Evolutionary experimentation through hybridization under laboratory condition in <Emphasis Type="Italic">Drosophila</Emphasis>: Evidence for Recombinational Speciation

Background  

Drosophila nasuta nasuta (2n = 8) and Drosophila nasuta albomicans (2n = 6) are a pair of sibling allopatric chromosomal cross-fertile races of the nasuta subgroup of immigrans species group of Drosophila. Interracial hybridization between these two races has given rise to new karyotypic strains called Cytorace 1 and Cytorace 2 (first phase). Further hybridization between Thailand strain of D. n. albomicans and D. n. nasuta of Coorg strain has resulted in the evolution of two more Cytoraces, namely Cytorace 3 and Cytorace 4 (second phase). The third phase Cytoraces (Cytorace 5 to Cytorace 16) have evolved through interracial hybridization among first, second phase Cytoraces along with parental races. Each of these Cytoraces is composed of recombined genomes of the parental races. Here, we have made an attempt to systematically assess the impact of hybridization on karyotypes, morphometric and life history traits in all 16 Cytoraces.     

Hybridization,transgressive segregation and evolution of new genetic systems in<Emphasis Type="Italic">Drosophila</Emphasis>

Introgressive hybridization facilitates incorporation of genes from one species into the gene pool of another. Studies on long-term effects of introgressive hybridization in animal systems are sparse.Drosophila nasuta (2n = 8) andD. albomicans (2n = 6)—a pair of allopatric, morphologically almost identical, cross-fertile members of thenasuta subgroup of theimmigrans species group-constitute an excellent system to analyse the impact of hybridization followed by transgressive segregation of parental characters in the hybrid progeny. Hybrid populations ofD. nasuta andD. albomicans maintained for over 500 generations in the laboratory constitute new recombinant hybrid genomes, here termed cytoraces. The impact of hybridization, followed by introgression and transgressive segregation, on chromosomal constitution and karyotypes, some fitness parameters, isozymes, components of mating behaviour and mating preference reveals a complex pattern of interracial divergence among parental species and cytoraces. This assemblage of characters in different combinations in a laboratory hybrid zone allows us to study the emergence of new genetic systems. Here, we summarize results from our ongoing studies comparing these hybrid cytoraces with the parental species, and discuss the implications of these findings for our understanding of the evolution of new genetic systems. This paper is dedicated to the memory of our teacher, Prof. N. B. Krishnamurthy.     

Inversion polymorphism in populations of Drosophila sulphurigaster albostrigata and Drosophila nasuta albomicans from Phuket,Thailand

Two species of the Drosophila nasuta subgroup of the Drosophila immigrans group, D. sulphurigaster albostrigata and D. nasuta albomicans were investigated in this study. Collections of both species were made from Phuket, Thailand. Both species have similar salivary chromosomes, with four autosomal arms and one sex chromosome arm, and both are highly polymorphic for paracentric inversions. D. s. albostrigata accounted for the majority of the isolines collected and exhibited the greater number of inversions. One inversion, C₁, was common to both species, indicating common ancestry.A non-random distribution of inversions was observed on the proximal end of chromosome II in both D. s. albostrigata and D. n. albomicans. An inter-collection comparison revealed that both rigid and flexible chromosomal polymorphism were operating in the two species, with a seasonal variation noted for one inversion in D. s. albostrigata. A non-random association of two inversions was observed in D. n. albomicans.Based on a comparison of the indices of crossing over, both D. s. albostrigata and D. n. albomicans were found to be more heterozygous than in previous studies, with D. n. albomicans appearing to have evolved further than D. s. albostrigata.Based on a thesis submitted for the degree of Ph. D. at the University of Queensland.     

Mitochondrial DNA evolution in theDrosophila nasuta subgroup of species

Summary TheDrosophila nasuta group consists of about 12 closely related species distributed throughout the Indo-Pacific region. They are of great interest because of their evolutionary idiosyncrasies including little morphological differentiation, the ability to intercross in the laboratory often producing fertile offspring, and substantial chromosomal evolution. Studies of metric traits, reproductive isolation, and chromosomal and enzyme polymorphisms have failed to resolve the phylogeny of the species. We report the results of a survey of the mitochondrial DNA (mtDNA) restriction patterns of the species. The phylogeny obtained is consistent with other available information and suggests thatD. albomicans may represent the ancestral lineage of the group. The amount of polymorphism in local populations (=1.0% per site) is within the typical range observed in other animals, includingDrosophila. The degree of differentiation between species is, however, low: the origin of the group is tentatively dated about 6–8 million years ago. This study confirms the usefulness of mtDNA restriction patterns for ascertaining the phylogeny of closely related species.     

Evidence for no sexual isolation between Drosophila albomicans and D. nasuta

Sexual isolation, the reduced tendency to mate, is one of the reproductive barriers that prevent gene flow between different species. Various species‐specific signals during courtship contribute to sexual isolation between species. Drosophila albomicans and D. nasuta are closely related species of the nasuta subgroup within the Drosophila immigrans group and are distributed in allopatry. We analyzed mating behavior and courtship as well as cuticular hydrocarbon profiles within and between species. Here, we report that these two species randomly mated with each other. We did not observe any sexual isolation between species or between strains within species by multiple‐choice tests. Significant difference in the courtship index was detected between these two species, but males and females of both species showed no discrimination against heterospecific partners. Significant quantitative variations in cuticular hydrocarbons between these two species were also found, but the cuticular hydrocarbons appear to play a negligible role in both courtship and sexual isolation between these two species. In contrast to the evident postzygotic isolation, the lack of sexual isolation between these two species suggests that the evolution of premating isolation may lag behind that of the intergenomic incompatibility, which might be driven by intragenomic conflicts.     

Inversion polymorphism in Southeast Asian populations of the Drosophila nasuta subgroup

Heterozygosity for chromosomal arrangements was investigated in four species of the nasuta complex. D. sulfurigaster albostrigata, D. albomicans and D. kohkoa are highly polymorphic whereas D. pulaua is monomorphic. Inversions were identified with the aid of photographic chromosome maps. The geographic distribution and frequencies of inversions detected and their possible phylogenies were discussed. Most inversions in D.s. albostrigata and D. albomicans occur on chromosome IIL. In D. kohkoa there is marked preponderance of inversions on chromosome III. Non-random association of certain inversions was noted. Variation in frequencies of inversions is interpreted as a result of adaptation to local ecogeographic conditions. Shared extant polymorphisms indicate phylogenetic relationships between species.The contents of this paper were incorporated in a Ph.D. thesis accepted by the University of Queensland in 1978. The author's present address is: Unit Genetik, Universiti Kebangsaan Malaysia, Jalan Pantai Baru, Kuala Lumpur, Malaysia.     

SIGNIFICANT ROLE FOR HISTORICAL EFFECTS IN THE EVOLUTION OF REPRODUCTIVE ISOLATION: EVIDENCE FROM PATTERNS OF INTROGRESSION BETWEEN THE CYPRINID FISHES,LUXILUS CORNUTUS AND LUXILUS CHRYSOCEPHALUS

Samples of Luxilus cornutus, Luxilus chrysocephalus, and their hybrids were collected along hypothesized routes of dispersal from Pleistocene refugia to examine the significance of geographic variation in patterns of introgression between these species. Patterns of allozyme and mitochondrial DNA (mtDNA) variation were generally consistent with those from previous studies. Tests of Hardy-Weinberg equilibrium revealed significant deficiencies of heterozygotes in all samples, indicating some form of reproductive isolation. Mitochondrial DNAs of each species were not equally represented in F₁ hybrids; however, this bias was eliminated when the two largest samples were excluded from the analysis. Backcross hybrids exhibited biased mtDNA introgression, as samples from Lake Erie (eastern) and Lake Michigan (western) drainages showed significant excesses of mtDNAs from L. chrysocephalus and L. cornutus, respectively, relative to frequencies of diagnostic allozyme markers. The extent and direction of allozyme and mtDNA introgression was quantified by calculating isolation index values from morphologically “pure” individuals of each species from each locality. Analysis of variance of these measures identified limited introgression of allozyme variants with no geographic pattern, but significant differences in direction of mtDNA introgression between drainages (i.e., postglacial dispersal route). Association between patterns of mtDNA introgression and dispersal route across the latitudinal width of the contact zone is best explained by genetic divergence during past isolation of ancestral populations from these drainages. These results identify a significant role for historical effects in the evolution of reproductive isolation and the process of speciation.     

Genetic and biochemical analysis of brown eye mutation in Drosophila nasuta nasuta and Drosophila nasuta albomicans

By analyzing the progeny of crosses involving brown eye mutants and the wild types in two members of Drosophila nasuta subgroup namely D. n. nasuta and D. n. albomicans we could show that the mutant gene is recessive, located in the chromosome 2 and the alleles of this gene are present at different loci. A study of fitness in the eye color mutants in comparison with the wild types revealed that D. n. nasuta mutant has higher viability at both 25 ± 1°C and ambient temperatures; while D. n. albomicans mutant has faster rate of development only at 25 ± 1°C. Quantitative analysis of eye pigments in the mutants revealed that there is biosynthesis of both pteridines and xanthommatins unlike in bw/bw of D. melanogaster, where only xanthommatins are synthesized. In both the species, the pteridine quantities in mutants are similar; whereas xanthommatin quantity in is 10 times higher than that of . Further, the F₁ progeny of intraspecific crosses (wild type X mutant) are found to have high amounts of pteridine, even when compared with parental wild type. This revised version was published online in July 2006 with corrections to the Cover Date.     

Introgressive hybridization and evolution of a novel protein phenotype: Glue protein profiles in thenasuta-albomicans complex ofDrosophila

Glue proteins are tissue-specific proteins synthesized by larval salivary gland cells ofDrosophila. InDrosophila nasuta nasuta andD. n. albomicans of thenasuta subgroup, the genes that encode the major glue protein fractions are X-linked. In the present study, these X-linked markers have been employed to trace the pattern of introgression ofD. n. nasuta andD. n. albomicans genomes with respect to the major glue protein fractions in their interracial hybrids, called cytoraces. These cytoraces have inherited the chromosomes of both parents and have been maintained in the laboratory for over 400–550 generations. The analysis has revealed that cytoraces withD. n. albomicans X chromosome show eitherD. n. nasuta pattern or a completely novel pattern of glue protein fractions. Further, quantitative analysis also shows lack of correlation between the chromosomal pattern of inheritance and overall quantity of the major glue protein fractions in the cytoraces. Thus, in cytoraces the parental chromosomes are not just differentially represented but there is evidence for introgression even at the gene level.     

Inheritance of chromosome length polymorphisms inCoprinus cinereus

The sexually compatible strains ofCoprinus cinereus 5302 and Dd 13 revealed chromosome length polymorphisms in their electrophoretic karyotypes. The dikaryon derived from two monokaryons contained a mixture of the two electrophoretic patterns. F₁ progenies were isolated by crossingC. cinereus 5302 and Dd 13 strains and it showed unique karyotypes. Chromosome length polymorphisms of both parental strains were inherited at random in the F₁ progenies. As a result, several novel electrophoretic karyotypes which had not been observed in either parental strains were found in the F₁ progeny. The rDNA probe hybridized with one chromosome in both parental strains, with two chromosomes in the hybridization pattern of both parental strains in the dikaryon, and with one to two chromosomes in the F₁ progenies. The relation between mating type and hybridization pattern has thus not been made clear in the case of F₁ progeny.     

The chromosomes of two Drosophila races: Drosophila nasuta nasuta and Drosophila nasuta albomicana V. Introgression and the evolution of new karyotypes

Reciprocal crosses were made between an Indian strain of D. n. nasuta (2n=8) and the Thailand strain of D. n. albomicana (2n=6). Hybrids were fertile. They were inbred for over four years. Later, the karyotypes of the hybrid populations were analysed. In the hybrid progeny of the cross between D. n. nasuta females and D. n. albomicana males, there were six types of kaotypes. Of these, only two types had a diploid content of chromosomes. They were males with 2n=7 and females with 2n= 8 , while others were aneuploids. This hybrid population is designated as Cytorace III. On the other hand, hybrid progeny of the reciprocal cross had 2n-8 in both males and females; and there was no karyotypic variation. This hybrid population is named as Cytorace IV. The composition of these new karyotypes of Cytorace III and IV have been presented and compared with those of Cytorace I and II reported by Ramachandra and Ranganath (1986).     

Inversion polymorphism in species of the Drosophila nasuta subgroup from Thailand

Thailand populations of three species of the D. nasuta complex have been analysed for the presence of paracentric inversions. D. albomicans and D. sulfurigaster albostrigata were collected from Phuket, Chiang Mai and the River Kwai, whilst D. kohkoa was found only in Phuket and the River Kwai. Chromosomal polymorphism was studied in respect to geographical distribution. The Phuket populations of all three species proved to be highly polymorphic by comparison with the River Kwai and Chiang Mai populations. The heterozygosity frequencies of inversions were calculated and the variations interpreted as a result of adaptation to local ecogeographical conditions. Shared polymorphisms revealed that D. kohkoa and D. s. albostrigata are more closely related to D. albomicans than they are to each other.Based on a thesis submitted for the degree of Ph.D. in the University of Queensland.     

Rapid Evolution of a Few Members of Nasuta-Albomicans Complex of Drosophila: Study on Two Candidate Genes, Sod1 and Rpd3

Drosophila nasuta nasuta (2n = 8) and D. n. albomicans (2n = 6) are morphologically identical, cross fertile and karyotypically dissimilar pair of chromosomal races belonging to nasuta subgroup of immigrans group of Drosophila. Interracial hybridization between these two races yielded karyotypically stabilized newly evolved Cytoraces with new combinations of chromosomes and DNA content, and are called nasuta-albomicans complex of Drosophila. Along with many other features, striking plasticity in the lifespan has been observed in the karyotypically stabilized members of nasuta-albomicans complex of Drosophila. These findings provide a strong background to understand any changes at the molecular levels. In view of this, we cloned and characterized Sod1 and Rpd3 in the members of nasuta-albomicans complex of Drosophila. The evolution of Sod1 and Rpd3 in D. n. nasuta and D. n. albomicans is contrasting with the other species of Drosophila, at the level of synonymous mutations, intron variation, InDels and secondary structure changes in protein. In the members of NAC of Drosophila there were synonymous changes, variations in intron sequences of Sod1, whereas, in Rpd3, synonymous, nonsynonymous, intron variation, and secondary structure changes in protein were observed. The contrasting differences in the levels of Rpd3 (and Sir2) proteins were also noticed among short-lived and long-lived Cytoraces. The Cytoraces have exhibited not only specific changes in Sod1 and Rpd3, but also show pronounced changes in the levels of synthesis of these proteins, which indicates rapid evolution of these Cytoraces in laboratory. Further these Cytoraces have become a model system to understand the process of anagenesis.     

THE GENETIC BASIS OF SEXUAL ISOLATION BETWEEN DROSOPHILA MELANOGASTER AND D. SIMULANS

The genetic analysis of sexual isolation between the closely-related species Drosophila melanogaster and Drosophila simulans involved two experiments with no-choice tests. The efficiency of sexual isolation was measured by the frequency of courtship initiation and interspecific mating. We first surveyed the variation in sexual isolation between D. melanogaster strains and D. simulans strains of different geographic origin. Then, to investigate variation in sexual isolation within strains, we made F₁ diallel sets of reciprocal crosses within strains of D. melanogaster and D. simulans. The F₁ diallel progeny of one sex were paired with the opposite sex of the other species. The first experiment showed significant differences in the frequency of interspecific mating between geographic strains. There were more matings between D. simulans females and D. melanogaster males than between D. melanogaster females and D. simulans males. The second experiment uncovered that the male genotypes in the D. melanogaster diallel significantly differed in interspecific mating frequency, but not in courtship initiation frequency. The female genotypes in the D. simulans diallel were not significantly different in courtship initiation and interspecific mating frequency. Genetic analysis reveals that in D. melanogaster males sexual isolation was not affected by either maternal cytoplasmic effects, sex-linked effects, or epistatic interaction. The main genetic components were directional dominance and overdominance. The F₁ males achieved more matings with D. simulans females than the inbred males. The genetic architecture of sexual isolation in D. melanogaster males argues for a history of weak or no selection for lower interspecific mating propensity. The behavioral causes of variation in sexual isolation between the two species are discussed.     

REPRODUCTIVE ISOLATION AND INTROGRESSION BETWEEN NOTROPIS CORNUTUS AND NOTROPIS CHRYSOCEPHALUS (FAMILY CYPRINIDAE): COMPARISON OF MORPHOLOGY,ALLOZYMES, AND MITOCHONDRIAL DNA

Hybrid zones in fluvial fishes may be heterogeneous from drainage to drainage. The comparison of data from morphology, allozymes, and mitochondrial DNA (mtDNA) indicates variability in the causes and degree of restriction of gene flow between Notropis cornutus and Notropis chrysocephalus. Allozyme marker loci show frequency-dependent introgression; i.e., the rarer species, whichever it is at a particular locality, tends to exhibit a higher proportion of introgressed alleles. Unlike allozymes, introgression of mtDNA haplotypes varies geographically. In westward-flowing Michigan drainages, N. cornutus mtDNA haplotypes are more common in F₁ hybrids and backcrosses, independent of parental frequencies. In eastward-flowing Michigan drainages, N. chrysocephalus mtDNA is more common in F₁ hybrids and backcrosses; this pattern may be due to local ecological effects or frequency-dependent introgression. Morphological data alone are not sufficient to distinguish all classes of hybrids. The lack of concordance of morphological, allozymic, and mtDNA introgression patterns implies operation of one or two factors: 1) geographically variable patterns of selection against different hybrid and backcross combinations or 2) genetic differences between Michigan populations inhabiting eastward- and westward-flowing drainage systems accumulated during historical isolation.     

Chromosomal polymorphism in species of the Drosophila nasuta complex in East and South East Asia

This paper is presented as a review of ten years collection of the D. nasuta complex from the Asian region by Dr. Wharton B. Mather. It incorporates the analysis of chromosomal polymorphisms with regard to their geographical distribution and then uses the polymorphisms to establish phylogenetic relationships between D. sulfurigaster albostrigata, D. albomicans and D. kohkoa.     

Production of wide hybrids and backcross progenies between Diplotaxis erucoides and crop brassicas

Intergeneric hybrids were produced between D. erucoides (), a wild species, and four cultivated species of Brassica, B. campestris, B. juncea, B. napus and B. oleracea, through embryo rescue. The hybrid nature of these plants was confirmed through morphological and cytological studies. Backcross pollinations with the pollen of the respective cultivars yielded BC progenies in the hybrids D. erucoides x B. juncea and D. erucoides x B. napus but not in D. erucoides x B. campestris and D. erucoides x B. oleracea. The hybrid D. erucoides x B. campestris was also used as a bridge species and crossed with B. juncea to raise the hybrid and backcross progenies. F₂ progenies were more amenable than f₁ hybrids for raising backcross progenies. Although D. erucoides is considered to be a close relative of B. campestris and B. oleracea, incompatibility barriers of this species with different cultivars do not reflect this relationship.     

Genomic analyses reveal natural selection on reproduction related genes between two closely related Populus (Salicaceae) species

Identifying the factors that cause reproductive isolation and their relative importance in species divergence is crucial to our understanding of speciation processes. In most species, natural selection is commonly considered to play a large role in driving speciation. Based on whole genome re-sequencing data from 27 Populus alba and 28 Populus adenopoda individuals, we explored the factors related to reproductive isolation of these two closely related species. The results showed that the two species diverged ~5–10 million years ago (Ma), when the Qinghai–Tibet Plateau reached a certain height and the inland climate of the Asian continent became arid. In highly differentiated genomic regions, the relative divergence (F_ST) and absolute divergence (d_xy) were significantly higher than the genomic background, θπ and shared polymorphisms decreased whereas fixed differences increased, which indicated that natural selection played a key role in the reproductive isolation of the two species. In addition, we found several genes that were related to reproduction that may be involved in explaining the reproductive isolation. Using phylogenetic trees resolved from haplotype data of Populus tomentosa and P. adenopoda, the maternal origin of P. tomentosa from P. adenopoda was likely to be located in Hubei and Chongqing Provinces.     

SDS-PAGE Pattern Polymorphism of X-Chromosomal Glue Proteins in Natural Populations of Two Drosophila nasuta Subgroup Species

For the first time, the glue protein patternpolymorphism in natural populations of D. n.nasuta and D. s. neonasuta has beenanalyzed by SDS-PAGE. The study involving 200 and 185isofemale lines comprising 2028 and 1900 individuals of D. n.nasuta and D. s. neonasuta, respectively,revealed the occurrence of eight variant glue proteinphenotypes in D. n. nasuta and seven in D.s. neonasuta. The number and frequency of variant phenotypes in differentpopulations of both species were found to vary. Analysisof glue protein patterns in the F₁ progeny ofcrosses involving parents with variant glue proteinphenotypes revealed that the polymorphic fractions areproduced by co-dominant genes located on the Xchromosome. More than 87% of the naturally inseminatedadult females were found to produce polymorphic progeny. The heterozygous female larvae were found toexceed the homozygotes in the isofemale line progeny ofmost of the populations.     

THE GEOGRAPHY OF MITOCHONDRIAL DNA VARIATION IN TWO SYMPATRIC SPARROWS

Geographic variation in mitochondrial DNA (mtDNA) restriction sites was studied in samples of two sympatric passerine birds, fox (Passerella iliaca) and song (Melospiza melodia) sparrows, collected at the same sites in the western United States. Different levels of variation and differentiation were observed in each species. In 46 fox sparrows taken at nine sites, five clones were observed, partitioned into two distinct east-west groups that meet at the Great Basin-Sierra Nevada interface; percent nucleotide divergence was 0.86 between groups and 0.08 within groups. An additional 43 individuals were examined using at least one of seven diagnostic endonucleases, and all supported the east-west groupings. Considering common mtDNA haplotypes as alleles, an F_ST of 0.50 was computed, which is an order of magnitude greater than that computed from allozyme comparisons (0.019); mtDNA analyses suggest little intergroup gene exchange. Compared to allozymic variation, analysis of mtDNA revealed a greater degree of population structuring and greater consistency with broad patterns of morphological variation. Fifteen clones were observed in 27 song sparrows taken at seven of the same sites at which fox sparrows were sampled; the percent nucleotide divergence averaged 0.27. There was no detectable geographic pattern to the variation, and no evidence of an east-west division as in the fox sparrow. However, the mosaic nature of mtDNA variation in song sparrows suggests limited gene exchange. Considering the 15 clones as alleles yielded an F_st of 0.24, which is reduced to 0.039 when corrected for sampling error. In spite of occupying the same geographic area, mtDNA analyses showed that the two species (or at least their mtDNA gene genealogies) have had different evolutionary histories.