Phylogeny and species limits in the Palaearctic chiffchaff Phylloscopus collybita complex: mitochondrial genetic differentiation and bioacoustic evidence*

Nucleotide sequences of the mitochondrial cytochrome b gene (1041 bp), analysis of vocalizations and behavioural evidence from zones of contact were used to reassess the species limits and phylogenetic relationships at the species and subspecies levels in the Phylloscopus collybita complex. A new classification is proposed which recognizes four biological species. Phylloscopus brehmii (Iberia) and P. canariensis (Canary Islands) are genetically and bioacoustically highly distinct. There is no mitochondrial gene flow between them or with P. collybita. The Mountain Chiffchaff P. sindianus (with subspecies sindianus and Iorenzii) is equally distinct genetically from southwest Asian subspecies of P. collybita (caucasicus, brevirostris). The status of the Siberian form tristis, which shares potentially synapomorphic characters with the Mountain Chiffchaff (ascending song notes, grey-brown adult plumage) but genetically closely resembles P. c. collybita and P. c. abietinus, remains uncertain. In two zones of secondary contact between taxa with “greenish” (P. collybita) and brownish plumage, hybridization is either unrecorded (caucasicus v lorenzii, Caucasus Mountains) or its extent is insufficiently known (abietinus v tristis, west of Ural Mountains). A phylogeny reconstructed from nucleotide sequences agrees with one based on song and some morphological characters in identifying P. brehmii as the oldest branch within the chiffchaff group. Of the remaining taxa, four fall into a clade with greenish plumage (P. collybita ssp.), two into one with brownish plumage (P. s. sindianus, P. s. lorenzii), while the position of P. canariensis with respect to these two clades is uncertain. Molecular and phenotypic phylogenies contradict each other regarding the affinities of P. (ssp.?) tristis.     

Effects of plant density and age on the mating system of Kandelia candel Druce (Rhizophoraceae), a viviparous mangrove species

Mating system is an important factor affecting gene flow and genetic structure of plant populations. Based on identification of chlorophyll-deficient offsprings on heterozygous individuals, selfing rates were estimated for Kandelia candel, a viviparous mangrove species. Outcrossing rates were significantly decreased when population was thinned, indicating that plant density plays an important role in plant reproductive features. Selfing rate of young population was significantly higher than that of mature population, due to pollen flow barriers in young population, created by dense leaves and twigs.     

Deep mitochondrial introgression and hybridization among ecologically divergent vole species

The completion of speciation is typically difficult to ascertain in rapidly diverging taxa but the amount of hybridization and gene flow in sympatry or parapatry contains important information about the level of reproductive isolation achieved. Here, we examined the progress in speciation between the Mediterranean (Microtus duodecimcostatus) and the Lusitanian pine vole (M. lusitanicus), which are part of the most rapid radiation of species known in mammals. These two Iberian pine voles are classified as separate species because of differences in morphology and ecology, but relatively many ambiguous individuals can be found in sympatric conditions. Our phylogenetic analyses of rangewide data from the mitochondrial cytochrome b gene (mtDNA) demonstrated high levels of diversity and a basal separation in two parapatric lineages. However, mtDNA affiliation was at odds with morphological classification or geographical distribution of the taxa. In contrast, statistical analyses of microsatellites (nucDNA) showed two clear genetic clusters in allopatry and sympatry generally matching morphological classification. This cytonuclear discordance over a large geographic area suggests historical introgression of mtDNA from M. duodecimcostatus to M. lusitanicus. There was statistical evidence for at least two recent hybrids in the sympatry zone but gene flow is apparently low given clear‐cut differences in nucDNA. Our results indicate a relatively advanced speciation process in these Iberian pine voles without fully established reproductive isolation. This situation enables use of combined population genomic and experimental approaches for the separation of patterns and mechanisms in the ongoing explosive diversification of these and other Arvicoline rodents in the future.     

Ecological divergence plays an important role in strong but complex reproductive isolation in campions (Silene)*

New species arise through the evolution of reproductive barriers between formerly interbreeding lineages. Yet, comprehensive assessments of potential reproductive barriers, which are needed to make inferences on processes driving speciation, are only available for a limited number of systems. In this study, we estimated individual and cumulative strengths of seven prezygotic and six postzygotic reproductive barriers between the recently diverged taxa Silene dioica (L.) Clairv. and S. latifolia Poiret using both published and new data. A combination of multiple partial reproductive barriers resulted in near‐complete reproductive isolation between S. dioica and S. latifolia, consistent with earlier estimates of gene flow between the taxa. Extrinsic barriers associated with adaptive ecological divergence were most important, while intrinsic postzygotic barriers had moderate individual strength but contributed only little to total reproductive isolation. These findings are in line with ecological divergence as driver of speciation. We further found extensive variation in extrinsic reproductive isolation, ranging from sites with very strong selection against migrants and hybrids to intermediate sites where substantial hybridization is possible. This situation may allow for, or even promote, heterogeneous genetic divergence.     

Can alternative mating tactics facilitate introgression across a hybrid zone by circumventing female choice?

Reproductive barriers and divergence in species’ mate recognition systems underlie major models of speciation. However, hybridization between divergent species is common, and classic mechanisms to explain permeable reproductive barriers rarely consider how an individual may attain reproductive success. Alternative mating tactics (AMTs) exist in various forms across animal taxa. Such tactics may allow poorer quality individuals to gain mating opportunities and facilitate introgression either through asymmetrical positive selection or by circumventing female choice altogether in areas of secondary contact. One such tactic is satellite behaviour in frogs, where silent males perch near advertisers in an attempt to intercept females. To test whether such satellite male tactics are context‐dependent and favoured by hybrids, we genotyped and quantified the morphology of 80 male spring peeper (Pseudacris crucifer) individuals involved in caller–satellite associations from a secondary contact zone between two intraspecific mitochondrial lineages. Irrespective of population, satellite behaviour was best predicted by size but not body condition. Within the contact zone, pure individuals showed a significantly greater probability of being active callers, whereas hybrids of one lineage were more likely to adopt the satellite tactic. We suggest that satellite behaviour in P. crucifer promotes introgression, breaks down reproductive isolating barriers and contributes to asymmetrical introgression in this secondary contact zone. AMTs may thus be an underexplored but important alternative to oft‐discussed causes of genetic discordance found in hybrid zones.     

HISTORICAL DIVERGENCE AND GENE FLOW: COALESCENT ANALYSES OF MITOCHONDRIAL,AUTOSOMAL AND SEX‐LINKED LOCI IN PASSERINA BUNTINGS

Quantifying the role of gene flow during the divergence of closely related species is crucial to understanding the process of speciation. We collected DNA sequence data from 20 loci (one mitochondrial, 13 autosomal, and six sex‐linked) for population samples of Lazuli Buntings (Passerina amoena) and Indigo Buntings (Passerina cyanea) (Aves: Cardinalidae) to test explicitly between a strict allopatric speciation model and a model in which divergence occurred despite postdivergence gene flow. Likelihood ratio tests of coalescent‐based population genetic parameter estimates indicated a strong signal of postdivergence gene flow and a strict allopatric speciation model was rejected. Analyses of partitioned datasets (mitochondrial, autosomal, and sex‐linked) suggest the overall gene flow patterns are driven primarily by autosomal gene flow, as there is no evidence of mitochondrial gene flow and we were unable to reject an allopatric speciation model for the sex‐linked data. This pattern is consistent with either a parapatric divergence model or repeated periods of allopatry with gene flow occurring via secondary contact. These results are consistent with the low fitness of female avian hybrids under Haldane's rule and demonstrate that sex‐linked loci likely are important in the initial generation of reproductive isolation, not just its maintenance.     

ELECTROPHORETIC CONFIRMATION OF INTERSPECIFIC HYBRIDIZATION IN AESCULUS (HIPPOCASTANACEAE) AND THE GENETIC STRUCTURE OF A BROAD HYBRID ZONE

Within a broad (>200 km wide) hybrid zone involving three parapatric species of Aesculus, we observed coincident clines in allele frequency for 6 of 14 electrophoretic loci. The cooccurrence of alleles characteristic of A. pavia, A. sylvatica, and A. flava was used to estimate genetic admixtures in 48 populations involving various hybrids between these taxa in the southeastern United States. High levels of allelic polymorphism (up to 40% greater than the parental taxa) were observed in hybrid populations and also in some populations bordering the hybrid zone. A detailed analysis of a portion of the hybrid zone involving A. pavia and A. sylvatica revealed a highly asymmetrical pattern of gene flow, predominantly from Coastal Plain populations of A. pavia into Piedmont populations of A. sylvatica. Computer simulations were used to generate expected genotypic arrays for parental, F₁; and backcross individuals, which were compared with natural populations using a character index scoring system. In these comparisons, hybrid individuals could be distinguished from either parent, but F₁ and backcross progeny could not be distinguished from each other. Most hybrid populations were found to include hybrids and one of the parental taxa, but never both parents. Three populations appeared to be predominantly hybrids with no identifiable parental individuals. Hybrids occurred commonly at least 150 km beyond the range of A. pavia, but usually not more than 25 km beyond the range of A. sylvatica. Introgression, suggested by genetically hybrid individuals and significant gene admixtures of two or more species in populations lacking morphological evidence of hybridization, may extend the hybrid zone further in both directions. The absence of one or both parental species from hybrid populations implies a selective disadvantage to parentals in the hybrid zone and/or that hybridization has occurred through long-distance gene flow via pollen, primarily from A. pavia into A. sylvatica. Long-distance pollen movement in plants may generate hybrid zones of qualitatively different structure than those observed in animals, where gene flow involves dispersal of individuals.     

INSIGHT INTO SPECIATION FROM HISTORICAL DEMOGRAPHY IN THE PHYTOPHAGOUS BEETLE GENUS OPHRAELLA

Speciation in phytophagous insects is commonly associated with shifts in host use. Using a phylogenetic framework to identify recently diverged taxa that have undergone a radical host shift, this study focuses on how reconstruction of the historical demography of a species, in conjunction with branching patterns between species, provides insight into mode of speciation. Analyses of mitochondrial cytochrome oxidase I sequences indicate that the leaf beetle Ophraella communa exhibits significant population structure, as shown by patterns of genealogical relationships among mitochondrial haplotypes and high F_ST-values. However, the absence of regional localization of old clades of haplotypes, negative Tajima's D, and unimodal rather than bimodal frequency distribution of the number of pairwise differences between sequences suggests an absence of long-term barriers to gene flow. Furthermore, we found no evidence of isolation by distance. This pattern of genetic variation is consistent with episodes of gene flow on a large geographic scale, perhaps owing to Pleistocene changes in climate. Ophraella communa and its sister species O. bilineata diverged during the early Pleistocene. The evidence of dynamic population structure in O. communa, potentially including episodic but massive gene flow, suggests that reproductive isolation evolved quite rapidly on a localized geographic scale, because speciation would probably have been reversed by gene flow if the evolution of reproductive isolation had been prolonged. That is, gene flow occasioned by range shifts during the Pleistocene would likely have interrupted speciation unless it occurred very rapidly. Sequence diversity implies a large effective population size (> 10⁶) in both O. communa and O. bilineata. However, a model based on a drastic bottleneck did not have a lower likelihood than a model with no bottleneck, simply because the time since speciation has been great enough for coalescence to a single ancestor that existed after the speciation event. Sequence diversity in itself, without reference to the time since speciation, cannot provide evidence on the demography of speciation.     

Extremely divergent host plant acceptance with gene flow between sympatric host races of the phytophagous ladybird beetle Henosepilachna diekei

Ecological divergence alone can prevent the majority of gene flow in the absence of other forms of reproductive isolation. Although the importance of ecological divergence in promoting reproductive isolation has been broadly recognized, its net impact on speciation has rarely been estimated in the wild. The phytophagous ladybird beetle Henosepilachna diekei Jadwiszczak & Wegrzynowicz includes two sympatric host races that are reproductively isolated solely by extreme specialization to either of the host plants Mikania micrantha Kunth (Asteraceae) or Leucas lavandulifolia Sm. (Lamiaceae) in West Java, Indonesia. To investigate the impact of differential host use as an isolating barrier, we carried out adult host acceptance tests and molecular population genetic analyses based on mitochondrial ND2 and nucleic ITS2 gene sequences using 13 wild populations of the host races, including four sympatric population pairs. Almost all individuals of these host races persistently accepted only the original host plant. We detected restricted but a degree of gene flow between these host races. A migration event occurred only in very recent time compared to their divergence time, indicating recent secondary contact of these host races in the surveyed area. These results reveal the remarkably large impact of host‐plant shift over almost the entire process of speciation and illustrate that ecological divergence has been maintained even under the presence of a certain degree of gene flow.     

Genetic analysis of a contact zone between two lineages of the ocellated lizard (Lacerta lepida Daudin 1802) in south‐eastern Iberia reveal a steep and narrow hybrid zone

Measuring the diffusion of genes between diverging taxa through zones of secondary contact is an essential step to understand the extent and nature of the reproductive isolation that has been achieved. Previous studies have shown that the ocellated lizard (Lacerta lepida Daudin, 1802) has endured repeated range fragmentation associated with the climatic oscillations of the Plio‐Pleistocene that promoted diversification of many different evolutionary units within the species. However, the oldest divergence within the group is estimated to have occurred much earlier, during the Miocene, around 9 Ma and corresponds to the split between the subspecies Lacerta lepida nevadensis Buchholz (1963) and Lacerta lepida lepida Daudin (1802). Although these two evolutionary units have documented genetic and morphological differentiation, most probably accumulated during periods of allopatry, little is known about patterns of gene flow between them. In this study, we performed a population genetic analysis of a putative area of secondary contact between these two taxa, using mtDNA and microsatellite data. We assessed levels of gene flow across the contact zone to clarify to what extent gene flow may be occurring. Hybridization between the subspecies was observed by the presence of genetically introgressed individuals. However, the overall coincidence of mitochondrial and multilocus nuclear clines and generally steep clines support the idea that this contact zone is acting as a barrier to gene flow. Taken together, these results suggest that L. l. lepida and L. l. nevadensis are in independent evolutionary trajectories and should be considered as two different species.     

EVOLUTIONARY IMPLICATIONS OF DNA DIVERGENCE IN THE DROSOPHILA OBSCURA GROUP

Using DNA–DNA hybridization, we have determined the degree of single-copy DNA (scDNA) divergence among eight species of the Drosophila obscura group. These include Old World and New World species as well as members of two subgroups. Contrary to classical systematics, members of the affinis subgroup are more closely related to American members of the obscura subgroup than are Old World species. The Old World species are not a monophyletic group. The degree of scDNA divergence among species is not necessarily correlated with morphology, chromosomal divergence, or ability to form hybrids. A unique pattern of hybrid formation was found: species separated by a ΔT_m of 6.5°C can form hybrids whereas species separated by a ΔT_m of 2.5°C cannot. As with other groups of Drosophila, the obscura group has discrete parts of the genome evolving at very different rates. The slow evolving fraction of the nuclear genome is evolving at about the same rate as mitochondrial DNA. The additional scDNA divergence accompanying the step from partial reproductive isolation (between North American pseudoobscura and the isolated Bogotà population) to full isolation is very small. The resolution of the technique was challenged by five closely related taxa with a maximum ΔT_m of 2.5°C separating them; the taxa were unambiguously resolved and the “correct” phylogeny recovered. Finally, there is some indication that scDNA in the obscura group may be evolving considerably slower than in the melanogaster subgroup.     

Distinctiveness in the face of gene flow: hybridization between specialist and generalist gartersnakes

Patterns of divergence and polymorphism across hybrid zones can provide important clues as to their origin and maintenance. Unimodal hybrid zones or hybrid swarms are composed predominantly of recombinant individuals whose genomes are patchworks of alleles derived from each parental lineage. In contrast, bimodal hybrid zones contain few identifiable hybrids; most individuals fall within distinct genetic clusters. Distinguishing between hybrid swarms and bimodal hybrid zones can be important for taxonomic and conservation decisions regarding the status and value of hybrid populations. In addition, the causes of bimodality are important in understanding the generation and maintenance of biological diversity. For example, are distinct clusters mostly reproductively isolated and co‐adapted gene complexes, or can distinctiveness be maintained by a few ‘genomic islands’ despite rampant gene flow across much of the genome? Here we focus on three patterns of distinctiveness in the face of gene flow between gartersnake taxa in the Great Lakes region of North America. Bimodality, the persistence of distinct clusters of genotypes, requires strong barriers to gene flow and supports recognition of distinct specialist (Thamnophis butleri) and generalist (Thamnophis radix) taxa. Concordance of DNA‐based clusters with morphometrics supports the hypothesis that trophic morphology is a key component of divergence. Finally, disparity in the level of differentiation across molecular markers (amplified fragment length polymorphisms) indicates that distinctiveness is maintained by strong selection on a few traits despite high gene flow currently or in the recent past.     

Genetic population structure of the paper wasp Polistes olivaceus (Hymenoptera: Vespidae) in Bangladesh

Dispersal triggers gene flow, which in turn strongly affects the ensuing genetic population structure of a species. Using nuclear microsatellite loci and mitochondrial DNA (mtDNA), we estimated the genetic population structure of the wasp Polistes olivaceus throughout Bangladesh. The level of population differentiation using nuclear markers (F _ST) appeared to be much lower than that estimated using mtDNA haplotype sequences (Ф_ST), even after correcting for effective population size differences between the two markers. These results suggest a philopatric tendency, in which gynes disperse less than males. We observed no isolation by distance among the study populations at either the nuclear or mtDNA level, suggesting nonequilibrium between gene flow and drift as a result of very frequent interpopulation movement. For the nuclear markers, an individual assignment test showed no genetically and geographically distinct groups. Instead, phylogenetic analyses as well as a minimum spanning network using mtDNA haplotypes consistently revealed two distinct lineages. The distribution of haplotypes indicated western populations with a single lineage and offered clear evidence for restricted gene flow across the Jamuna–Padma–Upper Meghna river system. Mismatch distributions exhibited a unimodal distribution, which along with a starlike haplotype network, suggested a population expansion in lineage I but not in lineage II. Overall, these results suggest that gene flow among populations of P. olivaceus was affected by both female philopatry and a major river system across Bangladesh.     

Characterization of a contemporaneous hybrid zone between two darter species (Etheostoma bison and E. caeruleum) in the Buffalo River System

Hybrid zones have long intrigued evolutionary biologists and provide a natural laboratory to explore the evolution of reproductive isolation (speciation). Molecular characterization of hybrid zone dynamics can provide insight into the strength of reproductive isolation as well as the underlying evolutionary processes shaping gene flow. Approximately one-third of darter species naturally hybridize making this species-rich North American freshwater teleost fish clade an ideal system to investigate the extent and direction of hybridization. The objective of this study was to use diagnostic microsatellite markers to calculate genetic hybrid index scores of two syntopic, but distantly related darter species, Etheostoma bison and Etheostoma caeruleum. A combination of hybrid index scores, assignment tests, and mitochondrial haplotype profiles uncovered mixed ancestry in approximately 6 % of sampled adult individuals, supporting contemporaneous hybridization that was previously undocumented in E. bison. Moreover, hybrids were not limited to the F₁ generation, but encompassed the entire suite of hybrid categories (F₁, F₂ and backcross hybrids). The low number of hybrids assigned to each hybrid category represents a bimodal hybrid zone, suggesting reproductive isolation is strong (but incomplete) and also advocates for the ability of hybrids to produce second-generation hybrids and backcross into both parental species, mediating introgression across species boundaries. To this end, cytonuclear profiles of the sampled parental species and hybrids were consistent with bidirectional gene flow, although there was an overall trend of asymmetric hybridization between E. caeruleum females and E. bison males. The spatiotemporal variation in hybridization rates and resulting cytonuclear patterns expanded on in this study provide a comparative genetic framework on which future studies can begin to elucidate the underlying processes that not only generate a mosaic hybrid zone, but maintain the distinctness of species in the face of gene flow.     

PATTERNS OF SPECIATION IN DROSOPHILA

To investigate the time course of speciation, we gathered literature data on 119 pairs of closely related Drosophila species with known genetic distances, mating discrimination, strength of hybrid sterility and inviability, and geographic ranges. Because genetic distance is correlated with divergence time, these data provide a cross-section of taxa at different stages of speciation. Mating discrimination and the sterility or inviability of hybrids increase gradually with time. Hybrid sterility and inviability evolve at similar rates. Among allopatric species, mating discrimination and postzygotic isolation evolve at comparable rates, but among sympatric species strong mating discrimination appears well before severe sterility or inviability. This suggests that prezygotic reproductive isolation may be reinforced when allopatric taxa become sympatric. Analysis of the evolution of postzygotic isolation shows that recently diverged taxa usually produce sterile or inviable male but not female hybrids. Moreover, there is a large temporal gap between the evolution of male-limited and female hybrid sterility or inviability. This gap, which is predicted by recent theories about the genetics of speciation, explains the overwhelming preponderance of hybridizations yielding male-limited hybrid sterility or inviability (Haldane's rule).     

Low reproductive isolation and highly variable levels of gene flow reveal limited progress towards speciation between European river and brook lampreys

Ecologically based divergent selection is a factor that could drive reproductive isolation even in the presence of gene flow. Population pairs arrayed along a continuum of divergence provide a good opportunity to address this issue. Here, we used a combination of mating trials, experimental crosses and population genetic analyses to investigate the evolution of reproductive isolation between two closely related species of lampreys with distinct life histories. We used microsatellite markers to genotype over 1000 individuals of the migratory parasitic river lamprey (Lampetra fluviatilis) and freshwater‐resident nonparasitic brook lamprey (Lampetra planeri) distributed in 10 sympatric and parapatric population pairs in France. Mating trials, parentage analyses and artificial fertilizations demonstrated a low level of reproductive isolation between species even though size‐assortative mating may contribute to isolation. Most parapatric population pairs were strongly differentiated due to the joint effects of geographic distance and barriers to migration. In contrast, we found variable levels of gene flow between sympatric populations ranging from panmixia to moderate differentiation, which indicates a gradient of divergence with some population pairs that may correspond to alternative morphs or ecotypes of a single species and others that remain partially isolated. Ecologically based divergent selection may explain these variable levels of divergence among sympatric population pairs, but incomplete genome swamping following secondary contact could have also played a role. Overall, this study illustrates how highly differentiated phenotypes can be maintained despite high levels of gene flow that limit the progress towards speciation.     

Attack of the clones: reproductive interference between sexuals and asexuals in the Crepis agamic complex

Negative reproductive interactions are likely to be strongest between close relatives and may be important in limiting local coexistence. In plants, interspecific pollen flow is common between co‐occurring close relatives and may serve as the key mechanism of reproductive interference. Agamic complexes, systems in which some populations reproduce through asexual seeds (apomixis), while others reproduce sexually, provide an opportunity to examine effects of reproductive interference in limiting coexistence. Apomictic populations experience little or no reproductive interference, because apomictic ovules cannot receive pollen from nearby sexuals. Oppositely, apomicts produce some viable pollen and can exert reproductive interference on sexuals by siring hybrids. In the Crepis agamic complex, sexuals co‐occur less often with other members of the complex, but apomicts appear to freely co‐occur with one another. We identified a mixed population and conducted a crossing experiment between sexual diploid C. atribarba and apomictic polyploid C. barbigera using pollen from sexual diploids and apomictic polyploids. Seed set was high for all treatments, and as predicted, diploid–diploid crosses produced all diploid offspring. Diploid–polyploid crosses, however, produced mainly polyploidy offspring, suggesting that non‐diploid hybrids can be formed when the two taxa meet. Furthermore, a small proportion of seeds produced in open‐pollinated flowers was also polyploid, indicating that polyploid hybrids are produced under natural conditions. Our results provide evidence for asymmetric reproductive interference, with pollen from polyploid apomicts contributing to reduce the recruitment of sexual diploids in subsequent generations. Existing models suggest that these mixed sexual–asexual populations are likely to be transient, eventually leading to eradication of sexual individuals from the population.     

The effect of mating system on growth of Arabidopsis lyrata in response to inoculation with the biotrophic parasite Albugo candida

The effects of variation in host reproductive systems on response to pathogens are not well understood. We inoculated individuals from outcrossing and inbreeding populations of North American Arabidopsis lyrata with Albugo candida (white blister rust) to test the effect of mating system and heterozygosity on disease response. We observed three host infection phenotypes, classified as fully resistant, partially resistant and fully susceptible. Overall, inbreeding populations had more susceptible and fewer partially resistant individuals than outcrossing populations, but the highest proportion of resistant individuals was found in two of the inbreeding populations. Mating system did not affect relative growth rate of inoculated plants, but there were strong effects of population and infection phenotype. We conclude that mating system per se does not determine the resistance of natural A. lyrata populations to infection by Albugo, but that the increased variability in responses among inbreeding populations may be due to reduced effective population size.     

On the evolution of heavy-metal tolerant populations in Armeria maritima: evidence from allozyme variation and reproductive barriers

The process of ecological differentiation leading to the evolution of heavy-metal tolerant populations in Armeria maritima was studied by comparing population genetic structure and pattern of gene flow between populations growing on heavy-metal contaminated against non-contaminated sites using allozyme markers. In addition the evolution of reproductive isolation among populations was studied by measuring pollen fertility in interpopulational hybrids. The allozyme data suggested that in A. maritima multiple independent evolutionary origins of heavy-metal tolerant populations have occurred in the absence of strong genetic bottlenecks. The pattern of gene flow among populations was consistent with the model of isolation by distance with considerable gene flow between neighbor populations, and no reduction of gene flow between tolerant and non-tolerant populations. Hence it appears that substantial gene flow has not hampered genetic differentiation, probably because of the high selection pressure for heavy-metal tolerance. The pattern of reproductive isolation among populations suggests that evolution of heavy-metal tolerant populations has not triggered the development of reproductive barriers against non-tolerant populations. However, partial reproductive isolation has occurred under geographic separation.