Genetic traces of hybrid zone movement across a fragmented habitat.

Theoretical and empirical studies suggest that the structure and position of hybrid zones can change over time. Evidence for moving hybrid zones has been directly inferred by repeated sampling over time, or indirectly through the detection of genetic footprints left by the receding species and the resulting asymmetric patterns of introgression across markers. We here investigate a hybrid zone formed by two subspecies of the Iberian golden-striped salamander, Chioglossa lusitanica, using a panel of 35 nuclear loci (31 SNPs and 4 allozymes) and one mitochondrial locus in a transect in central Portugal. We found concordant and coincident clines for most of the nuclear loci (n = 22, 63%), defining a narrow hybrid zone of ca. 6 km wide, with the centre positioned ca. 15 km south of the Mondego River. Asymmetric introgression was observed at another 14 loci. Their clines are displaced towards the north, with positions located either close to the Mondego River (n = 6) or further northwards (n = 8). We interpret these profiles as genetic traces of the southward displacement of C. lusitanica lusitanica by C. l. longipes over the wider Mondego River valley. We noted the absence of significant linkage disequilibrium, and we inferred low levels of effective selection per locus against hybrids, suggesting that introgression in the area of species replacement occurred under a neutral diffusion process. A species distribution model suggests that the C. lusitanica hybrid zone coincides with a narrow corridor of fragmented habitat. From the position of the displaced clines, we infer that patches of locally suitable habitat trapped some genetic variants that became disassociated from the southward moving hybrid zone. This study highlights the influence of habitat availability on hybrid zone movement.

Asymmetry in fitness-related traits of later-generation hybrids between two invasive species.

PREMISE: The importance of hybridization to invasion has been frequently discussed, with most studies focusing on the comparison of fitness-related traits between F1 hybrids and their parents and the consequences of such fitness differences. However, relatively little attention has been given to late-generation hybrids. Different fitness landscapes could emerge in later generations after hybrids cross with each other or backcross with their parents, which may play an important role in plant invasion and subsequent speciation. METHODS: In this study, artificial crosses were conducted to generate multiple generations, including F1, F2, and backcrosses between two invasive species: Cakile edentula (self-compatible) and C. maritima (self-incompatible). Putative hybrids were also collected in the sympatric zone and compared with their co-occurring parents for phenotypic and genetic differences. RESULTS: Genetic data provided evidence of hybridization happening in the wild, and phenotypic comparisons showed that natural hybrids had intermediate traits between the two species but showed more similarity to C. maritima than to C. edentula. The asymmetry was further identified in artificial generations for several phenotypic characters. Furthermore, backcrosses exhibited different patterns of variation, with backcrosses to C. maritima having higher reproductive output than their counterparts. CONCLUSIONS: Our results suggest that hybridization and introgression (backcrossing) in Cakile species is asymmetric and most likely to favor the proliferation of C. maritima genes in the mixed population and thus help its establishment, a finding that could not be predicted by characterizing F1 hybrids.

Conservation implications of asymmetric introgression and reproductive barriers in a rare primrose species.

BACKGROUND: Primula is a large genus of flowering herbs well known for their heterostyly. Currently few natural hybrids are known and reproductive barriers in this genus in the wild have received little attention. However, there is instance of hybridization between rare and widely-spread species, and conservation implications of such situation is poorly understood. In the present study, we investigated hybridization patterns and reproductive barriers between a wide spread species, Primula poissonii and a rare species P. anisodora, of which only three populations are currently known. RESULTS: Pollinator-mediated reproductive isolation was strong between parental species but not significant between hybrids and parental species. Hand pollination experiments showed significant reduction of both fruit- and seed-set for heterospecific pollination as compared with conspecific pollination for both parental species. Furthermore, hybrids had higher fruit- and seed-set when pollinated with P. anisodora pollen as opposed to P. poissonii pollen. Microsatellites identified backcrosses to P. anisodora in two of the three populations of P. anisodora, and additionally more individuals of P. anisodora showed introgression from P. poissonii than vice versa. CONCLUSIONS: These results provide evidence for potential genetic swamping of the P. anisodora populations, which could pose a serious threat for this locally endemic species.

Coexistence of two sympatric cryptic bat species in French Guiana: insights from genetic, acoustic and ecological data.

BACKGROUND: The distinction between lineages of neotropical bats from the Pteronotus parnellii species complex has been previously made according to mitochondrial DNA, and especially morphology and acoustics, in order to separate them into two species. In these studies, either sample sizes were too low when genetic and acoustic or morphological data were gathered on the same individuals, or genetic and other data were collected on different individuals. In this study, we intensively sampled bats in 4 caves and combined all approaches in order to analyse genetic, morphologic, and acoustic divergence between these lineages that live in the same caves in French Guiana. RESULTS: A multiplex of 20 polymorphic microsatellite markers was developed using the 454-pyrosequencing technique to investigate for the first time the extent of reproductive isolation between the two lineages and the population genetic structure within lineages. We genotyped 748 individuals sampled between 2010 and 2015 at the 20 nuclear microsatellite loci and sequenced a portion of the cytochrome c oxydase I gene in a subset of these. Two distinct, non-overlapping haplogroups corresponding to cryptic species P. alitonus and P. rubiginosus were revealed, in accordance with previous findings. No spatial genetic structure between caves was detected for both species. Hybridization appeared to be quite limited (0.1-4%) using microsatellite markers whereas introgression was more common (7.5%) and asymmetric for mitochondrial DNA (mtDNA). CONCLUSIONS: The extremely low rate of hybridization could be explained by differences in life cycle phenology between species as well as morphological and acoustical distinction between sexes in one or the other species. Taken together, these results add to our growing understanding of the nature of species boundaries in Pteronotus parnelli, but deserve more in-depth studies to understand the evolutionary processes underlying asymmetric mtDNA introgression in this group of cryptic species.

Genomic evidence for asymmetric introgression by sexual selection in the common wall lizard.

Strongly selected characters can be transferred from one lineage to another with limited genetic exchange, resulting in asymmetric introgression and a mosaic genome in the receiving population. However, systems are rarely sufficiently well studied to link the pattern of introgression to its underlying process. Male common wall lizards in western Italy exhibit exaggeration of a suite of sexually selected characters that make them outcompete males from a distantly related lineage that lack these characters. This results in asymmetric hybridization and adaptive introgression of the suite of characters following secondary contact. We developed genomewide markers to infer the demographic history of gene flow between different genetic lineages, identify the spread of the sexually selected syndrome, and test the prediction that introgression should be asymmetric and heterogeneous across the genome. Our results show that secondary contact was accompanied by gene flow in both directions across most of the genome, but with approximately 3% of the genome showing highly asymmetric introgression in the predicted direction. Demographic simulations reveal that this asymmetric gene flow is more recent than the initial secondary contact, and the data suggest that the exaggerated male sexual characters originated within the Italian lineage and subsequently spread throughout this lineage before eventually reaching the contact zone. These results demonstrate that sexual selection can cause a suite of characters to spread throughout both closely and distantly related lineages with limited gene flow across the genome at large.

Hybrid zone formation and contrasting outcomes of secondary contact over transects in common toads.

Much progress in speciation research stems from documenting patterns of morphological and genetic variation in hybrid zones. Contrasting patterns of marker introgression in different sections of the contact can provide valuable insights on the relative importance of various evolutionary mechanisms maintaining species differences in the face of hybridization and gene flow and on hybrid zone temporal and spatial dynamics. We studied species interactions in the common toads Bufo bufo and B. spinosus in France and northwestern Italy using morphological and molecular data from the mitochondrial and nuclear genomes in an extensive survey, including two independent transects west and east of the Alps. At both, we found sharp, coincident and concordant nuclear genetic transitions. However, morphological clines were wider or absent and mtDNA introgression was asymmetric. We discuss alternative, nonexclusive hypotheses about evolutionary processes generating these patterns, including drift, selection, long-distance dispersal and spatial shifts in hybrid zone location and structure. The distribution of intraspecific mtDNA lineages supports a scenario in which B. bufo held a local refugium during the last glacial maximum. Present-day genetic profiles are best explained by an advance of B. spinosus from a nearby Iberian refugium, largely superseding the local B. bufo population, followed by an advance of B. bufo from the Balkans, with prongs north and south of the Alps, driving B. spinosus southwards. A pendulum moving hybrid zone, first northwards and then southwards, explains the wide areas of introgression at either side of the current position of the contact zones.

Divergence history of the Carpathian and smooth newts modelled in space and time.

Information about demographic history is essential for the understanding of the processes of divergence and speciation. Patterns of genetic variation within and between closely related species provide insights into the history of their interactions. Here, we investigated historical demography and genetic exchange between the Carpathian (Lissotriton montandoni, Lm) and smooth (L. vulgaris, Lv) newts. We combine an extensive geographical sampling and multilocus nuclear sequence data with the approximate Bayesian computation framework to test alternative scenarios of divergence and reconstruct the temporal and spatial pattern of gene flow between species. A model of recent (last glacial period) interspecific gene flow was favoured over alternative models. Thus, despite the relatively old divergence (4-6 mya) and presumably long periods of isolation, the species have retained the ability to exchange genes. Nevertheless, the low migration rates (ca. 10(-6) per gene copy per generation) are consistent with strong reproductive isolation between the species. Models allowing demographic changes were favoured, suggesting that the effective population sizes of both species at least doubled as divergence reaching the current ca. 0.2 million in Lm and 1 million in Lv. We found asymmetry in rates of interspecific gene flow between Lm and one evolutionary lineage of Lv. We suggest that intraspecific polymorphism for hybrid incompatibilities segregating within Lv could explain this pattern and propose further tests to distinguish between alternative explanations. Our study highlights the importance of incorporating intraspecific genetic structure into the models investigating the history of divergence.

Mating system variation and assortative mating of sympatric bromeliads (Pitcairnia spp.) endemic to neotropical inselbergs.

PREMISE OF THE STUDY: The mating system is an important component of the complex set of reproductive isolation barriers causing plant speciation. However, empirical evidence showing that the mating system may promote reproductive isolation in co-occurring species is limited. The mechanisms by which the mating system can act as a reproductive isolation barrier are also largely unknown. METHODS: Here we studied progeny arrays genotyped with microsatellites and patterns of stigma-anther separation (herkogamy) to understand the role of mating system shifts in promoting reproductive isolation between two hybridizing taxa with porous genomes, Pitcairnia albiflos and P. staminea (Bromeliaceae). KEY RESULTS: In P. staminea, we detected increased selfing and reduced herkogamy in one sympatric relative to two allopatric populations, consistent with mating system shifts in sympatry acting to maintain the species integrity of P. staminea when in contact with P. albiflos. CONCLUSIONS: Mating system variation is a result of several factors acting simultaneously in these populations. We report mating system shifts as one possible reproductive barrier between these species, acting in addition to numerous other prezygotic (i.e., flower phenology and pollination syndromes) and postzygotic barriers (Bateson-Dobzhansky-Muller genetic incompatibilities).

Asymmetric Introgression and Cryptic Natural Hybridization between Two Species of Teucrium Section Polium (Lamiaceae) on the Balkan Peninsula.

In this work, we analyzed the morphology and genetic structure of Teucrium montanum, T. capitatum and their hybrid T. × rohlenae from three syntopic populations. A morphometric study showed that the parents and their hybrids exhibited continuous morphological variation, with the hybrid positioned exactly between the parents. Genetic analysis revealed that plants morphologically identified as T. × rohlenae are fertile hybrids that produce hybrid swarms dominated by later-generation hybrids. This suggests that introgression, rather than speciation, is the more likely outcome of hybridization between these plant species. The extent and direction of gene flow between the two species differed markedly between the three syntopic localities. At the Trilj locality, it was clearly unidirectional, with T. capitatum playing the dominant role. At the Sicevo locality, gene flow was slightly asymmetric, favoring the genetic background of T. capitatum, while at the Sliven site, it was completely asymmetric in the opposite direction. The extreme case of unidirectional gene flow was observed at the Trilj locality where plants morphologically identified as T. montanum could not be genetically distinguished from T. capitatum. This suggests that interspecific hybridization occurred long ago, leading to introgression and cryptic hybrids, blurring of species boundaries and generating evolutionary noise.

Different patterns of introgression in a three species hybrid zone among European cave salamanders.

Hybrid zones occur where genetically distinct populations meet, mate and produce offspring with mixed ancestry. In Plethodontid salamanders, introgressive hybridization is a common phenomenon, where hybrids backcross with parental populations leading to the spread of new alleles into the parental genomes. Whereas many hybrid zones have been reported in American Plethodontid salamanders, only a single hybrid zone has been documented in European plethodontids so far, which is located at the Apuan Alps in the Italian Peninsula. Here, we describe a previously unreported hybrid zone in the Northern Apennines involving all the three Plethodontid salamander species inhabiting the Italian Peninsula. We found 21 new Speleomantes sites of occurrence, from a hitherto unexplored area located at the boundaries between three Speleomantes species ranges. Using mitochondrial (Cytb and ND2 genes) and nuclear markers (two diagnostic SNPs at the NCX1 gene), we revealed a three-way contact zone where all the three mainland species hybridize: S. strinatii, S. ambrosii and S. italicus. We observed a strong mitonuclear discordance, with mitochondrial markers showing a conspicuous geographic pattern, while diagnostic nuclear SNPs coexisted in both the same populations and individuals, providing evidence of hybridization in many possible combinations. The introgression is asymmetric, with S. italicus mitogenome usually associated with S. a. ambrosii and, to a lesser extent, to S. strinatii nuclear alleles. This finding confirms that Plethodontid are a group of choice to investigate hybridization mechanisms and suggests that behavioural, genetic and ecological components may concur in determining the direction and extent of introgression.

Geographic variability of hybridization between red-breasted and red-naped sapsuckers.

Hybrid zones reveal the strength of reproductive isolation between populations undergoing speciation and are a key tool in evolutionary biology research. Multiple replicate transects across the same hybrid zone offer insight into the dynamics of hybridization in different environments, clarifying the role of extrinsic forces on the speciation process. Red-breasted and red-naped sapsuckers (Sphyrapicus ruber and Sphyrapicus nuchalis) have a long zone of contact over approximately 1,600 km from central British Columbia, Canada to central California, USA. We used Genotyping-by-Sequencing data from three independent sapsucker hybrid zone transects to compare hybridization dynamics between these species under variable geoclimatic conditions. We generated geographic clines of the genomic data to compare hybrid zone widths and used random forests models and linear regression to assess the relationship between climate and sapsucker ancestry along each transect. Our results show variation in the directionality of backcrossing, often indicative of moving hybrid zones. We note variable cline widths among transects, indicating differences in selection maintaining hybrid zone dynamics. Furthermore, random forests models identified different variables in close association with sapsucker ancestry across each transect. These results indicate a lack of repeatability across replicate transects and a strong influence of the local environment on hybrid zone dynamics.

Displaced clines in an avian hybrid zone (Thamnophilidae: Rhegmatorhina) within an Amazonian interfluve.

Secondary contact between species often results in the formation of a hybrid zone, with the eventual fates of the hybridizing species dependent on evolutionary and ecological forces. We examine this process in the Amazon Basin by conducting the first genomic and phenotypic characterization of the hybrid zone formed after secondary contact between two obligate army-ant-followers: the White-breasted Antbird (Rhegmatorhina hoffmannsi) and the Harlequin Antbird (Rhegmatorhina berlepschi). We found a major geographic displacement (∼120 km) between the mitochondrial and nuclear clines, and we explore potential hypotheses for the displacement, including sampling error, genetic drift, and asymmetric cytonuclear incompatibilities. We cannot exclude roles for sampling error and genetic drift in contributing to the discordance; however, the data suggest expansion and unidirectional introgression of hoffmannsi into the distribution of berlepschi.

Against the Odds: Hybrid Zones between Mangrove Killifish Species with Different Mating Systems.

Different mating systems are expected to affect the extent and direction of hybridization. Due to the different levels of sexual conflict, the weak inbreeder/strong outbreeder (WISO) hypothesis predicts that gametes from self-incompatible (SI) species should outcompete gametes from self-compatible (SC) ones. However, other factors such as timing of selfing and unilateral incompatibilities may also play a role on the direction of hybridization. In addition, differential mating opportunities provided by different mating systems are also expected to affect the direction of introgression in hybrid zones involving outcrossers and selfers. Here, we explored these hypotheses with a unique case of recent hybridization between two mangrove killifish species with different mating systems, Kryptolebias ocellatus (obligately outcrossing) and K. hermaphroditus (predominantly self-fertilizing) in two hybrid zones in southeast Brazil. Hybridization rates were relatively high (~20%), representing the first example of natural hybridization between species with different mating systems in vertebrates. All F1 individuals were sired by the selfing species. Backcrossing was small, but mostly asymmetrical with the SI parental species, suggesting pattern commonly observed in plant hybrid zones with different mating systems. Our findings shed light on how contrasting mating systems may affect the direction and extent of gene flow between sympatric species, ultimately affecting the evolution and maintenance of hybrid zones.

From refugia to contact: Pine processionary moth hybrid zone in a complex biogeographic setting.

Contact zones occur at the crossroad between specific dispersal routes and are facilitated by biogeographic discontinuities. Here, we focused on two Lepidoptera sister species that come in contact near the Turkish Straits System (TSS). We aimed to infer their phylogeographic histories in the Eastern Mediterranean and finely analyze their co-occurrence and hybridization patterns in this biogeographic context. We used molecular mitochondrial and nuclear markers to study 224 individuals from 42 localities. We used discordances between markers and complementary assignment methods to identify and map hybrids and parental individuals. We confirmed the parapatric distribution of Thaumetopoea pityocampa (Lepidoptera: Notodontidae) in the west and Thaumetopoea wilkinsoni in the east and identified a narrow contact zone. We identified several glacial refugia of T. wilkinsoni in southern Turkey with a strong east-west differentiation in this species. Unexpectedly, T. pityocampa crossed the TSS and occur in northern Aegean Turkey and some eastern Greek islands. We found robust evidence of introgression between the two species in a restricted zone in northwestern Turkey, but we did not identify any F1 individuals. The identified hybrid zone was mostly bimodal. The distributions and genetic patterns of the studied species were strongly influenced both by the Quaternary climatic oscillations and the complex geological history of the Aegean region. T. pityocampa and T. wilkinsoni survived the last glacial maximum in disjoint refugia and met in western Turkey at the edge of the recolonization routes. Expanding population of T. wilkinsoni constrained T. pityocampa to the western Turkish shore. Additionally, we found evidence of recurrent introgression by T. wilkinsoni males in several T. pityocampa populations. Our results suggest that some prezygotic isolation mechanisms, such as differences in timing of the adult emergences, might be a driver of the isolation between the sister species.

Past lake shore dynamics explain present pattern of unidirectional introgression across a habitat barrier.

Introgression patterns between divergent lineages are often characterized by asymmetry in the direction and among-marker variation in the extent of gene flow, and therefore inform on the mechanisms involved in differentiation and speciation. In the present study, we test the hypothesis that unidirectional introgression between two phenotypically and genetically distinct lineages of the littoral, rock-dwelling cichlid fish Tropheus moorii across a wide sandy bay is linked to observed differences in mate preferences between the two lineages. This hypothesis predicts bi-directional nuclear gene flow and was rejected by congruent patterns of introgression in mtDNA, AFLP and microsatellite markers, with admixture confined to the populations west of the bay. This pattern can be explained on the basis of habitat changes in the course of lake level fluctuations, which first facilitated the development of a symmetric admixture zone including the area corresponding to the present sand bay and then shaped asymmetry by causing local extinctions and cessation of gene flow when this area became once more inhabitable. This conforms with previous assumptions that habitat dynamics are a primary determinant of population-level evolution in Tropheus. In this respect, Tropheus may be representative of species whose preferred habitat is subject to frequent re-structuring.

Differential introgression of a female competitive trait in a hybrid zone between sex-role reversed species.

Mating behavior between recently diverged species in secondary contact can impede or promote reproductive isolation. Traditionally, researchers focus on the importance of female mate choice and male-male competition in maintaining or eroding species barriers. Although female-female competition is widespread, little is known about its role in the speciation process. Here, we investigate a case of interspecific female competition and its influence on patterns of phenotypic and genetic introgression between species. We examine a hybrid zone between sex-role reversed, Neotropical shorebird species, the northern jacana (Jacana spinosa) and wattled jacana (J. jacana), in which female-female competition is a major determinant of reproductive success. Previous work found that females of the more aggressive and larger species, J. spinosa, disproportionately mother hybrid offspring, potentially by monopolizing breeding territories in sympatry with J. jacana. We find a cline shift of female body mass relative to the genetic center of the hybrid zone, consistent with asymmetric introgression of this competitive trait. We suggest that divergence in sexual characteristics between sex-role reversed females can influence patterns of gene flow upon secondary contact, similar to males in systems with more typical sex roles.

Incomplete lineage sorting and introgression in the diversification of Chinese spot-billed ducks and mallards.

Incomplete lineage sorting and introgression are 2 major and nonexclusive causes of species-level non-monophyly. Distinguishing between these 2 processes is notoriously difficult because they can generate similar genetic signatures. Previous studies have suggested that 2 closely related duck species, the Chinese spot-billed duck Anas zonorhyncha and the mallard A. platyrhynchos were polyphyletically intermixed. Here, we utilized a wide geographical sampling, multilocus data and a coalescent-based model to revisit this system. Our study confirms the finding that Chinese spot-billed ducks and Mallards are not monophyletic. There was no apparent interspecific differentiation across loci except those at the mitochondrial DNA (mtDNA) control region and the Z chromosome (CHD1Z). Based on an isolation-with-migration model and the geographical distribution of lineages, we suggest that both introgression and incomplete lineage sorting might contribute to the observed non-monophyly of the 2 closely related duck species. The mtDNA introgression was asymmetric, with high gene flow from Chinese spot-billed ducks to Mallards and negligible gene flow in the opposite direction. Given that the 2 duck species are phenotypically distinctive but weakly genetically differentiated, future work based on genome-scale data is necessary to uncover genomic regions that are involved in divergence, and this work may provide further insights into the evolutionary histories of the 2 species and other waterfowls.

Hybridization, sex-specific genomic architecture and local adaptation.

While gene flow can reduce the potential for local adaptation, hybridization may conversely provide genetic variation that increases the potential for local adaptation. Hybridization may also affect adaptation through altering sexual dimorphism and sexual conflict, but this remains largely unstudied. Here, we discuss how hybridization may affect sexual dimorphism and conflict due to differential effects of hybridization on males and females, and then how this, in turn, may affect local adaptation. First, in species with heterochromatic sexes, the lower viability of the heterogametic sex in hybrids could shift the balance in sexual conflict. Second, sex-specific inheritance of the mitochondrial genome in hybrids may lead to cytonuclear mismatches, for example, in the form of 'mother's curse', with potential consequences for sex ratio and sex-specific expression. Third, sex-biased introgression and recombination may lead to sex-specific consequences of hybridization. Fourth, transgressive segregation of sexually antagonistic alleles could increase sexual dimorphism in hybrid populations. Sexual dimorphism can reduce sexual conflict and enhance intersexual niche partitioning, increasing the fitness of hybrids. Adaptive introgression of alleles reducing sexual conflict or enhancing intersexual niche partitioning may facilitate local adaptation, and could favour the colonization of novel habitats. We review these consequences of hybridization on sex differences and local adaptation, and discuss how their prevalence and importance could be tested empirically.This article is part of the theme issue 'Linking local adaptation with the evolution of sex differences'.

Asymmetric hybridization in Cordulegaster (Odonata: Cordulegastridae): Secondary postglacial contact and the possible role of mechanical constraints.

Two Cordulegaster dragonflies present in Italy, the Palaearctic and northern distributed Cordulegaster boltonii and the endemic to the south of the peninsula Cordulegaster trinacriae, meet in central Italy and give rise to individuals of intermediate morphology. By means of mitochondrial and nuclear markers and of Geometric Morphometrics applied to sexual appendages, we defined i) the geographical boundaries between the two species in Italy and ii) we determined the presence, the extent, and the genetic characteristics of the hybridization. Genetic data evidenced asymmetric hybridization with the males of C. trinacriae able to mate both interspecifically and intraspecifically. The results contrast with expectations under neutral gene introgression and sexual selection. This data, along with the morphological evidence of significant differences in size and shape of sexual appendages between the males of the two species, seem indicative of the role of mechanical constraints in intraspecific matings. The origin of the two species is dated about to 1.32 Mya and the hybridization resulted related to range expansion of the two species after Last Glacial Maximum and this led to the secondary contact between the two taxa in central Italy. At last, our results indicate that the range of C. trinacriae, a threatened and protected species, has been moving northward probably driven by climate changes. As a result, the latter species is currently intruding into the range of C. boltonii. The hybrid area is quite extended and the hybrids seem well adapted to the environment. From a conservation point of view, even if C. trinacriae has a strong genetic identity, the discovery of hybridization between the two species should be considered in a future species management.

Morphological versus genetic diversity of Viola reichenbachiana and V. riviniana (sect. Viola, Violaceae) from soils differing in heavy metal content.

Morphological characters, AFLP markers and flow cytometry were used to investigate the morphological and genetic variability and differentiation of Viola reichenbachiana and V. riviniana in non-metallicolous (NM) and metallicolous (M) populations. The aims were to clarify the taxonomic status of plants occurring in ore-bearing areas, to determine any relationship in V. reichenbachiana and V. riviniana from sites not polluted with heavy metals, and to examine the genetic variability and differentiation of M and NM populations of both species. Multivariate analyses based on morphological characters showed significant differences between V. reichenbachiana and V. riviniana from non-polluted sites, high levels of intra- and inter-population variability, and the occurrence of inter-specific hybrids. Plants from M populations showed hybrid characters but also fell within the range of V. riviniana or V. reichenbachiana. There were no significant differences in relative genome size between plants from polluted areas and V. riviniana from NM populations. Bayesian analysis of population genetic structure based on AFLP markers distinguished two main groups: V. reichenbachiana and V. riviniana together with the M populations. That analysis also revealed the occurrence of populations of inter-specific hybrids from non-polluted areas. Further Bayesian analysis of V. riviniana including NM and M populations separated all the studied M populations from NM populations. We conclude that plants forming the M populations are well adapted to a metal-polluted environment, and could be considered as stabilised introgressive forms resulting from unidirectional (asymmetric) introgression toward V. riviniana.