Introduction

Drosophila nasuta nasuta (2n = 8) consists of a pair of metacentric chromosomes (chromosome 2), two pairs of acrocentric chromosomes (in ♀, chromosome 3 and X, in ♂, chromosome 3 and a submetacentric Y chromosome) with a pair of basic dots (chromosome 4). D. n. albomicans (2n = 6) has two pairs of metacentric chromosomes (chromosome 2 and X3 in ♀, and Y3 in ♂) with a pair of long dots (chromosome 4). These are a pair of chromosomal races belonging to nasuta subgroup of immigrans group of Drosophila which are morphologically identical, cross fertile and karyotypically dissimilar (Wilson et al. 1969; Kitagawa et al. 1982). Interracial hybridization between these two races yielded karyotypically stabilized four newly evolved: Cytorace 1, (♂, 2n = 7; ♀, 2n = 6), Cytorace 2 (in both ♂ and ♀, 2n = 6), Cytorace 3 (♂, 2n = 7; ♀, 2n = 8), and Cytorace 4 (in both ♂ and ♀, 2n = 8), after F20–F50 generations through a phase of karyotypic mosaicism (Ramachandra and Ranganath 1986, 1990). Further, interracial hybridization among the newly evolved four Cytoraces, and the original parental races D. n. nasuta and D. n. albomicans, resulted in the formation of 12 new Cytoraces 5–16. All these karyotypically different recombined genomes were termed as nasuta-albomicans complex (NAC) of Drosophila (Ramachandra and Ranganath 1996). Based on the karyotypic homology, 16 Cytoraces were grouped under six types (Tanuja et al. 2003).To evolve these Cytoraces in nature it would have taken 1000s of years, whereas, in the laboratory it has taken a decade. During the evolution of these karyotypes some of the parental chromosomes were eliminated and some were retained. The members of NAC of Drosophila exhibited noticeable racial divergence in the earlier various studies such as, cytogenetics, morphometric, morphophenotypic, fitness, mating preferences, isozymes, dietary restriction (DR), and life span analysis (Ranganath 2002; Harini and Ramachandra 2003; Ranjini and Ramachandra 2009). Further studies on the members of NAC of Drosophila established the evolution of short-lived and long-lived Cytoraces in the laboratory (Ranjini and Ramachandra 2009). The lifespan of D. n. nasuta, D. n. albomicans, and two short-lived races, Cytorace 3 and Cytorace 15 extended significantly in response to DR when compared to the standard diet. On the other hand, two long-lived Cytoraces (in standard diet), Cytorace 9 and Cytorace 16 did not show any marked difference in their lifespan in response to dietary restriction. We have also reported the differential response to hormesis by laboratory evolved short-lived and long-lived Cytoraces of NAC of Drosophila (Ranjini and Ramachandra 2011). These members of NAC of Drosophila were not subjected for molecular analysis.

Oxidative stress has been widely implicated as an important factor in the aging process. In D. melanogaster, Sod1 and Sod2 comprise the total complement of Superoxide dismutase (Sod). Cu, Zn superoxide dismutase, Sod1 is an eukaryotic cytoplasmic enzyme that performs a protective function in the cell by scavenging superoxide radicals and dismutating them to hydrogen peroxide and molecular oxygen (McCord and Fridovich 1969). Removal of the superoxide radical by Sod and hydrogen peroxide by catalase and glutathione peroxidase prevents formation of the very reactive hydroxyl radical (OH), which is postulated to be responsible for much of the cellular damage (Seto et al. 1987). Mitochondrially localized manganese-binding superoxide dismutase (Sod2) is thought to play an important front-line defensive role against aging-related oxidative stress, because mitochondrial respiration is the principal source of reactive oxygen within cells (Kriby et al. 2002). They have reported that ablation of mitochondrial Sod2 through expression of a GAL4-regulated, inverted-repeat Sod2 RNA-interference transgene in an otherwise normal animal causes increased endogenous oxidative stress, resulting in the loss of essential enzymatic components of the mitochondrial respiratory chain and the tricarboxylic acid cycle, enhancing sensitivity to applied oxidative stress, causing early-onset mortality in young adults. Extensive genetic analysis of Sod1 has revealed the critical role of this enzyme in many diverse aspects of the biology of Drosophila and is present in all eukaryotes having fairly high rate of evolution and also conserved across a taxa, can be used for phylogenetic analysis (Kwiatowski et al. 1994). Several studies have shown that it is involved in biological process such as determination of adult lifespan and response to oxidative stress (Orr and Sohal 1994; Parkes et al. 1998; Sun and Tower 1999).

Another gene Rpd3, (http://flybase.org/) is a histone deacetylase protein coding gene from D. melanogaster involved in many unique biological processes such as negative regulation of cellular biosynthetic process, neuron differentiation, embryonic pattern specification, determination of adult lifespan, regulation of RNA metabolic process, coenzyme catabolic process and cellular respiration. Rogina and Helfand (2004) have reported that under DR conditions, activity of the histone deacetylase, Rpd3 is decreased and the dSir2-dependent pathway is activated indicating that Rpd3 and Sir2 are the key players in the lifespan extension and DR.

The members of NAC of Drosophila, short-lived and long-lived Cytoraces had shown differential responses to DR and oxidative stress. Therefore, we have made an attempt to clone and characterize two candidate genes, Sod1 and Rpd3, to find out any changes at gene and protein levels and to also understand the phylogenetic relationship of the members of NAC of Drosophila with other species of Drosophila.

Materials and Methods

Experimental Stocks

D. n. nasuta (Coorg, South India, 201.009).

D. n. albomicans (Okinawa, Texas collection, USA 3045.11).

Short-lived Cytoraces: Cytorace 3 (C3), and Cytorace 15 (C15) (Ramachandra and Ranganath 1990, 1996).

Long-lived Cytoraces: Cytorace 2 (C2), Cytorace 9 (C9), Cytorace 11 (C11), and Cytorace 16 (C16) (Ramachandra and Ranganath 1986, 1996).

Isolation and Cloning of Sod1 and Rpd3

Genomic DNA of D. n. nasuta, D. n. albomicans, Cytoraces 2, 3, 9, 11, 15, and 16 was isolated by using Berkeley Drosophila Genome Project (BDGP) protocol with slight modifications. The concentration and purity of genomic DNA was checked using a Biophotometer.

The two sets of primers used for Sod1 are as follows: Forward primers (1) 5′-ATGGTGGTTAAAGCTGTTTGCG-3′, (2) 5′-GTCGTCTGGTACACCCGTTA-3′, and Reverse primers (1) 5′-TAACGGGTGTACCAGACGAC-3′, (2) 5′-TTAGATCTTGGCGATGCCG-3′. The primers used for Rpd3 were: Forward primers (1) 5′-ATGCAGTCCCATAGTAAAAAAC-3′, (2) 5′-ATGCAATCTCATAGCAAAAAGC-3′, and Reverse primers (1) 5′-CTTGATCTTCTCCAGATA-3′, (2) 5′-TCAAATGTTGTTCTCCTTGGCG-3′. PCR amplifications were carried out with the various combinations of primers and except for the annealing temperature, all the other steps remained the same. After amplification, the sizes of PCR products were checked and cloned using InsTAclone™ PCR Cloning Kit from Fermentas (Catalogue No. K1214).

Analysis of Cloned Genes

Sequence analyses of cloned genes, Sod1 and Rpd3 were done by using CLUSTAL W, NCBI BLAST, ACCELRYS GENE, MEGA version 4 (Tamura et al. 2007), and DnaSP V5 (Librado and Rozas 2009). The Tajima’s test (Tajima 1989) was performed to distinguish whether DNA sequences are evolving randomly (neutrally) or evolving under a non-random process, including directional/balancing selection, demographic expansion or contraction, genetic hitchhiking or introgression by utilizing transition and transversion data and the ratio at first, second and third position of codon. In the Tajima’s test, p value less than 0.05 was used to reject the null hypothesis of equal rates between lineages.

Codon usage bias was analyzed in both the genes by using DnaSP v5. The analysis included Effective Number of Codons-ENC (Wright 1990) and Codon Bias Index (CBI) which is a measure of the deviation from the equal use of synonymous codons. CBI values range from 0 (uniform use of synonymous codons) to 1 (maximum codon bias), Scaled Chi Square (SChi2) (Shields et al. 1988) is a measure based on the Chi square statistics; i.e., based on the difference between the observed number of codons and those expected from equal usage of codons.

Assay on Rpd3 and Sir2 Proteins

Quantification of Rpd3 and Sir2 was done through ELISA by using primary antibodies—Rpd3 (dN-12): sc-30557 and—Sir2 (dF-16): sc-27476 (Santa Cruz Biotechnology, INC.). Sample preparation of both male and female flies (each 20) of Cytoraces 3, 15, 9, and 16 were fed with the standard diet (~15 mg of yeast) and restricted diet (~2 mg of yeast) was made by using the modified protocol of Yu et al. (1999). ELISA was performed by slight modification of protocol from http://www.abcam.com/. For ELISA experiment three replicate assessments were carried out with 40 flies (20 flies each for both male and female) of Cytoraces, 3, 15, 9 and 16. Standard curve was drawn from the data obtained from the serial dilutions with concentration on the x-axis versus absorbance on the y-axis. The concentration of test samples was obtained by plotting the optical density value perpendicular to the standard curve. Mean concentration ± Standard error was calculated using SPSS software and the graph was generated accordingly. One-Way ANOVA was carried out using SPSS software version 10.0 to understand the significance. A value of less than 0.05 was set as the minimum level of significance.

Results

Isolation and Cloning of Sod1 and Rpd3

In the present study, degenerate primers specific for Sod1 and Rpd3 were created by using the conserved sequences of 12 Drosophila species genomes and were PCR amplified. The amplified clones of Sod1 and Rpd3 were cloned and sequenced in all the members selected for the present study. The two exons of Sod1 were found interspersed with an intron and a number of deletions and insertions were found in the other members of NAC of Drosophila (Fig. 1a). Schematic representation of the organization of Rpd3 and the primers designed for amplifications are presented in Fig. 1b. The accession number of cloned sequences of Sod1 and Rpd3 of the above members under study and the 12 Drosophila species genome sequenced is presented in Table 1.

Fig. 1
figure 1

a Schematic representation of genomic organization of Sod1 in D. n. nasuta and other members of NAC of Drosophila. Filled blocks indicate insertion and empty blocks indicates deletion. The bold arrows indicate primer regions. The highlighted nucleotide in each member represents the substitution while treating D. n. nasuta as standard. b Schematic representation of organization of Rpd3. Arrows indicate the direction of primers used to clone Rpd3 in the members of NAC of Drosophila under study

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Table 1 List of Drosophila species and members of nasuta-albomicans complex (NAC) of Drosophila used for the analysis of Sod1 and Rpd3 with their respective NCBI GenBank accession numbers
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Analysis of Sod1

The synonymous substitutions in entire transcript of Sod1 among Drosophila stocks under study were analyzed (Supplementary Table 1). D. mojavensis showed the highest number of substitutions with all the Drosophila species. D. n. nasuta, D. n. albomicans and the Cytoraces showed the highest number of synonymous substitutions with D. obscura group, and the lowest with D. sechellia and D. yakuba of D. melanogaster subgroup. The maximum number of synonymous substitutions was seen in D. n. nasuta and D. n. albomicans as compared to C2, C9, C16. When C2, C9, and C16 were compared with C3, C11, and C15, only one synonymous substitution was observed. The nonsynonymous substitutions in entire transcript of Sod1 between Drosophila stocks under study was analysed (Supplementary Table 2). There were no nonsynonymous substitutions among D. n. nasuta, D. n. albomicans and Cytoraces under study; however, these races showed the highest number of nonsynonymous substitutions with D. ananassae and the lowest with D. grimshawi.

Figure 2a depicts the codon usage bias in Sod1 of all the Drosophila stocks under study. The highest Effective number of codons (ENC) and Scaled Chi square (Schi2) were observed in D. mojavensis and D. pseudoobscura respectively, while the highest Codon bias index (CBI) and G+C content at third codon position (G+C3s) were seen in D. yakuba. Among the members of NAC of Drosophila, ENC was the highest, while CBI, Schi2 and G+C3s were the lowest in C2, C9, and C16. Table 2 presents the Tajima’s relative rate test based on transitions and transversions change in Sod1 by using D. melanogaster as an outgroup. Transition and transversion changes were not observed between D. n. nasuta and D. n. albomicans. The highest numbers of bases in intron of Sod1 was observed in D. melanogaster species group ranging from 708 to 731 bp, and the lowest number of bases in intron were observed in D. obscura group (Table 3). Compared to D. n. nasuta, all these Cytoraces have shown insertions and deletions in intronic region as depicted in Fig. 1a.

Fig. 2
figure 2

Codon Usage bias in Sod1 (a) and Rpd3 (b) of all the 12 Drosophila species and 8 members of NAC of Drosophila. CBI Codon bias index, SChi2 Scaled Chi square, G+C3s = G+C content at third codon position

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Table 2 The equality of evolutionary rate between D. n. nasuta a and Drosophila species/races tested using D. melanogaster c as an outgroup in Tajima’s relative rate test in MEGA4 for Sod1
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Table 3 Total number of nucleotide bases lost and gained in the intron of Sod1 and Rpd3 in Drosophila species when compared with D. n. nasuta
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The multiple alignment of Sod1 protein for all the Drosophila species under study was obtained by Accelrys gene software (Fig. 3). There were no aminoacid changes between D. n. nasuta and D. n. albomicans. However, both of them showed maximum amino acid changes (22) with D. ananassae. Similar to D. n. nasuta and D. n. albomicans, D. pseudoobscura and D. persimilis being in the same group had not shown any aminoacid change. These aminoacids changes showed change in helix, sheet and band in D. n. nasuta when compared to other Drosophila species. The chemical and functional changes in the aminoacids display the secondary structure changes (data not shown).

Fig. 3
figure 3

Multiple alignment of Sod1 protein in 14 Drosophila species using Accelrys gene software. Dots in the alignment denote identical aminoacids in different species

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Analysis of Rpd3

The schematic representation of Rpd3 having four exons and three introns is presented in Fig. 1b. The synonymous substitutions in entire transcript of Rpd3 among Drosophila species under study is shown in Supplementary Table 3. D. willistoni showed the highest substitutions rate with all the Drosophila species. The total length of the Rpd3 in D. n. nasuta was 2138 bp and four insertions were seen in the intron of C9, while all other races showed similar intron length when compared to D. n. nasuta. D. n. nasuta, D. n. albomicans and Cytoraces under study showed the highest synonymous substitutions with D. wllistoni, and the lowest with D. grimshawi. When D. n. nasuta and D. n. albomicans were compared with the other members of NAC of Drosophila, C9, C15, and C16 showed 22, 1 and 2 bp synonymous substitutions respectively. The nonsynonymous substitutions in the transcript of Rpd3 between Drosophila species under study is presented in Supplementary Table 4. Among the members of NAC of Drosophila, C3, C15, and C16 showed 1, 3 and 2 bp substitutions respectively. The members of NAC of Drosophila showed the highest number of substitutions with D. mojavensis and the lowest with D. virilis.

Figure 2b depicts the codon usage bias in Rpd3 of all the members under study. The highest ENC was observed in D. willistoni and the lowest in D. ananassae, D. virilis, and D. ananassae showed the highest Schi2 and G+C3s. D. willistoni showed the lowest CBI and Schi2 than other Drosophila species. Among the members of NAC of Drosophila, all have shown almost similar codon usage values than other Drosophila species under study. Table 4 represents the data obtained by Tajima’s relative rate test based on transitions and transversions change in Rpd3 by using D. melanogaster as an outgroup. However, significant rejection of null hypothesis was seen between D. n. nasuta and other Drosophila species. Transition and transversion changes were not observed between D. n. nasuta versus D. n. albomicans, C2 and C11. The highest and the lowest number of bases in intron of Rpd3 were observed in D. virilis and D. grimshawi respectively than other Drosophila species. The intron length of D. n. nasuta was longer than D. obscura and D. grimshawi, but shorter than other Drosophila species under study (Table 3). Among the members of NAC of Drosophila, the intron length of C9 was four bases more (585 bp) than all other members of NAC of Drosophila (581 bp).

Table 4 The equality of evolutionary rate between D. n. nasuta and Drosophila species/Races tested using D. melanogaster c as an outgroup in Tajima’ relative rate test in MEGA4 for Rpd3
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As indicated in Table 5, the members of NAC of Drosophila under study had a shorter length of protein with 518 aminoacids compared to all other Drosophila species. D. n. nasuta and D. n. albomicans did not show any aminoacid changes between them. However, both of them showed more amino acid changes with D. mojavensis and less with D. virilis. Due to this, changes in helix, sheet and band were observed in D. n. nasuta when compared to other Drosophila species. The chemical and functional changes in the aminoacids display the secondary structure changes (data not shown). Three amino acid changes, A472 V, N485D and S492I were seen in C15. The split band was seen in turn region of secondary structure of Rpd3 in C15. In C16, two aminoacid changes, Q109P and R429G were noticed, due to which, in helix region, there was one bigger band and loss of one band, while, in turn region, one extra band was noticed (data not shown). There was only one aminoacid change, G506S in C3 which did not cause any secondary structure variation.

Table 5 Total number of amino acids in Rpd3 of D. n. nasuta, when compared to different Drosophila species
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Figure 4 represents evolutionary relationships of aminoacid sequence in Sod1 of all the members under study with Aedes aegypti as an outgroup. The members of D. melanogaster subgroup formed a separate lineage, where D. melanogaster, D. simulans and D. sechellia formed a single clade giving rise to sister clades comprising of D. erecta and D. yakuba. D. ananassae formed a sister clade with two members of D. obscura. This clade is then branched with D. willistoni. All the eight members of NAC of Drosophila formed separate clade branched with D. grimshawi, and they showed 72 % homology with A. aegypti. Figure 5 represents evolutionary relationships of aminoacid sequence in Rpd3 of all the members under study with A. aegypti as an outgroup. A big clade was formed comprising of D. melanogaster group, D. ananassae and D. obscura group. The Nei-Gojobori tree in track with D. willistoni formed two clades comprising of D. grimshawi and D. virilis, and another clade comprised of eight members of NAC of Drosophila, where D. n. nasuta, D. n. albomicans, C2, C9, C11and C16 shared single clade, and C3, and C15 formed separate clades. The members of NAC were closer to D. grimshawi, and they showed 89 % homology with A. aegypti. D. mojavensis with more number of aminoacid changes formed a distinct clade connected to both subclades.

Fig. 4
figure 4

The evolutionary distances were computed using the Nei-Gojobori method and are in the units of the number of aminoacid substitutions per site in Sod1. The evolutionary history was inferred using the Neighbor–Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Phylogenetic analyses were conducted in MEGA4

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Fig. 5
figure 5

The evolutionary distances were computed using the Nei-Gojobori method and are in the units of the number of aminoacid substitutions per site in Rpd3 per sequence. The evolutionary history was inferred using the Neighbor–Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Phylogenetic analyses were conducted in MEGA4

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Estimation of Rpd3 and Sir2 proteins

Mean concentration ± Standard error of Rpd3 and Sir2 protein in C3, C9, C15, and C16 from the three replicates was presented in Table 6. The mean concentration of Rpd3 protein in short-lived Cytoraces, C3 and C15 were significantly higher in control than DR (p < 0.005), whereas, there was no remarkable difference between control and DR in long-lived Cytoraces, C9 and C16 (Fig. 6a). The mean concentration of Sir2 protein in short-lived Cytoraces, C3 and C15 was higher in DR control (p < 0.005). However, there was no remarkable difference between control and DR dietary restriction in long-lived Cytoraces, C9 and C16 (Fig. 6b). In both Rpd3 and Sir2, there were no significant differences between the short-lived Cytoraces, C3 and C15 and long-lived Cytoraces, C9 and C16.

Table 6 Mean concentration ± Standard error of Rpd3 and Sir2 protein in C3, C9, C15, and C16 of nasuta-albomicans complex of Drosophila
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Fig. 6
figure 6

Mean ± S.E concentration of Rpd3 (a) and Sir2 (b) protein in two short-lived Cytoraces, 3, 15 and long-lived Cytoraces, 9 and 16 of NAC of Drosophila quantified through Enzyme-Linked Immunosorbent Assay (ELISA)

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Discussion

The role of hybridization in evolution has been debated for over a century. Hybridization and introgression have been neglected in evolutionary biology since the 1940s. The natural hybridization can affect the evolutionary history of the groups in which it occurs (Arnold 1996). The recombinant hybrids are less fit on average; some gene combinations may be fitter than the parents, even in the parental environment (Barton 2001). Recent molecular genetic studies indicate that hybridization is surprisingly frequent in natural populations and it may allow populations to regain traits that have been lost and possibly to replace damaged alleles with functional copies from related species (Rieseberg 2009). Hybrid or “recombinational” speciation is one of the suggested pathways by which new species might arise rapidly via hybridization between chromosomally or genetically divergent parental species (Ungerer et al. 1998). In the present study, the hybrids, karyotypically stabilized 700 generations old, cytoraces evolved through “recombinational” speciation (Harini and Ramachandra 2003) and their parents were analysed to check the molecular divergence if any in two genes Sod1 and Rpd3.

Sod1 and Rpd3 in Drosophila Species

Despite the large number of genetic and genomic resources, little is known concerning the phylogenetic relationships, ecology, and evolutionary history of all but a few species of nasuta subgroup of immigrans group of Drosophila (Katoh et al. 2007). In the present study, the evolution of Sod1 and Rpd3 in Drosophila lineage is very interesting. With respect to synonymous substitutions in Sod1, D. n. nasuta has shown closeness with D. melanogaster group, whereas Rpd3 showed consistent positioning of D. n. nasuta with virilis-repleta group as of nonsynonymous substitutions. Both Sod1 and Rpd3 data revealed the clustering of D. n. nasuta with virilis-repleta group with respect to transition and transversion, synonymous versus nonsynonymous substitutions, codon bias, and GC content at third position of codon suggesting the weak purifying selection and a large fraction of synonymous changes behaving as neutral. These findings indicate that selection is relaxed in the Drosophila lineage and strongly support the nearly neutral character of nonsynonymous mutations.

Coding Sequence of Sod1 and Rpd3

Throughout the Drosophila lineage, only one aminoacid deletion is observed in Sod1 of D. obscura group, whereas, in Rpd3 more InDel diversity have been observed across the species. D. n. nasuta has the less aminoacids than other species. Even though more number of InDels are observed in Rpd3, the substitution of aminoacid has not caused much change in the secondary structure and physiochemical features as compared to Sod1. This could be due to the substitutions of amino acid in the nonconserved regions of proteins that are nearly neutral or slightly positively selected (Freese and Yoshida 1965; Margoliashand Smith 1965; Zuckerkandl 1965).

Introns in Sod1 and Rpd3

Hawkins (1988) stated that in contrast to the vertebrates, which typically have large genomes and genes punctuated by large introns, dipterans have compact genomes with few and small introns. Kwiatowski et al. (1994) reported evolutionary favoring deletion of the second intron of Sod1 in the Drosophila lineage, and suggested that there is a general trend in the evolution of the Diptera favoring intron size reduction and even elimination. Similarly, Roy and Penny (2007) noted that certain ancient lineages underwent big bursts of intron gain while subsequent lineages experienced a precipitous decline in intron gain rates. The intron DR of Cu, Zn Sod1 in the two very closely related species, D. melanogaster and D. simulans found that nucleotide deletion/insertion is much more prevalent than substitution. In congruence with this, in the present study, reduction in intron length has been noticed in Drosophila lineage. The intron diversity places subgenus Sophophora before subgenus Drosophila. Surprisingly, D. pseudoobscura, D. persimilis and D. willistoni which forms closer clade with D. melanogaster with nucleotide substitutions at coding region have half the length of intron which is the smallest intron size as compared to all other Drosophila species under study indicating closeness of D. n. nasuta to these species. However, D. n. nasuta with the intron length of 408 bp falls after virilis-repleta group in the Drosophila lineage which is consistent with nucleotide substitution rate at coding region. The evolution of Rpd3 intron is very fascinating with respect to variations in length in different Drosophila species under study. The maximum length of intron was seen in D. virilis and all species under study except D. persimilis, D. pseudoobscura, and D. grimshawi showed an increased intronic length.

Evolutionary Dynamics of Sod1 and Rpd3 in Few Members of NAC of Drosophila

D. n. albomicans and other members of NAC of Drosophila have not shown any nonsynonymous substitutions in Sod1. Though synonymous substitutions are not seen between D. n. nasuta and D. n. albomicans, on comparison to Cytoraces, C2, C9, and C16 formed separate branching and C3, C11, and C15 formed another branch with specific synonymous substitutions. The C2, C9, and C16 with low CBI than any other Drosophila under study, have shown minimum codon bias. The data by Tajima’s relative test based on transition and transversion supported the neutral selection between D. n. nasuta and the members of NAC of Drosophila. With respect to Rpd3, C9 has shown the highest synonymous substitutions with other members, and, C3, C15, and C16 have shown prominent nonsynonymous substitutions with aminoacid changes which affected the secondary structure of Rpd3 protein. The Rpd3 of C3 and C9 showed more codon bias than any other Drosophila species under study. The Rpd3 of C9 is the one among NAC of Drosophila which is undergoing purifying selection.

Based on Sod1 phylogeny reported by Kwiatowski et al.(1994), within ~2.3 MY, number of synonymous substitutions in Sod1 between D. melanogaster and D. simulans are 11 and the difference in ENC, CBI and G+C3s are 3, 2.2 and 2 % respectively. There are no nonsynonymous and aminoacid substitutions between them. Interestingly, among the members of NAC of Drosophila C2, C9, and C16 as well as C3, C11, and C15 have acquired 6 and 5 synonymous substitutions at coding region when compared to D. n. nasuta and D. n. albomicans. The codon usage differences are more between D. n. nasuta, D. n. albomicans and members of NAC of Drosophila with 4, 8 and 1 % of ENC, CBI and G+C3s respectively, when compared to D. melanogaster and D. simulans.

On the other hand, the members of NAC of Drosophila have shown more substitutions at intron region and race specific deletions and insertions indicating the rapid divergence of intron within ~25 years as compared to D. melanogaster and D. similans. As per rough estimation, it is 4.78 synonymous mutations per MY which have occurred between the D. melanogaster and D. simulans, while it is 6.5 synonymous mutations within ~25 years in the Cytoraces. With respect to Rpd3, D. melanogaser and D. simulans have shown only 1–2 aminoacid changes with ~2.3 MY of divergence, while, within the span of ~25 years 1, 2, and 3 aminoacid changes are observed in C3, C16, and C15 respectively which is striking evident for rapid evolution.

Interestingly, as indicated by Bachtrog (2006), the most likely estimate of divergence time between D. albomicans and D. nasuta is about <0.5 MY. In both the genes understudy, no single nonsynonmous substitutions are observed between them. However, the hybrid cytoraces evolved by them have shown specific nonsynonymous substitutions and aminoacid change which is de novo in the context of molecular evolution.

According to Parmley and Hurst (2007) it is seductive to think that, a point mutation in a protein-coding exon that changes the DNA but not the protein sequence, would have no discernible fitness consequences. Indeed, even a decade ago such an assumption looked relatively sound. Since then, however, there has been a plethora of evidence to indicate that synonymous mutations can, indeed, have important fitness consequences, with over 40 genetic diseases now associated with such ‘‘silent’’ mutations (Chamary et al., 2006). Similarly, in the karyotypically evolved Cytoraces, the more number of synonymous mutations might be playing a role in fitness of Cytoraces.

These results are consistent with the hypothesis that a significant fraction of divergence at nonsynonymous and all noncoding sites has been driven to fixation by positive selection, and is in agreement with Andolfatto (2005). This could also be the reason where the prominent aminoacid substitutions in C15 and C16 have different effects on their lifespan.

Role of Rpd3 and Sir2 in the Regulation of Lifespan

Sirtuin 2 (Sir2) was the first gene to be found in budding yeast whose homolog in mammals is known as SIRT1 (SIR2L1 or Sir2α) and, since then, members of this highly conserved family have been found in nearly all organisms studied (Frye 2000). Sirtuins are hypothesized to play a key role in an organism’s response to stresses and to be responsible for the lifespan-extending effects of calorie restriction (Sinclair and Guarente 2006). Reduction of Rpd3 expression increases RNA levels of the histone deacetylase, Sir2, the direct overexpression extends lifespan (Rogina and Helfand 2004). In this study, the levels of Rpd3 which is higher in the control with standard diet has reduced in DR treated flies of C3 and 15 and the Sir2 levels has increased in them in DR treatment as compared to control. This implicates that higher Rpd3 levels in C3 and C15 is one of the main cause for their shorter lifespan. In contrast, low Rpd3 levels and no change in the Sir2 levels of long-lived C2 and C9 showed that they do not require additional stimuli from DR to increase Sir2 protein as it is already in adequate level in them for extended longevity. In addition to this, short-lived Cytoraces showed lower concentration of the endogenous levels of Sod than long-lived Cytoraces (Ranjini et al. 2011).

Thus, the members of NAC of Drosophila offer a unique opportunity to understand both evolutionary and molecular dynamics of aging, since they are the hybrid recombination products. The Cytoraces have exhibited not only specific changes in Sod1 and Rpd3, but also pronounced in the levels of synthesis of these proteins, which indicates rapid evolution of these Cytoraces in the laboratory. Further, these Cytoraces become a model system to understand the process of anagenesis.