REVIEWS The evolution of mutualisms: exploring the paths between conflict and cooperation E.A. Herre, N. Knowlton, U.G. Mueller and S.A. Rehner F rom the algae that help Mutualisms are of fundamental importance have a direct bearing on one of the power reef-building corals, in all ecosystems but their very existence central evolutionary questions to the diverse array of polposes a series of challenging evolutionary concerning mutualism: what faclinators that mediate sexquestions. Recently, the application of tors align the interests of partual reproduction in many plant molecular analyses combined with ners so that the relationships respecies, to the myriad nutritional theoretical advances have transformed our main mutually beneficial and symbionts that fix nitrogen and understanding of many specific systems, evolutionarily stable? aid digestion, and even down to thereby contributing to the possibility of a the mitochondria found in nearly more general understanding of the factors Current theory of conflict, all eukaryotes, mutualisms are that influence mutualisms. cooperation and constraint ubiquitous, often ecologically The potential for conflicts of dominant, and profoundly influeninterest to shape or destabilize E.A. Herre and N. Knowlton are at the Smithsonian tial at all levels of biological organmutualistic associations will deTropical Research Institute, Apartado 2072, Balboa, 1–6 ization . Although mutualisms pend on the extent to which the Republic of Panama (herrea@gamboa.si.edu); can be simply defined as reciprosurvival and reproductive interN. Knowlton is also at the Scripps Institute of cally beneficial relationships beests of the symbiont align with Oceanography, University of California, San Diego, tween organisms, they range from La Jolla, CA 92093-0202, USA (nknowlton@ucsd.edu); those of the host. Given that condiffuse and indirect interactions flicts of interest can occur even U.G. Mueller is at the Dept of Zoology, University of to highly integrated and coevolved within the genomes of single indiMaryland, College Park, MD 20742, USA (um3@umail.umd.edu); S.A. Rehner is at the Dept of associations between pairs of speviduals5,6,20, it seems unlikely that Biology, PO Box 23360, University of Puerto Rico, cies. Such mutualisms usually inthe interests of mutualists will Rio Piedras, San Juan, PR 00931, USA volve the direct exchange of goods ever be completely concordant. (attaboy@hotmail.com). and services (e.g. food defense and Although there is no general transport) and typically result in theory of mutualism, several facthe acquisition of novel capabiltors that can help align mutualities by at least one partner2,3. ists’ interests have been identified. Current theory5–8 suggests that mutualisms are best The passage of symbionts from parent to offspring (vertiviewed as reciprocal exploitations that nonetheless pro- cal transmission), genotypic uniformity of symbionts within vide net benefits to each partner. This view stresses the individual hosts, spatial structure of populations leading disruptive potential of conflicts of interests among the to repeated interactions between would-be mutualists, and erstwhile partners. Consequently, identifying factors that restricted options outside the relationship for both partinfluence the costs and benefits to each partner and quan- ners are thought to align interests and promote long-term tifying their influence constitute primary research objec- stability. Conversely, movement of symbionts between untives9. In particular, inquiry centers on the description of related hosts (horizontal transmission), multiple symbiconflicts of interest between partners and the attempt to ont genotypes and varied options are thought to unravel understand what mediates them10. This requires a clear them5–8,21–23. This framework is logically appealing, and many appreciation of the spatial, temporal and taxonomic con- cases appear to conform well with its predictions24,25. text in which these systems operate. Breakthroughs in However, it is worth scrutinizing why these factors are understanding have, and will, come precisely because of thought to reduce the potential for conflict among would-be the increased attention paid to the different ecological and mutualists and noting that those factors are often not indeevolutionary scales within which the mutualisms function. pendent. First, in the case of vertical transmission, both The expanding availability of a wide range of molecular symbiont and host benefit from successful reproduction by data has produced qualitative leaps in the types of infor- the host. Second, vertical transmission over many genermation available to researchers. This information can be ations will tend to reduce the genetic diversity of symbionts usefully combined with the results from field and laboratory by eliminating novel inputs to the symbiont community and studies. For example, genetic characterization of mutualists by providing a potential bottleneck at each generation11. has facilitated the unambiguous determination of the num- The resulting genetic homogeneity of symbionts within a ber and identity of interactants (e.g. genotypes and spe- host reduces selection for traits that increase betweencies), the degree and scale of their specificities and their symbiont competitive ability to the detriment of the host’s patterns of ecological transmission11–15. Similar approaches wellbeing and reproductive success5,6,23,25. Finally, vertical can also reveal the phylogenetic patterns of relationships transmission implies a continual interaction between host both between and within taxa of mutualists, and thus the and symbiont lineages. The absence of an independent extent to which speciation in hosts is tracked by speciation phase in a symbiont’s life cycle facilitates the evolution of in symbionts16–19, as well as the number of origins of particu- complete dependence, which reduces the evolutionary lar types of relationship11,16. Results from these studies viability of nonsymbiotic alternatives over the long term. TREE vol. 14, no. 2 February 1999 0169-5347/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0169-5347(98)01529-8 49 REVIEWS Identifying the players Box 1. Marine invertebrates and photosynthetic algae: the ecological significance of symbiont diversity Throughout the shallow tropical oceans, sessile animals often have symbiotic associations with photosynthetic, single-celled algae. Among the most spectacular and ecologically important are the associations formed between reef-building corals and dinoflagellates of the genus Symbiodinium. For many years, these symbionts were considered to be a single species, but physiological and genetic studies11,28 have revealed enormous, previously unsuspected, diversity. What was once considered a single species is now recognized as a group with at least three clades that, by extrapolation to free-living forms, are distinct at the family or ordinal level. These studies also revealed that there was no obvious concordance between host and symbiont phylogenies. Despite the growing appreciation of this cryptic diversity, it remained widely assumed that any single host formed an association with only one type of symbiont. However, in several ecologically dominant corals, it is now known that a single coral species and even single colonies are capable of hosting two or more types of symbiont28. Zonation of symbionts across the reef and within colonies appears to be related to levels of light. During adverse conditions, such as unusually high temperature, the mutualism between corals and algae can break down (‘coral bleaching’) in complex patterns that reflect this zonation. Thus, from the alga’s perspective, the host is more like a landscape composed of more and less suitable conditions than a uniformly hospitable environment28. From the coral’s perspective, horizontal transmission and complex mixtures of symbionts might provide short-term ecological flexibility to cope with fluctuating physical conditions that outweighs the possible costs of evolutionary conflicts among symbionts28. Many of the themes emerging from these studies of corals characterize other symbiotic systems as well11,26,27,29,35. Box 2. Figs and yuccas: model systems for understanding evolutionary conflicts There are over 700 species of figs (Ficus) described worldwide. The figs depend on minute pollinator wasps (Agaonidae) for continued sexual reproduction, and the wasps depend on the figs to complete their life cycle. Fossil evidence indicates that this relationship dates back at least 40 million years. In most cases, the relationship is overwhelmingly species-specific. In addition, recent molecular work suggests that the long evolutionary history of figs and their pollinators has been dominated by cospeciation between the two taxa19. Although in the long term the two mutualists depend completely upon one another, their reproductive interests are not identical. The fig benefits both from the production of viable seeds and from the production of female pollinator wasps that will potentially transfer the tree’s pollen to produce seeds in other trees. The wasps benefit only from the production of offspring (that necessarily come at the expense of approximately 50% of the potentially viable seeds). What prevents the shorter lived and much more numerous wasps from exploiting an ever greater number of seeds is still unanswered9,45. However, for most aspects that have been studied, the tree appears to be largely in control of the system9,45. It is interesting to compare the fig-wasp system with the yucca-moth system. Although there is the general dependence in both cases, there are instructive differences. The reproductive interests of individual female wasps are much more closely linked to their host than is the case with the moths, because the wasps tend to be trapped within the inflorescence they pollinate. Moreover, the female wasp offspring will carry pollen from the inflorescence in which they developed. In contrast, moths can pollinate and lay eggs in several different flowers, and their offspring are unlikely to provide the additional pollination service because they drop to the ground and emerge as adults much later10,38,39. The difference between the figs and yuccas in the degree to which their interests coincide with their partners is probably reflected in the much higher proportion of the fig seeds that support development of wasp offspring compared with the proportion of yucca seeds that support the development of the moth offspring. Nonetheless, not all mutualisms follow this pattern of vertical transmission with its proposed benefits. For example, many marine symbionts (Box 1) and mutualist associates of plants [e.g. pollinators (Box 2) and mycorrhizae] are horizontally transmitted, yet they are usually clearly beneficial. Moreover, vertical transmission does not guarantee benevolence (Box 3). Given these exceptions, it is important to determine the extent to which real systems conform to these patterns, and what factors are most responsible for determining conformity where it exists. 50 Determining the number and identities of the participants in mutualistic associations is a necessary first step for any evolutionary analysis, but it can be a surprisingly nontrivial task. Hosts and symbionts often lose characters found in their closest free-living relatives, or gain novel characters, making them difficult to distinguish and characterize taxonomically. The traditional solution for bacterial and fungal symbionts has been culturing. However, in some symbioses, what is successfully cultured does not necessarily reflect the actual community present in intact associations; and in other systems, symbionts cannot presently be cultured11,26–28. For these reasons, molecular analyses have played a critical role both in genetically characterizing isolated mutualists and in screening assemblages directly to assess the nature of symbiont communities. The resulting discoveries of stunning and unexpected diversity have transformed our understanding of mutualisms involving corals (Box 1), leaf-cutter ants (Box 4), and root symbionts26,27,29, among others. It is important to appreciate that symbiont diversity, cryptic and otherwise, can occur at different levels. At the level of different host species, different hosts can contain morphologically indistinguishable symbionts that are nevertheless quite distinctive both genetically and functionally. At the level of different individual hosts within a species, genetically different symbionts can be found in association with different host individuals (or populations). Even within individual host organisms, several distinct symbionts can be found12,26–28. The recognition that individual hosts can act as landscapes for communities of potentially competing symbionts (Box 1) raises the question of why competition among symbionts does not destabilize the mutualism, much as competition among parasites is believed to result in selection for increased virulence23,25. The ecological flexibility provided by symbiont diversity28,30 might play an important counterbalancing role. Patterns of ecological transmission and evolutionary association For patterns of transmission, it is useful to distinguish between transmission over ecological (generation to generation) and longer evolutionary (lineage to lineage) timescales. For example, systems dominated by strict vertical ecological transmission might be expected to produce concordant phylogenies between host and symbiont at all taxonomic scales, whereas in systems dominated by horizontal transmission, this outcome might be thought to be less likely. The explosion of systematic analyses using molecular techniques has generated phylogenetic reconstructions for one or both members of several speciose groups of mutualists. These studies show that patterns of transmission over ecological timescales do not necessarily translate into similar patterns at evolutionary timescales; available evidence suggests that all combinations of different patterns of ecological transmission and different degrees of phylogenetic concordance are found. Specifically, there are cases in which both evolutionary and ecological transmission appear to be predominantly vertical18. However, vertical evolutionary transmission (between lineages) is also found in cases in which ecological transmission is predominately horizontal (e.g. fig-pollinating wasps19, luminescent bacteria associated with deep-sea fish31 and sulfur oxidizing bacteria and some of their bivalve hosts14,32), apparently because vertical transmission is not the only mechanism that promotes cospeciation. Moreover, many intracellular bacteria (e.g. Wolbachia, TREE vol. 14, no. 2 February 1999 REVIEWS Box 3) show predominantly vertical transmission patterns at an ecological level, but this does not necessarily translate into phylogenetic patterns that are concordant with their hosts33. Presumably, this is because of sporadic cases of horizontal transfer between distantly related species. In an additional complexity, determining the extent to which co-cladogenesis is occurring will frequently depend on the taxonomic scale at which the question is asked14. For example, the phylogenetic relationships between some lineages of leaf-cutter ants show nearly perfect concordance with the relationships of their associated fungi. However, in some entire lineages the host phylogenetic relationships show essentially no correspondence with those of the fungi. In fact, there appear to be many lineages in which nonspecificity and noncongruence are the rule16,17 (Box 4). Unfortunately, for most mutualisms, we do not have adequate spatial and taxonomic sampling to determine the extent of concordance between host and symbiont lineages. Trajectories of costs and benefits Molecular data can provide a window on the taxonomic identities of mutualists, the structuring of their extant populations (e.g. patterns of spatial distribution and ecological transmission), their histories of phylogenetic associations and their evolutionary origins14,28,29,34, but provide relatively little information about the often rapid, and sometimes convoluted, evolutionary trajectories of costs and benefits received4,8. From studies that compare outcomes across several populations of mutualistic interactions between two species, we know that outcomes can vary among extant populations4,35,36. Several studies have documented that net costs and benefits can vary over relatively short timescales4,36 resulting from: (1) changes in the presence or abundance of influential third parties36,37; (2) variation in host densities that results in shifts in patterns of transmission24; (3) variation in resource availablility3,36; or (4) variation in physical conditions (Box 1). Furthermore, such studies raise questions concerning the degree of local adaptation in host and symbiont populations, such as whether hosts generally benefit most from local, presumably more highly coadapted symbionts. Moreover, in evolutionary time, comparisons across related taxa (particularly in cospeciating systems) can show different evolutionary outcomes that represent variations on a single theme of mutualistic interaction (e.g. leafcutters, figs, yuccas, ants, plants and lycaenid butterflies). Specifically, phylogenetic analyses reveal that parasitic lineages can be embedded in largely mutualistic groups and/or vice versa19,38,39. However, theory suggests that the species that parasitize mutualisms should not be the closest relatives to either partner38,39. Available evidence collected from figs (Ficus) and fig wasps (Agaonidae), and the yuccas (Yucca) and yucca moths (Tegeticula), supports this prediction19,38,39. Nonetheless, this proposition requires further testing. Mutualisms as model systems Mutualisms and rates of molecular evolution In those instances in which the host and mutualist cospeciate, the absolute times of divergence between pairs of cospeciating mutualists are effectively held constant. This allows a series of potentially instructive comparisons to be made in the accumulation of substitutions in homologous DNA sequences. First, comparisons can be made between the accumulation of substitutions at a given gene or set of genes in the ‘host’ and in the ‘symbiont’ (or parasite). TREE vol. 14, no. 2 February 1999 Box 3. Wolbachia and Buchnera: the implications of horizontal versus vertical transmission for the evolution of mutualism Theory suggests that vertical transmission selects for more benign relationships, and that symbionts transmitted vertically should generally have benign or even positive effects on their hosts. There is accumulating experimental and comparative support for this proposition. A classic example is the association found between aphids and their bacteria (Buchnera) that synthesize necessary amino acids for their hosts3,11,18,40. However, Wolbachia appears to be a maternally inherited endosymbiont that frequently has large negative effects on its host’s reproductive interests. At times, the bacteria distort the host’s sex ratio, often leading to all female broods, or produce reproductive incompatibility with other host individuals that do not carry the same strain of Wolbachia33. Superficially, these observations contradict the theoretical predictions. However, to assess the relevance of these observations, the timescales over which maternal transmission occurs and the magnitude of the negative effects of Wolbachia must be considered. Although most cases show that at an ecological timescale Wolbachia is transmitted vertically, there is clear phylogenetic evidence that Wolbachia ‘jumps’ from lineage to lineage; that is, whether its propagation is considered to be dominated by vertical transmission depends on temporal scale. In addition, Wolbachia can often have complex or little, if any, negative effect on its direct individual host44. Critical questions involve determining the actual routes and frequencies of horizontal transmission, as well as the magnitude of negative effects under real ecological situations, and then determining if there is a correspondence between ‘how bad the bugs are’ and ‘how much evolutionary jumping they can do’. Box 4. Fungus-growing ants and their fungi: phylogenetic transitions in patterns of symbiont acquisition The exclusively New World fungus-gardening ants in the tribe Attini (Formicidae) comprise over 200 described species, all obligately dependent upon the cultivation of fungus for food16,17,46. Ants in the leaf-cutter genera Acromyrmex and Atta are ecologically and economically important because of the vast quantities of foliage and flowers that they cut to culture the fungi in their often immense nests. Together with three additional genera, leaf-cutter ants are grouped into the monophyletic higher attines, which comprise about one-half of the species diversity of the tribe. Ants in the remaining seven genera of lower attines are less conspicuous, frequently cryptic and do not attack plants. The symbiotic associations of lower attine ants and their fungi are diverse: some species grow their fungi entirely on dead vegetable matter, some entirely on caterpillar frass and others on a mixed substrate that can even include seeds. Molecular data have been decisive in identifying the evolutionary origins and phylogenetic relationships of attine fungal symbionts. First, although most ant-associated fungi are members of the family Lepiotaceae (Agaricales; Basidiomycotina), phylogenetic analyses based on ribosomal DNA indicate that the fungus cultivated by several ant species in the lower attine genus Apterostigma is distantly related to all other attine fungi, and has been secondarily acquired long after the mutualism originated in the Amazon Basin approximately 50 million years ago16,17,46. Second, molecular analyses indicate that several distinct lepiotoid fungal lineages associated with lower attines are essentially identical to current free-living forms. Together with the apparent lack of morphological modification of many lower attine symbionts, these observations suggest the recent acquisition of novel symbionts from free-living stock46. Thus, as can be observed on both ecological and evolutionary scales, the presumably ancestral condition of repeatedly acquiring free-living fungi has been retained in some of the lower attines but appears to have been lost in the higher attines, which have developed longer-term associations with their generally more specialized symbionts. Second, comparisons can be made between the rates of accumulation of base changes between the symbionts and their free-living relatives. Depending on the attributes of the taxa available, these comparisons permit the evaluation of several factors that have been suggested to be important in influencing the rates of molecular evolution. Cospeciating mutualists often exhibit different generation times, different body sizes and metabolic rates, different effective population sizes and different degrees of sexual reproduction. Different taxa might also possess very different systems of DNA repair. These contrasts can be productively exploited. For example, Moran and colleagues have found that the aphid-associated Buchnera shows much faster rates of molecular evolution than do its 51 REVIEWS free living relatives, an observation that appears to oppose the idea that rates of evolution in mutualists should slow down18,40. A similar pattern has been found in lichens41. Mutualisms and the adaptive significance of sex Current theory regarding the adaptive value of sexual reproduction revolves around the ideas that sexual reproduction serves to: (1) maintain adaptation in the face of a constantly changing and potentially threatening biotic world and (2) remove deleterious mutations. Potentially, comparisons between groups of related species characterized either with or without sexual reproduction could be useful to assess the relative importance of these two proposed functions. For example, some groups of mutualists, such as dinoflagellates associated with marine invertebrates, fungi associated with attine ants, perhaps algae in some lichens, clavicipitaceous (i.e. smut-like) grass endophytes, and the fungal cultivars of fungus-gardening termites, are derived from free-living groups capable of both sexual and asexual reproduction. In each case, it appears that the balance between sexuality and asexuality has been shifted towards the latter. Interestingly, in the case of the endophytic fungi associated with grasses, the fungi appear to reduce the host’s tendency to reproduce sexually42, rather than the more typical reverse pattern43. There are several possible explanations for these patterns. For example, one school of thought suggests that ‘well integrated’ (e.g. intracelluar) symbionts are protected by their hosts from a menacing organic world of constantly evolving predators and parasites, and consequently do not ‘need’ sex43. An alternative, less benign, view of mutualisms suggests that mutualistic relationships are better characterized as a series of ongoing arms races. In this scenario, sex might be the critical element that allows one member to ‘keep up’, or if suppressed in one member has allowed the other to ‘get ahead’. Further progress in this area will depend on knowing the extent to which sex is actually absent, determining whether symbionts are represented by a single clone or are genetically heterogeneous, and estimating the phylogenies of the partners over various spatial and taxonomic scales. Ultimately, molecular data will play a crucial role in distinguishing among various possible interpretations. Conclusions Most organisms are involved either directly or indirectly in mutualistic interactions. However, there is no general theory of mutualism that approaches the explanatory power that ‘Hamilton’s Rule’ appears to hold for the understanding of within-species interactions. Underlying problems revolve around explicitly defining vague terms, such as ‘alignment of interest’, and employing biologically realistic currencies (i.e costs and benefits) at biologically relevant scales of organization. Ideally, all of these should be measurable and capable of being employed across radically different systems. For example, can the ‘conflict of interest’ and ‘costs and benefits’ within and between leafcutters that do or do not have vertically transmitted fungi be estimated and then compared with those values for corals that do or do not have vertically transmitted algae? We have implied that factors constraining ‘cheating’ or ‘defection’ are increasingly required because the interests of interacting species are not aligned. But can it be shown that increasingly stringent constraints (e.g. no options outside the relationship and/or increased host investment in symbiont control) operate in systems in which there are increasingly incongruent interests? 52 Ultimately, we cannot begin to determine whether there are any general principles or consistent patterns that characterize mutualisms if we misunderstand individual case studies. Ideally, for a number of cases, we need to identify and quantify the costs and benefits to each party, and to understand what factors influence variation in those costs and benefits. Importantly, we need to understand conflicts of interest and attempt to identify what factors maintain the alignment of interests. If there is nonalignment, what prevents the system from breaking down? To do this, it is crucial that we identify the mutualists, and understand their diversity, patterns of transmission and structuring at several spatial, temporal and evolutionary scales. This poses the monumental task of documenting basic, descriptive natural history for many distinct systems and coupling it with the often indispensable information that can increasingly be obtained from molecular approaches. Acknowledgements We thank Koos Boomsma and Jack Werren for stimulating discussion. 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(1998) The evolution of agriculture in ants, Science 281, 2034–2038 The cost of helping Robert Heinsohn and Sarah Legge T he study of cooperative Cooperative breeding in mammals, birds and remains poor. The approach to breeding in vertebrates fish has provided evolutionary biologists cooperative breeding has often aims to understand why with a rich framework for studying the been to compare the outcomes of some animals forgo indecauses and consequences of group-based philopatry and helping with the pendent reproduction and help reproduction. Helping behaviour is other options of dispersing to others to breed instead. Over the especially enigmatic because it often float or dispersing to breed3. Evalupast 30 years, the field has develentails an individual sacrificing personal ation of the final reproductive oped a rich set of theory1–3 and reproduction while assisting others in their rewards for each strategy leads to has been wracked by some major breeding attempts. The decision to help an ultimate understanding of why debates4,5. However, enough coothers to reproduce is affected by a particular decision was made7. operative species have been studimmediate and future costs analogous to Implicit in this approach is that ied in detail to establish common those of direct reproduction, but these the outcome reflects all the costs ground and to test theory. Indeed, components of the equation have usually and benefits of dispersal versus in a recent review of the field, been neglected. Recent research suggests nondispersal, and helping versus Emlen6 states that ‘the original that the type of benefit sought could nonhelping, but it does not lead determine the extent of help given. paradox of cooperative breeding to an appreciation of the nature of largely disappeared with the wideeach cost and benefit. Although spread confirmation that (1) helpwe have a large list of benefits to ers frequently do improve their Robert Heinsohn and Sarah Legge are in the Division of helping8, we still lack a cohesive Botany and Zoology, Australian National University, chances of becoming breeders…, framework that explains when Canberra, ACT 0200, Australia and (2) they frequently do obtain (robert.heinsohn@anu.edu.au; sarah.legge@anu.edu.au). they apply in various taxa or ecolarge indirect genetic benefits by logical circumstances. Less attenhelping to rear collateral kin’. With tion has been paid to the costs of identification of these direct and helping. indirect benefits to helpers, the original questions asked Consider the cooperatively breeding Seychelles warbler, by researchers would appear to be ‘largely answered’. Acrocephalus sechellensis. In an elegant study, Komdeur9 Despite this claim, some important questions remain un- showed that helpers much prefer to feed nestlings that are answered. In particular, our understanding of the varying more closely related to themselves; an important result level of helper contributions within and between species that emphasized the lability and adaptive nature of helping TREE vol. 14, no. 2 February 1999 0169-5347/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0169-5347(98)01545-6 53