Allorecognition and chimerism in an invertebrate model organism

The presence of highly specific histocompatibility reactions in colonial marine invertebrates that lack adaptive immune systems (such as the sponges, cnidarians, bryozoans, and ascidians) provides a unique opportunity to investigate the evolutionary roots of allorecognition and to explore whether homologous innate recognition systems exist in vertebrates. Conspecific interactions among adult animals in these groups are regulated by highly specific allorecognition systems that restrict somatic fusion to self or close kin. In Hydractinia (Cnidaria:Hydrozoa), fusion/rejection responses are controlled by two linked genetic loci. Alleles at each locus are co-dominantly inherited. Colonies fuse if they share at least one haplotype, reject if they share no haplotypes, and display transitory fusion if they share only one allele in a haplotype – a pattern that echoes natural killer cell responses in mice and humans. Allorecognition in Hydractinia and other marine invertebrates serves as a safeguard against stem cell or germline parasitism thus, limiting chimerism to closely related individuals. These animals fail to become tolerant even if exposed during early development to cells from a histoincompatible individual. Detailed analysis of the structure and function of molecules responsible for allorecognition in basal marine invertebrates could provide clues to the innate mechanisms by which higher animals respond to organ and cell allografts, including embryonic tissues.     

Allorecognition and chimerism in an invertebrate model organism

The presence of highly specific histocompatibility reactions in colonial marine invertebrates that lack adaptive immune systems (such as the sponges, cnidarians, bryozoans and ascidians) provides a unique opportunity to investigate the evolutionary roots of allorecognition and to explore whether homologous innate recognition systems exist in vertebrates. Conspecific interactions among adult animals in these groups are regulated by highly specific allorecognition systems that restrict somatic fusion to self or close kin. In Hydractinia (Cnidaria:Hydrozoa), fusion/rejection responses are controlled by two linked genetic loci. Alleles at each locus are co-dominantly inherited. Colonies fuse if they share at least one haplotype, reject if they share no haplotypes, and display transitory fusion if they share only one allele in a haplotype—a pattern that echoes natural killer cell responses in mice and humans. Allorecognition in Hydractinia and other marine invertebrates serves as a safeguard against stem cell or germline parasitism thus, limiting chimerism to closely related individuals. These animals fail to become tolerant even if exposed during early development to cells from a histoincompatible individual. Detailed analysis of the structure and function of molecules responsible for allorecognition in basal marine invertebrates could provide clues to the innate mechanisms by which higher animals respond to organ and cell allografts, including embryonic tissues.Key words: allorecognition, chimerism, invertebrate, innate immune system     

Model systems of invertebrate allorecognition

Nearly all colonial marine invertebrates are capable of allorecognition--the ability to distinguish between self and genetically distinct members of the same species. When two or more colonies grow into contact, they either reject each other and compete for the contested space or fuse and form a single, chimeric colony. The specificity of this response is conferred by genetic systems that restrict fusion to self and close kin. Two selective pressures, intraspecific spatial competition between whole colonies and competition between stem cells for access to the germline in fused chimeras, are thought to drive the evolution of extensive polymorphism at invertebrate allorecognition loci. After decades of study, genes controlling allorecognition have been identified in two model systems, the protochordate Botryllus schlosseri and the cnidarian Hydractinia symbiolongicarpus. In both species, allorecognition specificity is determined by highly polymorphic cell-surface molecules, encoded by the fuhc and fester genes in Botryllus, and by the alr1 and alr2 genes in Hydractinia. Here we review allorecognition phenomena in both systems, summarizing recent molecular advances, comparing and contrasting the life history traits that shape the evolution of these distinct allorecognition systems, and highlighting questions that remain open in the field.     

KIN INTERACTIONS IN A COLONIAL HYDROZOAN (HYDRACTINIA SYMBIOLONGICARPUS): POPULATION STRUCTURE ON A MOBILE LANDSCAPE

Many sessile colonial organisms intensively compete with conspecifics for growing space. This competition can result in either cooperative fusion or aggressive rejection between colonies, and some species have evolved highly polymorphic genetic systems that mediate the outcome of these interactions. Here we demonstrate the potential for interactions among close kin as the basis for the evolutionary maintenance of a genetically polymorphic allorecognition system in the colonial hydroid Hydractinia symbiolongicarpus, which lives on gastropod shells occupied by hermit crabs. Fusion between hydroids in the laboratory is restricted mainly to encounters between full siblings, whereas other encounters result in aggressive rejection. Natural selection acting on the costs or benefits of fusion between colonies could be responsible for the present maintenance of such a highly specific behavioral response, but only if encounters between fusible colonies still occur in contemporary populations. The large size of these hydroid populations and the mobility of the crabs should limit the potential for interactions among closely related hydroids on the same shell. However, RAPD polymorphisms among a large sample of hydroids from a population off the coast of Massachusetts indicate that genetically similar colonies are often found together on the same shell. Some genetic distances between colonies on the same shell were low relative to genetic distances between colonies on different shells or genetic distances between known full siblings from laboratory matings. We conservatively estimate that 2–18% of co-occurring colonies may be full sibling pairs. These observations suggest that encounters between genetically similar hydroids are common, despite the mobile nature of their habitat, and these encounters may provide frequent opportunities for natural selection to influence the evolution of cooperative and agonistic behaviors and their polymorphic genetic basis.     

The importance of life-history stage and individual variation in the allorecognition system of a marine sponge

In marine invertebrates with complex life cycles, it may often be the case that trade-offs and behaviors differ between adult and larval stages. In this study, I examined the effects of life-history stage on allorecognition system function in the sponge, Haliclona sp. For sedentary marine invertebrates, allorecognition systems allow individuals to distinguish between genetically similar and distinct tissue they may encounter and are thought to reduce costly tissue fusion with individuals other than self or kin. Although it was found that sessile adults fused preferentially with self-tissue and exhibited a functioning allorecognition system, free-swimming larvae fused equally with sibling and non-sibling larvae resulting in swimming chimeras capable of successful metamorphosis, suggesting a stage-activated allorecognition system. In addition, adult sponges differed significantly in the propensity of their larvae to fuse suggesting variation in parental strategies. Analysis of larval swimming behavior indicated that larvae aggregate and are capable of increasing their encounters with other larvae and perhaps their probability of fusing in nature. The pursuit of fusion at this motile stage, along with evidence of a functioning adult allorecognition system, suggests that larvae may not express a recognition system, or that factors other than relatedness such as benefits to larval or adult chimeras, are involved in larval fusion and a stage-activated allorecognition system. In addition, this study demonstrates the presence of variation among individuals in the allorecognition system's ontogeny in the sponge Haliclona sp.     

Allorecognition Triggers Autophagy and Subsequent Necrosis in the Cnidarian Hydractinia symbiolongicarpus

Transitory fusion is an allorecognition phenotype displayed by the colonial hydroid Hydractinia symbiolongicarpus when interacting colonies share some, but not all, loci within the allorecognition gene complex (ARC). The phenotype is characterized by an initial fusion followed by subsequent cell death resulting in separation of the two incompatible colonies. We here characterize this cell death process using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and continuous in vivo digital microscopy. These techniques reveal widespread autophagy and subsequent necrosis in both colony and grafted polyp assays. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and ultrastructural observations revealed no evidence of apoptosis. Pharmacological inhibition of autophagy using 3-methyladenine (3-MA) completely suppressed transitory fusion in vivo in colony assays. Rapamycin did not have a significant effect in the same assays. These results establish the hydroid allorecognition system as a novel model for the study of cell death.     

Allogeneic interactions in Hydractinia: is the transitory chimera beneficial?

The colonial marine hydroid, Hydractinia, exhibits four possible outcomes to allogeneic contacts: passive rejection, aggressive rejection, stable fusion and transitory fusion. In the special case of transitory fusion, Hydractinia colonies undergo tissue fusion, followed by tissue death at the original contact area, and colony separation. This type of rejection is different in several aspects from the rejection process that accompanies incompatible encounters. It has been suggested that in transitory fusion, the colonies gain immediate benefits from fusion, mainly due to size increase, without succumbing to costs associated with fusion (germ line parasitism). We report a long-term observation of repeated fusion and separation cycles in clones featuring transitory fusion that revealed a slow-down of specific growth rates following fusion, and recovery in growth rates following separation. Very rapid transfer of stained material between partners in transitory chimeras provides suggestive evidence that protection against germ line parasitism is far from being guaranteed by separation. Our data cast doubt as to whether the benefits considered for transitory fusion are sustainable and support the already made suggestion that fusion with self, rather than fusion with kin, has been the major selective force governing the evolution of allorecognition in colonial invertebrates.     

Kin or self‐recognition? Colonial fusibility of the bryozoan Celleporella hyalina

We estimated fusion frequency with respect to coancestry in the bryozoan Celleporella hyalina, whose briefly planktonic sexually produced larvae settle on algal substrata and proceed to form encrusting colonies by iterative budding. Frequency of fusion between paired colonies growing on an artificial substratum was positively correlated with coefficient of relatedness, with allorecognition ability increasing during the early stages of colonial growth after larval settlement. Parents repressed the growth of F1 progeny with which they had fused. The results are concordant with the Feldgarden-Yund model of selection for self-recognition, which regards fusion with kin as an inevitable source of error whose cost diminishes with increasing relatedness. Contrary to fusion compatibility, gametic compatibility is negatively correlated with coancestry, indicating a need for further research on the possibility of common or linked genetic control that has opposite effect at somatic and gametic levels.     

The social evolution of somatic fusion

The widespread potential for somatic fusion among different conspecific multicellular individuals suggests that such fusion is adaptive. However, because recognition of non-kin (allorecognition) usually leads to a rejection response, successful somatic fusion is limited to close kin. This is consistent with kin-selection theory, which predicts that the potential cost of fusion and the potential for somatic parasitism decrease with increasing relatedness. Paradoxically, however, Crozier found that, in the short term, positive-frequency-dependent selection eliminates the required genetic polymorphism at allorecognition loci. The 'Crozier paradox' may be solved if allorecognition is based on extrinsically balanced polymorphisms, for example at immune loci. Alternatively, the assumption of most models that self fusion is mutually beneficial is wrong. If fusion is on average harmful, selection will promote unconditional rejection. However, we propose that fusion within individuals is beneficial, selecting for the ability to fuse, but fusion between individuals on average costly, selecting for non-self recognition (rather than non-kin recognition). We discuss experimental data on fungi that are consistent with this hypothesis.     

EVOLUTION OF ALLORECOGNITION IN BOTRYLLID ASCIDIANS INFERRED FROM A MOLECULAR PHYLOGENY

Despite the functional and phyletic ubiquity of highly polymorphic genetic recognition systems, the evolution and maintenance of these remarkable loci remain an empirical and theoretical puzzle. Many clonal invertebrates use polymorphic genetic recognition systems to discriminate kin from unrelated individuals during behavioral interactions that mediate competition for space. Space competition may have been a selective force promoting the evolution of highly polymorphic recognition systems, or preexisting polymorphic loci may have been coopted for the purpose of mediating space competition. Ascidian species in the family Botryllidae have an allorecognition system in which fusion or rejection between neighboring colonies is controlled by allele-sharing at a single, highly polymorphic locus. The behavioral sequence involved in allorecognition varies in a species-specific fashion with some species requiring extensive intercolony tissue integration prior to the allorecognition response, while other species contact opposing colonies at only a few points on the outer surface before resolving space conflicts. Due to an apparent species-specific continuum of behavioral variation in the degree of intercolony tissue integration required for allorecognition, this system lends itself to a phylogenetic analysis of the evolution of an allorecognition system. We constructed a molecular phylogeny of the botryllids based on 18S rDNA sequence and mapped allorecognition behavioral variation onto the phylogeny. Our phylogeny shows the basal allorecognition condition for the group is the most internal form of the recognition reaction. More derived species show progressively more external allorecognition responses, and in some cases loss of some features of internal function. We suggest that external allorecognition appears to be a secondary function of a polymorphic discriminatory system that was already in place due to other selective pressures such as gamete, pathogen, or developmental cell lineage recognition.     

Experimental demonstration of the benefits of somatic fusion and the consequences for allorecognition

Allorecognition, the ability to distinguish “self” from “nonself” based on allelic differences at allorecognition loci, is common in all domains of life. Allorecognition restricts the opportunities for social parasitism, and is therefore crucial for the evolution of cooperation. However, the maintenance of allorecognition diversity provides a paradox. If allorecognition is costly relative to cooperation, common alleles will be favored. Thus, the cost of allorecognition may reduce the genetic variation upon which allorecognition crucially relies, a prediction now known as “Crozier's paradox.” We establish the relative costs of allorecognition, and their consequences for the short‐term evolution of recognition labels theoretically predicted by Crozier. We use fusion among colonies of the fungus Neurospora crassa, regulated by highly variable allorecognition genes, as an experimental model system. We demonstrate that fusion among colonies is mutually beneficial, relative to absence of fusion upon allorecognition. This benefit is due not only to absence of mutual antagonism, which occurs upon allorecognition, but also to an increase in colony size per se. We then experimentally demonstrate that the benefit of fusion selects against allorecognition diversity, as predicted by Crozier. We discuss what maintains allorecognition diversity.     

Differential effect of allorecognition loci on phenotype in Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa)

The allorecognition complex of Hydractinia symbiolongicarpus is a chromosomal interval containing two loci, alr1 and alr2, that controls fusion between genetically distinct colonies. Recombination between these two loci has been associated with a heterogeneous class of phenotypes called transitory fusion. A large-scale backcross was performed to generate a population of colonies (N = 106) with recombination breakpoints within the allorecognition complex. Two distinct forms of transitory fusion were correlated with reciprocal recombination products, suggesting that alr1 and alr2 contributed differentially to the allorecognition response. Specifically, type I transitory fusion is associated with rapid and persistent separation of allogeneic tissues, whereas type II transitory fusion generates a patchwork of continuously fusing and separating tissues.     

Genetic relatedness in groups of the humbug damselfish Dascyllus aruanus: small, similar-sized individuals may be close kin

Kin selection plays an important role in the evolution of social behaviour in terrestrial systems. The extent to which kin selection influences the evolution of social behaviour in marine systems is largely unexplored. Generally, it is considered that kin selection is irrelevant in marine systems, because it is assumed that the dispersing larval phase of marine organisms will break up kin associations. Here, we challenge this assumption and investigate the opportunity for kin selection in a coral reef fish: the humbug damselfish Dascyllus aruanus . This fish lives in groups composed of a large male and a number of smaller females and nonbreeders. We use 10 polymorphic microsatellite loci to assess the relatedness of 265 individuals from 35 groups. The mean coefficient of relatedness among group members is 0.01 ± 0.04, suggesting that individuals are not associated with close relatives. However, the distribution of pairwise relatedness of individuals within groups has an overabundance of positive values, and indicates that there might be 35 pairs of close relatives within groups. Further analyses reveal that close relatives likely are similar in size and small in size, suggesting that they might have recruited together. We conclude that it is possible for kin selection to operate in D. aruanus , but kin recognition will be a prerequisite for such selection. This study reveals that individuals can be associated with close relatives, and there is a hidden potential for kin selection, during certain parts of the life cycle of coral reef fishes.     

Estimating heritabilities and genetic correlations with marker-based methods: an experimental test in Mimulus guttatus

The calculation of heritabilities and genetic correlations, which are necessary for predicting evolutionary responses, requires knowledge about the relatedness between individuals. This information is often not directly available, especially not for natural populations, but can be inferred by using molecular markers such as allozymes. Several methods based on inferred relatedness from marker data have been developed to estimate heritabilities and genetic correlations in natural populations. Most methods use maximum-likelihood procedures to assign pairs or groups of individuals to predefined discrete relatedness classes (e.g., half sibs and unrelated individuals). The Ritland method, on the other hand, uses method of moments estimators to estimate pairwise relatedness among individuals as continuous values. We tested both the Ritland method and a maximum-likelihood method by applying them to a greenhouse population consisting of seed families of the herb Mimulus guttatus and comparing the results to the ones from a frequently used standard method based on half-sib families. Estimates of genetic correlations were far from accurate, especially when we used the Ritland method. However, this study shows that even with a few variable allozyme loci, it is possible to get qualitatively good indications about the presence of heritable genetic variation from marker-based methods, even though both methods underestimated it.     

Kin Discrimination in Protists: From Many Cells to Single Cells and Backwards

During four decades (1960–1990s), the conceptualization and experimental design of studies in kin recognition relied on work with multicellular eukaryotes, particularly Unikonta (including invertebrates and vertebrates) and some Bikonta (including plants). This pioneering research had an animal behavior approach. During the 2000s, work on taxa‐, clone‐ and kin‐discrimination and recognition in protists produced genetic and molecular evidence that unicellular organisms (e.g. Saccharomyces, Dictyostelium, Polysphondylium, Tetrahymena, Entamoeba and Plasmodium) could distinguish between same (self or clone) and different (diverse clones), as well as among conspecifics of close or distant genetic relatedness. Here, we discuss some of the research on the genetics of kin discrimination/recognition and highlight the scientific progress made by switching emphasis from investigating multicellular to unicellular systems (and backwards). We document how studies with protists are helping us to understand the microscopic, cellular origins and evolution of the mechanisms of kin discrimination/recognition and their significance for the advent of multicellularity. We emphasize that because protists are among the most ancient organisms on Earth, belong to multiple taxonomic groups and occupy all environments, they can be central to reexamining traditional hypotheses in the field of kin recognition, reformulating concepts, and generating new knowledge.     

An invertebrate histocompatibility complex

We have developed defined genetic lines of the hydroid Hydractinia symbiolongicarpus and confirmed earlier results showing that allorecognition is controlled by a single chromosomal region within these lines. In a large backcross population, we detected recombinants that display a fusibility phenotype distinct from typical fusion and rejection. We show that this transitory fusion phenotype segregates in a fashion expected of a single Mendelian trait, establishing that the chromosomal interval contains at least two genes that interact to determine fusibility. Using bulked segregant analysis, we have identified amplified fragment length polymorphisms (AFLP) cosegregating with fusibility, used these markers to independently confirm linkage of the two loci, and constructed a 3.4-cM map of an invertebrate histocompatibility complex.     

The biogeography of kin discrimination across microbial neighbourhoods

The spatial distribution of potential interactants is critical to social evolution in all cooperative organisms. Yet the biogeography of microbial kin discrimination at the scales most relevant to social interactions is poorly understood. Here we resolve the microbiogeography of social identity and genetic relatedness in local populations of the model cooperative bacterium Myxococcus xanthus at small spatial scales, across which the potential for dispersal is high. Using two criteria of relatedness—colony‐merger compatibility during cooperative motility and DNA‐sequence similarity at highly polymorphic loci—we find that relatedness decreases greatly with spatial distance even across the smallest scale transition. Both social relatedness and genetic relatedness are maximal within individual fruiting bodies at the micrometre scale but are much lower already across adjacent fruiting bodies at the millimetre scale. Genetic relatedness was found to be yet lower among centimetre‐scale samples, whereas social allotype relatedness decreased further only at the metre scale, at and beyond which the probability of social or genetic identity among randomly sampled isolates is effectively zero. Thus, in M. xanthus, high‐relatedness patches form a rich mosaic of diverse social allotypes across fruiting body neighbourhoods at the millimetre scale and beyond. Individuals that migrate even short distances across adjacent groups will frequently encounter allotypic conspecifics and territorial kin discrimination may profoundly influence the spatial dynamics of local migration. Finally, we also found that the phylogenetic scope of intraspecific biogeographic analysis can affect the detection of spatial structure, as some patterns evident in clade‐specific analysis were masked by simultaneous analysis of all strains.     

Detecting instability in animal social networks: genetic fragmentation is associated with social instability in rhesus macaques

The persistence of biological systems requires evolved mechanisms which promote stability. Cohesive primate social groups are one example of stable biological systems, which persist in spite of regular conflict. We suggest that genetic relatedness and its associated kinship structure are a potential source of stability in primate social groups as kinship structure is an important organizing principle in many animal societies. We investigated the effect of average genetic relatedness per matrilineal family on the stability of matrilineal grooming and agonistic interactions in 48 matrilines from seven captive groups of rhesus macaques. Matrilines with low average genetic relatedness show increased family-level instability such as: more sub-grouping in their matrilineal groom network, more frequent fighting with kin, and higher rates of wounding. Family-level instability in multiple matrilines within a group is further associated with group-level instability such as increased wounding. Stability appears to arise from the presence of clear matrilineal structure in the rhesus macaque group hierarchy, which is derived from cohesion among kin in their affiliative and agonistic interactions with each other. We conclude that genetic relatedness and kinship structure are an important source of group stability in animal societies, particularly when dominance and/or affilative interactions are typically governed by kinship.     

Polymorphism in the Immunoglobulin-like Domains of the Receptor Tyrosine Kinase from the Sponge Geodia Cydonium

Sponges [Porifera] are the phylogenetically oldest phylum of the Metazoa. They are provided with both cellular and humoral allorecognition systems. The underlying molecules are not yet known. To study allorecognition in sponges we first determined the frequency of graft rejection in a natural population of the marine sponge Geodia cvdonium. We then determined, for the first time at the molecular level, the degree of sequence polymorphism in segments of one molecule which may be related to sponge allorecognition and host defense: the Ig-like domains from the receptor tyrosine kinase [RTK]. Thirty six pairs of auto- and allografts were assayed, either by parabiotic attachment or insertion of grafts. All of the autografts fused, while only two allografts fused and 34 pairs were incompatibile. Rejection among the parabiotic allografts was characterized by the formation of a collagenous barrier, while the allografts that were inserted into the host underwent destruction. At the molecular level we first cloned to completion the 5′-end of sponge RTK, which displays a Pro-Ser-Thr-rich sequence; this is thought to act as a module of cell adhesion proteins. Then we analyzed RT-PCR products of amplification across the two Ig-like domains of RTK (about 500 bp), from two pairs of fusing sponges and one pair of rejecting sponges. High levels of polymorphism were recorded, including 18 nucleotide-substitution positions and a tri-nucleotide deletion, which translate into 13 polymorphic amino acid positions. Two of the six sponges were scored as heterozygotes. Among 9 informative polymorphic sites that were tested for linkage disequilibrium, 11 pairwise comparisons were found to be significant, implying the possibility of distinguishable alleles in this locus. To the best of our knowledge this is the first report of polymorphism in Ig-like domains of a receptor from invertebrates that may be associated with allorecognition. This data attests also that fusion in sponges is not confined to genetically identical individuals.     

Self/non-self Discrimination in Basal Metazoa: Genetics of Allorecognition in the Hydroid Hydractinia

Colonial basal metazoans often encounter members of their ownspecies as they grow on hard substrata, with the encounterstypically resulting in either fusion of close relatives or rejectionbetween unrelated colonies. These allorecognition responsesplay a critical role in maintaining the genetic and physiologicalintegrity of the colony. Allorecognition responses in basalmetazoans are controlled by highly variable genetic systems.The molecular nature of such systems, however, remains to bedetermined. Current efforts to identify the genes and moleculescontrolling allorecognition in basal metazoans have followedtwo pathways: identification of molecules differentially expressedin incompatible interactions, and positional or map-based cloningof allorecognition genes. Most studies following the first approachhave been performed with marine demosponges, while those followingthe second approach have centered on the cnidarian of the genusHydractinia. Here, I discuss the latter, focusing primarilyon the genetic control of allorecognition responses.