Trends among Institutional Animal Care and Use Committees and Animal Resource Programs for Providing Oversight and Animal Care during the (COVID)-19 Pandemic and Beyond.

To assess the impact of the COVID-19 pandemic on facility oversight and animal care at 2 y after declaration of the pandemic, we distributed a comprehensive survey to targeted groups within the broader animal research community in early 2022. A total of 265 surveys were returned (161 responses to IACUC questions, 193 responses to animal resource program [ARP] questions, and 89 responses to both). IACUC questions focused on the use of virtual interactions for IACUC activities and on remote work for IACUC support staff during and after the pandemic. ARP questions focused on remote work arrangements, altered work schedules, personnel shortages, and operational changes made during and after the pandemic. Results indicate that IACUCs readily adopted remote work options for staff and virtual meetings, changes that are now largely viewed as permanent, and are using modified methods for postapproval monitoring (PAM), while largely rejecting virtual semiannual facility inspections. This pattern reflects available guidance for virtual IACUC meetings, whereas new guidance for virtual semiannual facility inspections might have caused confusion and regulatory compliance concerns. Remote work options were available for some ARP staff, depending on job responsibilities, but were mostly eliminated by 2 y after the start of the pandemic. ARP staff expressed a strong desire for continued in-person presence of supervisors; this preference was not expressed by IACUC staff. Based on the timing of the survey (April 2022), substantial disruptions in the labor market had resulted in a considerable number of job openings and lack of entry level staff. Long-term assessment of effects on compliance and IACUC engagement would be useful given the widespread use of virtual IACUC meetings and other changes related to institutional and programmatic adoption of remote work options in ARPs.

Selection on genes associated with the evolution of divergent life histories: Gamete recognition or something else?

Gamete compatibility, and fertilization success, is mediated by gamete-recognition genes (GRGs) that are expected to show genetic evidence of a response to sexual selection associated with mating system traits. Changes in the strength of sexual selection can arise from the resolution of sperm competition among males, sexual conflicts of interest between males and females, or other mechanisms of sexual selection. To assess these expectations, we compared patterns of episodic diversifying selection among genes expressed in the gonads of Cryptasterina pentagona and C. hystera, which recently speciated and have evolved different mating systems (gonochoric or hermaphroditic), modes of fertilization (outcrossing or selfing), and dispersal (planktonic larvae or internal brooding). Cryptasterina spp. inhabit the upper intertidal of the coast of Queensland and coral islands of the Great Barrier Reef. We found some evidence for positive selection on a GRG in the outcrossing C. pentagona, and we found evidence of loss of gene function in a GRG of the self-fertilizing C. hystera. The modification or loss of gene functionality may be evidence of relaxed selection on some aspects of gamete interaction in C. hystera. In addition to these genes involved in gamete interactions, we also found genes under selection linked to abiotic stress, chromosomal regulation, polyspermy, and egg-laying. We interpret those results as possible evidence that Cryptasterina spp. with different mating systems may have been adapting in divergent ways to oxidative stress or other factors associated with reproduction in the physiologically challenging environment of the high intertidal. RESEARCH HIGHLIGHTS: Recent speciation between two sea stars was unlikely the result of selection on gamete-recognition genes annotated in this study. Instead, our results point to selection on genes linked to the intertidal environment and reproduction.

Cloning and Selfing Affect Population Genetic Variation in Simulations of Outcrossing, Sexual Sea Stars.

AbstractMany sea stars are well known for facultative or obligate asexual reproduction in both the adult and larval life-cycle stages. Some species and lineages are also capable of facultative or obligate hermaphroditic reproduction with self-fertilization. However, models of population genetic variation and empirical analyses of genetic data typically assume only sexual reproduction and outcrossing. A recent reanalysis of previously published empirical data (microsatellite genotypes) from two studies of one of the most well-known sea star species (the crown-of-thorns sea star; Acanthaster sp.) concluded that cloning and self-fertilization in that species are rare and contribute little to patterns of population genetic variation. Here we reconsider that conclusion by simulating the contribution of cloning and selfing to genetic variation in a series of models of sea star demography. Simulated variation in two simple models (analogous to previous analyses of empirical data) was consistent with high rates of cloning or selfing or both. More realistic scenarios that characterize population flux in sea stars of ecological significance, including outbreaks of crown-of-thorns sea stars that devastate coral reefs, invasions by Asterias amurensis, and epizootics of sea star wasting disease that kill Pisaster ochraceus, also showed significant but smaller effects of cloning and selfing on variation within subpopulations and differentiation between subpopulations. Future models or analyses of genetic variation in similar study systems might benefit from simulation modeling to characterize possible contributions of cloning or selfing to genetic variation in population samples or to understand the limits on inferring the effects of cloning or selfing in nature.

Diversity of Sea Star-Associated Densoviruses and Transcribed Endogenous Viral Elements of Densovirus Origin.

A viral etiology of sea star wasting syndrome (SSWS) was originally explored with virus-sized material challenge experiments, field surveys, and metagenomics, leading to the conclusion that a densovirus is the predominant DNA virus associated with this syndrome and, thus, the most promising viral candidate pathogen. Single-stranded DNA viruses are, however, highly diverse and pervasive among eukaryotic organisms, which we hypothesize may confound the association between densoviruses and SSWS. To test this hypothesis and assess the association of densoviruses with SSWS, we compiled past metagenomic data with new metagenomic-derived viral genomes from sea stars collected from Antarctica, California, Washington, and Alaska. We used 179 publicly available sea star transcriptomes to complement our approaches for densovirus discovery. Lastly, we focus the study on sea star-associated densovirus (SSaDV), the first sea star densovirus discovered, by documenting its biogeography and putative tissue tropism. Transcriptomes contained only endogenized densovirus elements similar to the NS1 gene, while numerous extant densoviral genomes were recovered from viral metagenomes. SSaDV was associated with nearly all tested species from southern California to Alaska, and in contrast to previous work, we show that SSaDV is one genotype among a high diversity of densoviruses present in sea stars across the West Coast of the United States and globally that are commonly associated with grossly normal (i.e., healthy or asymptomatic) animals. The diversity and ubiquity of these viruses in sea stars confound the original hypothesis that one densovirus is the etiological agent of SSWS.IMPORTANCE The primary interest in sea star densoviruses, specifically SSaDV, has been their association with sea star wasting syndrome (SSWS), a disease that has decimated sea star populations across the West Coast of the United States since 2013. The association of SSaDV with SSWS was originally drawn from metagenomic analysis, which was further studied through field surveys using quantitative PCR (qPCR), with the conclusion that it was the most likely viral candidate in the metagenomic data based on its representation in symptomatic sea stars compared to asymptomatic sea stars. We reexamined the original metagenomic data with additional genomic data sets and found that SSaDV was 1 of 10 densoviruses present in the original data set and was no more represented in symptomatic sea stars than in asymptomatic sea stars. Instead, SSaDV appears to be a widespread, generalist virus that exists among a large diversity of densoviruses present in sea star populations.

Nonspecific expression of fertilization genes in the crown-of-thorns Acanthaster cf. solaris: Unexpected evidence of hermaphroditism in a coral reef predator.

The characterization of gene expression in gametes has advanced our understanding of the molecular basis for ecological variation in reproductive success and the evolution of reproductive isolation. These advances are especially significant for ecologically important keystone predators such as the coral-eating crown-of-thorns sea stars (COTS, Acanthaster) which are the most influential predator species in Indo-Pacific coral reef ecosystems and the focus of intensive management efforts. We used RNA-seq and transcriptome assemblies to characterize the expression of genes in mature COTS gonads. We described the sequence and domain organization of eight genes with sex-specific expression and well known functions in fertilization in other echinoderms. We found unexpected expression of genes in one ovary transcriptome that are characteristic of males and sperm, including genes that encode the sperm-specific guanylate cyclase receptor for an egg pheromone, and the sperm acrosomal protein bindin. In a reassembly of previously published RNA-seq data from COTS testes, we found a complementary pattern: strong expression of four genes that are otherwise well known to encode egg-specific fertilization proteins, including the egg receptor for bindin (EBR1) and the acrosome reaction-inducing substance in the egg coat (ARIS1, ARIS2, ARIS3). We also found histological evidence of both eggs and sperm developing in the same gonad in several COTS individuals from a parallel study. These results suggest the occurrence of hermaphrodites, and the potential for reproductive assurance via self-fertilization. Our findings have implications for management of COTS populations, especially in consideration of the large size and massive fecundity of these sea stars.

Molecular evolution of mammalian genes with epistatic interactions in fertilization.

BACKGROUND: Genes that encode proteins associated with sperm competition, fertilization, and sexual conflicts of interest are often among the most rapidly evolving parts of animal genomes. One family of sperm-expressed genes (Zp3r, C4bpa) in the mammalian gene cluster called the regulator of complement activation (RCA) encodes proteins that bind eggs and mediate reproductive success, and are therefore expected to show high relative rates of nonsynonymous nucleotide substitution in response to sexual selection in comparison to other genes not involved in gamete binding at fertilization. We tested that working hypothesis by using phylogenetic models of codon evolution to identify episodes of diversifying positive selection. We used a comparative approach to quantify the evidence for episodic diversifying selection acting on RCA genes with known functions in fertilization (and sensitivity to sexual selection), and contrast them with other RCA genes in the same gene family that function in innate immunity (and are not sensitive to sexual selection). RESULTS: We expected but did not find evidence for more episodes of positive selection on Zp3r in Glires (the rodents and lagomorphs) or on C4BPA in Primates, in comparison to other paralogous RCA genes in the same taxon, or in comparison to the same orthologous RCA gene in the other taxon. That result was not unique to RCA genes: we also found little evidence for more episodes of diversifying selection on genes that encode selective sperm-binding molecules in the egg coat or zona pellucida (Zp2, Zp3) in comparison to members of the same gene family that encode structural elements of the egg coat (Zp1, Zp4). Similarly, we found little evidence for episodic diversifying selection acting on two other recently discovered genes (Juno, Izumo1) that encode essential molecules for sperm-egg fusion. CONCLUSIONS: These negative results help to illustrate the importance of a comparative context for this type of codon model analysis. The results may also point to other phylogenetic contexts in which the effects of selection acting on these fertilization proteins might be more readily discovered and documented in mammals and other taxa.

Positive selection on human gamete-recognition genes.

Coevolution of genes that encode interacting proteins expressed on the surfaces of sperm and eggs can lead to variation in reproductive compatibility between mates and reproductive isolation between members of different species. Previous studies in mice and other mammals have focused in particular on evidence for positive or diversifying selection that shapes the evolution of genes that encode sperm-binding proteins expressed in the egg coat or zona pellucida (ZP). By fitting phylogenetic models of codon evolution to data from the 1000 Genomes Project, we identified candidate sites evolving under diversifying selection in the human genes ZP3 and ZP2. We also identified one candidate site under positive selection in C4BPA, which encodes a repetitive protein similar to the mouse protein ZP3R that is expressed in the sperm head and binds to the ZP at fertilization. Results from several additional analyses that applied population genetic models to the same data were consistent with the hypothesis of selection on those candidate sites leading to coevolution of sperm- and egg-expressed genes. By contrast, we found no candidate sites under selection in a fourth gene (ZP1) that encodes an egg coat structural protein not directly involved in sperm binding. Finally, we found that two of the candidate sites (in C4BPA and ZP2) were correlated with variation in family size and birth rate among Hutterite couples, and those two candidate sites were also in linkage disequilibrium in the same Hutterite study population. All of these lines of evidence are consistent with predictions from a previously proposed hypothesis of balancing selection on epistatic interactions between C4BPA and ZP3 at fertilization that lead to the evolution of co-adapted allele pairs. Such patterns also suggest specific molecular traits that may be associated with both natural reproductive variation and clinical infertility.

Finding genes and lineages under selection in speciation.

What are the genes and traits that respond to selection and cause prezygotic reproductive isolation between species? This question has been hard to answer because genomes are large, the targets of selection may be scattered across the genome (Sabeti et al., ) and different genes may respond to the same selective pressure in different populations (Scheinfeldt et al., ). In this issue of Molecular Ecology, Weber et al. () use a clever comparative approach and leading-edge transcriptomic methods to identify the species and genes under positive selection for divergence between brittle stars (the echinoderm class Ophiuroidea) in the Ophioderma longicauda species complex. They found convincing evidence of positive or diversifying selection acting on two genes encoding ion channels that form part of the signal transduction cascade within the sperm in response to pheromones. Evidence for selection was concentrated in genes from one species (called C5, with internal fertilization and female parental care of brooded juveniles and not in the other species (called C3, with more conventional broadcast spawning and planktonic development of embryos and larvae). That analysis greatly extends the range of taxa, life history traits and molecules that are associated with positive selection in speciation. It also illustrates some of the current limitations on the application of RNAseq methods in the search for the targets of selection in nonmodel organisms like brittle stars. From both points of view, the new work by Weber et al. () has important implications for our understanding of speciation in the ocean.

Life-history predicts past and present population connectivity in two sympatric sea stars.

Life-history traits, especially the mode and duration of larval development, are expected to strongly influence the population connectivity and phylogeography of marine species. Comparative analysis of sympatric, closely related species with differing life histories provides the opportunity to specifically investigate these mechanisms of evolution but have been equivocal in this regard. Here, we sample two sympatric sea stars across the same geographic range in temperate waters of Australia. Using a combination of mitochondrial DNA sequences, nuclear DNA sequences, and microsatellite genotypes, we show that the benthic-developing sea star, Parvulastra exigua, has lower levels of within- and among-population genetic diversity, more inferred genetic clusters, and higher levels of hierarchical and pairwise population structure than Meridiastra calcar, a species with planktonic development. While both species have populations that have diverged since the middle of the second glacial period of the Pleistocene, most P. exigua populations have origins after the last glacial maxima (LGM), whereas most M. calcar populations diverged long before the LGM. Our results indicate that phylogenetic patterns of these two species are consistent with predicted dispersal abilities; the benthic-developing P. exigua shows a pattern of extirpation during the LGM with subsequent recolonization, whereas the planktonic-developing M. calcar shows a pattern of persistence and isolation during the LGM with subsequent post-Pleistocene introgression.

Sperm Bindin Divergence under Sexual Selection and Concerted Evolution in Sea Stars.

Selection associated with competition among males or sexual conflict between mates can create positive selection for high rates of molecular evolution of gamete recognition genes and lead to reproductive isolation between species. We analyzed coding sequence and repetitive domain variation in the gene encoding the sperm acrosomal protein bindin in 13 diverse sea star species. We found that bindin has a conserved coding sequence domain structure in all 13 species, with several repeated motifs in a large central region that is similar among all sea stars in organization but highly divergent among genera in nucleotide and predicted amino acid sequence. More bindin codons and lineages showed positive selection for high relative rates of amino acid substitution in genera with gonochoric outcrossing adults (and greater expected strength of sexual selection) than in selfing hermaphrodites. That difference is consistent with the expectation that selfing (a highly derived mating system) may moderate the strength of sexual selection and limit the accumulation of bindin amino acid differences. The results implicate both positive selection on single codons and concerted evolution within the repetitive region in bindin divergence, and suggest that both single amino acid differences and repeat differences may affect sperm-egg binding and reproductive compatibility.

Models of selection, isolation, and gene flow in speciation.

Many marine ecologists aspire to use genetic data to understand how selection and demographic history shape the evolution of diverging populations as they become reproductively isolated species. I propose combining two types of genetic analysis focused on this key early stage of the speciation process to identify the selective agents directly responsible for population divergence. Isolation-with-migration (IM) models can be used to characterize reproductive isolation between populations (low gene flow), while codon models can be used to characterize selection for population differences at the molecular level (especially positive selection for high rates of amino acid substitution). Accessible transcriptome sequencing methods can generate the large quantities of data needed for both types of analysis. I highlight recent examples (including our work on fertilization genes in sea stars) in which this confluence of interest, models, and data has led to taxonomically broad advances in understanding marine speciation at the molecular level. I also highlight new models that incorporate both demography and selection: simulations based on these theoretical advances suggest that polymorphisms shared among individuals (a key source of information in IM models) may lead to false-positive evidence of selection (in codon models), especially during the early stages of population divergence and speciation that are most in need of study. The false-positive problem may be resolved through a combination of model improvements plus experiments that document the phenotypic and fitness effects of specific polymorphisms for which codon models and IM models indicate selection and reproductive isolation (such as genes that mediate sperm-egg compatibility at fertilization).

Selection and demographic history shape the molecular evolution of the gamete compatibility protein bindin in Pisaster sea stars.

Reproductive compatibility proteins have been shown to evolve rapidly under positive selection leading to reproductive isolation, despite the potential homogenizing effects of gene flow. This process has been implicated in both primary divergence among conspecific populations and reinforcement during secondary contact; however, these two selective regimes can be difficult to discriminate from each other. Here, we describe the gene that encodes the gamete compatibility protein bindin for three sea star species in the genus Pisaster. First, we compare the full-length bindin-coding sequence among all three species and analyze the evolutionary relationships between the repetitive domains of the variable second bindin exon. The comparison suggests that concerted evolution of repetitive domains has an effect on bindin divergence among species and bindin variation within species. Second, we characterize population variation in the second bindin exon of two species: We show that positive selection acts on bindin variation in Pisaster ochraceus but not in Pisaster brevispinus, which is consistent with higher polyspermy risk in P. ochraceus. Third, we show that there is no significant genetic differentiation among populations and no apparent effect of sympatry with congeners that would suggest selection based on reinforcement. Fourth, we combine bindin and cytochrome c oxidase 1 data in isolation-with-migration models to estimate gene flow parameter values and explore the historical demographic context of our positive selection results. Our findings suggest that positive selection on bindin divergence among P. ochraceus alleles can be accounted for in part by relatively recent northward population expansions that may be coupled with the potential homogenizing effects of concerted evolution.

Incipient speciation of sea star populations by adaptive gamete recognition coevolution.

Reproductive isolation--the key event in speciation--can evolve when sexual conflict causes selection favoring different combinations of male and female adaptations in different populations. Likely targets of such selection include genes that encode proteins on the surfaces of sperm and eggs, but no previous study has demonstrated intraspecific coevolution of interacting gamete recognition genes under selection. Here, we show that selection drives coevolution between an egg receptor for sperm (OBi1) and a sperm acrosomal protein (bindin) in diverging populations of a sea star (Patiria miniata). We found positive selection on OBi1 in an exon encoding part of its predicted substrate-binding protein domain, the ligand for which is found in bindin. Gene flow was zero for the parts of bindin and OBi1 in which selection for high rates of amino acid substitution was detected; higher gene flow for other parts of the genome indicated selection against immigrant alleles at bindin and OBi1. Populations differed in allele frequencies at two key positively selected sites (one in each gene), and differences at those sites predicted fertilization rate variation among male-female pairs. These patterns suggest adaptively evolving loci that influence reproductive isolation between populations.

Shallow gene pools in the high intertidal: extreme loss of genetic diversity in viviparous sea stars (Parvulastra).

We document an extreme example of reproductive trait evolution that affects population genetic structure in sister species of Parvulastra cushion stars from Australia. Self-fertilization by hermaphroditic adults and brood protection of benthic larvae causes strong inbreeding and range-wide genetic poverty. Most samples were fixed for a single allele at nearly all nuclear loci; heterozygotes were extremely rare (0.18%); mitochondrial DNA sequences were more variable, but few populations shared haplotypes in common. Isolation-with-migration models suggest that these patterns are caused by population bottlenecks (relative to ancestral population size) and low gene flow. Loss of genetic diversity and low potential for dispersal between high-intertidal habitats may have dire consequences for extinction risk and potential for future adaptive evolution in response to climate and other selective agents.

Sea star populations diverge by positive selection at a sperm-egg compatibility locus.

Fertilization proteins of marine broadcast spawning species often show signals of positive selection. Among geographically isolated populations, positive selection within populations can lead to differences between them, and may result in reproductive isolation upon secondary contact. Here, we test for positive selection in the reproductive compatibility locus, bindin, in two populations of a sea star on either side of a phylogeographic break. We find evidence for positive selection at codon sites in both populations, which are under neutral or purifying selection in the reciprocal population. The signal of positive selection is stronger and more robust in the population where effective population size is larger and bindin diversity is greater. In addition, we find high variation in coding sequence length caused by large indels at two repetitive domains within the gene, with greater length diversity in the larger population. These findings provide evidence of population-divergent positive selection in a fertilization compatibility locus, and suggest that sexual selection can lead to reproductive divergence between conspecific marine populations.

Structure and evolution of the sea star egg receptor for sperm bindin.

Selection on coevolving sperm- and egg-recognition molecules is a potent engine of population divergence leading to reproductive isolation and speciation. The study of receptor-ligand pairs can reveal co-evolution of male- and female-expressed genes or differences between their evolution in response to selective factors such as sperm competition and sexual conflict. Phylogeographical studies of these patterns have been limited by targeted gene methods that favour short protein-coding sequences amplifiable by PCR. Here, I use high-throughput transcriptomic methods to characterize the structure and divergence of full-length coding sequences for the gene encoding the protein component of a large complex egg surface glycopeptide receptor for the sperm acrosomal protein bindin from the sea star Patiria miniata. I used a simple but effective method for resolving nucleotide polymorphisms into haplotypes for phylogeny-based analyses of selection. The protein domain organization of sea star egg bindin receptor (EBR1) was similar to sea urchins and included a pair of protein-recognition domains plus a series of tandem repeat domains of two types. Two populations separated by a well-characterized phylogeographical break included lineages of EBR1 alleles under positive selection at several codons (similar to selection on sperm bindin in the same populations). However, these populations shared the same alleles that were under selection for amino acid differences at multiple codons (unlike the pattern of selection for population divergence in sperm bindin). The significance of positively selected EBR1 domains and alleles could be tested in functional analyses of fertilization rates associated with EBR1 (and bindin) polymorphisms.

Extraordinarily rapid life-history divergence between Cryptasterina sea star species.

Life history plays a critical role in governing microevolutionary processes such as gene flow and adaptation, as well as macroevolutionary processes such speciation. Here, we use multilocus phylogeographic analyses to examine a speciation event involving spectacular life-history differences between sister species of sea stars. Cryptasterina hystera has evolved a suite of derived life-history traits (including internal self-fertilization and brood protection) that differ from its sister species Cryptasterina pentagona, a gonochoric broadcast spawner. We show that these species have only been reproductively isolated for approximately 6000 years (95% highest posterior density of 905-22 628), and that this life-history change may be responsible for dramatic genetic consequences, including low nucleotide diversity, zero heterozygosity and no gene flow. The rapid divergence of these species rules out some mechanisms of isolation such as adaptation to microhabitats in sympatry, or slow divergence by genetic drift during prolonged isolation. We hypothesize that the large phenotypic differences between species relative to the short divergence time suggests that the life-history differences observed may be direct responses to disruptive selection between populations. We speculate that local environmental or demographic differences at the southern range margin are possible mechanisms of selection driving one of the fastest known marine speciation events.

Next-generation studies of mating system evolution.

The specificity of mate selection can vary from wantonly indiscriminate to extraordinarily choosy, and depends in large part on molecules expressed on the surfaces of sperm and eggs. Understanding the evolution of this specificity of gamete recognition leads to important insights into the evolution of reproductive isolation and speciation. One productive area of research has focused on genes that encode gamete recognition proteins in broadcast-spawning marine invertebrates. These gene products are relatively accessible to biochemical and cellular analyses of expression and function, and they mediate almost all of the elements of mate selection and specificity between males and females of such species. However, genetic analyses of their evolution are currently limited to a few combinations of molecules and taxa, and may miss the broader view of adaptive responses to selection on mating specificity across many genes and many types of mating systems. A transcriptomic study shows how next-generation sequencing methods and analyses could relatively easily broaden such studies to more clades, deepen those studies to include more of the interacting molecular parts that mediate gamete recognition, and eventually lead to a more complete understanding of the molecular basis for mating system variation and its evolutionary response to selection.