HYBRID BREAKDOWN BETWEEN TWO HAPLODIPLOID SPECIES: THE ROLE OF NUCLEAR AND CYTOPLASMIC GENES

A central question in evolutionary biology concerns the population and genetic processes by which new species arise. Here, the genetic basis of hybrid breakdown between two haplodiploid species, Nasonia vitripennis and N. giraulti is investigated. Hybridization between the two species is normally prevented by microorganisms that cause bidirectional incompatibility. However, after elimination of microorganisms, F₁ hybrids females are readily produced (due to haplodiploidy, males develop from unfertilized eggs and are therefore not hybrids). F₁ hybrid females are viable and fecund, but recombinant (haploid) F₂ male offspring suffer from severe hybrid breakdown (larval and pupal mortality). This is typically interpreted as evidence for the existence of different coadapted gene complexes in the two species, which are broken up by recombination. F₂ recombinant eggs were rescued by fertilization with the complete chromosome complement from either species, supporting the view that hybrid lethality genes tend to be recessive. Negative epistatic interactions occur between nuclear genes of the two species, and between cytoplasmically inherited factors (cytoplasmic genes) of giraulti and nuclear genes of vitripennis. Interactions between nuclear genes and cytoplasmic genes are asymmetric. Experiments clearly demonstrate that the latter incompatibility is not due to maternal-effect genes, but to cytoplasmically inherited elements. Nuclear-mitochondrial interactions are possibly involved.     

THE INTERSPECIFIC ORIGIN OF B CHROMOSOMES: EXPERIMENTAL EVIDENCE

Abstract.— A centric fragment was generated during the introgression of a chromosome region from Nasonia giraulti into N. vitripennis. This neo B chromosome carries the N. giraulti or 123⁺ gene for wild‐type eye color. Using this phenotypic effect, the transmission of this chromosome was analyzed. The supernumerary chromosome showed less than Mendelian segregation rate in meiosis and some mitotic instability manifested as mosaic phenotype for eye color. However, transmission rate and mitotic stability increased over successive generations. The transmission rate through male gametogenesis was nearly 100%. These results support the interspecific hybridization model for B chromosome origin and reveal that problems in chromosome stability can persist for several generations after “foreign chromosomes” are introduced into a different species. We suggest that hybrid zones should be investigated as possible sites for neo‐B chromosome generation.     

Eleven microsatellite markers in Nasonia,Ashmead 1904 (Hymenoptera; Pteromalidae)

We designed primer sequences for 11 microsatellite markers in the jewel wasp Nasonia vitripennis. Most loci could be cross‐amplified in Nasonia longicornis and Nasonia giraulti, which make them amenable for linkage analysis in hybrid crosses. Eight loci were assigned to specific chromosomes. Additionally, 10 loci showed allelic variation in a Nasonia vitripennis field population. The observed number of alleles in this population ranged from two to seven, with observed heterozygosities from 0.0750 to 0.4750.     

Temperature stress increases hybrid incompatibilities in the parasitic wasp genus Nasonia

Hybrid incompatibilities, measured as mortality and sterility, are caused by the disruption of gene interactions. They are important post-zygotic isolation barriers to species hybridization, and much effort is put into the discovery of the genes underlying these incompatibilities. In hybridization studies of the haplodiploid parasitic wasp genus Nasonia, genic incompatibilities have been shown to affect mortality and sterility. The genomic regions associated with mortality have been found to depend on the cytotype of the hybrids and thus suggest cytonuclear incompatibilities. As environmental conditions can affect gene expression and gene interaction, we here investigate the effect of developmental temperature on sterility and mortality in Nasonia hybrids. Results show that extreme temperatures strongly affect both hybrid sterility (mainly spermatogenic failure) and mortality. Molecular mapping revealed that extreme temperatures increase transmission ratio distortion of parental alleles at incompatible loci, and thus, cryptic incompatible loci surface under temperature stress that remain undiscovered under standard temperatures. Our results underline the sensitivity of hybrid incompatibilities to environmental factors and the effects of unstable epistasis.     

When nuclear‐encoded proteins and mitochondrial RNAs do not get along,species split apart

The emergence of barriers to reproduction between two populations is one of the most important features of speciation. Among the mechanisms of reproductive isolation are incompatible interactions between gene products of the parental species that reduce the fitness of hybrid individuals. The accumulation of such incompatibilities is described by the Bateson–Dobzhansky–Muller model (BDM) 1 that provides a framework for understanding how genes can coevolve to stay compatible within populations and become incompatible between populations. Only a handful of such loci have been identified and characterized at the molecular level. In this issue of EMBO Reports, Jhuang and colleagues 2 show that BDM incompatibilities have accumulated between a nuclear‐encoded gene and a mitochondrial ribosomal RNA between two yeast species.     

Reproductive strategies under multiparasitism in natural populations of the parasitoid wasp Nasonia (Hymenoptera)

Parasitoid Nasonia wasps adjust their progeny sex ratio to the presence of conspecifics to optimize their fitness. Another trait under female control is the induction of offspring diapause. We analysed progeny sex ratios and the proportion of diapausing offspring of individual Nasonia females in host patches parasitized by two species, Nasonia vitripennis and Nasonia giraulti, in North American field populations using microsatellite fingerprinting. Both Nasonia species produced similar sex ratios on hosts that were co‐parasitized by their own species as by the other species, indicating that females do not distinguish between con‐ and heterospecific clutches. The sex ratios of the diapause and adult fractions of mixed broods from single females were not correlated. We found further indications that N. vitripennis females take the emergence time of the offspring into account in their sex allocation. The reproductive strategies of Nasonia under multiparasitism are largely adaptive, but also partially constrained by information.     

Homoploid hybrid origin of Yucca gloriosa: intersectional hybrid speciation in Yucca (Agavoideae,Asparagaceae)

There is a growing appreciation for the importance of hybrid speciation in angiosperm evolution. Here, we show that Yucca gloriosa (Asparagaceae: Agavoideae) is the product of intersectional hybridization between Y. aloifolia and Y. filamentosa. These species, all named by Carl Linnaeus, exist in sympatry along the southeastern Atlantic coast of the United States. Yucca gloriosa was found to share a chloroplast haplotype with Y. aloifolia in all populations sampled. In contrast, nuclear gene‐based microsatellite markers in Y. gloriosa are shared with both parents. The hybrid origin of Y. gloriosa is supported by multilocus analyses of the nuclear microsatellite markers including principal coordinates analysis (PCO), maximum‐likelihood hybrid index scoring (HINDEX), and Bayesian cluster analysis (STRUCTURE). The putative parental species share only one allele at a single locus, suggesting there is little to no introgressive gene flow occurring between these species and Y. gloriosa. At the same time, diagnostic markers are segregating in Y. gloriosa populations. Lack of variation in the chloroplast of Y. aloifolia, the putative maternal parent, makes it difficult to rule out multiple hybrid origins of Y. gloriosa, but allelic variation at nuclear loci can be explained by a single hybrid origin of Y. gloriosa. Overall, these data provide strong support for the homoploid hybrid origin of Y. gloriosa.     

High‐throughput olfactory conditioning and memory retention test show variation in Nasonia parasitic wasps

Most of our knowledge on learning and memory formation results from extensive studies on a small number of animal species. Although features and cellular pathways of learning and memory are highly similar in this diverse group of species, there are also subtle differences. Closely related species of parasitic wasps display substantial variation in memory dynamics and can be instrumental to understanding both the adaptive benefit of and mechanisms underlying this variation. Parasitic wasps of the genus Nasonia offer excellent opportunities for multidisciplinary research on this topic. Genetic and genomic resources available for Nasonia are unrivaled among parasitic wasps, providing tools for genetic dissection of mechanisms that cause differences in learning. This study presents a robust, high‐throughput method for olfactory conditioning of Nasonia using a host encounter as reward. A T‐maze olfactometer facilitates high‐throughput memory retention testing and employs standardized odors of equal detectability, as quantified by electroantennogram recordings. Using this setup, differences in memory retention between Nasonia species were shown. In both Nasonia vitripennis and Nasonia longicornis, memory was observed up to at least 5 days after a single conditioning trial, whereas Nasonia giraulti lost its memory after 2 days. This difference in learning may be an adaptation to species‐specific differences in ecological factors, for example, host preference. The high‐throughput methods for conditioning and memory retention testing are essential tools to study both ultimate and proximate factors that cause variation in learning and memory formation in Nasonia and other parasitic wasp species.     

Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA Methylation in Nasonia

Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia.     

Fast‐forward genetics by radiation hybrids to saturate the locus regulating nuclear–cytoplasmic compatibility in Triticum

The nuclear‐encoded species cytoplasm specific (scs) genes control nuclear–cytoplasmic compatibility in wheat (genus Triticum). Alloplasmic cells, which have nucleus and cytoplasm derived from different species, produce vigorous and vital organisms only when the correct version of scs is present in their nucleus. In this study, bulks of in vivo radiation hybrids segregating for the scs phenotype have been genotyped by sequencing with over 1.9 million markers. The high marker saturation obtained for a critical region of chromosome 1D allowed identification of 3318 reads that mapped in close proximity of the scs. A novel in silico approach was deployed to extend these short reads to sequences of up to 70 Kb in length and identify candidate open reading frames (ORFs). Markers were developed to anchor the short contigs containing ORFs to a radiation hybrid map of 650 individuals with resolution of 288 Kb. The region containing the scs locus was narrowed to a single Bacterial Artificial Chromosome (BAC) contig of Aegilops tauschii. Its sequencing and assembly by nano‐mapping allowed rapid identification of a rhomboid gene as the only ORF existing within the refined scs locus. Resequencing of this gene from multiple germplasm sources identified a single nucleotide mutation, which gives rise to a functional amino acid change. Gene expression characterization revealed that an active copy of this rhomboid exists on all homoeologous chromosomes of wheat, and depending on the specific cytoplasm each copy is preferentially expressed. Therefore, a new methodology was applied to unique genetic stocks to rapidly identify a strong candidate gene for the control of nuclear–cytoplasmic compatibility in wheat.     

HYBRIDIZATION LEADS TO SENSORY REPERTOIRE EXPANSION IN A GYNOGENETIC FISH,THE AMAZON MOLLY (POECILIA FORMOSA): A TEST OF THE HYBRID‐SENSORY EXPANSION HYPOTHESIS

Expansions in sensory systems usually require processes such as gene duplication and divergence, and thus evolve slowly. We evaluate a novel mechanism leading to rapid sensory repertoire expansion: hybrid‐sensory expansion (HSE). HSE occurs when two species with differently tuned sensory systems form a hybrid, bringing together alleles from each of the parental species. In one generation, a sensory repertoire is created that is the sum of the variance between parental species. The Amazon molly presents a unique opportunity to test the HSE hypothesis in a “frozen” hybrid. We compared opsin sequences of the Amazon molly, Poecilia formosa, to those of the parental species. Both parental species are homozygous at the RH2–1 locus and each of the four long wavelength sensitive loci, while P. formosa possess two different alleles at these loci; one matching each parental allele. Gene expression analysis showed P. formosa use the expanded opsin repertoire that was the result of HSE. Additionally, behavioral tests revealed P. formosa respond to colored stimuli in a manner similar or intermediate to the parental species P. mexicana and P. latipinna. Together these results strongly support the HSE hypothesis. Hybrid‐sensory repertoire expansion is likely important in other hybrid species and in other sensory systems.     

Segregation of male‐sterility alleles across a species boundary

Hybrid zones may serve as bridges permitting gene flow between species, including alleles influencing the evolution of breeding systems. Using greenhouse crosses, we assessed the likelihood that a hybrid zone could serve as a conduit for transfer of nuclear male‐sterility alleles between a gynodioecious species and a hermaphroditic species with very rare females in some populations. Segregation patterns in progeny of crosses between rare females of hermaphroditic Schiedea menziesii and hermaphroditic plants of gynodioecious Schiedea salicaria heterozygous at the male‐sterility locus, and between female S. salicaria and hermaphroditic plants from the hybrid zone, were used to determine whether male‐sterility was controlled at the same locus in the parental species and the hybrid zone. Segregations of females and hermaphrodites in approximately equal ratios from many of the crosses indicate that the same nuclear male‐sterility allele occurs in the parent species and the hybrid zone. These rare male‐sterility alleles in S. menziesii may result from gene flow from S. salicaria through the hybrid zone, presumably facilitated by wind pollination in S. salicaria. Alternatively, rare male‐sterility alleles might result from a reversal from gynodioecy to hermaphroditism in S. menziesii, or possibly de novo evolution of male sterility. Phylogenetic analysis indicates that some species of Schiedea have probably evolved separate sexes independently, but not in the lineage containing S. salicaria and S. menziesii. High levels of selfing and expression of strong inbreeding depression in S. menziesii, which together should favour females in populations, argue against a reversal from gynodioecy to hermaphroditism in S. menziesii.     

A multi‐locus approach resolves the phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups in Malaysia: evidence for distinct evolutionary lineages

A multi‐locus approach was used to examine the DNA sequences of 10 nominal species of blackfly in the Simulium subgenus Gomphostilbia (Diptera: Simuliidae) in Malaysia. Molecular data were acquired from partial DNA sequences of the mitochondria‐encoded cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA genes, and the nuclear‐encoded 18S rRNA and 28S rRNA genes. No single gene, nor the concatenated gene set, resolved all species or all relationships. However, all morphologically established species were supported by at least one gene. The multi‐locus sequence analysis revealed two distinct evolutionary lineages, conforming to the morphotaxonomically recognized Simulium asakoae and Simulium ceylonicum species groups.     

Recombinant hybrids retain heterozygosity at many loci: new insights into the genomics of reproductive isolation in Populus

The maintenance of species barriers in the face of gene flow is often thought to result from strong selection against intermediate genotypes, thereby preserving genetic differentiation. Most speciation genomic studies thus aim to identify exceptionally divergent loci between populations, but divergence will be affected by many processes other than reproductive isolation (RI) and speciation. Through genomic studies of recombinant hybrids sampled in the wild, genetic variation associated with RI can be observed in situ, because selection against incompatible genotypes will leave detectable patterns of variation in the hybrid genomes. To better understand the mechanisms directly involved in RI, we investigated three natural ‘replicate’ hybrid zones between two divergent Populus species via locus‐specific patterns of ancestry across recombinant hybrid genomes. As expected, genomic patterns in hybrids and their parental species were consistent with the presence of underdominant selection at several genomic regions. Surprisingly, many loci displayed greatly increased between‐species heterozygosity in recombinant hybrids despite striking genetic differentiation between the parental genomes, the opposite of what would be expected with selection against intermediate genotypes. Only a limited, reproducible set of genotypic combinations was present in hybrid genomes across localities. In the absence of clearly delimited ‘hybrid habitats’, our results suggest that complex epistatic interactions within genomes play an important role in advanced stages of RI between these ecologically divergent forest trees. This calls for more genomic studies that test for unusual patterns of genomic ancestry in hybridizing species.     

MITOTIC INSTABILITY IN HAPLOPAPPUS: STRUCTURAL AND GENIC CAUSES

Mitotic instability was found in an intraspecific hybrid of Haplopappus gracilis (Nutt.) Gray and in an interspecific hybrid of H. arenarius Benth. and H. aureus Gray. The latter cross was between distantly related species with different chromosome numbers and amounts of DNA. The intraspecific hybrid exhibited a partly recessive phenotype due to loss of a chromosome segment containing the wild type locus, and the interspecific hybrid showed abnormal developmental patterns for several morphological characters due to chromatin loss. Both hybrids were slower growing, smaller, and generally weaker than parental types. In both examples, this weakness was correlated with chromatin loss due to cleavage by cell wall formation across chromosome arms too long to separate properly at anaphase. This was caused by a very unequal translocation in H. gracilis and to a disparity in genome sizes in the interspecific hybrid. In both examples, the initial chromosome cleavage resulted in a breakage-fusion-bridge cycle that persisted into some BC, progeny of H. gracilis.     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.     

Allelic expression in intergeneric fox hybrids (Alopex lagopus × Vulpes vulpes). III. Regulation of the expression of the parental alleles at the Gpd locus linked to the X chromosome

The electrophoretic pattern of glucose-6-phosphate dehydrogenase (G6PD) was studied in 60 intergeneric fox hybrids (Alopex lagopus × Vulpes vulpes), 33 females and 27 males. It is shown that the structural gene for G6PD, designated Gpd, is located on the X chromosome in both Arctic and silver foxes. Analysis of G6PD patterns in the erythrocytes of hybrid females demonstrated that the phenotypic expression of parental alleles at the Gpd locus varied considerably: from 1:1 to the hemizygous manifestation of an allele of either the Artic or the silver fox. The expression of the parental allels at this locus is different in the various tissues of single female hybrids. It is suggested that the variable quantitative expression of the alleles at the Gpd locus in hybrid females is related to the presence of two cell populations having in an active state either the X chromosome of the Arctic fox or that of the silver fox. It is also proposed that the size of the two cell populations is largely affected by the different relationships between cells having different activated X-chromosomes among initiator (stem) cells from which various definitive organs and tissues develop. The number of initiator cells for erythroid tissue has been calculated to be five or six.     

The inheritance of tissue-specific lactate dehydrogenase isozymes in interspecific bass (Micropterus) hybrids

A combination of hybridization (in vivo and in vitro), immunochemical, and electrophoretic analyses reveals that both smallmouth bass, Micropterus dolomieui (Lacépède), and largemouth bass, M. salmoides (Lacépède), possess three homopolymeric lactate dehydrogenase (LDH) isozymes, A₄, B₄, and E₄. The retinal-specific E₄ isozymes of these two parental species possess different electrophoretic mobilities. The two bass species were hybridized to produce the interspecific F₁ hybrids. In addition, F₂ and F₃ hybrid generations were produced. The genetic data from these crosses indicate that the retinal-specific LDH isozyme is the product of a distinct nuclear gene (E locus) on an autosomal chromosome. This E gene appears to segregate independently of the gene for supernatant MDH. The LDH E gene is highly active in the bass neural retina and less active in other neural tissues. However, unlike in most teleosts, the bass LDH E gene also functions in such nonneural tissues as the heart and kidney.This research was supported by NSF grant GB 16425 to G. S. Whitt and by funds provided by the Illinois Natural History Survey to W. F. Childers.     

Molecular mapping of gene Gm-6(t) which confers resistance against four biotypes of Asian rice gall midge in China

The Chinese rice cultivar Duokang #1 carries a single dominant gene Gm-6(t) that confers resistance to the four biotypes of Asian rice gall midge (Orseolia oryzae Wood-Mason) known in China. Bulked segregant analysis was performed on progeny of a cross between Duokang #1 and the gall midge-susceptible cultivar Feng Yin Zhan using the RAPD method. The RAPD marker OPM06₍₁₄₀₀₎ amplified a locus linked to Gm-6(t). The locus was subsequently mapped to rice chromosome 4 in a region flanked by cloned RFLP markers RG214 and RG163. Fine mapping of Gm-6(t) revealed that markers RG214 and RG476 flanked the gene at distances of 1.0 and 2.3 cM, respectively. Another gall midge resistance gene, Gm-2, mapped previously to chromosome 4, is located about 16 cM from Gm-6(t), to judge by data from a segregating population derived from a cross between Duokang #1 and the Indian cultivar Phalguna that carries Gm-2. We developed a PCR-based marker-assisted selection kit for transfer of the Gm-6(t) gene into Ming Hui 63 and IR50404, two parental lines commonly used in hybrid rice production in China. The kit contains PCR primer pairs based on the terminal sequences of the RG214 and RG476 clones. Polymorphism between Duokang #1 and the hybrid parental lines was found at these markers after digestion of the PCR products with specific restriction endonucleases. The kit will accelerate introduction of gall midge resistance into hybrid rice in China. Received: 18 May 2000 / Accepted: 9 March 2001     

Genetics of a wing size difference between two Nasonia species

Very little is known about the genetics of morphological differences between species. This study investigates the genetic basis of a significant morphological difference between males of two closely related species of the parasitoid wasp Nasonia. One of the defining characters of species in the genus Nasonia is male forewing size. The forewings of Nasoniagiraulti males are 2.4 times larger than the forewings of Nasoniavitripennis males. Genetic analysis of hybrids between these species indicates that this difference is due to the effect of a few genes. Also discussed is the possible role of ‘pseudo linkage’ in analysis of F₂ hybrids. Pseudo linkage occurs when genes affecting a trait are linked to interacting hybrid lethal loci, and can lead to an overestimation of the number of regions involved in a phenotype. The large wing trait of N. giraulti was introgressed into a N. vitripennis background. Analysis of this introgression line indicates that 44% of the difference in wing size between the species is due to the presence of a single gene, or a few tightly linked genes, located on linkage group IV. Furthermore, the introgressed region appears to affect the width of the wing more strongly than the length. Indirect results suggest that this region affects wing cell size, rather than cell number. Results are consistent with the view that morphological and adaptive differences between species can have a simple genetic basis.