Patterns of reproductive isolation in three angiosperm genera

Analyses among animal species have found that reproductive isolation increases monotonically with genetic distance, evolves more quickly for prezygotic than postzygotic traits, and is stronger among sympatric than allopatric species pairs. The latter pattern is consistent with expectations under the reinforcement hypothesis. To determine whether similar trends are found among plant species, patterns of reproductive isolation (postpollination prezygotic, postzygotic, and "total" isolation) in three plant genera (Glycine, Silene, Streptanthus) were examined using data from previously published artificial hybridization experiments. In Silene, all measures of reproductive isolation were positively correlated with genetic distance. In contrast, in Glycine and Streptanthus, correlations between reproductive isolation and genetic distance were weak or nonsignificant, possibly due to the influence of biologically unusual taxa, variable evolutionary forces acting in different lineages, or insufficient time to accumulate reproductive isolation. There was no evidence that postpollination prezygotic reproductive isolation evolved faster than postzygotic isolation in Glycine or Silene. We also detected no evidence for faster accumulation of postmating prezygotic isolation between sympatric than allopatric species pairs; thus we found no evidence for the operation of speciation via reinforcement. In Silene, which included six polyploid species, results suggest that changes in ploidy disrupt a simple monotonic relationship between isolation and genetic distance.     

Evolution of reproductive isolation in plants

Reproductive isolation is essential for the process of speciation and much has been learned in recent years about the ecology and underlying genetics of reproductive barriers. But plant species are typically isolated not by a single factor, but by a large number of different pre- and postzygotic barriers, and their potentially complex interactions. This phenomenon has often been ignored to date. Recent studies of the relative importance of different isolating barriers between plant species pairs concluded that prezygotic isolation is much stronger than postzygotic isolation. But studies of the patterns of reproductive isolation in plants did not find that prezygotic isolation evolves faster than postzygotic isolation, in contrast to most animals. This may be due to the multiple premating barriers that isolate most species pairs, some of which may be controlled by few genes of major effect and evolve rapidly, whereas others have a complex genetic architecture and evolve more slowly. Intrinsic postzygotic isolation in plants is correlated with genetic divergence, but some instrinsic postzygotic barriers evolve rapidly and are polymorphic within species. Extrinsic postzygotic barriers are rarely included in estimates of different components of reproductive isolation. Much remains to be learned about ecological and molecular interactions among isolating barriers. The role of reinforcement and reproductive character displacement in the evolution of premating barriers is an open topic that deserves further study. At the molecular level, chromosomal and genic isolation factors may be associated and act in concert to mediate reproductive isolation, but their interactions are only starting to be explored.     

SPECIATION BY REINFORCEMENT OF PREMATING ISOLATION

The generation of premating isolation given partial or complete postzygotic isolation between populations is termed reinforcement or, in the case of complete isolation, reproductive character displacement. In this study we use computer simulations and a multilocus genetic model to reevaluate the theory of reinforcement. We consider the evolution of female preferences for a male secondary sexual trait. If the populations differ in mean female preference, there is direct selection on the preference for further divergence, which may be augmented by a correlated response to sexual selection on males. Two factors prevent divergence. First, if postzygotic isolation is not complete, gene flow can prevent divergence and lead to a hybrid swarm. This is the usual outcome whenever the average number of breeding adult offspring produced by a hybrid mating is sufficient to replace the parents. Second, one or the other population may become extinct because of the large number of hybrid matings it is involved in. The likelihood of extinction is lowered if population growth rates are high, if hybrids are inviable rather than infertile, or under some conditions when allopatric populations provide immigrants into the contact zone. Provided hybrid fitness is sufficiently low, there is a wide range of genetic and ecological conditions under which reinforcement rather easily occurs, and also a range under which it may occur because of stochastic effects on both the inheritance parameters and the population sizes.     

Along the speciation continuum: Quantifying intrinsic and extrinsic isolating barriers across five million years of evolutionary divergence in California jewelflowers

Understanding the relative roles of intrinsic and extrinsic reproductive barriers, and their interplay within the geographic context of diverging taxa, remains an outstanding challenge in the study of speciation. We conducted a comparative analysis of reproductive isolation in California Jewelflowers (Streptanthus, s.l., Brassicaceae) by quantifying potential barriers to gene flow at multiple life history stages in 39 species pairs spanning five million years of evolutionary divergence. We quantified nine potential pre‐ and postzygotic barriers and explored patterns of reproductive isolation in relation to genetic distance. Intrinsic postzygotic isolation was initially weak, increased at intermediate genetic distances, and reached a threshold characterized by complete genetic incompatibility. Climatic niche differences were strong at shallow genetic distances, and species pairs with overlapping ranges showed slight but appreciable phenological isolation, highlighting the potential for ecological barriers to contribute to speciation. Geographic analyses suggest that speciation is not regionally allopatric in the California Jewelflowers, as recently diverged taxa occur in relatively close proximity and display substantial range overlap. Young pairs are characterized by incomplete intrinsic postzygotic isolation, suggesting that extrinsic barriers or fine‐scale spatial segregation are more important early in the divergence process than genetic incompatibilities.     

Hybridization and reproductive isolation between diploid Erythronium mesochoreum and its tetraploid congener E. albidum (Liliaceae)

Polyploidy has played an important role in angiosperm diversification, but how polyploidy contributes to reproductive isolation remains poorly understood. Most work has focused on postzygotic reproductive barriers, and the influence of ploidy differences on prezygotic barriers is understudied. To address these gaps, we quantified hybrid occurrence, interspecific self‐compatibility differences, and the contributions of multiple pre‐ and postzygotic barriers to reproductive isolation between diploid Erythronium mesochoreum (Liliaceae) and its tetraploid congener Erythronium albidum. Reproductive isolation between the study species was nearly complete, and naturally occurring hybrids were infrequent and largely sterile. Although postzygotic barriers effected substantial reproductive isolation when considered in isolation, the study species’ spatial distributions and pollinator assemblages overlapped little, such that interspecific pollen transfer is likely uncommon. We did not find evidence that E. albidum and E. mesochoreum differed in mating systems, indicating that self‐incompatibility release may not have fostered speciation in this system. Ultimately, we demonstrate that E. albidum and E. mesochoreum are reproductively isolated by multiple, hierarchically‐operating barriers, and we add to the currently limited number of studies demonstrating that early acting barriers such as pollinator‐mediated isolation can be important for effecting and sustaining reproductive isolation in diploid‐polyploid systems.     

Extensive loss of Wnt genes in Tardigrada

Evolution of reproductive isolation is an important process, generating biodiversity and driving speciation. To better understand this process, it is necessary to investigate factors underlying reproductive isolation through various approaches but also in various taxa. Previous studies, mainly focusing on diploid animals, supported the prevalent view that reproductive barriers evolve gradually as a by-product of genetic changes accumulated by natural selection by showing a positive relationship between the degree of reproductive isolation and genetic distance. Haplodiploid animals are expected to generate additional insight into speciation, but few studies investigated the prevalent view in haplodiploid animals. In this study, we investigate whether the relationship also holds in a haplodiploid spider mite, Amphitetranychus viennensis (Zacher). We sampled seven populations of the mite in the Palaearctic region, measured their genetic distance (mtDNA) and carried out cross experiments with all combinations. We analyzed how lack of fertilization rate (as measure of prezygotic isolation) as well as hybrid inviability and hybrid sterility (as measures of postzygotic isolation) varies with genetic distance. We found that the degree of reproductive isolation varies among cross combinations, and that all three measures of reproductive isolation have a positive relationship with genetic distance. Based on the mtDNA marker, lack of fertilization rate, hybrid female inviability and hybrid female sterility were estimated to be nearly complete (99.0–99.9% barrier) at genetic distances of 0.475–0.657, 0.150–0.209 and 0.145–0.210, respectively. Besides, we found asymmetries in reproductive isolation. The prevalent view on the evolution of reproductive barriers is supported in the haplodiploid spider mite we studied here. According to the estimated minimum genetic distance for total reproductive isolation in parent population crosses in this study and previous work, a genetic distance of 0.15–0.21 in mtDNA (COI) appears required for speciation in spider mites. Variations and asymmetries in the degree of reproductive isolation highlight the importance of reinforcement of prezygotic reproductive isolation through incompatibility and the importance of cytonuclear interactions for reproductive isolation in haplodiploid spider mites.     

Evolution of postzygotic reproductive isolation in a guild of deceptive orchids

The evolution of reproductive barriers is of central importance for speciation. Here, we investigated three components of postzygotic isolation-embryo mortality, hybrid inviability, and hybrid sterility-in a group of food-deceptive Mediterranean orchids from the genera Anacamptis, Neotinea, and Orchis. In these orchids, pollinator-mediated isolation is weak, which suggests that postpollination barriers exist. Based on crossing experiments and a literature survey, we found that embryo mortality caused complete reproductive isolation among 36.3% of the species pairs, and hybrid inviability affected 55.6% of the potentially hybridizing species pairs. Hybrid sterility was assessed experimentally for seven species pairs. A strong reduction of fertility in all investigated hybrids was found, together with clear differences between male and female components of hybrid sterility. Postzygotic isolation was found to evolve gradually with genetic divergence, and late postzygotic isolation (i.e., hybrid inviability and sterility) evolved faster than embryo mortality, which is an earlier postzygotic isolation stage. These results reveal that intrinsic postzygotic isolation strongly contributes to maintaining species boundaries among Mediterranean food-deceptive orchids while establishing a prominent role for these reproductive barriers in the early stage of species isolation.     

Reciprocal gene loss following experimental whole-genome duplication causes reproductive isolation in yeast

Whole-genome duplication has shaped the genomes of extant lineages ranging from unicellular fungi to vertebrates, and its association with several species-rich taxa has fueled interest in its potential as a catalyst for speciation. One well-established model for the evolution of reproductive isolation involves the reciprocal loss of redundant genes at different loci in allopatric populations. Whole-genome duplication simultaneously doubles the entire gene content of an organism, resulting in massive levels of genetic redundancy and potential for reciprocal gene loss that may produce postzygotic reproductive isolation. Following whole-genome duplication, different populations can potentially change or lose gene function at different duplicate loci. If such populations come back into contact any F1 hybrids that are formed may suffer reduced fertility as some of the gametes they produce may not carry a full complement of functional genes. This reduction in hybrid fertility will be directly proportional to the number of divergently resolved loci between the populations. In this work, we demonstrate that initially identical populations of allotetraploid yeast subjected to mutagenesis rapidly evolve postzygotic reproductive isolation, consistent with the divergent loss of function of redundant gene copies.     

Divergent strategies in pre‐ and postzygotic reproductive isolation between two closely related Dianthus species

Quantifying the relative contribution of multiple isolation barriers to gene flow between recently diverged species is essential for understanding speciation processes. In parapatric populations, local adaptation is thought to be a major contributor to the evolution of reproductive isolation. However, extrinsic postzygotic barriers assessed in reciprocal transplant experiments are often neglected in empirical assessments of multiple isolation barriers. We analyzed multiple isolation barriers between two closely related species of the plant genus Dianthus, a genus characterized by the most rapid species diversification in plants reported so far. Although D. carthusianorum L. and D. sylvestris Wulf. can easily be hybridized in crossing experiments, natural hybrids are rare. We found that in parapatry, pollinator‐mediated prezygotic reproductive isolation barriers are important for both D. carthusianorum (0.761) and D. sylvestris (0.468). In contrast to D. carthusianorum, high hybrid viability in D. sylvestris (–0.491) was counteracted by strong extrinsic postzygotic isolation (0.900). Our study highlights the importance of including reciprocal transplant experiments for documenting extrinsic postzygotic isolation and demonstrates clearly divergent strategies and hence asymmetric pre‐ and postzygotic reproductive isolation between closely related species. It also suggests that pollinator‐mediated and ecological isolation could have interacted in synergistic ways, further stimulating rapid speciation in Dianthus.     

Asymmetry in reproductive isolation and its effect on directional mitochondrial introgression in the parapatric ground beetles <Emphasis Type="Italic">Carabus yamato</Emphasis> and <Emphasis Type="Italic">C. albrechti</Emphasis>

Speciation studies seek to clarify the origin of reproductive isolation, the various mechanisms working from mate recognition through postzygotic stages. Asymmetric effects of isolating barriers can result in asymmetrical gene introgression during interspecific hybridization. The flightless ground beetles Carabus yamato and C. albrechti are distributed parapatrically in Japan, showing repeated asymmetrical introgression of mitochondria from C. albrechti to C. yamato. This pattern suggests that reproductive isolation between these species is strong, but incomplete and asymmetric (i.e., weaker for the cross between a C. albrechti female and a C. yamato male). To test this hypothesis, we conducted interspecific mating experiments in the laboratory. The estimates of total reproductive isolation, which occurred mainly at the premating and postmating/prezygotic stages, were high (isolation index = 0.964 for C. yamato female × C. albrechti male and 0.886 for the reciprocal cross), supporting the hypothesis of strong, but incomplete isolation. However, the observed difference between the reciprocal crosses was not sufficiently large to conclude that it caused directional introgression of mitochondria. Instead, we found asymmetry in individual isolating barriers in the postmating/prezygotic stages that coincided with the prediction, perhaps resulting from morphological mismatch of heterospecific genitalia. Although this asymmetry was compensated for by an inverse asymmetry of isolation in the postzygotic stage, the contribution of these individual barriers to total isolation may change for our expectation when considering females mating with multiple heterospecific males.     

Postmating-prezygotic isolation is not an important source of selection for reinforcement within and between species in Drosophila pseudoobscura and D. persimilis

Abstract Most work on adaptive speciation to date has focused on the role of low hybrid fitness as the force driving reinforcement (the evolution of premating isolation after secondary contact that reduces the likelihood of matings between populations). However, recent theoretical work has shown that postmating, prezygotic incompatibilities may also be important in driving premating isolation. We quantified premating, postmating-prezygotic, and early postzygotic fitness effects in crosses among three populations: Drosophila persimilis, D. pseudoobscura USA (sympatric to D. persimilis ), and D. pseudoobscura Bogotá (allopatric to D. persimilis ). Interspecific matings were more likely to fail when they involved the sympatric populations than when they involved the allopatric populations, consistent with reinforcement. We also found that failure rate in sympatric mating trials depended on whether D. persimilis females were paired with D. pseudoobscura males or the reverse. This asymmetry most likely indicates differences in discrimination against heterospecific males by females. By measuring egg laying rate, fertilization success and hatching success, we also compared components of postmating-prezygotic and early postzygotic isolation. Postmating-prezygotic fitness costs were small and not distinguishable between hetero- and conspecific crosses. Early postzygotic fitness effects due to hatching success differences were also small in between-population crosses. There was, however, a postzygotic fitness effect that may have resulted from an X-linked allele found in one of the two strains of D. pseudoobscura USA. We conclude that the postmating-prezygotic fitness costs we measured probably did not drive premating isolation in these species. Premating isolation is most likely driven in sympatric populations by previously known hybrid male sterility.     

Quantifying patterns in the evolution of reproductive isolation

We present a likelihood-based statistical method for examining the pattern or rate of evolution of reproductive isolation. The method uses large empirical datasets to estimate, for a given clade, the average duration of two phases in the divergence of populations. The first phase is a lag phase and refers to the period during which lineages diverge but no detectable reproductive isolation evolves. The second is an accumulation phase, referring to the period during which the magnitude of reproductive isolation between diverging lineages increases. The pattern of evolution is inferred from the relative durations of these two phases. Results of analyses of postzygotic isolation data indicate significant differences among taxa in the pattern of evolution of postzygotic isolation that are consistent with predictions based on genetic differences among these groups. We also examine whether the evolution of postzygotic isolation is best explained by either of two models for the rate of accumulation: a linear model or a quadratic function as may be suggested by recent studies. Our analysis indicates that the appropriateness of either model varies among taxa.     

Accumulating postzygotic isolation genes in parapatry: a new twist on chromosomal speciation

Chromosomal rearrangements can promote reproductive isolation by reducing recombination along a large section of the genome. We model the effects of the genetic barrier to gene flow caused by a chromosomal rearrangement on the rate of accumulation of postzygotic isolation genes in parapatry. We find that, if reproductive isolation is produced by the accumulation in parapatry of sets of alleles compatible within but incompatible across species, chromosomal rearrangements are far more likely to favor it than classical genetic barriers without chromosomal changes. New evidence of the role of chromosomal rearrangements in parapatric speciation suggests that postzygotic isolation is often due to the accumulation of such incompatibilities. The model makes testable qualitative predictions about the genetic signature of speciation.     

ON THE COYNE AND ORR‐IGIN OF SPECIES: EFFECTS OF INTRINSIC POSTZYGOTIC ISOLATION,ECOLOGICAL DIFFERENTIATION,X CHROMOSOME SIZE,AND SYMPATRY ON DROSOPHILA SPECIATION

Coyne and Orr found that mating discrimination (premating isolation) evolves much faster between sympatric than allopatric Drosophila species pairs. Their meta‐analyses established that this pattern, expected under reinforcement, is common and that Haldane's rule is ubiquitous in Drosophila species divergence. We examine three possible contributors to the reinforcement pattern: intrinsic postzygotic isolation, dichotomized as to whether hybrid males show complete inviability/sterility; host‐plant divergence, as a surrogate for extrinsic postzygotic isolation; and X chromosome size, whether roughly 20% or 40% of the genome is X‐linked. We focus on “young” species pairs with overlapping ranges, contrasted with allopatric pairs. Using alternative criteria for “sympatry” and tests that compare either level of prezygotic isolation in sympatry or frequency of sympatry, we find no statistically significant effects associated with X chromosome size or our coarse quantifications of intrinsic postzygotic isolation or ecological differentiation. Although sympatric speciation seems very rare in animals, the pervasiveness of the reinforcement pattern and the commonness of range overlap for close relatives indicate that speciation in Drosophila is often not purely allopatric. It remains to determine whether increased premating isolation with sympatry results from secondary contact versus parapatric speciation and what drives this pattern.     

Reproductive isolation between two closely related hummingbird-pollinated neotropical gingers

Empirical estimates of the relative importance of different barriers to gene flow between recently diverged species are important for understanding processes of speciation. I investigated the factors contributing to reproductive isolation between Costus pulverulentus and C. scaber (Costaceae), two closely related hummingbird-pollinated understory Neotropical herbs. I studied broad-scale geographic isolation, microhabitat isolation, flowering phenology, overlap in pollinator assemblages, floral constancy by pollinators, mechanical floral isolation, pollen-pistil interactions, seed set in interspecific crosses, and postzygotic isolation (hybrid seed germination, greenhouse survival to flowering, and pollen fertility). Aside from substantial geographic isolation, I found evidence for several factors contributing to reproductive isolation in the sympatric portion of their geographic ranges, but the identity and relative strength of these factors varied depending on the direction of potential gene flow. For C. pulverulentus as the maternal parent, mechanical floral isolation was the most important factor, acting as a complete block to interspecific pollen deposition. For C. scaber as the maternal parent, microhabitat isolation, pollinator assemblage, mechanical floral isolation, and postpollination pollen-pistil incompatibility were important. Overall, prezygotic barriers were found to be strong, resulting in 100% reproductive isolation for C. pulverulentus as the maternal parent and 99.0% reproductive isolation for C. scaber as the maternal parent. Some postzygotic isolation also was identified in the F1 generation, increasing total isolation for C. scaber to 99.4%. The results suggest that ecological factors, including habitat use and plant-pollinator interactions, contributed to speciation in this system and evolved before extensive intrinsic postzygotic isolation.     

Genetic variation for postzygotic reproductive isolation between Caenorhabditis briggsae and Caenorhabditis sp. 9

The process of speciation is key to the origins of biodiversity, and yet the Caenorhabditis nematode model system has contributed little to this topic. Genetic studies of speciation in the genus are now feasible, owing to crosses between the recently discovered Caenorhabditis sp. 9 and the well-known C. briggsae producing fertile F(1) hybrid females. We dissected patterns of postzygotic reproductive isolation between these species by crossing eight isogenic strains of C. briggsae reciprocally with six strains of C. sp. 9. We determined that overall patterns of reproductive isolation are robust across these genetic backgrounds. However, we also quantified significant heritable variation within each species for interspecific hybrid incompatibilities for total adult progeny, egg-to-adult viability, and the percentage of male progeny. This demonstrates that intraspecific variation for interspecific hybrid incompatibility occurs despite extensive, albeit incomplete, reproductive isolation. Therefore, this emerging general phenomenon of variable reproductive isolation is not restricted to highly interfertile, early-stage incipient species, but also applies to species in the latest stages of the speciation process. Furthermore, we confirm Haldane's rule and demonstrate strongly asymmetric parent-of-origin effects (Darwin's corollary) that consistently manifest more extremely when hermaphroditic C. briggsae serves as maternal parent. These findings highlight Caenorhabditis as an emerging system for understanding the genetics of general patterns of reproductive isolation.     

Strength in numbers? Cytotype frequency mediates effect of reproductive barriers in mixed-ploidy arrays

When differentiated lineages come into contact, their fates depend on demographic and reproductive factors. These factors have been well-studied in taxa of the same ploidy, but less is known about sympatric lineages that differ in ploidy, particularly with respect to demographic factors. We assessed prezygotic, postzygotic, and total reproductive isolation in naturally pollinated arrays of diploid-tetraploid and tetraploid-hexaploid population mixes of Campanula rotundifolia by measuring pollinator transitions, seed yield, germination rate, and proportion of hybrid offspring. Four frequencies of each cytotype were tested, and pollinators consistently overvisited rare cytotypes. Seed yield and F1 hybrid production were greater in 4X-6X arrays than 2X-4X arrays, whereas germination rates were similar, creating two distinct patterns of reproductive isolation. In 2X-4X arrays, postzygotic isolation was near complete (3% hybrid offspring), and prezygotic isolation associated with pollinator preference is expected to facilitate the persistence of minority cytotypes. However, in 4X-6X arrays where postzygotic isolation permitted hybrid formation (44% hybrids), pollinator behavior drove patterns of reproductive isolation, with rare cytotypes being more isolated and greater gene flow expected from rare into common cytotypes. In polyploid complexes, both the specific cytotypes in contact and local cytotype frequency, likely reflecting spatial demography, will influence likelihood of gene exchange.     

POSTZYGOTIC ISOLATION OVER MULTIPLE GENERATIONS OF HYBRID DESCENDENTS IN A NATURAL HYBRID ZONE: HOW WELL DO SINGLE‐GENERATION ESTIMATES REFLECT REPRODUCTIVE ISOLATION?

Understanding speciation depends on an accurate assessment of the reproductive barriers separating newly diverged populations. In several taxonomic groups, prezygotic barriers, especially preferences for conspecific mates, are thought to play the dominant role in speciation. However, the importance of postzygotic barriers (i.e., low fitness of hybrid offspring) may be widely underestimated. In this study, we examined how well the widely used proxy of postzygotic isolation (reproductive output of F₁ hybrids) reflects the long‐term fitness consequences of hybridization between two closely related species of birds. Using 40 species‐specific single nucleotide polymorphism (SNP) markers, we genotyped a mixed population of collared and pied flycatchers (Ficedula albicollis and F. hypoleuca) to identify grand‐ and great grand‐offspring from interspecific crosses to derive an accurate, multigeneration estimate of postzygotic isolation. Two independent estimates of fitness show that hybridization results in 2.4% and 2.7% of the number of descendents typical of conspecific pairing. This postzygotic isolation was considerably stronger than estimates based on F₁ hybrids. Our results demonstrate that, in nature, combined selection against hybrids and backcrossed individuals may result in almost complete postzygotic isolation between two comparatively young species. If these findings are general, postzygotic barriers separating hybridizing populations may be much stronger than previously thought.     

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

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

Factors contributing to the accumulation of reproductive isolation: A mixed model approach

The analysis of large datasets describing reproductive isolation between species has been extremely influential in the study of speciation. However, the statistical methods currently used for these data limit the ability to make direct inferences about the factors predicting the evolution of reproductive isolation. As a result, our understanding of iconic patterns and rules of speciation rely on indirect analyses that have clear statistical limitations. Phylogenetic mixed models are commonly used in ecology and evolution, but have not been applied to studies of reproductive isolation. Here I describe a flexible framework using phylogenetic mixed models to analyze data collected at different evolutionary scales, to test both categorical and continuous predictor variables, and to test the effect of multiple predictors on rates and patterns of reproductive isolation simultaneously. I demonstrate the utility of this framework by re‐analyzing four classic datasets, from both animals and plants, and evaluating several hypotheses that could not be tested in the original studies: In the Drosophila and Bufonidae datasets, I found support for more rapid accumulation of reproductive isolation in sympatric species pairs compared to allopatric species pairs. Using Silene and Nolana, I found no evidence supporting the hypothesis that floral differentiation elevates postzygotic reproductive isolation. The faster accumulation of postzygotic isolation in sympatry is likely the result of species coexistence determined by the level of postzygotic isolation between species. In addition, floral trait divergence does not appear to translate into pleiotropic effects on postzygotic reproductive isolation. Overall, these methods can allow researchers to test new hypotheses using a single statistical method, while remedying the statistical limitations of several previous methods.