Environment-dependent costs and benefits of recombination in independently evolved populations of Escherichia coli*

Understanding of the causes by which reproductive isolation arises remains limited. We examine the role of adaptation in driving reproductive isolation among 12 Escherichia coli populations evolved in two different environments. We found that, regardless of whether parents were selected in the same or different environments, the average fitness of recombinants was lower than the expected, consistent with a prevailing influence of incompatibility between independently accumulated mutations. Exceptions to this pattern occurred among recombinants of some parents evolved in different environments. These recombinants were less fit than expected in the selective environment of one parent, but more fit than expected in the selective environment of the other parent. Our results indicate that both parallel and divergent adaptation can quickly lead to intrinsic genetic barriers contributing to the initial stages of speciation and show that these barriers can be complex, for example, depending on the environment in which recombinant offspring are tested.     

Ecologically dependent and intrinsic genetic signatures of postzygotic isolation between sympatric host races of the leaf beetle Lochmaea capreae

The fitness of hybrids might be compromised as a result of intrinsic isolation and/or because they fall between ecological niches due to their intermediate phenotypes (“extrinsic isolation”). Here, we present data from several crosses (parental crosses, F1, F2, and backcrosses) between the two host races of Lochmaea capreae on willow and birch to test for extrinsic isolation, intrinsic isolation, and environmentally dependent genetic incompatibilities. We employed a reciprocal transplant design in which offspring were raised on either host plant and their survival was recorded until adulthood. We also applied joint‐scaling analysis to determine the genetic architecture of hybrid inviability. The relative fitness of the backcrosses switched between environments; furthermore, the additive genetic–environment interaction was detected as the strongest effect in our analysis. These results provide strong evidence that divergent natural selection has played a central role in the evolution of hybrid dysfunction between host races. Joint‐scaling analysis detected significant negative epistatic effects that are most evident in the poor performance of F2‐hybrids on willow, indicating signs of intrinsic isolation. We did not find any evidence that genetic incompatibilities are manifested independently of environmental conditions. Our findings suggest the outcome of natural hybridization between these host races is mainly affected by extrinsic isolation and a weak contribution of intrinsic isolation.     

THE EVOLUTION OF STRONG REPRODUCTIVE ISOLATION

Felsenstein distinguished two ways by which selection can directly strengthen isolation. First, a modifier that strengthens prezygotic isolation can be favored everywhere. This fits with the traditional view of reinforcement as an adaptation to reduce deleterious hybridization by strengthening assortative mating. Second, selection can favor association between different incompatibilities, despite recombination. We generalize this "two allele" model to follow associations among any number of incompatibilities, which may include both assortment and hybrid inviability. Our key argument is that this process, of coupling between incompatibilities, may be quite different from the usual view of reinforcement: strong isolation can evolve through the coupling of any kind of incompatibility, whether prezygotic or postzygotic. Single locus incompatibilities become coupled because associations between them increase the variance in compatibility, which in turn increases mean fitness if there is positive epistasis. Multiple incompatibilities, each maintained by epistasis, can become coupled in the same way. In contrast, a single-locus incompatibility can become coupled with loci that reduce the viability of haploid hybrids because this reduces harmful recombination. We obtain simple approximations for the limits of tight linkage, and strong assortment, and show how assortment alleles can invade through associations with other components of reproductive isolation.     

Differential timing and control of noncrossover and crossover recombination during meiosis.

Unitary models of meiotic recombination postulate that a central intermediate containing Holliday junctions is resolved to generate either noncrossover or crossover recombinants, both of which contain heteroduplex DNA. Contrary to this expectation, we find that during meiosis in Saccharomyces cerevisiae, noncrossover heteroduplex products are formed at the same time as Holliday junction intermediates. Crossovers appear later, when these intermediates are resolved. Furthermore, noncrossover and crossover recombination are regulated differently. ndt80 mutants arrest in meiosis with unresolved Holliday junction intermediates and very few crossovers, while noncrossover heteroduplex products are formed at normal levels and with normal timing. These results suggest that crossovers are formed by resolution of Holliday junction intermediates, while most noncrossover recombinants arise by a different, earlier pathway.     

Along the speciation continuum in sticklebacks

Speciation can be viewed as a continuum, potentially divisible into several states: (1) continuous variation within panmictic populations, (2) partially discontinuous variation with minor reproductive isolation, (3) strongly discontinuous variation with strong but reversible reproductive isolation and (4) complete and irreversible reproductive isolation. Research on sticklebacks (Gasterosteidae) reveals factors that influence progress back and forth along this continuum, as well as transitions between the states. Most populations exist in state 1, even though some of these show evidence of disruptive selection and positive assortative mating. Transitions to state 2 seem to usually involve strong divergent selection coupled with at least a bit of geographic separation, such as parapatry (e.g. lake and stream pairs and mud and lava pairs) or allopatry (e.g. different lakes). Transitions to state 3 can occur when allopatric or parapatric populations that evolved under strong divergent selection come into secondary contact (most obviously the sympatric benthic and limnetic pairs), but might also occur between populations that remained in parapatry or allopatry. Transitions to state 4 might be decoupled from these selective processes, because the known situations of complete, or nearly complete, reproductive isolation (Japan Sea and Pacific Ocean pair and the recognized gasterosteid species) are always associated with chromosomal rearrangements and environment‐independent genetic incompatibilities. Research on sticklebacks has thus revealed complex and shifting interactions between selection, adaptation, mutation and geography during the course of speciation.     

Exogenous selection rather than cytonuclear incompatibilities shapes asymmetrical fitness of reciprocal Arabidopsis hybrids

Reciprocal crosses between species often display an asymmetry in the fitness of F₁ hybrids. This pattern, referred to as isolation asymmetry or Darwin's corollary to Haldane's rule, is a general feature of reproductive isolation in plants, yet factors determining its magnitude and direction remain unclear. We evaluated reciprocal species crosses between two naturally hybridizing diploid species of Arabidopsis to assess the degree of isolation asymmetry at different postmating life stages. We found that pollen from Arabidopsis arenosa will usually fertilize ovules from Arabidopsis lyrata; the reverse receptivity being less complete. Maternal A. lyrata parents set more F₁ hybrid seed, but germinate at lower frequency, reversing the asymmetry. As predicted by theory, A. lyrata (the maternal parent with lower seed viability in crosses) exhibited accelerated chloroplast evolution, indicating that cytonuclear incompatibilities may play a role in reproductive isolation. However, this direction of asymmetrical reproductive isolation is not replicated in natural suture zones, where delayed hybrid breakdown of fertility at later developmental stages, or later‐acting selection against A. arenosa maternal hybrids (unrelated to hybrid fertility, e.g., substrate adaptation) may be responsible for an excess of A. lyrata maternal hybrids. Exogenous selection rather than cytonuclear incompatibilities thus shapes the asymmetrical postmating isolation in nature.     

Variation in reproductive isolation across a species range

Reproductive isolation is often variable within species, a phenomenon that while largely ignored by speciation studies, can be leveraged to gain insight into the potential mechanisms driving the evolution of genetic incompatibilities. We used experimental greenhouse crosses to characterize patterns of reproductive isolation among three divergent genetic lineages of Campanulastrum americanum that occur in close geographic proximity in the Appalachian Mountains. Substantial, asymmetrical reproductive isolation for survival due to cytonuclear incompatibility was found among the lineages (up to 94% reduction). Moderate reductions in pollen viability, as well as cytoplasmic male sterility, were also found between some Mountain populations. We then compared these results to previously established patterns of reproductive isolation between these Mountain lineages and a fourth, widespread Western lineage to fully characterize reproductive isolation across the complete geographic and genetic range of C. americanum. Reproductive isolation for survival and pollen viability was consistent across studies, indicating the evolution of the underlying genetic incompatibilities is primarily determined by intrinsic factors. In contrast, reproductive isolation for germination was only found when crossing Mountain populations with the Western lineage, suggesting the underlying genetic incompatibility is likely influenced by environmental or demographic differences between the two lineages. Cytoplasmic male sterility was also limited in occurrence, being restricted to a handful of Mountain populations in a narrow geographic range. These findings illustrate the complexity of speciation by demonstrating multiple, independent genetic incompatibilities that lead to a mosaic of genetic divergence and reproductive isolation across a species range.     

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.     

Genomic scans reveal multiple mito‐nuclear incompatibilities in population crosses of the copepod Tigriopus californicus

The evolution of intrinsic postzygotic isolation can be explained by the accumulation of Dobzhansky‐Muller incompatibilities (DMI). Asymmetries in the levels of hybrid inviability and hybrid sterility are commonly observed between reciprocal crosses, a pattern that can result from the involvement of uniparentally inherited factors. The mitochondrial genome is one such factor that appears to participate in DMI in some crosses but the frequency of its involvement versus biparentally inherited factors is unclear. Here we assess the relative importance of incompatibilities between nuclear factors (nuclear‐nuclear) versus those between mitochondrial and nuclear factors (mito‐nuclear) in a species that lacks sex chromosomes. We used a Pool‐seq approach to survey three crosses among genetically divergent populations of the copepod, Tigriopus californicus, for regions of the genome that are affected by hybrid inviability. Results from reciprocal crosses suggest that mito‐nuclear incompatibilities are more common than nuclear‐nuclear incompatibilities overall. These results suggest that in the presence of very high levels of nucleotide divergence between mtDNA haplotypes, mito‐nuclear incompatibilities can be important for the evolution of intrinsic postzygotic isolation. This is particularly interesting considering this species lacks sex chromosomes, which have been shown to harbor a particularly high number of nuclear‐nuclear DMI in several other species.     

Sexual selection and assortative mating: an experimental test

Mate choice and mate competition can both influence the evolution of sexual isolation between populations. Assortative mating may arise if traits and preferences diverge in step, and, alternatively, mate competition may counteract mating preferences and decrease assortative mating. Here, we examine potential assortative mating between populations of Drosophila pseudoobscura that have experimentally evolved under either increased (‘polyandry’) or decreased (‘monogamy’) sexual selection intensity for 100 generations. These populations have evolved differences in numerous traits, including a male signal and female preference traits. We use a two males: one female design, allowing both mate choice and competition to influence mating outcomes, to test for assortative mating between our populations. Mating latency shows subtle effects of male and female interactions, with females from the monogamous populations appearing reluctant to mate with males from the polyandrous populations. However, males from the polyandrous populations have a significantly higher probability of mating regardless of the female's population. Our results suggest that if populations differ in the intensity of sexual selection, effects on mate competition may overcome mate choice.     

The nature of interactions that contribute to postzygotic reproductive isolation in hybrid copepods

Deleterious interactions within the genome of hybrids can lower fitness and result in postzygotic reproductive isolation. Understanding the genetic basis of these deleterious interactions, known as Dobzhansky-Muller incompatibilities, is the subject of intense current study that seeks to elucidate the nature of these deleterious interactions. Hybrids from crosses of individuals from genetically divergent populations of the intertidal copepod Tigriopus californicus provide a useful model in which to study Dobzhansky-Muller incompatibilities. Studies of the basis of postzygotic reproductive isolation in this species have revealed a number of patterns. First, there is evidence for a breakdown in genomic coadaptation between mtDNA-encoded and nuclear-encoded proteins that can result in a reduction in hybrid fitness in some crosses. It appears from studies of the individual genes involved in these interactions that although this coadaptation could lead to asymmetries between crosses, patterns of genotypic viabilities are not often consistent with simple models of genomic coadaptation. Second, there is a large impact of environmental factors on these deleterious interactions suggesting that they are not strictly intrinsic in nature. Temperature in particular appears to play an important role in determining the nature of these interactions. Finally, deleterious interactions in these hybrid copepods appear to be complex in terms of the number of genetic factors that interact to lead to reductions in hybrid fitness. This complexity may stem from three or more factors that all interact to cause a single incompatibility or the same factor interacting with multiple other factors independently leading to multiple incompatibilities.     

Lineages of Silene nutans developed rapid,strong, asymmetric postzygotic reproductive isolation in allopatry

Reproductive isolation can rise either as a consequence of genomic divergence in allopatry or as a byproduct of divergent selection in parapatry. To determine whether reproductive isolation in gynodioecious Silene nutans results from allopatric divergence or from ecological adaptation following secondary contact, we investigated the pattern of postzygotic reproductive isolation and hybridization in natural populations using two phylogeographic lineages, western (W1) and eastern (E1). Experimental crosses between the lineages identified strong, asymmetric postzygotic isolation between the W1 and the E1 lineages, independent of geographic overlap. The proportion of ovules fertilized, seeds aborted, and seeds germinated revealed relatively little effect on the fitness of hybrids. In contrast, hybrid mortality was high and asymmetric: while half of the hybrid seedlings with western lineage mothers died, nearly all hybrid seedlings with E1 mothers died. This asymmetric mortality mirrored the proportion of chlorotic seedlings, and is congruent with cytonuclear incompatibility. We found no evidence of hybridization between the lineages in regions of co‐occurrence using nuclear and plastid markers. Together, our results are consistent with the hypothesis that strong postzygotic reproductive isolation involving cytonuclear incompatibilities arose in allopatry. We argue that the dynamics of cytonuclear gynodioecy could facilitate the evolution of reproductive isolation.     

Accelerated mitochondrial evolution and "Darwin's corollary": asymmetric viability of reciprocal F1 hybrids in Centrarchid fishes

Reciprocal crosses between species can yield hybrids with different viabilities. The high frequency of this asymmetric hybrid viability ("Darwin's corollary") places it alongside Haldane's rule and the "large-X effect" as a general feature of postmating reproductive isolation. Recent theory suggests that reciprocal cross asymmetries can arise from stochastic substitutions in uniparentally inherited loci such as mitochondrial genomes, although large systematic differences in mitochondrial substitution rates can also contribute to asymmetries. Although the magnitude of asymmetry will be relatively insensitive to unequal rates of mitochondrial evolution in diverging species, we show here that rate asymmetries can have a large effect on the direction of viability asymmetries. In reciprocal crosses between species, the maternal parent with faster mitochondrial evolution will tend to produce less viable F(1) hybrids owing to an increased probability of mito-nuclear incompatibilities. We test this prediction using data on reciprocal hybrid viability and molecular evolution rates from a clade of freshwater fishes, Centrarchidae. As predicted, species with accelerated mitochondrial evolution tend to be the worse maternal parent for F(1) hybrids, providing the first comparative evidence for a systematic basis to Darwin's corollary. This result is consistent with the hypothesis that mito-nuclear incompatibilities can play an important role in reproductive isolation. Such asymmetrical reproductive isolation may help explain the asymmetrical mitochondrial introgression observed between many hybridizing species. However, as with any comparative study, we cannot rule out the possibility that our results arise from a mutual correlation with a third variable such as body size.     

Bacterial protoplast fusion: recombination in fused protoplasts of Streptomyces coelicolor

Summary Numerous recombinants arose when protoplasts of S. coelicolor were treated with polyethylene glycol and regenerated on non-selective solid medium. In six-factor crosses, recombination frequencies of more than 10% (up to 17%) were routinely observed. This recombination did not require either of the known sex factors, SCP1 and SCP2.The proportion of multiple crossover classes was much higher than amongst recombinants produced by conjugation between mycelia. Analysis of the spatial distribution of crossovers in double and quadruple crossover recombinants showed only a slight tendency for crossovers to occur closer together than randomly on the complete linkage group. This suggests that genomes brought together by protoplast fusion are complete, or nearly so (in conjugation, in contrast, one genome is represented by a comparatively short fragment). Individual colonies arising from fused protoplasts did not contain different parental genomes without recombinants, but recombinants often occurred without parentals. Several recombinant genotypes often occurred in the same colony, showing a segregation of some, only, of the parental alleles. Complementary genotypes, parental or recombinant, did not occur in the same colony. It is postulated that complete genomes of fused protoplasts usually become fragmented and that crossing-over, often repeated, occurs between the fragments, to generate haploid recombinants.Analysis of fusions between protoplasts of four different genotypes indicated that the average number of protoplasts fusing together was low, but nevertheless appreciable numbers of fusions involved three or four genomes. Crossing-over between them produced recombinants inheriting markers from three or four parents.The generation of nearly random populations of recombinants between two or more parent strains by protoplast fusion under the conditions described appears to have simple applications in industrial and academic strain construction.     

Mechanical and tactile incompatibilities cause reproductive isolation between two young damselfly species

External male reproductive structures have received considerable attention as a cause of reproductive isolation (RI), because the morphology of these structures often evolves rapidly between populations. This rapid evolution presents the potential for mechanical incompatibilities with heterospecific female structures during mating and could thus prevent interbreeding between nascent species. Although such mechanical incompatibilities have received little empirical support as a common cause of RI, the potential for mismatch of reproductive structures to cause RI due to incompatible species‐specific tactile cues has not been tested. We tested the importance of mechanical and tactile incompatibilities in RI between Enallagma anna and E. carunculatum, two damselfly species that diverged within the past ～250,000 years and currently hybridize in a sympatric region. We quantified 19 prezygotic and postzygotic RI barriers using both naturally occurring and laboratory‐reared damselflies. We found incomplete mechanical isolation between the two pure species and between hybrid males and pure species females. Interestingly, in mating pairs for which mechanical isolation was incomplete, females showed greater resistance and refusal to mate with hybrid or heterospecific males compared to conspecific males. This observation suggests that tactile incompatibilities involving male reproductive structures can influence female mating decisions and form a strong barrier to gene flow in early stages of speciation.     

Stereochemical Behavior of an NADH Model Compound Carrying l-Prolinamide at 3,5-Positions

Interspecific recombinants have been produced between Streptococcus cremoris H-61 and S. lactis J-1 by polyethylene glycol-induced protoplast fusion. All of the fusants obtained showed mixed physiological properties of the two parents, and possessed plasmids derived from both parents at random. Physiological properties of primary colonies were stably maintained among the progenies after the single-colony isolation procedure. Similarly, in most of the fusants the plasmid profiles of the primary colonies were stably maintained, but one lost 2 out of the 7 plasmid bands. However, there was no indication that plasmids from either one of the parents were preferentially lost. These results showed that interspecific genetic transfer occurred on chromosomal and plasmid DNA on the protoplast fusion and that the fusants obtained were not heterokaryons, but true recombinants.     

X‐AUTOSOME INCOMPATIBILITIES IN DROSOPHILA MELANOGASTER: TESTS OF HALDANE'S RULE AND GEOGRAPHIC PATTERNS WITHIN SPECIES

Substantial genetic variation exists in natural populations of Drosophila melanogaster. This segregating variation includes alleles at different loci that interact to cause lethality or sterility (synthetic incompatibilities). Fitness epistasis in natural populations has important implications for speciation and the rate of adaptive evolution. To assess the prevalence of epistatic fitness interactions, we placed naturally occurring X chromosomes into genetic backgrounds derived from different geographic locations. Considerable amounts of synthetic incompatibilities were observed between X chromosomes and autosomes: greater than 44% of all combinations were either lethal or sterile. Sex‐specific lethality and sterility were also tested to determine whether Haldane's rule holds for within‐species variation. Surprisingly, we observed an excess of female sterility in genotypes that were homozygous, but not heterozygous, for the X chromosome. The recessive nature of these incompatibilities is similar to that predicted for incompatibilities underlying Haldane's rule. Our study also found higher levels of sterility and lethality for genomes that contain chromosomes from different geographical regions. These findings are consistent with the view that genomes are coadapted gene complexes and that geography affects the likelihood of epistatic fitness interactions.     

Persistent postmating,prezygotic reproductive isolation between populations

Studying reproductive barriers between populations of the same species is critical to understand how speciation may proceed. Growing evidence suggests postmating, prezygotic (PMPZ) reproductive barriers play an important role in the evolution of early taxonomic divergence. However, the contribution of PMPZ isolation to speciation is typically studied between species in which barriers that maintain isolation may not be those that contributed to reduced gene flow between populations. Moreover, in internally fertilizing animals, PMPZ isolation is related to male ejaculate—female reproductive tract incompatibilities but few studies have examined how mating history of the sexes can affect the strength of PMPZ isolation and the extent to which PMPZ isolation is repeatable or restricted to particular interacting genotypes. We addressed these outstanding questions using multiple populations of Drosophila montana. We show a recurrent pattern of PMPZ isolation, with flies from one population exhibiting reproductive incompatibility in crosses with all three other populations, while those three populations were fully fertile with each other. Reproductive incompatibility is due to lack of fertilization and is asymmetrical, affecting female fitness more than males. There was no effect of male or female mating history on reproductive incompatibility, indicating that PMPZ isolation persists between populations. We found no evidence of variability in fertilization outcomes attributable to different female × male genotype interactions, and in combination with our other results, suggests that PMPZ isolation is not driven by idiosyncratic genotype × genotype interactions. Our results show PMPZ isolation as a strong, consistent barrier to gene flow early during speciation and suggest several targets of selection known to affect ejaculate‐female reproductive tract interactions within species that may cause this PMPZ isolation.     

The evolution of postzygotic isolation: accumulating Dobzhansky-Muller incompatibilities

Hybrid sterility and inviability often result from the accumulation of substitutions that, while functional on their normal genetic backgrounds, cause a loss of fitness when brought together in hybrids. Previous theory has shown that such Dobzhansky-Muller incompatibilities should accumulate at least as fast as the square of the number of substitutions separating two species, the so-called snowball effect. Here we explicitly describe the stochastic accumulation of these incompatibilities as a function of time. The accumulation of these incompatibilities involves three levels of stochasticity: (1) the number of substitutions separating two allopatric lineages at a given time; (2) the number of incompatibilities resulting from these substitutions; and (3) the fitness effects of individual incompatibilities. Previous analyses ignored the stochasticity of molecular evolution (level 1) as well as that due to the variable effects of incompatibilities (level 3). Here we approximate the full stochastic process characterizing the accumulation of hybrid incompatibilities between pairs of loci. We derive the distribution of the number of incompatibilities as a function of divergence time between allopatric taxa as well as the distribution of waiting times to speciation by postzygotic isolation. We provide simple approximations for the mean and variance of these waiting times. These results let us estimate. albeit crudely, the probability, p, that two diverged sites from different species will contribute to hybrid sterility or inviability. Our analyses of data from Drosophila and Bombina suggest that p is generally very small, on the order of 10(-6) or less.