Reproductive Isolation Research Articles

A new species of Diacheopsis (Myxomycetes) and a new habitat for myxomycetes.

We describe a new species, Diacheopsis resinae (Myxomycetes), collected from a microhabitat new for myxomycetes: stem wounds of coniferous trees (Norway spruce) where the resin is overgrown with a community of resinicolous fungi. The 80 known collections come from the Vosges (France), the Black Forest (Germany), Swabian Alp (Germany), and several localities in Denmark and Norway. Observations, but as well as metabarcoding of substrate samples with fungal (ITS [internal transcribed spacer]), bacterial (16S rDNA), and myxomycete (18S nuc rDNA) primers from eight trunks, revealed the new myxomycete to co-occur with resin-degrading ascomycetes (Infundichalara microchona, Lophium arboricola, Zythia resinae). The gram-negative bacterial genera Endobacter and Sphingomonas were found to be abundant in the substrate and may be a food source for the myxomycete. Fruit bodies were found mostly during the more humid winter season, with a peak in January/February. Partial sequences of two independent molecular markers (18S nuc rDNA, EF1α [elongation factor 1-alpha] gene) were obtained for 41 accessions, which form a monophyletic cluster in a two-gene phylogeny of Stemonititidales but do not group with other species of Diacheopsis, thus rendering this genus paraphyletic. The new species, although exclusively developing sessile sporocarps and morphologically undoubtedly falling into the genus Diacheopsis, is most closely related to species of Lamproderma, especially L. album, L. zonatum, and L. zonatopulchellum. Within D. resinae, three groups can be differentiated, which show nearly complete reproductive isolation, as judged from a recombination analysis of the two unlinked markers and the allelic combinations of the EF1α gene.

Mycorrhizal specialization toward each distinct Oliveonia fungus in two closely related photosynthetic Dactylostalix orchids

Abstract Although rhizoctonias from Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae are typical orchid mycobionts, orchid mycorrhizal fungi exhibit vast taxonomic and ecological diversity. This diversity stems from the high specificity of orchid mycorrhizal associations and the remarkable diversity of over 28,000 orchid species. The subtribe Calypsoinae is particularly notable for its diverse mycorrhizal partnerships, including rhizoctonias, ectomycorrhizal and saprotrophic non‐rhizoctonia fungi. However, the mycobionts within certain Calypsoinae lineages, such as the genus Dactylostalix, remain understudied. This study explores the physiological ecology of two photosynthetic Calypsoinae species, Dactylostalix ringens and Dactylostalix uniflora, to gain insight into potentially novel associations and their ecological implications. We analysed the mycorrhizal communities of both Dactylostalix species using high‐throughput ITS metabarcoding of root samples collected from multiple locations. Additionally, we measured the natural abundances of 13C and 15N isotopes in the leaves of the two Dactylostalix species and their co‐occurring autotrophic reference plants, as well as in fungal pelotons isolated from D. ringens, to assess the potential for partial mycoheterotrophy. Our findings revealed that D. ringens and D. uniflora form specialized mycorrhizal associations predominantly with distinct lineages of Oliveonia (Oliveoniaceae, Auriculariales), even in sympatric populations. Stable isotope analysis showed that both Dactylostalix species exhibited conflicting isotopic signals: elevated δ15N values, supporting partial mycoheterotrophy, but lower δ13C values compared to autotrophic plants, suggesting autotrophy. Peloton samples from D. ringens displayed only modest 13C enrichment relative to autotrophic references. These conflicting isotopic signals make it difficult to precisely determine whether both Dactylostalix species are autotrophic or partially mycoheterotrophic. Intriguingly, the 13C and 15N signatures of Dactylostalix species and their pelotons resemble those of many rhizoctonia‐associated orchids. This isotopic evidence implies a niche overlap with endophytic tendencies between rhizoctonias and Oliveonia, suggesting that potential endophytic traits may have facilitated the recruitment of Oliveonia as novel mycorrhizal partners. Furthermore, the mycorrhizal segregation between D. ringens and D. uniflora likely promotes their sympatric coexistence and may contribute to reproductive isolation through ecological specialization. Read the free Plain Language Summary for this article on the Journal blog.

Digest: Scarce pollen resources and asymmetric reproductive isolation.

What mechanisms cause asymmetries in reproductive success in crosses between closely related species that differ in floral style length? Feller et al. (2024) found that in five Phlox species, short-styled species produced smaller pollen grains than long-styled species. The smaller pollen of short-styled species lacked the resources to grow pollen tubes long enough to reach the ovules of long-styled species. This asymmetric pollen-style-length incompatibility may considerably affect patterns of gene flow among species.

Opening the species box: What parsimonious microscopic models of speciation have to say about macroevolution.

In the last two decades, lineage-based models of diversification, where species are viewed as particles that can divide (speciate) or die (become extinct) at rates depending on some evolving trait, have been very popular tools to study macroevolutionary processes. Here, we argue that this approach cannot be used to break down the inner workings of species diversification and that "opening the species box" is necessary to understand the causes of macroevolution, but that too detailed speciation models also fail to make robust macroevolutionary predictions. We set up a general framework for parsimonious models of speciation that rely on a minimal number of mechanistic principles: (i) reproductive isolation is caused by excessive dissimilarity between genotypes; (ii) dissimilarity results from a balance between differentiation processes and homogenizing processes; and (iii) dissimilarity can feed back on these processes by decelerating homogenization. We classify such models according to the main homogenizing process : (1) clonal evolution models (ecological drift), (2) models of genetic isolation (gene flow) and (3) models of isolation by distance (spatial drift). We review these models and their specific predictions on macroscopic variables such as species abundances, speciation rates, interfertility relationships or phylogenetic tree structure. We propose new avenues of research by displaying conceptual questions remaining to be solved and new models to address them: the failure of speciation at secondary contact, the feedback of dissimilarity on homogenization, the emergence in space of breeding barriers.

Incomplete barriers to heterospecific mating among Somatochlora species (Odonata: Corduliidae) as revealed in multi-gene phylogenies.

Mating between species occurs within many insect orders. The result of heterospecific mating depends upon the effectiveness of pre- and post-reproductive barriers. Incomplete reproductive barriers lead to introgression of DNA into one species or both. Intricate genital morphology among dragonflies provides little assurance of species specificity given that heterospecific mating or mating attempts have been observed among many species. The genetic consequence is unknown for many heterospecific matings. For example, Somatochlora species mating and genetic exchange have been hypothesized based on observational records and individuals with hybrid morphology. We investigate the potential of heterospecific mating between North American Somatochlora species as inferred from multi-gene phylogenies. We used mitochondrial genes (COI and ND3) and nuclear genes (EF1-α and ITS2) to construct phylogenies using maximum parsimony. Observation of non-monophyletic mtDNA lineages but monophyletic nDNA lineages between Somatochlora sister-species would indicate mtDNA introgression and suggest heterospecific matings. Our results highlighted three instances of non-monophyly of mtDNA clades in the following groups: (i) S. hineana + S. tenebrosa; (ii) S. kennedyi + S. forcipata + S. franklini; and (iii) S. calverti + S. provocans + S. filosa. Analysis of partitioned Bremer support indicates that mtDNA COI largely contributed to the non-monophyly of these species, thus suggesting mtDNA introgression resulting from heterospecific matings. Additionally, the topology resulting from the combined data analysis was concordant with previous taxonomic understanding of Somatochlora species groups. These multi-gene phylogenies of North American Somatochlora are the first, providing a foundation for future ecological and evolution studies and knowledge for effective decision-making and public policy, which is especially important for the endangered species, S. hineana.

Wait, What? What's Going On?- Pregnancy Experiences of Deaf and Hard of Hearing Mothers Who Do Not Sign.

Deaf and hard of hearing (DHH) women experience higher rates of reproductive healthcare barriers and adverse birth outcomes compared to their hearing peers. This study explores the pregnancy experiences of DHH women who do not sign to better understand their barriers and facilitators to optimal perinatal health care. Qualitative study using thematic analysis. Semi-structured, individual remote, or in-person interviews in the United States. Twenty-two DHH English speakers (non-signers) who gave birth in the United States within the past 5 years. Semi-structured interviews explored how DHH women experienced pregnancy and birth, including access to perinatal information and resources, relationships with healthcare providers, communication access, and their involvement with the healthcare system throughout pregnancy. A thematic analysis was conducted. The barriers and facilitators related to a positive perinatal care experience among DHH women. Five key themes emerged. For barriers, healthcare communication breakdowns and loss of patient autonomy highlighted DHH women's struggle with perinatal health care. In contrast, DHH participants outlined the importance of accessible health communication practices and accommodations, use of patient advocacy or self-advocacy, and assistive technologies for DHH parents for more positive perinatal care experiences. Perinatal healthcare providers and staff should routinely inquire about ways to ensure an inclusive and accessible healthcare experience for their DHH patients and provide communication accommodations for optimal care. Additionally, healthcare providers should be more aware of the unique parenting needs and resources of their DHH patients.

Hybrid incompatibility emerges at the one-cell stage in interspecies Caenorhabditis embryos.

Intrinsic reproductive isolation occurs when genetic differences between populations disrupt the development of hybrid organisms, preventing gene flow and enforcing speciation. 1-4 While prior studies have examined the genetic origins of hybrid incompatibility, 5-18 the effects of incompatible factors on development remain poorly understood. Here, we investigate the mechanistic basis of hybrid incompatibility in Caenorhabditis nematodes by capitalizing on the ability of C. brenneri females to produce embryos after mating with males from several other species. Contrary to expectations, hybrid incompatibility was evident immediately after fertilization, suggesting that post-fertilization barriers to hybridization originate from physical incompatibility between sperm and oocyte-derived factors rather than from zygotic transcription, which starts after the 4-cell stage. 19-22 Sperm deliver chromatin, which expands to form a pronucleus, and a pair of centrioles, which form centrosomes that attach to the sperm-derived pronucleus and signal to establish the embryo's anterior-posterior axis. 23,24 In C. brenneri oocytes fertilized with C. elegans sperm, sperm pronuclear expansion was compromised, frequent centrosome detachment was observed, and cortical polarity was disrupted. Live imaging revealed that defective polar body extrusion contributes to defects in mitotic spindle morphology. C. brenneri oocytes fertilized with C. remanei or C. sp. 48 sperm showed similar defects, and their severity and frequency increased with phylogenetic distance. Defective expansion of the sperm-derived pronucleus and unreliable polar body extrusion immediately after fertilization generally underlie the inviability of hybrid embryos in this clade. These results indicate that physical mismatches between sperm and oocyte-derived structures may be a primary mechanism of hybrid incompatibility.

NpCIPK6–NpSnRK1 module facilitates inter-subgeneric hybridization barriers in water lily (nymphaea) by reducing abscisic acid content

Abstract Pre-fertilization hybridization barriers are the main causes of inter-subgeneric hybridization challenges in water lily. However, the mechanism underlying low compatibility between pollen and stigma of water lily remains unclear. This study demonstrates that CBL-interacting protein kinase 6 (CIPK6) responded to the signaling exchange between incompatible pollen and stigma through interactions with SNF1-related kinase 1 (SnRK1) and promotes the accumulation of SnRK1 protein. Activated SnRK1 interacted with 9-cis-epoxycarotenoid dioxygenase 2 (NCED2) to promote its degradation, thereby inhibiting abscisic acid (ABA) synthesis. A decrease in ABA content in the stigma impaired the ABA-mediated removal of reactive oxygen species (ROS), ultimately resulting in the rejection of the incompatible pollen by the stigma. Our results highlight the essential role of the NpCIPK6–NpSnRK1–NpNCED2 module in conferring inter-subgeneric hybridization barriers in water lily by interfering with ABA synthesis and promoting ROS accumulation. This study offers valuable mechanistic insights into cellular signaling and reproductive barriers in water lily as well as across other biological contexts.

Increased evolutionary rate in the Z-chromosome of sympatric and allopatric species of Morpho butterflies.

Divergent evolution of genomes among closely related species is shaped by both neutral processes and ecological forces, such as local adaptation and reinforcement. These factors can drive accelerated evolution of sex chromosomes relative to autosomes. Comparative genomic analyses between allopatric and sympatric species with overlapping or divergent ecological niches offer insights into reinforcement and ecological specialization on genome evolution. In the butterfly genus Morpho, several species coexist in sympatry, with specialization across forest strata and temporal niches. We analyzed the genomes of eight Morpho species, along with previously published genomes of three others, to compare chromosomal rearrangements and signs of positive selection in the Z chromosome vs autosomes. We found extensive chromosomal rearrangements in Z chromosome, particularly in sympatric species with similar ecological niches, suggesting a role for inversions in restricting gene flow at a postzygotic level. Z-linked genes also exhibited significantly higher dN/dS ratios than autosomal genes across the genus, with pronounced differences in closely related species living in sympatry. Additionally, we examined the evolution of eight circadian clock genes, detecting positive selection in Period, located on the Z chromosome. Our findings suggest that the Z chromosome evolves more rapidly than autosomes, particularly among closely related species, raising questions about its role in pre- and post-zygotic isolation mechanisms.

Prezygotic barriers effectively limit hybridization in a rapid evolutionary radiation.

Hybridization is increasingly recognized as an important evolutionary process across the tree of life. In many clades, phylogenomic approaches have permitted unparalleled insight into the extent and frequency of hybridization. However, we continue to lack a deep understanding of the factors that limit and shape patterns of hybridization, especially in evolutionary radiations. In this study, we characterized patterns of introgression across Costus (Costaceae), a young evolutionary radiation of tropical understory plants that maintain widespread interfertility despite exhibiting strong prezygotic reproductive isolation. We analyzed a phylogenomic dataset of 756 genes from 54 Costus species using multiple complementary approaches - D-statistics, gene-tree-based tests, and phylogenetic network analyses - to detect and characterize introgression events throughout the evolutionary history of the radiation. Our results identified a moderate number of introgression events, including a particularly ancient, well-supported event spanning one of the deepest divergences in the clade. Most introgression events occurred between taxa or ancestral lineages that shared the same pollination syndrome (bee-pollinated or hummingbird-pollinated). These findings suggest that prezygotic barriers, including pollinator specialization, have been key to the balance between introgression and reproductive isolation in Costus.

Sex-specific expression of circadian rhythms enables allochronic speciation

Abstract Noctuid moths provide prime examples of species in various stages of allochronic speciation, where reproductive barriers are mediated by genetic divergence in daily or seasonal timing. Theory indicates that allochronic divergence might be one of the most plausible mechanisms of adaptive speciation, especially when timing is subject to divergent ecological selection. Here, we show that the validity of this theoretical expectation is entirely contingent on species characteristics of the mating system. Our analysis focuses on the moth Spodoptera frugiperda (Lepidoptera, Noctuidae), which occurs as two strains that differ in circadian reproductive activity. Unlike in generic models of assortative mating, where chronotypes diverge under mild assumptions, individual-based evolutionary simulations of the mating system and life cycle of S. frugiperda fail to recover allochronic diversification, even under conditions highly conducive to speciation. Instead, we observe that both chronotypes advance their activity schedule toward the early night, resulting in a rapid loss of allochronic variation. This outcome is caused by the fact that mating in S. frugiperda takes considerable time and potential mates are encountered sequentially, so that early males enjoy a systematic advantage. The undermining effect of male mate competition can be overcome when circadian genes evolve sex-specific expression, enabling early and late chronotypes to be maintained or even to diversify in sympatry. These results give new significance to sex differences in biological rhythms and suggest that species characteristics of the mating system and genetic architecture are key to understanding the scope for allochronic speciation across diverse species exhibiting variation in timing.

Sharp decline in male fertility in F2 hybrids of the female-heterogametic silk moth Bombyx.

Sexual selection drives rapid evolution of morphological, physiological, and behavioral traits, especially in males, and it may also drive the rapid evolution of hybrid male sterility. Indeed, the faster male theory of speciation was once viewed as a major cause of Haldane's rule in male-heterogametic XY taxa, but is increasingly being replaced by the genetic conflict hypothesis partly because it cannot explain the faster evolution of hybrid female sterility in female-heterogametic ZW taxa. The theory nonetheless predicts that there should be more genes for hybrid male sterility than for hybrid female sterility even in such taxa, but this remains untested. Thus, finding evidence for the faster male theory of reproductive isolation beyond the F1 generation in ZW systems still represents a challenge to studying the impact of sexual selection. In this study, we examined F2 hybrids between the domesticated silkworm Bombyx mori and the wild silk moth Bombyx mandarina, which have ZW sex determination. We found that although only females showed reduced fertility in the F1 generation, the F2 hybrid males had a significant reduction in fertility compared with the parental and F1 males. Importantly, 27% of the F2 males and 15% of the F2 females were completely sterile, suggesting the presence of recessive incompatibilities causing male sterility in female-heterogametic taxa.

Methyl Jasmonate (MeJA) Promotes the Self-Pollen Tube Growth of Camellia oleifera by Regulating Lignin Biosynthesis

Self-incompatibility (SI) poses a significant reproductive barrier, severely impacting the yield, quality, and economic value of Camellia oleifera. In this study, methyl jasmonate (MeJA) was employed as an exogenous stimulus to alleviate SI in C. oleifera. The research findings revealed that an exogenous dose of 1000 μmol·L−1 MeJA enhanced the germination and tube growth of C. oleifera self-pollen and greatly improved ovule penetration (18.75%) and fertilization (15.81%), ultimately increasing fruit setting (18.67%). It was discovered by transcriptome analysis that the key genes (CAD, C4H) involved in the lignin production process exhibited elevated expression levels in self-pistils treated with MeJA. Further analysis showed that the lignin concentration in the MeJA-treated pistils was 31.70% higher compared with the control group. As verified by pollen germination assays in vitro, lignin in the appropriate concentration range could promote pollen tube growth. Gene expression network analysis indicated that transcription factor bHLH may be pivotal in regulating lignin biosynthesis in response to MeJA, which in turn affects pollen tubes. Further transient knockdown of bHLH (Co_33962) confirmed its important role in C. oleifera pollen tube growth. In summary, the application of MeJA resulted in the stimulation of self-pollen tube elongation and enhanced fruit setting in C. oleifera, which could be associated with the differential change in genes related to lignin synthesis and the increased lignin content.

A Minimal Hybrid Sterility Genome Assembled by Chromosome Swapping Between Mouse Subspecies (Mus musculus).

Hybrid sterility is a reproductive isolation barrier between diverging taxa securing the early steps of speciation. Hybrid sterility is ubiquitous in the animal and plant kingdoms, but its genetic control is poorly understood. In our previous studies, we have uncovered the sterility of hybrids between musculus and domesticus subspecies of the house mouse, which is controlled by the Prdm9 gene, the X-linked Hstx2 locus, and subspecific heterozygosity for genetic background. To further investigate this form of genic-driven chromosomal sterility, we constructed a simplified hybrid sterility model within the genome of the domesticus subspecies by swapping domesticus autosomes with their homologous partners from the musculus subspecies. We show that the "sterility" allelic combination of Prdm9 and Hstx2 can be activated by a musculus/domesticus heterozygosity of as few as two autosomes, Chromosome 17 (Chr 17) and Chr 18 and is further enhanced when another heterosubspecific autosomal pair is present, whereas it has no effect on meiotic progression in the pure domesticus genome. In addition, we identify a new X-linked hybrid sterility locus, Hstx3, at the centromeric end of Chr X, which modulates the incompatibility between Prdm9 and Hstx2. These results further support our concept of chromosomal hybrid sterility based on evolutionarily accumulated divergence between homologous sequences. Based on these and previous results, we believe that future studies should include more information on the mutual recognition of homologous chromosomes at or before the first meiotic prophase in interspecific hybrids, as this may serve as a general reproductive isolation checkpoint in mice and other species.

Evolution of Sensory Receptors.

Sensory receptors are at the interface between an organism and its environment and thus represent key sites for biological innovation. Here, we survey major sensory receptor families to uncover emerging evolutionary patterns. Receptors for touch, temperature, and light constitute part of the ancestral sensory toolkit of animals, often predating the evolution of multicellularity and the nervous system. In contrast, chemoreceptors exhibit a dynamic history of lineage-specific expansions and contractions correlated with the disparate complexity of chemical environments. A recurring theme includes independent transitions from neurotransmitter receptors to sensory receptors of diverse stimuli from the outside world. We then provide an overview of the evolutionary mechanisms underlying sensory receptor diversification and highlight examples where signatures of natural selection are used to identify novel sensory adaptations. Finally, we discuss sensory receptors as evolutionary hotspots driving reproductive isolation and speciation, thereby contributing to the stunning diversity of animals.

Molecular phylogenetic relationships among lineages in the genus Carabus (Coleoptera: Carabidae) and radiation of the subgenus Apotomopterus

Abstract The genus Carabus (Coleoptera: Carabidae: Carabinae) is a group of predacious ground beetles found throughout Holarctic temperate zones. The diversification of this group is attributed not only to allopatric speciation but also to diversification in life-history traits and morphological traits that are involved in ecological adaptation and reproductive isolation. Precise phylogenetic reconstruction of Carabus is important to understanding the factors associated with species’ divergence in relation to such character divergence. Here, we analyse the phylogeny of Carabus among higher taxonomic groups and among species of the subgenus Apotomopterus, a hyper-diverse group endemic to East Asia. We used genome-wide sequence data from ultraconserved elements for phylogenetic reconstruction via maximum-likelihood and species-tree reconstruction methods. With regard to the relationships among higher taxonomic groups, the obtained phylogenetic trees resolved two structures with previously unresolved divergence. The subgenus Apotomopterus contained three lineages, i.e. the Vitalisi, Prodigus, and Sauteri groups. It showed diversification of the spinula, a sclerotized male genital structure, and variation in the female elytral notch (preapical emargination). These morphological traits may be involved in copulatory behaviour and potentially affect reproductive isolation, and their character state divergence may have facilitated species divergence of Apotomopterus.

Secondary contact zone and genetic introgression in closely related haplodiploid social spider mites.

How frequently hybridisation and gene flow occur in the contact zones of diverging taxa is important for understanding the speciation process. Stigmaeopsis sabelisi and Stigmaeopsis miscanthi high-aggression form (hereafter, S. miscanthi HG) are haplodiploid, social spider mites that infest the Chinese silver grass, Miscanthus sinensis. These two species are closely related and parapatrically distributed in Japan. In mountainous areas, S. sabelisi and S. miscanthi HG are often found in the highlands and lowlands, respectively, suggesting that they are in contact at intermediate altitudes. It is estimated that they diverged from their common ancestors distributed in subtropical regions (south of Japan) during the last glacial period, expanded their distribution into the Japanese Archipelago, and came to have such a parapatric distribution (secondary contact). As their reproductive isolation is strong but incomplete, hybridisation and genetic introgression are expected at their distributional boundaries. In this study, we investigated their spatial distribution patterns along the elevation on Mt. Amagi using male morphological differences, and investigated their hybridisation status using single-nucleotide polymorphisms by MIG-seq. We found their contact zone at altitudes of 150-430 m, suggesting that their contact zone is prevalent in the parapatric area, which is in line with a previous study. Interspecific mating was predicted based on the sex ratio in the contact zone. No obvious hybrids were found, but genetic introgression was detected although it was extremely low.

The genetic consequences of historic climate change on the contemporary population structure of a widespread temperate North American songbird

Studies of widely distributed species can inform our understanding of how past demographic events tied to historic glaciation and ongoing population genetic processes interact to shape contemporaneous patterns of biodiversity at a continental scale. In this study, we used whole-genome resequencing to investigate the current population structure and genetic signatures of past demographic events in the widespread migratory American goldfinch (Spinus tristis). Phylogenetic relationships inferred from whole mitochondrial genomes were poorly resolved. In contrast, a genome-wide panel of > 4.5 million single nucleotide polymorphisms (SNPs) strongly supported the existence of eastern and western populations separated by western mountain ranges and additional population structuring within the western clade. Demographic modeling estimated that the eastern and western populations diverged approximately one million years ago, and both populations experienced subsequent population bottlenecks during the last glacial period. Species distribution models showed a severe contraction of suitable habitat for the American goldfinch during this period, with predicted discontinuities that are consistent with multiple, isolated glacial refugia that coincide with present-day population structure. Low overall genetic differentiation between the eastern and western populations (FST ∼ 0.01) suggests ongoing gene flow accompanied divergence, and individuals with admixed genomic signatures were sampled along a potential contact zone. Nevertheless, outlier SNPs were identified near genes associated with feather color, song, and migratory behavior and provide strong candidates for further study of the mechanisms underlying reproductive isolation and speciation in birds.

A consequential one-night stand: Episodic historical hybridization leads to mitochondrial takeover in sympatric desert ant-eating spiders

Disentangling the genomic intricacies underlying speciation and the causes of discordance between sources of evidence can offer remarkable insights into evolutionary dynamics. The ant-eating spider Zodarion nitidum, found across the Middle East and Egypt, displays yellowish and blackish morphs that co-occur sympatrically. These morphs additionally differ in behavioral and physiological features and show complete pre-mating reproductive isolation. In contrast, they possess similar sexual features and lack distinct differences in their mitochondrial DNA. We analyzed both Z. nitidum morphs and outgroups using genome-wide and additional mitochondrial DNA data. The genomic evidence indicated that Yellow and Black are reciprocally independent lineages without signs of recent admixture. Interestingly, the sister group of Yellow is not Black but Z. luctuosum, a morphologically distinct species. Genomic gene flow analyses pinpointed an asymmetric nuclear introgression event, with Yellow contributing nearly 5 % of its genome to Black roughly 320,000 years ago, intriguingly aligning with the independently estimated origin of the mitochondrial DNA of Black. We conclude that the blackish and yellowish morphs of Z. nitidum are long-diverged distinct species, and that the ancient and modest genomic introgression event registered resulted in a complete mitochondrial takeover of Black by Yellow. This investigation underscores the profound long-term effects that even modest hybridization events can have on the genome of organisms. It also exemplifies the utility of phylogenetic networks for estimating historical events and how integrating independent lines of evidence can increase the reliability of such estimations.

Metabolomics reveals a key role of salicylic acid in embryo abortion underlying interspecific hybridization between Hydrangea macrophylla and H. arborescens.

Embryo abortion at the heart-shaped stage is the main reason for the failure of interspecific hybridization of hydrangea, and salicylic acid plays a key role during embryo abortion. Difficulties in obtaining seeds from interspecific hybridization between Hydrangea macrophylla and H. arborescens had severely restricted the process of breeding new hydrangea varieties. To clarify the cause of reproductive barriers, an interspecific hybridization was made between H. macrophylla 'Endless Summer' (female parent) and H. arborescens 'Annabelle' (male parent). The results showed that both parents' floral organs developed normally, 'Annabelle' had high pollen viability (84.83% at 8h after incubation), and the pollen tube could enter into the ovule of 'Endless Summer' at 72h after pollination. Therefore, the pre-fertilization barrier was not the main reason for the failure of interspecific hybridization. However, observation of the embryo development by paraffin sections showed that the embryo was aborted at the heart-shaped stage. In addition, salicylic acid (SA) content was significantly higher (fourfold, P < 0.01) at 21 days after pollination (DAP) as compared to that of 17 DAP, which means SA may be closely correlated with embryo development. A total of 957 metabolites were detected, among which 78 were significantly different. During the embryo abortion, phenylpropanoids and polyketides were significantly down-regulated, while organic oxygen compounds were significantly up-regulated. Further analysis indicated that the metabolic pathway was enriched in the shikimic acid biosynthesis pathway, which suggests that more SA was synthesized. Taken together, it can be reasonably speculated that SA plays a key role leading to embryo abortion underlying the interspecific hybridization between Hydrangea macrophylla and H. arborescens. The result is helpful to direct the breeding of hydrangea through distant hybridization.