BACKGROUND
gene duplication is one of the dominant driving forces in adaptive evolution of genome and genetic systems  <cit> .
duplicate genes are considered to be the raw materials and the primary mechanism for generation of novel gene functions  <cit> .
at least 15% of genes in human genome and 8% to 20% of drosophila melanogaster, caenorhabditis elegans, and saccharomyces cervisiae genomes are thought to arise from gene duplications  <cit> .
young duplicate genes will ultimately suffer one of three long-term fates:  one copy may lose gene function by nonfunctionalization/pseudogenization;  one copy may evolve a new beneficial function by means of neofunctionalization and the other retain the old; or  both duplicated copies may stably be maintained with daughter copy partitioning of ancestral gene function by subfunctionalization  <cit> .
many models have been proposed that pseudogenization could be the most common fate of duplicated genes  <cit> .
in addition, evidences for adaptive evolution of pseudogenes have been reported in many organisms, such as pseudogenes in  <dig> arabidopsis accessions  <cit>  and the rcsa gene in yersinia pestis <cit> .
similarly, the preservation of duplicated genes might be a by-product of neutral evolution  <cit> , or might be adaptive substitutions during or after fixation of duplicates  <cit> , indicating that selection for neofunctionalization is the mechanism to keep them  <cit> .
previous studies conducted in many organisms have widely reported that duplicate genes undergo adaptive evolution.
at the genome-wide level, the signatures of adaptive natural selection of young gene duplicates are found with high frequency in the human, macaque, mouse and rat genomes  <cit> .
furthermore, gene duplicates from drosophila pseudoobscura neo-x chromosome  <cit>  and a group of digestive protease encoding genes that are associated with recent, lineage-specific duplications in drosophila arizonae <cit>  are detected under adaptive evolution.
researches have been focused almost either on recent duplication events occurring in humans or other mammals involved in human diseases  <cit>  or on the duplication and adaptive evolution of single gene families, such as chalcone synthase genes and mads-box genes in plants  <cit> , fatty acid biosynthesis genes in bacteria  <cit> , or toll-like receptor genes in drosophila <cit> .
although gene gain and loss is estimated with a drosophila-wide perspective  <cit> , a systematic investigation of the genetic character and evolutionary pattern of young duplicate genes across the closely related drosophila species has not been reported.
sequencing of the genomes of the  <dig> worldwide drosophila species   <cit>  provides the opportunity to reveal the evolutionary genetics of recent duplications.
these species capture a range of evolutionary distances: closely related sister-species, such as d. simulans and d. sechellia or d. pseudoobscura and d. persimilis; distantly related species classified into different subgenera, such as sophophora and drosophila.
there are also cosmopolitan species such as d. melanogaster and d. simulans or highly restricted species such as d. sechellia, distributed in some specific geographic ranges  <cit> .
additionally, the diverse host preferences provide a way to connect recent duplications with ecological and environmental factors.
in this work, we conducted a genome-wide investigation of young duplicate genes across  <dig> drosophila species to uncover their evolutionary patterns.
RESULTS
young duplicate genes in  <dig> drosophila genomes
across the  <dig> drosophila genomes, a total of  <dig>  gene families were detected, including  <dig>  young duplicate gene families , suggesting that approximately  <dig> % of the total gene families included young duplicates.
in these young duplicate gene families, three types were defined based on their expansion patterns: species-specific expansions, lineage-specific expansions and complex expansions.
the species-specific young duplicate gene families clearly predominated  over the other two types of expansions.
on the other hand, uneven distribution of the species-specific young duplicate genes among different species, ranging from  <dig> to  <dig>  was also observed.
for example, d. melanogaster had the least family number , while the highest three values were found in d. willistoni , d. yakuba  and d. grimshawi , respectively .
this uneven distribution of the young duplicate genes was also found in the lineage-specific expansions and the complex expansions.
for example,  <dig> duplicate gene families were detected in lineages of d. persimilis and d. pseudobsura, which is approximately  <dig> - and  <dig> -fold greater than that in lineages of d. erecta and d. yakuba or d. sechellia and d. simulans, respectively.
also as expected, if there are more species  in a group of lineage-specific and complex expansions, fewer duplicate gene families were detected.
a corresponding species is involved in the relevant expansion events.
b corresponding species is not involved in the relevant expansion events.
c not all expansion events are showed because of space limitation.
interestingly, three young duplicate gene families were detected respectively in complex expansions occurring in  <dig> and  <dig> species .
although the six families were classified as the complex expansion type, species-specific duplication events also were found in all of these families , especially independent duplications after the species differentiation in many species.
for example,  <dig>   <dig> and  <dig> species-specific duplicate clades across all of the  <dig> species were detected in family  <dig> ,  <dig>  and  <dig> , respectively.
in the other  <dig> families,  <dig>   <dig> and  <dig> species-specific duplicate clades were distributed in  <dig>   <dig> and  <dig> 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  <dig>  times random repeats.
the chromosomal distribution was significantly non-random ; for example, chromosome  <dig> ,  <dig>  and x contained a mass of young duplicate genes .
figure  <dig> shows graphs representing these simulation results.
furthermore, the windows in figure  <dig> 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  <dig>  hotspot regions were found in all three types of young duplications, especially in species-specific expansions , e.g.
on chromosome  <dig> in d. grimshawi and on chromosome  <dig> 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 , 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 .
in complex expansions, few hotspot regions were detected along the chromosomes .
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  <dig>  different domains were found in  <dig> species, averaging  <dig> protein domains in each species .
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 duf <dig> in d. willistoni, annexin and dynein_ic <dig> in d. melanogaster, inositol_p and pap <dig> in d. yakuba, were high in one species but low  across the other  <dig> 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 ≥  <dig> species.
prominent examples of these protein domains were trypsin, p <dig> and wd <dig>  which were detected in  <dig>   <dig> and  <dig> species, respectively .
these proteins are all important in response to environmental stimuli  <cit> .
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  <dig> domains in  <dig> genomes.
interestingly, these high-frequency domains also had a large number of copies in the related species , 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  <dig> and  <dig> species .
furthermore, ank, egf, peptidase_m <dig> and peptidase_m17_n, which were all conserved and widespread domains in organisms for survival, exhibited high frequencies in  <dig> species .
in the shared expansion events of  <dig> species ,  <dig> of the top  <dig> protein domains such as histones, hsp <dig>  lys, co-occurred in  <dig> 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  <cit> , hsp <dig> protects cells from stress  <cit> , and lys  acts as a bacteriolytic enzyme by hydrolyzing bacterial cell walls  <cit> , 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  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  <dig> 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  <dig>  and  <dig> , respectively, which was significantly  larger than the genome-wide ka/ks  between ortholog pairs, suggesting relaxation of the functional constraints in the young duplicate gene families.
figure  <dig> shows that most of the gene pairs , including paralogs and orthologs, had ka/ks ratios less than  <dig>  demonstrating that most young duplicate genes were under purifying selection.
however, there were still  <dig> and  <dig> gene pairs with ka/ks ratios greater than  <dig> 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  <dig>  many values were just slightly greater than  <dig> and only few pairs were detected to have ka/ks ratios significantly greater than  <dig>
based on the strengths of boxes and whisker lines in species-specific expansions in figure  <dig>  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 , 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  or significant  differences .
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 .
in the same family, the dot above the trend lines  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  showed that most of the domains detected in the genes of upper dots, such as coesterase  <cit> , turandot  <cit>  and mip  <cit>  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  <dig> 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  <dig> generations/year and that the single nucleotide mutation rate was  <dig>  ×10- <dig> mutations per generation  <cit> .
furthermore, only ks values lower than  <dig>  were kept to avoid the saturation of nucleotide substitutions.
on the whole, the young duplication events occurred over a short span of time .
the duplication time of species-specific expansions fell in a range from  <dig>  mya  to  <dig>  mya   , indicating that all the species-specific expansions occurred within a short time.
moreover, most of our estimates of duplication time were less than the species divergence time reported by tamura  <cit> .
however, the divergence time of several closely related species previously reported, including d. simulans vs. d. sechellia  and d. pseudoobscura vs. d. persimilis  was slightly lower than their respective family duplication times .
similarly, higher duplication times in the four species were also found in lineage-specific expansions and complex expansions.
moreover, the lowest standard deviations of the duplication time were detected between these lineage species in lineage-specific expansions, which suggested that closely related species duplicated in close periods, especially the species pair d. persimilis vs. d. pseudoobscura .
in the six lineage species, there was a group of duplication times with more compact distribution and smaller values than those in species-specific expansions, which indicated that the expansion occurred over a more concentrated and closer period in lineage-specific expansions than in species-specific expansions.
it was clear that less species and closer relationships caused such results.
finally, although the highest standard deviations were found in complex expansions, especially in  <dig> species with a broader range for duplication time  and a larger standard deviation  than those in others, their distributions of duplication time were still in relative compact period.
this demonstrated that these duplicated genes in complex expansions might appear at relatively scattered time compared with duplicates in the other two types.
therefore, we might infer that drosophila species have consistently duplicated to adapt to environmental changes.
discussion
convergent evolution of young duplicate genes across the  <dig> 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  <cit> , such as, the specialized oxygen transport function of oxygen transport hemoglobins in jawed and jawless vertebrates  <cit>  and the similar substrate of apolipoprotein  in humans and hedgehogs  <cit> .
although there are many other theories could explain the evolutionary process of young duplicates, such as genomic drift proposed by nei  <cit> , convergent evolution might be more convincible for two evidences detected in our study.
in our study, the phylogenetic trees  and the chromosomal distributions  of young duplicate genes also provide evidence of convergent evolution.
six young duplicate families were found in complex expansions occurring in  <dig> or  <dig> species with many species-specific duplication clades across these  <dig> or  <dig> 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  <cit>  and independent convergent evolution has produced striking similarities between plant and animal histones  <cit> .
another example of similar genetic characteristics shared by distant species is the digestion function of lysozymes  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  <cit> .
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 <cit> .
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  <dig> 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  <cit> , but more evidence support the models of subfunctionalization or neofunctionalization, as the mechanisms for the preservation of duplicate genes under adaptive selection  <cit> .
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  <cit> , antibiotics  <cit> , weedicides  <cit>  or pesticides  <cit> , drugs or toxins  <cit> , extreme temperatures  <cit> , nutrient limitation  <cit>  and symbiosis between host and parasite  <cit> .
in this study, it was shown that the protein domains of trypsin, p <dig>  wd <dig> and pkinase in species-specific expansions, ank, egf, histone, hsp <dig> and lys in complex expansions occurred with high frequency across the  <dig> 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  <cit> , it is predominantly involved in diet and digestion.
the high frequency of trypsin across  <dig> drosophila species indicated that consistent and independent duplication for adaptation to specific dietary habits was due to their diverse ecosystems  <cit> .
for example, investigations of trypsin family conducted in various genomes, such as fruit fly  <cit> , mosquitoes  <cit>  and leaf-eating monkey  <cit> , have all indicated that adaption occurs in response to specific diets.
in particular, researches into the rapid diversification of trypsin genes in  <dig> drosophila species have provided insights into the ecological forces driving the adaptive evolution by comparing the relationship between duplications and host preference shifts  <cit> .
another protein domain shared between  <dig> drosophila species detected in this study was cytochrome p <dig> .
p <dig> comprise a superfamily of enzymes that occurs with a high degree of diversity in all organisms  <cit> .
among the various biological functions of p <dig>  we focused on the oxidation of xenobiotic compounds, which facilitates their excretion from the organism  <cit> .
abuse of insecticides has forced adaptive evolution in drosophila over an extremely short period.
a single p <dig> gene, cyp6g <dig>  is sufficient and necessary for ddt resistance  <cit>  and its cross-resistance to a wide range of other insecticides has also been identified in drosophila <cit> .
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  <cit> .
other expanded domains were also identified with roles in adaption to various ecological factors, especially stress.
for example, some sapk  mediate cellular responses to toxins and physical stress  <cit>  and tak <dig>  is a key regulator in response to diverse stimuli in adaptive immunity  <cit> , ankyrin proteins  play a role in stress responses and disease resistance both in animals  <cit>  and plants  <cit> , histones  <cit>  and hsp <dig> proteins protect cells from stress  <cit> , and lys proteins act as bacteriolytic enzymes by hydrolyzing cell bacterial walls  <cit> .
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  <dig>  some ka/ks ratios greater than  <dig> were also found both in species-specific and lineage-specific expansions , demonstrating that they were under adaptive selection.
furthermore, paralog gene pairs had higher ka/ks ratios than ortholog gene pairs across  <dig> drosophila species.
it can be concluded that the paralogs have higher frequency of adaptive evolution than the orthologs  <cit> .
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 <cit> , two immunity-related gene families, toll-like receptors and lysozyme in d. melanogaster <cit> , and metallothionein genes involved in metal tolerance  <cit> .
moreover, positive selection is a major force driving the evolution of male-specific recent duplicates on neo-x chromosome in d. pseudoobscura <cit>  and segmentally duplicated seminal fluid genes in d. melanogaster <cit> .
functional analysis of those gene families in which paralogs had higher ka/ks ratios than orthologs with ratios exceeding  <dig>  showed that adaptive evolution leads to species-specific and lineage-specific functional preference for each drosophila .
examples include the pdz-domain containing gene family in d. sechellia, stress-inducible humoral factor turandot  in d. yakuba and the pam <dig> family  in the d. pseudoobscura and d. persimilis pair.
combining the functional preference with the host preference of corresponding drosophila species  <cit> , it seems reasonable to infer that drosophila evolve for adaption to a given environment  <cit> .
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  <cit> .
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  <cit> .
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  <cit> .
in contrast, the pam <dig> proteins of d. pseudoobscura and d. persimilis play important regulatory roles in recruiting heat shock protein partners and responding to cold hardening  <cit> , 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 .
for example, annexin and dynein_ic <dig> in d. melanogaster, which are sperm-specific proteins  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  <cit> .
another example is alpha-amylase , a digestive enzyme of the two closely related species of the  <dig> drosophila, d. sechellia and d. simulans, for which genetic variation of duplicated amylase genes has been reported revealing adaptive evolution in drosophila <cit> .
consequently, adaptive evolution of drosophila species leads to young duplicate genes exhibiting specific function preference in response to ecological factors and environmental changes.
CONCLUSIONS
we identified  <dig>  young duplicate gene families across  <dig> drosophila species and detected three types of expansions in these gene families: species-specific, lineage-specific and complex expansions.
we found that the species-specific young duplicate genes predominated  over the other two types.
furthermore, we observed that, in the same gene family, independent species-specific expansions occurred in many species, even including  <dig> or  <dig> drosophila species, suggesting that these young duplicate genes were under convergent evolution after the drosophila species differentiation.
we also found that the functional preference of the young duplicate genes was mainly related to responses to environmental stimuli and biotic stresses, suggesting that adaptive evolution may play an important role in duplicate genes for adaption to ecological factors in drosophila species.
this work may help us to better understand the evolutionary patterns of young duplicate genes across  <dig> drosophila species.

 gene duplication is one of the dominant driving forces in adaptive evolution of genome and genetic systems. duplicate genes are considered to be the raw materials and the primary mechanism for generation of novel gene functions. at least 15% of genes in human genome and 8% to 20% of drosophila melanogaster, caenorhabditis elegans, and saccharomyces cervisiae genomes are thought to arise from gene duplications. young duplicate genes will ultimately suffer one of three long-term fates: one copy may lose gene function by nonfunctionalization/pseudogenization; one copy may evolve a new beneficial function by means of neofunctionalization and the other retain the old; or both duplicated copies may stably be maintained with daughter copy partitioning of ancestral gene function by subfunctionalization. many models have been proposed that pseudogenization could be the most common fate of duplicated genes. in addition, evidences for adaptive evolution of pseudogenes have been reported in many organisms, such as pseudogenes in arabidopsis accessions and the rcsa gene in yersinia pestis. similarly, the preservation of duplicated genes might be a by-product of neutral evolution, or might be adaptive substitutions during or after fixation of duplicates, indicating that selection for neofunctionalization is the mechanism to keep them. previous studies conducted in many organisms have widely reported that duplicate genes undergo adaptive evolution. at the genome-wide level, the signatures of adaptive natural selection of young gene duplicates are found with high frequency in the human, macaque, mouse and rat genomes. furthermore, gene duplicates from drosophila pseudoobscura neo-x chromosome and a group of digestive protease encoding genes that are associated with recent, lineage-specific duplications in drosophila arizonae are detected under adaptive evolution. researches have been focused almost either on recent duplication events occurring in humans or other mammals involved in human diseases or on the duplication and adaptive evolution of single gene families, such as chalcone synthase genes and mads-box genes in plants, fatty acid biosynthesis genes in bacteria, or toll-like receptor genes in drosophila. although gene gain and loss is estimated with a drosophila-wide perspective, a systematic investigation of the genetic character and evolutionary pattern of young duplicate genes across the closely related drosophila species has not been reported. sequencing of the genomes of the worldwide drosophila species provides the opportunity to reveal the evolutionary genetics of recent duplications. these species capture a range of evolutionary distances: closely related sister-species, such as d. simulans and d. sechellia or d. pseudoobscura and d. persimilis; distantly related species classified into different subgenera, such as sophophora and drosophila. there are also cosmopolitan species such as d. melanogaster and d. simulans or highly restricted species such as d. sechellia, distributed in some specific geographic ranges. additionally, the diverse host preferences provide a way to connect recent duplications with ecological and environmental factors. in this work, we conducted a genome-wide investigation of young duplicate genes across drosophila species to uncover their evolutionary patterns.  young duplicate genes in drosophila genomes across the drosophila genomes, a total of gene families were detected, including young duplicate gene families, suggesting that approximately % of the total gene families included young duplicates. in these young duplicate gene families, three types were defined based on their expansion patterns: species-specific expansions, lineage-specific expansions and complex expansions. the species-specific young duplicate gene families clearly predominated over the other two types of expansions. on the other hand, uneven distribution of the species-specific young duplicate genes among different species, ranging from to was also observed. for example, d. melanogaster had the least family number, while the highest three values were found in d. willistoni, d. yakuba and d. grimshawi, respectively. this uneven distribution of the young duplicate genes was also found in the lineage-specific expansions and the complex expansions. for example, duplicate gene families were detected in lineages of d. persimilis and d. pseudobsura, which is approximately - and -fold greater than that in lineages of d. erecta and d. yakuba or d. sechellia and d. simulans, respectively. also as expected, if there are more species in a group of lineage-specific and complex expansions, fewer duplicate gene families were detected. a corresponding species is involved in the relevant expansion events. b corresponding species is not involved in the relevant expansion events. c not all expansion events are showed because of space limitation. interestingly, three young duplicate gene families were detected respectively in complex expansions occurring in and species. although the six families were classified as the complex expansion type, species-specific duplication events also were found in all of these families, especially independent duplications after the species differentiation in many species. for example, and species-specific duplicate clades across all of the species were detected in family, and, respectively. in the other families, and species-specific duplicate clades were distributed in and 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 times random repeats. the chromosomal distribution was significantly non-random; for example, chromosome, and x contained a mass of young duplicate genes. figure  shows graphs representing these simulation results. furthermore, the windows in figure  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  hotspot regions were found in all three types of young duplications, especially in species-specific expansions, e.g. on chromosome in d. grimshawi and on chromosome 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, 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. in complex expansions, few hotspot regions were detected along the chromosomes. 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 different domains were found in species, averaging protein domains in each species. 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 duf in d. willistoni, annexin and dynein_ic in d. melanogaster, inositol_p and pap in d. yakuba, were high in one species but low across the other 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 ≥ species. prominent examples of these protein domains were trypsin, p and wd which were detected in and species, respectively. these proteins are all important in response to environmental stimuli. 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 domains in genomes. interestingly, these high-frequency domains also had a large number of copies in the related species, 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 and species. furthermore, ank, egf, peptidase_m and peptidase_m17_n, which were all conserved and widespread domains in organisms for survival, exhibited high frequencies in species. in the shared expansion events of species, of the top protein domains such as histones, hsp lys, co-occurred in 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, hsp protects cells from stress, and lys acts as a bacteriolytic enzyme by hydrolyzing bacterial cell walls, 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 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 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 and, respectively, which was significantly larger than the genome-wide ka/ks between ortholog pairs, suggesting relaxation of the functional constraints in the young duplicate gene families. figure  shows that most of the gene pairs, including paralogs and orthologs, had ka/ks ratios less than demonstrating that most young duplicate genes were under purifying selection. however, there were still and gene pairs with ka/ks ratios greater than 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 many values were just slightly greater than and only few pairs were detected to have ka/ks ratios significantly greater than based on the strengths of boxes and whisker lines in species-specific expansions in figure  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, 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 or significant differences. 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. in the same family, the dot above the trend lines 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 showed that most of the domains detected in the genes of upper dots, such as coesterase, turandot and mip 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 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 generations/year and that the single nucleotide mutation rate was ×10- mutations per generation. furthermore, only ks values lower than were kept to avoid the saturation of nucleotide substitutions. on the whole, the young duplication events occurred over a short span of time. the duplication time of species-specific expansions fell in a range from mya to mya, indicating that all the species-specific expansions occurred within a short time. moreover, most of our estimates of duplication time were less than the species divergence time reported by tamura. however, the divergence time of several closely related species previously reported, including d. simulans vs. d. sechellia and d. pseudoobscura vs. d. persimilis was slightly lower than their respective family duplication times. similarly, higher duplication times in the four species were also found in lineage-specific expansions and complex expansions. moreover, the lowest standard deviations of the duplication time were detected between these lineage species in lineage-specific expansions, which suggested that closely related species duplicated in close periods, especially the species pair d. persimilis vs. d. pseudoobscura. in the six lineage species, there was a group of duplication times with more compact distribution and smaller values than those in species-specific expansions, which indicated that the expansion occurred over a more concentrated and closer period in lineage-specific expansions than in species-specific expansions. it was clear that less species and closer relationships caused such results. finally, although the highest standard deviations were found in complex expansions, especially in species with a broader range for duplication time and a larger standard deviation than those in others, their distributions of duplication time were still in relative compact period. this demonstrated that these duplicated genes in complex expansions might appear at relatively scattered time compared with duplicates in the other two types. therefore, we might infer that drosophila species have consistently duplicated to adapt to environmental changes. discussion convergent evolution of young duplicate genes across the 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, such as, the specialized oxygen transport function of oxygen transport hemoglobins in jawed and jawless vertebrates and the similar substrate of apolipoprotein in humans and hedgehogs. although there are many other theories could explain the evolutionary process of young duplicates, such as genomic drift proposed by nei, convergent evolution might be more convincible for two evidences detected in our study. in our study, the phylogenetic trees and the chromosomal distributions of young duplicate genes also provide evidence of convergent evolution. six young duplicate families were found in complex expansions occurring in or species with many species-specific duplication clades across these or 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 and independent convergent evolution has produced striking similarities between plant and animal histones. another example of similar genetic characteristics shared by distant species is the digestion function of lysozymes 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. 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. 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 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, but more evidence support the models of subfunctionalization or neofunctionalization, as the mechanisms for the preservation of duplicate genes under adaptive selection. 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, antibiotics, weedicides or pesticides, drugs or toxins, extreme temperatures, nutrient limitation and symbiosis between host and parasite. in this study, it was shown that the protein domains of trypsin, p wd and pkinase in species-specific expansions, ank, egf, histone, hsp and lys in complex expansions occurred with high frequency across the 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, it is predominantly involved in diet and digestion. the high frequency of trypsin across drosophila species indicated that consistent and independent duplication for adaptation to specific dietary habits was due to their diverse ecosystems. for example, investigations of trypsin family conducted in various genomes, such as fruit fly, mosquitoes and leaf-eating monkey, have all indicated that adaption occurs in response to specific diets. in particular, researches into the rapid diversification of trypsin genes in drosophila species have provided insights into the ecological forces driving the adaptive evolution by comparing the relationship between duplications and host preference shifts. another protein domain shared between drosophila species detected in this study was cytochrome p. p comprise a superfamily of enzymes that occurs with a high degree of diversity in all organisms. among the various biological functions of p we focused on the oxidation of xenobiotic compounds, which facilitates their excretion from the organism. abuse of insecticides has forced adaptive evolution in drosophila over an extremely short period. a single p gene, cyp6g is sufficient and necessary for ddt resistance and its cross-resistance to a wide range of other insecticides has also been identified in drosophila. 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. other expanded domains were also identified with roles in adaption to various ecological factors, especially stress. for example, some sapk mediate cellular responses to toxins and physical stress and tak is a key regulator in response to diverse stimuli in adaptive immunity, ankyrin proteins play a role in stress responses and disease resistance both in animals and plants, histones and hsp proteins protect cells from stress, and lys proteins act as bacteriolytic enzymes by hydrolyzing cell bacterial walls. 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 some ka/ks ratios greater than were also found both in species-specific and lineage-specific expansions, demonstrating that they were under adaptive selection. furthermore, paralog gene pairs had higher ka/ks ratios than ortholog gene pairs across drosophila species. it can be concluded that the paralogs have higher frequency of adaptive evolution than the orthologs. 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, two immunity-related gene families, toll-like receptors and lysozyme in d. melanogaster, and metallothionein genes involved in metal tolerance. moreover, positive selection is a major force driving the evolution of male-specific recent duplicates on neo-x chromosome in d. pseudoobscura and segmentally duplicated seminal fluid genes in d. melanogaster. functional analysis of those gene families in which paralogs had higher ka/ks ratios than orthologs with ratios exceeding showed that adaptive evolution leads to species-specific and lineage-specific functional preference for each drosophila. examples include the pdz-domain containing gene family in d. sechellia, stress-inducible humoral factor turandot in d. yakuba and the pam family in the d. pseudoobscura and d. persimilis pair. combining the functional preference with the host preference of corresponding drosophila species, it seems reasonable to infer that drosophila evolve for adaption to a given environment. 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. 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. 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. in contrast, the pam proteins of d. pseudoobscura and d. persimilis play important regulatory roles in recruiting heat shock protein partners and responding to cold hardening, 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. for example, annexin and dynein_ic in d. melanogaster, which are sperm-specific proteins 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. another example is alpha-amylase, a digestive enzyme of the two closely related species of the drosophila, d. sechellia and d. simulans, for which genetic variation of duplicated amylase genes has been reported revealing adaptive evolution in drosophila. consequently, adaptive evolution of drosophila species leads to young duplicate genes exhibiting specific function preference in response to ecological factors and environmental changes.  we identified young duplicate gene families across drosophila species and detected three types of expansions in these gene families: species-specific, lineage-specific and complex expansions. we found that the species-specific young duplicate genes predominated over the other two types. furthermore, we observed that, in the same gene family, independent species-specific expansions occurred in many species, even including or drosophila species, suggesting that these young duplicate genes were under convergent evolution after the drosophila species differentiation. we also found that the functional preference of the young duplicate genes was mainly related to responses to environmental stimuli and biotic stresses, suggesting that adaptive evolution may play an important role in duplicate genes for adaption to ecological factors in drosophila species. this work may help us to better understand the evolutionary patterns of young duplicate genes across drosophila species. 
gene duplication supplies the raw materials for novel gene functions and many gene families arisen from duplication experience adaptive evolution. in our study, young duplicate gene families were identified across the drosophila species and three types of expansions, species-specific, lineage-specific and complex expansions, were detected in these gene families. our results suggest that convergent evolution acts on young duplicate genes after the species differentiation and adaptive evolution may play an important role in duplicate genes for adaption to ecological factors and environmental changes in drosophila. most studies of young duplicates have focused on mammals, especially humans, whereas reports describing their genome-wide evolutionary patterns across the closely related drosophila species are rare. this study reveals the evolutionary patterns of young duplicates across drosophila species on a genomic scale. interestingly, many independent species-specific expansions in the same gene family have been observed in many species, even including or drosophila species. our data also showed that the functional bias observed in these young duplicate genes was mainly related to responses to environmental stimuli and biotic stresses. our data showed that the species-specific young duplicate genes predominated over the other two types.
 gene duplication supplies the raw materials for novel gene functions and many gene families arisen from duplication experience adaptive evolution. most studies of young duplicates have focused on mammals, especially humans, whereas reports describing their genome-wide evolutionary patterns across the closely related drosophila species are rare. the sequenced drosophila genomes provide the opportunity to address this issue.  in our study, young duplicate gene families were identified across the drosophila species and three types of expansions, species-specific, lineage-specific and complex expansions, were detected in these gene families. our data showed that the species-specific young duplicate genes predominated over the other two types. interestingly, many independent species-specific expansions in the same gene family have been observed in many species, even including or drosophila species. our data also showed that the functional bias observed in these young duplicate genes was mainly related to responses to environmental stimuli and biotic stresses.  this study reveals the evolutionary patterns of young duplicates across drosophila species on a genomic scale. our results suggest that convergent evolution acts on young duplicate genes after the species differentiation and adaptive evolution may play an important role in duplicate genes for adaption to ecological factors and environmental changes in drosophila. young duplicationenvironmental factorconvergent evolutionadaptive evolution 