Postzygotic Barriers Research Articles

Dosage-sensitive maternal siRNAs determine hybridization success in Capsella.

Hybrid seed failure arising from wide crosses between plant species is a recurring obstacle in plant breeding, impeding the transfer of desirable traits. This postzygotic reproductive barrier primarily occurs in the endosperm, a tissue that nourishes the embryo and functions similarly to the placenta in mammals. We found that incompatible seeds show a loss of DNA methylation and chromatin condensation in the endosperm, similar to seeds lacking maternal RNA polymerase IV activity. This similarity is linked to a decrease in small interfering RNAs in the endosperm (sirenRNAs), maternal RNA polymerase IV-dependent short interfering RNAs that regulate DNA methylation. Several AGAMOUS-like MADS-box transcription factor genes (AGLs), key regulators of endosperm development, are targeted by sirenRNAs in cis and in trans. This finding aligns with the enrichment of AGL target genes among deregulated genes. We propose that hybrid seed failure results from reduced maternal sirenRNAs combined with increased AGL expression, leading to abnormal gene regulation in the endosperm.

The importance of reproductive isolation in driving diversification and speciation within Peruvian mimetic poison frogs (Dendrobatidae)

To explain how populations with distinct warning signals coexist in close parapatry, we experimentally assessed intrinsic mechanisms acting as reproductive barriers within three poison-frog species from the Peruvian Amazon belonging to a Müllerian mimetic ring (Ranitomeya variabilis, Ranitomeya imitator and Ranitomeya fantastica). We tested the role of prezygotic and postzygotic isolation barriers between phenotypically different ecotypes of each species, using no-choice mating experiments and offspring survival analysis. Our results show that prezygotic mating preference did not occur except for one specific ecotype of R. imitator, and that all three species were able to produce viable inter-population F1 hybrids. However, while R. variabilis and R. imitator hybrids were able to produce viable F2 generations, we found that for R. fantastica, every F1 hybrid males were sterile while females remained fertile. This unexpected result, echoing with Haldane’s rule of speciation, validated phylogenetic studies which tentatively diagnose these populations of R. fantastica as two different species. Our work suggests that postzygotic genetic barriers likely participate in the extraordinary phenotypic diversity observed within Müllerian mimetic Ranitomeya populations, by maintaining species boundaries.

One Genome, Multiple Phenotypes: Would Rhodnius milesi Carcavallo, Rocha, Galvão & Jurberg, 2001 (Hemiptera, Triatominae) Be a Valid Species or a Phenotypic Polymorphism of R. neglectus Lent, 1954?

Species of the Rhodnius genus have a complex taxonomy because the events of phenotypic plasticity and cryptic speciation make it difficult to correctly classify these vectors. During the taxonomic history of the genus, five synonymization events occurred. Additionally, some authors suggest that R. milesi possibly represent only phenotypic polymorphisms of R. neglectus. Thus, we analyzed the specific status of R. milesi in relation to R. neglectus using phylogenetic studies with the mitochondrial gene cytochrome B and the study of reproductive barriers. The phylogenetic reconstruction grouped R. milesi together with R. neglectus from different localities, demonstrating that these taxa represent the same species based on the phylogenetic species concept. Experimental crosses demonstrate the absence of pre- and postzygotic barriers under laboratory conditions. Additionally, when the hatch rates of crosses are compared to intraspecific crosses, it can be noted that they are high and very similar. Finally, the mortality rate of the hybrids does not indicate hybrid inviability, the absence of chromosome pairing errors does not indicate hybrid sterility, and the proportion between male and female hybrids demonstrates that Haldane’s rule was not acting. Therefore, we perform the formal synonymization of R. milesi with R. neglectus.

Seed quality and germination performance increase with cross-pollination in members of subtribe Orchidinae (Orchidaceae)

Abstract Over half of angiosperms have self-incompatibility systems to avoid self-pollination and inbreeding depression. Many members of Orchidaceae are self-compatible, but they may be prone to negative consequences of self-pollination. We investigated the effects of both within-flower (autogamy) and within-plant (geitonogamy) self-pollination, and cross-pollination on fruit set, seed quality, and germination rates in 11 related orchid taxa of subtribe Orchidinae. We hand-pollinated flowers and measured fruit set to estimate pre- and postzygotic barriers. We collected morphometric data on embryos and seeds and calculated the percentage of seeds with embryos and seed viability. We then calculated inbreeding depression values for each treatment for each taxon. Ten taxa produced fruits when self-pollinated, but germination rates and seed size increased with cross-pollination. Higher seed viability was correlated with larger embryos/seeds. However, geitonogamous pollination generally resulted in the lowest seed quality compared with autogamy and cross-pollination, with high values of inbreeding depression being detected. Only seven of the 11 taxa (Anacamptis papilionacea, Himantoglossum adriaticum, Ophrys apifera, Ophrys bertolonii, Orchis patens subsp. brevicornis, Orchis provincialis, and Serapias vomeracea) were confirmed as self-compatible. These results show that cross-pollination improves overall seed quality, and that the assessment of self-compatibility in orchids should be based on postfruiting stages.

Maintenance of a narrow hybrid zone between native and introduced red foxes (Vulpes vulpes) despite conspecificity and high dispersal capabilities.

Human-facilitated introductions of nonnative populations can lead to secondary contact between allopatric lineages, resulting in lineage homogenisation or the formation of stable hybrid zones maintained by reproductive barriers. We investigated patterns of gene flow between the native Sacramento Valley red fox (Vulpes vulpes patwin) and introduced conspecifics of captive-bred origin in California's Central Valley. Considering their recent divergence (20-70 kya), we hypothesised that any observed barriers to gene flow were primarily driven by pre-zygotic (e.g. behavioural differences) rather than post-zygotic (e.g. reduced hybrid fitness) barriers. We also explored whether nonnative genes could confer higher fitness in the human-dominated landscape resulting in selective introgression into the native population. Genetic analysis of red foxes (n = 682) at both mitochondrial (cytochrome b + D-loop) and nuclear (19,051 SNPs) loci revealed narrower cline widths than expected under a simulated model of unrestricted gene flow, consistent with the existence of reproductive barriers. We identified several loci with reduced introgression that were previously linked to behavioural divergence in captive-bred and domestic canids, supporting pre-zygotic, yet possibly hereditary, barriers as a mechanism driving the narrowness and stability of the hybrid zone. Several loci with elevated gene flow from the nonnative into the native population were linked to genes associated with domestication and adaptation to human-dominated landscapes. This study contributes to our understanding of hybridisation dynamics in vertebrates, particularly in the context of species introductions and landscape changes, underscoring the importance of considering how multiple mechanisms may be maintaining lineages at the species and subspecies level.

Heteroploid F1 hybrids generated from Coffea canephora × C. arabica interploidy crossing

Abstract Distant hybrids between two coffee species was considered promising for breeders to select novel genotypes highly resilient to changing global climate, despite pre- and or post-zygotic barriers. However, the different ploidy between C. arabica (2n=4x=44) and its diploid counterparts was another challenging barrier. The expected ploidy level for developed F1 hybrid was triploid. This study was aimed to identify ploidy level of F1 hybrids generated from Coffea canephora × C. arabica interploidy crossing. Ploidy determination was held using indirect methods, i.e. based on morphometric analysis of pollen and stomatal guard cells. Polar and equatorial diameter of 300 freshly collected pollen from each of six individuals of F1 hybrids were measured using ImageJ software. Stomatal frequency was measured as the number of guard cells on leaf area of 1 mm2. Pollen and stomatal guard cells of their diploid maternal and tetraploid paternal were also measured to be used as a standard for ploidy level determination. Generated datasets were immediately analysis statistically using t-Test procedure. The results shows that four of six F1 hybrid individuals had similar pollen size as well as stomatal density to diploid maternal. Meanwhile, another single individual was similar to tetraploid paternal. However, the rest of single individuals were similar to both of maternal and paternal. Based on those results, it could be inferred that there weres three different ploidy levels among F1 hybrid individuals, namely diploid, triploid, and tetraploid.

Ecological divergence despite common mating sites: Genotypes and symbiotypes shed light on cryptic diversity in the black bean aphid species complex.

Different host plants represent ecologically dissimilar environments for phytophagous insects. The resulting divergent selection can promote the evolution of specialized host races, provided that gene flow is reduced between populations feeding on different plants. In black bean aphids belonging to the Aphis fabae complex, several morphologically cryptic taxa have been described based on their distinct host plant preferences. However, host choice and mate choice are largely decoupled in these insects: they are host-alternating and migrate between specific summer host plants and shared winter hosts, with mating occurring on the shared hosts. This provides a yearly opportunity for gene flow among aphids using different summer hosts, and raises the question if and to what extent the ecologically defined taxa are reproductively isolated. Here, we analyzed a geographically and temporally structured dataset of microsatellite genotypes from A. fabae that were mostly collected from their main winter host Euonymus europaeus, and additionally from another winter host and fourteen summer hosts. The data reveals multiple, strongly differentiated genetic clusters, which differ in their association with different summer and winter hosts. The clusters also differ in the frequency of infection with two heritable, facultative endosymbionts, separately hinting at reproductive isolation and divergent ecological selection. Furthermore, we found evidence for occasional hybridization among genetic clusters, with putative hybrids collected more frequently in spring than in autumn. This suggests that similar to host races in other phytophagous insects, both prezygotic and postzygotic barriers including selection against hybrids maintain genetic differentiation among A. fabae taxa, despite a common mating habitat.

Extreme elevational migration spurred cryptic speciation in giant hummingbirds

The ecoevolutionary drivers of species niche expansion or contraction are critical for biodiversity but challenging to infer. Niche expansion may be promoted by local adaptation or constrained by physiological performance trade-offs. For birds, evolutionary shifts in migratory behavior permit the broadening of the climatic niche by expansion into varied, seasonal environments. Broader niches can be short-lived if diversifying selection and geography promote speciation and niche subdivision across climatic gradients. To illuminate niche breadth dynamics, we can ask how "outlier" species defy constraints. Of the 363 hummingbird species, the giant hummingbird (Patagona gigas) has the broadest climatic niche by a large margin. To test the roles of migratory behavior, performance trade-offs, and genetic structure in maintaining its exceptional niche breadth, we studied its movements, respiratory traits, and population genomics. Satellite and light-level geolocator tracks revealed an >8,300-km loop migration over the Central Andean Plateau. This migration included a 3-wk, ~4,100-m ascent punctuated by upward bursts and pauses, resembling the acclimatization routines of human mountain climbers, and accompanied by surging blood-hemoglobin concentrations. Extreme migration was accompanied by deep genomic divergence from high-elevation resident populations, with decisive postzygotic barriers to gene flow. The two forms occur side-by-side but differ almost imperceptibly in size, plumage, and respiratory traits. The high-elevation resident taxon is the world's largest hummingbird, a previously undiscovered species that we describe and name here. The giant hummingbirds demonstrate evolutionary limits on niche breadth: when the ancestral niche expanded due to evolution (or loss) of an extreme migratory behavior, speciation followed.

Comparative karyotype analysis of the red brocket deer (M. americana sensu lato and M. rufa) complex: evidence of drastic chromosomal evolution and implications on speciation process.

Chromosomal rearrangements are often associated with playing a role in the speciation process. However, the underlying mechanism that favors the genetic isolation associated with chromosomal changes remains elusive. In this sense, the genus Mazama is recognized by its high level of karyotype diversity among species with similar morphology. A cryptic species complex has been identified within the genus, with the red brocket deer (Mazama americana and Mazama rufa) being the most impressive example. The chromosome variation was clustered in cytotypes with diploid numbers ranging from 42 to 53 and was correlated with geographical location. We conducted an analysis of chromosome evolution of the red brocket deer complex using comparative chromosome painting and Bacterial Artificial Chromosome (BAC) clones among different cytotypes. The aim was to deepen our understanding of the karyotypic relationships within the red brocket, thereby elucidating the significant chromosome variation among closely related species. This underscores the significance of chromosome changes as a key evolutionary process shaping their genomes. The results revealed the presence of three distinct cytogenetic lineages characterized by significant karyotypic divergence, suggesting the existence of efficient post-zygotic barriers. Tandem fusions constitute the main mechanism driving karyotype evolution, following a few centric fusions, inversion X-autosomal fusions. The BAC mapping has improved our comprehension of the karyotypic relationships within the red brocket deer complex, prompting questions regarding the role of these changes in the speciation process. We propose the red brocket as a model group to investigate how chromosomal changes contribute to isolation and explore the implications of these changes in taxonomy and conservation.

Do genetic loci that cause reproductive isolation in the lab inhibit gene flow in nature?

The genetic dissection of reproductive barriers between diverging lineages provides enticing clues into the origin of species. One strategy uses linkage analysis in experimental crosses to identify genomic locations involved in phenotypes that mediate reproductive isolation. A second framework searches for genomic regions that show reduced rates of exchange across natural hybrid zones. It is often assumed that these approaches will point to the same loci, but this assumption is rarely tested. In this perspective, we discuss the factors that determine whether loci connected to postzygotic reproductive barriers in the laboratory are inferred to reduce gene flow in nature. We synthesize data on the genetics of postzygotic isolation in house mice, one of the most intensively studied systems in speciation genetics. In a rare empirical comparison, we measure the correspondence of loci tied to postzygotic barriers via genetic mapping in the laboratory and loci at which gene flow is inhibited across a natural hybrid zone. We find no evidence that the two sets of loci overlap beyond what is expected by chance. In light of these results, we recommend avenues for empirical and theoretical research to resolve the potential incongruence between the two predominant strategies for understanding the genetics of speciation.

Temperature and sex shape reproductive barriers in a climate change hotspot.

Climate change is altering species ranges and reproductive interactions in existing ranges, offering species new scope to mate and hybridize. The outcomes will depend on how environmental factors shape reproductive barriers across life stages, yet this is rarely assessed across the environments that species encounter in nature. We assess prezygotic and postzygotic barriers, and their dependence on temperature and parental sex, in species of a reef-building tubeworm (Galeolaria) from a fast-warming biodiversity hotspot in southern Australia. By replicating pure and reciprocal hybrid crosses across 5 temperatures spanning species' thermal ranges, we estimate thermal tolerance curves (defining niches) for crosses and reproductive isolation at each temperature. By also replicating crosses at 3 life stages, we partition the contributions of prezygotic barriers at fertilization, postzygotic barriers at embryogenesis, and postzygotic barriers at larval development to reproductive isolation. We show that barriers are weaker at fertilization and embryogenesis, but stronger and more temperature sensitive at larval development, as species diverge in thermal niche. Asymmetry of barriers between parental sexes, moreover, suggests a complex interplay between niche differentiation and maternal inheritance. Our findings point to a key role for temperature in reproductive isolation, but also challenges for predicting the fate of isolation in future climates.

Ecological disruptive selection acting on quantitative loci can drive sympatric speciation

The process of speciation generates biodiversity. According to the null model of speciation, barriers between populations arise in allopatry, where, prior to biology, geography imposes barriers to gene flow. On the other hand, sympatric speciation requires that the process of speciation happen in the absence of a geographical barrier, where the members of the population have no spatial, temporal barriers. Several attempts have been made to theoretically identify the conditions in which speciation can occur in sympatry. However, these efforts suffer from several limitations. We propose a model for sympatric speciation based on adaptation for resource utilization. We use a genetics-based model to investigate the relative roles of prezygotic and postzygotic barriers, from the context of ecological disruptive selection, sexual selection, and genetic architecture, in causing and maintaining sympatric speciation. Our results show that sexual selection that acts on secondary sexual traits does not play any role in the process of speciation in sympatry and that assortative mating based on an ecologically relevant trait forces the population to show an adaptive response. We also demonstrate that understanding the genetic architecture of the trait under ecological selection is very important and that it is not required for the strength of ecological disruptive selection to be very high in order for speciation to occur in sympatry. Our results provide an insight into the kind of scenarios in which sympatric speciation can be demonstrated in the lab.

Postzygotic barriers persist despite ongoing introgression in hybridizing Mimulus species.

The evolution of postzygotic isolation is thought to be a key step in maintaining species boundaries upon secondary contact, yet the dynamics and persistence of hybrid incompatibilities in naturally hybridizing species are not well understood. Here, we explore these issues using genetic mapping in three independent populations of recombinant inbred lines between naturally hybridizing monkeyflowers, Mimulus guttatus and Mimulus nasutus, from the sympatric Catherine Creek population. We discover that the three M. guttatus founders differ dramatically in admixture history, with nearly a quarter of one founder's genome introgressed from M. nasutus. Comparative genetic mapping in the three RIL populations reveals three new putative inversions, each one segregating among the M. guttatus founders, two due to admixture. We find strong, genome-wide transmission ratio distortion in all RILs, but patterns are highly variable among the three populations. At least some of this distortion appears to be explained by epistatic selection favouring parental genotypes, but tests of inter-chromosomal linkage disequilibrium also reveal multiple candidate Dobzhansky-Muller incompatibilities. We also map several genetic loci for hybrid pollen viability, including two interacting pairs that coincide with peaks of distortion. Remarkably, even with this limited sample of three M. guttatus lines, we discover abundant segregating variation for hybrid incompatibilities with M. nasutus, suggesting this population harbours diverse contributors to postzygotic isolation. Moreover, even with substantial admixture, hybrid incompatibilities between Mimulus species persist, suggesting postzygotic isolation might be a potent force in maintaining species barriers in this system.

Partial and asymmetrical reproductive isolation between two sympatric tropical shrub species: Cnidoscolus aconitifolius and C. souzae (Euphorbiaceae).

Reproductive isolation is conferred by several barriers that occur at different stages of reproduction. Comprehensive reviews on the topic have identified that barriers occurring prior to zygote formation are often stronger than those that occur afterward. However, the overrepresentation of temperate perennial herbs in the current literature precludes any generalization of this pattern to plants that present other life forms and patterns of distribution. Here, we assessed reproductive isolation barriers and their absolute contribution to reproductive isolation and asymmetry in Cnidoscolus aconitifolius and C. souzae, two closely related tropical shrub species that co-occur on the Yucatan peninsula. The reproductive barriers assessed were phenological mismatch, pollinator differentiation, pollen-pistil incompatibility (three pre-zygotic barriers), fruit set failure, and seed unviability (post-zygotic barriers). Reproductive isolation between the study species was found to be complete in the direction C. aconitifolius to C. souzae, but only partial in the opposite direction. One post-zygotic barrier was the strongest example. Most barriers, particularly the pre-zygotic examples, were asymmetrical and predicted the direction of heterospecific pollen flow and hybrid formation from C. souzae to C. aconitifolius. Both parental species, as well as the hybrids, were diploid and had a chromosome number 2n = 36. More studies with tropical woody perennials are required to fully determine whether this group of plants consistently shows stronger post-zygotic barriers.

Tracking hybrid viability across life stages in a natural avian contact zone.

Hybrid inviability is an important post-zygotic reproductive barrier between species, but emerging signs of reduced viability can be difficult to study across the lifespan of natural hybrids. We use a combination of long-term monitoring, extra-pair paternity, and mitochondrial DNA identification in a natural hybrid zone of Ficedula flycatchers to detect emerging signs of intrinsic hybrid inviability across their entire lifespan. We evaluate possible evidence of Darwin's corollary to Haldane's rule, predicting asymmetries in inviability between hybrids resulting from reciprocal crosses, due to incompatible genetic factors with sex-specific inheritance patterns. We found higher hatching failure among mixed-species pairs, possibly indicating early developmental impairments associated with specific parental genetic combinations. Adult hybrids had a higher basal mortality rate than both parental species and different age-specific mortality trajectories. There were signs of differences in age-independent mortality rates between the reciprocal hybrid crosses: hybrids with a pied flycatcher mother experienced slightly increased mortality later in life. Using an exceptional dataset with many natural hybrids tracked across life stages, we provide evidence for several emerging signs of reduced hybrid viability. Incompatibilities between alleles located on autosomes and uniparentally inherited factors such as Z-linked and/or mitochondrial genes are strong candidates underlying intrinsic hybrid dysfunction in this system.

Genetic enhancement of okra [Abelmoschus esculentus (L.) Moench] germplasm through wide hybridization.

The introgression of genetic material from one species to another through wide hybridization and repeated back-crossing, plays an important role in genetic modification and enriching the cultivated gene-pool with novel genetic variations. Okra (Abelmoschus esculentus [(L.) Moench)] is a popular vegetable crop with high dietary fibre and protein, rich in essential amino acids, lysine and tryptophan. The wild Abelmoschus genepool has many desirable traits like ornamental value, short internodal length, more number of productive branches, extended bearing, perennation tendency, reduced fruit length (more consumer preferred trait), high mucilage content (medicinal value), abiotic stress tolerances such as drought, high temperature and biotic stress resistances such as okra Yellow Vein Mosaic Virus (YVMV) and Enation Leaf Curl Virus (ELCV) diseases. The repeated use of elite breeding lines led to narrowing of the genetic base of the okra crop, one of the major factors attributed to breakdown of resistance/ tolerance to biotic stresses. YVMV and ELCV are the two major diseases, causing significant yield loss in okra. Hence, wide hybridization was attempted to transfer tolerance genes from wild species to the cultivated genepool to widen the genetic base. The screening of germplasm of wild Abelmoschus species at hotspots led to the identification of tolerant species (Abelmoschus pungens var. mizoramensis, A. enbeepeegeearensis, A. caillei, A. tetraphyllus and A. angulosus var. grandiflorus), which were further used in a wide-hybridization programme to generate interspecific hybrids with the cultivated okra. Presence of pre- and post-zygotic barriers to interspecific geneflow, differences in ploidy levels and genotype specific variations in chromosome numbers led to varying degrees of sterility in F1 plants of interspecific crosses. This was overcome by doubling the chromosome number of interspecific hybrids by applying Colchicine at the seedling stage. The 113 cross derivatives generated comprising amphidiploids in the F1 generation (30), F3 (14), one each in F2 and F4 generations, back cross generation in BC1F2 (03), BC1F3 (25), and BC2F3 (02), crosses between amphidiploids (27), multi-cross combinations (07) and inter-specific cross (between A. sagittifolius × A. moschatus subsp. moschatus) selfed derivatives at F8 generation (03) were characterized in the present study. Besides they were advanced through selfing and backcrossing. The amphidiploids were found to possess many desirable genes with a considerable magnitude of linkage drag. Majority of the wide cross derivatives had an intermediate fruit morphology and dominance of wild characters viz., hispid fruits, stem, leaves, tough fruit fibre, vigorous perennial growth habit and prolonged flowering and fruiting. The fruit morphology of three BC progenies exhibited a high morphological resemblance to the cultivated okra, confirming successful transfer of useful genes to the cultivated okra genepool. The detailed morphological characteristics of the various combinations of Abelmoschus amphidiploids and the genetic enhancement of the genepool achieved in this process is reported here.

Sperm-dependent asexual species and their role in ecology and evolution.

Sexual reproduction is the primary mode of reproduction in eukaryotes, but some organisms have evolved deviations from classical sex and switched to asexuality. These asexual lineages have sometimes been viewed as evolutionary dead ends, but recent research has revealed their importance in many areas of general biology. Our review explores the understudied, yet important mechanisms by which sperm-dependent asexuals that produce non-recombined gametes but rely on their fertilization, can have a significant impact on the evolution of coexisting sexual species and ecosystems. These impacts are concentrated around three major fields. Firstly, sperm-dependent asexuals can potentially impact the gene pool of coexisting sexual species by either restricting their population sizes or by providing bridges for interspecific gene flow whose type and consequences substantially differ from gene flow mechanisms expected under sexual reproduction. Secondly, they may impact on sexuals' diversification rates either directly, by serving as stepping-stones in speciation, or indirectly, by promoting the formation of pre- and postzygotic reproduction barriers among nascent species. Thirdly, they can potentially impact on spatial distribution of species, via direct or indirect (apparent) types of competition and Allee effects. For each such mechanism, we provide empirical examples of how natural sperm-dependent asexuals impact the evolution of their sexual counterparts. In particular, we highlight that these broad effects may last beyond the tenure of the individual asexual lineages causing them, which challenges the traditional perception that asexual lineages are short-lived evolutionary dead ends and minor sideshows. Our review also proposes new research directions to incorporate the aforementioned impacts of sperm-dependent asexuals. These research directions will ultimately enhance our understanding of the evolution of genomes and biological interactions in general.

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.

New species based on the biological species concept within the complex of Lariophagus distinguendus (Hymenoptera, Chalcidoidea, Pteromalidae), a parasitoid of household pests.

The pteromalid parasitoid Lariophagus distinguendus (Foerster) belongs to the Hymenoptera, a megadiverse insect order with high cryptic diversity. It attacks stored product pest beetles in human storage facilities. Recently, it has been shown to consist of two separate species. To further study its cryptic diversity, strains were collected to compare their relatedness using barcoding and nuclear genes. Nuclear genes identified two clusters which agree with the known two species, whereas the barcode fragment determined an additional third Clade. Total reproductive isolation (RI) according to the biological species concept (BSC) was investigated in crossing experiments within and between clusters using representative strains. Sexual isolation exists between all studied pairs, increasing from slight to strong with genetic distance. Postzygotic barriers mostly affected hybrid males, pointing to Haldane's rule. Hybrid females were only affected by unidirectional Spiroplasma-induced cytoplasmic incompatibility and behavioural sterility, each in one specific strain combination. RI was virtually absent between strains separated by up to 2.8% COI difference, but strong or complete in three pairs from one Clade each, separated by at least 7.2%. Apparently, each of these clusters represents one separate species according to the BSC, highlighting cryptic diversity in direct vicinity to humans. In addition, these results challenge the recent 'turbo-taxonomy' practice of using 2% COI differences to delimitate species, especially within parasitic Hymenoptera. The gradual increase in number and strength of reproductive barriers between strains with increasing genetic distance also sheds light on the emergence of barriers during the speciation process in L. distinguendus.

Embryo Rescue in Plant Breeding.

Embryo rescue (ER) techniques are among the oldest and most successful in vitro tissue culture protocols used with plant species. ER refers to a series of methods that promote the development of an immature or lethal embryo into a viable plant. Intraspecific, interspecific, or intergeneric crosses allow the introgression of important alleles of agricultural interest from wild species, such as resistance or tolerance to abiotic and biotic stresses or morphological traits in crops. However, pre-zygotic and post-zygotic reproductive barriers often present challenges in achieving successful hybridization. Pre-zygotic barriers manifest as incompatibility reactions that hinder pollen germination, pollen tube growth, or penetration into the ovule occurring in various tissues, such as the stigma, style, or ovary. To overcome these barriers, several strategies are employed, including cut-style or graft-on-style techniques, the utilization of mixed pollen from distinct species, placenta pollination, and in vitro ovule pollination. On the other hand, post-zygotic barriers act at different tissues and stages ranging from early embryo development to the subsequent growth and reproduction of the offspring. Many crosses among different genera result in embryo abortion due to the failure of endosperm development. In such cases, ER techniques are needed to rescue these hybrids. ER holds great promise for not only facilitating successful crosses but also for obtaining haploids, doubled haploids, and manipulating the ploidy levels for chromosome engineering by monosomic and disomic addition as well substitution lines. Furthermore, ER can be used to shorten the reproductive cycle and for the propagation of rare plants. Additionally, it has been repeatedly used to study the stages of embryonic development, especially in embryo-lethal mutants. The most widely used ER procedure is the culture of immature embryos taken and placed directly on culture media. In certain cases, the in vitro culture of ovule, ovaries or placentas enables the successful development of young embryos from the zygote stage to maturity.