TROPICS ACCELERATE THE EVOLUTION OF HYBRID MALE STERILITY IN DROSOPHILA

Understanding the evolutionary mechanisms that facilitate speciation and explain global patterns of species diversity has remained a challenge for decades. The most general pattern of species biodiversity is the latitudinal gradient, whereby species richness increases toward the tropics. Although such a global pattern probably has a multitude of causes, recent attention has focused on the hypothesis that speciation and the evolution of reproductive isolation occur faster in the tropics. Here, I tested this prediction using a dataset on premating and postzygotic isolation between recently diverged Drosophila species. Results showed that while the evolution of premating isolation was not greater between tropical Drosophila relative to nontropical species, postzygotic isolation evolved faster in the tropics. In particular, hybrid male sterility was much greater among tropical Drosophila compared to nontropical species pairs of similar genetic age. Several testable explanations for the novel pattern are discussed, including greater role for sterility‐inducing bacterial endosymbionts in the tropics and more intense sperm–sperm competition or sperm–egg sexual conflict in the tropics. The results imply that processes of speciation in the tropics may evolve at different rates or may even be somewhat different from those at higher latitudes.     

CYTOLOGICAL CHARACTERIZATION OF PREMEIOTIC VERSUS POSTMEIOTIC DEFECTS PRODUCING HYBRID MALE STERILITY AMONG SIBLING SPECIES OF THE DROSOPHILA MELANOGASTER COMPLEX

In accordance with Haldane's rule, hybridizations between species of the Drosophila simulans clade produce fertile females but sterile males. In this study, a comprehensive characterization was undertaken on the six types of F₁ males that were the result of the crosses between D. simulans, D. sechellia, and D. mauritiana. With the use of light and electron microscopy, it was shown that while each particular hybrid genotype exhibited a specific sterility phenotype, these phenotypes fell into two distinct classes. The two hybrid genotypes that possessed D. mauritiana X-chromosomes contained spermatogenic defects that caused arrests in premeiotic spermatogenic stages. The other four F₁ hybrids possessed postmeiotic spermatogenic defects. Nonsynchronous cell divisions, underdeveloped mitochondrial derivative-axonemal associations, and microtubule abnormalities were common to all of these hybrids. Each particular postmeiotically defective hybrid genotype demonstrated characteristically distinct profiles in sperm bundle number in addition to characteristic spermiogenic arrests in the furthest developed spermatids. These results in species hybrids contrast with the absence of significant differences in spermatogenic characters between species of this clade. In addition, by utilizing an attached-X cross, we investigated the influence of maternal effects and cytoplasmic factors on the sterility of D. simulans F₁ hybrids and found none. However, we discovered a strain of D. simulans (2119) that caused a large shift in sterility from postmeiotic to premeiotic when crossed to D. sechellia. This suggests that D. simulans is polymorphic for genes involving premeiotic and postmeiotic sterility and that the two types of sterilities between species may have a simple genetic basis.     

GENETICS OF SEXUAL ISOLATION AND COURTSHIP DYSFUNCTION IN MALE HYBRIDS OF DROSOPHILA PSEUDOOBSCURA AND DROSOPHILA PERSIMILIS

Despite the importance of sexual isolation to speciation, few studies have analyzed the genetic basis of interspecific mating discrimination, particularly using hybrid males. In this study, I investigated the genetic basis of sexual isolation using male hybrids of Drosophila pseudoobscura and D. persimilis. Hybrid male mating success was caused by interactions between the X-chromosome and autosomes (or Y-chromosome), and different arms of the X-chromosome contributed to mating success with females of each species. Further, although there was an X-chromosome component to mating success, its magnitude was not disproportionately large when compared with the proportion of the genome contained on this chromosome. Some hybrid males courted with an anomalously low intensity, so I simultaneously mapped the genetic basis of this “courtship dysfunction.” The courtship dysfunction was caused by an interaction between the left arm of the X-chromosome in D. persimilis with the autosomes or Y-chromosome from D. pseudoobscura. Anomalous courtship behavior in interspecific hybrids can obscure the conclusions of studies of the genetics of sexual isolation, so courtship intensity should be evaluated in all such investigations.     

DISSECTING THE GENETIC ARCHITECTURE OF F1 HYBRID STERILITY IN HOUSE MICE

Hybrid sterility as a postzygotic reproductive isolation mechanism has been studied for over 80 years, yet the first identifications of hybrid sterility genes in Drosophila and mouse are quite recent. To study the genetic architecture of F₁ hybrid sterility between young subspecies of house mouse Mus m. domesticus and M. m. musculus, we conducted QTL analysis of a backcross between inbred strains representing these two subspecies and probed the role of individual chromosomes in hybrid sterility using the intersubspecific chromosome substitution strains. We provide direct evidence that the asymmetry in male infertility between reciprocal crosses is conferred by the middle region of M. m. musculus Chr X, thus excluding other potential candidates such as Y, imprinted genes, and mitochondrial DNA. QTL analysis identified strong hybrid sterility loci on Chr 17 and Chr X and predicted a set of interchangeable autosomal loci, a subset of which is sufficient to activate the Dobzhansky–Muller incompatibility of the strong loci. Overall, our results indicate the oligogenic nature of F₁ hybrid sterility, which should be amenable to reconstruction by proper combination of chromosome substitution strains. Such a prefabricated model system should help to uncover the gene networks and molecular mechanisms underlying hybrid sterility.     

DEGREE OF SYMPATRY AFFECTS REINFORCEMENT IN DROSOPHILA

In a recent paper, Yukilevich (2012) showed that asymmetries between Drosophila species in the strength of premating isolation tend to match asymmetries in the costs of hybridization (inferred from asymmetries in the strength of postzygotic isolation and range sizes). The results provide novel evidence that the outcome of reinforcement can depend on the strength and frequency of selection against hybridization. Here, I reanalyze the data to demonstrate that another (unconsidered) factor, namely the quantitative degree of sympatry between species, also predictably affects reinforcement. Specifically, premating isolation is strongest at intermediate degrees of sympatry. This result complements, rather than challenges, those of Yukilevich (2012) . One possible explanation for this newly discovered pattern is that when the degree of sympatry is small, selection for avoidance of hybridization is rare, but when the degree of sympatry is large, homogenizing gene flow overcomes reinforcing selection. Thus, reinforcement may depend on the balance between selection and gene flow. However, the current work examined degree of sympatry, not gene flow itself. Thus, further data on gene flow levels in Drosophila is required to test this hypothesis, which emerged from the patterns reported here.     

THE GENETICS OF AN ISOLATING MECHANISM BETWEEN TWO SIBLING SPECIES OF DROSOPHILA

Matings between Drosophila simulans females and males of the sibling species D. mauritiana are of abnormally short duration. These rapid matings interrupt the transfer of sperm, leading to substantial reproductive isolation in interspecific as compared to intraspecific copulations. Genetic analysis of this behavior shows that it is influenced much more by the male than the female genotype, with genes from D. simulans being dominant. In males, shortened copulation is caused by interspecific divergence at a minimum of three loci, with one gene on each of the major chromosomes. This is an underestimate of the true number of loci affecting the trait, which could be much larger. The two autosomes have the largest effect, whereas that of the X chromosome is much smaller. The genetic architecture of copulation duration and the larger effect of male than female genotype suggest that females can detect and discriminate against differences in male genitalia.     

TEMPERATURE-BASED EXTRINSIC REPRODUCTIVE ISOLATION IN TWO SPECIES OF DROSOPHILA

Drosophila santomea and D. yakuba are sister species that live on the African volcanic island of São Tomé, where they are ecologically isolated: D. yakuba inhabits low-altitude open and semiopen habitats while D. santomea lives in higher-elevation rain and mist forest. To determine whether this spatial isolation reflected differential preference for and tolerance of temperature, we estimated fitness components of both species at different temperatures as well as their behavioral preference for certain temperatures. At higher temperatures, especially 28°C, D. santomea was markedly inferior to D. yakuba in larval survival, egg hatchability, and longevity. Moreover, D. santomea females, unlike those of D. yakuba , become almost completely sterile after exposure to a temperature of 28°C, and conspecific males become semisterile. Drosophila santomea adults prefer temperatures 2–3°C lower than do adults of D. yakuba . Drosophila santomea , then, is poorly adapted to high temperature, partially explaining its restriction to cool, high habitats, which leads to extrinsic premating isolation and immigrant inviability. Rudimentary genetic analysis of the interspecific difference in egg hatchability and larval survival showed that these differences are due largely to cytoplasmic effects and to autosomal genes, with sex chromosomes playing little or no role.     

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

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

EVIDENCE FOR DOBZHANSKY-MULLER INCOMPATIBILITES CONTRIBUTING TO THE STERILITY OF HYBRIDS BETWEEN MIMULUS GUTTATUS AND M. NASUTUS

Abstract Both chromosomal rearrangements and negative interactions among loci (Dobzhansky‐Muller incompatibilities) have been advanced as the genetic mechanism underlying the sterility of interspecific hybrids. These alternatives invoke very different evolutionary histories during speciation and also predict different patterns of sterility in artificial hybrids. Chromosomal rearrangements require drift, inbreeding, or other special conditions for initial fixation and, because heterozygosity per se generates any problems with gamete formation, F1 hybrids will be most infertile. In contrast, Dobzhansky‐Muller incompatibilities may arise as byproducts of adaptive evolution and often affect the segregating F2 generation most severely. To distinguish the effects of these two mechanisms early in divergence, we investigated the quantitative genetics of hybrid sterility in a line cross between two members of the Mimulus guttatus species complex (M. guttatus and M. nasutus). Hybrids showed partial male and female sterility, and the patterns of infertility were not consistent with the action of chromosomal rearrangements alone. F2 and F1 hybrids exhibited equal decreases in pollen viability (> 40%) relative to the highly fertile parental lines. A large excess of completely pollen‐sterile F2 genotypes also pointed to the segregation of Dobzhansky‐Muller incompatibility factors affecting male fertility. Female fertility showed a pattern similarly consistent with epistatic interactions: F2 hybrids produced far fewer seeds per flower than F1 hybrids (88.0 ± 2.8 vs. 162.9 ± 8.5 SE, respectively) and either parental line, and many F2 genotypes were completely female sterile. Dobzhansky‐Muller interactions also resulted in the breakdown of several nonreproductive characters and appear to contribute to correlations between male and female fertility in the F2 generation. These results parallel and contrast with the genetics of postzygotic isolation in model animal systems and are a first step toward understanding the process of speciation in this well‐studied group of flowering plants.     

UNIFICATION OF METHODS FOR ESTIMATING THE STRENGTH OF REPRODUCTIVE ISOLATION

Understanding the evolution of reproductive isolation is tantamount to describing the origin of species. Therefore, a primary goal in evolutionary biology is to identify which reproductive barriers are most important to the process. To achieve this goal, the strength of multiple forms of isolation must be compared in an equivalent manner. However, a diversity of methods has been used to estimate barrier strength, falling into several mathematically distinct categories. This study provides a unified method for calculating isolation that relates the amount of gene flow experienced by taxa to random expectations in a simple linear framework. This approach has three distinct advantages over previous methods: (1) it is directly related to gene flow, (2) it is symmetrical, such that measures in both the positive and negative range are comparable, and (3) it is equivalent between broad categories of reproductive isolation, allowing for appropriate comparisons. This linear formulation can be adjusted for use in all forms of isolation, and can accommodate cases in which null expectations for con‐ and heterospecific gene flow differ. Additionally, this framework can be used to calculate total reproductive isolation and the relative contributions of individual barriers.     

WOLBACHIA AND THE EVOLUTION OF REPRODUCTIVE ISOLATION BETWEEN DROSOPHILA RECENS AND DROSOPHILA SUBQUINARIA

Endosymbiotic bacteria of the genus Wolbachia are widespread among insects and in many cases cause cytoplasmic incompatibility in crosses between infected males and uninfected females. Such findings have been used to argue that Wolbachia have played an important role in insect speciation. Theoretical models, however, indicate that Wolbachia alone are unlikely to lead to stable reproductive isolation between two formerly conspecific populations. Here we analyze the components of reproductive isolation between Drosophila recens, which is infected with Wolbachia, and its uninfected sister species Drosophila subquinaria. Laboratory pairings demonstrated that gene flow via matings between D. recens females and D. subquinaria males is hindered by behavioral isolation. Matings readily occurred in the reciprocal cross (D. quinaria females × D. recens males), but very few viable progeny were produced. The production of viable hybrids via this route was restored by antibiotic curing of D. recens of their Wolbachia symbionts, indicating that hybrid offspring production is greatly reduced by cytoplasmic incompatibility in the crosses involving infected D. recens males. Thus, behavioral isolation and Wolbachia-induced cytoplasmic incompatibility act as complementary asymmetrical isolating mechanisms between these two species. In accordance with Haldane's rule, hybrid females were fertile, whereas hybrid males invariably were sterile. Levels of mtDNA variation in D. recens are much lower than in either D. subquinaria or D. falleni, neither of which is infected with Wolbachia. The low haplotype diversity in D. recens is likely due to an mtDNA sweep associated with the spread of Wolbachia. Nevertheless, the existence of several mtDNA haplotypes in this species indicates that Wolbachia have been present as a potential isolating mechanism for a substantial period of evolutionary time. Finally, we argue that although Wolbachia by themselves are unlikely to bring about speciation, they can increase the rate of speciation in insects.     

WIDESPREAD HOST‐DEPENDENT HYBRID UNFITNESS IN THE PEA APHID SPECIES COMPLEX

Linking adaptive divergence to hybrid unfitness is necessary to understand the ecological factors contributing to reproductive isolation and speciation. To date, this link has been demonstrated in few model systems, most of which encompass ecotypes that occupy relatively early stages in the speciation process. Here we extend these studies by assessing how host‐plant adaptation conditions hybrid fitness in the pea aphid, Acyrthosiphon pisum. We made crosses between and within five pea aphid biotypes adapted to different host plants and representing various stages of divergence within the complex. Performance of F1 hybrids and nonhybrids was assessed on a “universal” host that is favorable to all pea aphid biotypes in laboratory conditions. Although hybrids performed equally well as nonhybrids on the universal host, their performance was much lower than nonhybrids on the natural hosts of their parental populations. Hence, hybrids, rather than being intrinsically deficient, are maladapted to their parents’ hosts. Interestingly, the impact of this maladaptation was stronger in certain hybrids from crosses involving the most divergent biotype, suggesting that host‐dependent postzygotic isolation has continued to evolve late in divergence. Even though host‐independent deficiencies are not excluded, hybrid maladaptation to parental hosts supports the hypothesis of ecological speciation in this complex.     

SPECIATION IN THE EUROPEAN RABBIT (ORYCTOLAGUS CUNICULUS): ISLANDS OF DIFFERENTIATION ON THE X CHROMOSOME AND AUTOSOMES

Studies of gene flow between closely related taxa can provide insight into the genetic basis of speciation. To evaluate the importance of the X chromosome in reproductive isolation between subspecies of the European rabbit and to study the genomic scale over which islands of differentiation extend, we resequenced a total of 34 loci distributed along the X chromosome and chromosome 14. Previous studies based on few markers suggested that loci in centromeric regions were highly differentiated between rabbit subspecies, whereas loci in telomeric regions were less differentiated. Here, we confirmed this finding but also discovered remarkable variation in levels of differentiation among loci, with F_ST values from nearly 0 to 1. Analyses using isolation‐with‐migration models suggest that this range appears to be largely explained by differential levels of gene flow among loci. The X chromosome was significantly more differentiated than the autosomes. On chromosome 14, differentiation decayed very rapidly at increasing distances from the centromere, but on the X chromosome distinct islands of differentiation encompassing several megabases were observed both at the centromeric region and along the chromosome arms. These findings support the idea that the X chromosome plays an important role in reproductive isolation between rabbit subspecies. These results also demonstrate the mosaic nature of the genome at species boundaries.     

THE VARIABLE GENOMIC ARCHITECTURE OF ISOLATION BETWEEN HYBRIDIZING SPECIES OF HOUSE MICE

Studies of the genetics of hybrid zones can provide insight into the genomic architecture of species boundaries. By examining patterns of introgression of multiple loci across a hybrid zone, it may be possible to identify regions of the genome that have experienced selection. Here, we present a comparison of introgression in two replicate transects through the house mouse hybrid zone through central Europe, using data from 41 single nucleotide markers. Using both genomic and geographic clines, we found many differences in patterns of introgression between the two transects, as well as some similarities. We found that many loci may have experienced the effects of selection at linked sites, including selection against hybrid genotypes, as well as positive selection in the form of genotypes introgressed into a foreign genetic background. We also found many positive associations of conspecific alleles among unlinked markers, which could be caused by epistatic interactions. Different patterns of introgression in the two transects highlight the challenge of using hybrid zones to identify genes underlying isolation and raise the possibility that the genetic basis of isolation between these species may be dependent on the local population genetic make-up or the local ecological setting.     

SPECIES HYBRIDS IN THE LABORATORY BUT NOT IN NATURE: A REANALYSIS OF PREMATING ISOLATION BETWEEN DROSOPHILA ARIZONAE AND D. MOJAVENSIS

Understanding speciation relies critically on the identification of mechanisms responsible for maintaining species integrity (i.e., reproductive isolation) especially when closely related species are sympatric in nature. Studies of reproductive isolation in Drosophila often involve laboratory mating experiments that assume that patterns of mate choice in the laboratory are similar to those in the wild. Two sibling species, Drosophila arizonae and D. mojavensis , known to exhibit low levels of interspecific hybridization in the laboratory, but not in nature, were used in multiple-choice mating trials using various mating chamber designs as well as synthetic and natural media for developing larvae and courting adults. Sympatric populations were more sexually isolated than allopatric ones, consistent with past studies, and all experimental variables tested (chamber size, host plant presence and rearing substrates) had significant effects on levels of premating isolation between these species. Flies reared on cactus showed increased premating isolation versus those reared on synthetic laboratory food as did providing fermenting host plant tissue during mating trials. Also, surprisingly, smaller mating chambers led to an increase in premating isolation versus larger containers. The design of these types of mating trials is thus critical to understanding how mating behaviors in the laboratory are related to those in natural populations.     

ESTIMATING THE MATING BEHAVIOR OF A PAIR OF HYBRIDIZING HELICONIUS SPECIES IN THE WILD

Premating isolation between incipient species is rarely studied in nature, even though mating tests in captivity may give an inaccurate picture of natural hybridization. We studied premating barriers between the warningly colored butterflies Heliconius erato and H. himera (Lepidoptera) in a narrow contact zone in Ecuador, where hybrids are found at low frequency. Eggs obtained from wild-mated females, supplemented with eggs and young larvae collected from the wild, were reared to adulthood. Adult color patterns of these progeny were then used to infer how their parents must have mated. Likelihood was used to estimate both the frequencies of potential parental genotypes from adult phenotypes collected in the wild, and the degree of assortative mating from the inferred parents. The frequencies of parental genotypes varied across the hybrid zone, but our statistical method allowed estimates of hybrid deficit and assortative mating to be integrated across all sites sampled. The best estimate of the frequency of F₁ and backcross hybrid adults in the center of the hybrid zone was 10%, with support limits (7.1%, 13.0%; support limits are asymptotically equivalent to 95% confidence limits). Mating was highly assortative: in the center of the hybrid zone the cross-mating probability between H. erato and H. himera was only 5% (0.3%, 21.4%). Wild hybrids themselves mated with both pure forms, and the probabilities that they mated in any direction were not significantly lower than those among conspecifics. These results are consistent with earlier laboratory studies on mate choice, and suggest that selection against hybrids must be strong to prevent formation of a hybrid swarm. Unfortunately, the wide support limits on mating behavior precluded a measure of the strength of selection from these data alone. Our statistical approach provides a useful general method for estimating mate choice in the wild.     

THE EVOLUTION OF HYBRID INCOMPATIBILITIES ALONG A PHYLOGENY

The Dobzhansky–Muller model of speciation posits that defects in hybrids between species are the result of negative epistatic interactions between alleles that arose in independent genetic backgrounds. Tests of one important prediction from this model, that incompatibilities “snowball,” have relied on comparisons of the number of incompatibilities between closely related pairs of species separated by different divergence times. How incompatibilities accumulate along phylogenies, however, remains poorly understood. We extend the Dobzhansky–Muller model to multispecies clades to describe the mathematical relationship between tree topology and the number of shared incompatibilities among related pairs of species. We use these results to develop a statistical test that distinguishes between the snowball and alternative incompatibility accumulation models, including nonepistatic and multilocus incompatibility models, in a phylogenetic context. We further demonstrate that patterns of incompatibility sharing across species pairs can be used to estimate the relative frequencies of different types of incompatibilities, including derived–derived versus derived–ancestral incompatibilities. Our results and statistical methods should motivate comparative genetic mapping of hybrid incompatibilities to evaluate competing models of speciation.