Functional requirements driving the gene duplication in 12 Drosophila species

Young duplicate genes in 12 Drosophila genomes

Interestingly, three young duplicate gene families were detected respectively in complex expansions occurring in 11 and 12 species (Table 1). Although the six families were classified as the complex expansion type, species-specific duplication events also were found in all of these families (Additional file 1: Figure S1), especially independent duplications after the species differentiation in many species. For example, 15, 16 and 91 species-specific duplicate clades across all of the 12 species were detected in family 2,419, 7,827 and 8,177, respectively. In the other 3 families, 8, 11 and 145 species-specific duplicate clades were distributed in 8, 11 and 11 species, respectively. In addition, some lineage-specific duplications were also found in these families. All these results suggested that these duplicate gene families were likely to have been shaped by convergent evolution due to independent expansions in many species after the species differentiation.

Distribution of young duplicate genes on chromosomes

To explore the distribution of the young duplicate genes on the chromosomes, stochastic simulations were implemented using the observed gene numbers with 10,000 times random repeats. The chromosomal distribution was significantly non-random (P < 0.05); for example, chromosome 2 (2L & 2R), 3 (3L & 3R) and X contained a mass of young duplicate genes (Additional file 2: Figure S2). Figure 1 shows graphs representing these simulation results. Furthermore, the windows in Figure 1 with the observed number larger than the upper level of the confidence intervals correspond to hotspot regions on the chromosomes for the young duplications.

As shown in Figure 1, hotspot regions were found in all three types of young duplications, especially in species-specific expansions (Figure 1A), e.g. on chromosome 2 in D. grimshawi and on chromosome 3 in D. yakuba. In contrast, few hotspot regions were found in species such as in D. ananassae and D. melanogaster. Interestingly, some duplication hotspot regions were shared by more than one species in the species-specific expansions (marked by dash lines in Figure 1A), also suggesting convergent evolution of these genes among different species. However, none of shared hotspot region were detected in lineage-specific duplications, although the two species had similar trend lines which were generated by the observations and simulation numbers (Figure 1B). In complex expansions, few hotspot regions were detected along the chromosomes (Figure 1C).

Functional preference of young duplicate genes

To further reveal the genetic characteristics of the young duplications, the domains of the duplicates were detected using Pfam searches. Subsequently, the protein domains were counted in each species. For species-specific expansions, a total of 1,277 different domains were found in 12 species, averaging 106 protein domains in each species (Additional file 3: Table S1). Interestingly, approximately 84% of protein domains occurred only once or twice, suggesting that most domains were unique. However, the frequency of some protein domains, such as DUF1676 in D. willistoni, annexin and dynein_IC2 in D. melanogaster, inositol_P and PAP2 in D. yakuba, were high in one species but low (0 or 1) across the other 11 species, suggesting that these species-specific duplicate proteins might be driven by adaptive evolution in each species. Furthermore, some protein domains occurred in a lineage-specific manner, although they were detected in the species-specific expansion events. For example, the expansion of domains Gb3_synth and Gly_transf_sug shared by D. mojavensis and D. virilis, were greatly expanded only in these two species. A similar situation was also observed in the alpha-amylase domain, which occurred in two closely related Drosophila species, D. sechellia and D. simulans. Although different types and numbers of protein domains were examined in each species, we still found that approximately 4% of the domains appeared simultaneously in ≥ 6 species. Prominent examples of these protein domains were trypsin, p450 and WD40, which were detected in 12, 11 and 11 species, respectively (Additional file 3: Table S1). These proteins are all important in response to environmental stimuli [28, 29]. To investigate whether the high-frequency domains also occupied in large numbers in each species or vice versa, we examined the occurrence-frequency of the top 20 domains in 12 genomes. Interestingly, these high-frequency domains also had a large number of copies in the related species (Figure 2A), suggesting that these high-frequency duplicated proteins play an important role in the evolution of these species.

An identical approach was also used for the gene families of lineage-specific expansions and complex expansions. Our results showed that most gene families contained limited protein domains, although the number of the same domain was always different. However, some protein domains were still undergoing rapid expansion independently in many species, e.g. the six shared duplicate gene families in complex expansions occurring in 11 and 12 species (Table 1; Figure 2B and C). Furthermore, Ank, EGF, Peptidase_M17 and Peptidase_M17_N, which were all conserved and widespread domains in organisms for survival, exhibited high frequencies in 11 species (Figure 2B). In the shared expansion events of 12 species (Figure 2C), 12 of the top 20 protein domains such as histones, HSP70, Lys, co-occurred in 12 species. Numerous previous studies have shown that these protein domains are closely related to stress responses and pathogens in the environment, for example, histones are involved in stress responses [30], HSP70 protects cells from stress [31], and Lys (lysozyme) acts as a bacteriolytic enzyme by hydrolyzing bacterial cell walls [32], suggesting that these shared duplications play an important role in adaption to ecological factors and environmental changes in Drosophila.

Nonsynonymous and synonymous substitution between paralogs and orthologs

The ratio of nonsynonymous to synonymous nucleotide substitution (Ka/Ks) is considered as an important parameter indicating the strength of functional constraints. The smaller the Ka/Ks ratio is, the stronger the functional constraints are. The 12 Drosophila whole-genome data offer us unprecedented opportunity to explore the different selection pressure between paralogs and orthologs. Therefore, we examined Ka/Ks ratios for paralogs and orthologs in each duplicate gene family.

The average Ka/Ks between paralog gene pairs or ortholog gene pairs in these young duplicate gene families were 0.626 and 0.445, respectively, which was significantly (P < 0.05) larger than the genome-wide Ka/Ks (0.218) between ortholog pairs, suggesting relaxation of the functional constraints in the young duplicate gene families. Figure 3 shows that most of the gene pairs (91.2%), including paralogs and orthologs, had Ka/Ks ratios less than 1, demonstrating that most young duplicate genes were under purifying selection. However, there were still 229 and 82 gene pairs with Ka/Ks ratios greater than 1 for paralogs and orthologs, respectively, indicating that some young duplicate genes are driven by positive selection. However, in the gene pairs with Ka/Ks values exceeding 1, many values were just slightly greater than 1 and only few pairs were detected to have Ka/Ks ratios significantly greater than 1.

Based on the strengths of boxes and whisker lines in species-specific expansions in Figure 3, it was clear that Ka/Ks between paralogs had a broader dispersed distribution, larger median and quartile values than orthologs, indicating that paralogs had higher Ka/Ks than orthologs. Similar results were also obtained in lineage-specific expansions (Figure 3D), with the exception of D. sechellia vs. D. simulans and D. persimilis vs. D. pseudoobscura. To further ensure that the Ka/Ks of paralogs were significantly greater than those of orthologs, we conducted paired t-tests. Apart from four pairs, the other Ka/Ks ratios of paralogs and orthologs exhibited highly significant (P < 0.01) or significant (P < 0.05) differences (Additional file 4: Table S2). All the results showed that paralogs had significantly higher Ka/Ks than orthologs and indicated that paralogs are subject to weaker functional constraints and faster evolutionary processes than orthologs.

Linear analog was also performed between the mean Ka/Ks of paralogs and orhtologs (Additional file 5: Figure S3). In the same family, the dot above the trend lines (slope = 1) indicated that paralogs have higher evolutionary rates than orthologs. Interestingly, it was clear that some dots lay far away the trend lines. Detection of the protein domains of these dots (Additional file 4: Table S2) showed that most of the domains detected in the genes of upper dots, such as Coesterase [33, 34], Turandot [35] and MIP [36] were involved in stress responses. These results also suggested that the young duplicates result from adaption to the environment both in species-specific and lineage-specific expansions.

Evolutionary analysis of young duplicate genes across 12 Drosophila species

To detect the timing of recent duplication in each species, the Ks values were calculated. We adopted the common assumption that Drosophila species experienced about 10 generations/year and that the single nucleotide mutation rate was 5.8 ×10^-9 mutations per generation [37]. Furthermore, only Ks values lower than 1.0 were kept to avoid the saturation of nucleotide substitutions.

Convergent evolution of young duplicate genes across the 12 Drosophila species

Convergent evolution plays an important role in biological adaptation, by which distantly related organisms independently evolve similar structures or functions in order to adapt to similar environments or ecological niches [39], such as, the specialized oxygen transport function of oxygen transport hemoglobins in jawed and jawless vertebrates [40] and the similar substrate of apolipoprotein (a) in humans and hedgehogs [41]. Although there are many other theories could explain the evolutionary process of young duplicates, such as genomic drift proposed by Nei [42, 43], convergent evolution might be more convincible for two evidences detected in our study.

In our study, the phylogenetic trees (Additional file 1: Figure S1) and the chromosomal distributions (Figure 1) of young duplicate genes also provide evidence of convergent evolution. Six young duplicate families were found in complex expansions occurring in 11 or 12 species with many species-specific duplication clades across these 11 or 12 species. Interestingly, the phenomenon that the independent duplicates with similar function preference are under convergent evolution has also been previously reported both in animals and plants. For example, histone proteins are highly alkaline proteins in eukaryotic genomes which package DNA into nucleosomes [44] and independent convergent evolution has produced striking similarities between plant and animal histones [45]. Another example of similar genetic characteristics shared by distant species is the digestion function of lysozymes (Lys domain) in animals. Lysozymes are usually present in tears, saliva and other bodily fluids and have independently been recruited to the stomach and play important roles in enzyme functions across vertebrates [46]. Furthermore, some duplication hotspot regions were shared by more than one species across their chromosomes in species-specific expansions. Interestingly, conserved duplication hotspots have also been previously detected between D. melanogaster and D. simulans[47]. Similar function preference and identical hotspot regions arising from independent duplications suggest that the young duplicate genes have undergone convergent evolution which appears to have played an important role in the independent evolution of adaptive traits in 12 Drosophila species.

Adaptive evolution supported by functional bias of young duplicates

It is well-known that duplicate genes face three possible fates: pseudogenization, subfunctionalization and neofunctionalization. Pseudogenization is considered as the most common fate of duplicate genes [8–10], but more evidence support the models of subfunctionalization or neofunctionalization, as the mechanisms for the preservation of duplicate genes under adaptive selection [6, 15, 48, 49]. Many previous studies have shown that the duplicated genes could adapt to various conditions, in particular, genes encoding membrane or secreted proteins which are always involved in ecological stimuli or stress. For example, adaptive gene duplications have been found in response to biotic stress [50], antibiotics [51, 52], weedicides [53] or pesticides [54, 55], drugs or toxins [56], extreme temperatures [57, 58], nutrient limitation [59, 60] and symbiosis between host and parasite [61].

In this study, it was shown that the protein domains of trypsin, p450, WD40 and Pkinase in species-specific expansions, Ank, EGF, histone, HSP70 and Lys in complex expansions occurred with high frequency across the 12 Drosophila species. Interestingly, these young duplicates were also involved in different aspects of interactions with the environment. Trypsin is one of the largest families of secreted serine proteases found in the digestive system of vertebrates and invertebrates. Although it participates in many basic physiological processes [62–64], it is predominantly involved in diet and digestion. The high frequency of trypsin across 12 Drosophila species indicated that consistent and independent duplication for adaptation to specific dietary habits was due to their diverse ecosystems [27, 29]. For example, investigations of trypsin family conducted in various genomes, such as fruit fly [65], mosquitoes [66] and leaf-eating monkey [67], have all indicated that adaption occurs in response to specific diets. In particular, researches into the rapid diversification of trypsin genes in 12 Drosophila species have provided insights into the ecological forces driving the adaptive evolution by comparing the relationship between duplications and host preference shifts [65].

Another protein domain shared between 11 Drosophila species detected in this study was cytochrome p450 (CYP). P450 comprise a superfamily of enzymes that occurs with a high degree of diversity in all organisms [68]. Among the various biological functions of p450, we focused on the oxidation of xenobiotic compounds, which facilitates their excretion from the organism [69, 70]. Abuse of insecticides has forced adaptive evolution in Drosophila over an extremely short period. A single p450 gene, Cyp6g1, is sufficient and necessary for DDT resistance [28] and its cross-resistance to a wide range of other insecticides has also been identified in Drosophila[71, 72]. Furthermore, functional divergence and positive selection detected in mammalian CYP genes, provide insights into the adaptive selection of CYPs in response to high diversity of xenobiotics [73].

Other expanded domains were also identified with roles in adaption to various ecological factors, especially stress. For example, some SAPK (stress-active protein kinases, Pkinase) mediate cellular responses to toxins and physical stress [74] and TAK1 (transforming growth factor-β-activate kinase, Pkinase) is a key regulator in response to diverse stimuli in adaptive immunity [75], ankyrin proteins (Ank) play a role in stress responses and disease resistance both in animals [76] and plants [77], histones [30] and HSP70 proteins protect cells from stress [31], and Lys proteins act as bacteriolytic enzymes by hydrolyzing cell bacterial walls [32].

These observations indicate that shared young duplications reflect adaptive evolution of the Drosophila species to global ecological pressures.

Adaptive evolution contributes to specific functional preference

In this study, although most paralogs and orthologs of these young duplicate gene families had Ka/Ks ratios lower than 1, some Ka/Ks ratios greater than 1 were also found both in species-specific and lineage-specific expansions (Figure 3), demonstrating that they were under adaptive selection. Furthermore, paralog gene pairs had higher Ka/Ks ratios than ortholog gene pairs across 12 Drosophila species. It can be concluded that the paralogs have higher frequency of adaptive evolution than the orthologs [48]. Previous research has indicated that many genes families in Drosophila are driven by adaptive selection, such as, elastase/chymotrypsin, trypsin and astacin, which are all involved in digestive processes in D. arizonae[26], two immunity-related gene families, Toll-like receptors and lysozyme in D. melanogaster[24, 78], and metallothionein genes involved in metal tolerance [79, 80]. Moreover, positive selection is a major force driving the evolution of male-specific recent duplicates on neo-X chromosome in D. pseudoobscura[17] and segmentally duplicated seminal fluid genes in D. melanogaster[81].

Functional analysis of those gene families in which paralogs had higher Ka/Ks ratios than orthologs with ratios exceeding 1 (Figure 3) showed that adaptive evolution leads to species-specific and lineage-specific functional preference for each Drosophila (Additional file 3: Table S1). Examples include the PDZ-domain containing gene family in D. sechellia, stress-inducible humoral factor Turandot (Turandot domain) in D. yakuba and the Pam16 family (Pam16 domain) in the D. pseudoobscura and D. persimilis pair. Combining the functional preference with the host preference of corresponding Drosophila species [27], it seems reasonable to infer that Drosophila evolve for adaption to a given environment [82].

Interestingly, we found that D. sechellia only distribute in the Seychelle Islands in the Indian Ocean and prefer inhabiting Morinda citrifolia, the fruit of which contain nutrients such as alkaloids. Alkaloids are widely known that play an important role in inhibiting tumors by reducing microtubules disruption during mitosis [83]. Coincidently, PDZ proteins are involved in the interaction between syntrophin-associated serine/threonine kinase and microtubule-associated serine/threonine kinases and are recognized as tumor suppressors [84, 85]. Therefore, we speculated that duplication of PDZ in D. sechellia is closely associated with adaption to this unique habitat.

The D. yakuba species in Africa exhibited propagation of Turandot proteins which are adaptively resistant to high temperature, dehydration and UV irradiation [86, 87]. In contrast, the Pam16 proteins of D. pseudoobscura and D. persimilis play important regulatory roles in recruiting heat shock protein partners and responding to cold hardening [88–90], indicating superior adaptation of these species to their specific habitats situated in the regions of higher latitude in the Northern Hemisphere compared with other species.

Furthermore, other evidence of adaptive evolution in a single species or lineage species pairs was also detected in domain analysis (Additional file 3: Table S1). For example, annexin and dynein_IC2 in D. melanogaster, which are sperm-specific proteins (annexin X and cytoplasmic dynein intermediate chain) and absent in other species of the melanogaster species subgroup, support the hypothesis that male reproductive functions are driven by selective sweep and rapid molecular evolution [91]. Another example is alpha-amylase (Amy), a digestive enzyme of the two closely related species of the 12 Drosophila, D. sechellia and D. simulans, for which genetic variation of duplicated amylase genes has been reported revealing adaptive evolution in Drosophila[92].

Consequently, adaptive evolution of Drosophila species leads to young duplicate genes exhibiting specific function preference in response to ecological factors and environmental changes.

Identification of young duplicate genes

The 12 Drosophila genome sequences and annotations were downloaded from the Flybase Datebase [http://ftp.flybase.net/genomes/] [93], and the exact version for each species is shown in Table 1 (Additional file 4). An all-versus-all Blastn search with E-value (1.0e-40) was processed across all nucleotide coding sequences (CDSs) in 12 Drosophila species, then coverage of > 60% was used to divide the genes into gene families. Subsequently, Clustalw2.0 [94] was used for the pairwise alignment of the nucleotide sequences of genes in one family and the nucleotide diversity (π) was estimated with the Jukes and Cantor correction by MEGA v5.0 [95]. Young duplicate gene families were defined based on the following two conditions: (1) the number of family members was larger than the number of corresponding species in each family; (2) the highest identity of the paralogs in each family exceeded 90%.

To further analyze young duplicate gene families, three types of expansions were defined: species-specific expansions, lineage-specific expansions and complex expansions. Here, the species-specific expansions were denoted as young duplications occurring only in one species, while other species comprised ≤ 1 member or > 2 members but with less than 90% identity between paralogs. We also defined the latter two types as the young duplications of a gene family in species with a close (lineage-specific expansion) or distant (complex expansion) genetic relationship based on the species tree of the 12 Drosophila species [27]. Based on this principle, species-specific expansions were easily identified corresponding to each species. Furthermore, we defined the following six lineage-specific expansions: D. sechellia-D. simulans, D. yakuba-D. erecta, D. pseudoobscura-D. persimilis, D. melanogaster-D. sechellia-D. simulans, D. melanogaster-D. sechellia-D. simulans-D. yakuba-D. erecta and D. melanogaster-D. sechellia-D. simulans-D. yakuba-D. erecta-D. ananassae, and 14 complex expansions (Table 1 & Additional file 6: Table S4).

Sequence alignment and phylogenetic analysis

The amino acid sequences of the duplicate genes in each family were first aligned using the MUSCLE program with default options and then manually corrected using MEGA v5.0 [95]. The alignments were used to construct phylogenetic trees with 1,000 replicates using MEGA v5.0 based on the neighbor-joining (NJ) method [96]. NJ analysis was conducted using pairwise deletion of gaps and kimura-2 model (family3093, family9588 and family7827) or p-distance (family8177). Additionally, for the two families (family21 and family2419) with numerous members, NJ trees were constructed with default parameters and 1,000 bootstrap replicates in Clustalw2.0 [94].

Physical location and structural domains of the young duplicate genes

The hotspot regions for duplication events were examined by identifying the exact physical positions of young duplicate genes across the chromosomes. Detailed genome annotation information was available for the genes of D. melanogaster only. Thus, we performed Blast analysis using the CDSs of young duplicate genes in the other 11 genomes against the genome sequences of D. melanogaster to gain the position information.

We utilized the position information to process stochastic simulations with 10,000 random repeats by PERL script. Each chromosome was divided into a number of windows (1Mb/1 Window). We incorporated all genes of corresponding species into the given window of each chromosome for the species-specific duplicate gene families, and an identical approach was taken for the families of lineage-specific expansions and complex expansions. All young duplicate genes identified in this study were further examined for structural domains using the Pfam database [Pfam database 26.0, http://pfam.sanger.ac.uk/] [97] with E-value 1.0.

Calculation of nonsynonymous to synonymous ratio and estimation of duplication time of paralogs

CDSs in each young duplicate gene family were aligned according to alignments of protein sequences in Clustalw2.0 [94]. Subsequently, the nonsynonymous substitutions (Ka), synonymous substitutions (Ks) and ratio of nonsynonymous to synonymous substitutions (Ka/Ks) were calculated for paralog and ortholog pairs in each duplicate gene family using MEGA v5.0 [95].

The mean Ks values were calculated for paralog pairs in each species-specific duplicated gene family and then used to estimate the timing of duplications. The calculations were performed using a single nucleotide mutation rate of 5.8 × 10^-9 mutations per generation and it was assumed that Drosophila species experienced approximately 10 generations/year [37].