Stevan A. Springer

Coevolution of interacting fertilization proteins

Reproductive proteins are among the fastest evolving in the proteome, often due to the consequences of positive selection, and their rapid evolution is frequently attributed to a coevolutionary process between interacting female and male proteins. Such a process could leave characteristic signatures at coevolving genes. One signature of coevolution, predicted by sexual selection theory, is an association of alleles between the two genes. Another predicted signature is a correlation of evolutionary rates during divergence due to compensatory evolution. We studied female–male coevolution in the abalone by resequencing sperm lysin and its interacting egg coat protein, VERL, in populations of two species. As predicted, we found intergenic linkage disequilibrium between lysin and VERL, despite our demonstration that they are not physically linked. This finding supports a central prediction of sexual selection using actual genotypes, that of an association between a male trait and its female preference locus. We also created a novel likelihood method to show that lysin and VERL have experienced correlated rates of evolution. These two signatures of coevolution can provide statistical rigor to hypotheses of coevolution and could be exploited for identifying coevolving proteins a priori. We also present polymorphism-based evidence for positive selection and implicate recent selective events at the specific structural regions of lysin and VERL responsible for their species-specific interaction. Finally, we observed deep subdivision between VERL alleles in one species, which matches a theoretical prediction of sexual conflict. Thus, abalone fertilization proteins illustrate how coevolution can lead to reproductive barriers and potentially drive speciation.

ADAPTIVE GAMETE‐RECOGNITION DIVERGENCE IN A HYBRIDIZING MYTILUS POPULATION

Abstract Gamete-recognition proteins often evolve rapidly, but it is not known if their divergence occurs within species and corresponds with the evolution of reproductive isolation, or if divergence typically accumulates between already isolated lineages. We examined the evolution of a candidate gamete-recognition protein in several sympatric and allopatric populations of Mytilus blue mussels, species that hybridize in nature. Within a single species, Mytilus galloprovincialis, we found adaptive divergence of Lysin-M7, a sperm acrosomal protein that dissolves the egg vitelline envelope during fertilization. Mytilus galloprovincialis Lysin-M7 alleles group into two distinct clades (termed G and GD), and individual alleles in these clades are separated from each other by at least three and up to eleven amino-acid substitutions. Maximum-likelihood estimates of selective pressure (dN/dS = ω) implicate selection in the divergence between M. galloprovincialis Lysin-M7 clades, and within the GD clade. Exact tests of population differentiation indicate that the relative frequency of G and GD Lysin-M7 alleles differs significantly among M. galloprovincialis populations. Compared with allopatric Mediterranean samples, Lysin-M7 alleles in the GD clade are found at elevated frequency in samples from the East Atlantic and California, areas of secondary contact and hybridization between Mytilus species, and Australia, an area of unknown species composition. Adaptive divergence between the alleles most common in allopatry and those found at elevated frequency in samples from sympatry suggests that selection pressures acting in hybridizing populations, likely following Pleistocene secondary contact with M. edulis in the East Atlantic, drove the divergence of Lysin-M7 in M. galloprovincialis.

Extraordinary intraspecific diversity in oyster sperm bindin

In free-spawning invertebrates sperm–egg incompatibility is a barrier to mating between species, and divergence of gamete recognition proteins (GRPs) can result in reproductive isolation. Of interest are processes that create reproductive protein diversity within species, because intraspecific variants are potentially involved in mate choice and early speciation. Sperm acrosomes of the Pacific oyster Crassostrea gigas contain the protein bindin that bonds sperm to egg during fertilization. Oyster bindin is a single-copy gene encoding a diversity of protein variants. Oyster bindins have a conserved N-terminal region followed by one to five tandem fucose-binding lectin (F-lectin) domains. These repeats have diversified by positive selection at eight sites clustered on the F-lectins fucose binding face. Additional bindin variants result from recombination in an intron in each F-lectin repeat. Males also express alternatively spliced bindin cDNAs with one to five repeats, but typically translate only one or two isoforms into protein. Thus, positive selection, alternative splicing, and recombination can create thousands of bindin variants within C. gigas. Models of sexual conflict predict high male diversity when females are diverse and sexual conflict is strong. The amount of intraspecific polymorphism in male GRPs may be a consequence of the relative efficiency of local (molecular recognition) and global (electrical, cortical, and physical) polyspermy blocks that operate during fertilization.

Glycan Evolution in Response to Collaboration, Conflict, and Constraint

Glycans, oligo- and polysaccharides secreted or attached to proteins and lipids, cover the surfaces of all cells and have a regulatory capacity and structural diversity beyond any other class of biological molecule. Glycans may have evolved these properties because they mediate cellular interactions and often face pressure to evolve new functions rapidly. We approach this idea two ways. First, we discuss evolutionary innovation. Glycan synthesis, regulation, and mode of chemical interaction influence the spectrum of new forms presented to evolution. Second, we describe the evolutionary conflicts that arise when alleles and individuals interact. Glycan regulation and diversity are integral to these biological negotiations. Glycans are tasked with such an amazing diversity of functions that no study of cellular interaction can begin without considering them. We propose that glycans predominate the cell surface because their physical and chemical properties allow the rapid innovation required of molecules on the frontlines of evolutionary conflict.

Sexual selection by female immunity against paternal antigens can fix loss of function alleles

Humans lack the common mammalian cell surface molecule N-glycolylneuraminic acid (Neu5Gc) due to a CMAH gene inactivation, which occurred approximately three million years ago. Modern humans produce antibodies specific for Neu5Gc. We hypothesized that anti-Neu5Gc antibodies could enter the female reproductive tract and target Neu5Gc-positive sperm or fetal tissues, reducing reproductive compatibility. Indeed, female mice with a human-like Cmah(−/−) mutation and immunized to express anti-Neu5Gc antibodies show lower fertility with Neu5Gc-positive males, due to prezygotic incompatibilities. Human anti-Neu5Gc antibodies are also capable of targeting paternally derived antigens and mediate cytotoxicity against Neu5Gc-bearing chimpanzee sperm in vitro. Models of populations polymorphic for such antigens show that reproductive incompatibility by female immunity can drive loss-of-function alleles to fixation from moderate initial frequencies. Initially, the loss of a cell-surface antigen can occur due to drift in isolated populations or when natural selection favors the loss of a receptor exploited by pathogens, subsequently the same loss-of-function allele can come under sexual selection because it avoids being targeted by the female immune system. Thus, we provide evidence of a link between sexual selection and immune function: Antigenicity in females can select against foreign paternal antigens on sperm and rapidly fix loss-of-function alleles. Similar circumstances existed when the CMAH null allele was polymorphic in ancestral hominins, just before the divergence of Homo from australopithecines.

Oyster sperm bindin is a combinatorial fucose lectin with remarkable intra-species diversity

Sperm of the oyster, Crassostrea gigas, have ring-shaped acrosomes that, after exocytosis, bind the sperm to the egg vitelline layer. Isolated acrosomal rings contain proteins of various sizes: 35-, 48-, 63-, 75- and 88-kDa. These proteins, called bindins, have identical 24-residue signal peptides and conserved 97-residue N-terminal sequences, and they differ in mass because of the presence of between 1 and 5 tandemly repeated 134-residue fucose-binding lectin (F-lectin) domains. Southern blots suggest that oyster bindin is a single copy gene, but F-lectin repeat number and sequence are variable within and between individuals. Eight residues in the F-lectin fucose-binding groove are subject to positive diversifying selection, indicating a history of adaptive evolution at the lectins active site. There is one intron in the middle of each F-lectin repeat, and recombination in this intron creates many combinations of repeat halves. Alternative splicing creates many additional size and sequence variants of the repeat array. Males contain full-length bindin cDNAs of all 5 possible sizes, but only one or two protein mass forms exist in each individual. Sequence analysis indicates that recombination and alternate splicing create hundreds, possibly thousands, of different bindin sequences in C. gigas. The extreme within-species sequence variation in the F-lectin sequence of oyster bindin is a novel finding; most male gamete-recognition proteins are much less variable. In experimental conditions oyster eggs have poor polyspermy blocks, and bindin diversity could be an evolutionary response by sperm to match egg receptors that have diversified to avoid being fertilized by multiple sperm.

Beyond the phenotypic gambit: molecular behavioural ecology and the evolution of genetic architecture

Most studies of behaviour examine traits whose proximate causes include sensory input and neural decision‐making, but conflict and collaboration in biological systems began long before brains or sensory systems evolved. Many behaviours result from non‐neural mechanisms such as direct physical contact between recognition proteins or modifications of development that coincide with altered behaviour. These simple molecular mechanisms form the basis of important biological functions and can enact organismal interactions that are as subtle, strategic and interesting as any. The genetic changes that underlie divergent molecular behaviours are often targets of selection, indicating that their functional variation has important fitness consequences. These behaviours evolve by discrete units of quantifiable phenotypic effect (amino acid and regulatory mutations, often by successive mutations of the same gene), so the role of selection in shaping evolutionary change can be evaluated on the scale at which heritable phenotypic variation originates. We describe experimental strategies for finding genes that underlie biochemical and developmental alterations of behaviour, survey the existing literature highlighting cases where the simplicity of molecular behaviours has allowed insight to the evolutionary process and discuss the utility of a genetic knowledge of the sources and spectrum of phenotypic variation for a deeper understanding of how genetic and phenotypic architectures evolve.

Human-specific derived alleles of CD33 and other genes protect against postreproductive cognitive decline

Significance Most vertebrates die soon after they stop reproducing, but humans are an exception. Postreproductive humans care for offspring, assist in foraging, and communicate ecological and cultural knowledge, increasing the survival of younger individuals. Loss of cognitive capacity disrupts these benefits and burdens the group with the care of older members. We studied how the immunoregulatory receptor CD33 contributes to Alzheimer’s disease, a human-specific postreproductive condition. Surprisingly, a protective CD33 allele is derived and unique to humans, despite weak direct selection on older individuals. We identified several genes with derived alleles that protect against neurodegenerative disease and cerebrovascular insufficiency in old age. Selection by inclusive fitness may be strong enough to favor alleles that protect against cognitive decline in postreproductive humans. The individuals of most vertebrate species die when they can no longer reproduce. Humans are a rare exception, having evolved a prolonged postreproductive lifespan. Elders contribute to cooperative offspring care, assist in foraging, and communicate important ecological and cultural knowledge, increasing the survival of younger individuals. Age-related deterioration of cognitive capacity in humans compromises these benefits and also burdens the group with socially costly members. We investigated the contribution of the immunoregulatory receptor CD33 to a uniquely human postreproductive disease, Alzheimer’s dementia. Surprisingly, even though selection at advanced age is expected to be weak, a CD33 allele protective against Alzheimer’s disease is derived and unique to humans and favors a functional molecular state of CD33 resembling that of the chimpanzee. Thus, derived alleles may be compensatory and restore interactions altered as a consequence of human-specific brain evolution. We found several other examples of derived alleles at other human loci that protect against age-related cognitive deterioration arising from neurodegenerative disease or cerebrovascular insufficiency. Selection by inclusive fitness may be strong enough to favor alleles protecting specifically against cognitive decline in postreproductive humans. Such selection would operate by maximizing the contributions of postreproductive individuals to the fitness of younger kin.

Evolutionary Analysis and Classification of OATs, OCTs, OCTNs, and Other SLC22 Transporters: Structure-Function Implications and Analysis of Sequence Motifs

The SLC22 family includes organic anion transporters (OATs), organic cation transporters (OCTs) and organic carnitine and zwitterion transporters (OCTNs). These are often referred to as drug transporters even though they interact with many endogenous metabolites and signaling molecules (Nigam, S.K., Nature Reviews Drug Discovery, 14:29–44, 2015). Phylogenetic analysis of SLC22 supports the view that these transporters may have evolved over 450 million years ago. Many OAT members were found to appear after a major expansion of the SLC22 family in mammals, suggesting a physiological and/or toxicological role during the mammalian radiation. Putative SLC22 orthologs exist in worms, sea urchins, flies, and ciona. At least six groups of SLC22 exist. OATs and OCTs form two Major clades of SLC22, within which (apart from Oat and Oct subclades), there are also clear Oat-like, Octn, and Oct-related subclades, as well as a distantly related group we term “Oat-related” (which may have different functions). Based on available data, it is arguable whether SLC22A18, which is related to bacterial drug-proton antiporters, should be assigned to SLC22. Disease-causing mutations, single nucleotide polymorphisms (SNPs) and other functionally analyzed mutations in OAT1, OAT3, URAT1, OCT1, OCT2, OCTN1, and OCTN2 map to the first extracellular domain, the large central intracellular domain, and transmembrane domains 9 and 10. These regions are highly conserved within subclades, but not between subclades, and may be necessary for SLC22 transporter function and functional diversification. Our results not only link function to evolutionarily conserved motifs but indicate the need for a revised sub-classification of SLC22.

Duplicate abalone egg coat proteins bind sperm lysin similarly, but evolve oppositely, consistent with molecular mimicry at fertilization.

Sperm and egg proteins constitute a remarkable paradigm in evolutionary biology: despite their fundamental role in mediating fertilization (suggesting stasis), some of these molecules are among the most rapidly evolving ones known, and their divergence can lead to reproductive isolation. Because of strong selection to maintain function among interbreeding individuals, interacting fertilization proteins should also exhibit a strong signal of correlated divergence among closely related species. We use evidence of such molecular co-evolution to target biochemical studies of fertilization in North Pacific abalone (Haliotis spp.), a model system of reproductive protein evolution. We test the evolutionary rates (d N/d S) of abalone sperm lysin and two duplicated egg coat proteins (VERL and VEZP14), and find a signal of co-evolution specific to ZP-N, a putative sperm binding motif previously identified by homology modeling. Positively selected residues in VERL and VEZP14 occur on the same face of the structural model, suggesting a common mode of interaction with sperm lysin. We test this computational prediction biochemically, confirming that the ZP-N motif is sufficient to bind lysin and that the affinities of VERL and VEZP14 are comparable. However, we also find that on phylogenetic lineages where lysin and VERL evolve rapidly, VEZP14 evolves slowly, and vice versa. We describe a model of sexual conflict that can recreate this pattern of anti-correlated evolution by assuming that VEZP14 acts as a VERL mimic, reducing the intensity of sexual conflict and slowing the co-evolution of lysin and VERL.