Genetic analysis of female mating recognition between Drosophila ananassae and Drosophila pallidosa: application of interspecific mosaic genome lines

Drosophila ananassae and Drosophila pallidosa are closely related species that can produce viable and fertile hybrids of both sexes, although strong sexual isolation exists between the two species. Females are thought to discriminate conspecific from heterospecific males based on their courtship songs. The genetic basis of female discrimination behavior was analyzed using isogenic females from interspecific mosaic genome lines that carry homozygous recombinant chromosomes. Multiple regression analysis indicated a highly significant effect of the left arm of chromosome 2 (2L) on the willingness of females to mate with D. ananassae males. Not only 2L but also the left arm of chromosome X (XL) and the right arm of chromosome 3 (3R) had significant effects on the females' willingness to mate with D. pallidosa males. All regions with strong effects on mate choice have chromosome arrangements characterized by species-specific inversions. Heterospecific combinations of 2L and 3R have previously been suggested to cause postzygotic reproductive isolation. Thus, genes involved in premating as well as postmating isolation are located in or near chromosomal inversions. This conclusion is consistent with the recently proposed hypothesis that "speciation genes" accumulate at a higher rate in non-recombining genome regions when species divergence occurs in the presence of gene flow.     

Fine-scale genetic analysis of species-specific female preference in Drosophila simulans

Behavioural differences are thought to be the first components to contribute to species isolation, yet the precise genetic basis of behavioural isolation remains poorly understood. Here, we used a combination of behaviour assays and genetic mapping to provide the first refined map locating candidate genes for interspecific female preference isolating Drosophila simulans from D. melanogaster. First, we tested whether two genes identified as affecting D. melanogaster female intraspecific mate choice also affect interspecific mate choice; neither of these genes was found to contribute to species‐specific female preference. Next, we used deficiency mapping to locate genes on the right arm of the third chromosome for species‐specific female preference and identified five small significant regions that contain candidate genes contributing to behavioural isolation. All five regions were located in areas that would have low interspecific recombination, which mirrors the results of other behavioural isolation studies that used quantitative trait locus (QTL) mapping, but without the potential concern of bias towards regions of low recombination that QTL mapping may have. As this model system may be refined to the individual gene level using the same methodology, this initial map we provide may potentially serve as a ready template for the identification and characterization of the first behavioural isolation genes.     

Genetic studies of two sister species in the Drosophila melanogaster subgroup, D. yakuba and D. santomea

We performed genetic analysis of hybrid sterility and of one morphological difference (sex-comb tooth number) on D. yakuba and D. santomea, the former species widespread in Africa and the latter endemic to the oceanic island of S?o Tomé, on which there is a hybrid zone. The sterility of hybrid males is due to at least three genes on the X chromosome and at least one on the Y, with the cytoplasm and large sections of the autosomes having no effect. F1 hybrid females carrying two X chromosomes from either species are perfectly fertile despite their genetic similarity to completely sterile F1 hybrid males. This implies that the appearance of Haldane's rule in this cross is at least partially due to the faster accumulation of genes causing male than female sterility. The larger effects of the X and Y chromosomes than of the autosomes, however, also suggest that the genes causing male sterility are recessive in hybrids. Some female sterility is also seen in interspecific crosses, but this does not occur between all strains. This is seen in pure-species females inseminated by heterospecific males (probably reflecting incompatibility between the sperm of one species and the female reproductive tract of the other) as well as in inseminated F1 and backcross females, probably reflecting genetically based incompatibilities in hybrids that affect the reproductive system. The latter 'innate' sterility appears to involve deleterious interactions between D. santomea chromosomes and D. yakuba cytoplasm. The difference in male sex-comb tooth number appears to involve fairly large effects of the X chromosome. We discuss the striking evolutionary parallels in the genetic basis of sterility, in the nature of sexual isolation, and in morphological differences between the D. santomea/D. yakuba divergence and two other speciation events in the D. melanogaster subgroup involving island colonization.     

Genetic architecture of the cryptic species complex of Acanthocyclops vernalis (Crustacea: Copepoda). II. Crossbreeding experiments, cytogenetics, and a model of chromosomal evolution

Collectively, populations of Acanthocyclops vernalis, a species complex of freshwater copepods, are remarkably similar as to morphology and DNA content, despite variability in chromosome number. Reproductive isolation had been reported among some populations, but with each new investigation the species boundaries and factors that may influence them appeared less clear. To clarify the pattern of biological species within this group of populations, we adopted a comprehensive approach and examined patterns of reproductive isolation in populations for which morphology, chromosome number, DNA content, and 18S rDNA sequences are known. In this study we established nine isofemale lines from four sites in Wisconsin and performed 266 crosses. Crosses within and among these lines were used to relate the degree of reproductive isolation to chromosome differences and to construct a model to explain the origin and maintenance of chromosome number variability. Different gametic and somatic chromosome numbers were observed among specimens within some isofemale lines. In a few cases, gametes with different haploid numbers were produced by a single female. Matings within isofemale lines always produced at least some reproductively successful replicate crosses (produced viable, fertile offspring). Crosses between lines from the same site showed reduced success relative to within-line crosses. Crosses between populations from distant sites showed limited genetic compatibility, producing viable, fertile F1 offspring but infertile F2 adults. One cross between lines with different chromosome numbers (one with 2n = 8 and one with 2n = 10) produced fertile viable offspring, which reproduced for at least 60 generations. These hybrids had either eight or nine chromosomes in the third generation of inbreeding, and eight chromosomes after 20 generations. These hybrids also had reduced nuclear DNA contents at the third generation, a level that persisted through the 20th generation. Successful backcrosses between some hybrids and their parental lines further demonstrated the potential for genetic compatibility among forms with different chromosome numbers. We propose a model in which alterations due to Robertsonian fusions, translocations, and/or loss of chromosomal fragments generate heritable variation, only some of which leads to reproductive isolation. Hence, some of the criteria traditionally used to recognize species boundaries in animals (morphology, DNA content, chromosome number) may not apply to this species complex.     

Genetics of sexual isolation in females of the Drosophila simulans species complex.

Genetic analysis of hybrids between Drosophila simulans and D. sechellia shows that sexual isolation in females is caused by at least two genes, one on each major autosome, while the X chromosome has no effect. These results are similar to those of a previous study of hybrids between D. simulans and another sibling species, D. mauritiana. In this latter hybridization, each arm of the second chromosome carries genes causing sexual isolation in females, implying a total divergence of at least three loci. The genetic similarity between the D. simulans/D. mauritiana and D. simulans/D. sechellia hybridizations probably results from independent evolution and not phylogenetic artifacts, because the dominance relationships and behavioural interactions differ between the two hybridizations. The lack of an X-chromosome effect on sexual isolation contrasts with genetic studies of post-zygotic reproductive isolation, which invariably show strong effects of this chromosome.     

Observations which indicate heterochromatinization of an extra chromosome in the salivary gland cells of Drosophila hybrids

Salivary gland cells of Drosophila mulleri/D. arizonensis aneuploid male hybrids carrying 3 microchromosomes exhibited morphological features which indicate heterochromatinization of one of the small polytene chromosomes. The process apparently changes the chromosome surface producing a coating with a net-like structure and a strong affinity for lacto-acetic orcein. The possibility of a dosage compensatory mechanism operating to counteract the effect of the extra chromosome is discussed on the basis of previous data which indicated that the microchromosomes of these species have ribosomal cistrons and are controlled by regulatory mechanisms especially evident in hybrids.     

Restricted gene flow at specific parts of the shrew genome in chromosomal hybrid zones

The species and races of the shrews of the Sorex araneus group exhibit a broad range of chromosomal polymorphisms. European taxa of this group are parapatric and form contact or hybrid zones that span an extraordinary variety of situations, ranging from absolute genetic isolation to almost free gene flow. This variety seems to depend for a large part on the chromosome composition of populations, which are primarily differentiated by various Robertsonian fusions of a subset of acrocentric chromosomes. Previous studies suggested that chromosomal rearrangements play a causative role in the speciation process. In such models, gene flow should be more restricted for markers on chromosomes involved in rearrangements than on chromosomes common in both parent species. In the present study, we address the possibility of such differential gene flow in the context of two genetically very similar but karyotypically different hybrid zones between species of the S. araneus group using microsatellite loci mapped to the chromosome arm level. Interspecific genetic structure across rearranged chromosomes was in general larger than across common chromosomes. However, the difference between the two classes of chromosomes was only significant in the hybrid zone where the complexity of hybrids is expected to be larger. These differences did not distinguish populations within species. Therefore, the rearranged chromosomes appear to affect the reproductive barrier between karyotypic species, although the strength of this effect depends on the complexity of the hybrids produced.     

The genetics of reproductive isolation and the potential for gene exchange between Drosophila pseudoobscura and D. persimilis via backcross hybrid males

Hybrid male sterility, hybrid inviability, sexual isolation, and a hybrid male courtship dysfunction reproductively isolate Drosophila pseudoobscura and D. persimilis. Previous studies of the genetic bases of these isolating mechanisms have yielded only limited information about how much and what areas of the genome are susceptible to interspecies introgression. We have examined the genetic basis of these barriers to gene exchange in several thousand backcross hybrid male progeny of these species using 14 codominant molecular genetic markers spanning the five chromosomes of these species, focusing particularly on the autosomes. Hybrid male sterility, hybrid inviability, and the hybrid male courtship dysfunction were all associated with X-autosome interactions involving primarily the inverted regions on the left arm of the X-chromosome and the center of the second chromosome. Sexual isolation from D. pseudoobscura females was primarily associated with the left arm of the X-chromosome, although both the right arm and the center of the second chromosome also contributed to it. Sexual isolation from D. persimilis females was primarily associated with the second chromosome. The absence of isolating mechanisms being associated with many autosomal regions, including some large inverted regions that separate the strains, suggests that these phenotypes may not be caused by genes spread throughout the genome. We suggest that gene flow between these species via hybrid males may be possible at loci spread across much of the autosomes.     

Chromosome replacement in mixed populations of compound-2L; free-2R and standard strains of Drosophila melanogaster

Summary Crosses between compound-2L; free-2R (free-arm) and standard strains of Drosophila melanogaster produce two classes of inviable aneuploid hybrids in equal proportions: monosomic 2L and trisomic 2L. The lethal period for monosomics occurs during embryogenesis while the trisomics survive to late pupae. Since the hybrids are inviable, standard and free-arm strains within a mixed population remain genetically isolated. Genetic isolation in the absence of mating isolation offers an extreme example of unstable equilibrium. Relative fitness data indicate that an unstable equilibrium will be established between free-arm and standard strains at a ratio of 2.51. Indeed, in three cage experiments established at initial ratios of 31, free arms to standards, laboratory (Oregon R) or native (Okanagan S) standard strains were completely replaced in approximately 100 days by free-arm lines derived either from laboratory or from native genetic background. In contrast, one cage established at an initial ratio of 41 failed to show replacement and for 92 days remained at approximately the initial ratio. Subsequent genetic analysis of flies removed from this cage identified the presence of an anomalous strain through which genetic information was transferred reciprocally between the free-arm and standard lines. The second chromosomes carried by this strain consisted of a free-2R and a standard second on the right arm of which was attached a duplication for all of 2L. While the origin of the 2L·2R+2L chromosome was uncertain, genetic and cytological examinations revealed that it represented the reciprocal crossover product expected from an exchange that generated a F(2R). Additional crosses disclosed that the transmission frequency of the asymmetrical pair of second chromosomes, as well as their right-arm crossover products, was disproportionately in favor of the short arm. Since unequal transmission was invariably greater from female parents, this phenomenon was viewed as further evidence in support of the drag hypothesis.Supported by research grant A5853 from the National Science and Engineering Research council of Canada to D.G.H.     

Genetics of Male and Female Sterility in Hybrids of Drosophila pseudoobscura and D. persimilis

The genetic basis of male and female sterility in hybrids of Drosophila pseudoobscura-Drosophila persimilis was studied using backcross analysis. Previous studies indirectly assessed male fertility by measuring testis size; these studies concluded that male sterility results from an X chromosome-autosome imbalance. By directly scoring for the production of motile sperm, male sterility is shown to be largely due to an incompatibility between genes on the X and Y chromosomes of these two species. These species have diverged at a minimum of nine loci affecting hybrid male fertility. Semisterility of hybrid females appears to result from an X chromosome-cytoplasm interaction; the X chromosome thus has the largest effect on sterility in both male and female hybrids. This is apparently the first analysis of the genetic basis of female sterility, or of sterility/inviability affecting both sexes, in an animal hybridization.     

RFLP analysis to identify putative chromosomal regions involved in the anther culture response and callus formation of maize

Summary RFLP analysis was performed with anther culture-derived callus lines developed from the maize F₁ hybrids Pa91 x FR16 (PF), H99 x Pa91 (HP) and H99 x FR16 (HF). Relatively evenly spaced RFLP markers were selected to cover the maize genome with 52, 58 and 35 RFLP markers for the PF, HP and HF callus lines, respectively. The results from populations PF and HP combined with limited information from HF showed that six chromosomal regions on chromosomes 1, 2 (two regions), 3, 6 and 8 appear to be associated with the formation of embryo-like structures (ELSs) from microspores or the subsequent formation of regenerable callus from the ELSs. Regions at the end of the long arm of chromosome 2 and on the long arm of chromosome 8 appear to be associated with ELS formation, and the other regions appear to be associated with either ELS or regenerable callus formation or both. Certain regions that we have identified are the same as those found in other studies to be important for friable, embryogenic callus formation (chromosomes 1 and 3 and near the centromere of 2) and for ESL formation (chromosomes 1 and 3). This study has provided evidence for the genetic basis of the maize anther culture response and callus formation.     

‘Nucleolar dominance’ as observed in barley translocation lines with specifically reconstructed SAT chromosomes

Summary Diploid homo- and heterokaryotypes of barley translocation lines with only one satellite chromosome pair containing two nucleolus organizer regions (NORs) in opposite arms were found to show repressed nucleolus formation by the transposed NOR as evident from the formation of only micronucleoli. The same was true for auto-tetraploid homokaryotypes and for translocation lines with all NORs tandemly arranged into the same chromosome arm. When NORs were transposed to chromosomes without NOR in the standard karyotype, the normal pattern of nucleolus formation remained unaffected. The modified mode of nucleolus formation after the combination of all NORs in one chromosome pair is interpreted to be due to intrachromosomal nucleolar dominance analogous to interchromosomal nucleolar dominance observed in certain interspecific hybrids.     

The Genetics of Postzygotic Isolation in the Drosophila Virilis Group

In a genetic study of postzygotic reproductive isolation among species of the Drosophila virilis group, we find that the X chromosome has the largest effect on male and female hybrid sterility and inviability. The X alone has a discernible effect on postzygotic isolation between closely related species. Hybridizations involving more distantly related species also show large X-effects, although the autosomes may also play a role. In the only hybridization yet subjected to such analysis, we show that hybrid male and female sterility result from the action of different X-linked loci. Our results accord with genetic studies of other taxa, and support the view that both Haldane's rule (heterogametic F1 sterility or inviability) and the large effect of the X chromosome on reproductive isolation result from the accumulation by natural selection of partially recessive or underdominant mutations. We also describe a method that allows genetic analysis of reproductive isolation between species that produce completely sterile or inviable hybrids. Such species pairs, which represent the final stage of speciation, cannot be analyzed by traditional methods. The X chromosome also plays an important role in postzygotic isolation between these species.     

Genetic analyses of several Drosophila ananassae-complex species show a low-frequency major gene for parthenogenesis that maps to chromosome 2

Parthenogenetic strains of several species have been found in the genus Drosophila. The mode of diploidization in the eggs of females has been found to be post-meiotic nuclear fusion. The genetic basis for this parthenogenesis is not understood but is believed to be under the control of a complex polygenic system. We found parthenogenetic females in an isofemale strain (LAE345) of D. pallidosa-like collected in 1981 at Lae, Papua New Guinea, and established a parthenogenetically reproducing strain. Parthenogenetic strains of D. ananassae and D. pallidosa collected at Taputimu, American Samoa had also been established by Futch (1972). D. ananassae, D. pallidosa and D. pallidosa-like are very closely related species belonging to the ananassae complex of the ananassae species subgroup of the melanogaster species group. Using these three species, we found that more than 80% of females from parthenogenetic strains produced progeny parthenogenetically and that inter-specific hybrid females also produced impaternate progeny. In the present report, we demonstrate that the mode of parthenogenesis of D. ananassae appears to be the post-meiotic nuclear doubling of a single meiotic product, and that a major gene responsible for the parthenogenesis maps to the left arm of the second chromosome of D. ananassae. We also suggest that the genetic basis for parthenogenesis capacity may be identical among the three closely related species. We discuss the function of the gene required for parthenogenesis and its significance for the evolutionary process.     

Alteration of chromosome structure in ovarian nurse cells of Drosophila melanogaster by hybrid dysgenesis

The impact of hybrid dysgenesis on the chromosome structure of Drosophila melanogaster ovarian nurse cells was studied. In the examined lines and interlinear hybrids (including those yielded by dysgenic crosses in the P-M and I-R systems of hybrid dysgenesis), disturbed chromosome synapsis was revealed. The disturbance was somewhat similar to that observed in interspecific hybrids. Quantitative analysis showed that the mean frequency of nuclei with defective chromosome pairing ranged from 60.4 to 76%. FISH analysis of ovarian nurse chromosomes of Canton S x Berlin hybrids showed differences in the label localization in asynaptic homologs of arm 2L, which probably results in disrupted homolog pairing and reveal interlinear differences in localization of mobile genetic elements. Our results conform to Sved's model stating that hybrid dysgenesis is based on disorganization of the germline nuclear space.     

Dissection and cytological mapping of barley chromosome 2H in the genetic background of common wheat

We used gametocidal (Gc) chromosomes 2C and 3C(SAT) to dissect barley 2H added to common wheat. The Gc chromosome induces chromosomal breakage resulting in chromosomal aberrations in the progeny of the 2H addition line of common wheat carrying the monosomic Gc chromosome. We conducted in situ hybridization to select plants carrying structurally rearranged aberrant 2H chromosomes and characterized them by sequential C-banding and in situ hybridization. We established 66 dissection lines of common wheat carrying single aberrant 2H chromosomes. The aberrant 2H chromosomes were of either deletion or translocation or complicated structural change. Their breakpoints were distributed in the short arm (2HS), centromere (2HC) and the long arm (2HL) at a rough 2HS/2HC/2HL ratio of 2:1:2. We conducted PCR analysis of the 66 dissection lines using 115 EST markers specific to chromosome 2H. Based on the PCR result, we constructed a physical or cytological map of chromosome 2H that were divided into 34 regions separated by the breakpoints of the aberrant 2H chromosomes. Forty-seven markers were present in 2HS and 68 in 2HL. We compared the 2H cytological map with a previously reported 2H genetic map using 44 markers that were used in common to construct both maps. The order of markers in the distal region was the same on both maps but that in the proximal region was somewhat contradictory between the two maps. We found that the markers distributed rather evenly in the genetic map were actually concentrated in the distal regions of both arms as revealed by the cytological map. We also recognized an EST-marker or gene-rich region in the 2HL interstitial region slightly to the telomere.     

The genetic basis of behavioral isolation between Drosophila mauritiana and D. sechellia

Understanding how species form is a fundamental question in evolutionary biology. Identifying the genetic bases of barriers that prevent gene flow between species provides insight into how speciation occurs. Here, I analyze a poorly understood reproductive isolating barrier, prezygotic reproductive isolation. I perform a genetic analysis of prezygotic isolation between two closely related species of Drosophila, D. mauritiana and D. sechellia. I first confirm the existence of strong behavioral isolation between D. mauritiana females and D. sechellia males. Next, I examine the genetic basis of behavioral isolation by (1) scanning an existing set of introgression lines for chromosomal regions that have a large effect on isolation; and (2) mapping quantitative trait loci (QTL) that underlie behavioral isolation via backcross analysis. In particular, I map QTL that determine whether a hybrid backcross female and a D. sechellia male will mate. I identify a single significant QTL, on the X chromosome, suggesting that few major-effect loci contribute to behavioral isolation between these species. In further work, I refine the map position of the QTL to a small region of the X chromosome.     

Polymorphism and differentiation of rainbow trout Y chromosomes.

Most fish species show little morphological differentiation in the sex chromosomes. We have coupled molecular and cytogenetic analyses to characterize the male-determining region of the rainbow trout (Oncorhynchus mykiss) Y chromosome. Four genetically diverse male clonal lines of this species were used for genetic and physical mapping of regions in the vicinity of the sex locus. Five markers were genetically mapped to the Y chromosome in these male lines, indicating that the sex locus was located on the same linkage group in each of the lines. We also confirmed the presence of a Y chromosome morphological polymorphism among these lines, with the Y chromosomes from two of the lines having the more common heteromorphic Y chromosome and two of the lines having Y chromosomes morphologically similar to the X chromosome. The fluorescence in situ hybridization (FISH) pattern of two probes linked to sex suggested that the sex locus is physically located on the long arm of the Y chromosome. Fishes appear to be an excellent group of organisms for studying sex chromosome evolution and differentiation in vertebrates because they show considerable variability in the mechanisms and (or) patterns involved in sex determination.     

Genetic demonstration of mitotic recombination in cultured Chinese hamster cell hybrids

Chinese hamster ovary cell hybrids were constructed that are heterozygous for two markers, leuS and emtB, linked to the long arm of chromosome 2. In addition, the chromosome 2 carrying the wild-type leuS and emtB alleles contains, on its short arm, a homogeneously staining region (hsr) in which the gene encoding dihydrofolate reductase (dhfr) is amplified approximately 50-fold. This provides a convenient cytogenetic and biochemical means to distinguish the chromosome 2s from the different parents. Analysis of emetine-resistant segregants isolated from such hybrids identified three distinct classes of segregants. One rare class of segregants loses the wild-type leuS and emtB gene functions on the long arm of the hsr chromosome 2 (H-2) but retains the amplified dhfr genes on the opposite arm. Detailed genetic analysis of two such segregants that did not arise by chromosome loss or deletion revealed that new gene linkage relationships had been established on the H-2 chromosome in each, demonstrating that the segregation events in these cell lines involved mitotic recombination.     

Production of wheat-Psathyrostachys huashanica chromosome addition lines

Psathyrostachys huashanica Keng (2n = 14; N(h)N(h)) is an endangered wheat-related species, with a distribution in the Huashan region of central China. It has many agronomically promising characters including resistance to disease and drought and winter hardiness. We produced hybrids between common wheat as the female parent and P. huashanica as the male parent. From the offspring, we selected chromosome addition lines of common wheat carrying each of all seven chromosomes of P. huashanica. Four chromosomes (B, D, E and F) were recovered in disomic lines and three (A, C and G) in monosomic addition lines. These alien chromosomes were distinguished from each other by cytological analyses. Chromosome A was characterized by a 45S rDNA site in the subtelomeric region of the short arm. Chromosome B carried one 5S and one 45S rDNA sites co-localized in an interstitial region of the short arm, and the expression of the alien high-molecular-weight glutenin was observed in the endosperm of line B. Chromosome D had a 45S rDNA signal in the interstitial region of the long arm. Chromosomes C, E, and F were distinguished by the EST-SSR markers Ltc0464, Ltc0096, and Xcfe175, respectively. The homoeologous group of each alien chromosome was implied from the results above, and the utilization of these addition lines for wheat breeding was discussed.