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.     

Model systems for environmental signaling

Studies of environmental signaling in animals have focused primarilyon organisms with relatively constrained responses, both temporallyand phenotypically. In this regard, existing model animals (e.g.,"worms and flies") are particularly extreme. Such animals haverelatively little capacity to alter their morphology in responseto environmental signals. Hence, they exhibit little phenotypicplasticity. On the other hand, basal metazoans exhibit relativelyunconstrained responses to environmental signals and may thusprovide more general insight, insofar as these constraints arelikely traits derived during animal evolution. Such enhancedphenotypic plasticity may result from greater sensitivity toenvironmental signals, or greater abundance of suitable targetcells, or both. Examination of what is known of the componentsof environmental signaling pathways in cnidarians reveals manysimilarities to well-studied model animals. In addition to theseelements, however, macroscopic basal metazoans (e.g., spongesand cnidarians) typically exhibit a system-level capabilityfor integrating environmental information. In cnidarians, thegastrovascular system acts in this fashion, generating localpatterns of signaling (e.g., pressure, shear, and reactive oxygenspecies) via its organism-wide functioning. Contractile regionsof tissue containing concentrations of mitochondrion-rich, epitheliomuscularcells may be particularly important in this regard, servingin both a functional and a signaling context. While the evolutionof animal circulatory systems is usually considered in termsof alleviating surface-to-volume constraints, such systems alsohave the advantage of enhancing the capacity of larger organismsto respond quickly and efficiently to environmental signals.More general features of animals that correlate with relativelyunconstrained responses to environmental signals (e.g., activestem cells at all stages of the life cycle) are also enumeratedand discussed.     

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     

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 in the Colonial Marine Hydroid Hydractinia (Cnidaria/Hydrozoa)

The colonial marine hydroid Hydractinia has a sophisticatedallorecognition and effector system. Unlike many unitary organisms(i.e., vertebrates) which lack a current context for allorecognition,there is the potential for strong selection pressure for allorecognitionand response in Hydractinia. Hydractinia colonies use allorecognitionin intraspecific competition for two dimensional space; spaceis an absolute requirement for Hydractinia to successfully completeits life-cycle and thus interactions for space are of centralimportance for Hydractinia. Studies of the mechanisms, molecules,and genes involved in allorecognition in Hydractinia may contributeto our understanding of the evolution of allorecognition inthe metazoa.     

Evolution of sensory structures in basal metazoa

Cnidaria have traditionally been viewed as the most basal animalswith complex, organ-like multicellular structures dedicatedto sensory perception. However, sponges also have a surprisingrange of the genes required for sensory and neural functionsin Bilateria. Here, we: (1) discuss "sense organ" regulatorygenes, including; sine oculis, Brain 3, and eyes absent, thatare expressed in cnidarian sense organs; (2) assess the sensoryfeatures of the planula, polyp, and medusa life-history stagesof Cnidaria; and (3) discuss physiological and molecular datathat suggest sensory and "neural" processes in sponges. We thendevelop arguments explaining the shared aspects of developmentalregulation across sense organs and between sense organs andother structures. We focus on explanations involving divergentevolution from a common ancestral condition. In Bilateria, distinctsense-organ types share components of developmental-gene regulation.These regulators are also present in basal metazoans, suggestingevolution of multiple bilaterian organs from fewer antecedentsensory structures in a metazoan ancestor. More broadly, wehypothesize that developmental genetic similarities betweensense organs and appendages may reflect descent from closelyassociated structures, or a composite organ, in the common ancestorof Cnidaria and Bilateria, and we argue that such similaritiesbetween bilaterian sense organs and kidneys may derive froma multifunctional aggregations of choanocyte-like cells in ametazoan ancestor. We hope these speculative arguments presentedhere will stimulate further discussion of these and relatedquestions.     

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.     

Slow mitochondrial DNA sequence evolution in the Anthozoa (Cnidaria)

Mitochondrial genes have been used extensively in population genetic and phylogeographical analyses, in part due to a high rate of nucleotide substitution in animal mitochondrial DNA (mtDNA). Nucleotide sequences of anthozoan mitochondrial genes, however, are virtually invariant among conspecifics, even at third codon positions of protein-coding sequences. Hence, mtDNA markers are of limited use for population-level studies in these organisms. Mitochondrial gene sequence divergence among anthozoan species is also low relative to that exhibited in other animals, although higher level relationships can be resolved with these markers. Substitution rates in anthozoan nuclear genes are much higher than in mitochondrial genes, whereas nuclear genes in other metazoans usually evolve more slowly than, or similar to, mitochondrial genes. Although several mechanisms accounting for a slow rate of sequence evolution have been proposed, there is not yet a definitive explanation for this observation. Slow evolution and unique characteristics may be common in primitive metazoans, suggesting that patterns of mtDNA evolution in these organisms differ from that in other animal systems.     

The segmented urbilateria: a testable scenario

The idea that the last common ancestor of bilaterian animals(Urbilateria) was segmented has been raised recently on evidencecoming from comparative molecular embryology. Leaving asidethe complex debate on the value of genetic evidence, the morphologicaland developmental evidence in favor of a segmented Urbilateriaare discussed in the light of the emerging molecular phylogenyof metazoans. Applying a cladistic character optimization procedureto the question of segmentation is vastly complicated by theproblem of defining without ambiguity what segmentation is andto what taxa this definition applies. An ancestral segmentationmight have undergone many complex derivations in each differentphylum, thus rendering the cladistics approaches problematic.Taking the most general definitions of coelom and segmentationhowever, some remarkably similar patterns are found across thebilaterian tree in the way segments are formed by the posterioraddition of mesodermal segments or somites. Postulating thatthese striking similarities in mesodermal patterns are ancestral,a scenario for the diversification of bilaterians from a metamericancestor is presented. Several types of evolutionary mechanisms(specialization, tagmosis, progenesis) operating on a segmentedancestral body plan would explain the rapid emergence of bodyplans during the Cambrian. We finally propose to test this hypothesisby comparing genes involved in mesodermal segmentation.     

Expression of putative immune response genes during early ontogeny in the coral Acropora millepora

Background

Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals.

Methodology/Principal Findings

Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A.millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned.

Conclusions/Significance

Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are needed to further clarify emerging evidence of a complex innate immunity system in corals.     

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.     

The molecular basis of allorecognition in ascidians

The process of allorecognition consists of an ability to discriminate self from non-self. This discrimination is used either to identify non-self cells and reject them ("non-self histocompatibility") or to identify self cells and reject them (as in the avoidance of self-fertilization by hermaphrodites ("self incompatibility"). The molecular basis governing these two distinct systems has been studied recently in hermaphroditic ascidian urochordates. Harada et al. postulated two highly polymorphic self-incompatibility loci, Themis (A and B), that are transcribed from both strands, forward to yield sperm (s-) trans-membrane antigen, and reverse to yield the egg vitelline coat (v-) receptor. De Tomaso et al. characterized a candidate histocompatibility locus, encoding a highly variable immunoglobulin. Nyholm et al. isolated its candidate allorecognition receptor, fester. Only a minute similarity was found in the structure of the genes involved. It appears that ascidian harbor two very separate types of labeling and recognition genetic systems: one for self and the other for non-self.     

Compilation of All Genes Encoding Bacterial Two-component Signal Transducers in the Genome of the Cyanobacterium, Synechocystis sp. Strain PCC 6803

Bacteria have devised sophisticated signaling systems for elicitinga variety of adaptive responses to their environment, whichare generally referred to as the "two-component regulatory system."The widespread occurrence of the two-component systems in bothprokaryotes and eukaryotes implies that it is a powerful devicefor a wide variety of adaptive responses of cells to their environment.The two-component signal transducers contain one or more ofthree conserved and characteristic phosphotransfer signalingdomains, named the "transmitter, receiver, and alternative transmitter."The recently determined entire genomic sequence of Synechocystissp. strain PCC 6803 allowed us to compile systematically a completelist of genes encoding such two-component signal transductionproteins. The results of such an effort, made in this study,revealed that at least 80 ORFs were identified as members ofthe two-component signal transducers in this single speciesof cyanobacteria.     

Genetic and Molecular Analysis of the Love Song Preferences of Drosophila Females

The courtship songs of male Drosophila have been studied atthe behavioural, genetic and molecular levels. Less attentionhas been paid to the female's responses to these songs. Playbackexperiments are described which suggest that courtship songsare an important component of female mate choice. Some of theimplications of the behavioural responses of hybrid femalesbetween D. melanogaster and D. simulans are examined in thelight of theories concerning the mechanisms by which insectcommunication systems might evolve. The role of the period genein both male song production and in female song reception isconsidered, and the neural regions in the female which may beimportant for song integration are briefly discussed.     

EVOLUTIONARY GENETICS OF ALLORECOGNITION IN THE COLONIAL HYDROID HYDRACTINIA SYMBIOLONGICARPUS

Many sedentary, clonal marine invertebrates compete intensively with conspecifics for habitable space. Allorecognition systems mediate the nature and outcome of these intraspecific competitive interactions, such that the initiation of agonistic behavior and the potential for intergenotypic fusion depend strongly on the relatedness of the contestants. The dependence of these behaviors on relatedness, along with the extraordinary precision with which self can be discriminated from nonself, suggest that allorecognition systems are highly polymorphic genetically. However, allotypic specificity of this sort could be produced by any number of genetic scenarios, ranging from relatively few loci with abundant allelic variation to numerous loci with relatively few alleles per locus. At this point, virtually nothing is known of the formal genetics of allorecognition in marine invertebrates; consequently, the evolutionary dynamics of such systems remain poorly understood. In this paper, we characterize the formal genetics of allorecognition in the marine hydrozoan Hydractinia symbiolongicarpus. Hydractinia symbiolongicarpus colonizes gastropod shells occupied by hermit crabs. When two or more individuals grow into contact, one of three outcomes ensues: fusion (compatibility), transitory fusion (a temporary state of compatibility), and rejection (incompatibility, often accompanied by the production of agonistic structures termed hyperplastic stolons). Observed patterns of compatibility between unrelated, half-sib pairs, and full-sib pairs show that unrelated and half-sib pairs under laboratory culture have a very low probability of being fusible, whereas full sibs have a roughly 30% rate of fusion in experimental pairings. The genetic simulations indicate that roughly five loci, with 5–7 alleles per locus, confer specificity in this species. In ecological terms, the reproductive ecology of H. symbiolongicarpus should promote the cosettlement of kin, some of which should be full sibs, and some half sibs. Thus, there is potential for kin selection to play a major role in the evolution of the H. symbiolongicarpus allorecognition system. In genetic terms, this system conforms to theoretical predictions for a recognition system selected to distinguish among classes of kin, in addition to self from nonself.     

A Genome-wide Approach to Identify the Genes Involved in Biofilm Formation in E. coli

Biofilm forming cells are distinctive from the well-investigatedplanktonic cells and exhibit a different type of gene expression.Several new Escherichia coli genes related to biofilm formationhave recently been identified through genomic approaches suchas DNA microarray analysis. However, many others involved inthis process might have escaped detection due to poor expression,regulatory mechanism, or genetic backgrounds. Here, we screeneda collection of single-gene deletion mutants of E. coli named‘Keio collection’ to identify genes required forbiofilm formation. Of the 3985 mutants of non-essential genesin the collection thus examined, 110 showed a reduction in biofilmformation nine of which have not been well characterized yet.Systematic and quantitative analysis revealed the involvementof genes of various functions and reinforced the importancein biofilm formation of the genes for cell surface structuresand cell membrane. Characterization of the nine mutants of function-unknowngenes indicated that some of them, such as yfgA that geneticallyinteracts with a periplasmic chaperone gene surA together withyciB and yciM, might be required for the integrity of outermembrane.     

Different Expression Patterns of the Promoters of the NgrolB and NgrolC Genes during the Development of Tobacco Genetic Tumors

Hybrids (F1) between Nicotiana glauca and N. langsdorffii areprone to develop tumorous tissues on normaltype F1 tissues,namely, genetic tumors. To investigate the patterns of expressionof Ngrol genes during the development of genetic tumors, weperformed an analysis of transgenic genetic tumors that harboredthe promoters of the NgrolB and NgrolC genes fused to a reportergene for rß-glucuronidase (GUS) using a tumorization-redifferentiationsystem derived from F1 plants in vitro. Histochemical analysis of the expression of NgrolB-GUS in normal-typeF1 transgenic plants revealed GUS activity in meristematic zones,while in NgrolC-GUS transformed plants the activity was detectedmainly in the vascular systems of various organs. Tumorous tissues,which arose spontaneously as a consequence of aging or wereinduced by cutting, showed high levels of GUS expression underthe control of promoters of both the NgrolB and the NgrolC gene.Time course analysis during tumorization that followed cuttingof leaves of normal-type F1 plants showed clearly that NgrolB-GUSwas expressed in all dividing cells in the cut region after3 days. By contrast, the expression of NgrolC-GUS was detectedin organized tissues, such as procambium in teratomatous tumors,7–10 days after cutting treatment. During redifferentiationfrom genetic tumors to normal-type plants, the expression ofGUS under control of both Ngrol promoters decreased and expressionresembled that in normal-type tissues. These results suggestthe possibility that the Ngrol genes might be involved in formationof genetic tumors and, moreover, that the expression of NgrolBmight be linked to mitosis and while that of NgrolC might berelated to differentiation of tissues, such as the vascularsystem, in F1 plants. (Received January 19, 1996; Accepted March 24, 1996)