Darwin's 'mystery of mysteries': the role of sexual selection in plant speciation.

Sexual selection is considered one of the key processes that contribute to the emergence of new species. While the connection between sexual selection and speciation has been supported by comparative studies, the mechanisms that mediate this connection remain unresolved, especially in plants. Similarly, it is not clear how speciation processes within plant populations translate into large-scale speciation dynamics. Here, we review the mechanisms through which sexual selection, pollination, and mate choice unfold and interact, and how they may ultimately produce reproductive isolation in plants. We also overview reproductive strategies that might influence sexual selection in plants and illustrate how functional traits might connect speciation at the population level (population differentiation, evolution of reproductive barriers; i.e. microevolution) with evolution above the species level (macroevolution). We also identify outstanding questions in the field, and suitable data and tools for their resolution. Altogether, this effort motivates further research focused on plants, which might potentially broaden our general understanding of speciation by sexual selection, a major concept in evolutionary biology.

Repeatedly Northwards and Upwards: Southern African Grasslands Fuel the Colonization of the African Sky Islands in Helichrysum (Compositae).

The Afromontane and Afroalpine areas constitute some of the main biodiversity hotspots of Africa. They are particularly rich in plant endemics, but the biogeographic origins and evolutionary processes leading to this outstanding diversity are poorly understood. We performed phylogenomic and biogeographic analyses of one of the most species-rich plant genera in these mountains, Helichrysum (Compositae-Gnaphalieae). Most previous studies have focused on Afroalpine elements of Eurasian origin, and the southern African origin of Helichrysum provides an interesting counterexample. We obtained a comprehensive nuclear dataset from 304 species (≈50% of the genus) using target-enrichment with the Compositae1061 probe set. Summary-coalescent and concatenation approaches combined with paralog recovery yielded congruent, well-resolved phylogenies. Ancestral range estimations revealed that Helichrysum originated in arid southern Africa, whereas the southern African grasslands were the source of most lineages that dispersed within and outside Africa. Colonization of the tropical Afromontane and Afroalpine areas occurred repeatedly throughout the Miocene-Pliocene. This timing coincides with mountain uplift and the onset of glacial cycles, which together may have facilitated both speciation and intermountain gene flow, contributing to the evolution of the Afroalpine flora.

Morphological and environmental differentiation as prezygotic reproductive barriers between parapatric and allopatric Campanula rotundifolia agg. cytotypes.

BACKGROUND AND AIMS: Reproductive isolation and local establishment are necessary for plant speciation. Polyploidy, the possession of more than two complete chromosome sets, creates a strong postzygotic reproductive barrier between diploid and tetraploid cytotypes. However, this barrier weakens between polyploids (e.g. tetraploids and hexaploids). Reproductive isolation may be enhanced by cytotype morphological and environmental differentiation. Moreover, morphological adaptations to local conditions contribute to plant establishment. However, the relative contributions of ploidy level and the environment to morphology have generally been neglected. Thus, the extent of morphological variation driven by ploidy level and the environment was modelled for diploid, tetraploid and hexaploid cytotypes of Campanula rotundifolia agg. Cytotype distribution was updated, and morphological and environmental differentiation was tested in the presence and absence of natural contact zones. METHODS: Cytotype distribution was assessed from 231 localities in Central Europe, including 48 localities with known chromosome counts, using flow cytometry. Differentiation in environmental niche and morphology was tested for cytotype pairs using discriminant analyses. A structural equation model was used to explore the synergies between cytotype, environment and morphology. KEY RESULTS: Tremendous discrepancies were revealed between the reported and detected cytotype distribution. Neither mixed-ploidy populations nor interploidy hybrids were detected in the contact zones. Diploids had the broadest environmental niche, while hexaploids had the smallest and specialized niche. Hexaploids and spatially isolated cytotype pairs differed morphologically, including allopatric tetraploids. While leaf and shoot morphology were influenced by environmental conditions and polyploidy, flower morphology depended exclusively on the cytotype. CONCLUSIONS: Reproductive isolation mechanisms vary between cytotypes. While diploids and polyploids are isolated postzygotically, the environmental niche shift is essential between higher polyploids. The impact of polyploidy and the environment on plant morphology implies the adaptive potential of polyploids, while the exclusive relationship between flower morphology and cytotype highlights the role of polyploidy in reproductive isolation.

Utilizing paralogues for phylogenetic reconstruction has the potential to increase species tree support and reduce gene tree discordance in target enrichment data.

The analysis of target enrichment data in phylogenetics lacks optimization toward using paralogues for phylogenetic reconstruction. We developed a novel approach of detecting paralogues and utilizing them for phylogenetic tree inference, by retrieving both ortho- and paralogous copies and creating orthologous alignments, from which the gene trees are built. We implemented this approach in ParalogWizard and demonstrate its performance in plant groups that underwent a whole genome duplication relatively recently: the subtribe Malinae (family Rosaceae), using Angiosperms353 as well as Malinae481 probes, the genus Oritrophium (family Asteraceae), using Compositae1061 probes, and the genus Amomum (family Zingiberaceae), using Zingiberaceae1180 probes. Discriminating between orthologues and paralogues reduced gene tree discordance and increased the species tree support in the case of the Malinae, but not for Oritrophium and Amomum. This may relate to the difference in the proportion of paralogous loci between the data sets, which was highest for the Malinae. Overall, retrieving paralogues for phylogenetic reconstruction following ParalogWizard has the potential to increase the species tree support and reduce gene tree discordance in target enrichment data, particularly if the proportion of paralogous loci is high.

Relative performance of customized and universal probe sets in target enrichment: A case study in subtribe Malinae.

PREMISE: Custom probe design for target enrichment in phylogenetics is tedious and often hinders broader phylogenetic synthesis. The universal angiosperm probe set Angiosperms353 may be the solution. Here, we test the relative performance of Angiosperms353 on the Rosaceae subtribe Malinae in comparison with custom probes that we specifically designed for this clade. We then address the impact of bioinformatically altering the performance of Angiosperms353 by replacing the original probe sequences with orthologs extracted from the Malus domestica genome. METHODS: To evaluate the relative performance of these probe sets, we compared the enrichment efficiency, locus recovery, alignment length, proportion of parsimony-informative sites, proportion of potential paralogs, the topology and support of the resulting species trees, and the gene tree discordance. RESULTS: Locus recovery was highest for our custom Malinae probe set, and replacing the original Angiosperms353 sequences with a Malus representative improved the locus recovery relative to Angiosperms353. The proportion of parsimony-informative sites was similar between all probe sets, while the gene tree discordance was lower in the case of the custom probes. DISCUSSION: A custom probe set benefits from data completeness and can be tailored toward the specificities of the project of choice; however, Angiosperms353 was equally as phylogenetically informative as the custom probes. We therefore recommend using both a custom probe set and Angiosperms353 to facilitate large-scale systematic studies, where financially possible.

Allele Sorting as a Novel Approach to Resolving the Origin of Allotetraploids Using Hyb-Seq Data: A Case Study of the Balkan Mountain Endemic Cardamine barbaraeoides.

Mountains of the Balkan Peninsula are significant biodiversity hotspots with great species richness and a large proportion of narrow endemics. Processes that have driven the evolution of the rich Balkan mountain flora, however, are still insufficiently explored and understood. Here we focus on a group of Cardamine (Brassicaceae) perennials growing in wet, mainly mountainous habitats. It comprises several Mediterranean endemics, including those restricted to the Balkan Peninsula. We used target enrichment with genome skimming (Hyb-Seq) to infer their phylogenetic relationships, and, along with genomic in situ hybridization (GISH), to resolve the origin of tetraploid Cardamine barbaraeoides endemic to the Southern Pindos Mts. (Greece). We also explored the challenges of phylogenomic analyses of polyploid species and developed a new approach of allele sorting into homeologs that allows identifying subgenomes inherited from different progenitors. We obtained a robust phylogenetic reconstruction for diploids based on 1,168 low-copy nuclear genes, which suggested both allopatric and ecological speciation events. In addition, cases of plastid-nuclear discordance, in agreement with divergent nuclear ribosomal DNA (nrDNA) copy variants in some species, indicated traces of interspecific gene flow. Our results also support biogeographic links between the Balkan and Anatolian-Caucasus regions and illustrate the contribution of the latter region to high Balkan biodiversity. An allopolyploid origin was inferred for C. barbaraeoides, which highlights the role of mountains in the Balkan Peninsula both as refugia and melting pots favoring species contacts and polyploid evolution in response to Pleistocene climate-induced range dynamics. Overall, our study demonstrates the importance of a thorough phylogenomic approach when studying the evolution of recently diverged species complexes affected by reticulation events at both diploid and polyploid levels. We emphasize the significance of retrieving allelic and homeologous variation from nuclear genes, as well as multiple nrDNA copy variants from genome skim data.

Early evolution of reproductive isolation: A case of weak inbreeder/strong outbreeder leads to an intraspecific hybridization barrier in Arabidopsis lyrata.

Reproductive strategies play a major role in plant speciation. Notably, transitions from outcrossing to selfing may lead to relaxed sexual selection and parental conflict. Shifts in mating systems can affect maternal and paternal interests, and thus parent-specific influence on endosperm development, leading to reproductive isolation: if selfing and outcrossing species hybridize, the resulting seeds may not be viable due to endosperm failure. Nevertheless, it remains unclear how the switch in mating systems can impact reproductive isolation between recently diverged lineages, that is, during the process of speciation. We investigated this question using Arabidopsis lyrata, which recently transitioned to selfing (10,000 years ago) in certain North American populations, where European populations remain outcrossing. We performed reciprocal crosses between selfers and outcrossers, and measured seed viability and endosperm development. We show that parental genomes in the hybrid seed negatively interact, as predicted by parental conflict. This leads to extensive hybrid seed lethality associated with endosperm cellularization disturbance. Our results suggest that this is primarily driven by divergent evolution of the paternal genome between selfers and outcrossers. In addition, we observed other hybrid seed defects, suggesting that sex-specific interests are not the only processes contributing to postzygotic reproductive isolation.

Adaptive introgression: how polyploidy reshapes gene flow landscapes.

Rare yet accumulating evidence in both plants and animals shows that whole genome duplication (WGD, leading to polyploidy) can break down reproductive barriers, facilitating gene flow between otherwise isolated species. Recent population genomic studies in wild, outcrossing Arabidopsis arenosa and Arabidopsis lyrata indicate that this WGD-potentiated gene flow can be adaptive and highly specific in response to particular environmental and intracellular challenges. The mechanistic basis of WGD-mediated easing of species barrier strength seems to primarily lie in the relative dosage of each parental genome in the endosperm. While generalisations about polyploids can be fraught, this evidence indicates that the breakdown of these barriers, combined with diploid to polyploid gene flow and gene flow between polyploids, allows some polyploids to act as adaptable 'allelic sponges', enjoying increased potential to respond to challenging environments.

De Novo Mutation and Rapid Protein (Co-)evolution during Meiotic Adaptation in Arabidopsis arenosa.

A sudden shift in environment or cellular context necessitates rapid adaptation. A dramatic example is genome duplication, which leads to polyploidy. In such situations, the waiting time for new mutations might be prohibitive; theoretical and empirical studies suggest that rapid adaptation will largely rely on standing variation already present in source populations. Here, we investigate the evolution of meiosis proteins in Arabidopsis arenosa, some of which were previously implicated in adaptation to polyploidy, and in a diploid, habitat. A striking and unexplained feature of prior results was the large number of amino acid changes in multiple interacting proteins, especially in the relatively young tetraploid. Here, we investigate whether selection on meiosis genes is found in other lineages, how the polyploid may have accumulated so many differences, and whether derived variants were selected from standing variation. We use a range-wide sample of 145 resequenced genomes of diploid and tetraploid A. arenosa, with new genome assemblies. We confirmed signals of positive selection in the polyploid and diploid lineages they were previously reported in and find additional meiosis genes with evidence of selection. We show that the polyploid lineage stands out both qualitatively and quantitatively. Compared with diploids, meiosis proteins in the polyploid have more amino acid changes and a higher proportion affecting more strongly conserved sites. We find evidence that in tetraploids, positive selection may have commonly acted on de novo mutations. Several tests provide hints that coevolution, and in some cases, multinucleotide mutations, might contribute to rapid accumulation of changes in meiotic proteins.

How to Tackle Phylogenetic Discordance in Recent and Rapidly Radiating Groups? Developing a Workflow Using Loricaria (Asteraceae) as an Example.

A major challenge in phylogenetics and -genomics is to resolve young rapidly radiating groups. The fast succession of species increases the probability of incomplete lineage sorting (ILS), and different topologies of the gene trees are expected, leading to gene tree discordance, i.e., not all gene trees represent the species tree. Phylogenetic discordance is common in phylogenomic datasets, and apart from ILS, additional sources include hybridization, whole-genome duplication, and methodological artifacts. Despite a high degree of gene tree discordance, species trees are often well supported and the sources of discordance are not further addressed in phylogenomic studies, which can eventually lead to incorrect phylogenetic hypotheses, especially in rapidly radiating groups. We chose the high-Andean Asteraceae genus Loricaria to shed light on the potential sources of phylogenetic discordance and generated a phylogenetic hypothesis. By accounting for paralogy during gene tree inference, we generated a species tree based on hundreds of nuclear loci, using Hyb-Seq, and a plastome phylogeny obtained from off-target reads during target enrichment. We observed a high degree of gene tree discordance, which we found implausible at first sight, because the genus did not show evidence of hybridization in previous studies. We used various phylogenomic analyses (trees and networks) as well as the D-statistics to test for ILS and hybridization, which we developed into a workflow on how to tackle phylogenetic discordance in recent radiations. We found strong evidence for ILS and hybridization within the genus Loricaria. Low genetic differentiation was evident between species located in different Andean cordilleras, which could be indicative of substantial introgression between populations, promoted during Pleistocene glaciations, when alpine habitats shifted creating opportunities for secondary contact and hybridization.

Nested whole-genome duplications coincide with diversification and high morphological disparity in Brassicaceae.

Angiosperms have become the dominant terrestrial plant group by diversifying for ~145 million years into a broad range of environments. During the course of evolution, numerous morphological innovations arose, often preceded by whole genome duplications (WGD). The mustard family (Brassicaceae), a successful angiosperm clade with ~4000 species, has been diversifying into many evolutionary lineages for more than 30 million years. Here we develop a species inventory, analyze morphological variation, and present a maternal, plastome-based genus-level phylogeny. We show that increased morphological disparity, despite an apparent absence of clade-specific morphological innovations, is found in tribes with WGDs or diversification rate shifts. Both are important processes in Brassicaceae, resulting in an overall high net diversification rate. Character states show frequent and independent gain and loss, and form varying combinations. Therefore, Brassicaceae pave the way to concepts of phylogenetic genome-wide association studies to analyze the evolution of morphological form and function.

A novel allele of ASY3 is associated with greater meiotic stability in autotetraploid Arabidopsis lyrata.

In this study we performed a genotype-phenotype association analysis of meiotic stability in 10 autotetraploid Arabidopsis lyrata and A. lyrata/A. arenosa hybrid populations collected from the Wachau region and East Austrian Forealps. The aim was to determine the effect of eight meiosis genes under extreme selection upon adaptation to whole genome duplication. Individual plants were genotyped by high-throughput sequencing of the eight meiosis genes (ASY1, ASY3, PDS5b, PRD3, REC8, SMC3, ZYP1a/b) implicated in synaptonemal complex formation and phenotyped by assessing meiotic metaphase I chromosome configurations. Our results reveal that meiotic stability varied greatly (20-100%) between individual tetraploid plants and associated with segregation of a novel ASYNAPSIS3 (ASY3) allele derived from A. lyrata. The ASY3 allele that associates with meiotic stability possesses a putative in-frame tandem duplication (TD) of a serine-rich region upstream of the coiled-coil domain that appears to have arisen at sites of DNA microhomology. The frequency of multivalents observed in plants homozygous for the ASY3 TD haplotype was significantly lower than in plants heterozygous for ASY3 TD/ND (non-duplicated) haplotypes. The chiasma distribution was significantly altered in the stable plants compared to the unstable plants with a shift from proximal and interstitial to predominantly distal locations. The number of HEI10 foci at pachytene that mark class I crossovers was significantly reduced in a plant homozygous for ASY3 TD compared to a plant heterozygous for ASY3 ND/TD. Fifty-eight alleles of the 8 meiosis genes were identified from the 10 populations analysed, demonstrating dynamic population variability at these loci. Widespread chimerism between alleles originating from A. lyrata/A. arenosa and diploid/tetraploids indicates that this group of rapidly evolving genes may provide precise adaptive control over meiotic recombination in the tetraploids, the very process that gave rise to them.

Interspecific introgression mediates adaptation to whole genome duplication.

Adaptive gene flow is a consequential phenomenon across all kingdoms. Although recognition is increasing, there is no study showing that bidirectional gene flow mediates adaptation at loci that manage core processes. We previously discovered concerted molecular changes among interacting members of the meiotic machinery controlling crossover number upon adaptation to whole-genome duplication (WGD) in Arabidopsis arenosa. Here we conduct a population genomic study to test the hypothesis that adaptation to WGD has been mediated by adaptive gene flow between A. arenosa and A. lyrata. We find that A. lyrata underwent WGD more recently than A. arenosa, suggesting that pre-adapted alleles have rescued nascent A. lyrata, but we also detect gene flow in the opposite direction at functionally interacting loci under the most extreme levels of selection. These data indicate that bidirectional gene flow allowed for survival after WGD, and that the merger of these species is greater than the sum of their parts.

An empirical assessment of a single family-wide hybrid capture locus set at multiple evolutionary timescales in Asteraceae.

PREMISE: Hybrid capture with high-throughput sequencing (Hyb-Seq) is a powerful tool for evolutionary studies. The applicability of an Asteraceae family-specific Hyb-Seq probe set and the outcomes of different phylogenetic analyses are investigated here. METHODS: Hyb-Seq data from 112 Asteraceae samples were organized into groups at different taxonomic levels (tribe, genus, and species). For each group, data sets of non-paralogous loci were built and proportions of parsimony informative characters estimated. The impacts of analyzing alternative data sets, removing long branches, and type of analysis on tree resolution and inferred topologies were investigated in tribe Cichorieae. RESULTS: Alignments of the Asteraceae family-wide Hyb-Seq locus set were parsimony informative at all taxonomic levels. Levels of resolution and topologies inferred at shallower nodes differed depending on the locus data set and the type of analysis, and were affected by the presence of long branches. DISCUSSION: The approach used to build a Hyb-Seq locus data set influenced resolution and topologies inferred in phylogenetic analyses. Removal of long branches improved the reliability of topological inferences in maximum likelihood analyses. The Astereaceae Hyb-Seq probe set is applicable at multiple taxonomic depths, which demonstrates that probe sets do not necessarily need to be lineage-specific.

Pervasive population genomic consequences of genome duplication in Arabidopsis arenosa.

Ploidy-variable species allow direct inference of the effects of chromosome copy number on fundamental evolutionary processes. While an abundance of theoretical work suggests polyploidy should leave distinct population genomic signatures, empirical data remains sparse. We sequenced ~300 individuals from 39 populations of Arabidopsis arenosa, a naturally diploid-autotetraploid species. We find that the impacts of polyploidy on population genomic processes are subtle yet pervasive, such as reduced efficiency of purifying selection, differences in linked selection and rampant gene flow from diploids. Initial masking of deleterious mutations, faster rates of nucleotide substitution and interploidy introgression likely conspire to shape the evolutionary potential of polyploids.

Resolving the rapid plant radiation of early diverging lineages in the tropical Zingiberales: Pushing the limits of genomic data.

Many cases of rapid evolutionary radiations in plant and animal lineages are known; however phylogenetic relationships among these lineages have been difficult to resolve by systematists. Increasing amounts of genomic data have been sequentially applied in an attempt to resolve these radiations, dissecting their evolutionary patterns into a series of bifurcating events. Here we explore one such rapid radiation in the tropical plant order Zingiberales (the bananas and relatives) which includes eight families, approximately 110 genera, and more than 2600 species. One clade, the "Ginger families", including (Costaceae + Zingiberaceae) (Marantaceae + Cannaceae), has been well-resolved and well-supported in all previous studies. However, well-supported reconstructions among the "Banana families" (Musaceae, Heliconiaceae, Lowiaceae, Strelitziaceae), which most likely diverged about 90 Mya, have been difficult to confirm. Supported with anatomical, morphological, single locus, and genome-wide data, nearly every possible phylogenetic placement has been proposed for these families. In an attempt to resolve this complex evolutionary event, hybridization-based target enrichment was used to obtain sequences from up to 378 putatively orthologous low-copy nuclear genes (all ≥ 960 bp). Individual gene trees recovered multiple topologies among the early divergent lineages, with varying levels of support for these relationships. One topology of the "Banana families" (Musaceae (Heliconiaceae (Lowiaceae + Strelitziaceae))), which has not been suggested until now, was almost consistently recovered in all multilocus analyses of the nuclear dataset (concatenated - ExaML, coalescent - ASTRAL and ASTRID, supertree - MRL, and Bayesian concordance - BUCKy). Nevertheless, the multiple topologies recovered among these lineages suggest that even large amounts of genomic data might not be able to fully resolve relationships at this phylogenetic depth. This lack of well-supported resolution could suggest methodological problems (i.e., violation of model assumptions in both concatenated and coalescent analyses) or more likely reflect an evolutionary history shaped by an explosive, rapid, and nearly simultaneous polychotomous radiation in this group of plants towards the end of the Cretaceous, perhaps driven by vertebrate pollinator selection.

HybPhyloMaker: Target Enrichment Data Analysis From Raw Reads to Species Trees.

SUMMARY: Hybridization-based target enrichment in combination with genome skimming (Hyb-Seq) is becoming a standard method of phylogenomics. We developed HybPhyloMaker, a bioinformatics pipeline that performs target enrichment data analysis from raw reads to supermatrix-, supertree-, and multispecies coalescent-based species tree reconstruction. HybPhyloMaker is written in BASH and integrates common bioinformatics tools. It can be launched both locally and on a high-performance computer cluster. Compared with existing target enrichment data analysis pipelines, HybPhyloMaker offers the following main advantages: implementation of all steps of data analysis from raw reads to species tree reconstruction, calculation and summary of alignment and gene tree properties that assist the user in the selection of "quality-filtered" genes, implementation of several species tree reconstruction methods, and analysis of the coding regions of organellar genomes. AVAILABILITY: The HybPhyloMaker scripts, manual as well as a test data set, are available in https://github.com/tomas-fer/HybPhyloMaker/. HybPhyloMaker is licensed under open-source license GPL v.3 allowing further modifications.

Hybrids and horizontal transfer: introgression allows adaptive allele discovery.

Evolution has devised countless remarkable solutions to diverse challenges. Understanding the mechanistic basis of these solutions provides insights into how biological systems can be subtly tweaked without maladaptive consequences. The knowledge gained from illuminating these mechanisms is equally important to our understanding of fundamental evolutionary mechanisms as it is to our hopes of developing truly rational plant breeding and synthetic biology. In particular, modern population genomic approaches are proving very powerful in the detection of candidate alleles for mediating consequential adaptations that can be tested functionally. Especially striking are signals gained from contexts involving genetic transfers between populations, closely related species, or indeed between kingdoms. Here we discuss two major classes of these scenarios, adaptive introgression and horizontal gene flow, illustrating discoveries made across kingdoms.

Phylogenetic marker development for target enrichment from transcriptome and genome skim data: the pipeline and its application in southern African Oxalis (Oxalidaceae).

Phylogenetics benefits from using a large number of putatively independent nuclear loci and their combination with other sources of information, such as the plastid and mitochondrial genomes. To facilitate the selection of orthologous low-copy nuclear (LCN) loci for phylogenetics in nonmodel organisms, we created an automated and interactive script to select hundreds of LCN loci by a comparison between transcriptome and genome skim data. We used our script to obtain LCN genes for southern African Oxalis (Oxalidaceae), a speciose plant lineage in the Greater Cape Floristic Region. This resulted in 1164 LCN genes greater than 600 bp. Using target enrichment combined with genome skimming (Hyb-Seq), we obtained on average 1141 LCN loci, nearly the whole plastid genome and the nrDNA cistron from 23 southern African Oxalis species. Despite a wide range of gene trees, the phylogeny based on the LCN genes was very robust, as retrieved through various gene and species tree reconstruction methods as well as concatenation. Cytonuclear discordance was strong. This indicates that organellar phylogenies alone are unlikely to represent the species tree and stresses the utility of Hyb-Seq in phylogenetics.

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.