Predicting evolution with generalized models of divergent selection: a case study with poeciliid fish

Over the past century and half since the process of natural selection was first described, one enduring question has captivated many, "how predictable is evolution?" Because natural selection comprises deterministic components, the course of evolution may exhibit some level of predictability across organismal groups. Here, I provide an early appraisal of the utility of one particular approach to understanding the predictability of evolution: generalized models of divergent selection (GMDS). The GMDS approach is meant to provide a unifying framework for the science of evolutionary prediction, offering a means of better understanding the causes and consequences of phenotypic and genetic evolution. I describe and test a GMDS centered on the evolution of body shape, size of the gonopodium (sperm-transfer organ), steady-swimming abilities, fast-start swimming performance, and reproductive isolation between populations in Gambusia fishes (Family Poeciliidae). The GMDS produced some accurate evolutionary predictions in Gambusia, identifying variation in intensity of predation by piscivorous fish as a major factor driving repeatable and predictable phenotypic divergence, and apparently playing a key role in promoting ecological speciation. Moreover, the model's applicability seems quite general, as patterns of differentiation in body shape between predator regimes in many disparate fishes match the model's predictions. The fact that such a simple model could yield accurate evolutionary predictions in distantly related fishes inhabiting different geographic regions and types of habitat, and experiencing different predator species, suggests that the model pinpointed a causal factor underlying major, shared patterns of diversification. The GMDS approach appears to represent a promising method of addressing the predictability of evolution and identifying environmental factors responsible for driving major patterns of replicated evolution.     

Hydrologic regimes as potential drivers of morphologic divergence in fish

Fishes often exhibit phenotypic divergence across gradients of abiotic and biotic selective pressures. In streams, many of the known selective pressures driving phenotypic differentiation are largely influenced by hydrologic regimes. Because flow regimes drive so many attributes of lotic systems, we hypothesized fish exhibit phenotypic divergence among streams with different flow regimes. We used a comparative field study to investigate the morphological divergence of Campostoma anomalom (central stonerollers) among streams characterized by highly variable, intermittent flow regimes and streams characterized by relatively stable, groundwater flow regimes. We also conducted a mesocosm experiment to compare the plastic effects of one component of flow regimes, water velocity, on morphology of fish from different flow regimes. We observed differences in shape between flow regimes likely driven by differences in allometric growth patterns. Although we observed differences in morphology across flow regimes in the field, C. anomalum did not exhibit morphologic plasticity in response to water velocity alone. This study contributes to the understanding of how complex environmental factors drive phenotypic divergence and may provide insight into the evolutionary consequences of disrupting natural hydrologic patterns, which are increasingly threatened by climate change and anthropogenic alterations.     

Wave-induced abiotic stress shapes phenotypic diversity in a coral reef fish across a geographical cline

While morphological variation across geographical clines has been well documented, it is often unclear whether such changes enhance individual performance to local environments. We examined whether the damselfish Acanthochromis polyacanthus display functional changes in swimming phenotype across a 40-km cline in wave-driven water motion on the Great Barrier Reef, Australia. A. polyacanthus populations displayed strong intraspecific variation in swimming morphology and performance that matched local levels of water motion: individuals on reefs subject to high water motion displayed higher aspect-ratio fins and faster swimming speeds than conspecifics on sheltered reefs. Remarkably, intraspecific variation within A. polyacanthus spanned over half the diversity seen among closely related damselfish species from the same region. We find that local selection driven by wave-induced abiotic stress is an overarching ecological mechanism shaping the inter- and intraspecific locomotor diversity of coral reef fishes.     

Localised intraspecific variation in the swimming phenotype of a coral reef fish across different wave exposures

Wave-driven water flow is a major force structuring marine communities. Species distributions are partly determined by the ability to cope with variation in water flow, such as differences in the assemblage of fish species found in a given water flow environment being linked to swimming ability (based on fin shape and mode of locomotion). It remains unclear, however, whether similar assembly rules apply within a species. Here we show phenotypic variation among sites in traits functionally linked to swimming ability in the damselfish Acanthochromis polyacanthus. These sites differ in wave energy and the observed patterns of phenotypic differences within A. polyacanthus closely mirrored those seen at the interspecific level. Fish from high-exposure sites had more tapered fins and higher maximum metabolic rates than conspecifics from sheltered sites. This translates to a 36 % larger aerobic scope and 33 % faster critical swimming speed for fish from exposed sites. Our results suggest that functional relationships among swimming phenotypes and water flow not only structure species assemblages, but can also shape patterns of phenotypic divergence within species. Close links between locomotor phenotype and local water flow conditions appear to be important for species distributions as well as phenotypic divergence across environmental gradients.     

Shared and unique features of morphological differentiation between predator regimes in Gambusia caymanensis

When multiple groups of organisms experience similar environmental gradients, their patterns of differentiation might exhibit both shared and unique features. Here, we investigated the relative importance of three factors in generating body shape variation in a livebearing fish, Gambusia caymanensis, inhabiting the Cayman Islands: (i) shared patterns of divergent selection between predator regimes (presence/absence of piscivorous fish) driving replicated morphological differentiation, (ii) historical island effects yielding different morphologies across the three islands and (iii) unique effects of predation on morphological differentiation within each island. Shared effects of predation proved much more important than historical or unique effects. Populations coexisting with piscivorous fish exhibited larger caudal regions and smaller heads than conspecifics found in the absence of predatory fish. These results match a priori predictions, and mirror recent findings in a number of fish species, suggesting predation might often drive predictable morphological trends in disparate fishes. However, interestingly, the sexes achieved this morphological pattern through different means: head depth, caudal peduncle length and depth in males; head length, caudal peduncle depth in females. In G. caymanensis, we quantitatively confirmed that predation intensity represents a primary driver of body shape differentiation.     

Microevolutionary patterns in the common caiman predict macroevolutionary trends across extant crocodilians

Both extinct and extant crocodilians have repeatedly diversified in skull shape along a continuum, from narrow‐snouted to broad‐snouted phenotypes. These patterns occur with striking regularity, although it is currently unknown whether these trends also apply to microevolutionary divergence during population differentiation or the early stages of speciation. Assessing patterns of intraspecific variation within a single taxon can potentially provide insight into the processes of macroevolutionary differentiation. For example, high levels of intraspecific variation along a narrow‐broad axis would be consistent with the view that cranial shapes can show predictable patterns of differentiation on relatively short timescales, and potentially scale up to explain broader macroevolutionary patterns. In the present study, we use geometric morphometric methods to characterize intraspecific cranial shape variation among groups within a single, widely distributed clade, Caiman crocodilus. We show that C. crocodilus skulls vary along a narrow/broad‐snouted continuum, with different subspecies strongly clustered at distinct ends of the continuum. We quantitatively compare these microevolutionary trends with patterns of diversity at macroevolutionary scales (among all extant crocodilians). We find that morphological differences among the subspecies of C. crocodilus parallel the patterns of morphological differentiation across extant crocodilians, with the primary axes of morphological diversity being highly correlated across the two scales. We find intraspecific cranial shape variation within C. crocodilus to span variation characterized by more than half of living species. We show the main axis of intraspecific phenotypic variation to align with the principal direction of macroevolutionary diversification in crocodilian cranial shape, suggesting that mechanisms of microevolutionary divergence within species may also explain broader patterns of diversification at higher taxonomic levels.     

The effects of phenotypic plasticity and local adaptation on forecasts of species range shifts under climate change

Species are the unit of analysis in many global change and conservation biology studies; however, species are not uniform entities but are composed of different, sometimes locally adapted, populations differing in plasticity. We examined how intraspecific variation in thermal niches and phenotypic plasticity will affect species distributions in a warming climate. We first developed a conceptual model linking plasticity and niche breadth, providing five alternative intraspecific scenarios that are consistent with existing literature. Secondly, we used ecological niche‐modeling techniques to quantify the impact of each intraspecific scenario on the distribution of a virtual species across a geographically realistic setting. Finally, we performed an analogous modeling exercise using real data on the climatic niches of different tree provenances. We show that when population differentiation is accounted for and dispersal is restricted, forecasts of species range shifts under climate change are even more pessimistic than those using the conventional assumption of homogeneously high plasticity across a species' range. Suitable population‐level data are not available for most species so identifying general patterns of population differentiation could fill this gap. However, the literature review revealed contrasting patterns among species, urging greater levels of integration among empirical, modeling and theoretical research on intraspecific phenotypic variation.     

Subspecies delineation amid phenotypic,geographic and genetic discordance in a songbird

Understanding the processes that drive divergence within and among species is a long‐standing goal in evolutionary biology. Traditional approaches to assessing differentiation rely on phenotypes to identify intra‐ and interspecific variation, but many species express subtle morphological gradients in which boundaries among forms are unclear. This intraspecific variation may be driven by differential adaptation to local conditions and may thereby reflect the evolutionary potential within a species. Here, we combine genetic and morphological data to evaluate intraspecific variation within the Nelson's (Ammodramus nelsoni) and salt marsh (Ammodramus caudacutus) sparrow complex, a group with populations that span considerable geographic distributions and a habitat gradient. We evaluated genetic structure among and within five putative subspecies of A. nelsoni and A. caudacutus using a reduced‐representation sequencing approach to generate a panel of 1929 SNPs among 69 individuals. Although we detected morphological differences among some groups, individuals sorted along a continuous phenotypic gradient. In contrast, the genetic data identified three distinct clusters corresponding to populations that inhabit coastal salt marsh, interior freshwater marsh and coastal brackish–water marsh habitats. These patterns support the current species‐level recognition but do not match the subspecies‐level taxonomy within each species—a finding which may have important conservation implications. We identified loci exhibiting patterns of elevated divergence among and within these species, indicating a role for local selective pressures in driving patterns of differentiation across the complex. We conclude that this evidence for adaptive variation among subspecies warrants the consideration of evolutionary potential and genetic novelty when identifying conservation units for this group.     

The Rosy Region of Drosophila Melanogaster and Drosophila Simulans. I. Contrasting Levels of Naturally Occurring DNA Restriction Map Variation and Divergence

A 40-kb region around the rosy and snake loci was analyzed for restriction map variation among 60 lines of Drosophila melanogaster and 30 lines of Drosophila simulans collected together at a single locality in Raleigh, North Carolina. DNA sequence variation in D. simulans was estimated to be 6.3 times greater than in D. melanogaster (heterozygosities per nucleotide of 1.9% vs. 0.3%). This result stands in marked contrast to results of studies of phenotypic variation including proteins (allozymes), morphology and chromosome arrangements which are generally less variable and less geographically differentiated in D. simulans. Intraspecific polymorphism is not distributed uniformly over the 40-kb region. The level of heterozygosity per nucleotide varies more than 12-fold across the region in D. simulans, being highest over the hsc2 gene. Similar, though less extreme, variation in heterozygosity is also observed in D. melanogaster. Average interspecific divergence (corrected for intraspecific polymorphism) averaged 3.8%. The pattern of interspecific divergence over the 40-kb region shows some disparities with the spatial distribution of intraspecific variation, but is generally consistent with selective neutrality predictions: the most polymorphic regions within species are generally the most divergent between species. Sequence-length polymorphism is observed for D. melanogaster to be at levels comparable to other gene regions in this species. In contrast, no sequence length variation was observed among D. simulans chromosomes (limit of resolution approximately 100 bp). These data indicate that transposable elements play at best a minor role in the generation of naturally occurring genetic variation in D. simulans compared to D. melanogaster. We hypothesize that differences in species effective population size are the major determinant of the contrasting levels and patterns of DNA sequence and insertion/deletion variation that we report here and the patterns of allozyme and morphological variation and differentiation reported by other workers for these two species.     

AN EXPERIMENTAL STUDY OF COMPETITIVE PERFORMANCE IN BRASSICA RAPA (CRUCIFERAE)

Growth chamber experiments with rapid-cycling Brassica rapa were designed to estimate the signs and magnitudes of the genetic correlations for plant performance in each of three conditions: no-competition (isolated plants), intraspecific competition, and interspecific competition with Raphanus sativa. Biomass and flower number were highest in the no-competition treatment, intermediate under intraspecific competition, and lowest under interspecific competition. Significant among-family variation in biomass and flower number was found under each regime. The mean family performance (biomass or flower number) in the no-competition treatment was significantly positively correlated with the performance in only one of the competitive treatments (for biomass in the intraspecific treatment). For both biomass and flower number there was a significant positive correlation between family means in the intra- and interspecific regimes. These correlations were greater in magnitude than those for the comparison between no-competition and competition (intra- or interspecific) treatments. Our results suggest that the importance of traits affecting plant performance is environment-dependent; the performance of a family grown without competition was a poor predictor of performance with competition, while the performance of families grown under intra- and interspecific competition was positively correlated.     

Changing environmental gradients over forty years alter ecomorphological variation in Guadalupe Bass Micropterus treculii throughout a river basin

Understanding the degree of intraspecific variation within and among populations is a key aspect of predicting the capacity of a species to respond to anthropogenic disturbances. However, intraspecific variation is usually assessed at either limited temporal, but broad spatial scales or vice versa, which can make assessing changes in response to long‐term disturbances challenging. We evaluated the relationship between the longitudinal gradient of changing flow regimes and land use/land cover patterns since 1980 and morphological variation of Guadalupe Bass Micropterus treculii throughout the Colorado River Basin of central Texas. The Colorado River Basin in Texas has experienced major alterations to the hydrologic regime due to changing land‐ and water‐use patterns. Historical collections of Guadalupe Bass prior to rapid human‐induced change present the unique opportunity to study the response of populations to varying environmental conditions through space and time. Morphological differentiation of Guadalupe Bass associated with temporal changes in flow regimes and land use/land cover patterns suggests that they are exhibiting intraspecific trait variability, with contemporary individuals showing increased body depth, in response to environmental alteration through time (specifically related to an increase in herbaceous land cover, maximum flows, and the number of low pulses and high pulses). Additionally, individuals from tributaries with increased hydrologic alteration associated with urbanization or agricultural withdrawals tended to have a greater distance between the anal and caudal fin. These results reveal trait variation that may help to buffer populations under conditions of increased urbanization and sprawl, human population growth, and climate risk, all of which impose novel selective pressures, especially on endemic species like Guadalupe Bass. Our results contribute an understanding of the adaptability and capacity of an endemic population to respond to expected future changes based on demographic or climatic projection.     

Phenotypic plasticity and integration in response to flooded conditions in natural accessions of Arabidopsis thaliana (L.) Heynh (Brassicaceae)

Flood response is a crucial component of the life strategy of many plants, but it is seldom studied in non-flooded tolerant species, even though they may be subjected to stressful environmental conditions. Phenotypic plasticity in reaction to environmental stress affects the whole plant phenotype and can alter the character correlations that constitute the phenotypic architecture of the individual, yet few studies have investigated the lability of phenotypic integration to water regime. Moreover, little has been done to date to quantify the sort of selective pressures that different components of a plant's phenotype may be experiencing under contrasting water regimes. Genetic differentiation and phenotypic plasticity at the single-trait and multivariate levels were investigated in 47 accessions of the weedy plant Arabidopsis thaliana, and the relationship of plastic characters to reproductive fitness was quantified. Results indicate that these plants tend to be highly genetically differentiated for all traits, in agreement with predictions made on the basis of environmental variation and mating system. Varied patterns of apparent selection under flooded and non-flooded conditions were also uncovered, suggesting trade-offs in allocation between roots and above-ground biomass, as well as between leaves and reproductive structures. While the major components of the plants' multivariate phenotypic architecture were not significantly affected by environmental changes, many of the details were different under flooded and non-flooded conditions.     

Characterizing the contribution of plasticity and genetic differentiation to community‐level trait responses to environmental change

The match between functional trait variation in communities and environmental gradients is maintained by three processes: phenotypic plasticity and genetic differentiation (intraspecific processes), and species turnover (interspecific). Recently, evidence has emerged suggesting that intraspecific variation might have a potentially large role in driving functional community composition and response to environmental change. However, empirical evidence quantifying the respective importance of phenotypic plasticity and genetic differentiation relative to species turnover is still lacking. We performed a reciprocal transplant experiment using a common herbaceous plant species (Oxalis montana) among low‐, mid‐, and high‐elevation sites to first quantify the contributions of plasticity and genetic differentiation in driving intraspecific variation in three traits: height, specific leaf area, and leaf area. We next compared the contributions of these intraspecific drivers of community trait–environment matching to that of species turnover, which had been previously assessed along the same elevational gradient. Plasticity was the dominant driver of intraspecific trait variation across elevation in all traits, with only a small contribution of genetic differentiation among populations. Local adaptation was not detected to a major extent along the gradient. Fitness components were greatest in O. montana plants with trait values closest to the local community‐weighted means, thus supporting the common assumption that community‐weighted mean trait values represent selective optima. Our results suggest that community‐level trait responses to ongoing climate change should be mostly mediated by species turnover, even at the small spatial scale of our study, with an especially small contribution of evolutionary adaptation within species.     

Congruence of intraspecific variability in leaf traits for two co-occurring estuarine angiosperms

Studies seeking to identify sources of variability and trade-offs in leaf traits have done so by assembling large databases of traits, across species and time points. It is unclear to what extent interspecific patterns derived in such a manner apply to intraspecific variation, particularly at regional scales, and the extent to which interspecific patterns vary temporally. We tested the hypothesis that the leaf traits of two foundation species, the mangrove Avicennia marina and the eelgrass Zostera muelleri, would display similar patterns of intraspecific variability across gradients of latitude and estuarine condition, that match previously reported interspecific patterns, and that persist through time. We found intraspecific patterns of decreasing carbon to nitrogen ratio and mechanical elasticity, and increasing nitrogen content with latitude that were consistent between the two plant species, and with previously reported interspecific patterns for other groups of species. Specific leaf area, leaf toughness and total phenolics, by contrast, displayed species-specific patterns that varied markedly through time. Relationships between estuarine condition and leaf traits were highly variable temporally, and also displayed markedly different patterns of intraspecific variability between the two species. Our study highlights the considerable within-species variation in leaf traits that should be accounted for in regional to biome scale analyses. Although some intraspecific patterns mirrored those found across species, at global scales, the considerable variability in other leaf traits between species and through time highlights the need to better understand the drivers and constraints of this intraspecific variation.     

Intraspecific variation in tolerance of warming in fishes

Intraspecific variation in key traits such as tolerance of warming can have profound effects on ecological and evolutionary processes, notably responses to climate change. The empirical evidence for three primary elements of intraspecific variation in tolerance of warming in fishes is reviewed. The first is purely mechanistic that tolerance varies across life stages and as fishes become mature. The limited evidence indicates strongly that this is the case, possibly because of universal physiological principles. The second is intraspecific variation that is because of phenotypic plasticity, also a mechanistic phenomenon that buffers individuals’ sensitivity to negative impacts of global warming in their lifetime, or to some extent through epigenetic effects over successive generations. Although the evidence for plasticity in tolerance to warming is extensive, more work is required to understand underlying mechanisms and to reveal whether there are general patterns. The third element is intraspecific variation based on heritable genetic differences in tolerance, which underlies local adaptation and may define long-term adaptability of a species in the face of ongoing global change. There is clear evidence of local adaptation and some evidence of heritability of tolerance to warming, but the knowledge base is limited with detailed information for only a few model or emblematic species. There is also strong evidence of structured variation in tolerance of warming within species, which may have ecological and evolutionary significance irrespective of whether it reflects plasticity or adaptation. Although the overwhelming consensus is that having broader intraspecific variation in tolerance should reduce species vulnerability to impacts of global warming, there are no sufficient data on fishes to provide insights into particular mechanisms by which this may occur.     

Predicting ecological and phenotypic differentiation in the wild: a case of piscivorous fish in a fishless environment

Environmental variation drives ecological and phenotypic change. How predictable is differentiation in response to environmental change? Answering this question requires the development and testing of multifarious a priori predictions in natural systems. We employ this approach using Gobiomorus dormitor populations that have colonized inland blue holes differing in the availability of fish prey. We evaluated predictions of differences in demographics, habitat use, diet, locomotor and trophic morphology, and feeding kinematics and performance between G. dormitor populations inhabiting blue holes with and without fish prey. Populations of G. dormitor independently diverged between prey regimes, with broad agreement between observed differences and a priori predictions. For example, in populations lacking fish prey, we observed male‐biased sex ratios, a greater use of shallow‐water habitat, and larger population diet breadths as a result of greater individual diet specialization. Furthermore, we found predictable differences in body shape, mouth morphology, suction generation capacity, strike kinematics, and feeding performance on different prey types, consistent with the adaptation of G. dormitor to piscivory when coexisting with fish prey and to feeding on small invertebrates in their absence. The results of the present study suggest great potential in our ability to predict population responses to changing environments, which is an increasingly important capability in a human‐dominated, ever‐changing world. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 588–607.     

Disentangling biotic and abiotic drivers of intraspecific trait variation in woody plant seedlings at forest edges

In fragmented forests, edge effects can drive intraspecific variation in seedling performance that influences forest regeneration and plant composition. However, few studies have attempted to disentangle the relative biotic and abiotic drivers of intraspecific variation in seedling performance. In this study, we carried out a seedling transplant experiment with a factorial experimental design on three land‐bridge islands in the Thousand Island Lake, China, using four common native woody plant species. At different distances from the forest edge (2, 8, 32, 128 m), we transplanted four seedlings of each species into each of three cages: full‐cage, for herbivore exclusion; half‐cage, that allowed herbivore access but controlled for caging artifacts; and no‐cage control. In the 576 cages, we recorded branch architecture, leaf traits, and seedling survival for each seedling before and after the experimental treatment. Overall, after one full growing season, edge‐induced abiotic drivers and varied herbivory pressure led to intraspecific variation in seedling performance, including trade‐offs in seedling architecture and resource‐use strategies. However, responses varied across species with different life‐history strategies and depended on the driver in question, such that the abiotic and biotic effects were additive across species, rather than interactive. Edge‐induced abiotic variation modified seedling architecture of a shade‐tolerant species, leading to more vertical rather than lateral growth at edges. Meanwhile, increased herbivory pressure resulted in a shift toward lower dry matter investment in leaves of a light‐demanding species. Our results suggest that edge effects can drive rapid directional shifts in the performance and intraspecific traits of some woody plants from early ontogenetic stages, but most species in this study showed negligible phenotypic responses to edge effects. Moreover, species‐specific responses suggest the importance of interspecific differences modulating the degree of trait plasticity, implying the need to incorporate individual‐level responses when understanding the impact of forest fragmentation on plant communities.     

Incorporating intraspecific variation in tests of trait-based community assembly

Environmental filtering and niche differentiation are processes proposed to drive community assembly, generating nonrandom patterns in community trait distributions. Despite the substantial intraspecific trait variation present in plant communities, most previous studies of trait-based community assembly have used species mean trait values and therefore not accounted for intraspecific variation. Using a null model approach, I tested for environmental filtering and niche differentiation acting on three key functional traits??vegetative height, specific leaf area (SLA), and leaf dry matter content (LDMC)??in old-field plant communities. I also examined how accounting for intraspecific variation at the among-plot and individual levels affected the detection of nonrandom assembly patterns. Tests using fixed species mean trait values provided evidence of environmental filtering acting on height and SLA and niche differentiation acting on SLA. Including plot-level intraspecific variation increased the strength of these patterns, indicating an important role of intraspecific variation in community assembly. Tests using individual trait data indicated strong environmental filtering acting on all traits, but provided no evidence of niche differentiation, although these signals may have been obscured by the effects of dispersal limitation and spatial aggregation of conspecific individuals. There was also strong evidence of nonrandom assembly of individuals within single species, with the strength of environmental filtering varying among species. This study demonstrates that, while analyses using fixed species mean trait values can provide insights into community assembly processes, accounting for intraspecific variation provides a more complete view of communities and the processes driving their assembly.     

Universal scaling in the branching of the tree of life

Understanding the patterns and processes of diversification of life in the planet is a key challenge of science. The Tree of Life represents such diversification processes through the evolutionary relationships among the different taxa, and can be extended down to intra-specific relationships. Here we examine the topological properties of a large set of interspecific and intraspecific phylogenies and show that the branching patterns follow allometric rules conserved across the different levels in the Tree of Life, all significantly departing from those expected from the standard null models. The finding of non-random universal patterns of phylogenetic differentiation suggests that similar evolutionary forces drive diversification across the broad range of scales, from macro-evolutionary to micro-evolutionary processes, shaping the diversity of life on the planet.     

An experimental study of intraspecific variation, developmental timing, and heterochrony in fishes

Heterochrony is widely regarded as an important evolutionary mechanism, one that may underlie most, if not all, morphological evolution, yet relatively few studies have examined variation in the sequence of development. Even fewer studies have been designed so that intraspecific variation in the relative sequence of developmental events can be assessed, although this variation must be the basis for evolutionary change. Intraspecific variation in developmental ossification sequences was documented from the zebrafish (Danio rerio) by Cubbage and Mabee (1996) and from the Siamese fighting fish (Betta splendens) by Mabee and Trendler (1996), but a quantitative analysis of the patterns within this variation was not made. Here, we quantify the effect of rearing temperature on the sequence of ossification and characterize the levels and patterns of intraspecific variation in these fishes. For Danio, there were no temperature effects on the sequence of bone development across the cranium, cranial region development, cartilage versus dermal bones, or lateral line bone versus nonassociated bones. Likewise the level of variation in relative sequence (position) of ossification was low, about two ranks, across temperatures. At higher temperatures, we found higher levels of variation in iterated cranial bones and less in bones forming early in the sequence. No temperature effects on variation were found among regions, between lateral line-associated bones and nonassociated bones, between median and paired bones, or across the entire sequence, indicating concordant variability among the three temperatures. Individual bones with the highest levels of variability were not consistent among temperatures. Baseline patterns of intraspecific variation in Danio were compared to those of Betta. For both species, the level of intraspecific variation in sequence position was low and the variability of cranial bones was concordant. Individual bones with the highest levels of variability were not consistent between species. In both species, variation was widespread (distributed evenly across the sequence). We used comparisons (among regions, between dermal and cartilage bones, between lateral line-associated and other bones, between median and paired bones, between iterated and noniterated bones, between feeding-associated bones and others) to see which subsets were most variable and thus potentially useful in predicting high levels of evolutionary change. The only subset of bones that was significantly more variable than others was cartilage bones. If interspecific patterns are parallel to these intraspecific differences, cartilage bones would be expected to show higher levels of heterochrony. Although concordance across the cranial ossification sequence and among regions in Danio, Betta, and two other teleosts, Oryzias and Barbus, suggests an evolutionarily conserved pattern of ossification, identity in sequence position across taxa was not observed for any bone. Thus, variation existed in sequence position across temperatures and species. Intraspecific variation of this sort may influence the morphological outcome and evolutionary trajectories of species.