Latitudinal divergence in a widespread amphibian: Contrasting patterns of neutral and adaptive genomic variation

Stochastic effects from demographic processes and selection are expected to shape the distribution of genetic variation in spatially heterogeneous environments. As the amount of genetic variation is central for long‐term persistence of populations, understanding how these processes affect variation over large‐scale geographical gradients is pivotal. We investigated the distribution of neutral and putatively adaptive genetic variation, and reconstructed demographic history in the moor frog (Rana arvalis) using 136 individuals from 15 populations along a 1,700‐km latitudinal gradient from northern Germany to northern Sweden. Using double digest restriction‐site associated DNA sequencing we obtained 27,590 single nucleotide polymorphisms (SNPs), and identified differentiation outliers and SNPs associated with growing season length. The populations grouped into a southern and a northern cluster, representing two phylogeographical lineages from different post‐glacial colonization routes. Hybrid index estimation and demographic model selection showed strong support for a southern and northern lineage and evidence of gene flow between regions located on each side of a contact zone. However, patterns of past gene flow over the contact zone differed between neutral and putatively adaptive SNPs. While neutral nucleotide diversity was higher along the southern than the northern part of the gradient, nucleotide diversity in differentiation outliers showed the opposite pattern, suggesting differences in the relative strength of selection and drift along the gradient. Variation associated with growing season length decreased with latitude along the southern part of the gradient, but not along the northern part where variation was lower, suggesting stronger climate‐mediated selection in the north. Outlier SNPs included loci involved in immunity and developmental processes.     

Latitudinal divergence of common frog (Rana temporaria) life history traits by natural selection: evidence from a comparison of molecular and quantitative genetic data

The relative roles of natural selection and direct environmental induction, as well as of natural selection and genetic drift, in creating clinal latitudinal variation in quantitative traits have seldom been assessed in vertebrates. To address these issues, we compared molecular and quantitative genetic differentiation between six common frog (Rana temporaria) populations along an approximately 1600 km long latitudinal gradient across Scandinavia. The degree of population differentiation (QST approximately 0.81) in three heritable quantitative traits (age and size at metamorphosis, growth rate) exceeded that in eight (neutral) microsatellite loci (FST = 0.24). Isolation by distance was clear for both neutral markers and quantitative traits, but considerably stronger for one of the three quantitative traits than for neutral markers. QST estimates obtained using animals subjected to different rearing conditions (temperature and food treatments) revealed some environmental dependency in patterns of population divergence in quantitative traits, but in general, these effects were weak in comparison to overall patterns. Pairwise comparisons of FST and QST estimates across populations and treatments revealed that the degree of quantitative trait differentiation was not generally predictable from knowledge of that in molecular markers. In fact, both positive and negative correlations were observed depending on conditions where the quantitative genetic variability had been measured. All in all, the results suggest a very high degree of genetic subdivision both in neutral marker genes and genes coding quantitative traits across a relatively recently (< 9000 years) colonized environmental gradient. In particular, they give evidence for natural selection being the primary agent behind the observed latitudinal differentiation in quantitative traits.     

Phenotypic differentiation of the Solidago virgaurea complex along an elevational gradient: Insights from a common garden experiment and population genetics

Plant species distributed along wide elevational or latitudinal gradients show phenotypic variation due to their heterogeneous habitats. This study investigated whether phenotypic variation in populations of the Solidago virgaurea complex along an elevational gradient is caused by genetic differentiation. A common garden experiment was based on seeds collected from nine populations of the S. virgaurea complex growing at elevations from 1,597 m to 2,779 m a.s.l. on Mt. Norikura in central Japan. Population genetic analyses with microsatellite markers were used to infer the genetic structure and levels of gene flow between populations. Leaf mass per area was lower, while leaf nitrogen and chlorophyll concentrations were greater for higher elevations at which seeds were originally collected. For reproductive traits, plants derived from higher elevations had larger flower heads on shorter stems and flowering started earlier. These elevational changes in morphology were consistent with the clines in the field, indicating that phenotypic variation along the elevational gradient would have been caused by genetic differentiation. However, population genetic analysis using 16 microsatellite loci suggested an extremely low level of genetic differentiation of neutral genes among the nine populations. Analysis of molecular variance also indicated that most genetic variation was partitioned into individuals within a population, and the genetic differentiation among the populations was not significant. This study suggests that genome regions responsible for adaptive traits may differ among the populations despite the existence of gene flow and that phenotypic variation of the S. virgaurea complex along the elevational gradient is maintained by strong selection pressure.     

Phenotypic plasticity closely linked to climate at origin and resulting in increased mortality under warming and frost stress in a common grass

Phenotypic plasticity is important for species responses to global change and species coexistence. Phenotypic plasticity differs among species and traits and changes across environments. Here, we investigated phenotypic plasticity of the widespread grass Arrhenatherum elatius in response to winter warming and frost stress by comparing phenotypic plasticity of 11 geographically and environmentally distinct populations of this species to phenotypic plasticity of populations of different species originating from a single environment. The variation in phenotypic plasticity was similar for populations of a single species from different locations compared to populations of functionally and taxonomically diverse species from one environment for the studied traits (leaf biomass production and root integrity after frost) across three indices of phenotypic plasticity (RDPI, PIN, slope of reaction norm). Phenotypic plasticity was not associated with neutral genetic diversity but closely linked to the climate of the populations’ origin. Populations originating from warmer and more variable climates showed higher phenotypic plasticity. This indicates that phenotypic plasticity can itself be considered as a trait subject to local adaptation to climate. Finally, our data emphasize that high phenotypic plasticity is not per se positive for adaptation to climate change, as differences in stress responses are resulting in high phenotypic plasticity as expressed by common plasticity indices, which is likely to be related to increased mortality under stress in more plastic populations.     

Latitudinal variation in seed weight and flower number in Prunella vulgaris

Studies of seed-weight variation across altitudinal and latitudinal gradients have led to conflicting hypotheses regarding the selective value of this traint in relation to the length of the growing season. Growing-season length may also influence the evolution of seed number, and population differentiation in seed weight may be constrained by a negative genetic correlation between seed weight and seed number within populations. We examined variation in seed weight and an estimate of seed number (flower number) and the covariance of these traits among populations of Prunella vulgaris at five latitudes between northern Michigan and South Carolina. We measured seed weight and flower number in native habitats and in a common environment to determine the extent to which patterns observed in the field reflect genetic differentiation. We observed no genetically based variation in seed weight across the latitudinal gradient, although genetic variation among populations within a latitude was observed. In contrast to the lack of variation in seed weight, flower number increased clinally from northern Michigan to Tennessee in a common environment. Population mean flowering date in a common environment was successively later from north to south. Later-flowering individuals appear to achieve a larger size before flowering and consequently possess more resources for seed production. This difference may account for the greater flower production of late-flowering, southern populations. Independence of population mean seed weight and flower number across the latitudinal gradient suggests that population differentiation in seed weight has not been constrained by a trade-off between seed size and number within populations.     

Re-establishment of clinal variation in flowering time among introduced populations of purple loosestrife (Lythrum salicaria, Lythraceae)

Range expansion during biological invasion requires that invaders adapt to geographical variation in climate, which should yield latitudinal clines in reproductive phenology. We investigated geographic variation in life history among 25 introduced populations of Lythrum salicaria, a widespread European invader of North American wetlands. We detected a strong latitudinal cline in initiation of flowering and size at flowering, which paralleled that reported among native populations. Plants from higher latitudes flowered earlier and at a smaller size than those from lower latitudes, even when raised in a uniform glasshouse. Early flowering was associated with greatly reduced reproductive output, but this was not associated with latitudinal variation in abundance, and probably did not result from a genetic correlation between time to and size at flowering. As introduction to North America c. 200 years ago, L. salicaria has re-established latitudinal clines in life history, probably as an evolutionary response to climatic selection.     

Parallel flowering time clines in native and introduced ragweed populations are likely due to adaptation

As introduced species expand their ranges, they often encounter differences in climate which are often correlated with geography. For introduced species, encountering a geographically variable climate sometimes leads to the re‐establishment of clines seen in the native range. However, clines can also be caused by neutral processes, and so it is important to gather additional evidence that population differentiation is the result of selection as opposed to nonadaptive processes. Here, we examine phenotypic and genetic differences in ragweed from the native (North America) and introduced (European) ranges. We used a common garden to assess phenotypic differentiation in size and flowering time in ragweed populations. We found significant parallel clines in flowering time in both North America and Europe. Height and branch number had significant clines in North America, and, while not statistically significant, the patterns in Europe were the same. We used SNP data to assess population structure in both ranges and to compare phenotypic differentiation to neutral genetic variation. We failed to detect significant patterns of isolation by distance, geographic patterns in population structure, or correlations between the major axes of SNP variation and phenotypes or latitude of origin. We conclude that the North American clines in size and the parallel clines seen for flowering time are most likely the result of adaptation.     

Microsatellite and major histocompatibility complex variation in an endangered rattlesnake,the Eastern Massasauga (Sistrurus catenatus)

Genetic diversity is fundamental to maintaining the long‐term viability of populations, yet reduced genetic variation is often associated with small, isolated populations. To examine the relationship between demography and genetic variation, variation at hypervariable loci (e.g., microsatellite DNA loci) is often measured. However, these loci are selectively neutral (or near neutral) and may not accurately reflect genomewide variation. Variation at functional trait loci, such as the major histocompatibility complex (MHC), can provide a better assessment of adaptive genetic variation in fragmented populations. We compared patterns of microsatellite and MHC variation across three Eastern Massasauga (Sistrurus catenatus) populations representing a gradient of demographic histories to assess the relative roles of natural selection and genetic drift. Using 454 deep amplicon sequencing, we identified 24 putatively functional MHC IIB exon 2 alleles belonging to a minimum of six loci. Analysis of synonymous and nonsynonymous substitution rates provided evidence of historical positive selection at the nucleotide level, and Tajima's D provided support for balancing selection in each population. As predicted, estimates of microsatellite allelic richness, observed, heterozygosity, and expected heterozygosity varied among populations in a pattern qualitatively consistent with demographic history and abundance. While MHC allelic richness at the population and individual levels revealed similar trends, MHC nucleotide diversity was unexpectedly high in the smallest population. Overall, these results suggest that genetic variation in the Eastern Massasauga populations in Illinois has been shaped by multiple evolutionary mechanisms. Thus, conservation efforts should consider both neutral and functional genetic variation when managing captive and wild Eastern Massasauga populations.     

Signatures of diversifying selection at EST‐SSR loci and association with climate in natural Eucalyptus populations

Understanding the environmental parameters that drive adaptation among populations is important in predicting how species may respond to global climatic changes and how gene pools might be managed to conserve adaptive genetic diversity. Here, we used Bayesian F_ST outlier tests and allele–climate association analyses to reveal two Eucalyptus EST‐SSR loci as strong candidates for diversifying selection in natural populations of a southwestern Australian forest tree, Eucalyptus gomphocephala (Myrtaceae). The Eucalyptus homolog of a CONSTANS‐like gene was an F_ST outlier, and allelic variation showed significant latitudinal clinal associations with annual and winter solar radiation, potential evaporation, summer precipitation and aridity. A second F_ST outlier locus, homologous to quinone oxidoreductase, was significantly associated with measures of temperature range, high summer temperature and summer solar radiation, with important implications for predicting the effect of temperature on natural populations in the context of climate change. We complemented these data with investigations into neutral population genetic structure and diversity throughout the species range. This study provides an investigation into selection signatures at gene‐homologous EST‐SSRs in natural Eucalyptus populations, and contributes to our understanding of the relationship between climate and adaptive genetic variation, informing the conservation of both putatively neutral and adaptive components of genetic diversity.     

Large‐scale patterns in genetic variation,gene flow and differentiation in five species of European Coenagrionid damselfly provide mixed support for the central‐marginal hypothesis

Recently, an increased effort has been directed towards understanding the distribution of genetic variation within and between populations, particularly at central and marginal areas of a species’ distribution. Much of this research is centred on the central‐marginal hypothesis, which posits that populations at range margins are sparse, small and genetically diminished compared to those at the centre of a species’ distribution range. We tested predictions derived from the central‐marginal hypothesis for the distribution of genetic variation and population differentiation in five European Coenagrionid damselfly species. We screened genetic variation (microsatellites) in populations sampled in the centre and margins of the species’ latitudinal ranges, assessed genetic diversity (H_S) in the populations and the distribution of this genetic diversity between populations (F_ST). We further assessed genetic substructure and migration with Bayesian assignment methods, and tested for significant associations between genetic substructure and bioclimatic and spatial (altitude and latitude) variables, using general linearized models. We found no general adherence to the central‐marginal hypothesis; instead we found that other factors such as historical or current ecological factors often better explain the patterns uncovered. This was illustrated in Coenagrion mercuriale whose colonisation history and behaviour most likely led to the observation of a high genetic diversity in the south and lower genetic diversity with increasing latitude, and in C. armatum and C. pulchellum whose patterns of low genetic diversity coupled with the weakest genetic differentiation at one of their range margins suggested, respectively, possible range shifts and recent, strong selection pressure.     

Insight into the genetic bases of climatic adaptation in experimentally evolving wheat populations

Experimental populations evolving under natural selection represent an interesting tool to study genetic bases of adaptation. Evolution of genes possibly involved in adaptive response can be followed together with the corresponding phenotypic traits. Using experimental populations of hexaploid wheat, we studied the evolution of flowering time, a major adaptive trait that synchronizes the initiation of reproduction and the occurrence of favourable environmental conditions. During 12 generations, three populations were grown in contrasted environments (Vervins North France, Le Moulon near Paris, Toulouse South France) under the influence of natural selection, drift, mutation and recombination. Evolution of diversity at the major gene VRN-1 involved in wheat vernalization response has been analysed jointly with earliness estimated in controlled conditions. Whatever the population, rapid phenotypic changes as well as parallel genotypic variations were observed in the first seven generations, probably as the result of selection acting on this major gene which explains 80% of the trait variation overall. Different allelic combinations at physically unlinked copies of VRN-1 located on distinct genomes (A, B and D) were selected between populations. As theoretically expected, due to population differentiation, a high level of genetic diversity was maintained overall in generation 12. Surprisingly, in two populations out of three, the emergence of new alleles by mutation or migration, coupled with temporal variable selection or frequency-dependent selection, allowed to maintain within-population diversity despite local genetic drift and natural selection. This result may plead for an evolutionary approach of wheat genetic resource conservation.     

Life history variation in Campanula americana (Campanulaceae): population differentiation

Genetic variation in life history traits has important consequences for life-history evolution. Here we report the results of a greenhouse experiment investigating the broad sense genetic basis of variation in life history traits within and among five populations of Campanula americana distributed along a latitudinal gradient. The populations exhibit differentiation for a number of morphological traits (seed weight, number of branches, final plant size, number of capsules) and the phenological traits, days to emergence, days to bolting, the onset of flowering, and the duration of flowering. Families within populations differed only in days to emergence and seed weight. These results suggest that the life history differences among populations are genetically based. In addition, two life history types—winter annuals and biennials—have previously been reported from natural populations of Campanula americana. This experiment identified a third type—summer annuals from the Florida population.     

Local adaptation and oceanographic connectivity patterns explain genetic differentiation of a marine diatom across the North Sea–Baltic Sea salinity gradient

Drivers of population genetic structure are still poorly understood in marine micro‐organisms. We exploited the North Sea–Baltic Sea transition for investigating the seascape genetics of a marine diatom, Skeletonema marinoi. Eight polymorphic microsatellite loci were analysed in 354 individuals from ten locations to analyse population structure of the species along a 1500‐km‐long salinity gradient ranging from 3 to 30 psu. To test for salinity adaptation, salinity reaction norms were determined for sets of strains originating from three different salinity regimes of the gradient. Modelled oceanographic connectivity was compared to directional relative migration by correlation analyses to examine oceanographic drivers. Population genetic analyses showed distinct genetic divergence of a low‐salinity Baltic Sea population and a high‐salinity North Sea population, coinciding with the most evident physical dispersal barrier in the area, the Danish Straits. Baltic Sea populations displayed reduced genetic diversity compared to North Sea populations. Growth optima of low salinity isolates were significantly lower than those of strains from higher native salinities, indicating local salinity adaptation. Although the North Sea–Baltic Sea transition was identified as a barrier to gene flow, migration between Baltic Sea and North Sea populations occurred. However, the presence of differentiated neutral markers on each side of the transition zone suggests that migrants are maladapted. It is concluded that local salinity adaptation, supported by oceanographic connectivity patterns creating an asymmetric migration pattern between the Baltic Sea and the North Sea, determines genetic differentiation patterns in the transition zone.     

Genetic population structure in horse chestnut (Aesculus hippocastanum L.): effects of human‐mediated expansion across Europe

The geographical distribution of existing populations of horse chestnut (Aesculus hippocastanum L.) in Europe is determined by past demographic events during the Quaternary. In the present study we evaluate the imprints that northward expansions originated from common ancestry at southern Europe may have left on the present patterns of genetic variation for horse chestnut across the continent. Genetic diversity and levels of population structure in a European south–north gradient, ranging from the Balkans to the Scandinavian Peninsula, were determined with Amplified Fragment Length Polymorphism (AFLP) markers in 159 loci. A family of rarefaction techniques for the estimation of gene diversity was used to exclude potential confounding effects as a result of the unequal sample sizes. The results indicate that northern populations are not more genetically depleted than southern populations, thus suggesting that diversity for this species is not correlated with latitudinal distribution. Detailed hypotheses based on prediction models for different historical events associated with human‐mediated spread of cultivation are examined for a better understanding of the current genetic patterns of regional differentiation.     

Patterns of genetic diversity vary among shoot and root functional traits in Norway spruce Picea abies along a latitudinal gradient

Roots constitute a major segment of plant biomass, and variation in belowground traits in situ correlates with environmental gradients at large spatial scales. Local adaptation of populations maintains intraspecific genetic variation in various shoot traits, but the contribution of genetic factors to adaptation to soil heterogeneity remains poorly known. I established a common-garden experiment with three Norway spruce Picea abies populations sampled between 60° and 67° N in Finland, each represented by 13 or 15 maternal families, to determine whether belowground traits are as genetically differentiated among populations as those in the shoot along a collective latitudinal gradient of temperature and soil heterogeneity. Two growing season simulations enabled testing for among-population differences in phenotypic plasticity. I phenotyped 777 first-year seedlings from shoot to root to capture functional traits that may influence survival in the wild: autumn phenology, shoot growth, root system size, root architecture, root morphology and growth allocation. All traits exhibited within-population genetic diversity, but among-population differentiation ranged from strong in shoot traits to nonexistent in root system architecture and morphology that are scaled to root system size. However, latitudinal trends characterised root-to-shoot ratio and root tip-to-shoot ratio that account for among-population differences in aboveground growth. Overall trait variability was multidimensional with variable among- versus within-population trends: for example, phenology and shoot growth covaried across populations, but their association within individual populations was variable. Shoot growth correlated positively with root system size, but not with root architecture or morphology. Finally, the two higher-latitude populations exhibited greater phenotypic plasticity in shoot traits and growth allocation. The results demonstrate varying patterns of genetic variation in functional traits of Norway spruce in the boreal zone, suggesting simultaneous adaptation to multiple environmental factors. Functional traits that exhibit phenotypic plasticity, genetic diversity and little covariation will promote long-term survival of populations in fluctuating environments.     

Genomics of invasion: diversity and selection in introduced populations of monkeyflowers (Mimulus guttatus)

Global trade and travel is irreversibly changing the distribution of species around the world. Because introduced species experience drastic demographic events during colonization and often face novel environmental challenges from their native range, introduced populations may undergo rapid evolutionary change. Genomic studies provide the opportunity to investigate the extent to which demographic, historical and selective processes shape the genomic structure of introduced populations by analysing the signature that these processes leave on genomic variation. Here, we use next‐generation sequencing to compare genome‐wide relationships and patterns of diversity in native and introduced populations of the yellow monkeyflower (Mimulus guttatus). Genome resequencing data from 10 introduced populations from the United Kingdom (UK) and 12 native M. guttatus populations in North America (NA) demonstrated reduced neutral genetic diversity in the introduced range and showed that UK populations are derived from a geographic region around the North Pacific. A selective‐sweep analysis revealed site frequency changes consistent with selection on five of 14 chromosomes, with genes in these regions showing reduced silent site diversity. While the target of selection is unknown, genes associated with flowering time and biotic and abiotic stresses were located within the swept regions. The future identification of the specific source of origin of introduced UK populations will help determining whether the observed selective sweeps can be traced to unsampled native populations or occurred since dispersal across the Atlantic. Our study demonstrates the general potential of genome‐wide analyses to uncover a range of evolutionary processes affecting invasive populations.     

Genetic structure of a reef-building coral from thermally distinct environments on the Great Barrier Reef

Adaptation to localised thermal regimes is facilitated by restricted gene flow, ultimately leading to genetic divergence among populations and differences in their physiological tolerances. Allozyme analysis of six polymorphic loci was used to assess genetic differentiation between nine populations of the reef-building coral Acropora millepora over a latitudinal temperature gradient on the inshore regions of the Great Barrier Reef (GBR). Small but significant genetic differentiation indicative of moderate levels of gene flow (pairwise F _ST 0.023 to 0.077) was found between southern populations of A. millepora in cooler regions of the GBR and the warmer, central or northern GBR populations. Patterns of genetic differentiation at these putatively neutral allozyme loci broadly matched experimental variation in thermal tolerance and were consistent with local thermal regimes (warmest monthly-averages) for the A. millepora populations examined. It is therefore hypothesized that natural selection has influenced the thermal tolerance of the A. millepora populations examined and greater genetic divergence is likely to be revealed by examination of genetic markers under the direct effects of natural selection.     

Comparison of quantitative and molecular genetic variation of native vs. invasive populations of purple loosestrife (Lythrum salicaria L., Lythraceae)

Study of adaptive evolutionary changes in populations of invasive species can be advanced through the joint application of quantitative and population genetic methods. Using purple loosestrife as a model system, we investigated the relative roles of natural selection, genetic drift and gene flow in the invasive process by contrasting phenotypical and neutral genetic differentiation among native European and invasive North American populations ( Q _ST −  F _ST analysis). Our results indicate that invasive and native populations harbour comparable levels of amplified fragment length polymorphism variation, a pattern consistent with multiple independent introductions from a diverse European gene pool. However, it was observed that the genetic variation reduced during subsequent invasion, perhaps by founder effects and genetic drift. Comparison of genetically based quantitative trait differentiation ( Q _ST) with its expectation under neutrality ( F _ST) revealed no evidence of disruptive selection ( Q _ST >  F _ST) or stabilizing selection ( Q _ST <  F _ST). One exception was found for only one trait (the number of stems) showing significant sign of stabilizing selection across all populations. This suggests that there are difficulties in distinguishing the effects of nonadaptive population processes and natural selection. Multiple introductions of purple loosestrife may have created a genetic mixture from diverse source populations and increased population genetic diversity, but its link to the adaptive differentiation of invasive North American populations needs further research.     

Studying genetics of adaptive variation in model organisms: flowering time variation in Arabidopsis lyrata

Arabidopsis thaliana has emerged as a model organism for plant developmental genetics, but it is also now being widely used for population genetic studies. Outcrossing relatives of A. thaliana are likely to provide suitable additional or alternative species for studies of evolutionary and population genetics. We have examined patterns of adaptive flowering time variation in the outcrossing, perennial A. lyrata. In addition, we examine the distribution of variation at marker genes in populations form North America and Europe. The probability of flowering in this species differs between southern and northern populations. Northern populations are much less likely to flower in short than in long days. A significant daylength by region interaction shows that the northern and southern populations respond differently to the daylength. The timing of flowering also differs between populations, and is made shorter by long days, and in some populations, by vernalization. North American and European populations show consistent genetic differentiation over microsatellite and isozyme loci and alcohol dehydrogenase sequences. Thus, the patterns of variation are quite different from those in A. thaliana, where flowering time differences show little relationship to latitude of origin and the genealogical trees of accessions vary depending on the genomic region studied. The genetic architecture of adaptation can be compared in these species with different life histories.     

Phenotypic divergence of the common toad (Bufo bufo) along an altitudinal gradient: evidence for local adaptation

Variation in the environment can induce different patterns of genetic and phenotypic differentiation among populations. Both neutral processes and selection can influence phenotypic differentiation. Altitudinal phenotypic variation is of particular interest in disentangling the interplay between neutral processes and selection in the dynamics of local adaptation processes but remains little explored. We conducted a common garden experiment to study the phenotypic divergence in larval life-history traits among nine populations of the common toad (Bufo bufo) along an altitudinal gradient in France. We further used correlation among population pairwise estimates of quantitative trait (Q_ST) and neutral genetic divergence (F_ST from neutral microsatellite markers), as well as altitudinal difference, to estimate the relative role of divergent selection and neutral genetic processes in phenotypic divergence. We provided evidence for a neutral genetic differentiation resulting from both isolation by distance and difference in altitude. We found evidence for phenotypic divergence along the altitudinal gradient (faster development, lower growth rate and smaller metamorphic size). The correlation between pairwise Q_STs–F_STs and altitude differences suggested that this phenotypic differentiation was most likely driven by altitude-mediated selection rather than by neutral genetic processes. Moreover, we found different divergence patterns for larval traits, suggesting that different selective agents may act on these traits and/or selection on one trait may constrain the evolution on another through genetic correlation. Our study highlighted the need to design more integrative studies on the common toad to unravel the underlying processes of phenotypic divergence and its selective agents in the context of environmental clines.