THE EFFECT OF HABITAT ON PARENTAL AND HYBRID FITNESS: TRANSPLANT EXPERIMENTS WITH LOUISIANA IRISES

We performed transplant experiments with Louisiana irises to test the assumptions of three models of hybrid zone structure: the bounded hybrid superiority model, the mosaic model, and the tension zone model. Rhizomes of Iris fulva, I. hexagona, and F₁ and F₂ hybrids were planted at four sites in southeastern Louisiana in 1994. Wild irises grew at all four sites, but differed in genotypic composition among sites. The sites were characterized by (1) pure I. fulva plants; (2) I. fulva-like hybrids; (3) I. hexagona-like hybrids; and (4) pure I. hexagona plants. The sites differed significantly in light availability, soil moisture and chemical composition, and vegetation. Survival of transplants was high in all sites and did not differ significantly among plant classes. Iris hexagona produced significantly more leaf material than I. fulva at the I. hexagona and I. hexagona hybrid sites. The two species did not differ in leaf production at the I. fulva and I. fulva hybrid sites. Leaf production by both classes of hybrid was as great as, or significantly greater than, both parental classes in all sites. Iris hexagona rhizomes gained mass in the I. hexagona and I. hexagona hybrid sites, but lost mass in the I. fulva and I. fulva hybrid sites. Iris fulva rhizomes lost mass in all sites. There were no significant differences in rhizome growth among classes at the I. fulva site. At all other sites, F₁ rhizomes grew significantly more than all other classes except for I. hexagona at the I. hexagona hybrid site. There were no significant differences among classes in the production of new ramets. Overall blooming frequencies were 30% for I. fulva, 10% for F₁s, 3% for F₂s, and 0.7% for I. hexagona. Blooming frequency did not differ among sites for I. fulva, but significantly more F₁s bloomed at the I. hexagona site than at the I. fulva site. These results are inconsistent with all three models of hybrid zone structure. They suggest that once rhizomes become established, hybrids can reproduce by clonal growth as successfully as parents in all habitats, and can outperform them in some habitats. Clonal reproduction may ensure the long-term survival of early generation hybrids and allow the establishment of introgressed populations, despite the fact that F₁ hybrids are rarely produced in nature.     

Reproductive interactions between hybridizing irises: analyses of pollen-tube growth and fertilization success

Pollen-tube growth and seed siring ability were measured in crosses between the Louisiana iris species Iris fulva and Iris hexagona and their F₁ and F₂ hybrids. Flowers of the parental species were pollinated with self, outcross intraspecific, and interspecific pollen. Pollen-tube lengths were similar for all three pollen types in I. fulva, but in I. hexagona interspecific pollen tubes were longer than intraspecific pollen tubes. Pollen-tube lengths also differed for F₁ and F₂ flowers pollinated with I. fulva, I. hexagona, and hybrid pollen. For both hybrid classes I. fulva pollen tubes were the shortest while pollen tubes from I. hexagona and hybrids grew the furthest. Mixtures of genetically marked pollen were used to assess the seed siring ability of intra- and interspecific pollen in the parental species by varying the proportion of each pollen type in a replacement series design. For both species, the observed proportions of hybrid seeds were lower than the expected based on the frequency of each pollen type in the mixtures across all treatments. Flowers of I. fulva produced less than 10% hybrid progeny even when 75% of the pollen applied to stigmas was derived from interspecific flowers. The frequency of hybrid seed formation was somewhat greater in I. hexagona, but was still significantly lower than expected across all mixture treatments. Seed set per fruit remained constant across the mixture treatments for both species, but in I. fulva fruit set decreased with an increase in the proportion of interspecific pollen. The data indicate that both pre- and postfertilization processes contribute to discrimination against hybrid seed formation.     

HYBRID FITNESS IN THE LOUISIANA IRISES: ANALYSIS OF PARENTAL AND F1 PERFORMANCE

The assumption of hybrid inferiority is central to the two models most widely applied to the prediction of hybrid zone evolution. Both the tension zone and mosaic models assume that natural selection acts against hybrids regardless of the environment in which they occur. To test this assumption, we investigated components of fitness in Iris fulva, I. hexagona and their reciprocal F₁ hybrids under greenhouse conditions. The four cross types were compared on the basis of seed germination, vegetative and clonal growth, and sexual reproduction. In all cases, the hybrids performed as well as, or significantly better than, both of their parents. These results suggest that F₁ hybrids between I. fulva and I. hexagona are at least as fit as their parents. The results of this study are therefore inconsistent with the assumptions of both the tension zone and mosaic models of hybrid zone evolution.     

IRIS NELSONII (IRIDACEAE): ORIGIN AND GENETIC COMPOSITION OF A HOMOPLOID HYBRID SPECIES

Nuclear and chloroplast DNA variation was assayed for two populations of Louisiana irises (Bayou Teche and Young's Coulee) that demonstrated extreme morphological variation and for a sample of the putative hybrid species, Iris nelsonii. The genetic markers examined in this analysis were diagnostic for either Iris fulva. Iris hexagona, or Iris brevicaulis. These data demonstrated that the two morphologically variable populations were hybrid associations involving all three of these species and that all three of these species were involved in the origin of I. nelsonii. The distribution of genetic variation in I. nelsonii was significantly different from that present in either of the two hybrid populations. I. nelsonii demonstrated significantly fewer foreign markers than the two hybrid populations. This finding is in accord with the prediction that I. nelsonii is a hybrid species that has undergone stabilization with regard to genetic recombination and segregation. Although the genotypic makeup of I. nelsonii was significantly different from other parental and hybrid populations, individual plants from this species cannot be unequivocally differentiated from either I. fulva or certain hybrid genotypes. This reflects the paradoxical nature of genotypic variation in hybrid species. Thus, a hybrid species may include genotypes that overlap with both parental and contemporary hybrid populations. In the case of I. nelsonii it is necessary to utilize additional information (morphological, chromosomal, ecological) to identify plants belonging to this taxon. One hybrid population (Young's Coulee) is suggested as a paradigm for the progenitor population that gave rise to I. nelsonii.     

Integrating Bayesian genomic cline analyses and association mapping of morphological and ecological traits to dissect reproductive isolation and introgression in a Louisiana Iris hybrid zone

Hybrid zones provide unique opportunities to examine reproductive isolation and introgression in nature. We utilized 45,384 single nucleotide polymorphism (SNP) loci to perform association mapping of 14 floral, vegetative and ecological traits that differ between Iris hexagona and Iris fulva, and to investigate, using a Bayesian genomic cline (BGC) framework, patterns of genomic introgression in a large and phenotypically diverse hybrid zone in southern Louisiana. Many loci of small effect size were consistently found to be associated with phenotypic variation across all traits, and several individual loci were revealed to influence phenotypic variation across multiple traits. Patterns of genomic introgression were quite heterogeneous throughout the Louisiana Iris genome, with I. hexagona alleles tending to be favoured over those of I. fulva. Loci that were found to have exceptional patterns of introgression were also found to be significantly associated with phenotypic variation in a small number of morphological traits. However, this was the exception rather than the rule, as most loci that were associated with morphological trait variation were not significantly associated with excess ancestry. These findings provide insights into the complexity of the genomic architecture of phenotypic differences and are a first step towards identifying loci that are associated with both trait variation and reproductive isolation in nature.     

CPDNA INHERITANCE IN INTERSPECIFIC CROSSES AND EVOLUTIONARY INFERENCE IN LOUISIANA IRISES

Inheritance of chloroplast DNA haplotypes was determined for progeny from interspecific crosses involving Iris fulva and Iris hexagona. Polymerase chain reaction amplification of chloroplast DNA followed by restriction fragment length analysis of the amplification products was used to identify the haplotypes of 213 experimental hybrids. This analysis allowed a test for maternal, paternal, and biparental inheritance in the hybrid offspring. Two of the hybrid progeny possessed haplotypes that were combinations of those present in the pollen and seed parents. One of the offspring possessed only the paternal haplotype. The remaining 210 plants had the haplotypes characteristic of the maternal plant. Chloroplast DNA variation in iris populations has previously been used to infer not only introgressive hybridization between I. fulva and I. hexagona, but also the greater role of direct pollen transfer relative to seed dispersal as the avenue for interspecific gene flow. We reexamined the previous conclusions concerning the mode of introgressive hybridization between I. fulva and I. hexagona in light of the results from the chloroplast DNA inheritance analysis. The low level of paternal and biparental inheritance detected in this analysis suggests that previous analyses using the chloroplast DNA as a seed-specific marker were robust. Furthermore, data concerning barriers to hybridization between I. fulva and I. hexagona suggest that the probability of chloroplast DNA introgression via pollen is low.     

NATURAL HYBRIDIZATION BETWEEN IRIS FULVA AND IRIS HEXAGONA: PATTERN OF RIBOSOMAL DNA VARIATION

Genetic variation associated with the natural hybridization of Iris fulva and I. hexagona was investigated to test for the occurrence of introgression. These species have been viewed as a classic example of the process of introgressive hybridization (Anderson, 1949). However, more recent studies have concluded that there has not been an exchange of genetic material between I. fulva and I. hexagona (Randolph et al., 1967). Our analysis has involved the examination of both allopatric and parapatric populations of I. fulva and I. hexagona with reference to diagnostic ribosomal DNA markers. The pattern of variation in the parapatric population indicates the presence of the repeated backcrossing necessary to the process of introgressive hybridization. Indeed, in the region of parapatry, we suggest that localized introgression of ribosomal sequences has occurred into both I. fulva and I. hexagona. Significantly, we have also detected the presence of the diagnostic ribosomal markers from each species in allopatric populations of the alternate species. Our findings suggest that not only is introgressive hybridization presently occurring in parapatry between I. fulva and I. hexagona, but that past hybridization between these species has resulted in introgression into areas of allopatry.     

EFFECTS OF DIFFERENTIAL POLLEN-TUBE GROWTH ON HYBRIDIZATION IN THE LOUISIANA IRISES

To elucidate the importance of hybridization in evolution, it is necessary to understand the processes that affect hybridization frequency in nature. Here we focus on postpollination, prefertilization isolating mechanisms using two hybridizing species of Louisiana iris as a study system. We compared the effects of differential pollen-tube growth on the frequency of F₁ hybrid formation in experimental crosses between Iris fulva and Iris hexagona. Analyses of seed production in fruits from pure conspecific and heterospecific pollinations revealed that more seeds were produced in the top half than the bottom half of fruits for all four crosses. Heterospecific pollen was applied to flowers of each species at zero to 24 h prior to conspecific pollen, thereby giving a head start to the foreign pollen. Using diagnostic isozyme markers, the frequency of hybrid progeny was examined at the level of the whole fruit and separately for the top and bottom halves of fruits. In both species, the proportion of hybrid seeds per fruit increased significantly with increasing head starts, suggesting that differences in pollen-tube growth rates affect the frequency of hybridization. In I. fulva fruits, the increase in hybrid seeds occurred in both halves of the fruits, but in I. hexagona an increase was only detected in the top half of fruits. These findings are consistent with a model that assumes attrition of pollen tubes due to the greater length of I. hexagona styles. While pollen-tube growth rate appears to be the most important factor affecting hybridization frequency in I. fulva, both pollen-tube growth rate and pollen-tube attrition appear to be important in I. hexagona.     

POLLEN-TUBE COMPETITION,SIRING SUCCESS,AND CONSISTENT ASYMMETRIC HYBRIDIZATION IN LOUISIANA IRISES

Postpollination mechanisms can play an important role in limiting natural hybridization in plants. Reciprocal hand pollination experiments were performed to study these mechanisms in two species of Louisiana iris: Iris brevicaulis and I. fulva. Relative pollen-tube growth rates changed significantly through time, with I. fulva tubes increasingly outperforming I. brevicaulis tubes in both conspecific and heterospecific styles. However, this pattern of change in relative performance was a poor predictor of siring success: the majority of seeds sired by both maternal species was conspecific rather than hybrid. Experimental crosses and field studies show consistent asymmetric hybridization in Louisiana irises, with I. fulva being a more successful father and a more selective mother than both I. brevicaulis and a third species, I. hexagona. The cause of this pattern is not yet clear, but the pattern itself is unusual. Typically, short-styled species tend to be less successful in reciprocal crosses than long-styled relatives, but I. fulva has shorter styles than either I. brevicaulis or I. hexagona. The effects of pollen-tube competition, differential fertilization, and selective abortion in causing this pattern of asymmetric hybridization is discussed.     

ECOLOGICAL AND GENETIC ASSOCIATIONS IN AN IRIS HYBRID ZONE

Chloroplast DNA (cpDNA) markers and 12 nuclear (random amplified polymorphic DNA, or RAPD) markers were used to examine the distribution of genetic variation among individuals and the genetic and ecological associations in a hybrid iris population. Plants in the population occurred at various distances from the edge of a bayou in a relatively undisturbed mixed hardwood forest and in an adjacent pasture dominated by herbaceous perennials with interspersed oak and cypress trees. The majority of plants sampled possessed combinations of markers from the different Iris species. Genetic markers diagnostic for Iris fulva and I. brevicaulis occurred at high frequencies, whereas markers diagnostic for I. hexagona were infrequent. For the majority of the nuclear markers, significant levels of cytonuclear disequilibria existed because of intraspecific associations among the markers in both the pasture and the forest. The distribution of nuclear markers among individuals was bimodal; intermediate genotypes were absent and the majority of RAPD markers were associated with their intraspecific cpDNA haplotypes. Strong intraspecific associations existed among RAPD markers in the forest, but associations tended to be weaker in the pasture area. Ecological correlations were detected for all but one of the I. fulva and I. brevicaulis RAPD markers. The ecological associations of hybrids similar to I. brevicaulis resembled associations of I. brevicaulis parental genotypes, suggesting that these hybrid genotypes may be relatively fit in the same habitats. The hybrids similar to I. fulva, however, were distributed in habitats that were unique relative to the parental species. The patterns of genetic and environmental associations along with other available data suggest that (1) only advanced generation hybrids were present in the population; (2) formation of F₁ hybrids among Louisiana irises is rare, leading to sporadic formation of hybrid populations; and (3) selection and assortative mating have contributed to the formation of hybrid genotypes that tend to be similar to parental genotypes. The patterns of ecological and genetic associations detected in this population suggest that assortative mating and environmental and viability selection are important in the structuring and maintenance of this hybrid zone.     

Pollinator behaviour and the evolution of Louisiana iris hybrid zones

Pollinator preference may influence the origin and dynamics of plant hybrid zones. Natural hybrid populations between the red‐flowered Iris fulva and the blue‐flowered Iris brevicaulis are found in southern Louisiana. The genetic structure of these populations reflects a lack of intermediate genotypes. We observed pollinator behaviour in an experimental array with five plants each of I. fulva, I. brevicaulis, their F₁, and the first backcross generation in each direction, to obtain data on flower type preferences and transitions between flower types. The most abundant visitors were Ruby‐throated Hummingbirds (Archilochus colubris) and workers of the bumblebee Bombus pennsylvanicus. Hummingbirds visited I. fulva twice as often as I. brevicaulis and visited hybrids at intermediate frequencies. Bumblebee workers preferred the purple‐flowered F₁s and visited plants of I. fulva and the backcross to I. fulva more often than I. brevicaulis and its backcross. Overall, F₁ flowers were visited most frequently. Both hummingbirds and bumblebees visited nearest neighbours in almost 80% of the interplant movements. This meant that a majority of movements were between different flower types, rather than between plants of the same type. Findings from the present study suggest that pollinator preference is not a major causal factor for the lack of intermediate genotypes in natural iris hybrid populations. Instead, pollinator behaviour in our array promoted mixed mating between flower types belonging to different pollination syndromes. However, owing to predominant nearest‐neighbour visitation, the spatial distribution of parental and hybrid genotypes (in concert with pollinator behaviour) will have a strong influence on mating patterns and thus the genotypic structure and evolution of Louisiana iris hybrid zones.     

Determining population structure and hybridization for two iris species

Identifying processes that promote or limit gene flow can help define the ecological and evolutionary history of a species. Furthermore, defining those factors that make up “species boundaries” can provide a definition of the independent evolutionary trajectories of related taxa. For many species, the historic processes that account for their distribution of genetic variation remain unresolved. In this study, we examine the geographic distribution of genetic diversity for two species of Louisiana Irises, Iris brevicaulis and Iris fulva. Specifically, we asked how populations are structured and if population structure coincides with potential barriers to gene flow. We also asked whether there is evidence of hybridization between these two species outside Louisiana hybrid zones. We used a genotyping‐by‐sequencing approach and sampled a large number of single nucleotide polymorphisms across these species' genomes. Two different population assignment methods were used to resolve population structure in I. brevicaulis; however, there was considerably less population structure in I. fulva. We used a species tree approach to infer phylogenies both within and between populations and species. For I. brevicaulis, the geography of the collection locality was reflected in the phylogeny. The I. fulva phylogeny reflected much less structure than detected for I. brevicaulis. Lastly, combining both species into a phylogenetic analysis resolved two of six populations of I. brevicaulis that shared alleles with I. fulva. Taken together, our results suggest major differences in the level and pattern of connectivity among populations of these two Louisiana Iris species.     

Detecting adaptive trait introgression between Iris fulva and I. brevicaulis in highly selective field conditions

The idea that natural hybridization has served as an important force in evolutionary and adaptive diversification has gained considerable momentum in recent years. By combining genome analyses with a highly selective field experiment, we provide evidence for adaptive trait introgression between two naturally hybridizing Louisiana Iris species, flood-tolerant Iris fulva and dry-adapted I. brevicaulis. We planted reciprocal backcross (BC1) hybrids along with pure-species plants into natural settings that, due to a flooding event, favored I. fulva. As expected, I. fulva plants survived at much higher rates than I. brevicaulis plants. Backcross hybrids toward I. fulva (BCIF) also survived at significantly higher rates than the reciprocal backcross toward I. brevicaulis (BCIB). Survivorship of BCIB hybrids was strongly influenced by the presence of a number of introgressed I. fulva alleles located throughout the genome, while survivorship in the reciprocal BCIF hybrids was heavily influenced by two epistatically acting QTL of opposite effects. These results demonstrate the potential for adaptive trait introgression between these two species and may help to explain patterns of genetic variation observed in naturally occurring hybrid zones.     

NEUTRAL GENE FLOW ACROSS SINGLE LOCUS CLINES

We study the strength of the genetic barrier to neutral gene flow in a general one-locus, two-allele model that includes viability selection as well as fertility selection and premating isolation. We have separately considered adult migration and pollen migration. Our theoretical results suggest that selection against hybrid formation in the form of fertility selection or assortative mating is more effective in preventing introgression of neutral alleles than is reduction in hybrid viability. We argue that in experimental studies of introgression testing of F₁ hybrids is as important as testing of parental forms. To illustrate the utility of this approach, we estimate the strength of the barrier to neutral gene flow between Piriqueta caroliniana and P. viridis, between Iris hexagona and I. fulva, and between Chorthippus brunneus and C. biguttulus.     

The genetic architecture of reproductive isolation in Louisiana irises: pollination syndromes and pollinator preferences

In animal-pollinated plants, pollinator preferences for divergent floral forms can lead to partial reproductive isolation. We describe regions of plant genomes that affect pollinator preferences for two species of Louisiana Irises, Iris brevicaulis and Iris fulva, and their artificial hybrids. Iris brevicaulis and I. fulva possess bee and bird-pollination syndromes, respectively. Hummingbirds preferred I. fulva and under-visited both I. brevicaulis and backcrosses toward this species. Lepidopterans preferred I. fulva and backcrosses toward I. fulva, but also under-visited I. brevicaulis and I. brevicaulis backcrosses. Bumblebees preferred I. brevicaulis and F1 hybrids and rarely visited I. fulva. Although all three pollen vectors preferred one or the other species, these preferences did not prevent visitation to other hybrid/parental classes. Quantitative trait locus (QTL) mapping, in reciprocal BC1 mapping populations, defined the genetic architecture of loci that affected pollinator behavior. We detected six and nine QTLs that affected pollinator visitation rates in the BCIb and BCIf mapping populations, respectively, with as many as three QTLs detected for each trait. Overall, this study reflects the possible role of quantitative genetic factors in determining (1) reproductive isolation, (2) the pattern of pollinator-mediated genetic exchange, and thus (3) hybrid zone evolution.     

GENETIC INTERACTIONS AND NATURAL SELECTION IN LOUISIANA IRIS HYBRIDS

Hybridization between divergent lineages has long been assumed to give rise to unfavorable interactions between the parental genomes. These deleterious genetic interactions are further assumed to result in the production of hybrid offspring with decreased levels of viability and/or fertility. To test this assumption, we investigated the role of both nuclear and cytonuclear epistatic interactions in determining the frequencies of F₂ genotypes produced in crosses between two species of Louisiana iris, Iris fulva and I. brevicaulis. Overall, these crosses revealed a significant deficit of intermediate hybrid genotypes accompanied by an excess of parental-like genotypes, suggesting that genetic interactions may promote postmating reproductive isolation between these species. However, analyses of single and multilocus segregation patterns revealed a variety of negative and positive interactions between the genomes of the parental taxa at the nuclear and cytonuclear levels. Taken together, these results indicate that the traditional view that interactions between divergent genomes are always deleterious is an oversimplification. Rather, it seems likely that crosses between divergent lineages can lead to the production of both fit and unfit hybrid genotypes.     

THE GENETIC ARCHITECTURE OF REPRODUCTIVE ISOLATION IN LOUISIANA IRISES: HYBRID FITNESS IN NATURE

Negative epistasis in hybrid genomes commonly results in postzygotic isolation between divergent lineages. However, some genomic regions may be selectively neutral or adaptive in hybrids and thus may potentially cross species barriers. We examined postzygotic isolation between ecologically similar species of Louisiana Iris: Iris brevicaulis and I. fulva to determine the potential for adaptive introgression in nature. Line-cross analyses allowed us a general overview of the gene action responsible for fitness-related traits. We then used a QTL mapping approach to detect genomic regions responsible for variation in these traits. Although hybrid classes suffered reduced fitness for many traits, hybrid means were equivalent to at least one of the parental species in overall estimates of maternal and paternal fitness during the two years of the field study. The genetic architecture underlying the fitness-related traits varied across field site and year of the study, thus emphasizing the importance of the environment in determining the degree of postzygotic isolation and potential for introgression across natural hybrid zones.     

Environment-dependent performance and fitness of Iris brevicaulis, I. fulva (Iridaceae), and hybrids

We tested the relative fitness of two Louisiana Iris species (Iris brevicaulis and I. fulva) and their first-generation backcross hybrids in three experimental watering treatments: dry, field capacity, and flooded. Leaf area expansion rate, gas exchange (A(max), g(s), c(i)), and biomass at final harvest were measured for each species and hybrid class in all three environmental treatments. Fitness (based on total biomass) of the four genotypic classes differed significantly with environment. All genotypic classes performed most poorly in the dry treatment. The fitness ranking of genotypic class also changed across environments (significant genotypic class by treatment interaction) with hybrid genotype fitness shifting relative to parental genotypes. Integrating over all treatments, backcrosses to I. fulva showed the lowest fitness, whereas backcrosses to I. brevicaulis outperformed I. fulva. The differences in fitness were apparently achieved by a combination of differences in photosynthesis and allocation. In this system, hybrids are not necessarily less fit than their parents, and the relationship between hybrid and parental fitness is influenced by environmental conditions, lending support to the Hybrid Novelty model of hybrid zone evolution.     

Intimately linked or hardly speaking? The relationship between genotype and environmental gradients in a Louisiana Iris hybrid population

Several models of hybrid zone evolution predict the same spatial patterns of genotypic distribution whether or not structuring is due to environment-dependent or -independent selection. In this study, we tested for evidence of environment-dependent selection in an Iris fulva x Iris brevicaulis hybrid population by examining the distribution of genotypes in relation to environmental gradients. We selected 201 Louisiana Iris plants from within a known hybrid population (80 m x 80 m) and placed them in four different genotypic classes (I. fulva, I. fulva-like hybrid, I. brevicaulis-like hybrid and I. brevicaulis) based on seven species-specific random amplified polymorphic DNA (RAPD) markers and two chloroplast DNA haplotypes. Environmental variables were then measured. These variables included percentage cover by tree canopy, elevation from the high water mark, soil pH and percentage soil organic matter. Each variable was sampled for all 201 plants. Canonical discriminant analysis (CDA) was used to infer the environmental factors most strongly associated with the different genotypic groups. Slight differences in elevation (-0.5 m to +0.4 m) were important for distinguishing habitat distributions described by CDA, even though there were no statistical differences between mean elevations alone. I. brevicaulis occurred in a broad range of habitats, while I. fulva had a narrower distribution. Of all the possible combinations, I. fulva-like hybrids and I. brevicaulis-like hybrids occurred in the most distinct habitat types relative to one another. Each hybrid class was not significantly different from its closest parent with regard to habitat occupied, but was statistically unique from its more distant parental species. Within the hybrid genotypes, most, but not all, RAPD loci were individually correlated with environmental variables. This study suggests that, at a very fine spatial scale, environment-dependent selection contributed to the genetic structuring of this hybrid zone.