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.     

Incipient sexual isolation in thenasuta-albomicans complex ofDrosophila: No-choice experiments

Drosophila nasuta nasuta andDrosophila nasuta albomicans are cross-fertile races ofDrosophila. Hybridization between these races in the laboratory has given rise to new races (Cytoraces), among which karyotypic composition differs from one another and also from those of the parental races. In this study, we search for the evidence of incipient reproductive isolation among the parental races and four Cytoraces by assessing the fraction of no-matings, mating latency and copulation duration in all possible types of homo- and heterogamic crosses (N = 4184). In no-choice conditions, the latency time (time to initiation of copulation) is lower in homogamic crosses than in heterogamic crosses for both parental races and Cytoraces. Latency time and copulation duration are negatively correlated, whereas fraction of no matings is positively correlated with latency time. Thus these six closely related races of thenasuta-albomicans complex show the initiation of the earliest stages of pre-zygotic isolation, manifested as a tendency for matings to be initiated earlier and more often, and for a longer duration, among homogamic rather than heterogamic individuals.     

The chromosomes of two Drosophila races: D. nasuta nasuta and D. nasuta albomicans III. Localization of nucleolar organizer regions

In the two Drosophila races D. n. nasuta and D. n. albomicans the nucleolar-organizer regions (NORs) have been localized on metaphase chromosomes by a combined acid treatment and silver-staining technique. In both Drosophila races one NOR is present on the heterochromatic Y chromosomes and another on the microchromosomes. Such a NOR distribution has not yet been reported in Drosophila. It is suggested that this distribution has evolved from an original type with NORs on the X and Y chromosomes.     

Incipient sexual isolation in the nasuta-albomicans complex of Drosophila: mating preference in male-, female- and multiple-choice mating experiments

Interracial divergence is an important facet of speciation. Thenasuta-albomicans complex ofDrosophila with sixteen morphologically identical, karyotypically different but cross-fertile races is an excellent system to study a few dimensions of raciation.Drosophila nasuta nasuta, Drosophila nasuta albomicans, Cytorace 1, Cytorace 2, Cytorace 3 and Cytorace 4 of this subgroup have been subjected to male-, female- and multiple-choice mating experiments. Out of 8456 crosses conducted, 7185 had successful matings. The overall impression is that mating is far from random amongst these six closely related races of thenasuta-albomicans complex. The males ofD. n. albomicans, Cytorace 1 and Cytorace 4 in male-choice, the females of Cytorace 1 and Cytorace 2 in female-choice, and the males and females ofD. n. nasuta, D. n. albomicans, Cytorace 1 and Cytorace 4 against the males and females of Cytorace 2 in multiple-choice experiments, had significantly more homogamic matings than expected. Thus in this study of evolutionary experimentation on raciation under laboratory conditions, we have documented the initiation of preference for con-specific matings among closely related and independently evolving members of thenasuta-albomicans complex ofDrosophila.     

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.     

Estimation of inter-genotypic competitive ability of the parental races (Drosophila nasuta nasuta and D. n. albomicana) and of the newly evolved Cytoraces (I and II)

Interracial hybridization between D. n. nasuta (2 n = 8) and D. n.albomicana (2 n = 6) resulted in the formation of two new karyotypic strains denoted Cytorace I and Cytorace II. The karyotypes of each of these Cytoraces include chromosomal elements from both parental races (Ramachandra and Ranganath 1986a). The parental strains and the newly formed Cytoraces I and II were subjected to interspecific competition. The results reveal that all four experimental strains were competitively superior to the D. melanogaster tested strain. The study indicates certain degree of Cytogenetic divergence between parental and newly evolved genomes.     

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.     

ON THE ORIGIN OF INCIPIENT REPRODUCTIVE ISOLATION: THE CASE OF DROSOPHILA ALBOMICANS AND D. NASUTA

The nasuta subgroup of Drosophila consists of 12 known species classified within the immigrans group. D. nasuta and D. albomicans are two sibling species widely distributed throughout the Indo-Pacific tropics, which, although morphologically indistinguishable, have different meta-phase-chromosome configurations: chromosomes X and 3 are attached in D. albomicans, so that about 60% of its genes are sex-linked. Our experiments show that, at least in the laboratory, there is no sexual, mechanical, or gametic isolation between the two species. There is, however, hybrid “breakdown” expressed in three ways: 1) reduction in the number of F₂ hybrids produced per culture; 2) reduction in the fertility of F₂ (males) and F₃ (males and females) hybrid progenies; and 3) abnormal sex ratios in the progenies of crosses between strains of certain localities. In experimental populations, the karyotypes of both species are still present in substantial frequencies after 20 generations, although the frequencies of the two karyotypes vary depending on the geographic origin of the strains. Our results support the hypothesis that, in allopatry, the evolution of postzygotic isolation precedes that of prezygotic isolation. The mtDNA is polymorphic in both D. nasuta and D. albomicans and fairly similar between them. Assuming typical rates of mtDNA evolution, the two species would have diverged from each other about 500,000 years ago, whereas the African and Indian populations of D. nasuta (considered to be different subspecies by some authors) might have diverged some 350,000 years ago.     

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.     

The chromosomes of two Drosophila races: D. nasuta nasuta and D. nasuta albomicana

The microchromosomes of the totally cross fertile Drosophila races, D. nasuta nasuta and D. nasuta albomicana have been studied in nietaphase and polytene nuclei. In metaphase the microchromosome of D. n. albomicana is nearly five times longer than the homologous chromosome in D. n. nasuta. As shown by C-banding these length differences are mainly due to a massive addition of heterochromatin to the D. n. albomicana chromosome. In polytene nuclei these striking heterochromatin differences between the microchromosomes of the two Drosophila races cannot be observed. Analysis of the polytene banding pattern shows that the microchromosomes of both races differ by an inversion and by a duplication, present only in D. n, albomicana. The location and orientation of the duplicated regions in D. n. albomicana leads to a specific loop like chromosome configuration. On the basis of these differences within the Drosophila races studied it is assumed that the karyotype of D. n. albomicana is a more recent evolutionary product.     

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.     

The chromosomes of two Drosophila races: D. nasuta nasuta and D. n. albomicana

Heterochromatin distribution and differentiation in metaphase chromosomes of two morphologically identical Drosophila races, D. nasuta nasuta and D. n. albomicana, have been studied by C- and N-banding methods. — The total heterochromatin values differ only slightly between these races. However, homologous chromosomes of the two Drosophila forms show striking differences in the size of heterochromatin regions and there is an alternating pattern in D. n. nasuta and D. n. albomicana of chromosomes which contain more, or respectively less heterochromatin than their counterparts in the other race. — Three different N-banding patterns could be obtained depending on the conditions of the method employed: One banding pattern occurs which corresponds to the C-banding pattern. Another pattern is the reverse of the C-band pattern; the euchromatic chromosome regions and the centromeres are stained whereas the pericentric heterochromatin regions remain unstained. In the Y chromosomes of both races and in chromosome 4 of D. n. albomicana, however, the heterochromatin is further differentiated. In the third N-banding pattern only the centromeres are deeply stained. Furthermore, between the races, subtle staining differences in the pericentric heterochromatin regions can be observed as verified in F₁ hybrids. On the basis of C- and N-banding results specific aspects of chromosomal differences between D. n. nasuta and D. n. albomicana are discussed.Dedicated to Prof. W. Beermann on the occasion of his 60th birthday     

Satellite DNA of Drosophila nasuta nasuta and D. n. albomicana: Localization in polytene and metaphase chromosomes

The DNA from the two Drosophila nasuta races, D. n. nasuta and D. n. albomicana was investigated by CsCl density gradient centrifugation. D. n. nasuta has one major AT-rich satellite DNA sequence with a density of 1.664g/cm³, while D. n. albomicana has at least three satellites with densities of 1.674g/cm³, 1.665g/cm³ and 1.661 g/cm³. The isolated satellite sequences hybridize in situ to all heterochromatic regions of all metaphase chromosomes of both races. In polytene chromosomes the satellite sequences hybridize exclusively to the chromocenter. All chromosomal regions hybridizing with the satellites show also bright quinacrine fluorescence.     

Manipulation of Sod1 expression ubiquitously, but not in the nervous system or muscle, impacts age-related parameters in Drosophila

Superoxide dismutase 1 (SOD1) is an important antioxidant previously shown to impact life span in Drosophila. We examined the consequences of manipulating Sod1 expression throughout the body or in the nervous system or musculature on life span and age-related locomotor impairment (ARLI) in Drosophila. Ubiquitous overexpression of SOD1 extended life span but did not substantially forestall ARLI, whereas ubiquitous knock-down of Sod1 shortened life span and accelerated ARLI. Interestingly, neither overexpression of Sod1 nor expression of Sod1 RNAi in the nervous system or muscle altered life span or ARLI. Our studies suggest that the control of reactive oxygen species by SOD1 in tissues other than the nervous system and musculature support life span and ARLI in Drosophila.     

Divergence for tolerance to thermal-stress related traits in two Drosophila species of immigrans group

We investigated associations of the species specific divergence of thermal adaptations of Drosophila nasuta and Drosophila immigrans with seasonal as well as geographical changes in the relative abundance. In D. immigrans, cold resistance and egg-to-adult viability after a cold-shock have shown a positive cline with latitude, whereas no such clinal patterns were evident for heat resistance as well as egg-to-adult viability after heat stress. In contrast, basal heat tolerance and egg-to-adult viability after a heat-shock increased towards the equator i.e. from north to south in D. nasuta. Further, we also found species-specific divergence in seasonal climatic selection responses for heat as well as cold resistance i.e. warmer and higher humidity conditions of the rainy season have evidenced ∼2-fold higher heat tolerance of D. nasuta, but lower temperature as well as lower humidity conditions of the autumn significantly increased (∼3-fold) cold tolerance of D. immigrans. Geographical variations were significant for the absolute and relative hardening capacity for cold tolerance in D. immigrans, but for heat resistance of D. nasuta. Results of multiple regression analysis of changes in thermal tolerance level in latitudinal populations as a simultaneous function of climatic variables (T_ave and RH) have shown that average yearly relative humidity levels significantly affect heat tolerance of D. nasuta, but both T_ave and RH contribute to changes in the cold tolerance of D. immigrans. Thus, our results suggest that climatic selection responses for basal and hardened thermo-tolerance levels affect relative abundance of D. nasuta and D. immigrans across a latitudinal transect as well as seasons.     

Phylogeography,Interaction Patterns and the Evolution of Host Choice in Drosophila-Parasitoid Systems in Ryukyu Archipelago and Taiwan

Island biotas provide a great opportunity to study not only the phylogeographic patterns of a group of species, but also to explore the differentiation in their coevolutionary interactions. Drosophila and their parasitoids are exemplary systems for studying complex interaction patterns. However, there is a lack of studies combining interaction-based and molecular marker-based methods. We applied an integrated approach combining phylogeography, interaction, and host-choice behavior studies, with the aim to understand how coevolutionary interactions evolve in Drosophila-parasitoid island populations. The study focused on the three most abundant Drosophila species in Ryukyu archipelago and Taiwan: D. albomicans, D. bipectinata, and D. takahashii, and the Drosophila-parasitoid Leptopilina ryukyuensis. We determined mitochondrial COI haplotypes for samples representing five island populations of Drosophila and four island populations of L. ryukyuensis. We additionally sequenced parts of the autosomal Gpdh for Drosophila samples, and the ITS2 for parasitoid samples. Phylogenetic and coalescent analyses were used to test for demographic events and to place them in a temporal framework. Geographical differences in Drosophila-parasitoid interactions were studied in host-acceptance, host-suitability, and host-choice experiments. All four species showed species-specific phylogeographic patterns. A general trend of the haplotype diversity increasing towards the south was observed. D. albomicans showed very high COI haplotype diversity, and had the most phylogeographically structured populations, with differentiation into the northern and the southern population-group, divided by the Kerama gap. Differentiation in host suitability was observed only between highly structured populations of D. albomicans, possibly facilitated by restricted gene flow. Differentiation in host-acceptance in D. takahashii, and host-acceptance and host-choice in L. ryukyuensis was found, despite there being no differentiation in these two species according to molecular markers. Host choice assays show that L. ryukyuensis populations that have had more time to coevolve adapt their behavior to exploit the most suitable host – D. albomicans. L. ryukyuensis parasitoids on border ranges may, on the other hand, benefit from broader host-acceptance, that may facilitate adaptation to uncertain and variable environments. All results indicate that Drosophila-parasitoid populations in the Ryukyu archipelago and Taiwan have different evolutionary trajectories, and coevolve in a dynamic, complex, and local-specific way.     

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.