 (2003) 95–114 Cooperation and conﬂict in the evolution of individuality IV

BioSystems 69 (2003) 95–114

Cooperation and conflict in the evolution of individualityIV. Conflict mediation and evolvability inVolvox carteri

Richard E. Michod∗, Aurora M. Nedelcu, Denis RozeDepartment of Ecology and Evolutionary Biology, University of Arizona Tucson, AZ 85721, USA

Abstract

The continued well being of evolutionary individuals (units of selection and evolution) depends upon their evolvability, thatis their capacity to generate and evolve adaptations at their level of organization, as well as their longer term capacity fordiversifying into more complex evolutionary forms. During a transition from a lower- to higher-level individual, such as thetransition between unicellular and multicellular organisms, the evolvability of the lower-level (cells) must be restricted, whilethe evolvability of the new higher-level unit (multicellular organism) must be enhanced. For these reasons, understanding thefactors leading to an evolutionary transition should help us to understand the factors underlying the emergence of evolvability ofa new evolutionary unit. Cooperation among lower-level units is fundamental to the origin of new functions in the higher-levelunit. Cooperation can produce a new more complex evolutionary unit, with the requisite properties of heritable fitness variations,because cooperation trades fitness from a lower-level (the costs of cooperation) to the higher-level (the benefits for the group).For this reason, the evolution of cooperative interactions helps us to understand the origin of new and higher-levels of fitnessand organization. As cooperation creates a new level of fitness, it also creates the opportunity for conflict between levels ofselection, as deleterious mutants with differing effects at the two levels arise and spread. This conflict can interfere with theevolvability of the higher-level unit, since the lower and higher-levels of selection will often “disagree” on what adaptations aremost beneficial to their respective interests. Mediation of this conflict is essential to the emergence of the new evolutionary unitand to its continued evolvability. As an example, we consider the transition from unicellular to multicellular organisms and studythe evolution of an early-sequestered germ-line in terms of its role in mediating conflict between the two levels of selection, thecell and the cell group. We apply our theoretical framework to the evolution of germ/soma differentiation in the green algal groupVolvocales. In the most complex member of the group,Volvox carteri, the potential conflicts among lower-level cells as to the“right” to reproduce the higher-level individual (i.e. the colony) have been mediated by restricting immortality and totipotency tothe germ-line. However, this mediation, and the evolution of an early segregated germ-line, was achieved by suppressing mitoticand differentiation capabilities in all post-embryonic cells. By handicapping the soma in this way, individuality is ensured, butthe solution has affected the long-term evolvability of this lineage. We think that although conflict mediation is pivotal to theemergence of individuality at the higher-level, the way in which the mediation is achieved can greatly affect the longer-termevolvability of the lineage.© 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Levels of selection; Germ-line; Mutation; Green algae; Altruism; Group selection

∗ Corresponding author. Tel.:+1-520-621-7517/7509;fax: +1-520-621-9190.

E-mail address:[email protected] (R.E. Michod).

1. Evolvability, evolutionary transitions andfitness

The continued well being of evolutionary individ-uals depends upon their evolvability, which, in our

0303-2647/02/$ – see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.PII: S0303-2647(02)00133-8

96 R.E. Michod et al. / BioSystems 69 (2003) 95–114

view, includes not only their capacity to generate andevolve adaptations, but also as their capacity to diver-sify into more complex evolutionary units. To adaptevolutionary individuals must be units of selection,and, ever since Darwin, we have understood that a unitof selection must possess the following properties: thestruggle to survive; variation; and, heritability, so thatoffspring resemble parents. We may combine thesethree properties and simply say that a unit of selectionmust have heritable variation in fitness. There are avariety of such units in biology: genes, gene networks,prokaryote cells, eukaryotic cells (cells in cells), mul-ticellular organisms, and groups and societies. Howand why did these different kinds of evolutionaryunits arise?

In the present paper, we consider our theory forthe origin and evolution of new kinds of individuals(Michod, 1996, 1997, 1999; Michod and Roze, 1997,1999, 2000) from the perspective of evolvability.During a transition to a higher-level individual, suchas between unicellular and multicellular organisms,the evolvability of lower-level units (for example,cells) must be restricted, while the evolvability ofthe new higher-level unit (for example, the multi-cellular organism) must be enhanced. Consequently,understanding the factors leading to an evolutionarytransition should help us to understand the factorsunderlying evolvability.

We study the role of the process of conflict medi-ation in enhancing the evolvability of a new unit ofselection. As an application of our theory, we considera general feature of multicellular organisms, the sep-aration and specialization of germ and somatic-linesof cells, and apply our results to the evolution ofgerm/soma differentiation in the green algal group,Volvocales (Nedelcu and Michod, 2003). We use theterms “germ” and “soma” in the sense of there beingtwo kinds of cells in the multicellular group, cellsthat are specialized in contributing to the next gen-eration of individuals and cells that are specializedin vegetative functions and do not directly reproducethe next generation of individuals. Even organismsoften regarded as not having a germ-line, such asplants, have cells that are specialized in reproductiveand vegetative functions, and so meet our criteria ofreproductive specialization. Specialization of cells inreproductive and vegetative functions is an almostuniversal feature of multicellular life.

The basic problem in an evolutionary transition is tounderstand how a group of individuals becomes a newkind of individual, possessing the properties of herita-ble variation in fitness at a new level of organization—tantamount to evolvability of the new evolutionary in-dividual. During evolutionary transitions, preexistingindividuals form groups, within which interactionsoccur that affect the fitnesses of both the individualsand the group. For example, under certain conditions,bacteria associate to form a fruiting body, amoebaeassociate to form a slug, solitary cells form a colonialgroup, normally solitary wasps breed cooperatively,birds associate to form a colony, and some mammalsform societies. In addition, about 2 billion years ago,archaebacteria-like cells (destined to be the ancestorsof all eukaryotes) began alliances with other bacteriato form the first eukaryotic like cell.

Such associations and groups may persist and re-form with varying likelihood depending on propertiesof the group and the component individuals. Initially,group fitness is the average of the lower-level in-dividual fitnesses, but as the evolutionary transitionproceeds, group fitness becomes decoupled from thefitness of its lower-level components. Indeed, theessence of an evolutionary transition in individualityis that the lower-level individuals must “relinquish”their “claim” to fitness, that is to flourish and multiply,in favor of the new higher-level unit. This transfer offitness from lower to higher-levels occurs through theevolution of cooperation and mediators of conflict thatrestrict the opportunity for within-group change andenhance the opportunity for between-group change.Until, eventually, the group becomes a new evolu-tionary individual in the sense of being evolvable—possessing heritable variation in fitness (at the newlevel of organization) and being protected from theravages of within-group change by adaptations thatrestrict the opportunity for defection (Michod, 1999).

2. A model for the evolution of anearly-segregated germ-line

2.1. Conflict mediation

Key in the conversion of a group of cooperatorsto a new evolutionary individual is the evolution ofconflict mediators, genes that tweak development

R.E. Michod et al. / BioSystems 69 (2003) 95–114 97

and/or the ways in which the groups are organizedand by so doing tilt selection in favor of the groupand away from the level of the cell. Although theunderlying mechanisms may be diverse (germ-line,apoptosis, self-policing, mutation rate, determinategrowth, reproductive mode and propagule size), con-flict mediation serves to enhance the evolvability ofthe higher-level unit by restricting the evolvabilityof composite lower-level units. However, as we dis-cover below in the green algal group Volvocales,the way in which conflict mediation is accomplishedmay be short-sited and end up interfering withthe long-term evolvability of the new multicellularunit.

The steps involved in the origin of multicellular lifehave been discussed by a variety of authors (see, forexample,Maynard Smith and Szathmáry, 1995). Asmentioned earlier, the initial step was likely the for-mation of cell-groups leading to some kind of prim-itive colonial life. These cell groups could have beenformed in several ways, such as through aggregation,fragmentation, or single cell spore-like reproduction(Michod and Roze, 2000, 2001; Roze and Michod,2001). Single cell spore-like reproduction may havebeen accomplished simply by the failure of thedaughter cells to separate following cell division. Forexample, the multiple fission mode of reproduction involvocalean green algae discussed later is particularlywell suited to forming cell groups in this way. Alongwith the formation of cell groups, there was likely theevolution of cooperative functions which benefitedthe group. Cooperative interactions are fundamentalto the emergence of new individuals, as only cooper-ation exports fitness from the lower to the new higherlevel. However, the evolution of cooperation also setsthe stage for conflict. For multicellular organismsto emerge out of cell groups, conflict mediation isneeded to regulate the levels of selection conflicts in-herent in the initial structure of cell-groups (Michod,1999). The developmental programs and organiza-tional structures created through conflict mediationare the first emergent functions at the higher level.The evolution of a way of regulating conflict meansthat the group is no longer a collection of lower-levelunits, it is on its way to becoming a new higher-levelindividual. For example, in the case of the evolutionof reproductive specialization through a germ andsomatic-line of cells (considered here), the group is

Fig. 1. Multi-level framework for the origin of multicellular or-ganisms. The subscriptj refers to the number of cooperating cellsin a propagule;j = 0, 1, 2,. . . , N, where N is the total num-ber of cells in the offspring propagule group, assumed constantfor simplicity. The variablekj refers to the total number of cellsat the adult stage of propagules that start out withj cooperatingcells. The variableWj is the fitness of groupj, defined as the ex-pected number of propagules produced by the group, assumed todepend both on size of the adult group after development and itsfunctionality (or level of cooperation among its component cells)represented by parameterβ in the models later.

no longer divisible, because certain cells have specialfunctions.

2.2. Multi-level selection model

A multi-level selection approach begins by parti-tioning the total change in frequency of genotypesinto within and between-group components. Groupsare defined by a group property, usually the group fre-quency of a phenotype or genotype (or some otherproperty reflecting group composition). In our model,we assume that offspring groups are composed ofNcells as inFig. 1 (Michod, 1999; Michod and Roze,1999, 2000). The level of kinship among cells in theoffspring groups is determined byN.

During development, cells proliferate and die (pos-sibly at different rates depending on cell behaviour)to create the adult cell group. Cell behaviour is as-sumed to be determined by a single haploid geneticlocus with two alleles, C (for cooperative cells) andD for defecting cells. Deleterious mutation (from Cto D) may occur during cell division leading to theloss of cooperative cell functions (such as the propen-sity to become motile in aVolvoxcolony consideredlater) and a decrease in fitness of the adult group. The


adult group produces offspring groups of the nextgeneration. We have previously considered severaldifferent modes of reproduction according to howthe offspring group is produced: single cell (or sporereproduction), fragmentation and aggregation. In thepresent model, we consider only single cell reproduc-tion and assume that development starts from a singlecell (N = 1), as is the case inVolvox carteriand theother members of the volvocine lineage (seeFig. 5later).

The basic parameters of the model include develop-ment time,t, the within organism mutation rate fromC to D per cell division,µ, the effect of mutation onthe cell replication rate,b (b < 1 or b > 1 means uni-formly deleterious or selfish mutations, respectively),the deleterious effect of mutation on the cell group ororganism,β, and the propagule size,N. The parame-ter β measures the benefit of cooperation at the groupor organism level, and, hence, the deleterious effect ofmutation on group fitness. In addition, there is a pa-rameter that tunes the relative effect of group size onfitness.

To study how evolution may shape developmentand the opportunity for selection at the two levels oforganization, we assume a second modifier locus withtwo alleles M and m. The m allele encodes the ances-tral state (no mediation) while the M allele changesthe parameters of development and/or selection atthe primary cooperate/defect locus. In this way, wemay understand the evolution of developmental pro-grams that permit and enhance the evolvability ofthe group. The conflict mediator M allele may affectvirtually any aspect of the model, such as propagulesize,N (studied elsewhere,Michod and Roze, 1999,2000; Roze and Michod, 2001), adult size (whetherit is determinate or indeterminate, Michod and Liunpublished results), self-policing, programmed celldeath, development time and whether there is anearly-segregated germ-line. To study the evolution ofself-policing, we assumed the modifier affects the pa-rameters of selection at both levels,b andβ, reducingthe temptation to defect at some cost to the group(Michod, 1996; Michod and Roze, 1999). In the caseof the evolution of programmed cell death, we as-sumed the modifier directly decreases the replicationrate of mutant cells (Michod and Nedelcu, 2003). Inthe present paper, we study how the M allele maycreate an early-segregated germ-line.

2.3. Details of germ-line model

Using the two locus modifier model just out-lined, we have studied the evolution of a sequesteredgerm-line (Michod, 1996; Michod and Roze, 1997,1999). We assume that the modifier M allele cre-ates two separate lineages of cells within the group,a germ- and somatic-line, which may each undergoa different number of cell divisions and experience adifferent mutation rate. In our previous work, we as-sumed that the germ-line was sequestered as asinglecell set aside during thefirst cell division. Most organ-isms depart from this ideal, includingV. carteri, andsequester cells later in development. For this reason,we model the selective forces acting on the time ofsequestration,θ, and the number of cells sequestered,ν. In our previous work, we assumedθ = ν = 1.

A critical assumption concerns the definition of fit-ness in groups with and without germ/soma differ-entiation. In groups without separate germ and so-matic cells, all cells in the group perform reproductive(germ) and vegetative (somatic) functions. We assumefitness is proportional to group size, the number ofcells in the group,k, multiplied by a term represent-ing the functionality of the group, (1+ βf ), wherefis the frequency of cooperative cells in the group andβ is the benefit of cooperation discussed previously.This givesEq. (1) for the fitness of groups withoutgerm/soma differentiation.

W = k(1 + βf) (1)

In the groups with separate germ and somaticcells, there must be fewer cells available for somaticfunction, because some cells,ν, have been put asideto produce the germ-line cells. Consequently, theremust be a cost to sequestering theν cells; how shouldwe represent this cost during this early stage ofgerm/soma evolution?

We consider the initial phases of germ/soma evo-lution in which the presence of a differentiatedgerm-line is the only difference between the twokinds of groups (groups made up of undifferenti-ated cells and groups made up of differentiated germand somatic cells). For example, we assume that theresources available to the group remain essentiallyunchanged by germ/soma differentiation, except as aresult of changes in the numbers of cooperative andmutant cell types. The number of cell types change in


the differentiated group, because cooperative behav-iors are expressed only by somatic cells. By virtueof specializing in a single function, such as motility,somatic cells may be able to perform the vegetativefunctions better or for longer periods of time (after allsomatic cells do not have to take time to reproducethe group). These benefits of specialization are clearlyimportant and make it easier to evolve germ/somadifferentiation, but we ignore these effects here; theirconsequences seem obvious (in the sense that theywill make it easier to evolve germ/soma differentia-tion), and our interest lies elsewhere in the mediationof conflict between levels of selection brought aboutby the evolution of the germ-line.

To understand the cost of sequesteringν cells tocreate the germ-line, we think of theseν cells interms of two descendent populations of cells, accord-ing to the conditions either of their new existence asdifferentiated germs, or their prior existence as mem-bers of an undifferentiated cell group. In the case oftheir prior existence, theν cells would have givenrise to a descendent population of size, say,kν cellsundifferentiated with respect to reproductive or veg-etative functions. In the case of their new existenceas differentiated germs, the germ sample (theν cells)replicates for perhaps a different period of time witha different mutation rate, giving rise to a descendentpopulation of size, say,Kν germ cells. The cost ofthe germ-line can be seen as stemming from eitherthe newKν germ cells or the missingkν cells avail-able for vegetative (somatic) functions.

Following the definition of fitness in undifferenti-ated groups (Eq. (1)), we assume that fitness of thenewly differentiated group is fecundity multipliedagain by a term representing the functionality ofthe group (which has now changed because of thegerm-line cost). Fecundity is the number of gametes,taken to be proportional to size of the germ-lineKν.For concreteness, we think of the functionality asthe amount of resources accrued by the group forreproduction and we let fitness equal the total num-ber of gametes produced multiplied by the amount ofresources received by each gamete. We assume thatdifferentiation does not change the level of resourcesavailable to the group, taken to be proportional to theundifferentiated group size,k, except that there areKν new germ cells present, orkν somatic cells absent(depending on how the germ-line cost is interpreted).

When the cost of the germ-line is based on the newgerm cells, fitness equalsKν (k −Kν)(1+βfν)/Kν =(k − Kν)(1 + βfν), wherefν is now the frequency ofcooperating cells in the soma (instead of in the totalgroup), givingEq. (2).

Wν = (k − Kν)(1 + βfν) (2)

Eqs. (1) and (2)were used previously in our study ofgerm-line evolution when sequestration of a single celloccurred during the first cell division (Michod, 1996,1999; Michod and Roze, 1997, 1999, 2000). When thecost of the germ-line is based on the missing somaticcells, kν, fitness equalsKν(k − kν)(1 + βfν)/Kν =(k − kν)(1 + βfν) giving Eq. (3).

Wν = (k − kν)(1 + βfν) (3)

In both Eqs. (2) and (3), the number of gametescancels and fitness depends only on the amount ofresource. In our model, producing many low qualitygametes is the same as producing a few high qualityones. As shown later, depending upon how the cost ofa germ-line is paid (whether we useEqs. (2) and (3)),early sequestration of the germ-line may be advanta-geous or not.

As already mentioned, in many organisms with agerm-line, the germ-line is sequestered not during thefirst cell division but later in development. For ex-ample, in the green alga,V. carteri (considered inmore detail later), the precursors of the germ-line areformed after five cell divisions, but the germ-line issequestered only after the ninth cell division. We ex-tend our model to study the time of sequestration byintroducing two new parameters defined inFig. 2, thetime when the segregation occurs (θ) and the numberof cells sequestrated to form the germ-line (ν). In ourprevious model, the germ-line was differentiated fromthe soma at the beginning of the development, from

Fig. 2. Time of germ–soma segregation. Afterθ divisions,ν cellsare sampled to form the germ-line, each cell in the germ-linedivides tG more times. The number of cell divisions in the somais t andδ = t − tG −θ is the difference in number of cell divisionsbetween the germ-line and the soma.


one single cell so thatθ = 1, ν = 1 andtG was a freeparameter. Among the sample of cells sequestrated toform the germ-line at timeθ, there will be some Ccells and some D cells, the number of C cells beinggiven by a probability distribution. The technical de-tails underlying this distribution and its implicationsfor the model are given in theAppendix A.

Using two locus modifier techniques outlined earlierand discussed further inAppendix A, we have stud-ied the evolution of germ-line modifiers that sequesterν cells afterθ cell divisions for the two kinds of fit-ness functions (two kinds of germ-line cost) given inEqs. (2) and (3). In both formulations, fitness depends

Fig. 3. Evolution of germ-line segregation using fitnessEq. (2). In Eq. (2), we represent the cost of the germ-line by subtracting offthe number of resulting germ cells (after theν cells replicate fortG times), k − Kν. The germ-line modifier is characterized by threeparameters: the time of sequestration,θ, the number of divisions after sequestration,tG, and the number of cells sampled,ν. Germ-lineevolves for parameter values below the surfaces in all panels. The total number of replications in the germ istG + θ. The parameters forthe three slices in the two dimensional plots are given from top to bottom. For example, in panel (D), the top solid curve is forθ = 5and the bottom dotted curve is forθ = 20. Propagule sizeN = 1. Methods used to construct the graphs are given in theAppendix A.

upon the resources available to the germ-line, howeverin Eq. (2), the cost of the germ-line depends upon thenumber of gametes produced by the germ-line, whilein Eq. (3), the cost depends on the missing cells thatare no longer available for vegetative function.

3. Results

Results using fitnessEq. (2)are given inFig. 3 forthe following parameter valuesµ = µG = 0.003,β = 3, b = 1.05, t = 30 and no sex or recombination(r = 0). These parameter values have been explored


and discussed in our previous work and they are usedhere for comparison purposes. Please consult our pre-vious papers for discussion of the rationale for us-ing these parameter values (for example,Michod andRoze, 2000). The germ-line modifier evolves for pa-rameter values below the surfaces plotted inFig. 3. Inour previous work, the germ-line modifier was char-acterized by a single parameter,δ, the difference innumber of cell divisions between the germ-line and thesomatic-line. However, the germ-line modifier is nowcharacterized by three parameters: the time of seques-tration,θ, the number of divisions after sequestration,tG, and the number of cells sampled,ν. The differencein number of cell divisions between the germ-line andthe soma,δ = t − tG − θ, now depends on two of thegerm-line parameters in addition to the soma parame-

Fig. 4. Evolution of germ-line segregation using fitnessEq. (3). In Eq. (3), we represent the cost of the germ-line by subtracting off themissing somatic cells,k − kν. Germ-line evolves for parameter values below the surfaces in all panels. See legend toFig. 3. PropagulesizeN = 1. Methods used to construct the graphs are given in theAppendix A.

ter, t. The number of cell divisions after segregation ofthe germ-line,tG, is important in that it (in conjunc-tion with the number of cells sampled,ν) determinesthe size of the germ-line and ultimately the number ofgametes, that is this parameter determines fecundity(seeFig. 8 later).

The results inFig. 3 are straight forward; it is eas-ier for a germ-line to evolve (larger germ-lines maybe produced) the earlier the germ-line is sequestered(Fig. 3, especially panel (B)), the lower the numberof times it divides, and the fewer number of cells thatare sampled. This is because there are only advantagesto early segregation and low replication (in terms ofa lower effective deleterious mutation rate resultingfrom the fewer number of cell divisions), and the costof the germ-line is smaller the fewer cells that are


sampled. Recall, that in our formulation, fitness doesnot depend on the size of the germ-line directly (onlythrough resource availability), since the number of ga-metes cancels out in the formulation of fitness. Organ-isms following this model (usingEq. (2)) should forma germ-line by sequestering a single non-dividing cellduring the first cell division.

What about when the resources available to thegerm-line depend on the number of cells missing un-available for vegetative (somatic) function? The miss-ing cells are those that would have been formed by theν cells sequestered to form the germ cells. InFig. 4,we show the results for the same set of parameter val-ues as used inFig. 3, except usingEq. (3) instead ofEq. (2). In this case, there is a cost to the germ-line interms of missing somatic cells, and, so, there is an in-termediate optimum time for sequestration,θ (Fig. 4,especially panels (B) and (C)). Smallerθ is better interms of coping with the threat of deleterious muta-tion, however, there is a greater penalty to pay in termsof missing cells unavailable for somatic function.

4. Volvocalean green algae as a study case

4.1. The volvocalean green algal group

We use the volvocalean green algal group to testour model for the evolution of an early segregatedgerm-line. The volvocalean green algae compriseboth unicellular (Chlamydomonas-like) algae as wellas colonial forms in different stages of organizationaland developmental complexity (Fig. 5). Interestingly,both multicellularity and germ/soma separation haveevolved multiple times in this group. The differentlevels of organizational and developmental complex-ity are thought to be alternative stable states (Larsonet al., 1992). Nevertheless, despite their multiple andindependent acquisition of the multicellular state andgerm/some separation, none of these multicellularlineages attained high levels of complexity, as did thegreen algal ancestors of land plants, the charophytes.We believe that understanding this limited spurt of di-versification in complexity in this lineage will provideinsight into how a transition to multicellularity mayaffect the evolvability of the new multicellular unit(Nedelcu and Michod, 2003). We suggest here that,although a germ-line acts to mediate conflict between

Fig. 5. The volvocine lineage. A subset of colonial volvocaleangreen algae that show a progressive increase in cell number, vol-ume of extracellular matrix per cell, division of labor betweensomatic and reproductive cells, and proportion of vegetative cells.A: Chlamydomonas reinhardtii; B: Gonium pectorale; C: Pando-rina morum; D: Eudorina elegans; E: Pleodorina californica; F:V. carteri. Where two cell types are present, the smaller cells arethe vegetative/somatic cells, whereas the larger cells are the repro-ductive cells (gonidia). Images kindly provided by David L. Kirk.

the group and cell levels, and is thereby expected tocontribute to the evolvability of the new multicellulargroup, the way in which the germ and somatic-linesare created may nevertheless interfere with the longerterm evolvability of the lineage.

The basic morphological and developmental traitsof the volvocalean green algae appear to result fromthe interaction of conflicting structural constraints (im-posed by two very conserved anatomic traits) as wellas two strong selective pressures. The first structuralconstraint is the so-called “flagellation constraint”(Koufopanou, 1994) which leads to a trade-off be-tween reproduction and motility. This constraint hasa different structural basis than the one invoked in theorigin of metazoans (Margulis, 1981; Buss, 1987),although the end result is similar. In most green flag-ellates, during cell division the flagellar basal bodiesremain attached to the plasma membrane and flagella,and can act as centrioles (which is not the case inanimal cells); however, in volvocalean algae, due to acoherent rigid cell wall the position of flagella is fixedand thus, the basal bodies cannot move laterally andtake the position expected for centrioles during celldivision while remaining attached to the flagella (asthey do in other green flagellates). Consequently, celldivision and flagellar motility can take place simulta-neously only for as long as flagella can beat without


having the basal bodies attached (i.e. up to five celldivisions). The second constraint comes from theunique way of cell division in volvocalean green algae,namely, the multiple fission type of division termedpalintomy: cells do not double in size and then un-dergo binary fission; rather, cells grow about 2n -foldin volume, and then rapidly undergo a synchronousseries ofn divisions (under the mother cell wall).Because clusters, rather than individual cells, are pro-duced in this way, this type of division is suggestedto have been an important precondition facilitatingthe formation of volvocacean colonies (Kirk, 1998).

The two selective pressures that are thought to havefavored an increase in complexity in volvocalean al-gae are the advantages of a large size and the needfor motility (Bell, 1985; Kirk, 1998). Larger size isthought to be advantageous by allowing colonies toescape predators, move faster, maintain better ho-moeostasis or better exploit eutrophic conditions,while motility allows the colonies better access to theeuphotic/photosynthetic zone. Interestingly, as dis-cussed next, given the ancestral constraints just men-tioned, namely the flagellar constraint and the multiplefission type of cell division, it is difficult to achievethe two selective advantages simultaneously. Largersize via higher number of cells requires increased to-tal time for cell division and, when coupled with theflagellation constraint, this means decreased motility.

4.2. Germ/soma separation in Volvox

4.2.1. OverviewAs the volvocalean colonies increase in size and

number of cells, the number of cell divisions as well asthe size of the mature reproductive cell also increase.Due to the flagellation and palintomic constraints dis-cussed above, the motility of the colony during thereproductive phase is negatively impacted for longerperiods of time than are acceptable in terms of theneed to access the photosynthetic zone. For instance,in a V. carteri mutant with only 256 cells and nogerm/soma separation (discussed later), the flagellarmotility of the colony is negatively affected for asmuch 70% of the life cycle (i.e. 49–50 h in a 72-h lifecycle); its decreased motility might be responsible forthis mutant not being found in nature. In short, theneed for the colony to increase in size detracts fromthe motility of the colony.

This negative impact of the flagellation and palin-tomy constraints was likely overcome by a divisionof labor (cellular differentiation) in the colony: somecells became specialized for motility, while otherswere assigned to reproductive functions. The propor-tion of cells that maintain motility and become sterileis directly correlated with the total number of cellsin a colony: from none inChlamydomonas, Gonium,and Eudorina, to up to one-half inPleodorina andover 99% inVolvox(Larson et al., 1992). In the latter,the division of labor is complete: only the gonidia(the reproductive cells) undergo cleavage to formnew colonies; the somatic cells are sterile, terminallydifferentiated, and are thought to be genetically pro-grammed to undergo cellular senescence and deathonce the progeny was released from the parentalcolony (Pommerville and Kochert, 1981, 1982).

Volvox represents the most complex multicellularform in the volvocine lineage (Fig. 5). Germ/somaseparation evolved at least twice, but possibly severaltimes inVolvox(Kirk, 1999) and is realized differentlyamong the at least 18 recognized species ofVolvox(Desnitski, 1995). In someVolvoxspecies, includingV. carteri, the two types of cells are set apart by asym-metric divisions early in the embryonic development.Moreover, inV. carteri a difference in size, not a dif-ference in cytoplasmic quality, determines which path-way of differentiation a cell will follow (Kirk et al.,1993). It should be mentioned that our discussion lateris relevant only to this particular species ofVolvox.

4.2.2. A comparative viewMany multicellular organisms have a germ-line that

is segregated early in the development (for a list seefor example Table 1.1 inBuss, 1987). Among these,the nematode wormCaenorhabditis elegansand thegreen algaV. carterihave been characterized in detailin terms of their development. Interestingly, althoughthe number of somatic cells is small in both organ-isms (i.e. 959 cells versus 2000–4000, respectively),there are major differences between the two specieswith respect to the way in which germ-line is produced(Fig. 6) and the number of cell types (more than 20in the former and only 2 in the latter). What might bethe evolutionary reasons for such differences? Later,we describe features of development in the two lin-eages, contrast them, and point out the peculiarities ofgerm/soma separation inV. carteri.


Fig. 6. Two examples of early germ-line segregation. (A) Germ-line segregation in the nematode wormC. elegans; gray, black, and whiteellipses indicate germ-line blastomeres, primordial germ cells, and somatic blastomeres, respectively. P0 denotes the zygote; P1, P2, P3,are the germ-line blastomeres; P4 is the germ-line founder cell; Z2 and Z3 are primordial germ cells. Numerals indicate successive celldivisions. (B) Germ-line segregation in the green algaV. carteri; gray and white large ellipses denote totipotent and somatic blastomeres,respectively; gray, black, and white small ellipses indicate germ-line blastomeres, gonidia, and somatic initials/cells, respectively. Detailsforthe division pathways are shown only for one germ-line founder cell and one somatic blastomere. Numerals mark successive cell divisions.

The first four cell divisions during theC. elegansembryonic development are asymmetric (e.g.Seydouxand Schedl, 2001). The zygote, P0, as well as thesmaller cells resulting from the first three asymmet-ric divisions (i.e. P1–P3 inFig. 6A) act as stem cells:with every asymmetric cell division they produce an-other totipotent stem cell (they renew themselves) anda somatic blastomere. These stem cells, or germ-lineblastomeres, are the precursors of the germ-line. Thelast of these cells, P4, represents the founder cell (orprogenitor) of the germ-line. Ultimately, the P4 celldivides symmetrically to produce two primordial germcells (PGCs), Z2 and Z3, that migrate to the gonadand arrest mitosis until the larva hatches; later on, Z2and Z3 proliferate and undergo meiosis to producethe germ cells. What specifies the fate of the cellsfollowing the four asymmetric divisions is a set ofgerm-line granules, or P granules, which are presentin the zygote and are selectively distributed only to thegerm-line precursors and ultimately to the two PGCs(e.g.Seydoux and Schedl, 2001). The larger cells re-sulting from the first four asymmetric divisions act assomatic blastomeres or founder cells, and proliferate

to form various cell types, such as neurons, epithelial,muscle, gland cells, etc.; some of the divisions respon-sible for the 959 somatic cells in the adult take placeafter the larvae hatch.

In contrast toC. elegans, the first divisions inV.carteri are symmetrical (Fig. 6B). Up to the 16-cellstage, all the blastomeres are totipotent (if removedthey can create a new colony); after the fifth division,the 16 cells situated at the anterior pole of the embryowill take a distinct path relative to the 16 cells at theposterior pole. The first difference is that the sixthcell division is asymmetric in the 16 anterior cellsbut symmetric in the other 16 cells. What determineswhich cells will undergo an asymmetric division isnot well understood; physical cues (i.e. physical con-straints) associated with the number of cytoplasmicbridges and the position of cells in the embryo arethought to be involved in this process (Kirk, 1994).By having the first blastomeres functionally equiva-lent (i.e. totipotent) and by relying on spatial cues forinitiating the pathway leading to the differentiationof the germ-line,V. carteri differs from C. elegansbut resembles mammals (e.g.Anderson et al., 2001).


However, the further steps in the segregation of thegerm-line are rather unique inV. carteri.

The 16 cells predetermined to produce the germ-lineare totipotent, act as stem cells and undergo a seriesof three–four successive asymmetric cell divisions, ina manner similar to that of theC. elegansgerm-lineprecursors (germ-line blastomeres P0–P3) (Fig. 6).Nevertheless, the similarities stop here. In contrast toC. elegans, in V. carteri, the larger cell resulting fromthe asymmetric division is the one to act as a germ-lineblastomere. Furthermore, the asymmetric division isnot accompanied by a differential segregation of aspecific cytoplasmic component; what triggers the so-matic fate of the smaller cells inV. carteri is not thelack of distinct cytoplasmic components, as it is inC.elegans, Drosophila, andXenopus(e.g. Kloc et al.,2001; Mahowald, 2001; Seydoux and Schedl, 2001),but rather strictly a difference in cell size (Kirk et al.,1993). In addition, the large cells that result fromthe ninth division (and which are analogous to theC. elegans’s progenitor of the germ-line, P4) do notdivide to produce mitotically active primordial germcells, PGCs. Rather, these cells arrest mitosis (whilethe somatic blastomeres continue to divide for anothertwo–three cycles) and differentiate without furtherdivisions into single non-flagellated germ cells, go-nidia. Nevertheless, although each germ-line foundercell produces only one germ cell (gonidium), it isinteresting that there are up to 16 such cells in eachspheroid, as if there were 16 independent germ-lineseach producing a single germ cell. On the other hand,the smaller cells produced though asymmetric celldivisions in the anterior pole as well as the cells pro-duced by the 16 posterior blastomeres stop dividingwhile still in the embryo, and all differentiate into thesame type of cell, i.e. flagellated somatic cells.

5. Germ/soma separation and evolvability duringthe transition to multicellularity

We have discussed elsewhere how the transitionfrom unicellular to multicellular life requires the de-coupling of basic life-properties at the lower level andtheir re-coupling and re-organization in new ways atthe higher level (Nedelcu and Michod, 2003). More-over, we argued that some of the differences among theextant species, including differences in evolvability,

can be explained by the way in which the decouplingand re-coupling of these properties has been achievedduring the transition to multicellularity.

Two of the complex life-traits that become re-organized during the unicellular–multicellular transi-tion are immortality and totipotency. By “immortality”we mean the continued capacity for cell division andby “totipotency” we mean the capacity of a cell tocreate a new organism. In unicellular organisms, thesetraits are necessarily coupled at the cell level in thesense that they are both fully expressed in all cells.In multicellular organisms, however, the two traitsare re-organized within and between cell lineages.In species with defined germ and somatic-lines, thetwo traits are fully expressed only in the germ-line,whereas somatic cells obviously do not expresstotipotency and express continued replicative ability,that is immortality, to varying degrees. InV. carteri,immortality and totipotency are fully restricted to thegerm-line and somatic-lineages have no mitotic orreplicative potential (Nedelcu and Michod, 2003). Theun-coupling of immortality and totipotency provednot possible inV. carteri: these traits are express ei-ther together and fully (i.e. in the gonidia) or not at all(i.e. in the somatic cells). Immortality and totipotencyare thus still tightly linked inV. carteri, as they arein their unicellular ancestors. In support of this viewis the fact that “cancer-like” mutant somatic cells, inwhich immortality but not totipotency is re-gained,are missing inV. carteri. There are, however mutantforms ofV. carteri (discussed later) in which somaticcells re-gain both immortality and totipotency, but inneither of these mutants are the two traits expressedpartially or differentially (e.g. limited mitotic capacityor multipotency).

To ensure the emergence of individuality at thehigher level and the reproduction of the multicellu-lar individual (i.e. the heritability of the group-leveltraits), immortality and cell division have to bede-coupled from the reproduction of the lower-levelcells (previously unicellular individuals) and be co-opted for the reproduction of the group (the higher-level multicellular unit). Furthermore, in lineages witha separation between germ and soma, in somatic celllineages, cell division is not associated with repro-duction of the individual but rather became co-optedfor growth of the multicellular individual (Nedelcuand Michod, 2003).


V. carteri avoided the risks and potential conflictsof cell division in somatic cells by blocking it alto-gether and in this way achieved individuality at thecell-group level. In addition cell division was notco-opted for the post-embryonic growth of the mul-ticellular individual. We believe that this solution tothe mediation of conflicts at the lower level affectedthe long-term evolutionary adaptability of the lin-eage. Later, we investigate the possible reasons forundertaking this peculiar pathway. Our fundamentalpoint is that, although conflict mediation is pivotalto the emergence of individuality at the higher-level,the way in which the mediation is achieved duringthe transition in individuality can interfere with thelong-term evolvability of the lineage.

6. The evolution of a germ-line in Volvox carteri

6.1. The question

In many lineages, includingC. elegansfor instance,the germ cells are the descendants of a single germcell founder (e.g. P4 inC. elegans; Fig. 6A). In con-trast, as we discussed earlier, inV. carteri, the 16 germcells are formed from 16 independent germ-line blas-tomeres that stop dividing and differentiate directlyinto gonidia (Fig. 6B). Theoretically, the same 16 go-

Fig. 7. Two alternative scenarios for germ-line segregation inV. carteri. (A). The alternative that has been selected for inV. carteri:germ cells differentiate from 16 independent germ-line founder cells. (B). Our theoretical alternative: germ-line cells derive from a singlegerm-line founder cell. Symbols are as inFig. 6.

nidia could be produced from a single founder cell (asis the case inC. elegans) by dividing a total of fourtimes (1×24 = 16); this raises the question as to whyin V. carteri the germ cells differentiate from 16 in-dependent germ-line founder cells instead of only onegerm founder cell.

To address this issue, we envisioned forV. car-teri a developmental pattern similar to that ofC. el-egans, and asked what the consequences would be.Surprisingly, such a pathway would be identical tothe current one in terms of the numbers of gonidiaand somatic cells (Fig. 7). We envisioned that af-ter the fifth cell division (which is the time wherethe 16 germ-line blastomeres are determined inV.carteri), only one cell is sequestered, and would di-vide symmetrically a total of four times to produce16 gonidia; the other 31 cells undergo a path iden-tical to that of the 16 posterior cells inV. carteri,that is they divide symmetrically seven times to pro-duce somatic cells (Fig. 7). Interestingly, by havingonly one cell acting as a germ-line founder and 31(instead of 16) cells acting as somatic blastomeres,the same total of 3968 somatic cells (i.e. 31× 27 =3968) and 16 gonidia (i.e. 1× 24 = 16) would beproduced. Furthermore, under this scenario, asymmet-ric divisions are not required (see discussion later).Then, why was the former alternative favored overthe latter, especially when it requires the evolution


of a new trait, namely, the asymmetric type of celldivision?

6.2. Applying the model

Can the model on the evolution of an early seg-regated germ-line presented earlier help address thisquestion? We use our model to ask under which condi-tions would one or the other alternative diagrammed inFig. 7be selected for, so as to understand the “choice”thatV. carteri has made.

6.2.1. AssumptionsIn trying to understand the various selective factors,

we assume that 12 cell divisions is the highest numberof cell divisions possible under the multiple-fissionpalintomy type of cell division. In reality, this istrue for V. carteri. As discussed earlier, with themultiple-fission palintomy, to reproduce a colony with2n cells, a single reproductive cell must first grow 2n

before it dividesn times. Clearly, a single cell cannotgrow indefinitely in size (especially if it is a sphericalshape) without affecting its metabolic abilities (dueto changes of the surface/volume ratio). It appearsthat a 500–1000-fold increase in size is the most agonidia can stand without affecting its metabolism,and 212 cells is the highest number of viable cellsthat can result from such a gonidia. We do not have adetailed argument for this assumption, however thereare no palintomicVolvoxspecies in whichn is higherthan 12. Nevertheless, inVolvox species that have“escaped” the palintomic constraint,n can be higherthan 12. As a consequence, gonidia do not have togrow up to 2n and then dividen times, rather they onlydouble in size, divide, grow between divisions, divideagain and so on. Interestingly, in the non-palintomicspecies, gonidia differentiate late in development,suggesting a correlation between palintomy andearly-segregation of germ (i.e., the fast cell divisioncycles associated with palintomy may cause highermutation rates).

6.2.2. Cost of the germ-lineWe interpret the cost of the germ-line in terms of the

missing somatic cells that are unavailable for vegeta-tive functions (as expressed inEq. (3)). In V. carteri,such a cost is likely very important, due to the selec-tive pressures mentioned previously, namely, the need

for daily vertical migrations in the water column anda large body-size. The presence of a non-flagellatedgerm-line affects the fitness of the group by interferingwith the mobility of the group through decreasing thenumber of cells that participate in motility (i.e. by af-fecting the ratio of flagellated to non-flagellated cells).In addition, an early segregated and arrested germ-linedecreases the overall number of cells in the group andthus the body-size is affected.

The time of sequestration as well as the number ofcell divisions following the sequestration event influ-ence the cost of the germ-line both in terms of thetotal number of cells missing in the group as well asthe missing soma, that is missing flagellated cells. Weillustrate the trade-offs involved with three examples.First, if only one germ-line founder cell is sequesteredafter the first embryonic cell division (as in our ear-lier model) and continues to divide for the same 12times as the somatic-lineages, the total number of cellswould not be affected (i.e. 4096), but the flagellatedto non-flagellated cell ratio would be 1:1 and thus themotility of the group would be greatly affected. In thiscase, the cost of the germ-line is only in terms of thenumber of flagellated cells missing (and not overallcolony size), but the cost is very high (i.e. 2048 miss-ing motile cells). In addition, the germ cells wouldlikely accumulate the same number of mutations as thesomatic cells assuming mutation is dependent on thenumber of DNA replications. Second, if the foundercell segregated after the first cell division and under-went only four cell divisions to produce 16 gonidia(and then arrest mitosis while the somatic-lineagescontinue dividing for a total of 12 times) the groupwould be composed of only 2048 somatic cells and16 gonidia. In this case, the ratio increases in the fa-vor of the motile cells (2048:16), but the colony sizeis affected (2064 versus 4096 cells). The cost of hav-ing a germ-line segregating very early and arrestingmitosis after four cell divisions is very high in thisexample, both in terms of total number of cells (i.e.2032 missing cells) as well flagellated cells (i.e. 2048cells). However, germ cells would have undergone afewer number of cell divisions than the somatic cellsand would have fewer mutations as a result. Third,at the other extreme, if the founder cell is put asideonly after the eighth division and undergoes four addi-tional cell divisions to produce the 16 gonidia, the costwould only be in terms of the flagellated cells, which


is very low (i.e. 16 cells). Nevertheless, the germ cellswould be dividing for the same number of times as thesomatic cells.

A compromise may be reached between groupsize and percentage of flagellated cells by having thegerm-line segregate at an intermediate stage duringdevelopment and arresting mitosis earlier than in thesomatic-lineages, such that both the cost in terms ofmissing cells and the mutation levels are lower. Ifonly one founder-germ cell segregates after the fifthdivision (Fig. 7B) and undergoes a number of addi-tional four divisions to produce the 16 gonidia, thecost of germ-line in terms of total number of cellsis 112 cells (4096− 3984 = 112) and in terms offlagellated somatic cells is 128 cells (4096− 3968=128). In addition, there is a 25% decrease in thenumber of cell divisions in the germ cells relativeto somatic cells (9 versus 12), which might resultin a lower mutation level in the germ-line. How-ever, as discussed later, the same low cost can beachieved through either the scenario just discussedor the pathway thatV. carteri is actually following(Fig. 7A); why the latter was selected for is discussednext.

6.2.3. Parameters and valuesWe used two sets of values for the model’s param-

eters: first, the values used byV. carteri, and second,the values that correspond to the theoretical alter-native we envisioned inFig. 7B. In both cases, thesomatic cells divide for up to 12 times, thust = 12in our model. Likewise, the cost of the germ-lineis the same in both cases following that assumed inEq. (3) earlier; if all cells remained undifferentiatedand replicated 12 times, there would be 4096 cells inthe group, so there are 128 (i.e. 4096 – 3968) cellsmissing in the soma. We assume asexual reproduction(r = 0), a significant benefit of cooperation (comingfrom motility) β = 10, and that the mutation rates percell division are equal in the germ and soma,µ = µG.In addition, for each set of conditions, we used twovalues for the mutation rate,µ = 0.003 and an orderof magnitude higherµ = 0.03 (and, as already men-tioned, the number of missing somatic cells is thesame in both cases). The difference between the twosituations concerns the sampling of mutations and theresulting opportunities for conflict between levels ofselection.

The parameters that differ in value between thetwo cases are the time of sequestration (θ), the num-ber of germ-line cell divisions after sequestration(tG), and the number of cells sampled (i.e. germ-linefounders,ν). In the first case (Fig. 7A), there are16 germ-line founder cells (ν = 16) that segregatefrom the somatic-lineage after the ninth cell division(θ = 9) and arrest mitosis at that very point (tG = 0).In contrast, in the second case (Fig. 7B), there is onlyone germ-line founder cell (ν = 1) that segregatesearlier (θ = 5) and divides for an additional fourtimes (tG = 4). In both cases, the total number of celldivisions that gonidia undergo is nine, while the addi-tional number of cells divisions in the somatic-lineageis three (δ = 3).

6.2.4. PredictionsFig. 8 depicts the results of our model with both

sets of values (panels A and C versus panels B andD) and under two mutation rates (panels A and Bversus panels C and D). Interestingly, the model pre-dicts that a germ-line is easier to evolve (the germ-lineevolves for a greater range of parameter values) un-der the second set of parameter values, namely, thosecorresponding to a germ-line descending from a sin-gle germ-cell founder, the route not taken inV. carteri.In both cases, the model predicts that the germ-lineevolves easier when mutation is a threat, either be-cause mutations are selfish (b > 1) or frequent (a highmutation rate). When mutations are frequent, cooper-ation is harder to maintain, and so the within-groupadvantage for the mutant cells (represented by param-eterb) has to be lower. Under a lower mutation rate,a germ-line can evolve only when the within-groupadvantage of mutant cells is rather high, approaching1.2 in the first case, or 1.1 in the second case. Also,in the first case (Fig. 8 panel C), a germ-line evolvesonly if the number of cell divisions after segregationis very small, approximatelytG must be less than 2.These conclusions are mirrored inFig. 4. Panel C ofFig. 4 shows thattG (fecundity) can be higher withν = 1 andθ = 5 than withν = 16 andθ = 9, andthis explains the results ofFig. 8. It is always good todecreaseν (the initial number of germ cells), and it isgood to decreaseθ up to a certain point, after whichthere are not enough somatic cells (this is why we seea maximum onFig. 4C). So we can not say a prioriif ν = 1 andθ = 5 is better thanν = 16 andθ = 9


Fig. 8. Evolution of germ-line segregation inV. carteri. The main contrast in the figure is between a germ-line developed from 16 foundercells sequestered at cell division 9 (panels (A) and (C)) and a germ-line from a single founder cell sequestered at cell division 5 (panels(B) and (D)). The ordinate istG (as it is inFigs. 3 and 4) except expressed in terms of fecundity, that is the number of resulting gonidialgerm cells. For example, if a single cell is sequestered (ν = 1) to divide five times (tG = 5), fecundity is 16 gonidia. Likewise, if 16cells are sequestered (ν = 16) to divide no times (tG = 0), fecundity is 16 gonidia. The mutation rate is an order of magnitude higherin the bottom panels than in the top panels. In the region labeled “no cooperation” group living cannot be maintained. In the two otherregions, cooperative groups are maintained with or without a germ-line. All panels assume fitnessEq. (3) (cost of the germ-line resultsfrom missing somatic cells), equal mutation rate in germ and soma (µ = µG), no sex (r = 0), benefit of cooperationβ = 10, numberof cell divisions in somat = 12. RecalltG is the number of cell divisions in the germ-line after sequestration andb is the within-groupadvantage of mutants. Notingδ = t − tG, compare Figs. 6–1 (Michod, 1999), Fig. 1 (Michod and Roze, 1997), Fig. 6 (Michod and Roze,1999). Methods used to construct the graphs are given in theAppendix A.

(because of the non linear effect ofθ), butFigs. 4 and 8show that it should be better. However, the develop-mental biology ofVolvox carteridoes not agree withthis prediction of the model.

In summary, the model predicts that inV. carteri agerm-line is easier to evolve if mutant cells are selfish(but not so selfish that cooperation cannot be main-tained), or if the mutation rate is rather high (Fig. 8).Are any of these conditions met inV. carteri? More-over, the model suggests that the scenario based ona single germ-cell founder (Fig. 7B) should be easierto evolve than the one that has actually evolved inV.carteri (Fig. 7A). Then, why was the germ-line inV.carteri not formed under the conditions (i.e. a singlegerm-cell founder) that the model predicts are mostconducive for the evolution of a germ-line? Below wediscuss these issues.

7. Discussion

7.1. Selfish mutants

According to the model (Fig. 8), a germ-line ispredicted to evolve only when the advantage ofwithin-group mutations is rather high. This is consis-tent with the fact inV. carteri many mutations thataffect the somatic cells are selfish. In the somatic re-generator mutants, or Reg mutants, the somatic cellsstart out as small flagellated cells (wild type-like) andthen enlarge, loose flagella and re-differentiate intogonidia. A number of 39 mutants in four phenotypicclasses have been investigated, and all had muta-tions at the same locus,regA (Huskey and Griffin,1979). The gene affected in these mutants has beenshown to encode for an active repressor (Kirk et al.,


1999) that targets a number of at least 13 nucleargenes whose products are required for chloroplastbiogenesis (Choi et al., 1996; Meissner et al., 1999b).This finding suggests that the mechanism for theestablishment of a stable germ-soma separation inV. carteri is based on preventing the somatic cellsfrom growing (by repressing chloroplast biogenesisin these cells (Meissner et al., 1999a)) enough totrigger cell division. In another class of mutants, theGls/Reg mutants (Huskey and Griffin, 1979), all thecells (though fewer than in the wild-type, i.e. no morethan 128 or 256) act first as somatic cells and thenre-differentiate into reproductive cells; these mutantsresemble volvocacean species with no germ/somaseparation, such asEudorina. TheGls mutation hasbeen mapped to a gene,glsA, that encodes a proteinrequired for the asymmetric divisions responsible forthe segregation of germ-line and somatic blastomeres(Fig. 5A) (Miller and Kirk, 1999). Consequently, allcells are equal both in size and potential for differenti-ation, and undergo the ancestralChlamydomonas-likepathway of acting first as vegetative and then as re-productive cells (Tam and Kirk, 1991); it should bementioned that this mutation is only recovered on aregA− background such that the growth of somaticcells and thus their differentiation into reproductivecells is not suppressed.

The expression ofRegAandGls/Regmutations hasa profound effect on the higher-level unit, that is thecolony. While the somatic cells re-differentiate intogonidia, the spheroid is unable to maintain its motil-ity (and thus its position in the euphotic zone) formore than the duration of the first five cell divisions;however, the total number of cell divisions requiredto reach the final number of cells in the embryo isseven or eight, and eleven or twelve in the Gls/Regand Reg mutants, respectively. In conditions wheremotility and access to light are strong selective pres-sures, the higher-level is negatively affected by theoccurrence of these mutant cells at the lower-level,which are thus acting as “selfish” mutant cells. To ar-gue for the negative effects at the higher-level of thesetypes of selfish mutations in the environments wherewild-type forms ofVolvox are usually present is thefact that neither of the mutant forms are found as es-tablished populations in nature, although the Reg mu-tants occur spontaneously at a rather high rate (Kurnet al., 1978). Interestingly, however, when access to

light and the need for motility are not representing se-lective pressures (i.e. in lab settings or possibly shal-low waters), the fitness of these mutant forms proveshigher than the fitness of the wild-type (Koufopanouand Bell (1991)and our unpublished data).

7.2. Mutation rates

In our model, we used a value for the genome widemutation rate reported for unicellular organisms suchas yeast, bacteria, viruses (Drake, 1991), yet the modelstudies only a single locus assumed to represent thegenome wide functions involved in cellular functions.Our use of a genome wide rate in a single locus modelis clearly a leap, but it gives an idea of what we hopethe model represents. We have considered more real-istic mutation models elsewhere (Michod and Roze,2000; Roze and Michod, 2001). Concerning the highermutation rate assumed in panels C and D ofFig. 8, itis worth noting that volvocalean green algae seem tofeature levels of nucleotide substitution (as suggestedby the differences in branch length observed in phylo-genetic trees based on nuclear rRNA sequences (e.g.Friedl, 1997; Nakayama et al., 1998) that are higherthan those in other green algae as well as in their closerelatives, the land plants, which, incidentally, do nothave an early-defined germ-line.

7.3. Sixteen versus one germ-line founder

Although our model predicts that in both cases agerm-line is more likely to evolve if the mutationrate and the threat of selfish mutations are higher,these conditions are more relaxed under the scenarioin which gonidia descend from a single germ-line cellfounder (Fig. 8B), instead of 16 different founder cellsas is the case inV. carteri. Why is this scenario not theone that was selected for inV. carteri? Interestingly,although the end result, as far as the composition (i.e.number and type of cells) of the colony, is the sameunder both scenarios, two aspects are different and notrepresented in the model.

One aspect not represented in the model is asym-metric cell division. The second scenario (Fig. 7B)does not use or require asymmetric divisions. How-ever, the scenario that has been selected for inV. carteriinvolves asymmetric divisions; so, why did asymmet-ric division evolve inV. carteri? Asymmetric division


is considered a derived trait (Desnitski, 1992) in thevolvocalean green algal group. Asymmetric divisionsdo not occur in the less complexPleodorinaas well asmany species ofVolvox; in these cases, all gonidia aredeveloped from cells that were initially indistinguish-able from somatic cells, but then undergo enlargementand differentiation. InV. carteri, asymmetric divisionsand the segregation of the germ-line are not associatedwith the differential segregation of cytoplasmic com-ponents (Kirk et al., 1993). Rather, it has been shownthat the cells that remain above a threshold (i.e. 8�m)differentiate into germ-cells, whereas the cells that fallbelow the threshold will take the somatic path (Kirket al., 1993), regardless of the type of cell division. Toargue for this is also the fact that the formation of the2048 somatic cells produced by the 16 posterior blas-tomeres does not involve any asymmetric divisions.

Interestingly, in the Gls/Reg mutant, all of the celldivisions are symmetric; the cells follow the ances-tral Chlamydomonaspathway, acting first as vegeta-tive cells and then re-differentiating into reproductivecells. At the end of the seventh or eighth cell division(which marks the end of the embryonic cleavage inthis mutant form that only has 128 or 256 cells), allthe cells are about 4�m in size. At the equivalent timein development, the somatic initials in the wild-typeform are of the same size. However, due to the lesionpresent in theregAgene, the Gls/Reg mutant cells areable to grow post-embryonically and re-differentiateinto reproductive cells, whereas the wild-type somaticcells, in which theregAgene is active, are destined toremain small and terminally differentiated.

Thus, it is likely that under the scenario favoredby our model (Fig. 7B), subsequent to the seventh oreighth symmetric cell division, the cells would fallbelow the threshold size and would terminally differ-entiate into somatic cells; consequently, there wouldbe no reproductive cells to ensure the formation ofprogeny. This problem could be resolved by havingthe germ-line founder cells undergo fewer cell divi-sions, and by having more than one founder cells; forinstance, four instead of one germ-line founders thatwould undergo a number of only two additional di-visions would produce the same total number of 16gonidia (4× 22) and the critical cell size would notbe reached because the total number of cell divisionswould not exceed seven (5+ 2 = 7). However, theembryo would be short of 384 somatic cells (3×27 =

384). Under the selective pressure to achieve a largesize and high motility, such a decrease in the total num-ber of cells in the adult might be disfavored. Then, howcan the number of cells be increased while still produc-ing 16 germ cells? Asymmetric divisions are able to dothat by keeping the size of one (i.e. the germ cell blas-tomeres) of the two daughter cells above the threshold,while producing additional somatic blastomeres thatcontinue to proliferate below the threshold, and thusincrease the total number of somatic cells (Fig. 7A).Therefore, the path thatV. carteri undertook can beexplained as a consequence of the need to produce themaximum number of germ cells and reach the largestnumber of somatic cells, under the constraint of palin-tomy (i.e. no cell growth between cell divisions) andthe particular mechanism underlying somatic cell dif-ferentiation in this species (i.e. the expression ofregAwhen cell size falls below a threshold).

The other aspect that distinguishes the two alterna-tives for segregating a germ-line inV. carteri is con-cerned with the degree of relatedness among gonidia;the gonidia are less related to each other under thefirst scenario when compared to the second. It is pos-sible that by having the 16 germ cells deriving from16 independently segregated germ-line founder cells,an increase in genetic variance among the progeny isachieved. Because this species is reproducing mainlyasexually/clonally, such a pattern of producing thegerm cells might contribute to achieving genetic vari-ation in populations. On the other hand, it has beensuggested that one of the advantages of having an earlysegregating germ-line comes from the reduction in themutation level either by lowering the number of celldivisions (Buss, 1987) or by lowering the mutation rateper cell division (Michod, 1996, 1999; Michod andRoze, 2000). It is interesting to note that the way thatthe germ cells are produced inV. carteri ensures thatthe deleterious mutations that might occur during thelast four cell divisions (i.e. the sixth, seventh, eighth,and ninth) will be restricted to only one of the 16 germcells (Fig. 7A). In contrast, in the conventional wayof proliferating germ cells from only one germ-linefounder, deleterious mutations that would occur dur-ing the sixth division would be transmitted to half ora quarter of the progeny (depending on whether themutation affects both or only one of the DNA strands,respectively). In this light, the special way that thegerm-line is formed inV. carteri may be considered


as a means to allow the accumulation of non-lethalvariation, which is one of the premises for achievingevolvability.

8. Germ-line, conflict mediation and evolvability

Although an early-segregated germ-line and a somahave evolved inV. carteri, the soma was achieved in arather peculiar way.Volvoxwas not able to re-organizeimmortality and totipotency among its cell lineages;instead, inVolvox, both of these traits are entirely sup-pressed in the somatic cells. Moreover, the suppres-sion of these traits was achieved by acting on a singleprocess, namely cell division. Furthermore, the wayin which Volvoxsuppressed cell division was not byacting directly on the mitotic potential of the cells butrather indirectly by acting on a trait that was still verylinked to it, that is the growth of the cell (Nedelcuand Michod, 2003). By suppressing cell growth in so-matic cells, cell division is repressed and the potentialfor re-gaining immortality and totipotency (i.e. and forgaining access to the germ-line) is “under control.” Wesuggest that it is this type of conflict mediation affectedthe potential for further evolution in this lineage.

A direct implication is that “soma” inVolvoxdiffersfrom the soma of other multicellular organisms. Be-cause somatic cells do not divide, the post-embryonicgrowth and/or regeneration of the individual are notpossible; in addition, because the somatic cells un-dergo senescence and cell death at the age of 5 days(Pommerville and Kochert, 1981, 1982), the life spanof the higher-level individual is limited to the lifespan of the lower-level somatic cell. Due to its uniquetype of soma,Volvox is missing more than the abil-ity to grow, regenerate, or live longer (whose lackevidently does not constitute strong disadvantages inthe environment to which these algae are adapted,namely temporal aquatic habitats). Without a mitoti-cally active multipotent stem cell lineage or secondarysomatic differentiation there is less potential for celldifferentiation and further increases in complexity.However, somatic growth and differentiation are im-portant for the evolvability of a multicellular lineage.Without them,Volvoxdid not and will likely not attainhigher-levels of complexity.

In conclusion, we have tried to understand how de-velopmental processes are shaped during evolution-

ary transitions to increase the evolvability of the newhigher-level unit. In our formulation, evolvability ofthe new unit of selection (multicellular organism) de-pends on enhanced cooperative interactions amonglower-level units (cells). Conflict mediation, the pro-cess by which cooperative interactions are enhancedby reducing the temptation to defect, is instrumental increating a new evolutionary individual. Conflict me-diation increases the cooperativity of the group andheritability of fitness at the new level. Upon such pro-cesses does the continued evolvability of the new unitevolutionary unit depend. Nevertheless, conflict medi-ation, like other selective processes, can be short-sitedand in the case ofV. carteriappears to have interferedwith the long-term evolvability of the lineage.

Acknowledgements

We thank Laura Reed for discussions of modular-ity and evolvability and Lynne Trenery for readingthe manuscript and for discussions of the meaning ofcooperation.

Appendix A

The 2-locus 2-allele haploid dynamical system un-derlying this study has been introduced elsewhere(Michod, 1997, 1999; Michod and Roze, 1997). In thepresent study, we assume no recombination,r = 0.We consider two loci, the first locus controls cell be-havior (alleles C for cooperate and D for defect) andthe second modifier locus controls some aspect ofdevelopment or organization of the system (alleles mfor the ancestral state and M for the modified state).Here the modifier allele M creates a sequesteredgerm-line. There are four genotypes CM, Cm, DM,and Dm, referred to as genotype 1–4, respectively, inthe subscript notation later.

In an organism with a germ-line (cell groups com-ing from a CM zygote) there is a distribution of Cand D cells at the timeθ when the germ-line differ-entiates. We definek11,θ the number of CM cells atthe time θ, k31,θ the number of DM cells andk1,θ

the total number of cells (k1,θ = k11,θ, k31,θ) comingfrom a CM zygote.NC is the number of C cells inthe initial sample ofν cells which will give the germ-line and is given by a hypergeometric distribution


NC = H(k1,θ, ν, (k11,θ/k1,θ)). So we have for alli be-tween 1 andν,

Pi = Pr(NC = i) =

(k11,θ

i

) (k31,θν − i

)(

k1,θ

ν

) .

From our previous work (Michod, 1996; Michodand Roze, 1997), we know the values ofk11,θ, k31,θ,k1,θ at the time of the germ-line sample.

k11,θ = 2cθ(1 − µ)cθ,

k31,θ = µ2bcθ − 2cθ(1 − µ)cθµ

−1 + 2b−1 + µ,

andk1,θ = k11,θ+k31,θ. Hereµ is the mutation rate percell division from alleles C to D,c is the replicationrate of D cells (often assumed unity for simplicity andwithout loss of generality) andcb is the replication rateof mutant D cells. As we assume an infinite population,we assume thatPi is the frequency of CM groups thatwill give i C cells.

The ν cells in the germ-line sample go on and di-vide for a timetG to give the germ-line, and the muta-tion rate affecting the C allele isµG (a different rate,perhaps, if the germ-line precursor cells are treateddifferently). We callK11,i, K31,i andK1,i, the numberof C cells, the number of D cells and the total numberof cells in the germ-line of a CM individual whosesample hadi C cells. We have

K11,i = i × 2ctG(1 − µG)ctG,

K31,i = iµG2bctG − 2ctG(1 − µG)ctGµG

−1 + 2b−1 + µG

+(ν − i) × 2bctG,

andk1,i = k11,i + k31,i.We callk11,i, k31,i, andk1,i, the number of C cells,

the number of D cells and the total number of cellsin the soma of a CM individual whose sample for thegerm-line hadi C cells. We then have

k11,i = 2ct(1 − µ)ct − i × 2c(t−θ)(1 − µ)c(t−θ),

k31,i = µ2bct − 2ct(1 − µ)ctµ

−1 + 2b−1 + µ

− iµ2bc(t−θ) − 2c(t−θ)(1 − µ)c(t−θ)µ

−1 + 2b−1 + µ

− (ν − i)2bc(t−θ),

andk1,i = k11,i + k31,i.

The expression of fitness for a group initiated by aCM cell whose germ sample hadi C cells is then

W1,i = A

(1 + β

k11,i

k1,i

),

with A = 2ct(1−µ)ct+((µ2bct−2ct(1−µ)ctµ)/(−1+2b−1 + µ)) − K1,i, if the cost of the germ-line is interms of the new germ cells (Fig. 3), andA = k1,i, ifthe cost is in terms of number of missing somatic cells(Fig. 4). Note thatk1,i, is the number of somatic cellsafter the germ-line has been sequestered and takes intoaccount the cells that are missing.

To date, we have only studied the case of asexualreproduction, in which case the two-locus modifiermodel has four equilibria (given and discussed inTable 6–2 ofMichod, 1999). The first equilibriumand second equilibria do not interest us here as theycorrespond to no cooperation (C allele in frequencyzero) with the modifier allele at frequency unity orzero. The third and fourth equilibria correspond toa population polymorphic at the cooperate/defect lo-cus with the modifier M allele in frequency zero,or unity, respectively. We are interested in the casewhen the germ-line modifier allele M increases infrequency and the system undergoes a transition fromequilibrium three to equilibrium four. This transitioncorresponds to a transition in individuality, becausethe group of cells are no longer indivisible, somecells the somatic cells, can no longer produce thegroup. Instability of equilibrium three is determinedby the following eigenvalue condition

λ = (k22/k2)W2∑νi=0Pi(K11,i/K1,i)W1,i

< 1.

If λ < 1, when the M allele appears in the population,its frequency goes to fixation, and the system goesfrom equilibrium three to equilibrium four. It is thiscondition that leads to the results inFigs. 3, 4 and 8.

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Cooperation and conﬂict in the evolution of individuality IV. … · 2008-12-02 · BioSystems 69 (2003) 95–114 Cooperation and conﬂict in the evolution of individuality IV