Sea Squirt Immunity Gene Defies Expectations, Evolving Under Purifying Selection Not Diversifying Pressure

In the shallow waters of the world’s oceans, colonies of the tunicate Botryllus schlosseri wage a quiet chemical war over territory. When two colonies come into contact, they either fuse into a single chimera or mount a lethal rejection response, and the outcome of this encounter is dictated with complete accuracy by a single, extraordinarily polymorphic gene known as the Botryllus histocompatibility factor, or BHF. Because tunicates are the closest living relatives of vertebrates yet lack adaptive immunity, this gene has long fascinated immunologists as a living window into the evolutionary origins of self/non-self recognition. Now, a new study published in the journal Immunogenetics by Agniya M. Sokolova, Jacob Douek and Baruch Rinkevich of the Israel Oceanographic and Limnological Research institute has delivered a result that overturns the standard expectation for how such recognition genes should evolve: despite breathtaking nucleotide diversity and rampant length variation, BHF appears to be shaped primarily by purifying selection rather than the diversifying forces that govern its most famous counterparts in vertebrate immunity.

The expectations were not arbitrary. Genes involved in allorecognition, the ability to distinguish self from non-self among members of the same species, are classically assumed to be driven by balancing selection, the evolutionary force that maintains many different versions of a gene within a population. This pattern is abundantly documented in the major histocompatibility complex of vertebrates, in fungal heterokaryon incompatibility loci such as het-c, in plant self-incompatibility systems, and in the allorecognition genes of hydroids and sponges. Under this model, rare alleles enjoy a selective advantage, frequency-dependent dynamics keep many variants circulating, and the protein products of the gene diversify into ever more distinctive forms, each tuned to recognize a different repertoire of non-self partners. When the researchers set out to characterize BHF across natural and laboratory populations of B. schlosseri, they anticipated finding precisely these signatures.

Instead, they found something stranger. The team analyzed full-length BHF alleles from nineteen colonies, drawn both from laboratory-born stocks and wild populations, and uncovered thirty-three distinct alleles. At the DNA level, the locus is a riot of variation: nucleotide diversity is pronounced, alleles vary extensively in length, and the genealogy of the sequences shows clear evidence of intragenic recombination, with different segments of the gene telling contradictory evolutionary stories. Yet when the researchers translated the alleles into their protein products, the picture collapsed into remarkable uniformity. Those thirty-three alleles encode only seventeen distinct protein variants. The explosive divergence seen in the nucleotide sequence is largely silent at the level of the protein, a pattern that immediately suggested strong functional constraints keeping the encoded molecule stable even as its underlying blueprint drifts.

The geographic distribution of these alleles added another layer of intrigue. Highly divergent alleles, sequences so different from one another that they appear to have separated in the distant past, tend to coexist within the same individual colony, a hallmark of long-lived polymorphism. At the same time, identical alleles were found to be shared across continental-scale distances, a pattern difficult to reconcile with purely local evolution. In a species whose larvae disperse only over short ranges, such global allele sharing points to movement mediated by humans: B. schlosseri is a notorious fouling organism that hitches rides on ship hulls and in ballast water, and previous work by the same group and others has shown that historical navigation routes have left detectable footprints on the species’ seascape genetics. The new findings suggest that this human-mediated gene flow has effectively homogenized BHF alleles across ocean basins, mixing variant repertoires that might otherwise have diverged in isolation.

To probe the evolutionary forces at work, the researchers subjected the allele alignments to a battery of statistical tests designed to detect natural selection at individual codons. Contrary to the predictions of the diversifying-allele model, no evidence of balancing selection or directional positive selection emerged from the analyses. What the tests did reveal were signatures of purifying selection, the process by which natural selection removes harmful mutations and conserves protein function. In other words, the vast majority of the nucleotide variation in BHF is either silently synonymous or otherwise constrained from changing the protein it encodes, and the few amino-acid substitutions that do occur appear tolerated only within narrow limits. For a gene whose job is, by definition, to discriminate among an enormous diversity of non-self surfaces, this conservation of protein structure is genuinely counterintuitive.

The authors propose that the peculiar contrast between nucleotide and protein diversity at BHF arises from the combined effects of several processes acting simultaneously. Intragenic recombination continually shuffles DNA segments between alleles, generating new nucleotide combinations and inflating sequence diversity without necessarily altering protein sequences, particularly when crossovers occur in regions where the variants are functionally equivalent or where synonymous sites dominate. Human-mediated transport spreads the resulting allele repertoire globally, ensuring that any given colony can harbor partners for its most divergent alleles. Meanwhile, linkage to nearby loci that genuinely are under balancing selection could drag neutral and nearly neutral variation at BHF along for the ride, inflating apparent diversity at the locus without any direct selective diversification of the gene itself. This hitchhiking scenario echoes mechanisms documented in other systems, where polymorphisms maintained at one gene cast a long shadow of diversity across adjacent regions of the genome.

The technical groundwork for the study rested on methods refined over decades of research into the B. schlosseri fusion-rejection system, first described genetically by Abe Sabbadin in 1962 and later linked to a MHC-like gene system by Scofield and colleagues in 1982. The chromosomal location of the histocompatibility locus was resolved in 2005 by De Tomaso and coworkers, and the identity of BHF itself was established by Voskoboynik and colleagues in a landmark 2013 Science paper. Recombination analysis in the new study employed modern tools including RDP5, which screens sequence data for exchange events using multiple independent detection algorithms, while tests of codon-specific selection drew on maximum-likelihood frameworks that estimate synonymous and non-synonymous substitution rates across the gene. Exon- and intron-based phylogenies were compared to expose topological incongruence, the fingerprint of recombination, and predicted pairwise interaction outcomes were modeled on the assumption that colonies sharing at least one protein allele would fuse.

The broader significance of the finding extends beyond a single marine invertebrate. If extensive protein diversification is not a universal driver of allorecognition gene evolution, then the textbook equation of self/non-self recognition systems with runaway diversifying selection may need qualification. It raises the possibility that some recognition systems achieve functional diversity through means other than amino-acid innovation, for instance through the combinatorial pairing of a modest set of protein variants, or through differences in expression and regulation that are invisible to sequence-based analyses. It also cautions researchers studying other invertebrate allorecognition loci, from the Alr genes of hydroids to the aggregation factors of sponges, against assuming that polymorphism at the nucleotide level necessarily translates into adaptive protein diversification, since recombination, demographic mixing and linkage can each generate misleading signals.

For the field of comparative immunology, the BHF locus remains an indispensable touchstone precisely because it sits at the boundary between innate and adaptive immunity, in an organism whose blood-forming system has recently been shown to resemble that of mammals in surprising complexity. The new work does not diminish the gene’s predictive power over fusion and rejection; it simply reframes how that power is maintained. Purifying selection, recombination, and a globe-trotting lifestyle, it turns out, can conspire to keep a recognition gene both endlessly variable in its DNA and stubbornly conservative in its protein, a combination that ensures B. schlosseri will continue to puzzle and instruct immunologists for years to come.

Subject of Research: Evolutionary genetics of the Botryllus histocompatibility factor allorecognition locus in the colonial tunicate Botryllus schlosseri

Article Title: Intra-colony divergence and global allele sharing reflect purifying selection and recombination at the Botryllus histocompatibility factor locus

Article References: Intra-colony divergence and global allele sharing reflect purifying selection and recombination at the Botryllus histocompatibility factor locus. (n.d.). https://doi.org/10.1007/s00251-026-01413-2

Image Credits: AI Generated

DOI: 10.1007/s00251-026-01413-2

Keywords: Botryllus schlosseri, allorecognition, BHF, purifying selection, intragenic recombination, histocompatibility, tunicate immunity, balancing selection, polymorphism, self/non-self recognition, immunogenetics, gene flow