Designing eco-evolutionary experiments for restoration projects: Opportunities and constraints revealed during stickleback introductions.

Eco-evolutionary experiments are typically conducted in semi-unnatural controlled settings, such as mesocosms; yet inferences about how evolution and ecology interact in the real world would surely benefit from experiments in natural uncontrolled settings. Opportunities for such experiments are rare but do arise in the context of restoration ecology-where different "types" of a given species can be introduced into different "replicate" locations. Designing such experiments requires wrestling with consequential questions. (Q1) Which specific "types" of a focal species should be introduced to the restoration location? (Q2) How many sources of each type should be used-and should they be mixed together? (Q3) Which specific source populations should be used? (Q4) Which type(s) or population(s) should be introduced into which restoration sites? We recently grappled with these questions when designing an eco-evolutionary experiment with threespine stickleback (Gasterosteus aculeatus) introduced into nine small lakes and ponds on the Kenai Peninsula in Alaska that required restoration. After considering the options at length, we decided to use benthic versus limnetic ecotypes (Q1) to create a mixed group of colonists from four source populations of each ecotype (Q2), where ecotypes were identified based on trophic morphology (Q3), and were then introduced into nine restoration lakes scaled by lake size (Q4). We hope that outlining the alternatives and resulting choices will make the rationales clear for future studies leveraging our experiment, while also proving useful for investigators considering similar experiments in the future.

Inferring the evolution of reproductive isolation in a lineage of fossil threespine stickleback, Gasterosteus doryssus.

Darwin attributed the absence of species transitions in the fossil record to his hypothesis that speciation occurs within isolated habitat patches too geographically restricted to be captured by fossil sequences. Mayr's peripatric speciation model added that such speciation would be rapid, further explaining missing evidence of diversification. Indeed, Eldredge and Gould's original punctuated equilibrium model combined Darwin's conjecture, Mayr's model and 124 years of unsuccessfully sampling the fossil record for transitions. Observing such divergence, however, could illustrate the tempo and mode of evolution during early speciation. Here, we investigate peripatric divergence in a Miocene stickleback fish, Gasterosteus doryssus. This lineage appeared and, over approximately 8000 generations, evolved significant reduction of 12 of 16 traits related to armour, swimming and diet, relative to its ancestral population. This was greater morphological divergence than we observed between reproductively isolated, benthic-limnetic ecotypes of extant Gasterosteus aculeatus. Therefore, we infer that reproductive isolation was evolving. However, local extinction of G. doryssus lineages shows how young, isolated, speciating populations often disappear, supporting Darwin's explanation for missing evidence and revealing a mechanism behind morphological stasis. Extinction may also account for limited sustained divergence within the stickleback species complex and help reconcile speciation rate variation observed across time scales.

Evolution of stickleback spines through independent cis-regulatory changes at HOXDB.

Understanding the mechanisms leading to new traits or additional features in organisms is a fundamental goal of evolutionary biology. We show that HOXDB regulatory changes have been used repeatedly in different fish genera to alter the length and number of the prominent dorsal spines used to classify stickleback species. In Gasterosteus aculeatus (typically 'three-spine sticklebacks'), a variant HOXDB allele is genetically linked to shortening an existing spine and adding an additional spine. In Apeltes quadracus (typically 'four-spine sticklebacks'), a variant HOXDB allele is associated with lengthening a spine and adding an additional spine in natural populations. The variant alleles alter the same non-coding enhancer region in the HOXDB locus but do so by diverse mechanisms, including single-nucleotide polymorphisms, deletions and transposable element insertions. The independent regulatory changes are linked to anterior expansion or contraction of HOXDB expression. We propose that associated changes in spine lengths and numbers are partial identity transformations in a repeating skeletal series that forms major defensive structures in fish. Our findings support the long-standing hypothesis that natural Hox gene variation underlies key patterning changes in wild populations and illustrate how different mutational mechanisms affecting the same region may produce opposite gene expression changes with similar phenotypic outcomes.

Attempting genetic inference from directional asymmetry during convergent hindlimb reduction in squamates.

Loss and reduction in paired appendages are common in vertebrate evolution. How often does such convergent evolution depend on similar developmental and genetic pathways? For example, many populations of the threespine stickleback and ninespine stickleback (Gasterosteidae) have independently evolved pelvic reduction, usually based on independent mutations that caused reduced Pitx1 expression. Reduced Pitx1 expression has also been implicated in pelvic reduction in manatees. Thus, hindlimb reduction stemming from reduced Pitx1 expression has arisen independently in groups that diverged tens to hundreds of millions of years ago, suggesting a potential for repeated use of Pitx1 across vertebrates. Notably, hindlimb reduction based on the reduction in Pitx1 expression produces left-larger directional asymmetry in the vestiges. We used this phenotypic signature as a genetic proxy, testing for hindlimb directional asymmetry in six genera of squamate reptiles that independently evolved hindlimb reduction and for which genetic and developmental tools are not yet developed: Agamodon anguliceps, Bachia intermedia, Chalcides sepsoides, Indotyphlops braminus, Ophisaurus attenuatuas and O. ventralis, and Teius teyou. Significant asymmetry occurred in one taxon, Chalcides sepsoides, whose left-side pelvis and femur vestiges were 18% and 64% larger than right-side vestiges, respectively, suggesting modification in Pitx1 expression in that species. However, there was either right-larger asymmetry or no directional asymmetry in the other five taxa, suggesting multiple developmental genetic pathways to hindlimb reduction in squamates and the vertebrates more generally.

Freshwater Colonization, Adaptation, and Genomic Divergence in Threespine Stickleback.

The Threespine Stickleback is ancestrally a marine fish, but many marine populations breed in fresh water (i.e., are anadromous), facilitating their colonization of isolated freshwater habitats a few years after they form. Repeated adaptation to fresh water during at least 10 My and continuing today has led to Threespine Stickleback becoming a premier system to study rapid adaptation. Anadromous and freshwater stickleback breed in sympatry and may hybridize, resulting in introgression of freshwater-adaptive alleles into anadromous populations, where they are maintained at low frequencies as ancient standing genetic variation. Anadromous stickleback have accumulated hundreds of freshwater-adaptive alleles that are disbursed as few loci per marine individual and provide the basis for adaptation when they colonize fresh water. Recent whole-lake experiments in lakes around Cook Inlet, Alaska have revealed how astonishingly rapid and repeatable this process is, with the frequency of 40% of the identified freshwater-adaptive alleles increasing from negligible (∼1%) in the marine founder to ≥50% within ten generations in fresh water, and freshwater phenotypes evolving accordingly. These high rates of genomic and phenotypic evolution imply very intense directional selection on phenotypes of heterozygotes. Sexual recombination rapidly assembles freshwater-adaptive alleles that originated in different founders into multilocus freshwater haplotypes, and regions important for adaptation to freshwater have suppressed recombination that keeps advantageous alleles linked within large haploblocks. These large haploblocks are also older and appear to have accumulated linked advantageous mutations. The contemporary evolution of Threespine Stickleback has provided broadly applicable insights into the mechanisms that facilitate rapid adaptation.

An Ambidextrous STarfish-Inspired Exploration and Reconnaissance Robot (The ASTER-bot).

As more roboticists are turning to Nature for design inspiration, it is becoming increasingly apparent that multisystem-level investigations of biological processes can frequently lead to unexpected advances in the development of experimental research platforms. Inspired by these efforts, we present here a holistic approach to developing an autonomous starfish-inspired soft robot that embodies a number of key design, fabrication, and actuation principles. These key concepts include integrated and sequentially deployable magnetic tube feet for site-specific and reversible substrate attachment, individually addressable flexible arms, and highly efficient and self-contained fluidic engines. These individual features offer a level of synergistic motion control not previously seen in other starfish-inspired robots. For example, our bistable dome-like tube feet are capable of achieving high adhesion forces to ferrous surfaces and low removal forces. These tube feet are further integrated with a fluidic engine to drive the entire arm while maintaining the ability to accurately control the arm position with a 270° range of motion. Furthermore, the arm and fluidic engine are modular, allowing each of the five arms to be replaced in seconds or enabling the exploration of a variety of limb geometries. Through the incorporation of these different design elements, the ASTER-bot (named for its star-like body plan) is capable of locomotion on ferrous surfaces, above and below water, and on nonplanar surfaces. This article further describes the design, fabrication, and integration strategies and characterizes the energetic and locomotory performance of this pentaradial robotic prototype.

Evolution of static allometry and constraint on evolutionary allometry in a fossil stickleback.

Allometric scaling describes the relationship of trait size to body size within and among taxa. The slope of the population-level regression of trait size against body size (i.e. static allometry) is typically invariant among closely related populations and species. Such invariance is commonly interpreted to reflect a combination of developmental and selective constraints that delimit a phenotypic space into which evolution could proceed most easily. Thus, understanding how allometric relationships do eventually evolve is important to understanding phenotypic diversification. In a lineage of fossil Threespine Stickleback (Gasterosteus doryssus), we investigated the evolvability of static allometric slopes for nine traits (five armour and four non-armour) that evolved significant trait differences across 10 samples over 8500 years. The armour traits showed weak static allometric relationships and a mismatch between those slopes and observed evolution. This suggests that observed evolution in these traits was not constrained by relationships with body size, perhaps because prior, repeated adaptation to freshwater habitats by Threespine Stickleback had generated strong selection to break constraint. In contrast, for non-armour traits, we found stronger allometric relationships. Those allometric slopes did evolve on short time scales. However, those changes were small and fluctuating and the slopes remained strong predictors of the evolutionary trajectory of trait means over time (i.e. evolutionary allometry), supporting the hypothesis of allometry as constraint.

Cross-continental experimental infections reveal distinct defence mechanisms in populations of the three-spined stickleback Gasterosteus aculeatus.

Epidemiological traits of host-parasite associations depend on the effects of the host, the parasite and their interaction. Parasites evolve mechanisms to infect and exploit their hosts, whereas hosts evolve mechanisms to prevent infection and limit detrimental effects. The reasons why and how these traits differ across populations still remain unclear. Using experimental cross-infection of three-spined stickleback Gasterosteus aculeatus and their species-specific cestode parasites Schistocephalus solidus from Alaskan and European populations, we disentangled host, parasite and interaction effects on epidemiological traits at different geographical scales. We hypothesized that host and parasite main effects would dominate both within and across continents, although interaction effects would show geographical variation of natural selection within and across continents. We found that mechanisms preventing infection (qualitative resistance) occurred only in a combination of hosts and parasites from different continents, while mechanisms limiting parasite burden (quantitative resistance) and reducing detrimental effects of infection (tolerance) were host-population specific. We conclude that evolution favours distinct defence mechanisms on different geographical scales and that it is important to distinguish concepts of qualitative resistance, quantitative resistance and tolerance in studies of macroparasite infections.

Longer or shorter spines: Reciprocal trait evolution in stickleback via triallelic regulatory changes in Stanniocalcin2a.

Vertebrates have repeatedly modified skeletal structures to adapt to their environments. The threespine stickleback is an excellent system for studying skeletal modifications, as different wild populations have either increased or decreased the lengths of their prominent dorsal and pelvic spines in different freshwater environments. Here we identify a regulatory locus that has a major morphological effect on the length of stickleback dorsal and pelvic spines, which we term Maser (major spine enhancer). Maser maps in a closely linked supergene complex that controls multiple armor, feeding, and behavioral traits on chromosome IV. Natural alleles in Maser are differentiated between marine and freshwater sticklebacks; however, alleles found among freshwater populations are also differentiated, with distinct alleles found in short- and long-spined freshwater populations. The distinct freshwater alleles either increase or decrease expression of the bone growth inhibitor gene Stanniocalcin2a in developing spines, providing a simple genetic mechanism for either increasing or decreasing spine lengths in natural populations. Genomic surveys suggest many recurrently differentiated loci in sticklebacks are similarly specialized into three or more distinct alleles, providing multiple ancient standing variants in particular genes that may contribute to a range of phenotypes in different environments.

Predicting future from past: The genomic basis of recurrent and rapid stickleback evolution.

Similar forms often evolve repeatedly in nature, raising long-standing questions about the underlying mechanisms. Here, we use repeated evolution in stickleback to identify a large set of genomic loci that change recurrently during colonization of freshwater habitats by marine fish. The same loci used repeatedly in extant populations also show rapid allele frequency changes when new freshwater populations are experimentally established from marine ancestors. Marked genotypic and phenotypic changes arise within 5 years, facilitated by standing genetic variation and linkage between adaptive regions. Both the speed and location of changes can be predicted using empirical observations of recurrence in natural populations or fundamental genomic features like allelic age, recombination rates, density of divergent loci, and overlap with mapped traits. A composite model trained on these stickleback features can also predict the location of key evolutionary loci in Darwin's finches, suggesting that similar features are important for evolution across diverse taxa.

Threespine Stickleback: A Model System For Evolutionary Genomics.

The repeated adaptation of oceanic threespine sticklebacks to fresh water has made it a premier organism to study parallel evolution. These small fish have multiple distinct ecotypes that display a wide range of diverse phenotypic traits. Ecotypes are easily crossed in the laboratory, and families are large and develop quickly enough for quantitative trait locus analyses, positioning the threespine stickleback as a versatile model organism to address a wide range of biological questions. Extensive genomic resources, including linkage maps, a high-quality reference genome, and developmental genetics tools have led to insights into the genomic basis of adaptation and the identification of genomic changes controlling traits in vertebrates. Recently, threespine sticklebacks have been used as a model system to identify the genomic basis of highly complex traits, such as behavior and host-microbiome and host-parasite interactions. We review the latest findings and new avenues of research that have led the threespine stickleback to be considered a supermodel of evolutionary genomics.

Fitness maps to a large-effect locus in introduced stickleback populations.

Mutations of small effect underlie most adaptation to new environments, but beneficial variants with large fitness effects are expected to contribute under certain conditions. Genes and genomic regions having large effects on phenotypic differences between populations are known from numerous taxa, but fitness effect sizes have rarely been estimated. We mapped fitness over a generation in an F2 intercross between a marine and a lake stickleback population introduced to a freshwater pond. A quantitative trait locus map of the number of surviving offspring per F2 female detected a single, large-effect locus near Ectodysplasin (Eda), a gene having an ancient freshwater allele causing reduced bony armor and other changes. F2 females homozygous for the freshwater allele had twice the number of surviving offspring as homozygotes for the marine allele, producing a large selection coefficient, s = 0.50 ± 0.09 SE. Correspondingly, the frequency of the freshwater allele increased from 0.50 in F2 mothers to 0.58 in surviving offspring. We compare these results to allele frequency changes at the Eda gene in an Alaskan lake population colonized by marine stickleback in the 1980s. The frequency of the freshwater Eda allele rose steadily over multiple generations and reached 95% within 20 y, yielding a similar estimate of selection, s = 0.49 ± 0.05, but a different degree of dominance. These findings are consistent with other studies suggesting strong selection on this gene (and/or linked genes) in fresh water. Selection on ancient genetic variants carried by colonizing ancestors is likely to increase the prevalence of large-effect fitness variants in adaptive evolution.

Intercontinental genomic parallelism in multiple three-spined stickleback adaptive radiations.

Parallelism, the evolution of similar traits in populations diversifying in similar conditions, provides strong evidence of adaptation by natural selection. Many studies of parallelism focus on comparisons of different ecotypes or contrasting environments, defined a priori, which could upwardly bias the apparent prevalence of parallelism. Here, we estimated genomic parallelism associated with components of environmental and phenotypic variation at an intercontinental scale across four freshwater adaptive radiations (Alaska, British Columbia, Iceland and Scotland) of the three-spined stickleback (Gasterosteus aculeatus). We combined large-scale biological sampling and phenotyping with restriction site associated DNA sequencing (RAD-Seq) data from 73 freshwater lake populations and four marine ones (1,380 fish) to associate genome-wide allele frequencies with continuous distributions of environmental and phenotypic variation. Our three main findings demonstrate that (1) quantitative variation in phenotypes and environments can predict genomic parallelism; (2) genomic parallelism at the early stages of adaptive radiations, even at large geographic scales, is founded on standing variation; and (3) similar environments are a better predictor of genome-wide parallelism than similar phenotypes. Overall, this study validates the importance and predictive power of major phenotypic and environmental factors likely to influence the emergence of common patterns of genomic divergence, providing a clearer picture than analyses of dichotomous phenotypes and environments.

Inferred genetic architecture underlying evolution in a fossil stickleback lineage.

Inferring the genetic architecture of evolution in the fossil record is difficult because genetic crosses are impossible, the acquisition of DNA is usually impossible and phenotype-genotype maps are rarely obvious. However, such inference is valuable because it reveals the genetic basis of microevolutionary change across many more generations than is possible in studies of extant taxa, thereby integrating microevolutionary process and macroevolutionary pattern. Here, we infer the genetic basis of pelvic skeleton reduction in Gasterosteus doryssus, a Miocene stickleback fish from a finely resolved stratigraphic sequence that spans nearly 17,000 years. Reduction in pelvic score, a categorical measure of pelvic structure, resulted primarily from reciprocal frequency changes of two discrete phenotypic classes. Pelvic vestiges also showed left-side larger asymmetry. These patterns implicate Pitx1, a large-effect gene whose deletion generates left-side larger asymmetry of pelvic vestiges in extant, closely related Gasterosteus aculeatus. In contrast, reductions in the length of the pelvic girdle and pelvic spines resulted from directional shifts of unimodal, continuous trait distributions, suggesting an additional suite of genes with minor, additive pelvic effects, again like G. aculeatus. Similar genetic architectures explain shared but phyletically independent patterns across 10 million years of stickleback evolution.

Racial and Ethnic Disparities in Access to Emerging and Frontline Therapies in Common Dermatological Conditions: A Cross-Sectional Study.

OBJECTIVE: This study investigates possible race- and ethnicity-related disparities in the treatment of acne, atopic dermatitis, and psoriasis of newly approved treatments as well as existing therapies. METHODS: Aggregate level data was collected from patient medical records between 2013 and 2018. The odds ratio of patients who had been prescribed treatments for acne, atopic dermatitis, and psoriasis per racial and ethnic group were calculated using a 95% confidence interval after applying Bonferroni correction to account for multiple comparisons. RESULTS: Black patients with acne had statistically significant (p < 0.001) lower odds of receiving isotretinoin 0.26 [0.22-0.30], adapalene 0.72 [0.67-0.78], tazarotene 0.74 [0.64-0.86], and dapsone 0.39 [0.34-0.45] than white patients. The exceptions were tretinoin 1.28 [1.23-1.34] and benzoyl peroxide 3.00 [2.79-3.23] (p < 0.001). Hispanic patients with acne had statistically lower odds of receiving tretinoin 0.86 [0.79-0.95] (p < 0.001) compared to non-Hispanics. Black patients with atopic dermatitis were less likely to receive desonide 0.90 [0.78-0.93], tacrolimus 0.75 [0.68-0.83], pimecrolimus 0.71 [0.60-0.84], crisaborole 0.39 [0.26-0.57], dupilumab 0.42 [0.27-0.65]. The exception was hydrocortisone 2.50 [2.34-2.65] (p < 0.001). There was no statistically significant difference for Hispanics compared to non-Hispanics. Black patients with psoriasis had a lower likelihood of receiving cyclosporine 0.54 [0.35-0.83] and etanercept 0.65 [0.49-0.87]. CONCLUSIONS: This study demonstrates a racial and ethnic disparity in accessing newly approved and standard of care medical therapies for acne, atopic dermatitis, and psoriasis within the past three years.

Climate-driven habitat change causes evolution in Threespine Stickleback.

Climate change can shape evolution directly by altering abiotic conditions or indirectly by modifying habitats, yet few studies have investigated the effects of climate-driven habitat change on contemporary evolution. We resampled populations of Threespine Stickleback (Gasterosteus aculeatus) along a latitudinal gradient in California bar-built estuaries to examine their evolution in response to changing climate and habitat. We took advantage of the strong association between stickleback lateral plate phenotypes and Ectodysplasin A (Eda) genotypes to infer changes in allele frequencies over time. Our results show that over time the frequency of low-plated alleles has generally increased and heterozygosity has decreased. Latitudinal patterns in stickleback plate phenotypes suggest that evolution at Eda is a response to climate-driven habitat transformation rather than a direct consequence of climate. As climate change has reduced precipitation and increased temperature and drought, bar-built estuaries have transitioned from lotic (flowing-water) to lentic (still-water) habitats, where the low-plated allele is favoured. The low-plated allele has achieved fixation at the driest, hottest southernmost sites, a trend that is progressing northward with climate change. Climate-driven habitat change is therefore causing a reduction in genetic variation that may hinder future adaptation for populations facing multiple threats.

DNA fragility in the parallel evolution of pelvic reduction in stickleback fish.

Evolution generates a remarkable breadth of living forms, but many traits evolve repeatedly, by mechanisms that are still poorly understood. A classic example of repeated evolution is the loss of pelvic hindfins in stickleback fish (Gasterosteus aculeatus). Repeated pelvic loss maps to recurrent deletions of a pelvic enhancer of the Pitx1 gene. Here, we identify molecular features contributing to these recurrent deletions. Pitx1 enhancer sequences form alternative DNA structures in vitro and increase double-strand breaks and deletions in vivo. Enhancer mutability depends on DNA replication direction and is caused by TG-dinucleotide repeats. Modeling shows that elevated mutation rates can influence evolution under demographic conditions relevant for sticklebacks and humans. DNA fragility may thus help explain why the same loci are often used repeatedly during parallel adaptive evolution.

Shifting the Paradigm of Testosterone Replacement Therapy in Prostate Cancer.

Historically, testosterone and prostate cancer have been demonstrated to have a positive association leading providers to forgo testosterone replacement therapy (TRT) in men with concurrent histories of hypogonadism and prostate cancer. This paradigm has been gradually shifting with our evolving understanding of the relationship between testosterone and prostate cancer and the gaining popularity of the saturation model. Newer data suggests improved quality of life for men with hypogonadism after TRT leading to a more tempered view of the effects of this treatment and its risk in prostate cancer. As more reports emerge of TRT in men who have either undergone definitive treatment for prostate cancer or are on active surveillance, some providers see a role for TRT in these patients despite non-consensus in clinical guidelines. It is critical that we examine evidence currently available, while we await more rigorous data to emerge.

Management of advanced adenocarcinoma in Indiana Pouch urinary diversion.

Adenocarcinoma is a rare finding following urinary diversion with gastrointestinal segments. This report describes an 80-year-old woman with a history of bladder cancer who subsequently developed a pT4 adenocarcinoma 8 years following her radical cystectomy and Indiana Pouch continent urinary diversion. An en bloc resection of the pouch and affected small bowel was performed and the patient underwent conversion to an ileal conduit diversion. We use this case to highlight a mechanism for possible pathogenesis and the management of adenocarcinoma in urinary diversions including the need for regular surveillance and the surgical approach.