The effects of locus number,genetic divergence,and genotyping error on the utility of dominant markers for hybrid identification

In surveys of hybrid zones, dominant genetic markers are often used to identify individuals of hybrid origin and assign these individuals to one of several potential hybrid classes. Quantitative analyses that address the statistical power of dominant markers in such inference are scarce. In this study, dominant genotype data were simulated to evaluate the effects of, first, the number of loci analyzed, second, the magnitude of differentiation between the markers scored in the groups that are hybridizing, and third, the level of genotyping error associated with the data when assigning individuals to various parental and hybrid categories. The overall performance of the assignment methods was relatively modest at the lowest level of divergence examined (F_st ~ 0.4), but improved substantially at higher levels of differentiation (F_st ~ 0.67 or 0.8). The effect of genotyping error was dependent on the level of divergence between parental taxa, with larger divergences tempering the effects of genotyping error. These results highlight the importance of considering the effects of each of the variables when assigning individuals to various parental and hybrid categories, and can help guide decisions regarding the number of loci employed in future hybridization studies to achieve the power and level of resolution desired.     

Comparison of microsatellites and amplified fragment length polymorphism markers for parentage analysis

This study compares the properties of dominant markers, such as amplified fragment length polymorphisms (AFLPs), with those of codominant multiallelic markers, such as microsatellites, in reconstructing parentage. These two types of markers were used to search for both parents of an individual without prior knowledge of their relationships, by calculating likelihood ratios based on genotypic data, including mistyping. Experimental data on 89 oak trees genotyped for six microsatellite markers and 159 polymorphic AFLP loci were used as a starting point for simulations and tests. Both sets of markers produced high exclusion probabilities, and among dominant markers those with dominant allele frequencies in the range 0.1-0.4 were more informative. Such codominant and dominant markers can be used to construct powerful statistical tests to decide whether a genotyped individual (or two individuals) can be considered as the true parent (or parent pair). Gene flow from outside the study stand (GFO), inferred from parentage analysis with microsatellites, overestimated the true GFO, whereas with AFLPs it was underestimated. As expected, dominant markers are less efficient than codominant markers for achieving this, but can still be used with good confidence, especially when loci are deliberately selected according to their allele frequencies.     

Development of diagnostic microsatellite markers from whole‐genome sequences of Ammodramus sparrows for assessing admixture in a hybrid zone

Studies of hybridization and introgression and, in particular, the identification of admixed individuals in natural populations benefit from the use of diagnostic genetic markers that reliably differentiate pure species from each other and their hybrid forms. Such diagnostic markers are often infrequent in the genomes of closely related species, and genomewide data facilitate their discovery. We used whole‐genome data from Illumina HiSeqS2000 sequencing of two recently diverged (600,000 years) and hybridizing, avian, sister species, the Saltmarsh (Ammodramus caudacutus) and Nelson's (A. nelsoni) Sparrow, to develop a suite of diagnostic markers for high‐resolution identification of pure and admixed individuals. We compared the microsatellite repeat regions identified in the genomes of the two species and selected a subset of 37 loci that differed between the species in repeat number. We screened these loci on 12 pure individuals of each species and report on the 34 that successfully amplified. From these, we developed a panel of the 12 most diagnostic loci, which we evaluated on 96 individuals, including individuals from both allopatric populations and sympatric individuals from the hybrid zone. Using simulations, we evaluated the power of the marker panel for accurate assignments of individuals to their appropriate pure species and hybrid genotypic classes (F1, F2, and backcrosses). The markers proved highly informative for species discrimination and had high accuracy for classifying admixed individuals into their genotypic classes. These markers will aid future investigations of introgressive hybridization in this system and aid conservation efforts aimed at monitoring and preserving pure species. Our approach is transferable to other study systems consisting of closely related and incipient species.     

Species-specific SSR alleles for studies of hybrid cattails (Typha latifolia x T. angustifolia; Typhaceae) in North America

? Premise: Studies of hybridizing species are facilitated by the availability of species-specific molecular markers for identifying early- and later-generation hybrids. Cattails are a dominant feature of wetland communities, and a better understanding of the prevalence of hybrids is needed to assess the ecological and evolutionary effects of hybridization. Hybridization between Typha angustifolia and T. latifolia produce long-lived clones, known as Typha ×glauca, which are considered to be invasive. Although morphological variation in cattails makes it difficult to recognize early- and later-generation hybrids, several dominant, species-specific RAPD markers are available. Our goal was to find codominant, species-specific markers with greater polymorphism than RAPDs, to identify later-generation hybrids more efficiently. ? Methods: We screened nine SSR (simple sequence repeat) loci that were described from populations in Ukraine, and we surveyed 31 cattail populations from the upper Midwest and eastern USA. ? Key results: Seven SSR loci distinguished the parent taxa and were consistent with known species-specific RAPD markers, allowing easier detection of backcrossing. We used linear discriminant analysis to show that F(1) hybrid phenotypes were intermediate between the parent taxa, while those of backcrossed plants overlapped with the hybrids and their parents. Log(leaf length/leaf width), spike gap length, spike length, and stem diameter explained much of the variation among groups. ? Conclusions: We provide the first documentation of backcrossed plants in hybridizing cattail populations in Michigan. The diagnostic SSR loci we identified should be extremely useful for examining the evolutionary and ecology interactions of hybridizing cattails in North America.     

Maximum‐likelihood estimation of a hybrid index based on molecular markers

This paper elaborates on a hybrid index that utilizes information from genetic markers to quantify the genetic contribution of hybridizing species to individuals of unknown ancestry. Dominant markers will only lead to reliable and accurate estimates of hybrid index in later generation hybrids. In contrast, codominant markers can be fully resolved and their use is unproblematic. For both types of markers and allele frequencies that differ substantially between parental species (F_ST ≥ 0.17), a hybrid index based on 35–45 loci will have a nearly minimal confidence interval. Estimates of hybrid index are robust to modest errors in estimates of parental allele frequencies.     

Isozyme Evidence for Natural Hybridization in Phlomis (Lamiaceae): Hybrid Origin of the RareP.xmargaritae

Phlomisxmargaritae is a rare and sterile hybrid taxon foundin a single endangered population in southern Spain. It waspreviously described as a morphologically intermediate hybrid,putatively between Phlomis purpurea and P. composita. The presentstudy used allozymes as molecular markers to assess the hybrididentity ofP.xmargaritae . Ten putative loci from seven enzymesystems were resolved: five were monomorphic and fixed acrossall taxa studied and the rest (Aat-1, Aat-2, 6-Pgdh-2, Pgi andPgm) were polymorphic in at least one taxon. The two parentaltaxa are fixed for different alleles at 6-Pgdh-2 and show distinctallelic frequency differences for four other loci. Phlomisxmargaritaedisplays fixed heterozygous phenotypes for four of the fivepolymorphic loci, these being composed of combinations of thealleles found in the parental taxa. No unique alleles were detectedin P.xmargaritae. We conclude that this taxon is of hybrid originand confirm the identity of the parental taxa involved. It isfurther suggested that this population is composed of individualsthat are recent F₁hybrids that have not undergone backcrossingor introgression. Global conservation measures are necessaryfor the whole hybrid system in this location since further continuousassessment could reveal the evolutionary input of hybridizationin Phlomis.Copyright 2000 Annals of Botany Company Endangered species, genetic diversity, isozyme variation, hybrid species, Phlomisxmargaritae, plant conservation, population genetics, Lamiaceae, Spain.     

COLONY: a program for parentage and sibship inference from multilocus genotype data

Pedigrees, depicting genealogical relationships between individuals, are important in several research areas. Molecular markers allow inference of pedigrees in wild species where relationship information is impossible to collect by observation. Marker data are analysed statistically using methods based on Mendelian inheritance rules. There are numerous computer programs available to conduct pedigree analysis, but most software is inflexible, both in terms of assumptions and data requirements. Most methods only accommodate monogamous diploid species using codominant markers without genotyping error. In addition, most commonly used methods use pairwise comparisons rather than a full-pedigree likelihood approach, which considers the likelihood of the entire pedigree structure and allows the simultaneous inference of parentage and sibship. Here, we describe colony, a computer program implementing full-pedigree likelihood methods to simultaneously infer sibship and parentage among individuals using multilocus genotype data. colony can be used for both diploid and haplodiploid species; it can use dominant and codominant markers, and can accommodate, and estimate, genotyping error at each locus. In addition, colony can carry out these inferences for both monoecious and dioecious species. The program is available as a Microsoft Windows version, which includes a graphical user interface, and a Macintosh version, which uses an R-based interface.     

Similarity coefficients for molecular markers in studies of genetic relationships between individuals for haploid, diploid, and polyploid species

Determining true genetic dissimilarity between individuals is an important and decisive point for clustering and analysing diversity within and among populations, because different dissimilarity indices may yield conflicting outcomes. We show that there are no acceptable universal approaches to assessing the dissimilarity between individuals with molecular markers. Different measures are relevant to dominant and codominant DNA markers depending on the ploidy of organisms. The Dice coefficient is the suitable measure for haploids with codominant markers and it can be applied directly to (0,1)-vectors representing banding profiles of individuals. None of the common measures, Dice, Jaccard, simple mismatch coefficient (or the squared Euclidean distance), is appropriate for diploids with codominant markers. By transforming multiallelic banding patterns at each locus into the corresponding homozygous or heterozygous states, a new measure of dissimilarity within locus was developed and expanded to assess dissimilarity between multilocus states of two individuals by averaging across all codominant loci tested. There is no rigorous well-founded solution in the case of dominant markers. The simple mismatch coefficient is the most suitable measure of dissimilarity between banding patterns of closely related haploid forms. For distantly related haploid individuals, the Jaccard dissimilarity is recommended. In general, no suitable method for measuring genetic dissimilarity between diploids with dominant markers can be proposed. Banding patterns of diploids with dominant markers and polyploids with codominant markers represent individuals' phenotypes rather than genotypes. All dissimilarity measures proposed and developed herein are metrics.     

Genetics and morphology in a Borrichia frutescens and B. arborescens (Asteraceae) hybrid zone

Interspecific plant hybridization is a common and evolutionarily important phenomenon. Here, the results of a study of hybridization in the Florida Keys between two species of sea oxeye daisy, Borrichia frutescens and B. arborescens, are reported. Nuclear and chloroplast genetic loci, log-likelihood assignment tests, and maximum likelihood estimates of genealogical class frequencies were used to identify hybrid and parent genotypes, to investigate the utility of leaf and flower morphology for hybrid identification, and to study symmetry and degree of introgression between the species. Genetic analyses confirmed the identity of the hybrid and parent plants that were used for the morphological studies. Together, leaf and flower morphology can be used to identify hybrid and parental types with moderate accuracy (4% error rate). Population genetic analyses indicate that, in spite of a significant level of hybridization, pure B. frutescens and B. arborescens are persisting in the hybrid zone. Of the nonparentals, about 18% appear to be F(1) hybrids, over 50% F(2) hybrids, and the remainder backcrossed individuals but only with the B. frutescens parent. It is postulated that the hybrid zone in the Florida Keys is being maintained by a combination of positive assortative mating and clonal reproduction.     

CHARACTER DISCRIMINATORY POWER,CHARACTER-SET CONGRUENCE,AND THE CLASSIFICATION OF INDIVIDUALS FROM HYBRID ZONES: AN EXAMPLE USING STONE CRABS (MENIPPE)

Many investigators categorize individuals from hybrid zones to facilitate comparisons among genotypic classes (e.g., parental, F₁, backcross) for comparative studies in which components of fitness or geographic variation are being analyzed. Frequently, multiple character sets representing genetically independent traits are used to classify these individuals and various methodologies are employed to combine the classifications obtained from the different character sets. We adapted the principles of total evidence and taxonomic congruence (two formalized approaches used by systematists in formulating phylogenetic hypotheses) to address the problem of discriminating hybridizing species and classifying individuals from hybrid zones. As our model, we used two morphological (coloration and morphometric) and two molecular (allozyme and mitochondrial DNA restriction-fragment-length polymorphism) character sets that differentiate two stone crab species (Menippe adina and M. mercenaria). Using principal-components analysis, we determined that combining character sets and eliminating characters or character sets that did not have large eigenvector coefficients for the principal component that best separated the two species yielded the highest level of discrimination between species and allowed us to classify a broad range of morpho-genotypes as hybrids. For the stone crabs, three diagnostic allozyme loci and five diagnostic coloration characters best separated the species. The two character sets were not completely congruent, but they agreed in their classification of 50% of the individuals from the hybrid zone and rarely strongly disagreed in their classifications. Classification discrepancies between the two character sets probably represent variation between traits in interspecific gene flow rather than intraspecific, ecologically mediated variation. Our results support the assertions of previous investigators who espoused the benefits associated with using multiple character sets to classify individuals from hybrid zones and demonstrate that, if character sets are reasonably congruent and numerically balanced, combining diagnostic characters from multiple character sets (a total-evidence approach) can enhance discriminatory power between species and facilitate the assignment of hybrid-zone individuals to genotypic classes. On the contrary, classifying hybrid-zone individuals using character sets separately (a taxonomic-congruence approach) provides the opportunity to compare levels of introgression between species and to assess reasons for discordance among the data sets.     

A multiplicative-epistatic model for analyzing interspecific differences in outcrossing species

Epistasis may play an important role in evolution and speciation. Under multiplicative interactions between different loci, an analytical model is proposed to estimate genetic parameters at the individual locus level that contribute to interspecific differences in outcrossing species. The multiplicative epistasis model, inferred from a number of animal and plant experiments, suggests that genotypes at a pair of loci have genotypic values equal to the product of genotypic values at the two different loci. By considering the genetic property of outcrossing species (i.e., high polymorphisms) in the multilevel family structure analysis for an intra- and interspecific factorial mating design, a method is developed to provide estimates for allele frequencies and additive and dominant effects at individual loci in each of the two parental populations, the genotypic values of newly formed heterozygotes through species combination each with one allele from a parental population and the second from the other parental population, and the numbers of genetic factors that lead to species differentiation. Use of clones offers a tremendous power to test the adequacy of the model. However, the utilization of the model with species that cannot be cloned is also discussed. An example with interspecific hybrids of two forest tree species is used to demonstrate the model.     

Estimation of multilocus linkage disequilibria in diploid populations with dominant markers

Analysis of population structure and organization with DNA-based markers can provide important information regarding the history and evolution of a species. Linkage disequilibrium (LD) analysis based on allelic associations between different loci is emerging as a viable tool to unravel the genetic basis of population differentiation. In this article, we derive the EM algorithm to obtain the maximum-likelihood estimates of the linkage disequilibria between dominant markers, to study the patterns of genetic diversity for a diploid species. The algorithm was expanded to estimate and test linkage disequilibria of different orders among three dominant markers and can be technically extended to manipulate an arbitrary number of dominant markers. The feasibility of the proposed algorithm is validated by an example of population genetic studies of hickory trees, native to southeastern China, using dominant random amplified polymorphic DNA markers. Extensive simulation studies were performed to investigate the statistical properties of this algorithm. The precision of the estimates of linkage disequilibrium between dominant markers was compared with that between codominant markers. Results from simulation studies suggest that three-locus LD analysis displays increased power of LD detection relative to two-locus LD analysis. This algorithm is useful for studying the pattern and amount of genetic variation within and among populations.     

Inheritance and diversity of simple sequence repeat (SSR) microsatellite markers in various families of Picea abies

A large number of sequence-specific SSRs were screened by using electrophoresis on metaphore agarose gels with the bands visualized by ethidium bromide staining. Many SSRs appeared as codominant and many as dominant markers, with presence or absence of bands. A simple Mendelian inheritance pattern for most codominant and dominant SSR loci was found. For many codominant SSR markers, null alleles were detected. The proportion of dominant microsatellites detected in this study (close to 50 %) was much higher than that commonly reported in many other studies. A high proportion of dominant markers together with a high frequency of codominant markers with null alleles may represent two important limitations for the use of microsatellites in different studies. On the other hand, many polymorphic codominant SSR microsatellite markers were found to be highly repeatable, and can be used for population studies, seed certification, quality control of controlled crosses, paternity analysis, pollen contamination, and mapping of QTL in related families. In this paper, we report on the inheritance pattern and diversity of codominant and dominant SSR microsatellites in seven families of Picea abies sharing a common mother.     

A dispersal-dependent zone of introgressive hybridization between weakfish, Cynoscion regalis, and sand seatrout, C. arenarius, (Sciaenidae) in the Florida Atlantic

Five diagnostic codominant nuclear DNA markers and a diagnostic mitochondrial DNA marker were used to survey weakfish (Cynoscion regalis) and sand seatrout (C. arenarius), with particular focus on heretofore uncharacterized juvenile populations along the Florida (FL) Atlantic coast. Geographic and reproductive ranges of weakfish and sand seatrout were shown to overlap on the Atlantic coast along north and central FL. An active bidirectional zone of introgressive hybridization exists between these taxa, centered in the St Johns River, FL. Strong patterns of Hardy-Weinberg, linkage, and cytonuclear disequilibrium and a bimodal hybrid index distribution were observed for juvenile cohorts in the zone center, coupled with narrow (～240 km) concordant clines. Parental forms had disparate habitat preferences; hybrid forms occurred predominantly in intermediate habitats. All genetic data were consistent with the hypothesis that the C. arenarius-C. regalis hybrid zone is maintained by a dynamic equilibrium between continued interspecific gene flow and one or more opposing forces. Cytonuclear analyses indicated that parental forms mate assortatively in the zone but that mate recognition was imperfect. Ethological mating dynamics are likely stabilized by some form of endogenous or exogenous postfertilization selection against hybrids such that parental taxa will likely continue to evolve independently.     

A linkage map for the hybridising toads Bombina bombina and B. variegata (Anura: Discoglossidae)

Stable hybrid zones in which ecologically divergent taxa give rise to a range of recombinants are natural laboratories in which the genetic basis of adaptation and reproductive isolation can be unraveled. One such hybrid zone is formed by the fire-bellied toads Bombina bombina and B. variegata (Anura: Discoglossidae). Adaptations to permanent and ephemeral breeding habitats, respectively, have shaped numerous phenotypic differences between the taxa. All of these are, in principle, candidates for a genetic dissection via QTL mapping. We present here a linkage map of 28 codominant and 10 dominant markers in the Bombina genome. In an F2 cross, markers that were mainly microsatellites, SSCPs or allozymes were mapped to 20 linkage groups. Among the 40 isolated CA microsatellites, we noted a preponderance of compound and frequently interleaved CA-TA repeats as well as a striking polarity at the 5' end of the repeats.     

Early evolution in a hybrid swarm between outcrossing and selfing lineages in Geum

Although often considered as evolutionary dead ends, selfing taxa may make an important contribution to plant evolution through hybridization with related outcrossing lineages. However, there is a shortage of studies examining the evolutionary dynamics of hybridization between outcrossing and selfing taxa. On the basis of differential pollinator attractiveness, production and competitive ability of pollen, as well as levels of inbreeding depression, we predict that the early products of hybridization between outcrossing and selfing lineages will be F1s and first-generation backcrosses sired mainly by the outcrossing lineage, together with selfed F2s containing a limited genetic contribution from the outcrosser. These predictions were tested using amplified fragment length polymorphism and chloroplast markers to analyze the composition of a recent hybrid swarm between predominantly outcrossing Geum rivale and predominantly selfing Geum urbanum. In line with predictions, the hybrid swarm comprised both parental species together with F1s and first-generation backcrosses to G. rivale alone. Chloroplast data suggested that G. rivale was the pollen parent for both observed hybrid classes. However, there was no evidence for F2 individuals, despite the fact that the F1 was fully self-compatible and able to auto-pollinate. The pollen fertility of F1s was only 30% lower than that of the parental taxa, and was fully restored in backcross hybrids. Predicting future evolution in the hybrid swarm will require an understanding of the mating patterns within and among the mix of parental, F1 and backcross genotypes that are currently present. However, these results support the hypothesis that introgression is likely to be asymmetrical from selfing to outcrossing lineages.     

Environmental (in)dependence of a hybrid zone: Insights from molecular markers and ecological niche modeling in a hybrid zone of Origanum (Lamiaceae) on the island of Crete

The role of environment and the relative significance of endogenous versus exogenous selection in shaping hybrid zones have been crucial issues in the studies of hybridization. Recent advances in ecological niche modeling (ENM) offer new methodological tools, especially in combination with the genotyping of individuals in the hybrid zone. Here, we study the hybrid zone between the widely known spices Origanum onites and Origanum vulgare ssp. hirtum in Crete. We analyze the genetic structure of both parental taxa and their hybrid Origanum × intercendens using AFLP markers on 15 sympatric and 12 allopatric populations and employ ecological niche modeling and niche similarity tests to study their niche patterns. We complement these analyses with seed viability measurements. Our study revealed that the hybridizing taxa O. onites and O. vulgare ssp. hirtum and the resulting genotypic classes showed geographical and environmental niche similarities based on the predictions of ENMs and the subsequent similarity tests. The occurrence of the hybrid zone is not directly dependent on environmental factors which favor the fitness of the hybrid compared to the parental taxa, but rather on aspects such as historical factors and management practices, which may contribute to the localization and maintenance of the contact zone between parental species. Our results suggest that if a minimum required niche differentiation between genotypic classes is not achieved, environmental dependence might not have a prominent role on the outcome of the hybridization.     

Interspecific hybridization in natural populations ofCyrtandra (Gesneriaceae) on the Hawaiian Islands: Evidence from RAPD markers

Interspecific hybridization among Hawaiian species ofCyrtandra (Gesneriaceae) was investigated using randomly amplified polymorphic DNA (RAPD) markers. Thirty-three different primers were used to investigate interspecific hybridization for 17 different putative hybrids based on morphological intermediacy and sympatry with putative parental species. RAPD data provided evidence for the hybrid origin of all putative hybrid taxa examined in this analysis. However, the patterns in the hybrid taxa were not found to be completely additive of the patterns found in the parental species. Markers missing in the hybrid taxa can be attributed to polymorphism in the populations of the parental species and the dominant nature of inheritance for RAPD markers. Unique markers found within hybrid taxa require further explanation but do not necessarily indicate that the taxa are not of hybrid origin. The implications suggest that these interspecific hybridization events had, and continue to have, an effect on the adaptive radiation and conservation biology ofCyrtandra.     

Construction of a genetic linkage map in tetraploid species using molecular markers

This article presents methodology for the construction of a linkage map in an autotetraploid species, using either codominant or dominant molecular markers scored on two parents and their full-sib progeny. The steps of the analysis are as follows: identification of parental genotypes from the parental and offspring phenotypes; testing for independent segregation of markers; partition of markers into linkage groups using cluster analysis; maximum-likelihood estimation of the phase, recombination frequency, and LOD score for all pairs of markers in the same linkage group using the EM algorithm; ordering the markers and estimating distances between them; and reconstructing their linkage phases. The information from different marker configurations about the recombination frequency is examined and found to vary considerably, depending on the number of different alleles, the number of alleles shared by the parents, and the phase of the markers. The methods are applied to a simulated data set and to a small set of SSR and AFLP markers scored in a full-sib population of tetraploid potato.     

Molecular and morphological evidence reveals introgression in swarms of the invasive taxa Fallopia japonica, F. sachalinensis, and F. xbohemica (Polygonaceae) in the United States

Fallopia japonica (Japanese knotweed, Polygonaceae) is a well-known East Asian perennial that is established throughout the U.S. and Europe. Another congener, F. sachalinensis, and their hybrid, F. ×bohemica, also persist on both continents. Their invasive success is primarily attributed to their ability to spread via clonal growth. However, mounting evidence suggests invasion history and dynamics differ between continents and that sexual reproduction is more common than previously assumed. We used published morphological traits designed to distinguish the three taxa to characterize their distribution in 24 New England towns. We found continuous variation of all five traits, with 84% of our 81 individuals having at least one trait outside parental limits. Hierarchical cluster analysis, along with two chloroplast and one nuclear species-specific markers, suggests the presence of intercrossing, segregating hybrids, and likely introgression between F1 hybrids and F. japonica. Our markers also show the first evidence of bidirectional hybridization between parental taxa in the U.S., emphasizing the complex structure of populations in our region. This study is a first step toward unraveling the evolutionary forces that have made these taxa such aggressive invaders in the U.S. The data may also affect management strategies originally designed for largely monomorphic, clonal populations.