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.

Miocene phytolith and diatom dataset from 10.3Myo diatomite formation, Fernley, Nevada, USA.

Phytoliths are opal silica particles formed within plant tissues. Diatoms are aquatic, single-celled photosynthetic algae with silica skeletons. Phytolith and diatom morphotypes vary depending on local environmental and climatic conditions and because their silicate structures preserve well, the study of phytolith and diatom morphotypes can be used to better understand paleoclimatic and paleoenvironmental dynamics and changes. This article presents original data from an 820cm-deep stratigraphy excavated at the Hazen diatomite deposits, a high-elevation desert paleolake in the Fernley District, Northern Nevada, USA. The site has been studied for an assemblage of fossilized threespine stickleback, Gasterosteus doryssus, that reveal adaptive evolution. For this study, a total of 157 samples were extracted at 20 cm intervals covering approximately 24,500 years. After extraction, the samples were mounted on slides and viewed under 400-1000x light microscopy, enabling classification of 14 phytolith and 45 diatom morphotypes. Our data support paleoenvironmental reconstructions of the Hazen Miocene paleolake.

Convergence or redundancy: alternative views about the evolutionary genomics of character displacement.

An evolutionary debate contrasts the importance of genetic convergence versus genetic redundancy. In genetic convergence, the same adaptive trait evolves because of similar genetic changes. In genetic redundancy, the adaptive trait evolves using different genetic combinations, and populations might not share the same genetic changes. Here we address this debate by examining single nucleotide polymorphisms (SNPs) associated with the rapid evolution of character displacement in Anolis carolinensis populations inhabiting replicate islands with and without a competitor species (1Spp and 2Spp islands, respectively). We identify 215-outliers SNPs that have improbably large FST values, low nucleotide variation, greater linkage than expected and that are enriched for genes underlying animal movement. The pattern of SNP divergence between 1Spp and 2Spp populations supports both genetic convergence and genetic redundancy for character displacement. In support of genetic convergence: all 215-outliers SNPs are shared among at least three of the five 2Spp island populations, and 23% of outlier SNPS are shared among all five 2Spp island populations. In contrast, in support of genetic redundancy: many outlier SNPs only have meaningful allele frequency differences between 1Spp and 2Spp islands on a few 2Spp islands. That is, on at least one of the 2Spp islands, 77% of outlier SNPs have allele frequencies more similar to those on 1Spp islands than to those on 2Spp islands. Focusing on genetic convergence is scientifically rigorous because it relies on replication. Yet, this focus distracts from the possibility that there are multiple, redundant genetic solutions that enhance the rate and stability of adaptive change.

Repeated genetic divergence plays a minor role in repeated phenotypic divergence of lake-stream stickleback.

Recent studies have shown that the repeated evolution of similar phenotypes in response to similar ecological conditions (here "parallel evolution") often occurs through mutations in the same genes. However, many previous studies have focused on known candidate genes in a limited number of systems. Thus, the question of how often parallel phenotypic evolution is due to parallel genetic changes remains open. Here, we used quantitative trait locus (QTL) mapping in F2 intercrosses between lake and stream threespine stickleback (Gasterosteus aculeatus) from four independent watersheds on Vancouver Island, Canada to determine whether the same QTL underlie divergence in the same phenotypes across, between, and within watersheds. We find few parallel QTL, even in independent crosses from the same watershed or for phenotypes that have diverged in parallel. These findings suggest that different mutations can lead to similar phenotypes. The low genetic repeatability observed in these lake-stream systems contrasts with the higher genetic repeatability observed in other stickleback systems. We speculate that differences in evolutionary history, gene flow, and/or the strength and direction of selection might explain these differences in genetic parallelism and emphasize that more work is needed to move beyond documenting genetic parallelism to identifying the underlying causes.

Species divergence under competition and shared predation.

Species competing for resources also commonly share predators. While competition often drives divergence between species, the effects of shared predation are less understood. Theoretically, competing prey species could either diverge or evolve in the same direction under shared predation depending on the strength and symmetry of their interactions. We took an empirical approach to this question, comparing antipredator and trophic phenotypes between sympatric and allopatric populations of threespine stickleback and prickly sculpin fish that all live in the presence of a trout predator. We found divergence in antipredator traits between the species: in sympatry, antipredator adaptations were relatively increased in stickleback but decreased in sculpin. Shifts in feeding morphology, diet and habitat use were also divergent but driven primarily by stickleback evolution. Our results suggest that asymmetric ecological character displacement indirectly made stickleback more and sculpin less vulnerable to shared predation, driving divergence of antipredator traits between sympatric species.

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.

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.

(Non)Parallel developmental mechanisms in vertebrate appendage reduction and loss.

Appendages have been reduced or lost hundreds of times during vertebrate evolution. This phenotypic convergence may be underlain by shared or different molecular mechanisms in distantly related vertebrate clades. To investigate, we reviewed the developmental and evolutionary literature of appendage reduction and loss in more than a dozen vertebrate genera from fish to mammals. We found that appendage reduction and loss was nearly always driven by modified gene expression as opposed to changes in coding sequences. Moreover, expression of the same genes was repeatedly modified across vertebrate taxa. However, the specific mechanisms by which expression was modified were rarely shared. The multiple routes to appendage reduction and loss suggest that adaptive loss of function phenotypes might arise routinely through changes in expression of key developmental genes.

Male and female Anolis carolinensis maintain their dimorphism despite the presence of novel interspecific competition.

Natural selection favors sexual dimorphism that reduces resource competition between the sexes of the same species. However, niche partitioning among interspecific competitors should counter such divergence, as partitioning the niche results in smaller total niche widths for each individual species, leaving less room for the sexes to diverge. A straightforward (and long-standing) hypothesis emerges: species in competitor-rich ecological communities should show less sexual dimorphism than species in competitor-poor ecological communities. Here, we test this prediction using a well-documented natural experiment generated by the recent arrival of Anolis sagrei to a set of small islands in Mosquito Lagoon, Florida, containing Anolis carolinensis. Despite known interspecific habitat partitioning and rapid evolution in habitat-use traits by A. carolinensis in this system, sexual dimorphism between male and female A. carolinensis was not reduced as predicted on two-species islands relative to islands with only A. carolinensis. This is consistent with a small but growing body of empirical tests of the dimorphism-richness hypothesis that have been ambiguous in their support at best. A rethinking of the validity of this intuitive hypothesis is needed.

Copy number variation of a fatty acid desaturase gene Fads2 associated with ecological divergence in freshwater stickleback populations.

Fitness of aquatic animals can be limited by the scarcity of nutrients such as long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (DHA). DHA availability from diet varies among aquatic habitats, imposing different selective pressures on resident animals to optimize DHA acquisition and synthesis. For example, DHA is generally poor in freshwater ecosystems compared to marine ecosystems. Our previous work revealed that, relative to marine fishes, several freshwater fishes evolved higher copy numbers of the fatty acid desaturase2 (Fads2) gene, which encodes essential enzymes for DHA biosynthesis, likely compensating for the limited availability of DHA in freshwater. Here, we demonstrate that Fads2 copy number also varies between freshwater sticklebacks inhabiting lakes and streams with stream fish having higher Fads2 copy number. Additionally, populations with benthic-like morphology possessed higher Fads2 copy number than those with planktivore-like morphology. This may be because benthic-like fish mainly feed on DHA-deficient prey such as macroinvertebrates whereas planktivore-like fish forage more regularly on DHA-rich prey, like copepods. Our results suggest that Fads2 copy number variation arises from ecological divergence not only between organisms exploiting marine and freshwater habitats but also between freshwater organisms exploiting divergent resources.

Adding the third dimension to studies of parallel evolution of morphology and function: An exploration based on parapatric lake-stream stickleback.

Recent methodological advances have led to a rapid expansion of evolutionary studies employing three-dimensional landmark-based geometric morphometrics (GM). GM methods generally enable researchers to capture and compare complex shape phenotypes, and to quantify their relationship to environmental gradients. However, some recent studies have shown that the common, inexpensive, and relatively rapid two-dimensional GM methods can distort important information and produce misleading results because they cannot capture variation in the depth (Z) dimension. We use micro-CT scanned threespine stickleback (Gasterosteus aculeatus Linnaeus, 1758) from six parapatric lake-stream populations on Vancouver Island, British Columbia, to test whether the loss of the depth dimension in 2D GM studies results in misleading interpretations of parallel evolution. Using joint locations described with 2D or 3D landmarks, we compare results from separate 2D and 3D shape spaces, from a combined 2D-3D shape space, and from estimates of biomechanical function. We show that, although shape is distorted enough in 2D projections to strongly influence the interpretation of morphological parallelism, estimates of biomechanical function are relatively robust to the loss of the Z dimension.

Scale-dependent effects of host patch traits on species composition in a stickleback parasite metacommunity.

A core goal of ecology is to understand the abiotic and biotic variables that regulate species distributions and community composition. A major obstacle is that the rules governing species distributions can change with spatial scale. Here, we illustrate this point using data from a spatially nested metacommunity of parasites infecting a metapopulation of threespine stickleback fish from 34 lakes on Vancouver Island, British Columbia. Like most parasite metacommunities, the composition of stickleback parasites differs among host individuals within each host population, and differs between host populations. The distribution of each parasite taxon depends, to varying degrees, on individual host traits (e.g., mass, diet) and on host-population characteristics (e.g., lake size, mean host mass, mean diet). However, in most cases in this data set, a given parasite was regulated by different factors at the host-individual and host-population scales, leading to scale-dependent patterns of parasite-species co-occurrence.

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.

Character displacement in the midst of background evolution in island populations of Anolis lizards: A spatiotemporal perspective.

Negative interactions between species can generate divergent selection that causes character displacement. However, other processes cause similar divergence. We use spatial and temporal replication across island populations of Anolis lizards to assess the importance of negative interactions in driving trait shifts. Previous work showed that the establishment of Anolis sagrei on islands drove resident Anolis carolinensis to perch higher and evolve larger toepads. To further test the interaction's causality and predictability, we resurveyed a subset of islands nine years later. Anolis sagrei had established on one island between surveys. We found that A. carolinensis on this island now perch higher and have larger toepads. However, toepad morphology change on this island was not distinct from shifts on six other islands whose Anolis community composition had not changed. Thus, the presence of A. sagrei only partly explains A. carolinensis trait variation across space and time. We also found that A. carolinensis on islands with previously established A. sagrei now perch higher than a decade ago, and that current A. carolinensis perch height is correlated with A. sagrei density. Our results suggest that character displacement likely interacts with other evolutionary processes in this system, and that temporal data are key to detecting such interactions.

Microhabitat contributes to microgeographic divergence in threespine stickleback.

Since the New Synthesis, most migration-selection balance theory has predicted that there should be negligible differentiation over small spatial scales (relative to dispersal), because gene flow should erode any effect of divergent selection. Nevertheless, there are classic examples of microgeographic divergence, which theory suggests can arise under specific conditions: exceptionally strong selection, phenotypic plasticity in philopatric individuals, or nonrandom dispersal. Here, we present evidence of microgeographic morphological variation within lake and stream populations of threespine stickleback (Gasterosteus aculeatus). It seems reasonable to assume that a given lake or stream population of fish is well-mixed. However, we found this assumption to be untenable. We examined trap-to-trap variation in 34 morphological traits measured on stickleback from 16 lakes and 16 streams. Most traits varied appreciably among traps within populations. Both between-trap distance and microhabitat characteristics such as depth and substrate explained some of the within-population morphological variance. Microhabitat was also associated with genotype at particular loci but there was no genetic isolation by distance, implying that heritable habitat preferences may contribute to microgeographic variation. Our study adds to growing evidence that microgeographic divergence can occur across small spatial scales within individuals' daily dispersal neighborhood where gene flow is expected to be strong.

Repeatability of Adaptive Radiation Depends on Spatial Scale: Regional Versus Global Replicates of Stickleback in Lake Versus Stream Habitats.

The repeatability of adaptive radiation is expected to be scale-dependent, with determinism decreasing as greater spatial separation among "replicates" leads to their increased genetic and ecological independence. Threespine stickleback (Gasterosteus aculeatus) provide an opportunity to test whether this expectation holds for the early stages of adaptive radiation-their diversification in freshwater ecosystems has been replicated many times. To better understand the repeatability of that adaptive radiation, we examined the influence of geographic scale on levels of parallel evolution by quantifying phenotypic and genetic divergence between lake and stream stickleback pairs sampled at regional (Vancouver Island) and global (North America and Europe) scales. We measured phenotypes known to show lake-stream divergence and used reduced representation genome-wide sequencing to estimate genetic divergence. We assessed the scale dependence of parallel evolution by comparing effect sizes from multivariate models and also the direction and magnitude of lake-stream divergence vectors. At the phenotypic level, parallelism was greater at the regional than the global scale. At the genetic level, putative selected loci showed greater lake-stream parallelism at the regional than the global scale. Generally, the level of parallel evolution was low at both scales, except for some key univariate traits. Divergence vectors were often orthogonal, highlighting possible ecological and genetic constraints on parallel evolution at both scales. Overall, our results confirm that the repeatability of adaptive radiation decreases at increasing spatial scales. We suggest that greater environmental heterogeneity at larger scales imposes different selection regimes, thus generating lower repeatability of adaptive radiation at larger spatial scales.

Ecological factors and morphological traits are associated with repeated genomic differentiation between lake and stream stickleback.

The repeated evolution of similar phenotypes in independent populations (i.e. parallel or convergent evolution) provides an opportunity to identify genetic and ecological factors that influence the process of adaptation. Threespine stickleback fish ( Gasterosteus aculeatus) are an excellent model for such studies, as they have repeatedly adapted to divergent habitats across the Northern hemisphere. Here, we use genomic, ecological and morphological data from 16 independent pairs of stickleback populations adapted to divergent lake and stream habitats. We combine a population genomic approach to identify regions of the genome that are likely under selection in these divergent habitats with an association mapping approach to identify regions of the genome that underlie variation in ecological factors and morphological traits. Over 37% of genomic windows are repeatedly differentiated across lake-stream pairs. Similarly, many genomic windows are associated with variation in abiotic factors, diet items and morphological phenotypes. Both the highly differentiated windows and candidate trait windows are non-randomly distributed across the genome and show some overlap. However, the overlap is not significant on a genome-wide scale. Together, our data suggest that adaptation to divergent food resources and predation regimes are drivers of differentiation in lake-stream stickleback, but that additional ecological factors are also important. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.

Divergent Uses of "Parallel Evolution" during the History of The American Naturalist.

The mechanistic link between natural selection and parallel evolution is well established. Natural selection is the only known deterministic process that can regularly overcome chance and historical contingency to generate the evolution of similar characteristics in independent populations inhabiting similar environments. However, the ready inference of natural selection from parallel evolution has been established only relatively recently. Here, I review the use of "parallel evolution" in the first 125 years of The American Naturalist and show that there were other well-accepted definitions of the term through the history of the field. I discuss the legacy of those alternative ideas and how they helped to shape evolution and ecology as we know them today and finish by discussing a geometric use for "parallel" that may reduce terminological confusion.

Many-to-one form-to-function mapping weakens parallel morphological evolution.

Evolutionary ecologists aim to explain and predict evolutionary change under different selective regimes. Theory suggests that such evolutionary prediction should be more difficult for biomechanical systems in which different trait combinations generate the same functional output: "many-to-one mapping." Many-to-one mapping of phenotype to function enables multiple morphological solutions to meet the same adaptive challenges. Therefore, many-to-one mapping should undermine parallel morphological evolution, and hence evolutionary predictability, even when selection pressures are shared among populations. Studying 16 replicate pairs of lake- and stream-adapted threespine stickleback (Gasterosteus aculeatus), we quantified three parts of the teleost feeding apparatus and used biomechanical models to calculate their expected functional outputs. The three feeding structures differed in their form-to-function relationship from one-to-one (lower jaw lever ratio) to increasingly many-to-one (buccal suction index, opercular 4-bar linkage). We tested for (1) weaker linear correlations between phenotype and calculated function, and (2) less parallel evolution across lake-stream pairs, in the many-to-one systems relative to the one-to-one system. We confirm both predictions, thus supporting the theoretical expectation that increasing many-to-one mapping undermines parallel evolution. Therefore, sole consideration of morphological variation within and among populations might not serve as a proxy for functional variation when multiple adaptive trait combinations exist.

Contrasting effects of environment and genetics generate a continuum of parallel evolution.

Parallel evolution of similar traits by independent populations in similar environments is considered strong evidence for adaptation by natural selection. Often, however, replicate populations in similar environments do not all evolve in the same way, thus deviating from any single, predominant outcome of evolution. This variation might arise from non-adaptive, population-specific effects of genetic drift, gene flow or limited genetic variation. Alternatively, these deviations from parallel evolution might also reflect predictable adaptation to cryptic environmental heterogeneity within discrete habitat categories. Here, we show that deviations from parallel evolution are the consequence of environmental variation within habitats combined with variation in gene flow. Threespine stickleback (Gasterosteus aculeatus) in adjoining lake and stream habitats (a lake-stream 'pair') diverge phenotypically, yet the direction and magnitude of this divergence is not always fully parallel among 16 replicate pairs. We found that the multivariate direction of lake-stream morphological divergence was less parallel between pairs whose environmental differences were less parallel. Thus, environmental heterogeneity among lake-stream pairs contributes to deviations from parallel evolution. Additionally, likely genomic targets of selection were more parallel between environmentally more similar pairs. In contrast, variation in the magnitude of lake-stream divergence (independent of direction) was better explained by differences in lake-stream gene flow; pairs with greater lake-stream gene flow were less morphologically diverged. Thus, both adaptive and non-adaptive processes work concurrently to generate a continuum of parallel evolution across lake-stream stickleback population pairs.