Prezygotic Barriers Research Articles

A pollen expressed PME gene at Tcb1 locus confers maize unilateral cross-incompatibility.

A pollen expressed PME gene at Tcb1 locus confers maize unilateral cross-incompatibility.

Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a widely used model organism in genetics, evolution, and molecular biology. In recent years, it has also become a popular model organism to study problems related to speciation. The life cycle of yeast involves both asexual and sexual reproductive phases. The ease of performing evolution experiments and the short generation time of the organism allow for the study of the evolution of reproductive barriers. The efficiency with which the two mating types (a and α) mate to form the a/α diploid is referred to as the mating efficiency. Any decrease in the mating efficiency between haploids indicates a pre-zygotic barrier. Thus, to quantify the extent of reproductive isolation between two haploids, a robust method to quantify the mating efficiency is required. To this end, a simple and highly reproducible protocol is presented here. The protocol involves four main steps, which include patching the haploids on a YPD plate, mixing the haploids in equal numbers, diluting and plating for single colonies, and finally, calculating the efficiency based on the number of colonies on a drop-out plate. Auxotrophic markers are employed to clearly make the distinction between haploids and diploids.

Eco-Geography and Phenology Are the Major Drivers of Reproductive Isolation in the Royal Irises, a Species Complex in the Course of Speciation

The continuous nature of speciation implies that different species are found at different stages of divergence, from no- to complete reproductive isolation. This process and its underlying mechanisms are best viewed in incipient species. Moreover, the species complex can offer unique insight into how reproductive isolation (RI) has evolved. The royal irises (Iris section Oncocyclus) are a young group of species in the course of speciation, providing an ideal system for speciation study. We quantified pre- and post-zygotic reproductive barriers between the eight Israeli species of this complex and estimated the total RI among them. We tested for both pre-pollination and post-pollination reproductive barriers. Pre-pollination barriers, i.e., eco-geographic divergence and phenological differentiation were the major contributors to RI among the Iris species. On the other hand, post-pollination barriers, namely pollen-stigma interactions, fruit set, and seed viability had negligible contributions to total RI. The strength of RI was not uniform across the species complex, suggesting that species may have diverged at different rates. Overall, this study in a young, recently diverged group of species provides insight into the first steps of speciation, suggesting a crucial role of the pre-zygotic barriers.

Contrasting continental patterns of adaptive population divergence in the holarctic ectomycorrhizal fungus Boletus edulis.

In the hyperdiverse fungi, the process of speciation is virtually unknown, including for the > 20 000 species of ectomycorrhizal mutualists. To understand this process, we investigated patterns of genome-wide differentiation in the ectomycorrhizal porcini mushroom, Boletus edulis, a globally distributed species complex with broad ecological amplitude. By whole-genome sequencing 160 individuals from across the Northern Hemisphere, we genotyped 792 923 single nucleotide polymorphismsto characterize patterns of genome-wide differentiation and to identify the adaptive processes shaping global population structure. We show that B. edulis exhibits contrasting patterns of genomic divergence between continents, with multiple lineages present across North America, while a single lineage dominates Europe. These geographical lineages are inferred to have diverged 1.62-2.66 million years ago, during a period of climatic upheaval and the onset of glaciation in the Pliocene-Pleistocene boundary. High levels of genomic differentiation were observed among lineages despite evidence of substantial and ongoing introgression. Genome scans, demographic inference, and ecological niche models suggest that genomic differentiation is maintained by environmental adaptation, not physical isolation. Our study uncovers striking patterns of genome-wide differentiation on a global scale and emphasizes the importance of local adaptation and ecologically mediated divergence, rather than prezygotic barriers such as allopatry or genomic incompatibility, in fungal population differentiation.

Tissue Culture Techniques for the Production of Interspecific Hybrids in Poland: History and Achievements

Abstract Interspecific hybridization is a significant plant evolutionary process and, concomitantly, a frequently used method to broaden the genetic variability of species and genetically improve crops. However, in distant crosses, many prezygotic and postzygotic barriers are encountered that prevent free, uncontrolled gene flow between species. Therefore, various experimental methods exploiting tissue culture, such as in vitro fertilization, embryo rescue, and protoplast fusion, have been developed to raise hybrids that cannot be obtained naturally through conventional approaches. Professor Maciej Zenkteler of the Adam Mickiewicz University in Poznań pioneered the use of tissue culture in Poland to overcome barriers to sexual crossing. In the mid-1960s, he employed in vitro pollination in which pollen grains were directly applied onto the surface of exposed ovules as the primary method to bypass prezygotic barriers and in vitro culture of isolated ovules and developing embryos (embryo rescue) to overcome post-zygotic barriers. These approaches proved effective for species possessing numerous ovules and large placentas, such as representatives of the families Caryophyllaceae, Solanaceae, and Brassicaceae. Soon thereafter, these methods were extended to other Polish institutes and universities and applied for crop improvement, such as for the production of intergeneric hybrids between Salix and Populus species, resynthesis and broadening of the genetic variability of oilseed rape ( Brassica napus L.), and the generation of new interspecific hybrids of Allium . In the late 1970s, Professor Zenkteler was the first to undertake protoplast fusion. At that time, it appeared that no incompatibility could exist at the fusion level and that somatic hybridization would supersede the possibilities offered by sexual hybridization; however, the reality turned out to be slightly harsh. Nonetheless, successful regeneration of different interspecific hybrids of potato, tomato, or gentians has been achieved by Polish research groups thus far.

Trends in evolution of the Triatomini tribe (Hemiptera, Triatominae): reproductive incompatibility between four species of geniculatus clade

BackgroundThe geniculatus clade, composed by the rufotuberculatus, lignarius, geniculatus and megistus groups, relates evolutionarily the species of the genus Panstrongylus and Nesotriatoma. Several studies have shown that triatomine hybrids can play an important role in the transmission of Chagas disease. Natural hybrids between species of the geniculatus clade have never been reported to our knowledge. Thus, carrying out experimental crosses between species of the geniculatus clade can help to elucidate the taxonomic issues as well as contribute to the epidemiological knowledge of this group.MethodsExperimental crosses were carried out between species of the megistus and lignarius groups to evaluate the reproductive compatibility between them. A phylogenetic reconstruction was also performed with data available in GenBank for the species of the geniculatus clade to show the relationships among the crossed species.ResultsPhylogenetic analysis grouped the species of the geniculatus clade into four groups, as previously reported. In the interspecific crosses performed there was no hatching of eggs, demonstrating the presence of prezygotic barriers between the crossed species and confirming their specific status.ConclusionsIn contrast to the other groups of the Triatomini tribe, as well as the Rhodniini, there are prezygotic barriers that prevent the formation of hybrids between species of the megistus and lignarius groups. Thus, the geniculatus clade may represent an important evolutionary model for Triatominae, highlighting the need for further studies with greater sample efforts for this clade (grouping the 17 species of Panstrongylus and the three of Nesotriatoma).Graphical

Hybridization between closely related songbirds is related to human habitat disturbance.

Human habitat disturbances can promote hybridization between closely related, but typically reproductively isolated, species. We explored whether human habitat disturbances are related to hybridization between two closely related songbirds, black-capped and mountain chickadees, using both genomic and citizen science data sets. First, we genotyped 409 individuals from across both species' ranges using reduced-representation genome sequencing and compared measures of genetic admixture to a composite measure of human landscape disturbance. Then, using eBird observations, we compared human landscape disturbance values for sites where phenotypically diagnosed hybrids were observed to locations where either parental species was observed to determine whether hybrid chickadees are reported in more disturbed areas. We found that hybridization between black-capped and mountain chickadees positively correlates with human habitat disturbances. From genomic data, we found that (1) hybrid index (HI) significantly increased with habitat disturbance, (2) more hybrids were sampled in disturbed habitats, (3) mean HIs were higher in disturbed habitats versus wild habitats, and (4) hybrids were detected in habitats with significantly higher disturbance values than parentals. Using eBird data, we found that both hybrid and black-capped chickadees were significantly more disturbance-associated than mountain chickadees. Surprisingly, we found that nearly every black-capped chickadee we sampled contained some proportion of hybrid ancestry, while we detected very few mountain chickadee backcrosses. Our results highlight that hybridization between black-capped and mountain chickadees is widespread, but initial hybridization is rare (few F1s were detected). We conclude that human habitat disturbances can erode pre-zygotic reproductive barriers between chickadees and that post-zygotic isolation is incomplete. Understanding what becomes of recently hybridizing species following large-scale habitat disturbances is a new, but pressing, consideration for successfully preserving genetic biodiversity in a rapidly changing world.

Distribution patterns at different spatial scales reveal reproductive isolation and frequent syntopy among divergent lineages of an amphipod species complex in Western Carpathian streams

AbstractRecent discoveries of vast cryptic diversity among various aquatic invertebrates pose a challenge for understanding their ecology and biodiversity patterns, as well as for conservation. We studied the distribution, potential hybridization and habitat preferences of divergent lineages of the hyperdiverse Gammarus fossarum species complex (Crustacea: Amphipoda) in their contact zone in the Western Carpathians (Central Europe), to obtain insights into processes that may influence lineage coexistence. We initially screened over 60 sites to assess the regional distribution, confirming the presence of four lineages in streams of the study area. Two to three lineages were found to be syntopic at almost half the sites. Subsequently, nine such localities were studied in detail for the mesohabitat distribution (fast vs. slow water flow), pairing preferences, and temporal stability of the Gammarus community. A significant fraction of regional spatial variation was explained by the position in the dendritic river network, altitude, stream width, and anthropogenic stress. At the local scale, however, we did not find differences in distribution between mesohabitats with contrasting flow rates. Mixed precopulatory pairs in syntopy were extremely rare, even under highly imbalanced lineage ratios, confirming a strong prezygotic reproductive barrier between the studied lineages. This was congruent with species delimitation analyses using molecular markers, which confirmed the absence of any recent gene flow. The frequent and apparently temporally stable syntopy of these reproductively isolated Gammarus lineages warrants further research on the processes that may facilitate this coexistence, focusing on potential finer differences in their ecology, functional morphology, or biotic interactions.

Have female twisted-wing parasites (Insecta: Strepsiptera) evolved tolerance traits as response to traumatic penetration?

Traumatic insemination describes an unusual form of mating during which a male penetrates the body wall of its female partner to inject sperm. Females unable to prevent traumatic insemination have been predicted to develop either traits of tolerance or of resistance, both reducing the fitness costs associated with the male-inflicted injury. The evolution of tolerance traits has previously been suggested for the bed bug. Here we present data suggesting that tolerance traits also evolved in females of the twisted-wing parasite species Stylops ovinae and Xenos vesparum. Using micro-indentation experiments and confocal laser scanning microscopy, we found that females of both investigated species possess a uniform resilin-rich integument that is notably thicker at penetration sites than at control sites. As the thickened cuticle does not seem to hamper penetration by males, we hypothesise that thickening of the cuticle resulted in reduced penetration damage and loss of haemolymph and in improved wound sealing. To evaluate the evolutionary relevance of the Stylops-specific paragenital organ and penis shape variation in the context of inter- and intraspecific competition, we conducted attraction and interspecific mating experiments, as well as a geometric-morphometric analysis of S. ovinae and X. vesparum penises. We found that S. ovinae females indeed attract sympatrically distributed congeneric males. However, only conspecific males were able to mate. In contrast, we did not observe any heterospecific male attraction by Xenos females. We therefore hypothesise that the paragenital organ in the genus Stylops represents a prezygotic mating barrier that prevents heterospecific matings.

Overlaps in olfactive signalling coupled with geographic variation may result in localised pollinator sharing between closely related Ficus species

BackgroundIn brood site pollination mutualisms, pollinators are attracted by odours emitted at anthesis. In Ficus, odours of receptive figs differ among species and the specific pollinators generally only enter figs of their host species ensuring a pre-zygotic barrier to plant interspecific hybridisation. However, field observations recorded that, in Guangdong province in China, Valisia javana hilli, the local pollinator of F. hirta, entered and reproduced successfully in the figs of the closely related F. triloba on a regular basis. We propose that closely related Ficus species produce similar receptive fig odours. Under particular contexts of odours locally present, the receptive fig odours of non-host figs of a Ficus species may become attractive to pollinators of closely related Ficus species. We used the headspace technique to collect in situ receptive fig odours of F. triloba in a series of locations in China. Under controlled conditions, we tested the attraction of fig pollinating wasps from F. hirta and F. triloba to host figs and non-host figs in Y tube experiments.ResultsReceptive fig odours of F. triloba though different from those of F. hirta, were mainly composed of a same set of volatile organic compounds. When given the choice between receptive fig odours and air, the pollinating wasps were only attracted by their host’s odours. However, when given a choice between host and non-host figs the pollinators of F. hirta were equally attracted by the two odours while the pollinators of F. triloba tended to be more attracted by their host’s fig odours.ConclusionsReceptive fig odours vary geographically within species and the differentiation of receptive fig odours between closely related Ficus species is often incomplete. This allows localised or occasional pollinator sharing following different modalities. Cross stimulation when wasps are exposed simultaneously to odours of host and non-host species may be important. While occasional pollinator sharing may play a marginal role when wasp populations are robust, it may ensure the provisioning of new pollinators from the closest relative of a Ficus species if its pollinators go extinct.

The strength of reproductive isolating barriers in seed plants: Insights from studies quantifying premating and postmating reproductive barriers over the past 15 years.

Speciation is driven by the evolution of reproductive isolating barriers that reduce, and ultimately prevent, substantial gene flow between lineages. Despite its central role in evolutionary biology, the process can be difficult to study because it proceeds differently among groups and may occur over long timescales. Due to this complexity, we typically rely on generalizations of empirical data to describe and understand the process. Previous reviews of reproductive isolation (RI) in flowering plants have suggested that prezygotic or extrinsic barriers generally have a stronger effect on reducing gene flow compared to postzygotic or intrinsic barriers. Past conclusions have rested on relatively few empirical estimates of RI; however, RI data have become increasingly abundant over the past 15 years. We analyzed data from recent studies quantifying multiple pre- and postmating barriers in plants and compared the strengths of isolating barriers across 89 taxa pairs using standardized RI metrics. Individual prezygotic barriers were on average stronger than individual postzygotic barriers, and the total strength of prezygotic RI was approximately twice that of postzygotic RI. These findings corroborate that ecological divergence and extrinsic factors, as opposed to solely the accumulation of genetic incompatibilities, are important to speciation and the maintenance of species boundaries in plants. Despite an emphasis in the literature on asymmetric postmating and postzygotic RI, we found that prezygotic barriers acted equally asymmetrically. Overall, substantial variability in the strengths of 12 isolating barriers highlights the great diversity of mechanisms that contribute to plant diversification.

Contaminants of emerging concern may pose prezygotic barriers to freshwater mussel recruitment

Increased detection of contaminants of emerging concern (CECs) throughout the Laurentian Great Lakes Basin has raised concern about the impact CECs may have on aquatic life. In particular, the impacts of CECs on the highly imperiled freshwater mussels (family: Unionidae) have not yet been assessed. Contaminants may alter reproductive success during either the pre- or postzygotic stages of reproduction and lead to population declines. This study sought to address possible disruption of prezygotic processes while acknowledging the complexity of CEC mixtures in nature. River waters from sites with dominant urban and agricultural land use in the Milwaukee River Basin and Lower Maumee River Basin respectively were used as treatments during 21-day exposures of adult Lampsilis cardium, Lampsilis siliquoidea, and Eurynia dilatata. At the conclusion of exposures, the development of gonad tissue was histologically examined to address deviations from expected seasonal development as a barrier to recruitment. No abnormalities in development were found compared to control waters. Sperm densities were also examined; and, though no significant differences were found among treatments, lampsiline species sperm density tended to increase along the gradient of CEC mixture complexity while E. dilatata sperm density correlated positively with a lower concentration of pharmaceuticals, especially antibiotics, despite increased pesticide detection. Given the complexity of natural occurring CEC mixtures and variable unionid response, future work may utilize these data to elucidate combinations of CECs most likely to create impacts on gametogenesis which could lead to further population declines.

Hybridization and introgression are prevalent in Southern European Erysimum (Brassicaceae) species.

Hybridization is a common and important force in plant evolution. One of its outcomes is introgression - the transfer of small genomic regions from one taxon to another by hybridization and repeated backcrossing. This process is believed to be common in glacial refugia, where range expansions and contractions can lead to cycles of sympatry and isolation, creating conditions for extensive hybridization and introgression. Polyploidization is another genome-wide process with a major influence on plant evolution. Both hybridization and polyploidization can have complex effects on plant evolution. However, these effects are often difficult to understand in recently evolved species complexes. We combined flow cytometry, analyses of transcriptomic sequences and pollen tube growth assays to investigate the consequences of polyploidization, hybridization and introgression on the recent evolution of several Erysimum (Brassicaceae) species from the South of the Iberian Peninsula, a well-known glacial refugium. This species complex differentiated in the last 2 million years, and its evolution has been hypothesized to be determined mainly by polyploidization, interspecific hybridization and introgression. Our results support a scenario of widespread hybridization involving both extant and 'ghost' taxa. Several taxa studied here, most notably those with purple corollas, are polyploids, probably of allopolyploid origin. Moreover, hybridization in this group might be an ongoing phenomenon, as pre-zygotic barriers appeared weak in many cases. The evolution of Erysimum spp. has been determined by hybridization to a large extent. Species with purple (polyploids) and yellow flowers (mostly diploid) exhibit a strong signature of introgression in their genomes, indicating that hybridization occurred regardless of colour and across ploidy levels. Although the adaptive value of such genomic exchanges remains unclear, our results demonstrate the significance of hybridization for plant diversification, which should be taken into account when studying plant evolution.

Cross compatibility in intraspecific and interspecific hybridization in yam (Dioscorea spp.)

Yam (Dioscorea spp.) is a staple crop for millions of people in the tropics and subtropics. Its genetic improvement through breeding is being challenged by pre-zygotic and post-zygotic cross-compatibility barriers within and among species. Studies dissecting hybridization barriers on yam for improving the crossability rates are limited. This study aimed to assess the cross-compatibility, which yielded fruit set, viable seeds and progeny plants in an extensive intraspecific and interspecific crossing combinations in a yam genetic improvement effort to understand the internal and exogenous factors influencing pollination success. Cross-compatability was analyzed at the individual genotype or family level using historical data from crossing blocks and seedling nurseries from 2010 to 2020 at the International Institute of Tropical Agriculture (IITA). The average crossability rate (ACR) was lower in interspecific crossing combinations (6.1%) than intraspecific ones (27.6%). The seed production efficiency (SPE) values were 1.1 and 9.3% for interspecific and intraspecific crosses, respectively. Weather conditions and pollinator's skills are the main contributors to the low success rate in the intraspecific cross combinations in yam breeding. At the same time, genetic distance and heterozygosity played little role. Interspecific cross barriers were both pre-zygotic and post-zygotic, resulting from the evolutionary divergence among the yam species. Dioscorea rotundata had higher interspecific cross-compatibility indices than D. alata. Distant parents produced intraspecific crossbred seeds with higher germination rates compared to closest parents (r = 0.21, p = 0.033). This work provided important insights into interspecific and intraspecific cross-compatibility in yam and suggested actions for improving hybridization practices in yam breeding programs.

Patterns of genomic and allochronic strain divergence in the fall armyworm, Spodoptera frugiperda (J.E. Smith).

Speciation is the process through which reproductive isolation develops between distinct populations. Because this process takes time, speciation studies often necessarily examine populations within a species that are at various stages of divergence. The fall armyworm, Spodoptera frugiperda (J.E. Smith), is comprised of two strains (R = Rice & C = Corn) that serve as a novel system to explore population divergence in sympatry. Here, we use ddRADSeq data to show that fall armyworm strains in the field are largely genetically distinct, but some interstrain hybridization occurs. Although we detected F1 hybrids of both R‐ and C‐strain maternal origin, only hybrids with R‐strain mtDNA were found to contribute to subsequent generations, possibly indicating a unidirectional barrier to gene flow. Although these strains have been previously defined as “host plant‐associated,” we recovered an equal proportion of R‐ and C‐strain moths in fields dominated by C‐strain host plants. As an alternative to host‐associated divergence, we tested the hypothesis that differences in nightly activity patterns could account for reproductive isolation by genotyping temporally collected moths. Our data indicates that strains exhibit a significant shift in the timing of their nightly activities in the field. This divergence in phenology creates a prezygotic reproductive barrier that likely maintains the genetic isolation between strains. Thus, we conclude that it may be ecologically inaccurate to refer to the C‐ and R‐ strain as “host‐associated” and they should more appropriately be considered “allochronic strains.”

Reduced fertilization constitutes an important prezygotic reproductive barrier between two sibling species of the hybridizing Brachionus calyciflorus species complex

Brachionus calyciflorus is a species complex consisting of four recently described species. Although several lines of evidence support their species status, hybridization between two of the sibling species B. calyciflorus s.s. and B. elevatus has been inferred from both field and laboratory studies. In this study, we tested for the existence of prezygotic barriers between these species by performing two types of cross-fertilization experiments. In a ‘mate competition’ experiment we exposed mictic females to equal numbers of conspecific and allospecific males and demonstrate that intraspecific fertilizations occur at much higher frequencies than interspecific fertilizations, providing evidence for a strong prezygotic reproductive barrier. This result was consistent across numerous combinations of parental genotypes. In addition, by creating interspecific combinations of mictic females and males in a ‘no choice’ experiment, we found that interspecific fertilization success is independent of fertilization direction while it does seem to depend on maternal genotype. Our results demonstrate the existence of a strong prezygotic barrier that may play an important role in the maintenance of species boundaries. Yet, the observation of hybrids also shows a potential for gene flow between the species through hybridization.

Preventing inbreeding depression in Anacamptis coriophora (Orchidaceae) as a model of food-rewarding orchid

Pollination of deceptive orchids has enabled scientists to understand how these species avoid inbreeding depression by reducing the number of pollinator visits per inflorescence. In rewarding species, which receive a higher rate of visits per plant, geitonogamy is usually higher and therefore the risk of inbreeding increases. In this study, we assess the breeding system of the rewarding orchid A. coriophora, and the spatio-temporal changes in its fitness as well as variation in nectar content after pollination. We found that the species partially selects allogamous pollen if pollinia from the same stalk and other plants arrive to the stigma. Furthermore, when self-pollination occurs, despite successful fructification, seed viability is significantly lower than that of cross-pollinated plants. A. coriophora exhibits spatio-temporal variation in fitness that does not correlate with any plant feature. Moreover, nectar volume is reduced after pollination, but the sugar concentration is maintained. This study emphasizes how essential the pre-zygotic and post-zygotic reproductive barriers are for rewarding orchids to avoid inbreeding depression.

Small scale evolution

Small-scale evolution or microevolution concerns evolution at the intra-specific level or between closely related species. At the intra-specific level, it allows the analysis of the evolutionary forces at work: mutation, genetic drift, migration and selection. Moreover, because of the short evolutionary time, it is easier to identify the genetic basis of observed phenotypic differences. Most studies focus on current populations but more and more analyses are performed on ancient DNA. This provides important information for tracing the history of populations and also allows the reconstruction of phenotypes of individuals that disappeared several thousand years ago. In this short review, I present studies showing how pre-zygotic or post-zygotic barriers involved in species formation are set up using the example of the geographical barrier due to the formation of the Isthmus of Panama and that of the heterochromatin divergence in Drosophilidae. I also describe the different approaches that have been used to identify the genetic basis of well known phenotypic variations: candidate gene approach (about melanism in felines), QTL mapping (variation in the number of lateral bone plates in sticklebacks), association study (pigmentation in the Asian ladybird). Finally, I illustrate the key impact of natural selection with the iconic example of the evolution of the beak of Galapagos finches, and the role of certain developmental genes in its morphological diversification.

Reproductive biology of green-stemmed and red-stemmed Basella alba

Basella alba is an important, underutilised vegetable rich in vitamins and consumed in many parts of the world. However, the reproductive biology of this species is insufficiently known; therefore, this study was performed aiming to fill this knowledge gap. Although green-stemmed and red-stemmed Basella alba share a lot of reproductive attributes in common, the obtained results showed differences in their flower bud apex colour, anther colour, spike colour, days to 50% flowering, plant height at flower bud initiation and the mean number of flowers per spike. In both forms, the floral attributes favoured self-pollination. However, the possibility of cross-pollination among them due to the activities of pollinators such as ants, moths, butterflies and bees suggests a mixed mating system. Furthermore, both forms attained sexual maturity at varying periods, thus indicating the existence of a temporal prezygotic barrier between them and limiting the chances of their hybridisation. They could also be responsible for the ability to remain in their distinct forms even when they exist together in the same habitat.

Evaluating the legacy of multiple introductions of American martens on spatiotemporal patterns of genetic diversity

AbstractSpecies reintroductions are successful when established populations maintain both demographic stability and genetic diversity. Such a result may be obtained by ensuring both structural habitat connectivity and genetic connectivity among reintroduced and remnant populations. Nevertheless, prezygotic barriers such as assortative mating can prevent the flow of genetic material between populations, even when migration between populations is high. Limited gene flow may be particularly relevant for reintroductions that were sourced either from captive-bred populations or from disparate locations in the wild. American martens (Martes americana) have been reintroduced repeatedly in the Upper Midwestern United States in an effort to establish self-sustaining populations. We quantified levels of genetic diversity within and spatial genetic variance among four marten populations during two time periods separated by 10 years. Spatially informed and naïve discriminant analysis of principal components were used to assign individuals to populations. Results indicate that heterozygosity declined and inbreeding coefficients increased between the two collection periods, while genetic structure among populations also increased. Data are consistent with assortative mating contributing to reapportioning of genetic variation. Population assignment tests show that migration among populations is apparent, but admixture (based on cluster membership probabilities) is low and declined over time. Specifically, martens may be successfully dispersing between populations but a lack of admixture indicates a lack of reproductive contributions to genetic diversity by migrants. Because marten reintroductions in this region are well-documented and well-monitored, lessons can be derived from results to inform future reintroductions. We encourage a careful balance of supplementing genetic diversity via augmentation while avoiding translocation of animals from disparate populations that may result in reproductive isolation of migrants. In combination with the maintenance of a functionally connected landscape, this strategy would maximize the likelihood of a successful reintroduction in terms both of demography and genetics.