Speciation and development.

Understanding general principles about the origin of species remains one of the foundational challenges in evolutionary biology. The genomic divergence between groups of individuals can spawn hybrid inviability and hybrid sterility, which presents a tantalizing developmental problem. Divergent developmental programs may yield either conserved or divergent phenotypes relative to ancestral traits, both of which can be responsible for reproductive isolation during the speciation process. The genetic mechanisms of developmental evolution involve cis- and trans-acting gene regulatory change, protein-protein interactions, genetic network structures, dosage, and epigenetic regulation, all of which also have roots in population genetic and molecular evolutionary processes. Toward the goal of demystifying Darwin's "mystery of mysteries," this review integrates microevolutionary concepts of genetic change with principles of organismal development, establishing explicit links between population genetic process and developmental mechanisms in the production of macroevolutionary pattern. This integration aims to establish a more unified view of speciation that binds process and mechanism.

The role of conflict in shaping plant biodiversity.

Although intrinsic postzygotic reproductive barriers can play a fundamental role in speciation, their underlying evolutionary causes are widely debated. One hypothesis is that incompatibilities result from genomic conflicts. Here, I synthesize the evidence that conflict generates incompatibilities in plants, thus playing a creative role in plant biodiversity. While much evidence supports a role for conflict in several classes of incompatibility, integrating knowledge of incompatibility alleles with natural history can provide further essential tests. Moreover, comparative work can shed light on the relative importance of conflict in causing incompatibilities, including the extent to which their evolution is repeatable. Together, these approaches can provide independent lines of evidence that conflict causes incompatibilities, cementing its role in plant speciation.

The role of hybrid seed inviability in angiosperm speciation.

Understanding which reproductive barriers contribute to speciation is essential to understanding the diversity of life on earth. Several contemporary examples of strong hybrid seed inviability (HSI) between recently diverged species suggest that HSI may play a fundamental role in plant speciation. Yet, a broader synthesis of HSI is needed to clarify its role in diversification. Here, I review the incidence and evolution of HSI. Hybrid seed inviability is common and evolves rapidly, suggesting that it may play an important role early in speciation. The developmental mechanisms that underlie HSI involve similar developmental trajectories in endosperm, even between evolutionarily deeply diverged incidents of HSI. In hybrid endosperm, HSI is often accompanied by whole-scale gene misexpression, including misexpression of imprinted genes which have a key role in endosperm development. I explore how an evolutionary perspective can clarify the repeated and rapid evolution of HSI. In particular, I evaluate the evidence for conflict between maternal and paternal interests in resource allocation to offspring (i.e., parental conflict). I highlight that parental conflict theory generates explicit predictions regarding the expected hybrid phenotypes and genes responsible for HSI. While much phenotypic evidence supports a role of parental conflict in the evolution of HSI, an understanding of the underlying molecular mechanisms of this barrier is essential to test parental conflict theory. Lastly, I explore what factors may influence the strength of parental conflict in natural plant populations as an explanation for why rates of HSI may differ between plant groups and the consequences of strong HSI in secondary contact.

Genetic Architecture Underlying Nascent Speciation-The Evolution of Eurasian Pigs under Domestication.

Speciation is a process whereby the evolution of reproductive barriers leads to isolated species. Although many studies have addressed large-effect genetic footprints in the advanced stages of speciation, the genetics of reproductive isolation in nascent stage of speciation remains unclear. Here, we show that pig domestication offers an interesting model for studying the early stages of speciation in great details. Pig breeds have not evolved the large X-effect of hybrid incompatibility commonly observed between "good species." Instead, deleterious epistatic interactions among multiple autosomal loci are common. These weak Dobzhansky-Muller incompatibilities confer partial hybrid inviability with sex biases in crosses between European and East Asian domestic pigs. The genomic incompatibility is enriched in pathways for angiogenesis, androgen receptor signaling and immunity, with an observation of many highly differentiated cis-regulatory variants. Our study suggests that partial hybrid inviability caused by pervasive but weak interactions among autosomal loci may be a hallmark of nascent speciation in mammals.

Immunoglobulin genes, reproductive isolation and vertebrate speciation.

Reproductive isolation drives the formation of new species, and many genes contribute to this through Dobzhansky-Muller incompatibilities (DMIs). These incompatibilities occur when gene divergence affects loci encoding interacting products such as receptors and their ligands. We suggest here that the nature of vertebrate immunoglobulin (IG) genes must make them prone to DMIs. The genes of these complex loci form functional genes through the process of recombination, giving rise to a repertoire of heterodimeric receptors of incredible diversity. This repertoire, within individuals and within species, must defend against pathogens but must also avoid pathogenic self-reactivity. We suggest that this avoidance of autoimmunity is only achieved through a coordination of evolution between heavy- and light-chain genes, and between these genes and the rest of the genome. Without coordinated evolution, the hybrid offspring of two diverging populations will carry a heavy burden of DMIs, resulting in a loss of fitness. Critical incompatibilities could manifest as incompatibilities between a mother and her divergent offspring. During fetal development, biochemical differences between the parents of hybrid offspring could make their offspring a target of the maternal immune system. This hypothesis was conceived in the light of recent insights into the population genetics of IG genes. This has suggested that antibody genes are probably as susceptible to evolutionary forces as other parts of the genome. Further repertoire studies in human and nonhuman species should now help determine whether antibody genes have been part of the evolutionary forces that drive the development of species.

Asymmetric viability in reciprocal crosses of zebrafish Danio rerio and pearl danio Danio albolineatus.

Interspecies hybrids have long been studied to further understanding of speciation. Reciprocal crosses sometimes have asymmetric viability, a phenomenon termed 'Darwin's corollary to Haldane's rule'. It has been proposed that this asymmetry is caused by Dobzhansky-Muller incompatibilities between nuclear genes and cytoplasmic factors (e.g., maternal transcripts, mitochondrial genome). The molecular basis of this hypothesis has received little empirical investigation, presumably due to the lack of an appropriate model system. We report a case of extreme asymmetry in viability between reciprocal hybrids of zebrafish Danio rerio and pearl danio Danio albolineatus. Hybrids from D. rerio females × D. albolineatus males (n = 4 crosses) were viable, with 83.2 ± 9.6% surviving from fertilization to 5 days post-fertilization (dpf) and 80.1 ± 14.4% surviving from 5 to 21 dpf. Hybrids from D. albolineatus females × D. rerio males (n = 6 crosses) were inviable after embryonic development. These hybrids developed pericardial oedema at 1 dpf and only 37.2 ± 18.0% survived from fertilization to 5 dpf. Of the 595 larvae alive at 5 dpf, only one juvenile with stunted growth survived to 21 dpf. We propose that given the resources available for the D. rerio model system and the strong asymmetry in viability between reciprocal crosses, these hybrids will allow investigation of the molecular basis for Darwin's corollary to Haldane's rule.

Rampant Misexpression in a Mimulus (Monkeyflower) Introgression Line Caused by Hybrid Sterility, Not Regulatory Divergence.

Divergence in gene expression regulation is common between closely related species and may give rise to incompatibilities in their hybrid progeny. In this study, we investigated the relationship between regulatory evolution within species and reproductive isolation between species. We focused on a well-studied case of hybrid sterility between two closely related yellow monkeyflower species, Mimulus guttatus and Mimulus nasutus, that is caused by two epistatic loci, hybrid male sterility 1 (hms1) and hybrid male sterility 2 (hms2). We compared genome-wide transcript abundance across male and female reproductive tissues (i.e., stamens and carpels) from four genotypes: M. guttatus, M. nasutus, and sterile and fertile progeny from an advanced M. nasutus-M. guttatus introgression line carrying the hms1-hms2 incompatibility. We observed substantial variation in transcript abundance between M. guttatus and M. nasutus, including distinct but overlapping patterns of tissue-biased expression, providing evidence for regulatory divergence between these species. We also found rampant genome-wide misexpression, but only in the affected tissues (i.e., stamens) of sterile introgression hybrids carrying incompatible alleles at hms1 and hms2. Examining patterns of allele-specific expression in sterile and fertile introgression hybrids, we found evidence for interspecific divergence in cis- and trans-regulation, including compensatory cis-trans mutations likely to be driven by stabilizing selection. Nevertheless, species divergence in gene regulatory networks cannot explain the vast majority of the gene misexpression we observe in Mimulus introgression hybrids, which instead likely manifests as a downstream consequence of sterility itself.

Chromosome number is key to longevity of polyploid lineages.

Polyploidy is ubiquitous and often recursive in plant lineages, most frequently resulting in extinction but occasionally associated with great evolutionary success. However, instead of chromosome numbers exponentially increasing due to recurrent polyploidy, most angiosperm species have fewer than 14 chromosome pairs. Following genome duplication, diploidisation can render one copy of essential genes nonfunctional without fitness cost. In isolated subpopulations, alternate (homoeologous) gene copies can be lost, creating incompatibilities that reduce fitness of hybrids between subpopulations, constraining exchange of favourable genetic changes and reducing species fitness. When multiple sets of incompatible genes are genetically linked, their deleterious effects are not independent. The effective number of independently acting sets of incompatible loci in hybrids is limited by chromosome number and recombination. Therefore, species with many chromosomes are subject to a higher fitness penalty during diploidisation. Karyotypic changes, especially fusions, that reduce gene flow are normally fitness disadvantages, but during the diploidisation process, can increase fitness by reducing mixing of differentially diploidised alleles. Fitness penalties caused by diploidisation favour accelerated karyotypic change, with each change increasing barriers to gene flow, contributing to speciation. Lower chromosome numbers and increased chromosome fusions confer advantages to surviving the diploidisation process following polyploid formation, by independent mechanisms.

Ecological divergence in sympatry causes gene misexpression in hybrids.

Ecological speciation occurs when reproductive isolation evolves as a byproduct of adaptive divergence between populations. Selection favouring gene regulatory divergence between species could result in transgressive levels of gene expression in F1 hybrids that may lower hybrid fitness. We combined 58 resequenced genomes with 124 transcriptomes to identify patterns of hybrid gene misexpression that may be driven by adaptive regulatory divergence within a young radiation of Cyprinodon pupfishes, which consists of a dietary generalist and two trophic specialists-a molluscivore and a scale-eater. We found more differential gene expression between closely related sympatric specialists than between allopatric generalist populations separated by 1,000 km. Intriguingly, 9.6% of genes that were differentially expressed between sympatric species were also misexpressed in F1 hybrids. A subset of these genes were in highly differentiated genomic regions and enriched for functions important for trophic specialization, including head, muscle and brain development. These regions also included genes that showed evidence of hard selective sweeps and were significantly associated with oral jaw length-the most rapidly diversifying skeletal trait in this radiation. Our results indicate that divergent ecological selection in sympatry can contribute to hybrid gene misexpression which may act as a reproductive barrier between nascent species.

Role of seed germination in adaptation and reproductive isolation in Arabidopsis lyrata.

Seed germination is an important developmental and life history stage. Yet, the evolutionary impact of germination has mainly been studied in the context of dormancy, or for its role in reproductive isolation between species. Here, we aim to examine multiple consequences of genetic divergence on germination traits between two Arabidopsis lyrata subspecies: ssp. petraea (Eurasia) and ssp. lyrata (North America). Postdormancy germination time, a potentially adaptive trait, showed differentiation between the populations, and quantitative trait loci (QTL) mapping revealed that the trait variation is mainly controlled by two antagonistic loci. These QTL areas contain several candidate genes with known function in postdormancy germination in A. thaliana. The sequence variation of three genes was consistent with differential selection, and they also included fixed nonsynonymous substitutions with potential to account for the phenotypic differentiation. We further show that the divergence between the subspecies has led to a slight but significant reduction in hybrid germination proportions, indicating incipient reproductive isolation. Comparison of reciprocal F1 and F2 progenies suggests that Bateson-Dobzhansky-Muller incompatibilities likely act through uniparentally inherited factors. Examination of genomewide transmission ratio distortion further revealed that cytonuclear interactions cause substantial pregermination inviability in the hybrids. These results confirm that seed germination has adaptive potential beyond the dormancy stage and that hybrid seed inviability can be one of the first reproductive barriers to arise during divergence.

Experimental evolution of hybrid populations to identify Dobzhansky-Muller incompatibility loci.

Epistatic interactions between loci that reduce fitness in interspecies hybrids, Dobzhansky-Muller incompatibilities (DMIs), contribute genetically to the inviability and infertility within hybrid populations. It remains a challenge, however, to identify the loci that contribute to DMIs as causes of reproductive isolation between species. Here, we assess through forward simulation the power of evolve-and-resequence (E&R) experimental evolution of hybrid populations to map DMI loci. We document conditions under which such a mapping strategy may be most feasible and demonstrate how mapping power is sensitive to biologically relevant parameters such as one-way versus two-way incompatibility type, selection strength, recombination rate, and dominance interactions. We also assess the influence of parameters under direct control of an experimenter, including duration of experimental evolution and number of replicate populations. We conclude that an E&R strategy for mapping DMI loci, and other cases of epistasis, can be a viable option under some circumstances for study systems with short generation times like Caenorhabditis nematodes.

Evolutionary consequences of putative intra-and interspecific hybridization in agaric fungi.

Agaric fungi of the southern Appalachian Mountains including Great Smoky Mountains National Park are often heterozygous for the rDNA internal transcribed spacer region (ITS) with >42% of collections showing some heterozygosity for indels and/or base-pair substitutions. For these collections, intra-individual haplotype divergence is typically less than 2%, but for 3% of these collections intra-individual haplotype divergence exceeds that figure. We hypothesize that high intra-individual haplotype divergence is due to hybridization between agaric fungi with divergent haplotypes, possibly migrants from geographically isolated glacial refugia. Four species with relatively high haplotype divergence were examined: Armillaria mellea, Amanita citrina f. lavendula, Gymnopus dichrous and the Hygrocybe flavescens/chlorophana complex. The ITS region was sequenced, haplotypes of heterozygotes were resolved through cloning, and phylogenetic analyses were used to determine the outcome of hybridization events. Within Armillaria mellea and Amanita citrina f. lavendula, we found evidence of interbreeding and recombination. Within G. dichrous and H. flavescens/chlorophana, hybrids were identified but there was no evidence for F2 or higher progeny in natural populations suggesting that the hybrid fruitbodies might be an evolutionary dead end and that the genetically divergent Mendelian populations from which they were derived are, in fact, different species. The association between ITS haplotype divergence of less than 5% (Armillaria mellea = 2.6% excluding gaps; Amanita citrina f. lavendula = 3.3%) with the presence of putative recombinants and greater than 5% (Gymnopus dichrous = 5.7%; Hygrocybe flavescens/chlorophana = 14.1%) with apparent failure of F1 hybrids to produce F2 or higher progeny in populations may suggest a correlation between genetic distance and reproductive isolation.

Meiotic drive is associated with sexual incompatibility in Neurospora.

Evolution of Bateson-Dobzhansky-Muller (BDM) incompatibilities is thought to represent a key step in the formation of separate species. They are incompatible alleles that have evolved in separate populations and are exposed in hybrid offspring as hybrid sterility or lethality. In this study, we reveal a previously unconsidered mechanism promoting the formation of BDM incompatibilities, meiotic drive. Theoretical studies have evaluated the role that meiotic drive, the phenomenon whereby selfish elements bias their transmission to progeny at ratios above 50:50, plays in speciation, and have mostly concluded that drive could not result in speciation on its own. Using the model fungus Neurospora, we demonstrate that the large meiotic drive haplotypes, Sk-2 and Sk-3, contain putative sexual incompatibilities. Our experiments revealed that although crosses between Neurospora intermedia and Neurospora metzenbergii produce viable progeny at appreciable rates, when strains of N. intermedia carry Sk-2 or Sk-3 the proportion of viable progeny drops substantially. Additionally, it appears that Sk-2 and Sk-3 have accumulated different incompatibility phenotypes, consistent with their independent evolutionary history. This research illustrates how meiotic drive can contribute to reproductive isolation between populations, and thereby speciation.

Integrative genomic phylogeography reveals signs of mitonuclear incompatibility in a natural hybrid goby population.

Hybridization between divergent lineages generates new allelic combinations. One mechanism that can hinder the formation of hybrid populations is mitonuclear incompatibility, that is, dysfunctional interactions between proteins encoded in the nuclear and mitochondrial genomes (mitogenomes) of diverged lineages. Theoretically, selective pressure due to mitonuclear incompatibility can affect genotypes in a hybrid population in which nuclear genomes and mitogenomes from divergent lineages admix. To directly and thoroughly observe this key process, we de novo sequenced the 747-Mb genome of the coastal goby, Chaenogobius annularis, and investigated its integrative genomic phylogeographics using RNA-sequencing, RAD-sequencing, genome resequencing, whole mitogenome sequencing, amplicon sequencing, and small RNA-sequencing. Chaenogobius annularis populations have been geographically separated into Pacific Ocean (PO) and Sea of Japan (SJ) lineages by past isolation events around the Japanese archipelago. Despite the divergence history and potential mitonuclear incompatibility between these lineages, the mitogenomes of the PO and SJ lineages have coexisted for generations in a hybrid population on the Sanriku Coast. Our analyses revealed accumulation of nonsynonymous substitutions in the PO-lineage mitogenomes, including two convergent substitutions, as well as signals of mitochondrial lineage-specific selection on mitochondria-related nuclear genes. Finally, our data implied that a microRNA gene was involved in resolving mitonuclear incompatibility. Our integrative genomic phylogeographic approach revealed that mitonuclear incompatibility can affect genome evolution in a natural hybrid population.

Understanding Admixture: Haplodiploidy to the Rescue.

Hybridization has broad evolutionary consequences, from fueling or counteracting speciation to facilitating adaptation to novel environments. Hybridization and subsequent introgression appear widespread along the tree of life. However, our understanding of how distinct evolutionary forces shape admixed genomes and the fate of introgressed genetic variants remains scarce. Most admixture research in animals has focused on diploid organisms. We propose that haplodiploid organisms can help resolve open questions about the genomic consequences of hybridization in natural populations. The ploidy difference between haploid males and diploid females, the availability of genome-wide male haplotypes, and ongoing cases of admixture make haplodiploid organisms promising models to improve our knowledge with regards to the evolution of hybrid genomes.

When Two Rights Make a Wrong: The Evolutionary Genetics of Plant Hybrid Incompatibilities.

Hybrids between flowering plant species often exhibit reduced fitness, including sterility and inviability. Such hybrid incompatibilities create barriers to genetic exchange that can promote reproductive isolation between diverging populations and, ultimately, speciation. Additionally, hybrid breakdown opens a window into hidden molecular and evolutionary processes occurring within species. Here, we review recent work on the mechanisms and origins of hybrid incompatibility in flowering plants, including both diverse genic interactions and chromosomal incompatibilities. Conflict and coevolution among and within plant genomes contributes to the evolution of some well-characterized genic incompatibilities, but duplication and drift also play important roles. Inversions, while contributing to speciation by suppressing recombination, rarely cause underdominant sterility. Translocations cause severe F1 sterility by disrupting meiosis in heterozygotes, making their fixation in outcrossing sister species a paradox. Evolutionary genomic analyses of both genic and chromosomal incompatibilities, in the context of population genetic theory, can explicitly test alternative scenarios for their origins.

Cytonuclear incompatibility contributes to the early stages of speciation.

Genetic incompatibility is a hallmark of speciation. Cytonuclear incompatibilities are proposed to be among the first genetic barriers to arise during speciation. Accordingly, reproductive isolation (RI) within species should be heavily influenced by interactions between the organelle and nuclear genomes. However, there are few clear examples of cytonuclear incompatibility within a species. Here, we show substantial postzygotic RI in first-generation hybrids between differentiated populations of an herbaceous plant (up to 92% reduction in fitness). RI was primarily due to germination and survival, with moderate RI for pollen viability. RI for survival was asymmetric and caused by cytonuclear incompatibility, with the strength of incompatibility linearly related to chloroplast genetic distance. This cytonuclear incompatibility may be the result of a rapidly evolving plastid genome. Substantial asymmetric RI was also found for germination, but was not associated with cytonuclear incompatibility, indicating endosperm or maternal-zygote incompatibilities. These results demonstrate that cytonuclear incompatibility contributes to RI within species, suggesting that initial rates of speciation could be influenced by rates of organelle evolution. However, other genetic incompatibilities are equally important, indicating that even at early stages, speciation can be a complex process involving multiple genes and incompatibilities.

Mitochondrial-Nuclear Epistasis Impacts Fitness and Mitochondrial Physiology of Interpopulation Caenorhabditis briggsae Hybrids.

In order to identify the earliest genetic changes that precipitate species formation, it is useful to study genetic incompatibilities that cause only mild dysfunction when incompatible alleles are combined in an interpopulation hybrid. Such hybridization within the nematode species Caenorhabditis briggsae has been suggested to result in selection against certain combinations of nuclear and mitochondrial alleles, raising the possibility that mitochondrial-nuclear (mitonuclear) epistasis reduces hybrid fitness. To test this hypothesis, cytoplasmic-nuclear hybrids (cybrids) were created to purposefully disrupt any epistatic interactions. Experimental analysis of the cybrids suggests that mitonuclear discord can result in decreased fecundity, increased lipid content, and increased mitochondrial reactive oxygen species levels. Many of these effects were asymmetric with respect to cross direction, as expected if cytoplasmic-nuclear Dobzhansky-Muller incompatibilities exist. One such effect is consistent with the interpretation that disrupting coevolved mitochondrial and nuclear loci impacts mitochondrial function and organismal fitness. These findings enhance efforts to study the genesis, identity, and maintenance of genetic incompatibilities that precipitate the speciation process.

Interploidy hybridization barrier of endosperm as a dosage interaction.

Crosses between plants at different ploidy levels will often result in failure of endosperm development. The basis of this phenomenon has been attributed to parental gene imprinting of genes involved with endosperm development but a review of the data from maize indicates a dosage interaction between the contributions of the female gametophyte and the primary endosperm nucleus to early endosperm development. However, it is noted that parental imprinting is a non-mutational means that can alter dosage sensitive factors and therefore can contribute to this effect. Operationally, the genes determining ploidy hybridization barrier would qualify for Dobzhansky-Muller incompatibilities that prevent gene flow between species.

Rapid evolution of reproductive isolation between incipient outcrossing and selfing Clarkia species.

A major goal of speciation research is to understand the processes involved in the earliest stages of the evolution of reproductive isolation (RI). One important challenge has been to identify systems where lineages have very recently diverged and opportunities for hybridization are present. We conducted a comprehensive examination of the components of RI across the life cycle of two subspecies of Clarkia xantiana, which diverged recently (ca. 65,000 bp). One subspecies is primarily outcrossing, but self-compatible, whereas the other is primarily selfing. The subspecies co-occur in a zone of sympatry but hybrids are rarely observed. Premating barriers resulted in nearly complete isolation in both subspecies with flowering time and pollinator preference (for the outcrosser over the selfer) as the strongest barriers. We found that the outcrosser had consistently more competitive pollen, facilitating hybridization in one direction, but no evidence for pollen-pistil interactions as an isolating barrier. Surprisingly, postzygotic isolation was detected at the stage of hybrid seed development, but in no subsequent life stages. This crossing barrier was asymmetric with crosses from the selfer to outcrosser most frequently failing. Collectively, the results provide evidence for rapid evolution of multiple premating and postzygotic barriers despite a very recent divergence time.