Racial divergence in abdominal bristles among parental races and newly evolved cytoraces of nasuta-albomicans complex of Drosophila.

Cytoraces are the products of interracial hybridization between Drosophila nasuta nasuta and D. nasuta albomicans. These races differ from their parents in the chromosome composition, mating preference, certain fitness phenotypes and also a few morphophenotypic traits. Now, these cytoraces are passing through 330 generations. Racial divergence in the 4th and 5th abdominal bristles among the parental races and the newly evolved cytorace 1 and 2 is reported. The results revealed that the parental races have more number of bristles than newly evolved cytoraces. Thus, these cytoraces are evolved/evolving with reduced abdominal bristle number and better fitness.     

Does evolution reduce the body size? A study of the four members of newly evolved nasuta‐albomicans complex of Drosophila

Our long range interracial hybridization experiments between a pair of cross fertile races, Drosophila nasuta (2n = 8) and D.albomicans (2n = 6) have resulted in the evolution of two new karyotypic strains under laboratory conditions, which are named as Cytorace 1 and Cytorace 2. These Cytoraces harbor chromosomes from both parents. Here, we compare the body size of the parental races and newly evolved Cytoraces and the relationship between the body size and fitness. Analysis reveals that the parental races have reduced fertility and are larger in body size than newly evolved Cytoraces. Thus, the newly evolved Cytoraces show reduced body size and better fitness in the course of their evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

The speciation history of the Drosophila nasuta complex

The Drosophila nasuta subgroup of the immigrans species group is widely distributed throughout the South-East Asian region, consisting of morphologically similar species with varying degrees of reproductive isolation. Here, I report nucleotide variability data for five X-linked and two mtDNA loci in eight taxa from the nasuta subgroup, with deeper sampling from D. albomicans and its sister species D. nasuta. Phylogenetic relationships among these species vary among different genomic regions, and levels of genetic differentiation suggest that this species group diversified only about one million years ago. D. albomicans and D. nasuta share nucleotide polymorphisms and are distinguished by relatively few fixed differences. Patterns of genetic differentiation between this species pair are compatible with a simple isolation model with no gene flow. Nucleotide variability levels of species in the nasuta group are comparable to those in members of the melanogaster and pseudoobscura species groups, indicating effective population sizes on the order of several million. Population genetic analyses reveal that summaries of the frequency distribution of neutral polymorphisms in both D. albomicans and D. nasuta generally fit the assumptions of the standard neutral model. D. albomicans is of particular interest for evolutionary studies because of its recently formed neo-sex chromosomes, and our phylogenetic and population genetic analyses suggest that it might be an ideal model to study the very early stages of Y chromosome evolution.     

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

Interracial hybridization between Drosophila nasuta nasuta (2n=8) and D. n. albomicana (2n=6) has resulted in the evolution of two new karyotypic strains, called Cytoraces I and II. Males and females of Cytorace I have 2n=7 and 2n=6 respectively. The reconstituted karyotype is totally new in its composition, the chromosomes being drawn from both the parental races. The individuals of Cytorace II have 2n=6. Even though the chromosomes of the parental races are duly represented in the F₁, there is selective retention/elimination of certain chromosomes in the succeeding generations during which repatterning of the karyotype has taken place. Dynamics of each one of the parental chromosomes are presented and its implications re discussed.We dedicate this paper to the memory of the founder of our Department, the late Prof. M.R. Rajasekarasetty on the occasion of the Silver Jubilee of our Department     

Molecular phylogeny of the nasuta subgroup of Drosophila based on 12S rRNA, 16S rRNA and CoI mitochondrial genes, RAPD and ISSR polymorphisms

The nasuta subgroup is a cluster of morphologically almost similar forms with a wide range of geographic distribution. During the last three decades nature of inter-relationship among the members has been investigated at different levels of organization. The phylogenetic relationships of the members of the nasuta subgroup of the immigrans species group of Drosophila was made by employing Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats-PCR (ISSR-PCR) polymorphisms, mitochondrial 12S rRNA, 16S rRNA and Cytochrome C Oxidase subunit I (CoI) gene sequences. The phylogenetic tree generated by RAPD analysis is in nearly complete congruence with the classification based on morphophenotypic characters. The 12S and 16S rRNA genes were highly conserved across the nasuta subgroup and revealed only 3 and 4 variable sites respectively, of which only one site was informative. The CoI gene, on the other hand, revealed 57 variable sites of which 25 sites were informative. All the three species of orbital sheen complex were included in a major cluster in the phylogenetic trees derived from mitochondrial gene sequence data consistent with the morphophenotypic classification. The CoI analysis placed two species of frontal sheen complex, D. n. nasuta and D. n. albomicans in two different clades and this is inconsistent with morphological classification. The molecular clock suggested that divergence between the kohkoa complex and the albomicans complex occurred approximately 2.2 MYA, indicating recent evolution of the nasuta subgroup. The higher transition bias in the mitochondrial genes reported in the present study also suggested recent evolution of the nasuta subgroup.     

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.     

Electron microscopic band-interband pattern of polytene chromosomes in Drosophila nasuta albomicans. 2. Salivary gland chromosome 2L.

The band-interband pattern (division 28-52) of salivary gland chromosome 2L in Drosophila nasuta albomicans was studied by light (LM) and electron microscopy (EM) using squash preparations and surface-spread polytene (SSP) chromosome preparations, respectively. LM and EM maps were complied. Based on the digitized EM patterns of five homologous SSP chromosomes a computerized EM chromosome map was plotted. The EM pattern analysis showed a total number of 479 chromosome bands with an almost 83% increase compared with the LM analysis of squash preparations. By extrapolation of the data from 39% of the polytene genome analysed so far in D. n. albomicans, a total number of 2,926 chromosome bands was calculated. This is almost the same number of bands as was calculated earlier for Drosophila hydei using the same SSP chromosome preparation technique. The data in the literature concerning variations in the number of chromosome bands in different Drosophila species, the various chromosome preparation techniques adopted, and the different criteria used for the EM pattern analyses, are discussed.     

Phylogeny and evolution of the Drosophila nasuta subgroup based on mitochondrial ND4 and ND4L gene sequences

The sequences of the mitochondrial ND4 gene (1339 bp) and the ND4L gene (290 bp) were determined for all the 14 extant taxa of the Drosophila nasuta subgroup. The average A + T content of ND4 genes is 76.5% and that of ND4L genes is 83.5%. A total of 114 variable sites were scored. The ND4 gene sequence divergence ranged from 0 to 5.4% within the subgroup. The substitution rate of the ND4 gene is about 1.25% per million years. The base substitution of the genes is strongly transition biased. Neighbor-joining and parsimony were used to construct a phylogeny based on the resultant sequence data set. According to these trees, five distinct mtDNA clades can be identified. D. niveifrons represents the most diverged lineage. D. sulfurigaster bilimbata and D. kepulauana form two independent lineages. The other two clades are the kohkoa complex and the albomicans complex. The kohkoa complex consists of D. sulfurigaster sulfurigaster, D. pulaua, D. kohkoa, and Taxon-F. The albomicans complex can be divided into two groups: D. nasuta, D. sulfurigaster neonasuta, D. sulfurigaster albostrigata, and D. albomicans from Chiangmai form one group; and D. pallidifrons, Taxon-I, Taxon-J, and D. albomicans from China form the other group. High genetic differentiation was found among D. albomicans populations. Based on our phylogenetic results, we hypothesize that D. niveifrons diverged first from the D. nasuta subgroup in Papua New Guinea about 3.5 Mya. The ancestral population spread to the north and when it reached Borneo, it diversified sequentially into the kohkoa complex, D. s. bilimbata, and D. kepulauana. About 1 Mya, another radiation occurred when the ancestral populations reached the Indo-China Peninsula, forming the albomicans complex. Discrepancy between morphological groupings and phylogenetic results suggests that the male morphological traits may not be orthologous.     

Male accessory gland secretory proteins in nasuta subgroup of Drosophila: nature and SDS-PAGE patterns

Male accessory gland secretory proteins in seven members of Drosophila nasuta subgroup were analyzed by SDS-PAGE in combination with different staining techniques such as CBB-R250, Silver, PAS, PAS-silver and zinc-imidazole reverse staining. Based on coomassie blue patterns the protein fractions could be classified in to 3 major groups namely group I, group II as well as group III; with high molecular weight fractions falling into group I and low molecular weight fractions into group III. All the three groups of fractions are post-translationally modified by way of glycosylation and group III fractions are found to be highly glycosylated. Fractions of groups I and II when localized with silver stain and group III fractions when localized with PAS-silver stain appear yellow; suggesting that they are sialoglycoproteins. A 40 kD fraction of group II shows differential staining property with zinc-imidazole stain in closely related species namely D. n. nasuta and D. n. albomicans. Analysis of this protein fraction in F1 males of an interspecific cross revealed that it is synthesized by X-chromosomal gene.     

Male accessory gland secretions in hybrids of Drosophila nasuta nasuta and D. n. albomicans neither show luxuriance nor breakdown

Male accessory gland secretions, which have a role to play in reproduction have been investigated. The number of cells that make up the gland, the quantity of secretions synthesized and the influence of these secretions on fecundity of the female have been studied in D. n. nasuta, D. n. albomicans and their F1 progeny. The results revealed that the hybrid males show a trend towards D. n. nasuta in the synthesis of male accessory gland proteins and the fecundity of the female is influenced more by its genetic constitution rather than the quantity of accessory gland secretions.     

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.     

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.     

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.     

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.     

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).     

Comparative analysis of glue proteins in theDrosophila nasuta subgroup

The patterns of protein fractions from total salivary glands and from glue plugs were compared in seven members of the Drosophila nasuta subgroup by the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The glue protein patterns are member specific concerning the numbers and the electrophoretic mobilities of major and minor glue protein fractions. However, the major fractions of all subgroup members could be grouped into five SDS-PAGE domains according to the homologies of their electrophoretic mobilities, prominence of Coomassie blue staining, and PAS reaction. In all subgroup members, major fractions are involved in posttranslational modifications into larger protein molecules of the final glue. Quantitative estimations of the glue proteins in D. n. nasuta and D. n. albomicans reveal that they constitute between 55 and 60% of the total salivary gland proteins, whereas in D. melanogaster and in D. hydei the fraction is only 32 and 35%, respectively.     

Differential susceptibility of a few members of the nasuta-albomicans complex of Drosophila to paraquat-induced lethality and oxidative stress

The evolution of karyotypically stabilized short-lived (SL) and long-lived (LL) cytoraces in the laboratory have been established and validated through our previous lifespan studies. In the present investigation, we examined the possible reason(s) for the differential longevity among selected members of SL and LL cytoraces, employing the well known paraquat (PQ) resistance bioassay. Exposure of these races to varying concentrations of PQ revealed relatively higher resistance among LL cytoraces than SL cytoraces, as evident by the lower incidence of mortality. Biochemical analysis for endogenous markers of oxidative stress revealed that LL-2 cytorace exhibited lower reactive oxygen species (ROS) and lipid peroxidation (LPO) levels, higher activity levels of superoxide dismutase (SOD), and coupled with higher levels of reduced glutathione (GSH) compared with the levels found in SL-2 cytorace. These findings suggest that the higher susceptibility of SL cytoraces to PQ challenge may be, at least in part, related to the higher endogenous levels of oxidative stress markers. Although the precise mechanisms responsible for the longer longevity among LL cytoraces of the nasuta-albomicans complex of Drosophila merits further investigation, our data suggest that the relatively longer lifespan may be related to the status of endogenous markers that renders them more resistant towards oxidative-stress-mediated lethality, as evident in the PQ assay.     

Male accessory gland secretory proteins in a few members of the Drosophila nasuta subgroup

Male accessory gland secretory proteins in seven members of the Drosophila nasuta subgroup have been analyzed by SDS-PAGE. The study revealed remarkable simplicity in the patterns. The protein fractions, which migrate in three groups, could be categorized as major and minor. The number of major fractions varies from a maximum of eight to a minimum of four. Group I consists of high molecular weight fractions, and group III, low molecular weight fractions. Among different members analyzed, the variation with respect to pattern and the number of fractions are confined largely to group III protein fractions, while group I and II fractions are found to be conserved to a greater extent. These proteins are PAS positive and group III fractions are not sensitive to silver staining. Analysis of these tissue specific proteins in the F₁ and F₂ of interspecific crosses and backcross progeny as well as volume analysis revealed that a 26-kD fraction in D. n. nasuta follows an autosomal pattern of inheritance, while a 55-kD and a 25-kD fraction in D. n. albomicans and a 24-kD fraction in D. n. kepulauana follow an X-linked pattern of inheritance.