Single Cell RNA Sequencing Provides Clues for the Developmental Genetic Basis of Syngnathidae's Evolutionary Adaptations.

Seahorses, pipefishes, and seadragons are fishes from the family Syngnathidae that have evolved extraordinary traits including male pregnancy, elongated snouts, loss of teeth, and dermal bony armor. The developmental genetic and cellular changes that led to the evolution of these traits are largely unknown. Recent syngnathid genomes revealed suggestive gene content differences and provide the opportunity for detailed genetic analyses. We created a single cell RNA sequencing atlas of Gulf pipefish embryos to understand the developmental basis of four traits: derived head shape, toothlessness, dermal armor, and male pregnancy. We completed marker gene analyses, built genetic networks, and examined spatial expression of select genes. We identified osteochondrogenic mesenchymal cells in the elongating face that express regulatory genes bmp4, sfrp1a, and prdm16. We found no evidence for tooth primordia cells, and we observed re-deployment of osteoblast genetic networks in developing dermal armor. Finally, we found that epidermal cells expressed nutrient processing and environmental sensing genes, potentially relevant for the brooding environment. The examined pipefish evolutionary innovations are composed of recognizable cell types, suggesting derived features originate from changes within existing gene networks. Future work addressing syngnathid gene networks across multiple stages and species is essential for understanding how their novelties evolved.

Host genomic variation shapes gut microbiome diversity in threespine stickleback fish.

IMPORTANCE: A major focus of host-microbe research is to understand how genetic differences, of various magnitudes, among hosts translate to differences in their microbiomes. This has been challenging for animal hosts, including humans, because it is difficult to control environmental variables tightly enough to isolate direct genetic effects on the microbiome. Our work in stickleback fish is a significant contribution because our experimental approach allowed strict control over environmental factors, including standardization of the microbiome from the earliest stage of development and unrestricted co-housing of fish in a truly common environment. Furthermore, we measured host genetic variation over 2,000 regions of the stickleback genome, comparing this information and microbiome composition data among fish from very similar and very different genetic backgrounds. Our findings highlight how differences in the host genome influence microbiome diversity and make a case for future manipulative microbiome experiments that use host systems with naturally occurring genetic variation.

Ovarian transcriptional response to Wolbachia infection in D. melanogaster in the context of between-genotype variation in gene expression.

Wolbachia is a maternally transmitted endosymbiotic bacteria that infects a wide variety of arthropod and nematode hosts. The effects of Wolbachia on host biology are far-reaching and include changes in host gene expression. However, previous work on the host transcriptional response has generally been investigated in the context of a single host genotype. Thus, the relative effect of Wolbachia infection versus vs. host genotype on gene expression is unknown. Here, we explicitly test the relative roles of Wolbachia infection and host genotype on host gene expression by comparing the ovarian transcriptomes of 4 strains of Drosophila melanogaster (D. melanogaster) infected and uninfected with Wolbachia. Our data suggest that infection explains a small amount of transcriptional variation, particularly in comparison to variation in gene expression among strains. However, infection specifically affects genes related to cell cycle, translation, and metabolism. We also find enrichment of cell division and recombination processes among genes with infection-associated differential expression. Broadly, the transcriptomic changes identified in this study provide novel understanding of the relative magnitude of the effect of Wolbachia infection on gene expression in the context of host genetic variation and also point to genes that are consistently differentially expressed in response to infection among multiple genotypes.

The evolution of the testis transcriptome in pregnant male pipefishes and seahorses.

In many animals, sperm competition and sexual conflict are thought to drive the rapid evolution of male-specific genes, especially those expressed in the testes. A potential exception occurs in the male pregnant pipefishes, where females transfer eggs to the males, eliminating testes from participating in these processes. Here, we show that testis-related genes differ dramatically in their rates of molecular evolution and expression patterns in pipefishes and seahorses (Syngnathidae) compared to other fish. Genes involved in testis or sperm function within syngnathids experience weaker selection in comparison to their orthologs in spawning and livebearing fishes. An assessment of gene turnover and expression in the testis transcriptome suggests that syngnathids have lost (or significantly reduced expression of) important classes of genes from their testis transcriptomes compared to other fish. Our results indicate that more than 50 million years of male pregnancy have removed syngnathid testes from the molecular arms race that drives the rapid evolution of male reproductive genes in other taxa.

Evolution and developmental expression of the sodium-iodide symporter (NIS, slc5a5) gene family: Implications for perchlorate toxicology.

The vertebrate sodium-iodide symporter (NIS or SLC5A5) transports iodide into the thyroid follicular cells that synthesize thyroid hormone. The SLC5A protein family includes transporters of vitamins, minerals, and nutrients. Disruption of SLC5A5 function by perchlorate, a pervasive environmental contaminant, leads to human pathologies, especially hypothyroidism. Perchlorate also disrupts the sexual development of model animals, including threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio), but the mechanism of action is unknown. To test the hypothesis that SLC5A5 paralogs are expressed in tissues necessary for the development of reproductive organs, and therefore are plausible candidates to mediate the effects of perchlorate on sexual development, we first investigated the evolutionary history of Slc5a paralogs to better understand potential functional trajectories of the gene family. We identified two clades of slc5a paralogs with respect to an outgroup of sodium/choline cotransporters (slc5a7); these clades are the NIS clade of sodium/iodide and lactate cotransporters (slc5a5, slc5a6, slc5a8, slc5a8, and slc5a12) and the SGLT clade of sodium/glucose cotransporters (slc5a1, slc5a2, slc5a3, slc5a4, slc5a10, and slc5a11). We also characterized expression patterns of slc5a genes during development. Stickleback embryos and early larvae expressed NIS clade genes in connective tissue, cartilage, teeth, and thyroid. Stickleback males and females expressed slc5a5 and its paralogs in gonads. Single-cell transcriptomics (scRNA-seq) on zebrafish sex-genotyped gonads revealed that NIS clade-expressing cells included germ cells (slc5a5, slc5a6a, and slc5a6b) and gonadal soma cells (slc5a8l). These results are consistent with the hypothesis that perchlorate exerts its effects on sexual development by interacting with slc5a5 or its paralogs in reproductive tissues. These findings show novel expression domains of slc5 genes in stickleback and zebrafish, which suggest similar functions across vertebrates including humans, and provide candidates to mediate the effects of perchlorate on sexual development.

Leafy and weedy seadragon genomes connect genic and repetitive DNA features to the extravagant biology of syngnathid fishes.

Seadragons are a remarkable lineage of teleost fishes in the family Syngnathidae, renowned for having evolved male pregnancy. Comprising three known species, seadragons are widely recognized and admired for their fantastical body forms and coloration, and their specific habitat requirements have made them flagship representatives for marine conservation and natural history interests. Until recently, a gap has been the lack of significant genomic resources for seadragons. We have produced gene-annotated, chromosome-scale genome models for the leafy and weedy seadragon to advance investigations of evolutionary innovation and elaboration of morphological traits in seadragons as well as their pipefish and seahorse relatives. We identified several interesting features specific to seadragon genomes, including divergent noncoding regions near a developmental gene important for integumentary outgrowth, a high genome-wide density of repetitive DNA, and recent expansions of transposable elements and a vesicular trafficking gene family. Surprisingly, comparative analyses leveraging the seadragon genomes and additional syngnathid and outgroup genomes revealed striking, syngnathid-specific losses in the family of fibroblast growth factors (FGFs), which likely involve reorganization of highly conserved gene regulatory networks in ways that have not previously been documented in natural populations. The resources presented here serve as important tools for future evolutionary studies of developmental processes in syngnathids and hold value for conservation of the extravagant seadragons and their relatives.

Advancing human disease research with fish evolutionary mutant models.

Model organism research is essential to understand disease mechanisms. However, laboratory-induced genetic models can lack genetic variation and often fail to mimic the spectrum of disease severity. Evolutionary mutant models (EMMs) are species with evolved phenotypes that mimic human disease. EMMs complement traditional laboratory models by providing unique avenues to study gene-by-environment interactions, modular mutations in noncoding regions, and their evolved compensations. EMMs have improved our understanding of complex diseases, including cancer, diabetes, and aging, and illuminated mechanisms in many organs. Rapid advancements of sequencing and genome-editing technologies have catapulted the utility of EMMs, particularly in fish. Fish are the most diverse group of vertebrates, exhibiting a kaleidoscope of specialized phenotypes, many that would be pathogenic in humans but are adaptive in the species' specialized habitat. Importantly, evolved compensations can suggest avenues for novel disease therapies. This review summarizes current research using fish EMMs to advance our understanding of human disease.

QTL Mapping of Intestinal Neutrophil Variation in Threespine Stickleback Reveals Possible Gene Targets Connecting Intestinal Inflammation and Systemic Health.

Selection, via host immunity, is often required to foster beneficial microbial symbionts and suppress deleterious pathogens. In animals, the host immune system is at the center of this relationship. Failed host immune system-microbial interactions can result in a persistent inflammatory response in which the immune system indiscriminately attacks resident microbes, and at times the host cells themselves, leading to diseases such as Ulcerative Colitis, Crohn's Disease, and Psoriasis. Host genetic variation has been linked to both microbiome diversity and to severity of such inflammatory disease states in humans. However, the microbiome and inflammatory states manifest as quantitative traits, which encompass many genes interacting with one another and the environment. The mechanistic relationships among all of these interacting components are still not clear. Developing natural genetic models of host-microbe interactions is therefore fundamental to understanding the complex genetics of these and other diseases. Threespine stickleback (Gasterosteus aculeatus) fish are a tractable model for attacking this problem because of abundant population-level genetic and phenotypic variation in the gut inflammatory response. Previous work in our laboratory identified genetically divergent stickleback populations exhibiting differences in intestinal neutrophil activity. We took advantage of this diversity to genetically map variation in an emblematic element of gut inflammation - intestinal neutrophil recruitment - using an F2-intercross mapping framework. We identified two regions of the genome associated with increased intestinal inflammation containing several promising candidate genes. Within these regions we found candidates in the Coagulation/Complement System, NFkB and MAPK pathways along with several genes associated with intestinal diseases and neurological diseases commonly accompanying intestinal inflammation as a secondary symptom. These findings highlight the utility of using naturally genetically diverse 'evolutionary mutant models' such as threespine stickleback to better understand interactions among host genetic diversity and microbiome variation in health and disease states.

Developmental tuning of mineralization drives morphological diversity of gill cover bones in sculpins and their relatives.

The role of osteoblast placement in skeletal morphological variation is relatively well understood, but alternative developmental mechanisms affecting bone shape remain largely unknown. Specifically, very little attention has been paid to variation in later mineralization stages of intramembranous ossification as a driver of morphological diversity. We discover the occurrence of specific, sometimes large, regions of nonmineralized osteoid within bones that also contain mineralized tissue. We show through a variety of histological, molecular, and tomographic tests that this "extended" osteoid material is most likely nonmineralized bone matrix. This tissue type is a significant determinant of gill cover bone shape in the teleostean suborder Cottoidei. We demonstrate repeated evolution of extended osteoid in Cottoidei through ancestral state reconstruction and test for an association between extended osteoid variation and habitat differences among species. Through measurement of extended osteoid at various stages of gill cover development in species across the phylogeny, we gain insight into possible evolutionary developmental origins of the trait. We conclude that this fine-tuned developmental regulation of bone matrix mineralization reflects heterochrony at multiple biological levels and is a novel mechanism for the evolution of diversity in skeletal morphology. This research lays the groundwork for a new model in which to study bone mineralization and evolutionary developmental processes, particularly as they may relate to adaptation during a prominent evolutionary radiation of fishes.

Highly Reproducible 16S Sequencing Facilitates Measurement of Host Genetic Influences on the Stickleback Gut Microbiome.

Multicellular organisms interact with resident microbes in important ways, and a better understanding of host-microbe interactions is aided by tools such as high-throughput 16S sequencing. However, rigorous evaluation of the veracity of these tools in a different context from which they were developed has often lagged behind. Our goal was to perform one such critical test by examining how variation in tissue preparation and DNA isolation could affect inferences about gut microbiome variation between two genetically divergent lines of threespine stickleback fish maintained in the same laboratory environment. Using careful experimental design and intensive sampling of individuals, we addressed technical and biological sources of variation in 16S-based estimates of microbial diversity. After employing a two-tiered bead beating approach that comprised tissue homogenization followed by microbial lysis in subsamples, we found an extremely minor effect of DNA isolation protocol relative to among-host microbial diversity differences. Abundance estimates for rare operational taxonomic units (OTUs), however, showed much lower reproducibility. Gut microbiome composition was highly variable across fish-even among cohoused siblings-relative to technical replicates, but a subtle effect of host genotype (stickleback line) was nevertheless detected for some microbial taxa.IMPORTANCE Our findings demonstrate the importance of appropriately quantifying biological and technical variance components when attempting to understand major influences on high-throughput microbiome data. Our focus was on understanding among-host (biological) variance in community metrics and its magnitude in relation to within-host (technical) variance, because meaningful comparisons among individuals are necessary in addressing major questions in host-microbe ecology and evolution, such as heritability of the microbiome. Our study design and insights should provide a useful example for others desiring to quantify microbiome variation at biological levels in the face of various technical factors in a variety of systems.

A rich diversity of opercle bone shape among teleost fishes.

The opercle is a prominent craniofacial bone supporting the gill cover in all bony fish and has been the subject of morphological, developmental, and genetic investigation. We surveyed the shapes of this bone among 110 families spanning the teleost tree and examined its pattern of occupancy in a principal component-based morphospace. Contrasting with expectations from the literature that suggest the local morphospace would be only sparsely occupied, we find primarily dense, broad filling of the morphological landscape, indicating rich diversity. Phylomorphospace plots suggest that dynamic evolution underlies the observed spatial patterning. Evolutionary transits through the morphospaces are sometimes long, and occur in a variety of directions. The trajectories seem to represent both evolutionary divergences and convergences, the latter supported by convevol analysis. We suggest that that this pattern of occupancy reflects the various adaptations of different groups of fishes, seemingly paralleling their diverse marine and freshwater ecologies and life histories. Opercle shape evolution within the acanthomorphs, spiny ray-finned fishes, appears to have been especially dynamic.

Host Genotype and Microbiota Contribute Asymmetrically to Transcriptional Variation in the Threespine Stickleback Gut.

Recent studies of interactions between hosts and their resident microbes have revealed important ecological and evolutionary consequences that emerge from these complex interspecies relationships, including diseases that occur when the interactions go awry. Given the preponderance of these interactions, we hypothesized that effects of the microbiota on gene expression in the developing gut-an important aspect of host biology-would be pervasive, and that these effects would be both comparable in magnitude to and contingent on effects of the host genetic background. To evaluate the effects of the microbiota, host genotype, and their interaction on gene expression in the gut of a genetically diverse, gnotobiotic host model, the threespine stickleback (Gasterosteus aculeatus), we compared RNA-seq data among 84 larval fish. Surprisingly, we found that stickleback population and family differences explained substantially more gene expression variation than the presence of microbes. Expression levels of 72 genes, however, were affected by our microbiota treatment. These genes, including many associated with innate immunity, comprise a tractable subset of host genetic factors for precise, systems-level study of host-microbe interactions in the future. Importantly, our data also suggest subtle signatures of a statistical interaction between host genotype and the microbiota on expression patterns of genetic pathways associated with innate immunity, coagulation and complement cascades, focal adhesion, cancer, and peroxisomes. These genotype-by-environment interactions may prove to be important leads to the understanding of host genetic mechanisms commonly at the root of sometimes complex molecular relationships between hosts and their resident microbes.

Innate immune responses to gut microbiota differ between oceanic and freshwater threespine stickleback populations.

Animal hosts must co-exist with beneficial microbes while simultaneously being able to mount rapid, non-specific, innate immune responses to pathogenic microbes. How this balance is achieved is not fully understood, and disruption of this relationship can lead to disease. Excessive inflammatory responses to resident microbes are characteristic of certain gastrointestinal pathologies such as inflammatory bowel disease (IBD). The immune dysregulation of IBD has complex genetic underpinnings that cannot be fully recapitulated with single-gene-knockout models. A deeper understanding of the genetic regulation of innate immune responses to resident microbes requires the ability to measure immune responses in the presence and absence of the microbiota using vertebrate models with complex genetic variation. Here, we describe a new gnotobiotic vertebrate model to explore the natural genetic variation that contributes to differences in innate immune responses to microbiota. Threespine stickleback, Gasterosteus aculeatus, has been used to study the developmental genetics of complex traits during the repeated evolution from ancestral oceanic to derived freshwater forms. We established methods to rear germ-free stickleback larvae and gnotobiotic animals monoassociated with single bacterial isolates. We characterized the innate immune response of these fish to resident gut microbes by quantifying the neutrophil cells in conventionally reared monoassociated or germ-free stickleback from both oceanic and freshwater populations grown in a common intermediate salinity environment. We found that oceanic and freshwater fish in the wild and in the laboratory share many intestinal microbial community members. However, oceanic fish mount a strong immune response to residential microbiota, whereas freshwater fish frequently do not. A strong innate immune response was uniformly observed across oceanic families, but this response varied among families of freshwater fish. The gnotobiotic stickleback model that we have developed therefore provides a platform for future studies mapping the natural genetic basis of the variation in immune response to microbes.

Genetic evidence for monogamy in the dwarf seahorse, Hippocampus zosterae.

Syngnathid fishes (pipefishes, seahorses, and seadragons) exhibit a wide array of mating systems ranging from monogamy with long-term pair bonds to more promiscuous mating systems, such as polyandry and polygynandry. Some seahorses, including the dwarf seahorse Hippocampus zosterae, have been found to be socially monogamous. Although several seahorse species have also been shown to be genetically monogamous, parentage analysis has not yet been applied to the dwarf seahorse. We developed 8 novel microsatellites for the dwarf seahorse to conduct genetic parentage analysis to confirm that this species is indeed monogamous. Using 4 selected loci and a total of 16 pregnant male seahorses, with 8 collected in Florida and 8 sampled in Texas, we genotyped all of the offspring within each male's brood to determine the maternal contributions to each brood. We found a maximum of 4 alleles per locus segregating within each pregnant male's brood, a pattern consistent with each brood having exactly 1 mother and 1 father. These results support previous laboratory-based behavioral studies and indicate that the dwarf seahorse, H. zosterae, is genetically monogamous.

Functional similarity and molecular divergence of a novel reproductive transcriptome in two male-pregnant Syngnathus pipefish species.

Evolutionary studies have revealed that reproductive proteins in animals and plants often evolve more rapidly than the genome-wide average. The causes of this pattern, which may include relaxed purifying selection, sexual selection, sexual conflict, pathogen resistance, reinforcement, or gene duplication, remain elusive. Investigative expansions to additional taxa and reproductive tissues have the potential to shed new light on this unresolved problem. Here, we embark on such an expansion, in a comparison of the brood-pouch transcriptome between two male-pregnant species of the pipefish genus Syngnathus. Male brooding tissues in syngnathid fishes represent a novel, nonurogenital reproductive trait, heretofore mostly uncharacterized from a molecular perspective. We leveraged next-generation sequencing (Roche 454 pyrosequencing) to compare transcript abundance in the male brooding tissues of pregnant with nonpregnant samples from Gulf (S. scovelli) and dusky (S. floridae) pipefish. A core set of protein-coding genes, including multiple members of astacin metalloprotease and c-type lectin gene families, is consistent between species in both the direction and magnitude of expression bias. As predicted, coding DNA sequence analysis of these putative "male pregnancy proteins" suggests rapid evolution relative to nondifferentially expressed genes and reflects signatures of adaptation similar in magnitude to those reported from Drosophila male accessory gland proteins. Although the precise drivers of male pregnancy protein divergence remain unknown, we argue that the male pregnancy transcriptome in syngnathid fishes, a clade diverse with respect to brooding morphology and mating system, represents a unique and promising object of study for understanding the perplexing evolutionary nature of reproductive molecules.

A practical guide to methods of parentage analysis.

The use of molecular techniques for parentage analysis has been a booming science for over a decade. The most important technological breakthrough was the introduction of microsatellite markers to molecular ecology, an advance that was accompanied by a proliferation and refinement of statistical techniques for the analysis of parentage data. Over the last several years, we have seen steady progress in a number of areas related to parentage analysis, and the prospects for successful studies continue to improve. Here, we provide an updated guide for scientists interested in embarking on parentage analysis in natural or artificial populations of organisms, with a particular focus on computer software packages that implement various methods of analysis. Our survey of the literature shows that there are a few established methods that perform extremely well in the analysis of most types of parentage studies. However, particular experimental designs or study systems can benefit from some of the less well-known computer packages available. Overall, we find that parentage analysis is feasible and satisfying in most systems, and we try to provide a simple roadmap to help other scientists navigate the confusing topography of statistical techniques.

A microarray analysis of sex- and gonad-biased gene expression in the zebrafish: evidence for masculinization of the transcriptome.

BACKGROUND: In many taxa, males and females are very distinct phenotypically, and these differences often reflect divergent selective pressures acting on the sexes. Phenotypic sexual dimorphism almost certainly reflects differing patterns of gene expression between the sexes, and microarray studies have documented widespread sexually dimorphic gene expression. Although the evolutionary significance of sexual dimorphism in gene expression remains unresolved, these studies have led to the formulation of a hypothesis that male-driven evolution has resulted in the masculinization of animal transcriptomes. Here we use a microarray assessment of sex- and gonad-biased gene expression to test this hypothesis in zebrafish. RESULTS: By using zebrafish Affymetrix microarrays to compare gene expression patterns in male and female somatic and gonadal tissues, we identified a large number of genes (5899) demonstrating differences in transcript abundance between male and female Danio rerio. Under conservative statistical significance criteria, all sex-biases in gene expression were due to differences between testes and ovaries. Male-enriched genes were more abundant than female-enriched genes, and expression bias for male-enriched genes was greater in magnitude than that for female-enriched genes. We also identified a large number of genes demonstrating elevated transcript abundance in testes and ovaries relative to male body and female body, respectively. CONCLUSION: Overall our results support the hypothesis that male-biased evolutionary pressures have resulted in male-biased patterns of gene expression. Interestingly, our results seem to be at odds with a handful of other microarray-based studies of sex-specific gene expression patterns in zebrafish. However, ours was the only study designed to address this specific hypothesis, and major methodological differences among studies could explain the discrepancies. Regardless, all of these studies agree that transcriptomic sex differences in D. rerio are widespread despite the apparent absence of heterogamety. These differences likely make important contributions to phenotypic sexual dimorphism in adult zebrafish; thus, from an evolutionary standpoint, the precise roles of sex-specific selection and sexual conflict in the evolution of sexually dimorphic gene expression are very important. The results of our study and others like it set the stage for further work aimed at directly addressing this exciting issue in comparative genomics.