Combining molecular evolution and environmental genomics to unravel adaptive processes of MHC class IIB diversity in European minnows (Phoxinus phoxinus)

Host–pathogen interactions are a major evolutionary force promoting local adaptation. Genes of the major histocompatibility complex (MHC) represent unique candidates to investigate evolutionary processes driving local adaptation to parasite communities. The present study aimed at identifying the relative roles of neutral and adaptive processes driving the evolution of MHC class IIB (MHCIIB) genes in natural populations of European minnows (Phoxinus phoxinus). To this end, we isolated and genotyped exon 2 of two MHCIIB gene duplicates (DAB1 and DAB3) and 1′665 amplified fragment length polymorphism (AFLP) markers in nine populations, and characterized local bacterial communities by 16S rDNA barcoding using 454 amplicon sequencing. Both MHCIIB loci exhibited signs of historical balancing selection. Whereas genetic differentiation exceeded that of neutral markers at both loci, the populations' genetic diversities were positively correlated with local pathogen diversities only at DAB3. Overall, our results suggest pathogen‐mediated local adaptation in European minnows at both MHCIIB loci. While at DAB1 selection appears to favor different alleles among populations, this is only partially the case in DAB3, which appears to be locally adapted to pathogen communities in terms of genetic diversity. These results provide new insights into the importance of host–pathogen interactions in driving local adaptation in the European minnow, and highlight that the importance of adaptive processes driving MHCIIB gene evolution may differ among duplicates within species, presumably as a consequence of alternative selective regimes or different genomic context.     

Evidence for morphological and adaptive genetic divergence between lake and stream habitats in European minnows (Phoxinus phoxinus, Cyprinidae)

Natural selection drives local adaptation, potentially even at small temporal and spatial scales. As a result, adaptive genetic and phenotypic divergence can occur among populations living in different habitats. We investigated patterns of differentiation between contrasting lake and stream habitats in the cyprinid fish European minnow (Phoxinus phoxinus) at both the morphological and genomic levels using geometric morphometrics and AFLP markers, respectively. We also used a spatial correlative approach to identify AFLP loci associated with environmental variables representing potential selective forces responsible for adaptation to divergent habitats. Our results identified different morphologies between lakes and streams, with lake fish presenting a deeper body and caudal peduncle compared to stream fish. Body shape variation conformed to a priori predictions concerning biomechanics and swimming performance in lakes vs. streams. Moreover, morphological differentiation was found to be associated with several environmental variables, which could impose selection on body and caudal peduncle shape. We found adaptive genetic divergence between these contrasting habitats in the form of 'outlier' loci (2.9%) whose genetic divergence exceeded neutral expectations. We also detected additional loci (6.6%) not associated with habitat type (lake vs. stream), but contributing to genetic divergence between populations. Specific environmental variables related to trophic dynamics, landscape topography and geography were associated with several neutral and outlier loci. These results provide new insights into the morphological divergence and genetic basis of adaptation to differentiated habitats.     

Morphological divergence of lake and stream Phoxinus of Northern Italy and the Danube basin based on geometric morphometric analysis

Minnows of the genus Phoxinus are promising candidates to investigate adaptive divergence, as they inhabit both still and running waters of a variety of altitudes and climatic zones in Europe. We used landmark‐based geometric morphometric methods to quantify the level of morphological variability in Phoxinus populations from streams and lakes of Northern Italy and the Danube basin. We analyzed body shape differences of populations in the dorsal, lateral, and ventral planes, using a large array of landmarks and semilandmarks. As the species identification of Phoxinus on morphological characters is ambiguous, we used two mitochondrial genes to determine the genetic background of the samples and to ensure we are comparing homogenous groups. We have found significant body shape differences between habitats: Minnow populations inhabiting streams had a deeper body and caudal peduncle and more laterally inserted pectoral fins than minnows inhabiting lakes. We have also found significant body shape differences between genetic groups: Italian minnows had deeper bodies, deeper and shorter caudal peduncles, and a shorter and wider gape than both groups from the Danube. Our results show that the morphology of Phoxinus is highly influenced by habitat and that body shape variation between habitats was within the same range as between genetic groups. These morphological differences are possibly linked to different modes of swimming and foraging in the respective habitats and are likely results of phenotypic plasticity. However, differences in shape and interlandmark distances between the groups suggest that some (though few) morphometric characters might be useful for separating Phoxinus species.     

History,geography and host use shape genomewide patterns of genetic variation in the redheaded pine sawfly (Neodiprion lecontei)

Divergent host use has long been suspected to drive population differentiation and speciation in plant‐feeding insects. Evaluating the contribution of divergent host use to genetic differentiation can be difficult, however, as dispersal limitation and population structure may also influence patterns of genetic variation. In this study, we use double‐digest restriction‐associated DNA (ddRAD) sequencing to test the hypothesis that divergent host use contributes to genetic differentiation among populations of the redheaded pine sawfly (Neodiprion lecontei), a widespread pest that uses multiple Pinus hosts throughout its range in eastern North America. Because this species has a broad range and specializes on host plants known to have migrated extensively during the Pleistocene, we first assess overall genetic structure using model‐based and model‐free clustering methods and identify three geographically distinct genetic clusters. Next, using a composite‐likelihood approach based on the site frequency spectrum and a novel strategy for maximizing the utility of linked RAD markers, we infer the population topology and date divergence to the Pleistocene. Based on existing knowledge of Pinus refugia, estimated demographic parameters and patterns of diversity among sawfly populations, we propose a Pleistocene divergence scenario for N. lecontei. Finally, using Mantel and partial Mantel tests, we identify a significant relationship between genetic distance and geography in all clusters, and between genetic distance and host use in two of three clusters. Overall, our results indicate that Pleistocene isolation, dispersal limitation and ecological divergence all contribute to genomewide differentiation in this species and support the hypothesis that host use is a common driver of population divergence in host‐specialized insects.     

Influence of geography and environment on patterns of genetic differentiation in a widespread submerged macrophyte,Eurasian watermilfoil (Myriophyllum spicatum L., Haloragaceae)

The effects of geographic and environmental variables on the pattern of genetic differentiation have been thoroughly studied, whereas empirical studies on aquatic plants are rare. We examined the spatial genetic differentiation of 58 Myriophyllum spicatum populations distributed throughout China with 12 microsatellite loci, and we analyzed its association with geographic distance, geographic barriers, and environmental dissimilarity using causal modeling and multiple matrix regression with randomization (MMRR) analysis. Two genetic clusters were identified, and their geographic distribution suggested mountain ranges as a barrier to gene flow. The causal modeling revealed that both climate and geographic barriers significantly influenced genetic divergence among M. spicatum populations and that climate had the highest regression coefficient according to the MMRR analysis. This study showed that geography and environment together played roles in shaping the genetic structure of M. spicatum and that the influence of environment was greater. Our findings emphasized the potential importance of the environment in producing population genetic differentiation in aquatic plants at a large geographic scale.     

Morphological and genetic differentiation in anadromous smelt Osmerus mordax (Mitchill): disentangling the effects of geography and morphology on gene flow

Morphological analyses were combined with genetic analyses at nine microsatellite loci to examine the determinants of gene flow at 21 spawning locations of rainbow smelt Osmerus mordax along the east coast of Canada. Associations between morphology, geography and gene flow were examined using a computational geometric approach and partial Mantel tests. Significant barriers to gene flow and discontinuities in morphology were observed between Newfoundland and mainland Canada, as well as within Newfoundland samples. On regional scales, contrasting patterns were present with restricted gene flow between Newfoundland populations ( F _ST= c . 0·11) and high gene flow between mainland populations ( F _ST= c . 0·017). Within Newfoundland populations, geographic distance was significantly associated with gene flow ( r = 0·85, P < 0·001) contrasting mainland samples where gene flow was most associated with phenotypic divergence ( r = 0·33, P < 0·001). At large spatial scales, weak ( r = 0·19, P = 0·02) associations between gene flow and geographic distance were observed, and moderate associations were also observed between gene flow and morphology ( r = 0·28, P < 0·001). The presence of significant genetic isolation by distance in Newfoundland samples and the clear discontinuity associated with the Cabot Strait suggest geography may be the primary determinant of gene flow. Interestingly, the association between genetic and morphological divergence within mainland samples and overall, supports the hypothesis that gene flow may be moderated by morphological divergence at larger spatial scales even in high gene flow environments.     

Drivers of population genetic differentiation in the wild: isolation by dispersal limitation,isolation by adaptation and isolation by colonization

Empirical population genetic studies have been dominated by a neutralist view, according to which gene flow and drift are the main forces driving population genetic structure in nature. The neutralist view in essence describes a process of isolation by dispersal limitation (IBDL) that generally leads to a pattern of isolation by distance (IBD). Recently, however, conceptual frameworks have been put forward that view local genetic adaptation as an important driver of population genetic structure. Isolation by adaptation (IBA) and monopolization (M) posit that gene flow among natural populations is reduced as a consequence of local genetic adaptation. IBA stresses that effective gene flow is reduced among habitats that show dissimilar ecological characteristics, leading to a pattern of isolation by environment. In monopolization, local genetic adaptation of initial colonizing genotypes results in a reduction in gene flow that fosters the persistence of founder effects. Here, we relate these different processes driving landscape genetic structure to patterns of IBD and isolation by environment (IBE). We propose a method to detect whether IBDL, IBA and M shape genetic differentiation in natural landscapes by studying patterns of variation at neutral and non‐neutral markers as well as at ecologically relevant traits. Finally, we reinterpret a representative number of studies from the recent literature by associating patterns to processes and identify patterns associated with local genetic adaptation to be as common as IBDL in structuring regional genetic variation of populations in the wild. Our results point to the importance of quantifying environmental gradients and incorporating ecology in the analysis of population genetics.     

Quaternary climate instability is correlated with patterns of population genetic variability in Bombus huntii

Climate oscillations have left a significant impact on the patterns of genetic diversity observed in numerous taxa. In this study, we examine the effect of Quaternary climate instability on population genetic variability of a bumble bee pollinator species, Bombus huntii in western North America. Pleistocene and contemporary B. huntii habitat suitability (HS) was estimated with an environmental niche model (ENM) by associating 1,035 locality records with 10 bioclimatic variables. To estimate genetic variability, we genotyped 380 individuals from 33 localities at 13 microsatellite loci. Bayesian inference was used to examine population structure with and without a priori specification of geographic locality. We compared isolation by distance (IBD) and isolation by resistance (IBR) models to examine population differentiation within and among the Bayesian inferred genetic clusters. Furthermore, we tested for the effect of environmental niche stability (ENS) on population genetic diversity with linear regression. As predicted, high‐latitude B. huntii habitats exhibit low ENS when compared to low‐latitude habitats. Two major genetic clusters of B. huntii inhabit western North America: (a) a north genetic cluster predominantly distributed north of 28°N and (b) a south genetic cluster distributed south of 28°N. In the south genetic cluser, both IBD and IBR models are significant. However, in the north genetic cluster, IBD is significant but not IBR. Furthermore, the IBR models suggest that low‐latitude montane populations are surrounded by habitat with low HS, possibly limiting dispersal, and ultimately gene flow between populations. Finally, we detected high genetic diversity across populations in regions that have been climatically unstable since the last glacial maximum (LGM), and low genetic diversity across populations in regions that have been climatically stable since the LGM. Understanding how species have responded to climate change has the potential to inform management and conservation decisions of both ecological and economic concerns.     

Genetic and morphological differentiation patterns between sister species: the case of Onthophagus taurus and Onthophagus illyricus (Coleoptera, Scarabaeidae)

The present study investigated the morphological and genetic differentiation pattern between two sympatric dung beetle sister species, Onthophagus taurus and Onthophagus illyricus . The geometric morphometric approach coupled with the use of molecular markers allowed examination of the nature of interspecific relationships and an outline of the evolutionary and geographical processes that might have led to interspecific differentiation and the present-day partial sympatric and syntopic pattern of distribution. Geometric morphometrics failed to discrimininate the two species on the ground of external morphological traits, but revealed interspecific differences when the shape of the primary sexual traits was taken into account. The use of two different molecular markers (cytochrome oxidase subunit I gene and amplified fragment-length polymorphism) demonstrates that the two species are genetically well differentiated, forming two distinguishable lineages probably diverged during the Pliocene by allopatric speciation. No evidence of past or recent introgression and no support for hybridization were found, suggesting that sympatry was established subsequently, when speciation was accomplished. Both molecular markers and genital shape indicate that phenotypically intermediate individuals, despite their ambiguous appearance, are members of O. illyricus species rather than hybrids.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 197–211.     

Postglacial dispersal patterns and mitochondrial genetic structure of the Pyrenean desman (Galemys pyrenaicus) in the northwestern region of the Iberian Peninsula

The genetic structure of small semiaquatic animals may be influenced by dispersal across both rivers and land. The relative importance of these two modes of dispersal may vary across different species and with ecological conditions and evolutionary periods. The Pyrenean desman (Galemys pyrenaicus) is an endemic mammal of the Iberian Peninsula with a strong phylogeographic structure and semiaquatic habits, thus making it an ideal model to study the effects of river and overland dispersal on its genetic structure. Thanks to different types of noninvasive samples, we obtained an extensive sampling of the Pyrenean desman from the northwestern region of the Iberian Peninsula and sequenced two mitochondrial DNA fragments. We then analyzed, using an isolation‐by‐distance approach, the correlation between phylogenetic distances and geographical distances measured along both river networks and land to infer the relative importance of river and overland dispersal. We found that the correlations in the whole area and in a large basin were consistent with an effect of overland dispersal, which may be due to the postglacial colonization of new territories using terrestrial corridors and, possibly, a more extensive fluvial network that may have been present during the Holocene. However, in a small basin, likely to be less influenced by the impact of ancient postglacial dispersal, the correlations suggested significant overall effects of both overland and river dispersal, as expected for a semiaquatic mammal. Therefore, different scales and geographical regions reflect different aspects of the evolutionary history and ecology of this semiaquatic species using this isolation‐by‐distance method. The results we obtained may have crucial implications for the conservation of the Pyrenean desman because they reinforce the importance of interbasin dispersal for this species in the studied area and the need to protect the whole riverine ecosystem, including rivers, upland streams and terrestrial corridors between basins.     

The influence of landscape configuration and environment on population genetic structure in a sedentary passerine: insights from loci located in different genomic regions

The study of the factors structuring genetic variation can help to infer the neutral and adaptive processes shaping the demographic and evolutionary trajectories of natural populations. Here, we analyse the role of isolation by distance (IBD), isolation by resistance (IBR, defined by landscape composition) and isolation by environment (IBE, estimated as habitat and elevation dissimilarity) in structuring genetic variation in 25 blue tit (Cyanistes caeruleus) populations. We typed 1385 individuals at 26 microsatellite loci classified into two groups by considering whether they are located into genomic regions that are actively (TL; 12 loci) or not (NTL; 14 loci) transcribed to RNA. Population genetic differentiation was mostly detected using the panel of NTL. Landscape genetic analyses showed a pattern of IBD for all loci and the panel of NTL, but genetic differentiation estimated at TL was only explained by IBR models considering high resistance for natural vegetation and low resistance for agricultural lands. Finally, the absence for IBE suggests a lack of divergent selection pressures associated with differences in habitat and elevation. Overall, our study shows that markers located in different genomic regions can yield contrasting inferences on landscape‐level patterns of realized gene flow in natural populations.     

Fine‐grained adaptive divergence in an amphibian: genetic basis of phenotypic divergence and the role of nonrandom gene flow in restricting effective migration among wetlands

Adaptive ecological differentiation among sympatric populations is promoted by environmental heterogeneity, strong local selection and restricted gene flow. High gene flow, on the other hand, is expected to homogenize genetic variation among populations and therefore prevent local adaptation. Understanding how local adaptation can persist at the spatial scale at which gene flow occurs has remained an elusive goal, especially for wild vertebrate populations. Here, we explore the roles of natural selection and nonrandom gene flow (isolation by breeding time and habitat choice) in restricting effective migration among local populations and promoting generalized genetic barriers to neutral gene flow. We examined these processes in a network of 17 breeding ponds of the moor frog Rana arvalis, by combining environmental field data, a common garden experiment and data on variation in neutral microsatellite loci and in a thyroid hormone receptor (TRβ) gene putatively under selection. We illustrate the connection between genotype, phenotype and habitat variation and demonstrate that the strong differences in larval life history traits observed in the common garden experiment can result from adaptation to local pond characteristics. Remarkably, we found that haplotype variation in the TRβ gene contributes to variation in larval development time and growth rate, indicating that polymorphism in the TRβ gene is linked with the phenotypic variation among the environments. Genetic distance in neutral markers was correlated with differences in breeding time and environmental differences among the ponds, but not with geographical distance. These results demonstrate that while our study area did not exceed the scale of gene flow, ecological barriers constrained gene flow among contrasting habitats. Our results highlight the roles of strong selection and nonrandom gene flow created by phenological variation and, possibly, habitat preferences, which together maintain genetic and phenotypic divergence at a fine‐grained spatial scale.     

Diversification in the South American Pampas: the genetic and morphological variation of the widespread Petunia axillaris complex (Solanaceae)

Understanding the spatiotemporal distribution of genetic variation and the ways in which this distribution is connected to the ecological context of natural populations is fundamental for understanding the nature and mode of intraspecific and, ultimately, interspecific differentiation. The Petunia axillaris complex is endemic to the grasslands of southern South America and includes three subspecies: P. a. axillaris, P. a. parodii and P. a. subandina. These subspecies are traditionally delimited based on both geography and floral morphology, although the latter is highly variable. Here, we determined the patterns of genetic (nuclear and cpDNA), morphological and ecological (bioclimatic) variation of a large number of P. axillaris populations and found that they are mostly coincident with subspecies delimitation. The nuclear data suggest that the subspecies are likely independent evolutionary units, and their morphological differences may be associated with local adaptations to diverse climatic and/or edaphic conditions and population isolation. The demographic dynamics over time estimated by skyline plot analyses showed different patterns for each subspecies in the last 100 000 years, which is compatible with a divergence time between 35 000 and 107 000 years ago between P. a. axillaris and P. a. parodii, as estimated with the IMa program. Coalescent simulation tests using Approximate Bayesian Computation do not support previous suggestions of extensive gene flow between P. a. axillaris and P. a. parodii in their contact zone.     

Exploring the role of Micronesian islands in the maintenance of coral genetic diversity in the Pacific Ocean

Understanding how genetic diversity is maintained across patchy marine environments remains a fundamental problem in marine biology. The Coral Triangle, located in the Indo‐West Pacific, is the centre of marine biodiversity and has been proposed as an important source of genetic diversity for remote Pacific reefs. Several studies highlight Micronesia, a scattering of hundreds of small islands situated within the North Equatorial Counter Current, as a potentially important migration corridor. To test this hypothesis, we characterized the population genetic structure of two ecologically important congeneric species of reef‐building corals across greater Micronesia, from Palau to the Marshall Islands. Genetic divergences between islands followed an isolation‐by‐distance pattern, with Acropora hyacinthus exhibiting greater genetic divergences than A. digitifera, suggesting different migration capabilities or different effective population sizes for these closely related species. We inferred dispersal distance using a biophysical larval transport model, which explained an additional 15–21% of the observed genetic variation compared to between‐island geographical distance alone. For both species, genetic divergence accumulates and genetic diversity diminishes with distance from the Coral Triangle, supporting the hypothesis that Micronesian islands act as important stepping stones connecting the central Pacific with the species‐rich Coral Triangle. However, for A. hyacinthus, the species with lower genetic connectivity, immigration from the subequatorial Pacific begins to play a larger role in shaping diversity than input from the Coral Triangle. This work highlights the enormous dispersal potential of broadcast‐spawning corals and identifies the biological and physical drivers that influence coral genetic diversity on a regional scale.     

Variation in the digging apparatus of the subterranean silvery mole-rat, Heliophobius argenteocinereus (Rodentia, Bathyergidae): the role of ecology and geography

The skull of most subterranean tooth-digging rodents is markedly affected by their digging mode. In the present study, we investigated the cranial variation in a strictly subterranean, highly specialized Afrotropical tooth-digger, Heliophobius argenteocinereus (Bathyergidae, Rodentia), using a geometric morphometric approach and evaluated the effect of different factors on size and shape differences among four populations. No evidence for sexual dimorphism was found in skull size or shape. The cranial shape variation was large and influenced mainly by the type of habitat (miombo woodland versus farmland and grassland) and the latitudinal gradient. The dorsal side of the skull appears to be more plastic and adaptable to local environments, as well as more independent of size, than the ventral side. Only the shortening of the rostrum is presumably an adaptive process independent of size that leads to an increase of efficacy of the tooth-digging apparatus in Heliophobius , whereas the increase in the in-force and the more procumbent incisors both comprise size-related changes caused by ontogenetic allometric growth.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 822–831.     

Climatic drivers of leaf traits and genetic divergence in the tree Annona crassiflora: a broad spatial survey in the Brazilian savannas

The Cerrado is the largest South American savanna and encompasses substantial species diversity and environmental variation. Nevertheless, little is known regarding the influence of the environment on population divergence of Cerrado species. Here, we searched for climatic drivers of genetic (nuclear microsatellites) and leaf trait divergence in Annona crassiflora, a widespread tree in the Cerrado. The sampling encompassed all phytogeographic provinces of the continuous area of the Cerrado and included 397 individuals belonging to 21 populations. Populations showed substantial genetic and leaf trait divergence across the species' range. Our data revealed three spatially defined genetic groups (eastern, western and southern) and two morphologically distinct groups (eastern and western only). The east‐west split in both the morphological and genetic data closely mirrors previously described phylogeographic patterns of Cerrado species. Generalized linear mixed effects models and multiple regression analyses revealed several climatic factors associated with both genetic and leaf trait divergence among populations of A. crassiflora. Isolation by environment (IBE) was mainly due to temperature seasonality and precipitation of the warmest quarter. Populations that experienced lower precipitation summers and hotter winters had heavier leaves and lower specific leaf area. The southwestern area of the Cerrado had the highest genetic diversity of A. crassiflora, suggesting that this region may have been climatically stable. Overall, we demonstrate that a combination of current climate and past climatic changes have shaped the population divergence and spatial structure of A. crassiflora. However, the genetic structure of A. crassiflora reflects the biogeographic history of the species more strongly than leaf traits, which are more related to current climate.     

Morphologic and genetic variability in the Barbus fishes (Teleostei,Cyprinidae) of Central Italy

Italian freshwaters are highly biodiverse, with species present including the native fishes Barbus plebejus and Barbus tyberinus that are threatened by habitat alteration, fish stocking and invasive fishes, especially European barbel Barbus barbus. In central Italy, native fluvio‐lacustrine barbels are mainly allopatric and so provide an excellent natural system to evaluate the permeability of the Apennine Mountains. Here, the morphologic and genetic distinctiveness was determined for 611 Barbus fishes collected along the Padany–Venetian (Adriatic basins; PV) and Tuscany–Latium (Tyrrhenian basins; TL) districts. Analyses of morphological traits and mitochondrial DNA sequence data explored the natural and anthropogenic factors that have shaped their distribution ranges. Over 100 alien B. barbus were recorded in the Tiber basin (TL district) and Metauro basin (PV district). Comparisons of genetic and morphometric data revealed that morphometric data could identify alien B. barbus from native Barbus, but could not differentiate between B. tyberinus and B. plebejus. Genetic analyses revealed ~50 D‐loop mtDNA haplotypes and identified a distinct Barbus lineage present only in the Vomano River at the southern boundary of PV district. Demographic expansion and molecular variance analyses revealed a lack of geographic structuring across the sampling regions. While the contemporary B. plebejus distribution has been driven primarily by anthropogenic fish translocations, the dispersal of B. tyberinus has been via natural dispersion, including their crossing of the Apennine Mountains via temporary river connectivity. The results also revealed that the Barbus fishes of the mid‐Adriatic region of Europe have a complex pattern of local endemism. To conserve these patterns of genetic uniqueness, especially in the mid‐Adriatic basins, Barbus fishes should be managed by treating them as unique evolutionary units and ceasing translocations of all Barbus fishes between river basins.